Optimization of PCR amplification for B- and T-cell clonality analysis on formalin-fixed and paraffin-embedded samples.
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.
Bereczki
Laszlo
L
Laboratory of Tumor Pathology and Molecular Diagnostics, Institute for Biotechnology, Bay Zoltan Foundation for Applied Research, Szeged, Hungary.
Kis
Gyongyi
G
Bagdi
Eniko
E
Krenacs
Laszlo
L
D001402:B-Lymphocytes / Q000378:metabolism / Q000473:pathology*; D002999:Clone Cells; D004273:DNA, Neoplasm / Q000235:genetics; D005188:False Negative Reactions; D005189:False Positive Reactions; D005557:Formaldehyde; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics; D015334:Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor / Q000235:genetics; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D016393:Lymphoma, B-Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D016399:Lymphoma, T-Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D015636:Magnesium Chloride; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism / Q000473:pathology*; D019914:Taq Polymerase; D016707:Tissue Fixation
D004273:DNA, Neoplasm; D007143:Immunoglobulin Heavy Chains; D016692:Receptors, Antigen, T-Cell, gamma-delta; D015636:Magnesium Chloride; D005557:Formaldehyde; D019914:Taq Polymerase