A limited number of IgH-primers binding to framework region 1 is sufficient to detect the majority of mature small B-cell non-Hodgkin lymphomas on formalin-fixed paraffin-embedded tissue by PCR.
IGH gene rearrangement analysis by PCR is the widely accepted tool to determine clonality of B-cell lymphoid proliferations on formalin-fixed, paraffin-embedded tissue, but the results are often unsatisfying in terms of sensitivity. This is mainly due to poor quality DNA because of degradation and hence difficulties to amplify products of the needed length. Therefore, most previous attempts to determine clonality have depended on primers binding to framework region 3 thus producing amplification products of relatively short length. In order to improve clonality analyses, we have developed a sensitive monoplex PCR-protocol using primers binding to framework region 1 with extended cycling (42 cycles) and subsequent heteroduplex analysis. For comparison, multiplex reactions with alternative primers binding to framework region 1 according to the BIOMED-2 protocol were analyzed. By the two methods combined, we were able to detect clonality of 94% (16/17) of mature small B-cell non-Hodgkin lymphomas. The results suggest that PCR with primers binding to frame work region 1 may be the method of choice when assessing clonality of mature small B-cell non-Hodgkin lymphomas on formalin-fixed tissue.
Nelson
Dick
D
Department of Clinical Chemistry, Helsingborg Hospital, Helsingborg, Sweden.
Nelson
Axel
A
Hjorthagen
Lena
L
Sjövall
Elisabet
E
Ehinger
Mats
M
D017931:DNA Primers; D005557:Formaldehyde; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis / Q000235:genetics; D016393:Lymphoma, B-Cell / Q000175:diagnosis* / Q000235:genetics; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity