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.
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 two-colour 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.
van Tol
M J
MJ
Department of Paediatrics, Leiden University Medical Centre, The Netherlands.
Langlois van den Bergh
R
R
Mesker
W
W
Ouwerkerk-van Velzen
M C
MC
Vossen
J M
JM
Tanke
H J
HJ
D016026:Bone Marrow Transplantation; D002469:Cell Separation; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D018183:Transplantation Chimera / Q000235:genetics; D014960:X Chromosome; D014998:Y Chromosome