mTORC1 inhibition may improve T lymphocytes affected by aging.

BACKGROUND: Due to the increase of the elderly's population and related social and economic problems, it is very important to provide strategies on health. In this regard, induction of T lymphocytes responses, the most important cells of the immune system, may be a good approach. Among different agents considered as antiaging factors, mTORC1 pathway inhibitors are significant. So, the purpose of this study was to evaluate the effect of two mTORC1 inhibitors, Everolimus and Metformin, on age-related features of activated T cells. MATERIALS AND METHODS: Optimum doses of drugs was determined with evaluating the effect of treatments on IL-2 gene expression. T cells isolated from old and young mice were treated with drugs and PHA. IL-2 production was evaluated by ELISA. Also, the expression of CD28, PD-1, and KLRG-1, proliferation, and intracellular oxidative stress were assessed by flow cytometry-based assays, phenotyping, CFSE, and DCF-DA assay respectively. RESULTS: Both drugs increased IL-2 production in the T cells of old mice. Also, using drugs especially Metformin could improve age-related phenotypical markers and increase the proliferation of T cells of old mice significantly. In addition, Metformin and Everolimus reduced intracellular oxidative stress in aged cells. However, the effect of both drugs on the T cells of young mice wasn't significant or was in opposite to the results of old mice T cells. DISCUSSION: In line with studies noting mTOR inhibitors as antiaging drugs, Metformin and Everolimus may improve T cells affected from aging in vitro, and a decrease in intracellular oxidative stress may be one of their mechanism of function.

Group O blood donors in Iran: evaluation of isoagglutinin titers and immunoglobulin G subclasses.

This study aims to determine the most frequent titers of anti-A and anti-B (both presumed immunoglobulin [Ig]M and IgG) in Iranian group O blood donors and to compare these titer values with those found in other studies. In addition, alloantibody production and plasma levels of four IgG subclasses were compared between the high-titer and non-high-titer study groups. This study investigated anti-A and anti-B titers in 358 plasma samples. Based on these results, two study groups (high-titer and non-high-titer) were formed. Antibody detection tests were performed to detect unexpected antibodies to D, C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S, s, P1, Lea, and Leb. Four IgG subclasses were also evaluated through nephelometry assay. The most frequent titer obtained by room temperature and indirect antiglobulin tube tests was 256. The frequency of titers ≥512 was 31.5 percent. None of the cases showed unexpected RBC alloantibodies. IgG2 levels were significantly higher in the high-titer group. Evaluation of isoagglutinin titers in group O Iranian blood donors can provide insight into the frequency of isoagglutinin titers both within the Iranian population and as compared with other populations. A significant difference in IgG2 levels between the high-titer and non-high-titer groups was identified. More investigation needs to be conducted on the root cause of this finding. Immunohematology 2021;37:5-12 .This study aims to determine the most frequent titers of anti-A and anti-B (both presumed immunoglobulin [Ig]M and IgG) in Iranian group O blood donors and to compare these titer values with those found in other studies. In addition, alloantibody production and plasma levels of four IgG subclasses were compared between the high-titer and non­high-titer study groups. This study investigated anti-A and anti-B titers in 358 plasma samples. Based on these results, two study groups (high-titer and non­high-titer) were formed. Antibody detection tests were performed to detect unexpected antibodies to D, C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S, s, P1, Lea, and Leb. Four IgG subclasses were also evaluated through nephelometry assay. The most frequent titer obtained by room temperature and indirect antiglobulin tube tests was 256. The frequency of titers ≥512 was 31.5 percent. None of the cases showed unexpected RBC alloantibodies. IgG2 levels were significantly higher in the high-titer group. Evaluation of isoagglutinin titers in group O Iranian blood donors can provide insight into the frequency of isoagglutinin titers both within the Iranian population and as compared with other populations. A significant difference in IgG2 levels between the high-titer and non­high-titer groups was identified. More investigation needs to be conducted on the root cause of this finding. Immunohematology 2021;37:5­12 .

Establishing Blood Group Genotyping to Resolve ABO Discrepancies in Iran.

ABO discrepancies are recognized when the reactions obtained in the forward type do not "match" the reactions obtained in the reverse type. Discrepant results are often caused by a variant ABO gene. Molecular analysis is required to confirm the type of subgroups and discrepancy. In this study ABO genotyping was performed on a series of blood donors and patients to determine their definite blood groups. We examined 100 samples with ABO discrepancies from blood donors and patients referred to Tehran Blood Transfusion Center between October 2015 and August 2016. ABO genotyping was performed on all samples with allele specific PCR for differentiation of A, B and O alleles. Exon 6 and 7 of ABO gene were sequenced to confirm the results. The genotyping of donor/patients samples with discrepant results of ABO blood typing consisted of 61 cases of A2 and A2B, 3 cases of B 302 and 4 cases of Aw06. Genotyping of 6 samples that had extra antibody in their serum (AB blood group) confirmed the cell type reaction results. 6 samples that had shown a very weak reaction with anti-AB (similar to O blood group) and had no anti-A in their serum were genotyped as O 1 O 2. Blood group genotyping laboratory provides an efficient service for evaluation of ABO discrepancies and resolve the problems encountered in serology reactions.

HLA-DRB1*15:03 and HLA-DRB1*11: useful predictive alleles for alloantibody production in thalassemia patients.

BACKGROUND AND OBJECTIVES: Recently, thalassemia has been introduced as a chronic disease. In spite of prolonging life in thalassemia patients, the quality of their life has not significantly improved. One of the challenges that makes their quality of life poor is alloimmunisation which causes several complications to patients by restricting their options. Some individuals are more susceptible to developing an alloantibody than others. They are categorised as responders and non-responders. Determining responders before the first transfusion allows transfusion services to provide compatible blood and prevent alloimmunisation. The aim of the present study was to determine the relationship between HLA-DRB1*15:03, HLA-DRB1*11 and HLA-DRB1*09:01 alleles and alloimmunisation in Iranian thalassemia patients. MATERIALS AND METHODS: Antibody screening tests were performed by tube method, and HLA-DRB1 genotyping was determined by Sequence-Specific Primers (SSP-PCR) in 59 alloimmunised and 205 non-alloimmunised patients. HLA-DRB1 allele frequencies were compared between alloantibody-positive and -negative groups through the χ2 test. RESULTS: HLA-DRB1*15:03 allele frequency was significantly different between groups (P = 0·000, odds ratio (OR) = 4·193). There was a correlation between HLA-DRB1*11 and anti-K (P = 0·000, OR = 6·643). There was no association between HLA-DRB1*09:01 and alloimmunisation (P = 0·350). CONCLUSIONS: According to our results, detecting HLA-DRB1*15:03 and HLA-DRB1*11 alleles are useful in the pre-transfusion test and could determine responder patients and improve transfusion safety.

Four decades of National Blood Service in Iran: outreach, prospect and challenges.

OBJECTIVE: This study aims to compare the changes and progress made to Iranian Blood Transfusion Organization (IBTO) during 1974-2014 in order to identify the shortcomings. BACKGROUND: The History of Blood transfusion in Iran can be traced back to the 1940s. IBTO was established in 1974 as a national centralised organisation, supported by Iranian government for its budget and supplies and provides its products free of charge to both public and private hospitals. METHODS: The statistics have been derived from IBTO Statistical Center. Also related literature has been reviewed. RESULTS: From 1974 to 2014, donation per population has been increased about eight times. IBTO reached 100% voluntary blood donation in 2007, but the number of female blood donors in Iran is six times lower than average global rate. On one hand, the prevalence rate of Hepatitis B virus (HBV) and Hepatitis C virus (HCV) in blood donors was dropped in to one fifth and one third, respectively during past 8 years. On the other hand, irrational blood usage and lack of integrated blood stock management systems and non self-sufficiency on plasma-derivedmedicines are considered as main challenges of IBTO. CONCLUSION: Forty years since the establishment, IBTO managed to achieve considerable improvements in different fields but many challenges still remain, which need to be addressed urgently. Great gap between the number of male and female blood donors in Iran has to be filled. The improvement of knowledge and practice on patient blood management and use of alternatives are on the agenda of IBTO in next coming years.

Immunotherapy with tumor cell lysate-pulsed CD8α+ dendritic cells modulates intra-tumor and spleen lymphocyte subpopulations.

Using cellular adjuvants including dendritic cells (DCs) has provided a promising approach in immunotherapy of cancer. Our previous study showed that mice immunization with tumor cell lysate-pulsed DCs (TL-CD8α+DCs) could significantly suppress the tumor growth and increase mice survival. The aim of the present study was to investigate the impact of TL-CD8α+DC vaccine on intra-tumor and spleen lymphocyte subpopulations in tumor-bearing mice. ABalb/c mouse model of fibrosarcoma was used and changes in various lymphocyte subpopulations including CD4+, CD8+ and CD4+CD25+Foxp3+ T cells in mice immunized with TL-CD8α+ DCs were studied. The cytotoxic activity of the lymphocytes and tumor growth inhibitory rate were also measured. Immunotherapy with TL-CD8α+ DCs significantly enhanced both CD4+ and CD8+ lymphocytes, whereas decreased CD4+CD25+ Foxp3+ regulatory T cells as well as the tumor growth rate. There was also a decrease in the ratio of regulatory T cells to CD4+ and to CD8+ lymphocytes in both the tumor and spleen tissues as compared to that in the non-immunized control mice. Immunization with TL-CD8α+ DCs as well as CD8α+ DCs significantly increased the splenocytes cytotoxic activity by 45.1% and 18.2% of control, respectively. In conclusion, the current study indicated that TL-CD8α+ DCs can enhance tumor immunity against the fibrosarcoma by enhancing both the CD4+ and CD8+ lymphocytes and reducing regulatory T cells. This finding suggests the usefulness of TL-CD8α+DCs vaccine for cancer treatment.

Evaluation of apoptosis-related genes: Fas (CD94), FasL (CD178) and TRAIL polymorphisms in Iranian multiple sclerosis patients.

Multiple sclerosis (MS) is a central nervous system (CNS) demyelinating disease characterized by a relapsing-remitting course leading to progressive disability. Given the critical role of apoptosis-related genes in the maintenance of homeostasis in the immune privilege sites, mutations in these genes have a profound effect on occurring autoimmune diseases such as multiple sclerosis. In the current study, polymorphisms in the apoptosis-related genes: Fas _-670 A>G, FasL _-844C>T, FasLIVS2nt _124 A>G and TRAIL_1595C>T were analyzed in 107 Iranian patients suffering from MS and 112 unrelated healthy controls using PCR-RFLP method. Our results demonstrated that among Iranian patients with MS and controls being homozygous in Fas_670A/A, G/G and FasL_-844C/C, TT in the promoter region and homozygocity in the minor allele for FasLIVS2nt_124G/G and TRAIL_1595C/C, polymorphisms were not associated with the MS risk in Iranian patients when compared with normal controls. However, the Fas _-670G/G genotype had a borderline significantly increased frequency with secondary progressive MS type (X(2)=5.8, P=0.05). In conclusion, no statistical association was found between the Fas, FasL and TRAIL polymorphisms and the risk of MS in Iranian patients.

High expression of Fas ligand on cord blood dendritic cells: a possible immunoregulatory mechanism after cord blood transplantation.

BACKGROUND: Allogeneic cord blood transplantation is associated with less severe graft-versus-host disease (GVHD). Dendritic cells (DCs), as the most potent antigen-presenting cells of the immune system, play a central role in the development of GVHD. Because apoptosis induction is one of the known mechanisms that DCs use to regulate T-cell responses, we studied the immunostimulatory and apoptosis induction capacities of cord blood dendritic cells (CBDCs) and peripheral blood dendritic cells (PBDCs) to evaluate the mechanisms underlying the lower incidence of GVHD after cord blood transplantation. Presence of apoptosis-related markers Fas, Fas ligand (FasL), and CD40 and costimulatory molecules, along with the proportion of myeloid and lymphoid DCs subsets, were also measured on CBDCs and PBDCs. METHODS: Fresh CBDCs and PBDCs were isolated from cord and peripheral mononuclear cells as lineage-negative cells by using monoclonal antibodies against CD3, CD11b, CD14, CD16, CD19, CD56, CD34, and CD66b. DCs were cocultured with allogeneic T cells, and the effect of CBDCs and PBDCs on T-cell apoptosis and proliferation were determined through flow cytometric analysis and 3H-thymidine incorporation. RESULTS: Our findings showed that CBDCs markedly augment apoptosis of CD3+ T-cells. FasL expression on CBDCs was significantly higher than on PBDCs. However, there was no difference between Fas expression on CBDCs and PBDCs. Moreover, CBDCs were poor stimulators of allogenic T cells in mixed leukocyte reaction compared with adult peripheral blood DCs. They also displayed decreased expression of HLA-DR and CD86 molecules. The ratio of lymphoid DCs (CD11c-, CD123+) to myeloid DCs (CD11c+, CD123-) was also significantly higher in CBDCs compared with PBDCs. CONCLUSIONS: It seems that less severe GVHD after cord blood transplantation is due not only to a higher degree of immaturity of CBDCs, but also to delivery of apoptotic signals to the host T cells that recognize allo-MHC molecules on CBDCs in the early phase of immune response.

Hepatitis B virus genotype, HBsAg mutations and co-infection with HCV in occult HBV infection.

BACKGROUND: The association between mutations in the hepatitis B surface antigen (HBsAg) gene and the occurrence of occult HBV (OBI) in patients has not been studied adequately to determine if the two are correlated. The current study was aimed to investigate HBsAg mutations, the genotype of HBV and co-infection with HCV in OBI in the central part of Iran to determine any possible associations. MATERIAL AND METHODS: In this study, 3700 plasma samples were examined for the presence of HBsAg, anti-HBc and HBV-DNA. All HBsAg(-)/anti-HBc(+)/HBV-DNA(+) samples were regarded as OBI. The genotype of HBV was identified using Gap-PCR and RT-PCR was used to determine possible co-infection with HCV. Finally, direct sequencing was performed to analyse mutations within the surface antigen gene of HBV in occult versus acute HBV infection. RESULTS: Of the 3700 patient samples analysed, 352 (9.5%) cases were determined to be HBsAg(-)/anti-HBc(+) in which HBV-DNA was detected in 57 (16.1%), these latter patients were classified as OBI. All of the patients studied carried the D genotype. Direct sequencing of the S-gene from occult and acute HBV patients revealed one silent and one glycine to arginine mutation but the acute HBV patients showed an additional mutation (alanine to threonine). All the mutations were outside the range of the α-determinant. Furthermore, none of the OBI patients were co-infected with HCV. CONCLUSIONS: The absence of conformational mutations in the α-determinant of HBsAg confirmed that this antigen could be detected by commercial Elisa kits and therefore was not responsible for false negatives during blood screening. However, it can be concluded that suitable amounts of HBsAg were not expressed by HBV in the OBI patients to be detected by Elisa. Low level expression of HBsAg might be related to the D genotype of the virus. Furthermore, our results suggest that OBI is not related to co-infection with HCV.

Preparation of antibody against horseradish peroxidase using hybridoma technology.

BACKGROUND: Monoclonal antibody against horseradish peroxidase (HRP) has many applications which peroxidase anti-peroxidase. Peroxidase-antiperoxidase (PAP) complex formation is its most known and important usage. This complex is used in many immunohistochemical and immunocytochemical staining techniques. OBJECTIVE: The aim of this study was the preparation of anti-HRP monoclonal antibody through hybridoma technology. METHODS: The BALB/c mice were immunized by repeated injections of HRP. After the confirmation of their immunization by ELISA test, the spleen lymphocytes and SP2/0 myeloma cells were hybridized using PEG as fusing agent. The hybridoma cells were then selected by culturing in HAT medium. Identification and selection of anti-HRP producing clones were done by ELISA test on culture supernatants of the obtained clones. To acquire the monoclones, limiting dilution was performed twice and the effect of finally obtained antibodies on enzyme activity was investigated by a specific ELISA test. In vivo tumor induction method was used for production of concentrated antibody. At last class and subclass of the obtained antibodies were determined by Isostrip Kit. RESULTS: After seven rounds of cell fusions, 224 clones were obtained, from which, six ones were anti-HRP producers. Two clones (P1F11 and P2F6) with higher antibody secretion were selected and subcloned. Both derived hybridoma monoclones (P1F11D2 and P2F6F3) were producing antibodies from IgG1 subclass with kappa (Kappa) light chains which didn't affect the enzyme activity. The electrophoresis of ascetic fluid of tumor induced mice showed an obvious band in gamma (gamma) position. CONCLUSION: The obtained monoclonal antibodies are from IgG class and don't affect the enzyme activity, therefore it seems that they are suitable for PAP complex production.

Structural study on immunoglobulin G solution after pasteurization with and without stabilizer.

Pasteurization was investigated as a method of inactivating virus during the preparation of immunoglobulin for intravenous use. The effect of pH, protein concentration and the presence of protein stabilizers on the structure of immunoglobulin G (IgG) molecules during pasteurization was investigated using an immunoglobulin solution derived from a Cohn's fraction II preparation. Changes in the secondary and tertiary structure of IgG molecules as well as the degree of polymerization of protein were investigated using spectrophotometry, circular dichroism and size exclusion chromatography. Only slight changes in secondary and tertiary structure were observed after pasteurization in a 10 g L(-1) immunoglobulin solution at pH 4.5 and 5.5 in the absence of stabilizer and in a 50 g L(-1) immunoglobulin solution at pH 5.5 in the presence of glycine and sucrose or sorbitol. Concentrations of immunoglobulin solution below 20 g L(-1) were not denatured when pasteurized at pH 4.5 in the absence of stabilizers. High concentrations of immunoglobulin solution required stabilizers such as glycine and sorbitol or sucrose to prevent or reduce denaturation during pasteurization.

APAAP complex: production and usage in immunocytochemical and immunohistochemical staining.

BACKGROUND: The affinity of antibody to antigen, in addition to providing the possibility of measuring the antigen in tissue extracts through methods such as RIA (Radioimmunoassay) and EIA (Enzymimmunoassay) and possibility of isolating and analyzing dispersed cell colonies using flowcytometry, makes it possible to determine the site of antigen in tissues (Immunohistochemistry) or in cells (Immunocytochemistry). OBJECTIVE: Production of APAAP complexes and comparing them with similar foreign products to determine the site of antigen in tissues or in cells. METHODS: Secreted antibodies of the two hybridomas (A(1)G(9)G(3) and A(1)G(8)F(7) produced in our laboratory) were concentrated, purified and characterized. Then the monoclonal antibodies were mixed with alkaline phosphatase enzyme (ALP) to use in immunocytochemistry (ICC) and immunohistochemistry (IHC) staining. RESULTS: Both of the cell colonies had the potentiality of producing anti- alkaline phosphatase monoclonal antibody with high affinity. The complex from mAb and enzyme - for the third phase of APAAP technique - was very effective and its sensitivity was comparable to that of the similar foreign kit. CONCLUSION: Considering the high affinity of the mAb of the two hybridomas and the stability of the complex resulted from mixing mAb and the enzyme ALP for a long time, it is possible to use the obtained APAAP complex in the immunocyto (or histo) chemistry - as the third phase.

Pattern of factor VIII inhibitors in patients with hemophilia A in the north east of Iran.

This survey was conducted to evaluate coagulation factor VIII:C inhibitors among 102 hemophilia A patients from different cities of Khorasan province in north east of Iran in order to identify and characterize the pattern of inhibitor formation in these patients population. For this purpose, we randomly obtained plasma samples of 102 hemophilia A patients (44 patients with severe, 28 patients with intermediate and 30 patients with mild hemophilia A) and studied them using two tests: the APTT mix and Bethesda test were performed. In the whole group 20 patients (19.6%) factor VIII inhibitors were detected. These were in 11 patients with severe, five patients with intermediate and four patients with mild hemophilia A. None of patients with hemophilia A had previously been studied for the presence of an inhibitor, so there was no existing history of inhibitor evaluation.

HHV-8/KSHV during the development of Kaposi's sarcoma: evaluation by polymerase chain reaction and immunohistochemistry.

The human gamma-herpes virus-8 (HHV-8) was first described in AIDS-related Kaposi's sarcoma (KS) tumour samples. In this study, we report comparative studies on paraffin-embedded biopsies of AIDS-related KS (AKS) and endemic KS (EKS) with regard to HHV-8 content as evaluated using polymerase chain reaction (PCR) and immunohistochemistry. DNA was extracted either using Chelex-100 or using Qia-gene kit and was evaluated with the help of a semiquantitative PCR assay. The PCR detection of HHV-8 was more sensitive to the Chelex method than to Qia-gene. The threshold for PCR test sensitivity with the help of serial dilution of DNA was at the level of five plasmid ORF-26 regions, and DNA from 25 body cavity-based lymphoma-1 cells. The results expressed as virus load/actin unit showed progressively higher HHV-8 levels in late (nodular) cases, compared to those in early (patch/plaque) stages. Evaluation of HHV-8 DNA levels in tumour tissues, thus, indicates a correlation between virus load and KS stage. Double immunostaining of spindle cells (SC) in KS biopsies for CD34 and HHV-8/latency-associated nuclear antigen (LANA) showed an increase in double-positive SC in the lesions of nodular AKS and EKS cases, compared to that in plaque and patch stages. However, 10-15% of CD34+/LANA- SC cells were observed during the development from patch to nodular cases of AKS and EKS. Our results indicate that PCR analysis is a simple and sensitive diagnostic method for HHV-8 evaluation in KS tissues, processed for conventional histopathology.

The effect of cryopreservation on clonogenic capacity and in vitro expansion potential of umbilical cord blood progenitor cells.

BACKGROUND: Umbilical cord blood progenitor cells have been demonstrated to possess significant advantages over bone marrow in terms of proliferative capacity and immunologic reactivity. But the low number of hematopoeitic stem cells (HSC) is the most important limitation of its use. The ex vivo expansion of cord blood progenitor cells is the current strategy to overcome this problem. Furthermore, among the factors that enable successful cord blood transplantation is the ability to store and subsequently recover a sufficient number of viable cells. Since it would be costly to expand umbilical cord blood (UCB) progenitor cells, it is important to determine the feasibility and reproducibility of progenitor cell expansion after cryopreservation. We evaluated whether cryopreservation procedures might impair the clonogenic capacity and in vitro expansion of UCB. MATERIALS AND METHODS: We evaluated the cell viability, clonogenic capacity, CD34+38- content and in vitro expansion potential of progenitor cells from UCB (n = 10) separated mononuclear cells (MNC), before and after 1 month of cryopreservation by programmed rate freezing. RESULTS: Although cell viability decreased after cryopreservation (P < .05), there was no significant difference in CD34+ or CD34+38- absolute count, colonogenic capacity and in vitro expansion potential of cord blood progenitor cells (P > .05). CONCLUSIONS: Since the survival of CD34+ cells was greater than other elements, CD34+ cells seem more tolerant to cryopreservation than the other nucleated populations. Moreover in vitro expansion of UCB progenitor cells may be obtained following cryopreservation. Our results suggest that cryopreservation procedures do not impair the clonogenic capacity and in vitro expansion potential of cord blood stem/progenitor cells.

Production of monoclonal antibody against alkaline phosphatase.

BACKGROUND: Monoclonal antibody against alkaline phosphatase (Alp) has many applications which Alp-anti Alp complex (APAAP) formation is one of the most important ones. This complex is applicable in many immunohistochemical and immunocytochemical techniques such as diagnosis of various kinds of leukemias, lymphomas, skin diseases, kidney dysfunctions, etc. OBJECTIVE: Production of anti-Alp monoclonal antibody for utilization in APAAP complex. METHODS: After several arranged injections of Alp to Balb/c mice and determining the specific antibody titer by ELISA test, the spleen lymphocytes of immunized mice and Sp2/0 myeloma cells were fused using polyethylene glycol as fusing agent and hybridoma cells were cultured in HAT medium. Identification and selection of anti-Alp producing clones were done by performing ELISA test on supernatants of all resulting clones. Limiting dilution method was used to attain monoclones and the effect of obtained antibodies on enzyme activity was investigated by a specific ELISA test. For production of concentrated Ab, the hybridoma cells were injected to peritoneal cavity of mice and the produced ascetic fluids were collected. Finally class and subclass of the obtained antibodies were determined by Isostrip kit. RESULTS: After six rounds of fusion, 104 Hybridoma clones were obtained and two Anti-Alp producing clones (A_1G_8 and A_1G_9) were selected and subcloned. Both antibodies were IgG_1 with kappa (kappa) light chains. These antibodies did not affect the enzyme activity and the electrophoresis of ascetic fluids showed an obvious band in gamma (gamma) position. CONCLUSION: Because these antibodies are from IgG class and don't affect the enzyme activity, it seems that they are suitable for APAAP complex formation.

Haemorrhagic symptoms in patients with combined factors V and VIII deficiency in north-eastern Iran.

Of the six types of dual coagulation factors deficiency, combined factors V and VIII are the most common type, a few cases of this disease have been reported in different populations. This accounts for the relatively low number of cases reported so far. Our report, which included 19 patients, is the second largest group that has been reported from one centre in north-eastern Iran. The most frequent spontaneous bleeding symptoms were epistaxis and haemarthrosis, and the most frequent traumatic bleeding symptoms were bleeding after dental extraction and bleeding after cutting any part of the body. It seemed that dual coagulation FV and FVIII deficiency is as severe as single coagulation factor (V or VIII) deficiency.