Toshio Mazda

DISCRIMINATION OF HUMAN HLA-DRB1 ALLELES BY PCR-SSCP (SINGLE-STRAND CONFORMATION POLYMORPHISM) METHOD

A single‐strand conformation polymorphism (PCR‐SSCP) method has been adopted for discrimination of human HLA‐DRB1 alleles. This method enabled the detection of DN A polymorphisms including point mutations at a variety of positions in the DN A fragments of the HLA‐DRB1 gene. A total of 27 HLA‐DRB1 alleles from 172 healthy donors were analysed using a combination of PCR‐SSCP with group‐specific amplifications. Application of a small amount of amplified and denatured DNA to non‐denaturing electrophoresis followed by silver staining resulted in distinct banding patterns. Samples possessing a single allele in each amplification group showed two‐band patterns which correspond to the sense and antisense strands, while heterozygotes in the same group or a mixture of two single‐type samples showed four‐band patterns. All of the analysed alleles were discriminated in each DRB1 group. The method described here may be somewhat complicated for routine typing of HLA‐DRB1 alleles. However, it is useful in the screening of ‘new’ alleles as well as the donor‐recipient molecular matching of HLA class II genes for various purposes, e.g. selection of bone marrow transplant donors.

Predictive value of screening tests for persistent hepatitis C virus infection evidenced by viraemia : Japanese experience

In November 1989, Japanese Red Cross Blood Centres started screening for heaptitis C virus (HCV) with enzyme‐linked immunosorbent assay (Elisa) for the C100‐3 viral peptide as the first such nationwide programme in the world. Thereafter post‐transfusion non‐A non‐B hepatitis (PTNANBH) was reduced by 61–80%, but this was not as complete a success as our programme to prevent post‐transfusion hepatitis B by screening for high titer hepatitis B core antibody, which we began in the same period. In order to acquire more effective control of PTNANBH, the HCV core‐related antigen (GOR, N14) and second‐generation Elisa (Ortho2, Abbott2)and second‐generation antigen agglutination (PA, PHA) tests have been employed. Among 16,500 donors in 11 blood centers, 365 were serologically positive by at least one of these tests. Among these, HCV RNA was detected in 138 units and the remaining 227 were HCV RNA negatives. The effectiveness of these serological tests to detect HCV RNA‐positive status were analyzed. Passive haemagglutination and particle agglutination (PHA and PA) tests were highly effective to predict HCV viraemia among blood donors. Also, these tests can easily determine antibody titre. By either PHA or PA, all units with ≧212 agglutination titre (120 and 122 units) were HCV RNA positive and all agglutination‐positive units with serum alanine aminotransferase level higher than 35 Karmen units were HCV RNA positive. These results have formed the basis for implementing a more effective screening for HCV viraemia in blood donors, where effectiveness is defined as enhancing the protection of patients from post‐transfusion hepatitis C and providing higher quality information to achieve more effective donor counselling.

The novel acute phase protein, IHRP, inhibits actin polymerization and phagocytosis of polymorphonuclear cells

Objective and Design: In the present study, the involvement of the binding of IHRP (inter-alpha-trypsin inhibitor family heavy chain-related protein) and actin in phagocyte activity was investigated.¶Materials and Methods: The actin polymerization and the phagocytic activity of the polymorphonuclear (PMN) cells were studied in the presence of IHRP.¶Results: IHRP inhibited the polymerization of actin and the phagocytic activity of the PMN cells.¶Conclusion: 1) IHRP may bind to actin released from the damaged cells and suppress its toxic action by preventing the formation of actin fibril. 2) IHRP may bind to cell surface actin on PMN cells and inhibit their phagocytic activities. 3) From these results, IHRP may act as an anti-inflamma-tory protein.

Detection of Hpdel among Thais, a deleted allele of the haptoglobin gene that causes congenital haptoglobin deficiency

BACKGROUND: Congenital haptoglobin deficiency is a risk factor for anaphylactic nonhemolytic transfusion reactions in Japan. The deleted allele of the haptoglobin gene, Hpdel, which causes congenital haptoglobin deficiency, has also been observed in other Northeast Asian populations, such as Korean and Chinese persons. It has not been reported in several African and European‐African populations, however, or investigated in other countries.

Immunoglobulin E oligomers identified in blood components activate mast cells: relevance to anaphylactic transfusion reaction

BACKGROUND: In most cases of anaphylactic transfusion reaction, the mechanisms underlying its development are unclear. We found a donor whose transfused blood components were implicated in two cases of anaphylactic transfusion reaction, and we found that the donor plasma showed mast cell degranulation activity.

Impacts of papain and neuraminidase enzyme treatment on electrohydrodynamics and IgG-mediated agglutination of type A red blood cells.

The stability of native and enzyme-treated human red blood cells of type A (Rh D positive) against agglutination is investigated under conditions where it is mediated by immunoglobuline G (IgG) anti-D antibody binding. The propensity of cells to agglutinate is related to their interphasic (electrokinetic) properties. These properties significantly depend on the concentration of proteolytic papain enzyme and protease-free neuraminidase enzyme that the cells are exposed to. The analysis is based on the interpretation of electrophoretic data of cells by means of the numerical theory for the electrokinetics of soft (bio)particles. A significant reduction of the hydrodynamic permeability of the external soft glycoprotein layer of the cells is reported under the action of papain. This reflects a significant decrease in soft surface layer thickness and a loss in cell surface integrity/rigidity, as confirmed by nanomechanical AFM analysis. Neuraminidase action leads to an important decrease in the interphase charge density by removing sialic acids from the cell soft surface layer. This is accompanied by hydrodynamic softness modulations less significant than those observed for papain-treated cells. On the basis of these electrohydrodynamic characteristics, the overall interaction potential profiles between two native cells and two enzyme-treated cells are derived as a function of the soft surface layer thickness in the Debye-Hückel limit that is valid for cell suspensions under physiological conditions (approximately 0.16 M). The thermodynamic computation of cell suspension stability against IgG-mediated agglutination then reveals that a decrease in the cell surface layer thickness is more favorable than a decrease in interphase charge density for inducing agglutination. This is experimentally confirmed by agglutination data collected for papain- and neuraminidase-treated cells.

The effects of pH and agitation on platelet preservation

Platelet concentrates made with an initial pH of 7.85 or 6.85 by addition of alkali or acid were stored at 22°C on tumbler or horizontal agitators. The combination of a high pH and use of a tumbler rotator was associated with a 40 percent reduction in platelet count, fragmentation of platelets, release of lactate dehydrogenase, and a marked loss in response to adenosine diphosphate. Similar, but less striking changes occurred in acidified platelet concentrates stored on the tumbler rotator. Preservation was good for both alkaline and acidified concentrates stored on the horizontal agitator.

Electrophoretic mobility of red blood cells treated with standardized protease enzymes for use in blood group serology

Abstract The electrophoretic mobility of human red blood cells treated with protease enzymes (papain, bromelin and ficin) was measured as a function of enzyme concentration and electrolyte concentration of the medium. The results are analyzed via an electrophoretic mobility formula for “soft particles” (i.e. particles covered with a surface layer of polyelectrolytes). This mobility formula involves two parameters, the fixed charge density ( N ) and a parameter 1/λ characterizing “softness” of the cell surface layer. It is found that both parameters, plotted as a function of protease concentration, exhibit a steep change around the critical protease concentration at which the cell mobility decreases abruptly and the cells become agglutinable ( N decreases and 1/λ increases). The decrease in N accounts for the observed decrease in cell mobility. The increase in 1/λ suggests that the protease treatment leads to removal of glycoproteins from the surface layer, including not only their charged portions (sialic acid) but also their uncharged portions, making void spaces in the cell surface region.

A new solid‐phase assay system using magnetic force on blood group serology

Summary. A new solid‐phase assay system has been developed for the detection of irregular antibodies in plasma and serum specimens. By means of a magnetic indicator cell, we have established a sensitive and easy‐to‐handle antihuman globulin (AHG) test involving magnetic‐mixed passive hemagglutination (M‐MPHA). Comparative studies of M‐MPHA with conventional AHG tests demonstrated that this is sufficiently sensitive and specific to detect irregular antibodies to red blood cells. Elimination of centrifugation in this test seems to open the way toward new standardization of solid‐phase systems.

Standardization of Bromelin for Use in Routine One‐Stage Antibody Screening

Abstract. A simple standardization method for bromelin used in routine one‐stage antibody screening is described. Bromelin proteinase activity was assayed using casein as the substrate, and converted to units. The use of proteinase activity units for standardization of bromelin resolves differences between commercial preparations.