Multicentre trial of antepartum low-dose anti-D immunoglobulin.

Routine antenatal Rh immunoprophylaxis would substantially increase the use of anti-D Ig in the U.K. As availability of anti-D Ig is one factor influencing a decision to introduce routine antenatal prophylaxis, a trial was undertaken to test the efficacy of a lower dose of anti-D Ig than that used in earlier studies. RhD-negative primigravidae were randomized as controls or recipients of two doses of 250iu of anti-D Ig given at 28 and 34 weeks gestation. Blood samples were tested at delivery and at 6 months postpartum for the presence of immune anti-D, and again later if results were equivocal. Nine (1.5%) out of 595 control patients had immune anti-D at follow-up at 6 months and later; 4 (0.78%) of 513 treated women were immunized. It was concluded that, while two doses of 250iu of anti-D Ig may reduce alloimmunization, they are not as effective as two doses of 500iu in a previous trial.

Screening of blood donations for HIV-1 antibody: 1985-1991.

One hundred and ninety HIV-1 antibody positive donors have been detected, out of 14.85 million blood donations screened in the United Kingdom and the Isle of Man, since the start of testing in October 1985 to the end of March 1991. There were 145 men and 45 women, with an overall seropositivity rate of 0.001%. Records for new donors (ie, those donating for the first time) have been kept since February 1986 and 79 out of 1.9 million donations have been seropositive (58 men and 21 women); a rate of 0.004%. One hundred and forty-one (74.2%) of the 190 positive donors were found to have been exposed to a high risk of HIV infection. Twenty-four (13%) denied any exposure other than heterosexual intercourse with partners who were not considered to be at high risk. In six cases the partners were from countries where the main route of transmission is heterosexual. Seven donors (4%) attributed infection to some other cause. Eighteen (9.5%) have been lost to follow-up, are still being investigated or have not yet been interviewed. Combined tests which screen for both HIV-1 and HIV-2 antibodies were introduced in June 1990. The last 2.55 million donations (including 370,000 new donors) have been tested with these kits. One HIV-2 antibody positive donation had been confirmed in a new donor by the end of March 1991.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the sensitivity to complement-mediated lysis of erythrocytes (Inab phenotype) with a deficiency of DAF (decay accelerating factor).

No episodes of clinically significant in vivo haemolysis have been reported in individuals with a novel form of decay accelerating factor (DAF) deficiency (Inab phenotype), nor do functional in vitro assays for complement-mediated haemolysis show the extreme sensitivity to lysis characteristic of paroxysmal nocturnal haemoglobinuria (PNH) erythrocytes. DAF appears to be totally deficient in the Inab erythrocytes as judged by immunochemical and functional assays. Unlike PNH, the only other described DAF deficiency (where several other phosphatidylinositol (PI)-linked membrane proteins are also absent), the only protein lacking from Inab erythrocytes appears to be DAF. The Inab phenotype seems to be an inherited specific defect in DAF whereas PNH is an acquired defect in the mechanism of insertion of PI-linked proteins into cell membranes. These findings support the view that susceptibility of PNH erythrocytes to in vivo and in vitro complement-mediated haemolysis is not due simply to DAF deficiency but to either the combined lack of several membrane proteins or to deficiency of other regulatory proteins such as the membrane attack complex inhibitor/homologous restriction factor (MIP/HRF). The findings also raise questions as to the role of erythrocyte DAF.

HIV antibody screening of blood donations in the United Kingdom.

Routine screening of blood donations for anti-HIV commenced in the UK during October 1985 and by the end of February 1987 approximately 3.7 million donations had been tested. Seventy-two were confirmed anti-HIV positive, i.e. 0.002%. Of the anti-HIV-positive donors interviewed to date, the majority are young homosexual or bisexual men or intravenous drug abusers. Included in the study are data collected on approximately 470,000 donors giving blood for the first time. Twenty of these have been confirmed anti-HIV positive (0.004%), and 19 interviewed have admitted to being in risk categories. In 5 instances a positive anti-HIV donor was found negative on a previous occasion, and in 1 instance the products from the donation led to seroconversion in the recipients. The majority of anti-HIV-positive donors attending for blood donation did so because they did not consider that the self-exclusion categories specified in the leaflet issued to donors applied to them since homosexual activity or drug abuse was not currently being practised.

Test cells for use with anti-IgG sub-typing antisera in the antiglobulin test.

The preparation of test cells coated with specific IgG of known subclass is described. Such cells are required in the standardization of IgG subtyping reagents. At present, these test cells usually are prepared by coating cells with IgG myeloma paraproteins. However, these paraproteins may not be generally available and an alternative method is presented using more readily obtainable materials. Quantification of cell-bound IgG showed that the subclass did not affect the sensitivity of the antiglobulin test when using broad-spectrum anti-IgG and that the test cells produced had an optimal IgG coating. Reactions with subclass-specific antisera were however, considerably weaker that those obtained with broad-spectrum anti-IgG. A modified spin-layering technique for use with subclass-specific antisera is described.

Incidence of T activation in a hospital population.

The incidence of T activation in a hospital population has been studied. T poly-agglutinability likely to cause serological problems was found in only 1:10,000 but minor degrees of T activation occurred in 1:200. In a number of cases, severe infections were accompanied by T activation, but we were unable to establish any primary association between T activation and anaemia. The findings suggested that individuals whose erythrocyte sialic acid levels are slightly lower than normal are those who, when hospital patients, are likely to develop various degrees of T activation.

Reticulo-endothelial function in glomerulonephritis.

IgG- and C3b-coated erythrocytes were used to investigate splenic Fc-receptor function and hepatic C3b-receptor function by measurements of their blood clearance. Most patients with glomerular disease including those who were HLA, A1, B8, DR3 had normal Fc-receptor function. Occasional individuals with persistent glomerulonephritis had delayed clearance unrelated to disease activity. In rapidly progressive glomerulonephritis, the phenomenon of 'reticulo-endothelial blockade' was confirmed. C3b-receptor function was normal in most patients with persistent glomerulonephritis. A defect in splenic Fc-receptor function hepatic C3b-receptor function is unlikely to underlie persistent glomerulonephritis.

Quantitation of IgG on Erythrocytes: correlation of number of IgG molecules per cell with the strength of the direct and indirect antiglobulin tests.

The number of IgG molecules bound to the erythrocyte surface for a given agglutination score in the antiglobulin test was studied with several different examples of anti-D, anti-E, anti-c, anti-Kell, anti-Fya, anti-Jka and immune anti-A antisera. The serological scores show a significant correlation with the mean values for bound IgG molecules within a restricted range, although the number bound for a given score may vary by up to 20%. The limit of detection was 100-120 IgG molecules per cell and when over 1,000 were bound, the cells were completely agglutinated. Anti-Kell bound under low ionic strength saline conditions required a greater number of molecules for a given agglutination strength. The relatively low levels of bound IgG necessary to give strong agglutination make the direct antiglobulin test (DAT) less valuable for following the progress of auto-immune haemolytic anaemia (AIHA) than a quantitative test. The latter test does not, however, provide any additional information in AIHA cases with a negative DAT as in these the anaemia does not appear to be due simply to the number of bound IgG molecules. Detection of certain antibodies may not be achieved simply by increasing the sensitivity of the antiglobulin test when correctly performed.

The quantification of C3 fragments on erythrocytes: estimation of C3 fragments on normal cells, acquired haemolytic anaemia cases and correlation with agglutination of sensitized cells.

A sensitive method is described for the quantification of C3 fragments on erythrocytes. A radiolabelled monoclonal antibody, was used which was directed against a C3d determinant on all forms of cell bound C3. The number of C3 molecules on normal erythrocytes was estimated to be 420 +/- 140. The strength of the antiglobulin test increased from negative to 5+ over a range of only 850 C3 molecules (400-1250). A blood donor with a positive direct antiglobulin test was found to have 4800 molecules per cell whereas three cases of cold haemagglutinin disease with active haemolysis had from 16 000 to 52 020 C3 molecules per cell. This test has an application in the testing of acquired haemolytic anaemia cases with a positive direct antiglobulin test with C3 bound to the cells and in the standardization of sensitized cells used for testing antiglobulin reagents by various serological techniques.

Positive direct antiglobulin test in normal individuals. II.

Investigations into the IgG sub-types and number of molecules of IgG present on the red cells of 22 apparently normal healthy blood donors with positive direct antiglobulin tests are described. In all cases, the sub-type was IgG1 or IgG4, and none had more than 1000 mol of IgG per red cell. It is suggested that sequestration of IgG-coated cells only occurs when the number of IgG1 mol per cell reaches a certain level.

A quantitative antiglobulin test for IgG for use in blood transfusion serology.

A sensitive method has been developed using 125I-labelled anti-IgG which allows detection of IgG present on the red cell surface. By suitable absorption and correction for non-specific binding to trypsinized red cells, a reproducible, accurate method of quantitating IgG absorbed onto red cells is obtained. The number of IgG molecules present on normal cells was found to range from 5 to 90 with an average of 39. Studies of correlation of agglutination in the antiglobulin test with numbers of IgG molecules on the cell were also undertaken.

A rapid purification method for peanut anti-T lectin.

A novel method for the purification of the anti-T lectin from peanuts is described. The method is simple, easy to perform and requires no special reagents. The lectin is obtained in a highly purified from comparable to that prepared by affinity chromatography and large scale preparations are readily made. The lectin was shown by serological tests to have anti-T activity and is suitable for detection of minor T antigen activation on red cells.

Positive direct antiglobulin test in normal individuals.

65 normal, healthy people with a positive direct antiglobulin test (DAT) have been identified in a population of blood donors over a period of 14 years. 32 of them have been recalled for detailed study. A strong positive correlation with increasing age was noted, comparable to that seen in hospital patients with a positive DAT. No feature known to cause a positive DAT was identified in the healthy individuals, only 1 of whom went on to develop autoimmune haemolytic anaemia.

A simple method for th production of anti-C3 antiserum in rabbits.

A method is described for the production of an anti-C3+d specific antiserum in rabbits. The method does not require purification of C3 but relies on the binding of human C3 to the rabbit erythrocytes following activation of the alternative pathway. The antiserum produced is suitable for use as either a serological reagent or for immunoprecipitation.

Observations on the antiglobulin tests. II. C4 components on erythrocytes.

This paper describes anti-C4b(ii) antisera shich agglutinate cells from cases of autoimmune haemolytic anaemia, cold-acquired haemolytic anaemia, and certain test cells, using the antiglobulin test. It is suggested that these particular antisera possess activity against C4d absorbed on to erythrocytes. Methods of preparing test cells coated with C3d and C4d are given, together with methods for producing antisera in rabbits active against these components. The significance of these findings is discussed.

Preparation of test cells for the antiglobulin test.

Erythrocytes may be coated with blood group antibodies with or without reacting complement or sometimes apparently with complement alone. This may occur in vivo in such conditions as autoimmune acquired haemolytic anaemia, haemolytic disease of the newborn, or after transfusions of incompatible blood. It may occur in vitro also by the deliberate sensitization of erythrocytes during laboratory serological investigations. Blood group antibodies may be of immunoglobulin types gammaM, gammaA, or gammaG; we have never seen gammaD antibodies. The presence of these antibodies on the erythrocyte surface, together with complement components or the presence of complement components alone, may be detected by the direct antiglobulin test where sensitization occurs in vivo or by the indirect antiglobulin test where there is sensitization in vitro.