Optimization of PCR amplification for B-and T-cell clonality analysis on formalin-fixed and paraffin-embedded samples

Abstract

In many cases, particularly in retrospective studies, only formalin-fixed and paraffin-embedded (FFPE) tissue samples are available for molecular studies. DNA recovered from FFPE tissues generally consists of fragmented small target sequences with chemical alterations. Clonality analysis is not easy on FFPE samples, in fact, it requires even more experience than that of performed on fresh samples or is more complicated than most genomic PCR amplifications for somatic genes. In our study, we have performed a multi-parameter PCR evaluation investigating immunoglobulin heavy chain gene (IgH) and T-cell receptor gamma gene (TCRgamma) rearrangements on non-purified crude lysates of FFPE samples, in order to establish the significance of different variables on performance of PCR amplification. The results showed that a slight decrease in the concentration of primers in combination with a slight increase in MgCl2 and Taq polymerase concentrations, as well as the use diluted crude template and a standard amount of dNTPs can be the modifications of choice while adjusting IgH and TCRgamma clonality tests on poor quality DNA FFPE samples. Using our improved protocol, 74% (17/23) of the tested B-cell lymphomas and 68% (31/46) of the tested T-cell lymphomas demonstrated monoclonal PCR product, proving the applicability of our optimized method. Our experience may be of help during the optimization process in technically difficult cases as well as to determine which parameters and how should be changed to minimize false-negative and false-positive results.

Comparison of BIOMED-2 Versus Laboratory-Developed Polymerase Chain Reaction Assays for Detecting T-Cell Receptor-γ Gene Rearrangements

“…These findings are consistent with the need for optimizing the Mg 2ϩ concentration, which has been emphasized in a study involving B-and T-cell clonality analysis of FFPE tissues. 25 When many DNA targets are amplified simultaneously, those that are either smaller or more efficiently amplified can adversely affect the others by competing for reagents. 18,26 The annealing of the multi-V V11 primer could also have been adversely influenced by the AAA sequence at the 3Ј end.…”

PCR Analysis of IgH and TCR-γ Gene Rearrangements as a Confirmatory Diagnostic Tool for Lymphoproliferative Disorders

“…However, the advantage of molecular studies as complementary diagnostic techniques in various lymphoproliferative disorders has been discussed previously [25][26][27][28][29][30]. The use of such techniques for definite diagnosis in inconclusive cases of immunomorphological studies is arguable, and has been investigated just in some case-series studies.…”

Sensitivity for the Detection of a Clonally Rearranged Antigen Receptor Gene in Endoscopically Obtained Biopsy Specimens from Canine Alimentary Lymphoma

“…, Koichi OHNO 1) *, Yuko KOSHINO-GOTO 1) , Kazuyuki UCHIDA 2) , Kohji NOMURA 3) , Masashi TAKAHASHI 1) , Ko NAKASHIMA 1) , Yasuhito FUJINO 1) and Hajime TSUJIMOTO ABSTRACT. Canine alimentary lymphoma is currently diagnosed on the basis of findings of cytological or histopathological examination.…”

“…Furthermore, the quality of the sample is known to affect the result of histopathological diagnostic sensitivity [12], which may lead to a discrepancy between the findings of histopathology and PCR analysis. Five FFPE samples were included in this study; these samples had a potential risk of DNA fragmentation during the process of formalin fixation [1]. However, all samples presented a clear band with the C primer pair (approximately 130 bp).…”