Sta compact

Complete blood counts were measured with Unicel DxH800 Coulter Cell Analyzer (Beckman Coulter, USA) from K2EDTA samples. Serum LDH, creatinine, and ALT parameters were analyzed with AU 680 (Beckman Coulter, USA) spectrophotometrically. Ferritin levels were measured with a two-site immunoenzymatic assay in Access Analyzer (Beckman Coulter, USA). D-dimer parameter was quantitated with an immunoturbidimetric assay in 3.2% sodium citrated venous plasma (STA Compact, Diagnostica Stago, France). hs-CRP levels were measured nephelometrically (BN Prospec, Dade Behring, Germany). TSH, FT3, FT4, TPOAb, and TGAb parameters were measured by paramagnetic particle, chemiluminescent immunoassays in serum samples (DxI800, Beckman Coulter, USA). The reference range for TSH was 0.34–5.60 mU/L, for FT3 was 2.6–4.37 ng/L (0.061–0.103 pmol/L), and for FT4 was 0.61–1.12 ng/dl (0.144–0.26 pmol/L). TGAb was 0–115 IU/ml, and TPOAb was 0–34 IU/ml.
FSH and LH levels were also measured by paramagnetic particle, chemiluminescent immunoassays in serum samples (DxI800, Beckman Coulter, USA). Estradiol and testosterone levels were determined by electrochemiluminescence immunoassay (Modular Analytics E170, Roche Diagnostics, Germany).

Biochemical and hematological parameters were assayed for each child before and after treatment essentially as described previously.22 (link) Serum was used to investigate the lipid profile and parameters reflecting liver function, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin as well as creatinine reflecting kidney function (all by Modular P1600; Roche Diagnostic, Hitachi P; Germany). Viral markers, including both hepatitis B surface antigen and hepatitis C virus antibody, were analyzed using Architect i2000SR (Abbott Diagnostics, Abbott Park, IL) and found to be uniformly negative. Blood samples in tubes containing sodium citrate were used for analysis of coagulation tests, including prothrombin time and activated partial thromboplastin time, by an automated blood coagulation analyzer (STA Compact; Diagnostica Stago, Paris, France); 2 mL blood dispensed in EDTA tubes was analyzed for complete blood chemistry by using an automated blood hematology analyzer (Beckman Coulter LH785; Beckman Coulter, Miami, FL).
Aliquots of heparinized plasma collected at baseline and after treatment were stored at −76°C and analyzed for levels of total lipids and unsaturated fatty acids (FAs) as described.22 (link)

To relate the concentrations of UFH and LMWH added to PRP to target anticoagulant effects of heparin measured by factor Xa inhibitory activity (anti-Xa), PPP samples collected from the study horses were spiked with the drugs and frozen at −20°C until batch assay. Anti-Xa activities were measured using a commercial kit (Liquid anti-Xa, Diagnostica Stago) and an automated coagulation analyzer (STA Compact, Diagnostica Stago, Parsippany, NJ, USA). The assay is a one-step competitive inhibition assay, configured with a bovine factor Xa reagent and a chromogenic substrate of factor Xa. The color change of the reaction mixture is inversely proportional to the heparin concentration in the test plasma (expressed as units per milliliter anti-Xa). The assay calibrators contain known concentrations of UFH or LMWH in human plasma (STA-calibrator HBPM/LMWH, Diagnostica Stago). Routinely used target anti-Xa activities in horses (based on heparin therapy in humans) are 0.1–0.2 U/mL anti-Xa for prophylaxis and 0.3–0.7 U/mL anti-Xa for therapy (24 (link)).

Anti-Xa activity was measured by a chromogenic anti-factor Xa assay (Rotachrom Heparin, Diagnostica Stago S.A.S, Asnières-sur-Seine, France). Since apixaban calibrators were not commercially available at the time the assays were done, apixaban calibrators were prepared by crushing apixaban tablets and dissolving in DMSO, as per Barrett et al.
10 (link)
Results were calibrated against a standard curve developed from these calibrators (500–15.6 ng/mL). The standard curve remained linear down to 15.6 ng/mL, which was therefore considered as the lower limit of quantitation of the assay. Values below this limit were transposed to 15.6 ng/mL for the purpose of statistical analysis. All calibrators and patient samples were measured on a STA-Compact instrument (Diagnostica Stago S.A.S.). The anti-Xa assays were performed by Hemostasis Reference Laboratories (Hamilton, Ontario, Canada).

The anticoagulant activity (anti-Xa activity) of UFH and LMWH was measured in platelet-poor plasma (PPP) at the Comparative Coagulation Laboratory at Cornell University. The PPP was harvested from the supernatant of PRP after high-speed centrifugation (16,000 × g, Accuspin Micro, ThermoScientific, Rockford, IL, USA) for 5 min. The PPP was frozen at −20°C in a dedicated freezer and assays were performed in batch after each treatment for each group of horses with the other investigators remaining blinded as to the results. The PPP was thawed at 37°C in a water bath before analysis. The assay is configured with a bovine activated Factor X reagent added in excess to the test plasma and a chromogenic substrate of Factor Xa (Liquid anti-Xa, Diagnostica Stago, Parsipanny, NJ, USA). The assay is performed using the manufacturer’s automated coagulation analyzer (STA Compact, Diagnostica Stago). In this assay, residual uninhibited Factor Xa cleaves the chromogenic substrate so the inverse of the color change in the reaction mixture is proportional to the drug concentration in the test plasma. Results are expressed as U/mL anti-Xa, based on assay calibration with a standard containing known UFH or LMWH concentrations (STA-calibrators HBPM or LMWH, Diagnostica Stago).