Biomedical subjects

P Hakenberg

Publications and source records attributed to P Hakenberg.

5 recordsLinked to original sources

PubMed2000

HLA-DRB1,3,4,5 and -DQB1 allele frequencies and HLA-DR/DQ linkage disequilibrium of 231 German caucasoid patients and their corresponding 821 potential unrelated stem cell transplants.

Allelic matching within the HLA-DRB1 and -DQB1 loci significantly improves the clinical outcome of hematopoietic stem cell transplantation. Consequently, allelic typing of these loci is strongly recommended for the unrelated stem cell donor selection. In this study, the HLA-DRB1,3,4,5 and -DQB1 alleles of 231 patients and their corresponding 821 nonrandom potential stem cell donors were determined to define compatible donor/recipient pairs. Highly accurate HLA typing data were achieved by PCR-SSOP and a combination of group specific PCR-SSP and subsequent sequencing-based typing of nearly the whole second exon of each locus. The alleles DRB1*07, *09, and *10 were analyzed by PCR-reverse dot blot hybridization instead of sequencing. Additionally, DRB1 homozygosity was verified by temperature gradient gel electrophoresis. The identified 2104 HLA-DRB1 and HLA-DQB1 alleles as well as data on HLA-DRB3, -DRB4, and -DRB5 alleles were applied to a statistical program and absolute and relative delta values of DR/DQ linkages were calculated. The achieved data on the HLA-DRB1 allele distribution and on DR/DQ associations in terms of subtypes significantly ensure the typing reliability, since rare allele combinations will result in further investigations. Furthermore, detailed data on the DR/DQ allele associations allow estimations of the number of HLA-A, -B, and -DR matched unrelated stem cell donors necessary for the identification of DRB and DQB subtype identical donors.

Alleles↗

PubMed1998

NETCORD: a cord blood allocation network.

Several CB-specific prerequisites have to be taken into consideration to deal with the increasing number of stem cell transplants using multiple HLA mismatched umbilical cord blood (CB) specimens. Since CB represents a limited and expensive resource it demands an optimal allocation. Optimal CB allocation can not be optimal based solely on 'first come - first serve' basis, since frequently several CB specimens are simultaneously suitable for more than one patient. Furthermore, the immediate availability of CB gives rise to an above average proportion of urgent search requests for patients who need a transplantation urgently. Thus continuing reservation and release of a CB specimen has considerable influence on the stem cell transplant prospects of other patients. To avoid and/or solve allocation conflicts present in a much higher incidence than in the bone marrow donor setting a global co-ordinated allocation of cord blood specimens is of major advantage for these patients. Therefore, the CB allocation network NETCORD has been founded. Within NETCORD common rules for data representation and logistics were developed to make CB data comparable and interchangeable. The extended need for fast and reliable communication is met by modern communication technology based on Internet standards.

Blood Banks↗

PubMed1996

Hematopoietic transplant potential of unrelated cord blood: critical issues.

To date, hematopoietic stem and progenitor cells from human umbilical cord blood (CB) have been employed in approximately 90 allogeneic (56 sibling and 34 unrelated) matched and mismatched transplantations worldwide with easy and successful restoration of hematopoiesis. Requests for stem cell preparations from CB will continue to increase. Thus, as a pilot study, the examination and standardization of unrelated cord blood-derived stem cell preparations and banking as well as their biologic characterization were initiated. Up to October 1995, a total of 574 samples [mean volume 79 +/- 26 ml, total nucleated cells (NC) 8.5 +/- 5 x 10(8), BFU-E 9.5 +/- 8.6 x 10(5), CFU-GM 5.7 +/- 6.3 x 10(5), CFU-GEMM 1.6 +/- 1.9 x 10(5)] from cord-derived or placental-derived residual blood have been defined by hematologic, immunologic, and microbiologic criteria. These CB samples were collected from the umbilical cord vein immediately after vaginal full-term delivery (n = 450) or cesarean section (n = 124) and stored frozen in liquid nitrogen. Seven percent of all samples collected could not be considered for potential transplants because of volumes < 40 ml. Only 5.0 ml of a CB sample is required for routine laboratory testing, consisting of HLA class I typing, HLA class II typing by sequence-specific oligonucleotide probes (PCR-SSOP), ABO typing, sterility control, assessment of progenitor and stem cells by colony-forming and LTC-IC assays, and CD34+ status. To assess the potential problem of contaminating maternal cells, a PCR was performed on 7 representative samples. During the initial 6 months of the unrelated CB collection program, a median bacterial contamination rate of 18% (20% skin flora species, 80% perineal flora species) was encountered, which has since been reduced to < 1% through practical experience. With regard to viral infections, maternal sera was tested for HBsAg (0.6% positive), anti-HCV (0%), anti-HAV (IgG 18%, IgM 0%), anti-HIV-1-2 (0%), anti-EBV (IgG 98%, IgM 0%), anti-HTLVI-II (0%), anti-CMV (IgG 43%, IgM 0.4%), toxoplasmosis (46%) and syphilis (0%). In addition, all cord blood samples were tested by PCR for CMV infection. With regard to its clinical relevance, it is important that only 0.3% of all the samples were positive for CMV by this sensitive method. This may represent a critical advantage of CB grafts over bone marrow (BM) since, in contrast, > 40% of the unrelated BM donors have been identified to be positive for CMV. An additional advantage of CB is that since 20% of CB samples were collected from ethnic minorities, it appears possible to balance common HLA types and uncommon HLA types represented in this group. In summary, with the extensive practical experience of the obstetric collection team as well as the stem cell-processing laboratory, it appears feasible to obtain a 90% yield of unrelated CB-derived stem cell preparations for banking, which clearly should meet the medical and regulatory qualification criteria required for clinical transplantation. To test the feasibility of hematopoietic transplant potential of unrelated CB for adult patients, ex vivo expansion of CD34+-enriched stem/progenitor cell populations isolated from fresh or frozen CB was attempted in the presence of rh-IL-3, rh-IL-6, rh-EPO, rh-GM-CSF, and rh-SCF with or without fit 3. At varying time points (days 0, 2, 4, 7, 14, 21), the contents of these cultures were analyzed for the numbers of cells, CFC (BFU-E, CFU-GM, CFU-GEMM), and LTC-IC. In this setting, the increase of cells was 200-fold, that of CFC 70-fold, and most importantly that of LTC-IC was 4.5-fold after 7 days in culture in the presence of flt3. In conclusion, LTC-IC derived from CB can be maintained and considerably expanded ex vivo from highly enriched CD34 + CB cell populations from fresh or frozen CB samples.

ABO Blood-Group System↗

PubMed1993

Direct and indirect estimation of the sex ratio of mutation frequencies in hemophilia A.

Carrier detection tests were carried out in 119 families with hemophilia A by using the data obtained with current DNA techniques (e.g., RFLP analysis and direct identification of mutations), conventional carrier detection tests (e.g., factor VIII:C and von Willebrand factor antigen), and pedigree information. On the basis of this data, we estimated the sex ratio of mutation frequencies with three completely different methods and compared the results. Since the classical indirect method derived from Haldane is substantially influenced by reproductive fitness (f), the sex ratio of mutation frequencies was estimated for both f = .3 and f = .5, resulting in a male:female mutation ratio of 5.37 (95% confidence interval 2.16-13.02) and 3.26 (95% confidence interval 0.97-8.73), respectively. According to the equilibrium-independent indirect method formulated by Rosendaal et al., the male:female ratio was estimated to be 3.4 (95% confidence interval 1.18-8.81). Since current DNA techniques provide information on the grandparental origin of the patient's X chromosome, we were twice able to estimate directly the male:female mutation ratio as 15:1, by using the quotients of mutation origin (maternal grandfather/maternal grandmother and maternal grandfather/patient's mother, respectively). Application of the Fisher test shows that the male mutation rate is higher than the female rate (P = 8.55 x 10(-7). Since all three completely different approaches unequivocally showed a higher male than female mutation frequency, this should be considered to be an established fact in calculating the risk in hemophilia A families.

Adolescent↗

PubMedDate not supplied

Haematopoietic transplant potential of unrelated and related cord blood: the first six years of the EUROCORD/NETCORD Bank Germany.

To date, human umbilical cord blood (CB) has been employed successfully in well over 1000 allogeneic (unrelated and sibling) stem cell transplantations. Because of primary limitations in volume and cell numbers, over 90% of these transplantations were performed in children. Therefore requests for well standardised cord blood units of high quality are now increasing constantly. Examination and standardisation of unrelated and related cord blood stem cell preparations and banking as well as their biological characterisation was already initiated in Düsseldorf in 1992. Hitherto a total of 3236 CB samples with a mean volume of 89 +/- 25 ml, a mean total number of nucleated cells (NC) of 10 +/- 5 x 10(8) and a mean number of CFU-GM of 6 +/- 5 x 10(5) have also been validated by haematological, immunological and microbiological criteria. In addition to that, 97 directed CB donations of siblings with a clinical indication have been characterised and banked along the same lines. All CB units were collected from the umbilical cord vein immediately after vaginal full term delivery or caesarean section, then frozen and stored in liquid nitrogen. 1940 CB units were stored unseparated, the other 1296 were volume reduced using Hetastarch (HES) with a mean recovery of 85 +/- 13% of the nucleated cells, 86 +/- 12% and 84 +/- 13% for CFC and CD34+ cells, respectively. Only 5.0 ml of a CB sample is required for routine laboratory testing as there are HLA-class I typing, HLA-class II typing by sequence specific oligonucleotide probes (PCR-SOP), ABO typing, sterility control, assessment of progenitor and stem cells by colony forming assays, and CD34+ status as well as certain viral infections such as CMV, Hepatitis B, C, HIV, Parvo B19 by PCR technology before releasing the CB unit for transplantation. For apparent viral infections, maternal sera obtained at birth were tested for HBsAg, anti-HBc, anti-HCV, -HAV-(IgG, IgM), -HIV-1-2, -EBV- (IgG, IgM), -HTLVI-II, -CMV (IgM, IgG), toxoplasmosis and syphilis. Within the last three years a total of 4860 preliminary searches and 680 extended unit reports were submitted to the CB bank Düsseldorf by fax or World Wide Web. So far 68 unrelated and 3 related CB units were delivered. From these 70 have been transplanted in 30 different transplant centres world-wide. Until now the evaluation of the first 53 unrelated CB-transplantations was performed together with the EUROCORD transplant registry. Three patients were excluded from the analysis, since they received an unrelated CB-transplant for non-engraftment after previous allotransplants. The median patient age of these 50 patients was 5.0 years (range 0.3-44), the median weight 18 kg (range 4-70 kg). The majority of the patients transplanted for malignancies (66%) suffered from ALL (n = 19), AML (n = 7), CML (n = 4) and lymphoma (n = 2) with two third (75%) in an intermediate (2nd CR) or advanced stage of disease (> 2nd CR); 13 patients had metabolic diseases and immunodeficiencies and three had aplastic anaemia. All CB samples as well as the patients' blood samples were typed in Düsseldorf for HLA-class I by serology confirmed by PCR-SSP and by high resolution DNA typing for HLA-DRB1 and HLA-DQB1 alleles. 96% of the 50 patients receiving unrelated CB were mismatched at one or more HLA-antigens. 41 of the 50 patients transplanted with unrelated CB from Düsseldorf were evaluable for engraftment with an overall engraftment rate of 83%. According to the defined criteria of EUROCORD, 9 of the 50 patients were not evaluable for engraftment, since they died before day 60. The present median follow-up time is 14 months (1.4-38). The Kaplan-Meier estimate of survival at one year is 42 percent. The three paediatric patients after sibling CB transplantations (ALL, amegakaryocytic thrombocytopenia and CML) are alive with a follow-up time of 350, 379 days and 531 days. (ABSTRACT TRUNCATED)

Adolescent↗