Discussion
We describe an unusual presentation of hyperhemolysis in a beta thalassemia major patient who presented with high grade fever, bone pain and malaise. It was interesting to note that Day 2 DAT was negative for anti-IgG but showed 2+ for anti-C3d. A well performed DAT detects 100-500 molecules of bound IgG per red blood cell. A negative IgG autoantibody is a common feature in acute form of HS. There are several reasons which may explain a negative DAT in HS. They are (a) sensitization of the patient’s red cells with a small number of IgG molecules which is below the threshold for detection by a standard DAT (b) most standard DAT do not incorporate anti-IgA monoclonal reagent, thereby, missing out on the possibility of IgA mediated hemolysis (c) low affinity IgG autoantibodies being eluted from RBC during the washing process4. A super Coombs test, which incorporates an enhanced and more sensitive version of the standard DAT may be useful in cases where the standard DAT is negative.
It is also interesting to note that the negative anti-C3d on Day 15 repeated DAT in our patient could be attributed to the immunosuppressive corticosteroid therapy given to him.
The pathogenesis of HS is interesting. The mechanisms which may explain HS in our patient are: (a) macrophage hyperactivation (b) RBC alloimmunisation (c) complement regulation dysfunction (d) bystander hemolysis (e) suppression of erythropoiesis and (f) HLA alloimmunisation5. Macrophages are widely believed to be in a primed state in hemoglobinopathy syndromes. In response to allogeneic transfusion, macrophages become hyperactivated and bind to both host and donor red blood cells resulting in rapid destruction5. Interestingly, our patient had significant extramedullary hematopoiesis in the spleen resulting in hypersplenism. Since the splenic red pulp contains a large number of macrophages, therefore this group of patients are at considerable risk of developing HS.
Bystander hemolysis, a term coined by Petz and Garraty, is used to describe immune hemolysis of erythrocytes in the presence of recipient alloantibodies directed to antigen negative transfused red blood cells6. Besides RBC alloantibodies; HLA and plasma protein antigens from the transfused blood may react with the recipient’s antibodies contributing to complement regulation dysfunction, thereby leading to bystander hemolysis7. However, in this patient, we did not perform an HLA antibody screen, and facilities to measure levels of complement factors, C5b-9 to support our findings of complement dysfunction were unavailable.
Besides the above, RBC alloimmunization is also an important cause of hyperhemolysis. A negative antibody screen in our patient at Day 2 of transfusion is a feature commonly seen in the acute form of HS. Standard serological studies used may not be potent enough to detect low affinity alloantibodies against low frequency antigens. Antibody dependent cell mediated cytotoxicity (ADCC) may explain the mechanism of hemolysis in recipients with levels of alloantibody below the serological detection threshold8. Interestingly, anti-Cwwas subsequently detected in our patient’s serum on Day 15. This antibody including those of the Kidd or Rh system are often undetectable on pretransfusion testing but may increase rapidly in titer following a blood transfusion. Re-exposure of an antigen negative recipient to a red cell antigen positive transfusion could result in an anamnestic antibody response, precipitating hemolysis. Anti-Cw is an alloantibody directed against the Cw antigen (Rh 8), which is located on the RHCE gene. This would explain the initial presence of anti-C3d on Day 2 DAT in which, the anti-Cw causes activation of the complement. Cw is a low incidence antigen, and has been reported to be 1% and 2% in Blacks and Caucasians respectively9. Anti-Cw, usually an IgG antibody, may occur naturally or after an immune stimulation by blood transfusion such as seen in this patient. Unlike other Rh antigens, anti-Cw is usually not significant. Anti-Cw associated moderate-to-severe hemolytic disease of the fetus and newborn (HDFN) have been reported infrequently in case reports and thus may be of clinical significance in females of reproductive age10. However, literature on the significance of this antibody in acute and delayed hemolytic transfusion reaction is severely lacking.
Severe reticulocytopenia was also an additional feature seen in our patient. Thalassemia patients are known to have significantly lower serum levels of erythropoietin at steady state which may trigger neocytolysis during HS causing further destruction of erythroid precursors11. A bone marrow analysis would be helpful to confirm the presence of erythropoiesis suppression. Another mechanism which could also contribute to reticulocytopenia is increased peripheral consumption of reticulocytes by hyperactive macrophages in the presence of preserved bone marrow activity of erythropoiesis.
Management of HS is largely dependent on the severity of anemia and degree of hemolysis. Blood transfusion should be avoided in the milder form of HS as they are known to exacerbate hemolysis. Combination therapy of IVIg and steroids are synergistic in suppressing macrophage activation and shortening hemolysis through immodulatory effects12. Low dose IVIg at 0.4g/kg daily for 5 days and intravenous methylprednisolone 500mg daily for 2 days are recommended for the treatment of HS12. In severe forms of HS, red blood cell transfusion may be required but should be given in the presence of IVIg-steroid cover. Subcutaneous erythropoietin may have a role in direct stimulation of erythropoieis and preventing neocytolysis. Furthermore, since macrophage activation is an important mechanism in HS, anti-CD20 monoclonal antibody such as rituximab 375mg/m2 even at a single dose has shown response13. It is interesting to note that eculizumab, a C5 convertase inhibitor which inhibits complement activation is promising in the treatment of HS in sickle cell anemia and may also confer such benefit in thalassemia14. On another note, as hemolysis increases the risk of both arterial and venous thrombosis, it is imperative to ensure adequate anticoagulation prophylaxis is given during active HS. Splenectomy has a role in HS as it is known to resolve HS crisis in thalassemia15. However, our patient declined splenectomy due to personal reasons.