Sickle Trait Patients Research Articles

5576015 ROLE OF APOLIPOPROTEIN-1 GENE (APOL-1) AS A GENETIC PREDICTOR OF RENAL DYSFUNCTION AMONG SICKLE CELL DISEASE EGYPTIAN PEDIATRIC PATIENTS

Background: Sickle cell nephropathy (SCN) is linked with significant morbidity and mortality. SCN is first presented as glomerular hyperfiltration then it progresses to focal segmental glomerular sclerosis, and eventually, renal failure. SCN can be assessed by the presence of albuminuria, measuring estimated glomerular filtration rate (eGFR), abnormalities in kidney biopsy and/or imaging, and. SCN was linked to different gene polymorphisms such as the apolipoprotein-1 (APOL-1) gene two haplotypes; G1 (G1GM and G1G+ sub- haplotypes) and G2. Aim: Determination of the prevalence of APOL-1 among SCD Egyptian pediatric patients and its role as a genetic predictor of renal dysfunction in SCD pediatric patients. Methods: In this cross-sectional study, 69 patients with sickle cell anemia (SCA) and 28 patients with S/β thalassemia patients, and 3 sickle trait patients underwent full history taking, clinical examination, and laboratory tests including CBC, Hb electrophoresis, serum creatinine (and calculation of eGFR using “bedside Schwartz” formula), microalbuminuria, urine albumin/ creatinine ratio and genomic DNA analysis for detection of APOL-1 G1 SNPs S342G and I384M using PCR- RFLP analysis. Results: The prevalence of APOL-1 G1 variants among our patients was 44% (44 patients); 23% of them had G1GM sub-haplotype (10% of SCD patients) and 77% of them had G1G+ sub-haplotype (34% of SCD patients). Among the 17 cases of microalbuminuria, 9 cases (53%) had G1 variants. Hyperfiltration cases were 10 cases of which 7 cases (70%) had the G1 variants, 13 out of the 30 cases (43.3%) of mildly decreased eGFR had G1 variants and the two cases of mild to moderately decreased eGFR (100%) had G1 variant; the G1GM sub-haplotype. There was no significant association between the APOL-1 G1 variant in total and the chronic renal complications of SCD in the studied patients. There was no significant association between G1GM sub-haplotype and the earlier stages of chronic kidney disease as hyperfiltration and mildly decreased eGFR. A significant association was found between G1GM sub-haplotype and later stages of chronic kidney disease; as microalbuminuria (P=0.026) and mildly to moderately decreased eGFR (P=0.020). Conclusion: The prevalence of APOL-1 G1 variants in our study was 44% with a higher prevalence of G1G+ sub-haplotype (34%) than G1GM sub-haplotype (10%). There is a significant association between having G1GM sub-haplotype and chronic renal complications; as microalbuminuria and mildly to moderately decreased eGFR, thus, APOL-1 risk variant G1GM was associated with more severe stages of chronic kidney disease, indicating a more prominent association with SCN progression. Keywords: Sickle cell disease, nephropathy, albuminuria, estimated glomerular filtration rate, APOL-1, G1 variants.

Dynamic Sickling Assay: Using Enzymatically Induced Hypoxia to Assess Real-Time Sickling Scalable from Large Erythrocyte Populations to Individual Cells

Background: There is a pressing need for assays to assess both patient condition and to monitor the progress of Sickle Cell Disease (SCD) therapies. It would be beneficial for an assay to allow assessment of oxygen dissociation, hemoglobin polymerization and Red Blood Cell (RBc) sickling under both equilibrium (mimicking longer-term effects) and non-equilibrium (simulating in-vivo process) conditions. Also desirable is the CLIA-waived system suitable for use in clinical trials and ultimately for clinical management of patients on SCD therapies including those those targeting Hb oxygen affinity. Our proposed work introduces an RBC hypoxia-induced sickling assay as a clinical tool to assess SCD patient condition and to assist in development, monitoring, and optimization of SCD therapies. Methods: The system uses an enzymatic oxygen scrubbing system allowing for a wide range of deoxygenation rates and final hypoxia severity. Diluted blood samples were supplemented with Protocatechuic acid (PCA) and then with Protocatechuate 3,4-Dioxygenase (PCD) enzyme inducing hypoxia at a predetermined rate. The sample was injected into an anaerobic microfluidic chamber with the resulting Red Blood Cell (RBC) morphological changes observed through time-lapsed photography. Image analysis allows for identification and quantification of sickled RBC linked to different polymerization scenarios/mechanisms. An oxygen probe was used to track the rate of medium deoxygenation. Sickling profiles were constructed to represent a percent of sickled RBC in a total cell population measured every two seconds after the enzymatic reaction is initiated. Profiles were assessed though i) percent sickling at normoxia, ii) morphological Point of Sickling (mPoS) and iii) time delay of sickling both measured at 5% increase in sickling over the normoxia sickling baseline, iv rate of sickling, v) size of the sickling fraction and vi) Area under the Curve (AUC). The last four parameters are measured for each sickling fraction if more than one such is present. Results: In control experiments PCA/PCD system was shown to allow reliable and reproducible sample deoxygenation currently enabled in a range from seconds to hours. Comprehensive multidimensional profiles were constructed presenting fraction of sickled RBC as a function of a) time from enzymatic reaction initiation, and b) one or more of rate of oxygen consumption, end of reaction hypoxia level, or oxygen tension in the medium. Such profiling allows for fast optimization of assay parameters to target assay conditions most suitable for detection of desired effects.Significant changes in sickling profiles, with marked delay in sickling, were observed upon treatment of RBC with hemoglobin-modifying drugs like voxelotor.Sickling profiles of Sickle Trait (SCT) patients showed significant delay in sickling compared to SS-type SCD patients. The method shows the potential to differentiate SCT patients based on the risk of adverse conditions to occur due to the patient's trait status.By stopping the flow, the system allows tracking of morphological status of individual cells over the full course of medium deoxygenation. Individual cells show pronounced differences in their propensity to sickle, potentially correlated to individual cell Hb F content. Change in morphology of sickling cells from biconcave disk to crescent and then to holly leaf and granular forms had been observed upon progressively decreasing medium oxygen tension.RBC microparticle formation can be observed and tracked over time as an additional metric of erythrocyte response to developing hypoxia Conclusions: By using enzymatically induced hypoxia in a microfluidic environment, presented system allows comprehensive analysis of RBC sickling dynamics in a wide range of deoxygenation rates and conditions. It would find utility in SCD therapy development by offering a robust and high throughput method for assessing critical RBC response to therapies. With no need for cumbersome gas cylinders and low training requirements, a stand-alone system with single use disposable cartridges would also have utility in clinical trials both in the US and abroad, due to simplicity of use suitable for developing countries with limited infrastructure. When validated, the system can be adapted for Point of Care use either as complementary to other blood tests or in an independent monitoring mode.

Evaluation of Manual Hb A1C Determination Methods in Diabetic Patients with Sickle Cell Trait

Background and Aim HbA1c is an irreversible non-enzymatic glycation at one or both NH2- terminal valines of the β –chain. Sickle hemoglobin, or HbS, has a valine for glutamic acid substitution at position 6 of the β chain. The prevalence of sickle cell trait reported to be 2.5% in Basrah city. The hemoglobin A1C (A1C) test can lead to false outcomes resulting in over- treatment or under-treatment of diabetes in people with inherited hemoglobin variants. This study focus on the evaluation of sickle cell trait effect on HbA1c measurement by the minicolumn ion exchange resin methods and compare them with Biorad Variant (HPLC method).the one of methods certified by national glycohemoglobin standardization program (NGSP) Materials and Methods 60 diabetic patients identified with sickle cell trait were recruited in this study .Blood sample collect in EDTA tubes and were assessed for hemoglobin A1c using 3 methods commercially available and most fequently used for routine investigations. Stanbio Glycohemoglobin (Pre- Filâ), HUMAN Glycohemglobin HbA1-Test ( Fast Ion Exchange Resin Separation Method ) ,and the Bio-Rad VARIANT Hemoglobin A1c (HPLC ) method Results Mean age group for patient were 39.6 yrs with their mean blood glucose level of about 203 mg/dl , the average glycohemoglobin was 9.1% measured by Biorad Variant, compare to 7.57% and 7.99% for both Stanbio method and Human Method respectively, which show statistically significant difference (p<0.05). It also had been found that average difference between both manual methods (|Stanbio and Human) and Automated method (biorad variant HPLC) about (1.5, 1.15) . These difference show a significant statistical correlation with hemoglobin S level with r =0.856; p value<0.001. Conclusion This study demonstrate the significant difference in Glycated hemoglobin measurements between different methods commercially available in local markets which had been shown to give false negative results in sickle trait patients when compared to those methods standardized by NGSP system. And recommend that such methods to be avoided in patient with sickle cell trait.

A non‐electrolyte haemolysis assay for diagnosis and prognosis of sickle cell disease

Red blood cells (RBCs) from patients with sickle cell disease (SCD) lyse in deoxygenated isosmotic non-electrolyte solutions. Haemolysis has features which suggest that it is linked to activation of the pathway termed Psickle. This pathway is usually described as a non-specific cationic conductance activated by deoxygenation, HbS polymerisation and RBC sickling. The current work addresses the hypothesis that this haemolysis will provide a novel diagnostic and prognostic test for SCD, dependent on the altered properties of the RBC membrane resulting from HbS polymerisation. A simple test represented by this haemolysis assay would be useful especially in less affluent deprived areas of the world where SCD is most prevalent. RBCs from HbSS and most HbSC individuals showed progressive lysis in deoxygenated isosmotic sucrose solution at pH 7.4 to a level greater than that observed with RBCs from HbAS or HbAA individuals. Cytochalasin B prevented haemolysis. Haemolysis was temperature- and pH-dependent. It required near physiological temperatures to occur in deoxygenated sucrose solutions at pH 7.4. At pH 6, haemolysis occurred even in oxygenated samples. Haemolysis was reduced in patients on long-term (>5 months) hydroxyurea treatment. Several manoeuvres which stabilise soluble HbS (aromatic aldehydes o-vanillin or 5-hydroxymethyl, and urea) reduced haemolysis, an effect not due to increased oxygen affinity. Conditions designed to elicit HbS polymerisation in cells from sickle trait patients (deoxygenated hyperosmotic sucrose solutions at pH 6) supported their haemolysis. These findings are consistent with haemolysis requiring HbS polymerisation and support the hypothesis that this may be used as a test for SCD.

Rétinopathie drépanocytaire : analyse rétrospective portant sur 730 patients suivis dans un centre de référence

Sickle-cell disease is the most common genetic disease in the world, frequently complicated by potentially blinding retinal complications. The prevalence of sickle-cell retinopathy in patients followed in a referral center is presented in this study. The prevalence of proliferative sickle-cell retinopathy by Goldberg classification was determined via a retrospective study of primarily adult SS and SC sickle-cell disease patients and AS sickle trait patients followed in a single referral center for a mean period of 13 years. All patients underwent slit lamp examination and complete fundus examination. Seven hundred and thirty patients (mean age 32.5±10 years), consisting of 492 SS patients (67.4%), 229 SC patients (31.4%) and nine AS patients (1.2%), were included in the study. 54.6% of SC patients and 18.1% of SS patients had grade 3 to 5 proliferative sickle-cell retinopathy. The prevalence of severe forms of sickle-cell retinopathy was higher among SS men than among SS women (21.7% versus 15.5%; P<0.05). The high prevalence of sickle-cell retinopathy and the potentially severe complications associated with this disease justify screening and therapeutic management by a multidisciplinary team in the setting of a referral center.

Increased Red Cell Phosphatidylserine Exposure Correlates with Enhanced Thrombin Generation In Sickle Trait Patients with End Stage Renal Disease

AbstractAbstract 2665 Introduction:Phosphatidylserine (PS) exposure by red blood cells (RBCs) is increased in sickle cell disease, where it is thought to contribute to anemia, inflammation and activation of coagulation. RBCs from subjects with sickle cell trait (SCT) may also express more PS compared to controls. We recently reported a high prevalence of SCT in African Americans with end-stage renal disease (ESRD) - a state marked by anemia, profound inflammation and coagulation activation. No study has yet examined RBC PS exposure, inflammation or coagulation activation in African Americans patients with SCT and ESRD. Objective:To determine whether RBC PS exposure is elevated in ESRD patients with SCT receiving hemodialysis compared to patients with normal hemoglobin genotype, and whether markers of coagulation activation and inflammation are similarly elevated. The relationship between RBC PS exposure, thrombin generation and inflammation was also examined. Methods:We performed a cross sectional study of 10 patients with documented SCT and 9 race-matched controls. Both groups were receiving in center hemodialysis. The percentage of RBCs demonstrating PS exposure was determined by annexin-V binding and flow cytometry detection. Plasma thrombin/anti-thrombin (TAT) complexes and C-reactive protein (CRP) levels were determined by ELISA. Mann-Whitney U tests were used to examine differences between these variables among the groups. Medians and interquartile ranges (IQR) are shown. Spearman's correlation coefficient was used to assess the relationship among the variables tested. Results:Consistent with previous reports in ESRD patients, we found markedly elevated RBC PS exposure among both groups; however, patients with SCT had significantly elevated PS exposure compared to those without SCT (6.65%, [IQR: 5.1, 8.8] vs. 3.0%, [IQR: 2.1,4.2], p = 0.002). Furthermore, patients with SCT had higher TAT levels compared to those without SCT (16.1 ng/L [IQR: 11.9, 28.4] vs. 5.5 ng/L [IQR: 3.8, 8.6], p<0.0001). We also noted increased plasma CRP levels in those patients with SCT compared to control (33.7 mg/L [IQR: 22.8, 62.8] vs. 11.2 mg/L [IQR: 2.2, 19.5], p = 0.016). In patients with SCT, there was a striking correlation between PS exposure and TAT levels (r = 0.897, p<0.0001), but no correlation was noted between PS exposure and levels of CRP (r=-0.17, p = 0.63). Conclusions:These data suggest that patients with SCT trait in ESRD may be subject to an increased risk of thrombotic complications related to increased PS exposure on RBCs. Disclosures:No relevant conflicts of interest to declare.

Con: Exchange transfusion is not required for sickle cell trait patients undergoing cardiopulmonary bypass

SICKLE CELL TRAIT (SCT) is found mostly in those patients of West Indian and/or African-American origin, with an incidence of about 10%. 1 Ohashi Y. Akamatsu T. Hirata T. et al. Cardiac surgery using cardiopulmonary bypass in a patient with sickle-cell trait. Masui. 1996; 45: 1269-1271 PubMed Google Scholar This inherited condition leads to substitution of valine for glutamic acid in position 6 of beta-hemoglobin. In SCT, the typical levels of sickle hemoglobin (HbS) are between 30% to 50%. 1 Ohashi Y. Akamatsu T. Hirata T. et al. Cardiac surgery using cardiopulmonary bypass in a patient with sickle-cell trait. Masui. 1996; 45: 1269-1271 PubMed Google Scholar The author’s contention is that sickle trait patients do not need exchange transfusion before surgery requiring the use of cardiopulmonary bypass (CPB).

HB A2 Quantitation by Microcolumn-Precise Normal Range and Invalidity on the Quantitation in Presence of HBS

The reference range for Haemoglobin A2 (HbA2) by the microcolumn technique is not well defined. Various textbooks and inserts give different reference ranges. This leads to confusion in interpretation. Normal Saudi individuals were checked by the method, excluding all disease in them that can affect HbA2 level. The reference range obtained was 2.0-2.7 (mean+SD). This new range will decrease remarkably the overlap between normal, alpha thalassemia trait and beta thalassemia trait individuals. Also&gt;, and in spite of the claim by the manufacturer to the contrary, it was shown that presence of sickle haemoglobin, places the assay of Hb A2 significantly outside the normal range&gt; using a column that is supposed to be just for the purpose of accurate assay of Hb A2 in presence of HbS. The range in sickle trait patients was shown to be 2.5-3.4 (mean+SD).