An automated semiquantitative B and T cell clonality assay

Citation
Je. Miller et al., An automated semiquantitative B and T cell clonality assay, MOL DIAGN, 4(2), 1999, pp. 101-117
Citations number
40
Categorie Soggetti
Research/Laboratory Medicine & Medical Tecnology
Journal title
MOLECULAR DIAGNOSIS
ISSN journal
10848592 → ACNP
Volume
4
Issue
2
Year of publication
1999
Pages
101 - 117
Database
ISI
SICI code
1084-8592(199906)4:2<101:AASBAT>2.0.ZU;2-Y
Abstract
Background: Rearrangements of the antigen receptor genes in B and T cells g enerate products of unique length and sequence. Polymerase chain reaction ( PCR) assays are routinely used to identify clonal lymphocyte populations by detecting clonal V-J rearrangements or chromosomal translocations within t hese antigen receptor loci. Multiple primer sets are, however, required to detect the majority of clonal B- and T-cell malignancies. Products from the individual reactions must be analyzed separately to avoid misinterpretatio n. Moreover, small clonal populations remain difficult to identify. To addr ess these difficulties, we propose that an integrated fluorescence-based ap proach to clonal B- and T-cell detection would simultaneously identify both B- and T-cell neoplasia; increase amplicon resolution, analytic sensitivit y, and assay throughput; produce more comprehensive and semiquantitative da ta useful for evaluation of hematologic malignancies; and eliminate labor i ntensive agarose and polyacrylamide gel electrophoresis. Methods and Results: Samples were genomic DNA and cDNA. Differentially labe led primers were used to amplify regions diagnostic for B- and T-cell clona lity in a single plate with a single thermocycler program. Combined amplico n products underwent capillary electrophoresis for high resolution fraction ation and differential fluorescence detection and quantification. Data were automatically analyzed and archived. In a comparative analysis of a variet y of clinical samples, this automated and integrated B- and T-cell assay sh owed >94% agreement (33 of 35 results) with individual B- and T-cell PCR as says. Furthermore, this assay had an overall monoclonality detection rate o f 100%, and as little as 100 ng of sample DNA yielded complete B- and T-cel l clonality test results. The limit of detection was approximately 10(-2) c ells, and amplicons were sized to within 0.1 basepair. Serial dilutions of clonal B- and T-cell lines comprising a coded proficiency panel were identi fied and correctly ranked. Specificity was 100% as determined by analysis o f 18 control samples that were all negative for B- and T-cell clonality. Conclusions: Our data show that this automated and integrated B- and T-cell clonality assay system is a sensitive and specific tool useful for rapid i dentification of clonal lymphocyte populations and will likely have broad c linical applications.