Preanalytical stability of hemoglobin H inclusion body detection using rapid brilliant cresyl blue staining: a genotype-stratified stability study in EDTA blood stored at 4 °C.

INTRODUCTION: No evidence-based preanalytical guideline exists for EDTA blood storage before hemoglobin H (HbH) inclusion body detection. We evaluated the stability of rapid (30-minute) brilliant cresyl blue staining across α-globin and β-globin genotypes in blood stored at 4 °C. METHODS: Residual EDTA blood from 322 patients with microcytic hypochromic anemia was stored at 4 °C and tested on days 0, 2, 4, and 7 by 3 blinded medical technologists. Hemoglobin typing and genotyping followed inclusion body counting. Wilcoxon and Mann-Whitney U tests as well as intraclass correlation coefficient(2,1) were applied. RESULTS: Six α-globin genotype groups were identified, including HbH disease (n = 103), by β-globin genotype background. Non-HbH groups showed no meaningful inclusion bodies, confirming stable specificity. In HbH disease with a normal β-globin gene, 100% qualitative concordance was maintained at day 7, with no statistically significant quantitative change from day 0 (P = .28); the intraclass correlation coefficient(2,1) was 0.858. A transient increase at day 2 (P < .001) may have reflected in vitro HbH autoxidation during cold storage. Hemoglobin H disease co-inheriting HbE showed stable but markedly reduced counts throughout storage; a consistent pattern was observed in isolated cases co-inheriting β-thalassemia. DISCUSSION: EDTA blood stored at 4 °C maintains qualitative stability for HbH inclusion body detection for up to 7 days; HbE co-inheritance substantially reduces diagnostic sensitivity, with a similar pattern observed in isolated β-thalassemia co-inheritance.