Evaluating Whole Blood Clotting in vitro on Biomaterial Surfaces

Materials and Reagents

1. 3.0 ml vacuum blood tubes with no anticoagulant (BD Vacutainer, catalog number: 366703)
2. 24-well plate (Greiner Bio-One CELLSTAR, catalog number: 662-160)
3. 96-well plate (Greiner Bio-One CELLSTAR, catalog number: 687-100)
4. Standard pipette tips with a volume capacity of 20 μl and 1,000 μl (Eppendorf, catalog numbers: 22491911 and 22491351)
5. Deionized (DI) water
6. Human blood
7. Biomaterial surface (average top area ~25 mm²)
8. Vacuum line
9. Marker (from any commercial source)
10. 70% ethanol
11. PBS (see Recipes)
    

Equipment

1. Microplate reader (BMG LABTECH, FLUOstar Omega)
2. Horizontal shaker (VWR, catalog number: 97109-890)
3. Measurement pipettes (Eppendorf, catalog numbers: ES-20F and ES-1000F)
4. Timer (from any commercial source)
5. Tweezers (from any commercial source)
6. Bench
   

Software

1. Software Omega and MARS Data Analysis (BMG LABTECH, https://www.bmglabtech.com/reader-control-software/)
2. Software Microsoft Excel or Origin (OriginLab, https://www.originlab.com/)
   

Procedure

Note: All procedures are performed twice (using blood from two different donors) for three samples of each surface in the hood.

1. Biomaterial sterilization
   1. Place biomaterial surfaces in each well of a 24-well plate. Prepare one plate for each time point (15, 30 and 45 min) and label them on the lid with the corresponding time point.
   2. Add 500 μl of ethanol for 5 min at room temperature.
   3. Aspirate the ethanol and rinse the biomaterial surfaces three times with 500 μl of PBS.
   4. Aspirate the PBS and rinse the biomaterial surfaces once with 500 μl of DI water.
   5. Aspirate the DI water and let the biomaterial surfaces dry for at least 30 min.
      
      
      
2. Adding blood to the biomaterial surface
   1. Draw human whole blood in a vacuum tube with no anticoagulant.
   2. Immediately after the blood draw, carefully open the vacuum tube and pipette 7 μl of blood out of the tube and put onto each biomaterial surface (Figure 1).
   3. Close the lid and start counting the timer for each plate (15, 30 and 45 min).
      
      
      Figure 1. Whole blood on biomaterial surfaces. 7 μl of blood are placed onto each surface and allowed to clot for 15, 30 and 45 min. Group A and B are different biomaterial surfaces being studied.
      
      
      
3. Preparing the positive control (maximum hemoglobin release)
   1. Prepare a new 24-well plate with 500 μl of DI water in 3 wells.
   2. Add 7 μl of blood to each well with DI water (Figure 2A).
      
      
      Figure 2. Positive Control. A. the maximum hemoglobin release is obtained adding 7 μl of blood in 500 μl of DI water. B. 200 μl of each solution is transferred to a 96-well plate to read the absorbance.
      
      
   3. Place the plate on a horizontal shaker for 30 s (100 rpm).
   4. Move the plate to a bench and wait for 5 min to release the free hemoglobin.
   5. After 5 min, pipette 200 μl of the solution to a 96-well plate (Figure 2B).
   6. Read the absorbance using a microplate reader at a wavelength of 540 nm.
      
      
      
4. Transferring samples to release hemoglobin
   1. Prepare a new 24-well plate with 500 μl of DI water in each well.
   2. After 15 min, gently transfer the biomaterial surfaces from the first plate to a new well with DI water. Be careful while moving the biomaterial surfaces to not disturb the blood on the surface. The samples should under water after this step. If your samples are thicker, use more water in order to cover the whole with the blood.
   3. Place the plate on a horizontal shaker for 30 s (100 rpm).
   4. Move the plate to a bench and wait for 5 min to release the free hemoglobin (Figure 3).
      
      
      Figure 3. Different biomaterial surfaces, A and B, in DI water to release the free hemoglobin. The lysis of red blood cells occurs due to pressure change and hemoglobin is released in DI water.
      
      
      
5. Reading the absorbance
   1. After 5 min, pipette 200 μl of the “water + hemoglobin released” solution to a 96-well plate. Change the pipette tip for each solution (Figure 4).
   2. Read the absorbance using a microplate reader at a wavelength of 540 nm.
   3. Repeat Procedures C and D after 30 and 45 min for the second and third plates, respectively.
      
      
      Figure 4. Solutions from different biomaterial surfaces, A and B (water + hemoglobin released). 200 μl of each solution in Figure 3 is transferred to a 96-well plate to read the absorbance.

Data analysis

1. Absorbance values at 540 nm are read using a microplate reader. Export the data to an excel format file.
2. Any protocols involving blood are recommended to be performed at least twice using two different blood donors. However, in order to avoid donor-to-donor variability, only present the results obtained from one donor (Damodaran et al. 2013; Sabino et al., 2019).
3. Using Origin/Excel (or similar software), plot the mean/standard deviation of absorbance values from each group (Figure 5). Insert a line corresponding to the average absorbance measured for the positive control.
4. Conduct analysis of variance (ANOVA) for the experimental data using software Origin (or similar) at a 5% significance level (P < 0.05).
   
   
   Figure 5. Free hemoglobin concentration values measured in terms of absorbance for different biomaterial surfaces after 15, 30 and 45 min. The results were significantly different for all time points. Group B shows significant higher hemoglobin concentration, which indicates that these surfaces significantly delay whole blood clotting in comparison with group A.
   

Notes

This protocol can be applied to evaluate the whole blood clotting in vitro of any biomaterial surface that is stable underwater. If the sample floats in water, use double-sided tape to attach the sample to the well bottom. Sterilization process may vary depending on the biomaterial surface.

Recipes

1. PBS (10x)
   Dissolve 100 ml of 10x PBS in 900 ml of DI water and autoclave or filter sterilize
   

Acknowledgments

This work was supported by National Heart, Lung and Blood Institute of the National Institutes of Health under award number R01HL135505 and R21HL139208. This protocol was adapted from previous publications from our group (Leszczak et al., 2013; Sabino et al., 2019).

Competing interests

The authors declare no conflict of interest.

Ethics

All experiments were conducted in agreement with the National Institutes of Health's “Guiding Principles for Ethical Research”. Colorado State University Institutional Review Board approved the protocol (17-7195H, valid from 04/01/2017 to 03/31/2020) for blood isolation from healthy participants. Whole human blood was acquired through venipuncture from healthy individuals, and formal consents were obtained from the donors.

References

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2. Gorbet, M. B. and Sefton, M. V. (2004). Biomaterial-associated thrombosis: roles of coagulation factors, complement, platelets and leukocytes. Biomaterials 25(26): 5681-5703.
3. Leszczak, V., Smith, B. S. and Popat, K. C. (2013). Hemocompatibility of polymeric nanostructured surfaces. J Biomater Sci Polym Ed 24(13): 1529-1548.
4. Obstals, F., Vorobii, M., Riedel, T., de Los Santos Pereira, A., Bruns, M., Singh, S. and Rodriguez-Emmenegger, C. (2018). Improving hemocompatibility of membranes for extracorporeal membrane oxygenators by grafting nonthrombogenic polymer brushes. Macromol Biosci 18(3).
5. Sabino, R. M., Kauk, K., Movafaghi, S., Kota, A. and Popat, K. C. (2019). Interaction of blood plasma proteins with superhemophobic titania nanotube surfaces. Nanomedicine 21: 102046.
6. Simon-Walker, R., Cavicchia, J., Prawel, D. A., Dasi, L. P., James, S. P. and Popat, K. C. (2018). Hemocompatibility of hyaluronan enhanced linear low density polyethylene for blood contacting applications. J Biomed Mater Res B Appl Biomater 106(5): 1964-1975.