The importance of immunophenotyping for the proper diagnosis and management of patients with hematolymphoid neoplasia necessitates the development of guidelines for the appropriate performance of these techniques in the clinical laboratory. CLSI document H43-A2—Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline—Second Edition addresses issues of safety, specimen collection and transportation, sample preparation, immunofluorescent staining, instrument quality control, data acquisition, and data storage for the application of flow cytometry to the immunophenotypic analysis of these disorders. This document builds on CLSI document H42—Enumeration of Immunologically Defined Cell Populations by Flow Cytometry.

There has been a substantial expansion of the application of flow cytometry (FCM) in hematolymphoid neoplasia since the previous publication of this approved guideline as H43-A in 1998. Instrumentation has improved, routine use of four or more color FCM has expanded, and new applications in assessment of hematolymphoid malignancies are under constant development. Current classification systems rely upon immunophenotype for diagnosis, increasing the importance of clinical FCM in analysis of hematolymphoid neoplasia. In addition, the clinical utility of flow cytometric analysis in new disease categories, such as myelodysplastic syndrome, was established and prognostic immunophenotypic markers have been described. Flow cytometric immunophenotyping also plays a vital role in evaluation of patients for monoclonal targeted therapies. The document has therefore been revised to reflect these advances.

This document establishes performance guidelines for the flow cytometric immunophenotypic analysis of samples from patients with known or suspected hematolymphoid neoplasia. The World Health Organization (WHO) classification system relies upon morphology, clinical history, immunophenotype, and cytogenetics for diagnosis of hematolymphoid neoplasia. Therefore, immunophenotypic analysis of hematolymphoid neoplasia is crucial for the accurate diagnosis and classification of these complex malignancies. It is not within the scope of this document to recapitulate the criteria used to diagnose leukemias and lymphomas. Readers are referred to Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues (Jaffe ES, Harris N, Stein H, Vardiman JW, eds. Lyon, France: IARC Press; 2001). Because this document is intended primarily for laboratory workers in FCM, it cannot describe all the possible clinical situations in which flow cytometric analysis of leukemia or lymphoma is or is not appropriate. 

This document includes guidelines for phenotyping cases of acute and chronic leukemias, non-Hodgkin’s lymphomas, plasma cell neoplasms, and myelodysplastic syndromes. The subcommittee recognizes that most of the principles used to approach chronic lymphoid leukemias can also be applied to the study of lymphomas, so these problems are considered together. Special problems unique to the study of non-Hodgkin’s lymphomas are treated separately. 

At present, there are few agreed-upon standards for precision, accuracy, and interlaboratory comparability of leukemia analysis by FCM. Therefore, it is each laboratory’s responsibility to establish instrument performance criteria and staining characteristics for its own specific reagents.