Comparison of Giemsa and Hemacolor Staining Reveals Systematic Differences in Avian Leukocyte Profiles

Accurate identification of avian leukocyte fractions from blood smears depends strongly on the staining method. This study provides a comparative evaluation of Giemsa and Hemacolor staining, integrating qualitative and quantitative assessments based on paired samples from 39 Great Tits Parus major. Leukocyte profiles derived from Hemacolor-stained smears showed significantly higher heterophil counts and approximately 40% higher H:L ratios, whereas Giemsa-stained smears yielded higher lymphocyte proportions; eosinophil counts did not differ significantly between methods. These quantitative differences were consistent with microscopic characteristics: Hemacolor generated a lighter field with distinctly defined heterophilic granules, while Giemsa provided stronger nuclear contrast and facilitated lymphocyte identification. The consistent directional differences observed between staining methods indicate a systematic bias in leukocyte assessment. Because the two methods emphasize different cellular features, they produce non-interchangeable leukocyte profiles with implications for ecological and physiological interpretation. Accordingly, staining protocols should be explicitly reported, and a single method should be applied consistently within a dataset.