Reticulocyte analysis using light microscopy and two different flow cytometric procedures

Area of Science:

• Hematology
• Clinical Pathology
• Biomedical Engineering

Background:

• Reticulocyte counts are crucial indicators of bone marrow erythropoietic activity.
• Microscopic reticulocyte enumeration suffers from inaccuracy and poor reproducibility.
• Flow cytometry offers automated and improved quality for reticulocyte analysis.

Purpose of the Study:

• To determine the optimal thiazole orange (TO) concentration for EPICS Profile flow cytometer.
• To assess the correlation between microscopic and two flow cytometric reticulocyte counting methods.
• To evaluate the clinical utility of flow cytometry for routine reticulocyte analysis.

Main Methods:

• Comparison of three reticulocyte enumeration methods: microscopic (Brilliant Cresyl Blue) and two flow cytometric (FACSort, EPICS Profile) using thiazole orange (TO).
• Optimization of TO concentration for the EPICS Profile system.
• Correlation analysis between the tested methods and assessment of intra-assay variability.

Main Results:

• The optimal TO concentration for the EPICS Profile was found to be 0.1 mg/L.
• Good correlation (coefficients 0.82–0.87) was observed between microscopic and flow cytometric methods.
• Flow cytometry demonstrated significantly lower intra-assay coefficients of variation (6.3%–8.4%) compared to microscopy (27.5%).

Conclusions:

• Flow cytometry, particularly using thiazole orange, provides a reliable and reproducible method for reticulocyte counting.
• The optimized flow cytometric methods are suitable for routine clinical laboratory use.
• Automation via flow cytometry significantly enhances the accuracy and consistency of reticulocyte enumeration.