Clonal identities and multiple isotype transcripts in hematological diseases revealed by a single-strand conformation polymorphism analysis of the immunoglobulin heavy chain messenger signals.
A single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR)-amplified products of immunoglobulin (Ig) heavy chain rearrangements can be used to analyze B cell clonalities and clonal identities of B cells from different samples. However, the usefulness of the PCR-SSCP analysis is not fully assessed in B cell malignancies. For example, we did not know whether the PCR-SSCP method can be used to detect tumor-related clones in peripheral blood of patients with multiple myeloma and Waldenström's macroglobulinemia. In addition, because genomic DNA is used in the PCR-SSCP method, we could obtain no information about the isotype of the expanded B cell clone. In this study, we combined the reverse transcriptase (RT)-PCR of immunoglobulin heavy chain transcripts with an SSCP analysis and thus analyzed eight healthy individuals, five patients with B chronic lymphocytic leukemia, four patients with multiple myeloma and three patients with Waldenström's macroglobulinemia. Clonal B cell populations were detected as discrete bands in the RT-PCR-SSCP analysis that can be readily detected over the background of polyclonal rearrangements. Circulating tumor-related clones were detected in all but one peripheral blood sample from multiple myeloma and Waldenström's macroglobulinemia patients and B cell clones in peripheral blood and bone marrow from these patients showed a similar mobility on SSCP gel. Because the transcripts of different isotypes were separately analyzed, we could thus determine the isotype of B cell clones as well. When monoclonal Igs of different isotypes were detected in the individual samples, we analyzed the relationship of each monoclonal band by excising the band and then further analyzing it by a PCR-SSCP analysis. RT-PCR amplification in conjunction with the SSCP analysis is thus considered to be a useful method to detect and characterize the B cell clones in hematological diseases.
Shiokawa
S
S
Department of Clinical Immunology, Medical Institute of Bioregulation, Kyushu University, Beppu, Oita, Japan. sahusa@tsurumi.beppu.kyushu-u.ac.jp
Nishimura
J
J
Uike
N
N
Saburi
Y
Y
Suehiro
T
T
Yamamoto
K
K
D000368:Aged; D001402:B-Lymphocytes / Q000276:immunology*; D001854:Bone Marrow Cells / Q000276:immunology / Q000473:pathology; D017931:DNA Primers; D015342:DNA Probes; D005260:Female; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007132:Immunoglobulin Isotypes / Q000235:genetics*; D007075:Immunoglobulin M / Q000097:blood; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D009101:Multiple Myeloma / Q000235:genetics / Q000276:immunology*; D018807:Polymorphism, Single-Stranded Conformational; D012333:RNA, Messenger / Q000235:genetics; D012008:Recurrence; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D008258:Waldenstrom Macroglobulinemia / Q000235:genetics / Q000276:immunology*
D017931:DNA Primers; D015342:DNA Probes; D007143:Immunoglobulin Heavy Chains; D007132:Immunoglobulin Isotypes; D007075:Immunoglobulin M; D012333:RNA, Messenger