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 po...

<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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+clot+formation+under+diverse+clotting+conditions:+Comparing+turbidimetry+and+thromboelastography.">Fibrin clot formation under diverse clotting conditions: Comparing turbidimetry and thromboelastography.</a> Thrombosis Research. 187, 48-55 (2020).</li></ol>

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>&#8805;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>&#8805;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 &#8805; 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>&#8805;99.5% purity</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Millipore Sigma</td>
			<td>P9791</td>
			<td>&#8805;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>&#8805;99.5% purity</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Millipore Sigma</td>
			<td>71636</td>
			<td>&#8805;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, ...

Thromboelastography and turbidity assays are two simple and distinctive methods for clot characterization. These techniques together offer a more comprehensive understanding of how clotting variables impact clot features. Both of this assays, not only offer endpoint clot analysis but also attract fibrin clot formation over time, unlike many other clot characterization tools These techniques can help develop a physiologically relevant synthetic clot platform that can provide reliable diagnosis of the fibrinolytic state of thrombosis patients To monitor clot turbidity over time, use any commercially available spectrometer that has an absorbance range of 350 to 700 nanometers.Turn on the spectrometer and open the corresponding analysis software. Then select plate one and open the plate settings tab. Click ABS and kinetic to monitor a dynamic absorbance over time.Select 550 nanometers in the wavelength tab, then switch to the timing tab and adjust the total runtime to 60 minutes with an interval of 30 seconds, select the wells of interest by highlighting them. Pipette 140 microliters of PBS into one well of a UV transparent 96 well plate. Then add 10 microliters of thrombin and mix, immediately initiate clotting by adding 50 microliters of fibrinogen to the well and pipette up and down five times using only the first stop of the pipette taking care to avoid creating bubbles.Place the plate in the plate holder and click read in the software to start the turbidity reading. When the read has finished retrieve the turbidity data and obtain a turbidity tracing curve by plotting the absorbents change over time, derive maximum turbidity by taking the max absorbance value of the curve over time, then calculate 90%maximum turbidity by multiplying maximum turbidity by 0.9. Derive the time to maximum turbidity by computing the time from clot initiation to 90%maximum turbidity.Turn on the thromboelastography or TEG analyzer, and wait for the temperature to stabilize at 37 degrees Celsius, then open the TEG software and create an experiment name under the ID section. Click the TEG tab and follow the onscreen prompts to conduct an eTest test for all channels. Then place the lever back to the load position once all checks are complete.Click done in the information page and 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 five times to a fix the pin to the torsion rod then lower the carrier and press the cup downward into the base until it clicks.Pipette 20 microliters of thrombin solution into the TEG cup, then initiate clotting by adding 340 microliters of fibrinogen into the cup to obtain a 360 microliter clotting solution. Mix the contents of the cup by pipetting up and down five times. Slide the cup loaded carrier up move the lever to the read position and click start in the software to initiate the TEG reading.Once the reading is completed retrieve the parameters and obtain a TEG tracing curve by plotting amplitude over time. Elect MA as maximum amplitude, which is indicative of clot strain and TMA as time to maximum amplitude from the software. Representative turbidity and a TEG tracing curves of human and bovine fibrin clots at different fibrinogen levels are shown here.The 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. Maximum turbidity and time to maximum turbidity are the two parameters derived from turbidity, while maximum amplitude and time to maximum amplitude are derived from TEG. At a higher level of fibrinogen in the clotting solution all four values increase.Maximum turbidity is an optical measure of clot structure, which is indicative of fibrin fiber thickness and fibrin network density. While maximum amplitude is a mechanical measure that reflects absolute clot strain, taken together the two values provide complimentary insight about the clot micro structures. This procedure demonstrates a simplified clotting model to examine variables that mainly affect fibrin polymerization.This model can be customized by inclusion of additional clotting factors to study other parameters. The clotting model can be easily modified using clinical plasma samples to monitor clot formation and disillusion in the presence of antithrombotic drugs. Results could guide therapeutic management in patients.

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Research

JoVE Journal

Medicine

6.6K visualizações.  Indiana University School of Medicine. Os ensaios de tromboelastografia e turbidez são dois métodos simples e distintos para caracterização de coágulos. Essas técnicas em conjunto oferecem uma compreensão mais abrangente de como as variáveis de coagulação impactam as características do coágulo. Ambos os ensaios, não só oferecem análise de coágulos de ponto final, mas também atraem a formação de coágulos fibrinos ao longo do tempo, ao contrário de muitas outras ferramentas de caracterização de coágulos Essas...