CLSI H20—Reference Leukocyte Differential Count (Proportional) and Evaluation of Instrumental Methods describes a reference method for evaluating automated and semiautomated hematology instruments for their capability to perform an acceptable leukocyte (ie, WBC) differential count. CLSI H20 focuses on WBCs found in blood smears. CLSI H20 presents a detailed description of an acceptable manual (visual) WBC differential count, which serves as the reference method for the instrumental differential counter. The types of abnormalities for inclusion are outlined. A sample statistical method is also outlined, enabling the determination of the test method’s performance for qualitative, as well as quantitative, abnormalities.

CLSI H20-Ed3 replaces CLSI H20-A2, published in 2007. Several changes were made in this edition, including:

• Aligning terminology with international standards
• Providing updated consensus guidelines on qualifying new examiners to perform leukocyte differentials and on evaluating postexamination activities and clinical accuracy studies
• Expanding an alternative reference method approach (using multiparametric flow cytometric immunophenotyping procedures) to incorporate current and new practices and to reflect harmonization of the process 

– This approach uses recommended techniques for method calibration and validation, metrological traceability, and guidelines for cell identification of leukocytes normally found in the blood of healthy individuals.

CLSI H20 is intended for global use in medical laboratories to improve global harmonization of blood cell differential counts. This information might also be of interest to product manufacturers and regulatory agencies to aid in new instrument and assay development and the required validation and verification processes. The recommendations in CLSI H20 cover performance testing of leukocyte (WBC) differential counting. Only leukocytes found in healthy (nondiseased) individuals are covered. These cell types are neutrophils (segmented), neutrophils (band forms), lymphocytes (normal), lymphocytes (reactive forms), monocytes, eosinophils, and basophils. If a cell type is not identified, the system should appropriately flag it as abnormal, suspect, or unclassified or as nucleated red blood cells (NRBCs). 

Some devices group several cell types into a single category. For example, segmented and band neutrophils, eosinophils, and basophils could be combined as granulocytes. 

Reference interval criteria for flagging abnormal samples are not included in CLSI H20. The user should determine these criteria. Newer reference methods by immunophenotypic definition of healthy blood cells are intended for method calibration and validation studies. These newer reference methods are not intended for routine clinical practice.