Impact of hemodilution on flow cytometry based measurable residual disease assessment in acute myeloid leukemia.

Measurable residual disease (MRD) measured in the bone marrow (BM) of acute myeloid leukemia (AML) patients after induction chemotherapy is an established prognostic factor. Hemodilution, stemming from peripheral blood (PB) mixing within BM during aspiration, can yield false-negative MRD results. We prospectively examined hemodilution by measuring MRD in BM aspirates obtained from three consecutive 2 mL pulls, along with PB samples. Our results demonstrated a significant decrease in MRD percentages between the first and second pulls (P = 0.025) and between the second and third pulls (P = 0.025), highlighting the impact of hemodilution. Initially, 39% of MRD levels (18/46 leukemia-associated immunophenotypes) exceeded the 0.1% cut-off, decreasing to 30% (14/46) in the third pull. Additionally, we assessed the performance of six published methods and parameters for distinguishing BM from PB samples, addressing or compensating for hemodilution. The most promising results relied on the percentages of CD16dim granulocytic population (scarce in BM) and CD117high mast cells (exclusive to BM). Our findings highlight the importance of estimating hemodilution in MRD assessment to qualify MRD results, particularly near the common 0.1% cut-off. To avoid false-negative results by hemodilution, it is essential to collect high-quality BM aspirations and preferably utilizing the initial pull for MRD testing.

Analytical assay validation for acute myeloid leukemia measurable residual disease assessment by multiparametric flow cytometry.

BACKGROUND: Measurable residual disease (MRD) assessed by multiparametric flow cytometry (MFC) has gained importance in clinical decision-making for acute myeloid leukemia (AML) patients. However, complying with the recent In Vitro Diagnostic Regulations (IVDR) in Europe and Food and Drug Administration (FDA) guidance in the United States requires rigorous validation prior to their use in investigational clinical trials and diagnostics. Validating AML MRD-MFC assays poses challenges due to the unique underlying disease biology and paucity of patient specimens. In this study, we describe an experimental framework for validation that meets regulatory expectations. METHODS: Our validation efforts focused on evaluating assay accuracy, analytical specificity, analytical and functional sensitivity (limit of blank (LoB), detection (LLoD) and quantitation (LLoQ)), precision, linearity, sample/reagent stability and establishing the assay background frequencies. RESULTS: Correlation between different MFC methods was highly significant (r = 0.99 for %blasts and r = 0.93 for %LAIPs). The analysis of LAIP specificity accurately discriminated from negative control cells. The assay demonstrated a LoB of 0.03, LLoD of 0.04, and LLoQ of 0.1%. Precision experiments yielded highly reproducible results (Coefficient of Variation <20%). Stability experiments demonstrated reliable measurement of samples up to 96 h from collection. Furthermore, the reference range of LAIP frequencies in non-AML patients was below 0.1%, ranging from 0.0% to 0.04%. CONCLUSION: In this manuscript, we present the validation of an AML MFC-MRD assay using BM/PB patient specimens, adhering to best practices. Our approach is expected to assist other laboratories in expediting their validation activities to fulfill recent health authority guidelines.