Farklı şekillerde saklanan doku örneklerinde lenfoid hücre klonalitesinin moleküler yöntemlerle araştırılması
Özet
The detection of the B lymphoid cell clonality with molecular methods on paraffin embedded materials fixed in different solutions Summary Paraffin embedded tissue samples are used for routine pathology practice. Routine histopathology and immunohistochemistry methods may not be enough to differentiate benign reactive proliferations and suspicuous neoplastic lymphoid proliferations. In this occasion molecular methods are essential for differentiation. PCR is the most preferred molecular method for this purpose. Clonality of Ig heavy chain genes is used for diagnostic purposes in these occasions. In this study we used B-cell lymphoma cases which we know clonal Ig heavy chain gene rearrangements. We used multiplex PCR method with heteroduplex analysis. The effect of fixation solution type and block age on DNA quality, quantity and purity were analysed first at the beginning of the study. The DNA samples obtained from paraffin embedded blocks of 59 B-cell lymphoma cases. 42 of them were fixed in formaline, 7 in AFA, 6 in Holland solutions. Four cases have consultation blocks where the fixation status was unknown. 11 blocks were less than one year old, 24 were one year old, 12 were two years old, 6 were three and 6 were four years old. BIOMED-2 conrol primers were used for DNA quality PCR analysis. All the samples from AFA fixed tissues have high quality (<400 bp) DNA. However 92.8% of the formalin-fixed samples, 16.7% of Holland-fixed samples have high quality DNA. The success in determination of clonality results increased with the increase in DNA quantity and quality. Multiplex PCR method with heteroduplex analysis with BIOMED-2 Concerted Action BMH4-CT98-3936 IgH (VH-JH) ve IgH (DH-JH) primers was performed. 81,4% of B-cell lymhomas were found clonal with this method. The clonality was detected in 90.5% of formalin-fixed cases and 100% of AFA-fixed cases whereas no success was seen in Holland-fixed cases. Four cases (8%) were failed to give clonality results although they have good quality DNA. These cases were diagnosed as DLBCL, HCL, BL, FL. In conclusion, multiplex PCR method with heteroduplex analysis can reliably be used for the differentiation of suspicious lymphoid proliferations. This method is highly sensitive and specific. The most important point for the success of the method on paraffin embedded material is the fixative and the fixation status. AFA and formalin are suitable solutions for this purpose whereas Holland solution is not recommended. The block age up to five years does not seem to effect the DNA quality and the success of clonality analysis. Checking the quality of DNA samples before clonality analysis is essential for interpretation of the negative results. Key Words: B-Cell Clonality, Heteroduplex Analysis, Multiplex PCR