Kingdom National External Quality Assessment Research Articles

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.

PB1039 Evaluation of External Quality Assessment Material for Activated Clotting Time on Point of Care Devices: United Kingdom National External Quality Assessment Scheme for Blood Coagulation Pilot Studies

PB1039 Evaluation of External Quality Assessment Material for Activated Clotting Time on Point of Care Devices: United Kingdom National External Quality Assessment Scheme for Blood Coagulation Pilot Studies

PB1041 Evaluation of External Quality Assessment Material on the Renovated Point of Care Device, United Kingdom National External Quality Assessment Scheme for Blood Coagulation Studies

PB1041 Evaluation of External Quality Assessment Material on the Renovated Point of Care Device, United Kingdom National External Quality Assessment Scheme for Blood Coagulation Studies

Evaluation of Laboratories Supporting Invasive Bacterial Vaccine-Preventable Disease (IB-VPD) Surveillance in the World Health Organization African Region, through the Performance of Coordinated External Quality Assessment.

(1) Background: Laboratories supporting the invasive bacteria preventable disease (IB-VPD) network are expected to demonstrate the capacity to identify the main etiological agents of pediatric bacterial meningitis (PBM) (Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae) on Gram stains and in phenotypic identification. Individual reports of sentinel site (SSL), national (NL) and regional reference (RRL) laboratories participating in the World Health Organization (WHO)-coordinated external quality assessment, distributed by the United Kingdom National External Quality Assessment (EQA) Services (UK NEQAS) for Microbiology between 2014 and 2019 were analyzed. (2) Methods: The panels consisted of (1) unstained bacterial smears for Gram staining, (2) viable isolates for identification and serotyping/serogrouping (ST/SG) and (3) simulated cerebral spinal fluid (CSF) samples for species detection and ST/SG using polymerase chain reaction (PCR). SSLs and NLs tested for Gram staining and species identification (partial panel). RRLs, plus any SSLs and NLs (optionally) also analyzed the simulated CSF samples (full panel). The passing score was ≥75% for NLs and SSLs, and ≥90% for RRLs and NLs/SSLs testing the full panel. (3) Results: Overall, 63% (5/8) of the SSLs and NLs were able to correctly identify the targeted pathogens, in 2019; but there were challenges to identify Haemophilus influenzae either on Gram stains (35% of the labs failed 2014), or in culture. Individual performance showed inconsistent capacity, with only 39% (13/33) of the SSLs/NLs passing the EQA exercise throughout all surveys in which they participated. RRLs performed well over the study period, but one of the two failed to reach the minimal passing score in 2016 and 2018; while the SSLs/NLs that optionally tested the full panel scored between 75% and 90% (intermediate pass category). (4) Conclusions: We identified a need for implementing a robust quality management system for timely identification of the gaps and then implementing corrective and preventive actions, in addition to continuous refresher training in the SSLs and NLs supporting the IB-VPD surveillance in the World Health Organization, Regional Office for Africa (WHO AFRO).

External Quality Assessment Data for Investigation of von Willebrand Disease: Focus on Relative Utility of Contemporary Functional von Willebrand Factor Assays. The United Kingdom National External Quality Assessment Scheme (UK NEQAS) Experience.

Von Willebrand disease (VWD) is one of the most common hereditary bleeding disorders. Effective management of patients and their families depends on accurate diagnosis and subtype classification, and quality assurance including participation in proficiency testing programs is essential to ensure the accuracy of the panel of assays required to achieve this diagnosis. We report here findings from recent external quality assessment (EQA) exercises, as well as from a questionnaire about diagnostic practices employed by centers in the United Kingdom National Quality Assessment Scheme (UK NEQAS) performing von Willebrand factor (VWF) assays. Plasma samples from donors with VWD, "normal" donors, the International Society for Thrombosis and Haemostasis Scientific Subcommittee (ISTH SSC) plasma standard, and whole blood samples were sent to participants in the UK NEQAS BC program for VWF investigation. Calibration of lot#5 of the ISTH SSC plasma standard was shown to give improved comparability between the recovered value from an EQA exercise and the assigned potency for VWF activity assays. Diagnostic accuracy and precision amongst UK NEQAS participants was good, with an average 99% of centers reporting the correct interpretation for normal, type 1 and type 2 VWD samples, 100% diagnostic accuracy for centers performing FVIII binding assays, and good agreement amongst centers performing multimeric analysis. Genetic analysis of the VWF gene by specialist centers demonstrated errors in the genotyping process in one center, but also demonstrated failings in the interpretation of results in other centers. Despite evidence of good laboratory accuracy and precision in their assays, a questionnaire identified marked variation in diagnostic criteria employed, underlining the importance of guidelines to support the diagnosis of VWD.

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.

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.

A review of external quality assurance program performance PD-L1 for NSCLC

The Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) are accredited against International Standard (ISO/IEC) 17043 Conformity assessment – General requirements for proficiency testing. In 2017, the RCPAQAP began collaborating with the United Kingdom National External Quality Assessment Services for Immunocytochemistry and In-Situ Hybridisation (UK NEQAS ICC and ISH) for technical proficiency testing in PD-L1 for non-small cell lung cancer (NSCLC). RCPAQAP laboratories participating in the program has increased steadily over the last few years to 43 in 2021; 2018–2020 results will show that RCPAQAP participant results are comparable with results from the total number of 153 UK NEQAS participants.

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.

Pre-analytical variables in haemostasis: Findings from the United Kingdom National External Quality Assessment scheme for Blood Coagulation (UK NEQAS BC) haemolysis exercise.

Haemolysis is considered one of the major contributors of nonconformities and sample rejection in coagulation testing. Two lyophilized plasmas were distributed to 800 centres registered for prothrombin time (PT), activated partial thromboplastin time (APTT) and either Clauss fibrinogen or thrombin time (TT) in the UK NEQAS BC programme. The same pool of normal plasma was used to prepare both samples, to one of which red blood cell haemolysate was added to mimic haemolysis at 3g/L haemoglobin concentration. Participants were asked to complete a questionnaire about their laboratory approach to dealing with haemolysed samples, including strategies used to deal with different levels of haemolysis. Results for tests performed did not show great differences between the two samples. It should be noted that artificially constructed haemolysed samples may not behave in the same way as patient samples (ie, may not be commutable). However, the possibility of carrying out a large multicentre study for detection of haemolysis was demonstrated. Inconsistency in practice was observed with 226/551 (41%) of centres indicated they reject haemolysed samples solely on visual checks, and 163 (30%) using initial visual checks with further sample rejection evaluation by analyser flags. Furthermore, 333 (72%) of centres indicated that the level of haemolysis affects sample rejection decisions, while 132 (28%) stated it did not. Variability of responses for dealing with haemolysed samples reflects a lack of clear consistency in the pre-analytical area of sample processing.

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.

A review of external quality assurance program performance in non-gynaecological cytology and Pd-L1 For NSCLC

The Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) are accredited against International Standard (ISO/IEC) 17043 Conformity assessment – General requirements for proficiency testing. This standard requires evaluation and commentary of participant performance including variation between participants and comparisons between previous testing rounds. To implement these requirements for the Cytopathology non-gynaecological programs in 2019, methods from the Anatomical Pathology diagnostic programs were utilised. Methods for case selection, evaluation of participants and results from the first year will be discussed. In 2017, the RCPAQAP began collaborating with the United Kingdom National External Quality Assessment Services for Immunocytochemistry and In-situ hybridisation (UK NEQAS ICC and ISH) for PD-L1 for non small cell lung cancer (NSCLC) technical proficiency testing. In 2018 there were 22 RCPAQAP participants enrolled in this program and in 2019, enrolments increased to 40 participants. Results from 2018/19 will be discussed and comparisons with the broader UK NEQAS participants will be made. The RCPAQAP issued a PD-L1 for NSCLC interpretation pilot and results from this pilot will also be discussed along with other updates and developments for Anatomical Pathology and Cytopathology programs in 2020.

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.

The Challenges of Implementing a PD-L1 Proficiency Testing Program in Australia

Background. One important initiative that commenced at the Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) in 2017 was the collaboration with United Kingdom National External Quality Assessment Scheme (UK NEQAS) Immunocytochemistry (ICC) and In-Situ Hybridization (ISH) for the challenging implementation of a PD-L1 immunohistochemistry (IHC) proficiency testing program for non-small cell lung carcinoma (NSCLC). A RCPAQAP participant survey in 2016 showed that only eight laboratories were performing PD-L1 testing. The aim of the collaboration was to increase the sample size of the pilot program to provide meaningful results that could be reported back to RCPAQAP participants with appropriate recommendations. Other challenges of assessment included standardising the clinical cut-offs for positivity for each commercial assay, interpretation of laboratory developed tests (LDTs), using appropriate tissue to cover the critical interpretation points for each assay, interchangeability of clones and interpretation proficiency testing. Methods. The use of a ‘Gold Standard’ for each commercial assay was used as a baseline to compare participant results and tumour proportion score bin categories were implemented to harmonise interpretation across clones. Conclusions. The findings of the pre-pilot test suggest that the use of a clinically validated PD-L1 IHC assay performs better during assessment than adopting a laboratory developed test (LDT). The assessment committee also concluded that tonsil showed a better dynamic range of positivity than placenta. It was acknowledged that participants are limited by the platforms they have available and so it was suggested that validating an optimal method against the clinical assay and continual verification of the test may produce the expected result. The next big challenge is to extend proficiency testing from technical to interpretation. This is being implemented globally via the International Quality Network for Pathology (IQNPath) with participation through local External Quality Assurance programs, including RCPAQAP.

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...

Performance of an In-House Real-Time PCR Assay for Detecting Cytomegalovirus Infection among Transplant Patients from a Tertiary Care Centre

Background: Quantitative Cytomegalovirus (CMV) polymerase chain reactions are increasingly being used for monitoring CMV DNAemia in haematopoietic stem cell transplants and solid organ transplants. Objective: In this study, a commercial CMV viral load assay was compared with an in-house viral load assay. Materials and Methods: A total of 176 whole-blood samples were tested for CMV DNAemia using both assays. Results: Our evaluation showed a difference of 1 log10 copies/ml between the two assay systems in determining CMV viral loads in the clinical samples. Conclusion: The in-house viral load assay had a better correlation with clinical findings compared to the commercial assay. Quality assessment of these assays was done by the United Kingdom National External Quality Assessment Scheme (UKNEQAS), an external proficiency testing programme, and by the National Institute for Biological Standard and Control (NIBSC) standard. For UKNEQAS and NIBSC standards, the bias between the assays was 0.73 log10 and 0.85 log10, respectively. This difference is well within the acceptable range already reported in the literature.

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.