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1.  Performance of HbA1c Assay Methods: Good Enough? 
Clinical chemistry  2014;60(8):1031-1033.
PMCID: PMC4757849  PMID: 24938751
2.  The Effect of Increased Fetal Hemoglobin on Seven Common HbA1c Assay Methods 
Clinical chemistry  2012;58(5):945-947.
PMCID: PMC4754956  PMID: 22357875
HbA1c; fetal hemoglobin; HbF; interference; manufacturer claims; HPLC
3.  Sulfatide Analysis by Mass Spectrometry for Screening of Metachromatic Leukodystrophy in Dried Blood and Urine Samples 
Clinical chemistry  2015;62(1):279-286.
Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder caused by deficiency in arylsulfatase A activity, leading to accumulation of sulfatide substrates. Diagnostic and monitoring procedures include demonstration of reduced arylsulfatase A activity in peripheral blood leukocytes or detection of sulfatides in urine. However, the development of a screening test is challenging because of instability of the enzyme in dried blood spots (DBS), the widespread occurrence of pseudodeficiency alleles, and the lack of available urine samples from newborn screening programs.
We measured individual sulfatide profiles in DBS and dried urine spots (DUS) from MLD patients with LC-MS/MS to identify markers with the discriminatory power to differentiate affected individuals from controls. We also developed a method for converting all sulfatide molecular species into a single species, allowing quantification in positive-ion mode upon derivatization.
In DBS from MLD patients, we found up to 23.2-fold and 5.1-fold differences in total sulfatide concentrations for early- and late-onset MLD, respectively, compared with controls and pseudodeficiencies. Corresponding DUS revealed up to 164-fold and 78-fold differences for early- and late-onset MLD patient samples compared with controls. The use of sulfatides converted to a single species simplified the analysis and increased detection sensitivity in positive-ion mode, providing a second option for sulfatide analysis.
This study of sulfatides in DBS and DUS suggests the feasibility of the mass spectrometry method for newborn screening of MLD and sets the stage for a larger-scale newborn screening pilot study.
PMCID: PMC4737087  PMID: 26585924
4.  High-Throughput Assay of 9 Lysosomal Enzymes for Newborn Screening 
Clinical chemistry  2013;59(3):502-511.
There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the availability of treatments. Pilot studies have used tandem mass spectrometry with flow injection of samples to achieve multiplex detection of enzyme products. We report a multiplexing method of 9 enzymatic assays that uses HPLC-tandem mass spectrometry (MS/MS).
The assay of 9 enzymes was carried out in 1 or 2 buffers with a cassette of substrates and internal standards and 1 or 2 punches of a dried blood spot (DBS) from a newborn screening card as the source of enzymes. The pre–HPLC-MS/MS sample preparation required only 4 liquid transfers before injection into a dual-column HPLC equipped with switching valves to direct the flow to separation and column equilibration. Product-specific and internal standard–specific ion fragmentations were used for MS/MS quantification in the selected reaction monitoring mode.
Analysis of blood spots from 58 random newborns and lysosomal storage disease–affected patients showed that the assay readily distinguished affected from nonaffected individuals. The time per 9-plex analysis (1.8 min) was sufficiently short to be compatible with the workflow of newborn screening laboratories.
HPLC-MS/MS provides a viable alternative to flow-injection MS/MS for the quantification of lysosomal enzyme activities. It is possible to assay 9 lysosomal enzymes using 1 or 2 reaction buffers, thus minimizing the number of separate incubations necessary.
PMCID: PMC4737435  PMID: 23315484
5.  The Landscape of MicroRNA, Piwi-Interacting RNA, and Circular RNA in Human Saliva 
Clinical chemistry  2014;61(1):221-230.
Extracellular RNAs (exRNAs) in human body fluids are emerging as effective biomarkers for detection of diseases. Saliva, as the most accessible and noninvasive body fluid, has been shown to harbor exRNA biomarkers for several human diseases. However, the entire spectrum of exRNA from saliva has not been fully characterized.
Using high-throughput RNA sequencing (RNA-Seq), we conducted an in-depth bioinformatic analysis of noncoding RNAs (ncRNAs) in human cell-free saliva (CFS) from healthy individuals, with a focus on microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and circular RNAs (circRNAs).
Our data demonstrated robust reproducibility of miRNA and piRNA profiles across individuals. Furthermore, individual variability of these salivary RNA species was highly similar to those in other body fluids or cellular samples, despite the direct exposure of saliva to environmental impacts. By comparative analysis of >90 RNA-Seq data sets of different origins, we observed that piRNAs were surprisingly abundant in CFS compared with other body fluid or intracellular samples, with expression levels in CFS comparable to those found in embryonic stem cells and skin cells. Conversely, miRNA expression profiles in CFS were highly similar to those in serum and cerebrospinal fluid. Using a customized bioinformatics method, we identified >400 circRNAs in CFS. These data represent the first global characterization and experimental validation of circRNAs in any type of extracellular body fluid.
Our study provides a comprehensive landscape of ncRNA species in human saliva that will facilitate further biomarker discoveries and lay a foundation for future studies related to ncRNAs in human saliva.
PMCID: PMC4332885  PMID: 25376581
6.  Circulating tumor cell enumeration using a combination of EpCAM and Cell-surface vimentin based methods for monitoring breast cancer therapeutic response 
Clinical chemistry  2014;61(1):259-266.
Detection, isolation and enumeration of circulating tumor cells (CTCs) from cancer patients has become an important modality in clinical management of patients with breast cancer. Although CellSearch, an EpCAM based method that is used to isolate epithelial CTCs has gained immense importance, its inability to detect mesenchymal CTCs from breast cancer patients raises concerns for its utility as a clinical management tool.
To address this gap in technology, we recently discovered the utility of cell-surface vimentin (CSV) as a marker for detecting mesenchymal CTC from sarcoma tumors. Here in this study, we tested the sensitivity and specificity of detecting CTC from blood collected at a random time during therapy from each of the 58 patients with metastatic breast cancer utilizing 84-1 (mAb against CSV to detect epithelial mesenchymal transitioned CTC) and CellSearch methods. Also we tested the possibility of improving the sensitivity and specificity of detection using additional parameters including nuclear EpCAM localization and epithelial mesenchymal ratios.
CTC counts using CSV were significant in differentiating treatment responding (stable) and treatment non-responding (progression) populations in comparison to the CellSearch method. The results also indicated that a summation of CTCs detected from both methods with a threshold of 8 CTCs/7.5mL increased the specificity of CTC detection substantially in comparison with other tested combinations as determined by ROC curves.
Collectively, utilizing a summation of CellSearch and CSV methods provide new insights into using CTC enumeration to assess therapeutic response and thus provides a new approach to personalized medicine in breast cancer patients.
PMCID: PMC4360893  PMID: 25336717
Circulating tumor cells; Breast cancer; Vimentin; EMT
7.  Validation of DNA Methylation to Predict Outcome in Acute Myeloid Leukemia by Use of xMELP 
Clinical chemistry  2014;61(1):249-258.
Epigenetic dysregulation involving alterations in DNA methylation is a hallmark of various types of cancer, including acute myeloid leukemia (AML). Although specific cancer types and clinical aggressiveness of tumors can be determined by DNA methylation status, the assessment of DNA methylation at multiple loci is not routinely performed in the clinical laboratory.
We recently described a novel microsphere-based assay for multiplex evaluation of DNA methylation. In the current study, we validated and used an improved assay [termed expedited HpaII small fragment Enrichment by Ligation-mediated PCR (xMELP)] that can be performed with appropriate clinical turnaround time.
Using the xMELP assay in conjunction with a new 17-locus random forest classifier that has been trained using 344 AML samples, we were able to segregate an independent cohort of 70 primary AML patients into methylation-determined subgroups with significantly distinct mortality risk (P = 0.009). We also evaluated precision, QC parameters, and preanalytic variables of the xMELP assay and determined the sensitivity of the random forest classifier score to failure at 1 or more loci.
Our results demonstrate that xMELP performance is suitable for implementation in the clinical laboratory and predicts AML outcome in an independent patient cohort.
PMCID: PMC4384518  PMID: 25348669
9.  High-Sensitivity Troponin I and Amino-Terminal Pro–B-Type Natriuretic Peptide Predict Heart Failure and Mortality in the General Population 
Clinical chemistry  2014;60(9):1225-1233.
High-sensitivity cardiac troponin assays have potent prognostic value in stable cardiovascular disease cohorts. Our objective was to assess the prognostic utility of a novel cardiac troponin I (cTnI) high-sensitivity assay, independently and in combination with amino-terminal pro–B-type natriuretic peptide (NT-proBNP), for the future development of heart failure and mortality in the general community.
A well-characterized community-based cohort of 2042 participants underwent clinical assessment and echocardiographic evaluation. Baseline measurements of cTnI with a high-sensitivity assay and NT-proBNP were obtained in 1843 individuals. Participants were followed for new-onset heart failure and mortality with median (25th, 75th percentile) follow-up of 10.7 (7.9, 11.6) and 12.1 (10.4, 13.0) years, respectively.
When measured with a high-sensitivity assay, cTnI greater than the sex-specific 80th percentile was independently predictive of heart failure [hazard ratio 2.56 (95% confidence interval 1.88 – 3.50), P < 0.001] and mortality [1.91(1.49 – 2.46), P < 0.001] beyond conventional risk factors in this community-based cohort, with significant increases in the net reclassification improvement for heart failure. The prognostic utility of cTnI measured with a high-sensitivity assay goes beyond NT-proBNP, yet our data suggest that these 2 assays are complementary and most beneficial when evaluated together in identifying at-risk individuals in the community.
Our findings lay the foundation for prospective studies aimed at identification of individuals at high risk by use of a multimarker approach, followed by aggressive prevention strategies to prevent subsequent heart failure.
PMCID: PMC4692160  PMID: 24987112
10.  Seven Direct Methods for Measuring HDL and LDL Cholesterol Compared with Ultracentrifugation Reference Measurement Procedures 
Clinical chemistry  2010;56(6):977-986.
Methods from 7 manufacturers and 1 distributor for directly measuring HDL cholesterol (C) and LDL-C were evaluated for imprecision, trueness, total error, and specificity in nonfrozen serum samples.
We performed each direct method according to the manufacturer’s instructions, using a Roche/Hitachi 917 analyzer, and compared the results with those obtained with reference measurement procedures for HDL-C and LDL-C. Imprecision was estimated for 35 runs performed with frozen pooled serum specimens and triplicate measurements on each individual sample. Sera from 37 individuals without disease and 138 with disease (primarily dyslipidemic and cardiovascular) were measured by each method. Trueness and total error were evaluated from the difference between the direct methods and reference measurement procedures. Specificity was evaluated from the dispersion in differences observed.
Imprecision data based on 4 frozen serum pools showed total CVs <3.7% for HDL-C and <4.4% for LDL-C. Bias for the nondiseased group ranged from −5.4% to 4.8% for HDL-C and from −6.8% to 1.1% for LDL-C, and for the diseased group from −8.6% to 8.8% for HDL-C and from −11.8% to 4.1% for LDL-C. Total error for the nondiseased group ranged from −13.4% to 13.6% for HDL-C and from −13.3% to 13.5% for LDL-C, and for the diseased group from −19.8% to 36.3% for HDL-C and from −26.6% to 31.9% for LDL-C.
Six of 8 HDL-C and 5 of 8 LDL-C direct methods met the National Cholesterol Education Program total error goals for nondiseased individuals. All the methods failed to meet these goals for diseased individuals, however, because of lack of specificity toward abnormal lipoproteins.
PMCID: PMC4687457  PMID: 20378768
11.  Non–HDL Cholesterol Shows Improved Accuracy for Cardiovascular Risk Score Classification Compared to Direct or Calculated LDL Cholesterol in a Dyslipidemic Population 
Clinical chemistry  2011;57(3):490-501.
Our objective was to evaluate the accuracy of cardiovascular disease (CVD) risk score classification by direct LDL cholesterol (dLDL-C), calculated LDL cholesterol (cLDL-C), and non–HDL cholesterol (non–HDL-C) compared to classification by reference measurement procedures (RMPs) performed at the CDC.
We examined 175 individuals, including 138 with CVD or conditions that may affect LDL-C measurement. dLDL-C measurements were performed using Denka, Kyowa, Sekisui, Serotec, Sysmex, UMA, and Wako reagents. cLDL-C was calculated by the Friedewald equation, using each manufacturer’s direct HDL-C assay measurements, and total cholesterol and triglyceride measurements by Roche and Siemens (Advia) assays, respectively.
For participants with triglycerides <2.26 mmol/L (<200 mg/dL), the overall misclassification rate for the CVD risk score ranged from 5% to 17% for cLDL-C methods and 8% to 26% for dLDL-C methods when compared to the RMP. Only Wako dLDL-C had fewer misclassifications than its corresponding cLDL-C method (8% vs 17%; P <0.05). Non–HDL-C assays misclassified fewer patients than dLDL-C for 4 of 8 methods (P < 0.05). For participants with triglycerides ≥2.26 mmol/L (≥200 mg/dL) and <4.52 mmol/L (<400 mg/dL), dLDL-C methods, in general, performed better than cLDL-C methods, and non–HDL-C methods showed better correspondence to the RMP for CVD risk score than either dLDL-C or cLDL-C methods.
Except for hypertriglyceridemic individuals, 7 of 8 dLDL-C methods failed to show improved CVD risk score classification over the corresponding cLDL-C methods. Non–HDL-C showed overall the best concordance with the RMP for CVD risk score classification of both normal and hypertriglyceridemic individuals.
PMCID: PMC4684263  PMID: 21228254
12.  Immunoglobulins: Expanding the Role for Mass Spectrometry in Protein Biomarker Quantification 
Clinical chemistry  2014;60(8):1034-1035.
PMCID: PMC4674789  PMID: 24938750
13.  Association of 1,5-anhydroglucitol with diabetes and microvascular conditions 
Clinical chemistry  2014;60(11):1409-1418.
1,5-anhydroglucitol (1,5-AG) is inversely related to hyperglycemia and may be a useful indicator of short-term (1-2 week) hyperglycemia and glycemic excursions but its prognostic value is unclear. We sought to evaluate the associations of 1,5-AG with risk of diabetes and microvascular disease.
We measured 1,5-AG in blood samples from over 10,000 participants in the ARIC Study (baseline, 1990-1992) and characterized the independent associations with prevalent retinopathy, and with incident chronic kidney disease and incident diabetes during approximately 20 years of follow-up.
We found that 1,5-AG was associated with prevalent retinopathy, driven primarily by the strong association in persons with diagnosed diabetes: adjusted OR 11.26 (95%CI 6.17-20.53) for <6 ug/mL compared to 1,5-AG ≥10 ug/mL. This result remained significant after further adjustment for HbA1c (OR 4.85, 95%CI 2.42-9.74). In persons with diagnosed diabetes: low 1,5-AG (<6 ug/mL vs ≥10 ug/mL) was also associated with a greater than 2-fold increased risk of incident chronic kidney disease (adjusted HR 2.83, 95% CI 2.15-3.74) and remained significant after adjustment for HbA1c (HR 1.43, 95%CI 1.02-2.00). Nondiabetic participants with high 1,5-AG (≥ 10 ug/mL) had the lowest prevalence of retinopathy and lowest risk of kidney disease. In persons without diagnosed diabetes at baseline, 1,5-AG <10 ug/mL was also associated with incident diabetes (adjusted HR 2.29, 95%CI 2.03-2.58).
1,5-AG was associated with long-term risk of important microvascular outcomes, particularly in persons with diagnosed diabetes and even after adjustment for HbA1c. Our results suggest 1,5-AG may capture risk information associated with hyperglycemic excursions.
PMCID: PMC4215646  PMID: 25200356
14.  Effect of Plasma Protein Depletion on BNP-32 Recovery 
Clinical chemistry  2008;54(5):933-934.
PMCID: PMC4626888  PMID: 18443186
15.  Contamination of the Norepinephrine Prodrug Droxidopa by Dihydroxyphenylacetaldehyde 
Clinical chemistry  2010;56(5):832-838.
l-Threo-3,4-dihydroxyphenylserine (L-DOPS, droxidopa) is a norepinephrine (NE) prodrug under development to treat orthostatic hypotension. 3,4-Dihydroxyphenylacetaldehyde (DOPAL), an endogenous catecholaldehyde produced by enzymatic oxidative deamination of dopamine, is toxic to catecholaminergic neurons. Based on the observation of increasing plasma DOPAL after oral administration of L-DOPS to a patient, we examined whether other subjects also had DOPAL in their plasma after droxidopa administration, and whether droxidopa is contaminated with DOPAL.
Thirteen subjects took 400 mg droxidopa orally. We sampled venous blood at baseline and 1, 2, 3, 6, 24, and 48 h after drug administration and assayed L-DOPS, NE, and DOPAL by use of liquid chromatography with electrochemical detection (LC-ED). Droxidopa in acidic solution (20:80 mixture of 0.04 mol/L phosphoric acid:0.20 mol/L acetic acid) was vacuum centrifuged for 1 h at 30 °C and then assayed by LC-ED.
Droxidopa contained 0.01% DOPAL. At 6 h after droxidopa, all subjects had detectable DOPAL in plasma (1.89 nmol/L, P = 0.0001). Across the sampling times, plasma DOPAL correlated with plasma L-DOPS (r = 0.996). The mean increment in plasma DOPAL was more than 4 times that in plasma NE (0.39 nmol/L). In 2 patients with Parkinson disease and orthostatic hypotension, DOPAL was detected in plasma at baseline (0.12 nmol/L) and increased by about 70-fold after droxidopa. Vacuum concentration of droxidopa in the acid solution converted L-DOPS to DOPAL completely.
Droxidopa is contaminated with DOPAL. After oral droxidopa administration, DOPAL is detected in plasma of humans. Droxidopa is susceptible to extensive nonenzymatic conversion to DOPAL.
PMCID: PMC4578803  PMID: 20207766
16.  A Comprehensive Assay for CFTR Mutational Analysis Using Next-Generation Sequencing 
Clinical chemistry  2013;59(10):1481-1488.
Cystic fibrosis is a life-threatening genetic disorder that has been associated with mutations in the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene. Hundreds of CFTR mutations have been detected to date. Current CFTR genotyping assays target a subset of these mutations, particularly a mutation panel recommended by the American College of Medical Genetics for carrier screening of the general population. Fast sequencing of the entire coding sequence in a scalable manner could expand the detection of CFTR mutations and facilitate management of costs and turnaround times in the clinical laboratory.
We describe a proof-of-concept CFTR assay that uses PCR target enrichment and next-generation sequencing on the Ion Torrent Personal Genome Machine™ (PGM™) platform.
The scalability of the assay was demonstrated, with an average mean depth of coverage ranging from 500× to 3500×, depending on the number of multiplexed patient samples and the Ion Torrent chip used. In a blinded study of 79 previously genotyped patient DNA samples and cell lines, our assay detected most of the mutations, including single-nucleotide variants, small insertions and deletions, and large copy-number variants. The reproducibility was 100% for detecting mutations in independent runs. Our assay demonstrated high specificity, with only 2 false-positive calls (at 2184delA) found in 2 samples caused by a sequencing error in a homopolymer stretch of sequence. The detection rate for variants of unknown significance was very low in the targeted region.
With continued optimization and system refinements, PGM sequencing promises to be a powerful, rapid, and scalable means of clinical diagnostic sequencing.
PMCID: PMC4570467  PMID: 23775370
17.  Albuminuria Prevalence in First Morning Void Compared with Previous Random Urine from Adults in the National Health and Nutrition Examination Survey, 2009–2010 
Clinical chemistry  2013;59(4):675-683.
Albuminuria, defined as urine albumin/creatinine ratio (ACR) ≥30 mg/g, is a diagnostic component of chronic kidney disease (CKD). National estimates of ACR and CKD prevalence have been based on single random urine samples. Although 2 urine samples or a first morning void are known to produce different estimates of ACR, the impact of differing urine sampling schemes on nationally estimated rates of CKD is unknown.
In 2009–2010, the National Health and Nutrition Examination Survey (NHANES) participants provided 2 untimed urine samples for sequential ACR measurement: an initial random urine collected in the NHANES mobile examination center and a subsequent first morning void collected at home. Rates of albuminuria were calculated in the overall population and broken down by demographics, diagnosed diabetes and hypertension status, and estimated glomerular filtration rate (eGFR).
Overall, 43.5% of adults with increased ACR (≥30 mg/g) in a random urine also had increased ACR in a first morning urine. This percentage was higher among individuals ≥50 years old (48.9%), males (53.3%), participants with diagnosed diabetes (56.3%) and hypertension (51.5%), and eGFR <60 mL/min/1.72m2 (56.9%). The use of confirmed increased ACR (defined as the presence of ACR ≥30 mg/g in both samples taken within 10 days) to define CKD resulted in a lower overall prevalence (11.6%) than first morning urine (12.7%) or random spot urine only (15.2%).
ACR measured on random urine samples appears to overestimate the prevalence of albuminuria compared to first morning urine collections.
PMCID: PMC4562457  PMID: 23315482
18.  In Reply 
Clinical chemistry  2014;60(9):1236-1237.
PMCID: PMC4537528  PMID: 25037935
19.  The Potential Role of Oral Fluid in Antidoping Testing 
Clinical chemistry  2013;60(2):307-322.
Currently, urine and blood are the only matrices authorized for antidoping testing by the World Anti-Doping Agency (WADA). Although the usefulness of urine and blood is proven, issues remain for monitoring some drug classes and for drugs prohibited only in competition. The alternative matrix oral fluid (OF) may offer solutions to some of these issues. OF collection is easy, noninvasive, and sex neutral and is directly observed, limiting potential adulteration, a major problem for urine testing. OF is used to monitor drug intake in workplace, clinical toxicology, criminal justice, and driving under the influence of drugs programs and potentially could complement urine and blood for antidoping testing in sports.
This review outlines the present state of knowledge and the advantages and limitations of OF testing for each of the WADA drug classes and the research needed to advance OF testing as a viable alternative for antidoping testing.
Doping agents are either prohibited at all times or prohibited in competition only. Few OF data from controlled drug administration studies are available for substances banned at all times, whereas for some agents prohibited only in competition, sufficient data may be available to suggest appropriate analytes and cutoffs (analytical threshold concentrations) to identify recent drug use. Additional research is needed to characterize the disposition of many banned substances into OF; OF collection methods and doping agent stability in OF also require investigation to allow the accurate interpretation of OF tests for antidoping monitoring.
PMCID: PMC4545493  PMID: 24153253
20.  Maternal Methadone Dose, Placental Methadone Concentrations, and Neonatal Outcomes 
Clinical chemistry  2011;57(3):449-458.
Few investigations have used placenta as an alternative matrix to detect in utero drug exposure, despite its availability at the time of birth and the large amount of sample. Methadone-maintained opioid-dependent pregnant women provide a unique opportunity to examine the placental disposition of methadone and metabolite [2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP)], to explore their correlations with maternal methadone dose and neonatal outcomes, and to test the ability to detect in utero exposure to illicit drugs.
We calculated the correlations of placental methadone and EDDP concentrations and their correlations with maternal methadone doses and neonatal outcomes. Cocaine- and opiate-positive placenta results were compared with the results for meconium samples and for urine samples collected throughout gestation.
Positive correlations were found between placental methadone and EDDP concentrations (r = 0.685), and between methadone concentration and methadone dose at delivery (r = 0.542), mean daily dose (r = 0.554), mean third-trimester dose (r = 0.591), and cumulative daily dose (r = 0.639). The EDDP/methadone concentration ratio was negatively correlated with cumulative daily dose (r = 0.541) and positively correlated with peak neonatal abstinence syndrome (NAS) score (r = 0.513). Placental EDDP concentration was negatively correlated with newborn head circumference (r = 0.579). Cocaine and opiate use was detected in far fewer placenta samples than in thrice-weekly urine and meconium samples, a result suggesting a short detection window for placenta.
Quantitative methadone and EDDP measurement may predict NAS severity. The placenta reflects in utero drug exposure for a shorter time than meconium but may be useful when meconium is unavailable or if documentation of recent exposure is needed.
PMCID: PMC4543294  PMID: 21245372
22.  Cannabinoids in Exhaled Breath following Controlled Administration of Smoked Cannabis 
Clinical chemistry  2013;59(12):1780-1789.
Δ9-Tetrahydrocannabinol (THC), 11-nor-9-carboxy-THC (THCCOOH), and cannabinol (CBN) were measured in breath following controlled cannabis smoking to characterize the time course and window of detection of breath cannabinoids.
Exhaled breath was collected from chronic (≥4 times per week) and occasional (
THC was the major cannabinoid in breath; no sample contained THCCOOH and only 1 contained CBN. Among chronic smokers (n = 13), all breath samples were positive for THC at 0.89 h, 76.9% at 1.38 h, and 53.8% at 2.38 h, and only 1 sample was positive at 4.2 h after smoking. Among occasional smokers (n = 11), 90.9% of breath samples were THC-positive at 0.95 h and 63.6% at 1.49 h. One occasional smoker had no detectable THC. Analyte recovery from breath pads by methanolic extraction was 84.2%–97.4%. Limits of quantification were 50 pg/pad for THC and CBN and 100 pg/pad for THCCOOH. Solid-phase extraction efficiency was 46.6%–52.1% (THC) and 76.3%–83.8% (THCCOOH, CBN). Matrix effects were −34.6% to 12.3%. Cannabinoids fortified onto breath pads were stable (≤18.2% concentration change) for 8 h at room temperature and −20°C storage for 6 months.
Breath may offer an alternative matrix for testing for recent driving under the influence of cannabis, but is limited to a short detection window (0.5–2 h).
PMCID: PMC4537523  PMID: 24046200
Clinical chemistry  2013;59(11):1638-1648.
Since the mid-2000s synthetic cannabinoids have been abused as recreational drugs, prompting scheduling of these substances in many countries. To circumvent legislation, manufacturers constantly market new compounds; [1-(5-fluoropentyl)indol-3-yl]-(2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11), the fluorinated UR-144 analog, is one of the most recent and widely abused drugs, and its use is now linked with acute kidney injury. Our goal was to investigate XLR-11 metabolism for identification of major urinary targets in analytical methods and to clarify the origin of metabolites when one or more parent synthetic cannabinoids can be the source.
We incubated 10 μmol/L XLR-11 with pooled human hepatocytes and sampled after 1 and 3 h. Samples were analyzed by high-resolution mass spectrometry with a TOF scan followed by information-dependent acquisition triggered product ion scans with dynamic background subtraction and mass defect filters. Scans were thoroughly data mined with different data processing algorithms (Metabolite Pilot 1.5).
XLR-11 underwent phase I and II metabolism, producing more than 25 metabolites resulting from hydroxylation, carboxylation, hemiketal and hemiacetal formation, internal dehydration, and further glucuronidation of some oxidative metabolites. No sulfate or glutathione conjugation was observed. XLR-11 also was defluorinated, forming UR-144 metabolites. On the basis of mass spectrometry peak areas, we determined that the major metabolites were 2′-carboxy-XLR-11, UR-144 pentanoic acid, 5-hydroxy-UR-144, hydroxy-XLR-11 glucuronides, and 2′-carboxy-UR-144 pentanoic acid. Minor metabolites were combinations of the biotransformations mentioned above, often glucuronidated.
These are the first data defining major urinary targets of XLR-11 metabolism that could document XLR-11 intake in forensic and clinical investigations.
PMCID: PMC4537527  PMID: 24014837
Clinical chemistry  2009;55(4):684-697.
Personalized medicine is the provision of focused prevention, detection, prognostic, and therapeutic efforts according to an individual’s genetic composition. The actualization of personalized medicine will require combining a patient’s conventional clinical data with bioinformatics-based molecular-assessment profiles. This synergistic approach offers tangible benefits, such as heightened specificity in the molecular classification of cancer subtypes, improved prognostic accuracy, targeted development of new therapies, novel applications for old therapies, and tailored selection and delivery of chemotherapeutics.
Our ability to personalize cancer management is rapidly expanding through biotechnological advances in the postgenomic era. The platforms of genomics, proteomics, single-nucleotide polymorphism profiling and haplotype mapping, high-throughput genomic sequencing, and pharmacogenomics constitute the mechanisms for the molecular assessment of a patient’s tumor. The complementary data derived during these assessments is processed through bioinformatics analysis to offer unique insights for linking expression profiles to disease detection, tumor response to chemotherapy, and patient survival. Together, these approaches permit improved physician capacity to assess risk, target therapies, and tailor a chemotherapeutic treatment course.
Personalized medicine is poised for rapid growth as the insights provided by new bioinformatics models are integrated with current procedures for assessing and treating cancer patients. Integration of these biological platforms will require refinement of tissue-processing and analysis techniques, particularly in clinical pathology, to overcome obstacles in customizing our ability to treat cancer.
PMCID: PMC4529063  PMID: 19246616
Clinical chemistry  2014;60(5):765-772.
Human Herpesvirus-6 (HHV-6) latently infects a majority of adults. In about 1% of the population HHV-6 exists in a chromosomally integrated form (ciHHV-6) that resides in every somatic and germ cell and can be transmitted through the germ line. Patients with ciHHV-6 have been misdiagnosed and unnecessarily treated for active HHV-6 infection, sometimes with significant side effects, based on results from quantitative molecular HHV-6 tests.
A droplet digital PCR (ddPCR) assay was developed to identify ciHHV-6 in cellular patient specimens by precisely determining the ratio of HHV-6 to cellular DNA. We validated the assay on confirmed ciHHV-6 patient specimens and a cell line derived from a ciHHV-6 patient, and analyzed hematopoietic stem cell transplant patients suspected of ciHHV-6. We additionally evaluated whether the assay could be applied to stored plasma samples from a study of clinical correlates of HHV-6.
The ddPCR assay accurately identified ciHHV-6 in cellular samples (buffy coat, PBMCs), giving a ratio very close to 1 HHV-6/cell (1.02 ± .03 in FISH-confirmed samples). In stored plasma samples, the assay performance was set by design to have 100% sensitivity, which resulted in 82% specificity for ciHHV-6.
The possibility of ciHHV-6 is often overlooked in patients with detectable HHV-6 viral loads by quantitative PCR. Our ddPCR test provides rapid and accurate laboratory identification of ciHHV-6 from easily obtained cellular specimens. In addition, the assay provides excellent sensitivity and specificity using stored plasma samples, facilitating retrospective analysis of the clinical significance of ciHHV-6.
PMCID: PMC4511388  PMID: 24594780
Human Herpesvirus 6; ciHHV-6; digital PCR

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