Numerous applications in clinical medicine and forensic sciences depend on reliable data concerning the frequencies of human leukocyte antigen (HLA) genes and haplotypes. Assuming a Hardy-Weinberg ...equilibrium of the underlying population, these frequencies can be estimated from phenotype data using an expectation-maximization-algorithm also known under the name “gene counting.” We have refined this algorithm in order to cope with the heterogeneous resolution of HLA phenotypes frequently occurring in large datasets due to the structure of the HLA nomenclature. This was a prerequisite to analyze a set of 13,386 blood donors contributed by over 40 blood banks who were tested for HLA-DR when they volunteered to become marrow donors. This data set is still unique in the German national donor registry because their HLA-DR-typing was not biased by patient oriented searches or other strategies for selective typing. As a consequence of the size of the sample, the frequency estimates for the genes and the two- and three-locus haplotypes of HLA-A, HLA-B, and HLA-DR are of unprecedented precision and allow interesting projections concerning the efficiency and economic aspects of the development of a large donor registry in Germany.
The mechanisms by which newborns are at increased risk for invasive bacterial infections have been incompletely defined. A central element of innate immunity to bacterial infection is the ...neutrophil-a cell that contains cytoplasmic granules replete with antibiotic proteins and peptides. The activity of adult neutrophils against gram-negative bacteria is believed to depend to a significant degree on the presence in neutrophil primary (azurophilic) granules of the 55-kDa bactericidal/permeability-increasing protein (BPI), which binds with high affinity to bacterial lipopolysaccharides and kills gram-negative bacteria. In light of the importance of BPI to antibacterial host defense and to investigate possible factors underlying the risk of neonatal bacterial infections, we determined the relative content of BPI in the neutrophils of adults and newborns.
The cellular content of BPI was determined by Western blotting of neutrophils derived from full-term newborn cord blood (n = 21; mean gestational age: 38.6 weeks) and from adult peripheral blood (n = 22; mean age: 29 years). Extracellular levels of BPI in adult and newborn plasma were assessed by enzyme-linked immunosorbent assay. Neutrophil content of other azurophil granule markers also was assessed: myeloperoxidase by Western blotting and defensin peptides by acid-urea polyacrylamide gel electrophoresis and Coomassie staining. Acid extracts of newborn and adult neutrophils were analyzed for antibacterial activity against serum-resistant encapsulated isolate Escherichia coli K1/r.
The neutrophils of newborns contain at least threefold to fourfold less BPI per cell than adult neutrophils (67 +/- 13 ng per 10(6) cells vs 234 +/- 27 ng per 10(6) cells). The relative BPI-deficiency of newborn neutrophils apparently was not attributable to perinatal stress-related degranulation of intracellular BPI stores because: 1) newborn and adult neutrophils contained nearly identical amounts of 2 microbicidal constituents derived from the same primary (azurophil) granule compartment as BPI (the enzyme myeloperoxidase as well as defensin peptides), and 2) levels of extracellular BPI in newborn plasma, measured by enzyme-linked immunosorbent assay, represent only approximately 2% of cellular BPI content. As predicted by their lower BPI content, newborn neutrophil acid extracts demonstrated significantly lower antibacterial activity against E coli K1/r than did adult neutrophil acid extracts.
These data suggest that the neutrophils of newborns are selectively deficient in BPI, a central effector of antibacterial activity against gram-negative bacteria. BPI deficiency correlates with decreased antibacterial activity of newborn neutrophil extracts against serum-resistant E coli and could contribute to the increased incidence of gram-negative sepsis among newborns relative to healthy adults.neonatal sepsis, gram-negative bacteria, endotoxin, neutrophil, polymorphonuclear leukocyte, innate immunity, bactericidal/permeability-increasing protein, defensin, myeloperoxidase.
BACKGROUND: This prospective study was conducted to gain experience as to whether it is technically possible to produce autologous RBCs in additive solution from cord blood (CB), to optimize the ...blood supply for preterm infants.
STUDY DESIGN AND METHODS: CB was collected from 47 infants with a mean (± SD) birth weight of 1717 (± 699) g. Whenever possible, RBC components were prepared by standard centrifugation using a six‐bag system. All samples were put in sterility testing quarantine for 5 days, and a maximum storage of 14 days from collection to transfusion was specified. The babies were given either the autologous RBCs or standard allogeneic RBC concentrates, if autologous blood was not available.
RESULTS: In 81 percent of the samples, autologous RBC components could be processed (vol, 7‐87 mL; Hct, 31‐82%). But within the group of extremely low birth weight infants (body weight <1000 g), a mean CB net volume of only 37 mL was collected, and the RBC preparation was successful only in exceptional cases. Three CB samples (8.6%) tested positive in sterility testing. Of the 47 infants, 21 were treated with a total of 62 allogeneic and 4 autologous RBC transfusions. Most infants with a body weight over 1400 g did not need any RBC transfusion.
CONCLUSION: The preparation of autologous RBCs from the CB of preterm infants is technically possible in principle. However, major concerns must be raised as to whether such preparations are of benefit in ensuring safe care of neonates with blood components, with respect to the high rate of bacterial contamination and the limited availability in babies with low birth weight.
In Germany allotransplantation of bone marrow or peripheral blood stem cells is presently performed by 34 different teams operating more or less independently. Thus, strategies of immunogenetic donor ...search, use of the various tissue typing techniques and policy on acceptable HLA mismatches in related and unrelated settings may vary considerably from one transplant centre to another. This paper summarises the results of the first German consensus meeting on immunogenetic donor search for bone marrow/peripheral blood stem cell grafting. The main goal of the participating transplant physicians and immunogeneticists was to define national standards for the above issues.
Background and Objectives:
We describe the results of a comparative evaluation of a gel test (ID Micro Typing) and a micro‐column affinity test (MCAT, Cellbind Screen) for red cell antibody screening ...and identification under routine conditions. Materials and Methods: 3,000 serum samples of patients from the Mannheim University Hospital were tested in parallel by means of the gel test and the MCAT, using the low‐ionic‐strength‐saline indirect antiglobulin test and the protein G affinity technique, respectively. Test cells used were the same in all tests. In addition, we performed titration studies with all detected antibodies as well as with 59 frozen sera containing antibodies of known specificity. Results: A total of 154 antibodies (5.1%) were detected, 149 by gel test and 147 by MCAT. The overall sensitivity and specificity of the gel test was 96.8 and 96.5% and of the MCAT 95.5 and 97.2%. No significant differences between the gel test and MCAT were found when the titer scores of all 213 (fresh and frozen) antibodies were used to check the results. The mean scores for the gel test and the MCAT were 26.8 and 28.5, respectively. For anti‐Fya and anti‐Kell, a significantly higher titration score could be obtained in the MCAT, whereas anti‐Lua showed a significantly higher score with the gel test. Conclusion: For the screening of unexpected red blood cell antibodies, the MCAT is as sensitive as the gel indirect antiglobulin test. The sensitivity and specificity of the two systems are more or less the same although it seems that lgM antibodies are better detected by the gel test.
The present paper summarizes the results of the second German consensus meeting on immunogenetic donor search for allotransplantation of hematopoietic stem cells held in Essen in November 1999 under ...the auspices of the German Society for Immunogenetics (DGI) and the German Working Party for Blood and Marrow Transplantation (DAG-KBT). Immunogeneticists and transplant physicians from all over the country agreed to update the national standards for: (1) search strategy including the role of unrelated and extended family donor search after unsuccessful core family donor search, (2) histocompatibility loci to be typed, (3) histocompatibility typing techniques to be used (HLA serology vs DNA-based HLA typing, cellular tests, serum cross-match), and (4) acceptable HLA mismatches in the context of a defined underlying disease, donor type, and conditioning regimen.
Even after neutrophil counts return to near normal levels, patients undergoing myeloablative chemotherapy and bone marrow transplantation (BMT) are at risk for invasive bacterial infections, raising ...the possibility that their neutrophil function might be impaired. To assess potential qualitative defects in neutrophil function in patients undergoing BMT, we measured neutrophil content of the antimicrobial (poly)peptides BPI and defensins.
Neutrophil extracts were analyzed for content of BPI by Western blotting and ELISA and for defensin peptides by acid-urea polyacrylamide gel electrophoresis (PAGE). Antibacterial activity of neutrophil extracts was measured against Escherichia coli K1/r, a clinical isolate sensitive to synergistic killing by BPI and defensins.
Neutrophil extract BPI content on post-BMT days +20, +30, and +100 (169+/-35, 232+/-57, and 160+/-55 ng per 106 neutrophils, respectively) was similar to the neutrophil BPI content of normal controls (163+/-35 ng per 106 neutrophils). Neutrophil defensin content also did not vary during this time-span. Activity of neutrophil extracts against E. coli K1/r did not differ between BMT patients and controls.
At post-BMT days +20 to +100, neutrophils derived from engrafted marrow contain normal quantities of BPI and defensins. Any deficiencies of neutrophil function during this phase are not due to inadequate expression of these antimicrobial (poly)peptides but could reflect abnormalities in other aspects of neutrophil function.
On the basis of historical findings, 10% dimethylsulfoxide is still used to cryopreserve stem cells. We studied a final concentration of 5% dimethylsulfoxide in cryopreservation of cord blood-derived ...stem cells in autologous plasma. In our opinion, a final concentration of 5% dimethylsulfoxide in autologous plasma without further additives is sufficient for cryopreservation of cord blood stem cells in a banking routine.
Circulating maternal T lymphocytes were noted in the peripheral blood of six patients with severe combined immunodeficiency. Phenotypical analyses revealed the presence of both CD4 and CD8 subsets in ...some but not all cases. The maternal T cells could be stimulated by anti-TCR/CD3 mAb +/- rIL-2, but were virtually silent in the MLR and against the recall Ag purified protein derivative of tuberculin and tetanus toxoid, even in immunized patients engrafted with T cells from a responding mother. Using a panel of mAb against TCR V region gene encoded epitopes including V beta 5, V beta 6, V beta 8, V beta 12, and V alpha 2, we show that maternal T cells displayed a profoundly reduced TCR diversity, characterized by a lack of one or even several TCR V subsets in all six cases and a dramatic (5- to 25-fold) expansion of other TCR V subsets in three cases. In one patient analyzed, limited TCR diversity was also seen in T cells cultured from bone marrow and skin; restimulation experiments of these cells against cells expressing host MHC Ag were unsuccessful, as were attempts to exclusively allocate anti-host proliferative responses of maternal control T cells to the TCR V subsets that had undergone expansion in vivo. We conclude that a severely reduced TCR diversity is a common feature of maternal T cells engrafted in severe combined immunodeficiency patients. These novel findings provide a structural basis to understand the failure of these cells to protect the host from infections and may also help to understand their relative inefficiency to induce lethal, multi-organ, graft vs host disease. Moreover, as an experiment of nature, the reported phenomenon clearly illustrates the functional consequences in vivo of an insufficient TCR diversity.
Background and objectives: The very sensitive solid‐phase antiglobulin test is used widely in blood group serology. However, a disadvantage of the test is the sedimentation variability of indicator ...cells during centrifugation, which causes errors in the photometric evaluation. Materials and methods: To solve this problem a technique was developed which depends on the ‘length measurement of the absorption curve’ of the sedimented indicator cells. Results: 9,075 sera were tested for the presence of alloantibodies on the basis of the visual type of measurement and the newly developed technique. The agreement between the two evaluation systems was 99.94%. No false‐negative results were observed. Conclusion: The new method allows the complete automation of the solid‐phase antiglobulin test, enabling the analysis and evaluation of the solid‐phase microtiter plate assay to be more discriminating and safe.
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