Low measles vaccine failure in a school outbreak--Newfoundland.
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Biomedical subjects
Publications and source records attributed to F Stratton.
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During the summer, plasma samples from eight blood donors at this transfusion centre were found to have a bright orange colour. All donors appeared to be healthy and haematological tests showed no abnormality. Extracts of lipid from the plasma revealed the presence of the carotenoid canthaxanthin and other carotenoid metabolites which were identified by thin-layer chromatography and high-pressure liquid chromatography, light absorbtion and mass spectrometry. On questioning the donors it was found that they had been taking a course of 'Orobronze' (an oral 'bronzing' treatment containing canthaxanthin). Canthaxanthin was still detectable in the plasma several months after the donors had ceased taking the capsules. At present it seems that there would be no deleterious effects on healthy individuals taking orobronze. The full implications of the presence of canthaxanthin in plasma transfused to patients do, however, require further investigation of carotenoid metabolism in man.
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.
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 amount of antibody bound to cells in a low ionic strength solution (LISS) has been quantitated for several antibodies including anti-D, anti-c, anti-Kell, anti-Fya, and anti-Jka. With the exception of the Kell antibodies there was an enhancement of the rate of antibody uptake in LISS. For Rh antibodies the amount bound after a 5-min LISS incubation is comparable to that bound after 45 min in saline. For Kell antibodies a smaller amount was bound in LISS than in saline. The effect of the ratio of serum to cells was also studied, and with several antibodies there was an increase in the amount of antibody bound with a higher serum to cell ratio irrespective of suspending medium. For Kell antibodies this ratio appears to be of greater importance than the ionic strength for antibody detection. A modification to the LISS method to increase the serum to cell ratio is, therefore, presented.
The application of monoclonal antibodies to the quantification of blood group antigen sites on erythrocytes was examined. A second antibody technique using labelled anti-mouse IgG could not be used as it was not possible to predict the binding ratio between this and the monoclonal antibody. A series of monoclonal antibodies (R10, R18, BRIC 13, BRIC 14) to the erythrocyte sialoglycoprotein alpha (syn: glycophorin A) showed the number of antigen sites to be from 0.3 X 10(6) to 1.2 X 10(6) per erythrocyte and supported the conclusion that the Wrb antigen is located on this protein. An antibody with a specificity related to the Rh blood group system (R6A) showed 4.6 - 10.4 X 10(4) binding sites to be present on cells of phenotype cCDEe. On cells of phenotype -D- 1.24 X 10(4) binding sites were present but protease treatment increased the number of available sites to 1.3 X 10(5). An antibody to a Kell-related antigen (BRIC 18) recognized 2.5 - 5.9 X 10(3) sites per erythrocyte on cells of phenotype Kk. However, a similar number also appeared to be present on cells of the McLeod and Ko phenotypes although the affinity for the antigen on these cells was very much reduced. The potential of using monoclonal antibodies for this purpose and the value of this in the study of blood group systems has been demonstrated.
'Naturally occurring' anti-D has been studied in four Rh(D) negative males. The presence of this anti-D does not preclude successful deliberate immunization of Rh(D) negative volunteers; 'naturally occurring' anti-D does not provide a marker of non-responders to Rh immunization. 'Naturally occurring' anti-D is not necessarily the result of materno-fetal transfusion at birth.
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 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.
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 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.
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 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.
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After a single antigenic stimulus, not greater than 5.0 ml R2R2 red cells, anti-D was detected in 79% of D-negative volunteers increasing to 88% after subsequent spaced stimuli. The use of repeated antigenic stimuli at 2- or 4-weekly intervals to induce the immune response did not appear to increase the frequency of responders. The results obtained are compared with those of other workers and evidence is presented to suggest that the immunogenicity of red cells from different donors may have a role in determining the frequency of responders with detectable anti-D and is possibly associated with the R2 antigenic complex.
Twenty-nine Rho(D) negative male volunteers previously immunized with D-positive red cells have received series of antigenic stimuli repeated at intervals of 2 or 4 weeks. Although there is an individual variation in the magnitude of the anti-D response, the levels of anti-D achieved are, in general, higher than those obtained after stimuli spaced at intervals of several months. Moreover, certain volunteers whose plasma was not suitable for inclusion into pools for the preparation of anti-D immunoglobulin after spaced stimuli could donate regularly following the repeated stimuli. Whilst untoward clinical reactions did not occur as a result of the repeated stimuli five volunteers developed unwanted antibodies outside the Rh system and this may be related to the total dose of red cells injected.
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.
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