Establishment of reference intervals for kaolin-activated TEG and the STA compact automated analyzer for dogs and coagulation response in a canine model of endotoxemia

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EralpInanOya_2011_12_12.pdf (2.85 MB)

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2011

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http://dx.doi.org/10.22029/jlupub-11756

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ObjectiveThe first part of the study included method validation(intra-assay-repeatability, impact of hemolysis and lipaemia, effect of anticoagulant) and evaluation of reference intervals for dogs for kaolin-activated thromboelastography (TEG® 5000 Thrombelastograph, Haemonetics Corporation; formerly Haemoscope Corporation; Braintree, MA, USA) and the automated coagulation analyzer STA Compact (STA Compact®, Roche Diagnostics GmbH, Mannheim, Germany). In a second part of the study, the response of the coagulation system in a canine model of endotoxemia was evaluated. Material and MethodsBefore the establishment of reference intervals, repeatability and interferences were assessed. Investigated variables included the TEG values R, K, alpha, MA and G as well and the coagulation variables one stage prothrombin time (OSPT), activated partial thromboplastin time (aPTT), thrombin time (TT), fibrinogen, Factor VIII (FVIII), antithrombin (AT), protein C (PC), protein S (PS), resistance against activated protein C (APC-ratio), plasminogen, D-dimer, anti-FXa run on the coagulation analyzer STA Compact. Intra-assay repeatability for TEG variables was performed in 6 healthy dogs with duplicate measurements. The standard deviation required for calculation of the coefficient of variation (CV) was consistent with the root of the pooled variance estimate. The comparison between TEG results obtained with native whole blood or citrated whole blood was performed in 16 healthy dogs. For assessment of hemolysis on TEG results,17 healthy dogs were included. Hemolytic samples were prepared with two techniques: mechanical stress (HM) and as well as freeze and thawing (HF).The intra-assay repeatability for variables assayed with the STA Compact was assessed with 15 replicate measurements in a sample taken from one healthy dog. The intra-assay variation was estimated for variables mentioned above. To assess the influence of lipemia on measurements run on the STA Compact analyzer, samples with three grades of lipemia were prepared by adding Liquigen® (Liquigen®, Pfrimmer Nutricia GmbH, Erlangen, Germany) to the samples of three healthy dogs. The effect of hemolysis on these coagulation parameters was performed by treating whole blood in afreeze-thaw cycle and three grades of hemolysis were obtained.Establishment of reference intervals for TEG parameters and coagulation parameters measured on the STA Compact was performed with 56 healthy dogs. After evaluating the default settings of human assays applied on the STA Compact for canine specimens, some modifications of the test methods were needed. A standard curve has to be prepared with canine pooled plasma for PC, PS and FVIII. Also for both analyzer measurements the impact of sex on the results was established. In the second part of this investigation, the impact of endotoxemia on primary, secondary and tertiary hemostasis as well as TEG variables was evaluated in 10 healthy Mongrel dogs. A control group (n=5 dogs) and a treatment group (n=5 dogs) was created. Dogs in the treatment group received endotoxin (LPS) dissolved in sterile saline 0.9% which was administered intravenously at a dosage of 0.02 mg/kg. The control group received 0.2 ml/kg sterile saline 0.9%. Venous blood samples were collected before application of LPS or placebo (0 hour), 1, 4 and 24 hours after treatment.StatisticsResults were analyzed with the Graph Pad Prism (Graph Pad Software, San Diego, USA), Analyse-it Method evaluation (Analyse-it Method validation Edition version 2.12 - © 1997-2008, Analyse-it Software Ltd.) and BMDP statistical software (BMDP Statitical software Inc., 1440 sepulveda Blvd, Los Angeles, CA 90025 USA). Intra-assay variation for TEG variables was assessed by calculating the arithmetic mean and the pooled variance estimate, based on the differences in the duplicate determinations. For variables measured on the STA compact fifteen-run intra-assay repeatability for normal values was calculated from a sample of a healthy dog. The effect of anticoagulant and interferences was assessed by a paired t-test (or Wilcoxin signed rank sum test). For the establishment of the reference intervals, an Anderson Darling test was performed to verify the assumption of normality. In case of normal and log normal distribution of data, double sided reference intervals were obtained by calculating the mean±1.96 SD (standard deviation) so that 95% of the reference population wasincluded. If non-normal distribution of data was present, the non-parametric percentile method was applied. The 2.5 and 97.5 percentiles were calculated to obtain the 95% double sided reference interval. Data were depicted as histograms with the reference interval as well as the 90% confidence interval of the upper and lower reference limits. The influence of sex on the reference intervals was assessed by using an unpaired t-test or a comparable non-parametric test (Mann Whitney U test).The differences between control group and endotoxin treatment group were assessed with a two way analysis of varience and covariance with repeated measures.ResultsIntra-assay CVs for R; K; alpha; MA and G were 7.6%; 17.7%; 7.4%; 2.9% and 6.6% respectivley. Samples with hemolysis resulted in a significantlydecreased R value and decreased MA, G and alpha; value (P<0.001 to <0.0001). Furthermore, a significantly high K value was seen compared to the control group (P<0.01). There was no significant impact of anticoagulant on TEG variables.Inter-individual variation was higher in native samples than in citrated whole blood. Intra-assay CVs for the coagulation parameters OSPT, aPTT, TT, fibrinogen, FVIII, AT, PC, PS, APC-ratio, plasminogen, D-dimer and anti-FXa were 1.22%, 1.02%, 1.64%, 5.6%, 3.89%, 4.68%, 2.36%, 1.4%, 1.45%, 20.16%, 45.92% and 12.83%. FVIII-activity, antithrombin, proteinC, protein S, and APC-ratio were overestimated in haemolytic plasma, whereas fibrinogen, TT, and aPTT were underestimated. Lipemia resulted only in false high D-dimers.Reference intervals for the kaolin activated TEG were as follows: R=1.8-8,6min.; angle alpha=36.9-74.6 degrees; K=1.3-5.7 min.; MA=42.9-67.9 mm and G=3.2-9.6 Kdyn/cm2.Reference intervals for the STA Compact automated analyzer were as follows: OSPT=5.7-8.0 sec.; aPTT=10.0-14.3 sec.; TT=11.9-18.3 sec.; fibrinogen=1.3-3.1 g/l; AT=107.9-128.0 %; D-dimer=0.023-0.65 µg/ml; anti-FXa=0.04-0.26 IU/L; APC-ratio=2.0-3.0; PC=74.4-160.5 %; PS=75.5-118.9 % and FVIII=70.9-136.4 %. The results for PC, PS and FVIII have to be compared to a canine standard curve. There was no significant impact of sex on results of both analyzers. Second part of the study: The endotoxin-induced clinical signs included lethargy (n=5/5), diarrhea (n=4/5), vomitus (n=4/5) and abdominal pain (2/5). Regarding the evaluation of the impact of endotoxin on the coagulation process severe leukopenia (mean 2.5±0.7×109/l; P<.0001) and a significiant 2.2-fold increase in D-dimers (P=0.001) were observed at time point 1 hour. At time point 4 hours, a significant raise in body temperature (P=0.0006) and in OSPT (P=0.0042) was seen as well as a significant decrease in fibrinogen (P=0.0007), protein C (P=0.0021) and protein S (P=0.008). PLTs (P=.0284) and antithrombin (P=.0170) were lowest at time point 24 hours. TEG variables did not significantly differ between the groups. APC-ratio was higher in the sepsis group (P=0.0348), however, remained within the reference interval in 4/5 dogs.ConclusionThe data provided useful reference ranges for kaolin-activated TEG and for the STA Compact automated analyser but some human tests (e.g., Protein C, protein S and factor VIII) have to be modified. Regarding the impact of endotoxemia on the hemostatic system, the earliest indicator of sepsis-associated coagulation abnormalities were D-dimers which were followed by increased OSPT and decreased protein C. APC-ratio and TEG variables were not considered to be good screening variables in early sepsis/endotoxemia.

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