Impact of Staining Methods and Human Factors on Accuracy of Manual Reticulocyte Enumeration

Although peripheral blood reticulocyte enumeration reflects bone marrow functional integrity, which is important for differential diagnosis of hematological diseases, the factors affecting its accuracy have not been adequately addressed. Using 100 consecutive venous blood samples being processed with four supravital staining techniques [i.e., brilliant cresyl blue (BCB), new methylene blue (NMB), and BCB/NMB with Liu’s stain] for reticulocyte enumeration, two technologists (senior vs. junior) conducted microscopic counting. The results were compared with those obtained with an automated system (Sysmex XE-5000) that served as the standard. The aims of this study were to identify (1) the technique that gave the most reliable outcome, and (2) possible human factors (i.e., seniority, repeated counting) that may affect the counting results. Analysis showed least bias (i.e., deviation from automated counting) associated with BCB staining, followed by NMB. In addition, the senior observer exhibited a higher bias in counting compared with their junior counterpart. Repeated counting also correlated with a higher rate of bias. Nevertheless, inter-observer consistency was high (intraclass correlation coefficient >0.95) and inter-/intra-observer variations were non-significant (both p > 0.05). Our results supported the use of BCB stain for reticulocyte enumeration and the reliability of manual counting despite the involvement of human factors, which had negligible impacts on the final outcomes.