Quality control and sensitivity of polymerase chain reaction techniques for the assessment of immunoglobulin heavy chain gene rearrangements from fixed- and paraffin-embedded samples.
Frozen tissue is considered the gold standard if DNA is to be extracted for polymerase chain reaction (PCR) analysis. In molecular studies from paraffin-embedded material, only positive results are usually taken into account. Our goal was to evaluate both the sensitivity and the specificity of PCR techniques for immunoglobulin heavy chain (IgH) gene rearrangement according to various lengths and types of fixative before paraffin embedding. One set of studies compared IgH rearrangement in a case of mantle cell lymphoma tissue that had been fixed in 14 different ways before paraffin embedding and frozen tissue. Formalin fixation was found not to be deleterious for DNA, amplification being possible up to 15 days after fixation with good sensitivity. In contrast, the performance of PCR decreased for samples fixed in Bouin's liquid for longer than 6 hours or after 48 hours of incubation in a vacuum infiltration processor (in which Bouin's liquid-fixed and formalin-fixed samples are mixed). In addition, we undertook a retrospective study of 20 routinely processed B-cell lymphomas, with frozen formalin-fixed and Bouin's liquid-fixed tissues for each case. Of the 14 positive cases on frozen material, 13 were also clonal from paraffin-embedded tissues. Whatever the IgH locus analyzed, each time the adapted control was positive, results from paraffin-embedded material were identical to results obtained from frozen tissue. In this study, we showed that the use of paraffin-embedded tissue is efficient for the study of IgH gene rearrangement. Whenever adapted controls are used, it is even possible to assess negative results.
Camilleri-Broët
S
S
Service d'Anatomie Pathologique, Hôpital Hôtel Dieu, AP-HP, Paris, France.
Devez
F
F
Tissier
F
F
Ducruit
V
V
Le Tourneau
A
A
Diebold
J
J
Audouin
J
J
Molina
T
T
D017931:DNA Primers; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D006801:Humans; D016393:Lymphoma, B-Cell / Q000235:genetics* / Q000473:pathology; D020522:Lymphoma, Mantle-Cell / Q000235:genetics* / Q000473:pathology; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016707:Tissue Fixation
D017931:DNA Primers