Detection of circulating clonotypic cells among sorted PBMC B cells using single-cell in situ RT-PCR with patient-specific primers. (A) Single-cell RT-PCR using sorted B cells. Individual FMC63⁺ B cells were sorted directly into PCR tubes. Nested PCR first with consensus, and second with patient-specific primers was performed. The products had the expected size of 111 bp (Patient 1) or 160 bp (patient 4). M = molecular weight markers; Lanes 1 to 8 = sorted B cells; lanes 9,10 water controls. (B) In situ RT-PCR using sorted B cells. Row 1, CD19⁺ cells sorted from a peripheral blood of MM patient 11 and from a healthy donor underwent in situ RT-PCR amplification with patient 11 CDR2/CDR3-IgH–specific primers. Row 2, CD19⁺cells sorted from a peripheral blood of MM patient 12 and from a healthy donor underwent in situ amplification with CDR2/CDR3-IgH–specific primers for patient #12. Row 3, CD19⁺ cells sorted from a peripheral blood of MM patient 1 and from a healthy donor underwent in situ amplification with CDR2/CDR3-IgH–specific primers for patient 1. Row 4, CD19⁺ cells sorted from a peripheral blood of MM patient 5 and from a healthy donor underwent in situ amplification with CDR2/CDR3-IgH–specific primers for patient 5.