Anemia is a common clinicopathologic abnormality detected in avian species. It has been estimated that the frequency of anemia in birds, based on samples submitted for CBCs, is 12.7%.1 The initial diagnostic approach to anemia in both mammalian and avian patients is to first classify the anemia as regenerative or nonregenerative on the basis of the quantity of reticulocytes. For several mammalian species, reticulocyte counts are quantitatively assessed by use of automated analyzers. In contrast, avian erythrocyte regenerative responses are evaluated via semiquantitative assessment of polychromasia or estimation of reticulocyte percentages because the presence of nuclei in avian erythrocytes precludes the use of automated analyzers for quantitative reticulocyte counting.
Consequently, a problem associated with assessment of erythroid regeneration in birds is determining the best method for manually counting avian reticulocytes. There is general agreement that supravital stains such as NMB can be used to identify avian reticulocytes. However, investigators have reported2–4 detection of basophilic reticulum in a high percentage of avian erythrocytes following supravital staining; counting all cells containing any visible reticulum has yielded high reticulocyte percentages (some > 90%) in both healthy birds and birds with anemia induced via nutritional alterations. Clearly, more specific morphologic criteria to define reticulocytes are needed. Two commonly used classification schemes recommend counting either all cells with > 4 or > 5 distinct aggregates of reticulum; another scheme recommends counting all cells with a complete ring of reticulum that encircles the nucleus.5–7 This latter scheme also has been recommended in several current clinical textbooks, but as yet, a consensus on avian reticulocyte classification has not been reached.4,8,9 In addition, to the authors' knowledge, a quantitative correlation between polychromatophils and various reticulocyte morphologic types in avian species has not been published.
In studies performed throughout the 1900s and in recent years, a variety of methods for counting avian reticulocytes has been used, and often the reports fail to define the method of reticulocyte counting. Additionally, the terms reticulocyte and polychromatophil are sometimes used interchangeably. This lack of uniformity in avian reticulocyte assessment prevents comparison of results among studies and has been identified as a likely cause of apparent variation in reticulocyte counts in chickens.10 A similar lack of consistency in avian reticulocyte assessment exists among veterinary diagnostic laboratories. Many laboratories report only a semiquantitative estimate of polychromasia, whereas others report reticulocyte percentages (derived by use of various methods); few laboratories have established reference intervals for either variable.
The purpose of the study reported here was 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. Additionally, determination of a reference interval for polychromatophils in psittacines was undertaken.
Coefficient of variation
Immature erythroid cell
New methylene blue
Euthasol, Virbac Inc, Fort Worth, Tex.
0.5% new methylene blue stain, Ricca Chemical Co, Arlington, Tex.
7100 Aerospray stainer, Wescor Inc, Logan, Utah.
ADVIA 120, Bayer Diagnostics Division, Bayer Corp, Tarrytown, NY.
MedCalc for Windows, version 8.1.1, MedCalc Software, Mariakerke, Belgium.
SPSS for Windows, version 11.5, SPSS Inc, Chicago, Ill.
Fudge AM. Avian complete blood count. In: Fudge AM, ed. Laboratory medicine—avian and exotic pets. Philadelphia: WB Saunders Co, 2000;9–18.
Thrall MA. Hematology of birds. In: Thrall MA, ed. Veterinary hematology and clinical chemistry. Baltimore: Lippincott Williams & Wilkins Co, 2004;229–230.
Christie G. Haematological and biochemical findings in an anemia induced by the daily bleeding of ten-week-old cockerels. Br Vet J 1978;134:358–365.
Hawkey CM, Dennett TB. Normal and abnormal red blood cells. In: Hawkey CM, Dennett TB, Peirce MA, eds. Color atlas of comparative veterinary hematology. Ames, Iowa: Iowa State University Press, 1989;10–11.
Lucas AM, Jamroz C. Atlas of avian hematology. Agricultural monograph 25. Washington, DC: USDA, Government Printing Office, 1961;26–29.
Leighton FA. Morphological lesions in red blood cells from Herring Gulls and Atlantic Puffins ingesting Prudhoe Bay crude oil. Vet Pathol 1985;22:393–402.
Campbell TW. Avian hematology. In: Campbell TW, ed. Avian hematology and cytology. 2nd ed. Ames, Iowa: Iowa State University Press, 1995;3–19.
Alsaker RD, Laber J, Stevens J, et al. A comparison of polychromasia and reticulocyte counts in assessing erythrocytic regenerative response in the cat. J Am Vet Med Assoc 1977;170:39–41.
Deiss A, Kurth D. Circulating reticulocytes in normal adults as determined by the new methylene blue method. Am J Clin Pathol 1970;53:481–484.
Dacie SJV, Lewis SM. Preparation and staining methods for blood and bone-marrow films. In: Dacie SJV, Lewis SM, eds. Practical haematology. 6th ed. New York: Churchill Livingstone Co, 1984;58–59.
Seki N, Yassin MA, Cuevas LE, et al. Colour vision and proficiency in diagnostic microscopy. Trop Med Int Health 2005;10:433–434.