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
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. 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 1998-2007
– 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
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. 2002 Apr;69(4):258-71.
• 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. 2011 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. 2010
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. 2008 Oct;48(10):2229-
34.
• 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. 2002 May 1;99(9):3478-9.
• 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. 2013 Dec;48(4):292-3.
