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DAT negative autoimmune hemolytic anemia

Last author update: 28 November 2023
Last staff update: 28 November 2023

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PubMed Search: DAT negative autoimmune hemolytic anemia

Sarah Kesterson, M.D.
Evelyn M. Potochny, D.O.
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Cite this page: Kesterson S, George MR, Potochny EM. DAT negative autoimmune hemolytic anemia. website. Accessed April 15th, 2024.
Definition / general
  • The vast majority of cases of autoimmune hemolytic anemia (AIHA) can be separated into warm reacting, cold reacting, mixed type and paroxysmal cold hemoglobinuria (PCH), each with a classic pattern of direct antiglobulin test (DAT) positivity; however, some patients with clinical and laboratory evidence of AIHA have a negative DAT (Cohn: AABB Technical Manual, 20th Edition, 2020)
Essential features
  • Between 3% and 11% of hemolytic anemia cases clinically consistent with AIHA have a negative DAT (Blood Cells Mol Dis 2014;52:152)
  • High clinical suspicion of autoimmune hemolysis can guide additional testing to confirm the diagnosis and guide treatment
  • Clinical course of DAT negative AIHA is similar to DAT positive cases and similar therapies are utilized
  • Appropriately categorizing AIHA contributes to transfusion safety
  • Coombs negative autoimmune hemolytic anemia (AIHA)
  • AIHA associated with a negative DAT
  • DAT negative hemolysis
  • AIHA is the process in which shortened red blood cell (RBC) survival is caused by RBC destruction via an immune response, specifically by humoral autoantibody
  • RBC autoantibodies are directed toward the individual's own RBC antigens but given that these are usually high incidence antigens, the antibodies can bind not only to the individual's own cells but also those from a transfused blood component
  • Presence of autoantibody alone does not necessarily cause decreased RBC survival
  • It is not completely understood who will be clinically affected by autoantibodies (i.e., develop hemolytic anemia); the following may be involved (Harmening: Modern Blood Banking & Transfusion Practices, 7th Edition, 2018)
    • Thermal amplitude of antibody
    • IgG subclass of antibody
    • Amount of antibody bound to RBCs
    • Ability of antibody to fix complement
    • Individual's macrophage activity
    • Quantitative or qualitative change in band 3 and proteins 4.1 and 4.2 in the RBC membrane
  • If hemolysis does occur and the rate of RBC destruction outpaces that of RBC production, anemia may result
  • Intravascular and extravascular RBC destruction may be present
  • In the vast majority of cases, autoimmune hemolysis can be confirmed with a positive DAT
  • However, there are a minority of cases of AIHA with a negative DAT; these can often be explained by the following
    • IgG antibody concentration below the limit of detection of standard DATs
    • Low affinity IgG antibodies
    • Sensitization via IgA antibodies (not routinely demonstrated using commercial reagents)
    • Sensitization via warm reacting and monomeric IgM antibodies (not routinely demonstrated using commercial reagents, unless complement is also bound) (Blood 2017;129:2971)
Clinical features
  • Similar to DAT positive AIHA
  • Symptoms of anemia
    • Fatigue
    • Dyspnea
    • Pallor
  • Hemoglobin at presentation is often between 7 - 10 g/dL
  • 33% of cases may feature hemoglobin < 7 g/dL
  • Depending on coexisting conditions, may include
    • Leukocytosis (chronic lymphocytic leukemia)
    • Thrombocytopenia (Evan syndrome)
  • Absolute reticulocyte count is normally elevated
    • Coexisting conditions that limit reticulocyte response
      • Iron deficiency
      • Primary bone marrow disorder
      • Drug effect
      • Antibodies or viral effect against RBC precursors
    • Haptoglobin may be low
    • Lactate dehydrogenase (LDH) is high
    • Indirect bilirubin is high
    • Aspartate transaminase (AST) may be high
    • Mean corpuscular hemoglobin is high, reflecting increased reticulocytosis
    • Hemoglobin A1C may be unexpectedly low due to hemolyzed RBCs having limited time for glycation
    • Peripheral blood smear may show spherocytes
      • Pertinent negatives include absence of schistocytes (evidence against microangiopathic hemolytic anemias) and absence of sickle cells
  • References: Hematol Oncol Clin North Am 2022;36:315, Hematol Oncol Clin North Am 2022;36:307, Blood 2021;137:1283
  • Antibody screen is likely to be negative
  • DAT will be negative
  • There may be clinical suspicion by the patient's physician of AIHA despite a negative DAT, possibly due to the presence of anemia and reticulocytosis with no other obvious etiology
  • Peripheral blood smear may demonstrate spherocytes or microspherocytes
  • Hemolytic labs may suggest in vivo hemolysis; these include
    • Low haptoglobin
    • Elevated indirect bilirubin
    • Elevated LDH
    • Hemoglobinuria
  • Reference: Simon: Rossi's Principles of Transfusion Medicine, 5th Edition, 2016
  • DAT negative AIHA workup is not 1 test but a battery of tests used to evaluate potential etiologies
  • This workup typically starts with more sensitive tests to identify lower concentration or low affinity IgG and if negative, expands to additional studies
    • Each immunohematology reference lab (IRL) will have its own testing algorithm, often including
      • Repeat DAT with more sensitive reagents (depending on reagent, fewer than 300 - 500 IgG molecules per red cell may not be detected) (Blood 2017;129:2971)
      • Repeat DAT with an alternate method (e.g., gel card) and performed under differing temperatures
      • Repeat DAT testing with monospecific reagents (separate reagents for anti-IgG, anti-IgA, anti-IgM, anti-C3) (Eur J Haematol 2003;70:60)
      • Testing with enhancement media (e.g., polyethylene glycol (PEG) and polybrene [hexadimethrine bromide])
      • Elution with concentrated plasma / serum sample
      • Antibody detection with and without enzymes
  • Based on the suspected etiology of the negative DAT, many advanced testing options are available (Blood Cells Mol Dis 2014;52:152)
    • Identifying IgG that is present but not detectable necessitates more sensitive testing methods
      • Flow cytometry
      • Enzyme linked anti-IgG assay
      • Testing with enhancement media
        • Direct polybrene test
        • Direct polyethylene glycol test
        • Povidone (polyvinylpyrrolidone) augmented antiglobulin test
      • Eluate with papain treated red cells
      • Concentrated eluate assay
      • Radiolabeled anti-IgG
      • Column agglutination or gel test
      • Solid phase test
      • Complement fixation antiglobulin consumption test
      • Monocyte monolayer assay
    • Identifying low affinity IgG antibodies
      • DAT after 4 °C or low ionic strength saline red cell wash, to avoid the loss of antibody that can be seen with room temperature or 37 °C washing
      • Flow cytometry may also identify these antibodies
    • Isolated IgA or monomeric IgM warm reacting antibodies
      • Test with anti-IgA antisera
      • Test with anti-IgM antisera or IgM radioimmune antiglobulin test
      • This testing should help to identify the rare IgM antibodies that do not bind complement
      • Flow cytometry
    • Natural killer (NK) cell mediated hemolysis
      • 51Cr release assay (identifying NK cell destruction of autologous but not ABH matched allogeneic red cells)
Case reports
  • Similar to DAT positive AIHA
  • Corticosteroids (first line treatment)
  • Consideration of using rituximab as first line therapy in addition to corticosteroids
  • When refractory
    • Splenectomy
    • Rituximab
    • Other immunosuppressive drugs (azathioprine, cyclophosphamide, cyclosporine, mycophenolate mofetil)
  • Other therapies
    • Intravenous immunoglobulins
    • Danazol
    • Plasma exchange
    • Alemtuzumab
    • High dose cyclophosphamide
  • Novel therapeutics under further investigation include
    • Inhibitors of neonatal Fc receptor with FcRn targeting monoclonal antibodies
      • Nipocalimab
      • Rozanolixizumab
      • Orilanolimab
    • Inhibitors of cellular mediators of phagocytosis
      • Fostamatinib (splenic tyrosine kinase inhibitor)
    • Inhibitors of signal transducers
      • Rilzabrutinib (a reversible, covalent Bruton tyrosine kinase inhibitor)
  • Reference: Semin Hematol 2015;52:304
Sample assessment & plan
  • Assessment: John Doe is a 56 year old man, blood type A positive, who presented with lightheadedness and fatigue and was found to have anemia and clinical evidence of hemolysis (elevated LDH, decreased haptoglobin, indirect hyperbilirubinemia). An antibody screen was negative. Routine DAT was also negative. Peripheral blood smear demonstrated increased spherocytes.
  • Plan:
    • Due to clinical suspicion for autoimmune hemolytic anemia, specimen was sent out to our immunohematology reference laboratory, which demonstrated a warm reacting (IgG mediated) autoantibody following enhanced DAT testing
    • If red blood cell transfusion is required, A or O, Rh(D) positive or negative, full crossmatch compatible blood will be issued
    • Consider referral to hematology / oncology for management of this autoimmune hemolytic anemia
Differential diagnosis
Board review style question #1

A 7 year old boy is referred to pediatric hematology from a general pediatrician for new onset anemia with normal white blood cell (WBC) and platelet count after an upper respiratory infection. Iron studies are normal and there is no family history to suggest hemoglobinopathy, membranopathy or enzyme defect. He is found to be anemic with no other complete blood count (CBC) abnormalities. The pediatric hematologist is concerned about autoimmune hemolytic anemia and orders a direct antiglobulin test (DAT), which is negative and requests a peripheral blood smear, which shows increased spherocytes and polychromasia in the image above.

The pediatric hematologist then orders an enhanced DAT or DAT negative autoimmune hemolytic anemia workup. Which of the following is the most likely cause of the patient's apparent hemolysis and explanation for the negative DAT?

  1. Hereditary spherocytosis
  2. IgG antibody concentration below the limit of detection of standard DATs
  3. Lab error in the original DAT
  4. Microangiopathic hemolytic anemia
Board review style answer #1
B. IgG antibody concentration below the limit of detection of standard DATs. This likely represents a case of low level or low affinity IgG autoantibodies below the level of detection of a standard DAT. Answer A is incorrect because the child was previously healthy and there is no familial history of a membranopathy. Answer C, while possible, is unlikely because of the use of check cells for negative reactions at antihuman globulin phase in blood bank as a check and balance on negative results. Additionally, most DAT panels include 3 reagents: polyspecific IgG plus C3, monospecific IgG and monospecific C3. Even if there had been a problem with one of the reagents, the built in redundancy of the polyspecific reagent increases the likelihood of detecting any sort of reactivity in the DAT. Answer D is incorrect because there is no significant evidence to suggest microangiopathic hemolytic anemia; namely, there is no major increase in schistocytes, as compared to spherocytes.

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Reference: DAT negative autoimmune hemolytic anemia
Board review style question #2
A 55 year old woman with chronic lymphocytic lymphoma (CLL) presented with anemia and reticulocytosis, a negative routine direct antiglobulin test (DAT) for both complement and IgG, a negative red cell antibody screen, elevated lactate dehydrogenase (LDH), severely decreased haptoglobin and indirect hyperbilirubinemia. At this point, which of the following is the recommendation the blood bank medical director will most likely give to the consulting physician when contacted for assistance with evaluation for possible autoimmune hemolytic anemia (AIHA)?

  1. Peripheral blood smear
  2. Send out testing to immunohematology reference lab (IRL) for DAT negative AIHA
  3. Send out testing to IRL for Donath-Landsteiner testing
  4. Urinalysis
Board review style answer #2
B. Send out testing to the immunohematology reference lab (IRL) for DAT negative AIHA evaluation. Answers A and D are incorrect because at this point, finding spherocytes on a peripheral blood smear or evidence of hemoglobinuria on urinalysis are unlikely to provide any further information to confirm autoimmune hemolytic anemia, as there is already evidence of in vivo hemolysis. The consulting physician is likely to already have started corticosteroids for the treatment of presumed AIHA and confirmation of a DAT negative AIHA would support staying the course, as would clinical improvement with steroids. Answer C is incorrect because send out testing for a Donath-Landsteiner test would be of lesser value in this patient demographic. Unless the patient had known syphilis, paroxysmal cold hemoglobinuria (PCH) is unlikely in an elderly patient. It is most common in pediatric patients after a viral illness. It is confirmed with a positive Donath-Landsteiner test.

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Reference: DAT negative autoimmune hemolytic anemia
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