HbSS Patients In Steady State Research Articles

Protein S activity in sickle cell anaemia patients in steady state and crisis

Background: In sickle cell anaemia, there is an ever-present risk for haemostatic abnormalities which may result in reduced levels of naturally occurring coagulation inhibitors. Protein S is a Vitamin-K dependent, γ-carboxy glutamic acid-containing glycoprotein that potentiates the action of protein C when activated resulting in anticoagulation. This study measured serum Protein S activity in SCA patients during vaso-occlusive crisis and in steady-state, and this was compared with the activity in HbAA controls with a view to establishing if Protein S plays a role in VOC in SCA patients. Methods: This was an analytical prospective study comprised of 30 HbSS patients and 30 HbAA controls. Using ELISA method, Protein S levels were measured in the HbSS study group during crisis and in the HbAA control group; When the HbSS patients were in steady state, Protein S levels were also measured. Results: Protein S levels were highest in the HbAA control group (5.27+0.47 ng/ml) followed by the HbSS patients in steady state (5.08+0.56 ng/ml) and lowest in the HbSS patients in crisis (4.96+0.97 ng/ml). However, this difference was not statistically significant. Conclusions: Protein S levels are reduced in HbSS patients when compared with HbAA controls, and the levels are lower during a VOC compared with steady state.

Assessment of zinc level and its relationship with some hematological parameters among patients with sickle cell anemia in Abakaliki, Nigeria

Background: Sickle cell anemia (SCA) is an inherited hemoglobin (Hb) disorder with susceptibility for oxidative damage due to chronic redox imbalance in red blood cells (RBCs) which often results in hemolysis, endothelial injury, recurrent vaso-occlusive episodes, and derangement in hematological parameters. Zinc is an antioxidant which helps to reduce oxidative damage. This study aimed to assess the serum level of zinc and its relationship with some hematological parameters in patients with SCA (HbSS). Materials and Methods: This was a cross-sectional comparative study which involved HbSS patients in steady state with sex‐ and age‐matched HbAA control. Assay of Hb phenotype, serum zinc level, and some haematological parameters were done. Ethical approval was gotten from the institutional review board, and each participant gave informed written consent before recruitment into the study. Analysis of all data obtained was done using SPSS software, version 20. Results: Thirty adult patients with SCA and thirty sex- and age-matched controls with a mean age of 26.7 ± 7.6 years and 27.7± 5.3, respectively, were studied. There was a significant decrease in serum zinc level among patients with HbSS compared to those with HbAA (P = 0.038). Similarly, patients with HbSS had significantly lower Hb level, packed cell volume, and RBC count compared to HbAA control (P < 0.05). On the contrary, patients with HbSS had significantly higher white cell count and platelet count compared to HbAA individuals (P < 0.05). Correlation between serum zinc level and blood counts showed weak positive relationship between zinc level and Hb level (r = 0.04, P = 0.8) and weak negative relationship between serum zinc level and platelet count (r = −0.3, P = 0.1), as well as zinc and white blood cell (WBC) count (r = −0.2, P = 0.4). Conclusion: There was a significantly low level of zinc among patients with HbSS compared with HbAA controls. Patients with HbSS had zinc levels that weakly correlated with Hb level positively but weakly negatively correlated with platelet and WBC counts. Further studies are required on a wider scale to assess whether zinc supplementation may improve blood counts in patients with SCA.

Superoxide dismutase activity in sickle cell anemia patients during crisis and in steady State

INTRODUCTION: Sickle cell anemia is associated with intense oxidative stress, and optimal antioxidant levels are essential to prevent continuous oxidant tissue damage. The role of oxidant damage to red blood cells in sickle cell anemia (SCA) has been of interest in recent years, and some available results indicate that sickled red blood cells produce almost twice as much superoxide, hydrogen peroxide, and hydroxyl free radicals compared with normal red cells. To counter the effects of oxidants, red blood cells have evolved a number of self-sustaining activities of antioxidant defense enzymes including but not limited to superoxide dismutase (SOD). This study aimed to measure serum SOD activity in patients SCA patients (as a measure of antioxidant defense) during the crisis and in steady state and to compare this with the activity in HbAA normal controls. SUBJECTS AND METHODS: Eighty-eight participants were recruited into this case control study and comprised 34 HbAA controls, 12 HbSS patients in crisis, and 42 HbSS patients in steady state. Five milliliter of venous blood was collected from each participant and 3 ml was dispensed into plain tubes and centrifuged. The serum was used for the estimation of SOD activity. The remaining 2 ml was used for hemoglobin electrophoresis. RESULTS: SOD levels were highest in the HbAA control group (1.928 ± 0.051 U/mL) followed by the HbSS patients in steady state (1.844 ± 0.118 U/mL) and lowest in the HbSS patients in crisis (1.755 ± 0168 U/mL). This difference was statistically significant with P = 0.001. CONCLUSION: SOD levels are reduced during vaso-occlusive crisis.

Hematological parameters in Ghanaian sickle cell disease patients.

BackgroundEffective treatment and management of sickle cell disease (SCD) has been a challenge in Africa over the years. Hematological parameters are very useful profiles in the effective management of the disease. However, there is scarcity of studies on the hematological parameters of SCD in Ghana. This study aimed at determining hematological parameters among SCD patients with vaso-occlusion, those in the steady state as well as healthy controls at a teaching hospital in Ghana.MethodologyThis was a cross-sectional study involving a total of 628 subjects, including 148 HbAA controls, 208 HbSS patients in steady state, 82 HbSC patients in steady state, 156 HbSS patients in vaso-occlusive crises (VOC), and 34 HbSC patients in VOC. Venous blood sample was collected from all study participants. A full blood count was done within 2 hours of collection, and hemoglobin (Hb) concentration, packed cell volume, red blood cell (RBC) concentration, mean corpuscular Hb, mean cell volume, mean corpuscular Hb concentration, and white blood cells (WBC) and platelet (PLT) counts were recorded.ResultsWBC and PLT counts were significantly higher in both female and male patients with SCD, compared with their healthy counterparts (P<0.05). The level of WBC was, however, significantly higher in patients with HbSS VOC among the SCD patients (P<0.001). Levels of Hb, RBC, and hematocrit were significantly higher in the controls (P<0.001). There was no significant difference in mean cell Hb among male patients with SCD (P=0.274) and female patients with SCD (P=0.5410).ConclusionThe SCD patients had lower Hb and RBC than the controls; however, higher PLT and WBC are noted in various status of SCD, possibly reflecting spleen effect in these patients. Further studies are needed to confirm these findings.

Assessment of Bone Marrow Function in Sickle Cell Anaemia Patients Using Corrected Reticulocyte Counts

Introduction: Chronic hemolysis occurs in sickle cell anemia as a result of recurrent sickling and other abnormalities of the red blood cells including eryptosis. Exuberant reticulocytosis is anticipated to partially compensate for the resultant anemia. Sickle cell anemia patients may also have aplastic crisis, bone marrow (BM) infarction and erythropoietin deficiency which could lead to reticulocytopenia despite the anemia. High degree of reticulocytosis among asymptomatic infants with sickle cell anemia has been associated with an increased risk of death or stroke during childhood. Assessment of BM function in sickle cell anemia is important due to potential complications associated with both under-activity and hyperactivity. This study aimed at evaluating the erythropoietic function of the BM in steady state sickle cell anemia using corrected reticulocyte counts.Methods: This study was carried out at the hematology clinic in the University College Hospital, Ibadan. HbSS patients in steady state were recruited from the hematology clinic. Local ethical committee approval was obtained and all participants gave written informed consent. Patients with M. tuberculosis, Hepatitis B, HIV and P. falciparum infection were excluded. Peripheral blood samples were analyzed using Sysmex Ki-X21 for complete blood count (CBC) and standard point of care for serum electrolytes and liver function tests. The glomerular filtration rates were calculated using the Cockcroft-Gault formula. Reticulocyte counts were determined manually using fresh samples from K2 EDTA bottles and methylene blue stain. Two drops of stain were mixed with two to four volumes of anticoagulated blood and incubated at 37ºC for 15 minutes. Afterwards, the cells were re-suspended and blood films were made. Corrected reticulocyte count and reticulocyte production index were calculated. Participants were categorized according to corrected reticulocyte counts of greater than or less than 2.5%. Univariate and multivariate analyses were performed to determine variables associated with corrected reticulocyte count <2.5%.Results: 92 HbSS patients were recruited with a mean (SD) age of 19.6 (5.8) years. There was no correlation between age and eGFR (p-value: 0.227). Median (range) reticulocyte count, corrected reticulocyte count and reticulocyte production index were 5.5 (0.5 - 29.9), 3.3 (0.1 - 17.1) and 1.7 (0.2 - 8.6) respectively. 40 (43.5%) patients had corrected reticulocyte count <2.5% and 52 (56.5%) had a corrected count >2.5%. Those corrected reticulocyte count <2.5% were older (p: 0.013), taller (p: 0.041) and had higher aspartate transaminase (AST) levels (p: 0.006) than those with corrected counts >2.5% (Table 1). CBC parameters were not different when compared between both groups. Results of multivariate logistic regression analysis carried out showed that only AST was independently linked with corrected reticulocyte count <2.5% (R2: 0.172, p-value: 0.001) (Table 2).Table 1Factors Associated with Low Reticulocyte CountCorrected count<2.5%Corrected count>2.5%p-ValueAge (Mean, SD)21.4 (6.3)18.4 (5.0)0.013Gender (N, %)Male22 (55.0)28 (53.8)0.912Female18 (45.0)24 (46.2)Height (Mean, SD)1.6 (0.1)1.5 (0.1)0.041BMI (Mean, SD)18.7 (3.1)18.7 (3.0)0.753GFR (Mean, SD)64.3 (37.7)66.4 (29.3)0.453Bilirubin (Mean, SD)1.7 (1.1)1.9 (2.6)0.674AST (Mean, SD)22.5 (13.5)14.5 (6.6)0.006ALT (Mean, SD)13.4 (7.7)14.4 (11.1)0.876Table 2Independent Predictors of Corrected Reticulocyte Count <2.5%or95% CIp-ValueAge1.080.97 - 1.210.169Height19.80.11 - 366.100.259AST1.101.04 - 1.170.002Hemoglobin1.000.97 - 1.020.872R2: 0.172, p: 0.001Conclusion: Despite corrected reticulocyte count <2.5% in about half of the patients, there were similar hematological parameters and eGFR in both groups of patients. AST is a marker of hemolysis and low ALT rules out hepatic involvement. Since only 17.2% of the variability in BM response as assessed by corrected reticulocyte count could be accounted for by variables included in this study, there is a need to further evaluate the BM function of sickle cell patients to establish the causes of corrected reticulocyte count <2.5% in the setting of anemia, having ruled out erythropoietin as well as iron, folate or cobalamin deficiencies. This will aid the development of a functional algorithm for the individualized management of sickle cell disease patients with anemia. DisclosuresNo relevant conflicts of interest to declare.

Plasma levels of some coagulation parameters in steady state HBSC disease patients.

IntroductionSickle cell disease is a collection of autosomal recessive genetic disorders. It includes homozygous HbSS and double heterozygote combinations such as HbSC. Central and West Africa bears a significant burden of HbSC disease.MethodsProthrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen concentration (FC) and platelet count (PC) were determined in 41HbSC and HbSS patients in steady state along with 40 apparently healthy HbAA controls. One way ANOVA test was used to compare means; p values< 0.05 were considered statistically significant.ResultsThere was no significant difference in mean PT for the study groups (p = 0.192). Mean PC was highest in HbSS patients: 445.7 +/- 128.3 X 10 9/ L compared to HbSC: 330.0 +/- 97.7 X 10 9/ L andHbAA:245 +/- 77.7 X 10 9/ L (p= 0.000). Mean APTT was 28.1 +/- 3.8 seconds in controls,24.1 + /- 66 seconds in HbSS patients and 21.8 +/- 3.8 seconds in HbSC patients (p = 0.000). Mean FC in HbSS was 1.6 +/- 0.7 g/L, 3.2 +/- 0.6 g/L in HbSC and 2.9 +/- 0.4 g/L in HbAA (p =0.000).ConclusionA significant difference exists in PC, APTT and FC in HbSC patients compared to HbSS patients and HbAA controls. Elevated FC and shortened APTT may play a role in complications more characteristic of HbSC such as retinopathy and osteonecrosis. These suggestHbSC is not merely a milder form of HbSS; both diseases should be seen as different entities with regards to approaches for management.

Nitrite Levels Are Associated To Fetal Hemoglobin Concentration In Patients With Sickle Cell Anemia: Implications In Inflammatory Process

Introduction Sickle cell anemia (SCA) is a genetical hemolytic disorder defined as chronic inflammatory disease. Nitrite (NO-2) in SCA (HbSS) patients may be associated with the hemolytic process while fetal hemoglobin (Hb F) presents protective effect in these patients. Serum NO-2 interferes with the role of Hb F in reducing the hemolytic process contributing to vaso-occlusive crises. Aims The aim of this study was to evaluate the correlation of the serum levels of nitrite with fetal hemoglobin, low density lipoprotein cholesterol (LDL-C) and triglycerides levels in HbSS patients in steady-state of the Centro de Referência em Doença Falciforme de Itabuna (CERDOFI), Bahia, Brazil. Methods Forty-two patients diagnosed with SCA were included at baseline. Diagnosis was confirmed by high-performance liquid chromatography (HPLC). Serum levels of nitrite were performed using Griess reaction, and fetal hemoglobin, LDL cholesterol and triglycerides levels were measured by biochemical methods. The experimental protocol was approved by the Researcher Board Committee on Human Research, Gonçalo Moniz Research Center and informed consents were signed by patients. Results Our results showed a significant increase in NO-2 (p&lt;0.0001) in adult patients with SCA compared to the control group (healthy volunteers). Data demonstrated a negative correlation between nitrite and fetal hemoglobin (p=0.004, r=-0.627). No correlation was found between fetal hemoglobin or nitrite with LDL-Cl and triglycerides. Conclusion The results reinforce that the serum nitrite can predict the clinical disease course. Disclosures: No relevant conflicts of interest to declare.

The effect of sildenafil on the elasticity of erythrocytes in homozygous sickle cell disease

One of the features of homozygous sickle cell disease (HbSS) is the impaired elasticity of the erythrocyte membrane that could impede microcirculatory blood flow and cause hypoxia and tissue damage. We investigated the effect of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor that inhibits the breakdown of cyclic guanosine monophosphate (cGMP) resulting in vasodilatation, on the elasticity of HbSS erythrocyte. Blood samples from ten HbSS patients in steady state was exposed to different doses (5, 10, 20, and 40 μg/mL) of sildenafil and the elasticity of the erythrocytes measured at native hematocrit with the BioProfiler. An equal number of subjects with normal hemoglobin (HbAA) served as the control group. There was a marginal increase in elasticity with 5 μg/mL of the drug and this became significant (P<0.05) with the 10 μg/mL dose. Thereafter, gradual nonsignificant decreases were observed with the 20 and 40 μg/mL doses. A similar trend was observed for the control group. The elasticity values for the HbSS subjects at native hematocrit were significantly (P<0.05) less when compared with the corresponding concentrations for the HbAA controls. This was reversed at a corrected hematocrit of 45%. The result of this study shows that sildenafil caused an initial increase in erythrocyte membrane elasticity in both HbSS and HbAA subjects, and this later decreased with increasing concentration of the drug possibly due to the dual effect of cyclic adenosine monophosphate (cAMP).

The Relationship of Plasma Laminin Levels to Anemia and the Effect of Soluble Laminin on Sickle Red Cell Adhesion in SCD.

Sickle red cell (SS RBC) adhesion is believed to be a major factor in vaso-occlusion. SS RBCs adhere especially avidly to the extracellular matrix protein laminin (LAM), which is found in the subendothelial basement membrane as well as soluble in plasma. We asked (1) Whether the level of soluble LAM in plasma is related to chronic complications of SCD; and (2) Whether soluble LAM enhances the ability of SS RBCs to adhere to endothelial cells (ECs). We first established an ELISA assay to quantitate plasma LAM and found that plasma from all normal subjects tested had LAM levels ≤0.5 μg/mL, which was comparable to the lower limit of detection for our assay. However, plasma from HbSS patients in steady state had levels ranging from ≤0.5 to >8.1 μg/mL. Of 52 HbSS patients studied while in steady state, 5 had plasma LAM levels ≤0.5 μg/mL, 22 had levels >0.5 and <2.0 μg/mL, and 25 had levels between 2 and 8.1 μg/mL. We then compared these levels to a variety of clinical and laboratory endpoints. While patients with higher LAM levels (>3.0 μg/mL) did not have a higher frequency of stroke, acute chest syndrome, priapism, leg ulcers, or aseptic necrosis than patients with levels <1 μg/mL, there was an inverse relationship between plasma LAM level and hemoglobin (Hb) (p=0.004 by linear regression analysis). Patients with baseline (without hydroxyurea) Hb ≤8.5 g/dL (n=28) had mean plasma LAM levels of 3.44±0.42 (SEM) μg/mL, while patients with Hb >8.5 g/dL had LAM levels of 1.72±0.41 μg/mL (n=18, p=0.008). To evaluate whether soluble LAM contributed to SS RBC adhesion, we assayed adhesion using TNF-activated human umbilical vein endothelial cells in a graduated height flow chamber. When SS RBCs were first preincubated with physiologic amounts of LAM, we saw a marked increase in SS RBC adhesion to ECs, and this effect was related to the amount of LAM added. With addition of 5 μg/mL LAM, adhesion to ECs at 2 and 5 dynes/cm2 was increased by 20% and 46% over baseline, respectively. Addition of 10 μg/mL LAM enhanced adhesion at these shear stresses by a mean of 154% and 249%, respectively (n=4). Furthermore, SS RBCs from patients with lower plasma LAM levels had lower baseline adhesion in the absence of added LAM. We then incubated SS RBCs with ABO-compatible SCD plasmas with either high or low LAM levels, washed these cells, and tested them for their ability to adhere to ECs. Plasmas with high LAM (n=3, LAM 4.5–8.1 μg/mL) strongly enhanced adhesion by a mean of 71.9% over baseline at 2 dynes/cm2, while those with low LAM levels (0.4–0.6 μg/mL) enhanced adhesion by only 21.1%. Finally, in order to show that it was the LAM in these plasmas that enhanced SS RBC adhesion, rather than another factor elevated in parallel with LAM, we precleared high LAM plasma with monoclonal antibody reactive with LAMs-10,-11 and anti-murine Ig-Sepharose beads and then compared the activity of this plasma to the same plasma sham precleared with only Sepharose beads. Plasma LAM levels were also assayed to confirm removal of LAM by this procedure. Plasmas from which LAMs-10,-11 had been removed failed to enhance SS RBC adhesion, while the same plasmas subjected to sham preclears increased SS RBC adhesion to ECs by 76.9% at 2 dyne/cm2 (n=2). Thus, we conclude that plasma LAM can enhance SS RBC adhesion to ECs, and elevated levels of plasma LAM may contribute to vaso-occlusion. In addition, the inverse correlation of plasma LAM levels to Hb levels further suggests a relationship between SS RBC adhesion and the severity of anemia in SCD.

Blood mononuclear cells and platelets have abnormal fatty acid composition in homozygous sickle cell disease

Leukocyte adhesion to vascular endothelium contributes to vaso-occlusion and widespread organ damage in sickle cell disease (SCD). Previously, we found high expression of the adhesion molecules alphaMbeta2 integrin and L-selectin in HbSS individuals with severe disease. Since membrane n-6 and n-3 polyunsaturated fatty acids modulate cell adhesion, inflammation, aggregation and vascular tone, we investigated the fatty acid composition of mononuclear cells (MNC) and platelets of HbSS patients in steady state (n=28) and racially matched, healthy HbAA controls with similar age and sex distribution living in the same environment (n=13). MNC phospholipids of the patients had lower levels of docosahexaenoic acid (DHA, p<0.01) and increased arachidonic acid (AA, p<0.005) relative to HbAA controls. Similarly, platelets from HbSS patients had less eicosapentaenoic acid (EPA, p<0.05) and more AA (p<0.05) in choline phosphoglycerides (CPG), with reduced DHA (p<0.05) in ethanolamine phosphoglycerides. Platelet CPG had lower DHA levels in SCD patients with complications compared to those without (p<0.05). Reduced cell content of EPA and DHA relative to AA favours the production of aggregatory and proinflammatory eicosanoids that activate leukocytes and platelets. This facilitates inflammation, leukocyte adhesion, platelet aggregation and vaso-occlusion in SCD.

Plasma factor VII and thrombin-antithrombin III levels indicate increased tissue factor activity in sickle cell patients.

Although the mechanisms involved in the persistent clinical complications of sickle cell disease have not yet been fully delineated, previous studies suggest that sickle cell (HbSS) patients have a disposition to generate more thrombin and plasma in vivo than normal subjects. The reasons for the impaired regulation of haemostasis in HbSS patients is poorly understood. We report studies evaluating the extent to which in vivo coagulation and fibrinolysis are altered in HbSS patients in steady state. The concentrations of total factor VII (F(VII)t), factor VII zymogen (F(VII)z), thrombin-antithrombin III (TAT), fibrinopeptide A(FPA), and fibrin D-dimer in plasmas of 50 normal controls (HbAA) and 45 HbSS steady state patients, were measured using sensitive and specific enzyme-linked immunoassays. The average plasma concentration of F(VII)t, in sickle cell plasma was significantly lower than that of the control subjects (0.70 +/- 0.19 U/ml versus 1.16 +/- 0.41 U/ml), whereas F(VII)z in the patients and controls were 0.47 +/- 0.15 U/ml and 1.15 +/- 0.33 U/ml respectively, P < 0.001. Both measures of factor VII suggest a higher factor VII turnover in sickle cell disease. The mean concentration of TAT in the plasma of HbSS patients were significantly higher than those of HbAA controls (371 +/- 44 pM versus 42 +/- 2 pM) (P < 0.001), a difference that is strongly indicative of higher rates of in vivo thrombin generation by HbSS patients. Plasmas of HbSS patients had significantly higher concentrations of FPA compared to those of the control subjects (12.85 +/- 1.96 ng/ml versus 4.22 +/- 0.37 ng/ml) (P < 0.001). The D-dimer levels were also higher in the HbSS than control plasmas (1029.6 +/- 58.6 ng/ml versus 224.3 +/- 27.6 g/ml) (P < 0.001), with the patients' values being indicative of enhanced fibrinolysis. These results strongly suggest accelerated in vivo coagulation and fibrinolysis in HbSS patients even during steady state. They are consistent with the hypothesis that haemostasis is less tightly regulated in the HbSS patients than in HbAA controls. The altered regulation of haemostasis may contribute to the initiation of vaso-occlusive processes associated with sickle cell painful episodes.