The circulating blood inside our bodies has many functions including transporting O₂, CO₂, and delivering nutrients to the cells. This circulating blood is also a significant source of information on in vivo coagulation parameters, hypercoagulability, and alterations in fibrinolysis. Blood coagulation involving a blood fluid to become a solid clot is critical to stop bleeding when an injury occurs inside or outside of the body. However, abnormalities in blood coagulation such as hypercoagulability can cause excessive blood clots and vein blockage, leading to stroke (Kahn, 2003; Levine, 2005). Cancer (Falanga et al., 2013), infectious diseases such as HIV (Lijfering et al., 2008) and hepatitis, trauma (Rugeri et al., 2007), diabetes, and retinal vein occlusion (Kuhli-Hattenbach et al., 2010; Goren Sahin et al., 2018) etc. affect the coagulation stages and create serious complications, as well. For example, the tumor cells can cause thromboembolic complications due to activation of blood coagulation by producing procoagulants such as tissue factor (the primary activator of blood coagulation), releasing soluble factors such as thrombin that induce platelet activation, aggregation, expressing proteins regulating the fibrinolytic system and causing impairment in plasma fibrinolytic activity (Marinho and Takagaki, 2008). Therefore, accurate measurement and understanding of hemostasis including blood coagulation and fibrinolysis is highly demanded to study defects from sensing parameters in different disease models.

Blood coagulation and fibrinolysis are complex processes in which platelets, fibrins, enzymes, and a series of complex chemical reactions play a role. Since red blood cells (RBCs) also have significant functions in blood clotting and its disorders (Demiroglu, 1997; Yalcin et al., 2016; Litvinov and Weisel, 2017), the RBC rheology in different disease conditions has been widely studied (Ertan et al., 2017; Ugurel et al., 2017; Yalcin et al., 2017). For example, red blood deformability is controlled by calcium (Yalcin et al., 2016) and it directly affects the blood viscosity and clotting. In addition, the force provided by clotting is high enough for the 10 pN upper force limit needed for RBC aggregation triggered by Ca⁺ (Bernhardt et al., 2019). The coagulation process begins with vascular and platelet phase (primary hemostasis), continues with activation of the coagulation cascade (secondary hemostasis) and ends when the clot dissolves through the fibrinolytic system (fibrinolysis). In the primary hemostasis, after the endothelium cells (vascular inner surface) are injured, procoagulant subendothelial matrix (such as laminin, collagen, and fibronectin) will be exposed to initiate primary phase. Then, the platelets come in contact with the collagen that occurs on the surface of the vessel, and release of adhesive proteins such as von Willebrand factor (vWf) from the endothelial cells leads to adhesion of platelets to the exposed subendothelial matrix. Subsequently, the adhesion of platelets causes platelet activation which will be amplified by platelet activator thrombin generated by coagulation cascade. The secondary hemostasis includes the stages of consolidation of the platelet plug and formation of fibrin clot. Coagulation proteins are the main components of the coagulation system, leading to a complex sequence of reactions and conversion of soluble fibrinogen to insoluble fibrin strands.

Figure 1 illustrates blood coagulation process including enzymatic cascades and evolution of the related TEG signal, in different stages of coagulation. As shown in the Figures 1A,B, the enzymatic reaction is initiated by the intrinsic or extrinsic activators and finally leads to common pathway. At the beginning of the process, the complex form of the tissue factor and factor VII activates factor IX, which demonstrates that the intrinsic and extrinsic coagulation pathways are interconnected. Second, the remained process includes three consecutive phases: the initiation phase, an amplification phase, and the propagation phase. In the two last phases, both platelets and thrombin are actively contributing (Thiruvenkatarajan et al., 2014). In the intrinsic pathway, negatively charged surfaces stimulate blood clotting with factors XII, XI, IX, and VIII. In extrinsic pathway, release of tissue factor initiates blood clotting with factor VII. And finally, the common pathway is triggered by factor X and it results in fibrin generation with factor V, prothrombin, and fibrinogen. Produced soluble fibrins are converted to stable fibers through factor XIII. Figure 1C shows the mechanical resistance of clotting blood during coagulation process. To study the disorders in the coagulation cascade, time response of the mechanical changes of clotting blood are compared with the ranges recorded from healthy donor and patient bloods in databases. Thromboelastogram (TEG) and rotational thromboelastometry (ROTEM) have been widely used as rheometers adapted for clinical application of blood coagulation monitoring (Ganter and Hofer, 2008). To monitor blood coagulation, observing mechanical properties of the clotting blood is the most powerful method. Mechanical changes in the clotting blood (viscosity and shear resistance vs. time) are correlated to the different parameters in the coagulation process such as clotting times and clot formation. In fact, the blood clot formation is monitored when it is resisting an applied shear stress. In addition, the blood viscosity or shear resistance is affected by the shear rate and it should be monitored in physiological range of shear rates similar to those inside the body to prevent disturbance of the coagulation process by platelet aggregation (Heemskerk et al., 2002; Ranucci et al., 2014). Coagulation can be initiated by an intrinsic stimulator reagent such as ellagic acid/phospholipid (Bock et al., 1981), by the extrinsic stimulator reagent such as tissue factor (Camerer et al., 1996), or fibrinolysis can be stimulated by the platelet inhibitors such as cytochalasin D in combination with extrinsic stimulator (Nielsen et al., 2000).

In previous reviews (Ganter and Hofer, 2008; Harris et al., 2013a) various point of care (POC) devices for blood coagulation tests have been compared and their working mechanism of detection briefly discussed, while in this paper blood coagulation monitoring techniques covering recent emerged technologies (till 2019) from engineering perspective have been presented. Recent technical advances and developments has been covered by emphasis on the mechanisms of the blood coagulation measurements with different technologies in order to provide an insight to researchers on advantages of certain techniques in detecting disorders and future trends for clinical monitoring and treatments. Moreover, the role of nanomaterials for both detection and treatment has been discussed, and finally available commercial devices, future trends about monitoring of novel anticoagulant drugs, artificial intelligence in diagnosis, and monitoring for higher accuracy of measurements have been described. Therefore, both areas of academic and industrial sections have been covered which enables to fill the current gaps in the blood coagulation monitoring and possible developments for continuous hemostasis monitoring in the future.

Point-of-care (POC) tests for coagulation monitoring with the help of various activators, play an important role in detecting bleeding disorders and monitoring of anticoagulant drugs. Prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), Prothrombinase induced clotting time (PiCT) are the most useful tests to investigate patients with coagulopathies and drug monitoring (Kamal et al., 2007; Hussain, 2015). These tests are used to monitor safety and effectiveness of anticoagulants, and sensitive to the lack of many coagulation factors. PT test is used to investigate clotting blood from extrinsic and common pathways in which factors I (Fibrinogen), II (Prothrombin), V, VII and X contribute. APTT test is measuring clotting from intrinsic and common pathways. In summary, PT is sensitive to factors II, V, VII, and X deficiencies, whereas aPTT is sensitive to deficiencies of prekallikrein, high molecular weight kininogen (HMWK), factors XII, XI, IX, and VIII. In addition, PT and aPTT are also sensitive to the presence of circulating anticoagulants such as lupus (Rosner et al., 1987). To standardize PT values, International Normalized Ratio (INR) is used which is mathematically driven from PT to include reagent sensitivity and varying instrument to keep the patients within narrow therapeutic ranges. INR = (patient PT/patient MNPT) ^ISI represents the formula to calculate INR, where the MNPT is the mean normal PT, and the ISI is an international sensitivity index (a factor to adjust instrument and reagent sensitivity differences). The therapeutic or conventional INR ranges are between 2.0 to 3.0. INR levels above 4.9 are examined as critical values and increasing the risk of bleeding. As an alternative, a narrow INR range lower than 2.0 (1.5 to 1.9) is used to reduce bleeding risks and so improve the safety profile of anticoagulation (Secretariat, 2009; Ong et al., 2016; Guimarães et al., 2019).

The biggest appeal of POC devices is short measurement durations (25–45 min) compared to conventional coagulation tests (40–60 min). POC INR devices can also be used in pre-hospital emergency services to provide fast and reliable results for patients suspected of coagulation factor deficiencies (Niederdöckl et al., 2016). To perform the aforementioned coagulation tests, different technologies such as electrical impedance spectroscopy, micro/nanoelectromechanical (MEMS/NEMS) resonator based rheometers, optical and photoacoustic measurements, and microfluidic viscometers have been employed. In this regard, understanding working principle and mechanism of these techniques facilitates researchers in identifying proper setup for laboratory or clinical applications, current gaps, and designing future devices. Among these technologies, photoacoustic/ultrasound measurements (Huang et al., 2005a; Huang C.-C. et al., 2011; Scola et al., 2011; Das and Pramanik, 2019) and microfluidic platforms have been used for in/ex vivo tests which are very promising for translational medicine when actual monitoring of the drug/anticoagulant effects or detection of circulating blood clots (CBCs) is desired (Karpiouk et al., 2008; Jain et al., 2016a).

Table 1 lists different technologies including optical, electro-mechanical, photoacoustic, and electrical impedance spectroscopy for blood coagulation measurement, physical parameters, and their use in coagulation tests. Optical blood coagulation measurement techniques include laser speckle rheology (LSR), optical coherence elastography (OCE), fluorescent imaging, and surface plasmon resonance (SPR). While viscoelastic properties and shear modulus are correlated to the blood coagulation parameters in LSR (Tripathi et al., 2014) and OCE (Xu et al., 2019), the fluorescent intensity level and refractive index are measured for studying clotting parameters in fluorescent imaging (Dudek et al., 2010) and SPR (Hansson et al., 2007), respectively. The second category of the blood clotting measurement devices involve electromechanical technologies covering all types of MEMS/NEMS resonators, microfluidic viscometers, and ultrasound detection systems. The third category is photoacoustic detection based on applying a laser beam and detecting the emitted acoustic signal (thermoelastic wave). In this technology, a photoacoustic (PA) signal enables monitoring of the adherent thrombi and emboli in deep tissue (Juratli et al., 2016). As optoacoustic signals provide high accuracy measurements of physiologic variables at depths greater than the optical diffusion limit, it is a powerful tool for recording the physical changes of tissue by monitoring the effective absorption coefficient (μa), effective attenuation coefficient (μeff), and the thermoacoustic efficiency (Γ) parameters (Wang, 2009). The fourth and the most widely used technology for measuring clotting time in POC tests is electrochemical impedance spectroscopy technique since it is low cost and simple to implement. This technology employs integrated circuits such as electronic amplifiers and digital synthesizers to measure impedance change of clotting blood with high accuracy. Second category of this technology is electrochemical aptamer biosensors for thrombin measurement (Mir et al., 2006).

Growing field of nanomedicine in recent decades has created a need for the investigation of nanomaterials' biocompatibility including their anticoagulation properties. Anticoagulation properties of these nano materials such as metal nano particles (gold, silver, and platinum; Ehmann et al., 2015; Dakshayani et al., 2019), metal oxide nano particles of titanium oxide (Huang et al., 2003) and zinc oxide (Huang Z. et al., 2011), carbon nano materials of graphene oxide and carbon nanowires (Wang et al., 2012; Haung et al., 2013), and modified polymeric nano materials have been studied and they were shown to be used successfully in the treatment and detection of hemostasis diseases. Nano particles can specifically interact with the coagulation system or avoid to interact. Generally, this interaction can be in two different ways: contact with plasma coagulation factors or interaction with cells, such as epithelial cells, monocytes, and platelets (Matus et al., 2018).

Herein we review the usage of some nanomaterials for blood coagulation detection and treatment. Figure 3A shows the usage of different nanomaterials for wound healing. Polymeric nanoparticles, metallic nanoparticles, zinc oxide Nps, Nanocreia, and electrospun nanofibers have been used to stop bleeding and wound healing (Kalashnikova et al., 2015). For instance, electrospun N-Alkylated chitosan (NACS) fibers have been studied as an effective hemostasis agent. NACS can be used to stop bleeding by converting whole liquid blood into a gel immediately It has been explained that, the main mechanism of clotting by Ch-based hemostatic agent acts is by adhering and physically sealing the bleeding wound based on mainly the electrostatic interaction between positive charge of protonated amine group of Ch and negative charge of erythrocytes cell membranes. This fibers have been shown to be in favor of the activation of coagulation factors and platelets (Wang et al., 2018). Pillai et al. reported Chitosan hydrogel incorporated with nano Whitlockite [WH: Ca₁₈Mg₂ (HPO₄)₂(PO₄)₁₂] including approximately up to 20 wt% of the inorganic phase of human bone. WH consists of Ca²⁺, Mg²⁺, and PO4-3 ions which activate different coagulation factors involved in coagulation cascade. Figure 3B illustrates the mechanism of inducing hemostasis by Ch-nWH composite hydrogel, and its rapid and effective hemostasis in in vivo experiment (Muthiah Pillai et al., 2019). Figure 3D shows a hybrid of agar gel and thiol-coated silica nanoparticles immobilized with methylene blue, and the result of aPTT test to monitor heparin. Methylene blue is a Federal Drug Administration (FDA) approved contrast dye agent responding to heparin. This hybrid has a significant and dose-dependent increase in photoacoustic signal in the presence of both heparin and low molecular weight heparin (LMWH) (Wang et al., 2016). Responsive thrombus-specific theranostic (T-FBM) nanoparticles that could provide H₂O₂-triggered photoacoustic signal amplification have been shown to be used as an antithrombotic nanomedicine (Jung et al., 2018).

Sensing of thrombin activity assisted by nanomaterials is the another main application of them in coagulation monitoring (Lin et al., 2013). They have been used as synthetic biomarkers in Urine for non-invasive monitoring of intravascular thrombosis and cancer (Lin et al., 2013; Warren et al., 2014). To detect thrombin, thrombin-sensitive peptide substrates are conjugated with nanoparticles. For instance, thrombin-activated near-infrared fluorescence (NIRF) probe consists of a thrombin-cleavable peptide spacer and contained a terminal fluorescence reporter which is quenched when conjugated to a biocompatible delivery vehicle. Following thrombin addition, the probe is activated within minutes. The changes in the relative thrombin activity is measured through fluorescent intensity (Tung et al., 2002). In addition, different types of nanomaterials such as magnetic (Khurshid et al., 2017), metallic, and metal oxide nanomaterials have been employed for blood clot detection as explained in the electrochemical sensing.

Furthermore, there are many studies on treatment and imaging of acute thrombi by nanoparticles and nanotheranostics. In fact conjugation of the nanoparticles with chemical agents interacting with the blood coagulation elements enables treatment and imaging. Heparin conjugated nano particles is one example. Xiang et al. employed ROS-responsive heparin-DOCA/PVAX nanoparticles to inhibit thrombus formation (Xiang et al., 2019). In their work, by using nanoparticles that contain anti-inflammatory polymer (copolyoxalate containing vanillyl alcohol, PVAX) and anti-thrombotic heparin derivative deoxycholic acid (Hep-DOCA), they manipulated the inflammation-associated coagulation. Voros et al. reported TPA immobilization on iron oxide nanocubes to accelerate clot lysis (Voros et al., 2015). Xin Yu et al. used a novel fibrin-targeted paramagnetic nanoparticle contrast agent for high-resolution MRI characterization of human thrombus (Yu et al., 2000). McCarthy et al. developed magneto-fluorescent crosslinked dextran-coated iron oxide nanoparticle platform as a multifunctional nano agent for thrombus-targeted fibrinolytic therapy (McCarthy et al., 2012).

Moreover, there are some of the nano hemostasis materials commercially available or in the phase of FDA approval such as silver NPs (various FDA-approved silver-based formulations have been used for chronic wounds). For instance, Acticoat™ produces wound dressing consists of silver nanoparticle wich can be released to the wound. Electrospun nanofibers (Rolandi et al., 2014), synthetic fibrin polymers (Chan et al., 2015), and nanocarriers for drug delivery (Nethi et al., 2019) are other nanostructured materials which are in FDA approval phase. In addition, thrombin@Fe₃O₄ nanoparticles which has been used as a hemostatic agent in internal bleeding (Shabanova et al., 2018), PolySTAT, chitosan nanoparticles, and electrospun fibers are hemostasis materials approved by FDA. One commercial device for wound healing is SpinCare which is a portable electrospinning device and can produce nanofibers containing drugs such as antibiotics, as well as antibacterial compounds, collagen, silicon, and other substances that may facilitates healing a wound.

Commercial POCT devices for blood coagulation monitoring can be divided in two categories: (1) devices for standard diagnostic blood clotting tests such as PT/INR, and APTT tests, (2) devices which measure directly the viscoelastic properties of clotting blood. Table 2 lists some commercial coagulation analyzers. Hand-held devices to test blood clotting tests include Xprecia Stride™ Coagulation System (Piacenza et al., 2017) and CoaguChek (Plesch et al., 2008) which are working based on electrochemical sensing, Optical Coagulation Analyzer (OCG-102) which is working based on optical technology, and COAGMAX is a MEMS device. This portable devices allow monitoring of clotting status in homes, and also facilitate the rapid assessment of clotting status in all clinical scenarios including operating and emergency rooms. On the other hand, the second category of devices provide more information and coagulation parameters similar to TEG/Rotem devices. For instance, TEG-6S is a miniaturized version of TEG system which consists of a microfluidic cartridge and piezoelectric resonators (Gurbel et al., 2016), Thrombodynamics® Analyzer provides thrombodynamics analysis (TD) based on the analysis of thrombin distribution with optical and fluorescent imaging (Sinauridze et al., 2018), and Quantra™ Hemostasis Analyzer is a new developed device for evaluation of hemostasis providing clotting time, clot stiffness, platelet, and fibrinogen contributions to clot stiffness based on the ultrasonic technology (Ferrante et al., 2016; Baryshnikova et al., 2019). Figures 4A,B shows the commercial devices TEG-6S and Thrombodynamics® Analyzer respectively.

The TEG-6S is a fully automated diagnostic platform (see Figures 4A,B), a four-channel microfluidics cartridge is used for parallel analysis of 4 samples. The cartridge is loaded with (~0.4 ml volume) with a pipette. Reagents are dried and resident within each channel, and after moving blood by the microfluidic valve and bellow action they are mixed. After that, approximately 20 μl of mixed sample is transferred to the terminus of each microchannel, where clotting is monitored. Waste chamber is for the excess of the sample. Thrombodynamics® Analyzer provides thrombodynamics (TD) analysis (see Figures 4C,D), a new global coagulation test which measures the actual anticoagulant activity of the drug in a particular patient rather than concentration of heparin in the blood. In this system uses time-lapse video-microscopy to extract and quantify spatiotemporal parameters of fibrin clot growth/lysis. The clot growth curve is obtained from both optical and fluorescent imaging. As the Figure 4D shows, coagulation is initiated by an activator fixed in space and extends in a thin layer of non-stirred plasma. Clot size is measured through Light scattering while it increases in an area against time. Therefore, this system model imitate in vitro physiological and pathophysiological processes closer to in vivo conditions than other homogeneous global tests. The parameters extracted from clot growth curve are: Tlag (the time to the start of clot growth); Vi (initial) and V (stationary) spatial clot growth rates (the slopes of the clot size curve vs. time for the segments of 2–6 min and 15–25 min from the clot growth start for Vi and V, respectively); and CS (the clot size at 30 min after coagulation activation). Two other important parameters may also be measured: the maximum optical density of the formed clot (D), which characterizes the clot quality, and the time of appearance of spontaneous clots in the sample (Tsp) (Sinauridze et al., 2018). Competitive advantages of thrombodynamics test include: (1) it enables to evaluate the efficacy of anticoagulant drugs including newest version of direct oral anticoagulants (DOACs), (2) enables to evaluate efficacy of anti-platelet therapy, (3) enables to monitor and study thrombin generation.

Although recent developments in the development of blood coagulation analyzers are promising, still there are a high potential for developing novel monitoring and therapeutic technology in the future based on photoacoustic detection and nanotechnology. The aforementioned research papers showing the powerful methods of fluorescent imaging and photoacoustic detection is capable of in vivo measurements while there are some FDA approved dyes that can be used for heparin detection. On the other hand, microfluidic platforms which some are reported already in research papers have been patented and they may be produced in the future. Therefore, this review also help industry sectors for possible future products and trends which at the same time save the time and money for new start-up for developing new platforms based on this review.

Blood coagulation monitoring with high level of accuracy and reliability for anticoagulant drug dose adjustments (e.g., for heparin and warfarin), studying effects of the drugs, and checking the risks in surgeries for the patients is highly demanded. Viscoelastic assessments, optical (scattering and fluorescent imaging), and electrical impedance measurement are frequently used for both evaluating pharmacological treatments and diagnosing the blood coagulation abnormalities. Recent advances in microfluidic technology has enabled the researchers to simulate the blood coagulation process in physiological conditions and study the events in the molecular level. Moreover, the fluorescent imaging and targeting different particles with fluorescent probes in microfluidic channels facilitate understanding of the interactions and origin of defects with a remote, accurate, and multiplexed manner. Other platforms like centrifugal microfluidic devices can be used for future multiplexed analysis of blood as it facilitates the separation of different blood components. Moreover, we discussed different nanostructured materials for the detection and treatment blood coagulation purposes which helps developing future monitoring and control of hemostasis at the same time in in vivo condition. In this paper, we covered different technologies for coagulation sensing and their working principle, some recent commercial devices, and possibilities of continuous in vivo hemostasis monitoring.

MM, AE, and OY wrote the article, read, and approved the final version of the manuscript.