PHD theses : Medical Laboratory Science

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A New Simple And Cheap Method For The Eradication Of Transfusion — Induced Malaria In Sudan
(Neelain University, 2004) Mohamed Siddig Mohamed Ali
ABSTRACT Background: Transmission of malaria through blood transfusion is a genuine problem for the capability of plasmodia to survive in stored blood and the weakness of transfused patients. Systemic screening of blood donors is not a practical solution of the problem even by using an advanced technique as well as treatment of transfused patients after transfusion and/or prospective donors before donation. Therefore, an ideal way for such prevention could be to kill the parasite in donors’ blood before transfusion. Objective: To select the antimalarial drug of choice that can be routinely added to donors’ blood in vitro to kill malaria parasites within maximum three days. Material and Methods: Donors’ blood which was collected from 4484 blood donors has been screened for malaria parasite microscopically using Giemsa’ staining technique. Of these samples, only 30 (500ml of blood each) satisﬁed the inclusion criteria of this study. Each of these blood samples was subdivided equally into ten sub-samples to obtain a total of 300 sub- specimens. Three concentrations of each antimalarial drug (chloroquine, fansidar and quinine) were added to 30 specimens while 30 specimens (control) were leﬁ without adding antimalarial drug. Blood specimens were tested for parasite culture, platelets count, total leucocyte count, packed cell volume, lysis percentage, osmotic fragility, prothrombin time, activated partial thromboplastin time, sodium and potassium serum levels simultaneously on the day of collection. Thereafter, it was stored in the blood bank refrigerator (4°-6°C) and tested after 24 & 48 hours by the same laboratory procedures. Results: The reduction of malaria parasites numbers was found to be proportional to the concentrations of chloroquine, fansidar and quinine added to donors’ blood and to the storage period. Whereas, the control donors’ blood samples (without antimalarials) revealed stable number of the parasites even after 48 hours storage. Fansidar was highly effective aﬂer 24 hours storage followed by quinine and 48 hours storage revealed high effectiveness of fansidar followed by quinine and chloroquine. The optimal doses of the applied antimalarial drugs were generally safe to all constituents of the stored blood compared to the control blood samples. Conclusion and Recommendation: It was concluded that for eradication of transfusion induced malaria by in vitro processing of donors blood, fansidar is the best drug that can be used followed by quinine. So it was recommended to apply the optimal doses of these drugs to the components of the blood bags before phlebotomy.
GENOME WIDE SEARCH FOR VISCERALLEISHMANIASIS SUSCEPTIBILITY GENESIN SUDANESE POPULATION
(ALNEELAIN UNIVERSITY, 2005-10) MANAL AHMED MOHAMED AHMED FADL
ABSTRACT Sudan is one of the major foci of visceral leishinaniasis (VL) globally (the other two foci are Brazil and India). Familial clustering and ethnic differences suggest genetic factors may be involved in the infection. In this study a two stages genome-wide scan was employed using two independent sets of families from two villages: El-Rugab and Um—Salala. located 40 kilometers apart in the heart of the endemic area of eastem Sudan. These villages are inhabited by the Masalit ethnic group who migrated from westem Sudan in the 1980s. In the ﬁrst stage (= scanl) we genotyped 400 highly polymorphic microsatellite markers (approximately 10 cM apart) in 220 individuals from 38 multicase pedigrees (= scanl families) comprising 48 nuclear families (1 18 affected sibs). Scan] was followed by grid tightening strategy in which 35 additional markers were added close to regions that gave suggestive evidence for linkage of VL susceptibility in scan 1. These markers were typed in scanl families. ln the second stage (scan2). 20 markers from positive regions of linkage and the 35 additional markers were all typed in family set 2 (=scan2 families: 21 nuclear families, 48 affected sibs). ln scan 1, the multipoint analysis performed provided evidence for linkage of VL susceptibility to 5 regions on chromosome lp22, ]q3l.3. 5q34-35.3, 6q27 and l3q 31.1 (0.0002
Laboratory Diagnosis of Male Infertility among Sudanese Couples
(Alneelain University, 2006) Abdalmula Mohammed Abdalla
ln view of the increasing number of infertility among the couple male present in the Sudan .This study was conducted to essentially study the factors responsible for decreased male fertility, the impact of toxic and antioxidant elements on male reproductive function .and to introduce biochemical markers to be used in male infertility diagnosis . Group of 500 infertile male subjects were enrolled ;15O azoospermic 150 oligospermic patients, 100 patients with abnormal sperm motility and 100 patients with abnormal sperm morphology were studied, and compared with reference group of 100 fertile male. The fertility and thyroid hormones were measured by ELISA and trace elements in semen and serum/blood by AAS. Semen volume and liquefaction time in azoospermic and oligospermic patients was significantly reduced (p< .05 ). Signiﬁcant correlation was noticed between both FSH & LH with seminal volume , and blood Pb with liquefactions time in azoospermic patients .Signiﬁcant correlation was observed between seminal Zn and liquefaction time in both azoospermic and oligospermic subjects.(r < .05). Immunological infertility is higher in patient with abnormal sperm motility (9% lgC-3, 6% lgA) than other test groups. Testosterone in the test groups was significantly reduced , while FSH level was signiﬁcantly increased (p<.O5). Prolactin level was increased in azoospermia, and significantly increased in the other infertile groups (p<.O5). LH was significantly increased in both azoospermic and oligospermic patients (p<.O5) Significant correlation was noticed between ,LH and semen Cd in azoospermia , prolactin was signiﬁcantly correlated with Pb in both blood and semen, and with seminal Mn and Zn, LH with seminal Zn in oligospermic patients. Also correlation between, prolactin with blood Pb and FSH with blood Cd in abnormal sperm motility patients (r<.05), .The thyroid hormones showed conﬂicting levels in the infertile test groups, only T4 was slightly increased. In seminal plasma Cd level was significantly increased in both azoospermic and oligospermic patients (p < .05). Seminal Pb level was slightly elevated in infertile patients with azoospem1ia and oligospermia . Zn level was signiﬁcantly reduced in the infertile groups (p<.O5). Se , Mg & Ca were signiﬁcantly reduced in both azoospermic and oligospermic patients (p<.O5). Where as Mn, Ni, Co,Cr and Cu showed no signiﬁcant variations .ln blood Pb level was increased in the test groups, and Cd level was signiﬁcantly increased in azoospermia (p<.O5), and the other elements indicated conflicting level , _but within normal range when compared with control group. Pb level was significantly correlated in blood and semen of azoospermic and oligospermic subjects,Also Cd level was significantly correlated in both semen and blood of abnormal sperm motility patients (r<.O5). Seminal NAG was significantly reduced in the study groups , while citric acid was reduced in both azoospermic and oligospermic patients (p<.05).Semen fructose indicated conflicting level ,but within normal range .Significant correlation was noticed between NAG with both FSH and LH in azoospermia, and with prolactin and seminal Mg in oligospermia (r<.O5).2.7% of azoospermia had sertoli-syndrome , explained significant decreased in testosterone ,seminal NAG, citric acid Zn, and increased FSH. (P<.O5). 13% of the infertile have varcocele, recorded significant increased FSH,LH and decreased seminal NAG ,citric acid 8- Zn (p<. 0 5). 9.6% of the test groups had erecting and ejaculating problems. Characterized by signiﬁcant increased in prolactin and Mn level.7.2% of the test groups are smokers, explained significant increase of both Cd and Pb in blood and semen, and reduced semen volume, NAG and Zn level (p<.05) Treatment of adult male rats with Cd, Pb caused hair loss and enlargement of the testes. Followed by signiﬁcant increased of blood Cd , blood Pb, FSH and LH (p<.05), decreased testosterone and Zn (p< .O5).With blockage of spermatogenesis at seminiferous tubules level, maturation arrest ,and proliferation of the sertoli cell. Where as treatment with Zn, showed increased number of germs cells and developing spermatide .Mixing of Cd with Pb in one dose exacerbated the toxic action of this elements, caused decreased testosterone and Zn, increased FSH , LH and prolactin with deletion of the germ cell, and proliferation of the sertoli cells . While mixing of Zn with Cd and Pb , reduced the toxicity of this elements on fertility hormones, development of the germ cell, and proliferation of sertoli cells
SERO- DETECTION OF HEPATITICS B, HEPATITICS C AND SALMONELLA MICROORGANSMS AMONG SUDANESE SCHISTOMIAASIS PATEIEN IN GEZIARA
(Neelain University, 2006) MAZIN OMAR MOHAJER
This cross-sectional study was conducted to determine the relationship between schistosomiasis , salmonellosis, Hepatitis B, and Hepatitis C infections in Helate Mahajoub in Gezira Agricultural Scheme . ' Idrine and faecal samples were collected from 288 individuals and‘ inpyestigatcd for Schistosomaova using the modiﬁed Kato thick smear technique and the concentration technique. A serosurvey of 207 subjects was conducted to diagnose S.typlzi, S.paratypI1i A, and S.paratyplzi B using the Widal test technique. Hepatitis B surface antigen (HBsAg) and Hepatitis C antibodies were estimated using the immuno chromato- graphic technique (ICT). The scro-positivity of HBsAg and HCV antibodies were 21.7% and 9.1% respectively; while enteric fever showed a 30.9% sero- positivity. i The association established in this study may be explained by the immune status of the infected subjects studied
Isolation and Identification of Clostridium difficile and Other Clostridium Species Associated with Diarrheic Children and Their Susceptibility to Antimicrobial Agents
(Neelain University, 2007) Susan Abdul-laIeef Shareef
Clostridiznn species is part of the intestinal indigenous microbiota of young children and they can produce several endogenous infections Closlridimn difﬁcile frequently colonizes the "human large intestine when the normal colonic flora is disturbed by antibiotic therapy. The result of colonization may be asymptomatic, or it may lead to illness, ranging from mild diarrhea to pseudo-membranous colitis. C. diﬂiqile is a major nosocomial pathogen, responsible for up to 20% of cases of antibiotic- associated diarrhea in industrialized countries, and is an emerging problem in developing countries. The study concerned 552 children, aged l5 days and 8 years. 351 fecal samples were taken from diarrheic children, and (201) were taken from non- diarrheic patient children carrier of C. diﬁicile who were at risk in whom CDAD may develop after using chemotherapeutic agents. Sixty-two isolates of Clostridizun spp. were characterized by colony morphology on several media, spore shape and position, biochemical tests and GLC technique. They identiﬁed as: Closrridium septicum 5 (8.1 %), C. bifermenlans 1 1 (17.7 %), C. sordell/‘i4 (6.45 %), C. pe/fri/1gens5 (8. l %), C. novyi 8 (13 %), C. botulimmz 10 (16 %), C. sporogenes l3 (21 %), C. telani3 (4.8 %) and C. rerliu/n 2 (3.2 %). One strain of C10slria'izun difﬁcile (1.61 %) was detected on selective medium (CCFA). A total of 202 cooked meat carbohydrates selective (CMC+S) broth inoculated with fecal samples, were tested for detection of Closlridium
Molecular Genotyping of Methicillin Resistant Staphylococcus aureus in Khartoum Teaching Hospital
(Al Neelain University, 2007) Eidha Ali Awadh Bin Hameed
This is a cross—sectional study perfomied to detect the methicillin resistant Staphylococcus aureus (MRSA) isolates collected from Khartoum Teaching Hospital in the period from September 2005 to August 2007 by various molecular typing methods. A total of 48 S. aureus isolates were collected ﬁom clinical wound specimens in wards of surgery, orthopaedic, and burns. The number of MRSA was found to be 9 (l8.75%). The isolates were classiﬁed into 3 groups; group 1: S. aureus isolated from the surgical ward (28; 58.3%), group H: S. aureus isolated from the orthopaedic ward (14; 29.2%), and group IH: S. aureus isolated from the bums unit (6; 12.5%). All strains were studied for their susceptibility to commonly used antibiotics. The results revealed that the drug of choice for the treatment of nosocomial MRSA and MSSA is vancomycin. While multi-drug resistance was observed to be common among MRSA strains. Polymerase chain reaction (PCR) was used to amplify the sequence of S. aureus speciﬁc gene at (107 bp) all isolates. Methicillirt resistant (mec/1) gene yielded the amplicon for size at 1319 bp of 9 isolates, and coagulase (coa) gene produced ampliﬁcation products approximately at 500 bp (26/48), and sso bp (22/48). Two distinct PCR-restriction fragment length polymorphism (RFLP) pattems of coagulase gene exhibited among isolates of S. aureus; coaA and coaB. With Alul restriction enzyme digested, the product fragments were approximately at 190, 310 bp and 190, 390 bp with percentages of 54.2% (26/48) and 45.8% (22/48) respectively.
Isolation and ldentificationof Clostridium difficile and Other Clostridium Species Associated with Diarrheic Children and Their Susceptibility to Antimicrobial Agents
(Al Neelain University, 2007-09) Susan Abdul-lateefShareef
Clostridium species is part of the intestinal indigenous microbiota of young children and they can produce several endogenous infections Clostridium diﬁicile frequently colonizes the human large intestine when the normal colonic ﬂora is disturbed by antibiotic therapy. The result of colonization may be asymptomatic, or it‘ may lead to illness, ranging from mild diarrhea to pseudo-membranous colitis. C. dlﬂicile is a major nosocomial pathogen, responsible for up to 20% of cases of antibiotic- associated diarrhea in industrialized countries, and is an emerging problem in developing countries. The study concerned 552 children, aged 15 days and 8 years. 351 fecal samples were taken from diarrheic children, and (201) were taken from non- diarrheic patient children carrier of C. diﬁicile who were at risk in whom CDAD may develop after using chemotherapeutic agents. Sixty-two isolates of Clostridium spp. were characterized by colony morphology on several media, spore shape and position, biochemical tests and GLC technique. They identiﬁed as: Clostridium septicum 5 (8.1 %), C. bifermentans ll (17.7 %), C. s0rdellii4 (6.45 %), C. perfringens 5 (8. 1 %), C. novyi 8 (13 %), C. botulinum 10 (16 %), C. sporogenes 13 (21 %), C. tetani 3 (4.8 %) and C. tertium 2 (3.2 %). One strain of Clostridium diﬂicile (1.61 %) was detected on selective medium (CCFA). A total of 202 cooked meat carbohydrates selective (CMC+S) broth indcillated with fecal samples, were tested for detection of Clostridium I514 ll 1 VIII .,l it-l 14¢ ll 1- 1» Hr» I I diﬂicile using C. diﬂicile antigen detector (latex agglutination test); 14 (7 %) of them were positive. Fatty acid analysis by gas-liquid chromatography technique was used for detection of isocaproic (iC6) peak in height of Z 2mm, and also valeric and isovaleric acids. We found that 13 of them (93%) yielded height peaks of iC6 acid Z 2 mm, while one of them yielded height peak < 2mm. The isolates were tested against eight kinds of antimicrobial agents included: Metronidazole, Tetracycline, Vancomycin, Naldilic Acid, Trimethoprim, Erythromycin, Kanamycin, Gentamycin and Clindamycine. The inhibition zone diameter showed that the most effective antimicrobial agents was metronidazole, followed by erythromycin and Naldilic acid, while most of species revealed no inhibitory zone against trimethoprim. There were high levels of multi-drug resistance among the clostridia isolated. This may be attributed to the irrational use of these chemotherapeutic agents.
Isolation, Identiﬁcation and Molecular Characterization of Salmonella Species From different sources
(Al Neelain University, 2009-02) Abbas Hassan Mohammed
A total number of 733 samples were collected ﬁom different sources (clinical, food, poultry and water), and subjected to bacteriological examination for isolation of Salmonella spp. using enrichment and selective media. The percentages of the detection of the organism from the above sources were 10.5, 2.9,‘ 2.5 and 0.0 respectively. y From the 200 feacal specimens (100 from human and 100 from animals),'the organism was detected in 20% of the human specimens and only as 1% from the animal specimens. Food specimens revealed isolation of Salmonella spp as 10% and 1.85% from meat and milk specimens respectively, while percentages of 5.1 and 1.6 were obtained from chicken and eggs specimens and the raw shaworma revealed negative growth of the Salmonella spp. i Using the API method for conﬁnnation of the isolates gave unacceptable proﬁles with 66.7%, 44.4% and 66.7% for Salmonella spp isolated from clinical, food and poultry specimens respectively, (in all unacceptable proﬁle organisms the Salmonella spp was the ﬁrst suggested organism). 41.7% of the total specimens showed confirmation of the presence of the organism with identity of 99.9%. The API also showed that there are strong similarities in biochemical reactions of the Salmonella spp K. pneum. Ozaenae, E. coli 2, E. coli 1 and Hafnia alvei. The serological investigation revealed the identiﬁcation of Salmonella anatum from both poultry and food specimens, Salmonella allerton from both poulhy and human specimens and Salmonella enteritidis from both food and human specimens. Salmonella Kentucky, Salmonella albertslunal Salmonella abortus bovis, Salmonella tchad, Salmonella II. Other serotypes were detected in human specimens without detection for the common known pathogenic species of Salmonella typhi and Salmonella paratyphi. Food specimens also revealed the presence of Salmonella okerara, Salmonella Harrisonburg and Salmonella maiduguri. _ l All isolates ﬁom the different sources showed high sensitivity towards Arnykacin, Ciproﬂoxacine, Cefoperazone and Netilmicin antibiotics.‘ Most of the isolated Salmonella spp showed clear resistance toward Augmentin antibiotic. There were clear variations in the susceptibility of Salmonella spp toward different antibiotics according to their origin of source. The PCR product analysis indicated the presence of the gene associated with virulence in two of the two Salmonella enteritidis serovars with plasmid proﬁles containing molecular weights of 26, 424 and 51, 418 kb. The Salmonella enteritidis also showed the presence of virulence gene as single ampliﬁcation product of 460 bp.
ANTIMICROBIAL ACTIVITY AND CHEMICAL CHARACTERIZATION OF DIFFERENT FLORAL ORIGIN HONEY
(Neelain University, 2010) Mahasen Ahmed Wadi Hassan
ABSTRACT Objective: To evaluate the chemical characterization and antimicrobial activity of different ﬂoral origin honey samples against a wide range of bacterial strains , and to verify the nature of the active substance (s) . Methodology: Thirty —two different honey samples collected from different countries (raw and commercial honey) were tested in vitro for antibacterial activity of standard and clinical isolates using cup plate diffusion technique . The same clinical isolates were tested in vii-ro for some antibiotics sensitivity. Different honey concentrations, ethyl acetate extracts and water residue were assessed in vitro for antibacterial activity . Topical dressing of cutaneous leishmaniasis of 25 hamsters with natural honey was compared with other 25 group treated by Pentostam injection. Extraction of honey was carried out by petroleum ether and ethyl acetate. The extracts were subjected to thin layer chromatography Ethyl acetate extracts were analyzed by I-IPLC and GC-MS for chemical Characterization to fingerprint honey samples according to their ﬂoral origin. Results: All bacterial strains exhibited marked sensitivity to honey samples, even the most resistant bacterial strains to the commonly used antibiotics —MRSA and Pseudomonas spp. The clinical isolates showed remarkable resistance to the most antibiotics tested. Different honey concentrations inhibited bacterial growth even at low dilution 10%. Ethyl acetate extracts exhibited strong antibacterial activity while it is water residue showed no activity. Honey was found to be an effective treatment for the cutaneous leishmaniasis induced in hamsters as compared with control group which was treated by Pentostam injection. TLC of honey extracst resulted in different terpenoids and phenolic compounds. Analysis of honey samples by using HPLC and GC-MS revealed different phenolic compounds correlated with their botanical and geographical origin . Different honey samples showed different compounds according to their ﬂoral origin. Natural and commercial honeys samples exhibited potent antibacterial. Conclusion: The findings of the current study suggests that honey would be beneficial to control resistant bacterial strains without side effects. The active fractions (antibacterial of honey )may be avaluable source of future therapeutic. Effectiveness of honey dressing in treatment of leishmanial ulcers gives a promising trend in controlling such cases. GC-MS profile reﬂect the floral source , and forms abasis of an attempt to fingerprint honey samples. Much of the effectiveness of honey as antibacterial activity was attributed to phenolics ﬂavonoids, some of the volatile compounds , hydrogen peroxide normally present in honey due to the effect of some enzyme, the natural concentration and hygroscopic nature of honey, plus the cidic pH of honey. Honey can be used asanatural product without side effects.
Successful isolation, culturing, proliferation and transdifferentiation of stem cells obtained from Umbilical Cord blood and Wharton jelly
(Al-Neelain University, 2011) Hiba BadrEldin Khalil Ahmed Khalil
ntroduction Mesenchymal stem cells (MSCs) and Haemopoietic stem cell (CD34) could be isolated from human umbilical cord blood. In addition to that MSCs like, could also be isolated from Wharton jelly which represent a rich source of primitive cells, with the same properties of cord blood and bone marrow MSCs. Mesenchymal stem cells and haemopoietic stem cells have the capacity of self-renewal, proliferation and in viva or ex vivo differentiation. This study was conducted to compare stem cells from umbilical cord blood and Wharton jelly, focusing on isolation techniques, identiﬁcation and proliferation using various medium, (MesenCult and DMEM), and differentiation in vivo (albino rats) and ex viva (MethoCult medium), transdifferentiation into hepatocyte like cells and expression of CD34,CD45 and CD10. Materials and Methods Thirty female albino-rats age 5-6 week-old; weight 40-60 g, 20 were used for ‘human cells engraftment, whereas the remaining l0 served as negative controls. Twenty to fourty ml of 25 cord blood samples and length of 12 cm of 25 umbilical cords for Whaiton jelly samples were obtained from 25 newborns delivered after full tenn following ethical consent. The cord blood samples were grouped according to the time of collection after birth into three groups. Group one (G1) include samples processed after 5 - 7 hours, group (G2) after 3 - 9 hours and 9-13 hours for group three (G3), whereas Wharton jelly samples were grouped as G1 which include samples processed aﬁer 56 - 93 hours, G2 aﬁer 19 - 24 hours and 42 - 48 hours for G3 and time of trypsin digestion was 45 minutes for G1 and 30 minutes for G2 &G3.Ficoll Paque was used to obtain the mononuclear cells from cord blood samples while trypsin enzymatic treatment was used for Wharton jelly samples. Puriﬁcation of CD34 was done using RosetteSep and EasyScp techniques. The expression and count of CD34, CD45 and CD10 was detected by desired monoclonal antibodies and analyzed using tlowcytometric. The ex viva proliferation of MSCs was performed in DMEM at day 7 for G2 and day 14 for G1, whereas for MesenCult media it was performed at day 14 for G3. The differentiation process was done by CFU-F ﬁbroblast assay and hepatocyte growth factor protocol, and checked by monitored microscopic, immunocytochemistry and micro albumin assay. CD34 positive cells were cultured for 16 days in MethoCu]t media aﬁer which viable cells count, colonies count assays, and calculation of fold expansion and plating efﬁciency were done. In vivo engrattment and cell proliferation was tested using CD34 positive cells in experimental irradiated albino rats (n=l0) and CD34 improved by human MSCs (n=l0) infused by intravenous and intraperitoneal modality. Statistical methods The data was analyzed using SPSS 13, p.value considered statistically signiﬁcant below 0.05. Descriptive statistics (mean and standard deviation) obtained for numerical variables while frequency distribution and percent for categorical variables. Mean differences were tested by t-test or ANOVA. Results The results showed no signiﬁcant differences in the count of mononuclear cells isolated from cord blood and Wharton jelly samples based on mother age, weeks of gestation, length of umbilical cord and time of processing. The results also showed that the MesenCult media gave the best MSCs viable cells count and fold expansion compared with DMEM. According to the incubation time, DMEM produced the best viable cells count and fold expansion in 7 days in contrast to the 14 days incubation. A statistical signiﬁcant difference (p-value = 0.04) was observed when compared the MSCs viable cells count and fold expansion of cord blood group 1 and Wharton jelly group 2 in DMEM. The results also showed that the plating efﬁciency and colonies count of MSCs to form CFU-ﬁbroblast from Wharton jelly samples was higher with no signiﬁcant statistical difference (p-value % 0.2) than MSCs from cord blood samples. The activation of hepatocyte cell like obtained from the Wharton jelly samples was higher compared with MSCs obtained from umbilical cord blood. However, the difference turned to be statistically insigniﬁcant. Although CD10 was expressed by MSCs, we observed no signiﬁcant difference between cord blood and Wharton jelly samples. Although EasySep technique gave the best puriﬁcation count of CD34 and haemopoietic colonies count (CPU-E, BFU-E and CPU-GM) in ex vivo methoCult media, R0setteSep gave the best self renewal, proliferation into CD45 and engraﬁment in albino rats. In addition the combination of CD34 and MSCs gave the best engraftment and the best site for administration was the intravenous one. Conclusions According to the results, Wharton jelly was the best source of MSCs when as compared to cord blood. Regarding CD10 expression, the cord blood was the best compared to Wharton jelly. The best media for stem cell research was the MesenCult obtained from Stem Cell Technologies Company. The best technique for CD34 isolation was the positive selection conceming EasySep technique, although the best in vivo engraﬁment can be obtained by R0setteSep technique. MSCs look to have the same properties of bone marrow stroma, when it is combined with the haemopoietic stem cell (CD34) in case of in vivo engraftment.
INVESTIGATION OF ACTIVATION PRODUCTS IN MEDICAL LINEAR ACCELERATORS
(Al-Neelain University, 2011) Mohammed Khalil Saeed
Abstract Study of induced activity in a medical linear accelerator room was carried out on Clinac 2100C running 15 MV at Maggiore Hospital, Trieste, Italy. Mathematical model to calculate the induced dose rate has been derived and compared to measurements results. Both of experimental method and mathematical model present a good agreement. The experimental method was performed using filter papers and portable spectroscopy. The activation level reached its practical saturation value after a 30 min continuous irradiation, corresponding to 12000 MU at a dose rate of 240 MU/min. The filter paper method was first time used for the purpose of this measurement. Typical radionuclides produced in the treatment room were identified. The results obtained by this new method, consisting of filter paper, can represent a reliable tool. Moreover, the measurements uncertainty using portable spectrometer was decreased and determined to be 6.02%. In addition, the Clinac 2100C has been simulated with Monte Carlo code Geant4 and the neutron fluence, as a function of the neutron energy, has been calculated in the isocenter and outside the treatment room to estimate the equivalent dose to technologist and patients. The ambient dose equivalent for patient and radiotherapy technologist has been reported in this study using the above mentioned methods. The derived data using different field sizes have been used to evaluate the ambient dose equivalent from neutrons to a patient receiving radiation treatment. The maximum of annual ambient dose equivalent present for 15 MV photon beam is about 1.96 mSv for the technologists, in addition to 1.032 mSv/year received by them in the control room. The maximum of ambient dose equivalent received by patients for minimum field size present 1.79 mSv/Gy for 20 MV photon beam. These values represent neglected doses for technologists, but at same time cannot be ignored for patients, where they can represent a risk for healthy tissues and contribute to secondary malignancy insurgence.
Thesis submitted in fulfillment of the requirements of Ph.D. in Medical Parasitology (Molecular-Parasitology)
(Al-Neelain University, 2011) Elamin Abdulkareem Elamin
Abstract Introduction: In regions highly endemic for malaria, the prevalence of placental malaria ranges from 30% to 60% and has been associated with increased risk of adverse infant outcomes, particularly in primigravidae The study was conducted on mothers after delivery to detect hidden Plasmodium falciparum parasite, merozoite surface protein (msp1) & (msp 2) by PCR technique.75 pregnant women were enrolled in the study; the mean age of them was 26.30 ± 7.02. 5ml of venous blood were collected from the mothers after delivery. Materials and methods: Approximately, 5ml of venous and placental blood were obtained from 75 mothers after delivery, attended Omdurman Maternity Hospital, which is one of the largest maternity hospitals in the capital Khartoum. The mean age of the mothers was 26.3±7.02.The study was conducted during the period January to May 2009. Genomic DNA was extracted from peripheral and placental blood samples using modified phenol chloroform technique. The msp-1 allele (MAD20,) and msp-2 allele A1, A2, B1 and B2, P. falciparum primers were used for PCR. The PCR product was analyzed on 1.5% Agarose gel and visualized by gel documentation system after ethidium bromide staining. Results: The results revealed that the overall malaria detection rate in peripheral blood and placental blood using ICT was 10.7%. With the PCR (msp-1 alleles) the detection rate of malaria in peripheral blood was found to be 9.3%, while in placental blood the same technique showed a detection rate of 10.7%. For PCR (msp-2 alleles), the detection rate of malaria in peripheral blood was 12%, while in placental blood the same technique showed detection rate of 21.3% malaria. p The detection rate of the different combination of the positive and negative results in peripheral and placental blood by using msp-1 alleles revealed that the highest rate (84%) was reported among the peripheral negative & placental negative group while the lowest rate (4%) was reported among the peripheral positive and placental positive. The detection rate of the different combination of the positive and negative results in peripheral and placental blood by using msp-2 alleles revealed that the highest rate (72%) was reported among the peripheral negative & placental negative group while the lowest rate (5.3%) was reported among the peripheral positive and placental positive . When msp-1 and msp-2 alleles used in combined way for the detection of malaria parasite in peripheral and placental blood, they revealed that they were identical in 66.7% of examined samples, discordant in 10.7% of the samples, positive in peripheral blood and not in placental blood in 8% of the samples and positive in placental blood not in peripheral blood in 14.7% of the samples. Out of the 53 cases with no previous history of malaria when using the ICT, 14 were found negative for malaria and out of the 5 positive cases with previous history of malaria, 3 were found positive. For the msp-1 used in peripheral blood, out of 52 cases with no previous history of malaria, 16 were found positive for malaria and out of the 6 cases with previous history of malaria, 1 case was found positive for malaria. For the msp-1 used in placental blood, out of the 51 cases with no previous history of malaria, 16 were found positive for malaria and out of the 7 cases with previous history of malaria, 1 case was found positive for malaria. For the msp-2 used in peripheral blood, out of 51 cases with no previous history of malaria, 15 were found positive for malaria and out of the 7 cases with previous history of malaria, 2 cases were found positive for malaria. For the msp-2 used in placental blood, out of 46 cases of no previous history of malaria, 13 were found positive for malaria and out of the 11 cases with previous history of malaria, 4 cases were found positive for malaria. When using the msp-1 for peripheral blood, the result showed that the mean body weight among the negatives was 3.187 and it was 3.1833 among the positives. When using the msp-1 for placental blood the result showed that the mean body weight among the negatives was 3.1967 and it was 3.1000 among the positives. When using the msp-2 for peripheral blood the result showed that the mean body weight among the negatives was 3.1705 and it was 3.3286 among the positives. When using the msp-2 for placental blood the result showed that the mean body weight among the negative was 3.1786 and it was 3.1889 among the positives. Conclusion: In the vast majority of cases, some sequestered genotypes remain hidden, undetected in the peripheral circulation, indicating that analysis of peripheral parasites generates a partial picture of a P. falciparum infection. The cord blood must be collected from the umbilical cord to detected placental P. falciparum infection particularly in primigravidae الخلاصة مقدمة:- فى المناطق التى تنتشر فيها الملاريا بكثافة , تكون نسبة الاصابة للمشيمة حوالى 30% الى 60% وهذا يؤثر على الجنين بصورة مباشرة خاصة فى الحمل الاول . هذه الدراسة على السيدات بعد الولادة مباشرة لاكتشاف اقسومة البروتين السطحى لطفيل المنجلية المتصورة عن طريق تقنية تفاعل البلمرة السلسلى. المواد و الاساليب:- تم جمع حوالى 5 مل من دم الوريد و المشيمة من السيدات (75) موضوع الدراسة بعد الولادة مباشرة وذلك فى مستشفى امدرمان للولادة احد اكبر المستشفيات للولادة فى العاصمة الخرطوم خلال الفترة ما بين يناير و مايو من العام 2009.كان المتوسط العمرى للنساء 7.02 ± 26.3 . تم استخراج الحمض النووى من عينات الدم الطرفية و المشيمية بواسطة طريقة الفينول كلوروفورم المعدلة . كما تم استخدام البرايمرات الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 1و2 ف تقنية تفاعل البلمرة السلسلى , المستخلص من هذه التقنية تمت معالجته بحوالى 1.5 من الجل ومن ثم القراءة بواسطة التصوير الهلامى بواسطة الاشعة بعد تلطيخه بالاسيديوم بروميد. النتائج:- اظهرت نتيجة الفحص للدم الطرفى و المشيمى عن طريق التفاعلات المناعية الكروموتقرافية ان نسبة الاصابة كانت حوالى %10.7. بينما كانت نسبة الاصابة عندما تم استخدام تقنية تفاعل البلمرة السلسلى الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 1 هى %9.3 للعينات الطرفية. بينما كانت النتيجة فى عينات المشيمة هى%10.7 . بينما كانت نسبة الاصابة عندما تم استخدام تقنية تفاعل البلمرة السلسلى الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 2 هى %12 للعينات الطرفية . بينما كانت النتيجة فى عينات المشيمة هى %21.3 . اظهرت النتائج نسب مختلفة للمجموعات من حيث سلبية وايجابية النتيجة عندما تم استخدام تقنية تفاعل البلمرة السلسلى الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 1 حيث كانت اعلى نسبة عندما كانت النتيجة سلبية فى عينات الدم الطرفية و المشيمية وهى (%84). بينما كانت ادنى نتيجة عندما كانت النتيجة ايجابية فى الدم الطرفى و المشيمى وهى (%4). كما اظهرت النتائج نسب مختلفة للمجموعات من حيث سلبية وايجابية النتيجة عندما تم استخدام تقنية تفاعل البلمرة السلسلى الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 2 حيث كانت اعلى نسبة عندما كانت النتيجة سلبية فى عينات الدم الطرفية و المشيمية وهى (%72). بينما كانت ادنى نتيجة عندما كانت النتيجة ايجابية فى الدم الطرفى و المشيمى وهى (%5.3) . عندما تم استعمال تقنية تفاعل البلمرة السلسلى الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 1 و 2 للدم الطرفى و المشيمى كانت النسب , متماثلة فى %66.7 من العينات , مختلفة فى %10.7, كما كانت النسب ايجابية فى الدم الطرفى وسلبية فى المشيمى فى %8, وايجابى فى الدم المشيمى وسلبى فى الطرفى فى %14.7 . فى حوالى 53 من من ليس لهم اصابة سابقة بالملاريا وجد فقط 14 لهم نتيجة ايجابية ومن 5 كان لهم تاريخ اصابه بالمرض ظهر واحد فقط بنتيجة ايجابية. اما عندما تم استخدام تقنية تفاعل البلمرة السلسلى الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 1 للدم الطرفى فمن حوالى 52 من من لم يكن لهم تأريخ بالاصابة بالمرض كان هناك حوالى 16 نتيجة ايجابية وكان فى حوالى 6 من من كان لهم سابق اصابة بالملاريا فقط واحد ايجابى لنتيجة. اما عندما تم استخدام تقنية تفاعل البلمرة السلسلى الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 1 للدم المشيمى فمن حوالى 51 من من لم يكن لهم تأريخ بالاصابة بالمرض كان هناك حوالى 16 نتيجة ايجابية وكان فى حوالى 7 من من كان لهم سابق اصابة بالملاريا اظهرت النتيجة ان عينتين فقط ايجابيتين اما عندما تم استخدام تقنية تفاعل البلمرة السلسلى الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 2 للدم الطرفى فمن حوالى 51 من من لم يكن لهم تأريخ بالاصابة بالمرض كان هناك حوالى 15 نتيجة ايجابية وكان فى حوالى 7 من من كان لهم سابق اصابة بالملاريا اظهرت النتيجة ان عينتين فقط ايجابيتين. اما عندما تم استخدام تقنية تفاعل البلمرة السلسلى الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 1 للدم المشيمى فمن حوالى 46 من من لم يكن لهم تأريخ بالاصابة بالمرض كان هناك حوالى 13 نتيجة ايجابية وكان فى حوالى 11 من من كان لهم سابق اصابة بالملاريا اظهرت النتيجة ان 4 عينات ايجابية . عندما تم استخدام تقنية تفاعل البلمرة السلسلى الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 1 للدم الطرفى لمن كانت نتائجهم سلبية كان متوسط وزن الجنين هو 3.187 اما من كانت نتائجهم ايجابية فكان المتوسط هو 3.1833 .اما عندما استخدام تقنية تفاعل البلمرة السلسلى الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 1 للدم المشيمى لمن كانت نتائجهم سلبية كان متوسط وزن الجنين هو 3.1967 اما من كانت نتائجهم ايجابية فكان المتوسط هو 3.1000 . عندما تم استخدام تقنية تفاعل البلمرة السلسلى الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 2 للدم الطرفى لمن كانت نتائجهم سلبية كان متوسط وزن الجنين هو 3.1705 اما من كانت نتائجهم ايجابية فكان المتوسط هو 3.3286 .اما عندما استخدام تقنية تفاعل البلمرة السلسلى الخاصة باقسومة البروتين السطحى لطفيل المنجليات المتصورة 2 للدم المشيمى لمن كانت نتائجهم سلبية كان متوسط وزن الجنين هو 3.1786 اما من كانت نتائجهم ايجابية فكان المتوسط هو 3.1889
Successful isolation, culturing, proliferation and transdifferentiation of stem cells obtained from Umbilical Cord blood and Wharton jelly A thesis submitted in fulfillment for the requirements of the PhD degree in medical hematolo
(Al-Neelain University, 2011) Hiba BadrEldin Khalil Ahmed Khalil
Abstract Introduction Mesenchymal s tem cel ls ( MSCs) and Haemopoietic s tem cel l ( CD34) coul d be i solated from human umbilical cord blood. In addition to that MSCs like, could also be isolated from Wharton jelly which represent a rich source of primitive cells, with the same properties of cord blood and bone marrow MSCs. Mesenchymal s tem cel ls and haemopoietic stem cells have the cap acity of self-renewal, proliferation and in vivo or ex vivo differentiation. This s tudy was condu cted to compare s tem cells f rom umbilical cord bl ood a nd W harton j elly, focusing on i solation techniques, identification and proliferation using various m edium, (MesenCult a nd DMEM), and differentiation in vivo (albino r ats) and ex vivo (MethoCult medium), transdifferentiation into hepatocyte like cells and expression of CD34,CD45 and CD10. Materials and Methods Thirty female a lbino rats a ge 5 -6 week-old; weight 40 -60 g, 20 w ere us ed for hum an c ells engraftment, whereas the remaining 10 served as negative controls. Twenty t o f ourty ml of 25 cord blood samples and l ength of 12 cm of 25 umbilical cords for Wharton jelly samples were obtained from 25 newborns delivered after full term following ethical consent. XV The c ord bl ood s amples w ere gr ouped a ccording t o t he t ime of c ollection after bi rth into t hree groups. Group one (G1) include samples processed after 5 - 7 hours, group (G2) after 3 - 9 hours and 9-13 hours for group three (G3), whereas Wharton jelly samples were grouped as G1 which include samples processed after 56 - 93 hours, G2 after 19 - 24 hours and 42 - 48 hours for G3 and time of tr ypsin digestion was 45 m inutes for G 1 and 30 minutes for G 2 &G3.Ficoll P aque w as used to obtain the mononuclear cells from cord blood samples while trypsin enzymatic treatment was used f or W harton jelly s amples. P urification of C D34 was done us ing R osetteSep a nd EasySep techniques. The expression and count of CD34, CD45 and CD10 was detected by desired monoclonal antibodies and analyzed using flowcytometric. The ex vivo proliferation of MSCs was performed in DMEM at da y 7 for G2 and day 14 for G 1, whereas fo r MesenCult media it w as performed at day 14 for G3. The differentiation process was done by CFU-F fibroblast assay and hepatocyte growth factor protocol, and checked by monitored microscopic, immunocytochemistry and micro albumin assay. CD34 positive cells were cultured for 16 days in MethoCult media after which viable cells count, colonies count assays, and calculation of fold expansion and plating efficiency were done. In vivo engraftment and cell proliferation was tested using CD34 positive cells in experimental irradiated albino r ats (n=10) a nd CD34 i mproved b y hu man M SCs ( n=10) infused b y intravenous a nd intraperitoneal modality. XVI Statistical methods The da ta w as analyzed using S PSS 13 , p.value considered s tatistically significant below 0.05. Descriptive s tatistics ( mean and standard de viation) obtained f or num erical va riables w hile frequency distribution and percent for categorical variables. Mean differences were tested by t-test or ANOVA. Results The results showed no significant differences in the count of mononuclear cells isolated from cord blood and Wharton jelly s amples based on mother a ge, w eeks of gestation, length of um bilical cord and t ime of pr ocessing. The r esults also s howed t hat t he M esenCult m edia gave t he best MSCs viable cells count and fold expansion compared with DMEM. According to the incubation time, DMEM produced the best viable cells count and fold expansion in 7 days in contrast to the 14 days incubation. A statistical significant difference (p-value = 0.04) was observed when compared the MSCs viable cells count and fold expansion of cord blood group 1 and Wharton jelly group 2 i n DMEM. The results also showed that the plating efficiency and colonies count of MSCs to form CFU-fibroblast from Wharton jelly samples was higher with no significant s tatistical di fference (p-value = 0.2) than M SCs f rom c ord bl ood s amples. The activation of hepatocyte cell like obtained from the Wharton jelly samples was higher compared with MSCs obtained from umbilical cord blood. However, the difference turned to be statistically insignificant. Although C D10 w as expressed by MSCs, w e obs erved no s ignificant di fference between cord blood and Wharton jelly samples. XVII Although EasySep technique gave the best purification count of CD34 and haemopoietic colonies count (CFU-E, BFU-E and CFU-GM) in ex vivo methoCult media, RosetteSep gave the best self renewal, pr oliferation i nto C D45 a nd e ngraftment in albino r ats. In a ddition t he combination of CD34 and MSCs gave the best engraftment and the best site for administration was the intravenous one. Conclusions According to the results, Wharton jelly was the best source of MSCs when as compared to cord blood. Regarding CD10 expression, the cord blood was the best compared to Wharton jelly. The best m edia f or s tem cel l r esearch was t he M esenCult obtained from Stem C ell T echnologies Company. The best technique for CD34 isolation was the positive selection concerning EasySep technique, although the best in vivo engraftment can be obtained by RosetteSep technique. MSCs look t o ha ve t he s ame pr operties of bone m arrow stroma, w hen it is combined w ith t he haemopoietic stem cell (CD34) in case of in vivo engraftment. ملخص الدراسه المقدمه يمكن عزل الخلايا الجذعية الوسيطه و الخلايا الجذعية المكونه للدم من دم الحبل السري البشري والتي لديها القدره على الانتشار ، وتجديد الذات والتمايز إلى خلايا مختلفة في جسم الانسان, كما يمكن التعرف على هذه الخلايا ومميزاتها بالتجارب و البحوث العلميه داخل الانسان او الحيوان او عبر زراعتها في مزارع متخصصه بأستخدام اوساط متباينه . بالإضافة الى دم الحبل السرى هنالك مصدر اخر لهذه الخلايا و هو النسيج المحيط بالاوعيه الدمويه في الحبل السري ويسمى بالوارتون جلي و يمثل مصدرا غنيا للخلايا الوسيطه والتي لها نفس خصائص الخلايا الجذعيه المعزوله من دم الحبل السري ونخاع العظم. هدفت هذه الدراسة للمقارنة بين الخلايا الجذعية الوسيطه المستخلصه من دم الحبل السري والوارتون جلي باستخدام طرق مختلفه للعزل ومن ثم قياس قدرة هذه الخلايا على الانقسام وتجديد الذات و ذلك باستخدام نوعين من الوسائط هما من دم الحبل السري باستخدام تقنيتين CD كما هدفت الدراسه ايضا" الى عزل الخلايا الجذعيه 34 ، DMEM و MesenCult و من ثم قارنت القدره على الانقسام و التكاثر. RosetteSep و EasySep هما 9العينه و طرق البحث T 9T 9دم الحبل السري لاطفال حديثي الولاده و ذلك بعد اخذ الموافقه T 19T 9من T 19T شملت الدراسه 25 عينه دم تراوحت بين 20 الى 40 مل 9T . 9سم T 19T 9 ادنى بلغ 121 T 19T الاخلاقيه من ذويهم اضافه الى 25 عينه وارتون جل اخذت من حبال سريه لاطفال حديثى الولاده بطول 9T 9ابيض تراوحت اعمارهم بين 5 الى 6 اسابيع بوزن يتراوح بين 40 الى 60 جرام , 20 T كما شملت العينه استخدام ثلاثون فأر 1 29،اما ال 10 المتبقيين كانوا بمثابه ضوابط سلبيه. T 19استخدموا لزراعة الخلايا الجذعيه المكونه للدم المعزوله من الانسان T منهم 9 العينه, كما تم T 19T 29تجهيز T 29اء" علي زم ن T 9بن T 19T 9, المجموعه الاولى والثانيه والثالثه و ذلك T 19T 9تم تقسيم عينات الدم الى ثلاثه مجموعات T 9T 19T 29تجهيز T 29اء" علي زم ن T 9بن T 19T 9, المجموعه الاولى والثانيه والثالثه و ذلك T 19T ايضا" تقسيم عينات الوارتن جل الى ثلاثه مجموعات 9من عينات الدم الماخوذه T 19T 9احادية النواة1 T 19T 19استخدام محلول الفيكول لعزل الخلايا T 9كما تم T . 9 ووقت حفظ العينه في انزيم التربسين 1 T 19T العينه .29T 29 و استخدم انزيم التربسين لعزل الخلايا الشبيهه بالخلايا الوسيطه في عينات الوارتون جل T من الحبل السري XIX 9تم زراعة الخلايا الجذعيه الوسيطه المعزوله مسبقا" ف ي وس ط الدلبكو لفترة حضانه 14 يوم للمجموعه الاولى و 7 ايام T 9وقد تم ايضا" زراعة الخلايا في وسط المزنكلت لفترة حضانها 14 يوما للمجموعه الثالثه و ذلك لمعرفة T.12T 129T للمجموعه الثانيه قدرتها على الانقسام و الانتشار و التحول الى مستعمرات الفايبروبلاست. اما طرق تنقية الخلايا الجذعيه المكونه للدم فهي 9T 1 1T2T.2T 19TEasySep29T و1 RosetteSep 9T 2المخصصه لكل كاشف و تحليلها عبر T 19T 29الاجسام المضادة T 9الكواشف المناعيه ( سي دي 45 و 34 و 10 ) تم استخدام T 19T لمعرفة جهاز الانسياب الخلوي. اما عن معرفة الخلايا الجذعيه المكونه للدم, ومقدرتها على انشاء المستعمرات المكونه للدم فقد تم استخدام مزرعة الميزوكلت لفترة حضانه 16 يوما". 9شملت الدراسه عينة الفئران التي تم حقنها عبر وريد الذيل( 9 فئران) و جدار البطن ( 9 فئران) لاختبارمقدرة الخلايا المكونه للدم T الي الوصول الى النخاع و البدء بالانقسام, كما تم ايضا حقن بعض هذه الفئران بخلايا جذعيه مكونه للدم مدعومه بخلايا جذعيه 9T وسيطه.1 النتائج أسابيع T 9T الأم ،و عدد T 9T وفقا لعمر T 9T او الواتون جلي T 9T دم الحبل السري T 9T عينات T 9T المعزولة من T 9T الخلايا T 9T في عدد T 9T اختلاف كبير T 9T 9لم يكن هنالك T مزرعة المزنكلت اعطت نتائج لاعداد الخلايا الجذعيه الوسيطه مقارنة بمزرعة الدلبكو. أفضل عدد T 9T . معالجة العينه T 9T ووقت T 29T،29T الحمل أن T 9T أظهرت النتائج T 9T، اضافة الى ذلك T 9T. للخلايا الوسيطه تم حصده كان من مزرعة الدلبكو التي كانت فترة حضانتها 7 ايام بنسبه اعلى في وسط مزرعة المزنكلت مقارنة بخلايا دم الحبل T 9T المستعمرات T 29T 29عدد T المعزولة من الوارتون جيلى اعطت T 9T الخلايا 9كما اظهرت النتائج نجاح الخلايا الجذعيه الوسيطه و المكونه للدم في التحول الى خلايا شبيه بخلاي ا الكبد تملك نفس T.2T 29 يT السر دم T 9T 9مقارنة بعينات T لبروتينات الكبد T مقدرات الخلايا الكبديه , حيث اعطت الخلايا الجذعيه المعزوله من الوارتون جلى اعلي نسبه9 طريقه لعزل الخلايا المكونه للدم و اعطاء افضل عدد من T 9T أفضل T 9T 9 هي TEasySep9T 9T 9طريقة T كانت T 9T 9 ومن ناحية أخرى T.2T 29T الحبل السري للفئران كانت انجح من T 9T التي تم حقنها RosetteSep2T 9T 29. كما ان تطعيم الخلايا المكونه للدم المعزوله ب T المكونه للدم T 9T المستعمرات ايضا افضل النتائج كانت بعد الحقن الوريدي للخلايا المدعومه بالخلايا الجذعيه الوسيطه . EasySep خلايا الخلاصه وفقا للنتائج , فأن الوارتون جل يعتبر افضل مصدر للخلايا الجذعيه الوسيطه . في المقابل كانت الخلايا الجذعيه الوسيطه .CD المعزوله من دم الحبل السري قد اعطت افضل ظهور لمستضد ال 10 XX عل ى الرغ م م ن ا ن تقني ة ,(CD هي أفضل تقنية لعزل الخلايا المكونه للدم ( 34 EasySep اثبتت الدراسه ان تقنية قد اعطت افضل النتائج داخل الفئران وفقا لمعدل انتشار الخلايا المكونه للدم و عدد المستعمرات.
P.falciparum Immune Evasion Mechanisms: Possible Roles For Allelic Polymorphism and Antigenic Variation In Sudanese Isolates.
(ALNEELAIN UNIVERSITY, 2011-07) Habab Merghani Yassin Babiker
Abstract Genetic diversity within P. falciparum and the continuous variability of the parasite antigens, makes malaria an important global threat and it the reason behind failure to produce an effective vaccine against malaria. Objectives: This study aimed to determine and characterize Plasmodium falciparum genotypic variations and to detect probable genetic diversity of genotypically identical Pfalciparum isolates in Sudan and to determine whether there is any association between the presence of speciﬁc MSP-1 antibodies and the carriage of multiple P. falcinarum infections. Study design: It was cross-sectional, hospital-based and experimental study that was carried out in four localities, Kasab Rural hospital, Eddouiem, Gazira and Khartoum. One hundred and eighty six P. fulciparum infected patients were enrolled in the study. Methods: Nested polymerase chain reaction was carried out to genotyped study isolates using P. falciparum merozoite surface protein (PIMSP-1) gene speciﬁc primers. Genetic diversity of all isolates was characterized by random ampliﬁed polymorphic DNA (RAPD) analysis using thirteen arbitrary oligonucleotide primers. RAPD was also used to study the polymorphism of different isolates before and after in vitra cultures. ELISA test was performed to determine the prevalence of lgG antibodies against four rMSP-1 fragments (C-terminus, WELL, K1 DI & K1 Dll). Results: The |gG seroprevalence was 85.2, 23.8, 23.9 81 35.2 % to MSP-1,5, WELL, K1 DI & K1 Dll respectively. There was a gradual increase in overall antibody prevalence reaching a peak in adolescence and decrease thereafter. The difference was only age»speciﬁc for the C-terminal fragment. The three reported families of msp-Ialleles (K1, MADZO and R033) were observed among the studied isolates. The predominant allele was of Klvariant while R033 was less frequent. The majority of the patients had multiple parasite clones and multiplicity of infection (MOI) was found to be 1.82, this MOI was found to be higher in Kasab, Eddouiem and Khartoum and was signiﬁcantly associated with age and having malaria symptoms, but not with parasitemia and ﬁequency of infections. Patients with multiple infections had higher prevalence of antibodies to all antigen fragments studied, compared to those with single infection, but this was only signiﬁcant for MSP-119. RAPD primers recognized marked polymorphism and diﬁerent proﬁles among isolates with a .laccard’s Similarity Coefﬁcient ra.nged from 0.25 to 0.80. Different MSP-1 alleles showed different RAPD banding patterns, no certain genotype was found to be unique a certain banding pattem. Forty nine isolates were successfully in vitra cultured. RAPD analysis revealed that only 29 isolates showed different banding patterns before and after in vitro culture Conclusion: P. falciparum isolates ﬁ'om different parts of Sudan were genetically diverse and most of the infections were mixed with a high level of multiplicity of infection (MOI). This MOI extend the duration of the infection and delay the acquisition of protective immunity, making it diﬁicult to produce effective vaccines against these parasites and this is in turn increase the burden of the disease and poverty. Further longitudinal large size studies, including protein analysis and mono-clonal antibodies to identify functional antibodies are needed to evaluate the impact of this polymorphism on the immune response and in tum help in developing effective vaccine against malaria
Assessment of mosquito fauna and mosquito borne parasitic diseases in Merowe dam area
(Al Neelain University, 2011-11) Mohammed Medani Eltayeb Abdullah
Introduction Merowe dam is located in the Northern State of Sudan across the River Nile. It is situated between 16°-32° N and longitudes 30-32°E. The dam is a hydroelectric and irrigation scheme that may probably develop climatic and environmental changes which might have profound impact on the vectors and vector borne diseases in the area not only by the echo changes but also through the new labors and settlers. The present study aimed to identify the mosquito fauna in Merowe dam area, with special emphasis on seasonality, density, diversity, and distribution. The study aimed also to study the distribution of each Plasmodium species in the population of the area and to investigate the possibility of the presence of Wuchzmria hancroﬂi prevalence in the mosquitoes and population as well. Material and Methods: A total of 260 volunteers (64.2% of them were females; age ranged between 10 and 92 years) were selected by systematic random sampling method. Demographic and socioeconomic data were obtained using predesigned structured questionnaire. Blood samples were taken on filter papers for screening of Plasmodiimz spp and Wucheraria bnncrofti DNA using real time PCR. Adult mosquitoes were collected, during the dry and the wet seasons, using Madani’s Electric Trap (newly invented) for outdoor collection, whereas knock- down technique and Modiﬁed Medani's Aspirator were used for the indoor mosquito collection. Of these, 800 mosquitoes were identified as Culex (700 were Cx. qlainquefaciatus, 60 were Cx. pipiens, and 40 were Cx. univetatas), and examined, in pools of 20 samples each, for identification of Wucheraria bancrofti using real- time PCR. The remaining of the adults mosquitoes were used for the characterization of the mosquitoes Fauna in the area. Other 300 Culicinae mosquitoes were identiﬁed as fed-mosquitoes after eye inspection of the abdomen. Blood samples were collected from the abdomen of each fed-mosquito and processed for identification of its origin (human / un- human) using FOB methods. The larvae were randomly collected using dippers, larvae nets, and pipette. Some of the collected larvae were reared in emergency cages for further confirmation. Standard keys including software keys were used for adult and larvae identification. Results The analysis of the genomic DNAs obtained from 260 healthy volunteers, were positive for Plnsmodium spp in 135 cases (51.9%). Of these, P. falciparum spp was seen in 121 (89.9%), while P. vivrzx in only 2 cases (1.5%), and mixed infection was seen in 12cases (8.9%). The PCR analysis showed positive Wuchemrirz bancrofti in 3 cases out of 260, whereas the analysis of the 800 Culex mosquitoes showed the presence of Wucheraria bancroﬁi in 2 pools of Culex quinquefacintus. The result showed that Anopheles umbiensis, Culex quinquefncintus Cx. univittutus, Cx. Pipiens were the common species in the study area with variable prevalences with no significant variation . Blood meal analysis during dry season showed that 91.1% of Culex quinquefasciatus, and 75% of Culex pipiens were anthropophagic. During the wet season, the results showed 89.7% Culex quinquefasciatus, 88% Culex pipiens, and 100% Culex univittntus. Conclusion An. Dthali and Aedes agypti were identified in the area for the first time. The results of the study showed that An. arahiensis is the predominant anopheline species, whereas Cx. quinquefasciatus is the predominant Culicinae spp. Plasmodium falciparum is the predominant species detected in the study population, P.vivax has been identified for the first time including mixed infection with P. falcipurum in 8.9% of the study population. Wucheraria brmcrofti has been detected for the first time in both human subjects and Culex mosquito. Malaria positive cases were less among people who use a mosquito net and there is also a positive relation between fever and malaria detection. The above results strongly suggested slight, but evident, changing in the mosquito Fauna, arrival of new type of mosquitoes and their borne parasitic diseases that may require control preventive measures in the future.
Molecular Characterization of Breast Cancer in Sudanese Women Using PvuII, Xbal, and Hinfl Single Nucleotide Polymorphisms of ESR1 Gene
(Neelain University, 2012) Khalid Mohammed Adam Abd Allah Abo-Albasher
Abstract Background Breast cancer (BC) is the most frequently diagnosed and leading cause of cancer deaths among females world wide. It has been identiﬁed as the most common malignancy among Sudanese Women, accounting for 34% of all female cancers. The prolonged exposure to estrogen is well established risk factor for breast cancer. The effect of estrogen hormone on target tissues is mediated by its alpha receptor, through the binding of the hormone to the receptor Which in turn promote the proliferation and differentiation of mammary tissues. The estrogen receptor alpha is coded by ESRI gene which is genetically polymorphic. Aim The aim of this study was to evaluate the association between (Pvull, Xbal, and Hinfl) single nucleotide polymorphisms of the estrogen receptor alpha gene and BC risk, types, estrogen receptor status, and other risk factors such as mammographic density, family history, age, parity, and age at menarche, in Sudanese BC Women. Materials and Methods In an analytical case-control study, the three single nucleotide polymorphisms were genotyped in 139 BC cases and 139 age-matched breast cancer-free controls. A specimen of 3 ml of peripheral blood was drawn from each subject, from which the leukocyte DNA Was extracted using a modified salting out method, the purity and quantity of the extracted DNA was then assessed spectrophotometerically. The genotyping of the three SNPs was carried out by PCR-RFLP method using Pvull, Xbal, and Hinﬂ restriction enzymes, the products were then separated electrophoretically. The estrogen receptor statuses for 65 cases were determined on a freshly obtained breast tissues that were ﬁxed in formalin and embedded in paraffin according to standard histological procedures, the sections prepared are then stained using immunoperoxidase staining method. The mammographic densities of cases and controls were determined by assessing the mammogram of every subject by a radiologist and mammographam specialist independently. Data obtained were analyzed using SPSS, to calculate the frequency distribution for age, marital status, parity status, menopausal status, and age at menarche. The associations were calculated using chi square test. The unadjusted and adjusted logistic regression were used to calculate the odds ratio (Ors) and 95% conﬁdence intervals (CI) for assessing the association between a genotypic and allelic frequencies and BC. Hardy-Weinberg equilibrium online calculator was used to determine the deviation of genotypic frequencies from Hardy-Weinberg equilibrium. Results The results of BC cases showed a young mean age 46.5il0.4SD years, high frequencies of married women 97.1%, and parity 89.9%. The family history of breast or other type of cancers in Sudanese women, proved to be the strongest risk factor as compared to all factors such as age, mammographic density, age at menarche, parity, marital status, and menopausal status previously believed to be associated with BC, as those have family history of the disease showed an 11 folds increase in the risk of developing BC (OR: 11.8, 95%CI: 4-34.2, P-value .000) as compared to those have no family history. Breast cancer among Sudanese women also showed a higher frequency of the aggressive invasive ductal carcinoma (70.5%), with higher percentage of receptor positive status (63%). The results of minor allele frequency showed an association between the G allele of the Xbal SNP and the risk of BC (P-value = 0.014). Of all the three studied SNPs, rs9340799 (X12411) was the only one to show statistically signiﬁcant association with the risk of BC (P-value I 0.03), where the calculated odds ratios (OR) and 95% conﬁdence intervals (95%CI) using unadjusted logistic regression of the genotypic variants of this SNP showed that Xx genotype had (OR:l.6, 95%CI : 0.85-3.04), and xx genotype (OR= 2.6, 95%CI:l.23-5.46), while using the adjusted logistic regression, the Xx genotype showed (OR= 1.02, 95%CI: 0.75-1.4) (P-value = 0.000), and xx genotype showed (OR= 1.65, 95%CI: 0.95-2.87) (P-value = 0.01). The effect of xx genotype was evidently seen in parous women (OR=2.3, 95%CI:l.l-5.01) (P-value = 0.03). The association of genotypic variants of different SNPs with estrogen receptor status, showed a statistically signiﬁcant association between Xx variant of (Xbal) with positive receptor status (OR: 5.3, 95%CI: 1.8-15.8) (P-value = 0.002). The other two SNPs (Pvull and Hinfl) showed statistically signiﬁcant association only with mammographic density (P- value =0.000 and 0.01 respectively). Conclusion In conclusion, the ﬁndings of this study provide a persuasive indication of strong association between the homozygous recessive and heterozygous genotypes Xbal SNP and risk of BC in Sudanese women, and no clear association between the genotypic variants of the other two SNPs, namely, Pvull and Hinﬂ with the risk of developing the disease.
Assessment of Quality Assurance for Medical Laboratory Services in Sudan
(Neelain University, 2012) Siddig Bushra Mohamed
Abstract Clinical laboratory errors may arise mainly from the lack of awareness and adoption of the quality assurance program. Today, quality assurance is essential to meet the needs of clients and customers satisfaction (patients, clinical personnel and researchers), those who responsible for the care of those patients. This study was conducted in eight Sudanese states (Khartoum, Gazira, North Kordofan, White Nile, River Nile, Red Sea, Gadaref and Kassala state), during the period from 2009-2012, aimed to assess the current situation of quality assurance and factors affecting laboratory proﬁciency, as well as to identify gaps for implementing quality assurance program, of the international standard ISO 15189. in the ﬁrst experiment (phase one), 130 laboratories were randomly selected, and 520 samples of pathological lyophilized control sera well prepared were distributed to assess the performance to measure 4 parameters (Glucose, Urea, Uric Acid and Creatinine) as routine blood chemistry assay. (5 types of reagents were used, R (1, 2, 3, 4 and 5) and 3 types of equipments (fully automated, semi-automated and manual machine) in deferent laboratory levels A, B and C and deferent laboratory sectors Governmental, and Private based on the standard deviation index SDI (Z score) evaluation method. While in the second experiment (phase two) 200 samples of control sera were distributed in 6 laboratory Governmental and private, level A, B and C using the same reagents and equipments and every sample was tested 20 times. The results in phase one showed that poor perfomance was noticed as in glucose only 31 laboratories have succeeded (24%), while 99 have failed 76%. In Urea 56 laboratories were succeeded 43% while 74 were failed (57%). ln Uric Acid 33 laboratories were succeeded (25%) while 97 failed 75%. In Creatinine 71 laboratories succeeded (55%) while 59 were failed 45%. The implementation of full (IQC) 19%, full (EQA) 11%, full (SOPs) 8%, organizational chart in the laboratory l7%, Job description of laboratory personnel 3%, storage reagents at optimum temperature 15%, calibration of equipments 5%, sample rejection criteria 5%, request form including clinical remarks 3%, quality control sera in every run l2%, computer software for reporting archiving results 23% system of critical value 6%, enough laboratory space ll% air condition inside the laboratory 20%, waste disposal managements 5% and immunization of personnel against HBV & HCV 4%. Moreover, the second experiment (phase two) showed reagent (4) result an out of control reading in glucose and urea level compared with the other reagent which were showed variant readings which will considered clinically signiﬁcant. Low level of quality assurance and poor performance of laboratories were evident in selected states, in order to obtain reliable and accurate results.
Systemic Lupus Erythematosus Reactive Antibodies and Vitamin B1; Deﬁciency in Sudanese Psychiatric Patients
(Neelain University, 2013) Ezeldine Khalafalla Mohamed Abdelhabib
The contribution of vitamin B12 and folate deﬁciencies, as well as the systemic lupus erythematosus (SLE) to pathophysiology of psychiatric illnesses is well known worldwide, however it was not evaluated in Sudanese psychiatric patients. This study aimed to assess the association between the neuropsychiatric syndromes and the levels of both vitamin B1; and folic acid and SLE auto-reactive antibodies in Sudanese psychiatric patients. This study was a case control hospital based study, conducted during the period ﬁ'om December 2009» - November 2011 in the three major psychiatric hospitals in Khartoum state — Sudan, namely, Altigani Almahi psychiatric hospital, Taha Bashr psychiatric hospital and the psychiatry unit of Khartoum teaching hospital. -The study involved two groups: a test group of 100 psychiatric patients selected ﬁom the above-mentionedvhospitals and age/gender matched control group of 100 subjects with no past history of psychiatric illness. The medical history and clinical status of the patients were assessed by well trained psychiatric physician. Laboratory investigations, including a complete blood count (CBC), serum vitamin B1; ,serurn folate, anti nuclear antibodies (ANA), anti-double
Human Papilloma Virus in the Etiology of Head and Neck Cancers in Sudan; ‘ An immunohistochemical and molecular study
(Neelain University, 2013) Saadalnour Abusail Mustafa Bilal . -
Abstract Background Head and neck cancers (HN) are considered as ‘the sixth most common cause of death worldwide. In Sudan, HNCs represents 13.5% of all cancers. Tobacco_ use and alcohol consumption are the main risk factors for head and neck cancers. Other risk factor includes the viral infection, paiticularly with Human papilloma virus HPV which is responsible of about 20% of oral cancers, 70% of oropharyngeal cancers and 8% of other head and neck cancers. Aim _ The aim of this study was to investigate human papilloma virus in the etiology of head and neck cancers in Sudan, using immunohistochemistry and molecular methods. Material and methods This is a retrospective descriptive study conducted at Khartoum State during the period from January 2010 to December 2012. Two hundred tissue blocks were retrieved from different histopathology laboratories in Khartoum State. Of the 200 formalin ﬁxed paraffm wax processed tissue blocks, 150 were obtained from patients diagnosed with head and neck cancers and 50 samples were obtained ﬁom patients diagnosed with benign head and neck lesions. Infection with HPV was initially determined using pl61NI“A as a biomarker immunohistochemistry (IHC) methods, and then genotyping was subsequently assessed applying PCR. Tumor DNA was ampliﬁed using polymerase chain reaction (PCR) with high risk human papilloma virus consensus and multiplex primers. The expression of epidermal growth factor receptors (EGFR), P53 and CD5 as tumor markers were assessed in HNCs as well as in benign head and neck tumors using irnmunohistochemistry. The mean count of nucleolar organizer regions (NORs) was determined in head and neck cancers and benign head and neck tumors using silver impregnation method. Results Analysis of demographical characteristics showed that the age of the study population was ranging ﬁ'om 9 to 85 years with a mean age of 51 years. The male female ratio was 1.67:1. Approximately, 96% of HNCs were squamous cell carcinomas and 4% were adenocarcinomas. HPV was detected in 31/200 (15.5%) cases of head and neck tumors (28 (14%) cases malignant and 3 (1.5%) cases benign). Statistically, -HPV was signiﬁcantly associated with head and neck cancers, the p-value=0.032. HPV was detected in the age group 31-40 years. According to relation between HPV and lesion sites, the most affected sites were oral cavity, esophagus and larynx. The genotyping of HPV with PCR revealed the presence of high risk human papilloma virus HRHPV in six cases of head and neck cancers, including 3/6(50%) cases in the oral cavity and 3/6(50%) cases in the larynx. The detected I-IRHPV were type 16, 18 and 33. The expression of epidermal growth factor receptors EGFR was found in 84% of HNCS and 12% of benign head and neck tumors. Statistically there was signiﬁcant association between EGFR and head and neck cancers, the P-value= 0.000. In regard to the relation between P53 and head and tumors, we found that P53 was expressed in 19.3% of HNCs and 4% of benign head and neck tumors. Statistically, P53 was signiﬁcantly associated with HNCs, the P-value=0.009. CD5 expression was only found in 14.7% of HNCs and 8% of benign head and neck tumors. Statistically no signiﬁcant association between CD5 and HNCs. P-value=0.225. The association between nucleolar organizer region mean count (NORs) and head and neck cancers was statistically signiﬁcant. P-value=0.000, but no signiﬁcant association between (NORs) mean count and P16 expression in head and neck cancers. P-value=0.190. Conclusion In conclusion, the ﬁndings of this study provide strong association between HPV and head and neck cancers, particularly oral, esophagus and laryngeal cancers amongst Sudanese patients. On the other hand, when the conﬁrmation of the diaghosis of head and neck squamous cell carcinoma is required, the application of EGFR immunohistochemical method should be considered. The -use of AgNORs mean count analysis in head and neck tumors is recommended in Sudan, particularly for low-income patients.
Prevalence of Inherited Thrombophilia for the development of Thrombotic Cerebrovascular Accidents (CVAs) in Sudanese patients
(Neelain University, 2013) ABDELGADIR HAG ELAGIB OMER
English Abstract Prevalence of Inherited thrombophilia for the development of thrombotic Cerebrovascular Accidents (CVAs) in Sudanese patients Background: Cerebrovascular accidents (CVAs) represent a major cause of death and disability among middle-aged people. Cigarette smoking, alcohol intake, hypertension, diabetes mellitus, hyperlipidemia and heart disease are known predisposing factors for the development of thrombotic Cerebrovascular accidents (CVAs). In industrialized countries, one in six patients die in the ﬁrst months following ischaemic stroke, and half of survivors are permanently disabled despite best efforts to rehabilitate them and to prevent complications. Optimization of the early, and ongoing, management of patients with acute ischaemic stroke is pivotal to the reduction of both case fatality and long-term disability. Thrombotic cerebrovascular Accidents (CVAs) are an important cause of morbidity and mortality and marked disability in Sudanese patients, but data about possible causes is lacking here and in other developing countries. Fifty percent of stroke patients presenting to El-Shaab Teaching Hospital, the central referral neurology hospital, are <50 years. Patients with thrombotic cerebrovascular Accidents (CVAs) commonly end up with major disabilities (paralysis) so they became handicapped and consequently non- productive. A hypercoagulable state (thrombophilia) is an important cause of CVAs in patients <50 years. Presence of a group of auto-antibodies known as antiphospholipids (Lupus Anticoagulant and Anticardiolipin antibodies) together with deﬁciency of natural anticoagulants (PC, PS and ATII) is important known causes of thrombotic cerebrovascular Accidents (CVAs). The prevalence of inherited thrombophilias, factor V Leiden (FVL), prothrombin (PT- 202l0AG) and methylenetetrahydrofolate reductase (MTHFR) gene mutation and natural anticoagulants (PC, PS and ATIII) is not yet known. The objective of this study was to determine the prevalence of three major potential gene polymorphisms factor FV Gl69la (Leiden), prothrombin (PT-202l0AG) and methylenetetrahydrofolate reductase (MTHFR ) C677T) mutations together with auto- antibodies antiphospholipids (Lupus Anticoagulant and Anticardiolipin antibodies) deﬁciencies of natural anticoagulants (PC, PS and ATIII) as risk factors for the development of thrombotic Cerebrovascular Accidents (CVAs) in Sudanese patients. In addition to that other laboratory parameters are also evaluated. Early detection of predisposing factors of CVAs through regular screening of patients at risk can reduce morbidity and improve management. Following informed consent, 200 patients of both sexes and with an age of <50 years with conﬁrmed CVAs due to thrombosis were enrolled in the study. Comparable healthy controls (n= 300) age and sex matched were selected from medical staff and students. Full blood counts, PT, APTT, TCT, Protein C, Protein S, ATIII levels were conducted for patients and controls. Screening for anticardiolpin and mixing coagulation for antiphospholipid tests were also conducted (anti-phospholipids screening). Haematological, coagulation & immunological ﬁndings of patients and the controls were comparable. Antiphospholipids were found negative. PC, PS & ATIII, were signiﬁcantly lower in all patients compared to controls (p=0. 04), (u=0.04) and (p=0.02) respectively. Females were affected more than males in the study. PC, PS & ATIII were found low in female patients compared to female controls (p=0.0I), (p=0.03) & (p=0.0I). The commonest mutation was found to be FV Leiden (FVL) which was seen in 37 patients (37%), (33% heterozygous, 4% homozygous), followed by Prothrombin (PTH) which was seen in 4 patients (4%), (0% homomzygous, 4% heterozygous) followed by (MTHFR) which was seen in 2 patients (2%), (0% heterozygous, 2% homozygous). Control subjects showed negative results. Over all the inherited thrombophilia in all patients was 43%, out of I00 patients. Fiﬁy seven patients showed negative results, The genotype of our patients showed heterozygosity in 39 of them (39%), homozygosity in 4 (4%), the details are shown in table (3.3.6). In conclusion, PC, PS & ATIII deﬁciency and inherited thrombophilia could be a possible cause of increased incidence of thrombotic Cerebrovascular Accidents (CVAs) among Sudanese patients <50 years. The predominance of females in the study group could not be explained. Further studies are needed to verify our suggested explanation.