Cation Leak in Red Blood Cells of Patients with Wiskott-Aldrich Syndrome Leads to Non-Immunologic Hemolysis

Wiskott-Aldrich Syndrome (WAS) is a primary immunodeficiency that is associated with microthrombocytopenia. Autoimmunity is one of the defining characteristics of severe WAS, and can contribute to the decision to proceed to hematopoietic cell transplantation. Autoimmune Hemolytic Anemia (AIHA) is one of the most common autoimmune syndromes found in WAS. Even in the absence of AIHA, some patients with WAS have anemia, and this is often of unclear etiology.

We studied 29 patients with WAS, 6 of whom had had splenectomy, on 46 occasions. All patients were found to have anisopoikilocytosis. Common red blood cell (RBC) variants included stomatocytes, xerocytes, eccentrocytes, dacryocytes, spherocytes and target cells. Two WAS knockout mice whose blood smears were studied also had poikilocytosis, with dacryocytes predominating. The average hemoglobin in humans with WAS was 13.3 g/dL. In 19/46 measurements, hemoglobin was below the lower limit of normal for age. LDH was elevated in 1/43 measurements, and bilirubin was elevated in 7 of 46 measurements, 4 measurements being from a patient with molecularly diagnosed Gilbert’s syndrome. Average MCHC was 34.7 g/dL, with MCHC being elevated in 3/43 measurements. Despite these unremarkable screening assays, haptoglobin was decreased in 10/45 measurements and undetectable in 6 of these 10. The average plasma free hemoglobin and plasma free oxyhemoglobin were elevated to 21.4 and 14.7 mg/dL respectively. Analysis of osmotic fragility revealed average numbers near the lower end of the normal range, with fragility in 0.6 g/dL NaCl being abnormally low in 29/35 specimens and fragility in 0.65 g/dL NaCl being abnormally low in 9/35 specimens. A standard Coomb’s test was positive in 6/39 specimens. 4 such assays were positive for IgG, 1 for C3 and 1 for neither molecule despite a positive screening assay. Using enhanced methodology, 1/5 specimens was positive only with polyspecific serum. Overall, immune mediated hemolysis was detected by either method in 6/39 specimens. These data were interpreted to be consistent with a subclinical non-immune hemolysis in 33/41 specimens from 28/29 patients.

In order to evaluate these findings further, ektacytometry was performed in 5 specimens from 5 patients and intracellular cations were measured in 4 of these specimens. Ektacytometry revealed Omin of 135.8±5.1 mOsm/kg in patients with WAS and 156.4±5.3 mOsm/kg in control specimens (P=0.0002). Sodium and potassium concentrations in patients were 53.3±12.7 mmol/kg hemoglobin and 282.3±13.1 mmol/kg hemoglobin respectively. In control specimens, the concentrations were 4.4±10.5 mmol/kg hemoglobin and 297.5±14.4 mmol/kg hemoglobin (P=0.3196 and 0.1684).

These data are consistent with non-immune hemolysis in most patients with Wiskott-Aldrich syndrome. This hemolysis is associated with a xerocytic ekatacytometry curve, although cation concentrations in the small number of specimens tested only trended to statistical significance. This is the first intrinsic red cell defect described in patients with WAS, emphasizing that this disease truly affects all hematopoietic lineages and not only cells of the immune system. Whether the roles of the WAS protein in actin polymerization or signal transduction are involved in these abnormal functions remains unclear and requires additional research. These findings also have implications for clinical management of WAS as they indicate that hemolysis is commonly present without necessarily defining the autoimmune, severe clinical phenotype in these patients. As autoimmunity is often an important consideration in the decision to proceed to hematopoietic cell transplantation, these data should inform the decision-making process toward this and other therapeutic modalities for WAS.