Evaluation of the interference of platelets in reticulocyte counting by flow cytometry using thiazole orangeABSTRACT
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.
Key words:
reticulocyte count; flow cytometry; blood platelets
A) selection of the population of interest, R1, based on morphometric aspects for the construction of graph B (FL1 × anti-CD61PE/FL2); B) identification of the homogeneous population, R2, and possible interferents – R3 and R4; C) quantification of the percentage of positive cells in the upper right quadrant (stained with TO) present in the R2 region – graph drawn with the combination of gates in R1 and R2 regions.FC/TO: method of reticulocyte count by flow cytometry, using thiazole orange; SSC: side scatter; FSC: forward scatter.
Continuous lines: 95% binomial confidence interval; traced line: linear regression between the evaluated methods; Y = 0.827 + 0.97X; r2 = 0.92; r = 0.959.FC/TO: method of reticulocyte count by flow cytometry, using thiazole orange.
Continuous lines: 95% binomial confidence interval; traced line: linear regression between the evaluated methods; Y = 0.297 + 0.983X; r2 = 0.93; r = 0.964.FC/TO: method of reticulocyte count by flow cytometry, using thiazole orange.