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JAMA Diagnostic Test Interpretation
December 3, 2018

The Direct Antiglobulin Test for Evaluating Anemia

Author Affiliations
  • 1Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas
  • 2The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, Maryland
JAMA. Published online December 3, 2018. doi:10.1001/jama.2018.14317
Case

A 68-year-old woman with Rai stage 0 chronic lymphocytic leukemia (CLL) presented with a 2-day history of dyspnea on exertion. She reported no cough, chest pain, orthopnea, leg swelling, or viral-like symptoms. Her temperature was 36.8°C, heart rate 102/min, respiratory rate 22/min, and blood pressure 124/76 mm Hg. She had conjunctival pallor and scleral icterus, but no lymphadenopathy, hepatosplenomegaly, or ecchymosis. Results of initial laboratory testing are presented in the Table.

Table.  
Patient’s Laboratory Values
Patient’s Laboratory Values
Box Section Ref ID

How Do You Interpret These Results?

  1. The patient has cold agglutinin disease.

  2. The patient has pure red cell aplasia.

  3. The patient has undergone Richter transformation.

  4. The patient has warm autoimmune hemolytic anemia.

Discussion
Answer

D. The patient has warm autoimmune hemolytic anemia.

Test Characteristics

Autoimmune hemolytic anemia (AIHA) develops due to autoantibody production against surface antigens expressed on a patient’s red blood cells (RBCs). AIHA is subdivided based on immunoglobulin temperature reactivity—warm or cold. Warm IgG antibodies predominantly bind to RBC surface antigens at body temperature and cause warm AIHA, whereas cold IgM agglutinins bound to RBCs fix complement at temperatures below 37°C and can cause cold agglutinin hemolysis. These anti-RBC autoantibodies can be detected using the direct antiglobulin test (DAT), also called the Coombs test.1

To detect cold and warm autoantibodies through the conventional test tube method, the patient’s RBCs are first washed with saline to remove immunoglobulins and complement that are not bound to RBCs, leaving behind immunoglobulins and complement bound to RBCs.1 These RBCs are mixed with an antihuman globulin reagent, containing polyspecific rabbit anti-IgG and monoclonal murine anticomplement antibodies.2 In the presence of bound immunoglobulins and complement on the RBC surface, the antihuman globulins produce crosslinks between the patient’s RBCs, resulting in agglutination. The assay is semiquantitative, and results are generally reported on a scale of 0 to 4+ (0, no agglutination; 4+, solid agglutination). If the DAT is positive, it is repeated using monospecific antibodies to explicitly measure anti-IgG or anticomplement, usually C3d, reactivity.1

In a single-institution series of 100 patients with a positive DAT, only 29 patients had serologic evidence of hemolytic anemia, defined as hemoglobin lower than 12.3 g/dL, hematocrit lower than 37.4%, total bilirubin above 1.5 mg/dL, lactate dehydrogenase above 220 U/L, and haptoglobin lower than 6 mg/dL.3 Of note, the DAT is positive but not clinically significant in 7% to 8% of all hospitalized patients, even in those without evidence of hemolysis.1 False-positive results can occur due to spontaneous RBC agglutination or technical issues such as insufficient washing, overcentrifugation, or clotted specimens.1 The test tube method for the DAT is 43% sensitive and 87% specific for AIHA.4

During their lifetime, 7% to 10% of patients with CLL develop AIHA (warm, 90%; cold, 10%).5 The purported mechanism is polyclonal T-cell–dependent production of pathogenic antibodies, largely by nonmalignant B cells.6 A positive DAT can develop over the course of CLL, but actual AIHA is less common; in a prospective study of 777 CLL patients, the DAT had a positive predictive value of 28% and negative predictive value of 93% for AIHA.7 The 2018 Medicare midpoint reimbursement for a DAT is $6.65.8

Application of Test Results to This Patient

This patient has evidence of hemolysis (low haptoglobin, high lactate dehydrogenase, and reticulocytosis) and a positive IgG DAT, indicating warm AIHA. Warm AIHA can be primary or associated with lymphoproliferative disorders such as CLL, autoimmune diseases, viral infections, and drugs.3,9

Cold agglutinin disease occurs due to IgM autoantibodies. The presence of IgM autoantibodies is indicated by anti-C3d activity due to the complement-fixing property of IgM.1 This patient had a negative C3d DAT, indicating the absence of IgM autoantibodies. Pure RBC aplasia, characterized by severe anemia, normal white blood cell and platelet count, with decreased erythroid precursors in the bone marrow, occurs in less than 1% of CLL patients and is associated with reticulocytopenia.5 Richter transformation is the development of a more aggressive lymphoma and occurs in 3% of patients with CLL. Patients often develop lymphadenopathy and systemic symptoms rather than hemolytic anemia.10

What Are Alternative Diagnostic Testing Approaches?

DAT remains the primary test for determining whether an autoimmune hemolysis is present. Specialized testing, flow cytometry for RBC IgG and enzyme-linked anti-IgG assay, can detect IgG that is not measured by DAT, but it is rarely available in standard clinical laboratories. Thus, a definitive diagnosis of DAT-negative AIHA can often not be made.9 If DAT-negative AIHA is suspected, hematology consultation is recommended to guide further testing (if available) and empirical treatment.

Patient Outcome

The patient was diagnosed with CLL–associated warm AIHA and treated with oral prednisone (1 mg/kg per day). Because she was hemodynamically stable, no blood products were transfused. The patient received weekly follow-up with complete blood cell counts and markers of hemolysis. After 2 weeks, hemoglobin began improving (8.8 g/dL), and at 4 weeks, her hemoglobin level was 10.2 g/dL. Prednisone was tapered to 10 mg/d and discontinued after 5 months. At that time, her hemoglobin was 12.3 g/dL and hemolysis markers were negative. The patient remains in remission at her most recent follow-up, 9 months after completing therapy. Rituximab is a reasonable alternative therapy for patients with CLL-associated warm AIHA.

Box Section Ref ID

Clinical Bottom Line

  • The direct antiglobulin test (DAT) is a semiquantitative assay that detects the presence of bound anti–red blood cell (RBC) antibodies through RBC agglutination.

  • Warm autoimmune hemolytic anemia (AIHA) is primarily caused by IgG agglutinins that are active at body temperature and develops in 7% to 10% of patients with chronic lymphocytic leukemia (CLL).

  • The DAT has a low positive predictive value (28%) for warm AIHA in patients with CLL.

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Article Information

Corresponding Author: Srikanth Nagalla, MD, Department of Internal Medicine, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-8852 (srikanth.nagalla@utsouthwestern.edu).

Published Online: December 3, 2018. doi:10.1001/jama.2018.14317

Conflict of Interest Disclosures: None reported.

Additional Contribution: We thank the patient for sharing her experience and for granting permission to publish it.

Section Editor: Mary McGrae McDermott, MD, Senior Editor.
References
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Dearden  C, Wade  R, Else  M,  et al; UK National Cancer Research Institute (NCRI); Haematological Oncology Clinical Studies Group; NCRI CLL Working Group.  The prognostic significance of a positive direct antiglobulin test in chronic lymphocytic leukemia: a beneficial effect of the combination of fludarabine and cyclophosphamide on the incidence of hemolytic anemia.  Blood. 2008;111(4):1820-1826. doi:10.1182/blood-2007-07-101303PubMedGoogle ScholarCrossref
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Segel  GB, Lichtman  MA.  Direct antiglobulin (“Coombs”) test-negative autoimmune hemolytic anemia: a review.  Blood Cells Mol Dis. 2014;52(4):152-160. doi:10.1016/j.bcmd.2013.12.003PubMedGoogle ScholarCrossref
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Robertson  LE, Pugh  W, O’Brien  S,  et al.  Richter’s syndrome: a report on 39 patients.  J Clin Oncol. 1993;11(10):1985-1989. doi:10.1200/JCO.1993.11.10.1985PubMedGoogle ScholarCrossref
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