Patients that receive a T-cell depleted (TCD) hematopoietic stem cell transplantation (SCT) show higher risk of graft failure/rejection and of disease relapse than those that receive unmanipulated grafts. The purpose of the present investigation was to analyze the usefulness of chimaerism quantification in bone marrow (BM), peripheral blood (PB), and leukocyte lineages such as T lymphocytes (CD3+,both CD4+ and CD8+), B lymphocytes (CD19+) and myeloid cells (CD15+), for the early detection of graft failure/rejection episodes and disease relapse after TCD-PBSCT. Two of the ten (2/10) patients included in the study showed stable complete chimaerism (CC). The other 8/10 patients showed decreasing mixed chimaerism (MC) and 7 of them had either graft failure (n = 1)/rejection (n = 3) or disease relapse (n = 3). In two patients relapsed from chronic myeloid leukemia, MC was observed in BM and PB, with higher percentages of autologous cells in BM, as well as in leukocyte lineages, with higher percentages of recipient cells in the myeloid lineage than in lymphocytes. Combined analysis of chimaerism and minimal residual disease allowed early diagnosis of relapse and successful rescue therapy with donor leukocyte infusions (DLI), before the onset of hematological relapse. Chimaerism analysis allowed early diagnosis of incipient graft rejection in 3 patients. These patients showed MC both in BM and PB, with greater percentages of recipient cells in PB. Analysis of leukocyte lineages showed higher percentages of autologous cells in T lymphocytes (mainly CD8+) than in B or myeloid cells. Two of these patients were successfully treated with DLI and recovered normal PB counts and BM cellularity, as well as CC. The graft versus recipient hemopoiesis effect harbored by the donor immunocompetent cells infused seems useful forthe treatment of graft rejection, provided that an early diagnosis is made.

A 63-year-old woman complained of rectal bleeding of hemorroidal type and spontaneous emission of mucus in the feces for 2 months. Laboratory tests, tumoral markers included, were normal. A colonoscopy was performed revealing a 2-cm polypoid lesion ( Fig. 1A) with a mucoid and gelatinous material ( Fig. 2) coming out of it. Cytology and biopsies were inconclusive. An endoscopic ultrasonogram showed a cystic anechoic 20-mm lesion filled with some non-solid materials located in the submucosal layer of the rectal wall (Fig. 1B), without adjacent adenopathies, and a hypertonic internal anal sphincter. Histology showed mucosal thickening with dilated glands and the presence of inflammatory infiltrates, muscular fibers, and congestive vessels in the muscularis propria, findings that can be seen in rectal prolapse. These results, in addition to endoscopic and ultrasonographic findings, established the diagnosis.

Fluorescence in situ hybridization (FISH) has advantages over molecular techniques for the detection of minimal residual disease (MRD) during follow up of patients with haematological malignancies undergoing treatment. FISH is a fully quantitative technique and enables identification of the cell morphology, when it is performed on routine smears. However, one of its main disadvantages is its low sensitivity compared with that of other molecular approaches. MRD quantification by FISH can give false negative results due to the limited sensitivity of standard approaches (1-3%) in patients with low percentages of persistent ⁄ reappearing recipient cells after sex-mismatched allogeneic stem cell transplantation (SCT). In such cases, sequential FISH can be used to increase the sensitivity of MRD studies by specifically targeting recipient cells on routine smears previously used for chimaerism analysis. For this purpose, slides hybridized with sex-chromosome specific probes (CEP XY, Vysis Inc.) were washed in 0AE1% non-idet P-40 (NP-40) ⁄ 2x saline-sodium citrate (SSC) at room temperature for 1 min, dehydrated in an ethanol series (70-85-100%), denatured in 70% formamide ⁄ 2x SSC at 72°C for 20 s and hybridized again with the appropriate probe. In the example shown here, sequential FISH was used for the follow up of chimaerism (left) and MRD (right) in a Philadelphia (Ph) chromosome-positive chronic myeloid leukaemia (CML) patient in relapse after sexmismatched allogeneic SCT. Chimaerism was analysed using sex-chromosome specific probes (labelled red for the X chromosome and green for the Y chromosome) and MRD was subsequently analysed upon the same cells using probes for the BCR (red) and ABL (green) to detect the BCR-ABL fusion (D-FISH, Oncor Inc.). Donor cells (XX, left) showed the characteristic pattern of normal cells after FISH to detect the BCR-ABL fusion (right), while reappearing recipient cells (XY; left, arrow) showed the typical pattern of Ph-positive cells (right, arrow).