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

¹ Department of Haematology, The Canberra Hospital, Garran, Australia
² Australian National University Medical School, The Canberra Hospital, Garran, Australia
³ Department of Epidemiology, The Canberra Hospital, Garran, Australia
⁴ Department of Molecular Medicine, The Canberra Hospital, Garran, Australia
⁵ Department of Anatomical Pathology, The Canberra Hospital, Garran, Australia

Correspondence to:
Dr Dipti Talaulikar, Department of Haematology, The Canberra Hospital, Yamba Drive, Garran, Canberra, ACT 2606, Australia; dipti.talaulikar{at}act.gov.au

Background: Given its prognostic value, there is renewed interest in molecular staging in non-Hodgkin’s lymphoma (NHL) using immunoglobulin heavy and light chain (IgH, IgL) gene rearrangements.

Aims: 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 using two methods.

Methods: After manually extracting DNA from 13 FFDPE and 14 fresh frozen trephine biopsy specimens, 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 compared to fresh frozen samples, all of which generated amplicons up to 600 bp in size (p<0.001). Real-time amplification of the β-globin gene showed that the mean crossing threshold of fresh frozen samples was statistically lower than that of FFDPE samples (23.48 (95% CI 22.47 to 24.48) vs 33.64 (95% CI 32.15 to 35.12); p<0.001).

Conclusions: Although amplifiable DNA can be extracted from both 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.