On November 7, 2011, the permanent deferral from blood donation of men who have sex with men (MSM) changed in England, Scotland and Wales, to a 12-month deferral since last relevant sexual contact. This change was made following an evidence-based policy review by the Advisory Committee on the Safety of Blood, Tissues and Organs (SaBTO). The review concluded that the available evidence supported the introduction of a 12-month fixed period deferral and that the risks associated with a 12-month deferral of MSM were equivalent to a permanent deferral. The permanent deferral for MSM was introduced in 1985 in response to the spread of acquired immunodeficiency syndrome (AIDS) caused by HIV. The change was supported by new data on the level of compliance with the permanent deferral, advances in the testing and processing of donated blood, changes in the epidemiology of sexually transmitted infections (STIs) and improved scientific knowledge. This review discusses how the decision to change the deferral period was reached and highlights some of the remaining issues relating to this contentious matter.
MSM; Blood donation; Compliance; Risk; Policy
Anti-G antibodies are rarely found since anti-D, in combination with anti-C, are difficult to discriminate from anti-G antibodies in routine testing.
A 22-year-old, gravida-3, para-1, woman with blood group A Rh D neg ccddee and known antibody anti-Jk(b), gave birth to her second child. While anti-Jk(b) could not be detected at birth, a new anti-C was found. Antibody screening tests (IAT) were performed using gel cards and rare G positive rGr erythrocytes. Genotyping for RHD and RHCE was performed using PCR-SSP.
The child's blood group was A Rh D neg Ccddee. Genotyping revealed Cde/cde haplotypes. The erythrocytes of the new-born showed a positive direct antiglobulin test with IgG; anti-D and anti-C could be eluted. Erythrocytes with the rare phenotype rGr were reactive with the serum of the mother.
The presence of anti-D and anti-C in the eluate from then newborn's Ccddee erythrocytes proved anti-G or anti-G in combination with anti-D. When anti-C and anti-D are seen during a pregnancy, possibly anti-G is present. This observation is of relevance since women with anti-G can still develop anti-D and require rhesus prophylaxis.
Anti-G; Pregnancy; Antibodies; HDN
The risk of transfusion-transmitted infections has been greatly reduced by improvements in donor screening and testing. However, newly recognized blood-borne infectious agents can be threats to blood safety. In order to evaluate the prevalence some of these agents in blood donors, a systematic review was conducted. Data were obtained from published papers related to HGV, Torque Teno virus (TTV), HTLV, West Nile virus (WNV) and SEN virus (SEN-V). Based on these studies, the prevalence of HGV varied from 1 to 8.6% for anti-E2 and from 0 to 4.8% for HGV RNA. The prevalence of TTV DNA and HTLV-I varied from 2.7 to 79.5% and from 0.013 to 2.3%, respectively. The WNV-specific IgM antibody and WNV RNA are negative in blood donors. Prevalence rates of SEN-V in Iranian blood donors range from 23 to 90.8%. Consequences of these infectious agents for blood safety are different. Thus, the need to perform laboratory screening as well as effectiveness and efficiency of laboratory tests depend on pathogenicity level and epidemiological conditions of emerging infections. However, being prepared based on the current level of risk and interventions to reduce the risk can be effective in reducing the potential threat for blood supply.
Infectious diseases; Emerging; Blood supply; Iran
The detection of antibodies against the human leukocyte antigen (HLA) complex has become indispensable in every clinical practice. The development of solid-phase assays like the Luminex allows the standardized measurement of anti-HLA antibodies (HLAab) with high sensitivity, albeit the relevance for some clinical settings remains a matter of debate. In this review we aim to describe the principle of Luminex-based antibody detection, including two modifications that allow identifying solely complement-activating antibodies. We then describe three applications for Luminex: i) detection of HLAab preceding solid-organ transplantation and monitoring of donor-specific antibodies posttransplant as a risk factor for antibody-mediated rejection; ii) presence of HLAab in recipients as a risk for graft failure in hematopoietic stem cell transplantation, especially in haploidentical or mismatched transplantations; iii) role of HLAab in blood transfusion including refractory thrombocytopenia and selection of suitable platelet donors, transfusion-related lung injury after plasma transfusion, and immunization against HLA after red blood cell transfusion despite leukodepletion. Although the Luminex platform constitutes a potent technology for HLA antibody detection, some drawbacks require the well-educated analysis and interpretation of data in critical cases. In addition, Luminex has become an important tool to identify clinically relevant antibodies.
Luminex; HLA antibodies; Transfusion
RHD PCR of blood donors may be used to reveal weak D, partial D, DEL and chimeric D+/D− donors among presumed D-negative blood donors. Units donated by such donors pose a definite yet low risk for anti-D immunization of transfusion recipients. The frequency of DEL donors among D-negative donors is 1:350 to 1:2,000 in Europe and up to 1:5 in Asian countries. Different strategies for RHD PCR of blood donors have been used. Probably, the most cost-efficient implementation is replacement of sensitive D antigen testing with the indirect antiglobulin test by RHD PCR in pools which might even reduce total testing cost.
Antigen D; Rh blood group; Blood donors; Indirect antiglobulin test; DEL; Weak D; RHD PCR
The complement-dependent lymphocytotoxicity (CDC) method has been the classical technique to detect human leukocyte antigen (HLA) antibodies in sera of patients who are listed for kidney transplantation. Because of the drawbacks of CDC, such as low sensitivity and low resolution in characterizing antibody specificities, the more specific ELISA technology was introduced in the 1990s which utilizes solubilized HLA molecules instead of lymphocytes. During the last 10 years, the introduction of the Luminex-based single antigen bead (L-SAB) technology, which uses recombinant single HLA molecules, allows detection and characterization of HLA antibodies at greater sensitivity than CDC and ELISA. A drawback associated with this technique is that the interpretation of results is demanding and requires comprehensive experience in HLA antibody diagnostics. Herein we discuss the current role and value of L-SAB technology in the clinical management of sensitized kidney transplant recipients.
Luminex; Antibody; HLA; Kidney; Transplantation
Many modern therapies depend on platelet (PLT) transfusion support. PLTs have a 4- to 7-day shelf life and are frequently in short supply. In order to optimize the inventory PLTs are often transfused to adults without regard for ABO compatibility. Hemolytic reactions are infrequent despite the presence of ‘high titer’ anti-A and anti-B antibodies in some of the units. Despite the low risk for hemolysis, some centers provide only ABO identical PLTs to their recipients; this practice might have other beneficial outcomes that remain to be proven. Strategies to mitigate the risk of hemolysis and the clinical and laboratory outcomes following ABO-matched and mismatched transfusions will be discussed. Although the PLTs themselves do not carry the D antigen, a small number of RBCs are also transfused with every PLT dose. The quantity of RBCs varies by the type of PLT preparation, and even a small quantity of D+ RBCs can alloimmunize a susceptible D− host. Thus PLT units are labeled as D+/–, and most transfusion services try to prevent the transfusion of D+ PLTs to D– females of childbearing age. A similar policy for patients with hematological diseases is controversial, and the elements and mechanisms of anti-D alloimmunization will be discussed.
Platelets; ABO; Rh; Mismatch; Hemolysis; Alloimmunization; Antibody
With the introduction of the next generation sequencing (NGS) technologies, remarkable new diagnostic applications have been established in daily routine. Implementation of NGS is challenging in clinical diagnostics, but definite advantages and new diagnostic possibilities make the switch to the technology inevitable. In addition to the higher sequencing capacity, clonal sequencing of single molecules, multiplexing of samples, higher diagnostic sensitivity, workflow miniaturization, and cost benefits are some of the valuable features of the technology. After the recent advances, NGS emerged as a proven alternative for classical Sanger sequencing in the typing of human leukocyte antigens (HLA). By virtue of the clonal amplification of single DNA molecules ambiguous typing results can be avoided. Simultaneously, a higher sample throughput can be achieved by tagging of DNA molecules with multiplex identifiers and pooling of PCR products before sequencing. In our experience, up to 380 samples can be typed for HLA-A, -B, and -DRB1 in high-resolution during every sequencing run. In molecular oncology, NGS shows a markedly increased sensitivity in comparison to the conventional Sanger sequencing and is developing to the standard diagnostic tool in detection of somatic mutations in cancer cells with great impact on personalized treatment of patients.
NGS; HLA; Molecular oncology
Platelet concentrate transfusion is the standard treatment for hemato-oncology patients to compensate for thrombocytopenia. We have developed a novel platelet activation test in anticoagulated unprocessed blood (pac-t-UB) to determine platelet function in platelet concentrates and in blood of thrombocytopenic patients.
We have measured platelet activity in a platelet concentrate and in anticoagulated unprocessed blood of a post-transfusion thrombocytopenic patient.
Our data show time-dependent platelet activation by GPVI agonist (collagen related peptide; CRP), PAR-1 agonist (SFLLRN), P2Y12 agonist (ADP), and thromboxane receptor agonist (U46619) in a platelet concentrate. Furthermore, pac-t-UB showed time-dependent platelet activation in unprocessed blood of a post-transfusion patient with thrombocytopenia. Testing platelet function by different agonists in relation to storage show that 3-day-old platelet concentrates are still reactive to the studied agonists. This reactivity rapidly drops for each agonists during longer storage.
Pac-t-UB is a novel tool to estimate platelet function by different agonists in platelet concentrates and in unprocessed blood of thrombocytopenic patients. In the near future, we will validate whether pac-t-UB is an adequate test to monitor the quality of platelet concentrates and whether pac-t-UB predicts the bleeding risk of transfused thrombocytopenic patients.
Flow cytometry; Platelet, Platelet activation; Platelet concentrates; Platelet function; Platelet storage; Platelet transfusion; Platelets; Thrombocytopenia
Over the last 2 decades, cord blood (CB) has become an important source of blood stem cells. Clinical experience has shown that CB is a viable source for blood stem cells in the field of unrelated hematopoietic blood stem cell transplantation.
Studies of CB units (CBUs) stored and ordered from the US (National Marrow Donor Program (NMDP) and Swiss (Swiss Blood Stem Cells (SBSQ)) CB registries were conducted to assess whether these CBUs met the needs of transplantation patients, as evidenced by units being selected for transplantation. These data were compared to international banking and selection data (Bone Marrow Donors Worldwide (BMDW), World Marrow Donor Association (WMDA)). Further analysis was conducted on whether current CB banking practices were economically viable given the units being selected from the registries for transplant. It should be mentioned that our analysis focused on usage, deliberately omitting any information about clinical outcomes of CB transplantation.
A disproportionate number of units with high total nucleated cell (TNC) counts are selected, compared to the distribution of units by TNC available. Therefore, the decision to use a low threshold for banking purposes cannot be supported by economic analysis and may limit the economic viability of future public CB banking.
We suggest significantly raising the TNC level used to determine a bankable unit. A level of 125 × 107 TNCs, maybe even 150 × 107 TNCs, might be a viable banking threshold. This would improve the return on inventory investments while meeting transplantation needs based on current selection criteria.
Blood products; CD34+ cells; Collection efficiency; Cord blood; Cost-benefit analysis; Cost-effectiveness analysis; Cryopreservation; Health economics; Hematopoietic cell transplantation; Hematopoietic stem cells
Preoperative over-ordering of blood is common and leads to the wastage of blood bank resources. The preoperative blood ordering and transfusion practices for common elective general surgical procedures were evaluated in our university hospital to formulate a maximum surgical blood order schedule (MSBOS) for those procedures where a cross-match appears necessary.
We evaluated blood ordering practices retrospectively in all elective general surgical procedures in our institution over a 6-month period. Cross-match-to-transfusion ratios (C:T) were calculated and compared to current trust and the British Society of Haematology (BSH) guidelines. The adjusted C:T ratio was also calculated and was defined as the C:T ratio when only cross-matched blood used intraoperatively was included in the calculation.
541 patients were identified during the 6-month period. There were 314 minor and 227 major surgeries carried out. 99.6% (n = 226) of the patients who underwent major surgery and 95.5% (n = 300) of the patients having minor surgery had at least a group and save (G and S) test preoperatively. A total of 507 units of blood were cross-matched and 238 units were used. The overall C:T ratio was therefore 2.1:1, which corresponds to a 46.9% red cell usage. There was considerable variation in the C:T ratio, depending on the type of surgery performed. The adjusted C:T ratio varied between 3.75 and 37.
Compliance with transfusion policies is poor and over-ordering of blood products commonplace. Implementation of the updated recommended MSBOS and introduction of G and S for eligible surgical procedures is a safe, effective and cost-effective method to prevent preoperative over-ordering of blood in elective general surgery. Savings of GBP 8,596.00 per annum are achievable with the incorporation of updated evidence-based guidelines in our university hospital.
Cross-match/transfusion ratio; Transfusion; Surgery; Maximum surgical blood ordering schedule; MSBOS
The Mirasol pathogen reduction technology (PRT) system uses riboflavin and ultraviolet light and is currently approved and used in Europe for the treatment of platelets and plasma. Mirasol treatment is intended to reduce the infectious pathogen load and to inactivate leukocytes in blood products. Our objective was to evaluate buffy coat platelet concentrates (BCPCs) prepared with platelet additive solution (PAS) and treated with the Mirasol system and to examine the effects on platelet cell quality during storage.
26 BCPCs were prepared and split, creating 13 paired control and test units. The test units were treated with the Mirasol system and the platelet quality was assessed in all units over 7 days of storage.
All products met the incoming specifications for Mirasol treatment, and the pH of all Mirasol-treated BCPCs in PAS met the requirements of the Council of Europe guidelines throughout storage. Analysis of lactate production and glucose consumption rates, CD62p expression and cytokines indicates enhanced cellular metabolism in treated platelets, but the levels were within previously published ranges.
While Mirasol-treated BCPCs in PAS had increased metabolism and activation compared to controls, the results indicate that these units can be stored for 7 days with acceptable cell quality.
Pathogen reduction technology; Mirasol; Buffy coat platelets; PAS-IIIM
Xenotropic murine leukemia virus-related virus (XMRV) is a retrovirus that has recently been related to prostate cancers and chronic fatigue syndrome. Since other human-pathogenic retroviruses, such as HIV, human T-lymphotropic virus type I (HTLV-I) and –II, are known blood-transmitted pathogens, XMRV might present another hazard associated with products derived from in vitro cultures of human or animal origin, or blood component-based therapeutics. Here, we investigated whether XMRV was transmitted to individuals with hemophilia and frequent exposure to plasma-derived or recombinant clotting factors.
We used highly sensitive real-time PCR to test plasma samples from 127 consecutive individuals with hemophilia who consulted our hemophilia center either for treatment or for a standard check-up.
From the 127 hemophiliacs, 80 had prior contact to persons with either hepatitis B (n = 30), hepatitis C (n = 74) and/or HIV (n = 21), and 30 were currently being treated with plasma-derived and 97 with recombinant factor concentrates. None of the individuals tested positive for XMRV.
Independent of the ongoing discussion on whether the positive XMRV testing in initial reports was a result of reagent, sample, or tissue contamination, and whether XMRV is a real threat or a testing artifact, our data suggest that XMRV might not play an important role for hemophiliacs.
Hemophilia; Virus contamination; Plasma products; Virus safety; XMRV
Current notions in immunology hold that not only pathogen-mediated tissue injury but any injury activates the innate immune system. In principle, this evolutionarily highly conserved, rapid first-line defense system responds to pathogen-induced injury with the creation of infectious inflammation, and non-pathogen-induced tissue injury with ‘sterile’ tissue inflammation. In this review, evidence has been collected in support of the notion that the transfusion-related acute lung injury induces a ‘sterile’ inflammation in the lung of transfused patients in terms of an acute innate inflammatory disease. The inflammatory response is mediated by the patient's innate immune cells including lung-passing neutrophils and pulmonary endothelial cells, which are equipped with pattern recognition receptors. These receptors are able to sense injury-induced, damage-associated molecular patterns (DAMPs) generated during collection, processing, and storage of blood/blood components. The recognition process leads to activation of these innate cells. A critical role for a protein complex known as the NLRP3 inflammasome has been suggested to be at the center of such a scenario. This complex undergoes an initial ‘priming’ step mediated by 1 class of DAMPs and then an ‘activating’ step mediated by another class of DAMPs to activate interleukin-1beta and interleukin-18. These 2 cytokines then promote, via transactivation, the formation of lung inflammation.
Transfusion-related acute lung injury; TRALI; Innate immunity; Inflammation; Damage-associated molecular patterns; DAMPs
Immunoprophylaxis with IgG anti-D is a standard prevention of hemolytic disease of the fetus and newborn. Fetal Rhesus D (RhD) blood group genotyping from maternal plasma of RhD-negative pregnant women allows targeted prophylaxis with IgG anti-D in RhD-positive pregnancies only. We set up a reliable protocol for prenatal RHD genotyping.
153 pregnant Caucasian RhD-negative women were tested in the 27th week (range 7–38th week) of pregnancy. 18 of them were alloimmunized to the RhD antigen. The fetal RHD genotype was determined based on an automated DNA extraction and real-time polymerase chain reaction method. Intron 4 and exons 5, 7 and 10 of the RHD gene and the SRY gene were targeted.
The fetal RhD status and gender was 100% correctly predicted in all 153 pregnancies (55 RhD-positive males, 45 RhD-positive females; 23 RhD-negative males, 30 RhD-negative females).
The accuracy and applicability of our protocol for non-invasive fetal RhD determination allows the correct management of RhD-incompatible pregnancies. Our protocol could prevent unnecessary immunoprophylaxis in 53 of 153 cases. We therefore recommend that non-invasive fetal RHD genotyping is introduced as an obligatory part of prenatal screening.
Noninvasive RHD typing; Fetal DNA; Maternal plasma
Corneas needed for keratoplasty can be harvested using two techniques: whole globe enucleation and in situ excision of the corneoscleral disc. This study evaluates the rate of microbial contamination of the donor cornea organ culture medium according to the method of retrieval.
All donor corneas of our cornea bank received between January 1, 2001 and December 31, 2009 put into organ culture and microbio-logically tested were prospectively analyzed for microbial contamination of the organ culture medium.
2,805 donor corneas could be included in this study in total. 975 of them were retrieved by whole globe enucleation (group 1) and 1,830 by in situ corneoscleral disc excision (group 2). 15 corneas of group 1 (1.5%) and 46 corneas of group 2 (2.5%) showed a contamination of the organ culture medium. The difference was shown not to be statistically significant (p = 0.082).
The rate of microbial contamination in organ-cultured donor corneas does not seem to be dependent on the method of their retrieval.
Cornea donation; Whole globe enucleation; In situ corneoscleral disc excision; Organ culture; Microbiology
Since blood culture bottles are validated by the manufacturer for blood only, an additional validation for the use with fluids of tissue preparations is necessary.
Two 10-ml samples of cornea culture medium, histidine-tryptophan-ketoglutarate (HTK) solution, or Ringer solution at the end of femur head thermo-disinfection were given into blood culture bottles (BD BACTEC™ Plus Aerobic/F, Anaerobic/F for cornea culture medium and BD BACTEC™ Standard Aerobic/Anaerobic for HTK and Ringer solution) and subsequently spiked with 10–100 colony forming units (CFU) of bacteria or fungi (aerobic bacteria: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa; anaerobic bacteria: Clostridium sporogenes; fungi: Candida albicans, Aspergillus brasiliensis) according to the European Pharmacopoeia Chapter 2.6.1. Results: All tested bacteria and fungi could be detected in all solutions. All positive and negative controls were tested correctly. Compared to the positive controls, the microbial growth was delayed in the antibiotic-containing cornea culture medium, and negative in two cases of B. subtilis spiking. Conclusion: The use of BACTEC™ blood culture bottles seems to be a suitable method for microbiological testing of HTK solution, Ringer solution, and, with limitations, also for testing of the antibiotic-containing cornea culture medium.
Validation; Microbiology; Tissue
According to EU regulations (EU directive 2006/17/EC), blood specimens for virologic testing in the context of post-mortal tissue donation must be taken not later than 24 h post mortem.
To verify validity of NAT in blood specimens collected later, viral nucleic acid concentrations were monitored in blood samples of deceased persons infected with HIV (n = 7), HBV (n = 5), and HCV (n = 17) taken upon admission and at 12 h, 24 h, 36 h and 48 h post mortem. HIV and HCV RNA were quantified using Cobas TaqMan (Roche), HBV DNA was measured by in-house PCR.
A more than 10-fold decrease of viral load in samples taken 36 h or 48 h post mortem was seen in one HIV-infected patient only. For all other patients tested the decrease of viral load in 36-hour or 48-hour post-mortal samples was less pronounced. Specimens of 3 HIV- and 2 HBV-infected patients taken 24 h post mortem or later were even found to have increased concentrations (>10-fold), possibly due to post-mortem liberation of virus from particular cells or tissues.
Our preliminary data indicate that the time point of blood collection for HIV, HBV and HCV testing by PCR may be extended to 36 h or even 48 h post mortem and thus improve availability of tissue donations.
Post-mortal tissue donation; Virus safety NAT screening
We present the operational organization and daily workflow of our Hamburg model and the results of the years 2007–2011 concerning donation of corneas, musculoskeletal and, since 2010, cardiovascular tissues.
Each of the about 3,600 deceased every year undergoes an evaluation process by two coordinators on duty, the tissue coordinator and the family coordinator. All donation connected issues are carried out within the standardized protocols of a quality management system and documented in a special data base. Two catamnestic surveys evaluated the satisfaction of donor families retrospectively. The inclusion rate for cornea donation was 23% and for musculoskeletal donation 10%, with a decrease after the 75 years age restriction of musculoskeletal donors in 2011 defined by the contracting tissue bank German Institute for Cell and Tissue Replacement gGmbH (DIZG), Berlin.
Since 2007 1,268 corneas were explanted altogether, reflecting an increasing explantation rate from 156 (University Medical Center Hamburg-Eppendorf (UMC: 9) in 2007 up to 304 (UMC: 52) in 2011. Overall 173 musculoskeletal donors (5 years) and 11 cardiovascular donors (2 years) spent tissues. The consent rate was much higher. The evaluation of the families reflected a positive feedback for the guiding of the donation process.
Forensic institutes can act as an interface between donors and recipients without neglecting forensic investigations. They are uniquely positioned to recognize potential donors. In addition, the contact with a physician of the forensic institute may help families during the mourning phase.
Tissue donation; Tissue transplants; Forensic institute; Tissue donation results
Commercial available NAT systems are usually not validated for screening of post-mortem blood samples. NAT testing might be challenging due to inhibitory substances in the cadaveric blood sample that cause false-negative test results. Validation studies have to be performed to show the performance characteristics of the NAT assays for testing cadaveric blood.
A set of 32 post-mortem serum and plasma samples from cornea donors and 40 control samples from blood donors, serologically and NAT negative for all investigated parameters, were spiked with defined concentrations of WHO reference material and tested for HIV-1, HCV, HBV, and HAV by NAT using DRK Baden-Württemberg-Hesse CE PCR kits. Analytical sensitivity, analytical specificity and reproducibility/precision were validated and compared with each other in both groups of samples.
The analytical sensitivity was 100% for control and post-mortem specimens when spiked with virus standards at concentrations of 3 × level of detection (LOD). Invalid results did not occur. The analytical specificity rate for all assays was 100%. Intra-assay variation was analyzed as a function of sample material and sampling time post mortem. Values of % coefficient of variation (%CV) were comparable for serum and plasma but slightly higher for post-mortem samples especially for those samples collected more than 24 h post mortem.
Based on the presented validation, postmortem donor samples can be tested with the automated DRK Baden-Würtemberg-Hesse NAT system.
NAT; Post-mortem blood; Tissue donation; Validation; HCV; HIV; HBV; HAV
A safe look back of products requires their unique identification. Blood products are encoded in Germany with Eurocode since 1987. EU Directives 2004/23/EC und 2006/86/EC demanded unique identification and safe look back procedure also for tissues and cells. Eurocode IBLS e.V. and the DGTI working parties ‘Tissue Preparations’ and ‘Automation and Data Processing’ supplemented the already available Eurocode nomenclature for blood products with further data structures for tissue preparations and deliberated the federal authorities during the EU hearings. In result all EU member states can administer the coding system oneself, but have to take care about the ‘key code’ structure as defined and the common part at the begin of the ID number of the preparations. Eurocode today offers an EU-conform coding system considering various aspects of blood, tissue and cell preparations in an ISO-standardized form.
Eurocode; Tissue; Coding systems; Look back; European Directive 2006/86/EC