October 15, 2013 GME Morning report

Harmesh Naik, MD. AUTO IMMUNE HEMOLYTIC ANEMIA (AIHA) CLINICAL HEMOLYSIS INDICATORS

 Anemia  High reticulocyte counts  Elevated LDH level  Unconjugated (indirect) hyper bilirubinemia  Reduced haptoglobin level  Hemoglobinuria COMPARISON

Autoimmune hemolytic anemia Hereditary spherocytosis Hemolysis Hemolysis Spherocytes Spherocytes Splenomegaly Splenomegaly DAT positive DAT negative No Family history Family history TYPE OF ANTIBODY

 Auto antibody – generally seen in AIHA- detected generally by DAT  Allo antibody – generally seen after transfusion of donor RBCs containing antigen – detected generally by IAT

ANTI GLOBULIN TEST (COOMB’S TEST)

 Developed by Coombs in 1945  THE DIRECT ANTIGLOBULIN TEST (DAT) AND INDIRECT ANTIGLOBULIN TEST (IAT).

Zarandona J M , and Yazer M H CMAJ 2006;174:305-307 DIRECT COOMB’S TEST (DAT)

 Detects IgG or complement coating on surface of circulating RBCs  Detects In Vivo sensitization of RBCs  Useful in differentiating immune mediated hemolysis  Generally detects auto-antibodies in AIHA DIRECT COOMB’S TEST (DAT)

 Auto antibodies in AIHA are generally IgG antibodies with high affinity for human RBCs at 37*C 9body temperature)  As a result most of antibody is bound to RBCs and very little is free in plasma  So DAT can find RBC bound antibody effectively

INDIRECT COOMB’S TEST (IAT OR ANTIBODY SCREENING)

 Detects antibodies in serum (IgG antibodies in serum)  Detects in vitro sensitization of RBCs

COLD AUTO ANTIBODY

 Spontaneous agglutination after incubation of patient serum at 16*C  Generally an IgM antibody

POSITIVE DAT: NOW WHAT?

DAT +ve

No clinical Clinical hemolysis hemolysis

Investigate Stop cause POSITIVE DAT WITH HEMOLYSIS

DAT +ve AND Clinical hemolysis

Anti IgG Anti complement antibody antibody

Warm antibody Cold antibody AIHA AIHA

Eluate containing antibody agglutinates all RBCs DIRECT COOMB’S TEST (DAT): METHOD

 First done with poly specific antihuman globulin reagent  If positive  run it with monospecific reagent to detect type of coating antibody – IgG or IgA or IgM or C3C or C3D  If complement deposition – C3C or C3D is detected in absence of auto antibody – still need to rule out Cold antibody IgM or warm IgA (rare) or warm IgM (rare) or biphasic antibody  IgM is hard to detect because of removal during washing

DIRECT COOMB’S TEST (DAT): ELUTION

 Positive DAT – to identify warm antibody with specificity – separate antibody form RBC by elution  Eluate is tested with standard RBC panel  Most warm antibodies are non specific  Sometimes specific antibody can be identified (anti C or anti Rh or anti kell)  Cold IgM can be directed against anti I or Anti H  Biphasic are sometimes against anti P  Use specific blood products when indicated (example use C negative blood in anti C patient)

DAT AND IAT : COMMON RESULTS

DAT IAT Condition Comment + -- AIHA Auto-antibody + + AIHA Auto-antibody RBC bound and free -- + Prior transfusion Allo antibody Fetal to maternal transfer of RBCs RECENTLY TRANSFUSED PATIENT

 Positive DAT in patient may reflect binding of allo antibody (produced in transfused patient as result of antigen on donor RBCs) to donor RBCs still present in patient's circulation – suggests delayed transfusion reaction  Can be confused with auto antibody  Further characterization can determine if DAT positive antibody directed against donor RBCs or autolgous RBCs

ALLO IMMUNIZATION FROM PRIOR TRANSFUSION: UNEXPECTED ANTIBODY

+ve DAT • Transfusion of donor red • Allo antibody in serum can cells to a patient be detected by Indirect • Antigen on donor cells but • Allo antibody coats donor coomb’s test not present on recipient antigen positive RBCs but • Re transfusion of same triggers antibody formation not patients own RBCs donor antigen containing lacking antigen RBCs may trigger more • Results in positive DAT in antibody formation in 7-10 patient days and delayed reaction

Allo Hemolysis with immunization next transfusion TRANSFUSIONS IN AIHA PATIENT: INDICATIONS

 Chronic anemia in asymptomatic patients- do not need transfusion  Patients who are symptomatic at rest or cardiac patients with symptoms can be transfused to maintain clinically acceptable hemoglobin until hemolysis is treated  Transfused RBCs may have short survival and might be destroyed TRANSFUSIONS IN AIHA PATIENT: FINDING RIGHT BLOOD PRODUCT

 Most auto-antibodies react with all potential donor RBCs  Cross matching is difficult  Detection of coexisting allo antibody in patient’s serum (IAT) is time consuming and difficult  Serocompatible blood is found rarely if auto antibody is specific for certain blood group antigen  One must choose donor RBCs that are LEAST INCOMPATIBLE with patients serum in cross match testing TRANSFUSIONS IN AIHA PATIENT: WHO IS AT RISK OF REACTION

 Allo antibody is more likely to cause severe transfusion reaction than auto antibody  Patients with prior transfusions or history of pregnancy might contain allo antibodies – careful testing is necessary for this group  Never transfused or never pregnant patients are unlikely to have allo antibody – no need to delay transfusion for this group TRANSFUSIONS IN AIHA PATIENT: PRECAUTIONS

 Do not hold up transfusion in severely anemic symptomatic patient  Use LEAST INCOMPATIBLE blood product  Monitor patient very closely for transfusion reaction