National External Quality Assessment Service Research Articles

Evaluation of cryoprotein investigation using a digital external quality assurance scheme.

Background: Robust preanalytical and analytical processes are critical for the detection of cryoproteins. There is significant variation in practice in the detection, analysis and reporting. Results: A survey in 2018 of 137 laboratories participating in the UK National External Quality Assessment Service (UK NEQAS) (6) quality control program showed significant variation in the laboratory processes which highlighted the need for standardisation of the detection, analysis and reporting of cryoglobulins.Conclusion: The first available EQA scheme aiming to harmonise practice for cryoprotein testing has been developed by UK NEQAS and laboratories should participate in an appropriate EQA scheme to fulfil requirements for ISO accreditation.

Investigating the Current Harmonization Status of Tumor Markers Using Global External Quality Assessment Programs: A Feasibility Study.

The harmonization status of most tumor markers (TMs) is unknown. We report a feasibility study performed to determine whether external quality assessment (EQA) programs can be used to obtain insights into the current harmonization status of the tumor markers α-fetoprotein (AFP), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), cancer antigen (CA)125, CA15-3 and CA19-9. EQA sample results provided by 6 EQA providers (INSTAND [Germany], Korean Association of External Quality Assessment Service [KEQAS, South Korea], National Center for Clinical Laboratories [NCCL, China], United Kingdom National External Quality Assessment Service [UK NEQAS, United Kingdom], Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek [SKML, the Netherlands], and the Royal College of Pathologists of Australasia Quality Assurance Programs [RCPAQAP, Australia]) between 2020 and 2021 were used. The consensus means, calculated from the measurement procedures present in all EQA programs (Abbott Alinity, Beckman Coulter DxI, Roche Cobas, and Siemens Atellica), was used as reference values. Per measurement procedure, the relative difference between consensus mean for each EQA sample and the mean of all patient-pool-based EQA samples were calculated and compared to minimum, desirable, and optimal allowable bias criteria based on biological variation. Between 19040 (CA15-3) and 25398 (PSA) individual results and 56 (PSA) to 76 (AFP) unique EQA samples were included in the final analysis. The mean differences with the consensus mean of patient-pool-based EQA samples for all measurement procedures were within the optimum bias criterion for AFP, the desirable bias for PSA, and the minimum bias criterion for CEA. However, CEA results <8 µg/L exceeded the minimum bias criterion. For CA125, CA15-3, and CA19-9, the harmonization status was outside the minimum bias criterion, with systematic differences identified. This study provides relevant information about the current harmonization status of 6 tumor markers. A pilot harmonization investigation for CEA, CA125, CA15-3, and CA19-9 would be desirable.

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.

DS23 Efficiency and performance of slide stainers during Mohs frozen section processing

Abstract Mohs micrographic surgery (MMS) relies on good-quality, well-stained frozen sections for effective histological analysis. Slides should also be produced as quickly as possible. Automation of complementary devices in the laboratory ensures standardization and efficiency and improves the overall quality of performance. Our study evaluated the efficiency and performance of the Autostainer-XL (Leica) vs. the Linistat (Thermo Scientific) slide stainer within a busy MMS unit. The Autostainer-XL rapidly dries, loads and unloads slides automatically allowing the Mohs-trained biomedical scientist (BMS) to work in parallel on cryotomy. The Autostainer-XL stains slides with haematoxylin and eosin within 10 min and 50 s. In comparison, the Linistat was much slower and required the Mohs-trained BMS to fix the slides manually on a hotplate, place them in industrial methylated spirit, wash them in water and load individual slides into the Linistat, taking 16 min and 45 s to complete the staining process. Two blinded assessors retrospectively reviewed the same set of 50 Linistat-stained and 50 Autostainer-XL stained frozen section slides for consistency in staining. The scoring criteria were based on the modified UK National External Quality Assessment Service (NEQAS) Cellular Pathology Technique Mohs scheme assessment criteria. Each assessor allocated scores between 1 and 5 based on the scoring criteria. The mean quality scores were significantly higher for the Autostainer-XL compared with the Linistat (P &amp;lt; 0.05). In our MMS unit, up to 12 patients undergo MMS daily with an average of seven slides required per patient at the first stage, leading to a high processing burden within the Mohs laboratory, particularly at the first stage. The Autostainer-XL increased productivity, with the ability to load 30 slides per rack, while the Linistat was only able to load six slides per rack. In practice, within our MMS unit, the Autostainer-XL reduced the time taken to produce all slides at the first layer by more than 1 h when compared with the Linistat slide stainer. When surveyed, 100% (n = 8/8) of Mohs-trained BMS in the laboratory preferred the Autostainer-XL than the Linistat, with comments suggesting that it was easy to use, quick, precise and an important device that can withstand the demands of a busy Mohs laboratory. The Autostainer-XL offers improved efficiency and performance, with reduced staining times, an ability to process a larger number of slides and fully customisable automation when compared with the Linistat (Thermo Scientific) slide stainer.

Comparison of the Accuracy of NOVEOS vs ImmunoCAP in Two UK NEQAS Samples for Specific IgE: Impact of Cross-reactive Carbohydrate Determinants

Up to 30% of the general population develops IgE against cross-reactive carbohydrate determinants (CCDs) found in insect venoms and plants and as a result are at risk of receiving false positive test results from specific IgE (sIgE) immunoassays. ImmunoCAP utilizes a cellulose-based matrix that is known to contain CCD N-glycans that bind to anti-CCD IgE antibodies. This abstract reports on the analysis and resolution of discordant results between NOVEOS and ImmunoCAP in two recent United Kingdom based National External Quality Assessment Service (UK NEQAS) samples.

Results from the second WHO external quality assessment for the molecular detection of respiratory syncytial virus, 2019-2020.

External quality assessments (EQAs) for the molecular detection of human respiratory syncytial virus (RSV) are necessary to ensure the standardisation of reliable results. The Phase II, 2019-2020 World Health Organization (WHO) RSV EQA included 28 laboratories in 26 countries. The EQA panel evaluated performance in the molecular detection and subtyping of RSV-A and RSV-B. This manuscript describes the preparation, distribution, and analysis of the 2019-2020 WHO RSV EQA. Panel isolates underwent whole genome sequencing and in silico primer matching. The final panel included nine contemporary, one historical virus and two negative controls. The EQA panel was manufactured and distributed by the UK National External Quality Assessment Service (UK NEQAS). National laboratories used WHO reference assays developed by the United States Centers for Disease Control and Prevention, an RSV subtyping assay developed by the Victorian Infectious Diseases Reference Laboratory (Australia), or other in-house or commercial assays already in use at their laboratories. An in silico analysis of isolates showed a good match to assay primer/probes. The panel was distributed to 28 laboratories. Isolates were correctly identified in 98% of samples for detection and 99.6% for subtyping. The WHO RSV EQA 2019-2020 showed that laboratories performed at high standards. Updating the composition of RSV molecular EQAs with contemporary strains to ensure representation of circulating strains, and ensuring primer matching with EQA panel viruses, is advantageous in assessing diagnostic competencies of laboratories. Ongoing EQAs are recommended because of continued evolution of mismatches between current circulating strains and existing primer sets.

PB1854: NEXT GENERATION SEQUENCING APPROACHES IN CHRONIC LYMPHOCYTIC LEUKAEMIA: FINDINGS FROM EXTERNAL QUALITY ASSESSMENT

Background: Chronic lymphocytic leukaemia (CLL) represents a clinically heterogeneous, lymphoproliferative neoplasm resulting in chronic B-lymphocyte proliferation. Historically, large cytogenetic aberrations detected by fluorescent in situ hybridisation were identifiable in patients, enabling prognostic assessment. With the development of next generation sequencing (NGS), variants have been identified in several genes that are recurrently mutated in CLL, including TP53, ATM, BIRC3, NOTCH1 and SF3B1, with the prognostic implications of variants in these genes being reported. However, only TP53 variant status has been incorporated into routine clinical diagnostics to guide prognosis, risk stratification and appropriate therapeutic interventions based on findings indicating decreased survival and resistance to immunochemotherapy. Aims: The aims of this study were to assess NGS gene panel content and design approaches in CLL to determine levels of harmonisation amongst laboratories. Methods: The United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping has provided external quality assessment for use of NGS technologies in CLL since 2018. The programme routinely requests NGS panel information (gene content and exon coverage) and other methodological data from participating laboratories. Data returns from 2018-2021 relating to NGS gene content and panel analysis were evaluated. Results: In total, 127 genes were analysed across 34 laboratories, ranging from 1-82 genes analysed in the context of CLL per laboratory (median of 12 genes). Of the 34 laboratories evaluated, five analysed TP53 only, with four of these utilising a non-disease specific myeloid NGS panel. The most commonly analysed genes were TP53 (100% laboratories), SF3B1 (73.5%) and NOTCH1 (70.6%). For these genes, there was a high-level of standardisation in sequencing approaches; with 91.2% of laboratories sequencing coding exons 2-11 in TP53 and 100% laboratories sequencing ‘hotspot’ exon 34 of NOTCH1, accounting for >80% variants in NOTCH1 associated with CLL. For SF3B1, 87% laboratories sequenced exons 14-16, which are hotspots for variants. Other frequently analysed genes included MYD88 (61.8% laboratories), BTK (58.8%), ATM and PLCG2 (52.9%). Summary/Conclusion: The number of genes analysed in the context of CLL amongst laboratories was highly variable. Use of NGS genetic testing approaches in haemato-oncology aids in accurate diagnosis and prognosis, however, the cost of developing these panels is not insubstantial and as such, laboratories often design panels to encompass multiple lymphoid/haematological neoplasms. Whilst recommendations outline the need for TP53 testing in CLL, the lack of technical and clinical guidelines on minimal NGS panel content and bioinformatic analysis that provide additional prognostic information has resulted in a wide variation in bioinformatic analysis approaches. This inevitably results in wide disparity of data generated and subsequent clinical interpretation. Guidelines are urgently needed for the identification of minimal NGS panel content in CLL and in other mature lymphoid neoplasms, outlining key genes directly involved in the diagnostic, prognostic and theranostic outputs in order to standardise patient testing and care.

Detection of subarachnoid haemorrhage with spectrophotometry of cerebrospinal fluid- a comparison of two methods.

Spectrophotometric absorption curve analysis of cerebrospinal fluid (CSF) for oxyhaemoglobin and bilirubin is necessary to accurately diagnose subarachnoid haemorrhage (SAH) in patients with typical symptoms but with negative findings on X-ray examinations. In this study, we evaluated the performance of two methods for interpreting absorption curves; one method from the United Kingdom National External Quality Assessment Service (UK-NEQAS) and the other from the national quality assurance programme in Sweden (Equalis). Consecutive absorbance curves (n=336) were interpreted with two different methods, and their performance was compared to the diagnosis as stated in the patient records. The UK-NEQAS method displayed equal sensitivity to the Equalis method, but the specificity of the UK-NEQAS method was significantly higher than the Equalis method resulting in fewer false positive results. For UK-NEQAS, a positive predictive value (PPV) of 84.6% and a negative predictive value (NPV) of 99.7% were observed, whereas the Equalis method had a PPV of 27.5% and an NPV of 99.7%. The semi-automated method based on the guidelines from UK-NEQAS provides an efficient and correct interpretation of absorbance curves with short turn-around times. We propose using this method for the routine interpretation of CSF spectrophotometric curves.

Ultrasensitive molecular tests for Plasmodium detection: applicability in control and elimination programs and reference laboratories.

ABSTRACTObjective.To evaluate molecular tools to detect low-level parasitemia and the five species of Plasmodium that infect humans for use in control and elimination programs, and in reference laboratories.Methods.We evaluated 145 blood samples from patients who tested positive by nested polymerase chain reaction (nPCR), from asymptomatic individuals and from the WHO Global Malaria Programme/United Kingdom National External Quality Assessment Service. Samples were assayed using the genus-specific RealStar® Malaria PCR Kit 1.0 (alt-Gen; altona Diagnostics) and the RealStar® Malaria Screen & Type PCR Kit (alt-S&T; altona Diagnostics). The results from the molecular tests were compared with those from quantitative PCR (qPCR), nPCR and thick blood smear.Results.The levels of parasitemia ranged from 1 to 518 000 parasites/µL, depending on the species. Compared with nPCR, alt-S&T had a sensitivity of 100%, except for identifying P. falciparum, for which the sensitivity was 93.94%. All samples positive by alt-Gen were also positive by nPCR. When comparing alt-Gen to qPCR, the sensitivity was 100% for P. vivax, P. malariae and P. falciparum. For all Plasmodium species, the correlation between cycle threshold values of alt-S&T and alt-Gen compared with qPCR was significant (P < 0.0001, Spearman’s test), with r = 0.8621 for alt-S&T and r = 0.9371 for alt-Gen. When all Plasmodium species were considered, there was a negative correlation between the level of parasitemia and real-time PCR cycle threshold values (P < 0.0001). In this study, only 2 of 28 samples from asymptomatic individuals were positive by thick blood smear; however, all 28 of these samples were positive by alt-S&T.Conclusions.The alt-Gen and alt-S&T assays are suitable for detecting submicroscopic infections for distinct epidemiological purposes, such as for use in surveys and reference laboratories, and screening in blood banks, which will contribute to global efforts to eliminate malaria.

An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report.

Simple SummaryStandardization of flow-cytometric assessment of minimal residual disease in acute lymphoid leukemia (ALL) is necessary to allow concordant multicentric application of the methodology. This is a prerequisite for internationally collaborative trials, such as the AIEOP-BFM-ALL and the ALL IC-BFM trial. We developed and applied a comprehensive training and quality control program involving a large number of international laboratories within the I-BFM consortium to complement standardization of the methodology with an educational component as well as with persistent quality control measures to allow large ALL treatment trials which use multi-laboratory FCM-MRD assessments for risk stratification of pediatric patients with ALL.Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

The Use and Effectiveness of an Online Diagnostic Support System for Blood Film Interpretation: Comparative Observational Study.

BackgroundThe recognition and interpretation of abnormal blood cell morphology is often the first step in diagnosing underlying serious systemic illness or leukemia. Supporting the staff who interpret blood film morphology is therefore essential for a safe laboratory service. This paper describes an open-access, web-based decision support tool, developed by the authors to support morphological diagnosis, arising from earlier studies identifying mechanisms of error in blood film reporting. The effectiveness of this intervention was assessed using the unique resource offered by the online digital morphology Continuing Professional Development scheme (DM scheme) offered by the UK National External Quality Assessment Service for Haematology, with more than 3000 registered users. This allowed the effectiveness of decision support to be tested within a defined user group, each of whom viewed and interpreted the morphology of identical digital blood films.ObjectiveThe primary objective of the study was to test the effectiveness of the decision support system in supporting users to identify and interpret abnormal morphological features. The secondary objective was to determine the pattern and frequency of use of the system for different case types, and to determine how users perceived the support in terms of their confidence in decision-making.MethodsThis was a comparative study of identical blood films evaluated either with or without decision support. Selected earlier cases from the DM scheme were rereleased as new cases but with decision support made available; this allowed a comparison of data sets for identical cases with or without decision support. To address the primary objectives, the study used quantitative evaluation and statistical comparisons of the identification and interpretation of morphological features between the two different case releases. To address the secondary objective, the use of decision support was assessed using web analytical tools, while a questionnaire was used to assess user perceptions of the system.ResultsCases evaluated with the aid of decision support had significantly improved accuracy of identification for relevant morphological features (mean improvement 9.8%) and the interpretation of those features (mean improvement 11%). The improvement was particularly significant for cases with higher complexity or for rarer diagnoses. Analysis of website usage demonstrated a high frequency of access for web pages relevant to each case (mean 9298 for each case, range 2661-24,276). Users reported that the decision support website increased their confidence for feature identification (4.8/5) and interpretation (4.3/5), both within the context of training (4.6/5) and also in their wider laboratory practice (4.4/5).ConclusionsThe findings of this study demonstrate that directed online decision support for blood morphology evaluation improves accuracy and confidence in the context of educational evaluation of digital films, with effectiveness potentially extending to wider laboratory use.

Assessment of Standardization and International Scale (IS) Alignment Efforts of BCR-ABL1 Testing By the Chinese CML Alliance in an International External Quality Assessment Program

Sequential BCR-ABL1 mRNA level measurement by reverse transcription quantitative polymerase chain reaction (RT-qPCR) is embedded in management of chronic myeloid leukaemia (CML) patients.United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI) has been providing External Quality Assessment (EQA)/Proficiency Testing (PT) to 200 laboratories undertaking BCR-ABL1 RT-qPCR analysis since 2007.The Chinese CML Alliance (CCA), established in 2011 for the purpose of the standardization of BCR-ABL1 measurement includes 58 laboratories to date. Peking University People's Hospital (PUPH), the Chinese regional reference centre, has acquired conversion factors (CFs) for reporting BCR-ABLIS through sample exchanges with the Adelaide IMVS reference laboratory. Other members acquired their laboratory specific CFs thorough sample exchange with PUPH, and their CFs were regularly revalidated through testing distributed samples by PUPH.We reviewed performance of CCA laboratories in UK NEQAS LI's BCR-ABL1 Quantification program to evaluate if technical standardization initiatives and local result alignment to the International Scale (IS) (defined as BCR-ABLIS) increased accuracy and precision of BCR-ABL1 measurement by CCA labs.MethodsThis study was facilitated by Diaceutics and Novartis. 5 CCA laboratories were included in 3 EQA/PT send-outs provided by UK NEQAS LI. Six standard samples and one additional educational sample were tested by CCA laboratories, alongside up to 207 other international participants. Samples comprised two lyophilized cell line mixtures containing dilutions of BCR-ABL1 (e14a2) positive K562 cells in a background of BCR-ABL1 negative HL60 cells.For each send-out, CCA laboratories were assessed against the IS robust mean BCR-ABLIS for each sample tested and their proximity to the robust mean log reduction between two standard trial samples tested, providing information on CCA laboratory IS alignment and assay linearity.ResultMedian BCR-ABLIS sample levels in this study (calculated from all participant data) ranged from 6.1% to 0.002% (Figure 1). All five CCA laboratories used in-house based methods to measure BCR-ABL1; 3 based their in-house method on the Europe Against Cancer protocols. ABL1 was detected in all samples by the 5 centres with 4 achieving ABL1 levels >100,000. Additionally, 4/5 detected BCR-ABL1 transcript in all seven samples but 1/5 laboratories failed to detect any transcript in 1 sample (the sample that had the lowest level of transcript (median BCR-ABLIS = 0.002%; ~MR 4.7)).4/5 laboratories measured BCR-ABL1IS levels within +2 SD of the robust means for individual sample and log reduction analysis undertaken between samples for each send-out.DiscussionThis study shows that efforts by the CCA to standardize BCR-ABL1 measurement and align local results to the IS have been successful. Satisfactory results (within +2SD of the robust mean) were obtained in 4/5 laboratories providing reassurance that patient testing in these laboratories is accurate and precise. A root cause analysis undertaken in the laboratory with results >+2SD from the robust mean found a pre-analytical sample transposition error that when reanalyzed was within the acceptable range for both individual sample and log reduction results.The study showed that some CCA laboratories omitted the BCR-ABL1 standard curve, something not recommended in technical guidelines (Foroni et al., 2011) as erroneous results may occur due to unrecoqnized assay drift or suboptimal amplification. Standard curves for both ABL1 and BCR-ABL1 should always be run; however it is acceptable to run a standard curve each day for multiple runs but these must use the same master mix used for the sample quantification (Foroni et al., 2011).Future studies will include more laboratories in the CCA network to assess if the use of internationally recognized calibrants can achieve even greater standardization.Figure 1. All participant BCR-ABLIS results for the 7 samples tested. Blue markers = non CCA laboratories; red marker = CCA laboratories. Long horizontal black lines = IS median. Short horizontal black lines = range.*Ya-Zhen Qin andStuart Scott contributed equally to this study. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites.

Increasing numbers of travelers returning from endemic areas, migrants, and refugees have led to a significant rise in the number of imported malaria cases in non-endemic countries. Real- time PCR serves as an excellent diagnostic tool, especially in regions where experience in microscopy is limited. A novel fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) was developed and evaluated using 56 reference samples of the United Kingdom National External Quality Assessment Service (UK NEQAS) for molecular detection of malaria, including P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Species identification is based on single nucleotide polymorphisms (SNPs) within the genome where the MalLC640 probe binds, lowering the melting temperature in the melting curve analysis. The novel FRET-qPCR achieved 100% (n = 56) correct results, compared to 96.43% performing nested PCR. The high sensitivity, with a calculated limit of detection of 199.97 parasites/mL blood for P. falciparum, is a significant advantage, especially if low-level parasitemia has to be ruled out. Even mixed infections of P. falciparum with P. vivax or P. ovale, respectively, were detected. In contrast to many other real-time PCR protocols, this novel FRET-qPCR allows the quantitative and species-specific detection of Plasmodium spp. in one single run. Solely, P. knowlesi was detected but could not be differentiated from P. vivax. The turnaround time of this novel FRET-qPCR including DNA extraction is less than two hours, qualifying it for routine clinical applications, including treatment monitoring.

Factor VIII/IX inhibitor testing practices in the United Kingdom: Results of a UKHCDO and UKNEQAS national survey.

Inhibitor formation is the greatest challenge facing persons with haemophilia treated with factor concentrates. The gold standard testing methodologies are the Nijmegen-Bethesda assay (NBA) for FVIII and Bethesda assay (BA) for FIX inhibitors, which are affected by pre-analytical and inter-laboratory variability. To evaluate inhibitor testing methodology and assess correlation between self-reported and actual methodology. Methodology was evaluated using a survey distributed alongside a UK National External Quality Assessment Service Blood Coagulation external quality assurance (EQA) exercise for FVIII and FIX inhibitor testing. Seventy four survey and EQA exercise responses were received (response rate 63.2%), with 50 paired survey/EQA results. 47.1% (33/70) reported using the NBA and 42.9% (30/70) the BA for FVIII inhibitor testing. Review of FVIII inhibitor assay methodology demonstrated discrepancy (self-reported to actual) in 64.3% (BA reporting) and 27.6% (NBA reporting). Pre-analytical heat treatment was used by 32.4%, most commonly 56°C for 30minutes. Assay cut-offs of 0.1-1.0BU/mL were reported. EQA samples (acquired FVIII and congenital FIX) demonstrated titres and coefficients of variation (CV) of 3.1BU/mL (0.7-15.4BU/mL; CV=43%) and 18.0BU/mL (0-117BU/mL; CV=33%), respectively. No significant assay or laboratory factors were found to explain this variance, which could have resulted in change in management for 6 patients (5 misclassified high-titre FVIII inhibitors and 1 false negative for a FIX inhibitor). Heterogeneity was seen at each stage of assay methodology. No assay-related factors were found to explain variation in inhibitor titres. Further standardization is required to improve inhibitor quantification to guide patient care.

UPLC-MS/MS method for determination of retinol and α-tocopherol in serum using a simple sample pretreatment and UniSpray as ionization technique to reduce matrix effects.

Background Our goal was to develop a simple, rapid and precise ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of retinol and α-tocopherol in serum. Currently published LC-MS/MS methods either require complex extraction procedures (liquid-liquid or solid-phase) or do not meet desirable specifications for imprecision in serum (coefficient of variation [CV] <6.8% and 6.9%, respectively). Methods Sample preparation consisted of a simple protein precipitation with ethanol and acetonitrile. Stable isotope-labeled internal standards (IS) and a homemade calibration curve were used for quantification. The analysis was performed using an Acquity I-class Xevo TQ XS LC-MS/MS. Chromatographic runtime was 6.0 min using a reversed phase gradient elution. UniSpray (US) as an ionization technique was compared to electrospray ionization (ESI). Analytical validation included matrix effect, recovery and trueness compared to National Institute of Standards and Technology (NIST) standards and United Kingdom National External Quality Assessment Service (UK NEQAS) samples. Results Intra- and inter-run CVs were <4.9% for retinol and <1.7% for α-tocopherol, both complying with desirable specifications for imprecision. Bias compared to NIST standards was <3.1% for both compounds. The method was linear over the entire tested range. The lower limit of quantification (LLOQ) with US was lower than with ESI for both retinol (0.022 vs. 0.043 mg/L) and α-tocopherol (0.22 vs. 0.87 mg/L). Matrix effects were not significant (<15%) for retinol. However, for α-tocopherol matrix effects of on average 54.0% were noted using ESI, but not with US. Conclusions We developed a fast, precise and accurate UPLC-MS/MS method for the determination of retinol and α-tocopherol in human serum using a single-step sample pretreatment. Ionization using US eliminated the matrix effects for α-tocopherol.

PS1038 FLT3 TESTING IN THE RYDAPT ERA

Background:The approval of Rydapt (midostaurin) heralds the first targeted therapy in acute myeloid leukaemia (AML). Accurate detection of both FLT3 internal tandem duplications (ITD) and tyrosine kinase domain (TKD) variants is required to ensure that patients correctly receive the therapy. Additionally, recent European LeukaemiaNet AML guidelines now require the testing of a number of genetic markers including FLT3 to risk stratify patients.Aims:With the increased importance of FLT3 testing for the diagnosis, prognosis and treatment of AML, the aims of this study were to assess methodological variation and the quality of laboratory testing of FLT3 ITD variants.Methods:The United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI), has been providing external quality assessment (EQA) to laboratories undertaking FLT3 ITD testing by molecular genetic methods since 2011, and became IS0 17043 accredited in 2014. The programme currently has 130 active participants in 36 countries globally. Two lyophilised patient donated or cell line based samples are shipped to laboratories 3 times per annum and participants are asked to test the samples with their in‐house techniques, reporting both qualitative (ITD present/absent) and quantitative (mutation load/allelic ratio) data along with a number of methodological details.Results:Forty samples (including samples both positive and negative for the FLT3 ITD) have been issued to laboratories, in 20 separate distributions, since 2011. Positive samples have featured ITD sizes ranging from 30–126 bp, with mutation loads ranging from 10–90%.The most popular analysis methods used by participants were capillary electrophoresis (70.8%) and agarose gel electrophoresis (20.0%), with a small number of participant using next generation sequencing (NGS), melt curve analysis, acrylamide gel electrophoresis, microfluidic electrophoresis and Sanger sequencing. The majority of laboratories use laboratory developed (in‐house) tests (85.8%), with the remaining participants using commercially available assays.Concordance rates ranged from 83.3% to 100% (average 97.5%). Higher error rates were associated with low mutation load samples and larger ITDs (126 bp). High error rates were associated with gel electrophoresis and NGS based methods. Quantitatively, all laboratories were able to distinguish a high (median mutation load = 92%) and low (median mutation load = 11.7%) mutation load sample with a high degree of concordance (interquartile range = 2.5% and 3.7%, respectively), but variation in calculation methods used were identified.Summary/Conclusion:The quality of FLT3 testing was generally high, although a higher error rate was associated with low mutation load samples and large ITDs when participants used agarose gel or NGS based techniques. Gel electrophoresis is an inherently variable, outdated method and its use should not be encouraged. Standard NGS analysis software has difficulty in detecting large ITDs, as large insertions do not retain sufficient homology in the regions flanking the insertion to permit accurate alignment. Pair‐end based analysis approaches have been developed that can reliably detect larger insertions. The high degree of concordance seen in quantitative data was reassuring but could be improved further by standardising calculation methods.

Laboratory Performance on Reporting Monoclonal Gammopathy During Cerebrospinal Fluid Oligoclonal Banding Analysis from External Quality Assessment Surveys.

Cerebrospinal fluid (CSF) oligoclonal banding (OCB) analysis is a sensitive test used to mainly aid multiple sclerosis (MS) diagnosis. Monoclonal gammopathy is usually an incidental finding during CSF OCB analysis. The aim of this study was to assess laboratory performance on reporting monoclonal gammopathy pattern during CSF OCB analysis based on external quality assessment surveys. The CSF OCB surveys from the College of American Pathologists (CAP) from 2010 to 2015 were reviewed. The UK National External Quality Assessment Service (NEQAS) CSF OCB surveys from 2014 to 2017 were also reviewed. All monoclonal gammopathy patterns were confirmed by serum protein electrophoresis followed by immunofixation on a Sebia Hydrasys analyzer. There were 11 monoclonal gammopathy cases identified in the CAP OCB survey from 2010 to 2015. The average rate of CAP participants that correctly reported the pattern was 25.1% (range, 2.4%-66.7%). The most common pattern incorrectly reported was the systemic inflammation pattern, followed by the oligoclonal bands present/positive pattern. The NEQAS OCB survey from 2014 to 2017 had 4 monoclonal gammopathy cases and indicated a much higher number (average, 88.5%; range, 84.1%-90.8%) of participating laboratories to successfully detect monoclonal gammopathy. Monoclonal gammopathy is still an underrecognized pattern in the CSF OCB analysis by the CAP participating laboratories and warrants further education.

Lifestyle and demographic factors associated with human semen quality and sperm function

ABSTRACTIn this study, we sought to investigate the effect of lifestyle and demographic factors on classic and functional semen parameters. Three hundred and twenty-eight subjects who underwent semen analysis were recruited. Routine SA, sperm vitality, acrosome reaction (AR) assay and sperm DNA fragmentation index (DFI) were analyzed. Demographic and lifestyle information, including (1) BMI, (2) current smoking and alcohol drinking frequency, (3) sleep habits, (4) daily fluid intake, (5) weekly meat intake, (6) sports frequency, (7) trouser cell phone use, (8) age, and (9) abstinence time, were collected. Generalized additive models were used to analyze the possible non-linear association. The results showed that total sperm count (TSC) was significantly associated with age (P = 0.001), abstinence time (P = 0.001) and daily coffee intake (P = 0.044). Semen volume was significantly associated with age (P < 0.001) and daily coffee intake (P < 0.001). Sperm concentration was significantly associated with abstinence time (P = 0.011) and average sleep duration (P = 0.010). Sperm motility was significantly associated with age (P = 0.002) and daily juice intake (P = 0.001). Total motile sperm count was significantly associated with age (P = 0.003) and abstinence time (P = 0.009). DFI was significantly associated with age (P = 0.002), irregular sleeping habit (P = 0.008) and abstinence time (P = 0.032). The percentage of AR sperm was significantly associated with daily juice intake (P = 0.013). In conclusion, DFI and TSC were the most sensitive semen parameters for demographic and lifestyle features, whereas age had more influence on semen parameters than other demographic and lifestyle features.Abbreviations: BMI: body mass index; SA: semen analysis; AR: acrosome reaction; DFI: DNA fragmentation index; GAM: generalized additive model; TSC: total sperm count; TMC: total motile sperm count; IUI: intrauterine insemination; SCSA: sperm chromatin structure assay; SD: standard deviation; IQR: interquartile range; CBAVD: congenital bilateral absence of vas deferens; NEQAS: national external quality assessment service; HTF: human tubal fluid; HSA: human serum albumin.

Results of the UK NEQAS for Molecular Genetics reference sample analysis

AimsIn addition to providing external quality assessment (EQA) schemes, United Kingdom National External Quality Assessment service (UK NEQAS) for Molecular Genetics also supports the education of laboratories. As an enhancement...

RAS screening in colorectal cancer: a comprehensive analysis of the results from the UK NEQAS colorectal cancer external quality assurance schemes (2009\u20132016)

Evidence strongly indicates that extended RAS testing should be undertaken in mCRC patients, prior to prescribing anti-EGFR therapies. With more laboratories implementing testing, the requirement for External Quality Assurance schemes increases, thus ensuring high standards of molecular analysis. Data was analysed from 15 United Kingdom National External Quality Assessment Service (UK NEQAS) for Molecular Genetics Colorectal cancer external quality assurance (EQA) schemes, delivered between 2009 and 2016. Laboratories were provided annually with nine colorectal tumour samples for genotyping. Information on methodology and extent of testing coverage was requested, and scores given for genotyping, interpretation and clerical accuracy. There has been a sixfold increase in laboratory participation (18 in 2009 to 108 in 2016). For RAS genotyping, fewer laboratories now use Roche cobas®, pyrosequencing and Sanger sequencing, with more moving to next generation sequencing (NGS). NGS is the most commonly employed technology for BRAF and PIK3CA mutation screening. KRAS genotyping errors were seen in ≤10% laboratories, until the 2014–2015 scheme, when there was an increase to 16.7%, corresponding to a large increase in scheme participants. NRAS genotyping errors peaked at 25.6% in the first 2015–2016 scheme but subsequently dropped to below 5%. Interpretation and clerical accuracy scores have been consistently good throughout. Within this EQA scheme, we have observed that the quality of molecular analysis for colorectal cancer has continued to improve, despite changes in the required targets, the volume of testing and the technologies employed. It is reassuring to know that laboratories clearly recognise the importance of participating in EQA schemes.