Quantitation of mutant N-ras gene in clonal B-cell populations.

Bone marrow mononuclear cells from patient presentation samples were dual labeled with CD138-FITC/CD10-PE (Ai) and (Bi), CD138-FITC/CD45RO-PE (Aii) and (Bii), or CD138-FITC/CD126-PE (Aiii) and (Biii), FACS analyzed, and flow sorted for immunophenotypic subsets. One thousand cells from each quadrant 1 to 4 were subjected to PCR to amplify exon 2 of the N-ras gene and an 850-bp fragment of the 5′ UTR of the BCL6 gene. Products were cloned into a bacteriophage vector for quantitative plaque hybridization to determine the relative levels of mutant and wild-type alleles as shown. The quantitative data are presented as percentage of each cell population positive for an allelic mutation of the N-ras or BCL6 gene.