Monocyte-erythrocyte interaction in autoimmune haemolytic anaemia in relation to the number of erythrocyte-bound IgG molecules and subclass specificity of autoantibodies.

Monocyte-erythrocyte interaction in patients with autoimmune haemolytic anaemia (AIHA) was assessed by phagocytosis and rosette assays. In most patients, a relationship was observed between haemolysis and the phagocytosis of their own erythrocytes by allogenic peripheral monocytes. An evaluation of the number of immunoglobulins on patient erythrocytes and IgG subclasses of autoantibodies shows that in patients with only IgG1 antibody or with additional IgG2 or/and IgG4, phagocytosis was always observed when the number of erythrocyte-bound IgG molecules was above 2,000. On the other hand, in all patients where IgG3 was detectable, phagocytosis was observed even if the amount of IgG was as low as 230 molecules per erythrocyte. Similar observations were made in the rosette assay. Generally, the number of erythrocyte-bound IgG and the presence of phagocytosis were correlated with the degree of haemolysis, but there were exceptions, i.e. the amount of IgG and phagocytosis were high but there was no evidence of haemolysis, or where there was little IgG, no phagocytosis but haemolysis was present. Our data do not indicate that erythrocytes from AIHA are preferentially bound to autologous monocytes.

Fc receptors for IgG1 and IgG3 on human mononuclear cells--an evaluation with known levels of erythrocyte-bound IgG.

The Fc receptors on mononuclear cells were investigated by a rosette technique in which human erythrocytes were sensitized with a known number of molecules of anti-Rh antibodies (IgG1 or IgG3). The number of IgG molecules was quantitated by a radiometric antiglobulin test. The present quantitative evaluation reveals that (1) there is a logarithmic relationship between the proportion of rosettes and the amount of erythrocyte-bound immunoglobulin for both types of mononuclear cells, and for both subclasses; (2) similar percentage of rosettes can be obtained with fewer IgG3 than IgG1 molecules (about 1:4); (3) for a given number of erythrocyte-bound immunoglobulins a higher percentage of rosettes is observed with monocytes than with lymphocytes (ratios of about 3:1 for IgG1 and 5:1 for IgG3); (4) the minimum number of IgG3 molecules for adherence is 180-460 for monocytes, 520-1,300 for lymphocytes, whilst for IgG1 the numbers are 1,180-4,300 for monocytes and 3,400-14,200 for lymphocytes; (5) the minimum levels of sensitization by alloantibodies for adherence should be detectable by the antiglobulin test.

The quantification of erythrocyte antigen sites with monoclonal antibodies.

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.

Two individuals with elliptocytic red cells apparently lack three minor erythrocyte membrane sialoglycoproteins.

We have studied the erythrocytes of two individuals (P. L. and K. W.) who lack the Gerbich (Ge) blood-group antigen. The erythrocytes of P. L. and K. W. were not reactive with two monoclonal antibodies (NBTS/BRIC 4 and NBTS/BRIC 10) which reacted with normal erythrocytes. The membranes of P. L. and K. W. erythrocytes appeared to lack three minor sialoglycoproteins (beta, beta 1 and gamma). These three minor sialoglycoproteins were found to be associated with the cytoskeletons of normal erythrocytes. Approx. 10% of the erythrocytes of P. L. and K. W. were frankly elliptocytic. We suggest that one or more of the minor sialoglycoproteins may play a part in maintaining the discoid shape of the human erythrocyte.

Quantitation of antibody binding to erythrocytes in LISS.

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.

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.

Five-day storage of platelet concentrates. II. In-vivo studies.

Following in-vitro tests it was concluded that platelet concentrates stored for 5 days at 22 degrees C in polyolefin containers, coded PL732, should be as effective in clinical practice as similar concentrates stored in the standard PVC containers, coded PL146. These predictions have been confirmed by the following in-vivo tests; autologous survival studies in volunteers, determination of recovery, platelet increment calculations 1 and 24 h after transfusion and clinical appraisal after transfusion of haemorrhagic thrombocytopenic patients. Bacteriological cultures of the platelet concentrates were sterile after storage for more than 5 days. It can be concluded that the 5-day storage of platelet concentrates in these containers is a practical proposition.

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.