Clonality analysis of B-lymphoid proliferations using the polymerase chain reaction.
BACKGROUND
Polymerase chain reaction (PCR) based assays are becoming more reliable, simpler, and faster alternatives to traditional Southern blot hybridization (SBH) analysis for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements. However, a variety of technical approaches have been reported with markedly different results.


METHODS
We analyzed the frozen tissue of 147 neoplastic and hyperplastic lesions on which SBH had previously been performed. Semi-nested and single-step PCR methods were compared. Consensus primers to the joining segments and the framework region (FR) III of the variable segments of the IgH gene were used. All PCR products were analyzed by polyacrylamide gel electrophoresis (PAGE). Thirteen samples were re-analyzed using a denaturing gradient gel electrophoresis (DGGE) system.


RESULTS
The overall concordance between SBH and semi-nested PCR assays was 80.2%. In the non-Hodgkin's lymphoma (NHL) group, 75% of the cases with IgH rearrangements by SBH were found to be monoclonal by PCR. Regardless of type of lesion, 71.7% of the cases with IgH rearrangements by SBH were found to be clonal by PCR. The concordance between the semi-nested and single-step procedures was 87.1%. DGGE was helpful in clarifying the results for cases in which the PAGE analysis was difficult to interpret.


CONCLUSIONS
PCR analysis of IgH gene rearrangements was found to be an efficient technique for the initial determination of clonality in lymphoid proliferations. The single-step method had an advantage over the semi-nested method because of its simplicity and speed. The DGGE system was useful for the assessment of clonality in cases with equivocal results after PAGE. However, a combination of these techniques in specific cases may achieve higher specificity and sensitivity.
Lozano
M D
MD
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198-3135, USA.
Tierens
A
A
Greiner
T C
TC
Wickert
R S
RS
Weisenburger
D D
DD
Chan
W C
WC
D001402:B-Lymphocytes / Q000473:pathology*; D001483:Base Sequence; D015139:Blotting, Southern; D002455:Cell Division / Q000502:physiology; D002999:Clone Cells; D005069:Evaluation Studies as Topic; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains; D016393:Lymphoma, B-Cell / Q000235:genetics / Q000473:pathology; D008228:Lymphoma, Non-Hodgkin / Q000235:genetics* / Q000473:pathology*; D016399:Lymphoma, T-Cell / Q000235:genetics / Q000473:pathology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D007143:Immunoglobulin Heavy Chains