Summary:We prospectively studied the reconstitution of lymphocyte subpopulations in a group of 22 children, who survived disease-free at least 6 months after allogeneic BMT for a haematological malignancy. Absolute counts of total lymphocytes, B lymphocytes, T lymphocytes, and CD4 ϩ helper T lymphocytes reached the 5th percentile (p 5 ) of age-matched reference values within 6 months after BMT in 15, 17, 7 and 2 patients, respectively. In particular, CD4 ϩ helper T lymphocyte reconstitution was very slow. Unexpectedly, CMV reactivation had a profound positive influence upon the number of CD4 ϩ helper T lymphocytes in the children. In five patients, absolute B lymphocyte counts above the 95th percentile were reached from 6 months after BMT onwards, mimicking normal ontogeny. Unlike normal ontogeny, the percentages of helper T lymphocytes expressing the 'naive' CD45RA isoform were low and those expressing the 'memory' CD45RO isoform were high in the first 3 months after BMT, as described before. Thereafter, the CD45RA:CD45RO ratio slowly normalised. Also, CD7 expression was absent on up to 90% of T lymphocytes in the first months after BMT, and on a steadily decreasing percentage thereafter, as recently described in adults. However, the absolute counts of CD45RO ϩ /CD4ϩ and CD7 Ϫ /CD4 ϩ helper T lymphocytes did not change significantly. So, we found no evidence of peripheral expansion of previously primed donor-derived 'memory' T lymphocytes during the follow-up period which spanned 1-18 months after BMT. The absolute counts of 'naive' CD45RA ϩ helper T lymphocytes did not show a faster increase after BMT than in adults, despite the presumed presence of a non-involuted thymus in children. Bone Marrow Transplantation (2000) 25, 267-275. Keywords: absolute counts; bone marrow transplantation; CD45 isoforms; CD7; children; CMV; lymphocyte subpopulations BMT is increasingly used as a potential curative therapy for haematological and other malignancies, immunodeficiency diseases, and recently also for metabolic and autoimmune Correspondence: Dr E de Vries, Leiden University Medical Centre, Dept of Paediatrics, PO Box 9600, 2300 RC Leiden, The Netherlands Received 9 October 1998; accepted 28 September 1999 disorders.1-3 Unfortunately, infectious morbidity and mortality resulting from the period of immunodeficiency early post transplant are an important side-effect of BMT.Studies on immune reconstitution after allogeneic BMT have been performed extensively in adults. [4][5][6][7][8][9] The innate immune system (phagocytes) fully recovers in the first weeks to months after BMT, but complete functional reconstitution of the adaptive immune system (B and T lymphocytes) takes much longer. Therefore, infectious complications continue to be a problem for many months after BMT. 3,10 In particular, the reconstitution of CD4 ϩ helper T lymphocytes is very slow in adults. This could be related to decreasing thymic function with age. Presumably, reconstitution of the T cell compartment shortly after BMT is mainly provided by a peri...

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...