EuroFlow Standardised 8-Colour Flow Cytometry Achieves 10-5 MRD Sensitivity in B-Cell ALL
The EuroFlow consortium developed and validated a fully standardised 8-colour antibody panel for MRD detection in B-cell precursor ALL, achieving sensitivity comparable to quantitative PCR at 10-5 or better. In 377 follow-up samples, concordance with PCR-based MRD reached 98% when more than 4 million cells were acquired, including 97% concordance for samples below 0.01% MRD. This standardised approach enables reproducible, high-sensitivity flow-based MRD assessment across laboratories.
The original study
Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia.
- Authors
- Theunissen P, Mejstrikova E, Sedek L, van der Sluijs-Gelling AJ, Gaipa G, Bartels M, et al.
- Journal
- Blood
- Type
- Journal Article, Multicenter Study, Research Support, Non-U.S. Gov't
- PMID
- 27903527
Original abstract
A fully-standardized EuroFlow 8-color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients with a sensitivity of ≤10-5, comparable to real-time quantitative polymerase chain reaction (RQ-PCR)-based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients. Comparison with RQ-PCR-based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (≤10-5), if sufficient cells (>4 × 106, preferably more) are evaluated.