1. Preparation of phosphate buffer saline (PBS)
NOTE: PBS was used throughout this study as the described assays did not require the addition of calcium. It is important to note that when adding calcium, often utilized to re-calcify citrated blood products, PBS should be avoided as calcium is known to precipitate in phosphate buffers.
<ol>
	<li>Make a 0.01 M, pH 7.4 PBS buffer by mixing 137 mM sodium chloride, 1.8 mM potassium phosphate monobasic, 10 mM sodium phosphate dibasic and 2.7 mM potassium chloride in DI water.</li>
	<li>Verify buffer pH using a pH probe and adjust the pH using sodium hydroxide or hydrochloric acid as needed.</li>
	<li>Use this PBS for preparation of fibrinogen and clotting assays (unless otherwise specified). 
	NOTE: Buffer is suggested to reconstitute fibrinogen powder since rehydration in DI water can result in fibrinogen precipitation even at 37 &#176;C.</li>
</ol>
2. Preparation and storage of proteins
NOTE: Throughout the protocol the protein stock concentrations are prepared at different concentrations for turbidity and TEG to allow for the consistent ratio of salt, DI water, PBS and other residual factors in the final clotting solutions.
<ol>
	<li>Preparation and storage of fibrinogen 
	NOTE: Contaminants in commercially available fibrinogen include a significant amount of factor XIII, residual amounts of other clotting factors, storage buffer and salts. In the presence of calcium, factor XIII is known to crosslink the fibrin clot network. This effect contributes to a tightened clot structure and enhanced clot strength affecting fibrin characterization. Commercially available activity kits can be used to determine active factor XIIIa levels. To minimize variability caused by factor XIIIa, experiments should be designed with the exclusion of calcium or additional steps to remove factor XIIIa should be incorporated into this protocol. In addition, protein storage salt and buffer type should be assessed and if necessary dialysis can be carried out to transfer to a preferred assay working buffer.
	<ol>
		<li>Weigh and aliquot lyophilized fibrinogen powder (bovine or human) in 2 mL tubes at 20 mg of protein per tube and store aliquots at -20 &#176;C for up to 6 months.</li>
		<li>Allow fibrinogen aliquot to acclimate to RT (room temperature) for 10 minutes on the day of the experiment. Reconstitute fibrinogen by adding 600 &#181;L of PBS to the aliquot 20 min prior to use.</li>
		<li>Take 10 &#181;L of fibrinogen and dilute it with 190 &#181;L of PBS in a UV transparent 96-well plate or a cuvette. Determine fibrinogen concentration by taking absorbance at 280 nm via a commercial spectrometer and its software.</li>
		<li>Calculate fibrinogen concentration (mg/mL) using Beer&#8217;s law. Prepare 12 mg/mL (for turbidity assays) and 3.2 mg/mL (for TEG) fibrinogen stock solutions by further dilution with PBS (unless otherwise specified). 
		NOTE: Fibrinogen concentration determination (mg/mL) by Beer&#8217;s law: 
		<img alt="Equation 1" src="/files/ftp_upload/61519/61519eq1v2.jpg" /> 
		Molar extinction coefficient: &#603; = 513,400 L mol-1 cm-1 at 280 nm; Pathlength (L); Dilution factor (D); Molecular Weight (MW) = 340,000 Da. &#603; is derived by multiplying E0.1% = 1.51 (280 nm) (extinction coefficient, given by the supplier) with MW.</li>
	</ol>
	</li>
	<li>Preparation and storage of thrombin
	<ol>
		<li>Reconstitute lyophilized thrombin (bovine or human, 1000 U stock) in 200 &#181;L of deionized (DI) water to make 200 &#181;L of 5000 U/mL thrombin stock solution.</li>
		<li>Make 5 &#181;L (20 U/tube) aliquots of the solution and keep aliquots frozen at &#8722;20 &#176;C.</li>
		<li>Thaw thrombin aliquots at RT for 15 min on the day of experiment and make thrombin working solution by diluting it to 20 U/mL for turbidity assays and 18 U/mL for TEG with DI water (unless otherwise specified). 
		NOTE: Precautions should be taken to maintain enzyme activity which can be accomplished by maintaining enzymes on ice during thawing and use; however, no reduction in thrombin activity was observed when utilized directly after thawing at RT.</li>
	</ol>
	</li>
</ol>
3. Turbidity
<ol>
	<li>Use any commercially available spectrometer that has an absorbance range of 350 - 700 nm and a corresponding software to monitor clot turbidity over time (see Table of Materials).</li>
	<li>Turn on the spectrometer and open the corresponding analysis software.</li>
	<li>Select plate 1 and open plate settings tab. Click ABS mode and Kinetic to monitor a dynamic absorbance reading over time.</li>
	<li>Select 550 nm (or any value in the range of 350 &#8211; 700 nm) in the wavelength tab and adjust the total run time to be 60 min with an interval of 30 s in the timing tab. Select wells of interest for reading by highlighting the wells. Adjust other settings if needed. 
	NOTE: Selecting a wavelength at the lower end of the range (around 350 nm) brings a better sensitivity but absorbance might also exceed the detection limit of the spectrometer. The most commonly utilized wavelength for turbidity measurement is 405 nm in literature; however, this protocol uses 550 nm to ensure the dynamic turbidity values are within the detection limit for all experiments. The selected reading interval should be as short as possible to achieve the highest level of assay sensitivity. This will depend upon the spectrometer and number of wells being read during a given assay.</li>
	<li>Take a UV transparent 96-well plate. Pipette 140 &#181;L PBS in a well that is selected for reading. Add and mix 10 &#181;L of thrombin (20 U/mL) in the well. 
	NOTE: Do not use high binding assay plates to minimize nonspecific protein binding to the well surface. This could affect protein dispersion in the solution and result in high assay variability.</li>
	<li>Initiate clotting immediately by adding 50 &#181;L of fibrinogen (12 mg/mL) in the well to obtain a 200 &#181;L clotting solution with a final concentration of 3 mg/mL fibrinogen and 1 U/mL thrombin. Mix the contents in the well by pipetting up and down five times taking care to avoid the creation of bubbles in the solution as they will impact absorbance by scattering light. 
	NOTE: Use a multichannel pipette when running multiple clot samples on the same plate at the same time. Record time differences across wells and the time period prior to the first read by the instrument to offset clotting times.</li>
	<li>Place the 96-well plate in the holder and click Start in the software to start turbidity reading at RT. 
	NOTE: If carrying out the assay at an elevated temperature then the spectrometer, plate, and reagents must all be maintained at the desired temperature prior to clot initiation.</li>
	<li>Once completed, retrieve the turbidity data and obtain a turbidity tracing curve by plotting absorbance change over time in a plotting software.</li>
	<li>Derive TurbMax (maximum turbidity indicative of fibrin fiber thickness and fibrin network density) by taking the max absorbance value of the curve over time. 
	NOTE: Fibrin fiber mass/length ratio can be calculated from turbidity values using the equation provided in the following manuscript8.</li>
	<li>Calculate 90% maximum turbidity by multiplying TurbMax by 90%. Derive TurbTime by computing time from clot initiation to 90% maximum turbidity. 
	NOTE: The time to 90% maximum turbidity is a more reliable metric than time to absolute maximum turbidity as it better represents clot time by eliminating the highly variable final clot formation period. Additional clotting parameters such as clot onset time (time from start of test to when absorbance starts to increase), and clot formation rate (Vmax, the largest slope of the linear region in the turbidity tracing curve) can also be extracted from the turbidity tracings.</li>
</ol>
4. Thromboelastography (TEG)
<ol>
	<li>Turn on the thromboelastograph analyzer and wait for the temperature to stabilize at 37 &#176;C.</li>
	<li>Open TEG - software. Once logged in, create an experiment name under the ID section.</li>
	<li>Conduct an e-test for all channels by following the on-screen software prompts. Place the lever back to the load position once all checks are complete. 
	NOTE: A TEG e-test is required and should be conducted every time when using the instrument. TEG coagulation control assays (using TEG level 1 and level 2 controls) are required at the regular manufacturer suggested intervals when utilized for clinical samples.</li>
	<li>Click the TEG tab, input sample information for channels that will be used. Place a clear uncoated TEG cup in its corresponding channel. Slide the carrier up to the top and press the cup bottom 5-times to affix the pin to the torsion rod. Lower the carrier and press the cup downward into the carrier base until it &#8220;clicks&#8221;.</li>
	<li>Pipette 20 &#181;L of thrombin solution (18 U/mL) into the TEG cup. Initiate clotting immediately by adding 340 &#181;L of fibrinogen (3.2 mg/mL) into the TEG cup to obtain a 360 &#181;L clotting solution with a final concentration of 3 mg/mL fibrinogen and 1 U/mL thrombin in the cup. Mix the contents by pipetting up and down five times. 
	NOTE: Potential clotting factors or other components of interest should be added during this step being careful to always maintain a final volume of 360 &#181;L in the TEG cup.</li>
	<li>Slide the cup loaded carrier up, move the lever to the read position and click Start in the software to initiate the TEG reading.</li>
	<li>Once TEG is completed (after about an hour), retrieve TEG parameters and obtain a TEG tracing curve by plotting amplitude over time in a plotting software.</li>
	<li>Collect MA as TEGMax (maximum amplitude is indicative of clot strength) and TMA as TEGTime (time to maximum amplitude) from the software. 
	NOTE: MA is calculated by the software as the maximum amplitude at the time at which the amplitude has less than a 5% deviation over a 3-minute period of time. TMA is determined as the time from the maximum thrombus generation rate (near the split point) to the MA. Other parameters might also be useful to assess when performing the clot analysis. Some examples of these parameters include: R-time (the time from start of test to when amplitude reaches 2 mm), K (the time from the end of R to when amplitude reaches 20 mm), alpha (slope of line between R and K), and CLT (clot lysis time).</li>
</ol>
5. Fibrin characterization under different clotting conditions
NOTE: Perform fibrin characterization experiments by modulating a specific variable in the clotting solutions such as: fibrinogen and thrombin concentrations, ionic strength, pH, and total protein concentrations. Experimental preparations with these example variables are described in this section; however, other clotting factors and conditions of interest can be substituted as well. Carefully select a suitable buffer system taking into consideration each unique assay requirements. For turbidity and TEG assays, include a buffer only control to ensure an accurate background subtraction while analyzing the effect of these variables.
<ol>
	<li>Varying fibrinogen concentration (1, 2, 3, 4, 5 mg/mL)
	<ol>
		<li>Adjust step 2.1.4 to prepare the fibrinogen stock at different concentrations (4, 8, 12, 16, 20 mg/mL for turbidity assays and 1.1, 2.1, 3.2, 4.2, 5.3 mg/mL for TEG).</li>
		<li>Adjust step 3.6 to &#8220;add 50 &#181;L fibrinogen (4, 8, 12, 16, 20 mg/mL) into multiple wells of the 96 well-plate&#8221; for turbidity assays.</li>
		<li>Adjust step 4.5 to &#8220;add 340 &#181;L fibrinogen (1.1, 2.1, 3.2, 4.2, 5.3 mg/mL) into clear TEG cups&#8221; for TEG.</li>
	</ol>
	</li>
	<li>Varying thrombin concentration (0.1, 0.3, 0.6, 0.8, 1, 2.5, 5, 10 U/mL)
	<ol>
		<li>Adjust step 2.2.3 to prepare thrombin stock at different concentrations (2, 6, 12, 16, 20, 50, 100, 200 U/mL for turbidity assays and 1.8, 5.4, 10.8, 14.4, 18, 45, 90, 180 U/mL for TEG).</li>
		<li>Adjust step 3.5 to &#8220;add 10 &#181;L thrombin (2, 6, 12, 16, 20, 50, 100, 200 U/mL) into multiple wells of the 96 well-plate&#8221; for turbidity assays.</li>
		<li>Adjust step 4.5 to &#8220;pipette 20 &#181;L thrombin (1.8, 5.4, 10.8, 14.4, 18, 45, 90, 180 U/mL) into clear TEG cups&#8221; for TEG.</li>
	</ol>
	</li>
	<li>Varying ionic strength (0.05, 0.13, 0.14, 0.15, 0.16, 0.17 and 0.3 M)
	<ol>
		<li>Dissolve sodium chloride (21, 101, 111, 121, 131, 141, and 271 mM) along with 1.8 mM potassium phosphate monobasic, 10 mM sodium phosphate dibasic and 2.7 mM potassium chloride in DI water to make 0.01 M PBS solutions with varying ionic strengths.</li>
		<li>Adjust step 1.3 to use PBS made at different ionic strengths to prepare fibrinogen and clotting solutions for both turbidity and TEG assays.</li>
	</ol>
	</li>
	<li>Varying pH (5.8, 6.6, 7.3, 7.4, 7.5, and 8.0)
	<ol>
		<li>Dissolve sodium phosphate dibasic (0.7, 3.2, 7.7, 8.1, 8.4, 9.5 mM) and potassium phosphate monobasic (8.2, 6.0, 2.0, 1.7, 1.4, 0.5 mM) along with 2.7 mM potassium chloride and sodium chloride (153, 147, 138, 137, 136, 134 mM) to make 0.01 M PBS solutions with varying pH and a final ionic strength at 0.165 M.</li>
		<li>Verify buffer pH value via pH probe and adjust pH if needed.</li>
		<li>Adjust step 1.3 to use PBS made at different pH to prepare fibrinogen and clotting solution for both turbidity and TEG assays.</li>
	</ol>
	</li>
	<li>Varying albumin concentration (0, 20, 40, 50, 60, 80, 100 mg/mL)
	<ol>
		<li>Dissolve 2 g of lyophilized albumin in 500 &#181;L of PBS at RT for 20 min on the day of experiment.</li>
		<li>Determine albumin concentration using the same procedure mentioned in step 2.1.3 and 2.1.4 with a molar extinction coefficient of 43,800 L mol&#8722;1 cm&#8722;1 (at 280 nm) for albumin.</li>
		<li>Prepare albumin stock at different concentrations.</li>
		<li>Adjust step 3.5 to &#8220;Pipette 40 &#181;L PBS in a well that is selected for reading and add 10 &#181;L thrombin (20 U/mL)&#8221;. Adjust step 3.6 to &#8220;Initiate clotting immediately by adding 50 &#181;L fibrinogen (12 mg/mL) with 100 &#181;L albumin (0, 40, 80, 100, 120, 160, 200 mg/mL) into multiple wells to a final concentration of 3 mg/mL fibrinogen, 1 U/mL thrombin and 0, 20, 40, 50, 60, 80, 100 mg/mL albumin in wells.&#8221;</li>
		<li>Adjust step 4.5 to &#8220;Initiate clotting immediately by adding a mixture of 200 &#181;L albumin (36, 72, 90, 108, 144, 180 mg/mL) and 140 &#181;L 7.7 mg/mL fibrinogen into TEG cups to obtain a 360 &#181;L clotting solution with a final concentration of 3 mg/mL fibrinogen, 1 U/mL thrombin and 0, 20, 40, 50, 60, 80, 100 mg/mL albumin in TEG cups.&#8221;</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Beckman, M. G., Hooper, W. C., Critchley, S. E., Ortel, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venous+Thromboembolism.+A+Public+Health+Concern.">Venous Thromboembolism. A Public Health Concern.</a> American Journal of Preventive Medicine. 38, 495-501 (2010).</li><li>Goldhaber, S. Z., Bounameaux, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+embolism+and+deep+vein+thrombosis.">Pulmonary embolism and deep vein thrombosis.</a> The Lancet. 379 (9828), 1835-1846 (2012).</li><li>Weisel, J. W., Litvinov, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+fibrin+polymerization+and+clinical+implications.">Mechanisms of fibrin polymerization and clinical implications.</a> Blood. 121 (10), 1712-1719 (2013).</li><li>Weisel, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+assembly.+Lateral+aggregation+and+the+role+of+the+two+pairs+of+fibrinopeptides.">Fibrin assembly. Lateral aggregation and the role of the two pairs of fibrinopeptides.</a> Biophysical Journal. 50 (6), 1079-1093 (1986).</li><li>Tripathi, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+evaluation+of+whole+blood+prothrombin+time+(PT)+and+international+normalized+ratio+(INR)+using+a+Laser+Speckle+Rheology+sensor.">Clinical evaluation of whole blood prothrombin time (PT) and international normalized ratio (INR) using a Laser Speckle Rheology sensor.</a> Scientific Reports. 7 (1), 1-8 (2017).</li><li>Ryan, E. A., Mockros, L. F., Weisel, J. W., Lorand, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+origins+of+fibrin+clot+rheology.">Structural origins of fibrin clot rheology.</a> Biophysical Journal. 77 (5), 2813-2826 (1999).</li><li>Carr, M. E., Hermans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+and+Density+of+Fibrin+Fibers+from+Turbidity.">Size and Density of Fibrin Fibers from Turbidity.</a> Macromolecules. 11 (1), 46-50 (1978).</li><li>Carr, M. E., Shen, L. L., Hermans, J. A. N., Chapel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mass-Length Ratio of Fibrin Fibers from Gel Permeation and Light Scattering. 16, 1-15 (1977).</li><li>Gabriel, D. A., Muga, K., Boothroyd, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Effect of Fibrin Structure on Fibrinolysis. , 24259-24263 (1992).</li><li>Wolberg, A. S., Gabriel, D. A., Hoffman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+fibrin+clot+structure+using+a+microplate+reader.">Analyzing fibrin clot structure using a microplate reader.</a> Blood Coagulation and Fibrinolysis. 13 (6), 533-539 (2002).</li><li>da Luz, L. T., Nascimento, B., Rizoli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombelastography+(TEG):+practical+considerations+on+its+clinical+use+in+trauma+resuscitation.">Thrombelastography (TEG): practical considerations on its clinical use in trauma resuscitation.</a> Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine. 21 (1), 29 (2013).</li><li>Whitten, C. W., Greilich, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thromboelastography:+past,+present,+and+future+.">Thromboelastography: past, present, and future .</a> Anesthesiology: The Journal of the American Society of Anesthesiologists. 92 (5), 1223-1225 (2000).</li><li>Ranucci, M., Laddomada, T., Ranucci, M., Baryshnikova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+viscosity+during+coagulation+at+different+shear+rates.">Blood viscosity during coagulation at different shear rates.</a> Physiological Reports. 2 (7), 1-7 (2014).</li><li>Zeng, Z., Fagnon, M., Nallan Chakravarthula, T., Alves, N. 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The authors have nothing to disclose.

This protocol demonstrates the utilization of two distinct clot characterization tools testing a simplified fibrinogen/thrombin clot model using commercially available components. Both TEG and turbidity assays are easy to conduct. They not only provide end point clot examinations such as max clot formation (TurbMax and TEGMax) and clot formation times (TurbTime and TEGTime) but also assess the dynamic clot forming process. This makes TEG and turbidity valuable tools for clot ch...

Thrombosis is the pathological formation of a blood clot in the body that blocks blood circulation leading to high morbidity and mortality worldwide. There are 1 to 2 cases of venous thromboembolism and 2 to 3 cases of thrombosis-induced vascular diseases per 1000 people annually1,2. Presented here is a method leveraging thromboelastography (TEG)&#160;and turbidity to monitor clot formation under various clotting conditions. Fibrin(ogen) is the primary protein that is responsible for clot formation in the body. In the final steps of the coagulation cascade, fibrinopeptides are cleaved from fibrinogen by thrombin initiating the polymerization of insoluble fibrin monomers as the clot develops3,4. To understand clot formation in pathological thrombosis, it is necessary to characterize fibrin formation under diverse clotting circumstances. Multiple clot monitoring assays have been utilized to study fibrin clot formation in vitro. Prothrombin time (PT/INR) and activated partial thromboplastin time (aPTT) are two common clinical assays that measure the integrity of a specific coagulation pathway. However, they use time as the only variable that gives no indication of physical clot properties5. Electron microscopy allows visualization of the micro-structure of a completely formed fibrin clot but provides no information about the clot forming process itself6. Among all assays, turbidity assays and TEG offer the ability to track clot characteristics dynamically over time. These techniques enable the measure of comprehensive clotting profiles and therefore, provide some benefit over other fibrin clot characterization tools.
Specifically, turbidity assays (or clot turbidimetry) is widely used for research and clinical applications due to its simplistic implementation and the wide accessibility of spectrometers in research laboratories. This assay allows a dynamic measurement of light transmittance through a forming clot by taking individual repetitive readings at a defined wavelength (most commonly at a wavelength in the range of 350 &#8211; 700 nm)7. Temperature in the reading chamber can also be adjusted. As fibrin gel forms, the amount of light that travels through the protein network is reduced causing an increase in absorbance over time. Similarly, absorbance decreases when the clot network degrades. Turbidity assays can easily be multiplexed using a multi-well plate format to allow for high throughput sample screening in both 96- and 384-well plates. Several clot characteristics can be derived from a turbidity tracing curve (absorbance over time measurement) that include: maximum turbidity, time to maximum turbidity, time to clot onset, and clot formation rate (Vmax). A fibrin fiber mass/length ratio can also be derived from raw turbidity data to estimate fibrin fiber thickness8,9,10.
TEG is primarily utilized in the clinical setting to assess patients&#8217; hemostasis and clot lysis. It is also commonly used in surgical applications to determine when anti-fibrinolytic drugs or hemostatic blood products should be administered11,12. Clot formation occurs inside a TEG cup with all the clotting components being added to the cup prior to the initiation of the assay. The cup, with evolving clot, physically rotates against a pin that is inserted into its center and an electromechanical torsion sensor measures the increasing viscoelastic strength of the clot. This assay is typically carried out at the physiological temperature of 37 &#176;C; however, the temperature can be manually adjusted on the instrument. Maximum amplitude (MA), reaction rate (R), kinetics time (K), &#945;-angle (Angle), and time to maximum amplitude (TMA) are extracted by the TEG software from the dynamic TEG tracing. These values are typically compared with clinical normal ranges to assess a patient&#8217;s coagulation state. While TEG is not precisely a viscometer, as it measures clot strength in millimeter units, it does provide important viscoelastic clot data and functions as a valuable clinical decision making tool for physicians to decide to administer specific blood products and adjust therapeutic dosing13. When both TEG and turbidity assays are utilized together, they&#160;provide&#160;complementary clot characterization information as clot strength and kinetics are easily extracted from TEG and fibrin fiber thickness can be accessed by optical turbidity measurements.
As fibrin is a critical component of a blood clot, fibrin clot characterization under diverse clot formation conditions can provide valuable insight into how a specific variable contributes to the clot formation process and ultimate clot properties. Understanding this can provide guidance for thrombosis diagnosis and the development of therapeutics. To obtain a more representative fibrin clot characterization, plasma can be substituted to monitor clot formation as it resembles in vivo clotting conditions more closely than a simplified fibrinogen/thrombin model system. However, due to the intricate nature of the coagulation cascade, clot formation using plasma adds to the complexity, making it more difficult to isolate the impact of individual factors. Utilizing a simplified fibrinogen/thrombin model prevents the need to initiate the entire clotting cascade allowing for isolation of the final fibrin formation step. By including two major fibrin forming components (fibrinogen and thrombin), this setup creates a highly controlled clot formation condition. It is also important to note that while the simplified clot model is used here, this protocol can also be utilized to characterize more complex clots by including additional clotting factors. In this study, fibrin clot characterization using turbidity and TEG are carried out by varying fibrinogen and thrombin concentrations, ionic strength, pH, and total protein concentration in the clotting solution to mimic different in vivo clotting circumstances14. Details regarding these variations to the protocol have been included in Section 5.

<table><tbody><tr><td>96-Well Clear Flat Bottom UV-Transparent Microplate</td><td>Corning</td><td>3635</td><td>Non-treated acrylic copolymer, non-sterile</td></tr><tr><td>Albumin from human serum</td><td>Millipore Sigma</td><td>A1653</td><td>&amp;ge;96%, lyophilized powder</td></tr><tr><td>Arium Mini Plus Ultrapure Water System</td><td>Sartorius</td><td>NA</td><td>DI water source</td></tr><tr><td>Bovine serum albumin</td><td>Millipore Sigma</td><td>A2153</td><td>&amp;ge;96%, lyophilized powder</td></tr><tr><td>Disposable Cups and Pins for TEG 5000 (Clear)</td><td>Haemonetics</td><td>REF 6211</td><td></td></tr><tr><td>Fibrinogen, Bovine Plasma</td><td>Millipore Sigma</td><td>341573</td><td>contains more than 95% clottable protein</td></tr><tr><td>Fibrinogen, Plasmingogen-Depleted, Human Plasma</td><td>Millipore Sigma</td><td>341578</td><td>Contains &amp;ge; 95% clottable proteins.</td></tr><tr><td>Phosphate buffered saline</td><td>Millipore Sigma</td><td>P3813</td><td>Powder, pH 7.4, for preparing 1 L solutions</td></tr><tr><td>Potassium chloride</td><td>Millipore Sigma</td><td>60130</td><td>&amp;ge;99.5% purity</td></tr><tr><td>Potassium phosphate monobasic</td><td>Millipore Sigma</td><td>P9791</td><td>&amp;ge;98% purity</td></tr><tr><td>SevenEasy pH Meter</td><td>Mettler Toledo</td><td>S20</td><td></td></tr><tr><td>Sodium chloride</td><td>Millipore Sigma</td><td>71378</td><td>&amp;ge;99.5% purity</td></tr><tr><td>Sodium phosphate dibasic</td><td>Millipore Sigma</td><td>71636</td><td>&amp;ge;99.5% purity</td></tr><tr><td>SpectraMax M5 multi-detection microplate reader system</td><td>Molecular Devices</td><td>M5</td><td></td></tr><tr><td>TEG 5000 Thrombelastograph Hemostasis analyzer system</td><td>Haemonetics</td><td>07-022</td><td></td></tr><tr><td>Thrombin, Bovine</td><td>Millipore Sigma</td><td>605157</td><td></td></tr><tr><td>Thrombin, Human Plasma, High Activity</td><td>Millipore Sigma</td><td>605195</td><td></td></tr></tbody></table>

leveraging turbidity and thromboelastography for complementary clot characterization

Thrombosis is a leading cause of death worldwide. Fibrin(ogen) is the protein primarily responsible for clot formation or&#160;thrombosis. Therefore, characterizing fibrin clot formation is beneficial to the study of thrombosis. Turbidity and thromboelastography (TEG) are both widely utilized in vitro assays for monitoring clot formation. Turbidity dynamically measures the light transmittance through a fibrin clot structure via a spectrometer and is often used in research laboratories. TEG is a specialized viscoelastic technique that directly measures blood clot strength and is primarily utilized in clinical settings to assess patients&#39; hemostasis. With the help of these two tools, this study describes a method for characterizing an in vitro fibrin clot using a simplified fibrinogen/thrombin clot model. Data trends across both techniques were compared under various clotting conditions. Human and bovine fibrin clots were formed side-by-side in this study as bovine clotting factors are often used as substitutes to human clotting factors in clinical and research settings. Results demonstrate that TEG and turbidity track clot formation via two distinct methods and when utilized together provide complementary clot strength and fiber structural information across diverse clotting conditions.

The experiments shown in Figure 1 are representative turbidity tracing curves of human and bovine fibrin clots at different fibrinogen levels. Representative TEG tracing curves for fibrin clot formation at different fibrinogen levels are shown in Figure 2. Both tracing curves demonstrate that after a lag period following clot initiation, clot turbidity or clot amplitude increases over time and levels off at the end of clot formation. An endpoint value of maximum...

Watch this Scientific Journal Video about Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization at JoVE.com

Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization

Fibrin is responsible for clot formation during hemostasis and thrombosis. Turbidity assays and thromboelastograhy (TEG) can be utilized as synergistic tools that provide complementary assessment of a clot. These two techniques together can give more insight into how clotting conditions affect fibrin clot formation.

Thrombosis is a leading cause of death worldwide. Fibrin(ogen) is the protein primarily responsible for clot formation or&#160;thrombosis. Therefore, ...

leveraging-turbidity-thromboelastography-for-complementary-clot

Research

JoVE Journal

Medicine

6.7K Views.  Indiana University School of Medicine. Fibrin is responsible for clot formation during hemostasis and thrombosis. Turbidity assays and thromboelastograhy (TEG) can be utilized as synergistic tools that provide complementary assessment of a clot. These two techniques together can give more insight into how clotting conditions affect fibrin clot formation.

Video: Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Bioengineering

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

Immunology and Infection

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...

Combining the Chandler Loop and RT-FluFF for Ex Vivo Clot Lysis Monitoring

Tracking Fibrinolysis of Chandler Loop-Formed Whole Blood Clots Under Shear Flow in An In-Vitro Thrombolysis Model

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates.
The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.

Author Spotlight: Advancing Thrombolytic Testing by Integrating Flow Dynamics in In Vitro Models

In-vitro thrombolysis assays have often struggled to replicate in-vivo conditions whether in the model thrombus being digested or in the environment in which thrombolysis is occurring. Herein, we explore how coupling the Chandler loop and Real-Time Fluorometric Flowing Fibrinolysis Assay (RT-FluFF) is used for high-fidelity, ex-vivo, clot lysis monitoring.

Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

In Vitro Thrombosis Test for Ventricular Assist Devices

The risk of thrombosis remains a significant concern in the development and clinical use of ventricular assist devices (VADs). Traditional assessments of VAD thrombogenicity, primarily through animal studies, are costly and time-consuming, raise ethical concerns, and ultimately may not accurately reflect human outcomes. To address these limitations, we developed an aggressive in vitro testing protocol designed to provoke thrombosis and identify potential high-risk areas within the blood flow path. This protocol, motivated by the work of Maruyama et al., employs a modified anticoagulation strategy and utilizes readily available components, making it accessible to most laboratories conducting in vitro blood testing of VADs. We demonstrated the utility of this method through iterative testing and refinement of a miniature magnetically levitated pediatric VAD (PediaFlow PF5). The method has been effective in identifying thrombogenic hotspots caused by design and manufacturing flaws in early VAD prototypes, enabling targeted improvements before advancing to animal studies. Despite its limitations, including the absence of pulsatile flow and the influence of donor blood characteristics, this protocol serves as a practical tool for early-stage VAD development and risk mitigation.

We present a benchtop protocol to induce thrombosis in ventricular assist devices (VAD) within a recirculating test platform. This method serves to identify thrombogenic hotspots in the blood flow path and can help improve thromboresistance ahead of preclinical testing in animal models.

The risk of thrombosis remains a significant concern in the development and clinical use of ventricular assist devices (VADs). Traditional assessments of ...

Assessing Blood Hypercoagulation of EV-Rich Plasma

Determination of the Procoagulant Activity of Extracellular Vesicle (EV) Using EV-Activated Clotting Time (EV-ACT)

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. However, there is an urgent need for a bedside test to assess the procoagulant activity of EV in clinical settings. This study proposes the use of thrombin activation time of EV-rich plasma as a measure of EV's procoagulant activity. Standardized procedures were employed to obtain sodium-citrated whole blood, followed by differential centrifugation to obtain EV-rich plasma. The EV-rich plasma and calcium chloride were added to the test cup, and the changes in viscoelasticity were monitored in real-time using an analyzer. The natural coagulation time of EV-rich plasma, referred to as EV-ACT, was determined. The results revealed a significant increase in EV-ACT when EV was removed from plasma obtained from healthy volunteers, while it significantly decreased when EV was enriched. Furthermore, EV-ACT was considerably shortened in human samples from preeclampsia, hip fracture, and lung cancer, indicating elevated levels of plasma EV and promotion of blood hypercoagulation. With its simple and rapid procedure, EV-ACT shows promise as a bedside test for evaluating coagulation function in patients with high plasma EV levels.

Author Spotlight: Deciphering Coagulation Disorders in Traumatic Brain Injury Patients 

This protocol investigates the use of extracellular vesicle (EV)-rich plasma as an indicator of the coagulative ability of EV. EV-rich plasma is obtained through a process of differential centrifugation and subsequent recalcification.

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. ...