Hematology 425 Increased RBC Destruction Immune Extracorpuscular Defects

1. Hematology 425 Increased RBC DestructionImmune Extracorpuscular Defects Russ Morrison November 8, 2006

2. Immune Hemolytic Anemia (IHA) • In IHAs, RBC damage is the result of an immunologic event occurring on the surface of the RBCs • Sometimes it interaction of an antibody directly with the RBC membrane with or without complement • Sometimes the RBCs are involved as “bystanders” and absorb immune complexes, fix complement and are lysed by macrophages

3. Immune Hemolytic Anemia (IHA) • There are three types of IHAs • Autoimmune • Drug-induced immune • Alloimmune • Autoimmune HAs include warm-reactive antibody, cold-reactive antibody and paroxysmal cold hemoglobinuria (PCH)

4. Immune Hemolytic Anemia (IHA) • Drug-induced immune HAs may be divided according to the causative mechanism of RBC destruction • Immune complex-mediated • Hapten-mediated • Nonimmunologic protein reaction • Idiopathic autoimmune-mediated • Alloimmune HAs include hemolytic transfusion reactions and HDN

5. Autoimmune Hemolytic Anemia (AIHA) • AIHA is a complex clinical disorder characterized by premature RBC destruction due to autoantibodies bound to antigens on the RBC surface • Most cases are secondary to an altered state of immunity resulting in a loss of immune tolerance and self-recognition • The net result is RBCs damaged by the presence of autoantibodies

6. AIHA • Production of autoantibodies is normally kept in check by T-suppressor lymphocytes • In AIHA, this restraint is lost, resulting in the production of autoantibodies against RBC “self antigens” • AIHA is associated with CLL, lymphoma, immunoglobulin deficiency and autoimmune diseases (SLE is one), drugs and viral infections

7. AIHA - Diagnosis **diagnosis of AIHA depends on demonstration of antibody, complement, or both on the RBC surface** • AIHA may also be a primary disease and occurs in 1 in 80,000 persons in the general population each year • DAT (Coombs’ test) will identify antibodies and complement components on the surface of RBCs and is used as a screening test in the laboratory

8. AIHA - Diagnosis • Principle of the DAT • Net negative RBC surface causes RBCs to repel each other • IgG antibodies can not bridge the gap caused by this RBC repulsion • IgG antibodies, therefore, can not “connect” RBCs by bridging that gap to cause agglutination

9. AIHA - Diagnosis • Principle of the DAT • Polyspecific antihuman globulin (AHG) or Coombs’ antiserum has antibodies directed against the Fc portion of human IgG and complement. • When AHG is added to RBCs with IgG antibody or C fixed to the surface, agglutination of the RBCs occurs and can be seen macroscopically

10. AIHA - Diagnosis • Principle of the DAT 6. If agglutination is demonstrated, monospecific anti-IgG and anti-C3 are used to determine whether immunoglobulin or complement is bound to the RBCs

11. AIHA – Warm Reactive Abs • Warm-reactive AIHA is the most common form if AIHA • Accounts for approximately 70% of cases of AIHA • Two types of warm-reactive Abs exist • Primary (idiopathic) – cause is unknown • Secondary – associated with systemic autoimmune disease, CLL, lymphoma, viral infections and sometimes immune deficiency syndromes

12. AIHA – Warm Reactive Abs • Warm-reactive abs that cause HA are usually of the IgG type, 74% of the IgG1 subtype • The ag-ab reaction is maximized at 37C • Hemolysis is generally extravascular • RBCs in warm AIHA are removed from the circulation by phagocytic monocytes in the spleen which fix to the Fc receptors of the IgG bound to the RBC

13. AIHA – Warm Reactive Abs • A portion of the RBC membrane may be removed (causing spherocytosis) or the entire RBC may be engulfed • About 50% of WAIHA autoantibodies bind to the entire base Rh protein complex on the RBC surface • Some WAIHA is associated with IgM abs directed to high frequency glycophorin ags and have been known to be fatal

14. AIHA – Warm Reactive Abs • Some WAIHA has IgG and IgM autoabs present at the same time and are called mixed type WAIHA • igG is most likely responsible for extravascular hemolysis while the IgM component may lead to intravascular hemolysis • Patients with the mixed type WAIHA often have acute severe anemia that may become chronic

15. AIHA – Warm Reactive Abs • Clinical features are variable • Presentation is with a chronic hemolytic anemia, or occasionally jaundice • Splenomegaly may be present as can fever, pallor, hepatomegaly and tachycardia in severe cases • Anemia varies from mild to severe • PB smear shows increased polychromatophilic RBCs and spherocytes

16. AIHA – Warm Reactive Abs • Once hereditary spherocytosis has been ruled out, spherocytes suggest an immune hemolytic process • NRBCs can be observed in severe cases • Reticulocyte count is between 5 and 10% • Osmotic fragility is increased • Erythroid hyperplasia in the BM • Total bilirubin is mildly increased, as are urinary and fecal urobilinogen levels • Hapatoglobin levels are low yet hemoglobinuria is rarely seen • WBCs and neutrophils are often increased, platelets are usually normal

17. AIHA – Warm Reactive Abs • Demonstration of immunoglobulin and/or complement on the patient’s RBCs is necessary to make the diagnosis of WAIHA • Polyspecific DAT is used as the screening test and, if positive, monospecific (-IgG, -C) is used to detect the sensitization of the RBCs • Positive DAT nails the immune component of the HA

18. AIHA – Warm Reactive Abs • Prednisone has improved management and reduced mortality in these patients • Prednisone is administered for several months, then gradually tapered over 1 to 2 months • Relapses may occur and the patients should be followed up periodically • Splenectomy may be performed if long-term, high-dose prednisone is required to maintain a satisfactory Hgb level

19. AIHA – Warm Reactive Abs • With splenectomy, the primary organ removing the RBCs is removed • Splenectomy may not prove to be a cure and immunosuppressive drugs, intravenous immunoglobulin, or antilymphocytic globulin may be used.

20. Cold-Reactive Antibodies • Cold-reactive antibody AIHA occurs in 2 classes, primary or idiopathic (also called cold agglutinin disease) and secondary • Cold agglutinins are usually IgM, monoclonal abs and do not agglutinate RBCs at temperatures above 30C • As the temperature decreases, IgM binding to the RBCs increases • Thermal amplitude is the highest temperature at which agglutination is detected and will vary from patient to patient • Cold agglutinins cause a chronic HA that is accentuated by exposure to cold

21. Cold-Reactive Antibodies • Cold agglutinin disease refers to rare chronic AIHA in which autoantibody agglutinates human RBCs directly below normal body temperature (0-5C below) • Patients are usually middle aged or elderly and exhibit different combinations of hemolytic anemia, hemoglobinuria and peripheral vasocclusive phenomena resulting from exposure to cold

22. Cold-Reactive Antibodies • Agglutination of RBCs in the extremities leads to vascular obstruction and a clinical condition referred to as acrocyanosis or Raynaud’s phenomenon • Skin characteristically turns white, then blue with concurrent numbness or pain • Hct and reticulocyte counts are variable • Hemolysis is usually extravascular, but occasionally is intravascular

23. Cold-Reactive Antibodies • Secondary form of cold-reactive AIHA are associated with • Infections (Mycoplasma pneumonia, mono and other viral infections) • B-cell malignancies (CLL, lymphoma) • Autoimmune systemic disorders • Both types (primary and secondary) involve the formation of IgM antibodies against the Ii system of RBC antigens

24. Cold-Reactive Antibodies • Complement activation can be complete with brisk intravascular hemolysis or the activation sequence may stop at C3b allowing macrophages to interact leading to extravascular hemolysis • If C3b is inactivated by transformation to C3d, the RBCs will be protected since pagocytes do not have C3d receptors

25. Cold-Reactive Antibodies • A quick screen for cold-reactive antibodies may be performed by immersing an anticoagulated tube of blood in an ice bath • If cold agglutinins are present, gross autoagglutination occurs • Warm the blood to 37C and the agglutination goes away • PB smears made at RT show agglutination, while smears made at 37C do not

26. Cold-Reactive Antibodies • Remember that in Tom’s lectures we discussed these patients • Clumping of RBCs at RT can elevate the MCV, lower the RBC count and provide spurious results for MCH and MCHC • Warming the blood to 37C for 15 minutes before testing will usually provide accurate results • Warm blood smears may need to be prepared for accurate morphology

27. Cold-Reactive Antibodies • DAT is positive at 15-32C and usually positive for complement only • Cold agglutinin titer is positive and will usually be over 1:1000 • Treatment is as simple as avoidance of exposure to cold • Plasmapheresis may be used in severe cases, but provides only temporary benefit

28. Paroxysmal Cold Hemoglobinuria (PCH) • PCH is extremely rare cold-reactive AIHA • PCH is characterized by acute episodes of massive hemolysis that occur after exposure to cold • It is IgG ab that attaches to RBCs only at temperatures of less than 15C • The ab is directed against the P antigen • Complement is always fixed and binds to RBCs at low temperatures

29. PCH • This type of ab is referred to as “biphasic” antibody-antibody attachment • Fixation of complement occurs at low temperatures, but completion of complement sequence and RBC lysis occurs only after rewarming • Condition presents infrequently as a severe, acute, self-limited HA because individuals are rarely chilled to less than 15C

30. PCH • The cause of the ab in PCH is unknown • At body temperature, the ab is dissociated from RBCs which results in a negative DAT or positive only for complement • Shortly after exposure to cold, patients develop aching pains in the back and legs, abdominal cramps and headaches, followed by chills and fever • Hemoglobinuria with dark red or brown urine will be present after symptoms develop

31. PCH • Chronic anemia, increased reticulocytes, decreased Hgb, hemoglobinemia and hyperbilirubinemia may all be observed • Spherocytes are noted on the PB smear and phagocytized RBCs inside monocytes and neutrophils may be seen • DAT is often positive during a crisis as a result of coating of surviving RBCs with complement

32. PCH • This antibody, which binds only in the cold, is a nonagglutinating IgG with specificity for the P blood group antigen • The antibody can be demonstrated by incubating the patient’s fresh serum with RBCs at 4C and then warming the mixture to 37C, after which intense hemolysis will be observed

33. Drug-Induced HA • Usually a benign process, but severe and fatal cases have been reported • Drug-induced immune hemolysis is easily treated by avoiding the drug • Several types of DIIHA exist • Immune complex type • Hapten (drug absorbtion) mechanism • Nonimmunologic protein absorption • autoantibodies

34. Drug-Induced HA, IC • Immune complex type • Drug antibodies are formed • Drug-antidrug reactions form immune complexes • Immune complex is adsorbed onto the RBCs • Complement sequence is activated causing acute intravascular hemolysis, often with renal failure or thrombocytopenia

35. Drug-Induced HA, IC • RBCs are considered “innocent bystanders” • Commonly offending drugs are quinidine, phenacetin and stibophen • HA occurs after small doses, after short periods of administration or on readministration of a previously used drug • DAT shows complement only, serum is reactive only in the presence of the drug, RBC eluates are nonreactive

36. Drug-Induced HA • Drug Adsorbtion Mechanism (hapten) • Drug binds nonspecifically to the RBC and remains firmly attached • Patient develops drug to the drug-carrier complex which reacts with the cell-bound drug • Complement is usually not fixed • Penicillins and cephalosporins are implicated • DAT is positive with IgG • Patient’s serum is reactive with drug-treated RBCs only • RBC eluate also reacts only with drug-treated RBCs

37. Drug-Induced HA • Nonimmunologic Protein Adsorption • Cephalosporins are known to alter the RBC membrane so that numerous proteins, including IgG and complement are adsorbed onto the RBC surface • This phenomenon results in a positive DAT • Only rarely has HA of this type been reported • Serum and elutions are nonreactive

38. Drug-Induced HA • Autoantibodies • Aldomet and procainamide can cause gradual drug-induced AIHA • DAT is strongly positive with IgG only on the RBC surface • Serum and eluates react with unaltered homologous or autologous RBCs in the absence of the drug • Abs appear to be directed against the RBCs themselves • AIHA resolves after drug is withdrawn, but positive DAT may last up to 2 years

39. Drug-Induced HA Mechanism and Treatment • All of the mechanisms of DIIHA can be explained by a single theory which suggests that drug and/or drug metabolites interact with the RBC membrane and cause component immunogenic epitopes that are recognized as foreign by the patient’s immune system • These epitopes then elicit production of abs that react with the drug, drug-RBC complex or the RBC alone

40. Alloimmune Hemolytic Anemias 2 types of alloimmune hemolytic anemias exist • Hemolytic Transfusion Reactions • Hemolytic Disease of the Newborn (HDN)

41. Hemolytic Transfusion Reactions • Transfusion under the best conditions carries the risk of adverse reaction • Reactions are associated with significant morbidity and sometimes can be fatal • Most fatalities have involved human error • ABO mismatch • Administration of correctly cross-matched blood given to the wrong patient Transfusion reactions may be immediate or delayed

42. Hemolytic Transfusion Reactions Immediate • Symptoms begin within minutes or hours • Symptoms include chills, fever, urticaria, tachycardia, nausea, vomiting, chest and back pain, shock, anaphylaxis, pulmonary edema, congestive heart failure • Reactions may be hemolytic or febrile

43. Hemolytic Transfusion Reactions Immediate • Hemolytic transfusion reactions result from intravascular hemolysis, commonly due to ABO incompatibility or to destruction occurring in the macrophages of the spleen, liver and BM • Abnormal bleeding resulting from consumptive coagulopathy or DIC may occur in patients who have major intravascular hemolysis after incompatible transfusion

44. Hemolytic Transfusion Reactions Immediate • Clinical management should begin with immediate termination of the transfusion • Institution of measures to correct bleeding and correct shock as well as maintaining renal circulation to prevent tubular necrosis should be begun

45. Hemolytic Transfusion Reactions Immediate • Laboratory diagnosis of acute hemolytic transfusion reaction is based on evidence of hemolysis and of a blood group incompatibility • Hemoglobinemia, hemoglobinuria, or both are present • Bilirubin level is increased • Haptoglobin level is low • Urine should be examined for hemoglobin

46. Hemolytic Transfusion Reactions Immediate • Febrile reactions to transfusion may result from hemolytic reaction, sensitivity to leukocytes or platelets, bacterial contamination, or unknown causes

47. Hemolytic Transfusion Reactions Delayed • Delayed transfusion reaction may occur days or weeks after transfusion and may result in jaundice and anemia due to hemolysis • Development of previously undetected alloantibodies occurs 4-14 days after transfusion of apparently compatible blood • Often the patient has been alloimmunized by previous pregnancy or transfusion and ab levels were below detection threshold during compatibility testing

48. Hemolytic Transfusion Reactions Delayed • If blood is transfused containing an antigen that corresponds to recipient alloantibody, coating of the transfused RBCs with antibody will lead to hemolysis • Jaundice and lack of RBC increment are principal clinical signs • DAT is positive • Reactions are not as severe as acute

49. Hemolytic Transfusion Reactions • Hemolysis may also occur due to transmission of infectious diseases, malaria and babesia, hepatitis, HIV and CMV during transfusion

50. Rh and HDN • The D antigen of the Rh blood group system is the most important blood group antigen that may cause alloimmunization and HDN (though other blood group abs have been reported as the cause of HDN) • The presence or absence of the D antigen gives rise to the terms Rh positive (when D is present) and Rh negative, when absent