A novel modified semi-direct method for total leukocyte count in birds

The complete blood count and differential leukocytes count (DLC) are pessssrformed in non-mammalian species manually using several staining solutions and by direct or indirect hemocytometer counting. Some of these methods are time consuming and/or the solutions are not readily available to the clinician (e.g., Natt and Herrick's staining solution). A popular kit for non-mammalian indirect leukocyte count (Unopette 5877 kit) has recently become unavailable. Semi-quantitative leukocyte evaluation can be done on Romanowsky-stained blood smears, but is less accurate. This study evaluated a modified semi-direct method for total leukocyte count (MSDTLC) in avian blood, using a diluted commercially-available eosin-based dye. Blood samples were collected from 13 birds. Blood cell staining was evaluated microscopically by comparison of blood cells stained with the tested dye and with a Romanowsky stain. Leukocyte counts were calculated based on hemocytometer counting of cells stained with the tested dye with a DLC done in Romanowsky-stained blood smears. The precision of the MSDTLC was assessed by several methods. Dilution of the tested dye solution to 1:10 with distilled water showed the optimal results, allowing easy identification of heterophils and eosinophils. The agreement and correlation between counts was high (interclass absolute agreement correlation [ICC] 0.93, P<0.001 and r=0.872, P<0.001, respectively).The agreement and correlation between counts in each side of the hemocytometer were high (ICC 0.92, P<0.001 and r=0.940, P<0.001, respectively). However, results differed significantly (mean 22%, P<0.01). Differences were <21% in 56% of the counts. The MSDTLC using the readily-available diluted (1:10) tested eosin-based stain, performed well, with high precision, allowing reliable counting of both heterophils and eosinophils in avian blood samples.