Developing a Method of Fluorescent In-Situ Hydridization (FISH) to use on decalified Bone Marrow Transplants

Charanjit Sihota, Cellular Pathology

Very few studies have applied FISH to routinely fixed and paraffin wax embedded bone marrow trephines (BMT€™s).  This may be because of the acid based decalcification methods that are commonly used during the processing of BMT€™s, which may adversely affect the suitability of the samples for FISH analysis.

The purpose of this study was to develop and optimise a method of FISH to use on BMT€™s which had been fixed in acetic acid zinc formalin (AZF) and decalcified for a short period (6 hours) in formic acid.  This method of fixation and decalcification was hoped to have given favourable results in terms of FISH as the previous method of formalin fixation and EDTA decalcification had given unreliable results.  In this study various FISH runs were carried out with variations to the standard FISH protocol currently in place. Variations included altering sodium thiocyanate pre-treatment incubation times, protease digestion times, alteration of the denaturing temperature and time, variations in co-denaturing and pre-denaturing.

Once a new optimised FISH method had been developed, it was applied to 10 selected BMT patient cases each known to have a B-lymphoproliferative disorder.  The results showed that this newly devised method was overall unsuccessful as the majority of the results ranged between weak to negative staining.   Therefore it was not feasible to implement this newly developed protocol into routine practice to facilitate in disease diagnostics.