Sickle Cell Anemia (SCA), Hereditary Spherocytosis (HS) and Paroxysmal Nocturnal Hemoglobinuria (PNH) can share some clinical complications, such as leg ulcers and priapism, due to hemolytic processes. Intravascular hemolysis (IVH) results in the release of cell-free hemoglobin (Hb) and heme into the circulation, potentially causing depletion of nitric oxide, oxidative stress and inflammation. We compared the hemolytic profile and heme/Hb scavenging profiles of patients with SCA on hydroxyurea therapy (SCAHU; 15-30 mg/kg/day for ≥ 2 months) or not (SCA), hereditary spherocytosis (HS), splenectomized HS (SHS) and paroxysmal nocturnal hemoglobinuria not on eculizumab therapy (PNH). Blood samples were collected from patients and from healthy volunteers (CON). Plasma levels of heme, haptoglobin (Hp) and hemopexin (Hpx) were quantified by colorimetric assay or ELISA. Fetal hemoglobin (HbF) was determined in the SCA groups by HPLC. Reticulocytes (RET) and cell counts were determined using an automated flow cytometry hematology system and serum levels of lactate dehydrogenase (LDH) and unconjugated bilirubin (UCB) were quantified by a modular colorimetric automation system. As expected, plasma heme was significantly elevated in the SCA group, compared to the CON group and with intermediate values in the SCAHU group (57.4±9.3, 24.5±2.9, 43.2±4.5µM, respectively, p<0.05, N≥10), correlating significantly and negatively with HbF levels in the SCA patients (r=-0.94, p<0.01). Accordingly, RET and UCB were significantly increased in SCA, compared to CON, with SCAHU presenting intermediate values (RET: 16.2±1.4; 1.9±0.1; 11.5±1.2%; UCB: 2.1±0.7; 0.2±0.0; 1.44±0.2 mg/dL; respectively, p<0.01, N≥10), indicating increased red cell turnover and heme catabolism in SCA. No significant elevations in plasma heme were observed in the HS and SHS groups, compared to CON (35.3±3.8 and 34.5±10.0µM; P>0.05, N≥10), although RET and UCB levels were significantly elevated in HS (8.27±1.07 % and 1.72±0.57 mg/dL, P<0.01, N≥13), but not SHS (2.80±0.17 % and 0.99±0.66 mg/dL, N≥11), perhaps indicating a redistribution of extravascular hemolysis (EHV) to low-level IVH in SHS. Surprisingly, plasma heme was not significantly elevated in PNH individuals studied (36.5±4.0 µM, N≥10), even though concentrations of plasma LDH were the highest in this patient group (1196±305 U/L; compared to 182±8, 352±73, 407±38. 211±19 and 182±28 U/L for CON, SCA, SCAHU, HS, SHS, respectively, P<0.01; N≥10) and LDH correlated significantly with plasma heme in this group (r=0.905, P<0.01 for PNH). RET and UCB presented and intermediate values in PNH (5.35±1.21% and 0.84±0.18 mg/dL, P<0.05, N≥10, compared to CON). To investigate whether hemolytic profiles may be modulated by the Hb and heme scavengers, Hp and Hpx, we measured these proteins in the circulations of patients. Hp was significantly depleted in SCA, SCAHU, HS and PNH (1.0±0.4, 1.0±0.6, 37.8±0.16, 12.3±10.2, respectively, compared to 142.9±20.6 mg/dL in CON, p<0.05; N≥10), while SHS presented similar levels to those of CON (189.9±38.8 mg/dL, N=5). In contrast, somewhat surprisingly, Hpx levels varied considerably in each patient group, with a significant depletion only observed in the SHS group (12.2±0.3, 15.9±5.4, 15.0±3.8, 6.0±1.5, 1.5±0.4, 17.8±5.0mg/dL for CON, SCA, SCAHU, HS, SHS, PNH, respectively; P<0.05, SHS compared to CON, N≥10). As such, plasma heme was slightly lower in PNH than in SCA, in association with very high plasma LDH, suggesting that either IVH is slightly less severe or that Hb/heme scavenging is more efficient in the PNH population studied than in SCA. While EHV may be abolished by splenectomy in HS, intermediate heme levels may suggest that some IVH, or reduced heme catabolism, may still occur in some of these patients. Interestingly, depleted Hp stores were coupled with maintained (or even elevated) Hpx levels in SCA and PNH, indicating that hemolytic processes may stimulate Hpx production, potentially via a heme-oxygenase-1 (HO-1)-dependent pathway. Furthermore, significantly diminished levels of Hpx in SHS suggest a role for the spleen in Hpx maintenance, possibly due to the importance of this organ for HO-1 activity. In conclusion, while parameters suggestive of hemolysis can be observed in SCA, HS and PNH, specific profiles of hemolytic markers and heme catabolism indicate that hemolytic events may be modulated differently in these diseases.


Conran:Bayer AG: Research Funding. Almeida:Jassen & Cilag: Other: Currently employed with.

Author notes


Asterisk with author names denotes non-ASH members.