1. Mixed Type Autoimmune Hemolytic Anemia
Charles Stoudenmire, MD
2/7/14

2. Mixed AIHA
Combination of warm autoimmune hemolytic anemia and cold agglutinin disease
Can be idiopathic or secondary to lymphoproliferative disorders or SLE

3. Warm AIHA
Typically idiopathic or secondary to lymphoproliferative disorders, autoimmune disorders, or immunodeficiency
Secondary to IgG (rarely IgA or IgM) with broad specificity
Antibodies bind RBCs at 37°C
Splenic macrophages phagocytose IgG-coated RBCs (extravascular hemolysis)
Increase in serum bilirubin, but no hemoglobinemia or hemoglobinuria
Normal haptoglobin

4. Lab Testing (WAIHA)
Antibody screen and panels will show panagglutination
DAT positive for IgG, maybe C3d
Eluate shows panagglutination with IgG

5. Determining Compatibility (WAIHA)
Adsorption techniques
RBC phenotyping
Give phenotype-matched RBCs if possible
C, E, c, e, K, Jka, Jkb, Fya, Fyb, S, s (Johns Hopkins)
Least incompatible blood
Controversial
Transfusion carries risk of further hemolysis and volume overload

6. Treatment of WAIHA Steroids Rituximab IVIg Immunosuppressants
Splenectomy
Treat underlying illness
Plasma exchange if severe and acute

7. Cold Agglutinin Disease
Typically results from infection (transient) or lymphoproliferative disorder (chronic)
Secondary to IgM commonly directed against I/i blood group system
Moderate hemolytic anemia exacerbated by cold

8. CAD continued
Need high titer (>1:1,000) and thermal amplitude (reactive at >30°C) to be significant
IgM fixes complement, resulting in intravascular hemolysis
IgM typically dissociates at warmer temperatures
Hemoglobinemia, hemoglobinuria, hemosiderinuria, low haptoglobin
May have extravascular hemolysis if complement cascade not completed

9. Lab Testing (CAD) DAT positive for C3d Eluate non-reactive
Pre-warming (37°C) can prevent reactivity of autoantibody
Allows identification of alloantibodies
Crossmatch-compatible blood at 37°C
Blood warmer during transfusion
Rarely cold adsorption may be required

10. Treatment of CAD Cold avoidance Rituximab Interferon-alpha
Chlorambucil
Cyclophosphamide
Plasma exchange
Treat underlying illness

11. Mixed AIHA
Combination of warm autoimmune hemolytic anemia and cold agglutinin disease

12. MAIHA Often chronic course with periodic severe exacerbations
Hgb can drop below 5 g/dL
IgG autoantibody is usually the more clinically significant

13. Lab Testing (MAIHA) Screen and panels usually show panagglutination
DAT positive for IgG and C3d
Eluate shows panagglutination with IgG

14. Compatibility Adsorption at 37°C and 4°C, if possible
Otherwise donor units released as cross-match least-incompatible

15. Hillyer et. al. (Modified from Brecher ME (ed. ). 2005
Hillyer et. al. (Modified from Brecher ME (ed.) AABB Technical Manual, 15th ed. Bethesda, MD: AABB Press)

16. MAIHA and Rituximab Morselli et al. Blood 2002
62-year-old female presents with weakness and dyspnea
Anemic (Hgb 6.4 g/dL), 12.2% reticulocytes, elevated bilirubin and LDH, haptoglobin < 7 mg/dL
Positive for polyvalent serum and positive DAT for IgG and C3d
IgM cold agglutinin titer 1:1024

17. MAIHA and Rituximab cont.
Started on prednisone with increase in Hgb to 11 g/dL, reduction of positivity in direct and indirect antiglobulin tests, and reduction in cold agglutinin titer to 1:128
Steroids tapered with relapse of disease
Bone marrow cells positive for CD19 and 20
Rituximab started, with complete remission after 2 course (~2 months)
First time rituximab used for MAIHA

18. MAIHA and Rituximab other recent studies
Haller et al. Pediatric Transplant, 2010
Male infant with history of liver transplant at 2.5 months for congenital Factor VII def.
2 months post-transplant presented with pallor, jaundice, HS-megaly, tachypnea
Hgb 2.1 g/dL, elevated LDH, retic count, total bilirubin; low haptoglobin
RBC agglutination, platelet clumps, and spherocytes on peripheral smear
DAT strong for IgG, C3d, and IgM
Both IgG and IgM isolated, and serum levels high
Patient remained refractory to steroids and frequent transfusions
Rituximab started with remission one year after treatment

19. MAIHA and Rituximab other recent studies
Scaramucci et al. Blood Research. 2013
19-year-old female with thrombosis of portal and splenic veins, with partial occlusion of SMV
Hgb 3.9 g/dL with elevated LDH, indirect bilirubin, and retic. Count
DAT strongly positive for IgG and C3d
IAT positive at 4˚C and negative at 22 and 39˚C in presence of panagglutinin IgM
Idiopathic origin
Started on prednisone with remission, but 6 months after taper redeveloped AIHA.
Started on rituximab with remission; regularly monitored

20. MAIHA and Rituximab other recent studies
Gupta et al. Journal of Medical Case Reports. 2011
62-year-old Caucasian man with history of alcholism presented with dyspnea and confusion for 3 days
Hgb 4.5 g/dL; elevated LDH and retic count; low haptoglobin
DAT positive for IgG and C3d, “identified as cold agglutinins at 4˚C”
Refractory to steroids, so plasmapheresis started
Eventually started rituximab with marked improvement

21. Reevaluating the Incidence of MAIHA
Transfusion 2008
Institute for Transfusion Medicine- Charite-University Medicine, Berlin, Germany
Cited incidence of MAIHA from previous studies as 6-8% of AIHA
Is this accurate?

22. Methods
August
All in- and outpatients with detectable warm autoAbs
2192 patients
Warm Ab testing: DAT (anti-IgG, -IgA, -IgM, -C3d), eluate, serum indirect antiglobulin test
Defined presence of warm Ab if 2/3 above were met
If cold Abs present, RBCs warmed to 45C and washed at 40C before DAT and elution

23. Methods, cont.
Cold Ab testing: Hemagglutination and titration under strictly controlled temps.
37, 32, 30, 28, 24, 20, 4˚C
Added 5% suspension type O RBCs in saline to twofold serial dilutions of Abs
Incubated 2 hours; agglutination macroscopically observed

24. Table 2

25. Results 2192 patients with warm autoAbs
2147 patients without cold agglutinins detected at 20C
2 patients with cold agglutinins with amplitude of 24C
2 patients with cold agglutinins with amplitude of 30C

26. Patient 1 42-year-old woman with compensated AIHA for 17 years
No medication needed
Serum contained moderate warm and cold hemolysins
DAT invariably positive for C3d, infrequently positive for IgG
Eluate frequently positive with IgG
Warm Abs to platelet glycoproteins

27. Patient 1 cont. Hemolysis became significant on 2 occasions
Exposure to low temperatures (1998)
Pneumonia (2000)
Both times associated with acrocyanosis and hemoglobinuria
Eventually diagnosed with lupus-associated Evans Syndrome

28. Patient 2 70-year-old female with hemolysis and thrombocytopenia
DAT positive for IgG and C3d
Positive eluate with anti-IgG
Positive cold agglutinins with titer 1:256 at 4C and high amplitude (30C)
Antibodies to platelet glycoproteins
Treated with prednisolone and azathioprine with remission
Eventually diagnosed with lupus-associated Evans Syndrome

29. Discussion
Intra- and extravascular hemolysis can be associated with WAIHA and CAD
Antibodies in WAIHA can be IgM, and those in CAD can be IgG
“Mixed AIHA” may be a misdiagnosis in setting of cold agglutinins with high thermal range or warm agglutinin with strong agglutination.
Serologic tests must be performed under strictly controlled conditions

30. Discussion cont.
2 out of 2194 patients met serologic criteria for MAIHA with clinical evidence for hemolytic anemia (<0.1%)
The incidence of MAIHA is not as high as previously thought
Further studies are needed to assess relationship to SLE and overall incidence

31. Conclusion
Mixed type autoimmune hemolytic anemia incorporates both WAIHA and CAD
Behavior of autoantibodies may pose challenges in laboratory testing
Rituximab has promise as a therapeutic
The overall incidence of this disease may be lower than previously thought

32. References
Gehrs BC, Friedberg RC. Autoimmune hemolytic anemia. Am J Hematol Apr;69(4):
Gupta S, Szerszen A, Nakhl F, Varma S, Gottesman A, Forte F, Dhar M. Severe refractory autoimmune hemolytic anemia with both warm and cold autoantibodies that responded completely to a single cycle of rituximab: a case report. J Med Case Rep Apr 19;5:156.
Haller W, Hind J, Height S, Mitry R, Dhawan A. Successful treatment of mixed-type autoimmune hemolytic anemia with rituximab in a child following liver transplantation. Pediatr Transplant May;14(3):E20-5.
Hillyer, Christopher et al. Transfusion Medicine and Hemostasis: Clinical and Laboratory Aspects. 1st edition. Elsevier Inc. Burlington, MA. 2009
Mayer B, Yürek S, Kiesewetter H, Salama A. Mixed-type autoimmune hemolytic anemia: differential diagnosis and a critical review of reported cases. Transfusion Oct;48(10):
Morselli M, Luppi M, Potenza L, Tonelli S, Dini D, Leonardi G, Donelli A, Narni F, Torelli G. Mixed warm and cold autoimmune hemolytic anemia: complete recovery after 2 courses of rituximab treatment. Blood May 1;99(9):
Roback, John et al. AABB Technical Manual. 17th edition. AABB. Bethesda, MD. 2011
Scaramucci L, Giovannini M, Niscola P, Perrotti A, de Fabritiis P. Primary mixed-type autoimmune hemolytic anemia concomitant with acute splanchnic venous thrombosis of idiopathic origin in a young woman: an unexplained association. Blood Res Dec;48(4):292-3.