Sequential fluorescence in situ hybridization for the quantification of minimal residual disease in recipient cells after sex-mismatched allogeneic stem cell transplantation.

Abstract

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).