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 1
AUTOMMUNE HEMOLYTC ANEMA 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 2
Autoantibodies May result in RBC survival and acquired immune hemolytic anemia through: activation of complement system and/or removal within the reticuloendothelial system dentification of an autoantibody may explain decreased RBC survival in vivo f 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) 3
Direct Antiglobulin Test (DAT) 4
DAT evaluation Clinical picture Clinical history recent transfusion neonate, transplant, medication Reagent Polyspecific (anti-gg and anti-c3d) Method tube, gel, solid phase Further serological work up Antibody identification (if AT positive) Eluate 5
Causes of a Positive DAT Autoantibodies to intrinsic RBC antigens Alloantibodies recent transfusion HDFN Passively transfused antibodies VG or Rh immune globulin (Rhg) Drugs Nonspecifically adsorbed proteins Antibodies derived from passenger lymphocytes (solid organ or HPC transplant) 6
AHA CLASSFCATON Warm autoimmune hemolytic anemia Cold agglutinin disease Mixed type autoimmune hemolytic anemia Paroxysmal cold hemoglobinuria Drug-induced hemolytic anemia 7
WHEN CAN WE EXPECT HEMOLYSS? Antibody class and characteristics Concentration, antigen affinity, ability to fix complement, thermal amplitude gg antibodies are associated with extravascular hemolysis gm 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 8
RBC Destruction mmune 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. 9
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Warm Autoimmune Hemolytic Anemia (WAHA) ncidence: 1/100,000 adults DAT positive for gg DAT mostly gg only, but may be gg+c3 and rarely C3 only Rarely ga or gm Eluate and plasma: panagglutinin Antigen specificity: May have broad Rh specificity 11
WAHA Extravascular hemolysis Laboratory findings: Decreased H/H ncreased reticulocyte count ncreased LDH, decreased haptoglobin, increased bilirubin Urine negative for heme Disease associations B-cell neoplasia, lymphoproliferative, collagenvascular diseases 12
WAHA: Serologic findings Autoantibodies present in serum in 80% of patients Positive autocontrol 13
Differential: Antibody to a high frequency antigen Negative autocontrol 14
Further Work-Up of Positive DAT/AT 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!! 15
Warm autoimmune hemolytic anemia: mmunohematology tests Test Result Direct antiglobulin test, broad spectrum Positive Direct antiglobulin test, anti-gg Positive (90%) Red cells Direct antiglobulin test, anti-c3 Positive (30%) Eluate Antibody screen (serum, plasma) Positive - panagglutinating antoantibody Positive - panagglutinating autoantibody 16 Rh Rh Rh Complex Complex Complex Serum Rh Rh Rh Complex Complex Complex Eluate
Alloimmune hemolysis transfusion reaction due to anti-k: mmunohematology tests K K Test Result K K Direct antiglobulin test, broad spectrum Direct antiglobulin test, anti-gg Direct antiglobulin test, anti-c3 Eluate Antibody screen (serum, plasma) Positive Positive Negative Anti-K (eluted off of transfused, not self RBCs) Anti-K 17 Red cells K K Serum K K K Eluate
Elution Techniques Acid elution kit Digitonin plus acid Heat Freeze/thaw Ether 18
Warm autoadsorption of autoantibodies K Rh Complex K K K Rh Complex K Rh Complex K Rh Complex K The patient s serum: autoantibodies plus alloantibodies to K The patient s red cells: coated with autoantibodies 19
Warm autoadsorption 1) Wash and heat patient s cells (56 o C) to remove autoantibodies 2) Treat with ficin to enhance Rh complex exposure 20
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 21
Alloadsorption Can t do autoadsorptions in recently transfused patients Risk of transfused donor RBCs absorbing an alloantibody nstead perform alloadsorption Use reagent RBCs with known antigen specificity to rule out the majority of clinically significant antibodies 22
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, Jk a, Jk b, Fy a, Fy b, S, s 23
RBC phenotype challenges Monoclonal reagents Dissociate gg from RBC before testing with AT reagent Chloroquine diphosphate EGA Prediction of phenotype by DNA analysis 24
Transfusion Considerations When to release least incompatible RBCs? Requires medical director and/or ordering physician approval Realize blood bank may crossmatch units in LSS 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) ndications for transfusion include symptomatic anemia (compromise in cardiac or cerebral function) or unstable hematocrit 25
Warm AHA: Treatment Medication Steroids Decrease macrophage clearance of gg or C3b coated RBCs; eventually decrease antibody production VG Saturates Fc receptors on macrophages Rituximab Anti-CD20; mechanism complex (plasma cells are CD20 negative) Other immunosuppressive drugs (cytoxan, etc) Plasma exchange Large extravascular gg distribution decrease the efficacy of plasma exchange Splenectomy 50-60% response rate ncreased risk of sepsis lifelong after splenectomy 26
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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 28
Cold Agglutinin Disease Chronic Patients in 5 th -8 th decade of life Often have B-cell neoplasms, CLL, or Waldenstrom macroglobulinemia Monoclonal (gm) cold agglutinins Transient Younger patient population Abrupt onset, often secondary to infectious disease (Mycoplasma pneumoniae; EBV) Polyclonal cold agglutinins 29
Cold Agglutinin Disease is NOT: Raynaud s phenomenon Vasospasm at cool temperatures No associated autoantibody Cryoglobulinemia Monoclonal gms that self-associate or associate with gg and precipitate from solution May be polyclonal, may be ggs 30
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) or i (rarely Pr specficity) 31
Cold agglutinin disease (Antibodies preferentially bind at 4-20ºC) 4 o C 4 o C 4 o C 4 o C 4 o C 4 o 4C o C 4 o C 4 o C Low titer (1:4): common Moderate titer (1:64): infectious mono, mycoplasma pneumonia High titer (>1:512): cold agglutinin disease NO HEMOLYSS HEMOLYSS RARE HEMOLYSS COMMON 32
Typical panel seen with cold reacting antibodies Some automated techniques omit S phase Cold antibody may not be picked up until S crossmatch. 33
Cold Antibodies i = EBV Cord cells are i positive = Mycoplasma Adult cells are positive May lead to ABO discrepancy Test i,, A1 cells May need to prewarm sample Cold screen typically includes: Group A1, B, O, negative, and autocontrol cells f broad thermal range Prewarm technique Cold autoadsorption REST adsorption 34
Cold agglutinin disease: mmunohematology tests Test Direct antiglobulin test, broad spectrum Direct antiglobulin test, anti-gg Direct antiglobulin test, anti-c3 Eluate Result Positive Negative Strongly positive Negative Red cells 4 o C Antibody screen (serum, plasma) Positive - panagglutinating cold autoantibody if done at S Serum 35
Classic Complement Pathway gm 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 36
Cold Agglutinins: Pathogenesis of Autoantibody Formation mmune dysfunction Antigens sharing between infectious agent and RBC (antigen mimicry) nfection induced antigenic changes resulting in increased antigenicity 37
Cold Agglutinin Disease: Treatment Avoid cold exposure (cold weather, cold drinks) mmunosuppression: 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 AHA (Mixed AHA) Serologic findings characteristic of WAHA and has a cold agglutinin of high titer and thermal amplitude Both WAHA and CAD gg antibody is usually more pathogenic Patients often present with hemolysis 40
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Paroxysmal Cold Hemoglobinuria (PCH; Donath-Landsteiner) Biphasic gg autoantibody Binds in the cold, lyses in the warm DAT positive only for complement Antibody screen negative P specificity ntravascular hemolysis Often acute and severe 42
Syphilis Eder et al, mmunohematology, 21 (2): 2005. 43
Eder et al, mmunohematology, 21 (2): 2005. 44
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 45
Donath-Landsteiner Test Normal serum is a source of complement 46
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Proposed Unifying Theory of Drug-nduced Antibody Reactions 48
Drug-Associated mmune Hemolytic Anemia Hapten hypothesis (drug adsorption): Antibodies against a drug (PCN), which is RBC bound Negative antibody screen DAT positive for gg Eluate negative against panel RBCs Positive against drug coated RBCs mmune complex: Antibodies (gm) 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) 49
Drug-Associated mmune Hemolytic Anemia (2) Non-immunologic protein adsorption: Membrane modification Positive DAT without hemolysis Cephalothin, cisplatin Autoantibody induction (drug independent) nduction 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 50
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Drug nduced Hemolytic Anemia is NOT Caused by Daratumumab (Darzalex TM ) 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 52
Drug-Associated mmune 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 53
Case 1 59-year-old white male Diagnosis: Anemia (Hgb 6 g/dl; AHA suspected) No transfusion within 3 months Preliminary laboratory testing: ABO/Rh: O Rh-negative Direct antiglobulin test: Positive Polyspecific= 3+; gg= 3+; C3d= NEG Antibody screening test: Cell AT: 3+ Cell AT: 3+ Cell AT: 3+
Case 1: nitial Antibody Panel Rh-hr Kell Kidd Duffy Lewis MNSs P LSS Ficin Cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P 1 37 C AT 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 LSS Cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P 1 37 C AT 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 Jk a Jk b Fy a Fy b Le a Le b M N S s P 1 AT AT 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 nterpretation: 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+; gg= 2+; C3d= NEG Antibody screening test: Cell AT: 3+ Cell AT: 3+ Cell AT: 3+
Case 2: nitial Antibody Panel Rh-hr Kell Kidd Duffy Lewis MNSs P Albumin Ficin Cell D C E c e K k Jk a Jk b Fy a Fy b Le a Le b M N S s P 1 37 C AT 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 Jk a Jk b Fy a Fy b Le a Le b M N S s P 1 37 C 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 AT
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 Jk a Jk b Fy a Fy b Le a Le b M N S s P 1 AT AT 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 nterpretation: 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+ gg = 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 Jk a Jk b Fy a Fy b Le a Le b M N S s P 1 RT AT 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 Jk a Jk b Fy a Fy b Le a Le b M N S s P 1 RT AT 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
Case 3 nterpretation Cold autoantibody Anti-Jk a detected
AHA Take Home Messages Evaluate all pertinent clinical information Evidence of hemolysis? Transfusion or pregnancy history? s 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 mmunohematology Reference Lab for specialized tests 68
Acknowledgements Beth H. Shaz, MD, Chief Medical Officer-NYBC Sharing her slides Dalisay Charles-Pierre, Manager, NYBC mmunohematology Laboratory Sharing her case studies