Effects of hydroxyurea on blood rheology in sickle cell anemia: A two-years follow-up study

2.1 Patients

Twenty-eight adult SCA patients (37.0±11.6 yrs, 60.2±10.7 kg, 171.5±11.0 cm; 15 males/13 females; 50% with α-thalassemia) regularly followed by the Sickle Cell Center of Pointe-á-Pitre (Academic Hospital, Guadeloupe) were included in this study. At the beginning of the HU therapy all patients were at steady state, i.e., no blood transfusions in the previous three months and absence of acute episodes (infection, VOC, ACS, stroke, priapism) at least one month before inclusion into the study. Frequent hospitalized VOC (n = 14) and/or at least one ACS (n = 10) event in the preceding year were the main reasons to propose HU therapy to these patients. ACS and VOC were defined as previously described [11, 16]. For the other patients, HU therapy has been recommended because of frequent ambulatory sub-clinical VOC and persistent low hemoglobin levels. None of the participants had renal insufficiency, hepatic insufficiency or human immunodeficiency virus infection. Routine examination before starting HU therapy included the measurements of blood pressures (BP), mean hemoglobin saturation (by pulse oxymetry, SpO₂), heart rate (HR), collection of information regarding the presence of leg ulcers and frequencies of priapism. Patients were also questioned about chronic asthenia and the incidence of dyspnea during daily activities. The same parameters, as well as occurrence of the above complications (VOC, ACS), were prospectively recorded for 1 year and 2 years after the initiation of HU therapy. Before HU therapy (i.e., baseline) and at 6, 12 and 24 months of treatment, blood was collected for biochemical, hematological and hemorheological measurements. The study was performed in accordance with the Declaration of Helsinki, was approved by the IRB of the Academic Hospital of Pointe á Pitre (DRCI-CHUPAP 230514, Guadeloupe) and written informed consent was obtained from all patients.

2.2 Laboratory methods

Diagnosis of SCA was made by isoelectrofocusing (Multiphor II™ System, GE HEALTH CARE, Buck, UK), and cation-exchange high performance liquid chromatography (VARIANT™, Bio-Rad Laboratories, Hercules, CA, USA). Solubility test was also performed to confirm the presence of HbS. Genomic DNA was isolated from peripheral blood using standard desalting methods. Polymerase Chain Reaction technology (Gap-PCR) was used to detect 7 common α-thalassemia deletions including -α^3.7 and - α^4.2 alleles and triplication defects of the α-globin genes [26, 27].

2.3 Hematological and biochemical parameters

Hb concentration, hematocrit (Hct), MCV, mean corpuscular hemoglobin concentration (MCHC), RET, RBC, PLT, WBC and neutrophils counts were determined using a hematology analyzer (Max M-Retic, Coulter, USA). Lactate dehydrogenase (LDH) level was measured by standard biochemical methods at baseline, 1 year and 2 years of treatment.

2.4 Hemorheological parameters

All hemorheological measurements were carried out within 4 hours of the venipuncture to avoid blood rheological alterations, and after complete oxygenation of the blood [5]. Blood viscosity was determined at native Hct, at 25°C, and at a shear rate of 225 s^–1 using a cone/plate viscometer (Brookfield DVII+ with CPE40 spindle, Brookfield Engineering Labs, Natick, MA). RBC deformability was measured at 37°C and 30 Pa by laser diffraction analysis (ecktacytometry), using the Laser-assisted Optical Rotational Cell Analyzer (LORCA, RR Mechatronics, Hoorn, The Netherlands). The system has been described elsewhere in detail [5]. Briefly, 25 μl of prepared blood suspension was mixed with 5 ml polyvinylpyrrolidone (PVP; viscosity = 30 cP) and sheared into a Couette system made of glass. The diffraction pattern was analysed by the computer to calculate an elongation index reflecting RBC deformability [5]. RBC aggregation was determined at 37°C via syllectometry, (i.e., laser backscatter versus time, using the LORCA) after adjustment of the Hct to 40%. In addition, the RBC disaggregation threshold (i.e., RBC aggregates strength) was determined using a re-iteration procedure [5]. Seven pre-defined shear rates ranging between 7.5 s^–1 and 800 s^–1 were applied on the RBC suspension to locate the minimal shear rate needed to prevent RBC aggregation.

2.5 Statistical analysis

Values are expressed as means±SD. A One-Way Analysis of Variance (ANOVA) with repeated measurement was used to study the physiological, clinical and biological changes occurring with HU therapy. Post-hoc Tukey test was performed to locate the differences. McNemar test was used to test the effects of HU on different clinical parameters after 2 years of treatment. Pearson correlation was performed to test the associations between different variables. Significance level was defined as p < 0.05. Analyses were conducted using SPSS (v. 20, IBM SPSS Statistics, Chicago, IL).

3.1 Clinical data

Regarding the clinical course of the disease, HU was effective in 25 of the 28 patients after 1 year of treatment (two patients had ACS and one had VOC) and in 27 of the 28 patients after 2 years therapy (only one patient experienced a VOC episode). The number of VOC and ACS events decreased (p < 0.01 and p < 0.05, respectively) from 0.81±1.09 and 0.56±0.98 before treatment, respectively, to 0.16±0.52 and 0 after 1 year treatment and 0.04±0.20 and 0 after 2 years treatment. SpO₂ was improved compared to baseline values after 1 year and 2 years of HU therapy. HR was decreased after 2 years of HU therapy compared to baseline (Table 1). Blood pressures did not change with HU (Table 1). HU treatment decreased the proportion of patients with chronic asthenia and dyspnea from 64% and 93% to 0% and 21%, respectively (p < 0.001 for both). The frequency of patients with frequent priapism did not significantly change with HU (18% vs 4% before and after 2 years of therapy, respectively, p = 0.25) but the proportion of patients with leg ulcers tended to decrease (27% vs 13% before and after 2 years of therapy, respectively, p = 0.06).

Doses of HU were increased progressively and adjusted for each patient, based on the hematological parameters [30]. The mean doses of HU prescribed to the patients in this study are relatively low because some patients had early sign of hemato-toxicity leading physicians to decrease the doses. At 2 years of therapy, patients received 15.7±5.4 mg/kg/days of HU. HbF (Fig. 1A), Hb (Fig. 1B), MCV (Fig. 1C) and RBC deformability (Fig. 1I) increased at 6 months of HU treatment and remained elevated at 1 and 2 years after the beginning of therapy. RBC deformability at 2 years was also higher than at 6 months of HU therapy (Fig. 1I). WBC (Fig. 1D) and PLT (Fig. 1E) counts, LDH level (Fig. 1F) and RET count (data not shown; p < 0.001) were decreased at 6 months, 1 year and 2 years of HU therapy in comparison with baseline. MCHC was decreased after 2 years of HU therapy compared to baseline (data not shown, p < 0.05). RBC aggregates strength decreased below baseline values at 6 months but then the difference between baseline and 1 year/2 years of treatments was not significant (Fig. 1H). Blood viscosity (Fig. 1G) and RBC aggregation (data not shown) did not change with HU therapy.

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Fig.1

Effects of HU therapy on HbF level (1A), Hb level (1B), MCV (1C), WBC (1D) and PLT (1E) counts, LDH level (1F), blood viscosity (1G), RBC aggregates strength (1H) and RBC deformability (1I). HbF, Fetal hemoglobin; Hb, hemoglobin; MCV, mean cell volume; WBC, white blood cells; PLT, platelet; LDH, lactate dehydrogenase; RBC, red blood cell. Different from before treatment: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. Different from 6 months: ^$p < 0.05.

Since the highest changes in the biological values occurred at 6 months of HU therapy, we calculated the percentages of changes between baseline (i.e., before treatment) and 6 months of treatment. Subsequently, correlations were used to test associations between variables using these percentages of changes. Our results showed positive correlation between the changes in HbF level and RBC deformability (r = 0.46; p < 0.05) and MCV and RBC deformability (r = 0.56; p < 0.01). In addition, the changes in Hb level positively correlated with the changes in RBC deformability (r = 0.55; p < 0.01).