Influence of plasma interferences on screening coagulogram and performance evaluation of the automated coagulation analyzer sysmex® CS-2100i.

Background: Plasma interference is a problem for coagulation analyzers using a photo-optical detection method. Sysmex® CS-2100i is a fully-automated coagulation analyzer which has been developed to reduce this problem. Objective: To evaluate the influence of interferences on prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen analyzed by Sysmex® CS-2100i. The performance of this analyzer was also assessed in this study. Methods: Pooled plasma samples spiked with interfering substances including hemoglobin, bilirubin and lipid were used in the interference study. Real patients’ samples with these interferences were also tested. Control materials and pooled samples were used for precision, comparison, and carryover studies. Results: The PT, APTT, and fibrinogen could be analyzed in both plasma samples with interference added and real abnormal patients’ plasma samples. The deviation of PT, APTT, and fibrinogen levels was not obvious, except for an upward trend of fibrinogen level with increased hemoglobin. The highest %CV of PT, APTT, and fibrinogen were 1.24, 3.18, and 4.31, respectively for within-run and 2.32, 2.11, and 6.06, respectively for between-run precision analysis. The correlation coefficients of PT, APTT, and fibrinogen between Sysmex® CS-2100i and Sysmex® CA-1500 were 0.99, 0.99, and 0.98 with intra-class correlation coefficients of 0.99, 0.99, and 0.98 respectively. No significant carryover of specimen was observed. Conclusion: Sysmex® CS-2100i could analyze both samples with artificial interfering substance added and real abnormal patients’ samples. This machine also had an acceptable performance by our evaluation.

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.

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

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.

A 20-year-old female with impending foot gangrene: How to interpret her laboratory results.

A 20-year-old female presented with multi-system involvement. At first, she had acute cholecystis and was treated by cholecystectomy. After the operation she had neurological symptoms and was diagnosed with herpes encephalitis, confirmed by CSF serology. One month later she developed left foot arterial occlusion. Autoimmune hemolytic anemia with immune thrombocytopenia (Evan’s syndrome) and myocarditis were also detected concurrently. Her primary disease was suspected to be systemic lupus erythematosus. However, her antinuclear antibody assayed by the enzyme-link immunoassay (ELISA) method was negative twice but became positive by indirect immunofluorescence method. Other positive laboratory results were lupus anticoagulant, antineutroplil cytoplasmic antibody (ANCA), and anti proteinase 3 (anti-PR3). Their relevance will be discussed in details. She was given steroids, anticoagulants and underwent a femerofemoral bypass. Her clinical status improved afterwards.

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.

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.

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.