Comparison of intensity of platelet aggregation between patients receiving low and high aspirin dosage in post CABG patients.

Background: Aspirin used after coronary artery bypass graft surgery (CABG) improved patient survival and reduced graft thrombosis. However, individual variations in the antiplatelet effect of aspirin have been reported among CABG patients. Objective: To compare the intensity of platelet aggregation between patients receiving low and high aspirin dosage in post CABG patients. Methods: We prospectively studied the effect of aspirin dosage on platelet aggregation in 100 CABG patients. Oral aspirin was discontinued prior to CABG and re-started within 12 hours after CABG. Blood samples were collected and transferred to a laboratory prior to surgery then again on postoperative days two and eight for platelet aggregation test and platelet count within three hours after venipuncture. Results: One hundred patients (sixty five male and thirty five female patients) post coronary artery bypass graft (CABG) were evaluated for eligibility to enter the trial. The percentage of platelet aggregation was compared between low dose (<100 mg/day), and high dose (>100 mg/day) aspirin, at postoperative CABG days 2 and 8, which showed no significant difference for the platelet aggregation (p = 0.161 post CABG day 2 and p = 0.098 post CABG day 8). Conclusion: Low dosage aspirin should be used in post CABG patients because the intensity of platelet aggregation between patients post CABG receiving low and high aspirin dosage were not different, while the prophylactic effect of the low aspirin dosage in reducing the risk of cardiovascular events proved equally as effective as the high aspirin dosage.

Whole blood clotting time: Variation of practice in coagulation laboratory, members of Thailand national external quality assessment scheme.

Objective: To collect the necessary data for a perspective of Whole Blood Clotting Time (WBCT) practice in Thailand. Methods: In March 2007, 124 questionnaires were sent to laboratory members of the Thailand National External Quality Assessment Scheme (Thailand NEQAS) to obtain essential information about the WBCT practice. Results: From a dispatch of 124 questionnaires, 120 (96.77%) were returned. There were 101 (84.1%) hospitals performing WBCT in the laboratories and the mean number of WBCTs performed was 16.17 times/month. Eighty nine laboratories (88.11%) used the modified Lee-White methods. Seventy four laboratories (73.26%) used snake bite and other animal bites as the indication for WBCT. Thirty three laboratories (34.37%) had problems performing the WBCT. Conclusion: The WBCT methods among the practice of the Thailand NEQAS laboratory members were as various as problems concerning WBCT throughout Thailand. Their practice needs to be improved and standardized by proper education. It also emphasizes the need for an appropriate guidelines for WBCT in Thailand.

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.

An evaluation of B-type Natriuretic Peptide in Addition to Myoglobin, Creatine Kinase-MB, and Troponin I on the Emergency Department Patients with Acute Myocardial Infarction.

Objective: To assess the accuracy of B-type natriuretic peptide (BNP) in addition to myoglobin, creatine kinase-MB (CK-MB), and troponin I to diagnose patients with non ST-segment elevation myocardial infarction (NSTEMI) at the emergency department. Methods: During January to July 2007, a total of 100 patients with suspected acute myocardial infarction at the emergency department were included. 50 were classified as NSTEMI and 50 as non-NSTEMI according to the final hospital diagnosis. Blood samples for investigation of myoglobin, CK-MB, troponin I, and BNP analysis were collected in EDTA tubes concomitantly with routine blood specimens from the emergency department and measured by Biosite Triage Cardioprofiler Panel (Biosite Inc., San Diego, CA) Results: The diagnostic sensitivity of Myoglobin and BNP (cut-off value of 100 pg/mL) for acute myocardial infarction (AMI) was significantly higher than CK-MB and troponin-I at the emergency department (76 and 82 vs. 36 and 24 %, respectively, P< 0.001). BNP in addition to myoglobin, CK-MB, and troponin I improved the diagnostic sensitivity from 86% to 100%. The optimum cut-off point levels for myoglobin, CK-MB, troponin-I, and BNP were 150 ng/mL, 3.8 ng/mL, 0.15 ng/mL and 147 pg/mL respectively. Using the optimal cutoff point, the sensitivity was 96 % and specificity was 46 % in diagnosis for myocardial infarction. Conclusion: Multiple cardiac markers by use of quantitative point-of-care testing for myoglobin, CK-MB, troponin-I and BNP are useful for ruling out patients presenting to the emergency department with suspected NSTEMI.

A comparative study of portable monitors (CoaguCheck XS) for international normalized ratio (INR) determination with a laboratory-based system for control of oral anticoagulant treatment.

long-term warfarin therapy. Methods: 39 patients receiving long-term warfarin were eligible in this study. Parallel INR measurements were performed. Capillary INR (INR_C) measurements were determined with CoaguCheckÒ XS and venous INR (INR_V) were determined with standard laboratory methods. Results: We found an excellent correlation coefficient (r2 = 0.968, 95%CI = 0.82 – 0.99) between INR_V and INR_C among 39 patients receiving long-term warfarin. The mean difference between the two methods was 0.16 (p<0.0001). Although these differences were statistically significant, they were not clinically significant. In 97.4% of the INR parallel measurements the differences between the two methods were within 0.5 INR units. The Bland-Altman difference plot showed greater variation with increasing mean INR values. The coefficient of variation of CoaguCheckÒ XS was 1.07%. Conclusion: The CoaguCheckÒ XS was comparable in accuracy to a standard laboratory method. Its precision was good. It might be a suitable alternative to monitor INR values among patients receiving oral anticoagulants by increasing patient compliance with INR monitoring, and facilitating more frequent INR monitoring especially in highly educated patients.

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

Prevalence of 60 kda and 52 kda Ro/SS-A autoantibodies in anti-Ro positive Thai sera in Siriraj Hospital.

BACKGROUND: Anti-Ro antibody may directly react against either Ro60 or Ro52 or both antigens. To be more applicable for routine laboratory practice, the specific antigen type for antibody detection should be identified before test application. OBJECTIVE: Investigate the prevalence of 60 kDa and 52 kDa Ro/SS-A antibodies in Thai patients' sera in Siriraj Hospital. MATERIAL AND METHOD: Specimens for anti-Ro were requested between June and December 2005. They were tested with EUROLINE test kit for prevalence determination. The principle of the test is a qualitative in-vitro-assay that contains test strips coated with parallel lines of 14 highly purified antigens. Of 84 specimens requested for anti-Ro antibody, 76 were collected and tested with the EUROLINE test kits and eight were excluded due to inadequacy. RESULTS: The prevalence of anti-Ro60 and anti-Ro52 of all sera tested for anti-Ro by EUROLINE test kit were 30% (95% CI: 20-40%) and 26% (95% CI: 16-36%), respectively; and, those in anti-Ro positive Thai sera were 82% (95% CI: 68-96%) and 71% (95% CI: 54-88%), respectively. The prevalence of anti-Ro52 alone in anti-Ro positive Thai sera and all specimens requested for anti-Ro was about 18% (95% CI: 4-32%) and 7% (95% CI: 1-13%), respectively. The agreement and Kappa value between the two methods were 0.9 and 0.77, respectively. The study suggests that the test for anti-Ro detection should provide both Ro 60 and Ro 52 antigens. CONCLUSION: The prevalence of both anti-Ro 60 and anti-Ro 52 were quite common, therefore, the test for this specific antibody should provide both antigens for antibody detection.

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.

Analysis of serum ceruloplasmin levels in wilson disease.

C Nisarat Opartkiattikul, M.D. Department of Clinical Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. Siriraj Med J 2006;58: 610 E-journal: http:// www.sirirajmedj.com eruloplasmin is copper-containing protein of plasma which is synthesized mostly in the liver. Reduc tion in serum concentration of ceruloplasmin are diagnostically significant of Wilson disease Menkes disease, and nutritional copper deficiency.1 Indications for ordering ceruloplasmin are hepatitis-marker-negative liver disease in childhood or adolescence (suspected Wilson disease), neurodegenerative symptoms and signs of connective tissue disease in infants and small children (suspected Menkes disease), and hypochromic microcytic, iron-refractory anemia (suspected nutritional copper deficiency). At Siriraj Hospital, there are approximately twenty orders of ceruloplasmin level per month. Ceruloplasmin can be assayed either immunochemically (radial immunodiffusion, immunonephelometry or immunoturbidimetry) or functionally (copper oxidase activity). The latter assay measures only native, coppercontaining ceruloplasmin, whereas the formers measure both the intact molecule and, to varying degrees, apoceruloplasmin and proteolytic fragment. The oxidase activity method is more difficult to perform and less specific but it may provide better clinical information.2 Comparative analysis of serum ceruloplasmin levels in Wilson disease by oxidase activity method and immunonephelometric method reported in this study revealed a high correlation between the two methods. This result suggests for replacing the conventional oxidase activity method by immunonephelometric method which is simpler, automated and has a well accepted quality control. Even though the immunonephelometric method cannot report the ceruloplasmin result lower than 8 mg/ dL, this is of no clinical significance because the cut off level for diagnosis of Wilson disease is at 13mg/dL. REFERENCES 1. Kratz A, Lee-Lewandrowski E, Lewandrowski K. The plasma proteins. In: Lewandrowski K, ed. Clinical-laboratory management and clinical correla tion. Philadelphia, Lippincott, Williams & Wilkins, 2002:531-60. 2. Johnson AM, Rohlfs EM, Silverman LM. Protein. In: Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry, 3rd ed. Philadelpha, W.B.Saunders Co., 1999:477-540.

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.

An abnormal systemic and regional hypercoagulable state in patients with mitral stenosis.

Mitral stenosis still remains a major problem in Southeast Asia including Thailand. It contributes to the morbidity and mortality related to thromboembolism which was associated with the left atrial thrombus. However, the pathogenesis of left atrial thrombus in these patients is not completely understood. Therefore, the objective of this study was to investigate the coagulation and platelet activity including the function of the endocardium in the left atrium and peripheral circulation in patients with mitral stenosis who were free of left atrial thrombus and to compare those hematologic markers activity in the peripheral venous blood between the patients with mitral stenosis and the control. Thirty-six patients with moderate to severe mitral stenosis were included in the study. Most of the patients were in functional class II and 50 per cent had atrial fibrillation. Blood was obtained from the femoral vein, femoral artery, pulmonary artery and left atrium of these patients before heparin was administered to determine the value of various hematologic markers. In the control group, blood for determining the hematologic markers was collected only from the antecubital vein. The results of this study demonstrated that the levels of prothrombin activation fragment 1+2 (F1+2), thrombin-antithrombin III complex (TAT) and Beta-thromboglobulin (beta-TG) in the left atrium of the patients with mitral stenosis were significantly higher than those in the femoral vein and femoral artery, whereas the level of thrombomodulin was significantly lower in the left atrium compared with the femoral artery and vein. When comparing with the control group, the levels of TAT, plasminogen activator inhibitors-1 (PAI-1) from the peripheral vein were significantly higher and the level of thrombomodulin was also significantly lower in the patients with mitral stenosis. In conclusion, the present study demonstrated an abnormal hypercoagulable state of the left atrium and systemic circulation related to the abnormalities of coagulation, platelets and the endocardium which may cause the formation of left atrial thrombus in patients with mitral stenosis.

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.

Discordant cardiac troponin results in amphetamine - related rhabdomyolysis.

A case of a patient who developed amphetamine-related rhabdomyolysis and acute renal failure after an intestinal operation is reported. He initially had biochemical evidence of myocardial injury, with a concomitant increase in cardiac marker proteins CK-MB, cardiac troponin T (cTnT) and cardiac troponin I (cTnI) during the acute event. Following intensive treatment and improvement in renal function, levels of all myocardial marker proteins fell towards the normal range. Late in the course of the disease, however, there were re-elevations of CK-MB and cTnT, but not of cTnI, to levels exceeding 14 fold and 8 fold the upper limit of the reference range, respectively. Since, at present the possibility of re-expression of both CK-MB and cTnT in damaged and regenerating skeletal muscle can not be ruled out, the late occurrence of increased CK-MB and cTnT in our patient should not be interpreted as evidence of recurrent myocardial injury.

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.

Comparison of the efficacy of the weight-based heparin dosing nomogram and standard method.

Nowadays, heparin is extensively used in patients with various medical diseases without standardized dose adjustment. Previous studies revealed that delayed therapeutic level achievement led to suboptimal outcome. Objective: To determine the most effective method of heparin dose adjustment and factors affecting appropriate heparin dose. Methods: All patients requiring heparin treatment were monitored for APTT ratio in phase 1 (conventional method), then randomized into Standard and Weight-based method in phase 2 with the same manner of APTT ratio monitoring. Results: Student t-test for primary outcome favored the Standard dosing heparin nomogram in comparison with conventional method at 18 h ( p-value = .0006) and 24 h ( p-value = .03) and the Weight-based nomogram at 18 h ( p-value = .02). At 24 h, 21 of 28 patients ( 75%) of the Standard nomogram group achieved the therapeutic range, comparing with 14 of 28 patients (50%) in the Weight-based nomogram group and 15 of 39 patients ( 38%) in conventional group. Only age and body weight may affect the appropriate heparin dose. Conclusion: The Standard heparin nomogram was more effective and practical in heparin dose adjustment. Factors affecting appropriate heparin dose were age and body weight.

Prothrombin fragment 1+2 and fibrinogen in Thai type 2 diabetic patients.

Since the evidence for the hypercoagulable state in terms of prothrombin fragment 1+2 (F1+2) in Thai diabetic patients has never been reported, we studied plasma F1+2 levels in 68 type 2 diabetic patients and in 20 normal age-matched volunteers. Fibrinogen, D- dimer, glucose, HbA1C, cholesteroi, triglyceride, HDL-cholesterol and creatinine were also determined. It was found that the levels of F1+2 and fibrinogen in the diabetic patients were significantly higher than in the controls (p<0.001 and p<0.01 respectively), while D-dimer was detected positively in 17 out of 64 patients whereas none could be detected in the 20 healthy volunteers. A total of 23 out of 68 patients had higher levels of F1+2 than the normal range. When we compared the clinical characteristics, blood chemistry analysis and hypercoagulable markers of the diabetic patients between the groups of high F1+2 and normal F1+2, there were significantly higher numbers of positive D-dimer cases in the high F1+2 group compared with the normal F1+2 group (p=1.01). The correlation between F1+2 vs diabetic duration was 0.29 with p value less than 0.05. This study suggests that there are hypercogulable states in Thai diabetic patients.

Effect of endothelial cells from babies born to pre-eclamptic mothers on tissue factor activity of normal endothelial cells.

A technique to measure the tissue factor activity of endothelial cells was developed and the tissue factor activity of the endothelial cells was studied after stimulation with cultured media from the endothelial cells of babies born to severe per-eclampsia patients and from normal pregnancies. The results show no statistical differences. Media from the endothelial cells of the babies of pre-eclampsia patients induced the tissue factor activity to a mean level of 0.08 mU/ml, whereas that from the normal pregnancies stimulated the tissue factor synthesis to a mean level of 0.14 mU/ml. It may be concluded that there are no injurious substances released from the endothelial cells of babies that cause increased tissue factor synthesis from endothelial cells.

Assessment of the coagulation activation in normal pregnancy and severe pre-eclampsia using D-dimer levels.

D-dimer was measured in 20 normal non-pregnant women, 20 normal pregnant women and 20 severe pre-eclampsia patients. D-dimer was found to be 182 + 63 ng/ml (mean+ SD) in non-pregnant women, significantly higher at 1,355+ 279 ng/ml in normal pregnant women and 1,928+ 625 ng/ml in severe pre-eclampsia. No abnormalities were detected in a screening coagulogram. It is concluded that D-dimer may-be more useful than a screening coagulogram. For the early detection of the activation of the coagulation system that occurs in normal pregnancy and becomes elevated in severe pre-eclampsia.

Preoperative autologous blood collection and haemodilution with dextran.

Thirty patients, ASA class I-II with haemoglobin level > 12.5 gm percent, who underwent elective gynaecological surgery were included in the study. Control blood samples for haematocrit, haemoglobin, electrolytes, serum osmolarity, coagulogram and platelet function were investigated before the autologous blood was collected and haemodiluted with 3 percent dextran-40 solution. The cardiovascular responses, arterial blood gases, urine output and central venous pressure were also recorded. After the end of haemodilution, another blood samples (as study samples) were once collected for analysis. Haematocrit, haemoglobin, sodium and potassium showed to decrease significantly. However, the serum osmolarrity, coagulogram and platelet function had no any significant differences. All parameters were within normal limits. In conclusion, the autologous blood collection and haemodilution technique was suitable and possibly practical in ever elective surgical patients without any undesirable side effects.