Department of Hematology and Immunohematology

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    Assessment of Three Real-time Polymerase Chain Reaction Assays for Diagnosis of Hepatitis B, C and Human Immunodeficiency Virus Type-1 among Blood Donors in Khartoum State-Sudan
    (University of Khartoum, ) Elmoeiz Ali Elnagi Mohammed ; Ahmed Kamal Awadalla Bolad ; Hematology & Immunohematology
    Background: Careful screening of donated blood is mandatory and crucial. The tests that currently used for the detection of HBV, HCV and HIV-1 are primarily serological that use an enzyme-linked immuno-sorbent assay (ELISA). The ELISA test is cost-effective compared to molecular testing as it’s cheaper by 40%, however, it has some disadvantages such as missing of infected individuals during the serological window period and antibody cross-reactivity that may lead to false positive tests. Therefore, molecular techniques are more reliable and sensitive. In addition, molecular techniques have the capability to detect viral nucleic acid which minimizes the danger of transmitting serious diseases through blood transfusion. Objectives: The aim of this study was to assess the efficacy and the analytical performance of three commercial real-time PCR assays for diagnosis and quantification of HBV (DNA), HCV (RNA), and HIV-1 (RNA) in plasma samples from the blood donors of the central blood bank and national health laboratory in Khartoum state. Material and methods: This cross-sectional analytical study was conducted in Elzahrawi medical laboratory and Aliaa specialist hospital laboratory at Khartoum, during the period between 2014 and 2018. A total of 45896 plasma samples from apparently healthy donors were tested serologically by ELISA for the detection of HBV, HCV, and HIV-1. Out of these, 990 blood donors were randomly selected and included in this study. For each of HBV, HCV, and HIV-1, a total of 330 blood donors (including 200 serologically positive blood donors and 130 serologically negative blood donors as control) were tested by three different commercial real-time PCR assays that included genesig® real-time PCR Diagnostic assay, Sansure Quantitative Diagnostic assay, and RealMOD™ Green Real-time PCR Diagnostic assay. The statistical analysis of the data was performed and analyzed by SPSS version 23 using descriptive statistics, linear regression analysis & Pearson correlation coefficient, ANOVA, and post-hoc test. Results: The results of the three real-time PCR assays yielded a broad range of detection for all the three viruses and had specificities of 100%. The analytical sensitivity (the lower limit of detection) of the three real-time PCR assays for HBV DNA used in this study (genesig® real-time PCR advanced detection Kit, Sansure quantitative fluorescence diagnostic Kit, and RealMOD™ Green Real-time PCR diagnostic kit) were 250 copies/ml (56 IU/ml), 500 copies/ml (111 IU/ml), 500 copies/ml (111 IU/ml) respectively. For HCV RNA it was estimated to be <100 copies/ml (<40 IU/ml) for all three assays; and for HIV-1 RNA it was estimated to be 100 copies/ml for all three assays also. These assays showed similar specificity with excellent reproducibility and their results were also in agreement; however, non-significant underestimation of DNA levels could be present in the third assay (RealMOD™ Green Real-time PCR). Conclusion: In conclusion, the three real-time PCR assays gave an excellent agreement for 99.9% of the samples. In addition to that, they demonstrate a very good analytical sensitivity, great reproducibility, 100% specificity, broad linear quantitative range, and proved to be satisfactory for routine laboratory diagnosis of the three viruses (HBV, HCV, and HIV-1).
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    Association of Thrombophilias with Pregnancy Complications in North Kordofan, El-Obied City, Sudan Year 2014.
    (University of Khartoum, ) Elsafi, Siddig Safieldin Mohamed ; Babiker Ahmed Mohammed ; Department of Hematology and Immunohematology
    Background: Pregnancy complications are the leading causes of annual death for thousands women of reproductive age especially in developing countries. During pregnancy and puerperium, many changes affect the coagulation and fibrinolytic systems. Overall balance of haemostasis shifts towards hypercoagulability with growing evidence suggests that the pregnancy adverse complications are associated with inherited and acquired thrombophilias. So far, few similar studies have been conducted in Sudanese population. The aims of this study were to determine and compare the levels of haemostatic variables Activated protein C resistant (APCR), Lupus Anticoagulant (LA), Protein C (PC), Protein S (PS), Antithrombin III (ATIII), Homocysteine, blood coagulation factors (V, VIII, IX) and to examine the inherited thrombophilia Factor V Leiden (FVL), Prothrombin (PT), Mehtylenetetrahydrofolate reductase (MTHFR) in pregnant women with adverse pregnancy complications and healthy age-matched pregnant women. Methods: This is a hospital-based case-control study that included 134 women with one or more pregnancy complications e.g. preeclampsia, placental abruption, intrauterine growth restriction, stillbirth, venous thromboembolism and 125 healthy control group conducted at El-Obied Teaching Hospital, north Kordofan, Sudan over a period of three years (July 2014 to July 2017). Three blood samples (citrated, EDTA, clotted,) each 2.0 ml were collected from the study population. Citrated plasma for coagulation studies was obtained by mixing nine parts of whole blood with one part of 3.2 % tri-sodium citrate. Special assay protocol for an auto-analyzer from Dade Behring Company, Marburg, Germany, BCs was followed. Serum for homocysteine assay was separated and stored immediately at – 20C. Homocysteine assay was done using an Immunoassay analyzer, AXSYM System, from ABBOTT Company. EDTA sample was used for molecular analyses which have been done by strip assay protocol from Vienna Lab. Statistical analysis was performed using the SPSS version 17 unpaired t-test. Results: The (APCR) was increased (Test ratio = 0.88, normal ratio more than 2) in 13 patients with still birth compared to 4 controls (p=0.05) and in venous thromboembolism was increased in 4 patients and one control, the ratio was 0.86 (p=0.06). Acquired hyperhomocysteinaemia was significantly increased in 13 patients with preeclampsia (mean value = 10.2 mmol) and 5 controls (mean value = 7.4 mmol) p=0.01. Lupus Anticoagulant was detected in patients compared to controls as low positive ( 1.2 – 1.5 ratio) with Preeclampsia (5 to 1), Placental abruption (1 to 1), IUGR (3 to 1), Stillbirth (2 to 1 ) and VTE one high positive (> 2 ratio) to one low positive control. Protein C was increased in stillbirth with a mean value of 113% compared to 95% in control (p= 0.004). Protein S was reduced in study population (mean = 42%), normal range (60 -140%). The blood coagulation factors V, VIII and IX were increased relatively in patients. The mean concentrations % in patients compared to controls FV (132 / 114), FVIII (129 / 111) and FIX (115/ 107). Heterozygous mutation was detected in low frequency in patients and controls. FVL=1.8%, PT= 3.7% in patients compared to 0.00 % and 4% controls respectively. Conclusion: The acquired activated protein C resistant (APCR) and acquired hyperhomocysteinaemia were detected significantly in patients compared to control group. Blood coagulation factors V, VIII, IX were elevated relatively in patients than controls. The level of PS was significantly reduced in all patients as well as the control group without complications. The homozygous forms of inherited thrombophilia FVL, PT, were absent in this study. Only one homozygous MTHFR was detected in one patient with preeclampsia who lost her baby later. She had the highest homocysteine level in this study. Further studies with larger number for each criteria, acquired and inherited thrombophilia testing, treatment and prophylaxis definitely will improve the pregnancy outcomes.
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    Association of Il-16 Gene Polymorphism with Ischemic Stroke in Sudanese Patients
    (University of Khartoum, ) Ahmed, Haifa Alhaj Babiker ; Hassan Hussein Musa ; Haematology and immunehaematology
    Background: Stroke is the second leading cause of death after ischemic heart disease; IL-16 is identified as a pro-inflammatory cytokine that is a key element in the ischemic cascade after cerebral ischemia. In this study IL-16 gene was investigated for the first time in Sudanese ischemic stroke patients with special emphasis for detecting the rs11556218 T/G which was reported as more frequent in ischemic stroke. This polymorphism was reported to be associated with a series of inflammatory or autoimmune diseases, including Crohn's disease, allergic contact dermatitis, endometriosis, asthma, Graves' disease, and systemic lupus erythematosus (SLE). Objectives: this study aimed to investigate the association between the IL-16 gene polymorphisms and of ischemic cerebral stroke in Sudanese patients as well as the association of this polymorphism with different ischemic strokes risk factors. Material and Methods: This is a cross-sectional study conducted at the National Center for Neurological Sciences (NCNS), Khartoum, Sudan, during the period of March 2016 to September 2017. Structural questionnaire was designed to collect the demographic and clinical data concerning each participant. Hematological tests including platelets count and D-dimer were conducted. The genomic DNA was extracted from blood samples collected from the ischemic stroke patients and controls, and then IL-16 gene was amplified using polymerase chain reaction (PCR). Thereafter, 42 IL-16 PCR products were sent for sequencing to Macrogen Europe Laboratory and BGI-solutions Hong Kong Co. Ltd. Respectively. The data were analyzed using SPSS version (21), with reference p.value 5% (0.05) was considered to be statistically significant. The descriptive statistics were obtained for quantitative variables while (frequency, percent) for qualitative variables. The independent sample t-test used to test the difference between the quantitative variables among the two groups. The test of goodness of fit and the test of independency using chi-square were performed to test the relationship between qualitative variables. The sequencing results were analyzed using different bioinformatics soft-wares and tools. The obtained sequences, aligned using BioEdit- ClustalW software with a normal sequence from GenBank retrieved from National Center of Biotechnology Information (NCBI) were examined for the presence of polymorphisms. Results: One hundred and ten of ischemic stroke patients were included and 49 apparently healthy individuals were selected as controls. Males constituted 52.7% of the study population. The most affected age group was 65-82 years (48 patients), followed by 48-64 years (32 patients) constituting (44, 29%) respectively (P = 0.000). The studied risk factors associated with ischemic stroke have shown that hypertension and heart disease were significantly associated with ischemic stroke (P =0.000, 0.018) respectively. D.dimer was performed to 54 ischemic stroke patients, 83% have D. dimer ≥0.3 mg/l (P=0.000). High level of D.dimer was detected in 35.6% of hypertensive ischemic stroke patients while 8.9% of patients with heart diseases developed abnormal D.dimer results. The mean of platelets count was 231 for patients and 258 for control group (P = 0.059). The polymorphism rs11556218 T/G was detected in 75% of the patients (50% were having TG allele, GG allele was found in 25%). Mutation in IL-16 gene was associated with ischemic stroke risk factors including D. dimer (OR=3.5) and hypertension (OR=1.6). Moreover, it has significant relationship with ischemic stroke (OR=3). Conclusion: These findings indicated that the rs11556218 T/G can be used as indicator for ischemic cerebral stroke. Also this polymorphism is significantly associated with D.dimer and Hypertension.
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    Risk Factors of Coronary Thrombosis in Sudanese Patients Less than 50 Years Old
    (University of Khartoum, ) Makky, Maysaa EL Hussein EL Tayeb ; Maria Satti ; Haematology and Immunohaematology
    Background: The risk factors for coronary heart disease (CHD) can be classified into conventional risk factors and non-traditional or novel risk factors. Thrombophilia is defined as an increased risk for thrombosis. It is either inherited thrombophilia or acquired thrombophilia. The clinical manifestations of thrombophilia are venous thromboembolism at a young age, thrombosis at different sites and recurrent fetal loss. There is an observed rise in CHD in young people in Sudan in recent years. The aim of this study was to assess the relationship between thrombophilic factors (homocycteine, Protein C, Protein S, Antithrombin III , Factor VIII, body mass index, lipid profiles, and renal profiles) and coronary heart disease in patients younger than 50 years of age. Methods: This is an analytical case control study conducted in Khartoum state on 53 patients with coronary heart disease who are less than 50 years old, and 47 apparently healthy age and sex matched normal adult controls. In this study many expected risk factors were assessed including (protein C, protein S, and antithrombin-III levels, factor VIII, homocysteine, Total cholesterol (TC), Triglysride (TG), High density lipoprotein (HDL), Low density lipoprotein (LDL), Blood urea nitrogen (BUN), Creatinine and body mass index (BMI). Coronary heart disease events included acute coronary syndrome, ST elevated myocardial infarction, non ST elevated myocardial infarction and unstable angina. Results: A family history of coronary heart disease or thrombosis was found in 33% and 23% respectively. Cigarette smokers were 14 of the cases and 2 of the controls (P=0.002) . The mean of TC was 4.5472 mmol/l in cases with SD ±1.15 and 3.6511mmol/l with SD ±0.66 in controls (P = .000), mean of HDL was 0.8685mmol/l ( SD ±0.23) in cases, 0.9791mmol/l (SD ± 0.21) in controls (p= 0.015), LDL mean was 2.7794 mmol/l in cases and 2.0430 in controls withVI ±SD 1.07, SD ±0.63 respectively (p=.000) while the means of creatinine in cases and controls were 49.9528 umol/l (SD ±14.12) and 43.7851 umol/l (SD ±10.11 ) respectively (p=.015 ). All these factors showed significant association with CHD in the studied population (p< 0.05). Both TG and BMI (did not show significant difference. Protein C was low in 9/53 of the cases and 1/ 47 of the controls (P = 0.013). ATIII was low in 3 cases and non of controls (P = 0.109). elevated FVIII was found in 7cases versus 1 of the controls (P = 0.044) and homocysteine was high in 30 of the cases versus 17 of the controls (P = 0.032). protein S deficiency was not detected in cases or controls . Conclusion: Thrombophilic factors especially Protein C deficiency, Factor VIII or homocysteine elevation are causative factors for coronary heart disease. moreover cigarette smoking was found as significant risk factor for CHD in patients younger than 50 years old There were significant difference in the mean of TC, HDL, LDL and creatinine between cases and control.
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    Chronic ImmuneThrombocytopenia and Helicobacter Pylori infection in Sudanese Patients
    (University of Khartoum, ) Eshtyag Albushra Elawad ; Awad Elkareem Abass ; Department of Hematology and Immunohematology
    Background: Immune thrombocytopenia (ITP) is an autoimmune disease mediated by anti-platelet auto-antibodies. There is growing evidence that the eradication of H.pylori effectively increases the platelet count in a considerable proportion of H.pylori- infected ITP patients. This indicates that H. pylori has a causative role in this group of patients.The study aimed to determinesero-prevalence of H. pylori in chronic ITP patients and to compare the ITP characteristics in H.pylori infected and non-infected ITP patients. Method: Sixty ITP patients of at least 6|12 duration were studied. The following tests were doneon all patients: Complete blood count (CBC),blood cells morphology, IgG for H.pylori antibodies and anti platelet antibodies by ELISA, anti lewis antibodies by a serological method. All patients were screened for hepatitis B, C and HIV by ICT. The initial platelets count at diagnosis and bone marrow examination report performed by consultant hematologist were obtained from the patients notes. Results:The ages of the study population ranged between 2-74 years, there were 21 males and 39 females. Fifty seven patients already had corticosteroids treatment. Anti H.pyloriIgG, Anti-platelets, and anti-Lewis antibodies were detected in 63%, 58%, and 28% respectively. The platelet count at the time of diagnosis was significantly lowerthan that at the time of enrolling,( P < .00001). Moreover, a significantly lower platelets count at time of enrolling was found in H.pylorisero-positivethansero-negative ITP patients, (P= 0.00006).In ITP treated patients (57/60) there was a significant rise in platelets count after compared to the initial count,( P= 0.00001). In comparing of ITP characteristics between H.pylorisero-positive and sero-negative ITP patient; there was a significant difference as regard anti-platelets antibodiesstatus,(P= 0.0001), platelets count at time of enrolling(P=0.00006), and corticosteroid response, (P= 0.01). Conclusion: Anti H.pylori and anti-platelets antibodies were frequent in ITP patients. Reduction in the platelet count was observed in H.pylorisero-positiveITP patients more than sero-negative ones. In the majority of H.pylorisero-positive ITP patients; thrombocytopenia still persisted even in those patients who underwent corticosteroids treatment.