Medical Laboratory science

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    Investigating β-Thalassemia Major Cases Emerged After Obligatory Premarital Testing in Palestine
    (Al-Quds University, 2023-11-11) Mais Khader Yousef Al Shatleh; ميس خضر يوسف الشتلة
    β-Thalassemia major is a prevalent autosomal recessive disorder worldwide, affecting approximately 3-4% of the Palestinian population. Management of this condition poses significant challenges, particularly in resource-constrained settings. This study aims to investigate the factors contributing to the emergence of new cases of β-thalassemia major after the implementation of obligatory premarital screening, thereby informing strategies for prevention and intervention. Methods We conducted a cross-sectional case-series study that included all patients diagnosed with β-thalassemia born in or after 2010 in the West Bank. 69 eligible patients from 62 families were included in the study. The data used in this study were collected through a comprehensive questionnaire covering the demographic and medical information of each thalassemic child born after 2010 in the family, familial sociodemographic characteristics, background characteristics of the parents (fathers and mothers), premarital screening, and the knowledge, attitudes, and practices (KAP) of parents (mothers and fathers). In addition, we collected blood samples from 68 children and 62 parents for hematological assessment (complete blood count and hemoglobin electrophoresis). Results The largest proportion of emerging thalassemia cases were from the northern region and resided in rural localities. 71% of the cases were from families married before 2010. 56.5% of the parents reported undergoing premarital screening tests. The proportion of parents who underwent premarital screening differed significantly by type of locality, year of marriage, and age at marriage. In addition, investigating the self-reported results of the premarital screening tests of each couple, we have found that among 24 partners who did not get tested, 22 were married before 2010 and 19 had children with β-thalassemia major. Furthermore, among 12 couples who reported that the two partners were tested and were non-carriers, 4 couples had children with β-thalassemia major, 4 had children with sickle cell thalassemia, and 4 had children with thalassemia intermedia. Overall, the proportion of children with thalassemia major was lower by 20% among parents who married in/after 2010. On the other hand, comparison between the self-reported results of premarital screening and the results of the hematological assessment (mean capsular volume), a total of 24 out of 32 parents had discrepancies in their results. the hematological assessment also showed that 3 out of 62 parents had normal MCV, all of which had high HbS and were parents of children with sickle cell thalassemia. Also, 8 parents had both low MCV and high HbS and one parent had low MCV and high HbC and was a parent of two children with hemoglobin SC disease. Assessment of knowledge showed that all parents had adequate knowledge about thalassemia, 51.7% of the parents had poor overall attitudes, and 76.3% had poor attitude scores towards the termination of pregnancy. Furthermore, 47.4% of the parents had good overall practice scores. Knowledge scores were positively correlated with attitude scores (r=300; p-value=0.01) but not with practice scores (r=0.058; p-value=0.543). Attitude score towards prenatal diagnosis and overall practice scores were also positively correlated (r=0.271; p-value=0.003). Conclusions This study highlights the pressing need for proactive measures to address the prevalence of hemoglobinopathies in Palestine. Findings highlight the crucial role of Health Education and Awareness Programs, aimed at disseminating information about thalassemia, its inheritance patterns, and preventive measures. Additionally, the screening criteria should be revised to include screening for other hemoglobinopathies, and the screening must be performed in Designated Receptions Centers where trained staff perform and interpret the results in accordance with standard laboratory diagnostic protocols. Strengthening pre-marital counseling and screening services is imperative to ensure comprehensive coverage. Early detection, accurate carrier identification, and informed decision-making regarding marriage and family planning should be central in preventive efforts. Continuous training for healthcare professionals and community workers is crucial to effectively prevent and manage thalassemia.
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    Human Parvovirus B19 Epidemiology, Genotyping, Patients’ Hematological Presentation and Clinical Manifestations in Children in Southern Palestine
    (Al-Quds University, 2023-09-03) Miral Munther Badawi Abdo; ميرال منذر بدوي عبده
    Background: Human parvovirus (B19V) is a virus that is usually asymptomatic in healthy patients but can sometimes be symptomatic, especially in patients who have a previous underlying disease such as hematological and cardiopulmonary diseases. B19V targets the erythroid progenitor cells by attaching to the P antigen causing slapped cheek syndrome and may cause transient aplastic anemia. The aim of the study was to check the prevalence of B19V, the most common genotypes, its effect on CBC and the clinical symptoms especially transient aplastic crisis for the first time in Palestine. Methods: B19V was studied in children retrospectively from February 2014 to December 2022 in Caritas baby hospital in Bethlehem. Real-Time PCR for B19V was done on blood EDTA tube for 905 suspected patient samples out of which 28 patients tested positive for B19V (3%). The 28 B19V cases were compared with 32 B19V negative controls. Both groups were checked for social demographics, clinical symptoms, hematological parameters (CBC), and other lab tests (CRP, AST, and ALT). Moreover, genotyping was attempted on all RT-PCR positive B19V cases. This was done by amplifying B19V VP2 gene using VP2 special primers followed by Sanger sequencing. Results: As for social demographics, being a female or a male does not affect the chance of being infected with B19V, in addition to the age of the patient and the place of inhabitant. Regarding clinical manifestations some variables showed significant statistical association with B19V infection such as respiratory problems (p =0.046), malaise (p =0.005), and neurological issues (p =0.043); however, having fever, rash and anemia did not have any statistically significant association with B19V although 2 patients had severe anemia: one patient was diagnosed with pure red cell aplasia and another patient was diagnosed aplastic anemia. A statistically significant association was also found in certain CBC parameters, including WBC (p =0.003), platelets (p =0.018) and Hb (p =0.019, OD=9.6, 95% confidence interval: 1.45,63.5), notably patients with Hb < 10 mg/dl are 9.6 times more likely to test positive for B19V compared to Hb>10. CRP, AST, and ALT had no significant association with B19V, although all the B19V positive patients had CRP above 6 mg/dl. Of the patients analyzed, 40.7% of the cases had previous underlying disease which was significantly associated with B19V infection (p =0.003, OD=10.3, 95% confidence interval: 2 ,52.3), thus individuals with a previous underlying disease are 10.3 times more likely to test positive for the B19V compared to previously healthy patients. Moreover, 37% of the cases had coinfections of B19V and another microorganism. As for genotyping, of the 28 positive samples analyzed VP2 fragment was amplified from 5 samples (17.8%) that had high B19V viral load and the phylogenetic tree revealed that the most common genotype of B19V in Palestine is genotype 1.Conclusion: To conclude, this retrospective case-control study is the first study to check B19V in Palestine which provided valuable insights and more understanding about the virus in the country.
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    Evaluation of Western blot analysis for the detection of COVID-19 infected and vaccinated serum samples against different viral specific antigen preparations
    (Al-Quds University, 2023-01-15) Aseel Basem Sami Eqneiby; أسيل باسم سامي اقنيبي
    مقدمة:فيروس كورونا هوعبارة عن فيروس يصيب الجهاز التنفسي ويطلق عليه اسم المتلازمة التنفسية الحادة (SARS-Cov-2) , ينكون هذا الفايروس من جينوم RNA أحادي موجب الاتجاه (+ ssRNA) حيث يترواح حجمه بين ال 30 و ال 32 كيلو بايت تتضمن مجموعة من البروتينات السطحية والوظيفية , يقوم فيروس كورونا ب احداث المرض والوصول الى خلايا الجسم من خلال ارتباطه بالخلايا السطحية المعروفة ب اسم ACE2 والموجودة في خلايا الرئة , الهدف الرئيسي من هذه الدراسة هو استخدام الانتيجينات المختلفة لفيروس كورونا و التي تم الحصول عليها مخبريا لفحص وجود لاجسام المضادة داخل مجموعة من العينات الماخوذة من مجموعات مختلفة من المرضى ب استخدام تقنية ال Western-blot الطريقة: تم استخدام مجموعة من ال plaques تسمى ال M13 phase plaquesوالتي تحتوي على 12 منطقة من الاحماض الامينية ضد مجموعة من الامصال الإيجابية المجمعة ل مرضى COVID-19, تم استخدام مجموعتان من ال 12 amino acids والبروتينات المصنعة في تحليل ال Western-blot, كانت الاصال المستخدمة في هذه الدراسة ثلاثة أنواع : (20 عينة مصل COVID-19 موجبة ومطعمة ، 10 عينات مصل إيجابية غير ملقحة ، وبعض عينات المصل السلبية). استنتاج النتائج: بشكل عام استخدام phages M13 التي تحتوي على 12 حمضًا أمينيًا تم اختيارها باستخدام الأمصال المصابة بفيروس COVID-19 ؛ ثبت أنه أكثر تفاعلًا مع عينات مرضى ال COVID-19 المحصنة وحتى المصابة وغير الملقحة ، وهذا مقارنة باستخدام مستضدات البروتينات المؤتلف السنبلة ال s protein أو الغشائية ال m protein . وقد لوحظ بوضوح أن هذه الانتيجينات المختلفة لم تتفاعل سوى مع IgG وليس IgM ، مما يشير إلى فائدتها في الكشف عن الحالات التي تحتوي على عيارات عالية من الأجسام المضادة IgG وليس في حالات عدوى COVID-19 الحادة. يمكن أن تؤكد هذه النتيجة على إمكانية استخدام phages M13 التي تحتوي على 12 من الأحماض الأمينية في تقييم إجراءات التطعيم ضد COVID-19.
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    Identification of immunoreactive COVID-19 epitopes based on M13 phage display library
    (Al-Quds University, 2022-08-22) Tamer Shabana; تامر ابراهيم عيسى شبانه
    مقدمة: ظهر فايروس كورونا المستجد في اواخر العالم 2019 حتى يومنا هذا,حيث يعتبر علامة فارقة في تاريخ البشرية الإنسانية, وقد سبب الفايروس العديد من الازمات في العالم وإغلاق كامل مناحي الحياة في كل العالم هذا وقد سبب العديد من الاصابات وحصد الأرواح, حيث اعتبرت منظمة الصحة العالمية هذا الفايروس على انه وباء ويجب على كل دولة في العالم تطبيق الخطط والبروتوكولات الخاصة بها لتفادي اكبر قدر ممكن من الاصابات.يسبب فايروس كورونا المستجد التهاب رئوي حاد سرعان ما يتطور ويسبب حالات تخثر الدم وجلطات تؤدي الى الوفاة وفي خضم ذلك تعتمد الاستجابة ضد الفايروس على مناعة المصاب ومدى تفاعل جهاز المناعة مع الفايروس,تشكل الامصال والاجسام المضادة مركز اهتمام في هذه الدراسة حيث تم اخذ امصال مرضى مصابين في فايروس كورونا من مستشفيات وزارة الصحة الفلسطينية وتم تحديد من خلال تقنية المكتبة الخاصة بفيروس الفاج M13 ماهية وترتيب البروتينات الخاصة بفايروس كورونا المستجد لدى المرضى. طرق البحث: تقنية المكتبة الخاصة بفيروس الفاج M13 ساهمت وبشكل كبير في اكتشاف السطح الخاص بفايروس كورونا المستجد لدى المرضى وذلك بعد المرور بالعديد من الخطوات التأكيدية لوجود الاجسام المضادة في امصال المرضى حيث تم الاستعانة بتقنية الاليزا النقطية والفحوصات المصلية, وبعد ان تم التأكد من وجود الامصال, كان لا بد من وضع الامصال مع المكتبة الخاصة بفيروس الفاج M13 المتواجد داخل بكتيريا خاصة لمضاعفته, بعد ذلك ظهرت نتائج الارتباط مع الامصال وبينت ماهية وترتيب البروتينات على سطح فايروس كورونا المستجد. النتائج: اظهرت النتائج الاولية تكرار احماض امينية في جميع الفاج M13 بكميات كبيرة,تم اخذ هذا الترتيب وترجمته الى بروتين بإستخدام ادوات علمية موثوقة حيث اظهر في نهاية ذلك ان هذا الترتيب من الاحماض الامينة يُظهر ويشير الى نوعين من البروتينات الموجودة على سطح الفايروس المستجد ما يثير الاهتمام ايضاً انه تم تكرار هذا الترتيب بكميات لا يمكن تجاهلها وقد تشكل النسبة الاكبر من البروتينات على سطح فايروس كورونا المتواجد لدى المرضى في فلسطين. الاستنتاج: تُشكل الدراسة للبروتينات لدى مرضى مصابين في البلاد امراً مهماً لمعرفة ماهية وطبيعة الفايروس لمعرفة كيف يمكن للفايروس ان يهاجم جسم المرضى, تكرار الاحماض الامينية بتقنية فايروس الفاج M13 اظهرت نتائج مقبولة علمياً قد تتطور لتُعطي فحصاً خاصاً يكشف عن وجود الفايروس داخل البلاد.
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    Genotyping of Human Erythrocyte Antigens for Safe Blood Transfusion in Thalassemia Patients
    (Al-Quds University, 2022-05-23) Dorgam Muffed Ibraheem Yasin; ضرغام مفيد ابراهيم ياسين
    Management of β-thalassemia is a major challenge, especially in low resource countries. Blood transfusion is the mainstay treatment of patients with β-thalassemia major. However, blood transfusion is associated with several side effects including hemolytic and allergic reactions, iron overload, and transfusion-transmitted diseases. In this study, we assessed the biochemical, hematological, and hormonal parameters, estimated the prevalence of complications, studied the frequency of red blood cell alloimmunization and autoimmunization, and determined the genotype frequencies of blood group systems among transfusion-dependent β-thalassemia patients in the West Bank. Methods This study was conducted using 100 frequently transfused thalassemia patients. The patients were recruited through Thalassemia Daycare Units in five governmental hospitals patients with the highest transfusion frequency were selected for this study. A questionnaire was used to collect data regarding the basic characteristics of the patients. In addition, medical records were used to collect data regarding the complications of thalassemia among the patients. Blood samples were collected from the patients to measure biochemical, hematological, and hormonal parameters, in addition to screening and identification of antibodies and for DNA extraction. DNA samples were genotyped for Rhesus, Kell, Duffy, Kidd, MNS, Dombrock, Colton, Cartwright (Yt), Lutheran, Knops, Deigo, and Vel blood groups. Genotyping for blood groups was performed by sequence-specific primers (SSP)-PCR method. Results A total of 100 patients were included, 51% were males. The mean age among the patients was 21.9±10.9 years. The majority of the patients (60%) were recruited from Al Watani Hospital. The mean pre-transfusion hemoglobin level was found to be 7.89±0.99 g/dL and the mean serum ferritin level was 3670.42±3742.71 ng/dL. The results of liver function tests showed that 32%, 42%, and 34% had elevated ALT, ALP, and AST levels, respectively. Regarding the hormonal results,10% of the patients had subclinical hypothyroidism. The prevalence of growth hormone deficiency was 8%. Also, 8% of the patients had hypocalcemia and 70% had vitamin D deficiency. Elevated glucose levels were found among 15% of the patients. The most encountered complications were arthropathy (44%), hypogonadism (16%), and hepatic failure and delayed growth (7%). The genotyping results of the RHD blood group showed that 88% of the patients were RHD-positive whereas 7% were RHD-negative and 5% had no clear results. The allele frequencies of RHCE alleles were 0.440 and 0.560 for RHCE*C and RHCE*c, respectively, and 0.165 and 0.835 for RHCE*E and RHCE*e, respectively. Unexpectedly, for the Duffy blood group system, the null genotype (FY*02N.01/02N.01) was observed in 46% of the patients and the allele frequencies of FY*01 and FY*02 were 0.195 and 0.345, respectively. Furthermore, the allele frequencies of GYPA*M and GYPA*N were 0.585 and 0.405, respectively, and those of GYPB*S and GYPB*s were 0.275 and 0.725, respectively. The KEL*02, KEL*04, and KEL*07 allele frequencies were high among the patients in this study (0.920, 0.985, and 0.980, respectively). Furthermore, the allele frequencies of YT*A, LU*02, CO*01, KN*01, DI*B, DI*02.04, and VEL*01 were 0.940, 0.990, 0.990, 1.000, 0.980, 1.000, and 0.990. In addition, 2% of the patients had the Vel*01/-0.1 (Vel/Velnull) genotype. The rate of alloimmunization among patients was 8% and the most common antibodies were anti-E, anti-K and anti-D, and anti-C, respectively. The rate of autoimmunization was 5%. Conclusions The management of thalassemia should be based on internationally established guidelines. Understanding the frequencies of the major blood group systems other than the ABO and Rh systems is essential to provide accurate information regarding the local population’s requirements, reduce transfusion-related complications among frequently transfused patients, and facilitate the challenging task of providing antigen-negative blood for patients with multiple antibodies. Phenotyping of patients’ RBCs could have prevented the development of alloantibodies against Rh antigens C, E, and K. Furthermore, accurate testing for weak RhD among donors could also prevent alloimmunization against RhD antigens. The genotype and the allele frequencies observed among the sample of this study revealed several interesting findings that prompt further research. Keywords Alloimmunization, red cell genotyping, iron overload, beta-thalassemia, frequently transfused