1. Russ Morrison November 8, 2006
Hematology 425 Increased RBC Destruction Immune Extracorpuscular Defects
Russ Morrison
November 8, 2006
9/18/2018

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
9/18/2018

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)
9/18/2018

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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
9/18/2018

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
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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
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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.
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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
9/18/2018

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
9/18/2018

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
9/18/2018

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
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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
9/18/2018

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
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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
9/18/2018

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
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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
9/18/2018

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
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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
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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
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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
9/18/2018

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
9/18/2018

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
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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
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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
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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
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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
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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
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40. Alloimmune Hemolytic Anemias
2 types of alloimmune hemolytic anemias exist
Hemolytic Transfusion Reactions
Hemolytic Disease of the Newborn (HDN)
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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
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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
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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
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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
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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
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46. Hemolytic Transfusion Reactions
Immediate
Febrile reactions to transfusion may result from hemolytic reaction, sensitivity to leukocytes or platelets, bacterial contamination, or unknown causes
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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
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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
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49. Hemolytic Transfusion Reactions
Hemolysis may also occur due to transmission of infectious diseases, malaria and babesia, hepatitis, HIV and CMV during transfusion
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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
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51. Rh and HDN
The production of anti-D in the Rh negative woman and the passage of anti-D across the placenta into the circulation of the Rh positive fetus results in HDN
If the Rh positive father is homozygous, the children will all be D positive, if the father is heterozygous there will be a 50/50 chance that the fetus will be Rh positive and a victim of HDN
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52. Rh and HDN
Alloimmune HDN is a disease of the fetus and the newborn that is characterized by HA, hyperbilirubinemia and extramedullary erythropoiesis
Neonatal or intrauterine death occurs in about ¼ of these cases
Sever jaundice with the risk of brain damage (kernicterus) occurs in about one fourth of the children who survive
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53. Rh and HDN
IgG anti-D crosses the placenta and coats the fetal Rh positive RBCs
Coated RBCs form rosettes around macrophages, mainly in the spleen
RBC membrane is invaginated by pseudopodia from the phagocyte causing the RBC to lose membrane
Spherocytes form, erythrophagocytosis and lysis occur
Hemolysis causes anemia, splenomegaly and hepatomegaly
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54. Rh and HDN NRBCs greatly increase in the PB
Diffusely basophilic cells are present
Thrombocytopenia is common in severely affected babies and can lead to hemorrhagic problems
Unconjugated bilirubin passes back across the placenta and is cleared by the maternal liver until birth
After birth, bilirubin can not be conjugated by the infant’s immature liver and neuron cell death occurs
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55. Rh and HDN Laboratory Findings
Maternal blood group antibody screening is essential during pregnancy
If alloimmunization is diagnosed early, optimal management of the fetus can occur
The Rh negative mother who does not have anti-D should receive Rh immune globulin (RhoGam) at 28 weeks gestation and after delivery
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56. Rh and HDN
If the mother is alloimmunized, specificity and titer of maternal antibody should be determined
Antibody titers should be repeated at 18 weeks gestation and every 4 weeks thereafter until delivery
Measurement of bilirubin in amniotic fluid gives information relating to the severity of hemolytic disease occurring in the fetus
DAT, Hgb and bilirubin determinations may be performed from umbilical venous blood of the fetus
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57. Rh and HDN
Therapy
Intrauterine transfusion may be performed in cases of high fetal risk
Exchange transfusion after delivery is used to remove most of the coated and hemolyzing RBCs and to prevent further anemia and additional bilirubin overproduction
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58. Rh and HDN Prevention of Rh immunization
Rh immunization can be prevented by administration of RhIG to the Rh negative, unimmunized mother carrying an Rh positive fetus or delivering an Rh positive baby
RhIG must be given before a primary Rh immune response begins and dosage must be adequate to cover the volume of fetal-maternal bleed
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59. Erythroblastosis Caused by Other Blood Groups
ABO erythroblastosis is more common than Rh erythroblastosis and usually occurs in the first pregnancy
It produces hyperbilirubinemia, but rarely anemia
ABO hemolytic disease usually occurs when the mother is blood type O and the infant is type A or B and the mother has IgG anti-A or anti-B
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60. Erythroblastosis Caused by Other Blood Groups
Naturally occurring anti-A and anti-B is mostly IgM and does not cross the placenta, making this type of HDN milder than disease caused by anti-D
DAT results in ABO hemolytic disease are only weakly positive
Hemolytic disease of varying severity has been reported with other blood group antibodies
At delivery, cord blood should be tested for ABO, Rh and DAT
Cord blood findings plus the clinical appearance of the infant determine treatment
9/18/2018