Proficiency Testing Samples Research Articles

Antihuman Leukocyte Antigen–Specific Antibody Strength Determined by Complement-Dependent or Solid-Phase Assays Can Predict Positive Donor-Specific Crossmatches

N Context.—The association of circulating donor-specific antibody (DSA) strength with crossmatch results is of potential interest to predict allograft outcome. Objectives.—To systematically investigate the aforementioned association and to attempt to define a cutoff value for DSA strength that can predict a positive crossmatch result. Design.—We analyzed DSA strength and crossmatch results from the 2006 to 2008 proficiency testing samples of the American Society of Histocompatibility and Immunogenetics (n = 50). We further validated our findings in candidates for potential kidney transplant (n = 19). Results.—Proficiency test samples with positive antihuman globulin T-cell crossmatch results had significantly higher DSA strength, as assessed by Luminex (Austin,

Evaluation of comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry for the diagnosis of inherited metabolic disorders using an automated data processing strategy

Organic acidurias are a large group of inherited metabolic disorders (IMDs), commonly diagnosed by GC–MS analysis of organic acids in urine after acidic extraction and trimethylsilylation. In this study, a GC × GC-ToF-MS method has been optimized for the analysis of pathological metabolites in urine. An automated data processing strategy based on the use of mass spectra and GC retention times for the target search and quantification of pathological metabolites has been developed. Using this procedure, each unknown sample is automatically examined for the presence of markers of several diseases at the same time. The method has been applied for the analysis of 6 challenging proficiency testing samples from patients with IMDs (thymidine phosphorylase deficiency, mevalonic aciduria, hawkinsinuria, aromatic l-amino acid decarboxylase deficiency, propionic acidemia and medium-chain acyl-CoA dehydrogenase deficiency). Using the GC × GC-ToF-MS method, we were able to determine complete sets of markers for all the IMDs. The quality of the mass spectral matches for the pathological markers was higher than 800 (out of 1000).

Identification of a novel HLA‐DQB1 allele, DQB1*0326, in a College of American Pathologists 2009 survey specimen

We identified a novel DQB1*0326 allele from a proficiency test sample provided by the College of American Pathologists (CAP) medical society. This novel DQB1 allele was unexpectedly discovered by sequence-based typing method in an attempt to resolve a discrepant typing result between the CAP survey report and our laboratory report. This novel DQB1 allele is most similar to DQB1*030302 and DQB1*0311. DQB1*0326 has a nucleotide substitution resulting an amino acid change when compared with DQB1*030302 (M to L) and it differs from DQB1*0311 by one nucleotide variation causing an amino acid replacement (A to D).

Screening of degradation products, impurities and precursors of chemical warfare agents in water and wet or dry organic liquid samples by in-sorbent tube silylation followed by thermal desorption–gas chromatography–mass spectrometry

A standard method used by inspection teams of the Organisation for the Prohibition of Chemical Weapons (OPCW) for preparation of aqueous samples requires several extraction and derivatization steps. This results in tedious and time consuming on-site analysis. A simple thermal desorption–gas chromatography–mass spectrometry (TD–GC–MS) method was developed to analyse for a broad range of degradation products, impurities and precursors of chemical warfare agents (CWA) in water solutions and wet or dry organic liquid samples. The method is fast, sensitive, requires only microliter volumes of sample and enables the simultaneous determination of a wide range of compounds with widely differing polarity, volatility and reactivity. The applicability of the method was demonstrated by successful analysis of five OPCW Official Proficiency Test samples.

Identification ofEscherichia coliO157:H7 in a Proficiency Testing Program: An Update of Laboratory Performance

Objective: By analyzing and comparing results from a 2008 proficiency test (PT) event to results from a 2003 PT event, we assessed whether laboratories’ abilities to detect Escherichia coli ( E. coli ) O157:H7 had improved in the intervening 5 years. Methods: A proficiency test sample containing E. coli O157:H7 was mailed to participants enrolled in a PT program, and a survey about stool culture screening practices was distributed to laboratories that had submitted a result for the proficiency test sample. Results: In 2008, 85.3% of laboratories detected E. coli O157:H7, compared to 70% of laboratories in 2003. Also, 72.3% of laboratories now screen at least all bloody stools, compared to 49% of laboratories in 2003. Conclusions: Laboratories’ abilities to detect E. coli O157:H7 has improved substantially since 2003. However, many laboratories still fail to follow screening recommendations for Shiga toxin-producing E. coli .

Evaluation of in-house and commercial genotyping assays for molecular typing of hepatitis C virus in Hong Kong

This study aims to evaluate genotyping assays for hepatitis C virus (HCV). An in-house nucleic acid sequencing method is performed in parallel with the Roche Linear Array HCV genotyping test on 73 HCV-positive (66 clinical samples and seven proficiency testing quality control samples) and 12 HCV-negative samples (11 clinical samples and one proficiency testing sample). The performance of the in-house method was comparable with that of the Roche assay (concordance rate: 89.4%). Discordant results included four mixed infections missed by the in-house method, two false-negatives with the Roche assay, and three discrepant results. The in-house method exhibited a higher resolution (subtype vs. genotype level) at a lower running cost (25% of the commercial assay). The in-house method was also used to genotype 375 HCV clinical isolates to determine the genotypic distribution of HCV in Hong Kong between 2005 and 2008. A total of 441 (52.8%) clinical isolates proved to be genotype 1, which shows a poorer response to interferon therapy. Genotype 6 was the next most common (32.0%). Prevalence of genotypes 2 and 3 was 7.7% and 6.6%, respectively, and prevalence of genotypes 4 and 5 was 0.9% and 0%, respectively. Although the in-house nucleic acid sequencing method failed to detect a few cases of mixed HCV infection, its high resolution and low running cost make it suitable for surveillance and outbreak investigation.

Human Sperm Bioassay for Reprotoxicity Testing in Embryo Culture Media: Some Practical Considerations in Reducing the Assay Time

Human sperm assay (HSA) is a preferred in house quality control and proficiency test (PT) practiced in fertility laboratories. HSA is performed over varying durations, apparently without following set criteria. To better understand the assay time required for reprotoxicity testing in embryo culture media, we compared American-Association-of-Bioanalysts-(AAB-) administered HSA data to our own assay performed using PT samples obtained from AAB. Participating laboratories were required to culture sperm for 48 hours to determine media acceptability. Conclusions drawn from 48- and 24-hour observations were the same, suggesting that HSA could identify reprotoxic media in less time than required by AAB. Our assay revealed that changes in motility grade in adulterated media are significantly different from those in control media. Furthermore, grade changes can be identified earlier than differences in motility loss between samples. Analyzing motility and motility quality together provides a method for establishing an optimal time for HSA.

Preparation of Samples for Proficiency Testing of Pesticide Residue Analysis in Processed Foods

To conduct proficiency testing for the analysis of pesticide residues in processed foods, fortified samples of retort curry and pancake were examined. In the case of retort curry, heating and mixing were necessary at the time of preparation to provide a homogenous analytical sample. A mixture of 4 carbamates and 11 organophosphorus pesticides was spiked and 14 of them showed consistent results in the samples. In the case of pancake, 10 kinds of pesticides were added to the pastry. The prepared pastry was them cooked. The relative concentrations of most of the pesticides in the pancake were not affected and all the pesticides showed consistent results in the samples. These results showed that the two tested samples were suitable for proficiency testing.

Silver chemical vapor generation for atomic absorption spectrometry: minimization of transport losses, interferences and application to water analysis

A study of a transport process and interferences in the method of Ag chemical vapor generation (CVG) with AAS detection was performed. A modified externally heated quartz multiatomizer with a heated inlet arm was designed to minimize transport losses. Both transport efficiency and spatial distribution of a residual analyte in an apparatus were studied by means of 111Ag radioactive indicator. Compared to our previous study the apparatus design was significantly modified: removal of a spray chamber and heating of an inlet arm to 300 °C allowed us to reduce transport losses from 65% to 4% and thus to improve overall CVG efficiency up to 32.5%. Lower (ambient) or higher (700 °C) temperature was inferior to 300 °C due to either losses of Ag with aerosol droplets by deposition or trapping of decomposed Ag particles on a quartz surface, respectively. The interfering effect of hydride forming elements (As, Se) and some transition metals (Cu, Co, Ni, Au) was examined. No serious negative interference was observed up to 10 μg ml−1 level, with the exception of Au causing 18% signal depression already at 0.5 μg ml−1 level. Transmission electron microscopy and energy dispersive X-ray spectroscopy indicated mixed Ag and Au particles and formation of cross-linked structures was revealed. The upward calibration curvature was observed which was treated using power curve regression. The limit of detection under optimized conditions was 1.0 ng ml−1 and the method was also successfully tested using two proficiency testing samples of drinking water.

Rapid screening and confirmation of drugs and toxic compounds in biological specimens using liquid chromatography/ion trap tandem mass spectrometry and automated library search

Recent advances in liquid chromatography/tandem mass spectrometry (LC/MS/MS) technology have provided an opportunity for the development of more specific approaches to achieve the 'screen' and 'confirmation' goals in a single analytical step. For this purpose, this study adapts the electrospray ionization ion trap LC/MS/MS instrumentation (LC/ESI-MS/MS) for the screening and confirmation of over 800 drugs and toxic compounds in biological specimens. Liquid-liquid and solid-phase extraction protocols were coupled to LC/ESI-MS/MS using a 1.8-microm particle size analytical column operated at 50 degrees C. Gradient elution of the analytes was conducted using a solvent system composed of methanol and water containing 0.1% formic acid. Positive-ion ESI-MS/MS spectra and retention times for each of the 800 drugs and toxic compounds were first established using 1-10 microg/mL standard solutions. This spectra and retention time information was then transferred to the library and searched by the identification algorithm for the confirmation of compounds found in test specimens - based on retention time matches and scores of fit, reverse fit, and purity resulting from the searching process. The established method was found highly effective when applied to the analyses of postmortem specimens (blood, urine, and hair) and external proficiency test samples provided by the College of American Pathology (CAP). The development of this approach has significantly improved the efficiency of our routine laboratory operation that was based on a two-step (immunoassay and GC/MS) approach in the past.

Investigation of the identification point system adaptation in cocaine, benzoylecgonine and ecgonine methyl ester using a single quadrupole mass spectrometer

At present, no official criteria exist for drug identification using single quadrupole mass spectrometers although the European Union (EU) criteria for compound identification have been adopted. These criteria are evaluated with respect to the confirmation of cocaine and its metabolites by single quadrupole liquid chromatography/mass spectrometry (LC/MS) and problems are highlighted. Spiked samples, proficiency testing samples, certified reference materials and samples from real cases that had screened positive for cocaine derivatives by immunoassay were subjected to confirmation by LC/MS using single ion monitoring with in-source fragmentation. The EU criteria for compound identification were applied for the confirmation of cocaine, benzoylecgonine and ecgonine methyl ester. The use of the identification point (IP) system in spiked, proficiency testing samples and certified reference materials provided acceptable results in all cases while in some cases real positive samples did not provide acceptable results. Failure to meet the EU criteria was attributed to low fragmentation at the lower concentrations and the ion suppression effect while both factors affected compliance with the IP system. The identification of cocaine and its metabolites was considerably improved by using a combination of ammonium formate and formic acid as the LC mobile phase. It appears that poor in-source fragmentation in single quadrupole LC/MS and ion suppression may constitute a problem with drug identification when implementing the IP system in real samples, resulting in false negative results. Further investigation is needed for the use of such IP systems to be suitable for use in LC/MS methods.

Development and validation of a cellular biosensor detecting pesticide residues in tomatoes

Two of the most important categories of pesticides used in agricultural practice are organophosphates and dithiocarbamates. Their extensive and inappropriate use has rendered their reliable monitoring at trace levels more and more necessary. This study presents the construction of a rapid and sensitive cellular biosensor test based on the measurement of changes of the cell membrane potential of immobilized cells, according to the working principle of the Bioelectric Recognition Assay (BERA). The cells were immobilized by entrapment in a sodium alginate bead and directly applied in different pesticide dilutions and agricultural samples. The pesticides used were the organophosphate insecticide diazinon and the dithiocarbamate fungicide propineb. Two different cell types, N2a (neuroblastoma) and Vero (fibroblast) were used as the biosensory elements in order to investigate their differential response against the pesticides. In this way, we hoped to increase the selectivity of the assay. Based on the observed patterns of response, we demonstrate that the sensor can be used for the qualitative and, in some concentrations, quantitative detection of the pesticides with a high degree of reproducibility. The lowest detected concentration was 3nM. Finally, for the investigation of the effects of different pesticides on the accumulation of cytosolic Ca(2+), we conducted a fluorescent assay on N2a cells treated with tomato sample extracts, which were replicates of the E.U. proficiency test sample. The tomato samples were either organically grown or contained 14 different pesticides. The experimental results showed a higher increase of the intracellular Ca(2+) concentration in cells treated with non-organic samples compared to the cells treated with organic samples.

Validation of specificity in doping control: problems and prospects

Sir, The World Anti-Doping Agency (WADA) has been created in 1999 to improve and harmonize the global fight against doping [1]. To set the standards for laboratory anti-doping procedures around the world, WADA has developed the International Standard for Laboratories (ISL) [2]. According to the ISL, confirmation methods for non-threshold substances, which comprise the great majority of prohibited substances, must be validated. Specificity is one of the key analytical figures of merit for determining if such a method is fit for intended use. Its proper validation requires (Section 5.4.4.2.1 in [2]) that ‘‘The ability of the assay to detect only the substance of interest shall be determined and documented.’’ Experienced analysts will recognize that the word ‘only’ is of paramount importance here. The purpose of this Letter is to draw attention to the fact that, surprisingly, these methods often only comply with the requirement from which the word ‘only’ is removed. A case in point is the recently improved test for metandienone [3]. The authors seem to assume that specificity comes for free with the mass spectrometric identification and characterization of the substance of interest. However, logic dictates that specificity can only be ensured by also considering compounds with closely resembling mass spectra. This has been stressed by de Zeeuw [4] in an article aptly entitled ‘‘Substance identification: the weak link in analytical toxicology’’. Specifically, de Zeeuw reports 19 substances whose mass spectra closely resemble the spectrum of methamphetamine. One might argue that these substances will not be mistaken for methamphetamine, since a chromatographic separation step precedes mass spectrometric detection. However, from a scientific point of view, one cannot accept a rather vague assumption to stand proxy for a rigorous validation. Also from a practical point of view, a method being fit for purpose cannot be taken for granted. It follows that studies like the one reported in [3] can only lead to unsubstantiated claims with respect to specificity. This is a serious problem in practice because any lack of specificity remains unnoticed, unless specifically addressed. The proficiency testing (PT) program detailed in [1], for example, will not function as a safeguard since, quite naively, PT samples are not (systematically) designed to contain potential interferents! The correct state-of-the-art scientific approach is to search in an extensive library for candidate interferents, and subsequently scrutinize these candidates for co-elution. This search solves certain fundamental problems and also offers valuable benefits. For example, current selection of mass spectrometric signals for detection is commonly based on subjective ‘visual inspection’, see [3] for an illustration. This ad hoc approach could be replaced by a suitable objective (computerized) optimization strategy, which is the standard in fields like statistics and chemometrics. Method improvement and optimization easily come within reach.

A novel LC–ESI-MS method for the simultaneous determination of etravirine, darunavir and ritonavir in human blood plasma

The new potent combination of antiretrovirals etravirine, darunavir, and ritonavir requires a new bioanalytical method for clinical pharmacology investigations and potential therapeutic drug monitoring. The development and validation of a novel LC–MS method for the simultaneous quantification of the most recently FDA-approved protease inhibitor and non-nucleoside reverse transcriptase inhibitor is described. This novel method was developed and validated using a sub-2 μm particle column, and provides excellent chromatographic separation and peak shape for all three analytes and internal standard. The method was validated over the range of 0.002–2.0 μg/mL. Intra- and inter-day accuracy of all analytes ranged from 88 to 106%, and intra- and inter-day precision was <7%. Dilution of samples 2-, 5-, and 10-fold maintained accuracy and precision, using a sample volume as low as 10 μL. Finally, the applicability of the method was investigated with clinical samples and external quality assurance proficiency testing samples.

Laboratory Sanctions for Proficiency Testing Sample Referral and Result Communication: A Review of Actions From 1993–2006

The Clinical Laboratory Improvement Amendments of 1988 (CLIA) regulations for proficiency testing (PT) include prohibitions against intentional PT sample referral or result communication, and specify sanctions against laboratories that violate these regulations. There has been little published analysis of sanctions against clinical laboratories because of PT violations. To examine the application of principal sanctions as reported by the Centers for Medicare and Medicaid Services annually in the Laboratory Registry and to examine relevant aspects of judicial hearings and appeals in these cases. The Laboratory Registry was examined for all available years (1993-2006) to determine the incidence of application of principal sanctions for PT violations. In addition, the decisions from the US Department of Health and Human Services hearings and appeals were reviewed to better understand the judicial disposition of these cases. During the 14-year period examined, 78 laboratories received a principal sanction for a PT violation involving sample referral or result communication. During the same period, the number of laboratories in nonexempt states that would be expected to have participated in PT averaged 45 983. The interpretive meaning of the key terms intentional and referral, and the implications for sanctioned laboratories and their owners and operators are discussed. Applications of a principal sanction for a PT violation were rare during the period of this study. However, the consequences of the imposition of such a sanction are severe. Suggestions are offered on policies and practices to minimize the risk of a PT sample referral or result communication.

Laboratory sanctions for proficiency testing sample referral and result communication: A review of actions from 1993-2006

Abstract Context.—The Clinical Laboratory Improvement Amendments of 1988 (CLIA) regulations for proficiency testing (PT) include prohibitions against intentional PT sample referral or result communication, and specify sanctions against laboratories that violate these regulations. There has been little published analysis of sanctions against clinical laboratories because of PT violations. Objective.—To examine the application of principal sanctions as reported by the Centers for Medicare and Medicaid Services annually in the Laboratory Registry and to examine relevant aspects of judicial hearings and appeals in these cases. Design.—The Laboratory Registry was examined for all available years (1993–2006) to determine the incidence of application of principal sanctions for PT violations. In addition, the decisions from the US Department of Health and Human Services hearings and appeals were reviewed to better understand the judicial disposition of these cases. Results.—During the 14-year period examined, 78 ...

Therapeutic drug monitoring and drugs of abuse testing on the cobas® 6000 analyzer series: analytical performance under routine-like conditions

The analytical performance of the clinical chemistry module c 501 (cobas 6000 analyzer series) was evaluated for therapeutic drug monitoring and drugs of abuse testing using a spectrum of representative assays. Particular attention was paid to potential interactions between reagents using a simulated routine workload. Within-run and total imprecision were assessed using a selection of representative reagents. Deviation from a consensus mean was tested using samples from a proficiency testing scheme. Method comparison using routine samples was carried out against the MODULAR ANALYTICS SWA and COBAS INTEGRA 800 analysis systems. Total coefficients of variation (CV) ranged from 1.9% to 7.8% for individual drugs, and from 3.2% to 8.6% for drugs of abuse testing. Results from proficiency test samples were between 81% and 125% of the consensus mean for therapeutic drugs. Method comparisons (Passing-Bablok regression) showed overall good comparability to MODULAR ANALYTICS SWA and COBAS INTEGRA 800 systems, with slopes from 0.93 to 1.17 and correlation coefficients r > 0.98. Imprecision in a simulated routine run was tested using a total of 42 methods (10 therapeutic drug monitoring, 9 drugs of abuse testing, 3 enzymes, 12 substrates, 8 specific protein assays). Imprecision in the reference batch run ranged from 0.7% to 5.0% CV for therapeutic drug monitoring assays, except for digoxin (DIG) (7.3%), and from 0.9% to 7.7% for drugs of abuse testing. The CVs of general clinical chemistry and specific protein tests were within the expected limits of 2% and 4%. CV changes in the simulated routine run were within the expected limits for most assays. Negative DeltaCVs (> or = 2%) for DIG, digitoxin (DIGIT), cannabinoids (THC), and phencyclidine (PCP) may indicate improved performance when running these assays in a simulated routine operation. A positive DeltaCV (> or = 3%) was found for amphetamines (AMPHs). In conclusion, the cobas c 501 module seems to be well-suited for routine use as consolidated workstation. Except for a potential interaction with AMPH, as indicated by the positive DeltaCV, no significant interferences from different reagents could be observed during this study.

Role of subtyping in detecting Salmonella cross contamination in the laboratory

BackgroundWith the exception of M. tuberculosis, little has been published on the problems of cross-contamination in bacteriology laboratories. We performed a retrospective analysis of subtyping data from the National Salmonella Reference Laboratory (Ireland) from 2000–2007 to identify likely incidents of laboratory cross contamination.MethodsSerotyping and antimicrobial susceptibility testing was performed on all Salmonella isolates received in the NSRL. Phage typing was performed on all S. Typhimurium and S. Enteritidis isolates while multi-locus variance analysis (MLVA) was performed on selected S. Typhimurium isolates. Pulsed field gel electrophoresis (PFGE) using the PulseNet standard protocol was performed on selected isolates of various serovars.ResultsTwenty-three incidents involving fifty-six isolates were identified as likely to represent cross contamination. The probable sources of contamination identified were the laboratory positive control isolate (n = 13), other test isolates (n = 9) or proficiency test samples (n = 1).ConclusionThe scale of laboratory cross-contamination in bacteriology is most likely under recognized. Testing laboratories should be aware of the potential for cross-contamination, regularly review protocols to minimize its occurrence and consider it as a possibility when unexpected results are obtained.

EVALUASI HASIL UJI PROFISIENSI UNTUK KOMODITI AIR MINUM DALAM KEMASAN

KAN proficiency testing program on the bottled drinking water conducted on 2004 was participated by 46 testing laboratories. Participant laboratories were asked to test the sample for nitrat, nitrit, ammonium, sulphate Cl, Cn, F, F, Fe, Mn, Pb, Cu, Cd, As and Hg contents. Proficiency testing sample were prepared by provider, which also conduct homogeinity test for the Hg and Cu contents of the sample. As the sample was judged to be homogen, they sent to participant laboratory in 2 packs identified as sample KAN A and KAN B. Laboratory testing results were analyzed by statistical method, started on homogeinity of data, Dixon test and Z-score. The evaluation results shows that only sulphate, Cl, F, Fe and Cu contents could be analyzed among the other 14 parameters, which is could not be evaluated because of the in homogeinity of data provided by the laboratories. The inhomogeinity of data was caused by insufficiency of laboratory’s personnel and equipments. The evaluation of proficiency testing results for sulphate, Cl, F, Fe and Cu contents show only 1 satisfactory result among 5 accredited participants, and 9 satisfactory results among 39 non-accredited participants. The conclusion is based on the Z-score analysis of the evaluated parameters.

Comparison of Ion-Selective Electrode and Inductively Coupled Plasma-Mass Spectrometry to Determine Iodine in Milk-Based Nutritional Products

The performances of 2 official methods for iodine analysis based on inductively coupled plasma-mass spectrometry (ICP-MS) and the ion-selective electrode (ISE) method were compared for milk-based products. The aim of the study was to determine the performance characteristics of both methods to check the labeled concentration of iodine. Good precision was found for both methods with highest relative standard deviation of repeatability (RSD(r)) at 2.3 and 2.7% for ISE and ICP-MS, respectively. Intermediate reproducibility (RSD(iR)), single laboratory within 6 different days, was also good with the highest values at 7.3 and 8% by ISE and ICP-MS, respectively. Measurement uncertainty was estimated based on the RSD(iR) data, and it was concluded that both methods were capable of determining iodine concentrations within an uncertainty below +/- 20%. The accuracy of the methods was determined by analyzing certified reference materials, in-house proficiency test samples, and commercial products. Both methods returned similar results when applied on freshly opened samples. In samples that had been opened and kept exposed to air during storage, ISE returned lower iodine concentrations than ICP-MS. In commercial samples, the linear regression between both methods was ISE = 0.95 x ICP-MS -0.060 for freshly opened samples and ISE = 0.85 x ICP-MS + 0.069 for samples exposed to air. The tendency of ISE to return lower results than ICP-MS is explained by the fact that ISE is sensitive to iodide but does not measure iodine that may be bound organically to the matrix. This seems to be more pronounced in samples that were stored longer. Because in most countries iodine is labeled as total iodine, acceptance of an international standard based on the ICP-MS technique which takes all forms of iodine into account, is recommended. This would help to avoid any potential dispute on the accuracy of labeled iodine concentrations in finished products.