Variations in intraplatelet phospho-VASP expression due to pre-analytical sample preparations, illustration of a quality control issue in platelet pharmacology

Intraplatelet vasodilator-stimulated phosphoprotein [VASP] analysis is a commonly used laboratory approach for monitoring of the anti-platelet therapy with adenosine diphosphate [ADP] receptor blocking agents; however, it's testing in clinical laboratory needs a high level of experience and proficiency. The ability to recognize how the pre-analytical variations can change the results would be helpful for the interpretation of data from intraplatelet VASP analysis. The aim of this study was to describe the possible differences of intraplatelet phospho- VASP expression between washed and platelet rich plasma [PRP] samples, both at baseline levels and following experimentally induction of VASP phosphorylation. PRP and washed platelet samples were treated with different inducers of VASP phosphorylation, including forskolin [10 micro M], prostaglandin E1 [PGE1] [50 nM] and sodium nitro-prusside [SNP] [100 micro M]. Untreated PRP and washed platelet samples were also included in study as baseline controls. After labeling of platelets with either anti P-Serine157-VASP or anti P-Serine239-VASP, the samples were subjected to flow cytometric analysis to monitor the levels of intraplatelet phospho-VASP expression. Washed platelet samples tend to show increased expression of intraplatelet P-Serine[157]- VASP at baseline state and also more expression of P-Serine[157]-VASP and P-Serine[239]-VASP in response to forskolin and SNP, compared with PRP samples. Though, reduced levels of PGE1- induced VASP phosphorylation at both residues were detected for washed platelets. In this study we have provided some background information required for performing of intraplatelet VASP analysis on differently handled platelet samples and interpretation of the obtained results

[Comparison of three cord blood processing methods: hydroxyethyl starch, simple centrifugation and Sepax automated system]

Background: Different processing methods are being used to improve the quality of hematopoietic stem cell transplantation. Using hydroxyethyl starch, simple centrifugation and Sepax automation, this study was aimed to compare these three conventional methods.

Material and Methods: 90 cord blood samples were taken and processed by hydroxyethyl starch, simple centrifugation and Sepax automation methods. Then they were subjected to total nucleated cell [TNC] counting and CD34 positive counting as well as colony assay. Finally, all data were analyzed using one-way analysis of variance [ANOVA] and ps less than 0.05 were considered statistically significant.

Results: The TNC recoveries in hydroxyethyl starch, simple centrifugation and Sepax automation methods were 76%, 71% and 80%, respectively [p> 0.05]. The CD34+ cell recoveries in the Sepax automation and in the other two methods were 91% and 85%, respectively [p> 0.05]. Also, the colony assay recoveries were not significantly different among the three methods [p> 0.05].

Conclusion: No significant difference was seen in TNC number, CD34 positive counting and colony formation among the three different methods.

Frequency & specificity of RBC alloantibodies in patients due for surgery in Iran.

Background & objectives: Red blood cell alloimmunization is common in patients receiving multiple blood transfusions. Since the probability of repeat transfusion increases with longer life expectancy, it is important to study to which extent alloimmunized patients with a history of transfusion are prone to form alloantibodies after transfusion events. The aim of this study was to retrospectively analyze the alloimmunization against RBCs among transfused patients who were to undergo elective surgery in Tehran, Iran. Methods: A total of 3092 occasionally transfused patients, who were to undergo elective surgery, in four hospitals in Tehran were included in the study. For patients with alloantibodies, the data about sex, date of birth, history of transfusion, surgery, abortion and alloantibody specificity were collected. Results: Clinically significant alloantibodies were found in 30 patients. The presence of positive antibodies in the patients for whom cross-match had been done was one per cent. Most of them had surgery history or transfusion record during the preceding year. The three most frequent alloantibodies were anti-K (23.53%), anti- E (20.59%) and anti-c (17.56%). Interpretation & conclusions: The most common clinically significant alloantibodies identified in men and women were anti-K and anti-E, respectively. The most common causes of alloimmunization for men were surgery history and transfusion record and for women pregnancy.

Seroprevalence of Hepatitis B Virus infection and its risk factors in the west of Iran: a population-based study

Hepatitis B virus [HBV] infection is a serious global public health problem affecting billions of people globally. The lack of information of its seroprevalence among the general population is an obstacle for formulating effective policies to reduce the burden viral hepatitis. Therefore, this population based serological survey was conducted in Kurdistan province, where no epidemiological data was available to determine the prevalence and risk factors of HBV infection. 1613 healthy subjects were selected from all districts of Kurdistan province [in the western of Iran] using random cluster sampling. The subjects' age ranged from 6 to 65 years old. Serum samples were tested for HBcAb, HBsAg and anti HDV antibody. Screening tests were carried out by the third generation of ELISA. Various risk factors were recorded and multivariate analysis was performed. The prevalence of HBsAg and HBcAb in Kurdistan was before 0.80% [95% CI 0.44; 1.34] and 5.02% [95% CI 4.03; 6.17], respectively. None of HBsAg carriers had positive anti HDV antibody. Predictors of HBsAg or HBcAb in multivariate analysis were: older age and marriage. We did not find any significant differences between males and females. Our population based study suggests that intrafamilial HBV transmission plays a major role in HBV transmission in Kurdistan province. Furthermore, approximately 5% of general population in this province has prior exposure to HBV and less than 1% is HBsAg carriers. However, we could not find any case of HDV infection among them

Development of a novel three-dimensional biocompatible nanofibrous scaffold for the expansion and hepatogenic differentiation of human bone marrow mesenchymal stem cells

In this present study, we examined the differentiation potential of human bone marrow derived mesenchymal stem cells [hBMSCs] into hepatocytes on a three-dimentional [3D] nanofibrous scaffold formed by Poly [e-caprolactone] [PCL], collagen and polyethersulfone [PES]. The nanofiber was prepared by the electrospining technique. HBMSCs were isolated using combining gradient density centrifugation with plastic adherence. Flow cytometric analysis was used to identify the isolated MSCs. The performance of the cells on the scaffold was evaluated by scanning electron microscopy [SEM] and MTT assay. The hBMSCs were then cultured in a hepatic differentiation medium containing hepatocyte growth factor [HGF], oncostatin M [OSM] and dexamethasone [DEX] for up to 21 days. The results showed that the isolated hBMSCs expressed specific markers such as CD44, CD166, CD105 and CD13. The integrity of the MSCs was further confirmed by their differentiation potential to osteogenic and adipogenic lineages. Scanning electron micrographs and MTT analysis revealed that the cells adhered and proliferated well on the nanofibrous hybrid scaffolds. Immunocytochemical analysis of albumin and a-fetoprotein [AFP] showed the accumulation of these markers in the differentiated cells on the scaffold. Hepatocyte differentiation was further confirmed by showing expression of albumin, AFP and cytokeratin-19 [CK-19] at mRNA levels in differentiated cells. In conclusion, the evidences presented in this study show that the engineered scaffold is promising for maintenance of hepatocyte-like cells suitable for transplantation

correlation between the percent of CD3[-], CD56[+] cells and NK precursor function

The number and function of human natural killer [NK] cells are generally assessed to monitor the baseline of immune function, the effect of treatment, the progress of malignancy or metastases and diseases. NK cells recognise and kill target cells in the absence of prior sensitisation and are able to defend the host from infection of prevent the progression of a disease. Human NK cells express CD16 and CD56 which are [massively] being used as a major hallmark for the NK cell. The purpose of this study was to identify the unique subsets of peripheral blood mononuclear cells [PBMC] [%CD3[-] CD56[+] cells] by flow cytometry and to determine whether there is any correlation with functionally mature progeny of [NKp] precursor after five days of culture. The correlation was analysed using samples obtained from 120 Caucasian patietns. 20-30ml of whole blood was collected in sterile tube containing preservative free sodium heparin and a similar sample was obtained after five days. Maturation of NKp required the continuous presence of recombinant interleukin 2 [rIL-2], or interleukin 15 [rIL-15] and functional maturity of NK cells was determined by their ability to lyse target cells from the K562 cell line. The NK precursor frequency was measured by limiting dilution analysis [LDA], which the NKpf assay was set up with a range of cell dilution from 40,000 to 625 per 100 micro l/well in 96 [Eu-K562] was added and Eu release measured in culture supernatants using time-resolved fluorometry. The PBMC were set up in parallel cultures under various conditions. On day five cells were collected from culture plates and adjusted to 1x10 cells/ml and then mixed. The mixture was incubated and anti CD3 and anti CD56 were added. NK cells were enumerated in 120 patients by doubel staining with a combination of anti-CD[3]- and anti-CD56[+]. The results of these Immunophenotyping studies by flow cytometry showed no correlation between the NKpf [natural killer percursor frequency] and the percent of CD3[-]CD56[+] cell expressed after five days confirming that CD56 was inadequate as a unique marker for functional NK cells