Development of a technique for quantification of reticulocytes and assessment of erythrocyte regenerative capacity in birds

Objective - To develop a reticulocyte classification scheme, optimize an avian reticulocyte staining protocol, and compare the percentages of reticulocyte types with polychromatophil percentage in blood samples from birds. Sample Population - Blood samples from a red-tailed hawk and 31 ill birds. Procedures - A single blood sample obtained from a red-tailed hawk (Buteo jamaicensis) was used to optimize the staining protocol. For optimization of the staining protocol, 4 dilutions of whole blood with new methylene blue stain and 4 incubation times were evaluated. From samples submitted for avian CBCs, EDTA-anticoagulated whole blood samples from 31 ill birds were randomly selected and examined to compare polychromatophil and reticulocyte percentages. Reticulocyte staining was performed in all samples by use of a 1:3 (whole blood to new methylene blue) dilution with incubation for 10 minutes at room temperature (approx 22°C); reticulocytes were assessed as a percentage of 1,000 RBCs by 2 independent observers. In Wright-Giemsa-stained blood smears, a polychromatophil percentage was similarly determined. Results - 4 avian reticulocyte types were defined: ring-form reticulocytes, aggregate reticulocytes, and 2 subcategories of punctate reticulocytes. A reticulocyte-staining protocol was optimized. Interobserver and intraobserver variations in assessment of reticulocyte and polychromatophil percentages were not significant. A strong positive correlation (Spearman coefficient of rank correlation [p] = 0.978) was identified between the percentage of polychromatophils and the percentage of ring-form reticulocytes. Conclusions and Clinical Relevance - Results indicated that quantification of ring-form reticulocytes provides an accurate assessment of erythrocyte regenerative capacity in birds.