Markandey M. Tripathi

Assessing blood coagulation status with laser speckle rheology.

We have developed and investigated a novel optical approach, Laser Speckle Rheology (LSR), to evaluate a patients coagulation status by measuring the viscoelastic properties of blood during coagulation. In LSR, a blood sample is illuminated with laser light and temporal speckle intensity fluctuations are measured using a high-speed CMOS camera. During blood coagulation, changes in the viscoelastic properties of the clot restrict Brownian displacements of light scattering centers within the sample, altering the rate of speckle intensity fluctuations. As a result, blood coagulation status can be measured by relating the time scale of speckle intensity fluctuations with clinically relevant coagulation metrics including clotting time and fibrinogen content. Our results report a close correlation between coagulation metrics measured using LSR and conventional coagulation results of activated partial thromboplastin time, prothrombin time and functional fibrinogen levels, creating the unique opportunity to evaluate a patients coagulation status in real-time at the point of care.

Optical Thromboelastography to evaluate whole blood coagulation

Measurement of blood viscoelasticity during clotting provides a direct metric of haemostatic conditions. Therefore, technologies that quantify blood viscoelasticity at the point-of-care are invaluable for diagnosing coagulopathies. We present a new approach, Optical Thromboelastography (OTEG) that measures the viscoelastic properties of coagulating blood by evaluating temporal laser speckle fluctuations, reflected from a few blood drops. During coagulation, platelet-fibrin clot formation restricts the mean square displacements (MSD) of scatterers and decelerates speckle fluctuations. Cross-correlation analysis of speckle frames provides the speckle intensity temporal autocorrelation, g2 (t), from which MSD is deduced and the viscoelastic modulus of blood is estimated. Our results demonstrate a close correspondence between blood viscoelasticity evaluated by OTEG and mechanical rheometry. Spatio-temporal speckle analyses yield 2-dimensional maps of clot viscoelasticity, enabling the identification of micro-clot formation at distinct rates in normal and coagulopathic specimens. These findings confirm the unique capability of OTEG for the rapid evaluation of patients coagulation status and highlight the potential for point-of-care use.

Raman spectroscopy for clinical-level detection of heparin in serum by partial least-squares analysis

Abstract. Heparin is the most widely used anti-coagulant for the prevention of blood clots in patients undergoing certain types of surgeries including open heart surgeries and dialysis. The precise monitoring of heparin amount in patients’ blood is crucial for reducing the morbidity and mortality in surgical environments. Based upon these considerations, we have used Raman spectroscopy in conjunction with partial least squares (PLS) analysis to measure heparin concentration at clinical level which is less than 10 United States Pharmacopeia (USP) in serum. The PLS calibration model was constructed from the Raman spectra of different concentrations of heparin in serum. It showed a high coefficient of determination (R2>0.91) between the spectral data and heparin level in serum along with a low root mean square error of prediction ∼4u2009u2009USP/ml. It enabled the detection of extremely low concentrations of heparin in serum (∼8u2009u2009USP/ml) as desirable in clinical environment. The proposed optical method has the potential of being implemented as the point-of-care testing procedure during surgeries, where the interest is to rapidly monitor low concentrations of heparin in patient’s blood.

Elemental analysis of slurry samples with laser induced breakdown spectroscopy

Direct analysis of wet slurry samples with laser induced breakdown spectroscopy (LIBS) is challenging due to problems of sedimentation, splashing, and surface turbulence. Also, water can quench the laser plasma and suppress the LIBS signal, resulting in poor sensitivity. The effect of water on LIBS spectra from slurries was investigated. As the water content decreased, the LIBS signal was enhanced and the standard deviation was reduced. To improve LIBS slurry analysis, dried slurry samples prepared by applying slurry on PVC coated slides were evaluated. Univariate and multivariate calibration was performed on the LIBS spectra of the dried slurry samples for elemental analysis of Mg, Si, and Fe. Calibration results show that the dried slurry samples give a good correlation between spectral intensity and elemental concentration.

Clinical evaluation of whole blood prothrombin time (PT) and international normalized ratio (INR) using a Laser Speckle Rheology sensor

Prothrombin time (PT) and the associated international normalized ratio (INR) are routinely tested to assess the risk of bleeding or thrombosis and to monitor response to anticoagulant therapy in patients. To measure PT/INR, conventional coagulation testing (CCT) is performed, which is time-consuming and requires the separation of cellular components from whole blood. Here, we report on a portable and battery-operated optical sensor that can rapidly quantify PT/INR within seconds by measuring alterations in the viscoelastic properties of a drop of whole blood following activation of coagulation with thromboplastin. In this study, PT/INR values were measured in 60 patients using the optical sensor and compared with the corresponding CCT values. Our results report a close correlation and high concordance between PT/INR measured using the two approaches. These findings confirm the accuracy of our optical sensing approach for rapid PT/INR testing in whole blood and highlight the potential for use at the point-of-care or for patient self-testing.

Combustion Applications of Laser-Induced Breakdown Spectroscopy

The Laser induced breakdown spectroscopy (LIBS) has been applied to combustion product and flame diagnostics. In this chapter, combustion applications of LIBS in past 30 years were reviewed. The various issues including experimental parameters and data processing methods that are important to combustion applications were discussed. Possible other applications that are important to combustion have also been addressed.

Optical sensing of anticoagulation status: Towards point-of-care coagulation testing

Anticoagulant overdose is associated with major bleeding complications. Rapid coagulation sensing may ensure safe and accurate anticoagulant dosing and reduce bleeding risk. Here, we report the novel use of Laser Speckle Rheology (LSR) for measuring anticoagulation and haemodilution status in whole blood. In the LSR approach, blood from 12 patients and 4 swine was placed in disposable cartridges and time-varying intensity fluctuations of laser speckle patterns were measured to quantify the viscoelastic modulus during clotting. Coagulation parameters, mainly clotting time, clot progression rate (α-angle) and maximum clot stiffness (MA) were derived from the clot viscoelasticity trace and compared with standard Thromboelastography (TEG). To demonstrate the capability for anticoagulation sensing in patients, blood samples from 12 patients treated with warfarin anticoagulant were analyzed. LSR clotting time correlated with prothrombin and activated partial thromboplastin time (r = 0.57–0.77, p<0.04) and all LSR parameters demonstrated good correlation with TEG (r = 0.61–0.87, p<0.04). To further evaluate the dose-dependent sensitivity of LSR parameters, swine blood was spiked with varying concentrations of heparin, argatroban and rivaroxaban or serially diluted with saline. We observed that anticoagulant treatments prolonged LSR clotting time in a dose-dependent manner that correlated closely with TEG (r = 0.99, p<0.01). LSR angle was unaltered by anticoagulation whereas TEG angle presented dose-dependent diminution likely linked to the mechanical manipulation of the clot. In both LSR and TEG, MA was largely unaffected by anticoagulation, and LSR presented a higher sensitivity to increased haemodilution in comparison to TEG (p<0.01). Our results establish that LSR rapidly and accurately measures the response of various anticoagulants, opening the opportunity for routine anticoagulation monitoring at the point-of-care or for patient self-testing.

Blood coagulation profiling in patients using optical thromboelastography (OTEG) (Conference Presentation)

Impaired blood coagulation is often associated with increased postoperative mortality and morbidity in cardiovascular patients. The capability for blood coagulation profiling rapidly at the bedside will enable the timely detection of coagulation defects and open the opportunity for tailoring therapy to correct specific coagulation deficits Optical Thromboelastography (OTEG), is an optical approach to quantify blood coagulation status within minutes using a few drops of whole blood. The goal of the current study is to evaluate the diagnostic accuracy of OTEG for rapid coagulation profiling in patients. In OTEG, temporal laser speckle intensity fluctuations from a drop of clotting blood are measured using a CMOS camera. To quantify coagulation status, the speckle intensity autocorrelation function is measured, the mean square displacement of scattering particles is extracted, and viscoelastic modulus (G), during coagulation is measured via the generalized Stokes-Einstein relation. By quantifying time-resolved changes in G, the coagulation parameters, reaction time (R), clot progression time (K), clot progression rate (Angle), and maximum clot strength (MA) are derived. In this study, the above coagulation parameters were measured using OTEG in 269 patients and compared with standard mechanical Thromboelastography (TEG). Our results showed a strong correlation between OTEG and TEG measurements for all parameters: R-time (R=0.80, p<0.001), clotting time (R=0.78, p<0.001), Angle (R=0.58, p<0.001), and MA (R=0.60, p<0.001). These results demonstrate the unique capability of OTEG for rapid quantification of blood coagulation status to potentially improve clinical capability for identifying impaired coagulation in cardiovascular patients at the point of care.

Optical thromboelastography (OTEG) for diagnosis of hyperfibrinolysis in patients(Conference Presentation)

Fibrinolysis is a process that regulates the breakdown of a blood clot to enable wound healing and is an essential component of hemostasis. Abnormalities in the fibrinolysis pathway may cause hyperfibrinolysis, associated with the increased risk of life-threatening bleeding particularly following acute trauma or major surgery. Assessing fibrinolytic activity in bleeding patients at the bedside can enable the timely administration of fibrinolysis inhibitors to improve prognosis. Optical thromboelastography (OTEG), a novel technique to assess blood coagulation status, has the potential to quantify fibrinolysis in real-time at PoC. The goal of the current study is to test the accuracy of OTEG in quantifying fibrinolytic status of human blood. Fibrinolysis is activated by adding varying concentrations of tissue plasminogen activator (tPA), a known fibrinolysis activator. The blood sample is illuminated by laser light and the resultant speckle intensity autocorrelation curve is used to derive changes in clot viscoelastic modulus during coagulation. From the OTEG trace, the coagulation parameters, clotting time (R), clot progression time (K), maximum clot strength (MA), and clot lysis (LY%) are derived. Our results indicate that increased tPA (0-0.5μM/ml) activation causes dose-dependent increase in LY% measured with OTEG: For instance, the addition of 0.5μM/ml of tPA increased LY% from 14.0 % to 81.5%. OTEG measurements also show a strong correlation with standard-reference mechanical Thromboelastography (TEG) measurements (N = 15, R=0.87, p<0.05). These results demonstrate that OTEG can accurately evaluate fibrinolysis and may provide the capability for identifying hyperfibrinolytic patients at an increased risk of life-threatening hemorrhage.

Optical profiling of anticoagulation status (Conference Presentation)

Defective blood coagulation resulting from excessive procoagulant activity often leads to thrombotic disorders such as stroke and myocardial infarction. A variety of oral and injectable anticoagulant drugs are prescribed to prevent or treat life-threatening thrombosis. However, due to bleeding complications often associated with anticoagulant treatment, routine monitoring and accurate dosing of anticoagulant therapy is imperative. We have developed Optical thromboelastography (OTEG), a non-contact approach that utilizes a drop of whole blood to measure blood coagulation status in patients. Here, we demonstrate the capability of OTEG for rapidly monitoring anticoagulation in whole blood samples. OTEG monitors coagulation status by assessing changes in blood viscosity from temporal intensity fluctuations of laser speckle patterns during clotting. In OTEG a blood drop is illuminated with coherent light and the blood viscosity is measured from the speckle intensity autocorrelation curve, g2 (t). The metrics, clotting time (R+k), clot progression (angle) and maximum clot stiffness (MA) are then extracted. The aim of the current study was to evaluate the accuracy of OTEG in assessing anticoagulation status of common anticoagulants including heparin, argatroban and rivaroxaban status. A dose-dependent prolongation of R+k was observed in anticoagulated blood, which closely corresponded with standard-reference Thromboelastography (TEG) (r 0.87-0.99, P>0.01 for all cases). OTEG angle was unaltered by anticoagulation whereas TEG angle presented a dose-dependent diminution probably linked to clot rupture. In both OTEG and TEG, MA was unaffected by heparin, argatroban or rivaroxaban. We conclude that OTEG can accurately monitor anticoagulation status following treatment, potentially providing a powerful tool for routine monitoring of patients in the doctor’s office or in the home setting.