Autoimmune Hemolytic Anemia: Overview and Serologic Hemolytic Anemia: Overview and Serologic Case Studies ... AIHA CLASSIFICATION ... Donath-Landsteiner)

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    Autoimmune Hemolytic Anemia: Overview and Serologic Case Studies

    Helene DePalma, MS, MT(ASCP)SBB, CQA(ASQ)

    Associate Professor, CUNY-York College Clinical Laboratory Sciences Program

    Lead Technologist NYBC Transfusion Services

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    AUTOIMMUNE HEMOLYTIC ANEMIA

    Autoantibodies are directed against red blood cell membrane antigens Produced against a variety of self-antigens Follows a failure in the self/non-self

    discrimination mechanism of the immune system

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    Autoantibodies May result in RBC survival and acquired immune

    hemolytic anemia through: activation of complement system and/or removal within the reticuloendothelial

    system Identification of an autoantibody may explain

    decreased RBC survival in vivo If a patients RBCs are coated with autoantibody, the

    patient may present with: a serologic ABO discrepancy a positive Rh control a positive direct antiglobulin test (DAT)

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    Direct Antiglobulin Test (DAT)

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    DAT evaluation Clinical picture Clinical history

    recent transfusion neonate, transplant, medication

    Reagent Polyspecific (anti-IgG and anti-C3d)

    Method tube, gel, solid phase

    Further serological work up Antibody identification (if IAT positive) Eluate

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    Causes of a Positive DAT

    Autoantibodies to intrinsic RBC antigens Alloantibodies

    recent transfusion HDFN

    Passively transfused antibodies IVIG or Rh immune globulin (RhIg)

    Drugs Nonspecifically adsorbed proteins Antibodies derived from passenger lymphocytes (solid organ or HPC transplant)

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    AIHA CLASSIFICATION

    Warm autoimmune hemolytic anemia

    Cold agglutinin disease

    Mixed type autoimmune hemolytic anemia

    Paroxysmal cold hemoglobinuria

    Drug-induced hemolytic anemia

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    WHEN CAN WE EXPECT HEMOLYSIS? Antibody class and characteristics

    Concentration, antigen affinity, ability to fix complement, thermal amplitude

    IgG antibodies are associated with extravascular hemolysis IgM antibodies are associated with complement activation and

    potentially intravascular hemolysis

    Not all autoantibodies (or positive DATs) detected are associated with hemolysis or anemia

    1:1000-14,000 healthy blood donors have positive DATs 1:17-100 hospitals patients have positive DATs

    Evaluation of hemolysis should include: CBC, reticulocyte count, bilirubin, lactate dehydrogenase,

    haptoglobin; peripheral blood smear; and urinalysis

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    RBC Destruction

    Immune RBC destruction may produce either compensated or uncompensated anemia Compensated anemia: rate of RBC

    production will nearly equal the rate of RBC destruction.

    Uncompensated anemia: rate of RBC destruction exceeds the rate of RBC production.

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    Warm Autoimmune Hemolytic Anemia (WAIHA)

    Incidence: 1/100,000 adults DAT positive for IgG

    DAT mostly IgG only, but may be IgG+C3 and rarely C3 only

    Rarely IgA or IgM Eluate and plasma: panagglutinin

    Antigen specificity: May have broad Rh specificity

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    WAIHA

    Extravascular hemolysis Laboratory findings:

    Decreased H/H Increased reticulocyte count Increased LDH, decreased haptoglobin,

    increased bilirubin Urine negative for heme

    Disease associations B-cell neoplasia, lymphoproliferative, collagen-

    vascular diseases

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    WAIHA: Serologic findings

    Autoantibodies present in serum in 80% of patients

    Positive autocontrol

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    Differential: Antibody to a high frequency antigen

    Negative autocontrol

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    Further Work-Up of Positive DAT/IAT

    Elution Removes antibodies bound to patient RBCs, concentrates the antibodies and helps to determine

    specificity

    Adsorption Sponge patient RBCs are used to soak up antibodies in

    serum remaining antibodies may be alloantibodies

    These processes can take many hours!!

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    Warm autoimmune hemolytic anemia: Immunohematology tests

    Test Result

    Direct antiglobulin test, broad spectrum

    Positive

    Rh Rh Rh

    Eluate

    Red cells

    Rh Rh Rh

    Serum

    Direct antiglobulin test, anti-IgG Positive (90%)

    Direct antiglobulin test, anti-C3 Positive (30%)

    Eluate Positive - panagglutinating antoantibody

    Antibody screen (serum, plasma) Positive - panagglutinating autoantibody

    Complex Complex Complex

    Complex Complex Complex

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    Alloimmune hemolysis transfusion reaction due to anti-K: Immunohematology tests

    Test Result

    Direct antiglobulin test, broad spectrum

    Positive

    Eluate

    Red cells

    Serum

    Direct antiglobulin test, anti-IgG Positive

    Direct antiglobulin test, anti-C3 Negative

    Eluate Anti-K (eluted off of transfused, not self RBCs)

    Antibody screen (serum, plasma) Anti-K

    K

    K

    K

    K

    K

    K K K

    K

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    Elution Techniques

    Acid elution kit Digitonin plus acid Heat Freeze/thaw Ether

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    Warm autoadsorption of autoantibodies

    K K

    K

    K

    Rh

    K

    Complex

    Rh Complex

    Rh Complex

    Rh Complex

    K

    K

    The patients serum: autoantibodies plus alloantibodies to K

    The patients red cells: coated with autoantibodies

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    1) Wash and heat patients cells (56oC) to remove autoantibodies

    2) Treat with ficin to enhance Rh complex exposure

    Warm autoadsorption

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    Warm autoadsorption

    3) Add patients serum to an aliquot of treated cells; incubate, then remove serum

    4) Repeat with a new aliquot of patients treated red cells

    5) Remove serum for testing

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    Alloadsorption

    Cant do autoadsorptions in recently transfused patients

    Risk of transfused donor RBCs absorbing an alloantibody

    Instead perform alloadsorption Use reagent RBCs with known antigen specificity to rule

    out the majority of clinically significant antibodies

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    Autoantibodies and Alloantibodies

    Up to 30% of patients with RBC autoantibodies also have underlying alloantibodies

    Some centers phenotype or genotype patients

    with autoantibodies and provide phenotypically matched RBCs

    C, c, E, e, K, Jka, Jkb, Fya, Fyb, S, s

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    RBC phenotype challenges

    Monoclonal reagents Dissociate IgG from RBC before testing with IAT reagent Chloroquine diphosphate EGA

    Prediction of phenotype by DNA analysis

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    Transfusion Considerations When to release least incompatible RBCs?

    Requires medical director and/or ordering physician approval

    Realize blood bank may crossmatch units in LISS or albumin to obtain compatible crossmatch

    What to explain to clinician?

    Transfused RBCs, although crossmatch incompatible, will likely have a similar lifespan as the patients own RBCs (assuming alloantibodies have been ruled out)

    Indications for transfusion include symptomatic anemia (compromise in cardiac or cerebral function) or unstable hematocrit

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    Warm AIHA: Treatment Medication

    Steroids Decrease macrophage clearance of IgG or C3b coated

    RBCs; eventually decrease antibody production IVIG

    Saturates Fc receptors on macrophages Rituximab

    Anti-CD20; mechanism complex (plasma cells are CD20 negative)

    Other immunosuppressive drugs (cytoxan, etc) Plasma exchange

    Large extravascular IgG distribution decrease the efficacy of plasma exchange

    Splenectomy 50-60% response rate Increased risk of sepsis lifelong after splenectomy

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    Cold Agglutinin Disease

    Epidemiology: 1-2 per million incidence; typically in middle-aged or elderly patients

    Associations: Lymphoproliferative disorders

    Clinical features: hemoglobinuria and acrocyanosis after cold exposure

    Laboratory features: anemia, reticulocytosis, autoagglutination of cooled blood samples

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    Cold Agglutinin Disease

    Chronic Patients in 5th-8th decade of life Often have B-cell neoplasms, CLL, or

    Waldenstrom macroglobulinemia Monoclonal (IgM) cold agglutinins

    Transient Younger patient population Abrupt onset, often secondary to infectious

    disease (Mycoplasma pneumoniae; EBV) Polyclonal cold agglutinins

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    Cold Agglutinin Disease is NOT:

    Raynauds phenomenon Vasospasm at cool temperatures No associated autoantibody

    Cryoglobulinemia Monoclonal IgMs that self-associate or

    associate with IgG and precipitate from solution May be polyclonal, may be IgGs

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    Cold Agglutinin Titers

    Sample requirements Collect sample in a prewarmed tube Keep warm until clot retracts

    Titer is the highest serum dilution at which agglutination of RBCs in the cold is seen

    Less than 1 in 10 in healthy patients Greater than 1 in 10,000 in patients with cold

    agglutinin disease Thermal amplitude (more than titer) predicts

    severity of disease (>30C) I or i (rarely Pr specficity)

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