Clinical and Laboratory Standards Institute document H52-A2—Red Blood Cell Diagnostic Testing Using Flow Cytometry; Approved Guideline—Second Edition addresses the diagnostic RBC assays performed as fluorescence-based assays on a flow cytometry platform. Preferred and alternative testing procedures for fetomaternal hemorrhage detection, paroxysmal nocturnal hematuria screening, membrane defect anemia testing for hereditary spherocytosis, and nucleated RBC counting are reviewed. Preferred testing methods, points of validation and QC, and caveats of interpretation are discussed from the perspectives of laboratory practitioners, diagnostic test developers, and regulators. Where appropriate, this guideline integrates current statements of other relevant organizations, such as the International Council for Standardization in Haematology.

This document replaces the first edition of the approved guideline, H52-A, which was published in 2001. Several changes were made in this edition; chief among them is the revision of the document scope from restricted to fetomaternal hemorrhage (FMH) testing methods to a broader scope of all diagnostic assays on RBCs using FCM. These changes reflect both the expansion of diagnostic FCM testing using RBCs and the clinical need to provide guidelines for testing methods not previously covered by CLSI documents. Specifically, this revision expands the discussion of FMH testing to include preferred testing methodology relating to the diagnosis of paroxysmal nocturnal hemoglobinuria and nonimmune membrane-associated hemolytic anemias (hereditary spherocytosis, hereditary pyropoikilocytosis, and ovalocytosis). Additional diagnostic tools to further evaluate anemic conditions by the reliable quantitation of adult F-cells and nucleated RBCs are also included.

This document establishes performance guidelines for applying the science of flow cytometry (FCM) to RBC diagnostic testing. It provides guidelines for: 

• Specimen collection, handling, and storage 

• Procedures for calibrating instruments 

• Procedures for QC of blood samples 

Specific sections are dedicated to: 

• Paroxysmal nocturnal hemoglobinuria (PNH) 

• Diseases of RBC shape, including hereditary spherocytosis (HS) 

• Detection of fetomaternal hemorrhage (FMH) 

• Detection of nucleated RBCs (NRBCs) 

This document is intended for use by laboratory practitioners, in vitro diagnostic (IVD) device manufacturers concerned with quality laboratory medicine practice, and regulators responsible for clearance of new diagnostic devices and quality laboratory medicine practice. 

While there are many other RBC applications, particularly in the area of blood transfusion science or immunohematology, they will not be addressed in this guideline. In addition, it is beyond the scope of this document to establish general performance criteria and reference intervals. Therefore, it is each laboratory’s responsibility to establish instrument performance criteria and staining characteristics for its own specific reagents.