The research drew on samples collected between 5 and 26 January 2024 from Great Tits captured as part of a long-term study of physiological condition in central Poland. The dataset comprised 39 individuals, 27 males and 12 females, all of which were ringed with unique aluminum identification bands during processing. Great Tits were chosen because they are the focal species of the broader research program and were the most abundant birds captured during the winter sampling period; the sample size was determined by the number of individuals caught in that period rather than set in advance. Crucially, the design was paired: from each bird, two small blood samples of approximately 5 to 10 microliters were drawn from the ulnar vein to prepare smears, one of which was stained with Giemsa and the other with Hemacolor. This approach enables direct within-individual comparisons and increases statistical power, since each bird effectively serves as its own control.

Smears were stained using standard Giemsa and Hemacolor protocols, and leukocytes were identified and counted under oil immersion at 1000× magnification with an MBL-320T light microscope, following established avian hematological criteria. Counts were performed up to 100 cells per smear, and all counts were conducted twice for each slide by a single observer to allow repeatability assessment. The two stains produce visibly different preparations: according to the comparative overview presented in the paper, Hemacolor gives a lighter overall palette with magenta-to-violet nuclei against blue-gray cytoplasm and renders heterophil granules as distinct violet structures that are easier to recognize, while Giemsa produces a darker palette with strong red-violet nuclear contrast that facilitates lymphocyte identification. Representative photomicrographs accompanying the paper illustrate these differences, showing how the same cell types can appear more or less conspicuous depending on the reagent used.

Statistical analysis focused on nonparametric paired tests, chosen because the leukocyte proportions and ratio values did not meet the assumptions of normal distribution. The Wilcoxon signed-rank test, which accounts for both the direction and the magnitude of paired differences, was the primary analysis, and a conservative sign test assessing only the direction of change was run as a robustness check. All tests were two-tailed and performed in Statistica 13. The results were unambiguous. Hemacolor-stained smears showed significantly higher heterophil proportions and significantly higher heterophil-to-lymphocyte ratios than Giemsa-stained smears from the same birds, whereas Giemsa-stained smears showed significantly higher lymphocyte proportions. Eosinophil proportions, by contrast, did not differ significantly between the two methods.

The magnitude and consistency of the differences were striking. Heterophil proportions were higher in Hemacolor preparations in 37 of 39 individuals, and heterophil-to-lymphocyte ratios were higher in Hemacolor in the same 37 of 39 birds. Lymphocyte proportions were higher in Giemsa preparations in 36 of 39 individuals, with one bird showing identical values under both stains. Effect sizes were large: the effect size r calculated for the Wilcoxon signed-rank tests reached 0.83 for heterophils, 0.84 for lymphocytes, and 0.85 for the heterophil-to-lymphocyte ratio, all with p values below 0.000001, whereas eosinophils showed only a small effect size of 0.20. The sign test confirmed the same directional patterns, indicating that the observed shifts were not driven by a few extreme outliers but reflected a systematic tendency across nearly the entire sample.

Repeatability checks supported the reliability of the counting itself. Intraclass correlation coefficients calculated from the duplicate counts were high for the principal leukocyte variables, ranging from 0.95 to 0.96 for heterophils, from 0.89 to 0.95 for lymphocytes, and from 0.85 to 0.95 for heterophil-to-lymphocyte ratios, indicating high agreement between replicate counts. Repeatability was lower for eosinophils, with intraclass correlation coefficients of 0.42 to 0.60, which the authors attribute to the relatively low abundance of these cells in the examined smears. This pattern reinforces the interpretation that the observed differences between stains arise at the identification stage—because the same cells look different under different reagents—rather than from inconsistent counting or observer error.

The authors link these numerical biases to concrete staining properties described in their comparison of the two methods. Hemacolor renders heterophil granules in distinct violet tones that stand out clearly against the cytoplasm, making heterophils easier to recognize, whereas Giemsa produces paler, less conspicuous heterophil granules that can be less visible in dense cytoplasmic regions. Conversely, Giemsa’s strong nuclear contrast aids the identification of lymphocytes, whose high nucleus-to-cytoplasm ratio becomes especially salient under this stain. The paper also notes practical trade-offs beyond cell recognition: Hemacolor offers rapid and consistent staining, while modified Giemsa provides a more traditional, detail-enhancing chromatic profile, and the authors discuss the methods’ differing usefulness for archival purposes. Because certain leukocyte features, especially granule coloration in eosinophils, heterophils, and basophils, can vary subtly between the two techniques, the choice of stain directly shapes which cellular features a counter can rely on.

The ecological implications are considerable. Bird-derived physiological and hematological indicators are frequently applied in ecological studies addressing processes such as host-parasite interactions, disease dynamics, and responses to environmental stress, and the heterophil-to-lymphocyte ratio is widely interpreted as a proxy of physiological condition and environmental stress. If the ratio can shift systematically depending on staining protocol, then comparisons across studies, populations, or time periods that used different methods may be confounded by laboratory procedure rather than by biology. The authors emphasize that the bias they documented is directional and repeatable, which means it cannot be assumed to average out across datasets. Differences attributed to environmental conditions, habitat quality, or life-history variation may in part reflect methodological inconsistencies rather than true biological differences, a concern that is especially acute in studies comparing populations, assessing temporal trends, or synthesizing results across multiple datasets.

More broadly, the study serves as a reminder that seemingly mundane methodological choices can propagate into biological inference. The authors conclude that staining protocol should be treated as a genuine source of variation in avian hematological research, and they call for strict methodological consistency within studies and explicit reporting of staining protocols to ensure transparency and comparability across datasets. Mixing staining methods within a single dataset should be avoided, they argue, because it may introduce artificial variation unrelated to biological processes, and heterophil-to-lymphocyte ratios derived from different staining methods should not be treated as directly comparable. Because their paired design isolated the effect of the stain itself, the findings provide unusually clean evidence that the same blood can tell different quantitative stories depending on the chemistry applied to the slide. For a field that increasingly relies on leukocyte profiles as indicators of individual condition and population health, the message is clear: the stain on the slide is part of the measurement, and it should be documented, controlled, and reported as carefully as any other variable in the study.

Subject of Research: Methodological bias in avian blood smear staining and leukocyte profiling

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

Article References: Skwarska, J. (2026). Comparison of Giemsa and Hemacolor Staining Reveals Systematic Differences in Avian Leukocyte Profiles. Ecology and Evolution, 16(10), Article e74540. https://doi.org/10.1002/ece3.74540

Image Credits: AI Generated

DOI: 10.1002/ece3.74540

Keywords: avian hematology, Giemsa staining, Hemacolor staining, heterophil-to-lymphocyte ratio, Great Tit, blood smears, methodological bias, leukocyte profiles, ecological physiology, Romanowsky staining, wild birds, physiological condition