Limiting dilution analysis of freshly isolated PBMC from patient 1 and 19 using PSA. (A) Limiting dilution assay of PBMC from patient 1. The number of cells indicated in the figure were added to PCR tubes, reverse transcribed, and amplified in PSA with patient-specific primers. The transcripts in each tube were amplified in nested PCR using consensus primers to FR2 and Jh1 followed by a second amplification using patient-specific CDR2 and CDR3 primers. Of three tubes containing one cell, one was positive and two were negative suggesting an approximate concentration of the clonotypic cells in the blood of about 33%. The number of B cells as measured by flow cytometry for an aliquot of the same PBMC sample was 35%. This PBMC sample is the same as that described in Table 3, line 2, where clonotypic cells represented 18% of PBMC as measured by in situ RT-PCR on sorted B cells. (B) Limiting dilution of fresh PBMC from patient 19. IgH transcripts were amplified using VH3 family specific,40and Jh consensus primers, followed by patient-specific CDR2 and CDR3 primers in nested PCR. For this patient, 2 of 3 tubes were positive at the concentration of three cells per well (with 37% negative tubes at this concentration, the approximate frequency of clonotypic cells is one in every three PBMC, based on Poisson statistics), and 2 of 3 positive at one cell per well. By flow cytometry, patient 19 had 34% B cells in the PBMC samples used for the limiting dilution assay. (C) Clonotypic IgH transcripts at one cell per tube from patient 19. To more accurately determine the number of clonotypic cells, 96 tubes of one cell/tube were analyzed and 24 were positive (25%), as shown in the figure. With 75% negative tubes at this concentration, the chances of a given tube receiving more than one clonotypic cell are very low.