Description:FIELD OF THE INVENTION
The present invention relates to a system for real-time monitoring and quantitative assessment of coagulation kinetics of plasma and method thereof. More particularly, present invention relates to a Twin-interferometry (TIM) based optical system for analyzing coagulation of plasma from the aspects of anisotropy and directional dependent coagulation kinetics, to enable real-time monitoring and a quantitative assessment of coagulation kinetics and a method thereof.

BACKGROUND OF THE INVENTION
The coagulation of blood is a complex and dynamic phenomenon that plays a crucial role in haemostasis and wound healing. Haemostasis is a response of the body to a damaged blood vessel, which is essential to slow down, minimize and eventually stop the bleeding. Coagulation is the process by which flowing liquid blood plasma is converted to a soft, viscous gel entrapping the cellular components of blood including red cells and platelets and thereby preventing extravasation of blood. The process of coagulation that occurs in different pathways is associated with various coagulation factors. The coagulation factors are specialized proteins that are essential for the blood to coagulate normally. Understanding the basic mechanism of plasma coagulation is of great significance in the field of diagnosis and treatment.

In a nutshell, the blood coagulation occurs during vessels damage and comprises of two primary components: the formation of fibrin meshes across the wound and the aggregation of platelets across the mesh. In order to prevent excessive coagulation, Thrombin, a key enzyme in the coagulation cascade, primarily responsible for converting fibrinogen into fibrin, inherently involves a self-feedback loop that controls its own production when the amount of fibrin produced at the vessel wall exceeds a certain threshold. This control mechanism is driven by anticoagulant factors primarily Protein C and Protein S and a few others such as antithrombin, calcium ions, flow of blood, etc. that help to balance the coagulation cascade. Thus, investigating the dynamics involved in the coagulation cascade and modelling the entire process has a long history and continues to be an active topic of research.

A number of literature have been published including patent and non-patent documents in said domain. A non-patent literature by Ratto, N., Bouchnita, A., Chelle, P. et al., titled as “Patient-Specific Modelling of Blood Coagulation.”, published in 2021, discloses a methodological approach to patient-specific modelling of blood coagulation. It begins with conventional thrombin generation tests allowing the determination of parameters of a reduced kinetic model. Next, this model is used to study spatial distributions of blood factors and blood coagulation in flow, and to evaluate the results of medical treatment of blood coagulation disorders. This document attempts to study the process using complex equations. The main reason for having numerous complex equations is that the coagulation process involves many reaction rate constants as well as many unknowns. Therefore, a majority of theoretical models that describe the coagulation kinetics comprises of equations varying in number from a few to hundreds that present computational challenges and limitations for solution of the models.

On the other hand, addressing the process of coagulation with a simple and relatively lesser number of equations requires several assumptions and approximations that are difficult to validate.

In addition to reaction kinetic based theoretical models, numerous experimental approaches for studying and modelling the whole coagulation process have also been proposed in the literature. Prothrombin test, ultrasound, Rhinometry, optical coherent elastography based coagulation assessment, Laser Speckle correlation, microfluidic-based point-of-care coagulation sensing methods are a few of the techniques that are now utilized to quantify and assess the blood coagulation process. Highly controlled in vitro assays like standard PT/aPTT and thrombin generation tests, exhibit precise control over factors but lack physiological relevance and often only provide end-point data. Viscoelastic whole blood assays (TEG/ROTEM) offer a global assessment of clotting but are static and lack flow.

Another reference is made to a patent document US5167145A, titled as “Measurement of blood coagulation time using infrared electromagnetic energy”. This document relates to a method for recording the clotting time of whole blood by monitoring the transmission of infrared electromagnetic energy through a blood sample. It however does not address the challenge of providing precise, reliable, and artifact-free measurements of blood coagulation time, especially in a clinically relevant and user-friendly manner.

A non-patent literature by D. Maji, M. De La Fuente, E. Kucukal, et al., titled as “Assessment of whole blood coagulation with a microfluidic dielectric sensor, published in 2018, discloses dielectric microsensor, based on the electrical technique of dielectric spectroscopy for rapid, comprehensive assessment of whole blood coagulation. The main drawback of such complex ex vivo models, including perfusion chambers and microfluidic devices, is that these introduce controlled flow and surface interactions but are technically demanding. Each of the methods of experimental approaches provides unique insights but carries inherent limitations, necessitating a multi-pronged approach to fully understand this intricate biological process.

In order to obviate the drawbacks in the existing state of the art, there is a pressing need for a precise, reliable and cost-effective method that enables real-time monitoring and a quantitative assessment of coagulation kinetics of plasma to understand the complex and dynamic characteristics of coagulation of plasma.

OBJECT OF THE INVENTION
In order to overcome the shortcomings in the existing state of the art, the objective of the present invention is to provide a system for real-time monitoring and quantitative assessment of coagulation kinetics of plasma.

Yet another objective of the invention is to provide a Twin-interferometry (TIM)-based optical technique for studying and characterizing plasma coagulation under isothermal conditions.

Yet another objective of the invention is to provide a means to study fringe patterns and their correlation to the underlying coagulation dynamics.

Yet another objective of the invention is to provide a method for real-time monitoring and quantitative assessment of coagulation kinetics of plasma to include human plasma.

Yet another objective of the invention is to provide a method to extract certain significant coagulation parameters such as the half coagulation time, the rate of change of optical path length of the coagulating plasma, Nucleation dynamic changes in plasma parameters etc.

Yet another object of the present invention is to provide a precise, and cost-effective interferometric method to understand the dynamic characteristics of coagulation.

Yet another object of the present invention is to provide a method for detecting the rate of fringe shift as a function of refractive index variation to yield a quantitative assessment of coagulation kinetics.

SUMMARY OF THE INVENTION:
The present invention discloses a system for real-time monitoring and quantitative assessment of coagulation kinetics of plasma and method thereof. More particularly, present invention relates to a Twin-interferometry (TIM) based optical system for studying coagulation of plasma from the aspects of anisotropy and directional dependent coagulation kinetics in plasma under isothermal conditions.

The present invention discloses a Twin-interferometer that is designed to record the rate of shift in Michelson’s fringes caused by rate of change of optical path difference of coagulating plasma in two orthogonal directions. A pair of interferograms, referred to as the Twin- interferogram (TIG), are recorded for plasma samples with different CaCl2 concentration during its coagulation. The invention discloses a new technic for extracting various coagulation parameters from TIG, such as anisotropy, half coagulation time, constants governing the rate and dimensionality of coagulation, and their variation along distinct direction. TIG results as obtained in the invention are validated using polarizing optical microscopic (POM) and optical extinction techniques.

The system of the present invention comprises of a set of modules to include an optical module, data acquisition module, computing and analysis module, sample preparation module etc. The optical module comprises of the twin interferometer that is uniquely configured together in such a way that one of the arms is orthogonal to the other. The interferogram recorded along the two orthogonal directions, referred to as Twin- interferogram (TIG), furnishes the optical data or information pertaining to the presence of anisotropy and the rate of change of optical pathlength caused by rate of change of refractive index of coagulating plasma along the distinct directions of the optical beam. The data acquisition module enables data acquisition of the optical data from the optical module. The computing and analysis module assists in processing and analysis of optical data in the form of interferogram. The sample preparation module enables preparing the plasma sample that is to be analysed by the system.

The method of the present invention primarily comprises of steps of plasma sample preparation for analysis, arranging the arms of twin interferometer (TIM) of the system in required configuration, introducing the plasma sample into the TIM at the point of intersection of two optical beams from orthogonal arms of TIM and initiating coagulation, illuminating the TIM, recording interferograms and analysing the rate of fringe shift in twin interferogram (TIG), extracting significant parameters such as half coagulation time, coagulation rate constants, and their variation as a function of CaCl2 concentrations in two orthogonal directions are observed from TIG etc.

Accordingly, the present invention provides a precise, reliable and cost-effective system and a method thereof that enables real-time monitoring and a quantitative assessment of coagulation kinetics to understand the complex and dynamic characteristics of coagulation of plasma.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1(a) displays the schematic of the Twin-Interferometry based system.
Figure 1(b): displays flowchart depicting the working of Twin-Interferometry based system.
Figure 2 displays Twin -Interferogram of plasma sample injected with 0.04 M CaCl2 concentration (a) Recorded through the detector D1 (b) Recorded through the detector D2.
Figure 3 displays relative coagulation of plasma injected with various CaCl2 concentrations. (Solid line indicates the trend fitted to the observed data) (a) Extracted from the detector D1 (b) Extracted from the detector D2.
Figure 4 displays estimated t½ value for the plasma injected with various CaCl2 along the detectors D1 and D2.
Figure 5 displays Avrami plot for the plasma injected with various CaCl2 (a) For D1 (b) For D2.
Figure 6 displays a Twin interferogram of hot water cooling from 70°C to 28°C (a) Recorded through the detector D1 (b) Recorded through the detector D2.
Figure 7 displays Δt versus cooling time of water.
Figure 8 displays POM images of plasma injected with 0.0275 M CaCl2 concentration: captured at every 25 min interval during coagulation.
Figure 9 displays POM images of plasma injected with 0.04 M CaCl2 concentration: captured at every 10 min interval during coagulation.
Figure 10 displays digitally magnified POM image of plasma injected with 0.04 M CaCl2 concentration.
Figure 11 displays optically magnified POM image of plasma injected with 0.03 M CaCl2 subjected to the method of optical extinction.

DETAILED DESCRIPTION OF THE INVENTION WITH ILLUSTRATIONS AND EXAMPLES
While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.

Throughout the specification and claims, the following terms take the meanings explicitly associated herein unless the context clearly dictates otherwise. The meaning of “a”, “an”, and “the” include plural references. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The system, methods, and examples provided herein are only illustrative and not intended to be limiting.
The abbreviations used in the invention are represented in table 1 as below:
Table 1: Legend of abbreviations
S.no. Particulars Legend
1 Twin-interferometry TIM
2 Ethylenediamine tetra acetic acid EDTA
3 Twin- interferogram TIG
4 Polarizing optical microscopic POM

Some of the technical terms used in the specification are elaborated as below:
Plasma: Plasma refers to the liquid component of blood, specifically the straw-colored, viscous fluid that remains after blood cells (red blood cells, white blood cells, and platelets) are removed. It constitutes about 55% of the total blood volume. Plasma is essential for transporting various substances throughout the body, including nutrients, hormones, and waste products. It also plays a crucial role in maintaining blood pressure and supporting the body's immune system. In the present application the term plasma refers to the liquid component of blood of various animals including that of humans.
Coagulation kinetics: Coagulation kinetics in this document refers to Coagulation kinetics of blood. Blood coagulation, or clotting, is a complex process that involves the activation of a cascade of proteins and the formation of a fibrin mesh, ultimately stopping bleeding. This process is highly kinetic, meaning it involves a series of reactions with specific rates and mechanisms. Understanding the kinetics of coagulation is crucial for diagnosing and treating bleeding disorders. Some of the key aspects of blood coagulation kinetics are Cascade Activation, Factor Xa (FXa), Prothrombin Time (PT) etc.
Interferometry: Interferometry is a technique that measures the phase differences between two or more waves, usually light waves, to extract information about the system or object being studied. It leverages the phenomenon of wave interference, where waves combine to create a new wave with a different amplitude and phase. This interference pattern can be used to make precise measurements of distances, displacements, or other properties. It's a powerful tool for measuring changes in refractive index, small displacements, and surface properties, making it suitable for a wide range of biological and medical investigations.
Interferogram: The digitized form of the rate of change of the interference pattern due to variations in optical path length is called an interferogram. The terms interference pattern and interferogram have been used interchangeably in the document without affecting the essence/ significance of the invention.
Anisotropy: Anisotropy describes a material's property where its characteristics vary depending on the direction they are measured. In essence, it means that a material has different properties in different directions, as opposed to isotropy where properties are the same in all directions. Anisotropy in blood coagulation refers to the directional dependence of certain properties of blood clots, particularly in relation to their mechanical and structural features. This means that the clot's behavior and properties might differ depending on the direction in which they are measured or analyzed.
Coagulation factor: Coagulation factors are proteins in blood. They help form blood clots to stop bleeding when an individual has an injury. These proteins are also called clotting factors. There are 13 blood clotting factors, numbered I through XIII, are proteins that work together to form a clot and stop bleeding. They are involved in the coagulation cascade, a series of reactions that convert an inactive form of a protein into an active form, leading to the formation of a stable fibrin clot.
Isothermal conditions: An isothermal condition refers to a thermodynamic process where the temperature of a system remains constant throughout the process.
Half coagulation time: The half coagulation time of plasma, more accurately referred to as half of the activated partial thromboplastin time (aPTT), refers to the time it takes for plasma to clot half of its saturated clotting after the addition of reagents that activate the intrinsic and common coagulation pathways.
Nucleation: Nucleation is the initial formation of a stable unit cell during formation of crystal in a liquid or molten precursor solution . In plasma, nucleation and coagulation are key processes in particle formation and growth.

The reference numerals used in the present invention are tabulated below in table 2.
Table 2: Legend of Reference numerals
Ser No. Item description Reference signs/ numerals
1 System S
2 Optical module O
Light source O1
Optical beam splitter O2 or BS
Optical mirrors (O3) O3 or M
Beam expander O4
Optical detector O5 or D
Noise filter O6
Base O7
3 Data acquisition module A
Computing device A1
Interfacing tool A2
Recording tool A3
Controlling tool A4
4 Computing and analysis module C
Processing unit C1
Display unit C2
5 Sample preparation module SP
EDTA-coated vials SP1
Syringes for blood collection SP2
Cuvette SP3
coagulation agent solution SP4
Blood centrifuge device SP5
6 Power source P

Blood coagulation is a highly intricate biological process vital for stopping bleeding. It involves a precise interplay between platelets and numerous clotting or coagulation factors. These factors activate in a cascade, ultimately leading to the production of thrombin. Thrombin then converts fibrinogen into fibrin, forming a stable mesh that traps blood cells and seals the wound. This complex system is tightly regulated to ensure quick clot formation at injury sites without causing widespread, unwanted clotting. Disruptions to this balance can result in either bleeding disorders or dangerous clotting events.

The process of coagulation is characterized as occurring in two different pathways: the extrinsic pathways, which is triggered by an external vessel damage, and the intrinsic pathway, which is triggered by an internal vessel damage. These two pathways converge downstream to form a single pathway. All of these pathways are associated with various coagulation factors. The coagulation factors are specialized proteins that are essential for the blood to coagulate normally.

The factors involved in the extrinsic pathway are as follows. During vascular damage, a coagulation factor called factor VII emerges from the bloodstream via the external pathway and interacts with another factor called factor III or tissue factor, that is released by damaged cells outside the circulation. These two factors (factor VII and factor III) combine to form a complex known as factor VIIa, which then activates a factor X in the blood into factor Xa. The activated Xa combines with factor Va to form a complex called prothrombinase, which activates prothrombin into thrombin. The thrombin then converts soluble fibrinogen (also referred to as factor I) into insoluble fibrin fibres. The fibrin fibres form a mesh across the extrinsic pathway and induce the coagulation. This coagulation is further stabilized by factor XIII.

Coagulation factors associated with the intrinsic pathway are explained as follows. Internally damaged vessels generate negatively charged collagen on the damaged endothelium which activates the factor XII into XIIa, which then activates factor XI when it comes to contact with the endothelium. Further, the platelets present in the blood facilitate inducing factor VIII. This factor VIII combines with IX to produce an enzyme complex that activates factor X, which, along with factor Va, stimulates the production of thrombin, which eventually generates the fibrin fibres.

The occurrence of blood coagulation during vessels damage consists of two primary components: the formation of fibrin meshes across the wound and the aggregation of platelets across the mesh. To prevent undue coagulation, the thrombin inherently comprises of a self-feedback loop that checks its own production when the amount of fibrin produced at the vessel wall goes beyond a certain threshold. This control mechanism is driven by Protein C and Protein S by providing negative feedback on the coagulating cascade. Surplus thrombin activates thrombomodulin, which activates protein C, which then activates protein S, that degrades the factor Va and VIIIa to slow down the rate of generation of thrombin. Few other anticoagulant factors, such as antithrombin, calcium ions, flow of blood, etc. also help to balance the coagulation cascade.

STATEMENT OF THE INVENTION
The present invention discloses a system (S) for real-time monitoring and quantitative assessment of coagulation kinetics of plasma. The system comprises of a set of modules to include an optical module, data acquisition module, computing and analysis module, sample preparation module etc.
The details of the various modules of the present invention are presented in the following paragraphs and are illustrated in Fig. 1(a).

The optical module (O) of the present invention comprises of a plurality of interferometers for creating an interference pattern for analysing plasma. Each interferometer comprises of one light source (O1) for emitting optical beam towards sample of plasma, one optical beam splitter (O2) with mount, placed in the path of emitted light. The optical beam splitter is used for splitting said optical beam into two optical beams, one transmitted and one reflected.

The optical module further comprises of one pair of optical mirrors (O3) with their mounts for reflecting the two optical beams of light, comprising of one fixed mirror that reflects one of the optical beams back towards the optical beam splitter (O2) and one movable mirror that reflects the other optical beam back towards the optical beam splitter (O2) and whose position can be adjusted to change the path length of the optical beam. The optical module also comprises of one beam expander (O4) with mount, for expanding the optical beam for obtaining a broader optical signal in the form of interference pattern. It includes one optical detector (O5) for detection of said optical signal in the form of interference pattern formed by the beams on recombination. The module includes one noise filter (O6) in the form of pinhole with its mount for filtering optical noise from the optical signal and one base (O7) or platform for mounting all optical components.

The system further comprises of one data acquisition module (A) for data acquisition of the optical data from the optical module (O) that in turn comprises of one computing device (A1) for receiving said optical data from the optical detectors (O5), one interfacing tool (A2) for interfacing the optical detectors (O5) with said computing device (A1) and controlling the data acquisition rate and one recording tool (A3) for recording and storage of said optical data in the computing device (A1) of said data acquisition module (A).

The system further comprises of one computing and analysis module (C) that in turn comprises of one processing unit (C1) for processing and analysis of optical data in the form of interferogram, one display unit (C2) for display of analysis results.

The system also comprises of one sample preparation module (SP) for preparing plasma sample that is to be analysed by the system, comprising of a plurality of EDTA-coated vials (SP1) for collecting blood sample, a plurality of syringes (SP2) for blood collection and sample processing, a four-way transparent quartz cuvette (SP3) for holding the plasma sample in said optical module (O) during coagulation and analysis, Calcium chloride (CaCl₂) solution (SP4) of varied concentration for initiating coagulation reactions in blood sample and a blood centrifuge device (SP5) for extracting plasma from blood sample by centrifuging in the range of 5000-7000 RPM preferably 6000 RPM.

The system may also comprise of a power source (P) for providing power to said modules of the system (S).
The system is characterised in that the optical module (O) comprises of at least two interferometers also referred to as twin interferometer that are configured together in such a way that one of the optical beams passes orthogonal to the other in the sample. The two interferometers are independently adjusted to form an interference maximum, which is detected by two distinct optical detectors D1 and D2 (O51 and O52). The optical module (O) comprises of a third detector D3 that is used to monitor the intensity fluctuation of said optical beam due to thermal noise. The plasma sample of specific volume is taken in an optically flat four-way disposable square shaped glass cuvette (SP3), introduced in said Twin- interferometer at the point of intersection of two optical beams from the orthogonal arms of the interferometers. The optical module (O) comprising of twin-interferometer is optimally designed with all its components configured in a unique way to record the rate of shift in Michelson’s fringes in interferogram, caused by rate of change of optical path difference of coagulating plasma in two orthogonal directions. The interferogram recorded along the two orthogonal directions, referred to as Twin- interferogram (TIG), furnishes the information pertaining to the presence of anisotropy and the rate of change of optical pathlength caused by rate of change of refractive index of coagulating plasma along the distinct directions of the optical beam. The system (S) provides for extraction of various coagulation parameters from TIG, such as but not limited to anisotropy, half coagulation time, constants governing the rate and dimensionality of coagulation, and their variation along distinct direction and any directional dependent coagulation kinetics. Thereby the present invention provides for a precise, reliable and cost-effective system (S) that enables real-time monitoring and a quantitative assessment of coagulation kinetics to understand the complex and dynamic characteristics of coagulation of plasma.
The twin interferometer is optimally designed with all its components configured in a unique way such that the process of recording the rate of shift in Michelson’s fringes in interferograms comprises of steps as follows.
The optical beam from the light source (O1) is divided initially into two equal parts of by the beam splitter BS1 (O21). The transmitted beam from BS1 (O21) is split into two equal parts by a second beam splitter BS2 (O22).
The transmitted beam from BS2 (O22) is directed to detector D3 (O53) that monitors the intensity fluctuation of said optical beam due to thermal noise.
The reflected beam from BS2 (O22) is redirected by mirror M1 (O31) to another beam splitter BS3 (O23). The two resulting beams from BS3 (O23), one transmitted and one reflected are directed further back to BS3 (O23), using mirrors M3 (O33) and M4 (O34). These beams are overlapped at BS3 (O23), producing interference fringes or pattern that are detected by detector D1 (O51). The reflected beam from the initial beam splitter BS1 (O21) is divided by another beam splitter BS4 (O24). The two resulting beams are redirected back to BS4 (O24) via mirrors M2 (O32) and M5 (O35), where they interfere after passing through BS4 (O24) producing interference pattern. The resulting interference pattern is recorded by detector D2 (O52).

The optical expander (O4) is a convex lens with a short focal length and is placed such that interfered optical beams from each interferometer pass through it increasing the spatial extent of the interference fringes before they reach the optical detector (O5). It is used to broaden the laser beam. Expanding the fringes spatially helps to minimize or eliminate overlap between multiple fringe patterns, particularly those caused by ghost images resulting from reflections within the optical components.

The noise filter (O6) is placed such that the interfered optical beam emerging from the beam expander (O4) is directed towards the optical detector (O5) through it. The noise filter (O6) in the form of pinhole allows only a specific portion of the interference fringes to reach the detector. Thereby, it prevents the detector from being exposed to both bright and dark fringes at the same time, thereby reducing noise in the detected signal.

The optical detectors (O5) of the optical module (O) are detectors operating in their linear region to detect the intensity variation of Michelson’s fringes as the optical path length of the sample changes during the process of coagulation, such as reverse biased photo transistors. The optical beam is preferably laser beam in the range of 532 ± 2 nm produced by said optical source selected from a group comprising of He-Ne laser source, diode laser source, preferably He-Ne laser source that illuminates said twin interferometers.

A beam expander (O4) is a simple convex lens with a short focal length. It is used to broaden the optical laser beam. The interfered beams from each interferometer are passed through this beam expander (O4) to increase the spatial extent of the interference fringes before they reach the detector. Expanding the fringes spatially helps to minimize or eliminate overlap between multiple fringe patterns, particularly those caused by ghost images resulting from reflections within the optical components.

The interfacing tool (A2) of the data acquisition module for interfacing the optical detectors (O5) with the computing device (A1) and controlling the data acquisition rate are interfacing tools (A2) selected from Raspberry Pi, Arduino based drivers etc. preferably Arduino-based drivers. The recording tool (A3) for recording and storage of the optical data in the computing device (A1) of the data acquisition module (A) is selected from customised or open-source software selected from PuTTY, OpenSSH, KiTTY, MobaXterm etc. preferably an open-source PuTTY software.

The optical components in the TIM are set up in a vibration-free environment, aided by a passive vibration isolation table. The twin- interferometer is precisely adjusted so that two orthogonal optical beams pass through the same domain of the plasma sample in the cuvette (SP3).

The present invention provides a method for real-time monitoring and quantitative assessment of coagulation kinetics the steps of which are disclosed as follows. Fig. 1(b) illustrates the method of working of the invention in the form of flowchart. The plasma from blood sample is extracted using syringes (SP2) in an EDTA-coated vial (SP1) followed by arranging of the arms of twin interferometer (TIM) of optical module (O) of the system (S) for analyzing coagulation of plasma, in such a way that one of their arms is orthogonal to the other. A small volume, preferably 0.5 ml, of plasma sample is injected into an optically flat four-way disposable square shaped glass cuvette (SP3) of 3 ml volume, in the twin- interferometer at the point of intersection of two optical beams from the orthogonal arms of the twin interferometers. Further the method comprises of initiating coagulation of said plasma sample by injecting an equal volume of coagulation agent, preferably CaCl2, into the cuvette (SP3) containing the plasma sample in the TIM. The twin interferometer is illuminated using light source (O1) that emits optical beam towards plasma sample. splitting of said The optical beam is then split into two optical beams, one transmitted and one reflected.

The optical beams are then reflected and redirected towards specific optical beam splitters (O2), such that these beams recombine at the cuvette (SP3) to form an interference pattern. The twin interferometers are configured and adjusted independently to form an interference maximum, which is detected by two distinct optical detectors (O51 and O52) also referred as D1 and D2.The intensity fluctuation of optical laser beam due to thermal noise is monitored using a third detector (O53) also referred to as D3, The process of coagulation of plasma is monitored along the two orthogonal directions of the cuvette (SP3) in terms of fringe shift for the entire duration of the coagulation. The interferograms also referred to as Twin- interferogram (TIG) are recorded by the data acquisition module (A) along the two orthogonal directions, to obtain information pertaining to presence of anisotropy and the rate of change of optical path length caused by rate of change of refractive index of coagulating plasma along the distinct directions of the light beam.

The rate of plasma coagulation is determined by analyzing the rate of fringe shift in twin interferogram (TIG) by computing and analysis module (C), that is determined by the rate of change in the optical path length of plasma in the cuvette (SP3). The fringe intensity is plotted against the coagulation time by the processing unit (C1). Some significant parameters are extracted from the twin interferogram (TIG) obtained in the above steps and analysis carried out by the processing unit (C1) such as but not limited to anisotropy, half coagulation time, constants governing the rate and dimensionality of coagulation, their variation along distinct direction and other directional dependent coagulation kinetics. The analysis results are displayed on display device (C2) of said system (S).

The extraction of plasma from blood sample into EDTA-coated vials (SP1) is carried out by collecting blood sample in EDTA coated vials (SP1) from subject and centrifuging the collected blood sample at a speed in the range of 5000-7000 RPM, preferably 6000 RPM in a laminar flow unit or a blood centrifuge device (SP5) for a time interval in the range of 2-5 mins preferably 3 min to extract the plasma. The duration of coagulation ranges from 15 to 40 min depending on the CaCl2 concentration.

The system (S) finds application in the field of healthcare, research and development, materials science etc. for the purpose of study of coagulation time of human plasma, Prothrombin time etc. for clinical diagnostics and patient management, pre-operative assessment, trauma and critical care, liver disease management, drug discovery and development, biomaterial compatibility testing, fundamental coagulation research, characterizing complex biological fluids, developing novel biosensors for studying the kinetics of optically transparent polymer curing and solvent evaporation in materials science etc.

The present invention, therefore, provides for an optical Twin-interferometer based technique for extracting anisotropy and analyzing the coagulation of plasma under isothermal circumstances. Twin interferograms as part of the research work were obtained for diverse plasma samples undergoing induced coagulation at different CaCl2 concentrations. The fringe patterns and their correlation to the underlying coagulation dynamics are also disclosed in the invention. A few significant coagulation parameters such as half coagulation time, coagulation rate constants, and their variation as a function of CaCl2 concentrations in two orthogonal directions are studied, and the methodology for extracting these parameters is established by the invention. POM images validate the presence of anisotropy in plasma as shown by TIM. The periodic extinction of light shown by the locally oriented fibrin molecules in POM, for every 45° rotation of the sample, further substantiates the presence of anisotropy in plasma.

The present invention provides a method to extract certain significant coagulation parameters such as
(1) The half coagulation time, which is the time required to reach half of the saturated coagulation.
(2) The rate of change of optical path length of the coagulating plasma, optical path length in turn varies due to variation of refractive index as coagulation progress.
(3) Nucleation, which is the initial stage of coagulation in which a fibrin monomer is produced in aqueous medium, attracting other fibrin monomers to develop and create a polymer mesh with varying degrees of anisotropy.

The overall coagulation process is governed by two factors:
1) The tendency to form a monomer fibrin nucleus at the outset and
2) The mobility and the diffusivity of other fibrin monomers to reach the fibrin nucleus decrease as the density of the aqueous media increases, thereby slowing down the coagulation process. These parameters significantly influence some of the coagulation dynamics constants, such as dimensionality, crystallization rate, etc. according to Avrami theory and other phase transition theories.

The interferometric method of the present invention is precise and cost effective and can provide a large amount of insights into the dynamic characteristics of coagulation. Firstly, it enables real-time monitoring of the coagulation process as well as the dynamic changes in plasma parameters. Secondly, detecting the rate of fringe shift as a function of refractive index variation yields a quantitative assessment of coagulation kinetics.

EXAMPLES
The present invention shall now be explained with accompanying examples. These examples are non-limiting in nature and are provided only by way of representation. While certain language has been used to describe the disclosure, any limitations arising on account of the same are not intended. As would be seeming to a person skilled in the art, various working alterations may be made to the method in order to implement the inventive concept as taught herein. The figures and the preceding description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, the order of steps of method or processes of data flow described herein may be changed and is not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts need to be necessarily performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples.

The method of preparation of plasma and coagulation agent carried out experimentally towards development of the present invention is disclosed as follows. The blood samples were collected in Ethylenediamine tetra acetic acid (EDTA) coated vials from five different volunteers with their consent. The entire work, including blood collection, plasma extraction, and coagulation studies, was carried out in a laboratory setting, with all necessary precautions and safety protocols in accordance with established standards and procedures given by Government of India. The research and development for the present invention has been authorized by the ethical committee for human subject’s standards at Amrita Vishwa Vidyapeetham (Deemed to be University), Coimbatore campus, India.

The purpose of collecting blood in EDTA coated vessels is to inhibit coagulation by removing calcium from the blood. Most importantly, the EDTA does not distort or denaturize blood cells or proteins, making it suitable for the majority of haematological testing.

The collected blood was centrifuged at 6000 RPM in a laminar flow unit for 3 min to extract the plasma. The centrifuge essentially segregates the lighter plasma into an upper yellowish layer while the denser blood cells fall to the bottom. The segregated plasma in the EDTA-coated vessel is extracted with a single use syringe, and 0.5 ml of this extracted plasma is injected into an optically flat four-way disposable glass cuvette of 3 ml volume kept in the TIM. Since EDTA has removed calcium from the blood, an equal volume of CaCl 2 (0.5 ml) is injected into a cuvette containing 0.5 ml plasma in the TIM to initiate the coagulation process. CaCl2 essentially provides Ca, which acts as coagulation agent in this process. In order to vary the rate of coagulation, CaCl2 solutions of varying concentrations (0.02 M, 0.0225 M, 0.025 M, 0.0275 M, 0.03 M, 0.04 M and 0.05 M) were prepared by dissolving fused CaCl2 with 99% purity in doubly deionized water and used for this research work.

To avoid pre-analytics errors, all three detectors in TIM are enabled by single thread mode (started simultaneously) using computer code as soon as the plasma is injected in the cuvette. However, the coagulation process begins with the injection of CaCl 2 into the plasma which is monitored along the two orthogonal directions of the cuvette in terms of fringe shift for the entire duration of the coagulation. In the present invention, the coagulation time ranges from 15 to 40 min depending on the CaCl2 concentration in the current experiment.

The rate of fringe shift in this case is determined by the rate of change in the optical path length of plasma in the cuvette, which is determined by the rate of plasma coagulation. Furthermore, the constructed TIM setup revealed that the rate of coagulation varies along the different directions. The termination of fringe shift in Twin interferogram (TIG) shown as saturated line after final fringe as shown in Fig. 2(a) and 2(b) indicates termination of the coagulation. However, the occurrence of saturation (time at which coagulation stops or saturates) at different time intervals along the two orthogonal directions of the plasma establishes that the coagulation time is directionally dependent, proving anisotropy in plasma, which is further revealed by Polarizing Optical Microscopic study.

The technique of the present invention is specially designed to study and explore the coagulation mechanism and the anisotropic behavior of plasma during the coagulation process. According to an embodiment of the present invention, two Michelson interferometers are combined in such a way that one of their arms is orthogonal to the other. A laser beam of 532 nm produced by a 10 mW, He-Ne laser source illuminates both interferometers, as shown in Fig. 1(a). All optical components in the TIM are set up in a vibration-free environment, aided by a passive vibration isolation table. The beam splitters BS1, BS2, BS3, BS4 and mirrors M1, M2, M3 and M4 are used to split, reflect, and redirect the light beams in appropriate directions. In the beginning, two Michelson’s interferometers are independently adjusted to form an interference maximum, which is detected by two distinct detectors D1 and D2.

A third detector D3 is used to monitor the intensity fluctuation of laser beam due to thermal noise. The detectors typically operate in their linear region to detect the intensity variation of Michelson’s fringes as the optical path length of the sample changes during the process of coagulation, preferably reverse biased photo transistors. All the detectors are connected to computers through Arduino-based drivers, and the data is collected using the open-source PuTTY software.

The path of the light beam and the working of the optical module (O) is described as follows. The laser beam from a 10 mW source is initially divided into two equal parts of 5 mW each by the beam splitter BS1. The transmitted beam from BS1 is further split into two equal halves by a second beam splitter, BS2. The transmitted portion from BS2 is directed to a detector that monitors fluctuations in laser intensity due to thermal noise.
Meanwhile, the reflected beam from BS2 is redirected by mirror M1 to another beam splitter, BS3. The two resulting beams from BS3—one transmitted and one reflected—are further directed back to BS3 using mirrors M3 and M4. These beams then overlap at BS3, producing interference fringes that are detected by detector D1.

The reflected beam from the initial beam splitter BS1 is also divided by another splitter, BS4. The two resulting beams are redirected back to BS4 via mirrors M2 and M5, where they interfere after passing through BS4. The resulting interference pattern is recorded by detector D2.

Detectors D1 and D2 thus capture the interferograms generated by the Twin Interferometer Module (TIM). The plasma sample is placed at the intersection point where the two orthogonal beams from the respective interferometers pass through the sample in perpendicular directions. Changes in the optical path length due to coagulation within the plasma lead to shifts in the interference fringes in each interferometer.

The sampling rate and data acquisition are controlled by a computer program. The sample (Plasma) of specific volume is taken in an optically flat four-way disposable square shaped glass cuvette, introduced in the Twin- interferometer at the point of intersection of two laser beams from the orthogonal arms of the interferometers. The Twin- interferometry is precisely adjusted so that two orthogonal beam passes through the same domain of the sample in the cuvette. Variation in density of the sample during coagulation leads to the variation of optical path length in the sample, which eventually leads to shift in Michelson’s fringes in the corresponding interferometer. Conceptually, if the sample is isotropic, the rate of variation of optical path length along the two orthogonal directions is identical, which leads to an identical rate of shift in Michelson’s fringes in the two detectors. Whereas, if the sample is anisotropic, the rate of fringe shift is different along the orthogonal directions.

The interferogram recorded along the two orthogonal directions, referred to as Twin- interferogram (TIG), furnishes the information pertaining to the presence of anisotropy as well as the rate of change of optical pathlength caused by the rate of change of refractive index of coagulating plasma along the distinct directions of the light beam. Since the interferometry is sensitive enough to detect the optical path length variation of the order of half of the wavelength ( λ/2) of light, every ( λ/2 ) variation of optical path introduced by sample leads to one fringe shift in the interferogram.

POM technique is employed to study the anisotropic behaviour of plasma samples injected with CaCl2 solutions of concentrations varying from 0.020, to 0.050 M respectively. A few tens of microns thick plasma film with CaCl2 sandwiched between a transparent glass plate and a coverslip is held between a crossed polarizer and an analyzer in POM. The plasma film is equally distributed across the whole surface of the cover slip (10 mm x 10 mm). Essentially, the thickness of the film (few tens of micron) restricts the alignment of the formed fibrin molecules or fibrin mesh parallel to the region of the film. Since the diameter of a fibrin fibre is of the order of a few hundred nanometers, a film with a thickness of a few tens of micrometers and a surface area of 10 mm x 10 mm would easily accommodate the fibrin molecules for parallel alignment in the available space.

Images are captured at one-minute intervals for 3 hours using a 5MP camera connected to a computer. The process of capturing and storing of the images is controlled and automated by Xploview software. The sample is illuminated by green light, obtained using a green filter connected to the POM light source, for improved resolution.

The plasma extracted from blood is transparent in nature which is subjected to coagulation by adding CaCl2 solution of different concentration at ambient condition of 28° C. Plasma is normally composed of 91% to 92% of water and 8% to 9% of other bio molecules such as coagulants, Prothrombin, fibrinogen, albumin, globulins etc. The dispersion of these bio molecules in aqueous medium generally forms a self-assembling system that makes the plasma anisotropic in nature by exhibiting a semi-orientational and local translational ordering. Variation in plasma anisotropy during the coagulation causes variation in its associative parameters such as molecular ordering, density, refractive index, rate of refractive index, etc., along distinct directions.

These parameters are extracted from the TIG where the fringe intensity is plotted against the coagulation time (Fig. 2(a) and 2(b)). The following is the basic phenomenon involved in the process of coagulation. As CaCl2 solution of specific concentration is injected into the plasma, it propositionally supplies Ca2+ which activates the conversion of prothrombin into thrombin, which in turn activates the conversion of fibrinogen into fibrin monomers which eventually becomes fibrin fibres. In nutshell, the rate of plasma coagulation is determined by the rate of conversion of fibrinogen into fibrin or pro thrombin into thrombin. However, these processes are driven by the amount of Ca 2+ ions present in the plasma. The plasma with varying CaCl2 concentration showed variable coagulation rates, as shown in Fig. 3(a) and 3(b).As coagulation begins, the optical path length or refractive index of the sample begins to change with the rate of conversion of fibrinogen into fibrin, resulting in fringe shift. The time difference between two subsequent peaks (Δt) extracted from TIG indicates time taken to replace one bright or dark Michelson’s fringe by another or time taken to increase optical path length of the light through coagulating plasma by λ/2 m. Rate of variation of ‘Δt’ encapsulates the information regarding the rate of change of optical path length or the rate of plasma coagulation.

The relative coagulation “β”, defined as the ratio of ‘Δt’ to its maximum Δtmax i.e., β= Δt/Δtmax , plotted against the respective coagulation time for all the samples (along the two orthogonal directions) is shown in the Fig. 3(a) and 3(b). These figures illustrate that the variation of “β“ is different along the two orthogonal directions, indicating that variation of optical pathlength is not only influenced by the rate of formation of fibrin in the sample but also depend on how those fibrin monomers orients to form fibrin fibres. From experimental observation, the rate of optical path length variation is greater in one of the two orthogonal directions for all the samples, demonstrating that self-assembling fibrin monomers develop an average orientational ordering as the coagulation progresses, resulting in anisotropy in the sample. For all the samples, the relative coagulation in one of the orthogonal directions is always greater than the other. The relative coagulation along the direction along which the variation of “β”occurs slower is referred to as”β1”while the other, which is faster compared to the former, is referred to as “β2”. The variation of “β1”and“β2”(shown in Fig.3(a) and 3(b)) plotted against the coagulation time implies the following.

Variation of both β1 and β2 plotted against the coagulation time for all the samples are showing a typical “S” shaped sigmoidal trend.
Δt for all the samples are shorter in the beginning, increases as coagulation progress and saturates at the end of the coagulation, indicating that the rate of change of optical path length or refractive index is faster in the beginning, slower as time progresses and ultimately saturates, infers that the plasma coagulates at a faster rate in the beginning and rate falls as time progresses and eventually saturates. This process essentially advocates that the overall coagulation process is driven by heterogenous nucleation and growth kinetics such as the formation of a monomer fibrin nuclei in the beginning followed by the aggregation of the monomers and eventually becoming a fibrin mesh which do not change further. Moreover, the saturation is an indication for the complete transformation. As a result, the typical sigmoidal trend shown by coagulation of plasma (β vs coagulation time “t”) is fitted to an empirical relation.
β = 1− e− αtm (1)

Here, “β” represents the relative coagulation of plasma, “m” and “α” denotes the exponent and the coagulation rate constants, both of which are influenced by nature of nucleation and growth parameters, respectively. Linearization of the equation (1) for the two sets of β, corresponding to slower and faster coagulation rate, (log [-ln (1- β1)] = log α1 + m1 log t) and (log [-ln (1- β2)] = log α2 + m2log t) allows the evaluation of the constants m1, α1, m2, α2 as shown in the Fig. 5(a) and 5(b) respectively. The value of the constants obtained for plasma injected with various CaCl2 concentrations are listed in the Table 2.

The plots “β1 vs t” and “β2 vs t” illustrates that the trend of coagulation along two orthogonal directions is identical for all plasma samples injected with different CaCl2 concentrations, but the time taken to achieve the saturation differs, indicating the anisotropic behaviour of samples due to the self-assembling nature of fibrin monomer during the formation of fibrin network. Presence of anisotropy and its variation during coagulation are further validated by the POM technique.

Table 1: Estimated values of t1/2 from the detector D1 and D2.
Estimated- t 1/2 - (s)
Concentration
of CaCl2 (M) D1 D2
0.0200 454.7 362.1
0.0225 713.0 475.7
0.0250 1331.9 1123.0
0.0275 830.1 600.1
0.0300 655.3 551.2
0.0400 616.4 548.7
0.0500 609.9 558.1

The half coagulation time (t1/2) is retrieved as a standard index from the plots of β1 and β2 to estimate the rate of coagulation of all samples along two orthogonal directions of light beam. Although the mechanism that governs the entire coagulation process are complex and not well established, the variation of t1/2 values as shown in Table 1 plotted against CaCl2 concentrations injected into the plasma sample, as shown in Fig. 4, provides the coagulation dynamics with the following framework.

The t1/2 trend for all samples along two orthogonal directions is observed to be identical, but the coagulation rate for all samples along one of the orthogonal directions is observed to be faster than the other, indicating the presence of anisotropic tensor in the plasma samples. The plot further demonstrates that the value of t1/2 increases with increasing CaCl2 concentration at the beginning (for 0.02 M and 0.0225 M), reaches maximum at 0.025 M, then rapidly decreases as CaCl2 concentration is increased further. Therefore, the work on the present invention validates these outcomes and expands upon them especially by the trend of varying coagulation time against the CaCl2 concentration. The optimal value of t1/2 at 0.025 M in two orthogonal directions implies that there is a trade-off between two physical entities that drive the coagulation rate, one tending to slower the rate and the other tending to accelerate it. Interestingly, the maximum anisotropy is observed in plasma injected with three different CaCl2 concentrations, which are at 0.0225 M (lower) and 0.0275 M (higher) concentrations around the optimal concentration (0.025 M), indicating that the coagulation rate around this optimal concentration support the formation of fibrin monomers to have average directional orientation, which eventually leads to anisotropy in plasma. Further, the gradual decrease in anisotropy from a maximum to a minimum, almost negligible, for plasma samples injected with CaCl2 concentrations ranging from 0.0275 M to 0.05 M indicates that the process of coagulation or formation of fibrin monomers to become fibrin polymers in this range is driven by diffusion-controlled growth kinetics. Among all CaCl2 concentrations, the plasma sample injected with lowest concentration exhibits the fastest coagulation rate for both orthogonal directions as revealed from multiple experimental repetition. The observed decreasing trend of coagulation rate from optimal CaCl2 concentration to higher concentration agrees well with the published result. The results of anisotropy observed from this TIM method is verified by POM method as described in the following paragraphs.

Among numerous proposed theories in the prior art, classical nucleation based Avrami theory is commonly used to study and describe the nucleation and growth type of new thermodynamical phases transition in many bio molecules such as crystallization of fats, proteins, heam, bone minerals, etc. In the research work of the present invention, the usage of Avrami model is demonstrated to extract the coagulation constants (m1, α1, m2, α2) that govern the dimensionality of nucleation-growth and the coagulation rate of plasma injected with different CaCl2 concentrations. The formation of fibrin monomer in the aqueous plasma media is analogous to nucleation and growth type of phase transition in materials as proposed by Avrami model. During the coagulation, the nucleation of fibrin monomer initiates the formation of a new thermodynamic fibrin phase with lower free energy in the parent plasma media with higher free energy. Essentially, a thermodynamic nucleation process is driven by two components of Gibbs free energy: volume free energy, which supports the creation of stable nuclei, and surface free energy, which works against it. Thus, according to the standard expression, the total change in Gibbs free energy during phase transformation from fibrinogen to fibrin monomer is
ΔG = VΔGv +Sγs (2)
where V and S are the volume and surface area of the nuclei, ΔGv – the volume free energy during phase transformation, which is always negative, and γs – the interfacial energy between the transformed fibrin phase and parent plasma phase which is always positive. For a larger nucleus, the surface to volume ratio is small and volume free energy dominates, which eventually make the total Gibbs free energy negative and support for the nuclei growth. In the research work of present invention, the constants “m” (m1 and m2) and α (α1 and α2) are extracted from Avrami plots as shown in the Fig. 5(a) and 5(b), respectively. Here “α” depends on both nucleation and growth rates of fibrin and “m” depends on the dimensionality of the growth. Thus, according to the culmination of Avrami theory, the dimensionality of growth “m" (m = mN+mG) consists of two components: the nucleation component (mN) and the growth component (mG).

Table 2: Avrami constants extracted from the detector D1.
Concentration of CaCl2 (M)
D1
D2

m1 α1 x 10-5 R2 m2 α2 x 10-5 R2
0.0200 1.1
±0.1 3.0 ±
0.1 0.95 1.4 ±
0.2 3.7 ±
0.5 0.92
0.0225 1.5
±0.2 4.3 ±
0.4 0.92 1.0 ±
0.1 2.7 ±
0.2 0.94
0.0250 1.0
±0.1 3.0 ±
0.2
0.95 1.1 ±
0.1 3.6 ±
0.3 0.94
0.0275 1.1
±0.1 3.2 ±
0.3 0.91 1.1 ±
0.1 3.1 ±
0.2 0.93
0.0300 0.8 ±
0.1 2.7 ±
0.2 0.92 0.8 ±
0.1 2.4 ±
0.1 0.96
0.0400 1.1 ±
0.1 3.3 ±
0.3 0.93 0.7 ±
0.1 2.3 ±
0.2 0.92

0.0500 0.9 ±
0.1 2.8 ±
0.1 0.93 0.7 ±
0.1 2.2 ±
0.1 0.94

It is seen that the observed ‘m’ values for all the plasma samples injected with different CaCl2 concentrations along two orthogonal directions, as shown in Table 2, are close to one, implying that the nucleation component ‘mN’ is zero in all cases, indicating that coagulation follows a heterogeneous type of instantaneous nucleation, and the volume of the growth depends on time.

Among all the allowed permutations and combinations of mN and mG as allowed by Avrami theory, m = mN+mG, the only combination mN=0 and mG as m_G/2 only gives the overall ‘m’ value as one. According to Avrami theory, the mG in the expression (2) becomes m_G/2 for the cases of growth led by diffusion control mechanism with a volume that varies with time as V(t) αt^(m_G/2 ). Thus, the expression (2) becomes
m = mN +m_G/2 (3)
where, the allowed values for ‘mG’ in the expression (3) are 1, 2 or 3, which correspond to one-, two- or three-dimensional growth, as proposed. Therefore, the modified expression m = mN +m_G/2 for diffusion-controlled growth suggests that the experimentally observed ‘m’ value is one, only when mN= 0 corresponding to heterogeneous type instantaneous nucleation and mG = 2 corresponding to two-dimensional growth, which is essentially appropriate for the formation of fibrin mesh across the wound.

The TIM approach of the present invention is validated by studying density variation of an isotropic liquid, typically water, cooling from 65 °C to 28 °C. In this experiment, deionised water at 65 °C is taken in an optically flat four − way disposable cuvette and allowed to cool to room temperature in TIM. The shift in Michelson’s fringes due to density variation of cooling water which in turn vary the optical path length and refractive index along the two orthogonal directions is recorded and are shown in Fig. 6(a) and 6(b), respectively. The change in optical pathlength, which is proportional to time difference (Δt) between two subsequent fringes, is plotted against the cooling time as shown in Fig. 7 which illustrates the following observations.

The Δt versus cooling time curve obtained from the two orthogonal directions significantly coincides in the entire cooling process, demonstrating that the rate of change of optical path length along the two orthogonal directions is identical, implying that water is naturally isotropic. Further the Δt curve has an almost exponential tendency along the two directions, whereas the plasma exhibits a characteristic “S” shaped sigmoidal curve that is typically seen in crystallizing materials but not in non-crystallizing samples such as water. These results substantiate the sensitivity of TIM which can detect isotropic and anisotropic behaviour of samples.

POM study is employed to validate the results obtained from the interferometric technique. A plasma film sandwiched between a glass slide and a coverslip with specific CaCl2 concentration is subjected to POM analysis for three hours. POM images are taken every one minute by a 5 MP automated camera interfaced with a system. As an example, POM images of the plasma sample injected with 0.0275 M CaCl2 (one of samples shown high degree of anisotropy in TIM study) concentration captured at 10x optical magnification at every 25 min interval are shown in Fig. 8. In the beginning of coagulation, the first image (image captured at zeroth minute) shows only brown background with complete absence of green light as shown in Fig. 8. As coagulation progresses, the succeeding images taken at every 25-minute intervals shows the occurrence and expansion of several green stripes in the whole image (as seen in Fig. 8) demonstrating anisotropy in the sample. The degree of anisotropy, as demonstrated by TIM, is supported by the POM study. POM images of the samples injected with 0.0225, 0.025, and 0.0275 M concentration of CaCl2 show relatively more number of green stripes in the captured area, compared to the samples injected with other CaCl2 concentrations, which in turn validates the prediction of anisotropy using TIM. POM images of the plasma sample injected with 0.04 M CaCl2 (one of the samples shown moderate degree of anisotropy in TIM study) concentration captured at 10x optical magnification at every 10 min interval are shown in Fig. 9.

It is observed that the sample injected with 0.040 M CaCl2 concentration exhibits a statically negligible number of green stripes, showing that anisotropy is quenched in this sample as demonstrated by TIM, implying that fibrin growth in this sample is regulated by diffusion-controlled kinetics. In addition, the digitally magnified fibrin cluster as observed in POM images as shown in Fig. 10 is substantially agreeing with the schematic of fibrin structure reported so far.

Apart from the evidence for anisotropy obtained from TIM and sequence of POM images, the method of optical extinction on a POM image also validates the orientational ordering exhibited by fibrin monomers during the coagulation.

As per an embodiment of the method of this invention, the sample showing the homogeneous alignment of a cluster of fibrin molecules, appearing as prominent green stripes in POM image, are subjected to rotation.

As per the above method, one of the prominent green stripes shown in the POM, corresponding to the plasma injected with 0.03 M concentration of CaCl2, is subjected to 360° rotation in steps of 5°. Every 5° rotation of the prominent green stripe from initial position shows sequential drop of intensity and is completely extinguished at 45° as shown in Fig. 11, further the intensity of the stripe is periodically varying between maxima and minima for every 45° rotation during the entire 360◦ rotation. This periodic extinction of light shown by the green strip for every 45° rotation substantiates that the fibrin molecules are exhibiting a local orientational ordering in the coagulating plasma, resulting in anisotropy.

Therefore, the present invention provides a system comprising of an optical Twin-interferometer for extracting anisotropy and analysing the coagulation of plasma under isothermal circumstances. Twin interferograms were obtained for diverse plasma samples undergoing induced coagulation at different CaCl2 concentrations. A few significant coagulation parameters such as half coagulation time, coagulation rate constants, and their variation as a function of CaCl2 concentrations in two orthogonal directions are observed, and the methodology for extracting these parameters is disclosed. POM images validate the presence of anisotropy in plasma as shown by TIM. The periodic extinction of light shown by the locally oriented fibrin molecules in POM, for every 45° rotation of the sample, further substantiates the presence of anisotropy in the plasma.

The system and method of the present invention shall find application in the fields of healthcare, research and development, materials science etc. In the field of healthcare, it may find use in precisely measuring the clotting time of human plasma or prothrombin time during the clotting plasma for clinical diagnostics and patient management, pre-operative assessment, trauma and critical care, liver disease management etc. In the field of research and development it may find use in drug discovery and development, biomaterial compatibility testing, fundamental coagulation research etc. In the field of materials science and engineering, it may find use in characterizing complex biological fluids, developing novel biosensors, to study the kinetics of optically transparent polymer curing and solvent evaporation etc.  
, Claims:We claim:
1. A system (S) for real-time monitoring and quantitative assessment of
coagulation kinetics of plasma, said system comprising of: -
5
10
15
20
25
at least one optical module (O) comprising of a plurality of
interferometers for creating an interference pattern for analysing
plasma, said each interferometer comprising of:
• at least one light source (O1) for emitting optical beam
towards sample of plasma,
• at least one optical beam splitter (O2) with mount, placed in
the path of emitted beam of light, used for splitting said
optical beam into two optical beams, one transmitted and one
reflected,
• at least one pair of optical mirrors (O3) with their mounts, for
reflecting said two optical beams of light, comprising of: - -
one fixed mirror that reflects one of the optical beams
back towards the optical beam splitter (O2) and
one movable mirror that reflects the other optical beam
back towards the optical beam splitter (O2) and whose
position can be adjusted to change the path length of
the optical beam
• at least one beam expander (O4) with mount, for expanding
the optical beam for obtaining a broader optical signal in the
form of interference pattern,
• at least one noise filter (O6) in the form of pinhole with its
mount, for filtering optical noise from the optical signal,
• at least one optical detector (O5) for detection of said optical
signal in the form of interference pattern formed by the beams
on recombination and
41
• at least one base (O7) or platform for mounting all optical
components -
5
10
15
20
25 - -
at least one data acquisition module (A) for data acquisition of
optical signal/data from said optical module (O) comprising of:
• at least one computing device (A1) for receiving said optical
signal/ data from the optical detectors (O5),
• at least one interfacing tool (A2) for interfacing the optical
detectors (O5) with said computing device (A1) and
controlling the data acquisition rate and
• at least one recording tool (A3) for recording and storage of
said optical signal/ data in the computing device (A1) of said
data acquisition module (A)
at least one computing and analysis module (C) comprising of:
• at least one processing unit (C1) for processing and analysis
of optical data in the form of interferogram and
• at least one display unit (C2) for display of analysis results
at least one sample preparation module (SP) for preparing plasma
sample that is to be analysed by the system, comprising of:
• a plurality of EDTA-coated vials (SP1) for collecting blood
sample,
• a plurality of syringes (SP2) for blood collection and sample
processing,
• a four-way transparent quartz cuvette (SP3) for holding the
plasma sample in said optical module (O) during coagulation
and analysis,
• coagulation agent solution (SP4) of varied concentration for
initiating coagulation reactions in blood sample such as
Calcium chloride (CaCl₂) and
42
• a blood centrifuge device (SP5) for extracting plasma from
blood sample by centrifuging -
5
power source (P) for providing power to the said modules of the
system (S)
wherein -
10
15
20
25 - - - - -
said optical module (O) comprises of at least two interferometers,
also referred to as twin interferometer, that are configured together
in such a way that one of the optical beams referred to as arms is
orthogonal to the other,
said two interferometers are independently adjusted to form an
interference maximum, which is detected by two distinct optical
detectors D1 and D2 (O51 and O52),
said optical module (O) comprises of a third detector D3 (O53) that
is used to monitor the intensity fluctuation of said optical beam due
to thermal noise,
said plasma sample of specific volume is taken in an optically flat
four-way disposable square shaped glass cuvette (SP3), introduced
in said Twin- interferometer at the point of intersection of two optical
beams from the orthogonal arms of the interferometers,
said optical module (O) comprising of twin-interferometer is
optimally designed with all its components configured in a specific
way to record the rate of shift in Michelson’s fringes in
interferogram, caused by rate of change of optical path difference of
coagulating plasma in two orthogonal directions,
said interferogram recorded along the two orthogonal directions,
referred to as Twin- interferogram (TIG), furnishes the information
pertaining to the presence of anisotropy and the rate of change of
optical pathlength caused by rate of change of refractive index of
43
coagulating plasma along the distinct directions of the optical beam
and -
5
said system (S) provides for extraction of various coagulation
parameters from TIG, such as but not limited to anisotropy, half
coagulation time, constants governing the rate and dimensionality of
coagulation, and their variation along distinct direction and any
directional dependent coagulation kinetics
thereby providing a precise, reliable and cost-effective system (S) that
enables real-time monitoring and a quantitative assessment of
10
coagulation kinetics to understand the complex and dynamic
characteristics of coagulation of plasma.
2. The system (S) as claimed in claim 1, wherein said twin interferometer
is optimally designed with all its components configured in a specific
way such that the process of recording the rate of shift in Michelson’s
fringes in interferograms comprises of steps of: - dividing the optical beam from the light source (O1) initially into two
equal parts of by the beam splitter BS1 (O21), - splitting further of the transmitted beam from BS1 (O21) into two
equal parts by a second beam splitter BS2 (O22), - directing the transmitted beam from BS2 (O22) to detector D3 (O53)
that monitors the intensity fluctuation of said optical beam due to
thermal noise, - redirecting the reflected beam from BS2 (O22) by mirror M1 (O31) to
another beam splitter BS3 (O23), - directing further the two resulting beams from BS3 (O23), one
transmitted and one reflected, back to BS3 (O23), using mirrors M3
(O33) and M4 (O34),
44
- overlapping of these beams at BS3 (O23), producing interference
fringes or pattern that are detected by detector D1 (O51), - dividing the reflected beam from the initial beam splitter BS1 (O21)
by another beam splitter BS4 (O24), - redirecting the two resulting beams back to BS4 (O24) via mirrors M2
(O32) and M5 (O35), where they interfere after passing through BS4
(O24) producing interference pattern and - recording the resulting interference pattern by detector D2 (O52).
3. The system (S) as claimed in claim 1, wherein said optical expander (O4)
is a convex lens with a short focal length and is placed such that
interfered optical beams from each interferometer pass through it
increasing the spatial extent of the interference fringes before they reach
5
10
15
20
the optical detector (O5).
4. The system (S) as claimed in claim 1, wherein said noise filter (O6) is
placed such that the interfered optical beam emerging from the beam
expander (O4) is directed towards the optical detector (O5) through it
allowing only a specific portion of the interference fringes to reach the
optical detector (O5).
5. The system (S) as claimed in claim 1, wherein said optical detectors (O5)
are selected from detectors that operate in their linear region to detect
the intensity variation of Michelson’s fringes as the optical path length
of the sample changes during the process of coagulation, preferably
reverse biased photo transistors.
6. The system (S) as claimed in claim 1, wherein said optical beam is
preferably laser beam in the range of 532 ± 2 nm produced by said
optical source selected from a group comprising of He-Ne laser source,
diode laser source etc., preferably He-Ne laser source that illuminates
said twin interferometers.
45
7. The system (S) as claimed in claim 1, wherein said interfacing tool (A2)
is selected from Raspberry Pi, Arduino based drivers etc., preferably
Arduino-based drivers.
8. The system (S) as claimed in claim 1, wherein said recording tool (A3) of
5
10
15
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said data acquisition module (A) is selected from customised or open
source tools selected from PuTTY, OpenSSH, KiTTY, MobaXterm etc.,
preferably open-source PuTTY.
9. The system (S) as claimed in claim 1, wherein said optical components
in the TIM are set up in a vibration-free environment, aided by a passive
vibration isolation table.
10. A method for real-time monitoring and quantitative assessment of
coagulation kinetics, wherein steps of said method comprise of: - extracting plasma from blood sample using syringes (SP2) in an
EDTA-coated vial (SP1), - arranging the arms of twin interferometer (TIM) of optical module
(O) of the system (S) for analyzing coagulation of plasma, in such a
way that one of their arms is orthogonal to the other, - -
injecting a small volume, preferably 0.5 ml, of plasma sample into an
optically flat four-way disposable square shaped glass cuvette (SP3)
of 3 ml volume, in said twin- interferometer at the point of
intersection of two optical beams from the orthogonal arms of the
twin interferometers,
initiating coagulation of said plasma sample by injecting an equal
volume of coagulation agent, preferably CaCl2, into said cuvette
(SP3) containing said plasma sample in the TIM, - illuminating the twin interferometer using light source (O1) that
emits optical beam towards plasma sample, - splitting of said optical beam into two optical beams, one transmitted
and one reflected,
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- reflecting and redirecting of said optical beams towards specific
optical beam splitters (O2), such that these beams recombine at the
cuvette (SP3) to form an interference pattern, - configuring and adjusting said twin interferometers independently
5
10
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25
to form an interference maximum, which is detected by two distinct
optical detectors (O51 and O52) also referred as D1 and D2, - monitoring the intensity fluctuation of optical laser beam due to
thermal noise using a third detector (O53) also referred to as D3, - monitoring the process of coagulation of plasma along the two
orthogonal directions of the cuvette (SP3) in terms of fringe shift for
the entire duration of the coagulation, - recording interferograms by the data acquisition module (A) along
the two orthogonal directions, referred to as Twin- interferogram
(TIG), to obtain information pertaining to presence of anisotropy and
the rate of change of optical path length caused by rate of change of
refractive index of coagulating plasma along the distinct directions
of the light beam, - determining rate of plasma coagulation by analyzing the rate of
fringe shift in twin interferogram (TIG) by computing and analysis
module (C), that is determined by the rate of change in the optical
path length of plasma in the cuvette (SP3), - plotting fringe intensity against the coagulation time by the
processing unit (C1), -
extracting significant parameters from the twin interferogram (TIG)
obtained in the above steps and analysis by the processing unit (C1)
such as but not limited to anisotropy, half coagulation time,
constants governing the rate and dimensionality of coagulation, their
variation along distinct direction and other directional dependent
coagulation kinetics and
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- displaying the analysis results on display device (C2) of said system.
11. The method as claimed in claim 10, wherein said step of extracting
plasma from blood sample comprises of steps of: - collecting blood sample in EDTA coated vials (SP1) from subject and
5 - centrifuging the collected blood sample in the range of 5000-7000
rpm preferably at 6000 RPM in a laminar flow unit or a blood
centrifuge device (SP5) for a time interval in the range of 2-5 mins
preferably 3 min to extract the plasma.
12. The method as claimed in claim 10, wherein said duration of coagulation
ranges from 15 to 40 min depending on the concentration of coagulation
agent solution such as CaCl2.
13. The method as claimed in claim 10, wherein said method finds
application in the fields of healthcare, research and development,
materials science etc., for studying coagulation or Prothrombin time of
human plasma for clinical diagnostics and patient management, pre
operative assessment, trauma and critical care, liver disease
management, drug discovery and development, biomaterial
compatibility testing, coagulation research, characterizing complex
biological fluids, developing novel biosensors, for studying the kinetics
of optically transparent polymer curing and solvent evaporation etc.
Dated this the 22nd day of July 2025
To
The Controller of Patents,
The Patent Office, Chennai
________________________
Daisy Sharma
IN/PA- 3879
of SKS Law Associates
Attorney for the Applicant