Assessment of droplet digital polymerase chain reaction for measuring BCR-ABL1 in chronic myeloid leukaemia in an international interlaboratory study.

Measurement of BCR activator of RhoGEF and GTPase -ABL proto-oncogene 1, non-receptor tyrosine kinase (BCR-ABL1) mRNA levels by reverse transcription quantitative polymerase chain reaction (RTqPCR) has been critical to treatment protocols and clinical trials in chronic myeloid leukaemia; however, interlaboratory variation remains a significant issue. Reverse transcriptase droplet digital PCR (RTddPCR) has shown potential to improve testing but a large-scale interlaboratory study is required to definitively establish this. In the present study, 10 BCR-ABL1-positive samples with levels ranging from molecular response (MR)1·0 -MR5·0 were tested by 23 laboratories using RTddPCR with the QXDX BCR-ABL %IS kit. A subset of participants tested the samples using RTqPCR. All 23 participants using RTddPCR detected BCR-ABL1 in all samples to MR4·0 . Detection rates for deep-response samples were 95·7% at MR4·5 , 78·3% at MR4·7 and 87·0% at MR5·0 . Interlaboratory coefficient of variation was indirectly proportional to BCR-ABL1 level ranging from 29·3% to 69·0%. Linearity ranged from 0·9330 to 1·000 (average 0·9936). When results were compared for the 11 participants who performed both RTddPCR and RTqPCR, RTddPCR showed a similar limit of detection to RTqPCR with reduced interlaboratory variation and better assay linearity. The ability to detect deep responses with RTddPCR, matched with an improved linearity and reduced interlaboratory variation will allow improved patient management, and is of particular importance for future clinical trials focussed on achieving and maintaining treatment-free remission.

Current trends in flow cytometry automated data analysis software.

Automated flow cytometry (FC) data analysis tools for cell population identification and characterization are increasingly being used in academic, biotechnology, pharmaceutical, and clinical laboratories. The development of these computational methods is designed to overcome reproducibility and process bottleneck issues in manual gating, however, the take-up of these tools remains (anecdotally) low. Here, we performed a comprehensive literature survey of state-of-the-art computational tools typically published by research, clinical, and biomanufacturing laboratories for automated FC data analysis and identified popular tools based on literature citation counts. Dimensionality reduction methods ranked highly, such as generic t-distributed stochastic neighbor embedding (t-SNE) and its initial Matlab-based implementation for cytometry data viSNE. Software with graphical user interfaces also ranked highly, including PhenoGraph, SPADE1, FlowSOM, and Citrus, with unsupervised learning methods outnumbering supervised learning methods, and algorithm type popularity spread across K-Means, hierarchical, density-based, model-based, and other classes of clustering algorithms. Additionally, to illustrate the actual use typically within clinical spaces alongside frequent citations, a survey issued by UK NEQAS Leucocyte Immunophenotyping to identify software usage trends among clinical laboratories was completed. The survey revealed 53% of laboratories have not yet taken up automated cell population identification methods, though among those that have, Infinicyt software is the most frequently identified. Survey respondents considered data output quality to be the most important factor when using automated FC data analysis software, followed by software speed and level of technical support. This review found differences in software usage between biomedical institutions, with tools for discovery, data exploration, and visualization more popular in academia, whereas automated tools for specialized targeted analysis that apply supervised learning methods were more used in clinical settings.

Measurement of BCR-ABL1 by RT-qPCR in chronic myeloid leukaemia: findings from an International EQA Programme.

Sequential measurement of BCR-ABL1 mRNA levels by reverse transcription quantitative polymerase chain reaction (RT-qPCR) is embedded in the management of patients with chronic myeloid leukaemia (CML), and has played an important role in the remarkable improvement in patient outcomes seen in this disease. As a provider of external quality assessment (EQA) in this area, UK NEQAS for Leucocyte Immunophenotyping (UKNEQAS LI) has a unique perspective on the changing face of BCR-ABL1 testing in CML. To assess the impact of technical standardisation and the development of the International Scale (IS) upon the accuracy of BCR-ABL1 testing, we reviewed EQA trial data from 2007 to 2015. Comparison of participant results identified considerable variability at both high and low levels of disease, including therapeutically important decision points; however, results converted to the IS showed less variability compared to unconverted data sets. We also found that different methods of converting to the IS produce consistently different median results within UKNEQAS LI IS data sets. This data suggests that whilst the development of the IS has improved the comparability of results between centres, there is still the need for further improvement in the processes of converting raw results to the IS in order to fully realise the benefits of molecular monitoring of CML.

Monitoring of chimerism following allogeneic haematopoietic stem cell transplantation (HSCT): technical recommendations for the use of short tandem repeat (STR) based techniques, on behalf of the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping Chimerism Working Group.

Analysis of short tandem repeats (STR) is the predominant method for post-transplant monitoring of donor engraftment. It can enable early detection of disease relapse, level of engraftment and provide useful information on the graft-versus-host disease (GVHD)/graft-versus-tumour (GVT) effect, facilitating therapeutic intervention. Harmonization and standardization of techniques and result interpretation is essential to reduce the impact of laboratory variability on both clinical management and the results of multi-centre clinical trials. However, the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI) has highlighted significant issues inherent in STR testing that impact upon inter- and intra- laboratory variation. We present here consensus best practice guidelines and recommendations for STR chimerism testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 11 UK and Eire clinical laboratories. This document uses data obtained from the UK NEQAS LI Post-Stem Cell Transplant (SCT) Chimerism Monitoring Programme.

Quantification of cells with specific phenotypes I: determination of CD4+ cell count per microliter in reconstituted lyophilized human PBMC prelabeled with anti-CD4 FITC antibody.

A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) µL(-1) CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment.

European flow cytometry quality assurance guidelines for the diagnosis of primary immune deficiencies and assessment of immune reconstitution following B cell depletion therapies and transplantation.

Over the last 15 years activity of diagnostic flow cytometry services have evolved from monitoring of CD4 T cell subsets in HIV-1 infection to screening for primary and secondary immune deficiencies syndromes and assessment of immune constitution following B cell depleting therapy and transplantation. Changes in laboratory activity in high income countries have been driven by initiation of anti-retroviral therapy (ART) in HIV-1 regardless of CD4 T cell counts, increasing recognition of primary immune deficiency syndromes and the wider application of B cell depleting therapy and transplantation in clinical practice. Laboratories should use their experience in standardization and quality assurance of CD4 T cell counting in HIV-1 infection to provide immune monitoring services to patients with primary and secondary immune deficiencies. Assessment of immune reconstitution post B cell depleting agents and transplantation can also draw on the expertise acquired by flow cytometry laboratories for detection of CD34 stem cell and assessment of MRD in hematological malignancies. This guideline provides recommendations for clinical laboratories on providing flow cytometry services in screening for immune deficiencies and its emerging role immune reconstitution after B cell targeting therapies and transplantation.

Implementation of the updated NICE haematological cancers (NG47) improving outcomes guidelines across Specialist Integrated Haematological Malignancy Diagnostic Services (SIHMDS) in England: a UK NEQAS LI survey.

AIMS: Haematological malignancies represent a diverse group of diseases with complex diagnostic requirements. National Institute for Health and Care Excellence (NICE) Haematological Cancer: Improving Outcomes Guidance was published in 2003 and updated in 2016 (NG47), providing recommendations for service delivery including Specialist Integrated Haematological Malignancy Diagnostic Services (SIHMDSs). This survey assessed the implementation of NG47 guidelines, with a specific focus on implementation in relation to laboratory SIHMDS delivery. METHODS: A survey was issued to the 17 SIHMDSs identified in England. The questionnaire covered laboratory configuration, information systems, integrated reporting and multidisciplinary team (MDT) working recommendations. RESULTS: In the 10 responding SIHMDS, full implementation of recommendations was not achieved. Higher levels of implementation were reported in 'colocated' services compared with 'networked' SIHMDS. Increased guideline implementation was reported with longer duration since initial establishment of a SIHMDS and for laboratory based as opposed to clinical (MDT) reporting recommendations. CONCLUSIONS: Our survey highlights variable implementation of NICE guidance across SIHMDS, with likely inequity of access, standardisation and quality in haemato-oncology diagnostics. Provision of a more structured framework for guideline implementation could assist in increasing compliance to meet the goals of quality and equity of access to harmonised haemato-oncology diagnostics across the NHS in England. This would provide a basis for evaluating the clinical benefits and health economic impact of the SIHMDS model on patient care and outcomes.

Assessment of acute myeloid leukemia molecular measurable residual disease testing in an interlaboratory study.

The European LeukaemiaNet (ELN) measurable residual disease (MRD) working group has published consensus guidelines to standardize molecular genetic MRD testing of the t(8;21)(q22;q22.1) RUNX1::RUNX1T1, inv(16)(p13.1q22) CBFB::MYH11, t(15;17)(q24.1;q21.2) PML::RARA, and NPM1 type A markers. A study featuring 29 international laboratories was performed to assess interlaboratory variation in testing and the subsequent interpretation of results, both crucial to patient safety. Most participants in this study were able to detect, accurately quantify, and correctly interpret MRD testing results, with a level of proficiency expected from a clinical trial or standard-of-care setting. However, a few testing and interpretive errors were identified that, in a patient setting, would have led to misclassification of patient outcomes and inappropriate treatment pathways being followed. Of note, a high proportion of participants reported false-positive results in the NPM1 marker-negative sample. False-positive results may have clinical consequences, committing patients to unneeded additional chemotherapy and/or transplant with the attendant risk of morbidity and mortality, which therefore highlights the need for ongoing external quality assessment/proficiency testing in this area. Most errors identified in the study were related to the interpretation of results. It was noted that the ELN guidance lacks clarity for certain clinical scenarios and highlights the requirement for urgent revision of the guidelines to elucidate these issues and related educational efforts around the revisions to ensure effective dissemination.

Establishment and validation of a standard protocol for the detection of minimal residual disease in B lineage childhood acute lymphoblastic leukemia by flow cytometry in a multi-center setting.

Minimal residual disease detection, used for clinical management of children with acute lymphoblastic leukemia, can be performed by molecular analysis of antigen-receptor gene rearrangements or by flow cytometric analysis of aberrant immunophenotypes. For flow minimal residual disease to be incorporated into larger national and international trials, a quality assured, standardized method is needed which can be performed in a multi-center setting. We report a four color, flow cytometric protocol established and validated by the UK acute lymphoblastic leukemia Flow minimal residual disease group. Quality assurance testing gave high inter-laboratory agreement with no values differing from a median consensus value by more than one point on a logarithmic scale. Prospective screening of B-ALL patients (n=206) showed the method was applicable to 88.3% of patients. The minimal residual disease in bone marrow aspirates was quantified and compared to molecular data. The combined risk category concordance (minimal residual disease levels above or below 0.01%) was 86% (n=134). Thus, this standardized protocol is highly reproducible between laboratories, sensitive, applicable, and shows good concordance with molecular-based analysis.

Assessment of plasma cell myeloma minimal residual disease testing by flow cytometry in an international inter-laboratory study: Is it ready for primetime use?

BACKGROUND: Minimal/measurable residual disease (MRD) testing by flow cytometry (FC) has been proposed as a potential surrogate clinical endpoint in plasma cell myeloma (PCM) clinical trials. As a result, effort has gone into standardizing this approach on PCM patients. AIMS: To assess inter-laboratory variation in FC MRD testing of PCM patients in an independent inter-laboratory study. METHODS: A dilution series of five stabilized bone marrow samples manufactured to contain 0%, 0.1%, 0.01%, 0.001%, and 0.0001% neoplastic plasma cells (PCs) were tested blind, using standardized FC PCM MRD assays by 10 international laboratories. RESULTS: Laboratories' assays broadly adhered to the consensus guidelines; however, some deviations were identified in panel design, fluorochrome conjugates, and lysis reagents. Despite this, all laboratories that returned results detected neoplastic PCs down to 0.001% of leucocytes. 6/8 laboratories detected neoplastic PCs at a level of 0.0001%. Quantitative data returned by laboratories showed good consensus and linearity with increasing variation at lower levels of MRD. However, examples of analytical and post analytical error were identified. SUMMARY/CONCLUSION: Broadly standardized PCM MRD FC assays can attain the lower limit of detection (LOD) required by current and future clinical trials, an important consideration in establishing PCM MRD testing as a surrogate clinical marker in PCM clinical trials. Laboratories' assays showed good linearity, encouraging the prediction of survival based on log reduction in neoplastic PC populations in future clinical trials. However, the deviations from consensus guidelines identified in this study would suggest that if PCM MRD assays are further standardized interlaboratory variation could be reduced. © 2018 International Clinical Cytometry Society.

ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 4 - assay validation and quality assurance.

Over the past six years, a diverse group of stakeholders have put forth recommendations regarding the analytical validation of flow cytometric methods and described in detail the differences between cell-based and traditional soluble analyte assay validations. This manuscript is based on these general recommendations as well as the published experience of experts in the area of PNH testing. The goal is to provide practical assay-specific guidelines for the validation of high-sensitivity flow cytometric PNH assays. Examples of the reports and validation data described herein are provided in Supporting Information. © 2017 International Clinical Cytometry Society.

Laboratory Accuracy Improvement in the UK NEQAS Leucocyte Immunophenotyping Immune Monitoring Program: An Eleven-Year Review via Longitudinal Mixed Effects Modeling.

BACKGROUND: The United Kingdom National External Quality Assessment Service (UK NEQAS) for Leucocyte Immunophenotyping Immune Monitoring Programme, provides external quality assessment (EQA) to non-U.S. laboratories affiliated with the NIH NIAID Division of AIDS (DAIDS) clinical trials networks. Selected laboratories are required to have oversight, performance monitoring, and remediation undertaken by Immunology Quality Assessment (IQA) staff under the DAIDS contract. We examined whether laboratory accuracy improves with longer EQA participation and whether IQA remediation is effective. METHODS: Laboratory accuracy, defined by the measurement residuals from trial sample medians, was measured on four outcomes: both CD4+ absolute counts (cells/µL) and percentages; and CD8+ absolute counts (cells/µL) and percentages. Three laboratory categories were defined: IQA monitored (n = 116), United Kingdom/non-DAIDS (n = 137), and non-DAIDS/non-UK (n = 1034). For absolute count outcomes, the groups were subdivided into single platform and dual platform users. RESULTS: Increasing EQA duration was found to be associated with increasing accuracy for all groups in all four lymphocyte subsets (P < 0.0001). In the percentage outcomes, the typical IQA group laboratory improved faster than laboratories from the other two groups (P < 0.005). No difference in the overall rate of improvement was found between groups for absolute count outcomes. However, in the DPT subgroup the IQA group ultimately showed greater homogeneity. CONCLUSIONS: EQA participation coupled with effective laboratory monitoring and remedial action is strongly associated with improved laboratory accuracy, both incrementally and in the proportion of laboratories meeting suggested standards. Improvement in accuracy provides more reliable laboratory information facilitating more appropriate patient treatment decisions. © 2017 International Clinical Cytometry Society.

Current international flow cytometric practices for the detection and monitoring of paroxysmal nocturnal haemoglobinuria clones: A UK NEQAS survey.

BACKGROUND: Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired genetic disorder, with an incidence of approximately 1.3 new cases per million population per year. Evidence from the UK National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI) programme suggested major discrepancies on how PNH testing is undertaken. To investigate this we surveyed laboratories in the UK NEQAS LI PNH programme and report here the findings. METHODS: A questionnaire was distributed to all centres registered in UK NEQAS LI flow cytometry programmes (n = 1587). Comprising several subsections, it covered the majority of clinical flow cytometric practices. Participants completed a general section and then the subsections relevant to their laboratory repertoire. One subsection contained 34 questions regarding practices in PNH clone detection. RESULTS: A total of 105 laboratories returned results for the PNH section; the results demonstrated lack of consensus in all areas of PNH testing. Variation was seen in gating and testing strategies, sensitivity levels and final reporting of test results. Several incorrect practices were highlighted such as inappropriate antibody selection and failure to wash the red blood cells (RBCs) prior to analysis. CONCLUSION: Despite the availability of consensus guidelines there appears to be no agreement in the detection and monitoring of PNH. We found only fourteen centres using methods compatible with the International Clinical Cytometry Society guidelines. Of specific note we found that no two laboratories used the same method. This technical variation could lead to incorrect diagnoses, highlighting the need for better adoption and understanding of consensus practices. © 2016 International Clinical Cytometry Society.

Flow rate calibration. III. The use of stabilized biostandards to calibrate the flow rate and calculate absolute CD4+ T-cell counts.

BACKGROUND: We have previously reported a flow rate calibration method for the determination of absolute CD4(+) T-lymphocyte counts that removes the need for the addition of latex beads to each sample. However, a limitation with this approach is that a calibration factor (CF) needs to be applied to adjust for differences in viscosity between latex bead suspensions and biological specimens. We have also demonstrated the value of using stabilized whole blood samples in external quality assessment (EQA) studies; such samples have a stable absolute lymphocyte count for over 1 year, at 4 degrees C. It was successfully demonstrated that this material can be used as a flow rate biocalibration (FRB) material for use as a flow cytometric control to provide a sample with a known CD4(+) T-lymphocyte count. Such material has advantages over latex bead technology as it can act as a full process control as well as having the same matrix and viscosity characteristics as the test material, thus removing the need for a CF. METHODS: In this study, we have analyzed 268 consecutive normal, abnormal, and HIV(+) samples using FRB, incorporating the PanLeucoGating approach and compared this to the MultiSet method, defined as the predicate. RESULTS: Percentage similarity statistics revealed the following: 0-3,000 CD4(+) cells/mul mean percentage difference (MPD; bias) 1.2%, 95% CI of 5.6-8%; 0-200 CD4(+) cells/microl MPD of 1.25%, 95% CI of 11.63-14.13%; 201-500 CD4(+) cells/microl MPD of 1%, 95% CI of 4.6-6.6%. DISCUSSION: This study demonstrates that stabilized whole blood can be used for FRB. It has the advantage of being a full process control, in addition to costing less than latex beads with highly comparable results. As bench top flow cytometers are extremely stable, this is a low cost and robust alternative to bead based methods for generating absolute CD4 counts.

Flow rate calibration I: a novel approach for performing absolute cell counts.

BACKGROUND: Reports suggest that flow rate (FR) is constant on bench top flow cytometers. Therefore, if FR is constant, the volume acquired in a fixed time period will also be constant, enabling absolute leucocyte counting using flow rate calibration (FRC). METHODS: FR stability was ascertained on a standard FACSCalibur by counting TruCount beads suspended in phosphate buffered saline over 120 s. Studies using two lysing solutions (FACS lysing solution and PharM Lyse) and corresponding sample lysates established a lysing solution calibration factor (CF). Absolute CD4(+) T-lymphocyte counts on 10 peripheral blood samples determined using FRC were compared with the predicate method TruCount/MultiTEST, incorporating MultiSET software. Linearity studies were also performed at three different flow rates. RESULTS: A high degree of linearity over a wide range of counts (50 to >1,600 CD4(+) T lymphocytes/microl) at all three pressures was observed. Importantly, there was no significant difference from the predicate method when appropriate lysing solution CF was used. CONCLUSIONS: Using a simple calibration procedure and incorporation of an appropriate lysing solution CF, we show that FRC can easily be performed. The technical details that underpin this novel approach for absolute leucocyte enumeration are provided.

Flow rate calibration II: a clinical evaluation study using PanLeucoGating as a single-platform protocol.

BACKGROUND: CD4(+) T-lymphocyte enumeration is vital for monitoring disease progression in individuals positive for the human immunodeficiency virus (HIV), and as a result, there is a need to develop cost-effective protocols that provide accuracy, precision, and affordability. Recently, PanLeucoGating has been shown to fulfill these requirements; however, although comparable to state-of-the-art single-platform protocols (SP), there is still a requirement for an accurate total white cell count. To overcome this limitation, we recently developed a flow-rate based calibration method that enables the PanLeucoGating protocol to be used as a SP approach, and in this study show that this approach can be used for CD4(+) T-lymphocyte enumeration. METHODS: A total of 113 HIV samples were analyzed using three protocols: (a) state-of-the art SP bead-based method (MultiSet; predicate protocol), (b) PanLeucoGating protocol used as a dual-platform (DP) approach, and (c) the newly developed flow rate-based SP approach. We demonstrate that flow rate calibration can be achieved easily and that the method is highly comparable to the state-of-the-art SP method. RESULTS: A high correlation was observed between the predicate protocol and the SP PanLeucoGating approach over the whole range of CD4 counts tested (r(2) = 0.9928; bias 8 cells/microl), including the clinically relevant range (e.g., 0-200 CD4 cells/microl; bias 0 cells/microl). For batched samples, the cost of providing a CD4(+) T-lymphocyte count was reduced to approximately US $1. CONCLUSIONS: The SP PanLeucoGating is a cost-effective approach to CD4(+) T-lymphocyte enumeration that maintains accuracy and precision.

Perfect count: a novel approach for the single platform enumeration of absolute CD4+ T-lymphocytes.

BACKGROUND: The derivation of reliable CD4(+) T lymphocyte counts is vital for the monitoring of disease progression and therapeutic effectiveness in HIV(+) individuals. Flow cytometry has emerged as the method of choice for CD4(+) T lymphocyte enumeration, with single-platform technology, coupled with reference counting beads, fast becoming the "gold standard." However, although single-platform, bead-based, sample acquisition requires the ratio of beads to cells to remain unchanged, there is no available method, until recently, to monitor this. METHODS: Perfect Count beads have been developed to address this issue and to incorporate two bead populations, with different densities, to allow the detection of inadequate mixing. Comparison of the relative proportions of both beads with the manufacture's defined limits enables an internal QC check during sample acquisition. In this study, we have compared CD4(+) T lymphocyte counts, obtained from 104 HIV(+) patients, using TruCount beads with MultiSet software (defined as the predicated method) and the new Perfect Count beads, incorporating an in house sequential gating strategy. RESULTS: We have demonstrated an excellent degree of correlation between the predicate method and the Perfect Count system (r(2) = 0.9955; Bland Altman bias +27 CD4(+) T lymphocytes/microl). CONCLUSIONS: The Perfect Count system is a robust method for performing single platform absolute counts and has the added advantage of having internal QC checks. Such an approach enables the operator to identify potential problems during sample preparation, acquisition and analysis.