Liam Whitby

Reduction of intra‐ and interlaboratory variation in CD34+ stem cell enumeration using stable test material, standard protocols and targeted training

The European Working Group on Clinical Cell Analysis (EWGCCA) has, in preparation for a multicentre peripheral blood stem cell clinical trial, developed a single‐platform flow cytometric protocol for the enumeration of CD34+ stem cells. Using this protocol, stabilized blood and targeted training, the EWGCCA have attempted to standardize CD34+ stem cell enumeration across 24 clinical sites. Results were directly compared with participants in the UK National External Quality Assessment Scheme (NEQAS) for CD34+ Stem Cell Quantification that analysed the same specimens using non‐standardized methods. Two bead‐counting systems, Flow‐Count and TruCount, were also evaluated by the EWGCCA participants during trials 2 and 3. Using Flow‐Count, the intralaboratory coefficient of variation (CV) was ≤ 5% in 39% of the laboratories (trial 1), increasing to 65% by trial 3. Interlaboratory variation was reduced from 23.3% (trial 1) to 10.8% in trial 3. In trial 2, 70% of laboratories achieved an intralaboratory CV ≤ 5% using TruCount, increasing to 74% for trial 3; the interlaboratory CV was reduced from 23.4% to 9.5%. Comparative analysis of the EWGCCA and the UK NEQAS cohorts revealed that EWGCCA laboratories, using the standardized approach, had lower interlaboratory variation. Thus, the use of a common standardized protocol and targeted training significantly reduced intra‐ and interlaboratory CD34+ cell count variation.

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;

Flow cytometry to detect minimal residual disease is a sensitive and specific means to detect impending relapse in B lineage acute lymphoblastic leukemia (ALL). However as with all relatively sophisticated procedures assurance is needed that the methodology is widely applicable, and reproducible in different laboratories. In this paper, the UK ALL Flow MRD Group and the UK MRD steering group describe a four color flow protocol that was applicable to most patients and showed similarly high levels of concordance both between different laboratories and with MRD as detected molecularly. See related perspective article on page 748. 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.

Development and Evaluation of a Stabilized Whole-Blood Preparation as a Process Control Material for Screening of Paroxysmal Nocturnal Hemoglobinuria by Flow Cytometry

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disorder in which correct diagnosis is essential for effective patient management. Demonstration of deficiency of glycosylphosphatidylinositol (GPI)‐linked antigens from red cells and/or granulocytes by flow cytometry represents a highly specific diagnostic test for PNH. Currently, no external quality assessment (EQA) programme or reference material is available for whole‐blood PNH testing (red cells and leucocytes) by flow cytometry.

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

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).

ISHAGE Protocol: Are We Doing It Correctly?

Flow cytometric CD34+ stem cell enumeration is routinely performed to optimize timing of peripheral blood stem cell collections and assess engraftment capability of the apheresis product. While a number of different flow methodologies have been described, the highly standardized ISHAGE protocol is currently the most widely employed, with 204/255 (81%) international participants in the UK NEQAS CD34+ stem cell enumeration program indicating their use of this method. Recently, two laboratories were identified as persistent poor performers, a fact attributed to incorrect ISHAGE protocol usage/setup. This prompted UK NEQAS to question whether other laboratories were making similar errors and, if so, how this might affect individual EQA performance.

Flow rate calibration II: A clinical evaluation study using PanLeucoGating as a single‐platform protocol

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.

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.

Standardizing leucocyte PNH clone detection: An international study

Consensus and Practical Guidelines for robust high‐sensitivity detection of glycophosphatidylinostitol‐deficient structures on red blood cells and white blood cells in paroxysmal nocturnal hemoglobinuria (PNH) were recently published.

Current laboratory practices in flow cytometry for the enumeration of CD 4+ T-lymphocyte subsets

CD4+ T‐lymphocyte subset enumeration is routinely used for monitoring HIV disease progression, with approximately 300,000 tests performed annually in the UK alone. Technical variables can impact upon any laboratory test and therefore the final result obtained. Here, we report the findings of a survey questionnaire issued to 1,587 clinical flow cytometry laboratories to: (a) determine if the UK NEQAS for Leucocyte Immunophenotyping (UK NEQAS LI) lymphocyte subset external quality assessment (EQA) programme was suitable for current laboratory needs and practices; and (b) assess the impact of these responses on clinical practice where CD4+ T‐lymphocyte subsets analysis is undertaken. The survey covered areas not traditionally examined by EQA such as: staffing numbers, flow cytometer age and service intervals, plus six test specific sections covering: leukaemia immunophenotyping, CD4+ T‐lymphocyte subsets analysis (reported here), CD34+ stem cell testing, low level leucocyte enumeration, minimal residual disease testing and PNH testing.

Comparison of methodological data measurement limits in CD4⁺ T lymphocyte flow cytometric enumeration and their clinical impact on HIV management.

UK NEQAS for Leucocyte Immunophenotyping, an ILAC G13:2000 accredited External Quality Assessment (EQA) organization, with over 3000 international laboratories participating in 14 programmes, issues 2 proficiency testing samples of stabilized whole blood to 824 participants in the Immune Monitoring (lymphocyte subset) programme every two months. We have undertaken a study of 58,626 flow cytometric absolute CD4+ T lymphocyte count data sets from these laboratories over a 12‐year‐period (2001–2012) to determine counting method variation in data measurement limits and how this could influence the clinical management of HIV patients.