Six cases of immune hemolytic anemia attributed to donor-derived red cell antibodies after allogeneic bone marrow transplantation (BMT) are reported. In 2/6 cases, severe intravascular hemolysis was seen, 6/6 required increased red cell transfusion, and 1/6 was treated by plasma exchange. All recipients were receiving cyclosporine to prevent graft-v- host disease. Investigations showed that in each case, the donor lacked ABO or Rho(D) red cell antigens present in the recipient. The direct antiglobulin test was positive in 6/6. Relevant serum antibody (anti-A, four cases; anti-B, one case; anti-D, one case) was first detected one to three weeks after BMT. Eluates made from recipient red cells showed the same specificity as serum antibody. Maximum hemolysis occurred nine to 16 days after BMT, suggesting that active production of antibody by “passenger” donor lymphocytes was the likely mechanism of hemolysis, rather than passive transfer of antibody in the marrow infusion. Retrospective analysis of 21 consecutive cyclosporine-treated BMT patients receiving marrow lacking ABO or D antigens present in the recipient showed that (1) 15/18 patients tested had red cell antibody production against recipient red cell antigens; (2) despite the frequent presence of antibody specific for recipient red cell antigens, only 3/21 patients developed clinically significant hemolysis; (3) clinical hemolysis could not be predicted by donor or recipient red cell antibody titers. We conclude that although red cell antibody against recipient antigens is frequently produced after minor ABO and D mismatched BMT in cyclosporine-treated recipients, only 10% to 15% of cases develop clinically significant immune hemolysis. The data presented show that the most likely source of antibody is “passenger” donor lymphoid cells.

We combined the polymerase chain reaction (PCR) with oligonucleotide hybridization as a novel and sensitive technique to evaluate posttransplant chimerism. Specific oligonucleotides for hybridization were synthesized homologous to tandemly repetitive core sequences of regions with a variable number of tandem repeats (VNTRs). Polymorphisms at such loci result from allelic differences in the number of repeats. Primers flanking the repeat region of each of the corresponding VNTRs were used for amplification. Recipient and donor pretransplant DNA and recipient posttransplant DNA were amplified. The resultant fragments were analyzed after gel electrophoresis either by hybridization in-gel or after Southern transfer. To confirm our findings, we also performed standard assays of restriction fragment length polymorphisms (RFLPs). Evaluation of 13 selected cases indicated mixed chimerism (4), complete chimerism (5), recurrence of leukemia (2), and endogenous repopulation of hematopoiesis (2) after marrow transplantation. Sensitivity of the method was determined by mixing various proportions of recipient and donor DNA; the limit of detection of the minor component in a mixture was 0.1%. PCR data correlated with RFLP data in all cases except two in which PCR proved more sensitive than RFLP. PCR amplification of VNTRs combined with oligonucleotide hybridization is a novel technique for documenting posttransplant chimerism and has advantages over RFLP analysis: high sensitivity, use of small amounts of DNA (250 ng), ease of preparation of DNA, elimination of need for restriction enzymes, and the ability to complete studies in 2 days.

Twenty-nine of 172 patients (17%) who received an allogeneic bone marrow transplant (BMT) from histocompatible sibling donors for hematologic malignancies were mixed hematopoietic chimeras; ie, they had a mixture of donor and host hematopoietic or lymphohematopoietic cells at greater than or equal to 14 days after transplantation. Twenty- four of the 29 mixed chimeras (83%) have remained in continuous complete remission for up to 116 months (greater than 9 years) following BMT. Four of the 29 patients (14%) have had recurrent leukemia, and 7 of the 29 (24%) have had moderate or severe graft-v- host disease (GVHD). Twelve of these 29 patients have persisted as stable mixed chimeras for greater than or equal to 2 years after BMT, whereas other patients converted to all donor-type hematopoiesis. The incidence of mixed chimerism was independent of the pretransplant regimen, the donor or recipient age (less than 20 v greater than 20 years), remission status (first complete remission of acute leukemia and first chronic phase of chronic myelocytic leukemia v later stages of disease), and type of leukemia. Our data indicate that mixed hematopoietic chimerism is not rare after BMT for hematologic malignancies and that its presence is compatible with long-term disease- free survival. Prospective studies of mixed chimerism after BMT are warranted to achieve better understanding of its biologic importance.

Using age-fractionated erythrocytes, warm autoantibodies can be classified into two distinct categories, depending on their reactivity with reticulocyte-enriched (younger) or reticulocyte-poor (older) red cell fractions. The strength of the direct antiglobulin test (DAT) on the age-fractionated red cells of 24 patients indicated that 19 (79%) had an IgG warm autoantibody that reacted preferentially with older red blood cells. In 7 of these 19 patients (37%), the DAT was negative using reticulocyte-enriched red cell fractions. We have termed this preferential reactivity of warm autoantibodies with older red cells as type I. Five of the 24 patients studied (21%) had an IgG warm autoantibody that demonstrated no preference for young or older red cells. We have termed this pattern of warm autoantibody reactivity as type II. All 5 patients having type II warm autoantibodies had severe anemia. In contrast, 6 of 19 patients having type I warm autoantibody did not have clinical evidence of anemia when tested, and 11 of the 19 had only slight to moderate anemia. Additionally, our results using type I warm autoantibody raise questions regarding the blood group specificity of warm autoantibodies. The antigen recognized by type I warm autoantibody may be a cryptantigen. Rh specificity or relative Rh specificity, often associated with warm autoantibodies, may simply be a coincidental finding.

We have used the complement-fixing antibody consumption ( CFAC ) test to detect small concentrations of IgG on red blood cells from patients with hemolytic anemias that are not thought to be caused by an immune mechanism. Although patients with hereditary spherocytosis, pyruvate kinase deficiency, and mechanical hemolytic anemias generally had normal concentrations of IgG bound to their red cells (less than 25 molecules IgG per red cell), we found that 39/62 (63%) patients with sickle cell anemia had elevated values. These 39 patients had a mean of 195 and a maximum of 890 molecules of IgG per red cell. None of the patients had been transfused within the previous 90 days, and some had never been transfused. Direct antiglobulin tests were positive in only two instances and autoantibodies were not found in the serum of any patient. However, eluates from the red cells of 6 of 23 patients demonstrated antibody activity against all of a panel of normal red cells by the indirect antiglobulin test. There was no correlation between the number of IgG molecules on patients' red cells and the severity of their anemia, the incidence of painful sickle cell crises, the reticulocyte count, or with blood transfusion history. We conclude that further study of immunohematologic abnormalities in patients with sickle cell anemia is warranted, especially in view of previous reports in this population of patients with red cell autoantibodies, autoimmune hemolytic anemia, hemolytic transfusion reactions without detectable alloantibodies, and an association of some episodes of pain crises with immunologically mediated red cell destruction.