The Journal of ExtraCorporeal Technology No 56-2 | Page 12

Nalgene System , Rochester , NY ) and stored at �80 ° Cfor ELISA assay .
Measurements of platelet count
The whole bovine blood sample was collected in a sterile vacutainer tube containing 15 % EDTA K3 solution ( Covidien , Cardinal Health , 8881311446 ) and stored at 4 ° C . Platelet counts were assessed using Siemens Advia 2120i ( Siemens Medical Solutions , Inc ., Malvern , PA ) by a single optical cytometer after appropriate dilution of blood samples with ADVIA series RBC / PLT reagent . Platelets were counted from the signals of the common detector , and coincidence correction was made so that accurate counts were made over a wide range of cell types .
Assessments of platelet activation biomarkers
Markers for platelet activation were measured in duplicates by ELISA assay according to instructions provided by the manufacturer ( MyBioSource Inc ., San Diego , CA ) and at a 1:2 dilution . Collected plasma samples were used to measure four biomarkers of platelet activation : PF4 ( MBS741915 ), b-TG ( MBSO89872 ), P-selectin ( MBS734634 ), and GPIIb / IIIa ( MBS739298 ) at three different collection time points ( 5 min , 30 min , and 55 min ). Detection ranges and sensitivity for each kit are as listed : PF4 ( 25 – 500 ng / mL and 1.0 ng / mL ), b-TG ( 0.156 – 10 ng / mL and 0.05 ng / mL ), P-selectin ( 5 – 100 ng / mL and 1.0 ng / mL ), and GPIIb / IIIa ( 1 – 25 ng / mL and 0.1 ng / mL ). The values for standards and experimental samples were then analyzed using the ELISA analysis software ( BioTek ELISA Version 2.06 , BioTek , Winooski , VT ).
Statistical analysis
M . Sancheti et al .: J Extra Corpor Technol 2024 , 56 , 37 – 44 39 The values obtained from the ELISA assays were averaged across the five runs within each of the two coatings for each time point at 5 min , 30 min , and 55 min . The analysis methods used were descriptive statistics to quantify potential changes in biomarker values as determined by the ELISA assays . A standard deviation ( SD ) from the means was calculated and the individual values of each run were compared to the mean to see if they were within ± 2 SD of the mean . This method was used to determine whether any outlier values were found . Error bars were calculated as standard errors , and the results were represented as bar graphs . No statistical inference tests between two different coatings were used given the limited sample size and low statistical power . The descriptive statistics from this pilot study will be used to inform and power future study designs with adequate biological replicates .
Results Quantification of platelet counts
To establish a reliable in vitro protocol to measure platelet count post bypass using different coatings , we measured the range for inter-variation between bypass technical replicates for Trillium Ò Biosurface and Xcoating TM circuits , by running
Figure 2 . Quantification of Platelet counts in circulated bovine blood . ( A ) Bar graph presentation of platelet counts at different time points of bovine blood circulation using Trillium Ò Biosurface circuit . Data presents the average of five technical replicates using the same blood samples at 5 min , 30 min , and 55 min with no significant changes between time points . ( B ) Bar graph presentation of platelet counts at different time points of bovine blood circulation using Xcoating TM circuit . Data presents the average of five technical replicates using the same blood samples at 5 min , 30 min , and 55 min with no significant changes between time points . ( Mean ± SD , P < 0.05 , five technical replicates ).
the same bovine blood samples five times through each type of coated circuits ( Fig . 2 ). A new package of circuit was used for each technical replicate run ( five unused circuit packages for each type of coating ) and all five replicates were run during the same experimental day . Our data showed no significant increase or drop in platelet count at 5 min , 30 min , and 55 min for both coating types , with values at each time point falling within ± 2 SD of the mean for Trillium Ò Biosurface ( Fig . 2A ) and Xcoating TM ( Fig . 2B ).