Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

BACKGROUND: Fetal bovine serum (FBS) has been used to support the growth and proliferation of mammalian cells for decades. Owing to several risk factors associated with FBS, several trials have been conducted to evaluate substitutes to FBS with the same efficiency and the lower risk issues.METHODS: In this study, human platelet lysate (HPL) derived from activated human platelets was evaluated as an alternative to FBS due to the associated risk factors. To evaluate the efficiency of the preparation process, platelet count was performed before and after activation. The concentrations of several growth factors and proteins were measured to investigate HPL efficiency. HPL stability was studied at regular intervals, and optimal heparin concentration required to prevent gel formation in various media was determined. The biological activity of HPL and FBS was compared by evaluating the growth performance of Vero and Hep-2 cell lines.RESULTS: Result of platelet count assay revealed the efficiency of HPL preparation process. Growth factor concentrations in HPL were significantly higher than those in FBS, while the protein content of HPL was lower than that of FBS. Stability study data showed that the prepared HPL was stable for up to 15 months at −20℃. Ideal heparin concentration to be used in different media was dependent on calcium concentration. Results of cell viability assay showed that HPL was superior to FBS in supporting the growth and proliferation of Vero and Hep-2 cells.CONCLUSION: The HPL prepared by the mechanical activation of platelets may serve as an efficient alternative to FBS in cell culture process.

protective effect of silymarin on the suprarenal gland in chlorpyrifos intoxicated rats

Chlorpyrifos [CPF] is a widely used organophosphorous insecticide that induces toxic effects in man and animal through its inhibitory action on acetyl choline esterase enzyme. The present work aimed to evaluate the toxic effect of chlorpyrifos on the function and structure of the suprarenal gland and to investigate the possible protective effect of silymarin antioxidant against such toxicity. The study included twenty four adult male rats they were equally divided into four groups as follows: a control group [n=6] received the oral vehicle only [corn oil], a Silymarin group [n=6] received Silymarin in a dose of 6mg/kg b. w orally twice weekly for four weeks, the intoxicated group [n=6] received CPF 5mg/kg b. w, orally twice weekly for four weeks and the protection group [n=6] receivied silymarin orally 6mg/kg b. w. half an hour after chlorpyrifos, administration in a dose of 5mg/kg b. w. orally twice weekly for four weeks. By the end of the experiment, estimation of the following biochemical parameters was done: plasma choline esterase enzyme activity level, serum cholesterol, serum cortisol and serum testosterone. The suprarenal gland was examined by the light microscope using routine H and E stain and chromaffin stain reaction as well as by the Transmission Electron Microscope. The measured biochemical parameters showed significant variation in CPF group compared to the control. The chlorpyrifos intoxicated group revealed affection of the cells of the suprarenal cortex and medulla with variable degrees of degenerative changes. The protection group showed improvement of the levels of the biochemical parameters with partial restoration of the normal histological features of the suprarenal structure compared to the CPF intoxicated group. Accordingly, it was proved that silymarin is a reliable antioxidant that could protect against the toxic effect of chlorpyrifos on the suprarenal gland

Comparative study on the effect of some histological techniques on the quantitative morphometric analysis

Quantitative morphometric studies are commonly used nowadays in histological and pathological labs and researches. The aim of the study was to evaluate the possible effect of the histological techniques on the morphometric results and determination of correction coefficients of morphometric parameters in dependence on the histological procedure used. The organs and cells subjected to study were; human RBCs [lack of nuclei], albino rats liver cells [active cells] and albino rat uterus [for gross study and its muscles and fibers content]. Three techniques were selected; fresh cells [blood smear and liver cell print], frozen technique and paraffin sections fixed in neutral buffered formol [common histological technique]. Quantitative morphometric analysis parameters selected were diameter, perimeter, area and area percent. The obtained results were statistically analyzed by using student paired t-Test. The study showed statistically significant changes in quantitative morphometric results under the effect of histological techniques used. Frozen technique increased the morphometric results, while paraffin technique decreased them when compared with those of fresh data. Also the morphometric data of gross area and perimeter of organs affected by their fibrous elements. The study concluded that all quantitative morphometric results must be corrected by a coefficient factor which depends on the organ and technique used before analysis and evaluation of data

Evaluation of placental blood flow in pregnancy induced hypertension and pre-eclamptic patients [quantitative microscopic study]

A prospective controlled study to measure quantitatively placental and fetal blood volume as an area percent in normal pregnancy and in pregnancy complicated by induced hypertension [PIH], preeclampsia [PE] and correlate the findings with fetal outcome. It was conducted on 50 patients attended the labour ward of Bab El Shaaraya Maternity Hospital. They were divided into 2 groups. The control group consisted of 10 singletone full term normal pregnant patients. The study group consisted of 40 singletone, full term pregnant patients and their pregnancy is complicated either by PIH or PE. Twelve of them were pre-eclamptic, 8 patients had severe pre-eclampsia [SPE] and 20 patients had PIH. All patients were in active process of labour, fetal and maternal monitoring was performed. Mode of delivery, fetal weight and Apgar score were reported. Placental weight, diameter, thickness were recorded. Placental paraffin section were made, stained with Hx. and Eosin stain and examined for morphological changes. Then quantitative analysis of maternal and fetal blood as an area percent was done using Leica Quantimet 500+ Image Analysis System. Twenty different microscopic fields of lmm[2] surface area in each section were examined. Results of PIH showed a high significant increase [p < 0.005], while SPE showed highly significant reduction [p<0.001] in maternal blood area percent compared to the control placenta. There was no significant difference between PE maternal blood area percent and the control patients. There was a significant reduction in fetal blood area percent in PE and SPE patients compared to the control group, while no significant difference was noted between PIH patients and the control group. Placental weight of SPE patients was significantly lower than that of the control patients. The PIH and PE patients had non-significant difference in placental weights compared to the control. Newborns of PE and SPE patients showed a significant reduction of weight compared to control group newborns, but those of PIH patients were not significantly different in weight from the control group newborns

DNA flow cytometryn in colorectal carcinomas: Egyptian experience

The prognostic value of flow cytometric ploidy and S- phase measurements together with various morphological characteristics of the tumors were investigated in the study of 40 colorectal carcinomas. Repeated measurements of 324 paraffin embedded or fresh samples from the 40 tumors demonstrated the high reproducibility of ploidy and S-phase analysis. Tumor heterogeneity showing diploid and aneuploid cell population or aneuploid cell population with various ploidy values by measuring a mean of 4.6 samples/tumor was found in 40%, while the proportion of aneuploid tumors increased significantly with the grade but not with the stage of the tumors. The percentage of S- phase cells was correlated with the stage but not with the grade. It was found that staging, ploidy level, percentage of S-phase, histopathological types and grading had a prognostic value after a follow up of 24 months. Multivariate analysis using Cox's proportional hazard model revealed independent prognostic value for Dukes's stage and ploidy level

Rigid internal fixation in phalangeal hand fractures

Fifty-one patients with 64 recent fracture phalanges were treated with fixation using the mini fixation system of the ASIF group. There were 36 closed, and 28 open fractures. Fourty-two fractures were fixed with screws only, and twenty-two with mini plate and screws. Closed fractures fixed with screws gave 95.8% satisfactory results, while open fractures gave 61.1% satisfactory results. Closed fractures fixed with mini plate and screws gave 58.3% satisfactory results, while open fractures gave 10% satisfactory results