TY - JOUR
T1 - Analytical assay validation for acute myeloid leukemia measurable residual disease assessment by multiparametric flow cytometry
AU - Tettero, Jesse M.
AU - Dakappagari, Naveen
AU - Heidinga, Maaike E.
AU - Oussoren-Brockhoff, Yvonne
AU - Hanekamp, Diana
AU - Pahuja, Anil
AU - Burns, Kerri
AU - Kaur, Pavinder
AU - Alfonso, Zeni
AU - van der Velden, Vincent H.J.
AU - te Marvelde, Jeroen G.
AU - Hobo, Willemijn
AU - Slomp, Jennichjen
AU - Bachas, Costa
AU - Kelder, Angele
AU - Nguyen, Kevin
AU - Cloos, Jacqueline
N1 - Publisher Copyright:
© 2023 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.
PY - 2023/11
Y1 - 2023/11
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85173069800&partnerID=8YFLogxK
U2 - 10.1002/cyto.b.22144
DO - 10.1002/cyto.b.22144
M3 - Article
C2 - 37766649
AN - SCOPUS:85173069800
SN - 1552-4949
VL - 104
SP - 426
EP - 439
JO - Cytometry Part B - Clinical Cytometry
JF - Cytometry Part B - Clinical Cytometry
IS - 6
ER -