September-December 1998

Hemolysis Following Allogeneic Bone Marrow Transplantation: Discussion and Analysis of a Case

Though bone marrow transplantation (BMT) is rapidly expanding as a practical and worthwhile therapeutic modality, it poses new and interesting problems for transfusion services. For example, when formulating a strategy of transfusion support for the BMT recipient, the transfusion medicine professional must take into account the series of immunohematologic changes and complications that may arise in such patients. He or she must also apply techniques, methods, and approaches not routinely used in the general blood-banking environment.

The usual questions included in taking the patient’s history, such as transfusion history and previous pregnancies, are no longer enough since new conditions particular to BMT patients may play an important role in initiating and promoting hemolysis. Thus, the transfusion medicine professional must take pains to learn of the patient’s therapy, the donor’s history, differences in the antigenic make-up of patient and donor, and the patient’s clinical evolution, among other things. However, as more experience is gained in the fascinating field of BMT and as newer and more effective strategies for transfusion support of the BMT patient are developed, other factors that may affect the diagnosis and management of cases marked by clinically significant hemolysis will emerge.

In the present report of a case of hemolysis following allogeneic BMT, we address this new issue in transfusion medicine.

A 58-year-old woman with CML was treated with an allogeneic bone marrow transplant from an HLA matched unrelated donor (MUD). The patient’s blood was O (Rh) positive. Past surgical history revealed cholecystectomy at the age of 32. The patient’s RBC phenotype was D+, E+, C+, e+, c+, K =, Jk^a+, Jk^b+, Fy^a =, Fy^b+, M+, N+, S+, s+, P1=, Le^a=, Le^b+. Screening of the patient’s blood for RBC alloantibodies was negative.

The donor was A (Rh) positive. The donor’s RBC phenotype was D+, E+, C+, e+, c+, K+, Jk^a=, Jk^b+, Fy^a+, Fy^b+, M+, N=, S+, s+, P1+, Le^a+, Le^b=. Screening of the donor’s blood for RBC alloantibodies was negative. However, the donor had a history of an anti-Jk^a antibody.

Serologic screening for transfusion-transmitted diseases (HBsAg, HBcAb, HCVAb, HIV 1/2 Ab, p24 Ag, HTLV I-II Ab, RPR, and ALT) was unremarkable for both patient and donor. Screening for CMV antibodies by the latex method was nonreactive for the patient but reactive for the donor. Pretransplant restriction fragment length polymorphism (RFLP) studies on donor and patient blood revealed differences in their DNA, which made it possible to look for the donor’s and the patient’s DNA in samples taken from the patient after BMT and to assess and monitor engraftment and chimerism. After preparatory ablative chemotherapy for the patient, the donor bone marrow was infused into the patient. Following the transplant, the patient received a number of transfusions, intravenous IgG (IVIgG), granulocyte colony-stimulating factor (G-CSF), cyclosporine/prednisone (for prevention of graft-versus-host disease [GVHD]), ganciclovir, acyclovir, and prophylactic antibiotics.

After the bone marrow transplant, laboratory data evolved as shown in Table 1. Serologic screening of the patient’s blood for transfusion-transmitted diseases on post-BMT day 14 was reactive for HBcAB and for anti-HbsAg antibody. The patient had no history of hepatitis or hepatitis vaccination.

The patient was discharged from the hospital on post-BMT day 120 to be followed as an outpatient. RFLP studies done on post-BMT day 180 showed only donor bands, suggesting 100% engraftment.

One year later the patient had a recurrence. She was treated with chemotherapy, with the plan of infusing donor lymphocytes later on. Immunoelectrophoresis of the patient's serum on post-BMT day 546 demonstrated a monoclonal band, which on immunofixation was shown to contain IgG. Hemolysis resolved with prednisone and IVIgG. After the infusion of donor lymphocytes, the patient developed acute GVHD, for which she was treated.

The patient is still alive and in remission, 2 years after the bone marrow transplant.

*FWD, forward or erythrocytic typing; RVS, reverse or serologic typing; Neg., negative; A mf, group A mixed field.

Hemolytic anemias, in general, can be classified according to the mechanisms that lead to RBC destruction (Table 2). In this patient, we considered at first that the immunohemolysis we observed could have been due to any of several mechanisms including (a) passive transfer of anti-A, anti-B, and/or anti-D antibodies, other RBC alloantibodies, and/or antibodies to infectious agents, along with IVIgG; (b) transfusion of ABO-incompatible plasma in platelet concentrates; (c) autoimmune hemolytic anemia (AIHA); (d) reaction of the patient's anti-A antibodies against engrafting donor A-type RBCs; and (e) reaction of donor-derived anti-Jk^a antibodies against the patient’s Jk^a+ RBCs.

When patients receive anti-A, anti-B, anti-D, and/or other RBC alloantibodies containing IVIgG, some will develop a positive direct antiglobulin test (DAT), and some will even have hemolysis. The estimated half-life of IVIgG in the circulation is 21-24 days.

In a study by Lichtiger and Rogge (1), 126 of 143 lots of IVIgG tested (88%) were reactive for one or both isohemagglutinins (71% had anti-A + anti-B, 12% had anti-A, and 5% had anti-B). In 165 lots of IVIgG analyzed from 1986 to 1990, these same researchers also found that 81 lots analyzed (49%) had RBC alloantibodies (25% nonspecific, 23% anti-D, 8% anti-D + nonspecific, 1% anti-K, and 1% anti-D + anti-K). The rates of reactivity for antibodies to infectious agents were 29% for RPR, 39% for HIV-1, 96% for CMV, 97% for HBcAb, 100% for anti-HAV, and 100% for anti-HBsAg.

Screening of the IVIgG lot that our present patient received showed reactivity for anti-A3+, anti-B3+, anti-D2+, anti-HBsAg, anti-HAV, anti-CMV, and HBcAB. Therefore, IVIgG was probably the source of the anti-D detected on post-BMT day 7.

The presence of a reactive HBcAB combined with a reactive anti-HBsAg should be interpreted as evidence of previous infection with the hepatitis B virus in the past, with complete seroconversion. However, we know that (a) this patient was HbcAB negative before the BMT and (b) the IVIgG lot she received contained HBcAB and anti-HBsAg. More probably then, the source of the newly reactive HBcAB and anti-HBsAg in this patient was passive transfer along with the IVIgG. If correct, tests for these antibodies should have become negative in 3-4 weeks.

Another less likely explanation of these results would be that the patient had had hepatitis in the past with complete seroconversion and that the pre-BMT test result was a false-negative. This is extremely unlikely, however, for the HBcAB almost never disappears (2). However, it has been argued by Martin et al. that in rare cases HBcAB could fall to undetectable levels with chemotherapy or remote infection (3). These same reseachers have also described a case of reactivation of HBsAg and HBeAg after BMT accompanied by the disappearance of previously present anti-HbsAg (3). Even though their patient had had hepatitis, HBcAB was negative both before and after the bone marrow transplant.

A third possibility is that our patient acquired the anti-HBsAg through a nonrecorded vaccination and that the newly reactive HBcAB was a false-positive, a not uncommon finding (4).

It is well known that some patients receiving ABO-incompatible platelet transfusions will develop a positive DAT and/or hemolysis (5). This could explain our direct Coombs’ test results and elution of anti-A2+ detected on post-BMT day 60. However, our patient received only plasma-reduced type O platelets and type O RBCs, components that have an insignificant amount of anti-A and could not have caused the results we observed (6).

The development of immune-mediated hemolytic anemia is a recognized complication of allogeneic BMT (7-9). In most reported cases of post-BMT immune-mediated hemolytic anemia, alloimmunity was due to ABO or minor RBC antigen incompatibility between donor and recipient.

Autoimmune hemolytic anemia (AIHA) occurring after allogeneic BMT has been classified into 2 types: (a) early-onset (2-8 months posttransplant) usually due to a cold antibody (IgM) and (b) late-onset (6-18 months posttransplant) usually due to a warm antibody (IgG) (7). Post-BMT AIHA has been seen in association with monoclonal and oligoclonal Ig bands (7). It has been reported to occur in about 5% of all T cell-depleted allogeneic bone marrow transplant cases (8) and in less than 3% of non-T cell-depleted allogeneic bone marrow transplant cases (7). The specificity of the cold agglutinin is usually anti-I or anti-Pr (7). The warm autoantibody always has a nonspecific component (7) and sometimes an antibody against an antigen of the Rh system.

The results seen on post-BMT day 546 in our patient were probably due to post-BMT AIHA associated with a monoclonal band on serum immunoelectrophoresis.

AIHA directed against antigens in the ABO system has been estimated to represent about 0.13% of all AIHAs that occur in non-BMT patients (9). As with the other AIHA, it is more common after the age of 50 and occurs in association with infections, collagen diseases, pregnancy, lymphoid malignancies, carcinomas, acute leukemias, and myelodysplastic syndromes. Most of the cases reported had anti-A antibodies, but a few cases had anti-B and anti-H antibodies too. Sometimes the antibodies had anti-A activity in conjunction with anti-I activity.

Even though the results obtained at post-BMT day 60 in our patient could be taken to suggest AIHA, they more likely suggest hemolytic anemia of a type O host against type A emerging graft erythrocytes.

Anti-A in the patient’s plasma against donor A-type RBCs is an example of major ABO incompatibility. Major ABO incompatibility is present when the BMT recipient’s plasma is not compatible with the BMT donor’s RBCs, i.e., when the recipient is O and the donor is A, B, or AB; the recipient is A and the donor is B or AB; or the recipient is B and the donor is A or AB.

In cases of major ABO incompatibility, there is the risk of acute immune hemolysis at the time of marrow infusion and delayed immune hemolysis as the engrafted BFU-E (erythroid precursors) start producing type A RBCs. Delayed onset of hematopoiesis and graft failure have also been demonstrated (10-14).

In regards to acute immune hemolysis at the time of marrow infusion, stem cells do not express ABO antigens (12), but the RBCs infused with the harvested bone marrow do. Several studies have approached this problem of acute hemolysis by (a) decreasing the amount of isohemagglutinins in the recipient’s plasma or (b) reducing the number of RBCs in the infused marrow.

In the first case, antibodies from the recipient’s plasma can be removed by extracorporeal whole-blood or plasma immunoadsorption (14), by in vivo absorption with incompatible donor RBCs (15) or with A and B substances, or by plasma exchange. Immunoadsorption techniques, however, are less efficient than plasma exchange. Moreover, all of these techniques have the problem of antibody redistribution from the extravascular compartment and antibody rebound. In vivo adsorption with incompatible RBCs has caused severe hemolysis 6-10 days post-BMT in some cases, and is particularly dangerous. Steroids and immunosuppressive therapy help avoid the antibody-rebound phenomenon.

Instead, RBC depletion of the bone marrow product is now used by most transplant centers to prevent acute hemolysis. This is accomplished by methods including (a) gravity sedimentation (with hydroxyethyl starch, Plasmagel, Dextran, and Ficoll-Hypaque) (14) and (b) differential centrifugation in a cell processor. The latter method is faster and more efficient (75% of original nucleated cells and 57-83% of stem cells are recovered).

In our patient, acute hemolysis was not seen, as differential centrifugation of the donor bone marrow in a cell processor got rid of most donor RBCs.

In regards to delayed immune hemolysis, Sniecinski et al. (16) reported that hemolysis and delay in erythropoiesis beyond 170 days were more frequent in ABO-incompatible BMT patients who received cyclosporine-prednisone for GVHD prophylaxis (n=30) than in patients who received cyclosporine-methotrexate instead (n=28). Delayed immune hemolysis due to major ABO-incompatibility in a BMT recipient can be treated with O-type RBC transfusions, immunosuppression, and sometimes erythropoietin (17). However, nonimmune massive hemolysis has been reported when recombinant human erythropoietin and methylprednisolone are used to treat pure red cell aplasia after a major ABO-incompatible bone marrow transplant (18).

Delayed onset of hematopoiesis, especially erythropoiesis, has also been seen in some patients who have received major ABO-incompatible bone marrow transplants (19-21). After marrow infusion, circulating nucleated red cells appear in 7-10 days, and reticulocytes appear in 2-3 weeks. However, these times may be prolonged for patients receiving a transplant marked by major ABO incompatibility. In general, those patients with higher pretransplantation hemagglutinin titers have the longest delay in reticulocyte recovery, and reticulocytosis does not occur until there is a substantial reduction in titer to a level of £ 4 (12). A few reports have described delayed or failed engraftment of all cell lines (10,11) in patients with major ABO-incompatible bone marrow transplants. Bar et al. found an incidence of 20-27% of immunohematological complications (e.g., a >3-month delay in erythrocyte repopulation, red cell aplasia) among patients who received a lymphocyte-depleted major ABO-incompatible graft (19). However, Mehta et al. showed that ABO incompatibility increased RBC transfusion requirements in the first month posttransplant, while other factors such as diagnosis, conditioning regimen, acute GVHD, and age outweighed the influence of ABO incompatibility on RBC requirements beyond the first month (22).

We can explain the results we obtained at post-BMT day 60 in our patient as delayed hemolysis due to major ABO incompatibility (a group O host with a group A graft). Presumably, while the patient still had a high titer of isohemagglutinins in her plasma, there was no detectable production of group A graft-derived RBCs, although there may have been intramedullary hemolysis, as seen at post-BMT day 40 (elevated LDH and bilirubin). When the isohemagglutinin titer decreased and the patient’s serologic group was AB, the A graft-derived RBCs started emerging and adsorbed any remaining anti-A antibodies, as seen at post-BMT day 60. The transplant-derived erythropoiesis was delayed until post-BMT day 180, when erythrocytic (forward) typing became A. Until post-BMT day 180, the patient continued to be supported with O-type RBC transfusions, which would explain the mixed-field result of the forward typing. Although, the serologic group was A after post-BMT day 60, we do not know if the anti-B was coming from the graft or from the differentially absorbed host isohemagglutinins. The first possibility is more likely, but we would need immunoglobulin allotypes to prove it.

Cases of major Rh-incompatible BMT (patient anti-D antibodies against engrafting donor Rh+ RBCs) have also been described (23), and extravascular destruction of RBCs due to patient anti-D is in fact possible due to the presence of RBCs infused together with the marrow and of Rh+ RBCs from the engrafting marrow. After the ABO system, the most common antigen systems involved in post-BMT alloimmune hemolytic anemia are, in order of decreasing frequency, Rh (24), Kidd, M, N, and S; the incidence of post-BMT host antibodies directed against antigens different from the ABO and Rh systems is 2.1-8% (11). The risk for the development of post-BMT antibodies increases with CMV infection, GVHD, ABO incompatibility, and mild or incomplete preparative immunosuppressive regimens (11). In our patient, neither the anti-D antibody seen at post-BMT day 7 nor that seen at post-BMT day 546 can be explained by Rh incompatibility since both patient and donor were Rh positive.

Currently, the best approach to patients with major ABO-incompatible bone marrow transplants is to infuse RBC-depleted marrow and to anticipate possible delayed erythropoiesis and immune hemolysis, which can be followed with direct antiglobulin testing every 2-3 days. Supportive blood component therapy should be compatible with both recipient and donor blood types.

We therefore conclude that, in cases like the one described here, the patient should receive O-type RBCs, plasma-reduced O-type platelets (or A-type if O-type is not available), and A- or AB-type cryoprecipitate or fresh frozen plasma. In the case of the platelets, however, any non-O group platelets will be destroyed by the host's O plasma (about 30% of transfused platelets will be affected). The objective when we plasma-reduce any transfusion of platelets different from A or AB is to avoid hemolysis of the emerging A-type graft erythrocytes.

In addition, before the patient becomes immunosuppressed (10,12-14), all transfused blood components should be irradiated with 3.0 Gy of gamma radiation to prevent GVHD and leukocyte-reduced to prevent CMV infection (24) and foreign antigen sensitization.

Also, if BMT recipients develop alloantibodies, transfused blood components should be negative for the antigens against which the alloantibodies are directed. Our patient developed anti-Jk^a antibodies and should receive Jk^a— blood.

Minor ABO incompatibility may occur when the donor’s plasma is not compatible with the recipient’s RBCs, i.e., when the recipient is A, B, or AB and the donor is O; the recipient is B or AB and the donor is A; or the recipient is A or AB and the donor is B.

Bacigalupo has reported an increased incidence of GVHD in BMT patients with minor ABO incompatibility (25), but others could not confirm this (10). Hemolysis due to donor’s antibodies in donor plasma infusions and hemolysis as a delayed response to antibodies produced by the engrafting marrow ("passenger immunocompetent donor lymphocytes") have been reported (26). Cyclosporine, used to prevent GVHD, has been associated with this type of hemolysis. Hemolysis of host RBCs due to minor ABO-incompatible BMT donor lymphocytes has been reported to worsen with transfusion, due to hemolysis of transfused group O RBCs by obscure mechanisms (perhaps, e.g., by the innocent bystander mechanism). Methotrexate has been found to prevent this, probably due to its cytotoxicity to all cells, including donor B lymphocytes (27). Hemolysis due to minor Rh-incompatible transplantation (an Rh+ patient with an Rh— donor) has also been reported (28).

Hemolysis due to donor-derived anti-Jk^b antibodies has been reported (29), as has antigen-independent activation of anti-E antibodies in a graft 9 years after allogeneic BMT (30). In the latter case, the authors (30) hypothesized that the activation of anti-E antibodies after so long a time could have been due to desuppression of graft B cells or to activation of graft B cells by the Epstein-Barr virus.

In our patient, hemolysis of her RBCs due to donor-derived anti-Jk^a antibody would have been rare but possible. However, the patient’s direct antiglobulin test was negative when anti- Jk^a antibody was detected, and there were no signs of hemolysis (e.g., bilirubin, LDH, and haptoglobin levels were normal). We hypothesize that the anti-Jk^a alloantibody was probably produced by the donor bone marrow and could have been induced by the remaining host RBCs or by transfused Jk^a+ cells.

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Copyright 1998 The University of Texas M. D. Anderson Cancer Center, Houston, Texas