Autoimmune Hemolytic Anemia: Overview and Serologic...
Transcript of Autoimmune Hemolytic Anemia: Overview and Serologic...
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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 patient’s 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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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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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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Warm autoadsorption of autoantibodies
K K
K
K
Rh
K
Complex
Rh Complex
Rh Complex
Rh Complex
K
K
The patient’s serum: autoantibodies plus alloantibodies to K
The patient’s red cells: coated with autoantibodies
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1) Wash and heat patient’s cells (56oC) to remove autoantibodies
2) Treat with ficin to enhance Rh complex exposure
Warm autoadsorption
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Warm autoadsorption
3) Add patient’s serum to an aliquot of treated cells; incubate, then remove serum
4) Repeat with a new aliquot of patient’s treated red cells
5) Remove serum for testing
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Alloadsorption
•Can’t 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 patient’s 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:
•Raynaud’s 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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4oC
I
I
I
I
I 4oC
I
I
I
I
4oC I
I
I
4oC
I
I
I
I
I 4oC
I
I
I
I
4oC
I
I
I
I
I
4oC
I
I
I
I
I
4oC
I
I
I
I
I
Low titer (1:4): common
Moderate titer (1:64): infectious mono, mycoplasma pneumonia
High titer (>1:512): cold agglutinin disease
NO HEMOLYSIS HEMOLYSIS RARE HEMOLYSIS COMMON
4oC
Cold agglutinin disease
(Antibodies preferentially bind at 4-20ºC)
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Typical panel seen with cold reacting antibodies
• Some automated techniques omit IS phase
• Cold antibody may not be picked up until IS crossmatch.
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Cold Antibodies
• i = EBV – Cord cells are i positive
• I = Mycoplasma
– Adult cells are I positive
• May lead to ABO discrepancy – Test i, I, A1 cells – May need to prewarm sample
• “Cold screen” typically includes:
– Group A1, B, O, I negative, and autocontrol cells • If broad thermal range
– Prewarm technique – Cold autoadsorption – REST adsorption
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Cold agglutinin disease: Immunohematology tests
Test Result
Direct antiglobulin test, broad spectrum
Positive
Red cells
Serum
Direct antiglobulin test, anti-IgG Negative
Direct antiglobulin test, anti-C3 Strongly positive
Eluate Negative
Antibody screen (serum, plasma)
Positive - panagglutinating cold autoantibody if done at IS
4oC
I
I
I
I
I
I
I I
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Classic Complement Pathway • IgM binds RBCs activating the classic complement pathway
•Full assembly of membrane attack complex resulting in intravascular hemolysis
•Complement activation may not be sufficient to activate full MAC assembly, complement activation only to C3 –C3 coated RBCs
•Phagocytized by macrophages (extravascular hemolysis)
•Require 500-800 C3b molecules for clearance
•May have normal life span
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Cold Agglutinins: Pathogenesis of Autoantibody Formation
• Immune dysfunction •Antigens sharing between infectious agent and RBC (antigen mimicry)
• Infection induced antigenic changes resulting in increased antigenicity
Cold Agglutinin Disease: Treatment
•Avoid cold exposure (cold weather, cold drinks) • Immunosuppression: chorambucil, cyclophosphamide
•Treat the underlying illness! •Prednisone and splenectomy typically NOT effective
•Consider plasma exchange in severe cases
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Combined Cold and Warm AIHA (Mixed AIHA)
•Serologic findings characteristic of WAIHA and has a cold agglutinin of high titer and thermal amplitude
•Both WAIHA and CAD
• IgG antibody is usually more pathogenic – Patients often present with hemolysis
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Paroxysmal Cold Hemoglobinuria (PCH; Donath-Landsteiner)
•Biphasic IgG autoantibody –Binds in the cold, lyses in the warm –DAT positive only for complement –Antibody screen negative –P specificity
• Intravascular hemolysis –Often acute and severe
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PCH
• Diagnosis tricky due to temperature issues
– Sample must be collected in a prewarmed tube, and transported to the blood bank in a heel warmer or cup of warm fluid
– Clot must be allowed to retract at a warm temperature
– Serum is then evaluated for antibody
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Drug-Associated Immune Hemolytic Anemia
• Hapten hypothesis (drug adsorption): Antibodies against a drug (PCN), which is RBC bound
– Negative antibody screen – DAT positive for IgG
• Eluate negative against panel RBCs • Positive against drug coated RBCs
• Immune complex: Antibodies (IgM) against a drug/plasma protein immunogenic complex
– Most common mechanism of drug-induced hemolytic anemia
– DAT positive for complement only – Serum reacts with RBCs only in the presence of
the drug (ex. quinine, quinidine)
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Drug-Associated Immune Hemolytic Anemia (2)
•Non-immunologic protein adsorption: Membrane modification –Positive DAT without hemolysis –Cephalothin, cisplatin
•Autoantibody induction (drug independent) Induction of authentic antibodies against RBCs by the drug (methyldopa, fludarabine) –Positive DAT, with little hemolytic anemia –Stopping the drug leads to gradual
disappearance in autoantibody
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Drug Induced Hemolytic Anemia is NOT Caused by Daratumumab (DarzalexTM)
•Human monoclonal anti-CD38 to treat multiple myeloma
•CD38 is expressed on many cells, including RBCs
•Anti-CD38 interferes with antibody screening by direct binding to CD38 on RBCs
– Uniform reactions with all cells – Variable strength (1-3+) depending on method
•DAT results variable •Further testing: DTT-treated RBCs; genotyping
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Drug-Associated Immune Hemolytic Anemia
•Practical considerations:
–Stop potential offending drugs! –Convey any concerns about drugs to
reference laboratory •May need hospital pharmacy to supply offending drug for testing
Case 1 • 59-year-old white male • Diagnosis: Anemia (Hgb 6 g/dL; AIHA suspected) • No transfusion within 3 months • Preliminary laboratory testing:
– ABO/Rh: O Rh-negative – Direct antiglobulin test: Positive
Polyspecific= 3+; IgG= 3+; C3d= NEG – Antibody screening test:
Cell I IAT: 3+ Cell II IAT: 3+ Cell III IAT: 3+
Case 1: Initial Antibody Panel Rh-hr Kell Kidd Duffy Lewis MNSs P LISS Ficin
Cell D C E c e K k Jka Jkb Fya Fyb Lea Leb M N S s P1 37°C IAT AHG
1 + + 0 0 + 0 + + 0 0 + + 0 0 + 0 + + 0 3+ 4+
2 + + 0 0 + + + 0 + + 0 0 + + + 0 + + 0 3+ 4+
3 + 0 + + 0 0 + + + + + + 0 0 + + + 0 0 3+ 4+
4 + 0 + + 0 0 + 0 + + 0 0 0 + 0 + 0 + 0 3+ 4+
5 + 0 + + + 0 + + + 0 + 0 + 0 + 0 + 0 0 3+ 4+
6 0 0 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 0 3+ 4+
7 0 0 0 + + + + + 0 + 0 0 + + + + + + 0 3+ 4+
8 0 0 0 + + 0 + + 0 + + 0 + 0 + + + + 0 3+ 4+
9 + 0 0 + + 0 + + + 0 + 0 0 + 0 0 + + 0 3+ 4+
10 + 0 0 + + 0 + + 0 0 0 + 0 + + + 0 0 0 3+ 4+
Auto 0 3+ 4+
Case 1: Autoadsorbed serum tested with selected RBC panel
Rh-hr Kell Kidd Duffy Lewis MNSs P LISS Cell D C E c e K k Jka Jkb Fya Fyb Lea Leb M N S s P1 37°C IAT
1 + + 0 0 + 0 + + 0 0 + + 0 0 + 0 + + 0 0
2 + + 0 0 + + + 0 + + 0 0 + + + 0 + + 0 0
3 + 0 + + 0 0 + + + + + + 0 0 + + + 0 0 0
4 + 0 + + 0 0 + 0 + + 0 0 0 + 0 + 0 + 0 0
5 + 0 + + + 0 + + + 0 + 0 + 0 + 0 + 0 0 0
6 0 0 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 0 0
7 0 0 0 + + + + + 0 + 0 0 + + + + + + 0 0
8 0 0 0 + + 0 + + 0 + + 0 + 0 + + + + 0 0
9 + 0 0 + + 0 + + + 0 + 0 0 + 0 0 + + 0 0
10 + 0 0 + + 0 + + 0 0 0 + 0 + + + 0 0 0 0
Auto
All check cells OK
Case 1: Eluate Tested with RBC Panel
Rh-hr Kell Kidd Duffy Lewis MNSs P Eluate Last
Wash Cell D C E c e K k Jka Jkb Fya Fyb Lea Leb M N S s P1 IAT IAT
1 + + 0 0 + 0 + + 0 0 + + 0 0 + 0 + + 2+ 0
2 + + 0 0 + + + 0 + + 0 0 + + + 0 + + 2+ 0
3 + 0 + + 0 0 + + + + + + 0 0 + + + 0 2+ 0
4 + 0 + + 0 0 + 0 + + 0 0 0 + 0 + 0 + 2+ 0
5 + 0 + + + 0 + + + 0 + 0 + 0 + 0 + 0 2+ 0
6 0 0 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 2+ 0
7 0 0 0 + + + + + 0 + 0 0 + + + + + + 2+ 0
8 0 0 0 + + 0 + + 0 + + 0 + 0 + + + + 2+ 0
9 + 0 0 + + 0 + + + 0 + 0 0 + 0 0 + + 2+ 0
10 + 0 0 + + 0 + + 0 0 0 + 0 + + + 0 0 2+ 0
All check cells OK
Case 1 Interpretation:
• Warm autoantibody • Panagglutinin detected in plasma and eluate • No underlying alloantibody after autoadsorption
Case 2 • 68-year-old female • Diagnosis: CLL • Multiple RBC transfusions in the last month • Preliminary laboratory testing:
– ABO/Rh: A Rh-positive – Direct antiglobulin test: Positive
Polyspecific= 2+; IgG= 2+; C3d= NEG – Antibody screening test:
Cell I IAT: 3+ Cell II IAT: 3+ Cell III IAT: 3+
Case 2: Initial Antibody Panel Rh-hr Kell Kidd Duffy Lewis MNSs P Albumin Ficin
Cell D C E c e K k Jka Jkb Fya Fyb Lea Leb M N S s P1 37°C IAT AHG
1 + + 0 0 + 0 + + 0 0 + + 0 0 + 0 + + 0 3+ 4+
2 + + 0 0 + + + 0 + + 0 0 + + + 0 + + 0 3+ 4+
3 + 0 + + 0 0 + + + + + + 0 0 + + + 0 1+ 3+ 4+
4 + 0 + + 0 0 + 0 + + 0 0 0 + 0 + 0 + 1+ 3+ 4+
5 + 0 + + + 0 + + 0 0 + 0 + 0 + 0 + 0 1+ 3+ 4+
6 0 0 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 0 3+ 4+
7 0 0 0 + + + + + 0 + 0 0 + + + + + + 0 3+ 4+
8 0 0 0 + + 0 + + 0 + + 0 + 0 + + + + 0 3+ 4+
9 + 0 0 + + 0 + 0 + 0 + 0 0 + 0 0 + + 0 3+ 4+
10 + 0 0 + + 0 + + 0 0 0 + 0 + + + 0 0 0 3+ 4+
Auto + 0 0 + + 0 + 0 0 0 + 0 + + + 0 0 0 3+ 4+
Case 2: Alloadsorbed serum tested with selected RBC panel
Rh-hr Kell Kidd Duffy Lewis MNSs P Albumin Cell D C E c e K k Jka Jkb Fya Fyb Lea Leb M N S s P1 37°C IAT
1 + + 0 0 + 0 + + 0 0 + + 0 0 + 0 + + 0 0
2 + + 0 0 + + + 0 + + 0 0 + + + 0 + + 0 0
3 + 0 + + 0 0 + + + + + + 0 0 + + + 0 0 2+
4 + 0 + + 0 0 + 0 + + 0 0 0 + 0 + 0 + 0 2+
5 + 0 + + + 0 + + 0 0 + 0 + 0 + 0 + 0 0 2+
6 0 0 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 0 0
7 0 0 0 + + + + + 0 + 0 0 + + + + + + 0 0
8 0 0 0 + + 0 + + 0 + + 0 + 0 + + + + 0 0
9 + 0 0 + + 0 + 0 + 0 + 0 0 + 0 0 + + 0 0
10 + 0 0 + + 0 + + 0 0 0 + 0 + + + 0 0 0 0
Auto
All check cells OK
Case 2: Eluate Tested with RBC Panel
Rh-hr Kell Kidd Duffy Lewis MNSs P Eluate Last
Wash Cell D C E c e K k Jka Jkb Fya Fyb Lea Leb M N S s P1 IAT IAT
1 + + 0 0 + 0 + + 0 0 + + 0 0 + 0 + + 3+ 0
2 + + 0 0 + + + 0 + + 0 0 + + + 0 + + 3+ 0
3 + 0 + + 0 0 + + + + + + 0 0 + + + 0 3+ 0
4 + 0 + + 0 0 + 0 + + 0 0 0 + 0 + 0 + 3+ 0
5 + 0 + + + 0 + + 0 0 + 0 + 0 + 0 + 0 3+ 0
6 0 0 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 3+ 0
7 0 0 0 + + + + + 0 + 0 0 + + + + + + 3+ 0
8 0 0 0 + + 0 + + 0 + + 0 + 0 + + + + 3+ 0
9 + 0 0 + + 0 + 0 + 0 + 0 0 + 0 0 + + 3+ 0
10 + 0 0 + + 0 + + 0 0 0 + 0 + + + 0 0 3+ 0
All check cells OK
Case 2 Interpretation:
• Allo anti-E • Warm autoantibody • Panagglutinin detected in plasma and eluate
Case 3
• Diagnosis: Sepsis • Transfused 2 units a month ago • Preliminary laboratory testing: ABO/Rh: B Positive Direct antiglobulin test: Polyspecific = 2+ IgG = Negative C3d = 2+
Case 3: Serum tested with selected RBC panel
Rh-hr Kell Kidd Duffy Lewis MNSs P Albumin Cell D C E c e K k Jka Jkb Fya Fyb Lea Leb M N S s P1 RT IAT
1 + + 0 0 + 0 + + 0 0 + + 0 0 + 0 + + 2+ 2+
2 + + 0 0 + + + 0 + + 0 0 + + + 0 + + 2+ 1+
3 + 0 + + 0 0 + + + + + + 0 0 + + + 0 2+ 1+
4 + 0 + + 0 0 + 0 + + 0 0 0 + 0 + 0 + 2+ 1+
5 + 0 + + + 0 + + 0 0 + 0 + 0 + 0 + 0 2+ 2+
6 0 0 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 2+ 1+
7 0 0 0 + + + + + 0 + 0 0 + + + + + + 2+ 2+
8 0 0 0 + + 0 + + 0 + + 0 + 0 + + + + 2+ 2+
9 + 0 0 + + 0 + 0 + 0 + 0 0 + 0 0 + + 2+ 1+
10 + 0 0 + + 0 + 0 + 0 0 + 0 0 + + 0 0 2+ 1+
Auto 2+ 1+
All check cells OK
Case 3: REST adsorbed serum tested with selected RBC panel
Rh-hr Kell Kidd Duffy Lewis MNSs P Albumin Cell D C E c e K k Jka Jkb Fya Fyb Lea Leb M N S s P1 RT IAT
1 + + 0 0 + 0 + + 0 0 + + 0 0 + 0 + + 0 2+
2 + + 0 0 + + + 0 + + 0 0 + + + 0 + + 0 0
3 + 0 + + 0 0 + + + + + + 0 0 + + + 0 0 1+
4 + 0 + + 0 0 + 0 + + 0 0 0 + 0 + 0 + 0 0
5 + 0 + + + 0 + + 0 0 + 0 + 0 + 0 + 0 0 2+
6 0 0 0 + + 0 + 0 + 0 + + 0 + 0 + 0 0 0 0
7 0 0 0 + + + + + 0 + 0 0 + + + + + + 0 2+
8 0 0 0 + + 0 + + 0 + + 0 + 0 + + + + 0 2+
9 + 0 0 + + 0 + 0 + 0 + 0 0 + 0 0 + + 0 0
10 + 0 0 + + 0 + 0 + 0 0 + 0 0 + + 0 0 0 0
Auto
All check cells OK
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AIHA Take Home Messages • Evaluate all pertinent clinical information
– Evidence of hemolysis? – Transfusion or pregnancy history? – Is there a potential for alloantibodies in addition to the
autoantibody? – Diagnosis? – Medications?
• Determine if an underlying alloantibody is present • Obtain an RBC phenotype (or genotype for predicted
phenotype) • What’s in your toolkit?
– Elution kit, monoclonal reagents, enzymes, EGA • Know when to seek help!
– Send to an Immunohematology Reference Lab for specialized tests