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Warm autoantibody testing


Resident / Fellow Advisory Board: Giby V. George, M.B.B.S.
Editorial Board Member: Mrigender Singh Virk, M.D.
Adeyemi Sofoluwe, M.D.
Reggie Thomasson, M.S., M.D.

Last author update: 15 October 2024
Last staff update: 15 October 2024

Copyright: 2022-2025, PathologyOutlines.com, Inc.

PubMed Search: Warm autoantibody testing

Adeyemi Sofoluwe, M.D.
Reggie Thomasson, M.S., M.D.
Page views in 2025 to date: 528
Table of Contents
Definition / general | Essential features | Terminology | Pathophysiology | Clinical features | Laboratory | Case reports | Treatment | Sample assessment & plan | Differential diagnosis | Additional references | Practice question #1 | Practice answer #1 | Practice question #2 | Practice answer #2
Cite this page: Sofoluwe A, Thomasson R. Warm autoantibody testing. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/transfusionmedwarmautoantibodytest.html. Accessed April 29th, 2025.
Definition / general
  • Warm autoantibody is an antibody that binds to the patient's own red blood cells (RBCs) and reacts optimally at 37 °C (body temperature)
  • Identified in routine blood bank testing (e.g., type and screen, direct antiglobulin test)
  • Must determine if underlying alloantibodies are present, since they may be masked by the warm autoantibody
Essential features
  • Warm autoantibodies can be associated with autoimmune hemolytic anemia (AIHA), the incidence of which has been reported to vary from 1 to 3 per 100,000 patients per year (Hematology Am Soc Hematol Educ Program 2016;2016:690)
  • Increased incidence with age (J Pediatr Hematol Oncol 2007;29:792)
  • Considerable number of warm autoantibodies target Rh system antigens (BMJ Open 2015;5:e009085)
  • Glycophorin antigens are also targeted (BMJ Open 2015;5:e009085)
  • Warm autoantibodies often complicate blood bank testing by hindering the identification of RBC alloantibodies and delaying the selection of compatible units
  • New onset warm autoantibody may prompt clinicians to investigate for underlying disorders, such as lymphoproliferative disorders
Terminology
  • Warm reacting autoantibodies
  • IgG autoantibodies
Pathophysiology
  • Warm autoantibodies are due to primary (idiopathic) or secondary etiologies (e.g., B cell lymphomas [especially chronic lymphocytic leukemia], macroglobulinemias and autoimmune disorders [such as systemic lupus erythematosus])
  • Typically IgG subclass that can sometimes fix complement
  • Can lead to red cell destruction or decreased life span of red blood cells via extravascular hemolysis
Clinical features
  • Most warm autoantibodies are clinically silent and detected incidentally during preoperative screenings or blood donation assessments (Arch Pathol Lab Med 2015;139:1455, Haematologica 2017;102:e473)
  • Warm autoantibodies may cause hemolysis and anemia due to the removal of antibody and complement coated RBCs by the reticuloendothelial system (i.e., warm autoimmune hemolytic anemia [WAIHA])
  • Routine blood bank serological testing reveals the presence of warm autoantibodies; however, additional tests are needed to evaluate for the presence of any associated hemolysis
Laboratory
  • Primary screening
    • Warm autoantibodies can react with both self and nonself red blood cells demonstrating a panagglutination pattern potentially masking the presence of clinically significant alloantibodies
    • Must determine if underlying alloantibodies are present, especially in patients with history of transfusion, pregnancy and AIHA
    • Between 12% and 54% of patients with warm autoantibodies also have concurrent alloantibodies (Hematology Am Soc Hematol Educ Program 2022;2022:96)
  • Direct antiglobulin test
    • Screen using polyspecific antihuman globulin (AHG) to detect presence of antibody or complement coated RBCs
    • Positive result prompts further testing with monospecific anti-IgG or anti-C3d to identify the specific antibodies involved
    • Rare IgA RBC autoantibodies may not be detected by standard direct antiglobulin test (DAT)
    • Level of hemolysis may correlate with the strength of the DAT reaction and complement activity (J Pediatr Hematol Oncol 2007;29:792)
  • Elution
    • Eluates prepared from patient RBCs are a useful component of the serological workup of warm autoantibodies
    • Performed using a variety of methods including xylene, acid / glycine, heat, dithiothreitol plus enzyme (ZZAP), etc.
    • Since warm autoantibodies often react against Rh proteins, the eluate tends to be broadly reactive with all cells except the rare Rh null cell
  • Adsorption
    • Method is used to remove warm autoantibodies from serum or plasma in order to reveal the presence of any underlying alloantibodies
    • Autoadsorption uses the patient's own red cells as a substrate to remove warm autoantibodies
    • Alloadsorption uses phenotypically appropriate donor red cells as a substrate to remove warm autoantibodies
      • Used when the patient has been recently transfused (within 3 months) or when there is limited volume of patient RBCs available for testing
    • Various adsorption techniques exist using treated adsorbing cells (e.g., dithiothreitol + enzyme [ZZAP]) with or without the use of potentiators (e.g., polyethylene glycol [PEG])
      • Most reliable method to remove autoantibodies
    • Time consuming process
  • Dilution technique
    • Can be used to identify alloantibodies that are masked by autoantibodies (Ann Lab Med 2013;33:343)
    • Involves serial dilution of serum samples in saline until a 1+ reaction is observed in agglutination tests using LISS / Coombs cards
    • This method is used in labs without ZZAP or PEG capabilities, despite potentially less specific adsorption of warm autoantibodies
    • Dilution should only occur when alloantibody titers exceed autoantibody titers, emphasizing alloantibody detection in diluted serum
  • RBC phenotyping / genotyping
    • Phenotyping the patient's red blood cells can be used to provide antigen matched units if transfusion is needed
    • Also helpful to elucidate suspected alloantibody specificity and to help with selection of cells to be used in adsorptions
    • Phenotyping can be challenging due to the interference of autoantibodies coating the red blood cell surface; however, techniques do exist to help obtain accurate results
      • Direct agglutinating antisera
      • IgG antisera after elution of autoantibody (e.g., using ZZAP)
      • Reticulocyte separation by centrifugation
      • Flow cytometry
    • Genotyping can be performed in recently transfused patients or when there is serological interference from the autoantibodies that preclude phenotyping
  • Laboratory results indicative of hemolysis: normochromic normocytic anemia, polychromasia, reticulocytosis, spherocytosis, hemoglobinemia, decreased serum haptoglobin, increased serum total and unconjugated (indirect) bilirubin, increased lactate dehydrogenase, urinalysis may reveal hemoglobinuria and urobilinogen
Case reports
  • 5 year old girl with nausea, abdominal pain, jaundice and a hemoglobin of 5.5 g/dL who was diagnosed with a warm reactive IgM AIHA (Lab Med 2022;53:e83)
  • 23 year old woman who suffered from her first episode of acute and severe AIHA during SARS-CoV-2 and responded well to the treatment (Cureus 2021;13:e19118)
  • 32 year old man who presented with warm AIHA secondary to Epstein-Barr virus (EBV) reinfection (Cureus 2022;14:e26371)
  • 32 year old woman who presented with vague clinical presentation was found to have warm autoimmune hemolytic anemia and further immunological and inflammatory workup during and after hospitalization led to the diagnosis of systemic lupus erythematosus (Am J Case Rep 2021;22:e932965)
  • 62 year old woman with small lymphocytic lymphoma (phenotypic CLL) who developed symptomatic anemia 3 weeks after her fifth cycle of fludarabine, a T cell immunosuppressant (Vox Sang 1998;74:122)
Treatment
  • Transfusion support
    • Warm autoantibodies generally bind to all red blood cells leading to incompatible crossmatches
    • If no underlying alloantibodies are detected, then transfuse with ABO / Rh compatible RBCs
    • If underlying alloantibodies are detected, then select RBC units that are antigen negative for the corresponding alloantibody(ies)
    • If warm autoantibodies demonstrate specificity in the presence of ongoing hemolysis, then antigen negative blood may be considered (e.g., E negative blood in presence of autoanti-E)
    • Urgent transfusion needs should not be delayed due to an incompatible crossmatch
    • All crossmatch incompatible transfusions should occur slowly with more vigilant patient monitoring to assess for signs of transfusion reaction
    • Transfuse the smallest RBC volume possible to gain the best therapeutic benefit
  • Primary (idiopathic) AIHA are treated with corticosteroids (first line), intravenous immunoglobulin (IVIG), splenectomy and immunomodulatory therapies like cyclophosphamide and rituximab (Blood 2021;137:1283)
  • Secondary AIHA involves treating the underlying disorder
Sample assessment & plan
  • Assessment:
    • Jane Doe is a 45 year old woman with medical history significant for mild chronic hypertension and hypothyroidism, who presented with gradual onset fatigue, pallor and jaundice. She is otherwise generally healthy and has never received a blood transfusion. Her initial laboratory workup revealed a panreactive screen at 37 °C with positive autocontrol, positive DAT (IgG only), low hemoglobin, elevated bilirubin, increased reticulocyte count and spherocytes on peripheral blood smear.
  • Plan:
    • Perform an eluate and autoadsorption to further define autoantibody and investigation for any potential alloantibodies
    • Transfuse with ABO / Rh compatible RBCs if the patient shows signs of hemodynamic instability, severe symptomatic anemia (e.g., Hgb < 7 g/dL) or ongoing significant hemolysis
    • Do not delay transfusion if blood is needed urgently
    • Continue to monitor hemoglobin, bilirubin and reticulocyte count
    • Consider referral to hematology / oncology for further management and follow up
Differential diagnosis
  • Cold hemolytic anemia:
    • Cold agglutinin disease (CAD), a type of autoimmune hemolytic anemia where cold agglutinins bind to red blood cells at low temperatures, leading to hemolysis
    • Reacts best at < 30 °C
    • Autoantibodies against the I antigen are common, especially after Mycoplasma pneumoniae infection
    • Autoantibodies against the i antigen often appear after infectious mononucleosis
  • Paroxysmal cold hemoglobinuria (PCH):
    • Condition characterized by the sudden appearance of hemoglobin in the urine
    • Often associated with a viral illness
    • Caused by a biphasic IgG autoantibody that binds to the P antigen on RBCs at cold temperatures, fixing complement and leading to hemolysis when the RBCs are rewarmed in the body
    • Donath-Landsteiner test can be used to detect the presence of the implicated biphasic hemolysin
  • Drug induced hemolytic anemia:
    • Rare, largely unclear mechanism
    • Certain medications could lead to hemolysis and hence a positive DAT
    • There are 2 main types
      • Drug dependent antibodies
        • These antibodies only react in the presence of the drug (e.g., cefotetan and ceftriaxone)
      • Drug independent antibodies
        • These antibodies can react without the drug being present (e.g., fludarabine)
  • Autologous control (autocontrol):
    • Involves testing the patient's serum or plasma and autologous red cells under the same conditions as reagent red cells in antibody identification
    • Useful to help distinguish autoantibodies (positive autocontrol) from alloantibodies such as those targeting high incidence antigens (negative autocontrol), which can also appear as panreactive
  • Other causes of positive DAT:
Practice question #1
A 35 year old woman presents with fatigue, jaundice and dark urine. Laboratory tests reveal hemoglobin of 8 g/dL, elevated reticulocyte count and positive direct antiglobulin test (DAT) with IgG. Which of the following is the most likely diagnosis?

  1. Cold agglutinin disease
  2. Iron deficiency anemia
  3. Thalassemia
  4. Warm autoimmune hemolytic anemia
Practice answer #1
D. Warm autoimmune hemolytic anemia. The patient's symptoms (fatigue, jaundice, dark urine) and lab findings (low hemoglobin, elevated reticulocyte count, positive DAT with IgG) are indicative of hemolytic anemia. The presence of IgG on the DAT suggests warm autoimmune hemolytic anemia (AIHA), where the body's immune system produces antibodies that target red blood cells at body temperature. Answer B is incorrect because iron deficiency would not present with jaundice, dark urine or a positive DAT. Answer A is incorrect because cold agglutinin disease would typically reveal a DAT positive for complement binding instead of IgG. Answer C is incorrect because thalassemia is an inherited quantitative hemoglobinopathy and is not associated with a positive DAT.

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Reference: Warm autoantibody testing
Practice question #2
A patient with a history of systemic lupus erythematosus (SLE) presents with symptoms of anemia. The direct antiglobulin test (DAT) is positive for IgG and C3. Which of the following tests would be most helpful in confirming the presence of warm autoantibodies?

  1. Cold agglutinin titer
  2. Hemoglobin electrophoresis
  3. Indirect antiglobulin test (IAT)
  4. Serum ferritin
Practice answer #2
C. Indirect antiglobulin test (IAT). The IAT is used to detect antibodies in the patient's serum that can bind to red blood cells. This test is particularly useful in confirming the presence of warm autoantibodies, which are typically IgG antibodies that react at body temperature. The patient's history of SLE and positive DAT for IgG and C3 further supports the likelihood of warm AIHA. Answer A is incorrect because a cold agglutinin titers are used to diagnose cold agglutinin syndrome. Answer B is incorrect because hemoglobin electrophoresis is used to identify and quantify hemoglobin types including any variants. Answer D is incorrect because serum ferritin is used to measure iron containing proteins and diagnose conditions that affect iron levels.

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Reference: Warm autoantibody testing
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