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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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?

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

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

Table 2

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

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

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

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

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

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

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

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