How to Design and Validate a Clinical Flow Cytometry Assay

Introduction

The immunologic classification of leukemias and lymphomas was initially proposed in the mid-1980s. ¹ Since the Revised European-American Classification of Lymphomas ² and subsequent World Health Organization classifications, 345 immunophenotyping is accepted as a key required feature in accurate classification of lymphomas and is now routinely performed by multiparameter flow cytometry. However, a standardized clinical flow cytometry assay approved by the Food and Drug Administration (FDA) is not available for leukemia/lymphoma diagnosis. Most clinical flow cytometry assays are laboratory developed tests (LDTs), and the medical director of a clinical flow cytometry laboratory is responsible for their development and validation. ⁶ These assays are “high complexity tests” and are subject to regulation by Centers for Medicare and Medicaid Services, usually enforced by deemed entities such as the College of American Pathologists (CAP). The guidance provided in the current CAP laboratory general checklist COM.40350 for LDTs requires that for each LDT assay the laboratory must determine and document the analytical accuracy, precision, sensitivity, specificity, reportable range, and any other performance characteristic required to ensure analytical test performance, before clinical use of each LDT. The actual steps for development and validation of flow cytometry tests have been recently published by the Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical Laboratory Standards [NCCLS]) as Guidance H62. ⁷ These guidelines will likely form the basis for detailed checklist items for accreditation of clinical flow cytometry laboratories, and this review is primarily based on them.