Tissue factor expression of endothelial cell in response to atherosclerotic risk factors.

Objective: To study the effect of serum from patients with atherosclerotic risk factors on the synthesis of endothelial tissue factor. Methods: Serum from 30 diabetic patients, 30 hyperlipemic patients, 30 smokers and 30 normal serum were incubated with cultured endothelial cells from a human umbilical vein. The tissue factor of endothelial cells was measured using the assay that was developed in house after 24 hours incubation time. Results: Smokers’ serum can significantly cause the increase in endothelial tissue factor. The mean level of tissue factor induced by smokers’ serum is 1.12 microunits/cell whereas the mean level of tissue factor induced by diabetic serum, hyperlipemic serum and normal serum is 0.4, 0.48 and 0.2 microunit/cell, respectively. Conclusion: Smoking may increase the risk of thrombosis by increasing the tissue factor production of endothelial cells.

A survey of coagulation laboratory practice in Thailand: the first step to establish a National External Quality Assessment Scheme (NEQAS) for blood coagulation.

BACKGROUND: External quality assessment (EQA) is an essential component of laboratory quality assurance. In Thailand, there is no EQA program for coagulation tests at the national level. OBJECTIVE: To collect the necessary data in the first step to set up a National External Quality Assessment Scheme (NEQAS) and to assess the status of coagulation laboratory practice in Thailand. MATERIAL AND METHOD: Questionnaires were sent to hospitals to obtain information about the hospitals, their coagulation laboratory practice and EQA. RESULTS: From a dispatch of 220 questionnaires, 124 (56.4%) were returned. Of the 112 hospitals that had coagulation tests, all of them performed prothrombin time (PT), and 110 laboratories performed activated partial thromboplastin time (APTT) as well. Thirty eight percent of laboratories still used 3.8% sodium citrate as the anticoagulant for coagulation tests. The majority of laboratories (65%) reported normal control value with the patient results. Only 42% of coagulation laboratories established their own reference range. The denominators of PT ratio and APTT ratio calculations were derived from several sources apart from the mean of normal subjects. Seven of 112 (6%) laboratories participated in an EQA program. CONCLUSION: The present survey represents an overview of the current laboratory practice for coagulation tests in Thailand Improvement is necessary, and the survey results emphasize the need for establishing an EQA program in Thailand

Synthesis of von willebrand factor antigen in chlamydophila pneumoniae-infected endothelial cells.

Objective: The main objective of our study was to evaluate the response of endothelial cells infected with Chlamydophila pneumoniae (C. pneumoniae), by using von Willebrand factor (vWf) antigen as a marker for endothelial damage and dysfunction. Another objective was to evaluate the effect of cycloheximide on C. pneumoniae infectivity and vWf secretion from human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were harvested. After first passage, the HUVECs were inoculated with C. pneumoniae in three concentrations of cycloheximide (0, 1, and 2 µg/ml). At 24, 48, and 72 hours post-inoculation, supernatants from each HUVEC culture well were collected and measured for vWf antigen by sandwich ELISA as well as non-infected HUVECs were used as controls. C. pneumoniae infectivity was evaluated by indirect immunofluoresce technique and polymerase chain reaction. Results: The cycloheximide-treated HUVECs resulted in greater infection compared to the non-treated HUVECs. Means of vWf antigens from HUVECs infected with C. pneumoniae were not different from those of non-infected HUVECs. However, there was a significant change in vWf secretion when different concentrations of cycloheximide were used in the culture system. Conclusion: From our study, the results of vWf antigen secreted from HUVECs did not support the direct endothelial damage effect caused by C. pneumoniae infection. Therefore, vWf antigen is not a sensitive marker for this event. Furthermore, results from this study supported the infectious ability of C. pneumoniae on HUVECs and the importance of cycloheximide in improving the infectivity of this organism. However, the investigators had to use this substance cautiously, especially in protein synthesis study. Nevertheless, a non-variable dose of C. pneumoniae and the use of only one surrogate endothelial damage marker may limit the interpretation of this study.

Utilization of calculated low density lipoprotein cholesterol and measured low density lipoprotein cholesterol in Siriraj Hospital.

A study to determine the utilization of calculated low density lipoprotein (c-LDL) cholesterol and measured low density lipoprotein (m-LDL) cholesterol was conducted. The test results of total cholesterol, triglyceride, HDL-cholesterol and m-LDL-cholesterol from the same individuals aged > or = 18 years who had the tests done at the Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital during January to December 2004 were retrieved. The c-LDL-cholesterol level was computed using Friedewald formula. There were two data sets i.e. the m-LDL-cholesterol cut-off level derivation data set (784 subjects) and the m-LDL-cholesterol cut-off level validation data set (800 subjects). The study results revealed: 1) 2.6% of the subjects had blood triglyceride > 400 mg/dl hence c-LDL-cholesterol could not be computed, 2) the correlation between c-LDL-cholesterol levels and m-LDL-cholesterol levels from both data sets was very good (r > 0. 95, p < 0. 001), 3) the m-LDL-cholesterol levels were usually higher than c-LDL-cholesterol levels, 4) the m-LDL-cholesterol cut-off level derivation data set showed that m-LDL-cholesterol < 87, > 143, > 188, > 233 and > 254 mg/dl were highly correlated with c-LDL-cholesterol < 100, > or = 100, > or = 130, > or = 160 and > or = 190 mg/dl respectively, 5) an application of m-LDL-cholesterol cut-off levels derived from the m-LDL-cholesterol cut-off level derivation data set to the m-LDL-cholesterol cut-off level validation data set showed that m-LDL-cholesterol < 87, > 143, > 188, > 233 and > 254 mg/dl had accuracy in predicting c-LDL-cholesterol < 100, > or = 100, > or = 130, > or = 160 and > or = 190 mg/dl of 100%, 99. 7%, 100%, 100% and 100% respectively, 6) the use of m-LDL-cholesterol levels as a guide for initiating lipid-lowering agents based on cut-off values of c-LDL-cholesterol levels led to an overuse of lipid-lowering agents in 3.6% to 42.9% of the patients and 7) Nomogram for transforming m-LDL-cholesterol to c-LDL-cholesterol was developed as well as a formula for transforming m-LDL-cholesterol to c-LDL-cholesterol (c-LDL-cholesterol = 0.89 x m-LDL-cholesterol). Therefore, m-LDL-cholesterol assay has a very limited use in managing individuals with suspected or known dyslipidemia. The use of m-LDL-cholesterol level as a guide for management of abnormal LDL-cholesterol conditions leads to an overuse of lipid lowering medications and an enormous expense of m-LDL-cholesterol assay.

Evaluation of the activated partial thromboplastin time (aPTT) sensitivity to unfractionated heparin using three commercial reagents: Implication for therapeutic range.

At present, we are using the proposed therapeutic range for monitoring unfractionated heparin therapy which is the aPTT ratio of 1.5-2.5. However, the aPTT value is influenced by reagents and methods of detection. The College of American Pathologists and the American College of Chest Physicians recommended that site-specific validation of heparin therapeutic range should be established. The aim of this study was to determine the appropriate therapeutic range of unfractionated heparin therapy of our aPTT system by ex vivo study. For comparison, two other commercial reagents were also determined to observe the differences. Blood samples were drawn from 21 healthy blood donors who were not taking any medication and from other 24 patients suffering from either arterial or venous thrombosis, receiving continuous intravenous infusion of unfractionated heparin without concomitant oral anticoagulant therapy. Correlation coefficients between aPTT ratios and plasma heparin concentration varied between 0.722 (Actin FSL) to 0.817 (Actin FS). Calculated therapeutic ranges of aPTT ratios corresponding to the heparin level of 0.29 – 0.47 U/ml were 1.8 – 2.5, 1.9 - 2.5 and 2.7 - 4.6 for Actin FS, Actin FSL and Pathromtin SL, respectively. Therefore, the appropriate therapeutic range of our system obtained from this study might be aPTT ratio between 1.8 and 2.5 which is very closed to the ratio that we are using now.

The value of local ISI calibration in correcting the variability in INR determination.

We have compared the International Normalized Radio (INR) determination using the manufactures’ stated International Sensitivity Index (ISI) with an alternative method using local calibration of ISI with the calibrator plasma. It was found that the variability of the INR was less when the results were expressed by INR using local calibration of ISI. The results indicated that the local ISI calibration might reduce the variability in the INR determination.

Evaluation of home-made ELISA's for protein C and protein S antigenic assays.

Home-made enzyme-linked immunosorbent assays (ELISA) for protein C (PC) and protein S (PS) antigenic assays, using commercial antibodies, were set up in our laboratory. The latter can be used for the measurement of total PS and also free PS, after the precipitation of bound form. Here we describe the procedure for both PC and PS ELISA's, and their quality evaluation and cost. Intra- and inter-assay variation (n = 20) were calculated to be 7.3% and 8.1% for the PC ELISA and 10.2% and 10.1% for free PS ELISA. The accuracy of the tests assessed by external quality assurance of WHO International External Quality Assessment Scheme in Blood Coagulation (IEQAS) was satisfactory. The level of PC antigen in 50 healthy volunteers was 89 ฑ 18% and that of free protein S was 94 ฑ 16%. In conclusion the quality of the home-made ELISA's was acceptable while the cost was much cheaper than that of commercial ELISA kits.

The occurrence of granulation tissue at Vaginal vault from different suture materials of vault closure following hysterectomy.

A randomized double blind clinical trial was carried out in order to compare the occurrence of vaginal vault granulations between vaginal closure, using interrupted figure-of-eight sutures with chromic catgut and poliglecaprone 25 (MonocrylR). One hundred and twelve healthy patients who underwent total abdominal hysterectomy were randomly assigned to one of the two studied suture materials. Six weeks after the operation, the patients were examined blindedly whether there were vaginal vault granulations. The characteristics of both studied groups were comparable. The incidence of vaginal vault granulations in patients was 33.9%. The granulations occurred in 19.6% in the poliglecaprone 2. And 48.2% in the chromic catgut suture material group (P<0.01). In patients with vaginal vault granulation, 76.3% had abnormal vaginal discharge and/or bleeding, while only 6.7% of those without had (P<0.01). Closure of vaginal vault with poliglecaprine 25 should be employed in case of total abdominal hysterectomy because of decreasing the occurrence of vaginal vault granulations later on.

Accuracy of speculoscopy in detection of cervical neoplasia.

From May 1995 to June 1996, a total of 256 patients with associated gynecologic conditions requiring further cervical biopsy, cervical conization or total abdominal hysterectomy at Siriraj Hospital underwent examination for cervical neoplasia by speculoscopy without prior knowledge of the results of previous Pap smears. Compared with the histological results for the cervix, the accuracy, sensitivity, specificity, positive predictive value, negative predictive value, false positive rate and false negative rate of speculoscopy in the detection of cerical neoplasia were 82.0, 84.6, 79.2, 82.1, 81.9, 20.8 and 15.4 per cent respectively. When speculoscopy and Pap smear were combinded, sensitivity increased and the false negative rate decreased. In places where cervical cancer screening by Pap smear are not well established, speculoscopy could be an alternative method offering fair to good results compared with Pap smear.