(A) Clonal analysis by LM-PCR and I-PCR in purified CD3⁺ T cells and CD15⁺ granulocytes from 5 patients with ADA-SCID treated with GT. Nested PCR products were analyzed by electrophoresis on acrilamide gels with a 100-bp ladder as a marker (M). In Pt2, who received a low CD34⁺ cell dose and displayed poor myeloid engraftment (<0.1%), no RISs could be isolated from multiple samples of granulocytes, thus confirming the lack of potential contamination from T cells. The following representative time points are shown for LM-PCR in T cells: Pt1, 20 months; Pt2, 39 months; Pt3, 26 months; Pt4, 22 months; Pt5, 9 months. The following representative time points are shown for LM-PCR in granulocytes: Pt1, 47 months; Pt3, 33 months; Pt4, 22 months; Pt5, 12 months. The following representative time points are shown for I-PCR in granulocytes: Pt1, 5 months, Pt3, 26 months; Pt4, 4 months; Pt5, 3 months. (B) Evidence for common integrants identified in different cell subpopulations during long-term follow-up. Each RIS from Pt1, Pt2, Pt3, or Pt4 is identified by a unique number. The circles indicate the presence of a specific RIS detected after 6 months of follow-up at 2 or more different time points by random cloning in the indicated population. Numbers in red text refer to the number of independent RISs retrieved by random cloning for the time points in which ≥30 colonies were analyzed. The random cloning analysis in Pt1 (47 months) and Pt3 (33 months) T cells was not carried out because the material was not available. In Pt5, 1 common integration site was detected in T cells at 6 and 8 months after GT. The presence of common integrants was confirmed in selected cases by sequence-specific PCR, which allowed us to unequivocally track integrants in different lineages.