When a patient presents with anemia, a stepwise approach should be followed. The diagnosis of AIHA can be made with laboratory and clinical evidence of hemolysis and then determine the immune nature of hemolysis with DAT. Decreased RBC count, Hb and Hematocrit normo-macrocytic anemia, increased reticulocyte count, raised indirect bilirubin and LDH, reduced serum haptoglobin, and blood smear features with polychromasia or spherocytes, schistocyte and agglutination may be helpful for the physician to diagnosis of hemolysis as the cause of the anemia(5, 11).
In this patient’s medical history showed no hemolytic risk factors and her blood G6PD levels was reported normal. Laboratory findings is shown in this case report during the first 7 days of admission (Table 1). Increasing of serum LDH and decreasing of blood hemoglobin showed hemolytic anemia. Hemoglobin dropped consistently in spite of transfusion. In this patient, clinical evidence and laboratory findings indicate hemolytic anemia but DAT test was negative. Additionally, platelet count was normal and the peripheral blood smear on admission showed anisocytosis, normo-macrocytosis, polychromasia, some spherocytes and micro-spherocytes and no schistocytes that confirm extravascular immune hemolytic anemia in spite of negative DAT test.
Immune mediated hemolysis or G6PD deficiency have been known as two main causes of hemolytic anemia (12, 13). The precise incidence of presentation of patients that have an anemia compatible to warm-AIHA and a negative DAT is not known but has been estimated at 3–11% of all cases(14–16). Different causes for this finding included possible hemolysis by natural killer cells (NK cells) independent of antibody, presence of low affinity IgG that removed by preparatory washes protocols, sensitization below the threshold of detection of the commercial antiglobulin reagent (anti-human reagent potency), IgA or IgM autoantibodies, red cell sensitization by IgA alone, or rarely monomeric-IgM alone, that not accompanied by complement fixation, and therefore not detectable by a commercial polyclonal antiglobulin reagent. Due to these different possibilities, a negative DAT must be interpreted in conjunction with clinical findings. If clinical suspicion is high and research into non- immune causes is not justified, in addition more sensitive than the standard DAT protocol (microcolumn, solid phase, washings with cold or low-ionic salt solutions) may be negative but the patient response to steroid therapy as first line or rituximab as second-line treatment (9, 13, 17–19).
In this situation additionally to initial measures for treatment and resuscitation of the suspected cases of AIHA patient, pay special attention to immune mediated hemolysis and do not be misled by a negative coombs test. In these situation negative DAT did not confirm the diagnosis and DAT may be positive after several weeks of illness.