Administration of oral MMF was performed according to each patients prescribed regimen of twice-daily dosing at 9 am and 9 pm . Blood samples were collected predose (0 h) and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, and 12 h after both morning and evening administration of oral MMF. The precise sample time was recorded. Blood samples were drawn in K2-EDTA vacutainer tubes (4 mL Vacuette K2EDTA, Greiner Bio-One, Monroe, NC) and centrifuged for 10 min at 4°C (1800g). Plasma was separated into Cryovials and stored at −70°C until analysis.
Vacuette k2edta
Manufactured by Greiner
Sourced in Austria
Vacuette K2EDTA is a blood collection tube designed for the collection, transportation, and storage of whole blood samples. It contains the anticoagulant K2EDTA, which prevents blood clotting.
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5 protocols using vacuette k2edta
1
Pharmacokinetics of Oral Mycophenolate Mofetil
2
Equine Basal Glucose and ACTH Measurement
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Basal blood glucose concentrations were determined using lithium‐heparin blood (Vacuette LH, Greiner Bio‐One, Kremsmünster, Austria) immediately after sampling using a handheld veterinary glucometer (AlphaTRAK II, Zoetis, North Chicago, Illinois).
Blood for adrenocorticotropic hormone (ACTH) concentration measurement was collected in 6‐mL EDTA tubes (Vacuette K2EDTA, Greiner Bio‐One, Kremsmünster, Austria) and kept cool until centrifugation (within 8 hours). The separated plasma was frozen and stored at −80°C until shipment on dry ice to the diagnostic laboratory. Analyses were performed in duplicate using a chemiluminescent immunoassay (Immulite 2000 XPi, The Philip Leverhulme Equine Hospital, Liverpool, UK). All animals with seasonally increased basal plasma ACTH concentrations were excluded from the study. The seasonally adjusted ACTH cutoff concentrations used for the study were 89.4 pg/mL for horses sampled in October and 35.2 pg/mL for horses sampled in November and December (Adams A., Abstract, International Equine Endocrinology Summit, 2017).
Blood for adrenocorticotropic hormone (ACTH) concentration measurement was collected in 6‐mL EDTA tubes (Vacuette K2EDTA, Greiner Bio‐One, Kremsmünster, Austria) and kept cool until centrifugation (within 8 hours). The separated plasma was frozen and stored at −80°C until shipment on dry ice to the diagnostic laboratory. Analyses were performed in duplicate using a chemiluminescent immunoassay (Immulite 2000 XPi, The Philip Leverhulme Equine Hospital, Liverpool, UK). All animals with seasonally increased basal plasma ACTH concentrations were excluded from the study. The seasonally adjusted ACTH cutoff concentrations used for the study were 89.4 pg/mL for horses sampled in October and 35.2 pg/mL for horses sampled in November and December (Adams A., Abstract, International Equine Endocrinology Summit, 2017).
3
Quantifying Tac in Whole Blood
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Tac whole‐blood samples were collected using vacutainers with spray‐coated potassium EDTA acid (4 mL Vacuette K2EDTA; Greiner Bio‐One, Monroe, NC). The analysis was performed using liquid chromatography tandem mass spectrometry, as previously reported.20 Lower limit of quantification was 0.6 μg/L, with imprecision coefficients of 9.0% at 2.3 μg/L and 6.0% at 7.0 μg/L.
4
Factor X Measurement in Blood Samples
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Citrated blood, EDTA blood, and serum samples were obtained from 8 healthy volunteers in the research group. This study was approved by the Regional Ethics Committee (registration number 2018/1414/REK Vest). Citrated-blood [Vacuette 0.109 mmol/L (3.2%) (Greiner Bio-One GmBH, Kremsmünster, Austria)] was centrifuged at 2500g for 15 minutes and EDTA-blood [Vacuette K2EDTA (Greiner Bio-One)] and serum [Vacuette serum with clot activator (Greiner Bio-One)] at 2200g for 10 minutes. The serum tubes were kept idle for 30 minutes before centrifugation. In 3 of the volunteers, an additional sample was drawn in a plain serum glass tube [BD Vacutainer serum without additives (Becton Dickinson and company, NJ)], and these tubes were kept idle for 1 hour before centrifugation at 1400g for 12 minutes. Samples were aliquoted and frozen within 1 hour (1.5 hours for the serum tube without additives).
FX antigen was measured using ELISA (Zumutest Factor X; Hyphen Biomed, Neuville-sur-Oise, France) on a Versamax microplate reader (Molecular Devices, San Jose, CA). FX activity was measured with a clot method in seconds (Prothrombin time Quick reagent RecombiPlasTin; Instrumentation Laboratory, Bedford, MA) on ACL TOP 700 (Instrumentation Laboratory), calibrated to give results in IU/dL.
FX antigen was measured using ELISA (Zumutest Factor X; Hyphen Biomed, Neuville-sur-Oise, France) on a Versamax microplate reader (Molecular Devices, San Jose, CA). FX activity was measured with a clot method in seconds (Prothrombin time Quick reagent RecombiPlasTin; Instrumentation Laboratory, Bedford, MA) on ACL TOP 700 (Instrumentation Laboratory), calibrated to give results in IU/dL.
5
Comprehensive Hematology Profile in Rats
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All rats had 1.0 mL of whole blood collected via the sublingual vein into a Vacuette K2EDTA blood collection tube (Greiner Bio-One, Monroe, NC) 2 d before study start and on the day of study termination. Parameters were assessed using the Advia2120i hematology system (Siemens, Malvern, PA) and included red blood cell counts, red blood cell distribution width, absolute reticulocyte count, and counts of white blood cells, basophils, eosinophils, lymphocytes, neutrophils, and monocytes, measurement of hematocrit, hemoglobin, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet counts, and mean platelet volume. All analyses were completed by individuals who were blind to treatment groups.
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