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Clin Biochem Rev. 2004; 25(Suppl): S18–S128.
PMCID: PMC1934963

Proceedings of the 10th Asian Pacific Congress of Clinical Biochemistry in conjunction with the Australasian Association of Clinical Biochemists' 42nd Annual Scientific Conference

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S1 USING POINT OF CARE TROPONIN T TESTING TO DELIVER EVIDENCE BASED CARDIAC CARE IN RURAL SOUTH AUSTRALIA.

Introduction: A major obstacle to providing evidence based cardiac care in rural and remote communities is access to cardiac marker testing in the 60 minute time frame suggested by The American Heart Association and The American College of Cardiology.

The Integrated Cardiac Assessment Regional Network (iCARnet) was established in rural South East South Australia to address the inequity of cardiac health outcomes between metropolitan and rural areas.

Introduction of POCT for Troponin was mandatory if evidence based protocols established at Flinders Medical Centre were to be implemented. A 12-month clinical audit of the iCARnet network was undertaken to determine the impact of introducing Troponin POCT.

Method: Six Hospitals in the South East South Australia were given on-site training on how to perform POCT for Troponin, use of standardised cardiac marker protocols and performing quality control.

Clinical trials have shown increased benefit with an early invasive management strategy compared with a conservative management strategy for high-risk patients (troponin positive). We measured the impact of introducing POCT for Troponin by comparing time to angiography between the iCARnet sites and a comparable control population without access to Troponin POCT.

Results: Audit results showed that the introduction of iCARnet significantly reduced time to angiography (3.2 days vs 6.3 days, p=0.00001), improving work flow. Shortening times to angiography potentially improves long-term patient outcomes.

Conclusion: Troponin POCT facilitates the practice of evidence based cardiac care in rural emergency health facilities thereby helping to address the inequity in health services between urban and rural centres, particularly if implemented as part of a network. Troponin POCT performed by nursing staff in rural hospitals has enabled improved management of patients presenting with symptoms suggestive of Acute Coronary Syndrome.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S2 POINT-OF-CARE TESTING INFORMATICS: A RELIABLE SOLUTION?

Introduction: The use of information technology (IT) in the practice of modern laboratory medicine is now a pre-requisite to deliver good quality patient care. Nearly all hospitals and laboratories are now equipped with local-area-network (LAN) connecting networked computers and printers for the timely delivery of pathology reports. Through wide-area-network (WAN), various hospital laboratories can join to form a large network laboratory for better service provision with improved turnaround time of esoteric tests. On the other hand, requests for laboratory investigation can now be made electronically through ward computer terminals in a paperless manner. With this concept of bi-directional data transfer between clinical areas and central laboratory, point-of-care testing (POCT) analysers can now be connected to central laboratory for the purposes of quality assurance, result validation and documentation leading to its successful laboratory accreditation.

Methods: Two projects on the implementation of POCT connectivity in the New Territories East Cluster Hospitals in Hong Kong including (i) blood gas analysers using Bayer Rapidpoint 400 and Abbott i-STAT1; and (ii) glucometers using Abbott Precision Cx will be presented.

Results: POCT practice in Hong Kong has advanced to an international standard. However, we have experienced numerous difficulties throughout this journey, namely, acceptance of this new concept by clinicians & nurses, use of barcode scanning for data entry, interface-programming between instruments & our existing laboratory information system, and absence of a suitable external quality assurance programme. Most importantly, there are rooms for improvement in the programme design of the central server controlling all POCT instruments; otherwise, unwanted data may be resulted unexpectedly.

Discussion: Problems related to the acceptance and use of new instrument can be solved by continuous education while those related to instrument software can be handled by local customisation using a third party programme. Suggestions to programme design of the central server can be made to instrument vendors for future software upgrade.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S3 APPLICATION OF POINT-OF-CARE TESTING IN RAPID DETECTION OF RESPIRATORY PATHOGENS

Objective: Provide accurate and rapid laboratory evidence for diagnosis of respiratory infection and help doctors to rule out SARS patients.

Methods: Detect Streptococcus group A antigen, influenza virus antigens, respiratory syncytial virus antigen, mycoplasma pneumonia antibody IgM and legionella pneumonia antigen by enzyme immunoassay. Any test can be finished in 30 minutes.

Results: 1152 samples were detected among 879 patients in the past one and half year, 124 were positive, the infected rate was 14.1%. The samples for Strep. A Ag, Flu A+B Ag, RSV Ag, Mycoplasma pneumonia Ab and L. pneumonia Ag tests were 446, 266,116,227 and 97 respectively; positive rate were 11.9%, 11.7%, 21.6%, 5.3% and 3.1%. All age group people might be infected by above pathogens, the infected rate was 20.2% during SARS period, but only 12.0% in past one year before March 2003. No patients were diagnosed as SARS among positive group.

Conclusion: Enzyme immunoassay is a rapid and useful method to detect respiratory pathogens. These assays can provide doctors with the evidence of early diagnosis and proper treatment. At the present as no simple and rapid test kits are produced to be carried on in clinical laboratories for SARS early diagnosis, above tests can help doctors to exclude the diagnosis of SARS.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S4 EFFICACY OF POINT-OF-CARE TESTING

The key objective of point-of-care testing (POCT) is the rapid delivery of a test result to the requester, such that a clinical decision can be made and some form of action taken. If POCT is to be efficacious then this sequence should lead to an improved health outcome. The outcomes may be clinical, operational and/or economic in nature.

Classically POCT was first employed in order to resolve clinical decisions in the critically ill patient. However a systematic review of the literature indicates that there are a wide range of clinical settings in which POCT can be used effectively to improve outcomes. However it is also obvious that there are only a limited number of formal outcomes studies featuring the use of POCT; thus ‘absence of evidence of effect’ cannot be equated with ‘evidence of absence of effect’!

The main utilities of POCT for which there is evidence, and which are illustrative of the ways in which POCT can be used, include (i) risk stratification of patients admitted to the Emergency Room, (ii) ‘rule in’ or ‘rule out’ of diagnoses e.g. urinary tract infection in primary care, (iii) guiding therapy in chronic diseases, (iv) assessing the effectiveness of acute interventions, (v) monitoring chronic disease management including patient compliance, (vi) patient empowerment.

The clinical outcomes delivered POCT include a reduction in disease complications, improved patient satisfaction and improved patient morbidity. The operational outcomes are associated with more rapid decision making and include better use of staff time, greater patient convenience, improved use of hospital equipment and facilities. The benefits can translate into a reduction in the use of staff and facilities as a result of a reduction in need for clinic visits, reduction in length of stay and reduced re-admission rates.

Outcomes studies are an important feature of implementing POCT.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S5 CONCENTRATED OR DILUTE SPECIMENS FOR URINE DRUG TESTING: DOES IT MATTER?

Introduction: Many laboratories identify urine specimens submitted for drug screening as normally concentrated or dilute by creatinine analysis and/or specific gravity measurements. The objectives are: to discuss urine concentration and dilution, describe urine dilution analysis in the Correctional Service of Canada (CSC) drug-testing program and to present marijuana metabolite to creatinine ratios in the assessment of chronic marijuana users.

Methods: Urine specimens must have a creatinine value <20 mg/dL and specific gravity value ≤1.003 to be considered dilute in the CSC program. Marijuana monitoring included immunoassay screening for cannabinoids and analysing a series of urine specimens from each individual (GC-MS using deuterated internal standards) and calculating the cannabinoid to creatinine ratio for unchanged THC, Δ9-THC-COOH and 11-OH- Δ9-THC

Results: From 2000–02, 7.5% of the total CSC drug testing workload were dilute specimens. Each screening-negative dilute specimen was taken through the dilution protocol with lower screening and confirmation cut-off concentrations. Over 18% of the 6,110 dilute urine specimens confirmed positive (GC-MS) for one or more drugs when taken through the dilution protocol. Excretion profiles for unchanged THC and cannabinoid metabolites vary depending on whether one accounts for urine dilution and on the cannabinoid metabolite cut-off value by GC-MS.

Conclusion: One can reduce the false-negative rate for drugs of abuse screening in urine drug testing programs by incorporating lower screening and confirmation cut-off concentrations for dilute urine specimens. The long excretion half-life of THC-COOH is a major limitation when analysing single urine specimens versus a series of specimens for cannabinoids. Calculating unchanged THC and metabolite to creatinine ratios may improve the value of screening to determine if new marijuana or hashish use has occurred.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S6 IS THE TOXICOLOGY LABORATORY MEETING TODAY’S CHALLENGES?

The traditional role of the clinical toxicology laboratory is the support of the emergency department for the diagnosis and mangement of patients suspected of drug overdose. A modern toxicology service faces new challenges. The changing pattern of drug abuse requires development and implementation of new assays, and the resources available for laboratory funding continue to diminish. Moreoever, there is increasing demand for toxicology testings from other clinical services---obstetrics, pediatrics, addiction medicine, and pyschiatry---with requirements and expectations different from those of the emergency department.

This presentation will focus on effective utilization of available laboratory resources and expertise to provide clinically useful toxicology tests to support both the needs of the emergency department and those of other clinical services. Included in the discussion will be the selection of appropriate tests and the limitations and analytical issues relating to these tests.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S7 USE OF CYCLOSPORINE IN RENAL TRANSPLANTATION PATIENTS IN HONG KONG

Introduction: Cyclosporine is a lipophilic cyclic endecapeptide that exerts its immunosuppressive action through the inhibition of calcineurin. The more recent microemulsion formulation Neoral@ has been extensively used in renal transplantation patients in Hong Kong since its launch in the mid-90s. The cost of cyclosporine usage is substantial. Diltiazem, an antihypertensive agent, has antagonistic effect on cyclosporine metabolism rendering longer systemic availability of the immunosuppressant when they are co-administered.

Methods: A randomized, placebo-controlled, double-blind clinical study was carried out in 3 local hospitals. Renal transplantation patients receiving Cyclosporine were randomized into 2 treatment groups. Patients received Diltiazem according to their body weight and were followed up for 6 months. The primary outcome was a significant reduction in drug cost, followed by reduction in Cyclosporine dosage and blood level. Outcomes were analysed by intention to treat analyses.

Results: At 6 months post randomization, Diltiazem co-administration yield an estimated average cost-saving by 15% (the 95% confidence interval of the difference was HK$600 ± 517 (or AUS$105 ± 89) with no increase in adverse effects, complications, outpatient visits, or quality of life.

Conclusions: If the co-administration with Diltiazem to the nearly 1800 renal transplant recipients in the Hospital Authority, an estimated saving of HK$14.3 million (AUS$ 2.67 million) per annum, without adverse clinical effect, may be achieved.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S8 IMPACT OF PHARMACOGENETICS ON THERAPEUTIC DRUG MONITORING

Pharmacogenetics and its application to TDM is an emerging field. Gene test guided therapy could facilitate the selection of a drug and its dosage to which the patient best responds. A future goal may be a pharmacogenetic patient card. In this respect, polymorphisms of drug receptors (e.g. β2-adrenergic receptor), drug metabolising enzymes (TPMT, CYP3A5, CYP2D6, CYP2C19, UGT1A1, NAT2) and drug transporters (MDR1) are of interest. The CYP2D6 polymorphism plays an important role for the metabolism of antipsychotic and antidepressant drugs. In ultrarapid metabolisers due to a gene amplification a standard dose of these drugs leads to low plasma concentrations and a lack of therapeutic effect. Another well-investigated example is thiopurine-S-methyltransferase (TPMT) deficiency causing azathioprine intolerance accompanied with severe myelosuppression. Testing for TPMT phenotype/ genotype has already become a useful practice for a safer management of patients requiring thiopurine drugs. In the case of MDR1 the identified polymorphisms showed no or only a small effect on dose adjusted tacrolimus or cyclosporine blood concentrations. Conflicting results have been reported on the influence of MDR1 haplotypes on cyclosporine exposure. A polymorphism in CYP3A5 gene leads to a deficiency of the enzyme and may in part contribute to interindividual differences in CYP3A dependent drug clearance. Recent investigations have shown a significant difference in dose-adjusted tacrolimus blood levels between CYP3A5 expressors compared to non-expressors. CYP3A5 expressors may require larger tacrolimus doses to maintain adequate blood concentrations. In Caucasian stable renal transplant recipients this association, however, was less expressed for dose-adjusted cyclosporine blood concentrations. The methodological spectrum for genotyping is in a state of flux and ranges from conventional PCR based methods, sophisticated molecular haplotyping procedures, color multiplex hybridization assays (LightCycler) and denaturing HPLC methods to new chip technologies (e.g. AmpliChip). Drug disposition marker genotyping will be an additional future tool for individualisation of drug dosing in order to improve the efficacy and safety of pharmacotherapy.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S9 DIABETES AND METABOLIC SYNDROME IN CAUCASIAN AND INDIGENOUS AUSTRALIANS

The diabetes pandemic involves Australian and especially indigenous Australians, with ever earlier age of onset and a rising toll of microvascular and cardiovascular complications. In patients with typical symptoms, the diagnosis is made on a single unequivocally high blood glucose level. But in asymptomatic people the proper identification of diabetes and its precursors (IGT and IFG) requires a standard GTT. In Caucasians, selective case-finding in high risk subjects is recommended. In very high prevalence indigenous groups, whole population screening is likely to be effective. Interventions can reduce the progression from IGT to frank diabetes by 58%. The huge burden of end-stage renal failure can be reduced.

In preventive/screening programs, threshold values for fasting (or random) plasma glucose, are set low at >5.5 mmol/l to maximise case-finding by follow-up GTT. A low threshold also applies in the critical illness setting in coronary or intensive care units where there is a steep risk gradient for adverse outcomes with plasma glucose >6.5–8.0 mmol/l, and marked risk reduction occurs with intervention.

The metabolic syndrome is an important precursor for diabetes but is also a powerful cardiovascular, risk factor itself. The syndrome is a ‘blind spot’ for clinicians and biochemists because of the subtlety of its components, and the diversity of its definitions (at least 4 conflicting criteria: WHO, NCEP, EGIR, AACE). All of these definitions include (1) a measure of obesity; (2) abnormal glucose levels or insulin resistance; and (3) dyslipidaemia predominantly high triglyceride (>1.7 mmol/l) and low HDL-cholesterol (<1.0 mmol/l). In the clinical biochemistry setting, the visibility of the metabolic syndrome will improve if subtle glucose and lipid disturbances are highlighted, especially when seen in combination.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S10 DIABETES AND METABOLIC SYNDROME LESSONS LEARNT FROM HONG KONG

Asia has now taken on the centre stage in this global epidemic of diabetes and obesity where China and India will be the 2 leading countries contributing to more than 30% of the world population with diabetes. There is now confirmatory evidence showing that Asians develop cardiovascular risk factors at a lower degree of central or general obesity.

Visceral fat plays a pivotal role in the development of metabolic syndrome characterised by a constellation of cardiovascular risk factors, probably through fuel competition between glucose and free fatty acids. These risk factors include insulin resistance, dyslipidemia (high triglyceride and low HDL-C), hypertension, glucose intolerance, albuminuria and increased proinflammatory and prothrombotic markers. These risk factors interact to markedly increase the risk of cardiovascular and renal complications. In this regard, Asian diabetic patients have been shown to be at particularly high risk for developing renal failure.

The pathogenesis of this complex syndrome remains elusive but emerging evidence suggests a possible neuroendocrine origin including alterations in stress-related hormones and sex steroids which interact with genetic and environmental or lifestyle factors to enhance the deposition of visceral fat and increased lipolytic activities. In this global epidemic of diabetes, Asia is doubly hit by an aging population and a growing population of obese children and adolescents which will lead to development of cardiovascular and renal complications in their early middle-age.

Despite these worrying trends, optimal management of these risk factors can substantially reduce the onset of these cardio-renal complications through early diagnosis and multidisciplinary care. Given the silent nature of these conditions, a multi-targeted, multi-disciplinary yet well-coordinated program which aims to promote public awareness and professional education as well as to create an environment to promote healthy life style is urgently needed. Besides, there is also a need to change the current health care system to facilitate the delivery of high quality evidence based diagnostic and assessment services to allow early identification of these high risk subjects for intervention and treatment.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S11 PREVALENCE OF METABOLIC SYNDROME IN INDIA

Introduction: The last two decades have seen an explosive increase in number of people with diabetes globally. The prevalence of not only type 2 -Diabetes Mellitus but also Insulin Resistance (IR) has been reported to be significantly higher in Asian Indians because of prevailing metabolic and life style risk factors. This study is a part of our ongoing project on diabetes in northern India to examine the prevalence of metabolic syndrome in Indian families.

Methods: Study included 94 Indian families comprising of 94 diabetic subjects, their 100 first-degree diabetic relatives, 166 first-degree non-diabetic relatives and 196 non-diabetic spouses of diabetics and first-degree diabetic subjects, with no genetic predisposition to type 2 diabetes. Subjects were assessed for Metabolic Syndrome using NCEP-ATP-III criteria. Insulin Resistance, Z-score, Lipid Tetrad and Free Fatty Acid levels were also evaluated.

Results: Diabetics and their first degree diabetic relatives full filled the ATP-III criteria of Metabolic Syndrome, while the prevalence of Metabolic Syndrome was 30% and 19% in first degree non diabetics and spouses respectively. When diabetic and non-diabetic first-degree relatives were clubbed together, prevalence of Metabolic Syndrome was more than 50%. IR showed a positive correlation with W:H, TG, Total Cholesterol, LDL-Cholesterol and negatively correlated with HDL-Cholesterol in diabetic and non-diabetic subjects. FFA, Lipid Tetrad and Z-score were positively associated with diabetes and CHD risk. Obesity and Hypertension were identified as important risk factors of metabolic syndrome in Indian families.

Conclusion: Hence in view of the prevalence of Metabolic Syndrome in Indian families, therapeutic and preventive strategies are required to be directed for the management of various associated metabolic and modifiable life style factors.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S12 ROLE OF THE LABORATORY IN DIABETES MANAGEMENT

Diabetes mellitus is a chronic disease associated with significant morbidity and mortality. In Singapore, diabetes is the sixth major cause of death. National Health Surveys showed that the prevalence of diabetes mellitus among adults rose from 1.9% in 1975 and 4.7% in 1984 to 8.6% in 1992.

The laboratory plays a key role in the diagnosis and management of diabetes and its complications. The Diabetes Control and Complications Trial (DCCT) and UK Prospective Diabetes Study (UKPDS) demonstrated that optimisation of glycaemic control reduces the risk of microvascular complications.

Venous plasma glucose is recommended as the sole diagnostic criterion for diagnosing diabetes mellitus and screening high-risk individuals. Use of the oral glucose tolerance test remains controversial because of its poor reproducibility. Glycosylated haemoglobin (HbA1c) is an essential diabetic monitoring tool as it quantifies mean glycaemia over the preceding 2–3 months and is a measure of risk of complications in diabetic individuals.

Diabetic individuals should be monitored for development of complications. Microalbuminuria is a marker of early diabetic nephropathy. Lipids should also be checked in diabetic individuals as the Adult Treatment Panel (ATP III) of the National Cholesterol Education Program (NCEP) recognises the status of persons with diabetes to be equivalent to that of having established coronary heart disease.

Measurement of blood ketones, insulin and C-peptide may be useful in specific circumstances. Autoimmune markers, amylin, leptin and genetic markers have been identified in diabetic individuals but their routine use is currently not recommended.

As the global prevalence of diabetes increases, the laboratory will continue to play a major role in diagnosis and management. Participation in proficiency testing is vital to ensure validity and accuracy of laboratory indices critical in diabetes care.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S13 THE FUTURE OF LABORATORY MEDICINE: UNDERSTANDING THE NEW PRESSURES

Economical and new technological pressures strongly and equally challenge the future role of Laboratory Medicine. In this context, it is essential to take a broad view of the discipline and present to the administrators and decision makers the full spectrum of activities and organizational benefits Laboratory Medicine can provide. Medical laboratory professionals need to increase their efforts to discover and define the value of information coming out of the laboratory. The key to appreciating the importance and the true impact of diagnostic testing can only be achieved if the cost aspects are considered in the wider overall context of health economics and not within the more blinkered area of purely laboratory economics, where almost by definition every test represents a cost and its value is outside the scope of the laboratory practice. Presently, there are clear examples of clinical situations where a judicious choice and use of laboratory testing can significantly reduce the overall costs of treating the patient, accompanied frequently by a better overall clinical outcome for the patient. Advances in science and technology will continue to result in the introduction of more complex, expensive, and difficult-to-interpret tests. By integrating pathophysiologic rationale and preferences of the clinicians responsible for the care of the patient with valid and up-to-date clinical research evidence, Laboratory Medicine may promote the use of this new knowledge in a timely and responsible manner, contributing to provide better care more economically and helping the hospital to become more competitive.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S14 LABORATORY DIAGNOSIS OF MALARIA

Introduction: Even though described by Hippocrates as long ago as the 5th century BC, malaria still ranks among the major health and development challenges facing some of the poorest economies. Endemic in 101 countries, malaria affects an estimated 200–300 million people per year resulting in approximately 2 million deaths. There are four species of the genus plasmodium responsible for the malarial parasite infections that commonly infect humans, P.falciparum, P.vivax, P.malariae and P.ovale. The most important of these is P.falciparum because it can be rapidly fatal and is responsible for the majority of malaria related deaths.

Methods: In recent years a number of new techniques based on the “dipstick” format, have become available for the diagnosis of malaria. Other methods include the QBC II System, Becton-Dickinson’s Quantitative Buffy Coat (QBC) method and the polymerase chain reaction (PCR) which uses a non-isotopically labelled probe following PCR amplification. Additionally antibodies to malaria can be detected using enzymatic immunoassays or immunofluorescence techniques.

Results: The antibodies to the asexual blood stages appear days to weeks after the infection and may persist for months. Although useful in survey work or for screening blood donors and reducing wastage, they are of little value in the “acute” malaria situation. The highest density of malaria occurs in countries least able to afford sophisticated and expensive diagnostic tools and the established method of examining thick and thin blood films is still regarded by WHO as the “gold standard”. These and the methods outlined above will be discussed in this presentation.

Conclusion: Despite the problems associated with drug resistance, malaria is a curable disease if identified early enough and disease management through early diagnosis and prompt treatment is fundamental to malaria control.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S15 USE OF ANTIMALARIAL DRUGS IN AUSTRALIAN TRAVELLERS PRINCIPLES AND PRACTICALITIES

Australian Travellers are venturing to malaria risk areas in greater numbers than ever before, sometimes without even realizing it, and numbers of cases in returned travelers reflect this. Worldwide, the WHO’s Roll Back Malaria program has been disappointing. Resistance of the parasite to drugs is a problem which has largely not been matched by new agents. Coupled with this is a different sort of resistance, ie from consumers who have become more wary of using malaria tablets for fear of side effects or ‘hiding the symptoms’. This talk will be a practical one, looking at the principles of using drugs for malaria chemoprophylaxis, the drugs available and the circumstances where one may be preferred over another. The drugs available for treatment are also discussed with emphasis on standby treatment in travellers, and the role of newer agents such as the artemisinins.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S16 ETHICS IN LABORATORY MEDICINE: THE IFCC ETHICS FRAMEWORK.

Introduction: The IFCC Ethics Committee aims to increase awareness among Laboratory Professionals of ethical issues, to encourage the practice of Laboratory Medicine to the highest ethical standards, to develop position papers on appropriate ethics policies issues, to provide a voice for Laboratory Medicine on ethics policies and to link Laboratory Medicine, ethics and public interest.

Methods: A hierarchy of guiding principles is proposed. These principles have been chosen from a set of universal principles likely to achieve acceptance by all stakeholders, regardless of their belief system. This framework recognizes the cultural differences between nations and provides a mechanism to address supra-national issues of universal human ethics.

Results: There are four dimensions to the proposed fundamental guiding ethical principles:

  1. Autonomy (Self-determination and informed consent)
  2. Justice (Consistency with national and international law, and consistency with major ethical belief systems)
  3. Non-maleficence (Minimize or avoid doing harm)
  4. Beneficence (Balance societal needs against individual autonomy)

Proposals or issues requiring policy development can be considered and tested against this hierarchy of guiding principles. This comparison is intended to identify and highlight issues of difference or potential concern, which can then be discussed, refined or modified. The output of the process will be the development of policy and positions consistent with the above framework, acceptable to all participating stakeholders. The first two policy projects currently underway relate to Biobanks and Sample Storage, and to Forensic DNA testing.

Conclusions: Ethics in Laboratory Medicine has already been recognized by its inclusion in ISO 15189 (“Quality Management in the Medical Laboratory”). The IFCC Ethics Framework will assist and accelerate the integration of ethics with laboratory practice.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S17 APPLICATION OF TDM & PHARMACOGENOMICS TO OPTIMIZE TRANSPLANT IMMUNOSUPPRESSION

Cyclosporine and tacrolimus are calcineurin inhibitors (Ci) used in transplantation to suppress immune response to an allograft. Sirolimus and Everolimus are inhibitors of mTOR used in transplantation to suppress immune response to an allograft by a mechanism different from calcineurin inhibitors. Virtually every patient receiving an allograft transplant is regularly administered one or more of these drugs. The T-cell target of Ci’s is calcineurin, a calcium-dependent protein that catalyzes the phosphorylation of the nuclear factor to activate T cells (NFAT), a regulator of T cell gene transcription to generate interleukins and other chemokines that stimulate T cell proliferation. mTOR is a T cell protein that facilitates the transition of a T cell from the G to S1 phase of cell division, the first step in T cell proliferation.

Uptake of cyclosporine, everolimus, sirolimus and tacrolimus by gastrointestinal epithelial cells, and transport of these drugs into T cells, is actively controlled by p-Glycoprotein (pG), a member of the ATP dependent transporter superfamily. Cytochrome P450 3A4 (Cyp3A4), the primary enzyme involved the in vivo conversion of cyclosporine, everolimus, sirolimus and tacrolimus to inactive metabolites, is in the same coding region and is closely associated with pG in most cells.

Drugs commonly prescribed in transplant that interact extensively with pG and Cyp3A4 include antiarrythmics, antidepressants, antiepileptics, antifungals, antihypertensives, antiulceratives, and cholesterol lowering drugs. Grapefruit juice and St. John’s Wort also interact with pG and Cyp3A4. Knowledge of pG and 3A4 activity status pre-transplant, including donor status if a liver transplant, body weight, and concurrent medications to be delivered immediately post transplant determine the first dose of cyclosporine, everolimus, sirolimus and/or tacrolimus. Dose adjustments are then made based on blood concentration of cyclosporine, everolimus, sirolimus and/or tacrolimus. This presentation will review the algorithms commonly used for dose adjustments to achieve optimal outcome.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S18 LABORATORY MEDICINE TRAINING IN THE NEXT DECADE.

Introduction: During the last few decades there have been periodic concerns about the future of laboratory medicine. These have included a shortage of positions for graduates, too many pathologists or not enough pathologists. There have been many articles and meetings about the need for new skills in clinical pathology and how to avoid creating more “dinosaurs”.

Methods: Data about residency applications, job opportunities, laboratory budgets and the demographics of laboratory medicine professionals was obtained to help predict how training efforts should be focused in the future.

Results: Data indicates that there is a shortage of pathologists and clinical laboratory scientists in the U.S. that is likely to get worse. The median age of pathologists is > 55, yet the number of residency and other training positions is down. Additional evidence for the shortage of pathologists is the current high average number of job offers (over 4) for residents looking for positions. Simultaneously, the demands on laboratory medicine continue to increase. The increase in knowledge following the completion of the human genome project and related discoveries will challenge laboratory medicine with many more potential new tests and markers than ever before.

Conclusions: Obviously, we have a problem. We must address recruitment of young medical students and scientists to our field and assure that training is appropriate for the new challenges facing us. Approaches to refocusing training efforts in laboratory medicine include mirroring the changes in areas of specialization of academic clinical pathologists and changes in laboratory budget spending over the last decade. These two indicators, area of specialization and where the money goes, should provide us with directions toward training future clinical laboratory pathologists and scientists if we can recruit them!

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S19 EXPEDITING NEW TECHNOLOGIES AND PRODUCTS FROM THE BENCH TO THE BEDSIDE

We are at the threshold of great change in laboratory medicine. New enabling technologies are probing the genetic basis of disease. The focus is moving to disease prevention and targeted therapeutics. The important link between diagnostic tests and targeted therapies will grow. The value of diagnostic testing will be recognized for its significant role in disease prognosis and targeted therapies as well as disease confirmation, screening and monitoring. It is again a time of innovation. Clearly, it is just the beginning of the post-genomic era. In the near future, molecular-based tests will be introduced at an increasing rate. Not all will be practical or fit the criteria that will emerge for routine use but some have predicted that the number of diagnostic tests available to the clinician could grow from the thousand or so we have today to tens of thousands. Many new technologies and clinical products will result from the fundamental research associated with genomics and proteomics. With new technologies, tests will analyze complex protein systems rather than single proteins. There will be a need to catalog and characterize these proteins, compare variations in expression levels under different conditions, and study their interactions and identify their functional roles. Moving new technologies and products from the research bench to the clinical laboratory is a significant challenge. There are many factors that influence this process including regulatory, reimbursement and clinical utility as well as manufacturability and product performance. We will discuss these factors and the activities that the IVD industry and the laboratory community are using in the hopes of expediting the timeline of Bench to Bedside.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S20 STANDARDIZATION OF CLINICAL LABORATORIES IN CHINA

Introduction: After 1980, the Chinese government began standards work in health-care and set up the Chinese Committee of Clinical Laboratory Standards(CCCLS) in 1997. Since then, CCCLS has organized a group of experts to develop standard documents and actively participate in the international standards work on clinical laboratories.

Result: The first Session of CCCLS (1997–2002) have developed 28 standards, in which 20 standards were approved by Chinese government as National or Professional standards. The second Session (2002-) will not only continue and strengthen the efforts to developed standards, but will also help health authorities to set up reference systems in Clinical Laboratories in China. CCCLS has finished the translation of ISO documents (ISO15193, 15194, 15195 and ISO 17511, 18153) into Chinese and offered to the government as the candidates of National Standards. Recently, three task force groups have been set up to establish the reference systems on Blood Counting, Enzymatic Activity Concentration Analysis, and Blood Lipid Analysis. We hope that in the future 5 years there may be around 10 Reference Laboratories in China and some of them could participate in the International Reference Laboratory Network. We are pleased to announce that the National Centre for Clinical Laboratory has been accepted as the member by the Lipid International Laboratory Network.

Standardization in clinical laboratories is an international trend and activity. CCCLS would like to cooperate with International and Foreign Standards Organizations, for example, CCCLS is the active member of NCCLS and has translated some NCCLS documents into Chinese with the permission of NCCLS. We hope APFCB will take an important role in the development of standard documents in Asian-Pacific Area.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S21 FOLLOW UP ANALYSIS OF LABORATORY EXAMINATION IN 77 MEDICAL STAFF SUFFERING FROM SEVERE ACUTE RESPIRATORY SYNDROME (SARS)

Introduction: To study the results of serum laboratory examinations in 77 medical staff obtained severe acute respiratory syndrome(SARS) from hospital infection.

Methods: Routine blood test by STKS, lymphocyte subtypes by cytometry. 20 biochemistry markers by automatic biochemistry analyzer. Anti SARS-IgG by EIA. The patients were followed up 4 times. 120 normal medical staff were used as controls. Statistic method used one-way ANOVA(LSD).

Results: During the early period of SARS, the count of WBC and lymphocyte were significantly different. On the third month there were some abnormal rate of Lymphocyte subtypes in patients but there was no statistical significance. Some biochemistry markers(ALT, AST, LDH,γ-GT) increased during the early period of SARS, After the 3rd month they recovered and stable until 6th month. Glucose increased after one and a half months of SARS. In 4 times anti SARS-IgG was positive since the third month and still stable in 6th month. In controls that there was 1.67% positive.

Conclusion: WBC count and lymphocyte count in SARS early period there was value of helpful diagnosis. After 3 month suffering from SARS lymphocyte subtypes count there were not statistics Significance. In early period of SARS glucose metabolic was infected and liver was damaged. In all patients anti SARS-IgG was detected during the third month with SARS and still measurable in 6 months. 2 cases with weak positive anti SARS-IgG showed that recessive infection with SARS maybe exist.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S22 LABORATORY DIAGNOSIS OF AUTOIMMUNE LIVER DISEASES IN CHINA

Introduction: Autoimmune liver diseases could be classified as autoimmune hepatitis(AIH), primary biliary cirrhosis(PBC), and primary sclerosing cholangitis(PSC). Three subgroups also exist in autoimmune hepatitis. All these groups and subgroups are defined by the appearance of specific autoantibodies, i.e. Antinuclear antibody(ANA), Anti-smooth muscle antibody(ASMA), antibody against liver-kidney microsome(LKM), antibody against soluble liver antigen(SLA) for AIH, anti-mitochondrial antibody for PBC, and Anti-neutrophil cytoplasma antibody(ANCA) for PSC.

Methods: 5011 persons for annual physical examination were screened for autoantibodies associated with autoimmune liver diseases by indirect immunofluorescence, Western blot and ELISA. PBC specific M2 antibodies against immunodominant lipoy domains of PDC-E2, BCOADC-E2 and OGDC-E2 were assayed on AMA positive samples. The diagnosis of autoimmune liver disease for persons with high titer of autoantibodies and abnormal liver functions were confirmed by biopsy.

Results: the numbers of sera positive for ANA, ASMA, LKM, SLA, M2 and ANCA were 251, 126, 5, 104, 8 and 6. If plus abnormal liver functions, the numbers were changed to 69, 38, 2, 32, 4 and 1. The number of patients with AIH, PBC and PSC, after confirmed with liver biopsy were 21, 3 and 0.

Conclusions: Symptomless autoimmune liver disease is much more common in Chinese population than it has been supposed. The incidence of autoimmune liver diseases is estimated to be 4789 per million.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S23 PREVENTION AND TREATMENT OF HIV INFECTION IN CHINA

Introduction: There have been 840,000 known HIV-carriers, of whom 80,000 have developed full-blown AIDS since the first AIDS case in China was identified in 1985. HIV has gradually spread from groups with high-risk behaviors to the general population, and the effect of HIV infection has appeared on Chinese society.

Methods: To address the rapid spread of HIV/AIDS, the Chinese government developed and approved a number of strategies including the “China Medium & Long-term Plan on HIV/AIDS Prevention and Control (1998–2010)”, and “China HIV/AIDS Prevention and Control Action Plan (2001–2005)”. Government actions include advancing the work of prevention of and intervention into HIV, strengthening treatment and care of HIV infected patients through the “China CARES” project, and actively pursuing the effective management of HIV through efforts of the State and general society.

Results: The central government have allocated 1 billion Yuan (US$8120 million) specifically for the prevention and treatment of HIV since 2001 and additionally have invested 2.25 billion Yuan (US$272 million) to improve and upgrade blood stations in China’s central and western regions. Relevant departments have “fast tracked ” the registration of local companies researching, developing and producing HIV anti-retroviral drugs and have also approved the tax-free import of these medications. By 2002, Chinese companies were producing locally-made anti-retroviral drugs. Programs in the 127 counties where the “China CARES” project will be conducted were established in 2003, and integrated projects including antiretroviral treatment clinics, care and social support services will be implemented at these sites.

Conclusions: The Chinese government are making every effort to halt the STD/AIDS epidemic and to secure the national health and development of China.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S24 MOLECULAR EPIDEMIOLOGY OF THE SEVERE ACUTE RESPIRATORY SYNDROME

The outbreak of the severe acute respiratory syndrome (SARS) in 2003 has caused major public health concern. SARS is caused by a novel coronavirus, the SARS-coronavirus (SARS-CoV). Shortly after the outbreak started in Hong Kong, The Chinese University of Hong Kong established a Molecular SARS Research Group to study the molecular aspects, including the molecular epidemiology of SARS.

Our group is the first one in Asia to publicly release the complete genomic sequence of the SARS-CoV. We have demonstrated that even at the beginning of the SARS epidemic, there were already more than one SARS-CoV strain in Hong Kong. We have characterised the molecular genetic signatures of the SARS-CoV strains responsible for the Prince of Wales Hospital and Amoy Gardens outbreaks. We have also demonstrated a molecular epidemiological link between the Amoy Gardens and the late Taiwanese outbreaks.

As part of the Chinese SARS Molecular Epidemiology Consortium, we have studied the complete evolutionary history of SARS-CoV during the early, middle and late stages of the SARS epidemic. We believe that this information will be valuable for understanding the fundamental biology of this new virus and for the prevention or prompt control of future outbreaks.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S25 SERUM LD1, BLOOD CD3+, CD4+, CD8+ & NK CELL COUNTS AS PROGNOSTIC INDICATORS FOR SEVERE ACUTE RESPIRATORY SYNDROME (SARS): IMPLICATIONS FOR PATHOPHYSIOLOGY

Introduction: The pathophysiology of SARS is at present poorly understood. Blood leukocytes and lymphocyte subsets were reported to decrease, respectively, in 47% and up to 100% of 38 patients in Beijing, while advanced age and serum LD >300U/L have been associated with adverse clinical outcomes.

Methods: A retrospective analysis was performed on 109 confirmed SARS cases in two acute hospitals in Hong Kong. Serial blood samples were collected during hospitalisation for measuring complete blood picture including total haemoglobin (Hb), leukocyte and lymphocyte counts, serum LD and its isoenzymes, blood B-lymphocyte, CD3+, CD4+, CD8+, and natural killer (NK) cell counts. Multiple ROC curve comparison was used to analyse their prognostic values.

Results: Age >60 was found to be an independent prognostic indictor for death with AUC of 0.96 but not for ICU admission (AUC = 0.61). While serum LD could only achieve AUC of 0.68 for predicting death, LD1 >80U/ L was the best biochemical prognostic indicator with AUC of 0.84. Blood CD3+, CD4+, CD8+, and NK cell counts were promising immunological prognostic indicators for predicting ICU admission with AUC of 0.94, 0.91, 0.93, and 0.87, respectively.

Discussion: Apart from age, serum LD1 was the best prognostic indicator for predicting death compared to serum LD, Hb, leukocyte and lymphocyte counts. Its release could be originated from body tissues other than the myocardium consequent to cytokine and chemokine storm. Blood CD3+, CD4+, CD8+, and NK cell counts were superior prognostic indicators for predicting ICU admission in SARS patients compared to age, leukocyte count, and LD isoenzymes. Suppressed CD3+, CD4+, CD8+, and NK cell counts were implicated in SARS pathophysiology. Patients with increased serum LD1 should be closely monitored to ensure prompt management. Preparation for ICU admission could be planned ahead for patients with suppressed lymphocyte subsets.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S26 PLASMA INFLAMMATORY CYTOKINES AND CHEMOKINES IN SEVERE ACUTE RESPIRATORY SYNDROME

Introduction: Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel coronavirus, but its immunopathological mechasnisms have not been fully elucidated.

Methods: We investigated changes in plasma T helper (Th) cell cytokines, inflammatory cytokines, and chemokines in 20 adults (aged 21 – 58 years) and 7 children (0.3 – 17.5 years) diagnosed with SARS by cytometric bead array flow cytometry.

Results: Adult SARS patients showed significant elevation of Th1 cytokine interferon (IFN)-γ, inflammatory cytokines interleukin (IL)-1, IL-6 and IL-12 for at least two weeks after disease onset (all p < 0.05), but there was no significant elevation of inflammatory cytokine tumor necrosis factor (TNF)-α, anti-inflammatory cytokine IL-10, Th1 cytokine IL-2, and Th2 cytokine IL-4 (all p > 0.05). The chemokine profile demonstrated significant elevation of neutrophil chemokine IL-8, monocyte chemoattractant protein-1 (MCP-1), and Th1 chemokine IFN-γ-inducible protein-10 (IP-10) (all p < 0.05). Corticosteroid reduced significantly IL-8, MCP-1 and IP-10 concentrations from 5 – 8 days after treatment (all p < 0.001). Paediatric SARS patients showed similar but milder changes in cytokines and chemokines.

Discussion: Our observation confirmed the activation of Th1 cell-mediated immunity and hyperinnate inflammatory response in SARS through the accumulation of monocytes/macrophages and neutrophils.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S27 MOLECULAR DIAGNOSTICS FOR SARS

The most important measure for the termination of the Severe Acute Respiratory Syndrome (SARS) epidemic is the effective isolation of individuals infected with the SARS coronavirus (SARS-CoV). To achieve this goal, we need an accurate diagnostic test to identify these patients in the early phase of their illness. Although serological tests remain the gold standard of diagnosing SARS-CoV infection, antibody titres start to increase only after 2 weeks of infection. Therefore, they are not very useful for the purpose of early identification of SARS patients.

In view of this, our group developed a quantitative reverse-transcriptase polymerase chain reaction assay for the detection of SARS-CoV RNA in plasma and serum. The advantage of quantitative measurement of SARS-CoV RNA in serum is its direct reflection of the viral load of SARS patients while the viral RNA concentration in other samples, e.g. nasopharyngeal aspirate, would be affected by other factors, including the sample collection technique. Therefore, it would be particularly useful in comparing the efficacies of different anti-viral agents.

To evaluate the accuracy of the test, forty sero-positive SARS patients were studied. The sensitivities of the test for the detection SARS-CoV RNA in samples taken on admission, on day 7 and on day 14 of illness were 75%, 75% and 42%, respectively. On the other hand, none of the samples taken from 40 non-SARS patients showed positive result. The median viral RNA concentration taken on admission was 30 times higher in those patients subsequently required intensive care unit admission (p=0.002). This has indicated that the test has a relatively high sensitivity in the first week of illness and is prognostically useful in identifying those who are more likely to deteriorate.

With this test, we evaluated the effect of steroid on viral load. We have shown that there is a delayed clearance of SARS-CoV RNA in those patients randomized to the treatment of steroids.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S28 THE IFCC GLOBAL CAMPAIGN OF DIABETES MELLITUS

The IFCC has decided to focus on the Laboratory and Diabetes Mellitus and has therefore established a task force to address this theme.

The Task Force will provide scientific evidence on how to diagnose and monitor diabetes mellitus by laboratory parameters and it will use this evidence to educate the profession.

In more detail the Task Force will:

  1. Examine the best diagnostic strategy for diagnosing diabetes mellitus.
  2. Review and study the current use of laboratory test, and educate doctors and patients in interpretation of laboratory tests used in diagnosing and monitoring of diabetes mellitus.
  3. Provide quality specifications for analytes used to diagnose and monitor diabetes mellitus.
  4. Give recommendations on the minimum criteria for use of glucometers
  5. Try to associate with other groups or persons (including manufacturers) who will co-operate on the work to be done.

The lecture will present the activities of the Task Force, and will in some detail focus on the self-measurement of blood glucose and how clinicians and patients interpret results from glucose and HbA1c measurements.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S29 TOWARDS WORLDWIDE STANDARDISATION OF HbA1C ASSAYS

HbA1c has nowadays became one of the most important parameters in the treatment of patients with diabetes mellitus. It is widely used for both therapy monitoring, as well as target setting, and serves as a risk parameter for the development of diabetic complications. However, routine assays for HbA1c are not universally standardised. Therefore, the IFCC working group on HbA1c Standardisation took an effort to come up with a reference system for HbA1c. This sytem consists of 1) definition of the analyte as 1-ß-N-deoxy-fructosyl hemoglobin, 2) development of a reference method based on peptide mapping of hemoglobin fragments after proteolytic cleavage with Glu-C and 3) preparation of calibrators for this reference method. After the completion of these tasks, a Network of Reference Laboratories for HbA1c was started. These laboratories validated the reference method and performed intercomparison studies with existing standardisation schemes for HbA1c and manufacturers of routine HbA1c assays. Nowadays, the correlation with existing HbA1c schemes are known, and the Master Equation, describing this relation fixed (Clin Chem 50;2004:166–174). For manufacturers, yearly sets of reference materials with IFCC-assigned HbA1c-values are provided, which enables the manufacturers to calibrate their assays according to the new IFCC calibration. Prelimenary decision limits for the IFCC System (ref range 3–4%, treatment goal 5%, change of therapy advised >6%) were established.

For implementation of the IFCC Refererence System for HbA1c, a clinical implementation group was started in collaboration with IDF, ADA, EASD and the JDS. It was agreed on that the IFCC Reference Method should be introduced as anchor for all existing routine assays and local standardisation schemes and that one, unique and worldwide acceptable HbA1c numbering system should be introduced in the years to come. This new system could consist of a new name for the analyte, altough the name HbA1c is still preferred, while the reporting in % or fraction is under debate. The latest status of the work of the Clinical Implementation Group will be reported.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S30 HBA1C, FROM STANDARDIZATION TO CLINICAL DIAGNOSIS

The IFCC working group on Hb A1c standardization has published a Hb A1c reference method. The diagnostic criteria for Diabetes Mellitus (DM) have been adjusted over many years. The American Diabetes Association (ADA) and World Health Organization (WHO) guidelines differ in the reliance placed on fasting or 2 hour post glucose load glucose measurements, and the Oral Glucose Tolerance Test (OGTT) load (50g ADA versus 75g WHO) used in screening for gestational DM. Using Hb A1c is not currently recommended for screening and diagnosis of DM because of its previous lack of standardization and concern that it will miss DM diagnosed using fasting plasma glucose (FPG) or the OGTT.

Most people with DM will die due to cardiovascular disease (CVD). It is also recognized that people with impaired glucose tolerance (IGT) or impaired fasting glucose (IFG) are at greater risk of developing DM and CVD. Clinical studies are needed to establish: if Hb A1c can be a sole criterion to diagnose DM or confirm a diagnosis based on FPG or the OGTT; the role of Hb A1c as a risk predictor of CVD; if Hb A1c identifies a more advanced metabolic defect than either IGT or IFG; can Hb A1c be used as a measure of regression of IGT to normal GT.

Is there clinical validity in the argument that individuals with persistently normal Hb A1c levels have little or no risk of developing DM complications over time? If such complications occur before glucose rises to diabetic levels, then is it better to prevent IGT, not diabetes, to prevent complications and does Hb A1c have a role in recognizing this? The ability to identify such clinical significance will require a total error <2% for Hb A1c measurements.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S31 URINE MICROALBUMIN MEASUREMENTS IN DIABETES

Microalbuminuria is defined as an increased excretion of albumin above the reference range for healthy nondiabetic subjects, but which is undetectable by standard urinary albumin dipstick tests. It has also been defined as a urinary albumin excretion rate between 20 and 200 microgms per minute in an overnight or 24-hour sample on at least two of three occasions within a period of 6 months. This level of excretion has been found to be equivalent to 30 to 300 mg per 24-hr or 3 to 30 mgm per mmol creatinine. Recent studies suggest that the accuracy of routine clinical assays is limited firstly between the various immunometric methods used in clinical laboratories, which have wide limits of agreement. These findings suggest the need for global standardization of urine microalbumin assays. Furthermore these routine assays do not necessarily detect immunounreactive albumin, which is common in the urine of diabetic patients. There is strong evidence that the presence of microalbuminuria is prognostic for progression of kidney disease and that the higher levels of urine albumin excretion at baseline are predictive for more severe decrease in renal function as well as a faster decline in renal function over time. Clearly the accurate and precise measurement of urine albumin excretion rates in diabetic patients has important clinical implications. Increased attention from professional bodies is required to improve the standardization of this assay.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S32 TRENDS IN MOLECULAR BIOLOGY TECHNIQUES

Genome analysis using molecular biology tools has advanced significantly post the invention of Southern blots, Northern blots and PCR. The Australian Genome Research Facility, a Major National Research Facility, has established sequencing, genotyping, mutation detection and microarrays coupled with bioinformatic capabilities in order to capitalise on the plethora of genomic sequences that are being constantly being generated. The first genome sequenced in Australia, that of Leptospira was completed in 2003 in a collaborative effort between the Department of Microbiology, Monash University and AGRF. This year, AGRF with the National Institutes of Health, USA announced a partnership to sequence the genome of a small kangaroo species, the tammar wallaby. The biology and physiology of this animal have direct impact in the diary industry as well as showing the potential to translate findings into human clinical practice. Many researchers are now utilising microarrays and real time PCR as replacements for Northern blots. It is possible to isolate RNA from tissues of interest, generate cDNA libraries, sequence the clones, generate microarrays of any size from 50 to 15,000 clones and then test them in a comparative analysis. Microarrays do not rely on any underlying hypothesis or candidate gene approach. Microarrays can identify pathways of linked processes which otherwise would take years to ascertain. Positive differences can then be validated using Real Time PCR. At the DNA level, genetic polymorphisms, such as microsatellite markers or single nucleotide polymorphisms, are being utilised in family or population bases studies. Methods to analysis these genetic differences will be described.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S33 PLASMA DNA

Recently, much interest has been focused on the biology and diagnostic applications of cell-free DNA which is present in the plasma and serum of human subjects. Our group has discovered in 1997 that during pregnancy, the unborn fetus will release its DNA into the plasma of the pregnant woman. The concentration of such circulating fetal DNA increases with a progress in gestational age and such DNA molecules can be robustly analysed for non-invasive prenatal diagnosis. Examples of such applications include prenatal fetal RhD genotyping and the prenatal exclusion of beta-thalassaemia major. In addition, our group has also explored the diagnostic applications of such circulating DNA molecules for the detection and monitoring of cancer. In particular, we have demonstrated the feasibility of this approach for nasopharyngeal and hepatocellular carcinoma detection and monitoring.

In addition to circulating DNA, we have also shown that cell-free RNA is also present in the plasma. In particular, fetal and tumour-derived RNA is present in the plasma of pregnant women and cancer patients, respectively. We believe that further development in the field of plasma RNA might allow one to carry out non-invasive gene expression pro ling of the unborn fetus and a malignant neoplasm, with important implications for the future development of molecular diagnostics.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S34 NEW HORIZONS IN PHARMACOGENETICS: FROM LINKAGE DISEQUILIBRIUM MAPPING TO POPULATIONS

A great deal of effort and expense are being expended internationally in attempts to detect and utilize genetic polymorphisms contributing to susceptibility to complex human disease. Concomitantly, the technology for detecting and scoring single nucleotide polymorphisms (SNPs) has undergone rapid development, yielding extensive catalogues of SNPs across the genome. Population-based maps of the correlations amongst SNPs (linkage disequilibrium) are now being developed with the aim to accelerate the progress of complex human gene discovery. These genomic advances coincide with an increasing recognition of the importance of very large sample sizes for studying genetic effects. Together, these new genetic and epidemiological data hold renewed promise for the identification of susceptibility genes for complex traits such as response to therapy. This seminar will review the current state of knowledge about the structure of the human genome as related to SNPs and linkage disequilibrium, discuss the potential applications of this knowledge to mapping pharmacogenetic response genes, consider the issues facing whole genome association scanning using SNPs, and address the implications for genetic epidemiological investigations of pharmacogenetic phenotypes.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S35 GENOME-WIDE SCAN OF DISEASE GENES BY ASSOCIATION ANALYSIS USING 30,000 MICROSATELLITES.

Introduction: The "human genome diversity project" is conducted by common disease gene mapping by genome-wide association analyses using polymorphic genetic markers. For this purpose, SNP (single nucleotide polymorphism) markers are now being extensively and world-widely collected, and applied to disease mapping. However, SNP is generally bi-allelic, and so polymorphism is not considered to be extensive enough to localize disease genes efficiently on the human genome. Instead, we propose to use microsatellite which displays a high degree of polymorphism in repeat number of repetitious unit and so can serve as a more useful genetic marker for genome-wide mapping.

Methods: 30,000 polymorphic microsatellites were collected based on the genomic sequence of the entire human genome and subjected to genome-wide association analysis for mapping of common disease genes.

Results: The length of linkage disequilibrium with disease locus observed for microsatellite was found to be around 100 kb by our pilot study. This suggests that collection of one microsatellite per 100 kb throughout the entire human genome is enough for genome-wide mapping. Thus, the efficient method of genome-wide mapping is to first use microsatellites markers which enable to narrow down the critical region to 100 kb and thereafter to employ SNP markers within thus determined critical region for fine mapping to identify a causative gene. Based on this strategy, we collected 30,000 polymorphic microsatellite markers (one microsatellite per 100 kb) throughout the human genome and started genome-wide mapping of several complex diseases using them. As to rheumatoid arthritis, we have found 47 candidate regions for susceptible loci using 30.000 microsatellites in the genome-wide association study. By SNP association analysis of 7 candidate regions spanning approximately 100 kb among them, 7 susceptible loci for rheumatoid arthritis have been so far identified.

Conclusions: Microsatellite is a powerful genetic marker, well suited for genome-wide association mapping of susceptible loci for common or complex diseases with multifactorial genetic basis rather than SNP.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S36 EPIDEMIOLOGY OF VIRAL HEPATITIS

Among the various forms of viral hepatitis that have been identified, hepatitis B and C have particular global significance. They are widespread and occur at high prevalence in a number of areas, but more importantly they both establish a chronic state that can lead to liver damage and life-threatening complications such as liver cancer.

Although these two viral infections share some similarities in terms of their adverse outcomes, they have very distinct epidemiological characteristics. Hepatitis B infection becomes chronic mostly in people whose exposure is perinatally or in young childhood. It also can be prevented by a highly effective prophylactic vaccine which has been available for several decades.

On the other hand, chronic hepatitis C infection arises through exposure at any age and is transmitted virtually exclusively through blood contact. The patterns of hepatitis C transmission vary substantially around the world, with some countries having epidemics primarily driven by injecting drug use while others are probably more closely related to the use of non-sterile medical injecting equipment. Given these two contrasting epidemiological patterns the impacts of prevention have also been very different. Some countries that have implemented universal hepatitis B vaccination programs have started to show successes not only in the prevention of chronic infection, but also in reductions of liver cancer rates. On the other hand the harm reduction strategies that have been so successful for HIV prevention among injecting drug users in some countries seem to have had little or no impact on hepatitis C rates, largely because these epidemics began before harm reduction strategies were put in place.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S37 DIAGNOSIS OF HEPATITIS

To date, five major types of viral hepatitis are known to exist. All five types can produce acute hepatitis; whereas hepatitis B, C and D can present as chronic hepatitis. The clinical presentations and biochemical abnormalities resulting from acute or chronic infection with different types of viral hepatitis are similar. A definitive diagnosis requires testing for specific markers. While the presence of specific IgM antibodies generally indicates an acute infection, not all types of acute viral hepatitis produce a detectable level of IgM antibodies. For instance, antibody class identification offers little help in the diagnosis of hepatitis C infection. Although, IgM antibody is a useful marker for the diagnosis of acute hepatitis B infection, IgM antibody can also be found in a proportion of patients with chronic hepatitis B infection. Since hepatitis B carriers are not at an equal risk of complication and infectivity, specific markers for defining the level of virus carriage are needed for clinical management. The small secretary viral protein, e-antigen, has been regarded as a marker for differentiating active from silent carriage. However, it is now realized that the interpretation could be complicated by the presence of mutations at the pre-core region. In recent years, molecular techniques have been used increasingly in the diagnosis of viral hepatitis. The highly sensitive viral nucleic amplification technique is particular useful for shorten the false-negative window of conventional antibody assays when applied to screening for hepatitis C virus-contaminated blood donations.

The laboratory markers used for diagnosis of different types of viral hepatitis will be presented with emphases on the norms and exceptions in interpretation, as well as on the newer developments.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S38 TREATMENT OF HEPATITIS

The recent advances in the treatment of hepatitis B and C will be presented. The selection of patients, side effects and outcome of treatment of HCV with pegylated interferon and ribavirin will be presented. Results of treatment depend on the length of time of infection (acute versus chronic), genotype of HCV, co-infection with HIV, compliance and presence of cirrhosis. The role of liver transplantation in the treatment of end-stage liver disease due to HCV and the effect of post-liver transplant pegylated interferon and ribavirin will be discussed.

Advances in the treatment of hepatitis B include the development of nucleoside and nucleotide analogues and pegylated interferon. The timing of drug therapy has a significant impact on the success of treatment. Development of viral resistance to single agent lamivudine therapy is a common and influences outcome. Liver transplantation for end-stage liver disease due to HBV is effective and has excellent results when combined with HBV immunoglobulin prophylaxis and lamivudine therapy.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S39 HEPATOCELLULAR CARCINOMA

Primary hepatocellular carcinoma (HCC) is the fifth commonest malignancy in the world and the third commonest cause of cancer related mortality. There are well-defined risk factors, in particular, chronic viral hepatitis, exposure to aflatoxin and alcohol related cirrhosis. In developing countries it tends to affect younger patients with chronic hepatitis B whereas in developed countries it most commonly affects older patients with chronic hepatitis C.

By the time patients present with symptoms the prognosis is poor, therefore there is a need for early detection. Measurement of α-fetoprotein and ultrasound, at present, represents the most cost effective method of screening and increases the chances of earlier diagnosis, curative treatment and improving patient survival. There is a great interest in identifying novel HCC diagnostic markers. With the advent of proteomics, there is now the technology available to identify serum HCC related proteins which may aid diagnosis and predict treatment outcomes.

Treatment is dictated by the stage of presentation. Small tumours can be cured either by surgical resection or transplantation, or effectively treated by percutaneous ablation. The options for more advanced disease are poor, though there is some hope that chemotherapy combined with tumour embolisation will improve survival.

The ultimate goal, however, must be prevention, including the effective use and development of vaccines against viral hepatitis and education.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S40 UNCERTAINTY OF MEASUREMENTS IN LABORATORY MEDICINE THE EC - JRC VIEW

Various ISO standards and guides address the estimation of uncertainty of measurements in laboratory medicine. ISO 17511 is dealing with metrological traceability of values assigned to calibrators and control materials of IVDs. ISO 18153 deals with the values for catalytic concentration of enzymes assigned to calibrators, ISO 15193 with the requirements and layout of reference measurement procedures and ISO 15194 with the description of reference materials, both being part of the processes laid down in ISO 17511. Traceability, achieved by an unbroken chain of comparisons, is always related to an uncertainty of the comparative measurements adding to the uncertainty of the value assigned to the higher level calibrator. Another crucial document related to the estimation of measurement uncertainty, which is linked to the ISO Guides and standards above, is the ISO Guide to the Expression of Uncertainty in Measurement (GUM). The proper implementation of the GUM concept requires relatively large efforts in order to be able to identify all possible sources of measurement uncertainty and to allow correction for systematic measurement effects, the latter also related to an uncertainty. Applying the above mentioned guides and standards to well-defined analytes in a monoparametric traceability concept (e.g. only based on amount of substance or mass) is rather straightforward and should result, if correctly applied, in comparability of measurement results of different methods for a given sample within the stated uncertainty. However, for analytes without a reference measurement system, consisting of suitable reference materials, reference measurement procedures and not supported by reference measurement laboratories, formally correct but a too simplified application of a monoparametric traceability concept will not necessarily improve measurement comparability. Such a situation may even have adverse effects on the measurement comparability although formal traceability can be demonstrated, which is under afore-mentioned conditions not being effective.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S41 UNCERTAINTY OF MEASUREMENT - KEEP IT USEFUL

ISO/IEC 17025 has been the accreditation standard for Australian medical testing laboratories since 2002, but compliance with the Uncertainty of Measurement (UM) clause was delayed until January 2004. The Australasian Association of Clinical Biochemists recently convened a UM Working Group to recommend implementation guidelines for quantitative methods. The key points are:

  • Knowledge of the UM and traceability of routine methods is an essential part of the laboratory quality system and has clinical value.
  • UM is concerned with the routine performance characteristics of the test method, and not uncertainties associated with pre- or post-analytical factors.
  • The analyte(s), measurand(s), and analytical limitations of methods, particularly immunoassays, should be identified.
  • Long-term between-batch imprecision of internal quality control results, expressed as +/−2SD or +/−2CV, is the appropriate estimate of UM of routine methods.
  • Where clinically appropriate, the UM of a method should be assessed for fitness of clinical purpose(s) against an appropriate analytical goal based on the intra-individual biological variation of the analyte.
  • If the UM of a method does not meet its analytical goal, there may be a need to identify and modify procedures within the method that significantly contribute to the UM, or to change the method.
  • For a method where test results are clinically interpreted using decision values determined by a different method, then an estimate of bias or total error should also be made, and assessed against an appropriate analytical goal.
  • Relevant UM data should be readily accessible to clinical users.
  • For some clinical situations including selected UM information with patient reports may contribute to improving patient care.
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S42 REVIEW OF THE EXTERNAL QUALITY ASSESSMENT OF CLINICAL CHEMISTRY IN INDONESIA

The program of external quality assessment of clinical chemistry in Indonesia was started in 1979 by the Indonesian Association of Clinical Chemistry in collaboration with the Department of Health. This national program initially included seven parameters, and the data processing was done by Boehringer Mannheim. Now it covers seventeen parameters and the data processing is done by ourselves. 939 clinical laboratories from all twenty-six provinces of Indonesia in the year 2003 participated in this program. Technical guidance is performed regularly by the organiser. The laboratories involved are from the government hospitals (425), private hospitals and private laboratories (514). The three major automatic instruments used by participants are Cobas Mira, Hitachi and Beckman, while the three major manual ones are Boehringer Mannheim, Eppendorf and Merck. The automated instruments comprise 27% of the total instruments used in laboratories. The three major reagents used for the determination of glucose, the test most performed by participants, are Human, Randox and Roche. In evaluating the results, 81% of participants get VIS less than 200. The acceptable target value is obtained from the consensus value based on method. The program uses variant index system from WHO and is supervised by professionals in laboratory QC. It also has a good cooperation with the Committee of Analytical Quality, IFCC. Besides the above EQA program, there is another one which is organised by the Indonesian Association of Clinical Pathologists. It was done during 1990 until 1997, and started again in 2003 with 65 laboratories around Jakarta.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S43 CLINICAL LABORATORY ACCREDITATION IN INDIA

Over the years there has been a rapid expansion in the different branches of health care services. Consumer knowledge and expectations of health care have similarly improved with increasing demands for high quality care, including laboratory services. Especially in India, the last 1½ decades have seen significant growth in laboratory services. In spite of this growth, the clinical laboratories are often faced with the problem of wide inter-laboratory variation. Towards solving this problem, in the last few years, the National Accreditation Board for Testing & Calibration Laboratories (NABL) has been creating awareness on quality assurance activities with reference to accreditation. The objectives of NABL are to provide third party assessment of quality and technical competence. Three years ago NABL established links with International Bodies – Asia Pacific Laboratory Accreditation Cooperation and International Laboratory Accreditation Cooperation. This has imparted international recognition to NABL accredited laboratories. The ISO standard 17025 will be shortly replaced by ISO 15189 which is specific for medical laboratories. Participation in EQAS is a pre-requisite towards applying for accreditation. As accreditation concept is spreading fast, the clinical laboratories in India are expected to be marching ahead in the effective implementation of QA through adequate infrastructure, adequate staff with appropriate on-going training, SOP, adequate safety and waste disposal, documentation of quality system procedures, records & technical records, validation practices, IQC and EQAS. The laboratories are encouraged to incorporate multi-control QC rules towards detecting systematic (trends and shifts) and random errors. Currently accreditation is voluntary and, to date, 40 clinical laboratories have been accredited. Another 25 laboratories are in the process of gaining accreditation. It is expected that the number of accredited laboratories will increase in the future with rising awareness among consumers, physicians and clinical laboratories. Implementation of effective quality management certainly costs, but the benefits such as improved customer service, reduced wastage, etc outweigh the cost involved. The bottom line to be kept in mind is “Quality costs. Poor quality costs more”.

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S44 CLINICAL PRACTICE IS CHANGING: IMPLICATIONS FOR LABORATORY SERVICE PROVISION

Changing Clinical Practice: There are two main drivers for changing clinical practice in the UK public expectation and government policy. The public is becoming more knowledgeable about health and medicine and many individuals are willing to take greater responsibility for their personal health. Patients seek better quality information from doctors, services that are available locally and at times that suit their availability, and quicker diagnosis and treatment. Government policy centres on modernisation of services to include devolution of clinical practice from doctors to other competent healthcare professionals, often using evidence based guidelines and protocols. The impact of national and European legislation on working practices is also relevant.

Implications for Laboratory Service Provision: Laboratory service provision must be tailored to meet the needs of clinical practice. The increasing demand for central laboratory services continues with the greatest expansion occurring from the primary care sector, which expects rapid results together with interpretation. From within hospitals the demand for central laboratory services requires that more of the full laboratory repertoire is available on a 24/7 basis. Central services are being supplemented with a variety of devolved services that deliver point of care testing (POCT). POCT is increasingly available in hospital wards, clinics and theatres and in primary care centres. In addition POCT is extending into patient-centred ‘one-stop clinics’ into pharmacies and into the patient’s home.

Conclusions: The role of the laboratory is extending to include more outreach work. This involves ensuring quality in the services provided outside the laboratory, increased clinical liaison and clinical audit, and monitoring compliance with evidence based guidelines. The consultant clinical chemist has the responsibility to manage this extension as part of the clinical team.

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S45 MANAGEMENT OF CHANGE TO THE ORGANISATION OF CLINICAL LABORATORY SERVICES: THE UK NHS VIEW

In 1999 the Department of Health in England launched a Pathology Modernisation programme which culminated in the issuing in early 2004 of new guidelines for pathology services. These require laboratories to form single managed networks across wider health economies, reprofile the workforce and reconfigure services. There is no prescriptive model, but networks are expected to serve populations in excess of 1 million. Key steps to modernising services are: integrating pathology into wider service developments, whole system redesign, decreasing inappropriate variation and making effective use of new technology. Key goals are to improve quality and efficiency and deliver national clinical care targets with a focus on patients at a local level. A national Pathology board is overseeing the changes which are being driven through the 28 Strategic Health Authorities in England. Other NHS initiatives both drive and support the changes required in the way Pathology will be delivered. These include a new career progression framework for laboratory staff, and specific action to devolve appropriate clinical and laboratory services into primary care. Embracing new Point of Care technology and evolving new roles for practitioners will be crucial. A key element of this strategy will be to encourage the provision of some of these services by the private sector. The shift to primary care is being driven by national clinical guidelines for chronic disease management which stipulate the required pathology testing and also link the pay of general practitioners to achieving the targets. The change management agenda challenges many of the traditional views and practices of NHS pathology practitioners. We are seeing network formation gathering pace with a drive to standardising systems where appropriate, and a shifting balance between consolidation and distribution of services.

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S46 CLINICAL BENCHMARKING: ARE WE MEASURING THE RIGHT OUTCOMES

Benchmarking can be used in research and service development although it is more commonly used as a management tool. Performance evaluation is a necessary part of management decision making and it can broadly be split into an assessment of process and of outcomes. It is self evident that process needs to be optimised in order to achieve good outcomes, but one does not necessarily follow from the other. Economic analysis is associated with the use of resources, productivity is associated with the amount of service produced. Efficiency is concerned with the amount of service produced in relation to the resources used and effectiveness is associated with how well the service meets the stated needs. Quality is concerned with how well the service meets the patients’ needs.

Performance management in laboratory medicine is largely focussed on ‘process’. Regular performance management typically focuses on spending and workload, against targets set at the beginning of the year. Quality standards until recently have focused almost exclusively on analytical quality. Recent measures include assessment of interpretation and turnaround times, together with responses to questionnaires sent out to clinicians. Accreditation of services, and of individuals, together with clinical governance address certain quality measures. These are still largely process orientated.

Current benchmarking activities in laboratory medicine in Australia, the UK and the US predominantly focus on process, although there is some assessment of pre- and post analytical issues, e.g. within the Q-Probes initiative. Attempts have also been made in Australia to explore financial benchmarking, but largely directed to use of resources within the laboratory service, rather then looking at the impact of the laboratory on wider clinical resource utilisation.

Should benchmarking not focus on HOW the laboratory is used?

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S47 PERFORMANCE OF JAPANESE CERTIFIED ENZYME REFERENCE MATERIALS

Standardization in patient data between laboratories in clinical enzymology is very important. In order to minimize inter-laboratory differences, establishment of a reference system, including both reference measurement method and reference material, is indispensable. While the importance of global harmonization for enzymes is steadily being recognized, a national reference system in Japan has been already established and worked out effectively. The Japan Society of Clinical Chemistry (JSCC) published in 1994 the JSCC consensus methods measured at 37°C for 6 kinds of enzymes (AST, ALT, γ-GT, ALP, CK, LD). Since then, Asahi Kasei Pharma (AKP) has developed techniques, using genetic engineering, for the mass production of human enzymes to be used as reference material candidates. Human enzymes are preferred as enzymes sourced from other mammals may not always be appropriate in terms of reactivity and isozyme composition. Beyond development of mass production techniques for individual enzymes, AKP also succeeded in stabilizing a composition that includes all six enzymes, which is practically essential for a commercial product. This multi-enzyme preparation with a BSA (bovine serum albumin) matrix was certified by JCCLS (Japanese Committee for Clinical Laboratory Standards), as a Japanese certified enzyme reference material (JCERM) and put on the market in 1997. IVD (In Vitro Diagnostics) reagent manufacturers in Japan have developed their corresponding calibrators containing these recombinant enzymes, in which the values of enzyme activity are determined by using JCERM as the primary Reference Material. As a result, inter-laboratory differences have decreased remarkably to within 3%. JCERM, recently improved from frozen liquid to lyophilized powder in form, could potentially be accepted worldwide as an international reference material given its quality and practical convenience.

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S48 CHARACTERISTICS OF RECOMBINANT HUMAN CRP AS A CANDIDATE OF REFERENCE MATERIAL FOR CRP ASSAY

Introduction: For the immunoassay of the low-grade inflammation, namely high-sensitivity assay, accurate measurement of CRP is necessary on the basis of highly qualified reference material such as recombinant human CRP.

Methods: (1).

Results: Recombinant human CRP (rCRP) was secreted into culture supernatant of E. coli by co-expressing kil gene that has a function to secrete colicin E1 outside the cell. Five g of highly purified rCRP was produced from 180 L culture supernatant by ECH-Sepharose affinity chromatography using calcium-dependent adsorption and EDTA-dependent elution. The purified rCRP was indistinguishable from the native one with respect to calcium-dependent binding ability to phosphorylcholine, electrophoretic behaviour, N-terminal amino acid, and immunochemical properties. The molecular weight of rCRP monomer was determined to be 23059.7 Da by TOF/MS analysis.

Conclusions: Recombinant human CRP has the same protein structure as native one, indicating that rCRP has the potential as a reference material and/ or calibrator of high-sensitivity CRP assay.

Appendix: Dr. Kimberly et al. (2) evaluated five proposed secondary reference materials, including two diluted CRM470, two preparations of serum-based material with rCRP added, and one serum-based material with native human CRP. They concluded that that all materials performed similarity with regard to imprecision, linearity, and parallelism.

Reference

1. Tanaka T, Horio T, Matuo Y. Secretory production of recombinant human C-reactive protein in Escherichia coli, capable of binding with phosphorylcholine, and its characterization. Biochem Biophys Res Commun. 2002;295:163–166. [PubMed]
2. Kimberly Mary M, et al. Standardization of Immunoassays for Measurement of High-Sensitivity C-Reactive Protein. Phase I: Evaluation of Secondary Reference Materials. Clin Chem. 2003;49(4):611–616. [PubMed]
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S49 IFCC TRACEABLE REFERENCE MATERIAL AND REFERENCE MEASUREMENT PROCEDURE FOR HBA1C MEASUREMENT

Reference Material: Japanese Reference system for the measurement of HbA1c is maintained by using the matrix reference material JDS Lot 2 certified by the Japan Diabetes Society (JDS) in February 2001. JDS Lot 2 was prepared to have close reactivity to patient samples with regard to both of the IFCC reference method and KO500 HPLC/JSCC Designated Comparison Method (DCM); JDS Lot 2 is deep-frozen human Hb solution instead of the previous lyophilized reference material JDS Lot 1.

Reference Laboratory: The values of JDS Lot 2 were determined by the JSCC DCM calibrated with JDS Lot 1 to keep JDS value. The measurements were performed in three DCM reference laboratories, HECTEF Standard Reference Center (Dr. Masao Umemoto), Keio University School of Medicine (Dr. Izumi Takei) and Institute of Biopathological Medicine (Dr. Tadao Hoshino). Those laboratories are also IFCC HbA1c reference laboratories.

Compatibility: IFCC values are also shown in the certificate of JDS Lot 2 (5 levels), which were assayed by the IFCC reference laboratory network.

The JDS values (Y%) are converted to IFCC values (X%) by the following equation.

Y = 0.930 X + 1.70

Conclusions: JDS Lot 2 is useful to maintain the traceability to the IFCC reference system as well as the domestic reference system, and registered a JCTLM’s higher order reference material for HbA1c.

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S50 NOVEL MATRIX REFERENCE MATERIAL FOR BLOOD GASES MEASUREMENT

Introduction: The reference material for blood gas measurement contains bovine hemoglobin, and has been prepared to achieve certain pH, pCO2 and pO2 levels when gas-liquid equilibrium is established in the ampoule.

Methods: The values were measured at a temperature of 37 degree centigrade. The pH, pCO2 and pO2 were measured according to the IFCC reference method. For traceability NIST SRM186 was used for pH, and certified reference gases traceable to Japanese national standards were used. This material is intended to be used for verification of the accuracy of blood gas analyzers and for internal and external quality assurance. This material for blood gas measurement was certified by the Committee on Blood Gases/ Electrolytes of the Japan Society of Clinical Chemistry.

Results: Characteristics of this material was composed of bovine hemoglobin and solution having an electrolyte composition similar to plasma. Its properties are as follows: Ionic strength was 160 mmol/kg by calculation based on electrolyte concentrations, Base excess was 5mmol/L by pH-pCO2 nomogram method. Total hemoglobin was 12.5 g/dL by cyanmethemoglobin method. Methemoglobin was less than 5%. Certified values and uncertainty in three levels were determined at 7.25+/−0.03, 7.376+/−0.03 and 7.534+/−0.03 for pH in level 1, 2 and 3, 66.0+/−3.0, 47.5+/−2.5 and 28.8+/−2.0 for pCO2(mmHg) in level 1, 2 and 3 and 44.4+/−2.5 and 85.6+/−3/0 for pO2(mmHg) in level 1 and 2, respectively.

This product was delivered frozen with dry ice and should be stored and used under the following conditions: storage at less than −60 C with stability up to 6 months.

Conclusions: New Japanese blood gases traceability was established by IFCC reference method.

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S51 STANDARDIZATION OF URINE α1-MICROGLOBULIN IMMUNOASSAY

Introduction: α1-microglobulin (α1-m) is a low molecular weight glycoprotein, clinically used as a urine tubular marker. We have standardized a purified material by dry weight and absorption coefficient. However, a discrepancy was recently noted in the value between assay systems that led us to organize the standardization project.

Method: Purified material supplied from Fuji Rebio and 60 pathologic urines were measured by five commercially available assay systems. Western blots was performed using polyclonal antibody (DAKO). Evaluation was performed by ISO 17511 on traceability and ISO guide 35.

Result: The heterogeneity of α1-m in the calibrator was noted on western blots, consisting of a single monomeric band in one assay, mainly monomeric with faint dimeric band in three assays, and multiple bands in the rest. In pathologic urine, the heterogeneity was also observed. In measuring serially diluted material prepared by weighing for four consecutive times over two days, imprecision was allowable in the value within an actual clinical range. The ratio of measured value against assigned value on the material became constant in each assay system showing good proportionality between the systems. The same was true in pathologic urine. When the samples were measured on calibrators assigned from the material and one assay system taken as a tentative reference assay system (Denka), the regression line was y =0.996X + 0.97 (Dade Behring), y =0.996X + 0.97 (Eiken), y =0.981X − 0.08 (Fuji Rebio), and y =0.967X + 0.61 (Tomakomai), respectively

Conclusions: Despite of molecular heterogeneity in the calibrators of each assay system and urine, standardization of urine α1-m is possible. Introduction of internationally well-defined reference material is essential for completion.

Acknowledgments

We acknowledged the companies which participated in the project very collaboratively.

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S52 OUTCOMES STUDIES IN LABORATORY MEDICINE

Introduction: Outcomes studies endeavour to quantify the effect of interventions on health or on cost of healthcare. For diagnostic interventions, test attributes that may be examined include, in addition to availability of a test, the quality or timeliness of the test result. Outcome measures include mortality, morbidity and length of stay (LOS). The randomized controlled trial is the preferred study design for interventional studies, but such studies are less common for diagnostic interventions than for therapeutic ones. Sources of bias in outcomes studies include selection, lead-time and stage-migration biases.

Methods: I reviewed published outcomes studies in laboratory medicine identified by a variety of searches of PubMed and by hand-searching of Clinical Chemistry. Illustrative studies were selected for presentation. Because of claims that rapid tests for enterovirus markedly decreased LOS for aseptic meningitis, I reviewed LOS at the University of Virginia Hospital for aseptic meningitis when PCR testing of CSF for enterovirus was (2 periods) or was not (2 periods) available.

Results: Improvement of LOS often required modification of hospital procedures other than turnaround time of the test. Most forms of screening of adults, especially of the elderly, produce limited benefit in mortality, with absolute risk reductions ranging from 0 (PSA and mammography at age 90) to 0.5% (faecal occult blood tests in very healthy women at age 70). Simulation modelling illustrated that imprecision of glucose meters markedly impairs the ability to select the appropriate dose of insulin and may increase the frequency of hypoglycaemic episodes. At the University of Virginia, positive enterovirus results led to rapid discharge, but the mean LOS for patients with aseptic meningitis decreased by less than 3 hours.

Conclusions: Outcomes studies are becoming more common in laboratory medicine and can assess the analytical quality requirements for tests but they require careful attention to principles of study design and reporting. The demonstrated benefits of laboratory testing are, like benefits of most therapies, real but modest. I suggest that there is reason to proclaim the value of many tests, but that overstating of the benefits of testing or of therapies is both unwise and unethical.

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S53 INTERVENTIONS TO MODIFY TEST UTILISATION

The inappropriately excessive requesting of laboratory investigations has been well recognised as a waste of resources for many decades. It has been estimated that the degree of over-ordering in teaching hospitals is in the order of 20%. The factors that have lead to this situation include the automation of common tests, inadequate training of medical students and junior medical staff and a lack of detailed supervision by senior medical staff. Interventions reported range from to end-user surveys of specific tests, offering rewards, imposing rules to costs paid from clinical unit budgets. A study in which book vouchers were offered as a reward resulted in only temporary changes to ordering patterns. In another study, a non-pejorative questionnaire to requesters of serum electrophoresis surveying the reasons for ordering the test led to a 50% reduction in requests over one month. This strategy could be used to educate junior staff as well as control wastage. However this test would account for less than 1% of pathology expenditure in any inpatient unit. The tests that lend themselves to restriction by minimum retesting intervals similarly account for a small proportion of the costs. The majority of costs for pathology tests in a general medical unit would be accounted for by UEC, FBC, Coagulation studies and blood group/cross matching. There is evidence that the daily repeats of the first three groups accounts for much of the wastage. It is not possible for the laboratory to discriminate between patients who do, or do not, require daily testing. Computer based ordering systems which combine flags for over-frequent testing with a display of the previous results have been shown to be effective. The ability of a clinical unit to respond to price signals of costs appears to vary between the different clinical specialties.

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S54 QUALITY ASSURANCE OF INTERPRETATIVE COMMENTS

Introduction: ‘Cases for Comment’, an interpretative exercise in Clinical Biochemistry, began distribution by email in 1997. Comments made were broken down into components, and these were scored by peer review. After 100 Cases, this developed into a formal EQAS.

Methods: The EQAS is available through a web site accessible via the home page of UK NEQAS: around 23 Cases are distributed annually. Participants have 2 weeks to make a short interpretative comment on each Case. Whole anonymised comments are then scored on a scale from −1 to +3 by assessors working independently: the mean score then enables all comments to be ranked. A summary of each Case including background, and examples of low, median, and high scoring comments is made available through the web site. Participants are given the score for their own comment and their running score over previous comments compared with all participants.

Results: The Scheme has proved popular and there are currently more than 300 individual and group participants. Initially there was some opposition to comment scoring, but this is now quite well accepted. With time, the proportion of comments receiving zero or negative scores has markedly reduced. Several of the Cases have led to further internet discussion. The Cases are widely used as an educational resource, and a book is in preparation.

Conclusions: Scoring whole comments is undoubtedly more controversial than scoring components, but avoids the subjective element inherent in component selection and tests communication as well as interpretative skills. There has been an overall improvement in the standard of comments, and several difficult areas of interpretation and of ethics have been clarified. I believe that such schemes are as vital to improvements in laboratory performance as conventional analytical QA.

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S55 THE SITUATION OF LABORATORY MANAGEMENT IN CHINA

Introduction: This study is aimed to discuss the present situation, existing problems, and relevant measures concerning the management of clinical laboratories in China.

Methods: This study analysed the problems relevant to the quality management of medical science, based on experiences of ISO in developed countries in the management of clinical laboratories.

Results: Many problems still remain unsolved concerning the quality management of clinical laboratories. However, we have been doing something and will take some new actions.

Conclusion: Only legalized and standardized management may be a strong guarantee to the quality of clinical laboratory test.

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S56 MODY-THE CHINESE EXPERIENCE

In mass screening program in Taiwan, the rate of newly identified diabetes in children and adolescents was estimated to be 12.0 per 100,000. Of these cases, 54.2 % had type-2 DM, 9.5 % had type-1 DM and 8.7 % had drug-induced diabetes. Follow-up study disclosed that no more MODY individual was found in this population.

The prevalence of MODY2 and MODY3 in early-onset diabetes is around 3% and 5–10 %, respectively, in ethnic Chinese. The distinction between MODY and early onset type-2 diabetes may be difficult in certain situations, particularly in young adults. Recently, we have identified two patients with MODY3 had missense mutations in exon 3 of the HNF-1α gene (Y218C and R272H, respectively) in a region of the protein that corresponds to a predicted DNA binding domain. The P291fsinsC mutation, accounts for 20–25% of HNF-1αmutations in Caucasians, was not found in Chinese. Thus far, there are no hot spots for HNF-1αmutations in Chinese patients.

It has been suggested that the mutation in nucleotide 3243 of the mitochondrial DNA tRNA Leu (UUR) plays a role in the pathogenesis of diabetes mellitus. The prevalence of the mitochondrial gene A3243G mutation is around 0.48–2.7 % in early onset diabetic patients in Chinese.

Conclusions: The MODY2, MODY3 and mitochondrial gene A3243G mutations are occasionally found in Chinese patients with early-onset type-2 diabetes.

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S57 FAMILIAL HYPOCALCIURIC HYPERCALCAEMIA

The differential diagnosis between parathyroid-dependent and parathyroid-independent hypercalcaemia is a relatively simple matter, relying on the measurement of PTH and renal calcium handling. However, the differential diagnosis between primary hyperparathyroidism (PHPT) and familial hypocalciuric hypercalcaemia (FHH), both being parathyroid-dependent, is more difficult. Nevertheless, the distinction is important in order to avoid unnecessary and unsuccessful parathyroid surgery in FHH.

FHH is an autosomal dominant condition, resulting from an inactivating mutation in the calcium sensing receptor (CaR). CaR being the means by which circulating blood calcium is regulated by PTH, an inactivating mutation elevates the patient’s regulatory set-point for blood calcium leading to lifelong, usually asymptomatic, hypercalcaemia. Its hallmark is avid, PTH-mediated, renal calcium conservation. Although this may be assessed by a 24 hour urinary calcium, a more convenient method is to collect blood and a second voided sample of urine after an overnight fast with avoidance of calcium supplements or thiazide diuretics. The simultaneous measurement of blood PTH and urinary calcium excretion corrected for glomerular filtration (CaE) is useful in discriminating FHH from PHPT. We have found that most cases of FHH will have a PTH ≤ 1.5 times the upper limit of normal and a CaE of < 30 μmol/L GF. When FHH is suspected, measurement of blood calcium in family members should proceed.

To confirm the diagnosis of FHH when a familial pattern of hypercalcaemia is present, mutations of the CaR should be sought. By PCR amplification and gel purification of the coding region of the CaR followed by automated DNA sequencing, 8 separate single point missense or nonsense mutations have been found in our patients. Functional characterisation of the transfected mutations have confirmed their causative role in hypercalcaemia.

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S58 MEN 1 MOLECULAR BIOLOGY AND ASSOCIATED TUMOURS

Recent research has provided valuable insights into the molecular pathogenesis and clinical aspects of Multiple Endocrine Neoplasia Type 1 (MEN 1). MEN 1 is an autosomal dominant disorder resulting from loss of function of a tumour suppressor gene located on the chromosome 11q. The gene comprises 10 exons that encode a 610 amino acid product with the characteristics of transcriptional regulator. Genetic screening for the responsible mutation is available, although for 10–15% of affected families a causal mutation is not identifiable.

The classic endocrine abnormalities associated with MEN 1 are parathyroid, pituitary and pancreatic neoplasia. However, a much broader range of endocrine and non-endocrine abnormalities can develop.

  • Primary hyperparathyroidism (>95% of patients))
  • Enteropancreatic neuroendocrine tumours (~70%)
  • Pituitary adenomas (10–50%)
  • Adrenal macronodular hyperplasia in 10–30% of patients.
  • Bronchial carcinoid tumour and malignant thymoma (3–10%)
  • Multiple lipoma (~30%)
  • Cutaneous lesions (40–90%)
  • Enteropancreatic and thymic malignancy (25–30%)
  • Premature cardiovascular disease

The clinical management of MEN 1 remains controversial. Primary hyperparathyroidism is managed by subtotal parathyroidectomy. Despite optimal surgery hyperparathyroidism recurs in most patients necessitating repeat operative treatment. Enteropancreatic neoplasia is associated with a range of secretory (gastrinoma, insulinoma) and non-functioning tumours, some of which are malignant. Surgery is usually considered for large or rapidly growing lesions and insulinoma. Malignant transformation of pancreatic tumours remains an important cause of mortality in MEN 1. Pituitary tumours are mostly prolactin secreting adenomas and “non-functioning” adenomas. Dopamine agonists are first line therapy for the prolactinoma. Pituitary adenomas may be followed conservatively, however, in MEN 1 they appear more aggressive than in sporadic disease and surgical intervention is required in some cases.

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S59 INBORN (NEURO) METABOLIC DISORDERS: INDIAN SCENARIO

Introduction: Predilection of developing brain to metabolic insults due to Inborn Metabolic Disorders (IMDs) renders it mandatory for early diagnosis and medical intervention. IMDs, often with overlapping clinical symptoms, make it both a diagnostic and therapeutic challenge. The present paper is about our experience in diagnosis of IMDs in a neuropsychiatric set up and also the overview of IMDs in Indian scenario. Question being, what is the pattern and how serious is IMDs in health scenario of Indian subcontinent and what can be done.

Methods: Patients attending neuro-psychiatric clinics at our centre and outside referrals constituted the subjects for study. Laboratory investigations comprised screening tests for abnormal metabolites in urine and quantitative assay of both metabolite(s) and enzyme(s) activity in biological fluids (urine/ plasma/CSF/amniotic fluid) and tissues (RBCs/ WBCs/ Chorionic villi biopsies).

Results: Over a period of one decade (1994–2004), a total number of 338 cases of IMDs, comprising aminoacidopathies (n=68), lipidoses (n=88), carbohydrate metabolic disorders including mucopolysaccharidoses (n=175), porphyrias (n=2), mucolipidoses (n=5) were confirmed. Based on quantification of enzyme(s) activity in WBC’s lysate, we have been able to distinguish between the heterozygous carrier and the homozygous state. A rare bone disorder, characterized by late onset spondyloepi(meta)-physeal dystrophy, unique to India is identified. Case report(s), one each of Sandhoff’s and Mucolipidoses being rendered beneficial effects by Ayurvedic treatment, will also be presented.

Conclusion: There are evidences to suggest that Indian subcontinent has more IMDs both in numbers and varieties. Traditional Indian medicines, the ethnic diversity and large population of Indian subcontinent offer opportunities to identify hitherto unknown IMDs and also newer avenues to evaluate alternate medicines for some of these rare disorders.

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S60 ABC OF HDL: PARADIGM SHIFT IN MANAGING LIPID DISORDERS

There has been renewed interest in the protective role of high-density lipoprotein (HDL) against atherosclerosis and coronary heart disease (CHD). Previously supported by epidemiological evidence, new data have accrued from clinical trials and experimental studies testifying to the anti-atherogenic effects of raising HDL. This effect is mediated by both enhanced reversed cholesterol transport (RCT) and by direct mechanisms on the arterial wall that involve antioxidant, anti-adhesive and antiproliferative mechanisms. RCT involves the transportation of cholesterol from tissues via HDL, or apoB containing lipoproteins, to the liver for excretion in bile. The most compelling experimental evidence supporting the anti-atherogenic effects of HDL derives from work with transgenic animals.

Patients most at risk of cardiovascular disease due to impaired HDL metabolism are those with type 2 diabetes and the metabolic syndrome. The metabolic defect in these patients with insulin resistance relates to increased catabolism of HDL. This results principally from an expansion of the VLDL triglyceride pool size. The first clinical approach to raising low HDL cholesterol is to identify its cause. Correcting hypertriglyceridaemia usually increases low HDL cholesterol, but specific elevation in HDL cholesterol maybe required. HDL cholesterol targets for treatment are lacking, although it is generally agreed that HDL should be >1 mm per litre to lower risk. Lifestyle modifications in diabetic and metabolic syndrome patients should aim to decrease adiposity and improve insulin resistance. The most powerful pharmacological agents for raising HDL cholesterol are in decreasing order, niacins, fibrates, statins. Niacins may, however, impair glycemia in poorly controlled diabetics, although in combination with a statin it has been recently shown to decrease progression of CHD in subjects with low HDL. The HDL raising effect of statins is limited, so that the benefits in clinical trials do not appear to be mediated by effects of HDL metabolism. By contrast, studies with fibrates suggest that a significant proportion of the benefit on CHD is attributable to increase in HDL.

Novel therapies for raising HDL cholesterol include CETP inhibitors and PPAR-α/γ agonists. HDL-mimetic therapy is another promising approach, with recent data showing that the intravenous infusion of apoA1-Milano regresses experimental and human atherosclerosis. Molecular advances, including in particular the discovery of ATP-binding cassette transporters, will see the emergence of new therapeutic agents for testing in clinical trials. The new focus on therapeutic elevating in HDL cholesterol should be paralleled by refinements and new developments in biochemical methods for assessing HDL metabolism, including RCT.

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S61 THE ASIAN AND CHD RISK

Coronary heart disease (CHD) is growing in epidemic proportions in Asia. Although all ethnic groups have been affected, some appear to be at particularly high risk. The basis of these ethnic differences remains poorly understood. Recent studies involving Chinese, Malays and Asian Indians living in Singapore suggest that neither dietary, lifestyle nor genetic factors, when taken independently, are able to adequately explain ethnic differences in serum lipid profiles and CHD risk. According to the World Health Organization (WHO), it is estimated that mortality due to CHD, when combined with CHD associated diseases such as diabetes, obesity and cerebrovascular disease, accounts for > 50% of death in India and the Indian subcontinent. It has been established that Indians have a higher than average risk of CHD at an earlier age and poorer survival and this is impacted on by genetic, social and cultural factors. The Metabolic Syndrome typifies the new generation disorder which is prevalent in Asia. Several established risk factors that contribute to CHD are: elevated levels of plasma low density lipoprotein (LDL) cholesterol, low levels of plasma high density lipoprotein (HDL) cholesterol, smoking, diabetes, high blood pressure and stress. The emerging CHD risk factors include: inflammation, inflammatory cytokines, high sensitive C-reactive protein (hs-CRP), homocysteine, asymmetrical dimethylarginine (ADMA), oxLDL, isoprostanes and obesity. However, all of these factors together, including diabetes, do not explain the marked increase in CHD in certain ethnic Asian populations. The scenario is compounded by complex gene-environmental interactions as well as apparent clustering of many of these risk-factors. Several genetic variants in key candidate gene (apolipoprotein E, cholesteryl ester transfer protein, hepatic lipase, apolipoprotein A5, peroxisome proliferators activated receptor gamma) polymorphisms have been identified to modulate the association between environmental determinants and serum lipid concentrations in Asian ethnic groups. An improved understanding of the gene-environment interaction may enable therapeutic modalities like gene therapy to assist in CHD management.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S62 GENETICS OF LIPID DISORDERS

Plasma lipoproteins are important determinants of atherosclerosis, a disease of large arteries that underlies most deaths in industrialised countries. Genetic susceptibility plays a key role in atherosclerosis, particularly when clinical endpoints strike early in life. Some risk factors, such as dyslipidaemia, diabetes mellitus and hypertension, themselves have complex genetic determinants. Other risk factors, such as smoking, poor diet, physical inactivity and stress can modulate expression of the genetic susceptibility. Study of monogenic dyslipidaemias has revealed key metabolic mechanisms. The exemplar being the autosomal dominant form of familial hypercholesterolaemia whose characterisation led to the discovery of receptor-mediated endocytosis via the low-density lipoprotein (LDL)-receptor. This in turn led to the development of statin drugs, which decrease plasma LDL-cholesterol, and coronary heart disease (CHD) mortality. However, LDL-cholesterol reduction does not always prevent CHD, and many patients with CHD do not have elevated LDL-cholesterol as their primary dyslipidaemia. Early candidate-gene experiments have delineated the molecular basis of other monogenic dyslipidaemias. Recent positional cloning experiments have revealed novel genes, such as those for Tangier disease, β-sitosterolaemia, and autosomal recessive hypercholesterolaemia. Elucidating the pathways underlying the monogenic disorders of lipoprotein metabolism could provide new targets for novel intervention strategies and lead to additional downstream public-health benefits for CHD reduction.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S63 ANALYTICAL TECHNIQUES FOR MODIFIED LIPOPROTEINS

Atherosclerosis is a complex multifactorial disease, believed to be initiated by the uptake of modified low density lipoprotein (LDL) into the arterial wall. Much of the recent research on the pathogenesis of atherosclerosis has focuses on the involvement of oxidative stress and the possible role of oxidatively modified LDL. Oxidised LDL can be taken up by the scavenger receptors of macrophages to form foam cells and products resulting from oxidation of LDL have been detected in human atherosclerotic plaque.

A number of methods have been developed to measure the sensitivity of isolated LDL in response to ex vivo oxidation, or to measure the presence of oxidised LDL in plasma by ELISA. However, these assays have limitations and we have taken the approach of trying to detect specific biomarkers of oxidative damage in the circulation. F2-isoprostanes are formed by free radical oxidation of arachidonic acid and may represent specific markers of in vivo lipid oxidation. F2-Isoprostanes are formed in oxidised LDL and have been detected in plaque tissue. Both free and esterified isopostanes are present in plasma and free isoprostanes are excreted in urine. Methods including gas chromatography mass spectrometry (GCMS) and EIA will be discussed for the measurement of isoprostanes and the usefulness of these measures to determine oxidative stress in humans will be considered.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S64 TELEHEALTH TECHNOLOGY: REDUCING COSTS, & IMPROVING QUALITY OF LIFE FOR THE ELDERLY

Today’s technology now affords health care practitioners the opportunity to monitor patients vital signs while they are in their home and transfer the data to a variety of locations (e.g. hospital, home care agency, nurse or physician’s office) for review. Many times the technologies are referred to as a “Vital Sign Box” (VSB). This technology is a home monitoring system that allows patients and health care providers to work together to better manage conditions such as diabetes, congestive heart failure, pulmonary disease, etc. The VSB can monitor a patient’s pulse rate, temperature, cardiac and lung sounds, weight, blood pressure, O2 saturation as well as point of care testing. Some systems in addition to transmitting vital signs allow the practitioner and the patient to view one another and discuss results in real time (communication device). Web-based information technology has complimented this technology by affording healthcare providers the opportunity to review previous data (laboratory tests, vital signs, hospital encounters, etc.) from a variety of sources such as central repositories at home care agency, hospital information systems, laboratory information systems, physician offices, etc. The combination of these technologies can provide home health care agencies opportunities to reduce costs and at the same time improve the management of the patient.

The healthcare needs of the aged is a major concern for health care payers especially for those who have chronic conditions (e.g. diabetes and congestive heart failure), (Diabetes Care 26:1421–1426,2003). These Individuals utilize the more expensive healthcare facilities such as emergency departments and hospitals far more than others, which will result in an economic crisis. As the aged population continues to grow world wide, the need to manage and provide health care outside the traditional walls of the hospital becomes more evident. (Lehmann, CA “The future of home testing implications for traditional laboratories. Clin Chim Acta 323:31–36, 2002). This presentation will discuss the growing home health care market and the economic benefits of utilizing telehealth “vital sign technology” and “web-based POCT” in managing the aged population with such chronic diseases. It will present the outcomes of an on going study utilizing telehealth to manage home bound congestive heart failure patients.

This session is intended to describe the latest telehealth technology and its ability to perform and record vital signs and point of care testing of home patients. The economic impact of utilizing this technology in managing diabetes and congestive heart failure home patients will be described. The presentation will discuss the role of the clinical laboratory.

Learning Objectives:

  1. Participants will be able to describe the economic concerns of providing health care to a growing aged population.
  2. Participants will be able to list vital signs and POCTs available on telehealth technology
  3. Participants will be able to describe the benefits of providing health care outside the traditional walls of the hospital.
  4. Participants will be able to describe the possible roles of the clinical laboratory
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S65 HETEROPHILE ANTIBODIES AND OTHER PITFALLS IN IMMUNOASSAY TESTING

Driven by impressive technological and scientific innovation, (automated) immunoassay testing has gained widespread use as a specific and sensitive diagnostic tool in laboratory medicine. Interferences of heterophile or human anti-animal antibodies in immunoassays often go unnoticed or disregarded, to the detriment of patients. The term heterophile should be used for interfering antibodies when there is no history of medical treatment with animal immunoglobulins or other well-defined immunogens. However when there is such a history, human anti-animal antibodies should be used. The most common and important type of human anti-animal antibody is probably HAMA (human anti-mouse antibody) because of the increasing use of mouse monoclonal antibodies for therapeutic and imaging purposes. In two site immunoassays HAMA can interfere by bridging between the capture and the signal antibody, producing a false positive result. By preventing analyte-bindung to test-antibodies, false negative results could be attributable to HAMAs. Because of the great heterogeneity of human anti-animal antibody response to immunogens, response can be of different immunoglobulin classes, depending on the animal species involved, and can have anti-idiotype or anti-isotype specificity. Several strategies have been developed to eliminate anti-animal antibodies and antibody interferences on immunoassays. They can be removed by immunoextraction or by precipitation with PEG 6000. Blocking agents like polyclonal or polymerized IgG, mouse monoclonals or a mixture of those and IgG fragments (Fc, Fab, F(ab’)2) can be incorporated in the assay reagents or the sample itself. Another promising strategy is the use of chimeric monoclonal antibodies. Despite all the efforts in avoiding interferences, anti-animal antibodies are still an issue in immunoassay testing today. Besides those well characterised interferences, herbal remedies and even lifestyle could alter laboratory test results significantly. Although most diagnoses are based on laboratory test results, detailed information on patients’ history and lifestyle is indispensable to avoid any misclassification.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S66 WHAT CAN BETA-TRACE PROTEIN TELL IN OTORHINOLARYNGOLOGICAL DISEASES

Introduction: The ß-trace protein (BTP), a lipocalin, which recently has been identified as prostaglandin D-synthase, was found in human cerebrospinal fluid (CSF), aqueous humor, sperm und urine. BTP is a very specific and sensitive marker for CSF. To determine its clinical importance for hidden fistulas a prospective test study was undertaken to reveal the incidence of occult after endoscopic paranasal sinus surgery. In a second and different set up we evaluated BTP as diagnostic tool for the proof of perilymphatic fistulas in the middle ear.

Material and Methods: 69 patients undergoing routine endoscopic paranasal sinus surgery were included. Patients with an obvious intra- or post-operative CSF fistula were not included. 112 samples from intra-operative applied tamponades and 69 serum samples were analyzed using a nephelometric research assay for BTP (Dade Behring, Germany). Patients with an occult CSF fistula were followed-up after 6 months.

To determine the concentration of BTP in human perilymph, 20 postmortem samples taken with bores from the perforated footplate of the stapes under microscopic view.

Results: Beta-trace protein was found in ethmoid roof samples from two patients giving an incidence of occult CSF fistula of 2.9 %. Specimens from post-mortem examinations revealed a mean concentration of 49.1 ± 17.7 mg/L in CSF, 71.9 ± 29.3 mg/L in perilymph and 68.0 ± 21.7 mg/L in endolymph. Special attention must be paid the way of collecting samples

Conclusions: The results show that two of the main diagnostic fields could be covered by the BTP-test in ORL: the management of occult CSF leaks during and after surgery and the diagnosis of perilymphatic fistula, which is of great importance in many inner ear hearing disorders.

With nephelometry a fast and reliable method to determine ß-trace is given. Special attention must be paid to the way of collecting samples because of the high binding to packages.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S67 A NOVEL PANEL OF MARKERS TO DIAGNOSE STROKE

Abstract not supplied

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S68 PROTECTIVE IMMUNITY IN FILARIASIS - WITHDRAWN

Introduction: Epidemiological and immunological studies indicate the existence of a ‘putatively immune’ group of individuals in a filarial endemic area called as ‘endemic normals’. We have identified two native proteins (BmL3S2 & BmA 2b) and one recombinant protein BmALT-2 of Brugia malayi parasite as having immunoprophylactic potential based on their high seroreactivity with endemic normals and confirmed their ability to induce protective immunity in rodent model.

Methods : BmL3S2, a 70 kDa thiol protease was isolated from B.malayi infected larval extracts and BmA 2b, a 120kDa surface glycoprotein was isolated from adult worm extracts. BmALT-2 (Abundant larval transcript-2) is a recombinant protein expressed largely in L3 larvae. Two groups of jirds / mastomys susceptible to B.malayi infection were immunized with these proteins. Ten days after final dose the animals were used for in vitro, in situ and in vivo experiments to assess the protective immune response against B.malayi parasites.

Results: Jirds / mastomys immunized with BmL3S2 antigen showed 73–77% larvae and offered 73% protection against cytotoxicity against mf and L3 challenge infection. BmA 2b immunized mastomys showed more than 68% larvae and BmA 2 showed 88% reduction in the cytotoxicity against mf & L3 development of parasite to adult stage. Jirds immunized with rALT2 protein showed 76–84 % cytotoxicity against L3 larvae, which was significantly higher than that observed in animals immunized with only ALT2 DNA vaccine construct (48–56 %) or by bimodal vaccination i.e., primed by DNA vaccine and boosted by recombinant protein (64–67%).

Conclusions: More focused studies on immunological changes associated with the resistance induced by BmL3S2, BmALT-2 & rALT2 proteins and on longevity of protection offered would confirm their potential use as anti-filarial vaccines.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S69 VACCINE TO PEPTIDE ANTIGENS OF HIV

Introduction: Since sexual transmission of HIV-I occurs via mucosal surfaces, it is important to induce systemic and mucosal immunity at these sites. Mucosal immunity is normally hampered due to lack of proper delivery vehicle and poor transport of antigens across the epithelial barrier. In the present study, we have used non living microparticle vector co-entrapped with a plant lectin as an M cell target (portal of antigen entry at the mucosal surface) with peptide antigens derived from envelope, core and regulatory proteins of HIV-I serotype C (Indian strain).

Method: The mucosal (sIgA) and systemic (IgG) immune response was studied in mice of both inbred and outbred strains to compare the genetic restriction, if any exist, with and without lectin. The routes of immunization were combinational (primary with booster) i.e oral/oral, nasal/nasal etc. and single (without any booster) i.e oral, nasal, intramuscular and rectal. Different bleeds were assayed for antigen specific for sIgA titers in various washes (intestinal, nasal, rectal, and vaginal) and for IgG and IgA titres in sera. Antigen induced T- cell proliferative response was studied from the cells collected from spleen, lamina propria, peyer’s patches and cytokine levels of both CD4+ TH1 and TH2 subsets were estimated. Lastly, representative high titer sera and washes were tested for synctium inhibition assay in-vitro using laboratory adopted virus.

Results: The combinational routes of immunization failed to enhance the humoral response after four weeks while single route of immunization maintained both IgA and IgG levels in sera as well as sIgA in washes till 10th weeks. All the washes showed the presence SC component. Out of the four routes attempted, nasal route containing lectin showed highest peak antibody titres and T cell proliferation with cells obtained from systemic and mucosal areas. Cytokine profile showed high levels of IL-2, IFN-γ, IL-6 and TGF-β. Sera and washes showed 60–90% inhibition of synctium formation.

Conclusions: The study highlights the importance of microparticle delivery with a M cell target (lectin) using peptide antigens of HIV- I induced both systemic and mucosal immune response with no genetic restriction of immune response.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S70 ROLE OF ANTHRAX TOXIN IN THE PATHOGENESIS OF BACILLUS ANTHRACIS

Bacillus anthracis secrete three proteins, protective antigen (PA), lethal factor and edema factor that constitute anthrax toxin. Anthrax lethal toxin, the principal virulence factor associated with symptoms following infection with B. anthracis is a complex of protective antigen (PA) and lethal factor. Lethal toxin is lethal to several species of animals and some macrophage-like cell lines. PA, the central receptor-binding moiety acts as a delivery vehicle to translocate catalytic components of toxin to the cytosol of mammalian cells. The residue Asp683 of PA present in COOH-terminal region was critically important for binding to receptor. PA possesses two unique protease-sensitive sites. Mutation in the trypsin sensitive site of PA abolished its proteolytic activation with the concomitant loss of its ability to oligomerize and bind lethal factor. Similarly, mutation in the chymotrypsin sensitive site of PA failed to form channels in the membrane which is a prerequisite for translocation of catalytic moieties. A mutant PA protein, in which aminoacid residues (302–325) were substituted with the residues of amphipathic loop of iota-b toxin, assembled into hetero-heptameric structure with wild-type PA leading to inhibition of cellular intoxication. These observations suggest that PA mutants can be used as a therapeutic agent against anthrax toxin action in vivo.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S71 ASSOCIATION OF POLYMORPHISMS OF HUMAN SP-A1 AND SP-A2 GENES WITH ALLERGIC ASPERGILLOSIS AND PULMONARY TUBERCULOSIS IN INDIAN POPULATION

Introduction: A series of studies from our group, have established that lung surfactant protein A (SP-A) has an important role in strengthening the host defense against both allergic and invasive aspergillosis. SP-A also binds M. tuberculosis and enhances attachment and phagocytosis of M. tuberculosis by alveolar macrophages. In view of the importance of SP-A in host defense against aspergillosis and tuberculosis, structural and functional changes in SPA may affect the outcome of host-pathogen interaction.

Results: We investigated the relationship between SNPs in SP-A1 and SP-A2 genes and susceptibility to ABPA and pulmonary tuberculosis. In the present study, seven SNPs (4 exonic and 3 intronic) have been identified in the collagen regions of SP-A1 and SP-A2 genes in Indian population. Statistical analysis of these SNPs showed four of these SNPs have significant differences in their frequencies observed in ABPA patients vs. controls (SP-A1 C1416T p=0.118; OR = Infinity; SP-A2 T1492C p=0.034; OR = 4.8, SP-A2 G1649C p=0.031; OR = 4.2, SP-A2 A1660G p=0.058; OR = 7.0). Pulmonary tuberculosis patients also showed statistically significant differences in frequencies of four SNPs when compared with control population (SP-A1 C1416T p=0.000;O.R = 20.8, SP-A2 C1382G p=0.005; O.R = 3.7, SP-A2 G1649C p=0.010; O.R = 2.2, SP-A2 A1660G p=0.000; O.R = 12.3).

Discussion: It is interesting to note that three SNPs (SP-A1 C1416T, SP-A2 G1649C, SP-A2 A1660G) are commonly associated with patients of ABPA and pulmonary tuberculosis suggesting that dysfunction of SP-A2 caused by these SNPs may be one of the factors leading to increased risk of pulmonary infections. SP-A2 T1492C and SP-A2 C1382G in Intron 3 are distinctly associated with ABPA and pulmonary tuberculosis respectively. These two SNPs may be useful for identification of differential susceptibility of individuals to allergic bronchopulmonary aspergillosis and pulmonary tuberculosis.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S72 LUNG SURFACTANT PROTEINS IN INFECTIOUS DISEASES

Introduction: Pulmonary surfactant proteins, SP-A and SP-D, are immune molecules which can directly interact with pathogens and allergens, stimulate immune cells and manipulate cytokine and chemokine profiles during host’s immune response. Using an opportunistic fungal pathogen Aspergillus fumigatus (Afu), attempts have been made to understand participation of SPA and SP-D in the host immunity. Afu causes a systemic infection via lungs, called invasive aspergillosis (IPA) in the immunocompromised subjects. In the immunocompetent subjects, it can cause an allergic disorder, called allergic bronchopulmonary aspergillosis (ABPA).

Results: Therapeutic administration of these proteins in murine model of IPA can rescue mice from death. Treating mice, having ABPA, can suppress IgE levels, eosinophilia, pulmonary cellular infiltration and cause a marked shift from a pathogenic Th2 to a protective Th1 cytokine profile. Both SP-A gene deficient (AKO) and SP-D gene deficient (DKO) mice exhibited intrinsic hypereosinophilia and manifold increase in levels of IL-13, IL-5 and lowering of IFN-γ to IL-4 ratio suggesting a shift towards Th2 type in comparison to the wild type (WT) mice. These immunological changes could be reverted back on intranasal administration of native human SP-A, SP-D and rhSP-D to the respective KO mice. SP-D gene deficient mice were observed to be more susceptible than WT mice to Afu sensitization. On the contrary, AKO were more or less resistant to Afu sensitisation. SP-D and rhSP-D were effective in rescuing the Afu sensitized DKO mice while SP-A administered Afu sensitized AKO mice showed manifold elevated levels of IL-13 and IL-5 resulting in increased pulmonary eosinophilia and damaged lung tissue.

Discussion: These results highlight the potential of SP-A, SP-D and their recombinant forms, as novel therapeutics for lung allergy and infection. Formerly, Director Grade Scientist, IGIB & Scientist Emiritusm, CSIR

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S73 STANDARDIZATION: BENEFITS TO THE PATIENT

Standardization of laboratory methods aims at obtaining results that are as close as possible to the true value. If successful, determinations performed by various methods give comparable results, but poor assay precision may still cause variation between well-standardized methods.

Standardization requires availability of standards and reference methods. Standards should be homogeneous and pure but also correspond to the analyte occurring in biological fluids. This is possible only for low molecular weight substances, whereas most peptides and proteins occur in circulation as heterogeneous mixtures of variant forms. This is a problem for hormones and tumour markers. Nevertheless it is often possible to develop immunoassays that specifically measure, either together or separately, the clinically most relevant variants.

Adequate standardization is important for patient care especially when treatment decisions are made mainly or solely on the basis of a test result. This is crucial when tumour markers are used to monitor therapy of cancer patients but also in many other diseases. Tumour markers play especially important roles in the management of prostatic and testicular cancer and in choriocarcinoma because treatment of these is often based on measurement of tumour markers in serum performed before and after primary therapy. When first introduced, the large variation between assays for prostate specific antigen (PSA) caused much confusion when the same patient was monitored with various assays. International standards for PSA are now available and assays calibrated against these give comparable results. Treatment of testicular cancer is now very successful and even most patients developing a relapse are cured. Treatment of a relapse is often initiated solely on the basis on increasing marker levels in serum. Monitoring continues for many years, which increase the probability that different assays are used making standardization crucial. Treatment of growth hormone deficiency and acromegaly is based on determination of growth hormone (GH) and various GH assays give different results. Thus, decision limits for initiation of treatment need to be established separately for different assays. Appropriate assay standardization facilitates clinical decision-making and good patient care.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S74 STANDARDIZATION OF CARDIAC MARKER ASSAYS

Cardiac markers are measured by a number of different immunoassays using specific antibodies directed to the respective antigens. Lacking assay standardization, different results from different assays measuring the same marker may be obtained and this problem may cloud interpretations of reported data. Presently, there are no reference procedures for cardiac markers, certified reference materials should still be established, and, at least for cardiac troponins, the analyte in the patients’ blood is significantly different from newly synthesized protein. It is therefore clear that the problems of cardiac marker standardization will not be quickly solved. A number of projects are, however, underway under the auspices of IFCC and other organizations. The aim of this presentation is to reflect on some concepts related to the implementation of a metrologically correct measurement system, giving practical examples on how these concepts can be applied to immunoassays measuring cardiac markers.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S75 THE SUCCESSFUL IMPLEMENTATION OF THE IFCC ENZYME STANDARDISATION

Enzyme concentrations are usually not measured as mass concentrations but as catalytic concentrations, depending on the temperature, pH, buffer, ionic strength, presence of co-enzymes, activators, substrate etc. The peculiarity of these tests is that the analyte is defined by the measurement conditions. Therefore, standardisation is necessary to achieve comparable patient results and has been pursued for many years, but most often at a national level, leading to non-comparable results and requiring manufacturers to provide different kits for different countries. In 1998 the IFCC initiated a global reference system for enzymes, comprising

  1. reference measurement procedures,
  2. an international network of reference laboratories,
  3. reference materials (in co-operation with IRMM).

As of today, reference measurement procedures at 37°C are available for ALT, AST, CK, GGT and LDH. One for amylase is almost completed and one for ALP is in progress. Reference materials are available, except for AST. An unsolved problem is the future financing of the reference network. These procedures have become mandatory for value assignment to EQA samples in Germany, leading already to lower inter-laboratory CVs.

The benefit for manufacturers is that fewer types of kits need to be provided. Practical laboratory requirements, however, often mean that routine methods differ significantly from the reference procedures defining the analyte, causing problems with control materials and sometimes also patient samples. Examples are shorter pre-incubation and serum start instead of substrate start to increase the sample throughput. Sometimes liquid, ready to use reagents with a long shelf-life and calibration stability with some components added or changed are used.

Unfortunately standardisation of methods has not yet resulted in harmonisation of reference intervals even for equivalent reference populations. This is also required to make the standardisation clinically useful.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S76 PSA-STANDARDISATION, BENEFITS FOR MALE-PATIENTS

Prostate cancer is considered to be one of the main killers in males. In large population studies the risk for prostate cancer was estimated to be approx. 10 %, the incidence among malignancies was calculated to be 23 to 30 %, the mortality rate 11 %. Therefore screening, diagnosis and monitoring of patients by measurement of total PSA and free PSA in serum are commonly used especially in males between 40 and 80 years of age. In order to achieve this clinical objective immunoassays must give accurate, reliable and comparable results over space and time.

In 1999 the WHO accepted the 1st International PSA Reference Preparations submitted by the IFCC Working Group on PSA Standardization (Chair: T. A. Stamey, Stanford University, USA). The PSA target values of the preparations were assigned by amino acid analysis and mass spectrometry and are as follows:

WHO 96/668: 1 μg free PSA per vial

WHO 96/700: 1 μg total PSA per vial, 90 % PSA-ACT – 10 % free PSA

These preparations (reference materials) are intended to be used for calibration of immunoassays for total and free PSA and for validation of assays.

International collaborative studies demonstrated clearly that the use of WHO-IFCC PSA-ACT (90:10) reference material for calibration decreased the between-laboratory CVs of assay systems thus making patient results more comparable. However, it was also realized that some assays differ in their recognition of free and complexed PSA forms.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S77 VITAMIN D REQUIREMENTS FOR SKELETAL HEALTH

The Vitamin D endocrine system plays a primary role in the maintenance of calcium homeostasis as well as exerting a wider range of biological activities including regulation of cellular differentiation. It is well recognised that nursing-home residents with vitamin D insufficiency have increased risk of hip fracture. This low vitamin D status is associated with secondary hyperparathyroidism and raised biochemical markers of bone turnover. However there is continuing controversy over the level of serum 25-hydroxyvitamin D [25D] necessary for bone health. Evaluation of patient 25D levels against a reference range obtained from a reference population is invalid because such a range is only a measure of the sunlight exposure of the reference population. It does not necessarily reflect skeletal health. Currently investigators have assessed the relationship between 25D and biochemical variables reflecting calcium and bone homeostais as surrogate markers of skeletal health.

Biochemical markers of bone resorption and formation as well as parathyroid hormone often demonstrate an inverse relationship with 25D levels in an elderly population with low vitamin D status. The question remains as to the level of biochemical bone marker that reflects skeletal health. Large placebo controlled clinical trials have demonstrated a strong relationship levels between bone resorption markers above the premenopausal range and decreased bone density and increased risk of fracture. There is therefore a strong argument to define the appropriate level of 25D at which such biochemical markers are at their lowest levels.

A number of studies in the elderly, particularly women, have reported elevations of bone turnover markers and PTH at levels of 25D below 60 nmol/ L. It would be prudent to recommend a clinical critical level for 25D above 60 mol/L. Hypercalcaemia as a consequence of toxic vitamin D levels have been reported at 25D levels around 750 nmol/L. There is apparently a large therapeutic window for this pro-hormone. Monitoring of 25D can ensure that patients who would benefit from supplementation do not achieve toxic levels.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S78 REQUIREMENTS FOR VITAMIN A

The biochemistry, physiology and pathophysiology of vitamin A is reviewed. Vitamin A has multiple actions as retinol, retinal and retinoic acid in vision, epithelial integrity and cell differentiation. It exists in the serum as retinol, bound to RBP and is stored as retinyl esters. Carotenoids are ubiquitous and are divided into provitamin A (those which contain the β-ionone ring) and can be cleaved into retinal. Hyperabsorption of a variety of carotenoids results with hypercarotenoidaemia in xanthoderma but not hypervitaminosis A. The antioxidant and anti-cancer role of retinol and carotenoids is not supported by prospective clinical trials designed to reduce cancer and atherosclerosis, in a well nourished population. High dose vitamin A and carotene cannot be recommended except in person with low nutrient intake. The assay indications for serum retinol, retinyl esters and carotenoids are divided in the presentation with low or raised levels.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S79 NUTRITIONAL DEFICIENCIES CAUSED BY ENVIRONMENTAL CONTAMINATION OF HEAVY METALS WITH SPECIAL REFERENCE TO LEAD IN DEVELOPING COUNTRIES

Introduction: Essential trace metal interaction with heavy metals in humans is of current clinical significance. Health hazards due to environmental contamination of lead are a major problem in most parts of the world especially amongst nutritionally deprived. Lead in the environment easily enter the living system through inhalation, ingestion or dermal absorption and interferes with the uptake and utilization of micro minerals especially during growth and development. Population studies in India during 1997–2000 at St. John’s Medical College at Bangalore sponsored by The George Foundation has revealed that over 53% of children below 12 years of age had blood lead levels higher than 10mcg/dl (TGF International conference on Lead Poisoning, prevention and treatment, Bangalore 1999 & ACBICON-CMC Vellore 2000) and all of them were found to be anaemic. Studies during 2000–2002, NRCLPI recorded the most affected group for lead poisoning from amongst lead based Industrial workers and their family members (9th APCCB New Delhi 2002) accompanied by the symptoms of nutritional deficiencies. Work carried out at The National Referral Centre for Lead Poisoning in India (NRCLPI) demonstrated the beneficiary role of micro nutrients in preventing health hazards amongst children exposed to environmental sources of lead. NRCLPI noticed many disturbed biochemical mechanisms as a result of disturbed antioxidant status by the absorbed lead (IFFR-Lucknow, 2003, IFFR Chidambaram 2004). World over it is accepted that the blood lead level determination is sufficient for the evaluation of anaemia caused by lead poisoning.

Methods: However developed countries have complied with the global standards of regulating the environmental lead at work place in addition to blood lead evaluation using ESA 3010 B Lead analyzer. NRCLPI study has established and revealed high incidence of lead toxicity in most of the lead based industrial workers in India due to unacceptable blood lead levels (ILZDA-Bangalore 200, 2004).

Conclusion: NRCLPI has proposed a hypothesis, to protect children from the ill effects of environmental lead with timely nutritional intervention. Since the tissue threshold of lead depends upon the nutritional status and age, the form in which lead is absorbed from the environment is of great significance. In this regard NRCLI evaluated the lead in oriental medicines (IFCC Kyoto 2003), in environmental dust (Prague 2003) with the use of Niton XRF lead analyzer and lead in drinking water. NRCLPI studies with portable XRF monitoring environmental lead and the use of portable Led care of ESA Inc for the blood lead evaluation has provided supporting data in monitoring one of the major environmental hazards in developing countries. Evaluation of lead in the environment and nutritional status of the exposed made us to understand the problem and to prevent toxic and expensive chelation therapy.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S80 AN OVERVIEW OF IODINE DEFICIENCY DISORDERS IN THE ASIA PACIFIC REGION

The only known physiological function for the essential micronutrient iodine is to serve as a precursor in the synthesis of thyroid hormones. The distribution of iodine in soil and water is variable, generally being abundant in coastal regions and decreasing the further one travels inland. Severe deficiency is invariably present in the mountainous zones of the world. The major consequences of deficient or disordered iodine metabolism are a result of too little (hypothyroidism) or too much (hyperthyroidism) thyroid hormone and are collectively termed iodine deficiency disorders (IDD). Until recently, iodine deficiency was equated simply with endemic goitre, without recognition of the widely prevalent, subtle and devastating neurological and psychomotor consequences. Very severe iodine deficiency affecting people in remote, underdeveloped regions of the world, is associated with severe mental and physical impairment, termed endemic cretinism. Less severe and subtle forms of brain damage are more highly prevalent. The WHO says, “Iodine deficiency is the world’s most prevalent, yet easily preventable, cause of brain damage”. Where iodine is deficient IQ levels are, on average, 15 points below comparable iodine-replete populations. IDD affects over 740 million or 13% of the world’s population and 30% of the remainder are at risk of developing one or more of the consequences of IDD.

Many countries in the Asia Pacific region including, India, Bangladesh, Myanamar, Thailand, Cambodia, China, Hong Kong, Mongolia Laos, Malaysia, Indonesia, Papua New Guinea, Philippines, and Vietnam. have significant public health problems of IDD. Of major concern are recent reports of the re-emergence of iodine deficiency in Australia and New Zealand.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S81 AUTOIMMUNE THYROID DISEASE

Autoimmune thyroid disease (ATD) is common, affecting 5–10% of the population in iodine-sufficient areas. The epidemiology of ATD is well described, being more common in iodine-replete than iodine-deficient areas, more common in women than men and more common with increasing age. Although there is familial clustering of disease, there has been little success in identifying specific susceptibility genes. The pathogenesis of ATD is complex, and involves both humoral and cellular immune mechanisms. The principal autoantigens are the TSH receptor, thyroid peroxidase (TPO), thyroglobulin (Tg) and the sodium iodide symporter (NIS). Circulating TSH receptor antibodies (TRAb) are pivotal in the pathogenesis of Graves’ hyperthyroidism, but their role in the extrathyroidal manifestations of the disease is unclear. There is some evidence that TPO antibodies are cytotoxic, but the pathological role of antibodies to Tg and NIS is unclear.

In clinical practice, thyroid peroxidase antibody (TPOAb) testing by specific immunoassay is the most useful marker of thyroid autoimmunity, and has an important role in the management of subclinical hypothyroidism and post partum thyroid dysfunction. However, some patients with ATD are TPOAb negative but TgAb positive. TRAb are usually detected by a competitive binding assay which does not discriminate between stimulating and blocking antibodies. TRAb testing is useful in the diagnosis of Graves’ disease, but its utility in monitoring thionamide treatment and predicting remission of Graves’ disease is controversial. TRAb testing is indicated in pregnant women with a history of Graves’ disease, where high titres are associated with foetal and neonatal hyperthyroidism. In patients with differentiated thyroid cancer, TgAb should be measured routinely by immunoassy when serum Tg is requested. In antibody-positive patients with thyroid cancer, changes in antibody titre may have prognostic value.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S82 AUTOANTIBODY TESTING FOR COELIAC DISEASE

Coeliac disease (non-tropical sprue, gluten sensitive enteropathy) is an autoimmune disorder triggered by the ingestion of cereal proteins in genetically susceptible individuals. It is a common chronic, but treatable, disease with a prevalence of about 1% in Caucasian populations. It is also common in northern India and North Africa but considered to be rare in Chinese, Japanese and African-Caribbean people.

Coeliac disease has a strong association with other autoimmune disorders, particularly with type 1 diabetes and autoimmune thyroid disease. The diagnostic histological changes in the small bowel are an increase in intraepithelial lymphocytes, crypt hyperplasia and villous atrophy. It is however a multi-system disorder and the adult or child patient may initially present to a wide range of clinical specialities.

In the last 2 decades, serological tests have played an important role in the increased awareness of the high prevalence of the disease, in identifying subjects for confirmatory small bowel biopsy and in monitoring compliance with a gluten free diet.

The identification in 1997 of tissue transglutaminase-2 as the antigen against which the autoantibodies are directed in coeliac disease, has led to a greater understanding of the pathogenesis of the disorder and also to improved diagnostic tests. Enzyme linked immunoassays using recombinant human tissue transglutaminase as antigen have been shown to have high sensitivity and specificity for the detection of coeliac disease in selected samples and also in routine diagnostic practice. As with other ELISAs, high total IgA levels may lead to false positives. Coeliac disease has a high prevalence in subjects with selective IgA deficiency; the testing strategy must therefore ensure that such patients are identified and IgG-class antibodies used for their samples. An appropriate testing strategy will be discussed to enable the laboratory to play a full part in the diagnosis and monitoring of a disorder that is eminently treatable once the diagnosis has been considered and confirmed.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S83 CYTOKINE ASSAYS IN THE INVESTIGATION OF ALLERGIC ASTHMA

Introduction: T-helper type 2 (Th2) lymphocytes, inflammatory cytokines and chemokines have been implicated for the induction and maintenance of the inflammatory cascade in allergic asthma.

Methods: We compared, in plasma and whole blood culture supernatant, inflammatory cytokines interleukin (IL)-17, IL-18, IL-6, and IL-12; Th2 cytokines IL-10 and IL-13; chemokines regulated upon activation normal T cell expressed and secreted (RANTES), IFN-inducible protein-10 (IP-10), and monokine induced by IFN-gamma (MIG); and intracellular interferon-γ (IFN-γ) and IL-4 in Th cells of 41 patients with allergic asthma and 30 sex- and age-matched health subjects. Cytokines were measured by ELISA. Chemokines and intracellular cytokines were quantitated by immunofluorescence flow cytometry.

Results: Plasma IL-18, IL-12, IL-10, IL-13 concentrations were significantly higher in allergic asthmatic patients than control subjects (all p < 0.05). Plasma RANTES concentration was also significantly elevated in asthmatic patients (p < 0.01). Whole blood assay showed significantly decreased production of Th1 chemokine IP-10 and MIG by phytohaemagglutinin-activated peripheral blood mononuclear cells in patients (both p < 0.05). The percentage of IFN-γ producing Th1 cells was significantly higher in control subjects than asthmatic patients (p < 0.001), but the percentage of IL-4 producing Th2 cells did not differ (p > 0.05). Consequently, Th1/Th2 cell ratio was significantly lower in asthmatic patients than control subjects (p < 0.001).

Discussion: Allergic asthma is characterized by a Th2 predominance with elevation of both inflammatory and Th2 cytokines and chemokines.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S84 ROLE OF CHEMICAL MEDIATORS AND CYTOKINES IN ALLERGIC DISEASES

Allergic diseases (e.g., rhinitis, asthma and eczema) are characterized by IgE-mediated reaction to common allergens. They are primarily T-cell-mediated disorders with an intricate network between inflammatory cells and different types of soluble factors such as mediators, cytokines, chemokines and adhesion molecules. Th2-polarization with enhanced production of cytokines (i.e., IL-4, IL-5, IL-6, IL-13 and SM-CSF), which are involved in regulation of IgE, mast cells, basophils and eosinophils, is an key mechanism in the development of diseases. Mediators released by activated cells during allergic reactions are the major effectors in provoking the specific symptoms and non-specific hyperreactivity of allergic diseases. For example, many studies have demonstrated the occurrence of nasal symptoms within minutes after experimental and natural allergen exposure. This response has been described as the early phase reaction that is associated with a significant increase in and kinins. The infiltration and activation histamine, tryptase, PGD2, LTC4 of eosinophils are predominant condition during the late-phase reaction. The latter condition is found to be very common in the pathophysiology of patients with ongoing allergic rhinitis. Histamine was the first mediator of anaphylaxis to be discovered, and H1-antihistamines were the first drugs to be used in the pharmacological treatment of allergic disorders. Tryptase is one of the preformed neutral proteases in mast cell granules and can activate one of the protease-activated receptors expressed on the surface of endothelial and epithelial cells. Cysteinyl leukotrienes (CysLTs) are the lipid mediators, characterized as “slow reacting substance of anaphylaxis” (SRS-A) in the early investigations, play essential role in asthma and rhinitis. Understanding the mechanisms of cytokines and mediators released by activated cells and their pathophysiologic effect in allergic inflammation will aid development of more precisely targeted anti-inflammatory therapies in future.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S85 TUMOUR MARKERS: NOW AND IN THE FUTURE

Most of the tumor markers presently used were developed more than 20 years ago using immunological techniques. Antibodies obtained during the discovery phase were used to develop sensitive and specific immunoassays for determination of the markers in circulation. High sensitivity is essential because virtually all tumor markers occur at concentrations around 0.1 – 10 μg/L, which is about one million-fold lower than those of the major serum proteins. Markers are available for many but not all tumors but only a few markers are useful for early diagnosis. Therefore, new markers are intensly sought for by novel approaches, e.g., gene expression pro ling and proteomics. Over expression of certain sets of genes has been observed in virtually all tumors studied and this is associated with increased production of some tumor-associated proteins. However, only a few potentially useful new tumor markers have been found. Recently, several promising studies on the use of mass spectrometry for pro ling of serum proteins have been published. Although quite tumor-specific profiles have been described, the results have not been reproduced suggesting that the results are explained by over-training of the algorithms used. So far, only abundant proteins and peptides can be detected direct mass spectrometry of serum and the profiles identified reflect changes in common serum proteins rather than tumor-derived markers. However, by analysis of tumor tissue or extensive pre-fractionation of serum in combination with mass spectrometry new tumor-associated proteins will probably be identified. Detection of antibodies to tumor-associated proteins is another promising approach. It is likely that these methods will lead to development of new markers complementing those presently used. In the future, multiple markers will be used simultaneously and this requires development of algorithms utilizing clinical data, marker values, histopathology and imaging results. Establishment of such algorithms is one of the most important challenges in clinical chemistry.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S86 CURRENT TESTS AFP

Blood AFP is marker of hepatocellular carcinoma (HCC) and testicular and germ cells cancers (GCT). HCC usually presents at an advanced stage due to the lack of early symptoms. The screening of high risk subjects such as hepatitis B surface antigen positives has thus been advocated. AFP is used for this purpose, alone or with imaging techniques for the detection of small (<3 cm), asymptomatic HCC when it is at a potentially resectable stage. However, AFP suffers from poor sensitivity and specificity. Related to this are the wide range of cut-off levels used (10–500 ng/ml). Among the approaches used to improve the diagnosis of HCC are the separation of AFP isomers using lectins since AFP is a glycoprotein. AFP-L3, an important isomer, is useful, as is total AFP, for early detection, recurrence and metastasis; L3, additionally, is a marker of poor prognosis. Testing for the relatively HCC-specific Band +II using non-lectin isoelectric focussing separation has been shown to permit early diagnosis of HCC. Total AFP is also used in the monitoring chemotherapy response where an elevated AFP after therapy indicates local recurrence or metastases; AFP may be better than imaging techniques for this purpose. Circulating AFP mRNA has been proposed as a marker of micrometastases of HCC but is of doubtful value.

AFP should not be used for the screening of GCTs. However, pre-operative concentrations of AFP, chorionic gonadotropin and lactate dehydrogenase can confirm diagnosis and are used in the international germ cell tumour prognostic factor-based staging system. In addition, marker determinations should be made immediately after orchidectomy and prior to each treatment cycle. Serial marker measurements after orchidectomy contribute to patient management and to the monitoring for tumour recurrence.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S87 PLASMA FREE METANEPHRINES IN THE DIAGNOSIS OF PHAEOCHROMOCYTOMA

Introduction: Phaeochromocytoma is a relatively uncommon tumour. Diagnosis is commonly by measurement of catecholamines and/or metabolites on 24h urine collections. Alternatively, it has been proposed that plasma catecholamines should be used.

More recently it has been proposed that plasma free metanephrines have theoretical grounds for being superior diagnostically, but little has been published in the way of studies looking at “difficult” subjects who might be confused with phaeo patients eg ICW, Hypertension Clinic etc.

Methods: A study was performed looking at the relative diagnostic efficacy of plasma free metanephrines versus other urine and plasma catecholamine fractions in patients referred for investigation for possible phaeochromocytoma. The study was deliberately biased by including in the study population, patients from ICW, Hypertension Clinic, and Renal Haemodialysis.

Urine catecholamines and metabolites were by standard HPLC methods, whilst plasma free metanephrines were assayed by a modification of Eisenhofer’s method.

Results: Final study group comprised 22 patients with histologically confirmed phaeochromocytoma and “difficult” patients and controls.

Plasma free metanephrines were much superior to other catecholamine fractions for diagnosis of phaeo with ROC AUC >99%. Next best assay was urine metanephrines with ROC AUC 92%.

Conclusions: In this study designed to include subjects who might be confused with phaeochromocytoma patients, plasma free metanephrines proved superior to all other biochemical measures on plasma or urine, in identifying patients with phaeochromocytoma. Although it is a technically demanding assay, it appears to be the assay of choice for investigation of phaeochromocytoma.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S88 UTILISATION OF HUMAN SERUM TUMOUR MARKERS

In Malaysia, PSA, CEA, CA 19-9, CA 125, CA 15-3, AFP and hCG are often included in admission and executive screening profiles as well as wellness packages. Clinical practice guidelines on the utilisation of these serum tumour markers were produced in the hope that the misuse of tumour markers will decrease. Population screening for prostate cancer with PSA is not recommended. All males over the age of 40 years with a first degree relative with prostate cancer diagnosed at a young age (<60 years) may be screened by measuring PSA levels. PSA is useful in monitoring response to treatment and detecting recurrence early. CEA, CA 19-9, CA 125 and CA 15-3 should not be used in population screening for or diagnosis of colorectal, pancreatic, ovarian and breast cancer, respectively. CEA, CA 19-9 and CA 125 are useful in monitoring response to treatment and detecting recurrence early. CA 15-3 can be used in monitoring response of breast cancer patients to therapy but should not be used to detect recurrence since the benefit of early detection of recurrent disease remains to be determined. Chronic carriers of HBsAg, patients with chronic hepatitis C infection and liver cirrhosis may be screened for liver cancer using serum AFP and ultrasound once every 6 months. In this condition, AFP is useful in monitoring response to chemotherapy and early detection of recurrence. As about 50% of cases of choriocarcinoma follow a molar pregnancy, hCG can be used to screen this high risk group of patients. In choriocarcinoma, serum hCG is useful in determining prognosis, monitoring response to treatment and in the early detection of recurrence. AFP and hCG are not recommended as screening tests for germ cell tumours (GCTs) but may be helpful in the diagnosis of patients suspected of having GCTs and in monitoring response to treatment and in the early detection of recurrence. Pre-treatment concentrations of AFP, hCG and lactate dehydrogenase (LDH) are useful in staging GCTs and in determining prognosis of metastatic GCTs.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S89 STEPWISE SCREENING FOR THROMBOPHILIA

Thrombophilia occurs from very different biochemical, genetic and immunologic abnormalities.

Genetic Risk Factors: The most frequent risk factor is the resistance to activated protein C (Factor-V-Leiden mutation). The prothrombin gene variant G20210A is an other point-mutation elevating the risk. Antithrombin-III (ATIII)-, protein C (ProtC)- or protein S-deficiencies and homocystinuria caused by hereditary deficiency of the enzyme cystathione-ß-synthetase are also associated with arterial and venous thrombosis.

Other Risk Factors: Elevated factor VIII-activity (FVII-activity) is an independent risk factor for venous thromboembolism. Less well characterized disorders include elevated factor VII-, IX-, X-, and XI-levels, whereby especially certain combinations of elevated activities seem to increase the risk for thromboembolic events. Antiphospolipid antibodies (aPL) are also involved in the development of thromboembolic diseases. The so-called antiphospholipid antibody syndrome is characterized by severe and recurrent thromboembolic complications in brain, heart, lung, liver and by recurrent fetal loss or thrombocytopenia.

Laboratory Testing: A screening for thrombophilia is recommended in case of a juvenile thrombosis (< 45 a), recurrent thrombosis, familial thrombophilia and recurrent fetal loss. The screening for thrombophilia should include: activated partial thromboplastin time, prothrombin time, fibrinogen, ATIII, FVIII-, FIX-, FXI- and FXII-activities and homocysteine. The protein C pathway should be examined using appropriate kits screening the protein C system. APC-resistance, ProtC and ProtS S should only be measured in positive samples for identification and confirmation. Two types of tests exist for the detection of aPL: 1. lupus anticoagulant (LA) tests, in which the capacity of a plasma is tested to inhibit the clot-formation in-vitro, 2. specific ELISA. In summary, it should be stressed that currently, hypercoagulability disorders can be correctly diagnosed in approximately 80% of the patients suffering from thromboembolism using the appropriate laboratory tests.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S90 TRACEABILITY IN HAEMATOLOGICAL ASSAYS - WHY CELLS ARE DIFFERENT

The In vitro Diagnostic (IVD) Medical Device Directive of the EU requires that “the traceability of values assigned to calibrators and/or control materials must be assured through available reference measurement procedures and/or available reference materials of higher order”.

Although modern haematological analysers report around 50 parameters, internationally accepted haematological reference measurement procedures are available only for haemoglobin, haematocrit, erythrocytes, leucocytes, platelets and reticulocytes. Contrary to clinical chemistry, the typical procedure in many haematological measurements is counting and classifying cells. Differential blood count analysers classify cells by physicochemical properties such as resistance to lysis, light scatter, fluorescence, impedance, resistivity, enzyme activity etc. These properties cannot be metrologically traced to the subjective morphological properties that are currently used to classify subsets of cells by microscopy. In metrological words: the analytes are different. In some cases cell surface markers could be used to define cell subsets more objectively. Haemoglobin is the only haematological assay for which an internationally recognised reference material exists.

Another peculiarity of haematological assays concerns calibrators. Since viable cells change their characteristics rapidly and since they cannot be frozen nor lyophilised it becomes necessary to stabilise them. This process often dramatically changes the properties of the calibrator so that it no longer behaves like a patient sample, i.e. the calibrator is usually not commutable. Therefore, it is strongly recommended that values not be assigned to the calibrator by reference methods. Instead, values should be assigned by the manufacturer’s standing measurement procedure previously calibrated with the reference method using fresh blood samples. Non-commutability is also the reason why manufacturers usually do not provide traceable trueness control materials for internal quality control for haematological assays. Instead, the assigned values or ranges are instrument-specific.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S91 HYPERTENSIVE DISORDERS OF PREGNANCY: A LINK TO CARDIOVASCULAR DISEASE THROUGH THE METABOLIC SYNDROME

Hypertensive disorders of pregnancy (HDP) complicate 5 to 10% of all gestations and remain major causes of maternal and neonatal morbidity and mortality worldwide. These disorders include: preeclampsia (proteinuric hypertension), gestational hypertension (non-proteinuric) and chronic hypertension with or without superimposed preeclampsia. More severe events are attributable to preeclampsia which is responsible, in developed countries, for up to 15% of preterm births. Recent evidence suggests that preeclampsia occurs in two stages. The first stage develops very early in pregnancy and is characterized by alteration in placental perfusion secondary to abnormal implantation, defective trophoblastic invasion and remodelling of spiral arteries in the uteroplacental junction, increased cellular damage leading to oxidative stress and alteration in the production of endothelial mediators. The second stage is a consequent maternal systemic vascular dysfunction leading to multiple organ alterations.

In this presentation, we will review most recent knowledge on the pathophysiology of HDP, and in particular preeclampsia. We will discuss risk factors, biomarkers (including biochemical, haematological and echographic), genetic susceptibility, and preventive measures through clinical trials. We will also expose recent data on the long term risk of cardiovascular disease in these women. Findings from different groups suggest that HDP may uncover an increased susceptibility to cardiovascular disease later in life. Our research group studies the metabolic syndrome as a link between both entities, and observed an increased prevalence of metabolic syndrome in these women. This raises clinical considerations about long term cardiovascular risk assessment in women with prior HDP.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

S92 THE IFCC: CONTRIBUTIONS TO BETTER HEALTH

The main objectives of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) are to advance theory and practice in health services in order to support laboratory professionals, national societies, diagnostic industry and governmental and non-governmental organisations in providing citizens with meaningful and accurate laboratory test results for diagnosis, risk assessment and follow-up.

In the diagnostic process the pre-analytical, the analytical and the post-analytical phases are relevant for the outcome. Standardization of all these important steps improve the overall diagnostic quality and have an enormous socio-economic impact. In essence this concept results in integration of the diagnostic laboratory into Medicine. Considering laboratory diagnostics as a medical discipline and to support decision making by physicians at the wards diagnostic guidelines, standardization of analytical measurement procedures and interpretation of laboratory reports are essential. To reach these goals and to provide physicians with quality oriented laboratory reports the IFCC and its Divisions are continuously fostering this concept by multi-national, interdisciplinary projects. The Education and Management Division in collaboration with the Scientific Division are involved in setting-up evidence based diagnostic guidelines, training in new technologies, interpretation of test results, and establishment of reference systems for harmonization of measurements. Diagnostic algorithms, guidelines for the use of cardiac and bone markers, the reference systems for glycated hemoglobin and enzymes, reference materials for plasma proteins and PSA are examples for these achievements contributing for better health. IFCC educational activities provide the personnel in the diagnostic laboratory with diagnostic and analytical expertise thus being an adequate partner of the clinicians accepting responsibilities for making decisions.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O1 CONGENITAL ADRENAL HYPERPLASIA IN THE 21ST CENTURY AN 18TH CENTURY CLINICAL PRESENTATION

Introduction: We describe a 73 year old patient with undiagnosed classic simple virilising congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency.

Case History: A 73 year old man was admitted for a revision of his left total hip replacement. Postoperatively, the patient became febrile and shocked and was subsequently transferred to ICU. His condition deteriorated further and he had a cardiac arrest. Preoperatively, the patient had a sodium level of 132 mmol/L and the question of adrenal insufficiency was raised. Several investigations, including a short Synacthen test, (SST) were organized. However, a dose of hydrocortisone was given during SST interfering with the final cortisol level. The patient died 4 days postoperatively and a clinical diagnosis of cardiogenic shock secondary to perioperative myocardial infarction was made. Biochemical results on blood collected 2 days pre mortem showed a ACTH level of 1100 ng/L (10–50), cortisol 333 nmol/L, 17 hydroxyprogesterone 420 nmol/L (<7.0), and testosterone 22 nmol/L (F <3.5, M 10–35). At autopsy, virilisation of the external female genitalia, normal female internal genitalia and bilateral adrenal gland hyperplasia were noted. A normal female karyotype was confirmed by chromosome studies. Further DNA analysis showed a heterozygous T to A point mutation in the exon 4 of the 21-hydroxylase gene which results in amino acid substitution Ile 172 to Asn. A second mutation was not detected to date. This is a common mutation usually associated with simple virilising form of CAH.

Discussion: We present a case of a patient who lived as a male but was later found to be a female with classic simple virilising CAH at autopsy. This is an unusual presentation in the modern setting as most of the female patients with classic CAH are now investigated for their ambiguous external genitalia at birth.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O2 CARDIAC TROPONIN I PREDICTS ADVERSE HOSPITAL OUTCOME AFTER CORONARY ARTERY BYPASS GRAFT SURGERY

Introduction: Cardiac damage in coronary artery graft surgery (CABG) is an important contributor to post-operative cardiac dysfunction and delayed hospital discharge. While preoperative factors are known to contribute to poorer postoperative outcome, peri-operative damage has been harder to measure.

Methods: This prospective study of 300 patients having routine CABG was conducted to compare Cardiac Troponin I levels (cTnI) measured on the Abbott AxSYM at 6 and 24 hours postoperatively with ECG as predictors of adverse in-hospital outcome. Preoperative variables were also included in the calculations. Primary outcome indices were post-operative ICU stay and post-operative hospital stay. Outcome variables were stratified by tertiles of cTnI levels. Significance of differences of outcome variables across tertiles was examined.

Results: Multivariate analysis showed 24hr cTnI to be a significant predictor of increased postoperative ICU stay (p = 0.012) and postoperative hospital stay (p = 0.024). For 6hr cTnI corresponding significances were (p = 0.29 and 0.9). ECG was of no value (p = 0.39 and 0.47). Differences in 24hr cTnI levels were highly significant particularly for lowest vs. highest tertiles and allowed stratification of risk into ‘low’, (0–10 ug/L) ‘equivocal’ (10–20) ug/L and ‘high’ (>20 ug/L) categories.)

Discussion: Use of a single 24hr cTnI to quantify peri-operative myocardial damage identifies patients who are at greater risk of adverse hospital outcomes. This strategy should assist in allocation of patients to different management streams after CABG surgery.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O3 LABORATORY DIAGNOSTICS USING ADENOSINE DEAMINASE AND INTERFERON γ IN THE FACE OF A RE-EMERGENCE OF TUBERCULOSIS

Tuberculosis (TB) is a major health problem in many parts of the world including Singapore with 47 cases per 100,000 reported in 2000. Even developed countries are faced with the problem of re-emergence of TB. To shorten the time to a definitive diagnosis we have recently reviewed the performance of Adenosine Deaminase (ADA) and Interferon-gamma (IFNγ) on a cohort of hospitalized subjects requesting a pleural fluid analysis.

Fifty-five subjects (F:M 22:33) with an age range of 16 – 102 years (mean 64 years) were included. The diagnostic categories of the subjects included TB (14.5%), cancer (36%), liver failure (18%), post operative (7.3%), renal failure (6%), empyema (6%), heart failure (2%) and others (10%). ADA was measured using the Guisti method and IFNγ using an ELISA kit from Diaclone Research (France). ROC analysis of ADA reported 100% sensitivity and 95.7% specificity at a cut off of 27.1U/L for TB diagnosis. If polymorphonucleocytosis (PMN) cases (lymphocytes/PMN ratio >0.75) were excluded then sensitivity and specificity were both 100%. For IFNγ ROC analysis obtained a cut off of 153 pg/mL with a sensitivity of 75% and a specificity of 100%.

In this pilot study with small numbers, ADA proved to be superior in terms of ROC area under the curve. We now propose to offer ADA into clinical service giving a sensitive, rapid and inexpensive adjunct in the diagnosis of TB.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O4 proBNP – A BIOCHEMICAL MARKER OF CARDIAC IMPAIRMENT

Introduction: Natriuretic peptides are increasingly finding applications in both diagnosis and prognosis of patients suspected with heart failure, either due to systolic or diastolic dysfunction. ‘NT proBNP’ has been widely accepted as a marker of heart failure. The effect of renal function and other co-morbid states are still under evaluation.

Methods: Analysis for NT proBNP by chemiluminescence using Roche Elecsys 1010 (measured in pmol/L) in two hundred and twelve healthy individuals (136 males and 76 females) and 315 patients presenting to cardiac, respiratory and emergency room units. Detailed clinical and cardiac data were available in 110 patients.

Results: The mean values progressively increased with age in healthy population with wide standard deviation. Categorized by age in years (i) 40–55, (ii) 56–65, (iii) 66–75, the males had a mean of 32.37, 46.53 & 66.51 with SD 31.19, 42.94, 54.47 and the females had a mean of 28.8, 42.3, 54.59 with SD of 40.43, 39.82 & 59.40, respectively. In heart failure BNP has a good correlation with clinical severity independent of left ventricular systolic function.110 patients under NYHA classification had median of 32.90, 136, 339.20 & 1000 with a range of 4.5–500, 6–5690, 9–5000 & 66–8199 in the four classes respectively (Kruskal Wallis Test H=29.64, Df=3, p <0.0001).

Discussion: Though there is an excellent correlation in median levels with clinical severity of heart failure expressed as NYHA class, there is a wide overlap of the values. More systematic study with cut-off values will establish the role of BNP more clearly.

References

  • Current Problems in Cardiology; March & April 2003. Clinical Chemistry. 2003 2002 Feb;49:7. The Clinical Biochemist Review. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O5 REDUCTION IN OXIDATIVE STRESS, INFLAMMATION AND ENDOTHELIAL DYSFUNTION IN PATIENTS WITH NON-FAMILIAL HYPERCHOLESTEROLAEMIA TREATED WITH LOW DOSE STATIN

Introduction: Hypercholesterolaemia is associated with enhanced oxidative stress and endothelial dysfunction, both of which contribute to atherosclerosis. The link between endothelial dysfunction with oxidative stress and inflammation remains unclear. The aim of this study was to investigate the effects of low dose statin on oxidative stress, inflammation and endothelial function in patients with Non-Familial Hypercholesterolaemia (NFH).

Methods: 51 NFH patients (28 males, 23 females, mean±SD age=48.7 ±8.2 years) and 46 normolipaemic controls were recruited. Serum levels of thiobarbituric acid reacting substances (TBARS), oxidised LDL (oxLDL), soluble intercellular adhesion molecules (sICAM-1), highly sensitive c-reactive protein (hsCRP), interleukin 6 (IL6) and brachial artery endothelial-dependent flow mediated dilatation (FMD) were evaluated at baseline, 2 weeks, 3 and 9 months after 10mg/day atorvastatin treatment.

Results: At baseline, compared to controls, the NFH patients had higher levels of TBARS (median [95%CI]: 287 [160–439] vs 112 [50–221] nmol/ g, p<0.0001), oxLDL (56.8 [20.9–188.0] vs 27.8 [0.5–56.4] U/L, p<0.0001) and sICAM-1 (mean ± SEM: 1592.0 ± 73.2 vs 994.5 ± 21.2ng/mL, and lower FMD (mean ± SEM: 2.1 ± 0.9 vs 12.2 ± 3.6%, p<0.0001). TBARS and sICAM-1 levels were reduced by 2 weeks, with further reduction up to 9 months (p<0.0001 and <0.0001 respectively). FMD was increased after 2 weeks treatment, with further increment up to 9 months (p<0.001). Both hsCRP and IL6 levels were reduced at 3 months and 9 months (p<0.01 and p<0.0001 respectively). FMD was negatively correlated with TBARS (p<0.0001), sICAM-1 (p<0.0001) and IL6 (p<0.001).

Discussion: NFH patients have enhanced oxidative stress, inflammatory state and endothelial dysfunction. Low dose statin treatment leads to reduction in oxidative stress, low-grade systemic inflammation and improvement in endothelial dysfunction. Inflammation and oxidative stress are possibly associated with endothelial dysfunction.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O6 GENE POLYMORPHISMS AND HYPERTENSION IN A JAPANESE POPULATION

Introduction: There is a genetic predisposition for hypertension. For our understanding of the genetic factors of essential hypertension, gene polymorphisms have played a significant role as DNA markers in association and linkage studies. In northern Japan, there is a high incidence of hypertension and hypertensive cardiovascular diseases. Therefore, to clarify the relationship between DNA variations and hypertension, we studied the distribution of 16 gene polymorphisms in hypertensive and healthy subjects in the Aomori population in northern Japan.

Subjects and Methods: A total number of 856 Aomori residents were enrolled and 183 hypertensive patients (83 males and 100 females) and 193 healthy subjects (67 males and 126 females) were selected according to the diagnostic criteria in the present study. The genotypes of the gene polymorphisms were determined with electrophoretic patterns of the amplified products with specific primers before or after the adequate digestion procedures. The chi-square test was performed to detect the significant association.

Results: We found positive linkages between hypertension and 5 gene polymorphisms including angiotensinogen M235T (p=0.05), angiotensin-converting enzyme I/D (p=0.01), CYP11B2 T-344C (p=0.03), endothelial nitric oxide synthase Glu298Asp (p=0.04), and mitochondrial C16223T (p<0.01). However, there were no significant differences between the groups in distributions of gene polymorphisms including angiotensin type 1 receptor A1166C, CYP11B2 IC, 4 vasopressin V1a receptor variations, endothelial nitric oxide synthase 4b/a, Plasminogen activator inhibitor-1 4G/5G, aldehyde dehydrogenase-2 Glu487Lys, Y chromosomal Alu insertion/deletion, and mitochondrial C16362T.

Conclusion: These results suggest that the 5 gene variations might be genetic markers of hypertension in northern Japan.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O7 INTERLEUKIN-1 GENE POLYMORPHISMS AND ACUTE MYOCARDIAL INFARCTION IN CHINESE

Introduction: Interleukin-1 gene polymorphisms are contributory factors in inflammatory pathology. These polymorphisms are associated with the change of IL-1 levels and may perhaps play a important role in the development of acute myocardial infarction (AMI). By comparing the frequency of interleukin-1 gene polymorphisms in patients with AMI and healthy controls, to explore their association with AMI.

Methods: We studied the interleukin-1 gene polymorphisms, including IL-1A (−889C/T), IL-1B (−511C/T), IL-1B (+3953C/T), IL-1RN (+8006T/C), IL-1RN intron 2 VNTR, in 178 patients with AMI and in 190 controls, by polymerase chain reaction-restrict fragment length polymorphism technique.

Results: Allele (of IL-1RN intron 2 VNTR[IL-1RN*]) is always present with IL-1RN*C, allele C of IL-1RN(+8006T/C), both in patients with AMI and in controls. Carriers of IL-1RN*(or IL-1RN*C) had a significantly lower risk for AMI, odds ratio (OR) of 0.33 (95% confidence interval, 0.17 to 0.66), compared with noncarriers.

Conclusion: IL-1RN (+8006T/C) is probably in a complete linkage disequilibrium with IL-1RN intron 2 VNTR, whose genotypes were significantly associated with the development of AMI, while the IL-1RN*(or IL-1RN*C) may play a protective role.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O8 DOES PLASMA CHOLINESTERASE (BChE) PHENOTYPE PREDICT AYTPICAL AND FLUORIDE-RESISTANT BChE GENE MUTATIONS?

Introduction: Laboratory testing for scoline apnoea (SA) requires measurement of enzyme activity and detection of the atypical (A), fluoride-resistant (F), or other BChE variants. We compared phenotyping and genotyping methods.

Methods: Subjects: 126 with SA or a family history of SA. Reference interval (R.I.): 24 (21–50 years) with no apparent cause of low enzyme activity, A or F mutations. Phenotyping assay: bche enzyme activity before and after addition of dibucaine or fluoride. Genotyping assay: DNA polymerase chain reaction and restriction enzyme analysis for A, F, and kalow (k) bche variants.

BChE mutationBChE kIU/L R.I. 4.3–11.8 Mean (range)Dibucaine No. R.I. 79–84 Mean (range)Fluoride No. R.I. 86–91 Mean (range)
Heterozygous A: A, AK, AKK (n=47)5.1 (1.7–7.8)72.5 (63–79)87.5 (84–91)
Homozygous A: AA, AAK, AAKK (n=23)2.0 (0.6–6.1)21.1 (15–39)82.3 (75–90)
Heterozygous F: F-2, F-2K (n=5) AF-2K (n=2)3.6 (1.1–5.6)
2.1, 2.3
73.4 (63–79)
58, 57
79.6 (71–86)
78, 74
Negative A or F (n=49)
Positive K (n=34)
6.2 (1.9–11.4)
5.7 (0.9–10.6)
82.1 (78–86)
81.8 (78–86)
87.9 (82–93)
87.7 (82–93)

Phenotyping detected 68 of 72 (94%) patients with the A gene but only 4 of 7 (57%) with the F gene. The Fluoride No. was below the reference interval in 35 patients who did not carry the F gene. No A or F gene was detected for 10 of 49 (20%) patients with low enzyme activity (0.9–3.9 kIU/L), abnormal Dibucaine No. or Fluoride No.

Conclusions: Discordant genotyping/phenotyping results may be due to imprecision of enzyme measurement or the presence of other mutations. Phenotyping can predict A variants but genotyping or sequencing the BChE gene is necessary to predict the F-mutation.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O9 PYROSEQUENCING FOR THIOPURINE METHYL-TRANSFERASE (TPMT) VARIANTS.

Introduction: TPMT has an important role in the metabolism of thiopurine drugs such as azathioprine and mercaptopurine. Patients with TPMT deficiency are at very high risk of developing toxic side effects including profound and life threatening myelosuppression. Three TPMT gene variants (*2, G238C; *3A, G460A + A719G; *3C, A719G) account for most cases of TPMT deficiency. We developed Pyrosequencing based methods for these variants.

Methods: Biotin tagged PCR amplicons spanning exons 5, 7 and 10 of TPMT were captured on streptavidin-coated beads, followed by isolation of single-stranded DNA, hybridization to a sequencing primer and analysis by Pyrosequencing.

Results: Assay design was simple, and the generic sample processing/analysis involved enabled processing for TPMT and other genetic tests (eg HFE, apo E) on the same Pyrosequencing run. All genotypes were automatically and correctly assigned by the Pyrosequencing software. Most of the ~70 patient samples received for analysis were in context of prophylactic screening for variants. Four G460A and A719G heterozygotes were found; the G238C was not found among patient samples but was found in a screen of ~300 controls.

Conclusion: Thiopurine drugs are commonly used but there is a risk of severe side effects in a small proportion of patients. Laboratory monitoring for the most severe side effect, myelosuppression, consists of measuring blood counts after institution of drug therapy. An indication of the patient’s TPMT activity prior to drug therapy to identify at risk subjects would be more appropriate; however, since enzyme activity measurement is problematic, there has been a focus on DNA based methods. We have found Pyrosequencing to be more suitable than dHPLC or dideoxy sequencing for accurate yet rapid analysis of TPMT variants.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O10 THE POLYMORPHISMS OF OESTROGEN-METABOLIZING GENES AND BREAST CANCER RISK — A MULTIGENIC STUDY

Introduction: Endogenous oestrogen plays a very important role in the carcinogenesis and progression of breast cancer. The enzymes involved in the biosynthesis and metabolism of oestrogen have been proposed to contribute to this effect. To examine this hypothesis, we conducted a case-control study to investigate the relationship between polymorphisms of genes responsible for oestrogen biosynthesis (CYP17 and CYP19) and oestrogen sulphation of inactivation (SULT1A1), and the risk of breast cancer in Chinese women.

Methods: 213 pathology proven breast cancer patients and 430 matched controls were recruited. PCR-based RFLP and STRP assays were conducted to detect the mononucleotide transition of CYP17 MspA1 and SULT1A1 Arg213His and (TTTA) tandem repeat polymorphism of CYP19, respectively. Logistic regression analyses were used to determine the OR and 95% CI. The association between genotype and clinico-pathological features were also investigated.

Results: When the CYP17 A2 allele, CYP19 (TTTA)10 and SULT1A1 His allele were considered as the “putative high-risk” genotype, there was an increased risk of breast cancer with the number of high-risk genotypes in a dose-response effect (trend P=0.05), in that those carrying one high-risk genotype had an OR of 1.09 (95%CI 0.74–1.61) and two high-risk genotypes OR of 10.71(95%CI 1.24–92.48). In multivariate analysis, the SULT1A1 genotype remained the most significant determinant for breast cancer with the OR being 2.37 (95%CI 1.24–4.47), followed by CYP19 with the OR of 1.75 (95%CI 1.27–3.56). The (TTTA) 10 allele of CYP19 was associated with tumour size, and the His allele of SULT1A1 associated with status of lymph node metastasis.

Conclusions: This study supports the hypothesis that breast cancer can be initiated by oestrogen exposure. And estrogen-metabolizing genes are involved in this mechanism. Multigenic model was useful in discriminating individuals who are at higher risks of breast cancer.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O11 DETECTION OF ANTIGLIADIN ANTIBODIES IN COELIAC DISEASE - WITHDRAWN

Introduction: Coeliac disease is an immunologically mediated disease of small intestinal mucosa characterized by malabsorption and gluten intolerance. Gliadin is a toxic factor responsible for the damage of the intestinal mucosa in children. Recent studies showed that sera of coeliac patients contain antigliadin antibodies, with titers which is significantly correlation with biopsy results in all clinical stages of the disease. The aim of the study was to validate serological tests, using ROC to diagnose children younger than 16 years.

Methods: Study studied 138 children (aged 6 months to 16 years) in three clinical stages of the disease: 55 in acute phase, 49 in remission during gluten-free diet and 34 in disease relapse. Biopsy findings of the small intestinal mucosa of the patients were taken together with antigliadin antibodies of IgA and IgG class. Serological methods were validated on the basis of their sensitivity, specificity and positive and negative predictive values.

Results & Discussion: Biopsy was found to be positive in 61% of cases, with only one positive case in the remission group. Using IgA cut-off value of 7.7, with sensitivity was 86.9% (77.8–93.3% as 95% confidence intervals) and specificity was 96.3% (87.2–99.4%). Using IgG cut-off value was 25.7, with sensitivity was 90.5% (82.1–95.8%) and specificity was 83.3% (70.7–92.1%). The difference between ROC IgA/IgG areas was 0.064 (SE=0.028), p=0.023.

Conclusion: Results proved that serological methods significantly differ to such extent that their contemporary usage in monitoring of coeliac disease in children is more than justified.

References

  • Pratesi, et al. Scand J Gastroenterol. 2003;38:747–50. [PubMed]
  • Rostami, et al. Rom J Gastroenterol. 2003;12:101–6. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O12 AUTOANTIBODIES PROFILE IN DISCRIMINATION OF PATIENTS WITH LUPUS ERYTHEMATOSUS AND RHEUMATOID ARTHRITIS

Introduction: SLE and RA are recognized by American College of Rheumatology classification criteria and characterized with autoantibodies (AAb) against cell components. We investigated if AAb profile might differentiate clinical diagnose of active SLE/RA disease.

Methods: During one-year follow-up 37 patients with SLE and 18 with RA were re-examined. Standard laboratory parameters were analysed with AAb profile, set for nuclear antigens (ANA), dsDNA, ssA, ssB, Sm, scl-70, U1-nRNP, cardiolipin of IgA, IgG and IgM classes, IgG and IgM phosphatidylserine, and beta2-glycoprotein (enzyme-linked immunosorbent assay, Euroimmun AG, Luebeck, Germany). Data were analysed by chi-square test, t-test, logistic regression and odds ratio (OR) with 95% intervals using MedCalc software (Mariakerke, Belgium). Only p<0.01 was considered significant.

Results & Discussion: No difference in standard biochemical and haematological parameters was found between patient groups, except for leukocyte and monocyte counts (no clinical importance) and rheumatoid factor (71% positive in SLE and 24% in RA). Positive cases were found for twelve AAbs tested, except for IgM phosphatidylserine were all patients were negative. Only two AAbs were found positive in significantly different proportions in SLE/RA patients: ANA, 57% in SLE and 22% in RA, OR=4.6 (1.3–16.7), and dsDNA, only 27% in SLE, OR=8.5 (1.1–71.7). When multivariate regression method was used, only ANA was found significant: OR=6.6 (1.9–23).

Conclusion: Between thirteen AAbs tested, only well established ANA can differ patients with SLE and RA.

References

  • Egner W. The use of laboratory tests in the diagnosis of SLE. J Clin Pathol. 2000;53:424–32. [PMC free article] [PubMed]
  • Hiepe F, Dorner T, Burmester GR. Antinuclear antibody and extractable nuclear antigen-related diseases. Int Arch Allergy Immunol. 2000;123:5–9. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O13 CRP AND LACTATE IN THE DIAGNOSIS AND MONITORING OF TREATMENT OF BACTERIAL MENINGITIS IN CHILDREN

Introduction: The early and differential diagnosis of bacterial meningitis is very important to decrease high mortality and sequelae, especially with the bacterial culture positively less than 15% in Vietnam.

Methods: We measured CRP and lactate in serum and CSF to evaluate their significance in the differential diagnosis and the monitoring of treatment of children affected with bacterial meningitis (group 1, n=48), viral meningitis (group 2, n=32) and tuberculous meningitis (group 3, n=32). For the monitoring of bacterial meningitis, biochemical tests were performed before treatment, 7 days and 14 days after treatment. In addition to the measurement of CRP (turbidimetry) and lactate (technique of Noll), other biochemical and cytologic analysis of sera and CSF, serologic tests and PCR technique (DNA of Mycobacterium tuberculosis) were performed.

Results: The results showed that: 1) Serum CRP: significant difference between group 1 and normal group (p<0.001), between group 1 and group 2, group 3 (p<0.001); 2) Serum Lactate: significant difference between group 1 and normal group (p<0.001), between group 1 and group 2, group 3 (p<0.01); 3) CSF Lactate: significant difference between group 1 and normal group (p<0.001), between group 1 and group 2, group 3 (p<0.001); 4) Monitoring of treatment: the normalisation of CRP level after 14 days of treatment had a good prognostic value. After 7 days of treatment, the decrease of CSF lactate level of more than 50% compared to those before treatment showed good result. Serum lactate had no prognostic value.

Conclusion: CRP and lactate in sera and CSF proved to be sensitive in the early and differential diagnosis between bacterial meningitis and viral, tuberculous meningitis. They also had good prognostic value in the monitoring of bacterial meningitis in children.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O14 ASSOCIATION OF POLYMORPHISMS OF HUMAN SP–A1 AND SP-A2 GENES WITH ALLERGIC ASPERGILLOSIS AND PULMONARY TUBERCULOSIS IN INDIAN POPULATION - WIDTHDRAWN

Introduction: A series of studies from our group, have established that lung surfactant protein A (SP-A) has an important role in strengthening the host defense against both allergic and invasive aspergillosis. SP-A also binds M. tuberculosis and enhances attachment and phagocytosis of M. tuberculosis by alveolar macrophages. In view of the importance of SP-A in host defense against aspergillosis and tuberculosis, structural and functional changes in SPA may affect the outcome of host-pathogen interaction.

Results: We investigated the relationship between SNPs in SP-A1 and SP-A2 genes and susceptibility to ABPA and pulmonary tuberculosis. In the present study, seven SNPs (4 exonic and 3 intronic) have been identified in the collagen regions of SP-A1 and SP-A2 genes in Indian population. Statistical analysis of these SNPs showed four of these SNPs have significant differences in their frequencies observed in ABPA patients vs. controls (SP-A1 C1416T p=0.118; OR = Infinity; SP-A2 T1492C p=0.034; OR = 4.8, SP-A2 G1649C p=0.031; OR = 4.2, SP-A2 A1660G p=0.058; OR = 7.0). Pulmonary tuberculosis patients also showed statistically significant differences in frequencies of four SNPs when compared with control population (SP-A1 C1416T p=0.000; O.R = 20.8, SP-A2 C1382G p=0.005; O.R = 3.7, SP-A2 G1649C p=0.010; O.R = 2.2, SP-A2 A1660G p=0.000; O.R = 12.3).

Discussion: It is interesting to note that three SNPs (SP-A1 C1416T, SP-A2 G1649C, SP-A2 A1660G) are commonly associated with patients of ABPA and pulmonary tuberculosis suggesting that dysfunction of SP-A2 caused by these SNPs may be one of the factors leading to increased risk of pulmonary infections. SP-A2 T1492C and SP-A2 C1382G in Intron 3 are distinctly associated with ABPA and pulmonary tuberculosis respectively. These two SNPs may be useful for identification of differential susceptibility of individuals to allergic bronchopulmonary aspergillosis and pulmonary tuberculosis.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O15 A SERUM MARKER SCORE PREDICTS LIVER FIBROSIS IN CHRONIC HEPATITIS C PATIENTS.

Introduction: We aimed to develop a scoring system based on serum markers which is capable of staging liver fibrosis in hepatitis C patients without the requirement for a liver biopsy.

Methods: Ten candidate fibrosis markers were analysed in serum samples in a training set of 117 treatment naive hepatitis C patients. Multivariate logistic regression was used to develop a predictive score based on the most informative markers, whose performance was then assessed with an independent validation set of 104 hepatitis C patients. Fibrosis was staged on liver biopsies according to the METAVIR system, significant fibrosis was defined as F2, F3 or F4 [cirrhosis].

Results: A score ranging from 0.00 to 1.00 was computed from age, gender, bilirubin, γ-glutamyltransferase, α-2 macroglobulin and hyaluronic acid analyses. At a cut-point of 0.50, the score detected significant fibrosis in the validation set with a positive predictive value of 88% and negative predictive value of 65%. More advanced F3 or F4 fibrosis was excluded with a negative predictive value of 95%. For clinical application to detect significant fibrosis, we propose patients with scores ≥ 0.50 [40% of the validation set] would not require biopsy. Patients with scores below 0.5 [60% of the validation set] would require biopsy to exclude significant fibrosis, but not advanced F3 or F4 fibrosis, for which the negative predictive value of the score was 95%.

Conclusion: We have developed a score based on four serum markers which accurately predicted F2, F3 or F4 fibrosis and excluded advanced F3 or F4 fibrosis in hepatitis C patients.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O16 SERUM PARAOXONASE, HIGH DENSITY LIPOPROTEIN, CARDIVASCULAR RISK: A PARADOX AMONG SOUTH INDIANS???

Paraoxonase (PON), a component of high density lipoprotein (HDL), is a calcium dependent enzyme whose mechanism of action is incompletely elucidated. A cardio- protective role against oxidative damage has been attributed to this enzyme.

In this study we measured serum lipids and PON activity in a representative population from Mysore, South India and compared it with a tribal colony of Pani Yerawas from the same region and a sample population from Louisville Kentucky, USA. American sample had in general a low PON activity and exhibited a strong correlation with HDL. In contrast, the South Indian population had a three-fold higher PON activity and did not show any correlation with HDL. Also, triglycerides did not show negative correlation with HDL. The tribal sample had significantly lower cholesterol triglycerides and PON when compared with either the South Indian population or the American sample. The PON did not correlate with HDL. Interestingly there was no diabetes in this tribal population and no report of any cardiovascular diseases.

It is likely that PON activity is enhanced independently of HDL in the south Indian population. However this increase may not be due to genetic trait alone since a homogeneous sample from the same region having a different lifestyle had significantly lower values. Whether the increase is the result of an agricultural environment cannot be ruled out. Considering the increased genetic susceptibility of Indians to Coronary Artery Disease, South Indian population presents a paradox whether PON is a predictive protective factor of cardiovascular disease in a non-western population.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O17 PROTECTIVE ROLE OF ASCORBIC ACID ON OXIDATIVE DAMAGE IN HYPERCHOLESTEROLEMIA INDUCED ATHEROSCLEROSIS

Introduction: Atherosclerosis (Ath) poses a major threat to human life. Oxidative modification of LDL along with increased cholesterol has been implicated in the pathogenesis of Ath. This evidence prompted researchers to study the effects of antioxidants on atheroma formation, which by preventing or suppressing prooxidant states can act as antiatherogens. In light of the well-known antioxidant properties of ascorbate, we have studied the role of ascorbic acid (AA) on lipid profile, hypercholesterolemia (Hc) induced Ath and oxidative modification of LDL.

Methods: Rabbits were fed cholesterol (100 mg) and AA (0.5, 15 and 25 mg/100 g body weight/day) for different time periods. Serum lipid and lipid peroxidation levels were analysed. The descending aorta was analysed for histological changes. The binding and degradation of native and oxidized LDL was studied to elucidate the role of AA on the LDL receptors of macrophages. LDL isolated by ultracentrifugation from normal and hypercholesterolemic subjects was oxidized in presence and absence of ascorbic acid and its oxidation level studied.

Results: AA at higher doses for a longer duration caused significant amelioration of initial Hc, increased HDL-C and decreased lipid peroxidation level (p=0.0059). The severity of atherosclerotic lesions in the aorta of hypercholesterolemic rabbits was effectively prevented by ascorbic acid (p=0.05). Treatment of LDL isolated from hypercholesterolemic subjects with 80 μM ascorbic acid invitro significantly prevented oxidative modification (p=0.05).

Conclusion: These observations highlight the benevolent effects of ascorbic acid in the management of Ath. The effects pertain to control of Hc, increase in HDL-C, lowering of oxidation level leading to prevention of Ath.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O18 EFFECT OF GARLIC PEARLS ON OXIDIZED -LDL AND 8-ISO-PROSTAGLANDIN F IN PATIENTS OF ESSENTIAL HYPERTENSION

Background: Reactive oxygen species has been implicated in the pathogenesis of many diseases including hypertension. Some components of garlic are known to possess antioxidative properties and are believed to be protective against such diseases. Therefore, in the present study we investigated the effect of short-term garlic supplementation in essential hypertensive patients (EH) on indices of oxidative stress.

Methods: 20 patients of EH (Group I) and 20 age and sex matched normotensive controls were enrolled for the study. Both groups were given garlic pearls (GP) in a dose of 250 mg/day for 8 weeks. Baseline samples were obtained at start of the study and then after 8 weeks. Lipids and lipoprotein fractions, plasma oxidized low-density lipoproteins (ox-LDL), plasma and urinary 8-iso Prostaglandin F (8-iso-PGF), a biomarker of oxidative stress in vivo, and total antioxidant status (TAS) of these individuals were determined.

Results: We observed moderate hypercholesterolemia and significantly raised blood pressure in hypertensive patients as compared to the controls at baseline. Plasma ox-LDL and plasma and urinary concentration of 8-iso-PGF were significantly increased in EH group. Further, hypertensive patients had significantly low TAS as compared to the control group. After 8-wk GP supplementation, there was a significant decline in both systolic and diastolic blood pressure as well as in ox-LDL and 8-iso-PGF levels in EH patients. Further, a moderate increase in the TAS was also observed in this group compared to the controls.

Conclusion: These findings suggest that intake of GP may be beneficial in reducing blood pressure and oxidative stress in hypertensive individuals.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O19 GENETIC BASIS OF FAMILIAL HYPERCHOLESTEROLEMIA IN INDIA

Introduction: Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by multitude low-density lipoprotein receptor (LDLR) gene mutations. To-date over 10 different point mutations has been identified in different exons of the LDLR gene among Indian immigrants. Neither the prevalence nor the types of LDLR mutations causing FH in India are known. The purpose of this study was to define the spectrum of LDLR mutations causing FH in India.

Methods: Using PCR-based heteroduplex/single-strand conformation polymorphism analysis we scanned the entire 18 exons and promoter of the LDLR gene in 65 related and unrelated patients with clinical features of FH and 75 healthy controls. Heteroduplex analysis was performed to scan for mutations in part of exon 26 of the apoB-100 gene to exclude diagnosis of familial defective apolipoprotein B-100 (FDB).

Results: Scanning of LDLR gene revealed two novel insertion mutations (ins242G in exon 3 and ins397G in exon 4) in one patient each, and two missense mutations in exon 9- E387K in an FH family and L393R in a 39yr-old female. None of the subjects showed the presence of mutation in apoB-100 gene.

Conclusion: Four different mutations but no common mutations were identified in this study. In a highly heterogenous population like India with various ethnic groups, it is not unlikely that there may exist mutational heterogeneity of LDLR gene. Considering the fact that there exist various distinct communities in India (even in Mumbai), which has remained segregated for over centuries, it is likely that there might exist some community-based mutations. Also, it would be interesting to evaluate the role of non-LDLR, non-apoB locus ‘FH3’ in the PCSK9 gene that also causes autosomal dominant hypercholesterolemia.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O20 AN UNUSUAL CASE OF PRIMARY AMENORRHOEA IN A 17 YEAR OLD FEMALE

Introduction: A 17 year old female presented with primary amenorrhoea following late pubertal development. The only history of note was of bilateral inguinal herniae, featuring prominent masses, diagnosed at 2½ weeks of age. These were said to contain ovaries at operation.

Methods: Assays were Bayer ADVIA Centaur Testosterone, Progesterone, LH, FSH and Oestradiol, DPC Immulite 2000 Androstenedione and Dihydrotestosterone RIA following Celite Chromatography.

Results: Examination found a tall girl with stage 5 breast development, very early stage 2 pubic hair and a slightly rugose appearance of the labia. Initial screening results revealed a significantly elevated serum testosterone (reference interval 0.5–2.6 nmol/L)

LHFSHOestradiolTestosterone
23 U/L5 U/L<100 pmol/L12.3 nmol/L

Chromosome analysis revealed a 46XY karyotype. Further examination showed a 6cm vaginal passage and a right-sided gonad was seen at ultrasound. These indicated either a defect in the synthetic pathway to 5α-dihydrotestosterone or androgen resistance syndrome in a phenotypic normal female with a male genotype.

The androgen pathway was investigated by appropriate blood steroid measurements – progesterone 1.6 nmol/L, androstenedione 7.4 nmol/L and 5α-dihydrotestosterone 5.2 nmol/L.

Discussion: Steroid results indicated a normal androgen synthetic pathway thus excluding defects in the enzymes 17α-hydroxylase, 17-ketosteroid-reductase, 17,20-lyase and 5α-reductase. This patient is a genetic male with a defect in androgen sensing resulting in a normal female appearance. In retrospect the inguinal masses discovered in infancy were normal testes, not ovaries. Testosterone, LH and chromosome testing are essential in people presenting with primary amenorrhoea.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O21 A PROTEOMICS STUDY OF BREAST CANCER REVEALED ENHANCED EXPRESSION OF CK19 IN HER-2/NEU POSITIVE TUMOURS

The HER-2/neu oncogene encodes a 185 kD transmembrane protein (c-erbB2) that is phosphorylated upon ligand binding, promoting a signal transduction pathway and regulating cell growth and differentiation. C-erbB2 overexpression is strongly associated with advanced disease, metastasis and poor clinical outcome. To better understand the mechanism underlying the poor prognosis of breast tumours caused by HER-2/neu positivity, we adopted a proteomics approach, first separating the proteins found in frozen breast tumours versus those without HER-2/neu gene amplification, using two-dimensional gel electrophoresis and then identifying those proteins showing differential expression by peptide mass fingerprinting using matrix-assisted laser desorption ionization time of fight mass spectrometry (MALDI-TOF MS). Our results showed that cytokeratin 19 (CK19) was consistently overexpressed in HER-2/neu positive breast tumours. These results were further confirmed by Western blotting and immunoblotting analyses, as well as by semi-quantitative reverse-transcription-polymerase chain reaction (RT-PCR, p=0.024) and reverse-phase protein array (p=0.013). Immunohistochemical staining of microscopic sections from a previously constructed breast tumour tissue microarray (TMA) revealed that moderate to strong staining of CK19 was observed in 22 of the 27 HER-2/neu positive tumours (81.5%) and in 24 of 50 HER-2/neu negative tumours (48%) with significant difference (p=0.0068). These findings indicate that CK19, an intermediate fragment, is involved in the tumour proliferation and metastasis linked to HER-2/neu oncogene amplification in metastatic breast cancer and could possibly be a new supplementary prognostication biomarker of breast tumour progression.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O22 LOW FREE TO TOTAL PROSTATE SPECIFIC ANTIGEN (f/t PSA) RATIO IS NOT A GOOD DISCRIMINATOR OF CHRONIC PROSTATITIS AND PROSTATE CANCER: AN INDIAN EXPERIENCE.

Introduction: The effect of inflammation on f/t PSA ratio was retrospectively studied in patients with prostate cancer, BPH and chronic prostatitis.

Methods: 72 patients (19 prostate cancer, 15 BPH and 38 chronic prostatitis patients) with mean ± SD age of 66.28±10.3 years were selected. Routine urine analysis with culture, semen culture, Serum tPSA and fPSA, digital rectal examination (DRE) and transrectal ultrasound (TRUS) were done in all these patients. The diagnosis was confirmed histopathologically. The total and free PSA were analysed on Roche 1010 auto analyser.

Results: The patients with tPSA more than 2 ng/mL, DRE or TRUS findings suspicious for cancer and f/t PSA ratio less than 0.16 were chosen. The mean ± SD value for tPSA, fPSA and f/t PSA ratio were 17.23±11.8 ng/mL, 1.54±1.08 ng/mL and 0.095 in cancer, 7.0±3.9 ng/mL, 0.73±0.34 ng/mL and 0.10±0.032 in BPH and 12.63±11.93 ng/mL, 1.04±0.62 ng/mL and 0.089±0.040 in chronic prostatitis patients, respectively. One-way ANOVA with posthoc analysis showed tPSA and fPSA values were significantly different (p<0.028 and p<0.005), However, f/t ratio was not significantly different (p<0.279) in all these groups.

Discussion: Low f/t PSA ratio is used as a discriminator for BPH and prostate cancer in conjunction with DRE and TRUS findings; however, our results showed similar changes in chronic prostatitis. This change in f/t PSA ratio in prostatitis must be considered to interpret the results correctly.

References

1. Thakur V, et al. Disease Marker. 2004;10:41. in press.
2. Jung K, et al. J Urol. 1998;159:1595–8. [PubMed]
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O23 PROSTATE SPECIFIC ANTIGEN (PSA) AND PROSTATE CANCER RISK AMONG MEN PARTICIPATING IN A CANCER PREVENTION TRIAL

Introduction: PSA velocity defined as the rate of increase in total plasma PSA per year >0.75 μg/L/year, or an annual change of PSA >20% have been suggested as markers that accurately predict prostate cancer. To test this hypothesis we measured total PSA in a nested case-control study utilising serial PSA tests on men aged 43–81 years over an eleven year period.

Methods: Plasma samples were collected annually from participants prior to diagnosis, who were followed-up for prostate cancer at the end of 2000. Three controls were age matched to each prostate cancer case. Total PSA was measured on an Architect immunoassay analyser at baseline and annually for three years prior to the year of prostate cancer diagnosis.

Results: 59 prostate cancer cases were identified. Baseline PSA levels was significantly higher in men who subsequently developed prostate cancer than in controls; median PSA of 3.28 μg/L (range 0.36 to 112) and 0.73 μg/L (range 0.07 to 13.8), respectively. Using three consecutive PSA results the median PSA velocity for cases was 1.31 μg/L/year (range −3.73 to 38.13) and 0.05 μg/L/year (range −1.9 to 2.4) for controls. A PSA velocity >0.75 μg/L/year had a sensitivity of 59% and a specificity of 88% whereas, a PSA change >20% between analysis 2 and 3 had a sensitivity of 54% and a specificity of 63%. After adjusting for age, smoking status and alcohol intake, PSA velocity had a superior odds-ratio (OR=9.3, 95%CI 2.3 to 37.8) to a >20% increase in PSA level (OR=3.3, 95%CI 1.1 to 10.3) for detecting prostatic cancer.

Conclusions: Although PSA velocity >0.75μg/L/year was a more reliable indicator of prostate cancer than a 20% annual increase in PSA, the sensitivity was only 59%.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O24 PLASMA EPSTEIN-BARR VIRUS DNA CONCENTRATION ACCURATELY REFLECTS TUMOUR LOAD IN NUDE MICE IMPLANTED WITH NASOPHARYNGEAL CARCINOMA XENOGRAFTS

Introduction: Nasopharyngeal carcinoma (NPC) is closely related to Epstein-Barr virus (EBV) infection. Recently, plasma EBV DNA has been shown to be a good tumour marker for the diagnosis, monitoring and prognostication of NPC1,2. The rapid elimination of plasma EBV DNA after surgical resection of tumour is good evidence that plasma EBV DNA is released from the tumour cells3. The development of an animal model for studying this phenomenon would be very useful.

Methods: EBV positive (C666 and 2117) and negative (HK1) NPC xenografts are injected intraperitoneally into nude mice. The tumours were resected and weighed after 6 to 12 weeks. EBV DNA and human albumin DNA concentrations of the plasma and tumours were measured by real-time PCR.

Results: EBV DNA could be detected in the plasma of all mice implanted with EBV-positive xenografts. The tumours weighed from 0.05 to 0.54 g, and the plasma EBV DNA concentrations ranged from 464000 to 2330000 copies/mL There is a positive correlation between the plasma EBV DNA and the product of the weight and the EBV DNA content of the tumours (r = 0.693). However, in mice implanted with EBV-negative xenografts, only human albumin DNA but not EBV DNA could be detected in their plasma.

Discussion: Our findings show that EBV-positive NPC xenografts are able to release EBV DNA into the plasma of nude mice and the concentration of plasma EBV DNA reflects the tumour load. This new animal model would allow one to study further aspects of the release of tumour-associated nucleic acids into the circulation.

References

1. Lo YMD, et al. Cancer Res. 1999;59:1188–91. [PubMed]
2. Lo YMD, et al. Cancer Res. 2000;60:6878–81. [PubMed]
3. To EWH, et al. Clin Cancer Res. 2003;9:3254–9. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O25 PROGNOSTIC IMPLICATIONS OF MICROSATELLITE INSTABILITY IN GASTRIC CARCINOMA

Background: The aim of this study was to compare the MSI genotypes (MSS, MSI-L and MSI-H) with the clinicopathological variables and to identify some of the genetic differences between early (EGC) and advanced gastric carcinoma (AGC) as well as between the TNM stages.

Methods: The presence of MSI was examined using seven chromosomal arms (2p, 3p, 4q, 5q, 17p, 17q and 18q) with 13 microsatellite markers in 34 gastric carcinoma cases using tumour-normal paired samples. The data were analysed by genescan and genotype software, then was compared with the clinicopathological variables.

Results: There was a statistical significance found in the Borrmanns’ type (EGC vs AGC) and lymph node spread (Absent vs Present) compared with the MSI genotyping using the clinicopathological variables, and in the lymph node spread (Absent vs Present) compared with the RER genotyping. However, the MSI and RER genotypes had a similar significance in tumour location, differentiation, metastasis, and TNM stage. The frequency of the MSI according to each marker revealed a significant difference at D17S250 (p = 0.046) and D17S520 (p = 0.046) between EGC and AGC as well as at D17S520 (p = 0.038) and BAT26 (p = 0.042) between the TNM stages.

Conclusion: BAT26 and D17S520 might be useful loci for predicting a poor prognosis, Moreover D17S250 and D17S520 for the histological progression in a gastric carcinoma. Furthermore, this study suggests that MSI or RER genotyping of gastric carcinomas is a useful, independent tool for predicting diverse clinical courses.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O26 DEVELOPMENT OF SHORT MESSAGE SYSTEM (SMS)-BASED CRITICAL VALUE REPORTING SYSTEM

Introduction: Critical laboratory results are defined as test results which could provoke significant clinical outcomes of patients and should be managed as soon as possible. In our laboratory, laboratory personnel have reported critical results by calling to nurses and writing down test item, test result, reporting personnel, reporting time, and the person reported in laboratory information system (LIS). However, our conventional reporting system was subject to missed or delayed reporting due to the possibility of errors by laboratory personnel and nurses. To improve these shortcomings, we developed a SMS-based critical value reporting system.

Methods and Results: The LIS and the hospital information system (HIS) of Asan Medical Center were constructed as client server systems with NCR3600 as a server and with Oracle 8i as database. When patient’s test result is within the critical range, the LIS displays a specific flag. After confirming that the result is a real critical value, laboratory personnel make the data retrievable in the HIS by clinicians. At the same time, the information of the result including ‘test code, test result, patient’s identification number, patient’s name and ward, and doctor’s name’ is automatically transmitted to clinician’s pager or cellular phone. All these procedures were programmed by Powerbuilder and Visual C++.

Conclusions: We developed a critical value reporting system with SMS to prevent missed, delayed, or erroneous reporting to clinicians and to make the process of critical value reporting easier than before. With the introduction of SMS-based critical value reporting system, more prompt and improved patient care are anticipated.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O27 NOVEL DIAGNOSTIC TECHNOLOGY FOR ALLERGIC AND INVASIVE ASPERGILLOSIS

Introduction: International reference standards or WHO reference standards are not available for immunodiagnosis of allergic and invasive Aspergillosis. Availability of diagnostically relevant synthetic epitopes of the major allergens/ antigens of Aspergillus fumigatus will undoubtedly facilitate development of standardized diagnostic reagents. The present study established the diagnostic relevance of a synthetic epitope and suggested the possible use in development of nanotechnology-based sensors.

Results: An eleven amino acid synthetic peptide (P1) of Asp f 1 showed significant binding to specific IgG and IgE antibodies in sera of patients of allergic bronchopulmonary aspergillosis (ABPA) and inhibited IgG binding (89.10 ± 4.45 %) and IgE binding (77.32 ± 3.38 %) of the standardised diagnostic antigen of A. fumigatus (SDA). The P1 peptide induced specific IgE and IgG antibodies in mice and stimulated proliferation of PBMC’s of ABPA patients (Stimulation index: 5.132) with a Th2 type of cytokine profile similar to that of SDA. BIA core analysis also showed that peptide P1 has 100-fold higher binding with the purified IgG of ABPA patients’ (KD = 4.19 e−7 M) than with the purified IgG of normals (KD = 1.11 e−5 M). A 15-mer control peptide (Yersinia pestis) did not show any significant difference in binding with IgG fraction from patients (KD = 7.57e−5 M) and normal (KD = 3.62 e−5 M).

Discussion: Peptide based immunosensors can be developed using nanotechnology. Micro-cantilever and nano-mechanical cantilever sensors can detect very small biological and chemical entities by changes in surface stress or changes in the mechanical resonant frequency of the cantilevers. Immobilised diagnostically relevant epitopes such as P1 on the cantilever, as bioreceptors capture an antibody, the change in vibrational frequency of the cantilever can be detected and measured. This ultrasensitive method detects specific antigen or antibody in clinical samples.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O28 CLINICAL APPLICATIONS OF WHOLE BLOOD POLYMERASE CHAIN REACTION USING REAL TIME THERMOCYCLERS.

Introduction: We describe an innovative method for the detection of clinically significant SNPs. This method includes real time detection of PCR products generated directly from whole blood (WB-RTmPCR) and does not require DNA extraction. We have demonstrated the utility of this approach using the Roche LightCycler and Applied Biosystems’ 7700.

Methods: We performed a study comparing WB-RTmPCR with analysis of extracted DNA, also by real-time analysis for identification of mutations within the prothrombin (G20210A), factor V Leiden (G1691A) and haemachromatosis (G845A and C187G) genes.

Results: We obtained complete concordance for the prothrombin G20210A (87 GG, 7 GA), factor V Leiden G1691A (41 GG, 7 GA, 1 AA) and the haemachromatosis G845A (14 GG, 5 GA, 3 AA) and C187G (11 CC, 5 CG, 4 GG) mutations.

Discussion: We have demonstrated that this approach can be used successfully for melting curve analysis using FRET probes on the LightCycler, and the flurogenic 5’ nuclease assay (TaqMan) on the 7700. WB-RTmPCR has greatly streamlined work flow and sample processing and has additional benefits including reducing labour costs, test turnaround time and the potential for sample contamination. The methods have been used for genotyping on thousands of samples and have proved to be robust. The approach has applications for testing other clinically relevant genetic variants.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O29 POINT OF CARE TESTING: GLUCOMETER PROFICIENCY TESTING

Introduction: In 2002 Royal Perth Hospital enrolled in MediPro, the glucose proficiency testing program for MediSense glucometers. Our central laboratory is responsible for sample distribution, result submission and review of reports, for nearly 100 glucometers. To assist us, we developed some modifications to the program.

Reporting Results: We record our results electronically on a Word table, for clarity and to sort entries by location, identification or results. We fax results to the QAP Office, but would prefer to use e-mail.

Review: MediPro reports use the format developed by RCPA-AACB Chemical Pathology, with allowable limits of ± 10%, or ± 1.0 if <10mmol/L. We use our instrument group median instead of the overall median and apply review limits of ± 10% and action limits of ± 15% if ≥ 6.5mmol/L, or action limits of ± 1.0 if <6.5mmol/L

A summary page, showing histograms for all results and our instrument group results, with our site’s results overlaid, would aid report interpretation. On the review page, identification of meters with results outside the instrument group, rather than overall, limits would be preferable.

Follow-up: We circulate a monthly report summary to all users. Those with results outside review limits are contacted. The hospital’s technical service department provides a monthly report of meters inspected for faults. Our comparison studies using fresh human samples show differences between glucometer and laboratory autoanalyser glucose results to be <20%.

Outcomes: Development of a comprehensive nursing practice standard for operators. Management of common instrument failures. Decrease in the number of meters with results outside the action limits from an average of 3 per month (3.7%) in 2003 to one result only in the first four months of 2004 (<0.3%).

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O30 TOWARDS COMMON REFERENCE INTERVALS

Introduction: The current paradigm places the responsibility for establishing or validating reference intervals on individual laboratories. The AACB Working Party on Common Reference Intervals was established in 2003 to move towards common reference intervals for Australia and New Zealand. We describe problems with the current paradigm and steps required for common reference intervals.

Problems with the current paradigm: Unless methods and reference intervals are standardised, interpretation of results is difficult if patients have results from more than one laboratory. Placing results with varying reference intervals into common databases is difficult to accommodate. It is our experience that most laboratories do not perform reference interval validation to a high standard. The costs and difficulties of performing this task are high, particularly with the large number of assays offered in many laboratories. A recent survey of reference intervals for common analytes showed no correlation between assay results and reference intervals.

Acceptance of common reference intervals: Projects in Scandinavia, Spain and New Zealand are currently developing and implementing common reference intervals. Additionally common decision points are already accepted for many analytes, eg glucose, lipids, therapeutic drugs.

Steps towards common reference intervals: Common reference intervals can only be applied if any true between-method differences are not significant. Thus method standardisation and development of criteria for sharing intervals are fundamental to the process. Additional issues are the development of the correct interval for multiple laboratories and procedures for adoption.

Conclusion: The working party will develop policies and procedures to move towards the adoption of common reference intervals, where technically allowable, across Australia and New Zealand.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O31 LOW-RANGE ELEVATIONS OF TROPONIN I IN PATIENTS WITHOUT PREVIOUS ISCHAEMIC HEART DISEASE- EFFECT ON 180 DAY MORTALITY

Introduction: Troponin I is a biochemical marker of myocardial injury that has high prognostic significance in the setting of acute coronary syndromes. Frequently patients with non-cardiac disease also have mildly elevated troponin levels and it is unclear, whether they are also at increased risk of cardiovascular events.

Methods: We did a prospective case-control cohort study, where patients with mild elevations of troponin I (0.8–2 ug/L on the Abbott AxSYM) admitted for non-cardiac reasons were matched with patients without troponin I elevations. Patients with a history of ischemic heart disease (IHD) were excluded. Follow up was obtained at 6 month post discharge. The Simplified Acute Physiology Score (SAPS II) was used to control for severity of illness. Uni- and Multivariate Statistical analysis was done using the SAS program.

Results: 581 patients were screened. 261 patients without a history of IHD were evaluated. Chest pain was present in only 37 (22 cases, 15 controls, p=0.16). While cases were age and sex matched to controls, SAPS scores were different at baseline (34.8 vs. 22.6, p < 0.01). Ischemic ECG changes were more frequent in the cases (21.8 vs. 2.2%, p < 0.01). Significant independent predictors of 6-month mortality in multivariate analysis were heart rate p< 0.01, severe organ insufficiency p < 0.01, SAPS II p = 0.016, and age. Troponin I was not an independent predictor of either LOS or mortality at 6 months.

Discussion: In this case-control study mild troponin I elevation was not found to be an independent predictor of LOS or 6-month mortality. If these findings are confirmed, the frequent occurrence of mildly elevated troponin levels in sick patients might not need cardiological intervention, but rather treatment of the underlying condition.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O32 PREDICTIVE & PROGNOSTIC VALUES OF INCREASED CORD BLOOD AND NEONATAL URINARY F2-ISOPROSTANES IN ASPHYXIATED NEWBORNS

Introduction: Oxygen free radicals have been proven to play an important role in causing neuronal injury during perinatal asphyxia. We reported an increased umbilical cord arterial organic hydroperoxides and malondialdehyde in acidaemic newborns and in situations known to lead to intrapartum hypoxia. We demonstrated the elevated lipid peroxides correlated with xanthine, supporting hypothesis that hypoxia-reperfusion of foetal tissues during labour leads to free radicals formation via xanthine oxidase activation. We also showed F2-isoprostanes, a non-enzymatic catalysed oxygenation of arachidonic acid in membrane phospholipids, as a reliable marker for in vivo oxidative stress in foetuses.

Methods: We correlated cord blood and neonatal urinary lactate, organic acids and F2-isoprostanes to clinical evidence of severe asphyxia and subsequence neurodevelopmental handicap using GCMS validated.

Results & Discussions: The relationship between raised cord arterial 8-isoprostane and birth asphyxia in association with significant neonatal morbidity was confirmed. Serial changes in neonatal urinary 8-isoprostane and organic acids between neonates with poor and good outcomes were significantly different. Umbilical arterial 8-isoprostane and neonatal urinary 2,3-dinor-8-isoprostane were the biochemical discriminators between asphyxiated and non-asphyxiated infants, and also between those who were subsequently classified as having poor and good neonatal outcome. The best determinants in neonates between perinatal HIE and good neonatal outcome amongst infants asphyxiated at birth were cord arterial base excess and lactate. Elevated urinary F2-isoprostanes correlated with the prognosis of later neurodevelopmental handicap with significant sensitivity and specificity, indicating its clinical relevance.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O33 IS SUBCLINICAL HYPOTHYROIDISM A RISK FACTOR FOR CARDIOVASCULAR DISEASE? AN ANALYSIS FROM THE BUSSELTON HEALTH STUDY.

Introduction: Hypothyroidism is associated with hyperlipidaemia, but the effects of mild and subclinical hypothyroidism on lipid metabolism are unclear, and it is uncertain whether hypothyroidism increases the risk of cardiovascular disease.

Methods: Serum TSH and free T4 were measured on 2115 stored serum samples from the 1981 Busselton Health Survey (Western Australia). In a cross-sectional study, the results have been correlated with cardiovascular risk factors and the presence of cardiovascular disease in participants. In a longitudinal study, vital status and hospital admission for cardiovascular disease up to the end of 2001 have been determined from the WA Hospital Morbidity Data System.

Results: In females, serum TSH showed a strong positive correlation with total cholesterol (P<0.0001) and triglycerides (P<0.0001). In males, TSH was correlated with triglycerides (P=0.025), but not with cholesterol. Cholesterol was significantly higher in subjects with hypothyroidism (TSH>4mU/L) compared to euthyroid subjects (mean ± SD 6.34 ± 1.37 vs. 5.80 ± 1.17, P <0.001), as was serum triglycerides (1.65 ± 1.31 vs. 1.44 ± 1.03, P=0.032). Cholesterol and triglycerides were each significantly (P<0.01) higher in subjects with mild hypothyroidism (TSH 4.01–10 mU/L) than in unequivocally euthyroid subjects (TSH 2.0–4.0 mU/L). These differences persisted after adjustment for age, gender, BMI and diabetes.

Conclusions: Mild hypothyroidism is associated with significant elevations in serum total cholesterol and triglycerides. It remains to be determined to whether hypothyroidism is associated with increased cardiovascular risk in this population.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O34 THE PREVALENCE OF THROMBOPHILIC MUTATIONS IN TASMANIAN PATIENTS WITH VENOUS THROMBOSIS

Introduction: Venous thrombosis (VT) is a complex disease involving the interaction of genetic and environmental risk factors. It has been estimated that more than 60% of the predisposition to VT is attributable to genetic components. Factor V (FV), Prothrombin (PT) and Methylenetetrahydrofolate reductase (MTHFR) gene mutations have been implicated in increasing the risk of a venous thrombotic event. This study estimated the allelic frequency of three common FV, PT and MTHFR gene mutations in a healthy Tasmanian population and Tasmanian patients with a history of VT.

Methods: The thrombophilic mutations FV G1691A (Leiden) and PT G20210A were genotyped by a mutagenically-separated polymerase chain reaction and MTHFR C677T was genotyped using a polymerase chain reaction restriction fragment length polymorphism in 100 clinically diagnosed thrombotic patients and 120 controls

Results: The allelic frequencies of the FV, PT and MTHFR mutations were 0.5%, 5.0 % and 45.5% respectively in VT patients compared to 2.5%, 1.7% and 35.8% respectively in controls.

Discussion: An association of PT and MTHFR mutations with VT was established (PT: χ2 = 3.925, p = 0.047; MTHFR: χ2 = 4.240, p = 0.039) while the FV mutation was not shown to be associated with an increased risk for venous thrombosis (χ2=2.833, p=0.130; OR = 0.192, 95% CI = 0.023 – 1.622) in this study. The surprising absence of an association of the thrombophilic FV mutation with VT possibly strengthens the roles played by PT and MTHFR mutations in the pathogenesis of VT in the Tasmanian population. Clinicians investigating patients for hereditary causes of VT should be aware that FV mutations do not appear to play a major role in the causation of this disease in this population. It would be interesting to confirm this with a larger study size, and also to look at the incidence of confirmed FV-related VT in this cohort.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O35 RELATION BETWEEN LEPTIN AND INSULIN IN PATIENTS WITH TYPE II DIABETES MELLITUS

Introduction: Leptin and insulin are reported to control glucose metabolism therefore, a relationship between these two hormones should reveal the metabolic effect of these hormones on glucose metabolism. In this study, the relationship between leptin and insulin was evaluated in obese diabetes type II, BMI>30 kg/m2 (group I) and normal weight diabetes type II patients, BM<25kg/ m2 (Group II).

Study design: 49 subjected were studied. Of these, 32 subjects (4 male and 28 female) were group II and 17 subjects (8 male and 9 female) were group I. After measuring the weight and height and taking other information, a fasting blood sample was obtained from each individual and evaluated for levels of leptin, insulin, and HbA1c.

Results: The results obtained showed leptin, insulin and HbA1c levels of 5.16± μg/L and 19.07±14 μg/L, 6.75±1.2 μIU/mL and 10.4 μIU/mL and 9.38 ± 0.56% and 8.76 ± 0.36% in groups II and groups I, respectively.

Discussion: The results of this study show that plasma concentrations increased with the percentage of body fat and to body mass index. Plasma leptin in obese diabetes, in comparison to non-obese diabetes individuals, was three times higher (p=0.000).

The observation that leptin levels are elevated in proportion to body fat (r=0.211, p=0.245) conforms to the generally accepted idea that most obese individuals are resistant.

Statistical analysis indicates a direct correlation between fasting blood leptin and insulin (r=0.290 p=0.05) in group II, while this correlation is reverse in group I (r=−0.089 p=<0.05). The negative correlation of leptin with insulin in group I (obese diabetes) indicates that leptin may have a suppressive effect on the secretion of insulin in β cells.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O36 CHRONIC α-TOCOPHEROL REDUCES OXIDATIVE STRESS AND RESTORES VASCULAR DYSFUNCTION IN PATIENTS WITH ESSENTIAL HYPERTENSION

Background: Endothelins and nitric oxide have been implicated in the pathophysiology of hypertension and atherosclerosis. In the present study we have tested the hypothesis that oxidative stress could modulate and influence endothelin-2 (ET-2) and nitric oxide (NO) release in patients with essential hypertension (EH). In addition we have evaluated, whether chronic administration of α-tocopherol (vitamin E) supplementation could help in reducing vascular oxidative stress and improve these important indices of vascular dysfunction.

Methods: The study was conducted in 20 patients suffering from EH (Group I) and in 20 normotensive control subjects (Group II). Both groups received vitamin E capsules in a dose of 400mg/day for twelve weeks. Basal and 12-week follow-up blood samples were obtained from these subjects for determination of circulating plasma ET-2 levels. Systemic NO production was assessed as plasma nitrite (NO2) levels. Lipid peroxidation as malondialdehyde (MDA) production and the total antioxidant status (TAS) of these subjects was also determined.

Results: We observed significantly higher levels of plasma ET-2 and MDA in EH group. The NO production and TAS was significantly lower in the same group as compared to their control counterparts. Chronic treatment with vitamin E for 12 weeks significantly restored NO levels and TAS in EH patients, almost equal to the levels observed in the control group. Interestingly, vitamin E administration significantly attenuated plasma ET-2 levels in EH group.

Conclusion: These data suggest that ET-2 along with NO, play a major role in vascular dysfunction in hypertension associated with oxidative stress. This hypothesis needs to be validated in a large number of hypertensive individuals to reach a logical conclusion.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O37 AN AUTOMATED MICROBIOLOGICAL ASSAY FOR THE MEASUREMENT OF RIBOFLAVIN (VITAMIN B2) IN SERUM.

Introduction: Homocysteine is an independent risk factor for vascular disease such as stroke. Recent reports have suggested an inverse relationship between vitamin B2 and homocysteine in patients with the methylenetetrahydrofolate reductase C677T mutation. Since 12% of the population are homozygous and 42% heterozygous for this thermolabile mutation, adequate intake of riboflavin may provide some protection from hyperhomocysteinemia and ultimately vascular disease. Current methods for riboflavin are either non-specific or expensive.

Aim: To develop a sensitive, direct automated assay for the measurement of serum riboflavin.

Methods: A microbiological assay using a chloramphenicol resistant strain of L. casei as the test organism was developed. Addition of chloramphenicol and cycloheximide to the assay medium suppressed bacterial and yeast contamination and enabled the assay to be automated without recourse to aseptic procedures. A Gilson 222 robotic diluting system linked to a Gilford Stasar III spectrophotometer and controlled by an Epson PC was used for setting up and reading the assays. Using this equipment 120 tests per hour can be set up and following 24 hours incubation, can be read at 60 tests per hour.

Results: The optimum concentrations of nutrients for the organism were determined and an appropriate growth medium formulated. There was no significant difference in the growth response of the test organism to all three naturally occurring forms of vitamin B2. The new method was found to have a satisfactory analytical performance with linearity of 1, mean recovery of 101.4%, intra-assay and inter-assay precision of 4% and 9% respectively.

Discussion: A sensitive, automated, economical method for the assay of riboflavin has been developed. The method is sensitive to 5 nmol/L of riboflavin and has proven to be highly reproducible and is suitable for diagnostic purposes or population studies.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O38 ROLE OF INTESTINAL PARASITES IN THE DIETARY LYSINE REQUIREMENT OF CHRONICALLY UNDERNOURISHED INDIAN MEN

Introduction: We previously used the 24-h indicator amino acid balance method to show that the lysine requirement in undernourished Indian men from low socioeconomic and unsanitary environments is approx 50% higher than the requirement of well-nourished men. It is possible that this higher lysine requirement in persons with chronic undernutrition is due to environmental influences, including the presence of intestinal parasites. We assessed this possibility by using 24-h indicator amino acid balance at both the “normal” requirement for lysine intake and the higher requirement, before and after successful treatment to eradicate intestinal parasites in affected, undernourished men.

Design: 14 chronically undernourished men were studied before and after treatment for intestinal parasites, during each of two 7-d diet periods supplying either 30 (n = 7) or 45 (n = 7) mg lysine kg−1 d−1 from an L-amino acid diet. Twenty-four-hour indicator amino acid balance was estimated on day 6 by [13C] leucine tracer infusion.

Results: Before the parasite treatment, subjects were in neutral 24-h leucine balance at both lysine intakes. After the eradication of intestinal parasites, there was a significant (P < 0.001) improvement in 24-h leucine balances, which were positive at both lysine intakes.

Conclusions: Thus it appears that intestinal infestation with parasites may be one factor responsible for the higher lysine requirement observed in persons with chronic under nutrition.

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O39 TRANSFERRIN AND IRON COMPLEXES: THE KINETICS AND MECHANISM OF IRON (III) EXCHANGE

Introduction: The knowledge of kinetics and mechanism of iron up-take is important in view of the use of Fe (III) complexes to saturate transferrin for therapeutic purpose. This study has been concerned with the in vitro kinetic and mechanism of iron exchange between iron (III) complexes and apotransferrin.

Methods: The iron (III) complexes and chelators were synthesised in our laboratory. The purity of human apotransferrin and iron complexes were examined by FPLC/HPLC methods. The in vitro kinetic studies and mechanism of the reactions between apotransferin and Iron (III) hydroxypyranones and hydroxamic acids complexes were followed by the intensity of quenching fluorescence. The in vitro intestinal absorption of complexes was examined using everted gut sac method. Atomic absorption spectrometer and protein assay were performed to measure the mole fraction of iron/transferrin.

Results: The spectrofluorometric data showed that apotransferrin is able to remove iron from complexes. The follow up of the reactions revealed that the ternary complex of Iron-Chelate-Transferrin made this process favourable. Furthermore, the tendency of iron uptake was dependent on the complex concentrations. The speed of iron exchange was rapid (less than a minute). Everted gut sac results confirmed the iron absorption is facilitated by the character of the complexes.

Discussion: In spite of the tight binding of iron with iron chelators, apotransferrin could take out iron from the complexes. The process is based on the conformation of the chelator and the structure of the iron complex. Therefore, the lability and structure of iron chelator could be a key element for the design of iron complexes for iron absorption.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

O40 STUDY OF THE EFFECTS OF RAW GARLIC ON LIPIDS AND OTHER BLOOD BIOCHEMICAL FACTORS IN HYPERLIPIDAEMIC INDIVIDUALS

Background and Objectives: Atherosclerosis is one of the main factors of morbidity and mortality in developed countries. Coronary arterial atherosclerosis leads to angina pectoris and myocardial infarction. Garlic has been considered as one of the blood lipids lowering agents for ages, and various studies have been carried out that some of them confirmed this effect of garlic and some did not. The aim of this study was to evaluate the effect of raw garlic in human blood biochemical factors.

Materials and methods: This was a quasi-experimental study that carried out in 30 volunteer individuals with blood cholesterol higher than 245 mg/dL. Fasting blood samples were collected for biochemical tests. The volunteers consumed 5 g raw garlic twice a day for 42 days. Second fasting blood samples were collected and the individuals were told not to use any garlic for next 42 days after which the third fasting blood samples were collected and the biochemical factors was measured in a specialised pathobiology laboratory. Blood pressure and body weight were also measured in all three stages.

Results: The mean of total cholesterol and triglycerides in blood were reduced significantly 9.8% (P<0.01) and 13.9% (P<0.05) respectively after 42 days of garlic consumption. 42 days of no garlic consumption total cholesterol and triglycerides were 8.7% (P<0.01) and 16.1% (P<0.05) significantly less than the beginning of study respectively.

Other blood biochemical factors like FBS, HbA1C, urea, creatinine, uric acid, ALT, AST, ALP, HDL, LDL and also BMI and blood pressure were not change significantly during the study.

Conclusion: Garlic consumption alone can decrease serum lipids, but it cannot use as the main therapeutic agent for hyperlipidaemia. Garlic can be used in mild hyperlipidaemia or when the patients cannot tolerate the chemical drugs.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P1 BIOLOGICAL VARIATION OF N-TERMINAL PRO-BNP IN STABLE HEART FAILURE

Introduction N-terminal proBNP (proBNP) is a plasma biomarker for heart failure. We evaluated the within-individual biological variation (CVwi) of proBNP in patients with stable heart failure.

Methods Heart failure patients were enrolled during hospital admission. Samples were taken at home visit follow-up after 2 weeks, 3 months and 6 months. Stable disease was defined by the absence of death, heart transplantation, cardiac-related hospital admission or change in number of cardiac medications during 12 months follow up. ProBNP was measured using a Roche Elecsys 2010 analyser with all measurements for each patient performed in the same analytical run. Within-run imprecision of the assay was < 2.5% and between-run precision (CVa) was < 6%.

Results Data was available from 17 patients of which two were subsequently excluded as statistical outliers. The median of all proBNP results from the included patients was 2260 pg/mL (range: 380 to 10,440 pg/mL) and the average CVwi was 23% (range: 3 – 47%). With less stringent criteria for disease stability a higher average and maximum CVwi were obtained.

Conclusions This result gives a lower average CVwi in stable heart failure than has been described in healthy people (33%, Wu et al, 2004). The Roche proBNP assay is performing at the optimal level (CVa = 0.25 x CVwi) and the Critical Difference at the 95% confidence level is a 66% change in concentration. We note that these conclusions are based on average data with stringent criteria and higher variation may be found in some individuals with stable disease.

Acknowledgments

We thank Roche Diagnostics Australia for proBNP reagents.

Reference

  • Wu HB, et al. Am J Cardiol. 2003;92:628–631. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P2 DIAGNOSTIC PROCEDURES FOR HYPOURICEMIA

Introduction: Hypouricemia is defined as a serum urate level less than 2mg/dL (119umol/L). Primary hypouricemia may be caused by several purine metabolic disorders including mutations in SLC22A12 gene encoding human urate transporter. Secondary causes in different disorders are related to proximal tubular damage, use of uricosuric drugs etc. Hypouricemia and urinary uric acid investigation are sometimes overlooked. Therefore we have set up a diagnostic guideline for differential diagnosis.

Methods: Uric acid was quantified by a specific enzymic method and red cell enzyme with urinary purine nucleosides were measured by methods adapted to HPLC (1).

Results: A proposed scheme for the investigation of unexplained hypouricemia is a follows. Estimation of :1) excretion fraction of uric acid, 2) urinary xanthine, S-sulphocysteine, thiosulfate, 3) (deoxy)guanosine, (deoxy)inosine in urine, in positive cases performing an assay of purine nucleoside phosphorylase in erythrocytes. The evaluation of the patient’s case history with attention to urolithiasis, seizures and immunodeficiency is important. This flow chart allows the differentiation of a) hereditary renal hypouricemia, b) hereditary xanthinuria, c) molybdenum cofactor deficiency, d) purine nucleoside phosphorylase deficiency. Moreover, primary hyperuricemia can be excluded. Using this flow chart we have detected four new families of Czech origin with hereditary xanthinuria, one case of hereditary renal hypouricaemia and one patient with molybdenum cofactor deficiency from 3,200 serum and urine samples received during the last three years.

Discussion: The exclusion of primary hypouricemia allows one to search for the other causes. Available guidelines will help the early and cost-effective diagnosis of purine disorders with hypouricemia.

References

1. H.A. Simmonds et al. In: Techniques in diagnostic human biochemical genetics. F. Hommes, ed., Wiley-Liss, 1991:397–425.
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P3 HYPERHOMOCYSTEINEMIA IN IRANIAN PATIENTS WITH CORONARY ARTERY DISEASE

Introduction: An elevated plasma total homocysteine (tHcy) level has recently been established as an independent risk factor for coronary artery disease (CAD). The aim of this study was to evaluate plasma tHcy and lipid risk factors in a group of Iranians with coronary artery disease (CAD) and a control group.

Methods: In this case-control study, plasma tHcy in 50 patients (23 females, 27 males) with various degree of coronary stenosis, and 50 subjects (25 females, 25 males) with normal coronary angiograms, were determined by high performance liquid chromatography (HPLC). Lipids and apolipoprotein analysis were done by enzymatic and immunochemical methods respectively.

Results: Plasma tHcy in CAD patients (Mean ± SD, 20.6 ±13 μmol/L) was significantly higher than controls (13 ± 4 μmol/L, p = 0.001), and did not correlate with lipid risk factors but correlated with age.

Conclusion: There is a statistically significant difference in plasma tHcy levels between controls and patients with CAD. The mean plasma tHcy in the present study population are relatively high as compared to most other published reports. It seems that novel cardiovascular risk factors, including hyperhomocysteinemia are more common in some areas of Iran. This may be explained partly by the high rate of cardiovascular disease prevalence in the Iranian population.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P4 INDUCIBLE NITRIC OXIDE SYNTHASE (INOS) GENE POLYMORPHISM IN PREGNANCY INDUCED HYPERTENSION (PIH):- A PILOT STUDY IN NORTH INDIA.

Background: The preeclamtic placenta is associated with increased release of free radicals and cytokines which are known to regulate expression of INOS. INOS in turn is responsible for maintaining myometrial tone in normal pregnancy. The familial nature of PIH preeclamsia clearly indicates a significant genetic component. In the present study the candidate gene is INOS.

Aim: To study the etiopathological role of 1) nitric oxide (NO) and Superoxide dismutase (SOD), 2) Tumor necrosis factor (TNFα), & 3)INOS gene polymorphism in PIH.

Material & Method: Thirty (30) PIH patients and thirty (30) healthy pregnant women were selected for this study. They were screened at 28th, 36th weeks of gestation and just after delivery. Blood samples were analysed for NO, SOD, TNFα and INOS gene polymorphism.

Observation: In PIH patients at 36th weeks of gestation serum NO level significantly increased (p=007) whereas SOD activity decreased significantly (p=004). A double fold increase was observed in TNF level at 36 weeks in PIH patients (p=003) and then a decrease (p=001) after delivery. Two statistically significant single nucleotide polymorphisms (SNPS) were observed in PIH cases. These are A300G exon8 NOS2A and T274G exon16 NOS2A. These polymorphisms showed association with TNFα level and SOD activity.

Conclusion: PIH is a disease of multifactorial etiopathology. NO, TNFα, SOD activity and NOS2A polymorphism might play an intermingled role in the development of PIH.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P5 ANTI-CYCLIC CITRULLINATED PEPTIDE IN RHEUMATOID ARTHRITIS

Introduction: Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease. RA is diagnosed according to clinical manifestations. Serologic support is restricted to the determination of IgM rheumatoid factor (RF), which has a low specificity since it may be found in healthy elderly individuals, healthy immunised subjects, and patients with other autoimmune diseases or chronic infections. A new serological test, the anti-cyclic citrullinated peptide (anti-CCP) ELISA has been shown to have excellent specificity for the diagnosis of RA, especially in patients with early disease. The aim of this study was to determine the clinical utility of anti-CCP reactivity in rheumatoid arthritis patients.

Methods: Samples from 51 rheumatoid arthritis patients and 29 rheumatoid disease free controls were collected and assayed for IgM-RF & anti-CCP antibodies. Patient charts were reviewed for demographic information, clinical diagnosis, radiographic information, and other laboratory data.

Results: Anti-CCP level in the control group was noted to range from 0.0 to 17 units whereas in the RA patients it was from 0.0 to 332.7. There was a statistically significant difference between the control and the disease population (p< 0.05). The sensitivity and specificity of anti-CCP reactivity for the diagnosis of rheumatoid arthritis (RA) were 80.4% and 100% respectively, whereas for RF the same was 72.5% and 80.3%. From our data, we observed that there is a significant correlation between RF and anti-CCP (p<0.05). Significant correlation was observed between disease activity and the level of RF, but not with anti-CCP.

Conclusion: With their better specificity, anti-CCP antibodies can be useful in establishing the diagnosis of RA, but IgM RF is a better predictor of disease severity.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P6 ALTERATION OF APOLIPOPROTEINS AND LDL SIZE IN STATIN TREATED CARDIAC RISK PATIENTS

Introduction: Statins are used for treatment of hypercholesterolemia in humans. It blocks the HMG CoA to mevalonic acid reaction (1,2). Recent emerging evidence shows that subclasses of LDL, characterized by variations in density, size and chemical composition of LDL particles are also of clinical importance. It is well documented that the prevalence of small dense LDL is closely associated with high plasma levels of triglycerides and low plasma levels of HDL-cholesterol (3).

Methods: 36 Stain treated and 45 normal individual blood samples were collected and used for the following analysis. Fasting blood samples were collected and used for the following analysis of total cholesterol, triglycerides, direct VLDL, LDL, apolipoprotein AI, AII, B, CII, CIII and E. Mean LDL particle diameter was determined by density gradient polyacrlymide gel electrophoresis.

Results: A total of 81 subjects were recruited and the results of various parameters in the statin treated patients and control groups are shown in the table below.

ParameterStatin treated patients (36)Control (45)
Total cholesterol (mg/dl)95 ± 12140 ± 11
HDL (mg/dl)52 ± 746 ± 5
LDL (mg/dl)48 ± 8105 ± 9
Triglyceride (mg/dl)89 ± 11140 ± 12
Apo AI (mg/dl)131± 10122 ± 8
Apo B (mg/dl)47 ± 598 ± 7
Apo C II (mg/dl)5.4 ± 0.84.8 ± 0.4
Apo CIII (mg/dl)8.3 ± 0.68.7 ± 0.9
Apo E (mg/dl)4.2 ± 0.73.7 ± 0.5
LDL Particulate size (mg/dl)27.6 ± 3.225.8 ± 2.1

Conclusion: This study found that increase in HDL/Apo B, Apo CII/Apo C III ratio and LDL particle size reflects the efficacy of statin treatment.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P7 CORRELATION OF N-TERMINAL PRO-BRAIN TYPE NATRIURETIC PEPTIDE LEVEL AND ECHOCARDIOGRAPHIC PARAMETERS.

Introduction: N-terminal pro-brain type natriuretic peptide (NT-proBNP) has been identified as a promising biochemical marker for congestive heart failure (CHF). Recently, a NT-proBNP assay using an electrochemiluminescence method has been developed. We evaluated clinical utilities of NT-proBNP assay and its relationship with echocardiography

Methods: NT-proBNP was measured in eighty-four patients who underwent an echocardiographic examination. We compared NT-proBNP level with several echocardiographic parameters for left ventricular systolic function and cardiac chamber dimensions.

Results: NT-proBNP level was significantly increased as with decreasing left ventricular ejection fraction (LVEF). It was also positively correlated with the left ventricular mass, left ventricular mass index, left ventricular end diastolic dimension and left ventricular end systolic dimension. In patients with left atrial and ventricular enlargement, mean level of NT-proBNP was 7418.4 ± 9937.7 pg/mL, which was significantly elevated compared with the mean level of 354.3 ± 749.8 pg/mL in patients with normal cardiac chamber dimension (P< 0.001).

Conclusion: NT-proBNP showed a significant correlation with echocardiographic parameters, especially for systolic function and chamber enlargement. In short, NT-proBNP seemed to be a new promising alternative biochemical marker for CHF.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P8 SCREENING OF BUTYRYLCHOLINESTERASE (BCHE) GENOTYPE IN ORDER TO EVALUATE THE RISK OF PROLONGED APNEA AFTER SUCCINYLCHOLINE ADMINISTRATION

Introduction: Assay of serum BChE has several applications such as, diagnosis of intoxication with organophosphorous insecticides, neural affecting gases (war gases) and hepatic disease. In addition, determination of BChE genotypes and frequency is important. The individuals bearing abnormal genes of BChE may develop prolonged apnea after they receive succinylcholine. Therefore, frequencies of BChE genotypes determine special genotypes of BChE susceptibility to succinylcholine for evaluation of risk of prolonged apnea after receiving succinylcholine. We determined the BChE genotype frequency and risk of apnea after administration of succinylcholine in an Iranian population (Kermanshah).

Methods: we examined the frequency of BChE genotypes in 1553 volunteers and apparently healthy people from the Kermanshah population. By measurements of total BChE activity and percent of inhibition by dibucaine (dibucaine number DN), fluoride (FN) and Ro2 (Ro2N) are helpful in genotyping of individuals. SPSS was used for the statistical analysis.

Results: Reference range for serum total BChE activity was 4.6–16.5 U/ml (butyrylthiocholineIodide was substrate). The mean value for men (9.03) was significantly (p<0.05) higher than women (8.55U/ml). The frequency of four allele U,A,F,S were calculated as BChE1U=0.9826, BChE1A=0.01867, BChE1F=0.00934, BChE1S=0.00161. The frequency genotypes of BChE for UU, UA US UF and AA AF AS FF SS are 94.829%, 4.443% and 0.78% respectively.

Conclusion: Our study showed that in comparison with other studies the populations in west Iran (Kermanshah) can be considered as a population with high frequency of abnormal variants of BChE. Therefore, the measurement of BChE activity and genotyping would be advisable for any one who is to receive the muscle relaxant(succinylcholine).

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P9 STROMELYSIN-1 (MMP-3) GENE 5A/6A PROMOTER POLYMORPHISM IS ASSOCIATED WITH BLOOD PRESSURE IN A COMMUNITY POPULATION

Introduction: Vascular remodelling of large and small arteries contributes to the development of hypertension. Evidence suggests that stromelysin-1 (MMP-3) contributes to arterial remodelling. The expression of stromelysin-1 gene is partly regulated by a polymorphism in the promoter region of either five or six consecutive adenosine bases (5A/6A).

Methods: A cross-sectional study of 1,111 randomly selected community subjects (553 males and 558 females), age 27–77 years, who were assessed for conventional cardiovascular risk factors and the stromelysin-1 5A-1171/6A genotype.

Results: The association between stromelysin-1 genotypes and blood pressure was recessive and only with the 5A/5A genotype. Subjects with the 5A/5A genotype had a higher mean SBP (+4.2 mmHg) and DBP (+2.2 mmHg) compared to subjects with 5A/6A and 6A/6A genotypes combined. Subgroup analysis stratified by blood pressure medication status, revealed an independent association of 5A/5A genotype with SBP (+3.6 mmHg, P=0.001) and DBP (+2.0 mmHg, P=0.004) in subjects not on medication. Subjects with the 5A/5A genotype and taking medication had a higher mean SBP (+7.4 mmHg, P=0.02) and DBP (+2.7 mmHg, P=0.11) compared with 5A/6A and 6A/6A genotypes.

Conclusion: In this large randomly selected, cross-sectional population, the 5A/5A variant in the stromelysin-1 gene promoter was independently associated with increased blood pressure.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P10 ORAL CANCER: IT’S ASSOCIATION WITH TOBACCO, OXIDATIVE STRESS AND GENETIC SUSCEPTIBILITY

Aim and objective: Tobacco is known to generate free radicals resulting in alterations to antioxidant enzymes like, glutathione-ß-transferase (GST), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) as well as lipid peroxidation (LPx), total thiol and total antioxidant status (TAS). Therefore, it is of fundamental importance to evaluate the role of tobacco, antioxidant enzymes and oxidative stress markers in oral carcinogenesis.

Subjects and methods: 140 untreated oral cancer patients (PT) were enrolled for the study. Fifty healthy controls were further classified as smokers (WHT, n=25) and non-smokers (NHT, n=25). Blood and tissue samples were collected. The parameters were analyzed by spectrophotometric methods while gst-M1 genotype was analyzed using PCR amplification.

Results: WHT showed significantly low SOD (p=0.028) and thiol levels (p=0.033) while higher GPx (p=0.017) as compared to NHT. RBC GST, GR, SOD and CAT activities were significantly higher (p<0.05)) in PT as compared to WHT. GPx and thiol levels were significantly lower (p=0.025, p=0.0001, respectively) in PT as compared to WHT. Plasma GST values were significantly higher (p=0.002) and GR lower (p=0.05) in PT as compared to WHT. WHT with elevated levels of RBC GR, SOD, CAT, and lifetime tobacco exposure while lower plasma thiol levels showed higher risk of oral cancer development (p<0.05). GST, GR and SOD activities were significantly higher (p=0.005) while CAT and thiol levels were significantly lower (p<0.015) in malignant tissues than adjacent normal tissues. 63% of the PT had GST-M1 null genotype.

Conclusion: antioxidant enzymes showed risk of oral cancer development in WHT with higher antioxidant enzymes, higher lifetime tobacco exposure and lower oxidative stress markers. Individuals with GST-M1 null genotype may be at higher risk of oral cancer development.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P11 EFFECT OF AGE, SEX AND GLYCAEMIA ON BNP CONCENTRATIONS IN DIABETICS

Introduction: The Abbott Axsym B-type Natriuretic Peptide (BNP) assay is a new marker of heart failure. This study examines the effects of age, sex and both acute (plasma glucose) and chronic (HbA1c) glycaemic status on BNP measurements in a diabetic outpatient population.

Methods: 149 anonymised EDTA samples, with age and sex data available, on outpatients for HbA1c analysis (Roche Tinaquant) were analysed for glucose (Roche glucose oxidase on Roche 917) and BNP. Abbott suggests use of a 100 pg/mL cutoff and a lower limit of detection of 15 pg/mL. SPSS v12 (SPSS Corp) was used for logistic regression analysis.

Results: There were 89 women and 60 men with mean age 57.9 y (SD 14.2). Median glucose and HbA1c were 7.3 mmol/L and 7.0% respectively. The BNP results showed a skewed distribution (25, 50, 75, 95, 97.5, 99th percentiles: 0, 17.46, 83.35, 434.22, 1320.41, 2112.29 pg/mL). 48% of BNP measurements were undetectable. BNP was raised in 27% women and 17% men. Age prevalence of raised BNP rose from 12% (≤40y) to 38% (≥81y). In logistic regression analysis, increasing age was significantly associated with both detectable and raised BNP (odds ratios: 1.61 per decade, p 0.001; 1.43 per decade, p 0.025 respectively) while sex, HbA1c and glucose were not.

Discussion: Diabetic control does not appear to confound BNP levels. Other studies have shown both increasing age and female sex to be associated with increased BNP measurements (1) but this study supports the former only, suggesting that sex-stratified reference intervals are not required for the Abbott assay.

References

1. McCullough PA, Torbjorn O, Maisel AS. Rev Cardiovasc Med. 2003;4(2):72–80. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P12 HIGH CYCLOSPORIN CV’S IN THE RECIPIENTS OF KIDNEY TRANSPLANTS

Introduction: To ensure adequate immunosuppression while minimising toxicity, monitoring of cyclosporin concentrations in blood is an invaluable and essential aid in adjusting dosage This study was to emphasize issues related to intra-individual variability in cyclosporin pre-dose blood concentrations after transplantation.

Methods: 63 kidneys transplant recipients comprised of 17 female, 46 male with a mean age of 36 years (18–66 years), who provided 427 pre-dose blood samples, were studied. Cyclosporin was given orally and pre-dose blood levels were measured using a radioimmunoassay technique. The mean trough level and cyclosporin coefficient of variation (CV) were calculated for each patient. Clinical and laboratory data were recorded for calculation.

Results: The inter-patients variability in the pre-dose blood levels of cyclosporin ranged from 47 to 2700 ng/ml (mean: 261 ng/ml). Due to low exposure and high inter-individual variability (CV) in cyclosporin pre-dose blood levels, about 50% of the kidney transplant recipients experienced graft rejection. The mean intra-cyclosporin CV was 53% (range: 0.4%–269%) that seems higher than pioneer transplant center. To investigate the relationship between the frequency use of trough level measurements and selected outcomes, patients were divided into two groups according to their mean C0 (including two subgroups of C0 ≤ 200 and C0 >200 ng/ml) and cyclosporin coefficient of variation (including two subgroups of CV ≤ 50 and CV>50%). The results showed that the important risk factor predictive of poorer one year graft outcome was a low degree of cyclosporin exposure (≤200 ng/ml) and high cyclosprin CV’s (p = 0.001).

Discussion: To minimize the incidence of rejection, monitoring should follow a structured program. Targeting at sufficiently high mean cyclosporin levels with a low intra-individual variability may help to further improve long-termrenal allograft survival.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P13 THE EFFECT OF DIFFERENT THICKNESS OF THE SAME TISSUE SECTIONS ON THE COLORIMETRIC DETERMINATION OF COLLAGEN CONTENT-WITHDRAWN

Introduction: In clinical and experimental studies, it is important to measure the collagen content quantitatively in tissues for studying the development of fibrotic changes. A simple and reproducible method has been reported by López-de-León and Rojkind (1) routinely to estimate collagen content in 15 μm thick-paraffin-embedded sections. But there can be problems with studying 15 μm thick- sections The aim of this study is to evaluate the reliability of the colorimetric collagen measurement method with different tissue sections and staining procedures (2).

Methods: 40 Bile duct ligated-female Wistar Albino rats were used in this study. Collagen content was measured in 320 liver-tissue sections. Two different staining procedures were performed with 3, 6, 9 and 15 μm tissues. ANOVA and Pearson’s correlation were used in the statistical evaluation.

Results: Collagen contents of four different thicknesses of the same tissues showed a high correlation with each other (r mean= 0, 9896, p<0.01). The results of the different staining procedures are highly correlated (r mean= 0, 9638, p<0.01).

Discussion: This method is not affected by tissue thickness and different staining procedures.

References

1. López-de-León A, Rojkind M. A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded section. J Histochemical Cytochem. 1985;33:737–43. [PubMed]
2. Gascon-Barre, et al. Estimation of collagen content of liver specimens. J Histochem Cytochem. 1989;37:337–81. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P14 SIGNIFICANCE OF THE LABORATORY EXAMINATION TO THE PRIMARY BILIARY CIRRHOSIS DIAGNOSIS

Introduction: To investigate the clinical value of laboratory examinations in the diagnosis of primary biliary cirrhosis (PBC) according to abnormal parameters of serum biochemistry and immunity.

Method: 40 cases of PBC were selected in our in-patient department in our hospital. All the patients matched the diagnostic standard for PBC established by Heathcote. Serum aminotransferase (AST), alkaline phosphatase(ALP), gamma glutamyl transpeptidase (GGT), bilirubin(BIL), antinuclear Antibody (ANA), antimitochondrial antibody (AMA), anti-smooth muscle antibody (SMA), immune-globulin (Ig) estimations were evaluated.

Results: Aminotransferase level was slightly elevated. ALP and GGT increased to 10 or more times the upper limit of normal together with BIL and IgM. 32 (80%) cases were AMA positive, 12 (30%) cases were ANA positive, 8 cases were AMA negative, but with a high titer of ANA and SMA and high IgG concentration.

Conclusions: PBC is predominantly seen in middle-aged women. Typical symptoms are hepatosplenomegaly, jaundice, pruritus, and abdominal pain. Liver function tests indicate a lesser damage of hepatocytes and sevecholestasis in PBC. High level of immune-globulin and a positive autoimmune antibody (particularly AMA) reveal more autoimmune disease characters in PBC. Above all, the laboratory examinations can be done as the screening test for PBC because it is relatively harmless to the patient.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P15 GLYCOSAMINOGLYCAN SYNTHESIS IN HUMAN FETAL ORGANS.

Medical termination of pregnancy is allowed in India. Some patients have tubal ligation at the same time. Fetuses were obtained by hystrotomy. The object of the investigation was explained to the parents and permission to use the fetuses was obtained. Necessary clearance was obtained from the Ethical Committee of the Institute.

Generally speaking, the growth of a specific cell of an organ is achieved by infiltration into a connective tissue matrix and subsequent proliferation. One of the constituents of the matrix is glycoaminoglycans and this was measured in developing fetal organs from 11 to 20 weeks of gestation. Total mucoplysacharides measured as uronic acids in mg/g of fetal liver varied from 0.74 ± 0.16 at 13 weeks to 0.56± 0.07 at 20 weeks of gestation. In human fetal brain the unronic acid content in mg/g varied from 1.88 ± 0.20 at 13 weeks to 2.39 ± 0.11 at 20 weeks One of the enzymes needed for synthesis of mucopolysaccharide is glucosamine - 6 - phosphate synthesis. Its activity in fetal liver in umole glucosamine - 6 -phosphate - g protein/hr varied from 88.0±7.5 to 68.0 ± 6.1 In fetal brain the uronic acid varied from 0.20 ± 0.02 at 13 week to 0.11 ± 0.02 at 20 weeks in fetal brain glucosamine -6-phosphate synthesis activity varied from 11.5 ± 2.6 to 14.0± 0.7 at 20 weeks. It thought that the brain may not have much extra cellular space.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P16 PSEUDOHYPERKALAEMIA IN PATIENTS WITH EXTREMELY HIGH WHITE CELL COUNTS

Introduction: Pseudohyperkalaemia is commonly encountered in children presenting with acute lymphatic leukaemia (ALL) and very high white cell counts (WCC). Centrifugation of blood can easily disrupt the leukaemic cells and release potassium (K).

Methods: Since the beginning of 1999, 14 children with ALL have presented with WCC greater than 300 X 109/L. We found their first whole blood K result (measured as part of a “blood gas”) and the corresponding plasma K measurement, performed after centrifugation at either 3000 rpm for 10 minutes or 1500 rpm for 15 – 20 minutes if the patient was known to have a very high WCC. Results were only compared if the times between the specimens were less 20 minutes.

Results: Comparable results were obtained in six of the patients, as follows:

Initial WCC (x 109/L)Whole Blood K (mmol/L)Plasma K (mmol/L)
3323.73.8
3333.67.3
3803.45.8
7153.02.2
8124.09.8
9402.22.2

Conclusion: Plasma K’s measured following either normal or slow centrifugation in patients with very high white cell counts are highly variable and often falsely elevated. Measurement of whole blood K is much more reliable and must be used to monitor K in these patients.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P17 ANALYTICAL EVALUATION OF THE BAYER RAPIDPOINT 405 BLOOD GAS ANALYSER

Introduction: The Bayer Rapidpoint 405 blood gas analyser was evaluated for imprecision, paracetamol interference with glucose, and sample comparison on different samples and laboratory analysers. The analytes tested were pH, pCO2, pO2, THb, O2Hb, COHb, MetHb, HHb, iCa++, Na+, K+, Cl and glucose.

Methods: The comparative systems were Bayer Rapidlab 865 blood gas, Roche Hitachi Modular and Sysmex SE 9000 analysers. There were 56 arterial blood gas (ABG), 61lithium heparin plasma and 56 K2EDTA whole blood samples analysed for the sample comparison. Samples were obtained from within the hospital and selected to cover the widest possible measuring range.

Results: Acceptable precision was obtained for all the analytes at the different concentration levels (within-run CV range 0.04–5.90 %; between-run CV range 0.03–2.54 %). Paracetamol up to 483 mg/L produced no effect on the glucose analysis. No outliers were excluded in the sample comparison analysis and there were no aberrant unexplained results. Good correlations were obtained with the Rapidlab 865 with r2 values of 0.94 to 1.00 and Deming regression slopes of 0.91 to 1.08. Correlations with the Hitachi Modular analysers were acceptable (r2 values of 0.97 to 0.99; Deming regression slopes of 0.98 to 1.02), except for the Na+, which gave a mean bias of −7.1 mmol/L. This bias is related to the difference in ISE measuring systems and protein concentration in samples. The THb comparison with the Sysmex SE 9000 showed slight mean bias of 6.3 g/L, r2 0.99 and slope 0.96

Conclusions: From the data obtained in this study, the analyser showed good analytical performance. The analyser is easy to use for all level operators. There was an unanimous preference for it by the laboratory operators using the analyser during this study over the current Rapidlab 865.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P18 METHYLENE CHLORIDE EXPOSURE AND CARBOXYHAEMOGLOBIN LEVELS IN CABINET MAKERS

Introduction: Methylene Chloride (MeCl2) is a clear colourless volatile sweet smelling lipophilic solvent used as a constituent of wood varnishes and paints. Human exposure is mainly due to inhalation and its biotransformation by the hepatic mixed function oxidases (MFO) leads to formation of Carbon Monoxide (CO). Simultaneous exposure to MeCl2 and increased ambient CO results in undesirably increased carboxyhemoglobin (COHB) formation, which predisposes to carboxyhaemoglobinemia with the central nervous system as the primary target organ of toxicity.

Method: Ambient CO levels were determined in different parts of Ibadan, Nigeria and work place microenvironments of 100 cabinet makers (Test group) and 100 volunteers (controls) in non-furniture making occupations using a CO personal monitor. Questionnaires were administered and Carboxyhaemoglobin levels were determined in blood drawn from both groups by a differential spectrophotometric method.

Results: Ambient CO levels in Ibadan were observed to be between 4 and 52 ppm with a mean value of 20 ppm. Work environment CO levels were significantly higher in test subjects than controls at 5.2 ± 1.08 ppm and 2.08 ± 0.91 ppm respectively (P < 0.001). COHB in cabinet makers with mean working hours of 9.48 ± 2.9 per day was (3.95 ± 1.35%) while that of controls with mean working hours of 8.0 ± 0.8 per day was 1.08 ± 0.71% (P <0.001). Smoking however did not significantly affect COHB levels within the two groups (P > 0.05).

Discussion: It is imperative to replace Methylene Chloride for safer chemicals in wood varnishes and paints, and the use of protective gas masks and adequate ventilation should be mandatory whenever MeCl2 is used.

References

1. Verma A, et al. Carbonmonoxide-A putative neural messenger. Science. 1993;259:381–384. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P19 SERUM ANION GAP IN METABOLIC ALKALOSIS AND ACIDOSIS

Introduction: The serum anion gap is decreased in hyperchloremic acidosis and increased in diuretic -induced alkalosis The common thread observed in almost each instance of profoundly elevated anion gap values is a multi factorial pathogenesis that usually includes renal insufficiency, associated with a proven or likely cause of organic metabolic acidosis, or with exogenous phosphate intoxication

Methods: Anion gap is defined as ([Na]+[K]) − ([CL]+[HCO3]), where Na is sodium, K is potassium Cl is chloride, and HCO3 is bicarbonate. On the other hand, the anion gap is, in fact determined by the concentrations of unmeasured anions and unmeasured cations. All analyses were performed in the biochemistry laboratory a consisted of 75 normal volunteers, 92 patients with metabolic acidosis and 92 patients with metabolic alkalosis. Serum sodium and potassium were determined by flame photometry. Serum bicarbonate level was measured as total carbon dioxide content. Serum chloride level was determined by chlorimetric titration with silver ions.

Results: all patients with metabolic acidosis had a statistically significant increase in their mean anion gaps (29.4 ± 3.7 mEq/L), when compared with normal control volunteers (15.3 ± 2.4 mEq/L), P<0.05. Patients with metabolic alkalosis had a statistically significant increase in their mean serum anion gap (28.3 ± 4.5 mEq/L), when compared with normal control volunteers (15.3 ± 2.4 mEq/L), P<0.05.

Discussion: The results of our study confirm that metabolic acidosis is associated with a high anion gap. Also, the results of this study confirm that metabolic alkalosis is associated with a high serum anion gap.

References

1. De Keijzer MH, Koene RA, Baadenhuijsen H, Willems JL. The magnitude of the anion gap. Ned Tijdschr Geneeskd. 1999;143 (13):666–667. [PubMed]
2. Paulson WD. Effect of acute pH change on serum anion gap. J Am Soc Nephrol. 1996;7(2):357–363. [PubMed]
3. Oster JR, Gutierrez R, Schlessinger FB. Effect of hypercalcemia on the anion gap. Nephron. 1990;55:164–169. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P20 3 SIBLINGS WITH CONGENITAL DISORDER OF GLYCOSYLATION TYPE 1A WITH A MILD INTELLECTUAL PHENOTYPE

Introduction: The congenital disorders of glycosylation (CDG) syndromes are a clinically heterogenous group caused by deficiencies of enzymes required for protein glycosylation. CDG Type 1a is characterised by a deficiency of the cytosolic enzyme phosphomannomutase (PMM), which results in a diagnostic transferrin isoform pattern readily demonstrated by isoelectric focussing (IEF). Here we report 3 siblings (1 male & 2 females), in their mid-twenties, recently diagnosed with CDG 1a. They show mild mental retardation, cerebellar hypoplasia and ataxia. All have anti-thrombin III deficiency, osteoporosis and mild dysmorphic features. The sisters also have hypertrophic hypogonadism.

Methods: Transferrin Isoforms: Agarose IEF pH 5–7 of 2μL iron-saturated serum immunofixed with Dako anti-human transferrin, with Coomassie Blue / Silver stain. PMM: Leucocyte PMM analysis using linked kinetic spectrophotometry monitoring NADPH formation at 340nm. Mutation Analysis: Direct sequence analysis of PMM2 gene.

Results: IEF showed the typical Type 1 pattern with increased amounts of disialo- and asialo- transferrin, with no detectable monosialotransferrin. PMM activity ranged from <0.10 – 0.10 U/L (RR 0.30 – 1.6). All 3 siblings are compound heterozygotes for L32R and R141H mutations.

Discussion: IEF and PMM studies are consistent with CDG 1a, but the mild clinical phenotype is atypical. This may, in part, be explained by the L32H mutation, a mild mutation, which has 40–45% residual activity of PMM. Although clinical heterogeneity in CDG 1a is well documented, we believe that these siblings are among the mildest intellectually affected CDG 1a patients reported to date.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P21 GENETIC VARIATIONS OF MATRIX METALLOPROTEINASE-3 AND 12 PROMOTER REGIONS AND THEIR ASSOCIATIONS WITH SUSCEPTIBILITY TO ACUTE CORONARY SYNDROMES IN CHINA

Introduction: Matrix metalloproteinases (MMPs) are involved in plaque rupture, which is the main pathological cause of acute coronary syndromes (ACS). Recently, several genetic studies have demonstrated that MMP-3 5A/6A polymorphism and MMP-12 A/G polymorphism modify each transcriptional activity in allele specific manners. The aim of the study was to investigate the possible impact of these genetic polymorphisms on risk of ACS in Han Chinese population.

Methods: Using mismatch PCR primer, a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) was performed. The ACS group (51 patients) was compared with a control group comprising 100 healthy subjects without cardiovascular diseases.

Results: The prevalence of 5A/6A+5A/5A genotype (41.2% vs 24%) and 5A allele (22.5% vs 13%) of the MMP-3 gene was significantly more frequent in the ACS group than in control subjects (P<0.05). Logistic regression analyses revealed that the odds ratio of the 5A/6A+5A/5A was 2.217 (95%CI, 1.077 to 4.564, P=0.031). However, there was no difference in the frequencies of MMP-12 genotypes and A allele between patients and control subjects.

Conclusions: These findings indicate that the 5A/6A polymorphism of the MMP-3 gene is associated with risk of ACS, and might play an important role in the development of coronary atherosclerosis.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P22 DIAGNOSIS OF ALPHA1-ANTITRYPSIN DEFICIENCY BY TRYPSIN INHIBITOR CAPACITY, ISOELECTRIC FOCUSING AND PCR-RFLP (PCR-MEDIATED MUTAGENESIS TO CREATE NEW RESTRICTION ENZYME SITE)

Introduction: Alpha 1-antitrypsin (AAT) is a serine protease inhibitor. It serves as the major inhibitor of neutrophile elastase, a powerful proteolytic enzyme stored in neutrophile leukocytes. The structural gene locus encoding AAT is called Pi (proteinase inhibitor) and is located on chromosome14q32.1. AAT is coded by single gene. This gene was composed of five exons and four introns. It is well established that the AAT deficiency is related to many diseases including liver & lung diseases, rheumatoid arthritis, etc.

Material and Methods: In this study we investigated 53 patients with alpha1-antirypsin deficiency (AATD) for high risk alleles Z and S. Pi typing of α1 -antitrypsin was performed by Isoelectro Focusing (IEF) in a narrow pH gradient, 4.2–4.9.

All samples were analyzed for Trypsin Inhibitor Capacity (TIC), Polymearase chain reaction – Restriction Fragment Length Polymorphism or PCR-RFLP (PCR-mediated mutagenesis to create new restriction enzyme site).

Results And Discussion: The results clearly show that by TIC technique 50.08% of patients were AAT deficient. Serum AAT may be increased in various diseases but the IEF technique can detect 92.4 % of AAT deficient patients. While the PCR-RFLP technique can detect 100% of patients. In conclusion that mutation analysis for AAT high risk alleles should be carried out with a combination of IEF and PCR-RFLP.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P23 NOT ALL “MACROS” ARE THE SAME…

Introduction: Macroenzymes are not uncommonly found in the clinical laboratory, and are enzymes with higher relative molecular mass than normally seen in serum. Our laboratory provides specialised enzyme electrophoresis (EPG) services for further investigation of alkaline phosphatase (ALP), creatine kinase (CK) and lactate dehydrogenase (LD) abnormalities and has encountered a variety of different macro enzyme patterns. We present several cases to demonstrate that some macroenzymes are relatively easy to recognise, whilst others require additional procedures for identification.

Methods: ALP, CK and LD enzyme electrophoreses are routinely performed on commercial Titan Gel Isoenzyme kits (Helena Laboratories, Beaumont, TX), with comparison of patient sera and controls containing specific bands of known identity. Abnormal bands are further investigated by repeat EPG, following one or more additional steps, including polyethylene glycol (PEG) precipitation, trypsin digestion, or immunoprecipitation with a variety of specific antisera.

Results: The majority of macroenzymes observed are single atypical bands, comprising a complex of a single isoenzyme and a single immunoglobulin, most which have been well described in the literature. A number of more unusual patterns, however, have also been observed, with multiple atypical bands within the same serum EPG. Case 1 is a patient, later diagnosed with multiple myeloma, whose serum contains a single abnormal immunoglobulin, bound to more than one type of ALP isoenzyme. Case 2 is an otherwise clinically well female, with a persistently elevated total CK, whose serum contains a single CK isoenzyme binding several individual immunoglobulins of different classes.

Discussion: We include a brief, general discussion of the clinical context and implications of various macro enzymes.

References

  • Sturk A, Sanders G. Macroenzymes. Eur J Clin Chem Clin Biochem. 1990;28:65–81. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P24 EVALUATION OF ANTIHEPTOTOXIC EFFICACY OF PHYLLANTHIN FROM PHYLLANTHUS NIRURI

Natural products have always interested man since ancient times, particularly if they showed good biological activity. Phyllanthus niruri is a traditional remedy for a variety of diseases in Ayurveda. Phyllanthus niruri belongs to the family Euphorbiaceae with a common name Bhuiawla. It is predominantly used as a cure for liver disorders in India. Phyllanthus niruri is considered a most effective, natural, non-toxic remedy for Hepatitis-B. The principal constituent Phyllanthin was extracted from the leaves and isolated by a modified method. Effects of Phyllanthin on liver enzyme activities were studied on laboratory animals. Isolated Phyllanthin was given orally to pretreated carbon tetrachloride Sprague Dawley rats, 125 mg/kg and 250 mg/kg body weight for 5 and 15 days. Various biochemical parameters including Bilirubin, ALT, ALP, GGT were measured. Alkaline phosphate and bilirubin elevations indicateadisease of the liver integrity of cell membrane. Significant decreases in the enzyme activities were observed, p< 0.01 at the end of the experiment. Histopathological study was conducted to support the biochemical parameters. Results of this study revealed that Phyllanthin at 250 mg/kg from leaves of Phyllanthus niruri could afford a significant protective action in carbon tetrachloride induced hepatocellular injury.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P25 NEW JAPANESE ENZYME REFERENCE MATERIAL TRACEABILITY TO THE IFCC REFERENCE MEASUREMENT PROCEDURE

Introduction: Enzyme Reference Material (ERM) corresponds to the secondary calibrator on the extensive calibration hierarchy and metrological traceability to SI of ISO 18153. Japanese ERM Lot 4 was established for new material by lyophilized type according to the ISO18153.

Methods: Enzyme materials used were human recombinant types manufactured by Asahi Kasei Corporation, Tokyo, Japan. Measurements were AST, ALT, CK, GGT and AMY. Standard operating procedure (SOP) was used by the SOP for the measurement of catalytic concentration of enzymes by IFCC methods. Reference measurement laboratories (RML) were notified by ISO 15195. The certified values were determined by ISO Guide 35. The uncertainty of certified values was determined by the GUM. Measurement was performed by 27 RML, 2 replicate analysis and 3 vials of measuring sample.

Results: Certified values and uncertainty were determined 164 +/− 2 U/L for AST, 164 +/− 2 U/L for ALT, 458 +/− 5 U/L for CK, 423 +/− 4 U/L for LD, 151 +/− 2 U/L for GGT and 341 +/− 3 U/L for AMY, respectively. Stability of the ERM was determined by +/− 2 CV% which included measurement error for 24 months interval on the basis of the ISO Guide 33. Relative stability was estimated 2.4% for AST, 2.4% for ALT, 3.7% for CK, 3.8% for LD, 3.4% for GGT and 3.0% for AMY.

Conclusions: New Japanese ERM traceability was established by calibration hierarchy of ISO 18153. ERM Lot 4 is routinely available for lyophilized type which is maintained in long term stability.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P26 GLUTATHIONE S-TRANSFERASE ACTIVATION IN LIVER OF RATS EXPOSED TO SULPHUR MUSTARD

Introduction: Sulphur mustard (SM) has been frequently used as a vesicant chemical warfare agent. The mechanism of cell injury caused by SM is not well understood. Glutathione S-transferases (GSTs) are a major family of detoxification enzymes that play a vital role in protecting tissues against oxidative damage.

Methods: In this study, male Wistar rats (200–250 g body wt.) were randomly divided into seven groups as followed: group 1 control, group 2 sham that received DMSO (dimethyl sulfoxide used as solvent), group 3–7 experimental groups that received SM (1–80 mg/kg) by intraperitoneal injection only once. The rats were killed after 6 and 48 hours by ether. The liver of each group was removed and the GST content was measured by biochemical methods.

Results: The results show that the effect of SM is dose and time dependent. At low concentrations of SM, GST was increased, while at higher concentrations of SM, activity of GST was decreased after 6 and 48 hours of SM intoxication. A decrease of this activity after 48 hours was higher than 6 hours.

Conclusions: The data suggest that SM induced free radical generation and antioxidant depletion. Also, the oxidation of a cysteine SH-group in the active site of GST in liver is a likely cause of irreversible inactivation of this enzyme that induced oxidative stress and cell death.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P27 EFFECT OF METAL STRESS ON EXTRACELLULAR PROTEASE ACTIVITY OF PSEUDOMONAS FLUORESCENS ATCC 948

Introduction: Heavy metals are metals with density more than 5gm/cm2 redox active & bind to proteins and cause oxidative stress. This results in the formation of oxidized proteins, which often loose their function and undergo unfolding or conformational change and are unable to carry out the cellular tasks and biochemical reactions they are meant to perform. These oxidized proteins are removed by proteolysis i.e., by certain proteases. Heavy metal stress (Lead) has been shown to induce protease activity in Pseudomonas fluorescens. In the present study, we showed the direct link between oxidative stress caused by heavy metal stress and enhanced protease activity in Pseudomonas fluorescens. Protease was purified from the metal treated culture followed by its purification and characterization

Methods: The bacteria were grown in control as well as in the presence of heavy metals (Pb, Cu & Co) in citrate mineral media at 28°C with constant shaking at 200 rpm. Extracellular Protease activity was tested in the control and treated cultures. Protease from the lead treated culture was purified using anion exchange chromatography and characterized.

Results: The highest protease activity was found with exposure of culture to lead. The protease was purified to homogeneity and was found to be 33kDa metalloprotease. The purified protease was maximally active at 20°C and pH 6.0.

Discussion: The high activity of protease from lead treated culture (crude), to some extentconfirms that protease is necessary for the survival of bacteria when exposed to heavy metal stress.

References

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P28 INHIBITION OF PROTEINASE 3 (PR3) BY SURAMIN AND FCS: EFFECT OF PR3 AND SURAMIN ON CHO-CELLS

Introduction: Proteinase 3 (PR3) is a lysosomal protease that is stored in azorophilic granules, neutrophilic granulocytes and monocytes. A number of synthetic inhibitors for this proteinase are reported. Comprehensive studies on the inhibitory effect of suramin and heat inactivated Fetal calf serum (ΔFCS) on PR3 have not been reported. It is reported that PR3 is able to destroy the cytoskeletal integral proteins, but we could not find any reports that showed the effect of this protease on Chinese hamster ovary cells (CHO-cells) in culture medium. Suramin has proven to be useful as an antitumor drug, but there are not any reports on the effect of suramin on CHO-cells.

Methods: The effects of various concentrations of ΔFCS (from 0.5% up to 10%) and suramin (from 0.8 μM up to 100 μM) on PR3 and different concentrations of suramin (from 0.8 μM up to 1000 μM) on CHO-cells were investigated.

Results: Results showed that ΔFCS and suramin have an inhibitory effect on PR3 and these effects were related to increasing concentration. PR3 with the concentration of 2.2 U/ml did not have any effect on CHO-cells. Suramin between the concentration of 125 to 250 μM inhibits the cell growth for a week, but after that cells gain normal growth gradually, but with a concentration of less than 125 μM, cell growth was retarded for only a few hours. Suramin with a concentration of more than 500 μM inhibited the cell growth completely.

Conclusions: Although suramin reversibly inhibits the PR3 activity, in the concentration used in this project it has no long term effect on CHO-cells. Therefore it can be used in proteaseinvestigations. There are unknown components in ΔFCS which cause the inhibition of PR3 activity. These findinga are very important in PR3 production in culture medium. However CHO-cells are resistant to PR3 and suramin in low concentration

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P29 INFLUENCE OF THE TEMPERATURE AND PERIOD OF STORAGE TIME ON THE ENZYMATIC ACTIVITIES

Five enzymes ALP, ALT, AST, CK and LDH have been analyzed in already prepared “pool” serum, whose activity is influenced by the environment, the temperature and the period of storage time, in which they are kept. Commercial kits of reagents have determined the activity before and after containing the serums for the periods of 1, 2, 3, 5, 10 and 15 days at three different temperatures (18 °C, 4 °C, and −20 °C) with and without cryo protector 0,1% NaN3. The enzymatic activities were different, some were very active and others inactive. The activity of LDH and CK in a serum kept for 15 days on 4 °C and −20 °C without cryo protector. ALT is stable at 4 °C with 0,1% NaN3. ALP and AST kept activity at the temperature of −20 °C up to 15 day with cryo protector. In serum which has been frozen and thawed several times the activity of all enzymes are significantly different.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P30 PURIFICATION OF CATALASE FROM TILAPIA LIVER - WITHDRAWN

Introduction: Catalase (EC 1.11.1.6) is a tetrameric enzyme consisting of four identical tetrahedrally arranged subunits of 60 kDa that contains a single ferriprotoporhyrin group per subunit, and has a molecular mass of about 240 kDa. Catalase reacts very efficiently with H202to form water and molecular oxygen; and with H donors.

Methods: In this study catalase enzyme was purified from for the first time the liver of tilapia (Oreochromis niloticus). This purpose, tilapia liver is first homogenized and then centrifuged at 10.000 g. The enzyme extracts are dissolved in acetone and put in phenyl sepharose 6B for separation of the enzyme.

Results: Catalase specific activity was found as 52 U/mg protein in crude homogenate, whereas catalase specific activity was found as 7500 U/mg protein after purification with sepharose 6B chromatography. This value was 144 times higher than that of crude homogenate.

Conlusions: The catalase enzyme was purified successfully from tilapia. The optimum pH value for the catalase was found as 7.0. Catalase lost almost all of its activity in 96 hour when stored at 4 °C.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P31 ASSOCIATION OF GENE POLYMORPHISMS OF THE APOE-CI-CII GENE CLUSTER AND LDL-R GENE WITH CORONARY ARTERY DISEASE

Introduction: Coronary artery disease (CAD) is a multifactorial disorder that is thought to result from gene/environmental interactions. However little data are reported from China. We investigated the association of gene polymorphisms of the ApoE-CI-CII gene cluster and LDL-R gene on CAD and their interactions with alcohol drinking and smoking in the Hans Chinese.

Methods: A questionnaire survey of smoking and drinking behavior, dietary pattern, and anamnesis, was conducted among 203 CAD patients, and 365 controls. ApoE genotypes were identified by multi-ARMS, and the ApoCI promoter polymorphisms and AvaII polymorphisms of the ApoCII and LDL-R gene were detected by PCR-RFLP. The OR of susceptibility of the genes to CAD and the attributable proportions of interaction (API) for interactions with alcohol drinking and smoking were determined using multivariate logistic regression models.

Results: The frequencies of ApoE E4/3 genotype and the E4 allele in CAD were significantly higher than those in control group, P<0.01. A significant difference was also found for the ApoC1 H2 allele between groups, P<0.01. Significant differences for a de cit of Apo E3-H1 and excess of apo E4-H2 was found in the CAD by estimation of the haplotype frequencies. After control for possible confounding factors, the interaction among frequent smoking, Apo E4 and ApoCI H2 allele was significant, OR =18.3, API=57.3%. This was also shown among subjects with frequent alcohol intake: OR=12.7, API=43.5%, P<0.05. For the LDL-R and ApoCII-AvaII locus, no differences were found.

Conclusions: Both ApoE and ApoCI on chromosome 19q13.2 are the susceptibility loci for CAD, and their linkage disequilibrium may be responsible for the development of CAD. Alcohol drinking and smoking can modify these potential gene-gene interactions.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P32 CLONING AND EXPRESSION OF A NEW APOLIPOPROTEIN, APOLIPOPROTEIN M

Introduction: The purpose of our study was to clone and express a new apolipoprotein, ApoM, in order to construct a basis for the further study on the function of ApoM in atherosclerosis pathology.

Methods: Using isolated total RNA from normal adult mouse liver cells, cDNA encoding the mouse ApoM was amplified using the RT-PCR method. The PCR product was recombined into an expressed vector, pGEX-KG. After sequencing, the recombinant vector was used to transform competent expressive cells of E. coli, BL21, where the mouse ApoM gene was expressed. After being induced with IPTG, the expression products were analyzed using SDS-PAGE. The expressed fusion protein was purified through affinity chromatography.

Results: DNA sequencing showed that the cloned mouse ApoM gene sequence matches previously reported sequences. The recombinant vector pGEX-KG/ ApoM could be expressed efficiently in coli BL21. Expressed fusion protein existed in the form of inclusion bodies. The protein band was 54 000 on SDS-GAGE gel and its expression was about 35% of total bacteria protein in E. coli BL21.

Conclusions: The mouse ApoM was cloned successfully and expressed efficiently in E. coli BL21 which create a favourable condition for preparation of anti- ApoM antibody and studying the function of ApoM.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P33 STUDY ON THE ASSOCIATION OF SNPs OF THE CETP GENE WITH CORONARY HEART DISEASE

Introduction: To explore the influences of the TaqIB −629A/C polymorphism and the D442G mutation of the cholesteryl ester transfer protein (CETP) gene on serum lipids, apolipoproteins and CETP concentration, and their association with coronary heart disease.

Methods: Samples of peripheral blood white cells were extracted from 128 patients with CHD, and 247 unrelated controls, all of Chinese Han nationality. The gene variations in the CETP gene were determined by PCR-RFLP and subsequent agarose or polyacrylamide gel electrophoresis. The CETP concentration was determined using ELISA.

Results: (1) Compared with the control group, the frequencies of the B1 allele of CETP gene in CHD group was significantly higher (P<0.05). In each group, the serum CETP concentration and LDL-C levels was significantly higher in B1B1 genotype than in B2B2 genotype, but it was significantly lower for HDL-C levels (P<0.05). (2) For the –629A/C polymorphism, the frequencies of the C allele in CHD group was significantly higher than in controls (P<0.05). In the same group, the serum CETP concentration was significantly higher in B1B1 genotype than in B2B2 genotype, but it was significantly lower for HDL-C levels (P<0.05). (3) The frequencies of D442G mutation had no significantly different between the two groups. (4) After adjustment for major confounders including age, gender and body mass index, multiple logistic regression analysis showed that :the B1B1 genotype of CETP TaqIB polymorphism increased the risk of coronary heart disease significantly (OR=3.18, 95% CI:1.44–7.05); and the –629A/C polymorphism and D442G mutation was not in association with CHD significantly. There was a significantly higher risk for CHD in the haplotypes for B1B1 genotype and CC genotype, compared with the other haplotypes between the two SNPs.

Conclusions: The TaqIB and –629A/C polymorphisms may be highly correlated with coronary heart disease. The B1 allele and the C allele may be independent risk factor for CHD. CETP gene may be one of the genetic candidate genes for coronary heart disease among Chinese Han population.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P34 THE EFFECT OF C677T METHYLENE- TETRAHYDROFOLATE REDUCTASE GENOTYPE ON RED CELL N5METHYLTETRAHYDRO-FOLATE LEVELS IN STROKE PATIENTS.

Introduction: Elevated blood levels of homocysteine (tHcy) are an independent risk factor for atherothrombotic vascular disease, including stroke. Homocysteine is re-methylated back to methionine by methionine synthase, N5methyleneterahydrofolate reductase (MTHFR) acting on N5methyltetrahydrofolate (N5MTHF), which becomes a methyl group donor with cobalamin an essential cofactor.

Aim: To investigate the relationship between total red cell folate, red cell N5MTHF and the MTHFR genotypes in stroke patients.

Methods: The study included 120 consecutive patients presenting at hospital with acute stroke. The use of multivitamin supplements at hospitalisation was noted. Serum and red cell folate was measured by microbiological assay using a chloramphenicol resistant strain of Lactobacillus casei, Enterococcus faecalis and by the DPC-BioMediq Immulite™ 2000 analyser. Plasma tHcy, serum cobalamin and serum pyridoxal (vitamin B6) levels were measured and the C677T MTHFR genotypes were determined.

Results: There was no significant differences in blood levels of tHcy or vitamins according to MTHFR genotype in the overall patient group. But when patients taking vitamin supplements were excluded, total red cell folate and N5MTHF was significantly lower in patients with the TT genotype compared with CT or CC genotype.

Discussion: Irrespective of genotype, red cell folate levels were significantly lower when assayed microbiologically compared with the Immulite assay. Stroke patients with the TT compared with the CT and CC genotypes had substantially lower levels of total red cell folate (273 v 614 nmol/L) and N5MTHF (252 v 590 nmol/L). This difference was masked by the use of multivitamins and this may impact on our interpretation of the strength and independence of the association between elevated blood levels of tHcy and atherothrombotic risk reported in many epidemiological studies.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P35 A MOLECULAR GENETIC STUDY IN KOREAN PATIENTS WITH UDP-GALACTOSE-4-EPIMERASE (GALE) DEFICIENT GALACTOSEMIA

Introduction: GALE deficient galactosemia results from impairment of the human enzyme GALE which catalyses the interconversion of UDP-galactose and UDP-glucose. The human GALE gene is about 4 kb in size and is located on chromosome 1 (1p36). GALE deficient galactosemia has been known to be relatively frequent in Koreans as well as in Japanese. We investigated the molecular characteristics in Korean patients with GALE deficient galactosemia.

Methods: Human genomic DNA was isolated from peripheral blood and mutation analysis was performed by PCR followed by direct sequencing in 7 patients showing severely decreased enzyme activities. PCR-RFLP were done in 30 patients with moderately decreased GALE activities to investigate whether they have new mutations.

Results: Nine novel mutations including 8 missense mutations (A25V, R40C, D69E, E146K, T150M, R220W, V281I, and R335H) and one nonsense mutation (W336X) were detected. Six patients were compound heterozygotes and one patient had only one mutation detected. Among 30 patients, 17 patients had one of above mutations, but the others had none of them.

Discussion: We have identified 9 novel mutations in Korean patients with GALE deficient galactosemia associated with peripheral form. Among 9 mutations, the frequencies of V281I (9/30), R220W (6/30), and T150M (5/30) amino acid substitutions were relatively high. We could not find out any association between specific mutations and GALE enzyme activities, therefore further studies such as site-directed mutagenesis and mutated protein expression will be needed.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P36 NOVEL MUTATIONS OF THIAZIDE-SENSITIVE SODIUM CHLORIDE COTRANSPORTER IN 9 PATIENTS WITH HYPOKALEMIA AND HYPOMAGNESEMIA

Introduction: Gitelman’s syndrome is a variant of Bartter’s syndrome, characterized by impaired NaCl reabsorption in the distal convoluted tubule with hypokalemia, hypomagnesemia, and normocalcemic hypocalciuria. Recent advances in molecular genetics have demonstrated that the Gitelman syndrome results from mutations in the gene encoding the thiazide-sensitive Na-Cl cotransporter (TSC) which expresses in the distal convoluted tubule and has been shown to act to reabsorb NaCl. We took mutational analysis of TSC gene in 9 patients with hypokalemia and hypomagnesemia.

Methods: DNA was extracted from the peripheral blood of each subject. The 26 exons of TSC gene were amplified with specific primers and the products were directly sequenced with big dye-terminator method.

Results: We found 9 missense mutations including Thr180Lys, Arg261Cys, Asn406His, Gly439Ser, Leu623Pro, Met672Ile, Leu849His, Arg904Gln, and Arg955Gln. There were also cases of an insertion of 18bp in the 6th exon and deletion of C in the 16th exon. Three of 9 patients had double mutations.

Discussion: These results suggest that there is no mutational hot spot in the TSC gene. The 11 novel mutations might be responsible for the impairment of the NaCl reabsorption leading to the hypokalemia and hypomagnesemia in these patients. These novel mutations could bring new insights into the genotype-phenotype correlations among the hypokalemic tubulopathies.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P37 OPTIMISED PRIMER DESIGN FOR GENETIC TESTING OF ACUTE INTERMITTENT PORPHYRIA

Introduction: Acute intermittent Porphyria (AIP) is an inborn error of haem metabolism which may present with neurological symptoms. Screening of symptomatic patients by measuring porphyrins and precursors in urine, blood and faeces can usually identify patients with overt disease. However, with these methods, the detection of carriers or patients in remission can be difficult if not impossible. DNA based genetic studies are available for this purpose but since there are many known mutations for AIP, DNA sequencing is often required. To assist in the genetic diagnosis of AIP, we attempted to ascertain whether the task of DNA sequencing could be simplified by optimising primer design so that similar PCR conditions could be employed for all of the amplified PCR fragments.

Methods: Primers to cover all 14 exons of hydroxmethylbilane synthetase (reduced in activity in AIP) were prepared using the online database ExonPrimer http://ihg.gsf.de/ihg/ExonPrimer.html and with the following specifications: maximum fragment size, 600; optimal primer size, 25; optimal annealing temperature, 65 degrees centigrade. The primers were designed so that there was a 40 base pair separation from the intron-exon boundary and also a 40 base pair overlap between amplified fragments.

Results: It was found that using the above protocol for primer design, all PCR reactions required for sequencing could be carried out with identical magnesium concentrations and identical annealing temperatures and gave products suitable for sequencing.

Conclusions: It was concluded that by optimising primer design for hydroxmethylbilane synthetase, similar PCR conditions could be employed for all of the amplified fragments required for sequencing. By simplifying the process of DNA sequencing it is hoped that this will facilitate the genetic diagnosis of acute intermittent porphyria.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P38 RELATIONSHIP BETWEEN PARAOXONASE1 GENOTYPE AND CORONARY HEART DISEASE

Introduction: The aim of the study was to study the −108C/T, −162A/G and –909C/G polymorphisms in the promoter region of the human PON1 gene in subjects from the Hubei area and compare genotype frequencies and explore their influences on serum lipid levels and their relationship with CHD in 86 CHD and 128 normal people.

Methods: The genotypes of paraoxonase gene –108C/T, −162A/G and −909C/ G polymorphisms were assayed by PCR-RFLP. The gene polymorphisms have been confirmed by sequencing.

Results: (1) PON1-108C/T: The frequencies of the genotypes and alleles in CHD were 0.105, 0.547, 0.349 and 0.378, 0.622 respectively. The frequencies in controls were 0.250, 0.453, 0.297 and 0.477, 0.523 respectively. There was a significant difference in the genotype and allele frequency between the CHD group and controls. PON1-162A/G: The frequencies of the genotypes and alleles in CHD were 0.035, 0.105, 0.860 and 0.087, 0.913 respectively. The frequencies in controls were 0.047, 0.203, 0.750 and 0.148, 0.852 respectively. There was no significant difference in the genotype and allele frequencies between CHD group and controls. PON1-909C/G: The frequencies of the genotypes and alleles in CHD were 0.233, 0.558, 0.209 and 0.512, 0.488 respectively. The frequencies in controls were 0.258, 0.523, 0.219 and 0.520, 0.480 respectively. (2) PON1-108T/T genotype was a risk factor of CHD (OR=2.807, P=0.022). Genotypes PON1-162G/G and PON1-909G/G raised the risk of CHD. But the ORs of them were no difference. (3) PON1-108C/T, -162A/G and –909C/G were combined to analyze their association with CHD. The OR with one risk genotype was 1.812, P=0.244. The OR with two risk genotypes was 1.636, P=0.360. The OR with the risk genotypes was 3.556, P=0.079. There were no significance. That is to say that the increasing number of risk genotypes was not related to CHD. (4) The level of HDL-C and ApoAI was lower in the CHD group than in controls (P<0.05), but the level of TG, TC and LDL-C were not (P>0.05). PON1-108C/T: The level of HDL-C in CC homozygotes was lower significantly in the CHD group than in controls (P<0.05), there was no significant difference in TT homozygotes and TC heterozygotes between the two groups (P>0.05). The level of plasm lipids between two groups was no significantly different between PON1-162A/G and PON1-909C/G.

Conclusions: (1) The –108C/T polymorphism of PON1 may be associated with CHD in the Chinese. (2) The –162A/G and –909C/G polymorphisms of PON1 are not associated with CHD in the Chinese. (3) The PON1-108T/ T genotype may be a risk factor of CHD, and the PON1-108C/C genotype maybe a protective factor for CHD in the Chinese. (4) There is no cooperative effect among these risk genotypes.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P39 ESTABLISHMENT OF AN ASSAY FOR QUANTITATIVE RT-PCR OF HUMAN TELOMERASE REVERSE TRANSCRIPTASE (hTERT) GENE EXPRESSION

Introduction Telomerase is linked to cell immortalization. It may become a valuable marker of malignant transformation. We aimed to measure telomerase level by quantifying mRNA of its rate-limiting catalytic subunit hTERT, in colonic cancer cells, in a real time reverse transcription-PCR (RT-PCR) assay.

Methods Four primer pairs with corresponding TaqMan™ probes were examined; two for hTERT and one for each positive internal control (GAPDH and 18S RNA). RNA was extracted from cultured human colonic cancer cells and purified on silica-gel-based membrane. This was followed by reverse transcription, amplification in real time PCR, then quantification using standard curves of hTERT PCR products. Control samples without the addition of reverse transcriptase (-RT) were included. Effect of sample treatment with DNAse was examined. Specificity was confirmed with gel electrophoresis and sequencing of PCR products.

Results For all primer pairs, RNA concentration showed linearity with PCR copy number, up to 50 ng per μl of cDNA synthesis reaction volume. Analysis of four aliquots of cancerous colonic cells using each hTERT primer pair showed CV of 16.4% and 23.5%. Although all primer pairs were designed to avoid amplifying genomic DNA, one of the hTERT primer pairs consistently produced PCR products from -RT control. This did not occur after DNAse treatment.

Discussion A specific reproducible assay for hTERT mRNA has been established. One of the examined hTERT primer pairs amplified contaminating genomic DNA. Sample treatment with DNAse, which can potentially reduce RNA yield, is required when using this primer pair.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P40 DIFFERENTIAL MUTATION PATTERNS IN DNA BINDING DOMAIN OF P53 GENE IN SQUAMOUS CELL CARCINOMA OF OESOPHAGUS IN KASHMIR (INDIA), A HIGH INCIDENCE AREA

Introduction: The high incidence of oesophageal squamous cell carcinoma (ESCC) in Kashmir valley (India) has been attributed to an exposure to nitroso compounds (or their precursors), amines and nitrates present in the sun dried vegetables, hot salted tea (Noon Chai) and the pipe (Hukka) smoke. However, to date, no attempt has been made to study the presence of mutations in the tumours of patients with ESCC. In this study we have reported the incidence of mutations in exon 5–8 of the tumour suppressor gene p53 in patients with ESCC.

Methods: 55 patients including males (n=36) and females (n=19) with histologically proven ESCC were recruited for this study. DNA was isolated from tumour tissue by standard procedures. After amplification of exons 5–8 by the polymerase chain reaction, direct sequencing was performed on an ABI PRISM 3100 genetic analyser.

Results: Sequencing revealed the presence of known and unreported mutations in exons 5–7 (when compared to the IARC mutation database) in 36.36% tumours. Of the 20 mutations in 20 patients, 17 were single base substitutions (11 transitions + 6 transversions) and 3 were deletions. The 17 single base variations represented 12 missense mutations leading to amino acid substitutions, 2 nonsense mutations and 3 variations located in intron 6, one of which resulted in a splicing variant.

Conclusions: The frequency of mutations between males (9/36, 25%) and females (11/19, 57%) was significantly different (p=0.016) apparently putting females at a higher risk of carrying p53 mutations despite being affected with ESCC in smaller numbers than males. The pattern of distribution of mutations in Exons 5, 6, and 7 of p53 further suggested that the targets of mutation differed between males and females, an observation which would need further study of a larger set of samples from Kashmiri population.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P41 IMPROVED AND MODIFIED METHYLATION SPECIFIC PCR OF APC GENE PROMOTER IN SQUAMOUS CELL CARCINOMA OF OESOPHAGOUS

Introduction: Promoter hypermethylation of tumor suppressor genes (TSs) plays important role in the aetiology of cancers. Bisulfite treatment of DNA as used in methylation specific PCR (MSP) promotes DNA degradation and reduction of MSP sensitivity. We report here an improved MSP of TS gene; APC (Adenomatous Polyposis Coli) in esophagus cancer.

Methods: DNA was extracted from tissues of patients and blood of healthy donors, digested with Hind III and Bisulfite treated in agarose beads. Two step MSP PCR was conducted, with a primary amplification followed by secondary amplification.

Results: To improve PCR sensitivity primers were designed with maximum number of CpG dinucleotides. Using PCR enhancers (DMSO, 2 ME, BSA) with the digestion of DNA showed a profound improvement of PCR. Treatment of DNA in agarose beads led to the least DNA loss. Secondary amplification following primary amplification led to products either of unmethylated CpG islands or methylated CpG dinucleotide of APC promoter. Further analysis demonstrated that PCR was highly sensitive and specific without any non specific amplification. Forty percent of tumor samples were shown to be methylated.

Conclusion: Several factors improve MSP: 1) low temperature and short time of DNA treatment, 2) bisulfite treatment in agarose beads reduces loss of DNA, 3) enhancement of PCR by application of PCR enhancers along with digestion of DNA 4) and carrying out secondary amplification following to primary MSP which provides enough template for amplification and reduces non-specific product formation.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P42 A STUDY ON THE RELATIONSHIP BETWEEN PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ AND CARDIAC HYPERTROPHY

Introduction: To investigate the role of peroxisome proliferator-activated receptor γ in cardiac hypertrophy in vitro.

Methods: Hypertrophy in neonatal rat cardiac myocytes (MC) and cardiac non-myocytes (NMC) was established with angiotensin II (Ang II). mRNA expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was measured by reverse transcription-polymerase chain reaction (RT-PCR), proliferation of NMC was estimated with MTT assay and 3H-TdR uptake, the surface area of MC was analyzed by the aid of NIH Image J software, and the synthetic rate of protein in MC was detected by 3H-leucine incorporation.

Results: In conditions for hypertrophy, increases of surface area of MC, mRNA expression of ANP and BNP and 3H-leucine incorporation in MC and an increase of proliferation in NMC were detected, but not changes in mRNA expression of ANP and BNP in NMC. 15-deoxy-Δ12,14 prostaglandin J2 PGJ2) and pioglitazone, the representative natural and synthesized PPARγ (15d-activators, inhibited the changes above and reduced mRNA expression of ANP and BNP in NMC in a concentration dependent manner.

Conclusions: These results suggest that 15d-PGJ2 and pioglitazone inhibit cardiac hypertrophy induced by angiotensin II in vitro and PPARγ-dependent pathways may participate in the negative regulation process of cardiac hypertrophy.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P43 PROMOTING GENETIC REFERENCE MATERIAL PREPARATION FOR PCR: STRATEGIES FOR COVALENT ATTACHMENT OF DNA BEADS.

Introduction: Many academic societies are going to conduct quality management of molecular diagnostic tests. We tried to devise a good system for distribution of such reference materials.. Several covalent attachment chemistries were tested for the immobilization of DNA onto glass beads.

Methods: DNA was immobilized on Glass beads (Iuchi:2Z) of 2mm diameter, and dried. DNA from peripheral blood was extracted. A DNA bead was amplified by the PCR to determine ACE genotype and micro-satellites. Fragments were detected with ethidium bromide. There are several covalent attachment chemistries; Supra-molecules (Cycolo-dextrin, 18-Crown-6), Surfactants(Tween20, NP-40, Anhitol 24B), Glycerol, PEG and DMSO. These were tested for immobilization of DNA onto glass beads. Stability was examined by PCR amplifying it for three months by using several DNA beads stored at room temperature.

Results: The comparison was based on the ability of these chemistries to produce derivative DNA beads that give good hybridization signals. Supra-molecule (Cycolodextrin) gave us the best hybridization signals. Effect on addition became Supara-molecules, Surfactants, Glycerol, PEG and DMSO the result of the order without addition.

Discussion: As a point of the preparation of the DNA-beads reference material for PCR, which DNA turns to the low molecule, stable long-term, it is to be easy to be isolated. It is because the reason why Supar-molecular is good is related to the gentle combination interaction between the molecule. We further characterized stability of the derivative DNA beads.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P44 IN VITRO EFFECTS OF IL-13 AND IL-25 ON THE SURFACE EXPRESSION OF ICAM-1 AND ICAM-3 ON HUMAN EOSINOPHILS

Introduction: Allergic diseases such as asthma involve the recruitment and migration of eosinophils into inflamed tissues mediated by chemotatic cytokines and adhesion molecules. Over the past several years, it has become clear that interleukin (IL)-13 and a novel member of the IL-17 family, IL-25, are key mediators in the pathogenesis of allergic inflammation. We investigated the effects of IL-13 and IL-25 on the activation of human eosinophils with regard to the surface expression of adhesion molecules including intercellular adhesion molecule (ICAM)-1 and ICAM-3.

Methods: Purified human peripheral blood eosinophils were pre-activated with or without IL-5 for 30 minutes prior to treatment with either IL-13 or IL-25 for 17 hours. The surface expressions of ICAM-1 and ICAM-3 on eosinophils were then determined by immunofluorescent staining and flow cytometry.

Results: Unstimulated eosinophils expressed low levels of surface ICAM-1 and high levels of surface ICAM-3 on eosinophils. Both IL-13 and IL-25 alone could up-regulate the surface expression of ICAM-1 but down-regulate that of ICAM-3. IL-5 showed a synergistic effect with IL-13 but not with IL-25 on enhancing the surface expression of ICAM-1 and suppressing that of ICAM-3.

Discussion: Our findings suggest that IL-13 and IL-25 may play crucial roles in the pathogenesis of allergic inflammation by influencing the surface expressions of adhesion molecules on human eosinophils.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P45 ASSOCIATION BETWEEN MANNOSE-BINDING LECTIN POLYMORPHISM AND RHEUMATOID ARTHRITIS IN AN INDIAN POPULATION

Mannose-binding lectin (MBL) is a key element of innate immunity, with a structure similar to complement C1q. The polymorphisms of MBL gene affect the serum MBL level. It is proposed that MBL interactions with a galactosyl form of IgG found in rheumatic patients may lead to enhanced complement activation, an important mediator of the joint damage in rheumatoid arthritis (RA). We have compared the frequencies of allelic forms of MBL gene and their genotype frequencies, known to be associated with serum MBL level, in a group of hospital patients with severe RA and control subjects.

A total of 120 patients satisfying the American Rheumatism Association criteria were selected for blood collections. Samples from healthy unrelated individuals from North Indian population were used as controls. Genomic DNA was extracted from blood samples using standard procedures. Genotyping of codon 52, 54, 57 was performed by RFLP analysis and sequencing of gene specific PCR products that have been amplified by MBL exon 1 PCR primers. However, to obtain a complete description of the known MBL haplotypes, we have sequenced the promoter region and all the protein coding portion of the MBL gene. Our study population consisting of 100 healthy individuals and 120 patients with RA showed insignificant mutation in codon 52 (2% variation) or 57 (alleles C or D). However, the frequency of allele B (codon 54) in the patient group decreased with statistical significance, which is predicted to have high MBL level. Thus, the presence of low frequency of variant MBL alleles that is responsible for high MBL levels may result in enhancement of deleterious effects of complement-mediated inflammation.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P46 CD4/CD8 RATIO IN HEALTHY SUBJECTS CONSUMING A TRADITIONAL CHINESE HERBAL MEDICINE

Introduction: We investigated the immunomodulating effects of an innovative traditional Chinese herbal medicine (TCHM) that was derived from two centuries-old herbal formulae (Sang Ju Yin and Yu Ping Feng San) used for prevention and treatment of wan bing, a febrile disease with early presentations similar to Severe Acute Respiratory Syndrome (SARS).

Methods: Thirty-seven healthy adult volunteers were given the TCHM daily for 14 days. Blood samples were taken on Days 0, 15 and 29 for lymphocyte-subset analysis, complete blood count, differential count, renal and liver function tests, lactic dehydrogenase and creatine kinase assays. Each volunteer also completed a SF-36 questionnaire for health assessment. After 3 months, 23 volunteers agreed to participate in a control study with no TCHM treatment. Blood was taken again on Day 0, 15 and 29 for the same blood tests.

Results: Two volunteers withdrew on Day 2 due to headache and dizziness. All others (18 males, 17 females) remained well with no side effects. No study subject showed any changes in biochemical tests. There were no derangements in total white cells and lymphocytes, but the CD4/CD8 ratio of the T lymphocytes increased significantly from 1.31±0.50 on Day 0 to 1.41±0.63 on Day 15 (p<0.05), and returned to the initial level on Day 29. In the control study, there were no significant changes in the T lymphocyte CD4/CD8 ratio.

Discussion: The transient increase in CD4/CD8 ratio is likely due to the TCHM intake. We postulate that administration of the innovative TCHM may have beneficial immunomodulatory effects in preventing SARS.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P47 ACTIVATION OF P38 MAPK AND NF-ΚB IN IL-3, IL-5 AND GM-CSF INDUCED ADHESION AND CHEMOTAXIS OF HUMAN EOSINOPHILS

Introduction: Haematopoietic cytokines such as IL-3, IL-5 and GM-CSF influence eosinophil functions in allergic inflammation, but their action mechanisms are poorly understood.

Methods: We investigated the p38 mitogen activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signalling pathways for IL-3, IL-5 and GM-CSF induced adhesion, shape change and chemotaxis of human eosinophils using Western blot, gel shift assay, flow cytometry and chemotaxis assay.

Results: These cytokines could activate both p38 MAPK and NF-κB, and up-regulate the gene expression of several adhesion molecules including intercellular adhesion molecule-1 (ICAM-1), α6, β2 integrin (CD18) and CD44 of eosinophils. They could induce significant shape change and chemotaxis of eosinophils after 18-h incubation. Furthermore, IL-3 and IL-5 but not GM-CSF could enhance the adhesion of eosinophils onto airway epithelial BEAS-2B cells. Moreover, either the pharmacological inhibitor of p38 MAPK, SB 203580, or the inhibitor of proteasome for NF-κB, N-cbz-Leu-Leu-leucinal (MG-132), could significantly reduce the cytokine-induced shape change, chemotaxis and adhesion of eosinophils.

Discussion: We postulate that IL-3, IL-5 and GM-CSF regulate eosinophil adhesion and chemotaxis through a shared signalling pathway involving both p38 MAPK and NF-κB activities.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P48 IMMUNOMODULATORY EFFECTS OF YUN ZHI AND DANSHEN CAPSULES IN HEALTHY SUBJECTS - A RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDY

Introduction: The Chinese medicine Yun Zhi (Coriolus versicolor) (PSP) has been shown to modulate various immunological functions in vitro, in vivo and in human clinical trials. Danshen (Salvia miltiorrhiza) can benefit the microcirculation by its vasodilating activity conferring an anti-dementia effect. We investigated if regular oral consumption of Yun Zhi and Danshen could improve cell-mediated immunity in healthy subjects.

Methods: Ninety healthy subjects were recruited to take Yun Zhi (50 mg/kg body weight) plus Danshen (20 mg/kg) or placebo capsules daily for 4 successive months and, after a 2-month wash-out period, crossover to take placebo or Yun Zhi-Danshen capsules for 4 successive months. Flow cytometry was used to assess the ratio and absolute counts of blood lymphocyte subtypes and the concentration of T helper cell cytokines in whole blood culture supernatant. Gene expression of cytokines and cytokine receptors of peripheral blood mononuclear cells (PBMC) was analysed by cDNA expression array.

Results: Yun Zhi-Danshen could significantly elevate PBMC gene expression of interleukin (IL)-2 receptor, increase absolute count of T helper cell and ratio of CD4+ (T helper)/CD8+ (T suppressor and cytotoxic T) cells, and significantly enhance the ex vivo production of typical Th1 cytokine interferon-γ from PBMC activated with phytohemagglutinin and lipopolysaccharide (all p < 0.005). There were no adverse effects on liver and renal functions, muscle enzymes and biochemical bone profile.

Discussion: Regular consumption of Yun Zhi and Danshen may be beneficial for immunological functions by potential enhancement of cell-mediated immunity in healthy subjects.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P49 ELEVATION OF PLASMA SOLUBLE CYTOTOXIC T-LYMPHOCYTE-ASSOCIATED ANTIGEN-4 IN CHILDREN WITH ALLERGIC ASTHMA

Introduction: Cytotoxic T-lymphocyte-associated antigen (CTLA-4), expressed on the surface of activated T-cells, is a negative regulator affecting T-cell immune response in autoimmunity and allergy. We investigated the possible roles of circulating CTLA-4 in allergic asthma.

Methods: The plasma CTLA-4 concentration of 57 chronic asthmatic children treated or not treated with inhaled corticosteroid, 16 acute asthmatic children, and 26 sex and age matched control subjects was measured by enzyme-linked immunosorbent assay.

Results: Chronic asthmatic children not treated with inhaled corticosteroid had significantly higher plasma CTLA-4 than that of corticosteroid treated patients (p < 0.001) and control subjects (p < 0.035) [median (interquartile range): not steroid treated, 22.6 (17.0 – 26.3) ng/ml; steroid treated, 16.1 (12.0 – 19.7) ng/ml; control, 17.6 (14.9 – 21.9)]. In children with acute asthma, treatment with systemic steroid for 5 weeks significantly decreased plasma CTLA-4 (p < 0.01).

Discussion: We are the first investigators to observe elevated plasma CTLA-4 concentration in asthmatic children. Circulating CTLA-4 may play a crucial role in the pathogenesis of allergic asthma.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P50 INTRACELLULAR SIGNALLING MECHANISMS OF CYTOKINE-REGULATED CELL SURFACE EXPRESSION OF ICAM-1 ON HUMAN HMC-1 MAST CELLS

Introduction: Mast cells are the major effector cells of immediate hypersensitivity. We investigated their cytokine-regulated cell-surface expression of adhesion molecules.

Methods: HMC-1 cells of a human mast cell line were stimulated with different cytokines including stem cell factor (SCF), tumour necrosis factor (TNF)-α, interleukin (IL)-13, IL-18, and IL-25. The cell surface expression of intracellular adhesion molecule-1 (ICAM-1) was assessed using flow cytometry.

Results: SCF, TNF-α and IL-13 but not IL-18 and IL-25 could up-regulate the surface expression of ICAM-1 on HMC-1 cells. Moreover, SCF and TNF-α could synergistically up-regulate the ICAM-1 expression. PD098059 and SB203580, which are inhibitors of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), could suppress the SCF-induced ICAM-1 expression, the TNF-α-induced ICAM-1 expression, and the synergistic effect of SCF and TNF-α.

Discussion: SCF and TNF-α may act through the MAPK/ERK and p38 MAPK signaling pathways to regulate the cell surface expression of ICAM-1 on human mast cells. This mechanism provides a potential for the development of novel therapeutic strategies for treating allergy.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P51 ACTIVATION OF NF-κB AND P38 MAPK REGULATING THE RELEASE OF CYTOKINES AND CHEMOKINES FROM CO-CULTURE OF HUMAN EOSINOPHILS AND BRONCHIAL EPITHELIAL CELLS

Introduction: Trans-endothelial infiltration of eosinophils and their interaction with epithelial cells play a dominant role in asthma pathogenesis. We investigated eosinophilic activation of human bronchial epithelial cells enhancing the latter’s expression of cellular adhesion molecules, release of cytokines and chemokines, and morphological changes.

Methods: Inflammatory cytokines and chemokines in the co-culture supernatant of human eosinophils and BEAS-2B cells (from a human epithelial cell line), and cellular adhesion molecules on BEAS-2B cells, were measured by flow cytometry. NF-κB pathway-related genes were evaluated by cDNA expression array. NF-κB and p38 MAPK activities were assessed by ELISA and Western blot.

Results: Eosinophils were found to increase the release of cytokines (IL-6, IL-10 and TNF-α) and chemokines (RANTES, MIG, MCP-1, IL-8 and IP-10) by BEAS-2B cells, and up-regulate the corresponding genes therein. Moreover, eosinophils promoted the expression of adhesion molecule ICAM-1 on BEAS-2B cell surface, and enhanced NF-κB and p38 MAPK activities in BEAS-2B cells. It was also shown that lipopolysaccharide or TNF-α could further enhance eosinophil-mediated BEAS-2B cell activation and mediate eosinophil morphological change in co-culture. Both the NF-κB inhibitor, MG-132, and p38 MAPK inhibitor, SB203580, could inhibit the release of cytokines and chemokines in the co-culture supernatant and reduce the gene expression of cytokines and chemokines in BEAS-2B cells.

Discussion: Eosinophil-bronchial epithelial cell interaction may be crucial for the pathogenesis of allergic inflammation.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P52 REAL TIME QUANTITATIVE MEASUREMENT OF THE B LYMPHOCYTE STIMULATOR GENE EXPRESSION IN PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH AUTOIMMUNE DISEASES

Objective: To establish a specific FQ-PCR (fluorogenic quantitative RT-PCR) method for detecting the expression of B lymphocyte stimulator (BlyS) gene in peripheral blood mononuclear cells (PBMCs) and investigate the relationship between BlyS mRNA expression and autoimmune diseases.

Methods: Real-time quantitative reverse transcription (RT), based on fluorescent TaqMan methodology, was used to examine the specific expression of BlyS in 19 patients with autoimmune diseases (systemic lupus erythematosus, SLE; rheumatoid arthritis, RA), 20 subclinical patients with positive antinuclear Abs (ANA) but who did not meet the ACR criteria, 8 other diseased patients with negative autoantibodies but increased serum levels of immunoglobulins (Igshigh patients), and 20 healthy people.

Result: In 19 autoimmune disease patients, the BlyS mRNA expression ranged from 9.7×105–3.2×108 copies/μg RNA, and the mean value was 8.4±7.9×107 copies/μg RNA. In 20 subclinical patients the BlyS mRNA expression level was 1.3 ± 1.2x106 copies/μg RNA. The BlyS mRNA expression level in 8 other diseased patients, with negative autoantibodies but increased levels of serum immunoglobulins, was1.2±1.2×106 copies/ μg RNA. For 20 healthy people the BlyS mRNA expression level was 1.7±1.4×105 copies/μg RNA.

Conclusion: BlyS gene expression detected with FQ-PCR gave accurate, rapid results. The BlyS mRNA expression level was significantly elevated in autoimmune diseases, with the production of autoantibodies and higher serum IgG, and the BlyS mRNA might be used as a useful marker for early diagnosis of autoimmune diseases.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P53 PLASMA INFLAMMATORY CHEMOKINES IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

Introduction: Cytokines and chemokines have been implicated for mediating inflammation in systemic lupus erythematosus (SLE). We investigated the use of plasma inflammatory chemokines as markers of SLE disease severity.

Methods: Eighty SLE patients [78 women, 2 men; mean (SD) age of 37 (9) years] and 40 sex- and age-matched healthy subjects were studied. SLE was diagnosed according to criteria of the American College of Rheumatology, and disease severity assessed using the SLE Disease Activity Index (SLEDAI). Plasma inflammatory chemokines IFN-inducible protein-10 (IP-10), regulated upon activation normal T cell expressed and secreted (RANTES), monokine induced by IFN-gamma (MIG), monocyte chemo-attractant protein-1 (MCP-1), thymus and activation regulated chemokine (TARC), and neutrophil chemokine IL-8 were assayed by immunofluorescence flow cytometry.

Results: Compared to control subjects, SLE patients had significantly elevated plasma IP-10, RANTES, MIG, and MCP-1 [mean (interquartile range) of 2030 (1374–3294) vs 737 (597–864), 1642 (655–4364) vs 580 (296–1660), 516 (342–1067) vs 203 (159–250), 86 (55–132) vs 34 (22–47) pg/ml, respectively (all p<0.05); but there was no significant increase in TARC and IL-8. The SLEDAI of patients (median score 4, interquartile range 2–8) correlated positively with plasma IP-10, RANTES, MCP-1, IL-8 concentrations (Spearman r: 0.31–0.36, all p<0.05).

Discussion: Inflammatory chemokines contribute to SLE pathogenesis via their recruitment and trafficking of leucocytes (lymphocytes, neutrophils and monocytes) to organs and tissues of autoimmune injury. The plasma concentration of some newly identified inflammatory chemokines may serve as useful marker of SLE disease severity.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P54 IMUNOMODULATORY ACTIVITIES OF YUNZHI AND DANSHEN IN POST-TREATMENT BREAST CANCER PATIENTS A SELF CONTROLLED STUDY

Introduction: The Chinese medicine Yunzhi (Coriolus versicolor) has been shown to modulate various immunological functions in vitro, in vivo and in human clinical trials. Danshen (Salvia miltiorrhiza) can benefit the microcirculation by its vasodilating activity conferring an anti-dementia effect. The purpose of our clinical trial was to evaluate the combined immunomodulatory effects of Yunzhi and Danshen capsules in breast cancer patients after surgical treatment.

Methods: Eighty patients were recruited to take Yunzhi (50 mg/kg body weight) and Danshen (20 mg/kg) capsules daily for a total of 6 months. EDTA blood samples were collected every 2 months for the investigation of immunological functions. Flow cytometry was used to assess the percentages and absolute counts of human lymphocyte subsets in whole blood. Plasma concentration of soluble interleukin-2 receptor (sIL-2R) was measured by enzyme-linked immunosorbent assay.

Results: The ratio of T helper (CD4+)/T suppressor and cytotoxic lymphocytes (CD8+), and the percentage and the absolute counts of B lymphocytes were significantly elevated in patients with breast cancer after taking the Yunzhi-Danshen capsules for 4 months, while plasma sIL-2R concentration was significantly decreased (all p < 0.05).

Discussion: Yunzhi-Danshen capsules may be beneficial for promoting immunological functions in breast cancer patients.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P55 IMMUNOSUPPRESSIVE EFFECT OF LEFLUNOMIDE IN SYSTEMIC LUPUS ERYTHEMATOSUS: A PILOT OBSERVATIONAL STUDY

Introduction: We investigated the immunosuppressive effect of leflunomide, a novel antirheumatic drug, on chemokine expression in patients with systemic lupus erythematosus (SLE).

Methods: Eleven patients were enrolled and divided to take either leflunomide or placebo for 5 months. Blood samples were collected before and after 3 and 5 months of treatment. IFN-γ-inducible protein-10 (IP-10), monokine induced by IFN-γ (MIG), interleukin (IL)-8, regulated upon activation normal T-cell expressed and secreted (RANTES), and monocyte chemo-attractant protein-1 (MCP-1) in plasma and whole blood culture supernatant were assayed using flow cytometry.

Results: After leflunomide therapy, the SLE disease activity index (SLEDAI) had generally decreased. Plasma RANTES concentrations of leflunomide-treated patients were lower than those of the placebo group after 3 and 5 months of treatment (both p<0.05). However, relatively little changes were observed in other chemokines. For the whole blood assay with phytohaemagglutinin stimulation, RANTES concentrations of culture supernatant after 3 and 5 months of leflunomide were lower than those of the placebo group.

Discussion: Leflunomide showed a suppressive effect on RANTES expression, which may cause an inhibition on the activation and recruitment of T lymphocytes in SLE patients.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P56 ELEVATION OF PLASMA SOLUBLE T-CELL COUNTER RECEPTORS AND COSTIMULATORY MOLECULES IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

Introduction: The co-stimulatory interactions of the B7 molecules CD80 and CD86 on antigen presenting cells with their T-cell counter receptors CD28 and CTLA-4 regulate T-cell mediated immune responses in a reciprocal manner. We investigated the potential presence and clinical significance of the soluble forms of these co-stimulatory molecules and T-cell counter receptors (sCD80, sCD86, sCD28 and sCTLA-4) in blood plasma of patients with systemic lupus erythematosus (SLE).

Methods: Eighty SLE patients [78 women, 2 men; mean (SD) age of 37 (9) years] diagnosed according to criteria of the American College of Rheumatology, and 40 sex- and age-matched healthy subjects were studied. Plasma sCD28, sCTLA-4, sCD80 and sCD86 concentrations were measured by enzyme-linked immunosorbent assay.

Results: sCD28, sCTLA-4, sCD80 and sCD86 were ubiquitously present in plasma, with concentrations in patients significantly higher than those of control subjects [median (interquartile range) of 3.9 (2.3–4.7) vs 1.6 (1.3–1.9), 5.2 (4.1–7.3) vs 4.2 (3.4–5.7), 0.28 (0.18–0.43) vs 0.13 (0.11–0.21), and 1.8 (1.4–2.1) vs 1.4 (1.1–1.5) ng/ml respectively; all p< 0.01].

Discussion: The presence in the circulation of soluble co-stimulatory molecules and T-cell counter receptors may provide a means for T-lymphocytes to either enhance or inhibit their biologic effects through additional crossing-linking or competitive blocking to their cell-bound counterparts, thereby influencing the T cell-mediated immune responses.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P57 PREDICTION AND IDENTIFICATION OF AUTOEPITOPES OF PDC-E2 SPECIFIC CD8+CTL IN PRIMARY BILIARY CIRRHOSIS

Objective: The aims of this study were to identify autoepitopes of E2 subunit of pyruvate dehydrogenase complex (PDC-E2) specific CD8+CTL in primary biliary cirrhosis (PBC), which will contribute to development of a specific immunotherapy.

Methods: An online database SYFPEITHI was applied to predict HLA-A2.1 restricted epitopes which located in PDC-E2 30–50aa and 150–190aa where B-cell epitopes clustered with CD4+ T-cell epitopes. T2 cell line reconstitution and stabilization assay, induction of specific CTL lines from peripheral blood mononuclear cells (PBMCs) of patients with PBC and cytotoxicity of peptides-induced CTL were performed to screen the epitopes from those candidates.

Results: Five potential epitopes were predicted by the database. Of the 5 candidates, two peptides 159–167aa and 165–174aa, with high binding affinity to HLA-A2.1 molecules, could stimulate PBMCs from most HLA-A2.1 positive PBC patients to proliferate and peptide-induced CTL lines showed specific cytotoxicity.

Conclusion: This study indicates that peptides of KLSEGDLLA 159–167aa) and LLAEIETDKA (165–174aa) in the inner lipoyl domain of PDC-E2 are HLA-A2.1 restricted CD8+ CTL epitopes in PBC, which is important to quantitation of antigen specific CTL and design of inhibited peptides vaccine on the basis of epitopes recognized by CTL.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P58 FIVE YEARS EXPERIENCE WITH A GLOBAL MALARIA EDUCATIONAL PROGRAMME

Introduction: The morbidity and mortality caused by malaria poses an enormous burden on governments and health care facilities worldwide. For much of the world, the front-line diagnosis of this disease remains in the hands of the microscopist and microscopy is still the “gold standard” for the diagnosis and species identification of malaria.

Aim: To make use of emerging internet technology to distribute a concise, practical, malaria educational programme covering laboratory diagnosis, prophylaxis, treatment and a test and teach section, for healthcare professionals worldwide.

Methods: In 1998 a small group of Scientists from Western Australia began an on-line malaria information resource using Internet technology to bring the knowledge and experience of the authors to healthcare professionals worldwide. Recognising inequalities of access to the Internet, with many centres having limited or cost prohibitive access, a version of the malaria project was produced on CD-ROM and distributed, on request, free of charge courtesy of sponsors Abbott Diagnostics. With help from international colleagues, later versions of the CD were produced which included French and Spanish translations.

Results: The web site has received in excess of 500,000 visitors with more than 81 gigabytes of information down-loaded from the website between May 2001 and September 2003 (equivalent to about 83 million pages of text). The CD-ROMs have been requested and sent to thousands of institutions spanning 149 countries.

Discussion: Work from educators based at Royal Perth Hospital in Western Australia has managed to reach many thousands of “students” throughout the world. The on-line and CD-ROM programmes have proven to be invaluable tools in malaria education projects worldwide and could be a model on which other health education programmes are based.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P59 PROTEIN PROFILES TYPING OF SALMONELLA SPECIES BY SDS-PAGE TECHNIQUE

Introduction: The genus of Salmonella, one of the most important of the Enterobacteriacea family, can cause serious diseases in human and animals. Salmonella typhi and para-typhi can cause typhoidal and para-typhoidal infections, whereas non-typhoidal Salmonella species can cause non-typhoidal infections such as gastroenteritis and septicemia. The aim of this study was the typing of typhoidal and non-typhoidal Salmonella species by extraction of whole-cell proteins of strains using polyacrylamide gel electrophoresis (SDS-PAGE).

Materials & Methods: In this study, 112 Salmonella strains were Serotyped by Biomerix polyvalent and monovalent antisera. Whole-cell proteins of strains were also detected by the Taylor et al method. Gels were stained by coomassie Brilliant Blue and photographed through an orange filter. Rate flow (RF) of each protein band was also determined. Protein bands were detected by densitometery. The protein profile of each strain was compared with serotyping results.

Results: Of 112 Salmonella species, 62.2% were typhoidal Salmonella and 37.8% were non-typhoidal Salmonella. The most common serotypes were as follow: S. typhy 41.2%, S. paratyphy B 12.35, S. paratyphy C 8.7%, S.typhimurium 21.4%, S. entertidis 6.3%, S. arizona 4.2% and S. agona 2.1%. The results of serotyping were compared with the results obtained by SDS-PAGE. Many protein bands from major protein 205 KDa to minor protein 35 KDa were detected by SDS-PAGE and differentiated the strains well. Protein profiles of clinical strains were compared and showed some variations with results of serotyping and could divide them to 7 subgroups.

Conclusion: Our results show that extraction of whole-cell proteins of microorganisms including typhoidal and non-typhoidal Salmonella species by polyacrylamide gel electrophoresis could be used for identification and typing purposes.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P60

NOW ORAL PRESENTATION O14

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P61 SOLID LIPID NANOPARTICLE BASED INHALABLE DRUG DELIVERY SYSTEM AGAINST EXPERIMENTAL TUBERCULOSIS

The present study was designed to develop solid lipid nanoparticles (SLNs) as drug carriers for the reduction in dosing frequency as well as for direct pulmonary delivery against tuberculosis (TB), which otherwise demands prolonged chemotherapy.

SLNs of stearic acid encapsulating three front line antitubercular drugs (ATD), i.e. rifampicin, isoniazid and pyrazinamide were formulated. Drug loaded SLNs were aerodynamically characterized on seven stage Andersen Cascade Impactor and nebulized to guinea pigs for pharmacokinetic/chemotherapeutic evaluation.

The drug encapsulation efficiency ranged from 40–55% with a loading of 150–200mg drug/g formulation. Approximately 96% of the aerosols were respirable (≤6 μM) and the mass median aerodynamic diameter was 1.7 ± 0.1μM (geometric standard deviation = 1.9 ± 0.1μM) indicating favourable aerodynamics for bronchoalveolar drug delivery. A single nebulization of the formulation to normal and TB infected guinea pigs resulted in sustained drug levels in the plasma for 5 days and in the organs (lungs, liver and spleen) for 7 days, with an enhanced drug bioavailability as compared to aerosolized/oral free drug. In M. tuberculosis H37Rv infected guinea pigs, weekly nebulization of SLNs for 6 weeks produced equivalent clearance of bacilli with respect to conventional treatment 46 doses repeated administration of the SLNs was devoid of any biochemical hepatotoxicity. Solid lipid nanoparticles hold promise as a potential lipid based inhalable ATD carrier for a better management of TB.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P62 SPECIES IDENTIFICATION AND SUBTYPING OF THE MULTIPLE-BANDED ANTIGEN FOR UREAPLASMA BY POLYMERASE CHAIN REACTION

Introduction: Ureaplasma urealyticum is a recognized cause of nongonococcal urethritis, It has also been implicated in other genitourinary syndromes. There are two biovars and 14 serovars of U.urealyticum. The MBA (multiple- banded antigen) is the predominant antigen recognized during U.urealyticum infections and is probably an important virulence determinant. The aim of this study was to identify the MBA genes of U.parvum and U.urealyticum by PCR-based typing system.

Methods: Cervical and urethral swabs of 228 patients with genetic infection in STD Clinic were collected from each region hospital in Guangdong province to detect biotyping methods. The cultures were performed as soon as possible after receipt of specimens in the laboratory, while biovar and subtyping of MBA gene were performed by PCR. In this study, we designed 9 pairs oligonucleotide primers, targeting the 5’ ends of the MBA genes, to identify and subtype these Ureaplasma species. The 9 primer pairs could distinguish the two species, and subtypes within each species.

Results: These methods were used to identify and type U. urealyticum in 137 (60.1%) of 228 patients with genetic infection in STD clinic, among U.parvum (biovar 1) was detected in 40.3% and U.urealyticum (biovar 2) in 19.7%. A selection of 9 primer pairs used to identify and subtype, the results showed that serovars 3/14 (16.7%), 1 (14.0%), 6 (9.6%) and subtypes 2 (12.3%) and 1 (5.3%), 3(2.2%), respectively.

Conclusions: The PCR-based typing system will facilitate future studies of the relationship between individual Ureaplasma species or subtypes and human disease. The methods described are relatively rapid, practicable, and specific for serotyping isolates for identification of individual serovars in clinical specimens containing Ureaplasma species or subtypes.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P63 DETECTION OF LEGIONELLA PNEUMOPHILA IN WATER SYSTEMS AND CLINICAL SPECIMENS BY CULTURE AND POLYMERASE CHAIN REACTION

Introduction: Legionella pneumophila are a common cause of community-acquired respiratory tract infections and an occasional cause of nosocomial pneumonia after inhalation of contaminated water droplets from a variety of water sources. In the study, we are developing a PCR method for the detection of legionellae in respiratory samples, cooling tower water and lake water samples was evaluated and compared to culture.

Methods: Direct detection of legionella pneumophila, 20 cooling tower water samples and 20 water samples collected from 4 lakes (Liuhua, Yuexiu, Tianhe, Lihu) in Guangzhou city, and 7 bronchial washing and 15 sputum samples of patients with respiration tract infection in Zhongshan hospital were investigated by isolation culture and PCR assay.

Results: PCR tested positive for Legionella pneumophila in 3/20 (15%) bronchial washing and sputum samples of patients with respiration tract infection, while no Legionella pneumophila were isolated by culture method. The results from the 20 cooling tower water samples showed that the 11 (55.0%) positive Legionella pneumophila were detected by both culture and PCR assays, among 4 positive water samples were detected by PCR assay, but culture assay was negative. 13 of 20 (65%) water samples collected at 4 lakes were positive for Legionella pneumophila by PCR, but all of 20 water samples Legionella culture were negative. The results showed that 3 of 4 lakes demonstrated the presence of Legionella pneumophila contamination.

Conclusions: The PCR assay that is described demonstrates high sensitivity for routine detection of legionellae in every sample. The results suggest that Legionellae is commonly distributed in the water systems (including cooling towers and lake water) in Guangzhou.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P64 EFFECT OF HELICOBACTER PYLORI ON DUODENAL ENZYMES IN NON-ULCER DYSPEPSIA.

Introduction: It has been reported in the literature that H.Pylori is responsible for gastroduodenal disorders. It is possible that the activity of duodenal enzymes is affected by H.Pylori infection. Therefore, the aim of this study was to investigate alterations in duodenal enzymatic profile of non-ulcer dyspepsia (NUD) patients with H.Pylori infection.

Materials and Methods: Forty patients of NUD were selected and divided into two groups as study group (H.pylori positive) and control group (H.pylori negative). Estimation of activity of enzymes, lactase, sucrase, maltase, gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase was measured.

Results: Duration of symptoms was higher in the study group (p<0.01) as compared to the controls. Enzymatic analysis in the two groups did not show any significant change in any of the enzyme activity. Patients with lactose intolerance had low level of lactase enzyme as compared to lactose tolerant patients.

Conclusion: No significant difference in enzyme activities of duodenum was found in patients with H.Pylori infection as compared to the patients with no infection. Similar observation was obtained with inflammation and without inflammation when data of both the groups was pooled together.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P65 HBsAg CARRIERS WITH SERUM ANTI-HBc NEGATIVITY

An absence of the hepatitis B core antibody (anti-HBc) in hepatitis B surface antigen (HBsAg) carriers is quite unusual serologic findings, even if reported before that the absence of anti-HBc occurred almost exclusively in children who were infected perinatally. We found four cases (0.6%) of HBsAg carriers with serum anti-HBc negativity among 695 HBsAg-positive patients tested in Chung-Ang University Yongsan Hospital from Jan 2003 to Apr 2004. Of the four HBsAg carriers with serum anti-HBc negativity, two patients were HBsAg carriers with chronic hepatitis, one was in the early phase of acutely developed hepatitis and the last one was a HBsAg carrier infant born to a HBsAg carrier mother. Results of HBsAg were confirmed positive in two HBsAg carriers with chronic hepatitis after neutralization by HBsAg confirmatory kit provided by Abbott Diagnostics Division (Abbott Laboratories, Abbott Park, IL, USA), however, those were reactive nonconfirming in the other two patients in whom HBV DNA was detected by nested PCR and/or Cobas Amplicor HBV Monitor Test™ (Roche, Branchburg, NJ, USA). Hepatitis e antigen (HBeAg) was positive and serum ALT level was slightly elevated in one patient with chronic hepatitis who was persistently seronegative for anti-HBc for 6 months. Antibodies to HBeAg and HBsAg (anti-HBe and anti-HBs) were not detected in sera of all anti-HBc-negative HBsAg carriers. Thus, HBsAg carriers with anti-HBc negativity probably result from immune incompetency of the hepatitis B virus antigens or shedding of serologically undetectable quantities of complete and/or defective HBcAg from liver cell nuclei which contain HBcAg not detectable by immunofluorescence. It will be investigated whether anti-HBc will ultimately appear, hepatitis B virus (HBV)-derived liver damage has not yet occurred or a long time has passed since the last episode of liver damage subsided.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P66 CONFIRMATION OF PRESENCE OF HBSAG IN SAMPLES WITH LOW LEVEL HBSAG

Introduction: Hepatitis B surface antigen (HBsAg) is the most important serological marker of acute and chronic hepatitis B infection. Therefore, sensitivity of the currently used detection systems for HBsAg is of major importance for blood screening, diagnosis of HBV infection and therapy monitoring of HBV infected individuals. Highly sensitive detection systems, however, might have increased false positive results. This study was performed to investigate the distribution of the samples with low-level HBsAg and to confirm these low level HBsAg true positive.

Methods: HBsAg was tested by microparticle enzyme immunoassay (MEIA) for qualitative determination of HBsAg using AxSYM analyser (Abbott Laboratories, Abbott Park, IL, USA). The presence of HBsAg was confirmed by the neutralization assay with a specific antibody using HBsAg confirmatory kit (Abbott Laboratories, Abbott Park, IL, USA) and/or detection of HBV DNA. The presence of HBV DNA was evaluated by nested PCR and Cobas Amplicor HBV Monitor Test™ (Roche, Branchburg, NJ, USA).

Results: Among 695 HBsAg-positive patients tested in Chung-Ang University Yongsan Hospital from Jan 2003 to Apr 2004, 23 patients (3.1%) had HBsAg lower than 2.0 ng/mL (S/N value less than 10.0). The presence of HBsAg was confirmed by means of specific antibody neutralization in 8 out of 10 patients with HBsAg lower than 2.0 ng/mL. The results of anti-HBc were positive in 8 patients with confirmed positive HBsAg results, however, negative in 2 patients whose HBsAg confirmatory results were reactive nonconfiming. HBV DNA was detected only in one patient in the early phase of acutely developed hepatitis of whom HBsAg confirmatory results were reactive nonconfirming. Therefore, one patient was considered to be a false positive.

Conclusion: Patients with low level HBsAg in serum should not be neglected, when tested by AxSYM analyser, especially when anti-HBc was positive.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P67 PREVALENCE OF HBV & HCV WITH OTHER INFECTIOUS DISEASES AMONG BANGLADESHI POPULATION SEEKING OVERSEAS EMPLOYMENT

Introduction : Hepatitis B and C virus infections are responsible for major causes of mortality and morbidity around the world. In Bangladesh there is little research on prevalence of these diseases.

Methods : Prospective study carried out in NAFA Medical Centre, Dhaka, during the period of 1st March 2003 to August 31, 2003. A total of 4880 subjects, attended the laboratory for health fitness certificates within the study period, were selected. Gold standard laboratory tests were conducted for HBsAg, Anti-HCV, HIV, TPHA & VDRL.

Results : Among 4880 subjects, 252 were HBsAg positive and 39 were Anti-HCV positive. HIV was positive in only 09 subjects. TPHA & VDRL turned out to be positive in 121 & 109 subjects respectively. Prevalence of HBsAg were 5.16%, Anti-HCV 0.8%, HIV 0.18%, TPHA 2.48% & VDRL 2.23%.

Discussion : Results of the study represent a cross-sectional view of the prevalence of different infectious diseases among adult population of Bangladesh. Persistent high prevalence of HBV, HCV & HIV in our study population strengthen the view that equal importance need to be given in each of the viruses. Our HBsAg prevalence corresponds with 2–8% prevalence estimated for the region by the WHO.

References

1. WHO. Communicable / Infectious Disease: Hepatitis B. WHO Fact Sheet. 204. November 1998.
2. Gibney L. , Saquib N., Metzger J. et al. Human immunodeficiency virus, hepatitis B, C and D in Bangladesh Trucking industry: prevalence and risk factors. Int J Epidemiol. 2001;30 :878–84. [PubMed]
3. WHO. Introduction of hepatitis B vaccine into childhood immunization service. Geneva. 2001. (Rec 2000; 75 (3) : 18–19.)
4. WHO. Hepatitis C global prevalence (update). Wkly Epidemiol
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P68 ANTIBIOTIC RESISTANCE AND EVOLUTION OF ESCHERICHIA COLI IN A PATIENT BY LONG-TERM ANTIBIOTIC TREATMENT

Introduction: To study antibiotic sensitivities and characterization of β-lactamase activity with the same patient over a long time period whilst he was on prolonged antibiotic therapy.

Methods: Characterization of Extended-spectrum β-lactamases. Agar dilution method was used to determine the minimum inhibitory concentration (MIC) of several antibiotics. Qiagen Qiaprep spin Miniprep. The plasmids of clinical isolates were cloned into competent cells (K-12). Pulsed Field Gel Electrophoreses (PFGE). ESBLs genes were analysed by isoelectric focusing (IEF) assay and PCR initially. Genotypes were determined by sequencing.

Results: The MIC results showed a general increase in resistance to antibiotics tested from the earlier to the later isolates. The transformation was successful for transforming all of the 7 clinical isolates strains. PFGE: The digestion patterns with Not1 showed that seven clinical isolates were closely related. The Not1 digest showed the isolates have similar patterns each other. The last 5 clinical isolates have all got an activity at a high pH 9 which can be presumptively identified as AmpC, The last two clinical isolates have a weak activity with a PI of about 8.0, on the basis of this PI value these bands could be SHV type β-lactamases, All the isolates had a group of activities at the lower pH range of the gel (PH5), These can be presumptively identified as TEMβ-lactamases. Each isolate has the same OmpC sequence that distinct from other E. coli. isolates.

Conclusions: The date suggest that each isolate clearly originates from the same founder population but possesses a unique combination of attributes and mutations that leads to their characteristic antibiotic resistance.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P69 HYPOGLYCEMIC RESPONSES OF ANTITUBERCULAR TREATMENT

Isoniazid, Pyrazinamide, Ethambutol and Rifampicin are primary chemotherapeutic agent for the treatment of tuberculosis. Individually INH is known to affect Glycolysis and its overdose as in attempted suicide has been reported to produce occasional hyperglycaemia. So, it was thought of interest to see the effects of other drugs also on the homeostasis of Blood-Glucose in animals. The blood Glucose estimation was done by O. toludine method. Single graded dose of 1NH produced significant hyperglycaemia but Rifampicin and Ethambutol individually produced significant hypoglycaemia and Pyrazinamide, showed no effect on Blood Glucose level. With treatment of INH alone even the tolerance developed to its hyperglycaemic effect on daily administration for 4 weeks. Single graded dose of 1NH also didn’t produce and significant alteration in glucose tolerance but chronic administration for 4 weeks resulted in significant Glucose Tolerance. But when INH is given in combination of Ethambutol Pyrazinamide and Rifampicin a significant hypoglycaemia is reported in single graded dose and even on chronic administration for 4 weeks. The experiments were also carried out along with Adrenaline and Glibenclamide and confirmed our findings. These results indicate that ATT cause hypoglycaemic effects and a routine investigation of BGL is also suggested in-patients because co. existence of Diabetes and Tuberculosis is also not uncommon.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P70 CORRELATION BETWEEN SERUM LEVELS OF CHOLESTEROL AND HOMOCYSTEINE WITH OXIDATIVE STRESS IN HYPOTHYROID PATIENTS

Abnormalities in cholesterol and homocysteine metabolism have been reported in thyroid diseases. Since elevated levels of the both parameters are involved in atherogenesis and thyroid hormones are modulator of oxidative stress, the correlation between the parameters was assessed in hypothyroidism.

A total of 60 patients with thyroid dysfunction (30 hypothyroid and 30 hyperthyroid) and 30 apparently healthy sex and age matched individual as control group were included in this study. The mean age of hypothyroid, hyperthyroid and control groups were 43 ± 7.7, 39 ± 12 and 40 ± 7.9 years respectively. Homocysteine levels were measured by HPLC and those of thyroid hormones by Radioimmunoassay techniques and the other parameters with standard methods in Cobas mira autoanalyser.

The mean ± SD levels of homocysteine in hypothyroidism, hyperthyroidism and control groups were 17.09 ± 6.9, 7.79 ± 1.4 and 8.08 ± 1.9 μmol/L respectively. Marked correlation between increased level of creatinine and that of homocysteine was noticed (r=0.86, p=0.0001). Significant elevation in the serum levels of cholesterol, LDL-C and meaningful reduction in serum antioxidant status were observed in hypothyroid group (p<0.05 in all cases). The differences between those of control and hyperthyroid groups were not significant. Reverse and marked correlation between serum level of antioxidant capacity and that of homocysteine (r=−0.79, p=0.02), cholesterol (r=−0.93, p=0.02) and LDL-C (r=−0.83, p=0.001) were noticed. The correlation between serum levels of homocysteine, cholesterol and LDL-C with that of total antioxidant capacity in hypothyroid group suggests that enhanced production of free radicals may be an important contributing factor in abnormalities seen in homocysteine and cholesterol metabolism. Supplementary vitamin E and C and folic acid may be beneficial for hypothyroidism in prevention of atherosclerosis.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P71 POLYMORPHISMS OF APOLIPOPROTEIN A1-C3 GENES IN THE RISK ASSESSMENT OF CORONARY ARTERY DISEASE

Introduction: Genetic variants of APOA1-C3 have been associated with alterations in high density lipoprotein – cholesterol (HDL-C) and triglyceride (TG) levels in various population studies. Since low HDL-C and high TG are predominant risk factors for CAD in Indian scenario, we have elucidated the interrelationships of APOA1-C3 polymorphisms, lipid profile and coronary artery disease.

Methods: We recruited 167 angiographically proven CAD patients, 36 subjects with insignificant CAD and 229 controls samples from Northern India. Polymorphisms were studied by PCR-RFLP(1) and lipid profile by enzymatic kit.

Results: A significant association of rare ‘A’ allele (APOA1-75G/A polymorphism) with severity of CAD, lower HDL-C levels (in patients and controls) and lower apoA-I levels (in patients) was found. We came across a +40APOA1 G/A mutation associated with double vessel disease and lower levels of HDL-C and apoA-I levels. Rare ‘-‘ allele (APOA1 +83C/T and/or +84G/A polymorphism) was less prevalent in patients as compared to controls and was associated with lower levels of LDL-C (in controls). Rare S2 allele of APOC3 SstI polymorphism was associated with higher TG levels, but not with CAD and its severity.

Discussion: Screening for ‘A’ allele and +40G/A APOA1 mutant in the population would enable us to target the high-risk individuals in the population and would facilitate the prevention of CAD. Rare ‘-‘ allele carriers may be less prone to get the disease. Rare S2 allele may serve as marker for susceptibility to hypertriglyceridemia in the population.

References

1. Hum Genet. 1992;88:439–46. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P72 A COMPARISON OF APOLIPOPROTEINS A-I, A-II AND B, AND CHOLESTEROL FRACTIONS, IN CAPILLARY AND VENOUS SERUM

Introduction: The development of micromethods for the analysis of biochemical markers of chronic disease in capillary blood has considerable application in clinical epidemiology. Serum apolipoproteins (apo) A-I, A-II and B, and cholesterol fractions, are important biochemical markers of cardiovascular disease (CVD) risk. In this study, the analysis of apo A-I, apo A-II and apo B, and cholesterol fractions, was compared in capillary and venous serum.

Methods: Capillary (fingertip; 100 500 μL) and venous (median cubital) blood was collected simultaneously from 10 healthy adult subjects into serum tubes (Greiner Bio-one, Germany) following an overnight fast. Lipoprotein fractions in serum were separated by agarose gel electrophoresis, stained for cholesterol, and immunofixed for apo A-I, apo A-II and apo B.

Results: The immunofixation staining patterns of apo A-I, apo A-II and apo B in venous serum have been described in a corresponding abstract. Capillary serum from each subject showed identical immunofixation staining patterns for the apolipoproteins to those measured in the venous serum. There was also close agreement between the absolute values for total cholesterol, LDL-cholesterol, HDL-cholesterol and lipoprotein (a) cholesterol (when present) in the samples of capillary and venous serum.

Discussion: The collection of capillary blood is cheaper, safer, less traumatic and more suitable for mass screening in clinical epidemiological studies than venous blood collection. This study has shown minimal differences between capillary and venous serum for the qualitative and semi-quantitative analysis of apo A-I, A-II and B, and quantitative analysis of cholesterol fractions. Therefore, capillary (fingertip) blood appears to be suitable for epidemiological research into CVD risk requiring the analysis of multiple biochemical markers.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P73 LIPID PROFILES, TOTAL ANTIOXIDANT CAPACITY AND LIPID PEROXIDES IN CORONARY ARTERY DISEASE

Coronary artery disease (CAD) has been one of the most important causes of morbidity and mortality in the past two decades. It is known that oxidation of lipids and lipoproteins, increased levels of free radicals and reduction in antioxidant capacity plays an important role in the development of atherosclerosis and CAD. On the other hand several studies in different populations have shown that high lipoprotein (a) [Lp(a)] concentration is a risk factor for atherosclerosis. In this study alteration in the serum levels of lipid and lipoproteins (HDL-C, LDL-C, TG, TC), total antioxidant (TA), malondialdehyde (MDA), and Lp(a) were estimated in 60 male patients [age 31–70 years] with suspected CAD undergoing coronary angiography and results were compared with those obtained from sex and age matched apparently healthy individuals as control group (n=60). Serum level of MDA and TA were measured by spectrofluorometric and spectrophotometric methods respectively. Lp(a) level was measured by turbidimetric method. The results were analysed using SPSS program. Comparing with the control group beside the significant reduction in serum level of TA in patient group (1.1±0.2 vs. 1.3±0.2 mmol/L p<0.05), marked reduction in serum concentration of HDL-C (p<0.005) and significant elevation in the level of LDL-C (p<0.05), TG (p<0.005), TC (p<0.01) and MDA (5.4±1.7 vs. 2.9±0.6 nmol/ml p<0.05) was noticed. The level of Lp(a) in patients was also higher than control subjects (29.98±16.5 vs. 16.98±9.8 mg/dl p<0. 005). It was concluded that association of altered serum levels of lipid, lipoprotein, increased lp(a) and lipid peroxides, with reduced levels of TA in CAD patients could be a useful laboratory technique in confirmatory diagnosis of CAD.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P74 PREDICTION OF PIH IN PREGNANCY BY ESTIMATION OF SERUM TRIGLYCERIDES & MALONDIALDEHYDE IN 1ST TRIMESTER

Introduction: Pregnancy induced hypertension is most common medical problem of pregnancy associated with increased rate of maternal & foetal morbidity & mortality. Incidence of PIH is 8–10%. Recent information indicates that elevated lipids, lipid peroxidation products are found in PIH & they contribute to vascular endothelial cell dysfunction. We tried to associate levels of triglycerides, MDA to development of PIH.

Methods: Serum Triglycerides & Malondialdehyde were estimated in pregnancy between gestational age 18–20 weeks for 90 patients.

Triglycerides by GOD-PAP method. Malondialdehyde was estimated by TBRAS method.

Results: The values obtained were analysed using Pearson chi-square test, significant at 1% level for both triglycerides & Malondialdehyde.

Discussion: The mean of triglycerides value in normotensive women is 122.89 ± 22.85. The women who developed hypertension in later pregnancy is 215.62 ± 39.12. The chi square test is significant at 1% level suggesting that an increased levels of triglycerides at 18–20 weeks in patients develop hypertension in later pregnancy. The mean of MDA in normotensive women in 177.90 ± 20.3 and the mean of MDA for women who developed hypertension later is 353.68 ± 44.28. The chi square test is significant at 1% level suggesting association between levels of malondialdehyde & hypertension.

References

1. Carl A Hubell et al, lipid peroxidation in pregnancy new perspectives on pre-eclampsia Am. J. Obstet. Gynecol. 1989;161:1025–34. [PubMed]
2. Minoru Ishihara: studies on lipoperoxide of normal pregnant women & of patients with PIH. Clinica Chimica Acta. 1978:1–9. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P75 FAMILIAL DEFECTIVE APOLIPOPROTEIN B-100: DETECTION OF THE R3500W MUTATION IN HYPERCHOLESTEROLEMIC CHINESE

CY Chiu1 and TS Jap1,2

Introduction: Familial defective apolipoprotein B (apo B)-100 (FDB) is an autosomal dominant trait resulting in hypercholesterolemia. One of the FDB is caused by substitution of arginine by glutamine or tryptophan at codon 3500 of the apo B-100 gene. To characterize this apo B-100 defect in Chinese of Han descent, we screened hypercholesterolemic subjects in a region of 345 bp surrounding the apo B-100 gene codon 3500.

Methods: We isolated genomic DNA from peripheral blood samples of 31 hypercholesterolemic subjects (21 females and 10 males) and 50 normal subjects with no family history of hypercholesterolemia. The exon 26 of the apo B-100 gene was amplified by polymerase chain reaction (PCR). The 345 bp PCR products were sequenced with standard procedure. Restriction endonuclease (Nla III) digestion of PCR amplicons was used to confirm mutations detected by sequence analysis.

Results: We identified 2 patients with mutation in apo B-100 gene in codon 3500, resulting in a substitution of arginine by tryptophan. Both patients have strong family history of hypercholesterolemia. One patient was a 36 year-old woman with cholesterol level of 7.51 mmol/L, the other one was a 20 year-old gentleman with cholesterol level of 6.48 mmol/L.

Conclusions: We had identified two cases of familial defective apo B-100 with R3500W mutation in hypercholesterolemic Chinese.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P76 ANALYSIS OF ASSOCIATION BETWEEN APOLIPOPROTEIN E GENOTYPES, THE K VARIANT OF BUTYRYLCHOLINESTERASE (BCHE-K) AND ALZHEIMERS DISEASE (AD)

Introduction: Recent studies indicate that there is a synergic association between butyrylcholinesterase K Variant (BChE-K) and apolipoprotein E epsilon4 (APOE-epsilon4) in risk of Alzheimer’s disease (AD), whereas some studies, have been unable to confirm these findings.

Methods: We decided to conduct a study in 105 Alzheimer patients from Tehran, Iran and 129 age and sex matched controls test for can association between BChE K, APOE epsilon4, and AD by PCR RFLP methods.

Results indicate that distribution of BChE genotype in AD group shows significantly different compared to the control group (X2=12.2, df=2, p=0.002). In addition frequency of BChE K allele inAD group is significantly higher compared to control group 24% versus 12% (X2= 20.6, df=2, p<0.001) to an increased risk of AD in subjects posses this allele (OR=2.5,95%CI=1.64–3.8, p=0.001). Based on our fiddling this risk is increased from (OR=1.8,95%CI=o.85–3.95, p=0.05) in <75 years old to (OR=3.16,95%CI=1.41–7.1, p=0.001) in <74 year old subjects. In other hand the APOE-epsilon4 allele associated risk was found to declined from (OR=9.5,95%CI=3.74–24.1, p=0.001) in <75 years old subjects to (OR=1.36,95%CI= 0.49–4.1, p=0.58) in <74 years old subjects. Furthermore we found a very strong synergic association between BChE K and APOE-epsilon4 (OR=19.1,95%CI=428– 85.45, p<0.001). Although this risk factor decreased from (OR=36.2,95%CI=4.4–296, p=0.001) in subjects <75 year olds to (OR=6.2,95%CI=0.9–72.4, p=0.06) in subjects > 74 year olds.

Conclusion: In accordance with previous studies, we found strong synergic association between BChE K and APOE-epsilon4 in risk of AD. These risks of AD decrease with increase of age.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P77 ASSOCIATION BETWEEN APOLIPOPROTEIN E POLYMORPHISM AND ALZHEIMER’S DISEASE IN AN IRANIAN POPULATION

Introduction: Various studies have shown that E4 allele of apolipoprotein E (APOE) is a major risk factor for Alzheimer disease (AD). The association of APOE allele frequencies and AD remains unknown in populations from developing countries.

Methods: We examined the frequency of APOE genotype in 105 patients from Tehran with AD and Tehranian cognitively normal subjects of similar age and sex as control group by PCR-FFLP methods.

Results: The APOE E4 allele frequency was significantly higher in the AD group than in the control group (21% versus 6.2%, p<0.001). The odds ratio for AD in individuals with either one or two E4 copy was 4 (95% CI=2–7.8, p<0.001). In addition, the odds ratio (ORs) for APOE E4 heterozygous subjects were 3.2 (95% CI=1.6–7.8, p=0.001), and those for the APOE E4 homozygous subjects were 12.75 (95% CI=1.6–104, p=0.01). The ORS were not uniform across age groups, but were higher in women 5.3 (95% CI=2.5–10.9, p<0.001) than in men 3.9 (95% CI=1.2–12.4, p=0.01). The patient carrying one or two APOE E4 allele showed earlier age-at-onset (p<0.001). The APOE E2 allele frequency was lower in the AD group than in the control group (0.95% versus 2.7%, p=0.15), although it was not statistically significant. However, the ORs for E2 allele was found to be 0.34 (95% CI =0.78–1.8, p=0.21).

Conclusion: The APOE E4 allele increased the risk for AD in dose-dependent manner, and the APOE E4 conferred AD risk, that was both age and sex dependent in an Iranian population (Tehran). In addition, our results support the association between APOE.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P78 STUDY OF METRONIDAZOLE EFFECT ON LOWERING BLOOD SERUM LIPID

Introduction: Hyperlipidemia is one of the most important causes of atherosclerosis. Regarding the high prevalence of this disease in the world especially in Iran, finding suitable and effective treatments which can both control the level of blood lipids and solution should be the major aim. Metronidazole is the only drug that is used as an antibacterial and antiprotozoal agent. Researchers have found that oral dose of 750 mg/day has a suitable absorption, is widely spread in the tissues and reaches to a serum level of 4–6 g/ml. Despite its suitable efficiency, few clinical trials have been conducted in this regard. This study was designed as such to examine effect of Metronidazole on lowering serum lipids.

Methods: The present research was performed as a clinical trial without control, on 50 patients. The tests of LFT, BUN, creatinine, SGOT and SGPT were done on these subjects. Metronidazole was given with a daily dose of 750 mg for a week. 20 of these patients who had less complaint continued their drug consumption for another seven days. Lipid parameters such as total lipid, total cholesterol, and triglyceride were taken under investigation in both groups.

Results: Measuring the serum level of lipids indicated that the mean total serum lipid and total cholesterol decreased significantly compared to their level before using the drug (P<0.05). The findings also showed a similar decrease in serum TG level (P<0.05). In the patient who continues the drug consumption in the second week, the serum level of lipids under study decreased significantly compared to their level in the first week (P<0.01).

Discussion: Drug tolerance indicates appropriate action and metabolism in this regard. This can be a prognosis for the replacement of this drug with other in the treatment of hyperlipidemia. The significant increase of BUN shows the small changes in liver function; as if the drug should be used for a long time in order to treat the hyperlipidemia, requires a more accurate investigation. Altering the daily dose of the drug should also be considered.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P79 DETERMINATION OF SERUM HDL-C AND LDL-C IN STREPTOZOTOCIN-INDUCED DIABETIC RAT

Introduction: Lipoprotein has atherogenic and thrombotic properties, and increased serum lipoproteins and lipids concentration have been described in several studies as correlating with coronary heart disease. This study was undertaken to investigate the blood serum Lipoproteins profiles in normal and streptozotocin induced type 1 diabetic Rat.

Materials and Methods: Rat, aged 12–14 week and weighting between 24–35gr were used. Diabetes was induced by a intraperitoneal injection of buffered (0.15 molar citrate, PH=4.7) solution of streptozotocin at a dosage of 50 mg/ kg body weight. The animals were considered diabetic, if their blood glucose value were above 20 mmol/L. After injection LDL-C, HDL-C, triglyceride and cholesterol were determined by using Biochemical kits.

Results: The results show that there was a significant increase in serum cholesterol, triglycerides and LDL-C in streptozotocin induced diabetic rat, accompanied by a decrease in high density Lipoprotein cholesterol (HDL-C). Our results showed that in control groups LDL- cholesterol, HDL- cholesterol respectively were 3.26 ± 0.14 and 1.52 ± 0.08 mmol/L. Also, in induced diabetic rat were 5.92 ± 0.12 and 0.82 ± 0.04 mmol/L. In control group serum cholesterol and triglyceride, respectively were 2.37 ± 0.12 and 1.45 ± 0.11 mmol/L. In induced diabetic rat were 2.79 ± 0.13 and 3.69 ± 0.17 mmol/L.

Conclusions: The lipoprotein abnormalities in diabetes and relation of triglyceride, low density Lipoprotein cholesterol, cholesterol and high density Lipoprotein cholesterol was shown. Our results suggest that treatment of hyperlipidemia in diabetes helps improving the glycemic control. Also, our results showed that subject with diabetes lipoprotein metabolism differs from nondiabetic subject.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P80 LACK OF ASSOCIATION BETWEEN OXIDIZED LDL-CHOLESTEROL LEVELS AND HAPTOGLOBIN PHENOTYPES IN HEALTHY KOREAN SUBJECTS

Introduction: Haptoglobin (Hp) 2-2 has been known to be lower in antioxidative activity than Hp 1-1 and Hp 2-1. However, few reported the relationship between oxidative stress and haptoglobin phenotypes by using oxidized LDL. The relationship between haptoglobin phenotypes and oxidative stress was investigated in healthy adults.

Methods: The serum haptoglobin concentrations, albumin, uric acid, high sensitive C-reactive protein, iron profiles, lipid profiles, L-ascorbic acid, total antioxidant status (TAS) and oxidized LDL levels were measured and analysed in 200 healthy Korean men and women with Hp 2-1 and Hp 2-2.

Results: The serum concentrations of haptoglobin were significantly higher in Hp 2-1 than in Hp 2-2. But those of TAS and oxidized LDL did not show any statistical differences. Albumin and uric acid, two main contributors for TAS, show no difference between two groups. The L-ascorbic acid concentrations in serum showed lower values in Hp 2-2 than in Hp 2-1 for both sexes (P < 0.05).

Discussion: The concentration of L-ascorbic acid, the first line antioxidant, was lower in Hp 2-2 than in Hp 2-1, which did not significantly affect the TAS, and there was no significant difference in oxidized LDL for both groups in healthy adults.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P81 ENHANCED SUSCEPTIBILITY TO OXIDATION OF LDL AS RISK FACTOR FOR CORONARY HEART DISEASES

Atherosclerosis and coronary heart disease(CHD) is a major cause of morbidity and mortality in industrial countries. It has been shown that lipid peroxidation in addition to lipid abnormalities can also play an important laboratory evidence showing that the oxidation of LDL is an important index of antioxidant capacity of plasma. The aim of this study is to evaluate the susceptibility of LDL to oxidation in CHD patients and control group as risk factor in prompting atherosclerosis and CHD in patients. The study groups consisted of 12 males (age 40–60 years) with angiographically confirmed CHD as a patient group and the control group of consisted 15 healthy men that were seemed health and age matched with patients group. Blood samples were obtained after an overnight fast in tubes containing EDTA. LDL was isolated from plasma by sequential ultracentrifugation (rpm: 120000) and the kinetics of the oxidation of LDL fractions were performed in the presence of cu2+ at 234 nm. Comparing the results of cases with those of control showed that mean lag time of LDL oxidation were significantly lower than that of control group (60.5± 15.1 vs. 79.5±15.3 p<0.05).

These finding suggest that a short LDL oxidation lag time could be a useful index in determining LDL oxidation and progress of atherosclerosis in CHD patients.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P82 CETP GENE HAPLOTYPES ARE ASSOCIATED WITH PLASMA HDL-CHOLESTEROL LEVELS IN SEVERAL COMMUNITY POPULATIONS

Introduction: Cholesterol ester transfer protein (CETP) plays a major role in HDL metabolism. Three single nucleotide polymorphisms (SNPs) in the CETP gene, TaqIB, -2708G>A and –629C>A influence plasma HDL levels. We hypothesized that haplotypes constructed from these SNPs would be associated with significant variation in HDL levels and cardiovascular disease (CVD).

Methods: Haplotypes were determined in 3 populations from Western Australia; two cross-sectional studies of 1,111 and 1,578 randomly selected community based subjects who were assessed for cardiovascular risk factors and in 588 subjects with CVD.

Results: Multivariate haplotypic analysis showed a common haplotype (frequency 26–31%) of B2/A/A for TaqIB, -2708G>A and –629C>A polymorphisms was strongly associated with increased HDL levels in the three populations (p<0.000001, 0.03 and 0.0004, respectively). These associations were independent of sex, age, SBP, BMI, waist-hip ratio, cholesterol, diabetes mellitus, smoking and alcohol consumption. In a case control study, subjects with the B2/A/A haplotype had a decreased the risk of CVD with an odds ratio of 0.70 (p<0.00001).

Conclusion: This data demonstrates that the CETP haplotype B2/A/A for TaqIB, -2708G>A and –629C>A polymorphisms is independently associated with increased serum HDL levels in three separate populations. This haplotype is also associated with a highly significant reduction in clinical CVD.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P83 REFERENCE INTERVALS FOR SERUM APOLIPOPROTEINS A-I, A-II, B, C-II, C-III, E IN HEALTHY INDIANS AND EFFECT OF SEX, AGE, SMOKING, DRINKING AND LP(A) LEVEL

Aim: Study of lipoprotein & apolipoproteins in ethnic Indians vis a vis other ethnic groups have been matter of study for long. In the present study however, it is an attempt to study the same in Indian population in Indian settings. Are number of methods of estimation of serum apolipoproteins, out of which currently immunoturbidimetric assay method is to be more adaptable for routine automated laboratory estimation is adapted for the present study also. 82 healthy individuals were selected after screening them for normal lipid profile, X-ray chest, TMT, ECG and for diabetes. The age & sex difference, effect of drinking & smoking on all the parameter were studied.

Observation & results: The mean serum value for ApoA-1 was 111.61+- 19.06mg/dl ApoA-II was 27.12+-4.33 mg/dl, Apo B was 82.55+-16.47 mg/ dl, Apo C-II was 2.82+-2.07 mg/dl, ApoC-III was 6.90+-2.18 mg/dl, Apo E was 2.97+-0.92 mg/dl and Lp(a) was 16.91+-10.91 mg/dl. Effect of smoking & drinking was found to be non-significant. A two-tailed Person correlation coefficient & test of significance was worked out.

Conclusion: The reference intervals of Apolipoproteins & Lipoproteins determined in this interim analysis which is a part of more expanded study undertaken is presented. The values and results were compared with those of other workers. There was age & sex difference in most of the serum apolipoproteins and lipoproteins levels. The establishment of reference interval for apolipoproteins using commercially available reagent kit for automated analysers will help in a long way in assessing coronary heart disease particularly with hyperlipidemia.

Acknowledgements

Authors are thankful to Daiichi Pure Chemicals & Acurex Biomedical Pvt. Ltd. Diagnostics for providing assay kits & hope for their continued support

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P84 LP (A) AS INDEPENDENT RISK FACTOR FOR PREMATURE CAD IN INDIAN MALE POPULATION

Introduction: The study screened LP (a) as independent cardiac risk factor for premature CAD. Risk factor profile of 167 angio-graphically documented male CAD patients below the age of 40 yrs were compared with the control group consisting of 167 healthy similar profile subjects.

Methods: Levels of serum total cholesterol, HDL-C, TG, Apo A1, Apo B and lipoprotein (a) were measured on autoanalyser Beckman CX-4 using standard kits. LDL-C levels were calculated by Friedwald formula. Serum Homocysteine levels were measured on Abbott IMX system. Plasma Fibrinogen levels were measured on sysmex CA 50.

Results: Mean value in the study and control groups were: TCH 179.7± 53.6 vs. 174.3±52.6 mg/dl, LDL-C 112±59.4 vs. 101.8±54.6 mg/dl, HDL-C 40.5±7.6 vs. 41.9±7.3 mg/dl, Apo A1 106.6± 34.4 vs. 108.0 ±32.5 mg/dl, Apo B 116.3 ±57.9 vs. 113.5±46.2 mg/dl. LP(a) 43.2±21.6 vs. 27.3 ±15.4 mg/dl, TG 148.5 ±79.2 vs 144.7±74.3 mg/dl, FB 316.8±57.3 vs. 309.4± 52.0 mg/dl, HCY 13.2 ±5.7 vs. 11.9 ±5.3 μmol/l.

Discussion: Results indicates significantly higher levels of Lp(a) and LDL-C in the study group. There were no significant difference observed in TCH, HDL-C,TG,FB,APO A1,APO B, and HCY levels.

References

1. Graziani MS, et al. Eur J Clin Chem Clin Biochem. 1993 Mar;31(3):135–7. [PubMed]
2. Genest J, Jr, et al. Am J Cardiol. 1991 May 15;67(13):1039–145. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P84A STRESS-INDUCED EXPRESSION OF HO-1 IS REDUCED IN PATIENTS WITH CARDIOVASCULAR DISEASES

Introduction: Heme oxygenase is a stress-inducible cytoprotective enzyme. The beneficial role of this enzyme is well documented in rodents, but not in humans. The aim of the present study is to test the hypothesis that HO-1 protects cardiovascular system from oxidative stress and atherosclerosis in humans.

Methods: We compared the HO-1 expression in peripheral mononuclear cells (PMBCs) between patients with cardiovascular diseases (CVD group, n=32) and control subjects (n=9). We determined HO-1 mRNA by real time RT-PCR before and after incubation of PMBC with hemin, as an oxidative stress. We also evaluated the plasma levels of isoprostane and TNFalfa, as markers of oxidative stress and systemic inflammation, respectively, and the level of IL10 as HO-1 dependent anti-in amatory cytokine in patients.

Results: The Coefficient of variance of HO-1 mRNA measurement was less than 15%. Both basal and stimulated HO-1 mRNAs had nothing to do with age. Cardiovascular diseases include coronary heart disease, stroke and peripheral artery disease. Hemin-stimulated HO-1 expression was significantly lower in CVD group compared with control (0.97±0.3 vs. 4.4± 2, p<0.01), with no difference in the baseline (0.14± 0.05 vs. 0.3± 0.12). No difference was found either in the plasma levels of isoprosptane, TNFalfa, and IL10.

Conclusion: These results suggest that reduced capacity of HO-1 upregulation, but not a basal HO-1 expression or systemic basal oxidative or inflammatory status, is involved in the mechanism of cardiovascular disease.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P85 CHARACTERIZATION OF THE PRO-LIPOPROTEIN SIGNAL PEPTIDASE OF MYCOBACTERIUM TUBERCULOSIS H37RV

The gene encoding the type II signal peptidase, lspA gene of Mycobacterium tuberculosis, was identified by connected gene neighbourhood Bioinformatics approach and its putative function was characterized experimentally. We applied a 2-pronged strategy. Firstly, using the Bioinformatics approach we examined the neighbourhoods of selected genes belonging to some of the well-defined operons in various species of Mycobacteria. M. tuberculosis data was used as a starting point. The data so obtained was used for interpreting context-based expression and/or functional prediction. Secondly, we shortlisted some of the genes for functional characterization. Here we report the functional characterization of one such gene (lspA gene) of M. tuberculosis. This gene encodes for a putative pro-lipoprotein signal peptidase (SPase II) of M. tuberculosis. SPase II is involved in the removal of the signal peptide from prolipoproteins. The deduced amino acid sequence of the M. tuberculosis SPase II showed significant similarity with those of other known SPase II enzymes. In M. tuberculosis, the lspA gene is flanked by the isoleucyl-tRNA synthetase (ileS) gene and the pyrimidine biosynthetic (pyr) gene cluster. Functionality of Lsp in Escherichia coli was determined by using in vivo globomycin resistance assay, to show that expression of Lsp in E. coli increased the globomycin resistance. Globomycin is a cyclic peptide antibiotic that specifically inhibits the processing of prolipoprotein to mature lipoprotein. The ability of Lsp to complement the growth deficiency at the non-permissive temperature of E. coli strain Y815 carrying a temperature-sensitive mutation in its lsp gene for SPase II, was also checked.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P86 IODINE DEFICIENCY IN WOMEN ATTENDING AN ANTENATAL CLINIC IN MELBOURNE

Introduction: Women attending their first antenatal clinic (usually in the first trimester) were surveyed for thyroid status. As part of this project, urine iodine levels were measured. Iodine is required for the formation of thyroxine, and deficiencies appear to be re-emerging in the Australian population. The deleterious effect on intelligence of even a mild iodine deficiency can have serious consequences on the community.

Methods: We surveyed 400 volunteers from women attending the antenatal clinics at Box Hill Hospital for their first visit. Spot urines were analysed for iodine and creatinine. We received 177 urine specimens for iodine and 165 for creatinine. The samples were obtained throughout the calendar year of 2003.

Results: Both the spot urine iodine levels and the urine iodine/creatinine ratio showed a high proportion of women with iodine levels below the acceptable level.

Iodine/creatinine ratio
Median81
2.5–97.5 percentiles31–394
All results21–662
Severe deficiency < 20ug iodine/g creat0
Severe to moderate deficiency <50 ug iodine/g creat21%
Mild deficiency 50–99 ug iodine/g creat45%
Normal >100ug iodine/g creat34%

Discussion: It has been a concern for a few years that iodine intake in at least some regions of Australia may be inadequate --contamination of milk is less common and processed food manufacturers commonly use non-iodised salt. Our results show low urine iodine levels in our group of pregnant women with 66% of the women having a lower urine iodine level (corrected for creatinine) than the WHO recommendation.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P87 A MICROTITRE PLATE METHOD FOR MEASURING VITAMIN B6 IN SERUM.

Introduction: Vitamin B6 plays a vital role in many metabolic pathways and is essential for the breakdown and utilisation of proteins, carbohydrates and fats from food. B6 is a general term used to include three chemically, metabolically and functionally related forms: pyridoxine, pyridoxamine and pyridoxal. The major form of B6 in serum is pyridoxal-5-phosphate (P5P). For over 30 years, this laboratory has used a microbiological assay for measuring pyridoxal, using Lactobacillus casei as the test organism.

Aim: To adapt the currently used assay for pyridoxal to a microtitre plate system using the Tecan Genesis RSP 100 diluting system and the Tecan Sunrise plate reader.

Methods: A chloramphenicol resistant strain of L. casei was used as the test organism for the assay, removing the need for aseptic technique. Serum samples from 80 patients were assayed by the current microbiological assay on a Gilson 222 auto-diluting platform and by the new microtitre plate system on the Tecan. Both methods used the same test organism, standards and “in-house” prepared medium.

Results: There was good correlation between the methods (R2 0.98) with a mean of 39.4 nmol/L for the Gilson method and 39.1 nmol/L for the Tecan method.

Discussion: The microtitre plate method for vitamin B6 has been shown to give comparable results with a “macro” microbiological assay. The new method only requires 200 uL of reagent and after appropriate incubation, has the capacity to read 96 tests in 20 seconds. Conversely, the old method requires 5 ml of reagent and after a similar incubation, can only read one test per minute. Due to the speed of reading, the method would be ideally suited for institutional or nutritional studies.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P88 REVISED REFERENCE INTERVALS FOR PAEDIATRIC COPPER, ZINC AND SELENIUM USING ICP-MS IN NSW

Methods: Sample population was inpatients and outpatients without acute medical conditions at The Childrens’ Hospital Westmead. Patients with abnormal laboratory studies (ie. elevated WCC, LFTs, creatinine or CRP) were excluded from the reference population. Plasma copper, zinc and selenium were analysed on a VARIAN ICP-MS.

Discussion: Paediatric reference interval studies are challenging because venepuncture of healthy children is ethically contraindicated and sample volumes are small. We used ICP-MS to establish age-specific ranges. Laboratory monitoring of trace element status is important for children on TPN and/ or during prolonged illnesses.

Paediatric reference intervals tables 10-8-04

PLASMA COPPER

agensexumol/L 2.5 – 97.5 percentiles
0 – 6 months17M, F6.2 – 23.9
6 months – 5 yr56M, F13.3 – 43.8
5 – 12 yr23M, F14.7 – 32.3
12 – 18 yr16M, F14.7 – 24.2

PLASMA ZINC

agensexumol/L 2.5 – 97.5 percentiles
0 – 6 months17M, F10 – 26
6 months – 5 yr56M, F10.3 – 31.7
5 – 12 yr23M, F12 – 32
12 – 18 yr16M, F12 – 25

PLASMA SELENIUM

agensexumol/L 2.5 – 97.5 percentiles
0 – 6 months17M, F0.31 – 1.47
6 months – 5 yr56M, F0.61 – 2.27
5 – 12 yr23M, F0.72 – 1.82
12 – 18 yr16M, F0.58 – 1.57
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P89 REDEFINING TRADITONAL PROFILES IN CLINICAL BIOCHEMISTRY: INCOPORATING ANTIOXIDANT STATUS

Illness is inevitably linked with an abnormal biochemistry and alteration in the metabolism of nutrients and their by products. Clinical biochemistry has traditionally been used to provide information to confirm or refute a clinical diagnosis, to extend diagnosis by providing information about aetiology, to indicate the presence of iatrogenic complications, to provide prognostic information, to monitor the progress of a condition and to detect subclinical disease.

To improve on these established roles and take a more proactive part in the understanding and controlling of disease, standard biochemical profiles such as electrolytes and urea, liver, renal, bone haemopaietic, lipid, etc should be redefined by incorporating antioxidant status.

Antioxidant status predicts the existence or otherwise of oxidative stress. Oxidative stress has been implicated in a large number of disease states including coronary heart disease, cancer, neurologic damage, toxic states, exacerbation of infectious states etc. The contribution of oxidative stress to many pathologic states including metabolic disorders appears to be more widely recognized increasingly. Antioxidants have broad preventive and therapeutic applications in disease states since free radicals are not exclusive to any particular disease state but contribute to pathology of a large number of diseases.

By incorporating antioxidant status in traditional biochemical profiles, important subclasses yet unrecognised are likely to be uncovered within current classification systems. Oxidative classification of a disease and the ability to place an individual in a specific oxidatively defined sub-class should enable better understanding of disease states.

The oxidative approach may help to expand our knowledge of disease outcomes and prognosis, with the potential to predict treatment response in advance of initiation of therapy.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P90 ROLE OF SERUM GLUTATHIONE AS AN ANTIOXIDANT IN CORONARY ARTERY DISEASE (CAD)

Introduction: Lipids and lipoproteins are important risk factors for CAD but they do not account for the disease in 30 to 40% of the CAD population.

Methods: In order to find out the role played by non-lipid components in serum, such as antioxidants, a study was carried out on 250 CAD patients to see how their serum glutathione levels are related to an important CAD characteristic, MI [Myocardial infarction]. Serum glutathione was assayed by Beutler method, using DTNB reagent.

Results: The following results were obtained:

S.No.Serum level micro moles/dLMI group [n=128]Non-MI group [n=122]
i)Glutathione1.5302.35 [P<0.01]

Serum levels of glutathione were significantly lower in the MI group compared to the non-MI group CAD patients.

Conclusion: This study indicates the possibility of glutathione as an antioxidant operating in the period closer to the onset of clinical events, in CAD patients.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P91 2-(4-NITROPHENYLHYDROXYMETHYL)-6-METHOXY-1,2,3,4-TETRAHYDRONAPHTHALEN-1-OL AS A MODERATE INHIBITOR OF RETINOIC ACID METABOLISING ENZYMES.

Introduction: Cancers are essentially a derailment of cellular differentiation, therefore the possible beneficial effect of retinoic acid (RA) in prevention of cancer of the epithelial tissues has been investigated by several workers. In laboratory animals it has been demonstrated that RA inhibited the induction and even caused the disappearance of tumours. However, RA has only a small effect on established human epithelial cancer. A possible reason for this clinical failure could be the rapid in vivo metabolism of retinoic acid. RA could be metabolised through several pathways including hydroxylation at position 4 of the cyclohexenyl ring which is the most important route. Logically, inhibition of this hydroxylation would result in delay of degradation of RA, leading to higher tissue concentrations of RA and improved control of neoplastic differentiation. Ketoconazole a broad spectrum antifungal agent inhibits many mammalian cytochrome P-450-dependent enzymes and also competitively inhibits the cytochrome P-450-mediated metabolism of retinoic acid in vitro by hamster liver microsomes.

Methods: The inhibitory activity of 2-(4-Nitrophenylhydroxymethyl)-6-methoxy-1,2,3,4-tetrahydronaphthalen-1-ol (F68) synthesised in this research was examined against RA metabolism, in comparison with ketoconazole. Percentage inhibition was calculated from the conversion rate of the samples containing inhibitors to that of control samples which contained absolute ethanol instead of inhibitor. Ketoconazole was used as a standard. Due to the sensitivity of retinoic acid all assays were carried out in a dark room equipped with yellow light. The IC50 values were determined from a plot of % inhibition verses log of inhibitor concentration.

Results: The tetralone (F68) (IC50 = 5–10 μM) was about three fold more potent than the standard inhibitor ketoconazole (18.8 μM) against rat liver microsomal RA-metabolising enzyme(s).

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P92 SIMPLE BIOCHEMICAL MARKERS OF OXIDATIVE STRESS IN PREGNANCY

Micronutrients have been traditionally known to be increasingly required in pregnancy. Their roles as antioxidants in modulating oxidative stress in this state is poorly recognised.

Eighty (80) subjects, 40 pregnant, mean age 29.0 ± 4.4 years and 40 non-pregnant women, mean age 28.4 ± years were randomly selected after previously excluding other well known courses of oxidative stress. These subjects were selected from the out patient clinic of the Obstetrics and Gynaecology department of the University College Hospital (UCH) Ibadan.

The antioxidant profiles of both pregnant and control subjects including albumin, vitamin C, uric acid and bilirubin were determined in these subjects.

All the antioxidants varied in the same direction. Serum albumin, vitamin C, urate and bilirubin levels were all significantly decreased in the pregnant subjects compared with non-pregnant controls (P<0.05) in all cases.

These findings probably suggest the existence of oxidative stress in this state and increased requirement of antioxidant micronutrients not just for new tissue formation or raised metabolic rate but also to combat the raised free radical burden or ameliorate free radical toxicity.

These simple biochemical markers of oxidative stress have the advantage of being easily determined and incorporated into the routine of the gynaecological management plan of subjects and thus probably minimising maternal morbidity and mortality in the developing countries where antioxidant status is compromised.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P93 INVESTIGATION INTO THE ACCURACY OF NEONATAL BILIRUBIN MEASUREMENTS

Introduction: Neonatal jaundice is a common finding encountered in term newborns. Untreated, high concentrations of bilirubin can cause neurological problems. In sick or pre-term infants, kernicterus can occur at lower bilirubin concentrations, especially when pH or albumin concentration is low. Treatment usually consists of phototherapy, with exchange transfusion reserved for more serious cases.

The severity of jaundice is usually confirmed by the laboratory, and accurate bilirubin results are essential for correct treatment.

Most laboratories use the Australian Quality Assurance Programs (AQAP) to validate the accuracy and precision of their assays. Some analytes however suffer from matrix effects with quality control material, which makes it difficult to relate AQAP data to patient samples. Our laboratory regularly obtains low results in the AQAP programs.

Methods: Samples from 43 neonates and 32 adults with a range of elevated bilirubin values were measured by an automated and scaled down reference method of Doumas et al, on a Cobas FARA analyser. The assay was performed identically to the manual assay including a separate sample blank for each sample. The sample volume (10 μL) enabled the analysis of neonatal samples. The assay was calibrated against a pure standard and a commercial calibrator.

Results: Hitachi results were compared to the reference method using Passing-Bablok regression.

Neonatal samplesy = 1.01x + 0.5n = 43
Adult samplesy = 1.11x – 2.3n = 32

Conclusion: Results from QAP surveys may need careful interpretation, and it can be important to validate accuracy against a reference method using real samples. The practice of “calibrating” methods using AQAP control materials can lead to highly inaccurate measurements.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P94 SCREENING FOR NEONATAL HYPOGLYCAEMIA WITH ABBOTT MEDISENSE OPTIUM

Introduction: We evaluated the use of Abbott MediSense Optium (AMO) blood glucose meter in screening for neonatal hypoglycaemia.

Methods: 100 routine heel prick capillary blood samples on babies from the Neonatal Intensive Care Unit were split into two portions. Whole blood glucose was measured immediately using AMO (glucose dehydrogenase). The remaining portion in a sodium fluoride/oxalate micro-tube was sent on ice to the laboratory for plasma (pl) glucose analysis using the Beckman Coulter CX3 (glucose oxidase). Analyse-It Add-on for MS Excel was used for data analysis.

Results: Data ranged from whole blood glucose of 1.1–9.3 for AMO and pl glucose of 1.0–8.2 for CX3. Deeming regression gave a correlation coefficient (r) of 0.959, and the equation CX3 = 0.896*AMO + 0.102. The ability of the AMO to predict neonatal hypoglycaemia at different published cutoffs (1) is shown below, using a 100% sensitivity figure to allow “rule out”.

Predict pl glucose (mmol/L)Area under ROC curve (95% CI)AMO @ 100% sens (mmol/L)False Pos: True Pos
<2.60.990 (0.975–1.0)3.13: 15
<2.00.997 (0.990–1.0)2.43: 11
<1.10.990 (0.970–1.0)1.42: 2

Discussion: The Abbott Medisense Optium is a safe screening device for neonatal hypoglycaemia with a low number of false positives. The performance and cutoffs are similar to those previously reported using the Glucocard meter (2). The AMO is FDA approved for use in neonates. Use of this meter in neonatal units can reduce the demand for immediate conventional glucose analysis.

References

1. Green A and Morgan I. Neonatal and Clinical Biochemistry. ACB Venture Publishers 1993 1st Edition. p40.
2. Hawkins R, Stack J. Clin Biochem Revs. 1995;16:69.
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P95 THE CORRELATION BETWEEN PLASMA SOLUBLE CTLA-4 WITH AIRFLOW OBSTRUCTION AND ASSOCIATION OF CTLA-4 (+49A/G) POLYMORPHISM WITH SENSITISATION TO INHALANT ALLERGENS IN CHINESE ASTHMATIC CHILDREN

Introduction: Cytotoxic T lymphocyte antigen 4 (CTLA-4) is known to down-regulate the Th2 immune response. Recent studies have identified the association of A- to –G substitution at position +49 in exon 1 of the CTLA-4 gene with allergic diseases. We investigated the effect of this polymorphism on plasma soluble CTLA-4 concentration (sCTLA-4) in Chinese asthmatic children.

Methods: Two hundred thirty-six asthmatic children and 129 age- and sex- matched controls were recruited, with mean (SD) ages of 10.5 (3.7) and 11.2 (4.5) years, respectively. The patients underwent spirometry. Serum total IgE, allergen-specific IgE to common allergens, and plasma sCTLA-4 of all children were measured by enzyme immunoassay. Their CTLA-4 (+49A/G) was genotyped by restriction fragment length polymorphism.

Results: Expectedly, serum total IgE concentration was higher in asthmatic children than controls (mean (IQR) of 1022 (224–1046) vs 252 (15–233) kIU/ L, p<0.001), and more patients were atopic with ≥ 1 positive specific IgE test (OR 0.231, 95% CI 0.152 0.352; p<0.001). There was no difference in plasma sCTLA-4 between patients and controls (5.7 (3.0–8.4) vs 6.0 (3.6–8.0) ng/mL, p=0.481). However, sCTLA-4 was negatively correlated with age (r=−0.134; p=0.01) and FEV1/FVC ratio (r=−0.137; p=0.038). Genotyping showed that CTLA-4 (+49A/G) polymorphism was not associated with plasma sCTLA-4, serum total IgE concentration or asthma diagnosis. Children homozygous for A/A were more sensitised to dog (p=0.003) and cockroach (p=0.021) allergens than those with A/G and G/G genotypes.

Conclusions: Our study suggests that plasma sCTLA-4 concentration correlates with the extent of air flow obstruction in asthmatic children, and CTLA-4 (+49A/G) polymorphism is associated with sensitisation to indoor aeroallergens but not asthma or serum total IgE in Chinese children.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P96 RATE OF INCIDENCE AND CAUSES OF POSITIVE TESTS FOR REDUCING SUBSTANCES IN URINE OF CHILDREN.

Introduction: Inborn errors of metabolism is a general term applied to numerous genetic disorders pathology of which is usually attributed to excessive tissue storage or abnormally high circulating concentrations of a specific not degraded metabolic substance. Early diagnosis can prevent irreversible complications of some of these disorders. Errors in carbohydrate metabolism belong to this category of disturbances. Sugars of clinical interest are all reducing sugars.

Aim: The aim of this study was determination of the incidence rate and causes of positive Benedict’s test in hospitalised or outpatient children under 14 years of age. In addition, identification of interfering substances that could cause false positive results and the necessity of Benedict’s test prior to chromatography was other purposes of the study.

Method: 1473 urine samples were examined. Benedict’s test was done for detection of reducing substances and paper chromatography for identification of the specific reducing sugar and amino acids present in the urine.

Results: 59% of the samples were positive for reducing substances. Significant decrease in positive results occurred with restricted diet (free of fruits, vit. C, honey, drugs). Paper chromatography for sugars and amino acids were performed on positive samples. Lactose was the most frequently found (32%) sugar followed by galactose (24%).

In 54% there were no reducing sugars. The results also showed 36% cysteine and 19% other amino acids in positive samples.

No significant differences were observed in rate and degree of positive results with respect to sex and age of the patients.

Conclusion: The results obtained in this study indicate that important reducing sugars or amino acids may be detected even in weakly positive results of Benedict’s test, thus justifying follow-up studies of positive test for reducing substances in urine.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P97 HPLC ASSAY OF PTERINS IN CSF AND URINE USING A POLAR END-CAPPED COLUMN

Introduction: Assessment of tetrahydrobiopterin and neopterin in CSF, and primapterin in urine, is crucial in the diagnosis of rare autonomic disorders associated with neurotransmitter deficiencies. Assays are complicated by the lability of these analytes, their occurrence in multiple forms, interference by isoxanthopterin, and the difficulty of establishing paediatric reference ranges 1. These polar analytes also chromatograph poorly on conventional reverse phase columns. We investigated the suitability of a special chemistry polar end-capped column, designed for reverse phase separations of polar analytes.

Methods: CSF collected according to a published protocol 1, and random urines, were stored in foil at −70°. CSF, or urine diluted 1 in 20, was shaken with 1mg manganese dioxide per mL 0.05M phosphoric acid at 4°C for 40min. 50μL of supernatant was chromatographed on a Phenomenex Aqua C18 column with 5mM sodium phosphate pH3.2 at 1.3 mL/min. Fluorescence excitation at 360 nm was monitored at 440 nm.

Results: The four analytes plus 6-hydroxymethylpterin internal standard were baseline resolved in purely aqueous mobile phase. No interferences were observed in 50 CSF samples or 28 urine samples. Accuracy ranged from 5.5 to 10% against independent standards; precision from 4.7% to 9.2% between assays; and there was less than 12% difference between our results and those of a reference laboratory 1. The assay was sensitive to 1 nmol/L and linear to 1000 nmol/L.

Discussion: Using a C18 column with polar end-capping and a purely aqueous mobile phase, we obtain excellent resolution and quantitation of the main pterin analytes in CSF and urine. This gives accurate and reproducible results allowing the use of published reference values 1.

References

1. Hyland K, et al. Pediatric Research. 1993;34:10–14. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P98 PSEUDOPROTEINURIA AND URINE PROTEIN EPG INTERFERENCE BY A GELATIN BASED IV FLUID

Introduction: Whilst investigating severe hypoproteinaemia (total protein 36 g/L) in a 67 year old female, the presence of an interfering substance was suspected, due to a large discrepancy between total urine protein measurement, by a turbidimetric method (1.1 g/L) and by a dye binding method (8 g/L). A large, diffuse, strongly staining, unidentified band, was also present, on urine protein electrophoresis.

Review of other patients, with a similar, unidentified band on urine EPG, revealed intravenous Gelofusine® infusion, to be a common factor. A sample of the modified gelatine solution was analysed.

Methods: Protein measurements, in patient urine, as well as Gelofusine® aliquots, were performed on the Dade Dimension® Xpand™, by pyragallol red dye binding and by turbidimetry, following addition of benzethonium chloride, on the Modular Analytics SWA system (Roche Diagnostics Corporation, Indianapolis, IN, USA).

Protein EPGs were performed on the Paragon® Electrophoresis system (Beckman Coulter, Inc. Fullerton, CA 92835), and stained separately with Paragon Blue®, Coomassie Blue & Amido Black.

Results: The protein EPG profile, of the Gelofusine aliquot, was identical in appearance to the previously unidentified diffuse band, present in urine samples of patients who had received Gelofusine infusions. Gelofusine reacted with the dye binding method, whereas interference was absent with the turbidimetry method.

Discussion: Gelofusine contains modified gelatin polymers, of average MW 30 kD, sufficiently small to pass through the glomerular membrane into the urine. Interference, with Biuret and dye binding methods have been previously reported.1 Our study confirms urine EPG interference and necessity for turbidimetric protein measurement.

References

1. Hordijk W, et al. Pseudoproteinuria following gelatin infusion. Nephrol Dial Trnsplant. 1999;14:2782. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P99 URINARY PROTEIN ANALYSIS IN PRE AND POSTOPERATIVE CANCER PATIENTS

Introduction: The pre- and post-operative administration is very important. Urine is the precious material obtained without any surgical intervention. In this study, we separated the urinary proteins by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) in pre- and post-operative samples from patients to monitor renal dysfunction in response to surgical stress, and investigated the separated patterns in detail.

Methods: Urine samples were obtained from eight patients (P1–P8) with oesophageal or stomach cancer. Samples were collected before (day –1) and after operation (days 1–3, 5, 7, 10, 14 and 21 or 22) at the Tokyo Medical and Dental University Hospital. After the SDS-PAGE, the gels were stained by silver staining method. The separated urinary proteins in the gels were analyzed by the NIH image program and densitometric images were obtained. The images were divided into nine areas, each containing one major band, and the areas were classified as fractions (Frs.) A to I. The percentage of protein in each fraction was calculated from the densitometry pattern of each lane. The percentages in each fraction were also expressed as a fraction concentration, relative to timed urinary total protein concentration.

Results: The percentage of protein in Frs. E, F and G increased on postoperative days 1–7; conversely, the percentages in Frs. A, B, C, H and I decreased. In particular, the fraction percentages of Frs. E, F and G peaked within two days of operation. The changes in these three urinary proteins were similar to changes in serum CRP. In light of the postoperative concentration increases in most fractions, it was considered that renal damage had occurred.

Conclusions: We suggest that analysis of proteins in urine using SDS–PAGE is valuable in non-invasive monitoring of renal dysfunction following surgical stress.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P100 CELLULOSE ACETATE MEMBRANE ELECTROPHORESIS IN THE ANALYSIS OF URINARY PROTEINS IN PATIENTS WITH TUBULOINTERSTITIAL NEPHRITIS

Introduction: In this study, a rapid and highly sensitive colloidal silver staining reagent suited for use with cellulose acetate membranes. We then analysed urinary total protein concentrations and urinary protein fractions by classifying the samples according to the presence of acute or non-acute interstitial nephritis. The relation between urinary protein fractions and complications of interstitial nephritis was also analysed.

Methods: 3.2 μL from the urine samples were applied to the tip of the membrane. After electrophoresis, the urinary proteins were fixed and staining in freshly prepared with colloidal silver solution for 20 minutes with continuous shaking. Densitometry at 505 nm was carried out as described above. After electrophoresis, each track was overlayed with cellulose acetate strips impregnated with 15 specific antisera. Immunofixed strips were washed in 0.9% (w/v) NaCl solution and stained with colloidal silver solution.

Results: Concentration of the sample was not necessary if there was a concentration of more than 5 mg/L in one band. The urinary electrophoretic patterns of the 14 samples show clear fractionation and various electrophoretic patterns. With this method, 15 urinary proteins (pre albumin, albumin, α1-microglobulin, α1-antitrypsin, α2-macroglobulin, haptoglobin, retinol binding protein, transferrin, β2 - microglobulin, IgA, IgG, κ- and λ light chains, cystatin C, lysozyme) were identified clearly. We believe that it will be useful to analyse the change in each percentage over time as the disease progresses or remits. The urinary protein fraction percentages were also obtained by densitometry.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P101 SIGNIFICANCE OF FREE KAPPA AND FREE LAMBDA RATIO IN AMYELOID AND MYELOMA PATIENTS

Introduction: Serum pre light chain measurement is useful in any disease that leads to abnormal monoclonal or polyclonal light – chain production. Multiple myeloma in diagnosis is based on the presence of excess monoclonal plasma cells in the bone marrow, monoclonal immunoglobulin in serum or urine and osteolytic bone lesions. Primary systemic amyeloidosis is a protein conformation disorder characterised by the deposition of monoclonal free light chain fragment as amyeloid deposits.

Methods: 71 patients’ plain blood samples were collected and used for free kappa and free lambda analysis, of these 30 were myeloma patients, 19 amyeloidosis patients and 22 normal individuals.

Results: Normal individuals free kappa (7.7 ± 3.5 mg/l) and free lambda (13.9 ± 8.1 mg/l). Free kappa/free lambda ratio of normal individual .55 ± 1.32.

Conclusion: This study concludes that free kappa by free lambda ratio is very much useful in diagnosis, treatment and monitoring of amyeloid and myeloma patients.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P102 CLONING AND EXPRESSION OF THE NUCLEAR ENVELOPE PROTEIN GP210 AUTOANTIBODIES GENE PURIFICATION AS WELL AS CLINICAL APPLICATION OF THE RECOMBINANT PROTEIN

Introduction: To express and purify nuclear pore complex protein gp210 gene in E. coli. To develop a simple assay to detect gp210 autoantibodies.

Methods: Stable expression nuclear pore complex protein gp210 gene in E coli is obtained by its being cloned into the PET-30a vector and tronsformated into E coli. This fusion protein was purified by NAT chromatography and identified by SDS-PAGE and Western blot.

Results: The relative molecular mass of expressed product was 69KD as predicted. There is a single special strip on SDS-PAGE gel. Of 40 patients with PBC, autoantibodies against gp210 were detected in 15 of 40 (77.5%) patients, but in none of the 10 control sera in Western blot.

Conclusion: Using the method of gene engineering, we obtain recombinant polypeptide containing the predominant autoepitope. It provides test reference for clinical diagnosis of the PBC.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P103 IMMUNOSUPPRESSANT DRUG MONITORING DO CONSENSUS GUIDELINES HELP?

The routine monitoring of drugs used in organ transplantation is a growth area in many therapeutic drug monitoring laboratories. However, the complexity of clinical practices often outstrips the skills of many generalist chemistry laboratories. As exemplified by the cyclosporin (CsA) experience, there has been widespread adoption of immunoassays that do not attain recommended guidelines (1). A fundamental recommendation was to only use methods that are specific for parent CsA (without major CsA-metabolite bias). However, the majority of laboratories internationally have adopted the least specific methods available. This begs the question as to who takes responsibility for the quality of tests delivered in our clinical laboratories? Should accreditation authorities play a role? Should clinical staff take responsibility for laboratory methods offered? Should regulatory authorities only approve ‘acceptable’ products? Or should laboratory scientists exercise their professional responsibility and apply better method selection criteria? One would hope that the latter (at least) should apply, or risk this role being usurped by others. Laboratories without such expertise, eg. general chemistry environments, should strongly consider recommendations of others with expert knowledge in the field and adopt practices as published in the scientific literature. A recent Australian & New Zealand approach was to form a key body (including representatives from each State and NZ) and provide a peer-reviewed consensus guideline for CsA monitoring to assist all laboratories (2).

References

1. Morris RG, Lam AK. Cyclosporin monitoring in Australasian - survey of laboratory practices in 2000. Ther Drug Monit. 2002;24(4):471–478. [PubMed]
2. Morris RG, Ilett KF, Tett SE, Ray JE, Fullinfaw RO, Cooke R, Cook S. Cyclosporin monitoring in Australasia – Update of consensus guidelines 2002. Ther Drug Monit. 2002;24(6):677–688. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P104 A SCREENING PROCEDURE FOR DRUGS IN SOLID SPECIMENS WITH ELECTROSPRAY-IONIZATION TANDEM MASS SPECTROMETRY

Introduction: Technically, it is difficult to employ a single method to determine all the drugs of abuse due to differences in the chemical nature of the drugs. GC/MS procedures with different extraction, derivatization and chromatographic conditions were traditionally employed to determine different groups of drugs. In this report, flow-injection electrospray-ionisation mass spectrometry (ESI-MS) was employed to detect drugs of abuse in solid specimens.

Methods: Drugs in tablet, capsule, crystal or powder forms were dissolved in methanol and mixed with Heroin-D9 (internal standard). An Agilent LC/MSD (Trap) equipped with autosampler was employed to inject 10 μL of samples without chromatographic separation. The ESI-MS was operated in positive ion, scan mode (m/z 100 to m/z 500). Total analysis time is 1.3 min per sample. Mass and MS/MS spectra were obtained for each drug standards to generate spectra library.

Results: More than 80 samples were screened and confirmed with GC/MS procedures. Drugs detected included the following: Heroin, codeine, 6-acetylmorphine, 6-acetylcodeine, cocaine, MDA, MDMA, methamphetamine, ketamine, diazepam, triazolam, unitrazepam, lorazepam, zolpidem and zolpiclone. Carryover rates between samples were usually less than 2%.

Discussion: The ESI-MS screening procedure is simple, fast and accurate. No chemical derivatization was required.

Reference

  • Weinmann W, Svoboda M. Fast screening for drugs of abuse by solid phase extraction combined with flow-injection Ionspray-Tandem mass Spectrometry. J Anal Toxicol. 1998;22:319–328. [PubMed]
Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P105 DETERMINATION OF THEOPHYLLINE AND ITS METABOLITES USING LC-TANDEM MASS SPECTROMETRY (LC-MS/MS)

Introduction: HPLC methods for the measurement of theophylline and its metabolites required restraints of caffeine containing food and beverages or they required a chromatographic separation time of 30 minutes or longer. We described a rapid and simple LC-MS/MS method for the determination of theophylline and its metabolites including 1,3-dimethyluric acid (13DMU), 1-methylxanthine (1MX), 3-methylxanthine (3MX) and 1-methyluric acid (1MU).

Methods: A serum sample was mixed with 100 μL of internal standard solution (1 μg of theophylline-1,3-15N2) and 100 μL of methanol. After mixing and centrifugation, the clear supernatant was evaporated to dryness, and the reconstituted residue was analysed by LC-MS/MS. After separation on a RP C18 analytical column with an isocratic mobile phase consisting of methanol/0.05% acetic acid aqueous solution (70:30) at a flow rate of 0.25 mL/min, the analytes were ionised in the electrospray negative ionisation interface.

Results: The interfering compounds, such as caffeine and 37DMX, were barely detected in negative mode, and other compounds, such as 17DMU, could be successfully separated using HPLC. The calibration data obtained for concentrations from 0.625 to 20 μg/mL of theophylline and from 0.063 to 2 μg/mL for the metabolites showed a linear and reproducible curve in the observed analytical ranges. All the compounds showed good recoveries except theophylline, and the results showed satisfactory within-run and between-day precision with less than 10% of CVs. Comparison with the fluorescent polarisation immunoassay (FPIA) method for plasma theophylline showed close concordance over the entire range of evaluated concentrations (y = 1.008x + 0.1455. y=LC-MS/MS, x=FPIA, r2=0.980, n=93).

Discussion: We developed a high throughput and simple LC-MS/ MS method for the simultaneous measurement of theophylline and its metabolites without interferences from caffeine metabolites. This method could be used successfully for the determination of theophylline and its metabolites in routine therapeutic drug monitoring.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P106 MEASUREMENT OF DIAZEPAM LEVELS BY EMIT

Introduction: The measurement of diazepam for therapeutic purposes is not easily rationalised. However, diazepam levels are requested in certain clinical settings such as drug dependency and detoxification units. An easily accessible automated method is desirable. We have assessed the use of the Emit® tox ™ serum benzodiazepine assay for the quantitative measurement of diazepam on the Hitachi 917 autoanalyser and compared the results with those obtained by HPLC.

Methods: Specimens received from patients attending drug dependency and detoxification facilities were tested for diazepam using Emit® and HPLC. The Emit® chemistry was performed as per manufacturer’s instruction except that a five point calibration set was prepared by 1:1 dilution of the low and medium calibrators. The HPLC analysis was performed following extraction into hexane/ethylacetate 70%/ 30% ethylacetate, evaporation and reconstitution. The diazepam and it metabolites are measured by photodiode array at 240nm against methylclonazepam as internal standard using an ODS column with aqueous phosphate/acetonitrile mobile phase.

Results: Emit® correlates with HPLC r= 0.94 but overestimates the amount of diazepam (slope = 0.42) because the immunoassay cross-reacts the metabolites, principally nordiazepam. When the diazepam and nordiazepam by HPLC are summated, the correlation has a near unity slope. Clobazam interferes with the Emit® immunoassay.

Discussion: The Emit® serum benzodiazepine assay is calibrated against diazepam but is not intended to be specific for diazepam. It can be used as a convenient method for the measurement of diazepam on an autoanalyser where the reported levels will approximate the sum of diazepam and its major active metabolite, nordiazepam. This test is useful in the clinical context of managing and monitoring cases of benzodiazepine abuse and dependence.

Clin Biochem Rev. 2004; 25(Suppl): S18–S128.

P107 SIMULTANEOUS DETERMINATION OF AMPHETAMINE-LIKE DRUGS, KETAMINE AND ITS METABOLITES IN URINE SAMPLES BY GC-MS

Introduction: Amphetamine-like drugs and ketamine are drugs of abuse with different CNS effect. Recently, there has been an increase of ketamine and MDMA abuse associated with “Rave” culture. More than 50% of the abusers in rave party used both drugs together. A procedure for the simultaneous detection