Leucocyte depletion of neocyte-rich red blood cell concentrates by use of immunomagnetic beads.

In a series of pilot experiments we depleted neocyte-rich red blood cell concentrates of contaminating leucocytes by use of immunomagnetic beads. The beads were coated with monoclonal antibodies directed against the leucocyte common antigen CD 45. The number of cells isolated depended on the dose of beads which also corresponded to the decrease in leucocytes in the neocyte concentrates. The required ratio of beads to target cells was approximately 25:1, the depletion rate of leucocytes was about 96.0-99.8%.

SLP assay: a rapid assay for detection of red blood cell antibodies in the serology of pregnancies.

A rapid manual test for the detection of red cell antibodies called SLP assay has been developed and compared with the sensitivity of the antiglobulin assay. Acid-soluble proteins (SLP) from human leukocytes cause aggregation of human red blood cells. SLP represents a group of proteins consisting of 5 fractions of different positively charged macromolecules, which are able to reduce the negative charge of the red cells. Reduction of the negative charge results in a nonspecific hemagglutination of different strengths, depending on the SLP fractions used. This hemagglutination can be reversed by neutralizing the SLP with heparin. In the case of blood group-antibody-mediated aggregation the hemagglutination is nonreversible despite neutralization with heparin and remains stable for several hours. Because of the high sensitivity of the SLP assay all blood group antibodies from the IgM type as well as from the IgG type are detectable even in low concentrations. The sensitivity of the SLP assay is comparable to the antiglobulin assay.

A comparison of a solid phase IRC assay and the PSIFT for detection of antibodies to platelets.

A rapid solid phase indicator red cells assay (IRCA) for detection of platelet antibodies was developed and its sensitivity compared with PSIFT. Platelets were attached to the surface of polystyrene microtitre plate wells by means of a sodium carbonate buffer and centrifugation. Uncovered areas were blocked by a gelatin blocking buffer. After serum incubation bound platelet-specific antibodies were made visible by anti-IgG-coated indicator red cells and a brief centrifugation. A positive result, meaning the presence of an anti-platelet antibody was indicated by red cell adherence over the reaction surface. In the absence of serum antibodies to platelets the indicator red cells formed a pellet. The IRCA showed a high sensitivity; the anti-platelet antibody Thrombocyte was detectable until a dilution of 1:1,600 whereas the same antibody in the PSIFT could only be detected until a dilution of 1:400.

Serum soluble interleukin-2 receptor levels of normal blood donors.

Following T cell activation a part of the IL-2-receptor (sIL-2-R) was released from the T cell membrane. Elevated serum levels of sIL-2-R were observed in some pathological conditions. The serum of healthy blood donors (n = 228) was investigated by ELISA to see whether a correlation existed in normal donors between the age or the sex of the donor and the sIL-2-R. The serum levels found were in the range between 5 and 398 pM. No correlation was found between the different ages or the sex of the donors.

A simple and safe method for single HLA-antigen-typing by a solid phase assay.

A rapid solid phase assay for detection of single HLA-antigens on platelets was developed. The platelets were attached to the surface of polystyrene microtitre plate wells by means of a sodium carbonate buffer and centrifugation. Uncovered areas were blocked by a gelatin blocking buffer. After incubation with commercially available anti-HLA-sera the bound anti-HLA-specific antibodies directed against HLA-antigens present on the platelets were made visible by anti-IgG-coated indicator red cells and a brief centrifugation. A positive result, meaning the presence of an HLA-antigen, was indicated by a slight red cell adherence over the reaction surface. In the absence of the HLA-antigen no binding occurred and the indicator red cells formed a small red disc-like pellet.

[A new procedure for lymphocyte depletion of thrombocyte concentrates with immunomagnetic beads]. / Neues Verfahren zur Lymphozytendepletion von Thrombozytenkonzentraten durch immunmagnetische Beads.

In a series of pilot experiments we depleted platelet concentrates from contaminating leukocytes by use of immunmagnetic beads. The beads were coated with monoclonal antibodies directed against the leukocyte/lymphocyte antigens CD 2, CD 19 or CD 45. The amount of isolated cells depended on the dose of the used beads and was analogous to the observed decrease of leukocytes in platelet concentrates. The total amount of necessary beads for a complete leukocyte depletion depends on the grade of leukocyte contamination, which shows a high variation among different platelet preparations.

Use of stroma for the detection of blood-group antibodies by ELISA.

A micro enzyme-linked immunosorbent assay (ELISA), using lyophilized stroma as carrier of red blood cell antigens, which stay stable longer than usual, using intact erythrocytes, was developed for determination of blood-group antibodies in the AB0, Kell and Lewis-systems. Stroma being fixed on microtiter plates was incubated with antisera and peroxidase-conjugated anti-human globulin. The transformation of the substrate added was determined photometrically. A binding of antibodies to the stroma could be demonstrated up to an antibody dilution of 1:1024 for the ABO-system, of 1:512 for the Kell-system and of 1:64 for the Lewis-system. By standardization of this method the quantitative determination of antibodies becomes possible without being restricted by the limited stability of intact erythrocytes.

Genetic markers in alcoholism: no association with HLA.

We carried out a study on 63 patients suffering from alcoholism in order to determine the frequency of 27 HLA antigens. In comparison to healthy blood donors no significant deviation of HLA distributions in alcoholics was found. The data on alcoholic patients with physical consequences such as cerebral seizures, liver cirrhosis and polyneuropathy failed to identify an association with HLA.

HLA antigens in schizophrenia, major depressive disorder, and schizoaffective disorder.

We carried out a study with 150 patients suffering from endogenous psychosis in order to confirm earlier findings of an association between HLA and various psychiatric syndromes. Our sample consisted of 107 schizophrenics, 25 patients with recurrent endogenous major depressive disorder, and 18 schizoaffectives. A significant excess of HLA B27 was found among schizophrenics with a family history of endogenous psychosis. This association is consistent with reported earlier studies. The preliminary data on the schizoaffective sample suggest a positive association with HLA B7. The data on patients with endogenous major depressive disorder failed to identify an association with HLA.

Platelet counting with a laser nephelometer.

A method for platelet counting is described, based on the Laser nephelometric principle. Experimental results are reported, together with the practical considerations for the standardisation and correlation of the method, and for application of the method in the routine biochemical laboratory. Venous blood, taken with EDTA-NaCL solution according to Schulz et al (1071, Z. Klin. Chem. Klin. Biochem. 9, 329-333) is used. The blood is centrifuged at 100 g for 10 min and 10 microliter of the supernatant is added to 3000 microliter of a suspending medium (dilution 1:80); 300 microliter platelet suspension are read in a nephelometer cuvet or tube against blank. The number of platelets per liter blood are determined with the aid of a standard curve. The sensitivity and reproducibility of the method, and the correlation with the "electronic coulter counting" method are satisfactory.

Human tonsil lymphocytes in the transfer of cell-mediated immunity.

Peripheral blood lymphocytes have the ability to transfer cellular immunity. In this study, we investigated whether human tonsil lymphocytes could transfer cellular immunity. Human tonsil lymphocyte ultrafiltrates, after separation through gel filtration, showed five fractions in Sephadex-G-10, as well as four in Sephadex-G-25,4. The extracts and the fractions have characteristic biochemical as well as biological properties. The biological activity of ultrafiltrates from human tonsil lymphocytes was compared with the activity of ultrafiltrates prepared from human peripheral blood lymphocytes, for identification studies of a 'Transfer Factor' from human tonsils. Ultrafiltrates prepared from human peripheral blood lymphocytes (Ascher et al., 1976) and tested, showed similar biochemical and biological properties.

Neutralising effect of histone fractions from human leukocyte nuclei on heparin.

Histone fractions from human leukocyte nuclei can interact with heparin to form complexes measurable by means of laser nephelometry. Histone fractions have not all the same affinity to bound heparin and the lysinrich histone fraction cannot interact with heparin. The formation of histone/heparin complex depends not on the temperature between 20 degrees--37 degrees C or on the pH between 7--8.2. The precision of the method was estimated. The significance of the histones/heparin interactions for an assay method for control to the biological activity of heparin preparations and a possible biological role of these interactions in vivo was briefly discussed.

Stimulatory effect of histone on glutathione reductase of human erythrocytes.

Glutathione reductase of hemolyzates, as well as intact erythrocytes from normal individuals can be activated by the addition of human leukocyte nuclear histones and histone subfractions f1 and f3. Intact erythrocytes incubated with histone show also in their hemolyzates a high glutathione reductase activity as compared with such not treated with histone cells. The degree of stimulation of glutathione reductase by histones is comparable to that caused by flavin adenosine diphosphate (FAD). Histones and FAD show a competitive stimulatory effect on glutathione reductase activity. ATP shows an inhibitory effect on glutathione reductase activity, which can be reversed by addition of histone.

Inhibitory effect of histone on the peroxidase activity of the Hp/Hb complex.

The human leukocyte nuclear histones can bind to haptoglobin (Hp), and thus interfere with subsequent binding of Hb. The Hp/Hb complex possesses peroxidase activity, but a Hp/histone complex does not. The inhibitory effect of histone molecules depends directly on the Hp and Hb, as well as the histone concentrations. The biological significance of the complex Hp/Hb as a measure of intravascular hemolysis and the interference of histones in this assay is briefly discussed.

Effect of histones on the intracellular metabolism of ascorbic acid.

Basic proteins are able to control the intracellular metabolism of ascorbic acid in human erythrocytes. The stimulating effect of ascorbic acid expressed by means of cellular peroxidase reactions of intact erythrocytes depends on the ascorbic acid and basic protein concentrations as well as on the structure of the histone molecule. Leukocyte and calf thymus histones can stimulate the intracellular metabolism of ascorbic acid in human erythrocytes, in contrast to the protamines which show an inhibitory effect in the above mentioned intracellular peroxidase metabolic system.

Alcohols-histones metabolic interactions in intact human erythrocytes.

Alcohols have a stimulating effect on the intracellular peroxidatic reactions of intact human erythrocytes. This effect depends directly on the molecular weight of alcohol and the H2O2 concentration. The human leukocyte nuclear histones, flavin-adenine-dinucleotide (FAD) and nicotinamide-adenine-dinucleotide (NAD) inhibit the peroxidatic reactions during the metabolism of alcohols in intact human erythrocytes. The participation of erythrocyte catalase, the possible metabolic pathway and the inhibitory effect of human leukocyte nuclear histones, FAD and NAD on the intracellular metabolism of alcohols is discussed.

[Blood group specific properties of leucocyte nuclei (author's transl)]. / Blutgruppenspezifische Eigenschaften der Leukozytenkerne

The absorbing and precipitating capacity of leucocyte nuclear suspensions derived from conserved blood of various blood groups was examined with anti-A-, anti-B-, anti-H, anti-M-, anti-N-, and anti-CDE-, and anti-D-sera as well as with anti-AHP- and Evonymus-extracts. Nuclei of blood group A absorbed anti-A, but not anti-B; nuclei of blood group B absorbed anti-B, but not anti-A. Nuclei of blood group 0 absorbed anti-H, but neither anti-A nor anti-B. Anti-M-, anti-N-, anti-CDE-, and anti-D-sera showed no absorption effect after incubation with leucocyte nuclei of corresponding blood groups. A nuclei were strongly precipitated by anti-AHP; B- and 0-nuclei were markedly precipitated by Evonymus-extract. B- and 0-nuclei showed only a weak precipitation with anti-AHP-extract; A-nuclei were only slightly precipitated by Evonymus-extract. Hence, leucocyte nuclei possess blood group specific properties, which, according to the present studies, are limited to certain blood group systems.