This prospective study was carried out to assess the usefulness of five laboratory tests in the diagnosis of hereditary spherocytosis (HS), based on the correlation of erythrocyte membrane protein defects with clinical and laboratory features, and also to determine the membrane protein deficiencies detected in Argentina. Of 116 patients and their family members tested, 62 of them were diagnosed to have HS. The specificity of cryohemolysis (CH) test was 95.2%, and its cut-off value to distinguish HS from normal was 2.8%. For flow cytometry, cut-off points of 17% for mean channel fluorescence (MCF) decrease and 14% coefficient of variation (CV) increase showed 95.9% and 92.2% specificity, respectively. Both tests showed the highest percentages of positive results for diagnosis. Either CH or flow cytometry was positive in 93.5% of patients. In eight patients, flow cytometry was positive only through CV increase. Protein defects were detected in 72.3% of patients; ankyrin and spectrin were the most frequently found deficiencies. The CV of the fluorescence showed significantly higher increases in moderate and severe anemia than in mild anemia (p = 0.003). Severity of anemia showed no other correlation with tests results, type of deficient protein, inheritance pattern, or neonatal jaundice. CH and flow cytometry are easy methods with the highest diagnostic accuracy. Simultaneous reading of mean channel fluorescence (MCF) decrease and CV increase improve diagnostic usefulness of flow cytometry. This test seems to be a reliable predictor of severity. The type of detected protein deficiency has no predictive value for outcome. Predominant ankyrin and spectrin deficiencies agree with reports from other Latin American countries.
We studied 31 children with hemolytic anemia, or with positive family history for hereditary spherocytosis (HS), to assess the reliability of capillary blood samples for the diagnosis. HS was diagnosed in 20 patients. Cryohemolysis (CH) was positive in 94% and eosin-5'-maleimide flow cytometry in 90% of them, whereas flow cytometric osmotic fragility was positive in 94%. Capillary blood sampling showed to be useful for the diagnosis. Simultaneous use of these three tests allows confirming diagnosis in 100% of patients. The use of very small blood volumes (300 µl) allows an earlier diagnosis in neonates and small infants.
Patients with Gram-negative nosocomial pneumonia have high plasma concentrations of lipopolysaccharide, interleukin-6, and tumor necrosis factor-alpha, but the antibiotic therapy evaluated did not significantly modify these concentrations.
Aquaporin-1 (AQP1) is the membrane water channel responsible for changes in erythrocyte volume in response to the tonicity of the medium. As the aberrant distribution of proteins in hereditary spherocytosis (HS) generates deficiencies of proteins other than those codified by the mutated gene, we postulated that AQP1 expression might be impaired in spherocytes. AQP1 expression was evaluated through flow cytometry in 5 normal controls, 1 autoimmune hemolytic anemia, 10 HS (2 mild, 3 moderate, 2 severe, and 3 splenectomized), and 3 silent carriers. The effect of AQP1 inhibitors was evaluated through water flow-based tests: osmotic fragility and hypertonic cryohemolysis. Serum osmolality was measured in 20 normal controls and 13 HS. The effect of erythropoietin (Epo) on AQP1 expression was determined in cultures of erythroleukemia UT-7 cells, dependent on Epo to survive. Independent of erythrocyte size, HS patients showed a lower content of AQP1 in erythrocyte membranes which correlated with the severity of the disease. Accordingly, red blood cells from HS subjects were less sensitive to cryohemolysis than normal erythrocytes after inhibition of the AQP1 water channel. A lower serum osmolality in HS with respect to normal controls suggests alterations during reticulocyte remodeling. The decreased AQP1 expression could contribute to explain variable degrees of anemia in hereditary spherocytosis. The finding of AQP1 expression induced by Epo in a model of erythroid cells may be interpreted as a mechanism to restore the balance of red cell water fluxes.
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