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

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  • 1 Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616.
  • | 2 Veterinary Medical Teaching Hospital, University of California, Davis, CA 95616.
  • | 3 Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616.

Abstract

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 [ρ] = 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.

Abstract

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 [ρ] = 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.

Contributor Notes

Presented in abstract form at the 41st Annual Meeting of the American Society of Veterinary Clinical Pathology in conjunction with the 57th Annual Meeting of the American College of Veterinary Pathologists, Tucson, Ariz, December 2006.

Address correspondence to Dr. Johns.