Abstract
Introduction: The reticulocyte count by flow cytometry (FC) - an automated counting method - can present errors due to the presence of interfering factors, contributing to a slight increase in results. However, automated methods have large advantages over the manual method, taken as reference, what justifies efforts to improve their quality. Objective: Evaluate platelet interference with the reticulocyte count by FC, using thiazole orange (TO) (FC/TO). Materials and methods: The method of reticulocyte count by FC/TO and a modified automated equivalent method, which excluded CD61-positive cells (platelets) from analysis (FC/TO/MOD), were compared to the manual method. Conclusion: Results were analyzed according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) to assess interchangeability between the methods, by linear regression analysis and paired t-test. The exclusion of interfering fragments from result analysis by the modified method produced results in closer proximity to those of the reference method.
Highlights
The reticulocyte count by flow cytometry (FC) – an automated counting method – can present errors due to the presence of interfering factors, contributing to a slight increase in results
These cells stained with the monoclonal antibody (R4) that presented the same morphometric characteristics of erythrocytes (R1 region – FSC × SSC) were excluded in the production of the fluorescence 1 (FL1) × FL2 scatter plot; just cells of the R2 region were selected for the analysis of reticulocyte percentage
The comparison between results of FC methods and the manual method was made by graphs produced by the Excel program, presenting confidence intervals (CI) of 95% for binomial variables, which represent the standard error of the reference method
Summary
The reticulocyte count by flow cytometry (FC) – an automated counting method – can present errors due to the presence of interfering factors, contributing to a slight increase in results. The Clinical and Laboratory Standards Institute (CLSI) and the International Committee for Standards in Haematology (ICSH) produced the document H44-A2 – Methods for Reticulocyte Counting (Automated Blood Cell Counters, Flow Cytometry, and Supravital Dyes); approved guideline, second edition[1] –, that comprises a set of technical norms for reticulocyte counting and the quality control for its implementation. This protocol, as well as other authors[2,3,4], points to the interferences that may occur with automated enumeration, including those caused by the presence of leukocytes and platelets. Robinson et al (1998)(6) suggest that thiazole orange (TO), a fluorescent dye used in automated methods of reticulocyte and reticulated platelets count, would bind, in a non-specific form, to the granules present in platelets
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