External Proficiency Testing Scheme Research Articles

Predicting flow cytometry crossmatch results from single-antigen bead testing.

The aim of this study was to devise an algorithm that would predict flow cytometry crossmatch (FCXM) results using single-antigen bead (SAB) mean fluorescent intensity (MFI) levels using samples received through the National External Quality Assurance Scheme (NEQAS) 2B external proficiency testing scheme between 2019 and 2023. A total of 159 serum samples were retrospectively screened using LABScreen Single Antigen Class I and II (SAB), and 40 peripheral blood samples were human leucocyte antigen (HLA) typed with LABType SSO. Donor-specific antibodies were identified for each cell-serum combination tested, and cumulative MFI values were calculated for each test before correlating the screening result with the consensus crossmatch results for this scheme. HLA Class I MFIs were combined to predict the T cell crossmatch. For the B cell crossmatch prediction, two options were considered: (i) HLA Class II MFI values alone and (ii) HLA Class I+Class II MFIs. Receiver operating characteristic analysis was carried out to identify the combined MFI threshold that predicted NEQAS consensus results with the greatest sensitivity and specificity. HLA Class I combined MFI >5000 predicted T cell crossmatch results with 96% sensitivity, 100% specificity, 100% positive predictive value (PPV) and 92% negative predictive value (NPV). For B cell results, HLA Class I+Class II combined MFIs >11,000 gave the best model, showing 97% sensitivity, 82% specificity, 96% PPV and 85% NPV. However, for samples with only HLA Class II sensitization, combined MFIs >13,000 improved the B cell crossmatch predictions: 92% sensitivity, 95% specificity, 96% PPV and 91% NPV. Using this model, combined MFI can be used to predict the immunological risk posed by donor-specific antibodies when it is not possible to carry out an FCXM.

The history and evolution of HLA typing external proficiency testing schemes in UK NEQAS for H&I.

The UK National External Quality Assessment Service (NEQAS) provide an external proficiency testing (EPT) service for clinical laboratories. UK NEQAS for Histocompatibility and Immunogenetics (H&I) has been providing EPT schemes for over 45years and has grown during this time to provide 19 EPT schemes. Accurate human leucocyte antigen (HLA) typing is critical to support safe clinical services, including transplantation, therefore high quality, relevant EPT schemes are required as part of a laboratory's quality assurance. This article reviews the development of the HLA typing EPT schemes, from the first HLA phenotyping scheme in 1975, via the first HLA genotyping scheme in 1992, through to the introduction in 2017 of HLA third field assessment results from next-generation sequencing technology. In addition, the introduction of EPT schemes to cover HLA associated diseases and pharmacogenetic reactions, including HLA-B27, HLA*B*57:01 and HLA-DQ for coeliac disease are discussed. The accuracy of laboratory EPT results for HLA phenotyping are >96% (2018-2022), HLA genotyping >99% (2020-2022), HLA-B27 testing >99% (2018-2022) and B*57:01 testing >99% (2017-2022). However, for HLA genotyping for coeliac disease 22%-46% of laboratories made errors in 2020-2022. On investigation, the high rate of unsatisfactory performance was attributed to laboratories lacking specific knowledge to interpret HLA genotyping results and accurately report HLA types for coeliac disease. A misleading commercial kit insert was also identified. The assessment of scheme results has uncovered several issues which have been addressed with the intention of educating participants and improving clinical services. The UK NEQAS for H&I EPT schemes have evolved over the past four decades to reflect changes in HLA typing technology, laboratory clinical practice and to cover post-analytical interpretative elements of HLA typing.

Concordance with established tests and reproducibility of results obtained with a new single antigen chip array for HLA antibody detection (HISTO SPOT® HLA AB)

The purpose of this study was to analyze performance of a new single antigen chip array system (HISTO SPOT® HLA AB) developed for HLA antibody detection and compare with results obtained using single antigen Luminex-based systems and serum samples from the Eurotransplant external proficiency testing scheme. Results were analysed from 11 independent Eurotransplant laboratories using HISTO SPOT® HLA AB utilising the Eurotransplant external proficiency testing (EPT) sera and these were compared to published results from 67 labs using the Luminex-based technologies. In addition, QC results from different batches of the test were analysed. Generally, concordance of results with the results from the Luminex technique was good. With the Luminex tests more consensus results and more questionable results were found than with the HISTO SPOT® HLA AB test. Within the HISTO SPOT® HLA AB testing group we found a discrepancy rate from the consensus of 2.9% for the EPT sera which is far below the 25% allowed to pass the quality test and only slightly higher than for the Luminex single antigen tests with 1.2%. The average global coefficient of variation (CV) of the mean signal (raw data) for the HISTO SPOT® HLA AB test was 13% which is lower than the values reported for Luminex tests in the literature. The average global CV for the signal/background ratio was higher with 28%. In the present study, the mean signal is the best parameter to compare results between labs and the new HISTO SPOT® HLA AB test is at least as good in terms of signal reproducibility as the Luminex tests. In conclusion, the HISTO SPOT® HLA AB test is a good alternative to be used in addition or instead of the Luminex tests in clinical labs.

Validation of a hydride generation atomic absorption spectrometry methodology for determination of mercury in fish designed for application in the Brazilian national residue control plan

In the present study, a method for the determination of mercury (Hg) in fish was validated according to ISO/IEC 17025, INMETRO (Brazil), and more recent European recommendations (Commission Decision 2007/333/EC and 2002/657/EC) for implementation in the Brazilian Residue Control Plan (NRCP) in routine applications. The parameters evaluated in the validation were investigated in detail. The results obtained for limit of detection and quantification were respectively, 2.36 and 7.88 μg kg−1 of Hg. While the recovery varies between 90–96%. The coefficient of variation was of 4.06–8.94% for the repeatability. Furthermore, a comparison using an external proficiency testing scheme was realized. The results of method validated for the determination of the mercury in fish by Hydride generation atomic absorption spectrometry were considered suitable for implementation in routine analysis.

Validation of a method for the determination of tributyltin in seawater by stir bar sorptive extraction–liquid chromatography tandem mass spectrometry

The main objective of this work is to develop an accurate, sensitive, simple and cost-effective method for the determination of tributyltin species (as cationic form) in seawater at ultra-trace levels. The method is based on the use of stir bar sorptive extraction (SBSE) followed by solvent desorption and liquid chromatography tandem mass spectrometry (SD–LC–MS/MS (QqQ)) analysis, operating in positive electrospray ionisation (ESI) and in the selected reaction monitoring (SRM) mode. Extraction and LC–MS/MS parameters were accurately optimised to achieve the highest recoveries and to enhance the analytical characteristics of the method. The different parameters that affect the extraction procedure, the chromatographic and spectrometric conditions, and the surrogate selection were evaluated. In contrast with previously proposed methods, the proposed method involves a simplified sample treatment. Quality parameters such as linearity, accuracy in terms of trueness and precision, uncertainty and specificity were examined with good results. The evaluation of two surrogates (tri-n-propyltin chloride and tripentyltin chloride) was also carried out. The limit of detection (LOD) and limit of quantification (LOQ) obtained were 0.8 and 2.5ngL−1, respectively. Precision, expressed as relative standard deviation (%RSD), was lower than 16%, and the determination coefficient (R2) was higher than 0.990 with a residual deviation for each calibration point lower than ±25%. Mean recoveries were between 92% and 102%. The accuracy of the method was also studied by participating in an external proficiency testing scheme. After validation, the method was applied to the analysis of all tributyltin species found in natural seawater samples. The method meets the requirements of the United States Environmental Protection Agency (US-EPA).

Human Leukocyte Antigen Proficiency Testing for Central and Eastern Europe: A Summary of Over 10 Years of Activity

The first trial of human leukocyte antigen (HLA) Proficiency Testing for Central and Eastern Europe (formerly Proficiency Testing of HLA class I Typing for Central and Eastern Europe) was organized in 1999 with 5 Polish institutions that were involved in HLA typing. Since then, 46 HLA laboratories in 13 countries have participated in our external proficiency testing schemes. The workshop conducted by our institution meets the expectations of the European Federation for Immunogenetics to provide data on the reliability of histocompatibility testing. Our standardization workshop currently covers serological typing of HLA class I A and B loci and DNA typing at the low- and high-resolution level of HLA class I (A, B, C) and class II (DRB1, DQB1) loci. It has >30 HLA laboratories participating each year. Thirty-two laboratories in 9 countries participated in the last session, the 15th workshop in 2009. Twenty-three laboratories submitted results of HLA class I typing by serology; 28 participated in HLA class I DNA typing and 28 participated in HLA class II DNA typing, including 5 and 7 laboratories that sent results of high-resolution typing for class I and class II specificities, respectively. Continuous improvement is seen with respect to both serology and DNA typing, although some doubtful results are still reported.

Documenting assay quality in TDM

Key goals in providing therapeutic drug monitoring (TDM) services are consistency and accuracy of results. These concepts are often misunderstood, with a disproportionate emphasis placed on assay reproducibility, rather than calibration issues. Whilst TDM has been delivered using immunoassay kits, there has been some logic in developing assay-related target ranges locked into the calibrators supplied by diagnostic companies. However, with a growing number of laboratories turning to the use of high-performance liquid chromatography with mass-spectrometric detection (HPLC/MS) for TDM services, preparation of accurate calibrators assumes more importance, as the responsibility for calibrator accuracy usually rests with individual centres. The availability of HPLC/MS has also highlighted the differences between chromatographic and immunoassays for a number of analytes, including naturally occurring compounds. These quality issues, calibration accuracy and reproducibility of the methodology, will be discussed in the context of assay validation and documentation of performance to satisfy regulatory standards. Illustrations will draw on data from external proficiency testing schemes for a number of drug classes, and the development of assays for infrequently measured analytes. It will be concluded that there are increasing demands on laboratories to show that their measurements of drugs and endogenous compounds are consistent between laboratories, and that they provide clinicians with reliable information on which to base dose changes or diagnoses. It is the responsibility of laboratories to show that they have addressed these issues by documenting the processes involved.

Optimisation and critical evaluation of a collision cell technology ICP-MS system for the determination of arsenic in foodstuffs of animal origin

The determination of arsenic ( 75As) was studied using an ICP-MS equipped with collision cell technology (CCT). Different mixtures of gases (He and H 2) were tested using HCl conditions and a He flow rate of 4 mL min −1 was found to be suitable for the removal of the poly-atomic spectral interference [ 40Ar 35Cl] +. Trueness of the optimised method has been evaluated in both standard and CCT modes on six certified reference materials in foodstuffs of animal origin and on three external proficiency testing schemes (FAPAS). The results obtained generally coincided with the certified values, except for CCT mode in some categories of samples (meat, mussels and milk powder), for which a positive bias on results was observed due to the formation of poly-atomic interferences within the collision cell. The main interferences were studied and their contributions estimated. [ 58Fe 16O 1H] + and [ 74Ge 1H] + were the most significant interferences formed in the cell. Finally, different parameters (e.g. hexapole and quadrupole bias voltage, nebuliser gas flow) were optimised to try to attenuate these interferences.

A HPLC–mass spectrometric method suitable for the therapeutic drug monitoring of everolimus

We report here the validation of an HPLC–electrospray–tandem mass spectrometry method for the quantification of everolimus, an immunosuppressant drug. Whole blood samples (100 μl) were extracted by protein precipitation which involved sample pre-treatment with zinc sulphate followed by acetonitrile (containing internal standard, 40- O-(3′-hydroxy)propyl-rapamycin). HPLC was performed using a step-gradient at a flow rate of 0.6 ml/min on a Waters TDM C 18 column (10 mm × 2.1 mm I.D.) with a resultant chromatographic analysis time of 2 min. Mass spectrometric detection by selected reaction monitoring (everolimus m/ z 975.5 → 908.3; internal standard m/ z 989.5 → 922.3). The assay was linear from 0.5 to 40 μg/l ( r 2 > 0.994, n = 11). The inter- and intra-day analytical recovery and imprecision for quality control samples (1.25, 12.5 and 30 μg/l) were 93.4–98.2% and <10.7%, respectively ( n = 10). At the lower limit of quantification (0.5 μg/l) the inter- and intra-day analytical recovery was 94.4–95.8% with imprecision of <14.1% ( n = 10). The absolute recovery of everolimus (6.5 μg/l) and internal standard (12.5 μg/l) was 96.5 and 88.3%, respectively ( n = 3). A comparison of our method against the mean of all HPLC methods for a series of samples from an external proficiency testing scheme revealed good correlation as shown by the regression analysis: y = 0.973 x + 0.301 ( r 2 = 0.986, n = 71). In conclusion, the method described is suited to the current requirements for therapeutic drug monitoring of everolimus.

Determination of chromium, iron and selenium in foodstuffs of animal origin by collision cell technology, inductively coupled plasma mass spectrometry (ICP-MS), after closed vessel microwave digestion

The determination of chromium ( 52Cr), iron ( 56Fe) and selenium ( 80Se) isotopes in foodstuffs of animal origin has been performed by collision cell technology (CCT) mode using an inductively coupled plasma mass spectrometry (ICP-MS) as detector after closed vessel microwave digestion. To significantly decrease the argon-based interferences at mass to charge ratios ( m/ z): 52 ( 40Ar 12C), 56 ( 40Ar 16O) and 80 ( 40Ar 40Ar), the gas-flow rates of a helium and hydrogen mixture used in the hexapole collision cell were optimised to 1.5 ml min −1 H 2 and 0.5 ml min −1 He and the quadrupole bias was adjusted daily between −2 and −15 mV. Limits of quantification (LOQ) of 0.025, 0.086 and 0.041 mg kg −1 for Cr, Fe and Se, respectively, in 6% HNO 3 were estimated under optimized CCT conditions. These LOQ were improved by a factor of approximately 10 for each element compared to standard mode. Precision under repeatability, intermediate precision reproducibility and trueness have been tested on nine different certified reference materials in foodstuffs of animal origin and on an external proficiency testing scheme. The results obtained for chromium, iron and selenium were in all cases in good agreement with the certified values and trueness was improved, compared to those obtained in standard mode.

Simultaneous Analysis of Cadmium, Lead, Mercury, and Arsenic Content in Foodstuffs of Animal Origin by Inductively Coupled Plasma/Mass Spectrometry after Closed Vessel Microwave Digestion: Method Validation

A method validation of the total analysis of cadmium (Cd), lead (Pb), arsenic (As), and mercury (Hg) in foodstuffs by inductively coupled plasma/mass spectrometry (ICP/MS) after closed vessel microwave digestion is presented. Due to the lack of reference method for ICP/MS techniques in food and, based on the project of the European Committee of Normalization (CEN/TC 275/WG 10), the Agence Française de Normalisation (AFNOR) guidelines NF V03-110 were used for the evaluation of this method based on 2 steps, sample preparation and multielement detection. Several criteria considered as compulsory (linearity, specificity, precision under repeatability conditions, and trueness) have been estimated and discussed, in addition to intermediate precision reproducibility, the limit of detection, and the limit of quantification. Furthermore, a comparison with in-house methods using electrothermal atomic absorption was performed using an external proficiency testing scheme and food samples. The results indicated that this method could be used in the laboratory for the routine determination of these 4 cumulative toxic metals in foodstuffs with acceptable analytical performance.

HLA‐B*8102*, a new allele found in an external proficiency testing scheme

A new human leukocyte allele HLA-B*8102 has been identified in a cell provided by the UCLA International Cell Exchange scheme. The nucleotide sequences of exons 2 and 3 of the new allele are identical with HLA-B*8101. Four nucleotide differences, however, are observed in exon 1, three of them representing non-synonymous base changes.