Donald R. Branch

In vitro determination of red cell alloantibody significance using an assay of monocyte-macrophage interaction with sensitized erythrocytes.

Summary One hundred and forty‐eight red cell alloantibodies, of specificities generally considered to be of clinical significance, were studied in vitro for their ability to induce phagocytosis of sensitized red cells by allogeneic mononuclear phagocytes. Results indicate that only 53% of the alloantibodies studied mediated significant phagocytosis in vitro. The percentages for each blood group system were as follows: Kell, 73%; Jka, 32%; Jkb, 67%; D, 75%; E, 60%; Fya, 62%; Yta, 25%; Ge, 22%; and Vel, 25%. Significant phagocytosis was independent of the strength of the indirect antiglobulin test. The percentage of anti‐Jka and anti‐Fya mediating significant phagocytosis was increased when fresh complement was added during the sensitization procedure and/or red cells homozygous for the antigen in question were used. The in vivo clinical significance or lack of significance was documented for nine alloantibodies; five caused haemolysis and four did not. Those causing in vivo haemolysis mediated in vitro phagocytosis by monocyte‐macrophages whereas the antibodies that did not result in haemolysis showed no increased in vitro phagocytosis. Autologous monocytes were more reliable than random allogeneic monocytes in that phagocytosis was increased over that obtained using allogeneic monocyte‐macrophages with two of four alloantibodies having documented clinical significance. The use of target red cells homozygous for the antigen in question, the addition of fresh complement in the antibody sensitization procedure, and use of autologous and allogeneic monocyte‐macrophages appear necessary for optimal results.

Correlation of in vivo alloantibody significance or insignificance with an in vitro monocyte-macrophage phagocytosis assay.

the pancytopenia could be due to an infection-induced remission ofALL, as was mentioned by de Vaan & van Oostrum (1982). However. it seems reasonable to speculate that the first aplastic phase was itself pre-leukaemic state of ALL, because the patient developed ALI, 4 months after the aplastic phase via a recovery phase, d~ reported in childhood ALL. Aplastic presentation as pre-leukaemia state of ALL should be suspected not only in children but also in adults.

A New Elution Procedure Using Chloroform, a Nonflammable Organic Solvent

A new method for eluting red cell antibodies using chloroform has been shown to be effective. The method is similar to ether and xylene techniques but can be completed within 10 min after adequate cell washing. Comparison studies using ether, xylene and chloroform showed that antibodies eluted by chloroform yielded equivalent titration scores. Antibodies within the Ss blood group system were easily eluted using chloroform but not using ether. Also, the chloroform method yielded informative eluates when prepared from red cells of patients with warm antibody autoimmune hemolytic anemia, drug‐induced immune hemolytic anemia, hemolytic disease of the newborn caused by ABO or Rh fetal‐maternal incompatibility, or from patients having a positive direct antiglobulin test as a result of alloantibodies stimulated by recent transfusion (‘delayed transfusion reaction’). The advantages of chloroform elution are: (1) chloroform is nonflammable; (2) the eluate is readily obtained from the top layer after centrifugation; (3) no residual solvent remains in the eluate, and (4) the method is rapid.

Unmasking of Kx antigen by reduction of disulphide bonds on normal and Mcleod red cells

Summary We have investigated the effect of dithiothreitol (DTT) treatment of human red cells upon the Kx blood group antigen. At low concentrations of DTT (≤2 mM) there is enhancement of the Kx antigen concomitant with the complete denaturation of the Jsa and Jsb antigens of the Kell blood system. This unmasking of the Kx antigenic site is near maximal using 2 mM DTT. At this concentration of DTT, only the Jsa and Jsb antigens are completely denatured; all other Kell system antigens tested (K, k, Kpb, Ku) are essentially unaffected. These results argue against the Kx antigen serving strictly as a carbohydrate precursor substance involved in a sequential biosynthetic pathway of Kell blood group antigens. Also, McLeod red cells, after treatment with DTT, were found to contain Kx antigen, although in much lower density than normal red cells, indicating that, although not a typical carbohydrate precursor substance, Kx may, nevertheless, be essential for the serological expression of Kell related antigens. It is hypothesized that the Kx structure and the Kell blood group antigen structure are two separate subunits associated in a quaternary conformation involving at least one interchain S‐S bond. Our results should allow for a clearer understanding of the relationship between the serological expression of the Kx antigen and the serologically observed reactivity of the Kell blood group antigens of individuals having normal, Ko and McLeod phenotypes.

High-titer, high-thermal-amplitude cold autoagglutinin not associated with hemolytic anemia.

Abstract. An example of an unusual cold autoagglutinin is reported. The antibody was monoclonal IgMKappa able to fix complement, and, in the presence of albumin, had both a high titer (>4,096 at 4 °C) and a wide thermal range (4–37 °C). The patient was closely followed over a 3‐year period with no evidence of hemolysis ever documented, despite a persistently positive direct antiglobulin test and the presence of the cold autoagglutinin. In contrast to previous reports regarding cold‐agglutinin disease, this case demonstrates that in vivo hemolysis is not always associated with cold autoagglutinins that in vitro show a high thermal range in the presence of albumin.

Human granulocytes lack red cell Kx antigen

Summary We have investigated the presence or absence of the red cell Kx antigen on human granulocytes by measuring specific uptake of anti‐Kx using three techniques: direct measurement by 125I‐staphylococcal protein A (125ISPA) and avidin‐biotin‐complex (ABC) immunoperoxidase staining and also, an indirect measurement using granulocyte adsorption of anti‐Kx, Our results with all three methods indicate that the Kx antigen is not present on normal human granulocytes. Prior to adsorption of the anti‐Kx serum with purified, pooled, normal human granulocytes, 11 of 21 (53%) of normal granulocytes were non‐reactive by 125I‐SPA and 16 of 20 (80%) by ABC. This pattern of reactivity was shown to be due to contamination of our anti‐Kx serum with an antibody to a granulocyte‐specific antigen unrelated to the Kx antigen. After adsorption, there was no diminution in the reactivity of the adsorbed anti‐Kx compared to the unadsorbed antiserum against red cells which express strong Kx antigen, i.e. Ko and DTT‐modified normal human red cells, by either serologic or 125I‐SPA techniques. Likewise, reactivity with McLeod red cells, which have weak expression of the Kx antigen, was not changed using either the unadsorbed or adsorbed anti‐Kx. The adsorbed anti‐Kx was nonreactive with all 12 normal donors’granulocytes tested by 125I‐SPA and with 10 normal donors’granulocytes tested by ABC. Furthermore, granulocytes from a Ko individual were nonreactive using either unadsorbed or adsorbed anti‐Kx. These studies indicate that Kx antigen is not present on normal human granulocytes. Further, additional adsorption studies using granulocytes from a boy with X‐linked chronic granulomatous disease (CGD) indicated that these granulocytes also do not possess the Kx antigen. In contrast to previous reports, these data suggest that Kx antigen is most probably a red cell‐specific antigen and that the red cell Kx antigen has no direct relationship to the biochemical defect in CGD.

The red cell antigens A, B, D, U, Ge, Jk3 and Yta are not detected on human granulocytes

We report the inability to detect the following red blood cell antigens on human granulocytes: A, B, D, U, Gerbich (Ge), JkaJkb (Jk3) and Cartwright (Yta). To study each antigen, granulocytes were purified on density gradients, fixed in glutaraldehyde, and the uptake of specific antisera measured using two direct immunological techniques: 125I‐staphylococcal protein A (125I‐SPA) binding and avidin‐biotin‐complex (ABC) immunoperoxidase staining. Glutaraldehyde fixation was shown not to affect the antigenicity when the antisera were tested using red blood cells. Using three anti‐A, three anti‐B and three anti‐A,B antisera, our 125I‐SPA results of 47 tests with granulocytes from group A individuals and 39 tests with granulocytes from group B individuals indicate that A or B antigens are not expressed on human granulocytes. Tests using ABC were also negative with 37 and 36 granulocytes from group A or B individuals, respectively. In addition, no positive results using 125I‐SPA were obtained with granulocytes from individuals having antigen positive red cells when tested with two anti‐D (number of tests performed (n= 22), three anti‐Ge (n= 22), three anti‐U (n= 20), two anti‐Jk3 (n= 17), and three anti‐Yta (n= 25); control anti‐NA1 or ‐NB1 antisera were invariably positive. Also, using these antisera, no positive results were obtained by ABC except with one anti‐Yta antiserum which was positive with one of seven granulocytes tested. This anti‐Yta was also positive with three of 10 granulocytes by 125I‐SPA. This activity was shown to be due to a granulocyte‐specific antibody; adsorption of the antiserum with human granulocytes removed all activity against granulocytes but did not reduce the activity against red cells. Thus, our results are in agreement with recent reports which demonstrated the absence of the A, B and D antigens on human granulocytes. However, we have been unable to confirm previous reports which indicated the