The effect of decalcification solutions on kappa and lambda in situ hybridization

Abstract

Consistently reliable in situ hybridization (ISH) staining and microtomy on decalcified formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies has been a significant challenge. This study evaluates five decalcification solutions’ decalcification cutting and kappa/lambda ISH staining characteristics. Five random benign tonsils and one femoral head were decalcified at intervals from 0·5 to 4 hours in five decalcification solutions. Tonsil sections were stained for kappa and lambda by ISH. Stain characteristics were blindly graded and compared to non-decalcified tonsil sections using hierarchical linear model (HLM) analysis. The femoral head was cored with a standard bone marrow core biopsy needle, and cutting adequacy was evaluated at the same time intervals. Specimens were blinded and judged as ‘acceptable’ (A), ‘cutting not optimal’ (NO), or ‘unacceptable’ (U) by an experienced histotechnologist. Decalcification solutions Formical-2000, Formical-4, and Immunocal demonstrated approximately equivalent quality in staining (average score = 7·642, 7·952, and 7·805, respectively) through-out 4 hours. Both ethylenediaminetetraacetic acid (EDTA) and Nitrical showed significant drop-off in stain performance. Four solutions had acceptable cutting performance at 1 hour of decalcification, but Immunocal was slower, with adequate cutting obtained at 2 hours. Formical-2000, Formical-4, and Immunocal demonstrated acceptable, similar staining performance, but Immunocal required longer decalcification times. These solutions should be considered as leading candidates for use in histology laboratories.