Performance evaluation of thrombomodulin, thrombin-antithrombin complex, plasmin-α2-antiplasmin complex, and t-PA: PAI-1 complex.

BACKGROUND: To conduct a comprehensive performance evaluation of a fully automated analyzer for measuring thrombomodulin (TM), thrombin-antithrombin complex (TAT), plasmin-α2-antiplasmin complex (PAP), and t-PA: PAI-1 complex (tPAI-C). METHODS: According to the Clinical and Laboratory Standards Institute (CLSI) EP05-A2, EP06-A specifications, TM, TAT, PAP, and tPAI-C were analyzed to evaluate intraassay variability and interassay variability, linear range, carryover rate, reference range, sample stability, and interferences. RESULTS: The intraassay variability and interassay variability of the four factors were all below 5%. The carryover rates were below 1%. Linear verification analysis revealed correlation coefficients of 0.998-0.999. The recommended reference ranges of TM, TAT, and PAP were appropriate for our laboratory, whereas the reference of tPAI-C should be established by each laboratory. Stability assessment revealed that TM is stable for 2 days at room temperature but lacks stability at colder temperatures. In contrast, TAT is stable for 5 days at 4°C and -20°C but has poor stability at room temperature. PAP and tPAI-C are stable for 3 days at all three temperatures. The measurement of TM, TAT, PAP, and tPAI-C is not altered by the presence of 510 mg/dL hemoglobin, 1490 FTU triglycerides, or 21.1 mg/dL conjugated and free bilirubin. CONCLUSION: The determination of TM, TAT, PAP, and tPAI-C using a high-sensitivity chemiluminescence analyzer performs well in terms of precision, carryover rate, linear range, and interference. Thus, this method is suitable for the detection of these substances in clinical specimens.

Biological Variations of Lupus Anticoagulant, Antithrombin, Protein C, Protein S, and von Willebrand Factor Assays.

The results of lupus anticoagulant (LA), antithrombin (AT), protein C (PC), and protein S (PS) testing, and the values of von Willebrand factor antigen (VWF:Ag) are important in diagnosis and therapeutic monitoring of thrombosis and hemostasis diseases. Till now, no published study has focused on the biological variations in LA testing, and only a few studies have examined the biological variations of AT, PC, PS, and VWF:Ag. With the latest fully automated instruments and improved reagents, the analytical, within-subject, and between-subject biological variations were estimated for these five coagulant parameters in a cohort of 25 apparently healthy subjects. Blood specimens were collected at 8:00 am, 12:00 pm, and 4:00 pm on days 1, 3, and 5. The analytical biological variation (CV(A)) values of all the parameters were less than 3%. The within-subject biological variation (CV(W)) and between-subject biological variation (CV(G)) values of the LA normalized ratio were 4.64 and 6.83%, respectively. No significant differences were observed in the intraday and interday biological variations of LA tests, or in AT, PC, PS, and VWF:Ag values. Additionally, the utility of the conventional population-based reference intervals of the five coagulation parameters was evaluated by the index of individuality, and data on CV(W) and CV(A) were used to calculate the reference change value to identify the significance of changes in serial results from the same individual.

Moderate-intensity exercise improves the thromboelastography coagulation index in children with severe hemophilia A.

This pilot study explored the effect of moderate-intensity exercise on factor VIII (FVIII) activity and global hemostatic status of the children with severe hemophilia A. Eleven children aged 6 to 15 years with severe hemophilia A participated in a moderate-intensity exercise test by using Recumbent Cross Trainer (NuStep, T5XR) for at least 10 min after reaching the target heart rate or until volitional exhaustion within a safety framework. Blood samples were collected pre and postexercise for plasma FVIII: C and thromboelastography (TEG) parameters and coagulation index. The average duration of exercise was 11.8 min (10-13 min). There was no report on bleeding events or adverse symptoms requiring termination of the exercise test. The average FVIII activity of the 11 children was 0.66 (0.5-0.8) IU/dl before and 0.93 (0.5-2.3) IU/dl after exercise. The increase of FVIII in the 11 children as a group was not statistically significant (P = 0.052). There were significant changes of TEG measurements, with shortening of R (P < 0.05), and increase in K decrease (P < 0.05), alpha angle (P < 0.05), maximum amplitude (P < 0.05), and coagulation index (P < 0.01). Among the 11 children, the relative coagulation index increase after exercise was greater than 50% in seven (63.6%), less than 20% in three (27.3%), and less than 10% in one (9.1%). TEG analysis showed that the global hemostatic function for the children with severe hemophilia A can be enhanced after moderate-intensity exercise.

[Research of the efficiency of different lupus anticoagulant assays].

OBJECTIVE: To provide valuable test by evaluating the efficiency of lupus anticoagulant assays based on different principles. METHODS: 370 citrated samples were recruited, LA were tested by dRVVT, SCT, APTT-Actin, APTT-Actin FSL and APTT-SS respectively, and 1:1 mixing tests before incubation were studied in all the recruited subjects. For LA testing, the results of 1:1 mixing test were expressed as index of circulating anticoagulant (ICA), and the confirm test results were expressed as percent of correction (C) and normalized ratio (NR). The reference interval (RI)/Cut-off values of all the assays were established, and the efficiency of all the assays were further studied. RESULTS: The RI/Cut-off values of all assays were different. The sensitivity of dRVVT were the highest among all the assays (70.3%-93.1%), and the sensitivity of APTT (<55.0%) were the lowest. The sensitivity of APTT-SS (47.5%-53.5%) were higher than APTT-Actin (32.7%-39.6%) and APTT-Actin FSL (29.7%-36.6%). The specificity of dRVVT, SCT and three APTT were 91%-100%, 80.6%-100%, and 85.1%-98.5%, respectively. The specificity of SCT confirm test were 100%. Sensitivity of all the five methods were decreased in the weak positive LA. The sensitivity of dRVVT and SCT were more than 50.0%, however, the sensitivity of APTT-Actin and APTT-Acitn FSL were less than 16.0%. The sensitivity of APTT-SS were 23.7%-30.5%. Except the APTT-SS, dRVVT and SCT were correlated with thrombotic risk in APS patients (OR: 5.562-9.240, P<0.05). CONCLUSIONS: Every Laboratory should establish local LA RI/Cut-off values. dRVVT is the best assay for LA test, and APTT-SS method is superior to the other APTT method. The different test combinations of dRVVT and SCT are correlated with thrombotic risk in APS patients.

Procoagulant imbalance aggravated with falling liver function reserve, but not associated with the presence of portal vein thrombosis in cirrhosis.

OBJECTIVES: Hypercoagulability, hemodynamic changes, and endothelial injury are the three major contributors to the development of thrombosis. However, the role of hypercoagulability in portal vein thrombosis (PVT) in liver cirrhosis is still controversial. The aim of this study is to elucidate the relationship between procoagulant imbalance and PVT in patients with liver cirrhosis. METHODS: This study included 151 patients with cirrhosis with (n=20) or without PVT (n=131). Levels of procoagulant factor (FVIII) and anticoagulants [protein C (PC), protein S (PS), and antithrombin (AT)] were measured. Procoagulant imbalance was also evaluated using a thrombin generation test with/without Protac and the results were expressed as Protac-induced coagulation inhibition percentage (PICI%). The lower the PICI% value, the greater the procoagulant imbalance. RESULTS: The levels of PC (P<0.001), PS (P<0.05), and AT (P<0.001) decreased progressively from Child-Pugh A to C in all patients, whereas the levels of FVIII did not alter with the severity of cirrhosis (P>0.05), which indicated the balance tilting toward procoagulation in liver cirrhosis. Similarly, the PICI% values decreased from Child-Pugh A to C (P<0.001). However, there were no differences in the levels of PC, PS, AT, FVIII or PICI% between patients with and without PVT (P>0.05), even after stratification by Child-Pugh classification (P>0.05). CONCLUSION: Procoagulant imbalance is not associated with the presence of PVT in patients with cirrhosis, although the imbalance worsens with the severity of cirrhosis.

Biological and analytical variations of 16 parameters related to coagulation screening tests and the activity of coagulation factors.

To accurately estimate longitudinal changes in individuals, it is important to take into consideration the biological variability of the measurement. The few studies available on the biological variations of coagulation parameters are mostly outdated. We confirmed the published results using modern, fully automated methods. Furthermore, we added data for additional coagulation parameters. At 8:00 am, 12:00 pm, and 4:00 pm on days 1, 3, and 5, venous blood was collected from 31 healthy volunteers. A total of 16 parameters related to coagulation screening tests as well as the activity of coagulation factors were analyzed; these included prothrombin time, fibrinogen (Fbg), activated partial thromboplastin time, thrombin time, international normalized ratio, prothrombin time activity, activated partial thromboplastin time ratio, fibrin(-ogen) degradation products, as well as the activity of factor II, factor V, factor VII, factor VIII, factor IX, and factor X. All intraindividual coefficients of variation (CVI) values for the parameters of the screening tests (except Fbg) were less than 5%. Conversely, the CVI values for the activity of coagulation factors were all greater than 5%. In addition, we calculated the reference change value to determine whether a significant difference exists between two test results from the same individual.

Improved glomerular filtration rate estimation using new equations combined with standardized cystatin C and creatinine in Chinese adult chronic kidney disease patients.

OBJECTIVES: The newly developed glomerular filtration rate (GFR)-estimating equations developed by the CKD-EPI Collaboration and Feng et al. (2013) that are based on standardized serum cystatin C (ScysC), combined/not combined with serum creatinine (Scr), require further validation in China. We compared the performance of four new equations (CKD-EPIcys, CKD-EPIcr-cys, Fengcys, and Fengcr-cys equations) with the CKD-EPI creatinine equation (CKD-EPIcr) in adult Chinese chronic kidney disease (CKD) patients to clarify their clinical application. DESIGN AND METHODS: GFR was measured using the dual plasma sampling (99m)Tc-DTPA method (mGFR) in 252 adult CKD patients enrolled from four centres. Scr and ScysC were measured by standardized assays in a central laboratory. Each equation's performance was assessed using bias, precision, accuracy, agreement, and correct classification of the CKD stage. RESULTS: The measured GFR was 46 [25-83] mL/min per 1.73 m(2). The CKD-EPIcys, CKD-EPIcr-cys and Fengcys equations provided significantly higher accuracy (P15: 38.9%, 39.7%, and 38.9%) than the CKD-EPIcr equation (29.8%). The CKD-EPIcr-cys and Fengcr-cys equations presented higher precision (IQR of the difference, 16.4 and 17.3 mL/min per 1.73 m(2), respectively) and narrower acceptable limits in Bland-Altman analysis (56.6 and 50.8 mL/min per 1.73 m(2), respectively) than single marker-based equations. The CKD-EPIcr-cys equation achieved the highest overall correct proportion (61.5%) in classification of CKD stages. CONCLUSIONS: Combining ScysC and Scr measurements for GFR estimation improves diagnostic performance. The Scr-ScysC equation showed better performance than equations based on either marker alone. The CKD-EPIcr-cys equation showed the best performance for GFR estimation in Chinese adult CKD patients.

Development of the personalized criteria for microscopic review following four different series of hematology analyzer in a Chinese large scale hospital.

BACKGROUND: A generally accepted guideline ("41 rules") published by the International Consensus Group for Hematology Review (ICGHR) can not be suitable for all the laboratories because the facility type, laboratory requirements, sample volume, review rate, turn around time, instrument model and characters etc. are quite different from each other, which may cause a higher workload for microscopy review or lead to false or misleading results. Therefore, we decided to develop the personalized review criteria for 4 series of hematology analyzers in the same hospital, and describe all the implement procedures in detail. METHODS: The total 1770 blood samples were collected from Peking Union Medical College Hospital. Referring to the suggested criteria by international consensus group for hematology review ("41 rules"), the personalized review criteria for 4 series of hematology analyzers including Siemens Advia 2120, Sysmex XE-2100, Sysmex XT-1800i and Sysmex XS-800i were established and validated by adjusting the rules in order to reduce the false positive rate and keep the false negative acceptable by clinical. RESULTS: Using the "41 rules", high review rates of 37.94%, 35.56%, 33.44% and 37.94% were got respectively in Siemens Advia 2120, Sysmex XE-2100, Sysmex XT-1800i and Sysmex XS-800i. Three false positive rules mainly were observed in all of 4 analyzers: white blood cell < 3 × 10(9)/L or >30 × 10(9)/L, platelet < 100 × 10(9)/L or > 1000 × 10(9)/L and immature granulocyte. Specialized rules were observed in different series of analyzers, atypical/variant lymphs flag were found mainly in Sysmex XE-2100, Aniso-RBC were found mainly in Sysmex XT-1800i, flag of "immature granulocyte" mainly in Sysmex XS-800i, Micro-RBC, Macro-RBC and Aniso-RBC mainly in Siemens Advia 2120. Rules of immature granulocyte, blast, and NRBC flag would be mainly triggered by hematology malignant tumor. We could not delete these rules due to the risk of false negative of serious disease, other rules were deleted or revised. After continually optimizing to the rules, we finalized the criteria suitable for Siemens Advia 2120, Sysmex XE-2100, Sysmex XT-1800i and Sysmex XS-800i in our laboratory. The false negative rates were 2.94%, 2.86%, 3.10% and 2.78%, the review rates were 31.07%, 30.00%, 30.01% and 30.09%, and there was no hematology malignant tumor missed. Validated by 547 samples, the false negative rates of our optimized rules were 0.37%, 0.55%, 0.55%, and 0.91% respectively. CONCLUSION: The criteria can be based on the criteria established by International Consensus Group for Hematology Review but must be optimized according to the different requirements.

Expression of matrix metalloproteinase-1 mRNA in peripheral blood mononuclear cells of systemic lupus erythematosus patients and its relationship with atherosclerosis.

BACKGROUND: Matrix metalloproteinase-1 (MMP-1) plays an important role in atherosclerosis. This study was to examine expression of MMP-1 mRNA in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE), and to explore its relationship with atherosclerosis in SLE. METHODS: Fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to examine the expression of MMP-1 mRNA in PBMCs in 80 SLE patients, including 39 prone to atherosclerosis (Group A) and 41 unprone to atherosclerosis (Group B). Meanwhile, 30 patients who were free of cardiovascular diseases and 30 healthy individuals were selected as disease and normal control group (Groups C and D). The changes of MMP-1 gene expression were analyzed by differences of cycle threshold (DeltaCt), with the following formula: DeltaCt = Ct(target) gene - Ct(reference) gene. RESULTS: The expression level of MMP-1 mRNA in Group A was significantly higher than that of group B (DeltaCt = 8.64 +/- 2.43 vs DeltaCt = 12.09 +/- 2.26, t = 6.588, P < 0.01). The expression level of MMP-1 mRNA of SLE patients was significantly higher than that of Group C (DeltaCt = 10.41 +/- 2.90 vs DeltaCt = 12.29 +/- 2.51, t = 3.135, P < 0.01) and Group D (DeltaCt = 10.41 +/- 2.90 vs DeltaCt = 12.48 +/- 1.69, t = 3.675, P < 0.01). CONCLUSIONS: In comparison to disease and control group, expression of MMP-1 mRNA in PBMCs of SLE patients was significantly elevated, and significant difference of MMP-1 mRNA expression was also found between SLE patients prone and unprone to atherosclerosis, indicating that expression of MMP-1 mRNA may be correlated with the pathogenesis and activity of atherosclerosis in SLE.