TY - JOUR
T1 - Validation of a 12-color flow cytometry assay for acute myeloid leukemia minimal/measurable residual disease detection
AU - Wang, Sa A.
AU - Jorgensen, Jeffrey L.
AU - Hu, Shimin
AU - Jia, Fuli
AU - Li, Shaoying
AU - Loghavi, Sanam
AU - Ok, Chi Young
AU - Thakral, Beenu
AU - Xu, Jie
AU - Medeiros, L. Jeffrey
AU - Wang, Wei
N1 - Publisher Copyright:
© 2023 International Clinical Cytometry Society.
PY - 2023/9
Y1 - 2023/9
N2 - Background: Acute myeloid leukemia (AML) minimal/measurable residual disease (MRD) by multicolor flow cytometry is a complex laboratory developed test (LDT), challenging for implementation. We share our experience in the validation of a 12-color AML MRD flow cytometry assay to meet stringent regulatory requirements. Methods: We worked under the guidelines of the CLSI HL62 publication, illustrated the details of the validation process that was tailored to uniqueness of AML MRD, and tested its clinical validity in 61 patients. The “trueness” was determined by correlating with concurrent molecular genetic testing and follow-up bone marrow examinations. Results: Under assay specificity, we shared the details of panel design, analysis, and criteria for interpretation and reporting. The assay accuracy was assessed by testing known positive and negative samples and correlating with molecular genetic testing and follow-up bone marrow examination. The limit of detection (LOD) and limit of quantification (LOQ) were validated to a level between 0.01% and 0.1%, varied from the leukemia-associated immunophenotypes (LAIP) and the numbers of events obtained for analysis. Assay linearity, precision and carry over studies all met acceptable criteria. In the clinical validity test, the concordance was 93%, specificity 98% and sensitivity 83%. The most challenging aspects of the assay were the discrimination of pre-leukemic cells (persistent clonal hematopoiesis) or underlying myelodysplastic clones from AML MRD with immunophenotypic switch or subclone selection. Conclusion: The validation met all criteria and obtained FDA IDE (investigational device exemption) approval. This study provides ample technical and professional details in setting up the AML MRD flow cytometry assay and illustrates through the example of the “fit for purpose” validation process. We also highlight the need for further characterization of abnormal blasts bearing the potential for AML relapse.
AB - Background: Acute myeloid leukemia (AML) minimal/measurable residual disease (MRD) by multicolor flow cytometry is a complex laboratory developed test (LDT), challenging for implementation. We share our experience in the validation of a 12-color AML MRD flow cytometry assay to meet stringent regulatory requirements. Methods: We worked under the guidelines of the CLSI HL62 publication, illustrated the details of the validation process that was tailored to uniqueness of AML MRD, and tested its clinical validity in 61 patients. The “trueness” was determined by correlating with concurrent molecular genetic testing and follow-up bone marrow examinations. Results: Under assay specificity, we shared the details of panel design, analysis, and criteria for interpretation and reporting. The assay accuracy was assessed by testing known positive and negative samples and correlating with molecular genetic testing and follow-up bone marrow examination. The limit of detection (LOD) and limit of quantification (LOQ) were validated to a level between 0.01% and 0.1%, varied from the leukemia-associated immunophenotypes (LAIP) and the numbers of events obtained for analysis. Assay linearity, precision and carry over studies all met acceptable criteria. In the clinical validity test, the concordance was 93%, specificity 98% and sensitivity 83%. The most challenging aspects of the assay were the discrimination of pre-leukemic cells (persistent clonal hematopoiesis) or underlying myelodysplastic clones from AML MRD with immunophenotypic switch or subclone selection. Conclusion: The validation met all criteria and obtained FDA IDE (investigational device exemption) approval. This study provides ample technical and professional details in setting up the AML MRD flow cytometry assay and illustrates through the example of the “fit for purpose” validation process. We also highlight the need for further characterization of abnormal blasts bearing the potential for AML relapse.
KW - accuracy
KW - AML MRD
KW - laboratory developed test
KW - multi-color flow cytometry
KW - sensitivity
KW - specificity
KW - validation
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U2 - 10.1002/cyto.b.22140
DO - 10.1002/cyto.b.22140
M3 - Article
C2 - 37605812
AN - SCOPUS:85168466615
SN - 1552-4949
VL - 104
SP - 356
EP - 366
JO - Cytometry Part B - Clinical Cytometry
JF - Cytometry Part B - Clinical Cytometry
IS - 5
ER -