Flow cytometry reticulocyte counting using acridine orange: validation of a new protocol

Contagem de reticulócitos por citometria de fluxo utilizando acridine orange: validação de um novo protocolo

Karina Augusta Viana Maria das Graças Carvalho Luci Maria Sant'Ana Dusse Aline Caldeira Fernandes Renato Sathler Avelar Danielle Marquete Vitelli Avelar Beatriz Carvalho Claudia Maria Franco Ribeiro Lis Ribeiro do Valle Antonelli Andrea Teixeira Olindo Assis Martins FilhoAbout the authors


Currently, the reticulocyte counting is a challenge for clinical laboratories in Brazil, mainly for the ordinary ones, which still use the manual method. This method has some limitations, since it consists of a laborious method, time consuming, with low accuracy.


This study has developed and evaluated the performance of a New Laboratory Protocol for flow cytometry (FC) reticulocytes counting using acridine orange (AO) as dye, aiming to standardize a more precise, easy, fast implementation, and low cost protocol. After standardization of the New Protocol (FC/AO), it was compared with the manual method. The results were analyzed according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS), now known as Clinical and Laboratory Standards Institute (CLSI), to evaluate the interchangeability of methods in linear regression analysis and paired t test, besides other quality control tests.


Based on these results concerning to the correlation between the methods and the tests related to quality control, we can admit that FC/AO for reticulocyte counting shows undeniable advantages when compared to the preexisting manual method.

reticulocyte counting methods; flow cytometry; acridine orange; manual reticulocyte count

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