Validation of a candidate international harmonisation protocol‐compliant measurement procedure for reticulocyte counting using an erythrocyte gating strategy excluding the platelet component

The International Harmonisation Protocol (IHP) for Reticulocyte Percentage (%Retic) in CLSI-H44-A2 is a microscopic procedure performed on a new-methylene-blue-stained blood film (NMB-IHP). However, IHP-compliant Measurement Procedures (MPs) based on flow cytometry (FCM) are recommended for practical and accurately assigned reference values. Thiazole orange (TO) for nucleic acid staining and CD235a immunostaining for erythrocyte gating are currently widespread for MP using FCM (TO/CD235a-MP); however they have not been validated using NMB-IHP. We recently developed MP using FCM with anti-CD45/CD41a/CD61 antibodies, that are utilized for excluding platelets from the un-nucleated blood cell fraction to select erythrocytes (TO/CD41a/CD61-MP). The aim of this study is to validate TO/CD41a/CD61-MP for measurement of reticulocytes as an IHP-compliant MP. First, TO/CD235a-MP was validated as an IHP-compliant MP by comparing it with NMB-IHP. Then TO/CD41a/CD61-MP was compared with TO/CD235a-MP. The practical utility of TO/CD41a/CD61-MP was evaluated using XN-2000™(Sysmex) and Celltac G + ™(Nihon Kohden) hematology analyzers to assess the accuracy of different nucleic acid staining methods. We first confirmed that TO/CD235a-MP was consistent with NMB-IHP, then evaluated TO/CD41a/CD61-MP using TO/CD235a-MP. Regression analysis demonstrated consistency between TO/CD41a/CD61-MP and TO/CD235a-MP, thereby establishing TO/CD41a/CD61-MP as IHP-compliant MP. In the accuracy assessment, the correlation coefficients to TO/CD41a/CD61-MP were observed to be 0.97 for both hematology analyzers with %Retic ranging from 0.0 to 8.2. TO/CD41a/CD61-MP serves effectively as an IHP-compliant MP. This study provides the first empirical validation of FCM-based MPs.