Warm autoimmune hemolytic anemia (AIHA) is a rare type of anemia caused by the reaction of auto-antibodies with red blood cell (RBC) surface antigens at body temperature, leading to the hemolysis of RBCs . The direct antiglobulin test (DAT) (also known as the direct Coombs test) was first described by Coombs, Mourant, and Race in 1945  and is still an essential assay for the diagnosis of AIHA. The DAT can detect RBC-bound IgG and complement (C3), which opsonize RBCs. However, 5–10% of patients with AIHA are DAT negative . The principal reasons for DAT-negative AIHA are: (a) RBC-bound IgG being below the threshold of detection by standard methods; (b) low affinity of IgG; and (c) RBC-bound IgA or rarely IgM . To achieve greater sensitivity, alternative DAT methods have been developed, such as the micro-column (gel), solid-phase, polybrene, and flow cytometry methods, as well as enzyme-linked antiglobulin, immunoradiometric, and mitogen-stimulated tests . Moreover, because washing RBCs with room temperature saline causes the low-affinity antibodies to be eluted from RBCs, washing RBCs with cold saline or with low ionic strength saline (LISS) may be effective in detecting low-affinity antibodies.
Because the prevalence of DAT-negative AIHA is low, the relative proportions of each of the aforementioned reasons for DAT-negative AIHA have not been clarified. Moreover, a diagnostic algorithm for DAT-negative AIHA has not yet been established.
In this issue of the journal, Kamesaki and Kajii  report a detailed laboratory analysis of patients with DAT-negative AIHA and propose a classification of DAT-negative AIHA using a combination of several DAT methods. They studied patients with undiagnosed hemolytic anemia who showed negative results using the routine tube method of DAT (TM-DAT), who were further investigated with anti-IgA and anti-IgM sera and the column agglutination method of DAT (CM-DAT), and with immunoradiometric quantitation of RBC-bound IgG. According to the results, they proposed a classification of DAT-negative AIHA into 5 groups as follows: (1) positive CM-DAT after washing RBCs with phosphate-buffered saline (PBS) and RBC-bound IgG in the immunoradiometric assay over the cutoff value (the cutoff value of RBC-bound IgG is 78.5 IgG molecules/RBC ); (2) negative CM-DAT and RBC-bound IgG in the immunoradiometric assay over the cutoff value; (3) negative CM-DAT after washing RBCs with PBS, but positive TM- or CM-DAT after washing RBCs with LISS and RBC-bound IgG in the immunoradiometric assay under the cutoff value; (4) negative CM-DAT after washing RBCs with PBS, but positive TM- or CM-DAT after washing RBCs with LISS and RBC-bound IgG in the immunoradiometric assay over the cutoff value; and (5) positive TM-DAT with anti-IgA or anti-IgM sera. The estimated relative fractions of patients with DAT-negative AIHA resulting from RBC-bound IgA or IgM, low-affinity IgG autoantibodies, and RBC-bound IgG autoantibodies under the threshold level of positive DAT were approximately 5, 15, and 80%, respectively.
The study by Kamesaki and Kajii  indicated that most cases of DAT-negative AIHA were due to RBC-bound IgG being below the threshold of detection by standard methods, followed by cases of low-affinity IgG and RBC-bound IgA or IgM. When AIHA patients are TM-DAT negative, the next step may be to evaluate RBC-bound IgG by an immunoradiometric method, then with low-affinity IgG autoantibodies or with anti-IgA or IgM sera. However, there are many other DAT methods, and it is a challenge to choose which combination of methods to use. Moreover, we should keep in mind that these tests for DAT-negative AIHA have a low predictive value, and thus the results must be interpreted based on the clinical status of the patients.
Nevertheless, Kamesaki and Kajii’s  work in this issue casts a new light on DAT-negative AIHA. This classification of DAT-negative AIHA can reduce misdiagnosis rates and may be useful in predicting the prognosis of each DAT-negative AIHA type. Further development in this field will be expected.