Date of Award

1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Stephen D. Gaunt

Abstract

Automated analyzers have revolutionized diagnostic hematology in mammalian species. These commercial instruments utilize flow cytometric technology to enumerate blood cell concentrations. Because of the nuclei present in most non-mammalian erythrocytes and thrombocytes, these instruments are unable to calculate leukocyte counts in birds, amphibians, reptiles, and fish. These investigations sought to determine if three commonly used methodologies in flow cytometry could sufficiently differentiate avian erythrocytes, leukocytes, and thrombocytes, and ultimately form a basis for performing total leukocyte counts. Fluorescein isothiocyanate (FITC) and thiazole orange (TO) were used to stain samples of whole, erythrocyte-, and leukocyte-enriched chicken blood. Although fluorescent microscopic and flow cytometric results obtained using both stains suggested a higher propensity for these dyes in leukocytes and thrombocytes, the difference in fluorescence intensity with erythrocytes was not sufficient to assess their concentration. Furthermore, leukocytes stained with FITC were found consistently in the large erythrocyte peak in cell sorting experiments. Cell counts performed on a population of cells defined by higher TO staining correlated poorly with manual total leukocyte counts. Chicken blood cells were also examined for reactivity with anti-spectrin, anti-vimentin, and anti-$\beta$-tubulin antibodies. Leukocytes demonstrated a higher non-specific staining with secondary antibodies. The inclusion of normal serum as a blocking step essentially eliminated this reactivity. The non-specific staining was not detected by flow cytometry. Although these investigations verified that standard flow cytometric techniques may be utilized to analyze avian leukocytes, sufficient differentiation of these cells from erythrocytes was not achievable for quantitative purposes. Methods with increased sensitivity of fluorescence detection or improved specificity of leukocyte staining are needed to develop a system by which this important diagnostic evaluation can be automated in non-mammalian hematology.

Pages

162

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