Simultaneous detection of X and Y chromosomes by two-colour fluorescence in situ hybridization in combination with immunophenotyping of single cells to document chimaerism after sex-mismatched bone marrow transplantation

Abstract

Summary:A powerful approach to documenting engraftment after allogeneic BMT is the quantification of the degree of chimaerism in distinct haematopoietic cell lineages. This cannot be achieved by the recently developed, quantitative, modifications of PCR amplification of highly polymorphic DNA markers, unless this technique is applied to separated cell populations. Here, we report the development of a new method, in which cells are simultaneously characterized by enzymatic immunophenotyping and identified for their origin by twocolour fluorescence in situ hybridization with X and Y chromosome-specific DNA probes (XY-FISH/immunostaining). The method enables the rapid, reliable and quantitative analysis of chimaerism within distinct cell lineages after sex-mismatched BMT, without the requirement for cell separation techniques. This is illustrated by investigation of the pattern of chimaerism in patients receiving a sex-mismatched BMT for the treatment of primary immunodeficiencies. The results obtained with the quantitative XY-FISH/immuno staining method show a good correlation with the data generated by the semi-quantitative analysis of PCR amplified minisatellites in FACS-sorted cell fractions. In addition, XY-FISH/immunostaining was successfully applied to detect materno-fetal engraftment of T cells in a SCID patient. Keywords: BMT; chimaerism; XY-FISH; immunophenotyping Investigation of haematopoietic chimaerism after allogeneic BMT is relevant to the documentation of early engraftment, graft rejection, leukaemic relapse as well as the occurrence of incomplete engraftment, ie mixed or split chimaerism. [1][2][3][4] In general, two different strategies are pursued to investigate the pattern of chimaerism within distinct haematopoietic cell lineages. First, the cell (sub)populations of interest are isolated by FACS-sorting or immunomagnetic bead fractionation after labelling surface membrane antigens selectively expressed on these cells. The donor and/or recipient origin of the isolated cells can be determined by PCR amplification of highly polymorphic DNA markers, such as mini-and micro-satellites. [5][6][7] Second, in the case of sex-mismatched BMT, cells are simultaneously characterized for their lineage affiliation and donor or recipient origin through the combination of immunophenotyping and detection of the presence of the X or Y chromosome by in situ hybridization. [8][9][10][11][12] This method generates quantitative data, can be completed within 2 days, and requires only a small number of cells. The main disadvantages are that its use is limited to sex-mismatched BMT and, more importantly, that hybridization with a single chromosomal probe leaves room for speculation concerning the origin of some cells, due to the inevitable imperfection of the in situ hybridization procedure. The latter problem can be circumvented by the simultaneous usage of X-and Y-specific DNA probes, and by the application of the presence of two sex chromosomal signals as the scoring criterion in any cell. [13][14][15] In the curren...

Graft Versus Host Disease in Intestinal Transplantation

“…Fluoresence in situ hybridization (FISH) for X and Y chromosomes was used when possible in cases of sex mismatch between donor and recipient to document donor-cell infiltration in tissue (14). Percentage of donor cells in peripheral blood was determined by immunostaining for donor HLA markers when available, or as a percentage of recipient HLA negative cells (15) when appropriate antibodies for donor HLA were not available.…”

Early determinants of long-term T-cell reconstitution after hematopoietic stem cell transplantation for severe combined immunodeficiency

“…Chimerism of the different cell lineages was determined by XY-fluorescence in situ hybridization (FISH) or by polymerase chain reaction (PCR) amplification of short tandem repeats as previously described. 19,20 TRECs and telomere lengths DNA was purified from CD4 ϩ or CD8 ϩ T cells using the QIAamp Blood Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Signal joint TREC content of these fractions was quantified using real-time PCR as described previously.…”

HLA-identical haematopoietic stem cell transplantation for acute leukaemia in children: less relapse with higher biologically effective dose of TBI

“…(for two consecutive determinations) and of platelet engraftment as the first day at which blood platelets rose to 50 Â 10 9 /l (untransfused). Engraftment of different donor cell lineages was documented by XY-FISH analysis in patients with opposite sex donor 29 or by analysis of variable number of tandem repeats 30 and later on of CA repeats 31 with PCR technology.…”