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Published on: May 19, 2020
Validation of a candidate international harmonisation protocol-compliant measurement procedure for reticulocyte
Yutaka Nagai1,2,3, Tomohiro Takeda2, Hiromichi Matsushita1,4
1Department of Laboratory Medicine, Keio University School of Medicine, Tokyo, Japan.
Abstract:
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.
Insights
This study validates a new flow cytometry method for measuring reticulocyte percentage, confirming its accuracy and compliance with international standards. The thiazole orange/CD41a/CD61-MP offers a reliable alternative for clinical diagnostics.
Area of Science:
- Hematology
- Clinical Chemistry
- Biotechnology
Background:
- The International Harmonisation Protocol (IHP) for Reticulocyte Percentage (%Retic) relies on microscopic methods (NMB-IHP).
- Flow cytometry (FCM) offers practical advantages for accurate reticulocyte measurement.
- Existing FCM methods (TO/CD235a-MP) lack validation against NMB-IHP.
Purpose of the Study:
- To validate a novel FCM-based Measurement Procedure (MP) using thiazole orange and anti-CD45/CD41a/CD61 antibodies (TO/CD41a/CD61-MP) as an IHP-compliant method.
- To compare the TO/CD41a/CD61-MP with established NMB-IHP and TO/CD235a-MP.
- To assess the practical utility and accuracy of TO/CD41a/CD61-MP on automated hematology analyzers.
Main Methods:
- Validated TO/CD235a-MP against NMB-IHP.
- Compared the novel TO/CD41a/CD61-MP with the validated TO/CD235a-MP using regression analysis.
- Assessed the accuracy of TO/CD41a/CD61-MP on Sysmex XN-2000™ and Nihon Kohden Celltac G+™ analyzers.
Main Results:
- TO/CD235a-MP was confirmed to be IHP-compliant.
- Regression analysis showed strong consistency between TO/CD41a/CD61-MP and TO/CD235a-MP.
- High correlation coefficients (0.97) were observed between TO/CD41a/CD61-MP and automated analyzers for %Retic (0.0-8.2).
Conclusions:
- The TO/CD41a/CD61-MP is validated as an IHP-compliant method for reticulocyte measurement.
- This study provides the first empirical validation of FCM-based MPs for reticulocyte counting.
- The TO/CD41a/CD61-MP demonstrates practical utility and accuracy for clinical applications.
