Researchers identified 6 categories for the classification of patients who tested negative on the direct antiglobulin test (DAT) for autoimmune hemolytic anemia (AIHA), a group of disorders that includes cold agglutinin disease (CAD). The classification system could help predict outcomes in affected patients.

The 6 categories are: tube DAT-negative, low-affinity immunoglobulin (Ig) G, double DAT-negative, IgA- or IgM-positive, low-affinity IgM, and non-AIHA classifications.

The investigators observed that albumin concentration and platelet count in patients with DAT-negative AIHA significantly predicted 1-year survival rate. The low-affinity IgG group demonstrated the highest platelet counts and albumin concentrations, improved responses to steroids, and an increased 1-year survival rate, compared to patients in the other 5 categories.

Continue Reading

“When treating a patient with DAT-negative hemolysis, atypical AIHA should be considered and tested in reference laboratories, especially before treatment,” the authors recommended.

Previously, the same researchers developed a diagnostic algorithm to classify DAT-negative AIHA based on red blood cell (RBC)-bound IgG levels. “The aim of the present study was to assess the clinical utility of our diagnostic algorithm and classification system and determine the characteristics of each subgroup in DAT-negative AIHA,” they wrote.

Read more about CAD diagnosis

The researchers enrolled 637 patients between 2015 and 2019 at the Center for Community Medicine at Jichi University in Tochigi, Japan. Due to a lack of data or survey completion, the researchers performed laboratory testing and classification in 382 of the patients.

The investigators identified 3 sorting groups based on DAT results: double DAT-negative tube method (TM) and column method (CM) tests, negative TM-DAT and positive CM-DAT, and double DAT-positive.

Double DAT-positive results strongly indicated warm AIHA. When TM-DAT was negative and CM-DAT was positive, RBC-bound IgG levels and CM-DAT results following RBC washing procedures delineated between tube DAT-negative AIHA and low-affinity IgG-type AIHA.

Lastly, when TM-DAT and CM-DAT were both negative, RBC-bound IgG levels above the cutoff value of 78.5 IgG molecules/RBC helped to confirm double DAT-negative AIHA. The investigators also assessed for the presence of IgA and IgM antibodies.

If all tests were negative and the RBC-bound IgG levels were below the cutoff value of 78.5 IgG molecules/RBC, the patient was suspected of non-AIHA categorization.

Of the 122 patients with negative TM-DAT tests, researchers classified 71 as idiopathic, which included 12 DAT-negative AIHA, 25 low-affinity IgG-type AIHA, 19 double DAT-negative AIHA, 12 non-AIHA, 2 IgA- or IgM-positive, and 1 low-affinity IgM-type AIHA.

The investigators followed up with the patients’ physicians after 1 year to ascertain disease course, final clinical diagnosis, and 1-year survival rates. In total, the clinicians diagnosed 126 patients with TM-DAT-negative AIHA, including 122 with warm AIHA, 2 with mixed type, 1 with CAD, and 1 with paroxysmal cold hemoglobinuria.

Among these 126 patients, laboratories classified 16% with DAT-negative AIHA, 35% with low-affinity IgG-type AIHA, 25% with double DAT-negative AIHA, 6% with IgA- or IgM-positive AIHA, 0.8% with low-affinity IgM-type AIHA, and 17% as non-AIHA. Using this data as a reference, the researchers determined that the use of their algorithm by immunohematology reference laboratories could diagnose patients with DAT-negative AIHA into 1 of the 6 categories with 97% sensitivity and 84% specificity.


Kamesaki T. Diagnostic algorithm for classification and characterization of direct antiglobulin test-negative autoimmune hemolytic anemia with 1-year clinical follow-up. Transfusion. Published online October 19, 2021. doi:10.1111/trf.16709