Heteroduplex analysis of VDJ amplified segments from rearranged IgH genes for clonality assessments in B-cell non-Hodgkin's lymphoma. A comparison between different strategies.
OBJECTIVE
The main difficulty of PCR-based clonality studies for B-cell lymphoproliferative disorders (B-LPD) is discrimination between monoclonal and polyclonal PCR products, especially when there is a high background of polyclonal B cells in the tumor sample. Actually, PCR-based methods for clonality assessment require additional analysis of the PCR products in order to discern between monoclonal and polyclonal samples. Heteroduplex analysis represents an attractive approach since it is easy to perform and avoids the use of radioactive substrates or expensive equipment.


METHODS
We studied the sensitivity and specificity of heteroduplex PCR analysis for monoclonal detection in samples from 90 B-cell non Hodgkin's lymphoma (B-NHL) patients and in 28 individuals without neoplastic B-cell disorders (negative controls). Furthermore, in 42 B-NHL and in the same 28 negative controls, we compared heteroduplex analysis vs the classical PCR technique. We also compared ethidium bromide (EtBr) vs. silver nitrate (AgNO(3)) staining as well as agarose vs. polyacrylamide gel electrophoresis (PAGE).


RESULTS
Using two pair consensus primers sited at VH (FR3 and FR2) and at JH, 91% of B-NHL samples displayed monoclonal products after heteroduplex PCR analysis using PAGE and AgNO(3) staining. Moreover, no polyclonal sample showed a monoclonal PCR product. By contrast, false positive results were obtained when using agarose (5/28) and PAGE without heteroduplex analysis: 2/28 and 8/28 with EtBr and AgNO(3) staining, respectively. In addition, false negative results only appeared with EtBr staining: 13/42 in agarose, 4/42 in PAGE without heteroduplex analysis and 7/42 in PAGE after heteroduplex analysis.


CONCLUSIONS
We conclude that AgNO(3) stained PAGE after heteroduplex analysis is the most suitable strategy for detecting monoclonal rearrangements in B-NHL samples because it does not produce false-positive results and the risk of false-negative results is very low.
González
M
M
Service of Hematology, University Hospital of Salamanca, Paseo de San Vicente, 58-182, Salamanca, 37007, Spain. margondi@gugu.usal.es
González
D
D
López-Pérez
R
R
García-Sanz
R
R
Chillón
M C
MC
Balanzategui
A
A
Mateos
M V
MV
Alaejos
I
I
Langerak
A W
AW
Orfão
A
A
Van Dongen
J J
JJ
San Miguel
J F
JF
D001402:B-Lymphocytes / Q000737:chemistry / Q000473:pathology*; D015139:Blotting, Southern; D002999:Clone Cells; D004396:Coloring Agents; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004996:Ethidium; D005188:False Negative Reactions; D005189:False Positive Reactions; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D005803:Genes, Immunoglobulin; D020180:Heteroduplex Analysis; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D008206:Lymphatic Diseases / Q000235:genetics / Q000473:pathology; D008228:Lymphoma, Non-Hodgkin / Q000235:genetics / Q000473:pathology*; D014411:Neoplastic Stem Cells / Q000737:chemistry / Q000473:pathology*; D012680:Sensitivity and Specificity; D016622:Silver Staining; D013194:Staining and Labeling / Q000379:methods*; D045567:VDJ Recombinases
D004396:Coloring Agents; D007143:Immunoglobulin Heavy Chains; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases; D004996:Ethidium