A High‐Sensitivity 10‐Color Flow Cytometric Minimal Residual Disease Assay in B‐Lymphoblastic Leukemia/Lymphoma Can Easily Achieve the Sensitivity of 2‐in‐10⁶ and Is Superior to Standard Minimal Residual Disease Assay: A Study of 622 Patients

Abstract

BackgroundFlow‐cytometric minimal residual disease (FC‐MRD) monitoring is a well‐established risk‐stratification factor in B‐lymphoblastic leukemia/lymphoma (‐B‐ALL) and is being considered as a basis for deintensification or escalation in treatment protocols. However, currently practiced standard FC‐MRD has limited sensitivity (up to 0.01%) and higher false MRD‐negative rate. Hence, a highly sensitive, widely applicable, and easily reproducible FC‐MRD assay is needed, which can provide a reliable basis for therapeutic modifications.MethodsA 10‐color high‐event analysis FC‐MRD assay was studied for the evaluation of MRD status at postinduction, (PI; day‐35), postconsolidation, (PC; day‐78), and subsequent follow‐up time‐points (SFU) in bone marrow samples from pediatric B‐ALL.ResultsOne‐thousand MRD samples (PI‐62.2%; PC‐26.5%; and SFU‐11.3%) from 622 childhood B‐ALL patients were studied. High‐event analysis was performed with median 4,452,000 events (range, 839,000 to 8,866,000 events) and >4 million events in 71% samples. MRD was measurable in 43.2% of PI‐samples, in 29.4% PC‐samples, and in 32.7% SFU‐samples. To simulate comparison with standard FC‐MRD, we reanalyzed MRD results gating only first 500,000 and first 1000,000 events in 122 PI‐MRD positive samples with MRD levels <0.02%. Of these samples gated for 500,000 events and 1000,000 events, 32% and 21.3% were found to be falsely MRD‐negative, respectively.ConclusionsWe report an easily reproducible high‐sensitivity 10‐color FC‐MRD assay with the sensitivity of 2‐in‐106 (0.0002%). It allowed the detection of low‐level MRD in samples, which could have been reported negative using the standard FC‐MRD with limited event analysis. Thus, this high‐sensitivity MRD‐methodology can provide a reliable basis for therapeutic modifications in B‐ALL. © 2019 International Clinical Cytometry Society

Post-induction Measurable Residual Disease Using Multicolor Flow Cytometry Is Strongly Predictive of Inferior Clinical Outcome in the Real-Life Management of Childhood T-Cell Acute Lymphoblastic Leukemia: A Study of 256 Patients

“…MFC-MRD was performed in BM aspirate samples at an early time-point i.e., at the end of induction (post-induction, PI; day 35-40) and at a later time-point i.e., the post-consolidation (PC-MRD, day 78-83). BM samples for MRD assessment were processed using Euroflow bulk-lysis protocol (20) and an 10-color antibody-panel. In brief, the cell suspension was prepared by bulk erythrocyte lysing with ammonium chloridebased lysing reagent, and the cells were stained with the 11-antibody 10-color MRD panel (Supplementary Table S2).…”

“…(9,(17)(18)(19). On the contrary, MFC-MRD has advantages like wider availability, easy processing, and is relatively inexpensive (9,19,20). Most importantly, it has a very low turn-around-time making it suitable for quick therapeutic decisions (20).…”

“…On the contrary, MFC-MRD has advantages like wider availability, easy processing, and is relatively inexpensive (9,19,20). Most importantly, it has a very low turn-around-time making it suitable for quick therapeutic decisions (20). Hence, MFC-MRD can be easily adaptable in the routine clinical practice for T-ALL management, especially in the LMIC with limited resources.…”

Lossless Compression of Cytometric Data

“…However, things have changed, as lossless compression algorithms (LCAs) nowadays can achieve significant space savings, without severely impacting user experience. On the other hand, long‐term storage remains fairly expensive, and data sets keep growing in size, in terms of parameters and events .…”

Minimal Residual Disease Detection in Acute Lymphoblastic Leukemia

“…MRD phenotypes will show markers similar to relapsed leukemia cells saved for reference from original diagnostic cells or can be identified through profile databases in the case of secondary ALL. In contrast to the four-color flow cytometers, which have the capacity to measure MRD up to the important diagnostic level of 0.01%, 8-12 color flow cytometers can normally measure MRD levels up to 0.001% cells or 1 MRD cell in 100,000 cells in a bone marrow specimen with even higher sensitivity [43,53].…”