Safety of the blood supply.
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Bacterial Infections↗
Biomedical subjects
H G Klein
Publications and source records attributed to H G Klein.
Overexpression of human lecithin cholesterol acyltransferase leads to hyperalphalipoproteinemia in transgenic mice.
Lecithin cholesterol acyltransferase (LCAT) is a key enzyme which catalyzes the esterification of free cholesterol present in plasma lipoproteins. In order to evaluate the role of LCAT in HDL metabolism, a 6.2-kilobase (kb) fragment consisting of 0.851 and 1.134 kb of the 5'- and 3'-flanking regions, as well as the entire human LCAT gene, was utilized to develop transgenic mice. Three different transgenic mouse lines overexpressing human LCAT at plasma levels 11-, 14-, and 109-fold higher than non-transgenic mice were established. Northern blot hybridization analysis demonstrated that the injected 6.2-kb fragment contained the necessary DNA sequences to direct tissue specific expression of the human LCAT gene in mouse liver. Compared to age- and sex-matched controls, total cholesterol and HDL cholesterol levels were increased in all 3 transgenic mice lines by 124-218 and 123-194%, respectively, while plasma triglyceride concentrations remained similar to that of control animals. Fast protein liquid chromatography analysis of transgenic mouse plasma revealed marked increases in high density liposportin (HDL)-cholesteryl ester and phospholipid as well as the formation of larger size HDL. Thus, the majority of the increase in transgenic plasma cholesterol concentrations was due to accumulation of cholesteryl ester in HDL consistent with enhanced esterification of free cholesterol in mouse HDL by human LCAT. Plasma concentrations of apoA-I, apoA-II, and apoE were increased in high expressor homozygote mice who also demonstrated an accumulation of an apoE-rich HDL1. Like the mouse enzyme, human LCAT was found to be primarily associated with mouse HDL. Our studies demonstrate a high correlation between plasma LCAT activity and total as well as HDL cholesterol levels establishing that in mice LCAT modulates plasma HDL concentrations. Overexpression of LCAT in mice leads to HDL elevation as well as increased heterogeneity of the HDL lipoprotein particles, indicating that high levels of plasma LCAT activity may be associated with hyperalphalipoproteinemia and enhanced reverse cholesterol transport.
Animals↗
In vitro expression of structural defects in the lecithin-cholesterol acyltransferase gene.
Classic LCAT deficiency (CLD) and fish eye disease (FED) are two clinically distinct syndromes, associated with defects in the lecithin-cholesterol acyltransferase (LCAT) gene resulting in total (CLD) or partial (FED) enzyme deficiency. In order to investigate the underlying molecular mechanisms that lead to different phenotypic expression in CLD and FED, LCAT mutants associated with either CLD (LCAT147, LCAT156, and LCAT228) or FED (LCAT10, LCAT123, LCAT158, LCAT293, LCAT300, and LCAT347) were expressed in vitro in human embryonic kidney 293 cells and characterized with respect to LCAT expression and enzyme activity. Evaluation of mutant LCAT gene transcription by Northern blot analysis demonstrated LCAT mRNA of normal size and concentration. Although all constructs gave rise to similar intracellular LCAT mass, the amount of enzyme present in the media for LCAT147, LCAT156, and LCAT300 was reduced to less than 10% of normal, suggesting that these mutations disrupted LCAT secretion. Western blot analysis of cell culture media containing wild type or mutant LCAT demonstrated the presence of a single normal-sized band of 67 kDa. The ability of the different enzymes to esterify free cholesterol in high density lipoprotein-like proteoliposomes (alpha-LCAT-specific activity) was reduced to less than 5% of normal for CLD mutants LCAT147 and LCAT228 and FED mutants LCAT10, LCAT123, LCAT293, and LCAT347, whereas that of LCAT156, LCAT158, and LCAT300 ranged from 45 to 110% of control. Although most FED mutant LCAT enzymes retained the ability to esterify free cholesterol present in alpha- and beta-lipoproteins of heat-inactivated plasma, esterification was undetectable in all CLD mutants (LCAT147, LCAT156, and LCAT228). In contrast, all mutant enzymes retained the ability to hydrolyze the water soluble, short-chained fatty acid substrate p-nitrophenolbutyrate. In summary, our studies establish the functional significance of nine LCAT gene defects associated with either FED or CLD. Characterization of the expressed LCAT mutants identified multiple, overlapping functional abnormalities that include defects in secretion and/or disruption of enzymic activity. All nine LCAT mutants retained the ability to hydrolyze the water-soluble PNPB substrate, indicating intact hydrolytic function. Based on these studies we propose that mutations in LCAT residues 147, 156, 228 (CLD) and 10, 123, 158, 293, 300, and 347 (FED) do not disrupt the functional domain mediating LCAT phospholipase activity, but alter structural domains involved in lipid binding or transesterification.
Blotting, Northern↗
The effect of donor red cell sedimentation rate on efficiency of granulocyte collection by centrifugal leukapheresis.
BACKGROUND: Granulocyte collection relies on the use of a red cell-sedimenting agent and is influenced by poorly defined intrinsic donor characteristics. The donor's baseline red cell (erythrocyte) sedimentation rate (ESR), a readily measurable intrinsic donor variable, may influence the effectiveness of red cell-sedimenting agents and affect the granulocyte yield. STUDY DESIGN AND METHODS: The in vitro and in vivo effects of 6-percent hydroxyethyl starch on the donor ESR were prospectively studied in 67 granulocytapheresis procedures with a blood cell separator, and the findings were correlated with granulocyte collection efficiency (GCE). A relationship that predicts the GCE on the basis of the donor ESR was then derived and tested. RESULTS: The 3.7-fold mean increase in the donor ESR measured after in vitro addition of hydroxyethyl starch approximated the 3.3-fold increase measured when it was administered to the donors. Higher baseline ESR correlated with larger in vitro and in vivo hydroxyethyl starch-induced increases in ESR and predicted more efficient cell collections and greater cell yields. Furthermore, both ESR and GCE, which varied significantly among donors, remained constant for a given donor undergoing repeat procedures. The results could be summarized by a simple predictive formula that relates the GCE (%) to the ESR (mm/hour): GCE = 1.3ESR + 45. The GCE and yield as predicted by the formula were accurate within 10 percent of the observed values in six subsequent procedures. CONCLUSION: In granulocyte harvests using a blood cell separator with hydroxyethyl starch as the sedimenting agent, 1) both ESR and GCE vary widely among donors yet may remain relatively constant for a given donor; 2) the baseline ESR correlates with hydroxyethyl starch-modified ESR and with GCE; and 3) it may be possible to predict GCE and yield from the donor's baseline ESR.
Blood Donors↗
Collection and use of circulating hematopoietic progenitor cells.
Although lymphocytes and monocytes are becoming increasingly important in transfusion therapy, peripheral stem cells have been responsible for the recent explosive interest in harvesting mononuclear cells from the peripheral circulation. Despite their low concentration in peripheral blood and the consequent difficulty in cell collection, circulating hematopoietic progenitor cells are collected and used almost routinely. These mononuclear cells, possessing the capacity for hematopoietic reconstitution and the potential for definitive therapy of a variety of disorders, have been the focus of recent intense interest in transfusion medicine.
Adult↗
Cellular gene therapy.
During the last decade, molecular genetic techniques have been used increasingly to transfer human genes into mammalian cells, to correct and enhance cell function, and finally to treat human disease. Despite the current obstacles to developing even the simplest therapeutic strategy, gene therapy promises to have an almost unlimited future. The ability to collect specific blood cells in large numbers, to manipulate their expansion, growth, and differentiation in vitro, and also to cryopreserve these cells for later use has been central to the early developments in gene therapy. This article reviews the major concepts involved in blood cell-based gene therapy, a model for all somatic cell gene therapy.
Animals↗
1-Deamino-8-D-arginine vasopressin (DDAVP) increases platelet membrane expression of glycoprotein Ib in patients with disorders of platelet function and after cardiopulmonary bypass.
1-deamino-8-D-Arginine vasopressin (DDAVP) shortens the bleeding time in some patients with platelet dysfunction and decreases blood loss in some cardiopulmonary bypass patients. We studied platelet membrane glycoproteins in patients with von Willebrand disease (vWD), disorders of platelet function, and in cardiopulmonary bypass patients after infusion of 0.3 microgram/kg of DDAVP. Platelets from 8 cardiopulmonary bypass patients, receiving DDAVP immediately after surgery, were compared to those of 14 patients not receiving DDAVP. We also studied 12 patients with vWD, and 8 patients with platelet dysfunction receiving DDAVP. Fixed platelets, stained with monoclonal fluorescein (FITC)-labeled antibodies directed against GPIb (CD42b antigen), GPIb/IX, GPIIb/IIIa (CD41a antigen), CD63 antigen (a platelet activation protein), and P-selectin (CD62 antigen) were studied by flow cytometry. Binding of CD42b monoclonal antibody (MoAb) and anti-GPIb/IX to platelets from both groups of bypass patients increased during the 18-20 hr after surgery, but the group receiving DDAVP showed the greater increase (P = 0.032). Platelets from patients receiving DDAVP for vWD or for platelet dysfunction, had increases in CD42b MoAb and anti-GPIb/IX binding (P < 0.01) that coincided with shortening of their bleeding time. No changes were seen in binding of other antibodies. When platelets from normal donors were incubated with DDAVP for 20 hr, there were increases in platelet surface CD42b MoAb binding, while immunogold-stained transmission electron micrographs of permeabilized platelets demonstrated decreases in cytoplasmic CD42b MoAb binding. DDAVP increases platelet membrane GPIb expression in a variety of patients and may account for improvement in hemostasis seen in some studies. Redistribution of GPIb from the cytoplasm to the membrane may account for this increased expression.
Adult↗
Cellular gene therapy: an overview.
Recombinant DNA technology has provided the tools for gene-based cellular therapies. The increasing ability to characterize disease in molecular terms affords the scientific rationale for gene therapy, while advances in cell biology, cytokine physiology, and gene delivery have made somatic cell gene therapy feasible. In 1990, the first successful human gene therapy was performed to correct severe combined immunodeficiency resulting from an inherited disorder, adenosine deaminase deficiency. Peripheral blood lymphocytes were used to deliver a gene product as temporary therapy. Today more than a dozen clinical protocols, using a variety of cell types and gene vectors, are in different stages of development. Some promise to be curative. Potential target illnesses include a variety of inherited genetic diseases, as well as such acquired disorders as cancer and AIDS. Although basic problems relating to gene transfer efficiency and expression, assay systems, and quality assurance remain, the technology to collect large numbers of cells, to purify cell subsets, to gene-modify these cells, and to expand them for human infusion suggest practical new strategies for cellular gene therapy.
Acquired Immunodeficiency Syndrome↗
Transfusion of blood components to persons infected with human immunodeficiency virus type 1: relationship to opportunistic infection.
BACKGROUND: Although a recent study reported shorter survivals in human immunodeficiency virus type 1 (HIV-1)-infected patients who received transfusions, no study has analyzed the relationship in such patients between the frequency of opportunistic infection and transfusion. STUDY DESIGN AND METHODS: Records of 196 HIV-1-infected subjects with CD4 lymphocyte counts below 250 cells per mm3 were reviewed to determine if there were more opportunistic infections in patients who previously received transfusions than in those who received no transfusions. The decline in CD4 cells was also compared, and the frequency of transfusion reactions and red cell alloantibodies was assessed. RESULTS: The frequency of cytomegalovirus (CMV), wasting, and bacterial infections (p < 0.01), but not the frequency of Pneumocystis carinii pneumonia (PCP) (p > 0.2), was significantly increased in the patients who had previously received transfusions, and this effect was independent of CD4 count, race, or risk factor. The frequency of CMV infection, but not of PCP, was also related to the number of units of blood received (p < 0.01). Significant bacterial infections occurred primarily in persons with CMV infection, of whom there were more in the transfusion cohort. When analyzed separately in the group of patients without CMV infection, the frequency of bacterial infection was unrelated to transfusion. The death rate in those who received transfusions was also greater than that in patients who had never received a transfusion. None of the 130 patients who received red cell transfusions developed red cell alloantibodies. CONCLUSION: The higher incidence of certain infectious complications in HIV-1-infected patients who received transfusions indicates that the relationship of transfusion to virus activation in patients with acquired immunodeficiency syndrome and the potential benefits of modifying the preparation of blood components should be investigated further.
AIDS-Related Opportunistic Infections↗
Effect of amphotericin B and fluconazole on platelet membrane glycoproteins.
BACKGROUND: Fever, chills, and reduced platelet recovery may result when platelets are transfused simultaneously with amphotericin B. Amphotericin B reportedly increases the pitting of membranes in stored platelets. STUDY DESIGN AND METHODS: The effects of amphotericin B and another antifungal agent, fluconazole, on platelet membrane glycoproteins (GP) were examined by the incubation of split aliquots of fresh and stored platelet concentrates (PCs) with these drugs for 3 days in storage bags. To determine the effect of storage, PCs were stored for 5 days, and aliquots removed on Days 1 through 5 were placed in platelet storage bags with 4 micrograms per mL of amphotericin B for 2 to 6 hours. Membrane glycoprotein expression was assessed by flow cytometry with fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed against the following antigens: GPIb (CD42b), CD63 (an activation protein), P-selectin (CD62), and GPIIb/IIIa (CD41a). RESULTS: Amphotericin B produced a concentration-dependent decrease in the surface binding of CD42b MoAb with no consistent changes in the binding of CD41a, CD63, or CD62 MoAbs after a 3-day exposure. Stored but not fresh PCs showed decreased binding of MoAb CD42b after a 6-hour exposure to amphotericin B (4 micrograms/mL). Fluconazole produced no changes. When the binding of MoAb CD42b to permeabilized platelets was used to measure total platelet content, amphotericin B (4 micrograms/mL) decreased MoAb CD42b binding to a similar degree in fresh and stored platelets. Inhibition of aggregation to ADP and collagen and ADP and epinephrine was seen in stored but not fresh PCs. CONCLUSION: Therapeutic levels of amphotericin B resulted in partial loss of total platelet GPIb in fresh and stored PCs, but decreased surface expression of platelet membrane GPIb only in stored platelets. This difference between fresh and stored platelets may be related to the limited reservoir of GPIb available for redistribution to the membrane in the previously stored PCs and may account for the decreased recovery of transfused platelets observed in some patients receiving amphotericin B.
Amphotericin B↗
The hospital cost (fiscal year 1991/1992) of a simple perioperative allogeneic red blood cell transfusion during elective surgery at Duke University.
We sought to determine the actual cost to Duke University Medical Center of a perioperative red blood cell transfusion. A recent audit at Duke University Medical Center determined the base average direct and indirect hospital costs for providing a unit of red blood cells. The Transfusion Service's base cost for providing an allogeneic unit of red blood cells was $113.58. To obtain the actual hospital cost of transfusing a unit of red blood cells in the perioperative period, associated costs were calculated and added to the Transfusion Service's base cost. These associated costs included compatibility tests on multiple units per each unit transfused in the perioperative period, performing ABO and Rh typing and antibody screening on samples from patients who were not subsequently transfused, compatibility tests on units not issued, handling costs of units issued but not used, physically administering the blood, and the cost of the recipient contracting an infectious disease or developing a transfusion reaction. These associated costs increased the cost of transfusing an allogeneic unit of red blood cells in the perioperative period to $151.20. Perhaps the techniques described in the study can be used to quantify cost/benefit ratios associated with future changes in transfusion practice.
Blood Banks↗
Oxygen carriers and transfusion medicine.
The US blood supply is once again expanding (14 million units a year) and annual estimated whole blood and red blood cell (RBC) transfusion now exceeds 12 million units. The observed increase in total transfusions and units transfused per surgical procedure may result from more aggressive therapies, an aging population, and improved access to health care. While autologous blood collection has grown 20-fold in the past decade, autologous blood still accounts for < 8% of transfusions and is unlikely to replace much more of the allogeneic transfusion needs. Although safer than ever, allogeneic blood still transmits infectious disease (HIV:1 in 225,000 units, hepatitis:1 in 3300 units, HTLV I/II:1 in 50,000 units) and poses additional immunologic and non-immunologic risks. Allogeneic RBCs are probably underutilized because of safety concerns. While the cost of a unit of RBCs has been estimated at $150, costs are substantially higher in some areas and blood processing (filtration, gamma irradiation, washing) add additional expense. The narrowing margin between supply and demand, and repeated regional blood shortages argue for the value of safe, effective oxygen carriers.
Blood Substitutes↗
Mononuclear cell adoptive immunotherapy.
In contrast with red cells, platelets, and granulocytes, hemotherapy using lymphocytes and monocytes is only beginning to be explored. Blood banks and transfusion services, the traditional sources of expertise in sterile cell processing, storage, and transfusion, have played an integral role in the early technical development of mononuclear cell adoptive immunotherapy, and are logical partners in its future. This article reviews the clinical experience with this novel transfusion therapy.
Abortion, Habitual↗
Transfusion support of hematology and oncology patients. The role of recombinant hematopoietic growth factors.
Recombinant DNA technology has made more than a dozen human cytokines available to treat patients with hematologic and oncologic disorders. These small glycoproteins mediate cell growth and differentiation and regulate complex cellular networks by autocrine, paracrine, and endocrine mechanisms. Direct infusions of hematopoietic cytokines can decrease the requirement for transfusion in many patients with hematologic disorders and malignancies and permit more aggressive myelosuppressive therapy. Stimulation of blood donors with cytokines may enhance collection of a variety of transfusion components from granulocytes to peripheral blood progenitor cells. Using combinations of recombinant growth factors can effect ex vivo cell expansion and differentiation. Such cellular components are currently being studied in immunotherapy of cancer, in molecular approaches to inherited and acquired genetic abnormalities, and in bone marrow transplantation. In the future, this technology may be applied to producing transfusion components.
Blood Component Transfusion↗
Immunologic aspects of blood transfusion.
For 20 years, clinical reports and laboratory observations have suggested that allogeneic blood transfusion effects important changes in the recipient's immune response. The seminal clinical studies involved dose-dependent improvement in renal allograft survival in patients transfused with allogeneic whole blood, red blood cells, and buffy coat preparations. Subsequently, a burgeoning, but unclear literature proposed that allogeneic blood transfusion decreases survival or tumor-free survival of patients who undergo operations for a variety of different malignancies. Similar studies suggest that the risk of postoperative infection increases when patients receive allogeneic blood. Transfusion reportedly improves some patients with Crohn's disease. In summary, these findings have been interpreted as evidence for an immunosuppressive effect of allogeneic blood transfusion. A small prospective study showed that paternal buffy coat infusion decreases the rate of fetal loss in a subset of women with recurrent abortion. These data suggest induction of "tolerance." Laboratory studies confirm changes in lymphocyte subsets, lymphocyte activation, natural killer cell activity, antigen-presenting function, and phagocytic cell function in patients and animals that receive allogeneic blood. The clinical relevance of these observations remains controversial. Allogeneic leukocytes induce expression of latent cell-associated viruses (human immunodeficiency virus, cytomegalovirus, Epstein-Barr virus), suggesting further immune-mediated adverse effects of transfusion. The mechanisms and clinical importance of these observations have become areas of intense interest and investigation for transfusion medicine.
Abortion, Habitual↗
An overview of current management.
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Adult↗
Management of autoimmune disorders.
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Autoimmune Diseases↗
Management of hematological disorders and cancer.
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ABO Blood-Group System↗