In-utero coxsackievirus B4 infection of the mouse thymus.

Type B coxsackievirus (CV-B) infections are involved frequently in the triggering of several autoimmune diseases such as myocarditis, dilated cardiomyopathy, pericarditis, pancreatitis, type 1 diabetes, encephalitis, thyroiditis or Sjögren's syndrome. Serological and virological evidence suggests that maternal infections during pregnancy can play a role in the appearance of these diseases in offspring. The current study aims to explore the effect of an in-utero CV-B infection on the fetal thymus, the central site for programming immunological self-tolerance. In this perspective, female Swiss albino mice were inoculated intraperitoneally or orally with the diabetogenic CV-B4 E2 strain at gestational days 10 or 17. Offspring were killed at different post-inoculation times, and their thymuses were analysed for evidence of infection and alterations in thymic T cell subsets. In-utero CV-B infection of the thymus was demonstrated during the course of vertical transmission, as attested by viral RNA and infectious virus detection in most analysed samples. No histopathological changes were evident. Thymic T cells were not depleted, despite being positive for viral RNA. As evidenced by flow cytometry analysis, CV-B infection of the fetal thymus induced significant changes of thymic T cell populations, particularly with maternal inoculation at gestational day 10. Altogether, these findings suggest that CV-B infection of the fetal thymus may play an important role in the genesis of autoimmune diseases.

The Duffy blood group system in the Tunisian population.

BACKGROUND: Tunisia was described to as genetically heterogenous. Besides the 1% native Berber, the genetically influence of the Europeans seems much larger than that of sub-Saharan populations. Due to their ethnic variability, blood group variants have the potential to support population analyses. The aim of this study was to estimate the Duffy blood group system in this mixed population with enhanced characterization of samples with aberrant expression. MATERIALS AND METHODS: Standard serological testing for the Duffy antigen was done for 105 Tunisian blood donors. Samples with altered Fy expression underwent DNA sequencing of the DARC, RHD and RHCE genes. RESULTS: The Fy(a-b+) was the most common phenotype identified in the Tunisian population (38.1%). Five samples with Fy(a-b-) phenotype were determined as FY*02N.01/FY*02N.01 by a homozygous occurrence of the FY*B-67C>T alteration. Another three individuals exhibited a Fy(b+(w))Fy(x) expression, confirmed by a FY*A/FY*02M.01 (n = 1) and a FY*02M.01/FY*02M.01 (n = 2) genotype. RHD and RHCE sequencing (n= 8) revealed altered alleles observed in black populations in 5 samples. One individual with FY*02M.01/FY*02M.01 have the silent 165C>T nucleotide substitution each in the RHD and RHCE gene. DISCUSSION: The composition of blood group variants determined in this study confirms the genetically proximity of Tunisia to Europe. The small sub-Saharan genetic influence was approved by a limited number of variant samples associated with the black population.

[Clinico-biological and immunohaematological profile of patients with ß-thalassemia in Tunisia: about 26 cases]. / Profil clinico-biologique et immunohématologique des patients atteints de ß-thalassémie en Tunisie : à propos de 26 cas.

AIM OF THE STUDY: To study the clinical and biological profile of ß-thalassemic patients in our region, reflecting the quality of their care. PATIENTS AND METHODS: A retrospective study (2010-2011) on 26 ß-thalassemic patients followed in the pediatrics service at CHU Farhat Hached Sousse, Tunisia. Epidemiological, clinical and biological data were collected from medical records and transfusion files of patients. The transfusion protocol adopted was to maintain a hemoglobin level>10g/dL by regular transfusions every 3-4 weeks. Iron chelation therapy, in order to maintain serum ferritin<1500ng/mL, was introduced when serum ferritin exceeded 800-1000ng/mL. RESULTS: The mean age of patients at diagnosis was 15 months. The clinical impact of anemia had resulted in failure to thrive in 54% of patients and facial dysmorphism in 23%. The average transfusion requirement was estimated at 311.02mL/kg/year with 6 cases of hyperconsumption. The immunohaematological monitoring showed the appearance of anti-RBC alloimmunization in one patient and 4 cases of autoimmunization. Poor adherence of chelation therapy was 62% and causing 5 cases of cardiac complications, 4 cases of liver injury and 14 cases of endocrine complications. CONCLUSION: Improving the therapeutic care of ß-thalassemic children requires better monitoring of transfusion recovery and improved adherence to chelation therapy.

RHD positive among C/E+ and D-negative blood donors in Tunisia.

PURPOSE OF THE STUDY: The aim of this study was to investigate RHD alleles among Tunisian blood donors with D-negative phenotype and positive for C and/or E antigen. PATIENTS AND METHODS: A total of 100 D-negative and C/E+ samples were analyzed by RHD genotyping using an initial test for RHD exon 10. In case of a positive reaction, further molecular investigations including real time quantitative PCR, allele specific PCR and nucleotide sequencing were done to elucidate the RHD involved mechanisms. RESULTS: Seventy-five percent of the studied samples lacked the RHD gene. Twenty-three percent carried the hybrid RHD-CE-D alleles (16 RHD-CE(3-7)-D, 5 RHD-CE(4-7)-D, 1 RHD-CE(4-8)-D, 1 RHD-CE(3-8)-D) and 2% were weak D (1 weak D type 1 and 1 weak D type 5). CONCLUSION: Our study proved the high frequency of RHD gene among serologically D-negative samples, positive for C and/or E antigen. Thus achieving systematically RHCE phenotyping in all transfusion centers on the Tunisian territory and considering blood donated from D-negative C/E+ persons as D-positive will be recommended to reduce anti-D allo-immunization.

Anti-D auto-immunization in a patient with weak D type 4.0.

We report the case of a 56-year-old patient with blood group O+C-c+E-e+K-, followed for a myelodysplasic syndrome and treated by regular pheno-identical and compatible (RBCs) transfusion since December 2007. In June 2009, a positive crossmatch was found with 2 RBCs O+C-c+E-e+K-. A positive anti-body screening with a positive autocontrol was detected and anti-D was unidentified in the patient's serum. The DAT was positive (IgG) and elution identified an anti-D. The following assumptions were then made: it could be a partial D phenotype with anti-D alloantibodies or RH: 1 phenotype with an anti-D auto-antibodies. Molecular analysis by multiplex PCR and sequencing have depisted a weak D type 4.0 phenotype. In October 2009, over three months of RH:-1 RBC transfusion, the antibody screening and DAT (IgG) remained positive, and an eluate made from the patient's erythrocytes contained an anti-D. All these funding confirmed the autoimmune nature of the anti-D. This case report illustrates the importance of a well-conducted and immunohematological laboratories test in order to distinguish between auto- or allo-immune of anti-D in a RH: 1 poly-transfused patients. This distinction is of great importance for transfusion support.

RHD*weak partial 4.0 is associated with an altered RHCE*ce(48C, 105T, 733G, 744C, 1025T) allele in the Tunisian population.

BACKGROUND AND OBJECTIVES: D is the most immunogenic blood group antigen. About 1% of whites carry an altered RHD allele leading to quantitative or qualitative changes in the antigen D expression. T201R and F223V encoded by 602C>G and 667T>G are specific amino acid substitutions of the weak D type 4 cluster of African origin, comprising the alleles RHD*09.01, RHD*09.02, RHD*09.03, RHD*09.04 and RHD*09.05. The purpose of this study was to estimate the presence of these RHD genotypes in the Tunisian population. MATERIALS AND METHODS: Ethylenediaminetetraacetate blood samples from 907 D+ and 93 D- blood donors were tested for markers 602G and 667G by allele-specific primer-polymerase chain reaction (PCR-ASP). Samples with positive reactions were re-evaluated by DNA sequencing for RHD and RHCE exons 1-10 and adjacent intronic sequences. RESULTS: Among 907 D+ samples, 19 individuals were identified to harbour the RHD*weak partial 4.0 allele. RHCE sequencing post-haplotype-specific extraction (HSE) revealed an altered RHCE*ce(48C, 105T, 733G, 744C, 1025T) in those samples. The linkage of the RHCE polymorphisms to one haplotype was proven by DNA sequencing post-HSE. CONCLUSION: The RHD*weak partial 4.0 allele syn. RHD*09.03 was estimated to occur 1 in 47 among D+ Tunisians. There was no evidence for other RHD alleles included in the weak D type 4 cluster.

[System RH: screening of partials D with RHD/RHCE hybrid gene]. / Système RH : dépistage de D partiels avec RHD/RHCE gène hybride.

AIM OF THE STUDY: The determination of the RhD phenotype is important in transfusion medicine. However, the complexity of the expression of the D antigen is the cause of the discrepancies observed between two serological determinations and the omission by serology of some variants that can be cause alloimmunization. Therefore, it is important to known in a population the RHD alleles responsible for partial D and weak D phenotype. The aim of the study was the screening of partial D with RHD/RHCE gene hybrid by PCR-multiplex. MATERIALS AND METHODS: Our study involved 308 blood donors from Tunisian Sahel (269 D positive and 39 D negative). We used the multiplex PCR assay to amplify specific exons of the RHD gene 3, 4, 5, 6, 7, 9 and 10. Further molecular investigations were carried to characterize the RHD variants that were detected by the multiplex. RESULTS: In the 269 D positive samples, one case showed the absence of amplification of exons 4 and 5 of RHD gene. This variant was identified by PCR-SSP on weak D type 4. None of the RHD exons were amplified from DNA of 39 D negative samples in favor of a total deletion of the RHD gene. CONCLUSION: We have no found any partial D variant with RHD/RHCE gene hybrid. Results in D negative samples showed that RHD gene deletion is the most frequent mechanism of D negative phenotype in the Tunisian population.

[Platelet-leukocyte aggregates as a marker for platelet activation in platelet concentrates]. / Complexes leucoplaquettaires en tant que marqueur d'activation dans les concentrés plaquettaires standards.

OBJECTIVES: Several in vitro laboratory tests to assess the quality control of platelet concentrates (PC) are available. Some of them have a good correlation with the platelet recovery index. To assess the quality control of standard PC prepared in our blood bank, we measured the blood gas and the degree of platelet activation. MATERIALS AND METHODS: SPC were prepared by the PRP method. Fifty-five SPC (45 SPC at day one of storage and 20 SPC at day five of storage) were analysed. Blood gas (pH, PO(2), PCO(2) and bicarbonate concentration) in the SPC were measured by blood gas automate. Platelet activation profile were determined by measuring the percentage of platelet expressing the CD62p (% CD62) and the percentage of platelet-leukocyte aggregate (% PLA). RESULTS: The pH values of all studied SPC were comprised between 7.0 and 7.6. SPC at day 1 of storage have a significantly higher pH than those at day 5 of storage (7.5+/-0.05 versus 7.3+/-0.14; p<0.001). The % CD62p were higher in SPC at day five compared to the SCP at day one without reaching a statistical significance (28.4+/-15% versus 24.3+/-9.7%, p=0.052). The percentage of PLA were higher in SPC at day one compared to SCP at day five although this difference is not statistically significant (22.2+/-7.5% versus 17.9+/-8.0%; p=0.23). CONCLUSION: Preparation and storage procedure adopted in our centre did not significantly affect the quality SPC. Our study is the first to assess the PLA in PC. Studies assessing the PLA are warranted to appreciate the clinical impact of this parameter.

Prevalence of C282Y and H63D mutations in the haemochromatosis (HFE) gene in Tunisian population.

The studies of the HFE mutations: H63D and C282Y in North African populations have revealed the extreme rarity or even the absence of the C282Y mutation. We have examined 1140 chromosomes (570 Tunisian people) for the presence of the two HFE mutations by PCR-RFLP analysis. We have found that the allele frequencies are, respectively, 15.17% (+/-2.1%) for the H63D and 0.09% (+/-0.17%) for the C282Y. These results are consistent with the worldwide spread of the H63D mutation and the north European restriction of the C282Y. This study will be completed by determining whether homozygote trait for H63D and associated risk factors (beta thalassémia) can lead to iron overload in Tunisia.

[Hereditary sideroblastic anemia: a rare diagnosis]. / Anémie sidéroblastique héréditaire: une maladie rare.

Hereditary sideroblastic anemia is a very rare disease recessive and X-linked that affect heme biosynthesis by deficit or decreased of delta aminolevulinic acid synthase (ALAS) activity. We report a case of a six-month-old boy, admitted in the hospital for anemic syndrome. The hemogram showed anemia (hemoglobin: 4.5 g/dL), frankly hypochronic microcytic and a regenerated (mean corpuscular hemoglobin concentration: 26 g/dL, mean cell volume: 53 fl, reticulocytes: 10 x 10(9)/L) with red cells morphologic disorders in smears (anisopoikylocytosis) without attack of the other lineages; white blood cells: 11 x 10(9)/L (neutrophils: 64% and lymphocytes: 35%); platelets: 350 x 10(9)/L. Examination of bone marrow showed an important erythroid hyperplasia (about 69%) with dyserythropoiesis. Perls stain revealed intense siderosis with 90% of ringed sideroblasts and a large number of siderocytes. Exploration of ALAS2 and ABC7 genes on the DNA of the infant was not found abnormalities. Treatment with pyridoxine corrects moderately the anemia. By the way, we proposed to remind that iron deficiency, inflammatory syndrome and thalassemia are the common microcytic anemia. However, it's mandatory to explore other causes if diagnosis is not solved.

[Quality control of platelet concentrates: experience of Sousse (Tunisia) blood centre]. / Contrôle de qualité des concentrés plaquettaires: expérience du centre de transfusion de Sousse (Tunisie).

The aim of this prospective study was to control the quality of platelet concentrates prepared in Sousse blood centre: standard platelet concentrates (SPC) and apheresis platelet concentrates (APC) in order to detect anomalies and apply corrections. The quality control included three parameters: pH, volume, cells count: platelets, residual white blood cells (WBC) and red blood cells (RBC). The SPC pH was measured with pH meter, samples were obtained at the end of shelf-life by the destructive method. The control of SPC cells count was determined with a Beckman coulter, samples were collected by the stripping method. The APC quality control was assessed in the same conditions than those described for SPC but samples were collected by the transfer method. Quality control results were compared to Europe standards. During a period of six months (July-December 2002), 475 SPC (16% of the production) and 36 APC (60% of the production) were controlled. More than 95% of the SPC meet standards in regard to pH, volume and residual WBC count. However, the number of platelets and residual RBC meet standards respectively in 58% and 42% of SPC. All the APC meet standards for the three quality parameters. In conclusion, in order to improve the quality of our SPC, in regard to the number of platelets and residual RBC, two actions are respectively necessary to satisfy requirements: collecting the appropriate volume of whole blood and controlling the separation technique.

[Fever-shivers reaction and standard platelet concentrates transfusion: a prospective study]. / Réaction frissons-hyperthermie et transfusion de concentrés plaquettaires standard: étude prospective.

Fever-shivers reaction (FSR) is the most frequent transfusion immediate incident related to platelet transfusions. The aim of our prospective study was to assess the frequency of the different immediate incidents, especially the frequency and the causes of the FSR, observed during the transfusion of standard platelet concentrates (SPC). For each FSR, analysis of causes included: a bacterial culture of the implicated SPC, a blood culture and HLA antibody screening (lymphocytotoxicity assay) among the patients. In the study period, 34 patients were followed during 74 transfusions. Ten immediate incidents were noted; FSR: N = 8, erythema-urticaria: N = 1 and nausea-vomit: N = 1. The FSR was observed in 6 patients who received 56 SPC. Analysis of causes of this reaction revealed that: HLA antibodies were present in one patient; bacterial contamination was not found neither among the patients nor in the implicated SPC, and the risk of the FSR occurrence rose with increased storage time of the SPC transfused. Indeed, a significant difference was noted between the mean age of the SPC implicated in the FSR and the mean age of those not implicated (P = 0,0028). In conclusion, the FSR is a frequent incident observed during SPC transfusions. In the majority of cases, the cause of this reaction was not identified. Further studies will be necessary to better understand the physiological mechanisms of the FSR.

[The utility of class I HLA antibodies screening in a Tunisian obstetric department in order to select good HLA typing reagents]. / Intérêt du dépistage des anticorps anti-HLA de classe I dans un service obstétrical tunisien pour la sélection de bons réactifs de typage HLA.

In order to reduce the cost of the serologic class I HLA typing, we have established a screening program for HLA antibodies among obstetric patients. Class I HLA antibodies were identified by standard microlymphocytotoxicity test using a panel of 100 lymphocytes. In the study period, carried out between January 2000 and June 2002, 817 women were tested, most of them (62%) during the last trimester of pregnancy and in the other cases after delivery. These patients, aged between 18 and 45 years, were in the majority (88%) multiparous. From the total number of 817 maternal serum samples screened, 194 specimens (23,74%) tested positive for HLA antibodies. Thirty eight HLA specificities were characterised in 110 maternal sera of which 62 contained monospecific HLA antibodies. HLA-A2 and HLA-B51(5) were the most common specificities characterised in this study. HLA alloimmunization was present since the first pregnancy. There was no statistically significant linear correlation between HLA alloimmunization and the number of pregnancies. Fifty maternal serum samples (6,11%) could be used as HLA typing reagents. Of these 50 sera, 36 had monospecific HLA antibodies. These useful antisera covered a total of 30 specificities: 8 HLA-A and 22 HLA-B. The cost of self - screening for useful antisera as estimated at dollars 62/mL, the personnel was not considered. However, the lowest cost of commercial HLA typing sera is approximately dollars 400/mL. Our study showed the utility and the net economic advantage to use maternal sera as reagents in our new HLA laboratory with limited budget.

[Role of ALAT level in preventing post-transfusional hepatitis. Prospective study of 1,180 blood donors in Tunisia]. / Intérêt du dosage de l'ALAT dans la prévention des hépatites post transfusionnelles. Etude prospective à propos de 1180 donneurs de sang du sahel tunisien.

In Tunisia, regular serological tests for prevention of blood transmitted hepatitis consist in the research of HBS antigen and HCV antibodies. Our purpose in this study is to estimate the prevalence of hypertransaminasemia in blood-donors and to determinate to what extent it could prevent blood-transmitted hepatitis. Therefore we have assessed ALAT sera level in 1180 blood-donors. It rate is considered elevated if higher than twofold the normal rate (> N = 40 Ul/l). Donors with high ALAT level were summoned three months later after their blood gift to undergo clinical examination and a new serological test, researching seroconversion of HBS Ag and HCV antibodies. With regarding to estimation of residuel HCV infection risk, we were based on M.P Busch's data. Hpertransaminasemia was modified in 134 individuals (11.5%). Only 67 had replied to our summons. Alcoholism was involved in one case. Smoking was found in most of male donors. We had discovered neither weight excess nor drug or medicines consumption which could explain increasing ALAT. New serological list had revealed seroconversion for HCV antibodies in ELISA but with undeterminated profile in Immunoblot (anti NS5 solely). PCR was not carried out. Residual infection risk being considered, use of hypertransaminasemia detection in blood donors should prevent nearly 1.67 blood transmitted hepatitis per million transfusions units. However if we consider shortage in blood derivates in Tunisia, such a decision should be comprehensively weighted numerous blood donors will be moved aside.