Optimized Method for Reticulocyte Counting: Simple, Accurate, and Comparable to Flow Cytometry

Abstract

Background: Reticulocytes are immature red blood cells with RNA, spending the final stages of their maturation in the peripheral blood. The number of reticulocytes in the peripheral blood is the salient evidence of the effectiveness of bone marrow to produce red blood cells. Currently, reticulocyte count is done manually or automatically in clinical laboratories. Difficulties and limitations of these approaches, including imprecision, poor reproducibility and laboriousness are clear. This study used modification in manually staining method to achieve better counting.

Materials and Methods: The reticulocyte counts of 30 samples were obtained from patients referred to Afzalipour hospital in Kerman, Iran. The patients aged between 0-3 years old (median age= 1.4 years). Samples were investigated for reticulocyte count by three different methods, including conventional, modified, and flow cytometry methods. Modified staining method is used in the optimized method which helps operator to countg reticulocyte better than conventional method.

Results: Analysis of the results showed a reasonable agreement. Sphericity assumption was checked by Kolmogorov-Smirnov and Mouchley analyses. Repeated measurement analysis was performed using Greenhouse-Geisser correction (F=16.6, df=1.105) according to pairwise comparisons with Bonferroni adjustment. Mean differences for flow cytometry, optimized and common methods were 3.88, 3.59, and 2.46 were significant at the 0.05 level, respectively.

Conclusion: The proposed optimized method was simple method, highly reliable, and comparable to flow cytometry. Due to the ease of implementation, this method did not need specialized training or the use of complex devices so it is affordable.

Key words: Blood Stains, Flow Cytometry, Reticulocyte, Reticulocyte Count, Staining and Labeling