The distribution of HLA antigens on human corneal tissue.

Frozen sections of human corneas, as well as cultured cells derived from the epithelial, stromal, and endothelial layers, were examined for class I (HLA-A, B, C) and class II (HLA-DR) histocompatibility antigens using mouse monoclonal antibodies in an indirect immunofluorescence assay. On frozen sections, class I antigens were readily detected on corneal epithelium and keratocytes. Class I antigens were not detected on endothelial cells on frozen sections of adult corneas, but were identified on endothelial cells from some individuals less than 2 years of age. Class II antigens were not detected on corneal epithelial, stromal or endothelial cells on frozen sections. However, HLA-DR-positive dendritic cells were seen in corneal epithelium and were more numerous near the limbus. HLA-DR was expressed by cuboidal cells in the basal layer of conjunctival epithelium from several infants. Cultured cells derived from corneal epithelium, stroma, and endothelium consistently expressed class I but not class II antigens.

Modulation of HLA antigen expression on corneal epithelial and stromal cells.

Human corneal epithelial cells and stromal fibroblasts in culture were incubated with gamma interferon or with medium conditioned by phytohemagglutinin (PHA)-stimulated mononuclear cells. The corneal cells were placed into suspension, assayed for class I (HLA-A,B,C) and class II (HLA-DR) antigens by indirect immunofluorescence, and analyzed with flow cytometry. Epithelial cells treated for 5 days with conditioned medium (CND-M) did not exhibit an increase in class I or an induction of class II antigen expression, although a trend toward increased class I antigen expression was present. Epithelial cells treated for 5 days with 250-500 U/ml of gamma interferon did not demonstrate an increase in class I but did show an induction of class II antigen expression; again, however, a trend toward increased class I antigen expression was present. Stromal fibroblasts treated for 3-5 days with CND-M exhibited an increase in class I antigen expression, but stromal fibroblasts treated for 1-5 days with CND-M did not show an induction of class II antigen expression. Stromal fibroblasts incubated for 1-5 days with 250-750 U/ml of gamma interferon demonstrated both an increase in class I and an induction of class II antigen expression. These data suggest that host lymphokines may intensify the process of corneal graft rejection by augmenting class I antigen expression on allogeneic cells. Moreover, the induction of class II antigen expression by host lymphokines on cells in transplanted corneal tissue may lead to host sensitization and subsequent allograft rejection.

Laser densitometric analysis of class I HLA antigen expression by corneal epithelium.

Laser densitometric analysis of immunoperoxidase stained tissue was used to quantitate class I HLA (HLA-A,B,C) antigen expression by human corneal epithelium. Frozen sections of human donor corneas stored in modified McCarey-Kaufman medium for less than 24 hr were evaluated for class I HLA antigen by an indirect immunoperoxidase technique using a monoclonal antibody reactive against a class I HLA antigen determinant. Photomicrographs of stained epithelium taken under standardized conditions were evaluated by laser densitometry. Measurements from peripheral and central corneal epithelium on the same tissue section were compared. Total stain, stain density, and stain intensity were higher for peripheral than for central corneal epithelium, indicating that class I HLA antigen expression is greater for peripheral than for central epithelium.

HLA typing of cultured amniotic fluid cells.

HLA typing was performed on 18 cultures of human amniotic fluid cells using cytotoxicity and absorption technics. Confirmation of antigen assignments was obtained in nine of ten instances, where HLA typing also was performed on cord blood. Three major problems were encountered in performing these studies: (1) complement cytotoxicity, (2) false-positive reactions, and (3) false-negative reactions. False-positive and false-negative reactions occurred more frequently with sera defining HLA-B locus specificities than with sera defining HLA-A locus specificities. Absorption studies were helpful in making antigen assignments when false reactions occurred. Preliminary studies suggest that the frequency of false-positive reactions can be decreased by absorbing HLA typing sera with antigen-negative amniotic fluid cultured cells, buffy coat, or platelets. Accurate antigen assignment is difficult when parental HLA types are unavailable.

The role of hematopoietic growth factors in transfusion medicine.

Hematopoietic growth factors have already had an enormous impact on transfusion practice by eliminating or reducing the need for red blood cell transfusions in a variety of anemic states characterized by an absolute or relative decrease in erythropoietin. In addition, GM-CSF and G-CSF have stimulated the production of autologous neutrophils in febrile neutropenic patients in whom granulocyte transfusions had been considered ineffective. With the discovery of c-Mpl ligand and the promising results obtained with IL-11 and IL-3, a combination of growth factors that successfully stimulate platelet production may soon be identified. This first era in the clinical application of hematopoietic growth factors has been characterized largely by treatment of the patient to stimulate production of autologous cells or to enhance the ability of transplanted hematopoietic progenitor cells to repopulate the patient. The use of G-CSF to increase the yield of granulocytes harvested by apheresis procedures and to mobilize peripheral blood stem cells in allogeneic donors has initiated a new era in which the cell donor is treated to enhance cell production and enhance the repopulating ability of hematopoietic progenitor cells. As our understanding of hematopoiesis grows, scientists will be able to identify growth factors to overcome or correct deficient hematopoiesis. Increasingly, component transfusions will be reserved for life-threatening situations in which endogenous cell production cannot be stimulated or cell production will be too slow to prevent life-threatening events.

Donor origin Rh antibodies as a cause of significant hemolysis following ABO-identical orthotopic liver transplantation.

A group A, D-positive patient underwent orthotopic liver transplantation from a group A, D-negative (cde/cde) donor. Anti-D and -E were eluted from the recipient's red cells and were found in the recipient's serum 13 days later, at which time significant hemolysis developed. These Rh antibodies appear to he secondary to passive transfer of sensitized donor lymphocytes, a rare finding following liver transplantation.

Assessment of blood administration procedures: problems identified by direct observation and administrative incident reporting.

BACKGROUND: Adverse events in blood administration frequently involve the identification of transfusion recipients or components. This report details the results of an investigation of the efficacy of direct observation and that of a hospital-wide incident-reporting system in detecting standard operating procedures (SOPs) for deviations in blood administration. STUDY DESIGN AND METHODS: A process-driven audit form targeting 19 blood administration steps was developed for direct observation monitoring of blood administration. Over 18 months, 202 transfusions were observed in selected hospital locations. Data from this audit were compared with data collected from the incident reporting system. RESULTS: Through direct observation, 334 events were identified for a rate of 1.65 SOP deviations per transfusion. The incident reporting system identified 52 adverse events. Deviations were categorized as being related to the patient or component information, transfusion, patient monitoring, record documentation, and ordering or delivery of the component. Fifty-five percent of the events detected with direct observation related to identification of the patient or component, compared with 17 percent of incident reports. Using direct observation, 9 percent of transfused patients had wristband identification deviations. Such SOP deviations were not detected with the incident reporting system. Transfusion SOP deviations represented 15 percent of direct observation reports and 38 percent of incident reports. Direct observation identified deviations in monitoring practices and record documentation not detected by incident reporting. CONCLUSION: Direct observation appears to be an effective means for identifying deviations related to patient identification, patient monitoring, and record documentation.

Autologous and allogeneic red cell survival studies in the presence of autoanti-AnWj.

Chromium survival studies were performed with AnWj-positive allogeneic blood in a patient with autoanti-AnWj. 99mTc-labeled autologous RBCs that had depressed AnWj expression had normal survival (77% [94.7% 51Cr equivalent]) at 24 hours, whereas 51Cr-labeled allogeneic AnWj-positive cells had 76 percent survival at 24 hours and 55 percent survival at 7 days. These studies suggest that the specificity of the autoantibody may have implications for transfusion therapy when the development of such autoantibodies is associated with decreased antigen expression on the patient's cells.

Differentiation in human chorionic villus cultures: hCG and HLA expression.

The present report describes methods to separate, culture, and study syncytio-cytotrophoblast and mesenchymal core of the first-trimester human chorionic villus. The cultured outer layer cells (syncytio-cytotrophoblast) are multinucleated, pleomorphic, and active in the formation of human chorionic gonadotrophin (hCG). The mesenchymal core cells are more fibroblast-like in appearance, do not show multinucleation, and have less hCG in their culture media. Both cultured cell types express HLA (ABC) Class I histocompatibility antigens but not HLA (DR) Class II antigens. These and previous studies from this laboratory postulate different embryonic origins: (1) Syncytio-cytotrophoblast cultures of chorionic villus derive from differentiated trophoblast and preserve multinucleation as well as hCG hormone function. (2) Cells cultured from the chorionic villus core originate from extraembryonic mesenchyme. (3) Amniocytes (AF cells) cultured from amniotic fluid resemble the multipotential and early-stage trophoblast, retaining pleomorphism, multinucleation, and lacunae formation as well as production of hCG, progesterone, oestrogen, basement membrane glycoprotein, and Type IV collagen. These cell types cultured from the chorionic villus and amniotic fluid provide a means for in vitro study of specific embryonic cell lineages.

Immunological characteristics and clinical significance of anti-H in the Ah phenotype.

A red cell survival study with 51Chromium-labeled A1 cells was performed in a patient with the Ah phenotype whose serum contained IgM anti-H but not anti-A1. A1 cells were agglutinated weakly at 37 degrees C in the crossmatch, but not in the antiglobulin test. One hour after the survival study was begun, 67 percent of injected cells had been destroyed, and only 20 percent of the labeled cells remained after 24 hours. This study suggests that Ah individuals should be transfused only with Ah or Oh red cells.

Anti-JMH identified in serum and in eluate from red cells of a JMH-negative man.

Anti-JMH was identified in the serum of an 80-year-old JMH-negative man. Before transfusion, his direct antiglobulin test was weakly positive with polyspecific reagents, anti-C3 and anti-IgG. An eluate prepared from his red cells contained anti-JMH. Chromium-51-labeled JMH-positive cells which were weakly incompatible in vitro appeared to survive normally. Following transfusion with three JMH-positive units, the patient's hematocrit increased from 20.7 percent to 32.1 percent.

Human erythrocyte acetylcholinesterase bears the Yta blood group antigen and is reduced or absent in the Yt(a-b-) phenotype.

The Cartwright (Yt) blood group antigens have previously been shown likely to reside on a phosphatidylinositol-linked erythrocyte membrane protein. In this study, an unusual individual whose red blood cells (RBCs) were of the previously unreported Yt(a-b-) phenotype were used, along with normal Yt(a+) cells, to investigate serologically and biochemically the relationship of the Yta antigen to known phosphatidylinositol-linked erythrocyte proteins. Yt(a-b-) RBCs expressed normal amounts of various phosphatidyl-inositol-linked proteins except acetylcholinesterase. Further, human anti-Yta reacted with acetylcholinesterase in immunoprecipitation and immunoblotting studies. Thus, acetylcholinesterase is now identified as the protein bearing the Yt blood group antigens.