Comparative Study of Alkaline Solutions in the Bluing Step on Hematoxylin and Eosin Staining Quality in Renal Tissue: An Experimental Study
Keywords:
Hematoxylin and eosin staining, Lithium carbonate, Staining quality, Histopathology, Renal tissueAbstract
Objective: To compare the effectiveness of three alkaline solutions: ammonium hydroxide, lithium carbonate, and potassium hydroxide in the bluing step on Hematoxylin and Eosin (H&E) staining quality in renal tissue, evaluating nuclear staining, cytoplasmic staining, color contrast, and comparing costs and safety profiles.
Methods: A factorial experimental design with completely randomized arrangement was conducted. H&E staining was performed on 135 porcine renal tissue blocks using three alkaline solutions at 0.05% concentration in the bluing step with five different time intervals (3 dips, 10 dips, 30 seconds, 1 minute, and 2 minutes). Quality assessment was conducted by five blinded experts using a 4-point scoring system. Data were analyzed using two-way ANOVA, Tukey’s HSD test, and Intraclass correlation coefficient. Statistical significance was set at p<0.05.
Results: Lithium carbonate 0.05% provided the best nuclear staining quality (mean score 2.78±0.15 at 3 dips) compared to ammonium hydroxide (2.67 ± 0.29) and potassium hydroxide (2.22±0.19) with statistical significance (p<0.001, η² = 0.15). For cytoplasmic staining, lithium carbonate showed superior results (p<0.001, η² = 0.09) with time intervals showing no significant effect (p=0.112). Regarding color contrast between nucleus and cytoplasm, lithium carbonate and ammonium hydroxide provided good and consistent results, while potassium hydroxide showed a declining trend with increased time (p<0.001, η² = 0.12). Quality grading revealed that lithium carbonate achieved the highest proportion of “excellent” grade at 93.3% for nuclear staining compared to ammonium hydroxide 84.4% and potassium hydroxide 40.0% (p<0.001). Cost analysis showed lithium carbonate had a usage cost of only 0.50 baht per procedure with superior safety advantages including odorless, non-volatile properties, and easy storage. Inter-rater reliability was excellent (ICC=0.86-0.91).
Conclusions: Lithium carbonate 0.05% is a suitable alternative for the bluing step, providing superior staining quality, enhanced safety, and acceptable usage cost compared to commonly used ammonium hydroxide solutions. It can be applied in histopathology laboratories to improve diagnostic quality and enhance occupational safety.
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