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

Abstract

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.

Multicentric standardization of minimal/measurable residual disease in B‐cell precursor acute lymphoblastic leukaemia using next‐generation flow cytometry in a low/middle‐level income country

“…The performance of some laboratories was variable during the study, reflecting non-strict adherence to SOPs over time (Figure S7) and indicating the need for continuous quality assessment, complemented by educational programmes on the standardization of methods even for experienced laboratories, as previously demonstrated. 12,13 Our results showed that standardization resulted in a greater accuracy in the MRD evaluation of BCP-ALL in different laboratories and on different MFC platforms, as previously described, 14 and also that MFC reproducibility is possible in a multicentre study if the laboratories adhere to the proposed SOPs. Comparability between samples will also be essential to enable automated analysis using the EuroFlow database.…”

Reliable Flow-Cytometric Approach for Minimal Residual Disease Monitoring in Patients with B-Cell Precursor Acute Lymphoblastic Leukemia after CD19-Targeted Therapy

“…MFC-MRD detection was performed with an 11-color panel (10 antibodies and DNA-stain Syto41, Thermo Fisher Scientific, Waltham, MA, USA) generally, according to the I-BFM-FLOW network guidelines [ 26 ], but with an adjustment for possible CD19 loss. Bone marrow aspirates were stained according to the manufacturer’s instructions, with appropriate amounts of directly conjugated monoclonal antibodies (MoABs) used for the sample cellularity.…”

“…Lymphoblasts were considered leukemic if they represented a distinct population with leukemia-associated phenotypes and lymphoid light-scatter parameters. As per the I-BFM-FLOW network guidelines [ 26 ] and in accordance with the EuroFlow standardized approach [ 28 ], we defined a minimum of ten clustered leukemic lymphoblasts to consider a sample as MRD positive (lower limit of detection—LOD). The proportion of the leukemic cell population was presented as the percentage of Syto41-positive cells.…”

Low-intensity therapy cures over 40 % of children with rapid Flow-MRD responding ALL: the ALL-MB 2008 trial results

“…MRD was assessed by MFC in three laboratories (two in Russia and one in Belarus) according to well-harmonized approach [40] based on AIEOP-BFM-ALL-MRD-Flow study group guidelines [42], as previously described [40]. All three laboratories use the MFC methodology based on standard analyses, and had participated in AIEOP-BFM QA [43] system as well as in intragroup proficiency tests [40]. 4-9-color MFC was used to evaluate the expression of antigens commonly used for MRD detection in BCP-ALL: CD19, CD10, CD34, CD45, CD20, CD38, CD58, and CD11a [40].…”