Abstract
Hereditary Spherocytosis (HS) is the most common congenital hemolytic anemia in individuals, with a prevalence of about 1:2000–1:3000 in the Caucasian population. The clinical manifestations of HS are highly heterogeneous with variable hemolytic anemia, jaundice, reticulocytosis, splenomegaly, and gallstones. The mode of inheritance is autosomal dominant in 75% of cases, due to mutations of ankyrin (ANK1), band 3 (SLC4A1), and β-spectrin (SPTB) gene; the remaining 25% include recessive HS forms, mostly due to compound heterozygosity for defects in the genes encoding α-spectrin (SPTA1), or protein 4.2 (EPB42), and de novo The deficiency/dysfunction of any of them results in a loss of surface area leading to the typical spheroidal, osmotically fragile cells. The diagnostic workflow is based on clinical and family history, and on laboratory investigations with different sensitivity and specificity. The ektacytometric analysis is considered a gold standard tool for HS diagnosis, and the profiles observed in this pathology, are typically bell-shaped. Indeed, two distinct profiles may be observed (HS1 and HS2); nonetheless, the clinical and biochemical significance of these differences, and their correlation with the clinical phenotype of HS remain poorly understood. We analyzed a large cohort of not splenectomized HS patients (N=204), focusing on their ektacytometric curves and their hematologic and biochemical features. We observed that HS2 patients were significantly younger than HS1 at the time of the study (10 yrs, range 0.1-71, versus 22 yrs, range 0.1-73, p=0.002). Moreover, HS2 group showed a higher number of severe cases and lower number of mild cases compared to HS1 (13.7% versus 2.6% and 17.6% versus 36.6%, respectively, p=0.004). In particular, in HS2 patients, Hb level and MCHC values were significantly lower, and RDW values were higher compared to HS1 (p=0.004, p<0.001, and p=0.034, respectively). As regards laboratory findings, we observed a greater number of HS2 patients with positive NaCl osmotic fragility test on fresh blood, (82.3% versus 60.1%, p=0.007), while no significant differences were found considering the other osmotic fragility tests, including EMA-binding test, or RBC membrane protein defects, although spectrin defect was more frequent in HS2 group of patients and band 3 in HS1 group. In the attempt to identify which parameter could influence the differences observed at ektacytometric analysis in the two groups, we analysed the correlations between ektacytometric parameters and laboratory and hematologic data. In both groups, a significative association was observed between spherocytes and Omin, as well as between AGLT and EMA-binding with EImax, and with AUC; conversely, a negative correlations were observed between AGLT, GLT, Pink test and Omin, and between spherocytes number with EImax. Interestingly, only in the HS1 group, we observed a negative correlation between MCHC and all osmoscan parameters related to osmolality (Omin p=0.013, Ohyper p<0.001, and Omax p=0.003). Moreover, this parameter, which is closely associated with the suspicion of HS, was elevated in only 45% of all HS cases, specifically in 53% of HS1 and 26% of HS2 cases. These findings confirm that MCHC cannot be regarded as a distinctive marker for the differential diagnosis of HS, contrary to previous assumptions, and that the reduced cell density is indeed associated to the rightward shift of the entire osmoscan curve observed in HS2—previously attributed solely to alterations in hydration state (Ohyper)—without necessarily implying an increase in cell volume or a reduction in the number of spherocytes.
