CLS 3311 Advanced Clinical Immunohematology

1. CLS 3311Advanced Clinical Immunohematology Autoimmune Hemolytic Anemia

2. Hemolytic Anemia Auto Immune Antibody Specificity Drop in hemoglobin and hematocrit due to shortened red cell survival. Antibody directed against self antigens on the patients red blood cells Most react with high incidence antigens such as anti-e or anti-I. Auto-immune Hemolytic Anemia

3. Auto-immune Hemolytic Anemia • Mixing patient serum with most donor, reagent and self red blood cells will cause: • Agglutinationor • Coating with antibody and/or Complementor • Cell lysis (or a combination of all three!!) • Auto antibodies can “hide” or mask the presence of clinically significant allo antibodies Categories: • Warm autoimmune hemolytic anemia (70%) • Cold autoimmune hemolytic anemia (18%) • Drug induced hemolytic anemia (2%)

4. Auto-immune Hemolytic Anemia First step: Rule out Other Causes Of Hemolysis such as: • Hereditary spherocytosis • Hemoglobinopathy • Sickle cell crisis • Infusion of hemolyzed RBC’s • Infusion of toxic substances • Need to ASSESS current patient status

5. ASSESSMENT of Patient Status Patient History • Transfusion and pregnancy history • Patient Medication History Laboratory Tests • DAT using bothpolyspecific (anti-IgG, -C3d) and monospecific antisera (anti-IgG) • Antibody Identificationon both serum and eluate. Compare results of each. • Tests to search for additional clinically significant, unexpected antibodies such as an autoadsorption.

6. Cold Autoimmune Hemolytic Anemia • Table 21-1, page 439 Harmening compares Benign to Pathological cold auto antibodies. Be comfortable with this table. Benign cold auto-antibody • Tend to have a low titer (<4) • Tendency to react at 25oC or optimally at 4oC • Specificity usually: anti-I • May be detected only using enzyme treated cells • Typically good Complement activators (IgM) • Only seen when usingPolyspecific AHG reagent.

7. Cold Hemagglutinin DiseasePathological Cold Auto Antibodies • Tend to have ahightiter (>1000 at 4oC) • Broad thermal range of reactivity up to 32oC • Specificity: anti-I, anti-P, etc. • May be detected only using enzyme treated cells • Typically good Complement activators (IgM)

8. Cold Hemagglutinin DiseasePathologic Cold Auto Antibody • Chronic • Idiopathic: No known cause • Acute/transient secondary to infectious disease or condition • Mycoplasma pneumoniae or Infectious Mono • Paroxysmal Cold Hemoglobinuria • Table 21-6, page 443 Harmening

9. Cold Hemagglutinin DiseasePathologic Cold Auto Antibody Chronic: • Often seen in elderly patients • Tends to be associated with Lymphoma, CLL, Waldenstroms macroglobulinemia • Increased Erythrocyte Sedimentation Rate (ESR) • Raynaud’s syndrome and hemoglobinuria seen during cold weather

10. Cold Hemagglutinin DiseasePathologic Cold Auto Antibody Acute/Transient • Often secondary to lymphoproliferative disease (lymphoma) or Mycoplasma pneumonia infections (anti-I specificity) • Also seen in patients with Infectious Mononucleosis with anti-i specificity. • Transient: When infectious process is complete auto antibodies fade.

11. Cold Hemagglutinin DiseasePathologic Cold Auto Antibody Paroxysmal Cold Hemoglobinuria • Auto-Antibody with anti-P specificity • Antibody is a Biphasic IgG class antibody that binds complement at cold temperatures and lyses red cells at warm temperatures • Donath-Landstiener test is confirmatory for PCH (Refer to P Blood Group Lecture)

12. Effects of Cold AutoAntibodies on Laboratory Testing Forward ABO Grouping • If cells are heavily coated with auto-antibody, they may spontaneously agglutinate on forward grouping. How is this corrected? • Pre-warming all reagents and patient samples. • In extreme cases may need to chloroquin treat patient red cells to remove auto-antibody from cell surface.

13. Effects of Cold AutoAntibodies on Laboratory Testing ABO Reverse Grouping • Cold auto antibody can agglutinate Reagent red cells due to testing at room temperature How is this corrected? • Pre-warming serum and reagent RBC’s • High titer antibody? May need to auto absorb patient serum. Rh Typing • If Weak D testing is required may get False Positive using polyspecific AHG: correct using Monospecific AHG.

14. Effects of Cold AutoAntibodies on Laboratory Testing Antibody Identification • Auto-anti-I will agglutinate: • Patients own cells and all adult panel cells • Fails to agglutinate or only weakly agglutinates cord blood cells • Confirm that it is indeed anti-I • Look for clinically significant allo antibody that can be masked by the cold auto antibody. • Pre-warm serum • Start testing at 37oC • Use monospecific anti-IgG

15. Effects of Cold AutoAntibodies on Laboratory Testing Compatibility Testing • Look for incompatibility due to clinically significant allo Antibody that may be masked by the cold auto antibody. • Pre-warm serum • Start testing at 37oC • Use monospecific anti-IgG • Loss of reactivityat 37oC then donor cells are suitable for transfusion. In other words, if after 37oC incubation there is no more agglutination then you can safely transfuse those donor cells.

16. Effects of Cold AutoAntibodies on Laboratory Testing Direct Antiglobulin Test (DAT) • Positive with cold auto antibodies • Only Complement (C3 components) is present because these are IgM antibodies • Use Monospecific: Is it IgG or C’ coating the RBC? Test with both anti-C3d and anti-IgG AHG to differentiate DAT specificity. • Eluate: Not usually performed because it is only Complement coating the red blood cell.

17. Warm Autoimmune Hemolytic Anemia CLINICAL CHARACTERISTICS • Most common AIHA • Moderate to severe hemolytic anemia • More likely to have an anemia that requires txn. • Frequently hemolysis has an acuteonset with increased reticulocytes and spherocytes (indicating extra-vascular hemolysis) • 80% of cases involve IgG class auto-Antibody

18. Clinical Characteristicsof WARM Auto Antibodies • May activate complement: some do, some don’t. • Usually have enhanced reactivity with enzyme treated cells (Ficin, Papain) • Antibody Specificity is usually against a high incident antigen, frequently involving the Rh system such as anti-e. • Here is a great article on Compatibility Testing in WAIHA’sby George Garratty from the University of Southern California. http://www.cbbsweb.org/cbgarrattyaiha.html

19. Effects of WARM Auto Antibodies on Laboratory Testing • ABO Grouping - usually not a problem • Rh Typing - can be a problem with IgG coated red blood cells. • Use monoclonal anti-D or Chemically Modified anti-D • Can Chloroquin treat red blood cells to remove antibody and perform Weak D. Must be careful because chloroquin can denature some Rh antigens.

20. Effects of WARM Auto Antibodies on Laboratory Testing Direct Antiglobulin Test • Positive in the following combinations: • Approx. 67% have both IgG and C’ coating • Approx. 20% have IgG only coating • Approx. 3% have C’ only coating

21. Effects of WARM Auto Antibodies on Laboratory Testing Antibody Identification Patient History: Very important • Pregnancy and transfusion history may indicate possible presence of an allo antibody in addition to the auto antibody. • Transfusion: May also indicate presence of donor red blood cells if transfused within last 120 days. Patient phenotype is very important and hard to perform with donor cells present. • Medications: Some medications have been implicated in Warm AHIA.

22. Effects of WARM Auto Antibodies on Laboratory Testing Antibody Identification • Is it Necessary to identify the Warm Auto Antibody? • It’shelpfulto know the specificity of the auto-antibody, BUT this may not bepossible or practical. • MUST KNOWif there is an underlying unexpected clinically significant allo antibody. • Warm auto-antibodies tend to be more difficult to adsorb out of patient serum than the cold auto-antibodies. May want to treat patient cells with ZZAP prior to Auto Adsorption with patient serum.

23. Effects of WARM Auto Antibodies on Laboratory Testing Antibody Identification • May take multiple adsorptions to remove sufficient antibody to enable detection of allo antibody. • CANNOT do AUTO absorption if the patient has been transfused in the last 120 days. Why? • Can do an adsorption with allogeneic red cells. May need to do multiple adsorptions with various Rh phenotypes (R1R1, rr, R2R2). Must also vary clinically significant blood groups phenotypes, too, such as Kell, Kidd and Duffy.

24. Warm Autoimmune Hemolytic Anemia Selection Of Blood For Transfusion • Transfusion of patients with WAIHA is usually NOT recommended. Why? • Transfused cells are usually destroyed as rapidly as the patients own cells. • When transfusion is unavoidable, usually must transfuse with Least Incompatible Blood. • Patient is generally transfused with small volumes, to maintain O2 carrying capacity, until hemolysis diminishes or other therapy can effect a more lasting benefit.

25. Drug-InducedImmune Hemolytic Anemia's • Most drugs are probably capable of binding loosely, or firmly, to circulating cells, which can lead to an immune response. • Antibodies can be formed to the drug itself or to the drug plus membrane components. • When an antibody is formed against the combination of the drug and membrane it may recognize primarily the drugorprimarily the membrane. • Figure on next slide illustrates these options.

26. AABB Technical Manual, 14th Edition, Page 440

27. Drug-InducedImmune Hemolytic Anemia's Four Mechanisms • Drug-Dependent Antibodies reactive with Drug-Coated Red Cells • Other Drug-Dependent Antibodies: “Immune Complex” Mechanism • Drug-Independent Antibodies: Autoantibody Production • Nonimmunologic Protein Adsorption

28. Drug InducedImmune Hemolytic Anemia's Drug-Dependent Antibodies Reactive with Drug-Coated Red Cells • Drug binds firmly to the RBC membrane and antibody is mainly directed against the drug itself. Drug Adsorption mechanism. • DAT: Strongly positive with IgG. May have some C’. • IAT: Non reactive. Unless using drug coated RBCs. • Eluate: Is reactive with drug coated red cells, only. • Medications: Penicillin and Cephalosporins.

29. Figure 20-2. The drug adsorption mechanism. The drug binds tightly to the red cell membrane proteins. If a patient develops a potent anti-drug antibody, it will react with the cell-bound drug. Such RBCs will yield a positive result in the DAT using anti-IgG reagents. Complement is usually not activated and lysis is primarily extravascular in nature. Penicillin-G is the prototype drug. AABB Technical Manual, 14th Edition, Page 442

30. Drug-Dependent Antibodies Reactive with Drug-Coated Red Cells Penicillin • Approx. 3% of patients receiving high dose penicillin (millions of unit per day) intravenously develop a positive DAT. • Occasionally they develop hemolytic anemia • Hemolysis develops gradually and can become life threatening if etiology is unrecognized. • Discontinuation of drug stops process. May take weeks for hemolysis to stop completely.

31. Drug-Dependent Antibodies Reactive with Drug-Coated Red Cells Cephalosporins • Approx 4% receiving 1st or 2nd generation cephalosporins develop a positive DAT. • Prevalence and severity of cephalosporin induced immune hemolysis appears to be increasing. • Discontinue drug and process stops. May take weeks for hemolysis to stop completely.

32. Drug InducedImmune Hemolytic Anemia's Other Drug-Dependent Antibodies: “Immune Complex” Mechanism • Drugs that do NOT bind well to the red cell membrane. • Previous theory implied drug/anti-drug immunecomplex formation: Never Proven! But we still use the “immune complex” designation to describe this mechanism. • May cause acute intravascular hemolysis and be difficult to demonstrate serologically. • Antibody can be either IgG or IgM

34. Other Drug-Dependent Antibodies: “Immune Complex” Mechanism • DAT: C’ may be only globulin easily detected on the red cells • IAT (and Eluate): Non reactive. Unless using drug coated RBCs. • Acute intravascular hemolysis with hemoglobinemia and hemoglobinuria. Renal failure is common. • Severe hemolytic episodes may recur after initial exposure, even with very small doses of the drug.

35. Drug InducedImmune Hemolytic Anemia's Drug-Independent Antibodies: Autoantibody Production • The druginitially induces an immune response that results in the formation of an auto antibody directed against the red cell. The antibody has NO reactivity with the drug. • Some appear indistinguishable from WAIHA • DAT: Strongly positive with IgG • IAT and Eluate: react strongly with all cells tested (without drug coating) • Medication:-methyldopa, L-dopa, etc.

36. Nonimmunologic Protein Adsorption • Cephalosporins (primarily cephalothin) • Cephalothin (Keflin) alters the red cell membrane so that it adsorbs proteins (albumin, IgA, IgG, IgM, and C’ components) in a non specific and non immunologic manner. The red cell membrane becomes “sticky”. • DAT and IAT: Positive with most AHG reagents. • Panel and Eluate: No pattern, reacts with everything at AHG phase.