Analysis of PNH-type cells after the second PBSCT. (A) High-sensitivity flow cytometry detected small populations of CD55⁻CD59⁻cells in both granulocytes and red blood cells at the development of the second LGF as well as in those obtained 6 and 12 months later, but did not detect PNH-type cells in the donor or in the recipient before the second PBSCT. The numbers denote the proportion of PNH-type cells in CD11b⁺ granulocytes or glycophorin A⁺ RBCs. (B) D1S80 allelic patterns of sorted GPI-AP⁻ granulocytes, GPI-AP⁺ granulocytes, and CD3⁺ lymphocytes. The polymerase chain reaction (PCR) products were subjected to 8% polyacrylamide gel electrophoresis and visualized by silver staining. (C) Nucleotide sequences of PIGA exon 2 in DNA from PNH-type granulocytes obtained 6 and 12 months after the second PBSCT. (D) A schematic illustration for ARMS-PCR is shown. Primer positions for the first, second are shown by short arrows. A black box and adjacent lines represent exon 2 and introns, respectively. (E) Amplified products of control PCR (the upper gel) and ARMS-PCR (the lower gel) were electrophoresed in 12.5% polyacrylamide gel and visualized by the silver staining. A pMD20-T vector containing the mutated exon 2 fragment was used as a positive control for ARMS-PCR. The template DNA derives from a plasmid containing the mutated exon 2 in lane 1, donor BM in lane 2, donor PB in lane 3, recipient BM in lane 4, recipient PB in lane 5, and BM from healthy individuals in lanes 6 to 11. PCR with a 5′ primer specific to the nucleotide sequence upstream of the mutated sequence amplified a 261 bp fragment from DNA of the donor and all healthy individuals.