A-124 Optimizing Reticulocyte Analysis in Anemic Patients: A 1:3 Dilution versus Manual Counting for Flag Resolution

Abstract Background The RET Abn Scattergram flag on the Sysmex XN-Series hematology analyzer can complicate reticulocyte assessment, especially in anemic patients with hemoglobinopathies. While Sysmex recommends running a 1:5 dilution to resolve this flag, this dilution lowers the RBC count below the acceptable threshold in anemic patients. The alternative, a manual reticulocyte count, is labor intensive and lacks precision, with CV ranging from 15-45% in the literature. An automated solution would be preferrable. This study evaluates the efficacy of a 1:3 dilution to measure reticulocyte counts, comparing undiluted, 1:3 diluted, and manual reticulocyte counts to determine its reliability in resolving the flag. Methods Thirty patient samples flagged for RET Abn Scattergram abnormalities were analyzed on the Sysmex XN-20. Each sample underwent a complete blood count (CBC) and reticulocyte analysis using a 1:3 dilution, alongside manual reticulocyte counting with New Methylene Blue (NMB) staining. Dilutions were prepared by mixing whole blood with Sysmex DCL and analyzed in secondary sampling mode. Reticulocyte percentages were obtained directly from the analyzer, while absolute counts were calculated. For manual counting, NMB-stained blood samples were examined under oil immersion at 100x magnification. Reticulocytes were identified by their dark-stained reticular material and counted as a percentage of 1,000 erythrocytes. Results The 1:3 dilution showed a strong correlation with undiluted reticulocyte results (r² = 0.9331, Slope = 1.0278). Correlations between automated (undiluted and 1:3) and manual counts were slightly lower (r² = 0.8574 and r² = 0.7836, respectively), but the differences were not deemed clinically significant. Notably, the 1:3 dilution successfully cleared the RET Abn Scattergram flag in 87% (26/30) of cases, while 13% (4/30) retained the flag. Conclusion Our findings suggest that a 1:3 dilution effectively maintains the RBC count >0.50x106/ul and does not significantly alter reticulocyte results compared to undiluted analysis (r² = 0.9331). The 1:3 dilution cleared the RET Abn Scattergram flag in 87% of cases, indicating these automated results are reportable without resorting to a manual reticulocyte count. We observed a weaker correlation between automated and manual reticulocyte counts, which may be due to the imprecision and subjectivity of this method. Of note, there was no clinically significant difference in results across all three methods. Further research is needed to evaluate the possibility that reporting the undiluted reticulocyte count may be adequate in this setting. Given the time-intensive nature and low precision of a manual reticulocyte count, use of a 1:3 dilution is an acceptable alternative for measuring reticulocyte counts in anemic patients.