Viscoelastic coagulation monitoring systems and methods for hidradenitis suppurativa detection

Abstract

Viscoelastic coagulation monitoring systems having two glass discs mounted on flexible plastic arms for receiving a sample of blood therebetween, and an analyzer configured to measure a coagulation response of the sample of blood to determine whether the sample of blood indicates a hypercoagulable state in a HS patient are provided. The system may cause movement of a first glass disc relative to a second glass disc at a sufficient speed to cause coagulation of the sample of blood, and the analyzer may measure various parameters of the coagulation response including, e.g., Clotting Time (CT), Clot Formation Time (CFT), Alpha Angle (a-angle), Maximum Clot Firmness (MCF), Amplitude at 10 and 20 minutes (A10, A20, respectively), Lysis Index at 30 and 45 minutes (LI30 and LI45, respectively), indicative of a hypercoagulable state in a HS patient.

Description

Atty. Dkt. No. 225871-031001VISCOELASTIC COAGULATION MONITORING SYSTEMS AND METHODS FOR HIDRADENITIS SUPPURATIVA DETECTIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to EP Patent Appl. No. 24386129.1, filed November 15, 2024, the entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] This technology generally relates to blood coagulation monitoring systems and more particularly to systems and methods for monitoring viscoelastic coagulation to detect hidradenitis suppurativa (HS).BACKGROUND

[0003] The process by which the body prevents blood loss is referred to as coagulation. Coagulation involves the formation of a blood clot (thrombus) that prevents further blood loss from damaged tissues, blood vessels or organs. The formation of a blood clot is a complicated process involving a first system comprised of cells called platelets that circulate in the blood and serve to form a platelet plug over damaged vessels and a second system based upon the actions of multiple proteins (called clotting factors) that act in concert to produce a fibrin clot. These two systems work in concert to form a clot and disorders in either system can yield disorders that cause either too much or too little clotting.

[0004] Platelets serve three primary functions: (1) sticking to the injured blood vessel (a phenomenon called platelet adherence); (2) attaching to other platelets to enlarge the forming plug (a phenomenon called platelet aggregation); and (3) providing support for the processes of the coagulation cascade (molecules on the surface of platelets greatly accelerate several key reactions). When a break in a blood vessel occurs, substances are exposed that normally are not in direct contact with the blood flow. These substances (primarily collagen and attached multimeric von Willebrand factor) allow the platelets to adhere to the broken surface. Once a716396068v1 1Atty. Dkt. No. 225871-031001platelet adheres to the surface, it releases chemicals that attract additional platelets to the damaged area, referred to as platelet aggregation. These two processes are the first responses to stop bleeding. The protein-based system (the coagulation cascade) serves to stabilize the plug that has formed and further seal up the wound.

[0005] The support role of the platelet to the coagulation cascade is provided, in part, by one of the components on the outside of a platelet, called phospholipids, which are required for many of the reactions in the clotting cascade. The goal of the cascade is to form fibrin, which will form a mesh within the platelet aggregate to stabilize the clot. All of the factors have an inactive and active form. Once activated, the factor will serve to activate the next factor in the sequence until fibrin is formed. The coagulation cascade takes place at the site of a break in, e.g., a blood vessel that has the platelet aggregate. Fibrin forms a mesh that, in concert with the platelets, plugs the break in the vessel wall. The fibrin mesh is then further stabilized by additional factors which cross-linkup the clot (much like forming an intricate network of reinforced strands of fibrin).

[0006] Currently, thromboelastography (TEG) is the accepted clinical standard for testing the efficiency of whole blood coagulation. As an example, the related U.S. Pat. No. 8,450,078, entitled “Portable Coagulation Monitoring Device and Method of Assessing Coagulation Response” (herein incorporated in its entirety) discloses a portable coagulation monitoring device typically comprising glass plates used to diagnose trauma-related coagulopathies in the field.

[0007] Hidradenitis Suppurativa (HS) is a chronic inflammatory condition primarily affecting apocrine-gland-rich regions of the body, namely axillary and groin areas. HS manifests as recurrent painful inflammatory lesions, which may lead to the formation of draining tunnels (dTs). Research on pathogenesis of HS has been focused on the characteristics of the recurrent inflammatory responses which are mainly led by the overproduction of tumor necrosis factor alpha (TNFa), interleukin (IL)-17 and IL-1. Although these cytokines are pro-inflammatory, they are also described to orchestrate the activation of the coagulation cascade, a phenomenon known as immunothrombosis. Immunothrombosis is characterized by primed platelets and endothelium which are activated, while natural anticoagulant pathways and fibrinolysis are716396068v1 2Atty. Dkt. No. 225871-031001inactivated. In many systemic inflammatory diseases, a thrombotic tendency has been observed. Previous studies aimed at investigating an association between the inflammation in HS and prothrombotic / hypercoagulable state, using standard coagulation tests (STCs), have not yielded any statistically significant results.

[0008] In view of the foregoing drawbacks of previously known systems and methods, there exists a need for viscoelastic coagulation tests (VCT) that assess coagulation in whole blood from the beginning of clot formation to clot lysis, e g., to thereby identify the association between inflammation and prothrombotic status in the context of HS.SUMMARY

[0009] In accordance with one aspect, a device for assessing Hidradenitis Suppurativa (HS) is provided. The device may be configured to receive a sample of blood, cause coagulation of the sample of blood, and monitor coagulation results to determine whether the sample of blood indicates a hypercoagulable state in a HS patient. For example, the device may comprise a test cartridge configured to receive a sample of blood, and an analyzer unit configured to receive the test cartridge and cause coagulation of the sample of blood. The test cartridge may comprise a pair of plates configured to define a capillary therebetween, the capillary sized and shaped to receive the sample of blood.

[0010] Each of the plates of the pair of plates may be suspended via a flexible arm, such that, when the test cartridge is received by the analyzer unit, the analyzer unit may be configured to drive movement of a first plate of the pair of plates via the corresponding flexible arm relative to a second plate of the pair of plates at a predetermined speed sufficient to cause the sample of blood to coagulate and bind to the first and second plates to thereby induce movement of the second plate. The device further may comprise an optical sensor configured to optically detect interaction between the pair of plates during coagulation, the interaction indicative of the coagulation results.

[0011] For example, the device may be configured to measure a vascular endothelial growth factor (VEGF) of the coagulation results, wherein an increased amount of VEGF is indicative of the hypercoagulable state in the HS patient. Additionally, or alternatively, the device may be716396068v1 3Atty. Dkt. No. 225871-031001configured to measure an intercellular adhesion molecule-1 (ICAM-1) of the coagulation results, wherein a decreased amount of ICAM-1 is indicative of the hypercoagulable state in the HS patient. Further, the device may be configured to measure a vascular-cell adhesion molecule- 1 (VCAM-1) of the coagulation results, wherein a decreased amount of VCAM-1 is indicative of the hypercoagulable state in the HS patient. In addition, the device may be configured to measure a clot formation time (CFT) of the coagulation results, wherein a decreased amount of CFT is indicative of the hypercoagulable state in the HS patient.

[0012] The device further may be configured to measure an alpha angle of the coagulation results, wherein an increased alpha angle is indicative of a velocity of clot formation and the hypercoagulable state in the HS patient. Moreover, the device may be configured to measure a clot firmness (CF) of the coagulation results after a predetermined time period after a start of clot formation of the sample of blood, wherein an increased CF is indicative of the hypercoagulable state in the HS patient. In addition, the device may be configured to measure a maximum clot firmness (MCF) of the coagulation results before the clot dissolves, wherein an increased MCF is indicative of the hypercoagulable state in the HS patient.

[0013] In accordance with another aspect, a method for assessing Hidradenitis Suppurativa (HS) is provided. The method may comprise: receiving a sample of blood; causing coagulation of the sample of blood; and monitoring coagulation results to determine whether the sample of blood indicates a hypercoagulable state in a HS patient. For example, receiving the sample of blood may comprise receiving the sample of blood within a capillary defined between a pair of plates of a test cartridge, each plate of the pair of plates suspended via a flexible arm. The method further may comprise inserting the test cartridge within an analyzer unit. Accordingly, causing coagulation of the sample of blood may comprise driving, via the analyzer unit, movement of a first plate of the pair of plates via the corresponding flexible arm relative to a second plate of the pair of plates at a predetermined speed sufficient to cause the sample of blood to coagulate and bind to the first and second plates to thereby induce movement of the second plate. Additionally, monitoring the coagulation results may comprise optically detecting, via an optical sensor, interaction between the pair of plates during coagulation, the interaction indicative of the coagulation results. Further, driving movement of the first plate of the pair of716396068v1 4Atty. Dkt. No. 225871-031001plates may comprise driving, via the analyzer unit, movement of the first plate in an oscillating manner relative to the second plate.

[0014] In some embodiments, monitoring the coagulation results may comprise measuring a vascular endothelial growth factor (VEGF) of the coagulation results, such that an increased amount of VEGF may be indicative of the hypercoagulable state in the HS patient. Additionally, or alternatively, monitoring the coagulation results may comprise measuring an intercellular adhesion molecule- 1 (ICAM-1) of the coagulation results, such that a decreased amount of ICAM-1 may be indicative of the hypercoagulable state in the HS patient. Additionally, or alternatively, monitoring the coagulation results may comprise measuring a vascular-cell adhesion molecule- 1 (VCAM-1) of the coagulation results, such that a decreased amount of VCAM-1 may be indicative of the hypercoagulable state in the HS patient. Additionally, or alternatively, monitoring the coagulation results may comprise measuring a clot formation time (CFT) of the coagulation results, such that a decreased amount of CFT may be indicative of the hypercoagulable state in the HS patient. Additionally, or alternatively, monitoring the coagulation results may comprise measuring an alpha angle of the coagulation results, such that an increased alpha angle may be indicative of a velocity of clot formation and the hypercoagulable state in the HS patient. Additionally, or alternatively, monitoring the coagulation results may comprise measuring a clot firmness (CF) of the coagulation results after a predetermined time period after a start of clot formation of the sample of blood, such that an increased CF may be indicative of the hypercoagulable state in the HS patient. Additionally, or alternatively, monitoring the coagulation results may comprise measuring a maximum clot firmness (MCF) of the coagulation results before the clot dissolves, such that an increased MCF may be indicative of the hypercoagulable state in the HS patient.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 illustrates an exemplary viscoelastic coagulation monitoring (VCM) system in accordance with some embodiments.

[0016] FIG. 2 illustrates an exemplary test cartridge of the VCM system of FIG. 1 in accordance with some embodiments.716396068v1 5Atty. Dkt. No. 225871-031001

[0017] FIG. 3 A illustrates an exemplary method of thromboelastography using the VCM system of FIG. 1.

[0018] FIG. 3B is a graph showing exemplary variables measured by the VCM system that characterize the clot.

[0019] FIG. 4 is an exemplary graph illustrating a trace showing the proportional movement of an un-driven arm of the VCM cartridge of the VCM system plotted against test time.

[0020] FIG. 5 is a study flow chart of enrolled HS patients and comparators.

[0021] FIG. 6 is a series of plots of comparative variables measured by the VCM system between HS patients and matched comparators.

[0022] FIG. 7 is an exemplary graph illustrating clot formation measured by the VCM system for a patient with HS and hypercoagulable state.

[0023] FIG. 8 is a series of scatterplots illustrating variables measured by the VCM system and the total AN count, represented as the sum of abscesses and inflammatory nodules.

[0024] FIG. 9 illustrates serum concentrations of TNFa, VEGF, ICAM-1, VCAM-1 and PDGF-A and correlation with variables measured by the VCM system.DETAILED DESCRIPTION

[0025] Embodiments of this technology are directed to exemplary viscoelastic coagulation monitoring systems and methods for providing viscoelastic coagulation tests (VCT) to assess coagulation in whole blood from the beginning of clot formation to clot lysis, to thereby detect hidradenitis suppurativa (HS) in a blood sample of a patient. Pre-clinical activation of coagulation has never been studied in HS.

[0026] The viscoelastic coagulation monitoring (VCM) system described herein may be designed for in-vitro diagnostic use by trained professionals in an acute care setting and in a laboratory environment. Moreover, the VCM system may provide a semi-quantitative indication of the coagulation state of fresh, whole blood samples to assist in the assessment of clinical716396068v1 6Atty. Dkt. No. 225871-031001hemostasis. For this purpose, the VCM system may record clot firmness as the whole blood sample clots, retracts, and lyses in real time, such that the VCM system may analyze clotting time, clot formation time, alpha angle, maximal clot firmness, and / or fibrinolysis of a patient’s blood sample. These evaluation tests may indicate, e.g., to a doctor, whether the patient’s blood is clotting normally, too slowly, or too rapidly. The output of the VCM analyzer may comprise a qualitative graphical representation and several numeric parameters describing the curve quantitatively. Moreover, the output of the VCM analyzer may be evaluated together with the patient’s medical history, the clinical picture, and, if necessary, further coagulation tests.

[0027] FIG. 1 illustrates an exemplary viscoelastic coagulation monitoring (VCM) system. The VCM system may be constructed similar to U.S. Patent No. 11,768,210, assigned to the assignee of the instant application, the entire contents of which are incorporated herein by reference. For example, as shown in FIG. 1, the VCM system may include a VCM analyzer, a VCM test cartridge, a VCM heater plate, a VCM disposable syringe, a VCM stylus, a VCM system check cartridge, and VCM quality control materials.

[0028] FIG. 2 illustrates an exemplary VCM test cartridge. The VCM test cartridge may comprise two glass discs / plates mounted on flexible plastic arms. The discs are held parallel to each other by the flexible plastic arms and form a capillary sized and shaped to hold the test sample of blood. The glass discs trigger clotting of the blood sample via contact activation. In addition, each of the flexible arms may have an engagement feature at its end, which interacts with the VCM analyzer when the VCM test cartridge is inserted into the VCM analyzer.

[0029] Referring again to FIG. 1, the VCM heater plate may be used to warm the VCM cartridges prior to running a test. This may be required as the speed of sample clotting may be affected by temperature. Further, the VCM system may be designed to ensure that all samples are run at 37 °C, equivalent to body temperature. It may be important to pre-heat VCM cartridges on the VCM heater plate before running a test to ensure that the tests run at the correct temperature. Failure to pre-heat the VCM cartridges may result in slower clot times. In some embodiments, the VCM heater plate may be sized and shaped to heat up to four VCM cartridges at a time.716396068v1 7Atty. Dkt. No. 225871-031001

[0030] FIG. 3 A illustrates an exemplary method of thromboelastography using the VCM system. The VCM system uses a variation on standard thromboelastography to measure the hemostasis of a blood sample. For example, the VCM system measures the changes in the viscoelastic properties of a blood sample as a clot forms, retracts and / or lyses, and converts these measurements to data describing the characteristics of the clot. As shown in FIG. 3A, when a test runs, a motor in the VCM analyzer causes one of the flexible arms in the VCM cartridge to oscillate back and forth, and optical sensors in the VCM analyzer may measure the movement of both the arm being driven by the VCM analyzer and the un-driven arm. As the blood sample starts to clot, it attaches the two surfaces of the glass capillary to each other, which causes the undriven arm of the VCM cartridge to start to follow the movement of the driven arm. The extent to which the un-driven arm follows the movement of the driven arm is proportional to the strength of the clot. As the clot becomes stronger, the strength of the attachment between the two surfaces of the capillary increases, which increases the movement of the un-driven arm. When the clot lyses, the strength of the attachment decreases, and thus, the movement of the un- driven arm decreases.

[0031] The VCM analyzer software may calculate the movement of the un-driven arm as a proportion of the movement of the driven arm, and may displays this data in real-time in the form of a mirrored trace, as shown in FIG. 3B. As shown in FIG. 3B, the trace shows the proportional movement of the VCM arm plotted against test time. The VCM analyzer software may use this data to calculate a series of parameters to characterize the clot, as shown in FIG. 3B. For example, exemplary parameters are shown in Table 1 copied below.Table 1716396068v1 8Atty. Dkt. No. 225871-031001&&

[0032] In accordance with one aspect, the VCM system may be integrated in clinical practice for diagnosis and follow-up of HS as immunothrombosis participates in HS, which may be screened by abnormalities in viscoelastic coagulation monitoring. Accordingly, the VCM system may surrogate clinical improvement.

[0033] Full-blown pro-coagulant phenomena like pulmonary fibrosis and deep venous thrombosis are not often in HS, whereas it has not been reported that HS patients have abnormal coagulation times. As a consequence, the study of coagulation activation as described herein was performed at a pre-clinical level in order to decipher if HS has the characteristics of immunothrombosis. Specifically, the analysis was done using the VCM system described herein, which provides a dynamic and global analysis of hemostasis, from the formation to the lysis of the clot.

[0034] In this study, the VCM system was used to investigate the changes of clotting formation in HS in the aim to assess features of immunothrombosis, as described in further detail below. Specifically, a comparison between blood samples collected from 30 HS patients and 29 healthy subjects, with no known acute or chronic inflammatory disease was conducted.716396068v1 9Atty. Dkt. No. 225871-031001Specifically, these blood samples were analyzed using the VCM system. A separate analysis was performed amongst those 30 aforementioned patients and twelve healthy controls, evaluating the platelets, aPTT, INR and fibrinogen.

[0035] As a result, the parameters CFT, alpha angle, A10, A20, and MCF were shown to be significantly greater in patients’ than in controls’ blood samples. The standard coagulation assessments did not retrieve any statistically significant differences among the two cohorts. There was evident positive correlation between the A10, A20, and MCF with AN count, whereas AN count had a negative correlation with the CFT. Accordingly, these results indicate a hypercoagulable state in HS patients when the blood coagulation analysis is performed with the VCM system in comparison to the SCTs. The inflammation in HS does not seem to affect the time of the initiation phase of coagulation or the fibrinolysis; however, it does affect the stages of the clot formation and stabilization.

[0036] The study was conducted between February 2024 and June 2024. Participants were enrolled from the outpatient Department of Immunology of Infectious diseases of ATTIKON University Hospital in Athens, Greece (approval of the Ethics Committee EBD11 / 10-01-2019). Participants were patients with HS and matched comparators. All individuals provided written informed consent before participation in the study.

[0037] The inclusion criteria for patients with HS were: a) adults (age > 18 years old) of either sex; b) written informed consent, c) diagnosis of HS based on early onset after puberty, presence of subcutaneous nodules in skin areas rich in apocrine glands and compatible history of recurrent pus drainage from the affected areas; d) at least one HS lesion of Hurley stage II or III. Exclusion criteria were: a) any medical history of intake of oral or systemic anticoagulants or of aspirin (low- or high-dose) or of clopidogrel or of vitamin K; b) any medical history of deep venous thrombosis or pulmonary embolism or arterial embolism; c) any medical history of inflammatory diseases including but not limited to psoriasis, rheumatoid arthritis, systemic lupus erythematosus or inflammatory bowel disease, d) any medical history of increase of international normalized ratio (INR.) or of prolongation of activated partial thromboplastin time (aPTT) or of primary or secondary thrombocytosis; and e) any intake of non-steroidal anti-inflammatory drugs the last 15 days. Healthy comparators should meet all the following criteria: a) adults (age > 18716396068v1 10Atty. Dkt. No. 225871-031001years old) of either sex; b) written informed consent; and c) 1 : 1 matching with the HS study participants according to the Charlson’s Comorbidity Index (CCI), age and sex. Healthy comparators should also not meet any of the exclusion criteria of the HS participating patients.

[0038] For the time since HS onset was recorded, INs, abscesses, and dTs were counted. The Hurley stage of each skin lesion was also recorded. The inflammatory lesion count (AN) was calculated as the sum of INs and abscesses. The international HS4 score (IHS4) was calculated by providing one point for each IN, 2 points for each abscess, and 4 points for each dTs. All patients and healthy comparators were subject to one study visits where they were subject to blood collection for analysis with the VCM system and for the measurement of platelet counts, coagulation times, and blood levels of vascular endothelial growth factor (VEGF).Patients who were advised to come back for follow-up visits after 12 weeks were subject to a second analysis with the VCM system. During this second visit, skin lesions were also recorded and the IHS4 score was calculated. These patients were split into improved and non-improved is at least 55% of IHS4 score was achieved or not.

[0039] As described above, the VCM system is a non-invasive device designed to provide a semi-quantitative indication of the coagulation state of the fresh whole blood sample at the point of care. The VCM system (made available by Entegrion, Inc. of Durham, North Carolina) was set up and tested at least once daily using the system check cartridge, according to the manufacturer’s instructions. A total of 10 ml of whole blood was collected from participants after venipuncture of one forearm vein under aseptic conditions. Immediately upon collection 0.5 ml of the untreated raw specimen was inserted into one VCM cartridge pre-heated at 37 C and inserted into the VCM Analyzer. The total analysis lasted for one hour and the following parameters were captured: Clotting Time (CT), Clot Formation Time (CFT), Alpha Angle (a- angle), Maximum Clot Firmness (MCF), Amplitude at 10 and 20 minutes (A10, A20, respectively), Lysis Index at 30 and 45 minutes (LI30 and LI45).

[0040] FIG. 4 illustrates exemplary representative analysis and definitions for each variable, where Alpha = Alpha-angle; A10 = Amplitude at 10 minutes; A20 = Amplitude at 20 minutes; CT = Clotting Time; CFT = Clotting Formation Time; LI30 = Lysis Index at 30 minutes; LI45 = Lysis Index at 45 minutes; and MCF, Maximum Clot Firmness. As shown in FIG. 4, CT is the716396068v1 11Atty. Dkt. No. 225871-031001time from the beginning of the test until the time when an amplitude of 1% above the baseline is achieved. The CFT is the time between 1% amplitude and 10% amplitude of the clotting signal. The Alpha angle is defined as the angle between the time axis and the tangent to the clotting curve through the 1% amplitude point. In other words, the Alpha angle describes the kinetics of clotting. The MCF is the measure of the firmness of the clot and therefore the clot quality, e.g., it is the maximum amplitude that is reached before the clot is dissolved by fibrinolysis and the clot firmness falls again. The A10 and A20 represent the clot firmness and the amplitude at 10 and 20 minutes after clot time. The LI30 and LI45 are the amplitude of the clot at 30 and 45 minutes after clot time as a percentage of the MCF. The normal values of each parameter are provided.

[0041] The remaining blood was then poured into one EDTA tube (BD Vacutainer), into one heparin-coated tube, and into one sterile tube. The EDTA tube was used for the measurement of the total platelet count using one Coulter hemocytometer. The heparin-coated tube was centrifuged and INR, aPTT, and fibrinogen were measured. More precisely, Thromborel® S (made available by Siemens of Munich, Germany) was used for the determination of INR, and Multifibren® U (made available by Siemens of Munich, Germany) for fibrinogen’s measurement. The sterile tube was centrifuged and concentrations of tumor necrosis factoralpha (TNFa; made available by Diaclone of Besan^on, France), vascular endothelial growth factor (VEGF, made available by Thermo Fisher Scientific of Waltham, Massachusetts), platelet- derived growth factor-A (PDGF-A, made available by R&D Systems of Minneapolis, Minnesota), vascular-cell adhesion molecule- 1 (VCAM-1, made available by ELK Biotechnology of Wuhan, China) and intercellular adhesion molecule-1 (ICAM-1, made available by ELK Biotechnology of Wuhan, China) were measured by an enzyme immunosorbent assays. The lower limit of detection was 25 pg / ml for TNFa; 303 pg / ml for VEGF, 156 pg / ml for PDGF-A, 0.79 ng / ml for VCAM-1; and 78.13 pg / ml for ICAM-1.

[0042] The parameters generated by the VCM system (“VCM variables”) were presented as box plots and 95% confidence intervals (Cis). Comparisons between patients and healthy comparators were done by the Mann-Whitney U test. The variables which were different between patients and comparators were further analyzed within the patient group by Spearman’s rank of order correlations with the count of the skin lesions and with the measured serum716396068v1 12Atty. Dkt. No. 225871-031001cytokines. The total VCM variables which are abnormal in each participant were calculated using the as normal reported ranges. Comparisons between patients and comparators were done by Pearson’s correlation. The odds ratio and 95% Cis for patients presenting with at least one abnormal VCM variable was calculated using Mantel and Haenszel statistics. The number of abnormal VCM variables during the second visits and the change for the first visits was also recorded. Comparisons between responders and non-responders were done by the binomial test. Any p-value less than 0.05 was considered statistically significant.

[0043] As a result, 72 patients and 105 healthy comparators in total were screened for study inclusion; 30 patients and 29 comparators were enrolled, as shown in the study flow chart of FIG. 5, where CCI = Charlson’s comorbidity index; HS = hi dradenitis suppurativa; and NSAIDs = non-steroidal anti-inflammatory drugs. Both groups did not differ in age, sex, comorbidities, absolute platelet count, INR and blood fibrinogen levels, as shown in Table 2 copied below, illustrating the demographics of study participants, where CCI = Charlson’s Comorbidity Index; dT = draining tunnel; HS = hidradenitis suppurativa; IHS4 = international HS4 score; IL = interleukin; IN = inflammatory nodule; INR = international normalized ratio; n = number of patients; Q = quartile; and SD = standard deviation.Table 2716396068v1 13Atty. Dkt. No. 225871-031001

[0044] The differences between HS patients and matched comparators for the VCM variables are shown in FIG. 6, which compare VCM variables between HS patients and matched comparators (where Alpha = Alpha-angle; A10 = Amplitude at 10 minutes; A20 = Amplitude at 20 minutes; CT = Clotting Time; CFT = Clotting Formation Time; LI30 = Lysis Index at 30 minutes; L145 = Lysis Index at 45 minutes; MCF = Maximum Clot Firmness; and the p-value of comparison is provided in each panel), and in FIG. 7, which illustrates a diagram generated by the VCM system for a patient with HS and hypercoagulable state. As shown in FIG. 6, CFT was significantly lower in HS patients and alpha, MCF, A10 and A20, and MCF were significantly higher in HS patients. Overall, nine patients with HS (30%) had at least abnormal at least one of these five VCM variables altered compared to the normal values; abnormality was either decrease of CFT and / or increase of MCF, alpha, A10, A20, and MCF; nil comparator had VCM variable abnormalities (0%) (p of comparison 0.002). The number of abnormal VCM variables was greater in patients with severe HS than in patients with mild or moderate HS, as shown in Table 3 copied below. As shown in Table 3, the degree of severity is defined by the IHS4 score. The altered VCM variables are either decrease of CTF or increase in any of alpha, A10, A20, and MCF. The p-value of the linear-to-linear association is 0.024.Table 3716396068v1 14Atty. Dkt. No. 225871-031001

[0045] The analysis was focused on patients with HS, studying the existence or not of a correlation between the five prevailing abnormalities in VCM variables and the lesion counts of HS. No statistically significant correlation was found between any of the studied VCM variables and the years since HS onset (data not shown). As shown in FIG. 8, which are scatterplots of VCM variables and the total AN count, e.g., sum of abscesses and inflammatory nodules (where A10 = Amplitude at 10 min; A20 = Amplitude at 20 min; CFT = Clotting Formation Time; MCF = Maximum Clot Firmness, and the Spearman’s correlation co-efficient (rs) and the respective p- value are provided for each panel), significant correlations were found between the absolute AN count and the VCM variables, but not between the absolute dT count and the VCM variables (data not shown). More precisely, the AN count was negatively correlated with CTF whereas the AN count was positively correlated with the A10, A20, and MCF.

[0046] Follow-up VCM variables measurements were repeated in 10 patients; six patients experienced less than 55% decrease of the IHS4 score from the first visit; four patients experienced at least 55% decrease from the first visit. Decrease of the number of abnormal VCM variables was found in one patient (16.7%) and in three patients (75%) respectively (p: 0.004 by the binomial test; odds ratio 5.00; 95% Cis 0.6-339.05; p: 0.089 by the Mantel and Haenszel test).

[0047] The concentrations of TN Fa, VEGF and PDF-A did not differ between patients with normal and abnormal VCM variables findings. However, patients had blood VEGF higher than healthy comparators. The concentrations of VC AM- 1 were lower than comparators and the concentrations of ICAM-1 were lower in patients with abnormal VCM variables findings than in patients with normal VCM findings. VCAM-1 levels were positive correlated with CFT and negatively correlated with alpha angle, as shown in FIG. 9, which illustrates serum concentrations of TNFa, VEGF, ICAM-1, VCAM-1, and PDGF-A and correlation with VCM variables findings, where HS = hidradenitis suppurativa; n = number of patients; ICAM = intracellular adhesion molecule; TNF = tumor necrosis factor; VCAM = vascular cell adhesion molecule; VEGF = vascular endothelial growth factor; w / o = without; and the arrows indicate716396068v1 15Atty. Dkt. No. 225871-031001the statistically significant comparisons. No other significant correlations were found between the other cytokines and VCM variables measurements.

[0048] Accordingly, this is the first study to provide findings of pre-clinical activation of the coagulation in HS. Patients do not have abnormal findings in platelets or coagulation times. However, 30% are presenting abnormal VCM variables which are attenuated when HS improves. HS is a chronic inflammatory disorder and secondary increases of the absolute platelet counts are described. Patients with secondary thrombocytosis from HS were excluded from the study and no differences in INR and fibrinogen were found with comparators. This suggests that the described abnormal VCM variables are indicative of pre-clinical activation of the coagulation cascade in HS.

[0049] Data on the blood levels of mediators involved in the platelet, endothelium and vascular interaction in HS are missing. It is shown that keratinocytes coming from HS lesions produce increased amounts of VEGF compared to keratinocytes coming from healthy and non- lesional skin. This is compatible with our finding of increased blood VEGF in HS patients. However, this study is the first to report down-regulation of 1CAM-1 and VCAM-1 in HS. Both these mediators are decreased when there is disruption of vascular integrity. This seems to happen in HS, and the VCM variables measurements appear to mirror this abnormality.

[0050] The VCM system technique in comparison to the clinically utilized laboratory evaluation of coagulation, encompasses the interaction of the coagulation factors with red blood cells, platelets, other blood components, and vascular endothelium. In whole, this method provides a global evaluation of the kinetics of the thrombus (e g., initiation, formation, stabilization, and lysis). The abnormal features of coagulation in HS shown by the VCM system are: a) decrease of CFT which indicates increased rate of initial clot formation; b) increase alphaangle (degrees) which indicates increased velocity of clot formation; c) increase of A10 and A20 representing stable clot firmness 10 and 20 minutes after start of clot formation; and d) increase of MCF reflecting high clot quality since MCF is a measure of the maximum amplitude that is reached before the clot is dissolved by fibrinolysis. These VCM variables were increased with HS severity and with the formation of AN lesions, but not of dTs. This is aligned with the716396068v1 16Atty. Dkt. No. 225871-031001hypothesis that immunothrombosis is a component of the pathogenesis of HS since Ans, and not dTs, are the active pro-inflammatory skin lesions.

[0051] The results argue in favor of further research towards the introduction of the VCM system in the monitoring of HS. The odds for abnormal VCM variables are greater in HS and may assist diagnosis; whereas, abnormal VCM variables seem to resolve upon improvement, thereby making the VCM system an effective tool to surrogate treatment responses.

[0052] While various illustrative embodiments of the invention are described above, it will be apparent to one skilled in the art that various changes and modifications may be made therein without departing from the invention. The appended claims are intended to cover all such changes and modifications that fall within the true scope of the invention.716396068v1 17Atty. Dkt. No. 225871-031001ReferencesRevuz J. Hidradenitis suppurativa. J Eur Acad Dermatol Venereal 2009; 23: 985-98.Frew JW. Hidradenitis suppurativa is an autoinflammatory keratinization disease: a review of the clinical, histologic, and molecular evidence. JAAD Int 2020; 1(1): 62-72.Frew JW, Hawkes JE, Krueger JG. A systematic review and critical evaluation of inflammatory cytokine associations in hidradenitis suppurativa [version 1; peer review: 2 approved, 1 approved with reservations], FlOOOResearch 2018; 7:1930.Giamarellos-Bourboulis EJ. Keratinocytes as drivers of hidradenitis suppurativa inflammation: need for priming. Br J Dermatol 2023; 188(3): 318-319.Aksu K, Donmez A, Keser G. Inflammation-induced thrombosis: mechanisms, disease associations and management. Curr Pharm Des 2012; 18: 1478-1493.Miller IM, Ahlehoff O, Ibler K et al. Hidradenitis suppurativa may not be associated with venous thromboembolia: Results from a large Danish cross-sectional study.J Dermatol Set 2016; 81(1): 61 - 63.Miller IM, Johansen ME, Mogensen UB et al. Coagulation status in hidradenitis suppurativa: a danish population- and hospital-based cross-sectional study. Dermatology 2015; 231(2): 119- 126.Amelio GS, Raffaeli G, Amodeo I et al. Hemostatic evaluation with viscoelastic coagulation monitor: a nicu experience. Front Pediatr 2022; 10: 910646.Revuz JE, Jemec GB. Diagnosing Hidradenitis Suppurativa. Dermatol Clin 2016; 34(1): 1-5.Zouboulis CC, Tzellos T, Kyrgidis A et al. Development and validation of IHS4, a novel dynamic scoring system to assess hidradenitis suppurativa / acne inversa severity. Br J Dermatol 2017; 177: 1401-9.Kimball AB, Sobell JM, Zouboulis CC et al. HiSCR (Hidradenitis Suppurativa Clinical Response): a novel clinical endpoint to evaluate therapeutic outcomes in patients with716396068v1 18Atty. Dkt. No. 225871-031001hi dradeniti s suppurativa from the placebo-controlled portion of a phase 2 adalimumab study. J Eur Acad Dermatol Venereal 2016; 30(6): 989-94.Kimball AB, Jemec GB, Yang M et al. Assessing the validity, responsiveness and meaningfulness of the Hidradenitis Suppurativa Clinical Response (HiSCR) as the clinical endpoint for hidradenitis suppurativa treatment. Br J Dermatol 2014; 171(6): 1434-42.Jones D, Banerjee A, Berger PZ et al. Inherent differences in keratinocyte function in hidradenitis suppurativa: evidence for the role of IL-22 in disease pathogenesis. Immunol Invest 2018; 47(1): 57-70.Singh V, Kaur R, Kumari P, Pasricha C, Singh R. ICAM-1 and VCAM-1; gatekeepers in various inflammatory and cardiovascular disorders. Clin Chim Acta 2023 548: 117487Panigada M, Meli A, Scotti E et al. Viscoelastic Coagulation monitor as a novel device to assess coagulation at the bedside, a single-center experience during the COVID-19 pandemic. ASAIO J 2021; 67(3): 254-262.Entegrion. VCM Operators Manual. For the near-patient semi-quantitative viscoelastic measurement of coagulation in whole blood. Durham, NC: Entegrion, Inc 2018.716396068v1 19

Claims

Atty. Dkt. No. 225871-031001WHAT TS CLAIMED:

1. A device for assessing Hidradenitis Suppurativa (HS), the device configured to: receive a sample of blood;cause coagulation of the sample of blood; andmonitor coagulation results to determine whether the sample of blood indicates a hypercoagulable state in a HS patient.

2. The device of claim 1, wherein the device comprises:a test cartridge configured to receive a sample of blood; andan analyzer unit configured to receive the test cartridge and cause coagulation of the sample of blood.

3. The device of claim 2, wherein the test cartridge comprises a pair of plates configured to define a capillary therebetween, the capillary sized and shaped to receive the sample of blood.

4. The device of claim 3, wherein each of the plates of the pair of plates are suspended via a flexible arm, and wherein, when the test cartridge is received by the analyzer unit, the analyzer unit is configured to drive movement of a first plate of the pair of plates via the corresponding flexible arm relative to a second plate of the pair of plates at a predetermined speed sufficient to cause the sample of blood to coagulate and bind to the first and second plates to thereby induce movement of the second plate.

5. The device of claim 4, wherein the analyzer unit is configured to drive movement of the first plate in an oscillating manner relative to the second plate.

6. The device of claim 4, further comprising an optical sensor configured to optically detect interaction between the pair of plates during coagulation, the interaction indicative of the coagulation results.

7. The device of claim 1, wherein the device is configured to:6396068v1 20Atty. Dkt. No. 225871-031001measure a vascular endothelial growth factor (VEGF) of the coagulation results, wherein an increased amount of VEGF is indicative of the hypercoagulable state in the HS patient.

8. The device of claim 1, wherein the device is configured to:measure an intercellular adhesion molecule-1 (ICAM-1) of the coagulation results, wherein a decreased amount of ICAM-1 is indicative of the hypercoagulable state in the HS patient.

9. The device of claim 1, wherein the device is configured to:measure a vascular-cell adhesion molecule-1 (VCAM-1) of the coagulation results, wherein a decreased amount of VCAM-1 is indicative of the hypercoagulable state in the HS patient.

10. The device of claim 1, wherein the device is configured to:measure a clot formation time (CFT) of the coagulation results,wherein a decreased amount of CFT is indicative of the hypercoagulable state in the HS patient.

11. The device of claim 1 , wherein the device is configured to:measure an alpha angle of the coagulation results,wherein an increased alpha angle is indicative of a velocity of clot formation and the hypercoagulable state in the HS patient.

12. The device of claim 1, wherein the device is configured to:measure a clot firmness (CF) of the coagulation results after a predetermined time period after a start of clot formation of the sample of blood,wherein an increased CF is indicative of the hypercoagulable state in the HS patient.

13. The device of claim 1, wherein the device is configured to:6396068v1 21Atty. Dkt. No. 225871-031001measure a maximum clot firmness (MCF) of the coagulation results before the clot dissolves,wherein an increased MCF is indicative of the hypercoagulable state in the HS patient.

14. A method for assessing Hidradenitis Suppurativa (HS), the method comprising: receiving a sample of blood;causing coagulation of the sample of blood; andmonitoring coagulation results to determine whether the sample of blood indicates a hypercoagulable state in a HS patient.

15. The method of claim 14, wherein receiving the sample of blood comprises receiving the sample of blood within a capillary defined between a pair of plates of a test cartridge, each plate of the pair of plates suspended via a flexible arm, the method further comprising:inserting the test cartridge within an analyzer unit,wherein causing coagulation of the sample of blood comprises driving, via the analyzer unit, movement of a first plate of the pair of plates via the corresponding flexible arm relative to a second plate of the pair of plates at a predetermined speed sufficient to cause the sample of blood to coagulate and bind to the first and second plates to thereby induce movement of the second plate, andwherein monitoring the coagulation results comprises optically detecting, via an optical sensor, interaction between the pair of plates during coagulation, the interaction indicative of the coagulation results.

16. The method of claim 15, wherein driving movement of the first plate of the pair of plates comprises driving, via the analyzer unit, movement of the first plate in an oscillating manner relative to the second plate.

17. The method of claim 14, wherein monitoring the coagulation results comprises measuring a vascular endothelial growth factor (VEGF) of the coagulation results, and6396068v1 22Atty. Dkt. No. 225871-031001wherein an increased amount of VEGF is indicative of the hypercoagulable state in the HS patient.

18. The method of claim 14, wherein monitoring the coagulation results comprises measuring an intercellular adhesion molecule- 1 (ICAM-1) of the coagulation results, wherein a decreased amount of ICAM-1 is indicative of the hypercoagulable state in the HS patient.

19. The method of claim 14, wherein monitoring the coagulation results comprises measuring a vascular-cell adhesion molecule-1 (VCAM-1) of the coagulation results, wherein a decreased amount of VCAM-1 is indicative of the hypercoagulable state in the HS patient.

20. The method of claim 14, wherein monitoring the coagulation results comprises measuring a clot formation time (CFT) of the coagulation results,wherein a decreased amount of CFT is indicative of the hypercoagulable state in the HS patient.

21. The method of claim 14, wherein monitoring the coagulation results comprises measuring an alpha angle of the coagulation results,wherein an increased alpha angle is indicative of a velocity of clot formation and the hypercoagulable state in the HS patient.

22. The method of claim 14, wherein monitoring the coagulation results comprises measuring a clot firmness (CF) of the coagulation results after a predetermined time period after a start of clot formation of the sample of blood,wherein an increased CF is indicative of the hypercoagulable state in the HS patient.

23. The method of claim 14, wherein monitoring the coagulation results comprises measuring a maximum clot firmness (MCF) of the coagulation results before the clot dissolves, wherein an increased MCF is indicative of the hypercoagulable state in the HS patient.6396068v1 23

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