38 M . Sancheti et al .: J Extra Corpor Technol 2024 , 56 , 37 – 44
Figure 1 . Schematic of the laboratory-based ( in vitro ) Cardiopulmonary Bypass Circuit .
Recent advances in bypass technologies have created biocompatible surfaces to potentially decrease platelet activation during CPB , therefore , reducing the number of complications after surgery . Heparin-coated circuits are widely used because they seem not to cause excessive platelet activation as compared to non-coated circuits [ 5 ]. Another commonly used circuit poly-2-methoxyethyl acrylate ( PMEA ) has a protective water layer that allows proteins to retain their conformation and reduce adherence to foreign surfaces [ 6 ].
While this has been the focus of research and advancement in cardiothoracic surgery , there is minimal scientific and datadriven evidence that establishes the biocompatibility of these circuits or their potential benefits in reducing platelet activation during CPB ; hence , there has yet to be a reliable and standardized laboratory protocol that compares these biocompatible circuits in vitro with respect to their effects on platelet function and activation [ 7 ].
Much of our evidence of biocompatibility comes from platelet activation studies done during clinical studies with patients undergoing surgery with limited sample size and multiple independent variables [ 8 – 10 ]. In this study , we aimed to 1 ) establish a well-controlled and standardized in vitro protocol to measure surface markers of platelet activation at different time points during the bypass in the laboratory setting using commercially available Enzyme-Linked Immunosorbent Assay ( ELISA ) kits , and 2 ) determine the intra-coating variability across technical replicates for two types of coated circuit that are commonly used in CPB to establish the required technical replicate within each independent biological replicate .
Materials
and methods Bovine blood collection
Bovine blood was obtained via venipuncture from a local slaughterhouse and treated with 30,000 IU of heparin in the collection bucket to reduce clotting during sample transportation . Eight to nine ( 8 – 9 ) liters of bovine blood were used for one run of CPB . An activated clotting time was accepted and maintained above 480 s after heparin administration [ 11 ]. The circuit was not connected to a living system ; hence no extra heparin was added during the experimental runs of CPB . Experiments with CPB were started within 1 – 2 h and completed within 8 – 9 h post-blood collection .
CPB circuit setup
One bovine blood donation was used for five technical replicates of experimental CPB runs using Trillium Ò Biosurface ( 541T , Medtronic Inc ., Minneapolis , MN ) with a heparinized surface . Another bovine blood donation was used for five technical replicates of experimental CPB runs using Xcoating TM ( 3CX * FX25RWC , Terumo Cardiovascular , Ann Arbor , MI ), which is a PMEA-coated circuit [ 12 ]. The laboratory simulated circuit included a pump , arteriovenous ( AV ) loops , tubing , reservoirs , oxygenators , arterial line filters , a heat exchanger ( 16-02-85 , SORIN Group Deutschland GmbH , Müchen , Germany ), and a roller pump ( Stockert S3 Roller Pump with S3 Console 10-60-00 , SORIN Group Deutschland GmbH , Müchen , Germany ). All parts of the circuit were replaced before each technical replicate trial ( Fig . 1 ).
During the experimental runs , bovine blood was mixed with crystalloid ( Normosol-R ) to prime and de-air the CPB circuit . The amount of crystalloid used was calculated to maintain a hematocrit of 23 – 27 %. Blood was circulated at a rate of 4 L / min and was mixed with crystalloid for the first 5 min of the run . Blood samples were collected at baseline ( 5 min after initiation of CPB at 37 ° C ), 30 min after initiation of CPB at 32 ° C , and 55 min after initiation of CPB at 37 ° C . The circuit run was completed in 60 min . At each desired time point , 1 mL of blood samples were collected using K2 EDTA-coated Vacutainer tubes ( catalog number 367863 ; Becton Dickinson , San Diego , CA ). Collected blood samples were centrifuged at 2500 RPM ( Microfuge Lite 367121 , Beckman Coulter , Inc ., Palo Alto , CA ) for 15 min . Separated plasma samples were aliquoted ( 500 lL ) into small CryTubes ( Thermo Scientific