Mazzi et al. Supplementary Material.

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SUPPLEMENTARY MATERIALS AND METHODS

HC analysis by STR. HC analysis by STR was performed using the AmpFISTR Profiler Plus (Applied Biosystems) according to the manufacturer’s recommendations, using 2-5ng genomic DNA extracted by the EZI DNA blood kit (Qiagen). The kit amplifies nine tetranucleotide polymorphisms in the following loci: D3S1358, vWa, FGA, TH01, TPOX, CSF1P0, D5S818, D13S317, D7S820 and the amelogenin gene. PCR products were separated using an automatic sequencing device (Automatic Sequencer 377, Perkin Elmer), according to the manufacturer’s recommendations. Results were analyzed using the Genescan software (Applied Biosystems). For quantitative determination of donor (D)- or host (H)-specific signals, the peak areas of interest were evaluated as a fraction of the total area under the peaks present in the sample.

HC analysis by HLA tying using PCR-sequence specific oligonucleotide probing (SSO). Patients transplanted from donors mismatched for HLA-A,B,C,DRB,DQB1 were subjected to HC analysis by 2-digit genomic typing of these loci, using the Dynal RELITM SSO kit (Dynal-Biotech, Invitrogen Corporation), according to the manufacturer’s recommendations. Briefly, 100ng of genomic DNA, extracted by the QIAmp DNA blood minikit (Qiagen), was amplified using biotinylated locus-specific primers and hybridized to an array of immobilized probes on a nylon strip, in the semi-automatic hybridization device AutoRELI 48 (Dynal-Biotech, Invitrogen Corporation). Positive signals were read by scanner image capturing (Dynal-Biotech, Invitrogen Corporation). On the basis of patient and donor HLA typing, informative H- and/or D-specific probes were identified and analyzed for signal intensity (SI) colorimetrically or by visual assessment. Colorimetric analysis was performed only on samples for the determination of the standard curve (supplementary Figure 1). The percent relative intensity (RI) of each probe was assigned by normalizing against SI obtained for each probe in the presence of 100% donor or of 0% donor, according to the formula RI = (SI x% - SI 0% / SI 100% - SI 0%) x 100. Mean %RI was calculated by dividing the sum of RIs of all H- or D-specific probes by the total number of these probes. The visual ranges were corrected on the basis of comparison between the signal intensity of host and donor-specific probes, as “full donor” (100% D = 0% H), “minimal host” (1-5% H = 95-99% D), “significantly weaker host than donor” (5-10% H = 90-95% D), “weaker host than donor” (10-30% H = 70-90% D), “almost equal host/donor” (70-30% H = 30-70% D), according to the colorimetric RIs resulting from the standard curve (supplementary Figure 1) .

HC analysis by HLA-typing using PCR-sequence specific priming (SSP). Patients transplanted from donors mismatched at the HLA-DPB1 locus were subjected to HC analysis by 4-digit HLA-DPB1 typing using the Olerup SSP™ kit (GenoVision-Quiagen), according to the manufacturer’s recommendations. Briefly, 24ng of genomic DNA, extracted by the QIAmp DNA blood minikit (Qiagen), was used for each of 48 mixes of DPB1 allele-specific amplifications. On the basis of patient and donor HLA typing, informative H- and/or D-specific mixes were identified. In hypo-cellulated samples with low extraction yields of genomic DNA, only the informative patient- and donor-specific PCR reactions were carried out. PCR products were analyzed on a 2.5% agarose gel, and specific bands were evaluated visually. Visual SI was scored on the basis of comparison between the intensity of donor- and host-specific bands, and classified as minimal (1-10% H), intermediate (10-30% H) or strong (30-70% H).

Construction of a standard curve for HC analysis. Peripheral blood (PB) from two haploidentical healthy sisters was mixed at defined percentages of white blood cells (WBC) and analyzed for specific HLA or STR signals as described above. HLA typing of the two sisters was HLA-A*02,*11; B*41,*51; Cw*15,*17; DRB1*07,*11; DQB1*02,*03; DPB1*0301,*0402 and HLA-A*11,*24; B*18,*51; Cw*07,*15; DRB1*1101,*1104; DQB1*03; DPB1*0301,*0402; respectively. Since the two sisters were identical for HLA-DPB1, for this locus, PB from one of the sisters was mixed with PB from an unrelated healthy individual typed as DPB1*1001,*1101 at the same defined WBC ratios used for analysis of the other loci.


SUPPLEMENTARY FIGURE 1. Standard curve for HC analysis by SSO. Shown is the mean relative intensity (RI) of 5 host (H)-specific (black bars) or 4 donor (D)-specific (grey bars) probes at different H to D WBC ratios, as described in the supplementary Materials and Methods. The derived visual ranges of percentages H/D are given below the x-axis. Note the linear increase of %RI in the range between 1 and 30% host cells, and the significantly less marked increase thereafter (40-90% host cells).


SUPPLEMENTARY FIGURE 2. HC follow-up by SSO, STR or FISH for the Y chromosome in transplants TX#26 and 27. Shown is the percentage of HC determined by the three methods on monthly BM aspirates after transplantation. Open and filled circles indicate the absence or presence of Y chromosome positive metaphases in FISH analysis, respectively. The two dashed lines indicate the time of two consecutive morphological disease relapses (12% and 7% blasts in BM aspirate, respectively), with intermittent complete remission. Arrows indicate a positive HC in SSO (white arrows) or STR (black arrows), in isolated single positive (SP) analysis (short vertical arrows) or in SP with concomitant suspect (CS; long oblique arrow).