A comparative study of the quality of DNA obtained from fresh frozen and formalin-fixed decalcified paraffin-embedded bone marrow trephine biopsies using two different methods.

Abstract

Introduction and aims: Given its prognostic value, there is renewed interest in molecular staging in on-Hodgkin's Lymphoma (NHL) using immunoglobulin heavy and light chain (IgH, IgL) gene rearrangements. In this study, we have used two methods to compare the efficiency of DNA amplification from fresh frozen and formalin fixed decalcified paraffin embedded (FFDPE) bone marrow trephines for use in molecular staging.

Methods: After manually extracting DNA from 14 FFDPE and 13 fresh frozen trephine biopsies, two methods were used to test for amplifiability: use of the amplification control master mix supplied in the In vivo Scribe Immunoglobulin heavy chain (IgH) Clonality kit, which creates 5 amplicons between 96 -600 base pairs (bp); and real-time amplification of the β globin gene.

Results: Using the first method, the mean maximum length of amplicons generated from FFDPE trephines was statistically lower at 300 bp as compared to fresh frozen samples, all of which generated amplicons up to 600 bp in size (p<0.0001). Real-time amplification of the β globin gene showed that the mean crossing threshold of fresh frozen samples was statistically lower than FFDPE samples [23.48 (95% CI 22.47, 24.48) vs. 33.64 (95% CI 32.15, 35.12); p<0.0001].

Conclusions: Although amplifiable DNA can be extracted both from fresh-frozen and FFDPE trephine samples for IgH/ IgL analysis, freshly frozen specimens are superior as a source of template DNA, especially for higher base pair PCR products.