R.J. Sokol, D.J. Booker, R. Stamps, S. Jalihal, and B. Paul

Autoimmune hemolytic anemia, in which the direct antiglobulin test (DAT) is negative or weakly positive, may be due to low-affinity autoantibodies. We describe two such cases. An 8-year-old male presented with weight loss, jaundice, a hemoglobin of 33 g/L, reticulocytes of 306 x 10⁹/L, and haptoglobin of < 0.1 g/L. The DAT was negative. After washing the red blood cells (RBCs) with saline at 4°C, the DAT was positive for IgG and an eluate contained an IgG3 autoantibody, thus confirming a diagnosis of autoimmune hemolytic anemia (AIHA). Red cell transfusions and corticosteroids were given with eventual complete recovery. A 73-year-old male had a hemoglobin of 89 g/L and haptoglobin of < 0.1 g/L. The DAT was initially negative but was positive for IgG using cold-washed (4°C) RBCs; it was also positive with unwashed cells in the DiaMed system and an eluate contained IgG1 autoantibody. AIHA was therefore confirmed and prednisolone started but continued hemolysis necessitated splenectomy before full recovery occurred. Although RBCs may be strongly sensitized with low-affinity autoantibodies in vivo, the IgG is easily removed when RBCs are washed at room temperature for a DAT. The DiaMed system that uses unwashed RBCs overcomes this problem, but cold washing the RBCs at 4°C must be used when preparing eluates. Immunohematology 1997;14:115-118.

C.L. Barron and M.B. Brown

The use of polyethylene glycol (PEG) to enhance the adsorption of warm autoantibodies on red blood cells (RBCs) was evaluated in our laboratory in an effort to reduce the time and cost associated with routine differential adsorptions. Sera from 19 patients with warm autoantibodies were tested. Fourteen of these sera contained alloantibodies or additional autoantibody specificities underlying the dominant autoantibody. The sera were differentially adsorbed using equal volumes of serum, reagent RBCs, and PEG for 15 minutes at 37°C. The PEG/serum mixture was harvested and used for testing. Six drops of the PEG/serum mixture were tested against reagent RBCs for 15 minutes at 37°C. An antiglobulin test was then performed using anti-IgG. The PEG adsorption technique took a total of 10 hours to completely eliminate autoantibody reactivity in all 19 samples. The reference method required a total of 59.5 hours to adsorb the autoantibodies in all 19 samples. Two weak alloantibody specificities (anti-K, anti-Jkb), known to be present in the serum, were not detected in the PEG tests. Four specificities were weaker with the PEG adsorbed serum. All other alloantibody specificities (13) were detected with equal or greater strength in the PEG adsorbed serum. The use of PEG to enhance the adsorption of autoantibodies should be considered as an option to reduce the time and cost of labor-intensive differential adsorptions. Laboratories should be cautioned that weak alloantibodies may not be detected using this method. Immunohematology1997;13:119-122.

J.D. Bellamy, D.J. Booker, N.T. James, R. Stamps, and R.J. Sokol

Complement has a complex role in immune mediated red blood cell (RBC) destruction and usually induces extravascular hemolysis of C3b-coated RBCs by erythrophagocytosis and by acting synergistically with cell-bound immunoglobulins. A sensitive two-stage enzyme-linked direct antiglobulin test (ELDAT) was developed and used to measure RBC-bound C3b and C3d in 120 healthy adult individuals and in 60 patients suffering from a variety of conditions, including warm- and cold-type autoimmune hemolytic anemia, neoplasia, and collagen diseases. The results were compared with those of standard agglutination tests employing polyclonal and monoclonal antiglobulin reagents. Small amounts of C3b and C3d were detected on RBCs of the healthy individuals only by the ELDAT and probably reflected the continuing low-grade activation of complement necessary for the maintenance of homeostasis of a variety of physiological systems. The quantity did not vary with age or gender. In the patients, increased amounts of RBC-bound C3b and C3d were relatively common and probably resulted from autoantibody activity, immune-complexes, and nonspecific adsorption. There was no association between positive ELDAT results and the presence of active hemolysis. The ELDAT was far more sensitive than the agglutination tests for detecting RBC-bound C3b and also for C3d if the monoclonal reagent was employed. Immunohematology1997;13:123-131.

W.J. Judd, E.A. Steiner, and P.C. Knafl

The recently FDA-licensed anti-IgG gel test for pretransfusion antibody detection requires crossover validation before implementation. Six hundred coded samples sent for routine pretransfusion tests were used to compare GEL (ID-MTS, Ortho Diagnostic Systems Inc., Raritan, NJ) with Löw and Messeter's low-ionic-strength saline (LISS). There were 456 GEL-LISS-, 97 GEL+LISS+, 45 GEL-LISS+, and 2 GEL+LISS- tests. The 144 positive tests involved 157 antibodies; 67 of these (cold auto, anti-M, -Le, etc.) were considered harmless with respect to transfusion management. GEL-LISS+ tests included seven samples containing potentially significant antibodies (assumed from specificity): anti-K(4), -Jk^a, -Fy^b, and -S. Two potentially significant antibodies (anti-C and -D) were GEL+LISS-. Sensitivity and specificity for potentially significant antibodies were 92% and 96% for GEL, and 98% and 90% for LISS, respectively. The seven GEL-LISS+ samples associated with potentially significant antibodies were from six patients. Five of these antibodies, all detected in immune-suppressed patients, reacted predominantly as agglutinins in LISS. None of these seven antibodies were detected reliably by polyethylene glycol and LISS-additive tube methods. In light of the immune status of the patients with GEL-LISS+ agglutinins with specificity normally considered potentially significant, and because other valid methods did not detect these antibodies, their clinical importance is questionable. Excluding these questionable antibodies, GEL has the same sensitivity and better specificity than LISS. GEL is a valid method for pretransfusion antibody detection. Immunohematology1997;13:132-135.

C.J. Hunter and M.D. Ziebol

A sample was submitted to a reference lab from a 27-year-old Asian female, gravida 4 para 1, for antibody identification. Anti-Jk3 with an IgM component was identified. Subsequently, the antibody was eluted from the infant's cord and venous red blood cells. Normal bilirubin and hematocrit levels ruled out hemolytic disease of the newborn (HDN). Anti-Jk3 has been implicated in two cases of mild HDN. In this case, this noncomplement-binding antibody caused a positive direct Coombs test without clinical signs of HDN. Immunohematology 1997;13:136-137.

M.C. Zago-Novaretti, C.R. Jorge, E. Jens, P.E. Dorihiac-Llacer, and D.A.F. Chamone

A 12-year-old Caucasian male with cystinosis received a kidney from his mother, whose red blood cells typed as group O, D+, E-. Her serum contained an anti-E with an IgG1 titer of 16 (score 31). The recipient's type was group O, D+, E+, with a negative antibody screen in the pretransplant period. The recipient and donor Rh phenotypes were most likely DCcEe and Dccee, respectively. Because the recipient's mother had no transfusion history, she was probably immunized by the fetal red blood cells of her one pregnancy (the recipient). The kidney had been immediately perfused with saline after removal from the donor. No acute or delayed hemolysis was observed clinically or in laboratory tests performed immediately after the transplant and at 7, 15, and 30 days after the transplant. Antibody screens were still negative at 6 months. In this case, anti-E was not present in the transplanted kidney in sufficient concentration to cause hemolysis of the recipient's red blood cells and transplanted lymphocytes did not synthesize sufficient anti-E to be detectable or to cause hemolysis. Immunohematology 1997;13:138-140.