Deficiencies of natural anticoagulant proteins including antithrombin (AT), protein C (PC) and protein S (PS) are important causes of inherited thrombophilia. This study aimed to report on the practical experience gained in performing genetic analyses of a large cohort of patients with AT, PC and PS deficiencies and to relate this knowledge to clinical application. We genotyped a large cohort of 709 unrelated patients with AT (231), PC (234) and PS (244) deficiencies referred to us by physicians throughout Germany. Mutations were detected by direct sequencing and multiplex ligation-dependent probe amplification (MLPA). The highest mutation detection rate (MDR) was found for the SERPINC1 gene (83.5%), followed by the PROC (69%) and PROS1 (43%) genes. Even at AT activities close to the normal range (75%), the MDR was 70%. Contrastingly, for PC and PS deficiencies, the MDR dropped significantly and mildly lowered to subnormal values. At PS activities >55% for PS no mutations were detected. Mutation profiles of all three genes were similar with the highest prevalence for missense mutations (63-78%), followed by nonsense (7-11%), splice-site mutations (7-13%), small deletions (1-8%), small insertions/duplications (1-4%) and large deletions (3-6%). In conclusion, genetic testing is a useful diagnostic tool for diagnosing thrombophilia. Based on our data, genetic analysis for patients with AT deficiency is indicated for all subnormal activities. In contrast, genotyping is not advisable for PC activities >70% and for PS activities >55%.
Pluripotent stem cells of hematopoiesis and lymphopoiesis are among the CD34+ cells in blood or bone marrow. After granulocyte-colony stimulating factor (G-CSF) treatment, 1% to 2% of the mononuclear cells in blood are CD34+ cells, which can be procured by leukapheresis. We investigated the potential of CD34+ blood cells for reconstituting hematopoiesis and lymphopoiesis after allogeneic transplantation. HLA- identical sibling donors of 10 patients with hematologic malignancies were treated with G-CSF (filgrastim), 5 microgram/kg subcutaneously twice daily for 5 to 7 days. CD34+ cells were selected from the apheresis concentrates by immunoadsorption, concomitantly the number of T cells was reduced 100- to 1,000-fold. After transplantation, five patients received cyclosporine A for graft-versus-host disease (GvHD) prophylaxis (group I); five patients additionally received methotrexate (group II). G-CSF and erythropoietin were given to all patients. Mean numbers of 7.45 x 10(6) CD34+ and 1.2 x 10(6) CD3+ cells per kilogram were transplanted. In group I, the median times of neutrophil recovery to 100, 500, and 1,000 per mm3 were 10, 10, and 11 days, respectively. Group II patients reached these neutrophil levels after 10, 14, and 15 days, respectively. Platelet transfusions were administered for a median of 18 days in group I and 30 days in group II, and red blood cells for 9 and 12 days, respectively. Between day 30 and 60, lymphocytes reached levels of 353 +/- 269 cells per mm3. The median grades of acute GvHD were III in group I and I in group II. Two patients in group I died from acute GvHD. Two leukemic relapses occurred in group II. Complete and stable donor hematopoiesis was shown in all patients with a median follow up of 370 (45 to 481) days. Allogeneic blood CD34+ cells can successfully reconstitute hematopoiesis and lymphopoiesis. Reduction of T cells by CD34+ blood cell enrichment and cyclosporine A alone might not be sufficient for prophylaxis of severe acute GvHD.
Objectives: Human platelet alloantigen (HPA) typing has potential clinical relevance in a variety of contexts. We can improve methods for HPA genotyping by complementing our knowledge of the DNA sequence polymorphisms of HPA genes and experience with various DNA-based HPA typing techniques. Methods: A newly available DNA polymerase, AmpliTaq Gold (Perkin Elmer), provided in an inactive state and activated by heat, makes it possible to perform a hot start polymerase chain reaction (PCR) in order to prevent nonspecific amplification during the setup of PCR. To establish a practical procedure for HPA-1, 2, 3 and 5 genotyping, we applied the AmpliTaq Gold for a hot start PCR and employed 8 pairs of published sequence-specific primers (SSP). A simple simultaneous genotyping of these 4 HPA systems could be rapidly achieved with high specificity. Results: The HPA gene frequencies observed in 126 randomly selected German blood donors were 0.82 and 0.18 for HPA-la and lb, 0.92 and 0.08 for HPA-2a and 2b, 0.63 and 0.37 for HPA-3a and 3b and 0.90 and 0.10 for HPA-5a and Sb, respectively. Conclusion: Using our hot start PCR-SSP procedure with AmpliTaq Gold a simple, rapid and reproducible genotyping for HPA-1, 2, 3 and S systems could be achieved.
Summary:introduced relevant changes in the management of the harvest of haemopoietic cells by cytapheresis. 3 It is well-recognized that neither CFU-GM nor the total Determinations of committed haemopoietic progenitor cells, namely CFU-GM (colony-forming unitpopulation of CD34-expressing cells themselves are representing the truly repopulating haemopoietic cells. 4,5 granulocyte/macrophage) and of CD34-expressing haemopoietic cells as assessed by multiparameter flow Nonetheless, there is agreement that the haemopoietic short-term recovery phase is correlated to the numbers of cytometry are routine diagnostic tools in haemopoietic cell therapy. Generally, the tests are used to optimise CFU-GM transplanted 6-8 and, with even more significance, to the numbers of CD34-expressing cells transplanted. 9 the timing and management of cytapheresis and to assess the engraftment potential of the harvested cells.Notably, this correlation of cells transplanted with the days to haemopoietic short-term recovery is not a linear one. Both measurements, however, are at best surrogate markers, as an adequate routine test which effectively Whereas almost all patients having received the high cell dose, eg a cell dose exceeding the respective threshold assesses the short-and long-term repopulating haemopoietic cell is not available. Nonetheless, cell threshold dose, will recover quickly, there is a huge variability in recovery time among the patients having received a cell doses have been established. Above these thresholds rapid engraftment is almost invariable but below these dose lower than the threshold dose. We have speculated that this variability is not due to the biology, but that it thresholds the outcome is variable. In this study we have focussed on the imprecision in counting haemopoietic may be due to the imprecision of counting those haemopoietic cells to be transplanted when present at low numbers. cells, as assessed as CFU-GM and as CD34-expressing cells. The data on both tests have been analysed from Therefore, we have analysed the data on both CFU-GM colony counting and on flow cytometric counting of CD34-six European institutions. The coefficient of variation in CFU-GM colony counting was about 30%, whereas the expressing cells as determined in the routine setting at six experienced European institutions. A total number of 632 coefficient of variation in flow cytometric counting of CD34-expressing cells was about 10%. These data sugflow cytometric determinations of CD34-expressing cells and of 1460 determinations of CFU-GM colonies were gest that the technical imprecision in enumerating progenitor cells, particularly CFU-GM, at low levels, might available for analysis. make a major contribution to the clinical variability observed after transplantation of sub-threshold progenitor cell dose.Materials and methods Keywords: CD34 cells; CFU-GM; laboratory assessment; imprecision Each of the six clinical institutions enrolled in this study is active in autologous and/or allogeneic reinfusion or transplantation of haemop...
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