Evaluation of residual disease detection by 10-colour flow cytometry using Navios, Navios EX and Kaluza C versus real-time quantitative polymerase chain reaction
Abstract
Background: Measurable residual disease (MRD) analysis is critical in acute lymphoblastic leukaemia (ALL) management protocols. Real-time quantitative polymerase chain reaction (RT-qPCR) is the accepted gold standard in MRD testing. Multi-parameter flow cytometry has emerged as a comparative testing modality. We evaluated the concordance of our centre’s 10-colour flow cytometry (Navios and Navios EX) with RT-qPCR (ABI 7500 Fast Real-Time PCR) for MRD detection. Aim: To investigate the concordance of MRD detection using flow cytometry and RT-qPCR; and, separately, to compare immunoglobulin heavy chain (IgH) rearrangement PCR with RT-qPCR. Setting: The National Health Laboratory Services Flow Cytometry and Somatic Cell Genetics Units at Charlotte Maxeke Johannesburg Academic Hospital. Methods: Flow cytometry results between March 2020 and June 2023 were compared with RT-qPCR results in follow-up samples of patients with a diagnosis of B-cell acute lymphoblastic leukaemia (B-ALL), with one of the following genetic lesions: t(1;19), t(12;21) or t(9;22). Results: A total of 74 bone marrow aspirate samples were eligible for inclusion. The flow cytometry assay showed a low sensitivity (13%) but a high specificity (98%) when compared with RT-qPCR. The positive and negative predictive values were 75% and 71% for flow cytometry versus qPCR, below the acceptable levels of 80% and 90%, respectively. Compared with RT-qPCR, the immunoglobulin heavy (IgH) assay demonstrated higher sensitivity (43%) and positive predictive value (91%), although sensitivity remained below levels for clinical utility. Conclusion: The flow cytometry assay at our centre exhibited significantly lower sensitivity compared to PCR-based methods for the detection of MRD in follow-up cases. Among the PCR techniques, RT-qPCR demonstrated superior sensitivity for MRD detection in B-ALL. Contribution: The research demonstrates the limitations of the current flow cytometry assay compared with RT-qPCR and identifies the need for improved MRD-tailored approaches, particularly in resource-limited settings where molecular testing may not be readily available.