shahram samiee

T helper 1 (TH1) and TH2 lymphocytes are the most important components of the immune system affected by blood transfusion. This study aimed`` to evaluate the effect of blood transfusion on gene expression of transcription factors related to the development of TH1, TH2, TH17 and regulatory T cells (Tregs). In this cross-sectional study, 20 patients diagnosed with abdominal aortic aneurysms requiring surgical repair were studied from January 2018 to August 2020. We utilized real-time PCR to evaluate the expression of transcription factor genes associated with TH1, TH2, TH17, and Treg, namely T-box-expressed-in-T-cells (T-bet), GATA-binding protein 3 (GATA-3), retinoid-related orphan receptor (RORγt), and fork head box protein 3 (Foxp3), respectively. The sampling occurred before anesthesia, 24- and 72 hours post-transfusion, and at the time of discharge. The results showed that the T-bet gene expression, compared to the time before transfusion, was significantly decreased 24 hours after blood transfusion and upon discharge while GATA3 genes exhibited a significant reduction both 24 and 72 hours after the transfusion, as compared to the pre-transfusion levels and the time of patient discharge. The Foxp3 gene demonstrated an increase at all study stages, with a notable surge, particularly 72 hours after red blood cell (RBC) transfusion. Conversely, the expression of RORγt gene, consistently decreased throughout all stages of the study. RBC transfusion in abdominal aortic aneurysm patients altered the balance of transcription gene expression of TH1, TH2, TH17, and Treg cells.

Due to the presence of platelet antigen polymorphisms, human platelet membrane glycoproteins can be identified as an alloantigen or autoantigen. The aim of this study was to determine the frequencies of human platelet antigens (HPAs)‑1 to‑5 and‑15 in Turkmen blood donors and establish a panel of accredited HPAs negative donors as well as an HPA‑typed platelet donor registry.

HPA‑1 to‑5 and‑15 typing was performed by the polymerase chain reaction‑sequence‑specific primer techniques on 80 unrelated Turkmen donors who were referred to Aq‑Qala Blood Transfusion Center in Golestan Province from September 2018 to October 2019.

The frequencies of HPA phenotypes were determined as follows: HPA‑1aa: 92.5%, HPA‑1ab: 7.5%, HPA‑2aa: 77.5%, HPA‑2ab: 20.0%, HPA‑2bb: 2.5%, HPA‑3aa: 75.3%, HPA‑3ab: 50%, HPA‑3bb: 11.2%, HPA‑4aa: 100%, HPA‑5aa: 78.5%, HPA‑5ab: 21.5%, HPA‑15aa: 41.2%, HPA‑15ab: 56.2% and HPA‑15bb: 17.5%.

Determining the genotype of HPAs that play an important role in platelet refractory can improve the management of alloimmunization due to the incompatibility of HPAs between the recipients and donors. Therefore, the registration process for national platelet donors can help patients accelerate and improve the quality of transfused platelets.

Umbilical cord blood (UCB) was used to source hematopoietic stem cells in the past. Despite the apparent advantages of UCB transplantation, virus reactivation poses a considerable danger in allogeneic hematopoietic stem cell transplantation (HSCT). Human Parvovirus B19 is regarded as a potential threat to UCB contamination. This study aimed to evaluate the prevalence of parvovirus B19 in cord blood donors by Semi-Nested PCR. This study is the first large-scale report of the B19 DNA in cord blood donors in Iran.

A total of 691 umbilical cord blood were collected under standard procedure. Then, DNA from buffy coat and plasma were extracted, and semi-nested PCR was performed for all samples.

Two out of 691 samples (0.29%) indicated viremia in plasma and buffy coat.

In this line, designing and validating a quantitative PCR assay for detection, quantification, and discrimination of Human B19 DNA genotypes of cord blood donors is necessary to enhance the safety of this source of stem cells.

Age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly. Although it has been shown that Y402H polymorphism in the CFH gene was strongly associated with AMD in the Iranian population, there were no data on other single nucleotide polymorphisms (SNPs), which have the most significant association with AMD. This study aimed to investigate hot point regions in exon 10 and intron 9.

One hundred and sixty-six AMD patients and 69 controls were recruited. Their blood was collected in the tubes containing EDTA. Then, DNA was extracted from the blood, and its quality was evaluated. Primers were designed for intron 9 and exon10 sequencing. A viral polymorphisms analysis software named CEQ was used for the analysis of putative polymorphisms.

We noticed three polymorphisms in study cases: rs7535263 and C66379A in intron 9 and rs2274700 in exon 10. Based on the McNamara’s test (rs7535263 and rs2274700) and the Phi and Cramer’s test (C66379A), a significant difference was found between the control and patient groups regarding rs7535263 and rs2274700 polymorphisms.

We found a synonymous or silent mutation, A473A, rs2274700 in exon 10 in 85% of patients. From two intronic SNPs, just rs7535263 showed association with the disease in studied patients living in Gilan Province, Iran. Although no significant relationship was found between controls and patients regarding the C66379A allele, it would be important that no other sources have reported C66379A polymorphism in AMD yet.

Human platelet antigens (HPAs) are glycoproteins on the platelet surface that a single nucleotide mutation in the coding region gene could lead to the variation of different HPA polymorphisms. These antigens have shown variation among different races and may trigger immune responses during blood transfusion and pregnancy. Genotyping of HPAs is useful for managing these reactions and establishing a platelet registry to decrease platelet transfusion reactions. This study aimed to compare allelic and genotype frequencies of human platelet antigens in the Azeri ethnicity by TaqMan Real-time and polymerase chain reaction with sequence-specific primers (PCR-SSP) methods. DNA was extracted from the whole blood of 100 Azeri blood donors in the Ardabil Blood Transfusion Center. Genotyping of HPA-1 to -5 and -15 was performed by TaqMan Real-time PCR, and PCR-SSP and consistency of results were evaluated. The results of PCR-SSP and TaqMan Real-time PCR showed complete consistency. The allele frequencies were 91.5% and 8.5% for HPA-1a and -1b; 88% and 12% for HPA-2a and -2b; 58% and 42 % for HPA-3a and -3b; 100% for HPA-4a; 91% and 9% for HPA-5a and -5b; 56.5% and 43.5% for HPA-15a and -15b alleles. Not incompatibility was detected in HPAs genotyping by PCR-SSP and TaqMan Real-time PCR so that real-time PCR can be used as a robust and quick method for HPA genotyping. We found differences between Azeri blood donors and previously reported HPAs alleles’ frequency in other ethnicities in the country. This fact highlights the need for a platelet registry to recruit platelet donors from different ethnicities and increase the number of donors by using faster methods.

Platelet activation occurs for a variety of reasons from the moment of sampling to that of injection. Activation of the platelets leads to activation of mitochondria. They move toward the membrane, sprouting and shedding freely from it, leading to the release of pro inflammatory mediators and mtDNA. Therefore, mtDNA level is considered as one of the parameters of platelet activity. The aim of this study was to compare mtDNA levels during storage.

This experimental study was performed on 22 PRP products. We obtained consent of all donors referring to Iran Innovation Center of Iranian Blood Transfusion Organization. Samples were taken from the PRP bags on day 1, 3 and 5. Then, the fold change of mtDNA was assessed by Real time PCR. The results were analyzed by paired t-test and the P value of less than 0.05 was considered significant.

The rate of mtDNA decreased 0.8-fold on day 3 to 1, 0.6-fold on day 5 to 3, and 0 .49 on day 5 to 1. These changes were significant on days 5 to 1 (p= 0.003) and they were not significant on days 3 to 1 (p= 0.4). 

Conclusions  

The amount of mtDNA increased on the first day, and then decreased during the storage of  PRP products.

Human platelet antigens (HPAs) are part of platelet GP complexes have the potential to contribute to the autoantibody production. Moreover, these antigens demonstrate different patterns of distribution on different ethnic groups and variation in some types of diseases. This study was objected to determine the incidence of HPA-1 to -5 and -15 polymorphisms in the Iranians suffering from primary Immune thrombocytopenic purpura (ITP). 

In this case-control investigation, 30 patients by definite primary ITP were randomly selected and enrolled in the study. HPA genotyping was performed implicating by the Single Specific Primer PCR (SSP-PCR). For the control group, data of recently published gene polymorphism among Iranian Blood donors were deployed for comparison. 

The incidence of HPA-1 to -5 and -15 polymorphisms in the Iranian patients with primary ITP was found to be: HPA-1a/1a: 0.933, HPA-1a/1b: 0.067, HPA-2a/2a: 0.133, HPA-2a/2b: 0.867, HPA-3a/3a: 0.2, HPA-3a/3b: 0.533, HPA-3b/3b: 0.267, HPA-4a/4a: 1, HPA-5a/5a: 0.967, HPA-5a/5b: 0.330, HPA-15a/15a: 0.166, HPA-15a/15b: 0.667 & HPA-15b/15b: 0.167.  

This study provides special new data on the distribution of HPA allele among the Iranians ITP patients.Furthermore, it might useful toccharacterize understanding more presizely about ITP and HPA distribution. However, further studies concerning platelet immunology are needed to do help on best practice on management of immune diseases triggered by platelet antibodies.

Recent progress in understanding the pathogenesis of premature ventricular contraction (PVC)-induced cardiomyopathy (PIC) has suggested a key role for inflammation. The aim of this study was to evaluate the expression of messenger RNAs (mRNAs) and the protein production of interleukin-6 (IL-6), IL-10, tumor necrosis factor alpha (TNF-α) and interferon-γ (IFN-γ) and two circulating micro-RNAs related to inflammation and cardiovascular disease; miR-155 and miR-146. The study population was comprised 25 patients with PIC and 25 patients with normal left ventricular ejection fraction despite frequent PVCs. TNF-α, IL-6, IL10, and IFN-γ levels were evaluated in peripheral blood mononuclear cells (PBMCs) by flow cytometry and their mRNAs were assessed by real time PCR. We analyzed circulating levels of these cytokines by enzyme linked immunosorbent assay (ELISA). Two circulating micro-RNAs, miR-155 and miR-146a, were also investigated. The flow cytometry findings showed that the median fluorescence intensity (MFI) of antibodies reacted with the IL-6 and TNF-α were higher in PIC group than the control group (P-value<0.001). In ELISA, the levels of IL-6 (P-value <0.001) and TNF-α (P-value <0.001) and in RT-PCR the relative expression levels of IL-6 (P-value <0.001) and TNF-α (P-value <0.001) were significantly higher in the PIC group. The relative expression levels of miR-155 and miR-146a were not significantly different between 2 groups (P-value>0.05). In our patients with PIC, there was an elevation in the expression levels of IL-6 and TNF-α in PBMCs. This finding may provide further insights into the inflammatory pathways involved in PIC.

The aim of this study was to report the results of the use of real-time polymerase chain reaction (PCR) for the diagnosis of hepatitis B virus (HBV) infection in cornea donors at the Central Eye Bank of Iran. Between 2014 and 2016, all cornea donors that had negative screening serologic results for hepatitis B (HB) surface antigen, HB surface antibody (Ab), hepatitis C virus Ab, human immune deficiency virus Ab, human T-cell leukemia virus Ab, and syphilis, and positive serology for HB core Ab were subjected to real-time PCR with a detection limit of 400 IU/mL to identify HBV DNA. Positive results for HBV DNA were considered occult HBV infections in these donors. Over the 3-year period, 122 out of 10448 cornea donors had negative screening serologic tests outside of HB core Ab. Of which, 90 cases were subjected to real-time PCR. Occult HBV was detected in 11 cases (12.2%), resulting in the rejection of the corresponding corneas. The remaining 79 cases (87.8%) had negative results for HBV DNA and the corresponding corneas were used for transplantation. Implementation of PCR for the detection of occult HBV in cornea donors is necessary to not only increase the security level of cornea donation but also minimize the rejection rate of donors that have isolated HB core Ab reactivity.

With the improvement of quantitative molecular hepatitis C virus (HCV) RNA assays, the usefulness of these assays has been indicated for management of HCV infection. Recently, various real time assays with different methodology and performance characteristics have been introduced..
This study aimed at designing, developing, and evaluating an in-house 1 step TaqMan real time-polymerase chain reaction (RT-PCR) assay for detection and quantification of HCV-RNA..
The primers and probe were selected from a highly conserved region of the HCV genome, which allowed the detection of 4 common HCV genotypes in Iran. Using 4 quantification standards from 101 IU/µL to 104 IU/µL and clinical specimens, the current study determined analytical sensitivity, linear range, precision, analytical and clinical specificity, and validity of the assay. Data was analyzed by the SPSS statistical software (version 16)..
The sensitivity of the assay with 95% probability, determined by probit analysis, was 15 IU/µL. The assay showed a linear range of 101 IU/µL to 104 IU/µL (R2 = 0.989). The coefficient of variation for intra and inter assay precision of the assay, based on threshold cycle value, ranged from 0.24 to 0.4, and from 1.94 to 3.19, respectively. The analytical and clinical specificity was 100%. No bias in relation to concentration between the results of 29 HCV RNA positive clinical specimens, simultaneously tested by artus HCV LC RT-PCR reagents and in-house reagents, was observed in the method comparison..
The in-house 1 step TaqMan Real Time RT-PCR assay showed acceptable performance characteristics. Our study presents the robustness and cost-effectiveness of the method for detection and quantification of HCV RNA..

CD27 is a biomarker associated with both T-cells and B-cells activation. Plasma soluble CD27 (sCD27) was identified as a marker of disease outcome in Human Immunodeficiency Virus (HIV) infection. Testing of plasma sCD27 represents a good tool to monitor the change of immune activation during HIV infection.We sought to analyses role of Hepatitis C Virus (HCV) and also GB Virus type C (GBV-C) co-infections on HIV-related immune activation, through measuring sCD27 plasma levels.Blood samples from a total of 86 patients with HIV infection were taken. Plasmas were analyzed for HCV using serologic test and GBV-C by reverse transcriptase polymerase chain reaction (RT-PCR). CD4+ and CD8+T-cell counts were evaluated by CD3/CD4+ and CD3/CD8+ double staining of whole blood followed by flow cytometric analysis. Then Cross-sectional comparison of sCD27 plasma levels was carried out among patients: HIV (n=20), HIV/ GBV-C (n=14), HIV/ (HCV) (n=26) and HIV/HCV/GBV-C (n=26).Plasma level of sCD27 was higher in HIV/HCV/GBV-C patients as compared to HIV mono-infected patients (p= 0.006) and based on results there was significant differences in the plasma levels of sCD27 between HIV-infected individuals with and without HCV coinfection (P=0.017) and also correlation between sCD27 and percent of CD4+T-cells was in highest level among HIV/HCV co-infected patients group [r= -0.59 (p=0.001)]. High levels of sCD27 among HIV/HCV patients argues in favor of sCD27 plasma level determination for monitoring of clinical features among HIV/HCV coinfected patients.

In recent years, the confidential unit exclusion (CUE) option has been used to increase blood safety at blood transfusion centers in several countries. The epidemiologic characteristics of diseases and demographic characteristics of patients vary in different countries; therefore, we investigated whether the CUE option is useful in Iran. In this study, we determined the prevalences of hepatitis B virus (HBV) and hepatitis C virus (HCV) in CUE-positive and CUE-negative units, as well as the efficacy of the CUE option. The aim of this study was to evaluate the efficacy of the CUE option in reducing the prevalences of HBV and HCV in blood units.Patients and All donors were tested for the HCV antibody (anti-HCV) and hepatitis B surface antigen (HBsAg). Supplemental tests were performed to confirm the presence of viruses in the units that tested positive. In total, 2000 units (1000 CUE-positive units and 1000 CUE-negative units) were tested using the nucleic acid testing (NAT) method. The prevalence of infectious markers was estimated in all demographic subgroups. The prevalences of HBV and HCV markers were higher in donors who opted for CUE than in those who did not. The CUE option had low sensitivity (21.5%) and positive predictive value (PPV; 20.9%) for the markers. Most of the donors who opted for CUE for the first time were men with low levels of education. The CUE option has low sensitivity and PPV, and its effectiveness in reducing the transmission of infectious diseases through window-period units is minimal. The CUE process can be continued in Iran because Iran is geographically located in a region where HBV is endemic; however, higher levels of education are necessary to make this process effective.

Dendritic cells (DCs) are ideal accessory cells in the field of gene therapy. Delivery of DNA and siRNA into mammalian cells is a useful technique in treating various diseases caused by single gene defects. Selective gene silencing by small interfering RNAs (siRNAs) and antisense oligodeoxynucleotides (ODN)s is an efficient method for the manipulation of cellular functions. An efficient, functional delivery system with no toxicity problems would be attractive. We compared two commercially available cationic lipids, Lipofectamine and FuGENE6, in the delivery of both siRNA and antisense ODNs into mice spleen-derived DCs. Cellular uptake was measured by the means of fluorescein-labelled non-silencing siRNA and antisense ODNs as a model system using flow cytometry. Cytotoxicity of the two delivery systems was compared with propidium iodide and annexin-V staining, and quantified with flow cytometry. The efficiency of our oligonucleotide delivery systems was compared by measuring CD40 expression by flow cytometry. CD40 expression in DCs was 38%. After siRNA transfection by Lipofectamine, CD40 expression decreased to 13%, and after transfection by FuGENE6, it decreased to 18%. The difference was statistically significant. CD40 down regulation in DCs transfected with the two different antisense sequences by Lipofectamine was 21% and 23%, and down regulation after transfection by FuGENE6 was 19% and 18%, respectively. The differences were not statistically significant. The effects of siRNA and antisense ODNs were specific. Lipofectamine was a more potent delivery system in siRNA effect, followed by FuGENE6. There was no significant difference between Lipofectamine and FuGENE6 as a delivery system of antisense ODNs.

Interferon producing cells have an important role in controlling HIV. The interferon producing cell count was evaluated in order to study the possibility of immune modulating qualification of HGV co-infection in HIV patients’ safety. This descriptive study was performed on 83 HIV positive patients with CD4+ cell count >400 to evaluate the interferon producing cell count. These 83 patients were categorized in 4 groups based on their current infection status. Patients infected with HCV and HGV (HGV+/HCV+), patients infected with HGV but not with HCV (HGV-C+/HCV-), patients infected with HCV but not with HGV (HGV-/HCV+) and patients not infected by HGV and HCV (HGV-/HCV-). Interferon producing cell analysis was performed by flow cytometric method and the presence of -HGV RNA by using RT-PCR and PCR-Elisa. Interferon producing cell count in HGV co-infected individuals revealed a wider range of numbers however there was no significant difference in the interferon producing cells count between HIV infected patients with and without HGV co-infection. HGV/HCV co-infection doesnt have an effective role on increasing of production or rotation of interferon producing cell in HIV infected patients with relatively intact immune system.

Hepatitis virus infections are common in patients suffering from human immunodeficiency virus (HIV) infection due to similar transmission routes of these viruses. Hepatitis virus infections lead to impaired survival in HIV positive patients. In contrast to the expectations, some studies indicate that HGV infection slows down the progression and eventually death of AIDS patients. The aim of this study was to estimate the frequency of active HGV infection among HIV infected patients in Tehran. Flow cytometric analysis of lymphocyte subpopulations was performed on blood samples of 103 patients with HIV infection referring to the central laboratory of Iranian blood transfusion organization. Their plasma samples were analyzed for the presence of HGV RNA by RT-PCR. HGV Positive cases were confirmed by PCR-ELISA. Of 103 HIV-positive patients, 16 (15.5%) were HGV RNA-positive. It was apparent that the frequency of HGV in patients with CD4 count above 500 cell/μl is higher. Sucha frequency is not high compared with those found in other studies. The high frequency of active HGV infection in patients with asymptomatic HIV infection may be due to the loss of active HGV infection in patients with advanced HIV infection.