Methods for non-invasive detection of skin pathologies, transplantation complications and risk thereof

Abstract

The present invention relates to methods for non-invasive detection of transplantation complications (e.g., rejection, infection, vascular failure, inflammation) or the risk thereof after tissue / organ / cell transplantation. The present methods allow for early, effective, and continuous monitoring of transplant health in a pigment-agnostic manner.

Description

[0001] METHODS FORNON-INVASIVE DETECTION OF SKIN PATHOLOGIES, TRANSPLANTATION COMPLICATIONS AND RISK THEREOF

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 555,195, filed on February 19, 2024. The entire contents of the foregoing are incorporated herein by reference.

[0004] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under grant Nos. W81XWH-17-1-0437 and W81XWH-17-1-0440 awarded by the Department of Defense, Grant No. EEC 1941543 awarded by the National Science Foundation, and Grant Nos. EB028782 and AH71958 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] The present invention relates to the technical field of skin pathologies and organ / tissue / cell transplantation, and specifically to methods allowing detection of transplantation complications (e.g., rejection, infection, vascular failure, inflammation).

[0008] BACKGROUND

[0009] Transplantation includes, e.g., autologous transplantation, allogeneic transplantation, and xenotransplantation. Autologous transplantation is a procedure where a patient's own healthy cells or tissues are collected, processed, and then transplanted back into the same patient. By contrast, allogeneic transplantation refers to the transfer of an organ / tissue / cell from a donor to a recipient (e.g., for the purpose of replacing the recipient’s damaged or absent organ / tissue / cell). The major barrier for transplantation between genetically non-identical individuals is the recipient's immune system, which will treat the transplanted organ / tissue / cell as an “invader” and immediately or chronically reject it, causing transplantation complications (e.g., rejection, infection, vascular failure, inflammation). Early detection of such transplantation complications is critical in the context of transplant surgery, where early intervention can significantly impact patient outcomes. For example, if the immune response or rejection is detected early, the chance of transplant rejection or failure may be minimized with the use of immunosuppressant agents or the adjustment of immunosuppressive regimens.

[0010] SUMMARY

[0011] The present invention provides methods for non-invasive detection of the presence or absence of skin pathologies, transplantation complications (e.g., rejection, infection, vascular compromise, inflammation), or the risk of developing transplantation complications. The present methods allow for effective and continuous monitoring of transplant status with minimal discomfort for a transplant patient.

[0012] In one aspect, the disclosure is related to a method for noninvasively determining the risk of developing transplantation complications in a test area in a subject, comprising: (a) measuring a first temperature at a first time point in a first region of said subj ect using a temperature measurement device that does not substantially apply any one of themial energy, positive pressure, and negative pressure to said first region of the subject, wherein the first region is within the test area.

[0013] In some embodiments, the method further comprises: (b) providing a control temperature; (c) determining, based on the difference between the first temperature and the control temperature, the risk of developing of transplantation complications.

[0014] In one aspect, the disclosure is related to a method for treating transplantation complications at a test area in a subject, comprising: (a) measuring a first temperature at a first time point in a first region of said subject using a temperature measurement device that does not substantially apply any one of thermal energy-, positive pressure, and negative pressure to said first region of the subject, wherein the first region is within the test area.

[0015] In some embodiments, the method further comprises: (b) providing a control temperature; (c) determining, based on the difference between the first temperature and the control temperature, the risk of developing of transplantation complications; and (d) applying an immunosuppressive agent to the subject if the risk of developing transplantation complications is high. In some embodiments, step (b) comprises measuring a control temperature at a control region of said subject using the temperature measurement device, wherein the control region is outside the test area.

[0016] In some embodiments, step (b) comprises providing a historical control temperature.

[0017] In some embodiments, the method further comprises: measuring a second temperature at a second time point in the first region, and determining, based on temporal temperature changes in the first region, the risk of developing of transplantation complications.

[0018] In some embodiments, the method further comprises: measuring a third temperatures at a third time point in the first region, and detennining. based on temporal temperature changes in the first region, the risk of developing of transplantation complications.

[0019] In some embodiments, the temperature measurement device comprises an infrared sensor or a near-infrared sensor.

[0020] In some embodiments, the temperature measurement device comprises an infrared gun or a forward-looking infrared (FLIR) camera.

[0021] In some embodiments, the temperature measurement device comprises a thennistor, a resistance temperature detector, a thermocouple, and / or a semiconductor-based temperature sensor.

[0022] In some embodiments, the test area is an area on the subject’s skin.

[0023] In some embodiments, the subject’s skin is Fitzpatrick skin type IV-VI.

[0024] In some embodiments, the first temperature is an average temperature of the test area.

[0025] In some embodiments, step (a) comprises measuring a peripheral temperature and a center temperature of the test area.

[0026] In some embodiments, step (a) comprises calculating the average temperature of the test area by taking the average of the peripheral temperature and center temperature.

[0027] In some embodiments, step (c) comprises determining that the risk of developing of transplantation complications is high when the temperature difference is at least 1-5 °C. In some embodiments, step (c) comprises determining that the risk of developing of transplantation complications is high when the temperature difference is at least 0.2-0.4 °C.

[0028] In some embodiments, the method further comprises measuring a blood oxygen level and / or a tissue oxygen level.

[0029] In some embodiments, the first temperature is obtained within 48 hours after transplantation.

[0030] In some embodiments, the first temperature is obtained within 24 hours after transplantation.

[0031] In some embodiments, the method comprises determining the risk of developing of transplantation complications within 72 hours after transplantation.

[0032] In some embodiments, the transplantation comprises a skin graft (e.g., full thickness skin graft, partial thickness or split skin graft).

[0033] In some embodiments, the transplantation comprises vascularized composite allotransplantation (VC A).

[0034] In some embodiments, the transplantation comprises solid organ transplantation.

[0035] In some embodiments, the method further comprises obtaining a biopsy from the subject to assess transplantation complications via histological analysis.

[0036] In some embodiments, the biopsy is only obtained after the risk of developing of transplantation complications is determined to be high based on temperature analysis.

[0037] In some embodiments, the method further comprises using surgical exploration, CT scan, and / or doppler ultrasound analysis to assess transplantation complications.

[0038] In some embodiments, the temperature measurement device does not contact the subject's skin.

[0039] In some embodiments, the method comprises training a machine learning model using a set of training data and determining the risk of developing of transplantation complications using testing data.

[0040] In some embodiments, the training data comprises temperatures at multiple locations within the test area. In some embodiments, the training data comprises temperatures at multiple time points.

[0041] In some embodiments, the immunosuppressive agent is selected from cyclosporine, a steroid such as methylprednisolone, antithymocute globulin, alemtuzumab, clazakizumab, imlifidase, carfilzomib, tocilizumab, mTOR inhibitors, leflunomide, plasmapheresis with IVIG, rituximab, bortezomib. eculizumab, azathioprine. FK-506, or 15 -deoxy spergualin.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0043] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0044] DESCRIPTION OF DRAWINGS

[0045] FIGS. 1A-1E show the detection of transplantation complications (e.g.. rejection, infection, vascular failure, inflammation) using an infrared detector in a mouse model. FIG. 1A shows the study design. FIG. IB shows pictures of the transplantation site. FIG. 1C shows infrared images of the transplantation site. FIG. ID shows temperature difference data as detected by infrared gun. FIG. IE shows temperature difference data as detected by FLIR one camera.

[0046] FIGS. 2A-2C show data analysis regrading the temperature difference data. (A) Temperature differences between graft and native skin of POD 1 and POD2 were combined for AUC analysis; (B) AUC analysis of specificity / sensitivity of temperature using the IR gun in detecting rejection; (C) AUC analysis of specificity / sensitivity of temperature using the FLIR camera in detecting rejection;. **** / °°°° p < 0.COOL

[0047] FIGS. 3A-3D show comparison of IR data with clinical assessment and histology assessment. (A-i) Representative clinical and temperature images on POD1. (A-ii) Representative clinical and temperature images on POD3. (A-iii) Representative clinical and temperature images on POD7. (B) Histological analysis, light microscopy, xlO (scale bar 250 pm), H&E staining, (i) Representative images on POD1. (ii) Representative images on POD3. (iii) Representative images on POD7.

[0048] (C) Analysis of clinical assessment scores and histology' grading (i / ii / iii) and blinded microscopic Banff evaluation (iv / v). (D) Association between temperature assessment, clinical rejection score and histological Banff score (daily ROC curve based AUC of individual data points for each type of assessment versus POD curve).

[0049] * p < 0.0332; ** / °° p < 0.0021; *** p < 0.0002.

[0050] FIGS. 4A-4C show the translation of thermal assessment to clinical applications. (A) Use of thermal assessment of VC A transplants can be combined with other data to make a risk assessment of the chance of early rejection based upon which further diagnostics can be recommended or, alternatively, to refrain from diagnostics. (B) Potential of thermal assessment in longitudinal timeline of VCA transplantation and postoperative care. (C) Potential applications of thermal assessment.

[0051] FIGS. 5A-5B show residual plot and QQ-plot for both (A) IR gun and (B) FLIR images.

[0052] FIGS. 6A-6B show- daily area under curve (AUC) curves of IR-gun analysis of pigmented allogeneic transplant (FIG. 6A) and non-pigmented allogeneic transplant (FIG. 6B).

[0053] FIGS. 7A-7B show daily area under curve (AUC) curves of FLIR camera analysis of pigmented allogeneic transplant (FIG. 7A) and non-pigmented allogeneic transplant (FIG. 7B).

[0054] FIGS. 8A-8C show histology evaluation of graft muscle biopsies at day 3, longitudinal and cross-section, H&E staining, x20 (scale bar 100 um) (A) At POD 1 muscle samples in the rejection groups show' mild to moderate signs of ischemia with myocyte size variation (§) and myocyte damage (*). (B) At POD 3, signs of rejection are seen with moderate edema (#), myocyte damage (*), and inflammation (±). (C) By POD 7, severe ischemic changes are seen with early necrosis (f ).

[0055] FIGS. 9A-9C show' histology evaluation of graft muscle biopsies at day 7, longitudinal and cross-section, H&E staining, x20 (scale bar 100 um) (A) Syngeneic transplants (n = 12) show no signs of rejection, normal architecture, and presence of skin adnexa on POD 7 (Banff 0). (B) In contrast, both pigmented allogeneic (n = 9) and (C) non-pigmented allogeneic (n = 11) show immune cell infiltration (*), apoptotic bodies (#), microthrombi (±), full-thickness skin necrosis (f) with severe loss of architecture (§) (Banff IV) in all replicates on POD 7.

[0056] FIGS. 10A-10C show transcutaneous temperature readings in a pig transplant model. (A) Temperature (°C) measured from the flap of each experimental pig. (B) Temperature (°C) measured from the abdominal control skin of each experimental pig. (C) Difference in temperature (°C) between each graft and their respective control site. Each animal is represented by a different line color with the autologous grafts illustrated by dotted lines and the neck flaps with induced ischemia represented by solid lines. Neck Flap; neck flap isolation with induced ischemia, Auto; autologous VRAM flap transplantation to the neck.

[0057] FIG. 11 shows the temperatures of the graft sites in a pig transplant model.

[0058] DETAILED DESCRIPTION

[0059] Immune responses against the transplanted organ / tissue / cell often cause transplant failure or transplant rejection and in some cases the need to apply or adjust immunosuppressive therapies or to remove the transplanted organ / tissue / cell from the recipient or acceptor. An early detection capability is critical in the context of transplant surgery', where early intervention can significantly impact patient outcomes. For example, if the immune response or rejection is detected early, the chance of transplant rejection or failure may be minimized with the use of immunosuppressant drugs. Therefore, there is a critical need for non-invasive detection and prediction methods that can be used in the early detection of allograft rejection. Early diagnosis of acute rejection is essential for the immunological management of transplant patients, affecting comorbidity, chronic rejection, and risk of complete graft failure.

[0060] Transplants involving skin (e.g., VCAs) are especially high-risk due to the immunogenic nature of skin tissue, and acute rejection episodes occur in 89% of patients. Traditionally, diagnosis relies on serial biopsies and clinical observation. Biopsies are risky and painful, while visual assessment of the skin can be imprecise and subjective, especially in pigmented skin where early signs of rejection, such as erythema, are less apparent. Early detection of changes in graft health can lead to prompt treatment, reducing the severity of rejection episodes and potentially avoiding complete graft failure. By developing additional non-invasive and objective methods, VCA's surface-level accessibility can be leveraged for more effective early detection and monitoring. Currently, rejection in tissue transplantation (e.g., Vascularized Composite Allotransplantation (VCA)) is typically diagnosed based on clinical assessment based on visual observation (e.g., for signs such as erythema) and / or histology assessment. For visual observation, skin pigmentation can pose challenges for physicians to diagnose pathologies. In VCA. this increases the difficulty of diagnosing rejection by clinical observation, which could be improved by noninvasive monitoring, thereby completely avoiding or aiding in guiding location for invasive diagnostics. For histology assessment, serial biopsies are an invasive tool to diagnose rejection and cause discomfort and potential harm to patients. Therefore, there is a need for non-invasive detection and prediction methods that can be used in the early detection of transplantation complications (e.g., rejection, infection, vascular failure, inflammation).

[0061] Described herein are non-invasive detection and prediction methods that can be used in the early detection of skin pathologies (e.g., cellulitis, vasculopathy), and transplantation complications (e.g., rejection, infection, vascular failure, inflammation), based on thermal assessment. For example, an infrared (IR) gun can be used for point measurements of temperature. Forward-looking IR (FLIR) imaging technologies can also be used. The methods described herein offer a reliable tool to use across various skin pigmentation levels. Both IR guns and FLIR cameras record the graft surface temperature by analyzing the emitted IR from the graft in the 8 to 14 microns wavelength range. Predicated on the thermodynamic principles of heat transfer from the blood circulation to the graft, these measurements sen e as an indirect measure of skin perfusion and, consequently, graft viability with correlation to early stages of graft rejection. Traditional non-invasive methods include ultrasound and MRI techniques, and often involve blood flow assessment, visual markers after intravenous injection, or stiffness measurements. In comparison, the thermal assessment (e.g., based on IR) approach described herein is fast, portable, quantitative, and particularly valuable in resource-limited settings due to its straightforward application and cost-effectiveness, thereby addressing a critical gap in skin diagnostics and reconstructive transplant surgery. The present methods utilize thennal assessment techniques (e.g., IR gun, FLIR imaging, thermistors, resistance temperature detectors, thermocouples, semiconductor-based temperature sensors) in detection of skin pathologies, transplantation complications (e.g., rejection, infection, vascular failure, inflammation) or the risk thereof in a pigmentation-agnostic manner. The present methods can be more effective in early detection than clinical assessments and / or histology assessments. In some cases, clinical assessments and / or histology assessments cannot detect rejection until Post Operative Day (POD) 3, especially in pigmented skin. In some cases, the present methods can show significant differences between rejection and non-rejection groups as early as POD 1, irrespective of skin pigmentation. Without wishing to be bound by theory, the methods described herein can detect decreased temperature in the test area (e.g., an area on the patient’s skin) as a sign of rejection, because rejection often cause impaired microcapillary perfusion and therefore disrupted heat distribution in the test area. As rejection is a multifactorial process, combining measurements such as temperature, heart rate, oxygenation and other chemical measurements (e.g., oxidized rmtochondna levels) can further increase sensitivity and specificity, and guide physicians in clinical decision-making (FIG. 4A).

[0062] The methods described herein can improve patient outcomes and postoperative care. The methods can be used to guide the clinical decision-making process and minimize invasive, costly, and time-consuming diagnostic tools for patients. The present methods can offer assessment independent of skin pigmentation, and offer a more inclusive approach to clinical care. The present methods do not rely on expensive and / or high-accuracy equipment. The detection methods can be low- cost, do not require extensive training, and may be appropriate for home care use. The methods described herein offer an objective, quantifiable, non-invasive, easy-to-use, and quick adjunct tool for early rejection detection in a pigment-agnostic manner.

[0063] Detecting Skin Pathologies, Transplantation Complications or the Risk Thereof

[0064] Transplantation includes, e.g., autologous transplantation, allogeneic transplantation, and xenotransplantation. Autologous transplantation (autotransplantation, or autograft) is a procedure where a patient's own healthy cells or tissues are collected, processed, and then transplanted back into the same patient. Allogeneic transplantation (allo-transplantation, or allograft) refers to the transfer of an organ / tissue / cell from a donor to a recipient (e.g., for the purpose of replacing the recipient’s damaged or absent organ / tissue / cell). Xenotransplantation refers to the transfer of an organ / tissue / cell from an animal to a human recipient. Allografts can either be from a living or cadaveric source. Examples of organs that can be transplanted are the heart, kidneys, liver, lungs, pancreas, intestine, and thymus. Examples of tissues include bones, tendons (both referred to as musculoskeletal grafts), cornea, skin, heart valves, nerves, and veins. Examples of cells include stem cells, bone marrow cells, and immune cells (e.g., CAR-T cells).

[0065] Skin-containing transplantations, which play a crucial role in reconstructive surgery, exemplify the challenges at the intersection of skin pathology and transplant medicine. Vascularized Composite Allotransplantations (VCAs), autotransplantations, free flap transfers, and sentinel skin flaps, while innovative, are often hindered by the difficult}' in early detection of complications when using subjective clinical observations, especially in pigmented skin. Far from being a challenge unique to VCAs, such disparities are representative of a broader issue in the field of transplantation and medical diagnostics in general. Amongst others, race and ethnicity greatly determine the chance of referral for transplant evaluation, being added to the waiting list, and receiving a transplant. Recent attempts to address challenges with pigmented patients have sometimes included adding more invasive procedures, placing a greater burden on the patient. For example, the first black patient to receive a face transplant underw ent additional mucosal biopsies, which w ere not typically required for other patients. Considering these observations, inadequate diagnostic tools and sluggish technological development contribute to discriminatory practices and non-invasive alternatives may be found to prevent unnecessary procedures in all patients.

[0066] By focusing on thermal parameters, this disclosure provides methods that are agnostic to skin pigmentation. The methods can be used for effective, non-invasive early detection tool for graft rejection, suitable across different skin types, to facilitate early, accessible, and straightforward intervention irrespective of skin pigmentation, leading to improved clinical outcomes and more equitable healthcare.

[0067] The methods include determining a temperature of a test area (e.g., an area on the patient’s skin). The temperature of an object, such as human body, can be determined by methods known in the art. Tw o common methods can include: using a contact thermometer having a probe which comes into physical contact with the measured object, or using a non-contact thermometer that measures infrared (IR) or near-infrared (NIR) radiation exchange between its radiation detector and an object (target).

[0068] An exemplary appropriate temperature measuring device can comprise one or more of thermistors, resistance temperature detectors, thermocouples, semiconductorbased temperature sensors, and the like. The temperature measuring device can also comprise a controller, a storage device, and / or a powder source. Some exemplary appropriate temperature measuring devices are described in U.S. Patent No. 5,178,464 and U.S. Application Publication Nos. US2024 / 0216168A1 and US2005 / 0094705A1. The temperature of the tested area can be determined by a thermistor. The temperature of the tested area can be determined by an infrared (IR) measuring device, such as an IR gun. Other temperature measuring devices can be used.

[0069] Infrared (IR) and near-infrared (NIR) imaging technologies offer distinct advantages in transplant monitoring. While IR thermography provides surface temperature mapping, NIR imaging extends this capability to deeper tissue layers, potentially uncovering subdermal processes that precede visible clinical signs. An infrared thermometer relies on thermal radiation exchange between an object (target) and a sensor. If an object is warmer than sensor, the latter's temperature increases upon its exposure to a target. The sensor’s temperature increase may be used for the target’s temperature measurements.

[0070] The present methods use handheld pyrometers, which can operate on the principles of noncontact infrared (IR) thermometry (FIG. 1A). These devices, characterized by their ergonomic design and rapid response thermopile sensors, offer real-time point-based temperature readings with high precision. Ideal for swift clinical evaluations, they utilize wavelength modulation for accurate thermal detection in diverse surgical settings. In addition to point-based techniques, FLIR imaging systems with incorporated quantum well infrared photodetectors (QWIPs) for enhanced thermal sensitivity can be used to generate comprehensive thermographic maps. Thermal maps facilitate granular analysis of thermal gradients across the entire region of interest as well as the healthy surrounding tissue, offering insights into the nuanced (patho)physiological responses. Region of interest selection for integration analysis will be included. The present methods can use anon-contact IR gun measurement device for skin areas with adjustable inter-laser distances with options of preselection by physicians to provide anatomical mapping for patients or nurses.

[0071] The use of IR technology for temperature measurement, while straightforward, has surprisingly not played a larger role in clinical practice, nor have temperature profiles of transplant organs been extensively studied. One reason for this may be that it is only in recent years that this technology has achieved affordability, accuracy, and compactness for medical use. Both IR gun and FLIR camera offer significant advantages in the <$500 price range, where the gun provides higher accuracy for point measurements where FLIR camera allows spatial coverage of the graft at some loss in accuracy. Additionally, FLIR cameras can also require significant postprocessing to obtain an average temperature of the whole graft. In some cases, similarly priced IR gun ($350) and FLIR imaging camera ($400) can show a similar trend of changes in temperature for POD 0-7. However, in some cases, the FLIR image-based analysis did not reach significance in early graft rejection analysis, possibly because the resolution and / or noise of the FLIR. One camera (±3°C) that was used was insufficient to capture the small-scale differences between allogenic and syngeneic grafts. For instance, the multiple comparisons test showed a mean temperature difference of at least 0.27°C between the allogeneic groups compared to the syngeneic group on all the Post Operative Days (PODs). The necessary precision of temperature measurement may be pathology-dependent. The FLIR One smartphone thermography has been used successfully, however, some applications will require higher precision. Especially in patient populations that have limited access to medical resources, or that are not only vulnerable but also frequently hospitalized, improvements in non-invasive, low-cost IR technology can greatly increase the quality of life. Such accessible, low-cost detection of other clinical pathologies could be facilitated in pigmented as well as non-pigmented skin using these assessment techniques.

[0072] The temperature measuring device can have a minimal temperature sensitivity of 0.05 °C, 0.1°C, 0.15°C, 0.2°C, 0.25°C, 0.3°C, 0.35°C, 0.4°C, 0.45°C, or 0.5°C. The temperature measuring device can have a minimal temperature sensitivity of 0.1 °C. The temperature measuring device can have a minimal temperature sensitivity of 0.2°C. The temperature measuring device can have a minimal temperature sensitivity of 0.3°C. The temperature measuring device can have a minimal temperature sensitivity of 0.4°C. The temperature measuring device can have a minimal temperature sensitivity of 0.5°C. The temperature measuring device can have a minimal temperature sensitivity’ of 1°C. This level of sensitivity can be crucial for detecting subtle, yet clinically significant, thermal variations indicative of early - stage graft rejection or inflammation. For other applications lower or higher sensitivity may be sufficient or required.

[0073] The present methods can include determining a temperature within a test area (“test area temperature”) and a control temperature. The test area can be an area on the patient's skin. The test area can be the area where the transplant was received. The border of the test area can overlap with the border between donor tissue and recipient tissue. The test area can be within the area where the transplantation is received. The border of the test area can be surrounded by the border between donor tissue and recipient tissue. The control temperature can be a temperature within a control area. The control temperature can be the temperature of the healthy skin surrounding the test area, an adjacent area, a contralateral area, or any other area on the body. The control temperature can be a representative temperature from a cohort of healthy control skin.

[0074] The test area can be an area beneath the patient’s skin. The test area can be the area where the transplant was received. The border of the test area can overlap with the border between donor tissue and recipient tissue. The test area can be with the area where the transplantation is received. The border of the test area can be surrounded by the border between donor tissue and recipient tissue.

[0075] One temperature monitoring device can be used to detennine both the test area temperature and the control temperature. Two or more temperature monitoring devices can be used to determine the test area temperature and the control temperature. One temperature monitoring device can include two or more temperature sensors.

[0076] Two temperature sensors can be disposed at different locations on or near a patient’s skin. For example, a first temperature sensor can be disposed in the test area to provide a temperature of the test area (“test area temperature”), and a second temperature sensor can be disposed outside of the test area to provide a control temperature. Two or more temperature sensors are disposed in the test area to provide an average temperature of the test area. For example, a first temperature sensor can be disposed at the center of the test area to determine a center temperature and a second temperature sensor can be disposed at the peripheral of the test area to determine a peripheral temperature. The temperature of the test area can be determined based on the center temperature and the peripheral temperature. The temperature of the test area can be determined by taking the average of the center temperature and the peripheral temperature. The temperature of the test area can be determined by taking a weighted average of the center temperature and the peripheral temperature.

[0077] Temperatures at multiple locations within the test area can be measured, and the test area temperature is determined based on the temperatures at multiple locations within the test area. For example, temperatures at multiple locations within the test area can be measured, and the test area temperature determined by taking the average of the temperatures at multiple locations within the test area. Temperatures at multiple locations within the test area can be measured, and the test area temperature can be determined by taking the median of the temperatures at multiple locations within the test area. In cases where some temperatures may be less informative than others, a clustering procedure may be implemented to remove the outliers (e.g., the highest temperature and / or the lowest temperature). The temperature of the test area can be determined by (1) removing the outlier temperatures from the temperatures at multiple locations within the test area and (2) taking the average of the remaining temperatures at multiple locations within the test area. Multiple locations can be tested to map temperature across the test area; this can be accomplished by using or moving devices, or by using multiple devices at the same time.

[0078] Temperatures at multiple locations outside the test area can be measured, and the control temperature is determined based on the temperatures at multiple locations outside the test area. Temperatures at multiple locations outside the test area can be measured, and the control temperature is determined by taking the average of the temperatures at multiple locations outside the test area. Temperatures at multiple locations outside the test area can be measured, and the control temperature can be determined by taking the median of the temperatures at multiple locations outside the test area. In cases where some temperatures may be less informative than others, a clustering procedure can be implemented to remove the outliers (e.g., the highest temperature and / or the lowest temperature). The control temperature can be determined by (1) removing the outlier temperatures from the temperatures at multiple locations outside the test area and (2) taking the average of the remaining temperatures at multiple locations outside the test area. Multiple locations can be tested to map temperature across the control area; this can be accomplished by using or moving devices, or by using multiple devices at the same time.

[0079] The presence and / or the risk of skin pathologies and / or transplantation complications (e.g., rejection, infection, vascular failure, inflammation) can be determined based on the temperature of the test area and the control temperature. Skin pathologies, transplantation complications or the risk thereof can be determined based on the difference between the temperature of the test area and the control temperature. For example, if the temperature of the test area is significantly lower than the control temperature, transplant health is poor and there is a high chance of developing complications and / or rejection. The present methods can determine that a high chance of developing complications and / or rejection is present when the temperature difference between the temperature of the test area and the control temperature exceeds a threshold value. The threshold value can be 0.5-10°C, 0.5-6°C, 4-6°C, 4- 8°C, 1-2°C, 1.2-2°C, or 1.2-1.6°C. The threshold value can be 0.5-3°C, 1-3°C, 1-2°C, 1-1.6°C, or 1.4-1.6°C. The threshold value can be 1.4-1.6°C. The threshold value can be 4-6°C. The threshold value can be 0.2-0.4°C. The threshold value can be 1.45°C. It should be noted that this threshold is dependent on the device type and specifics used, location of sensing, and other operational factors. The threshold value can also be varied to adjust the accuracy of diagnosis, trading off type I (false positive) vs type II (false negative) errors. The present methods can determine that a high chance of developing complications and / or rejection is present when the test area temperature is lower than the control temperature by more than 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C, 1°C, 1.1 °C, 1.2°C, 1.3°C, 1.4°C, 1.5°C, 1.6°C, 1.7°C, 1.8°C, 1.9°C, 2°C, 2.1°C, 2.2°C, 2.3°C, 2.4°C, 2.5°C, 2.6°C, 2.7°C, 2.8°C, 2.9°C, 3°C, 3.1°C. 3.2°C, 3.3°C, 3.4°C, 3.5°C, 3.6°C, 3.7°C, 3.8°C, 3.9°C, 4°C, 4.1°C, 4.2°C, 4.3°C. 4.4°C, 4.5°C, 4.6°C. 4.7°C, 4.8°C, 4.9°C. 5°C, 5.5°C, 6°C, 6.5°C, 7°C. 7.5°C, 8°C, 8.5°C, 9°C, 9.5°C, or 10°C. The present methods can determine that a high chance of developing complications and / or rejection is present when the test area temperature is lower than the control temperature by more than 0.5-10°C, 0.5-6°C, 0.5-3°C, 1-3°C, 1-2°C. 1-1.6°C, or 1.4-1.6°C. The present methods can determine that a high chance of developing complications and / or rejection is present when the test area temperature is lower than the control temperature by more than 1.4-1.6°C.

[0080] The temperature sensors can be used to measure temperature changes over time. The temperature monitoring device can include a storage device that can store temperature data over time. The temperature monitoring device can include a controller (e.g.. a processor) that can determine the difference between the test area temperature and the control temperature. The temperature monitoring device can include a controller that can measure the rate of change in temperature over time. This rate of change or derivative of temperature with respect to time can be used as an additional metric or threshold to indicate the presence of rejection or failure.

[0081] The present methods can include the below steps.

[0082] First, a test area is chosen based on the operated field on the patient’s skin.

[0083] Second, a thermal assessment device is used to obtain temperatures of the test area and surrounding healthy skin. Exemplary thermal assessment devices are pointbased infrared guns (Digisense, Cat. N° 20250-07) and FLIR thennal images (FLIR ONE® Pro - iOS). For devices with point-based measurements (e.g., IR gun), measurements of the center and periphery of the test area, and measurements of the surrounding healthy skin (control measurements) are taken, at least three measurements each. For thennal mapping devices (e.g., FLIR camera), the test area is selected to determine an average intensity of pixels (range 0-255). The pixel intensity’ is scaled to the temperature range of the flap (transplanted tissue). The same is repeated for the control skin area.

[0084] Third, the thennal data from step three is inputted into visualization software (e.g., Excel for IR gun and Teledyne software for FLIR images).

[0085] Fourth, the visualization software is used to generate an average test area temperature, which is then compared against a control temperature from surrounding healthy (control) skin. If the temperature difference is at least 1.45°C, the present methods can determine that the graft is undergoing rejection. It should be noted that this threshold is dependent on the device type and specifics used, location of sensing, and other operational factors. It is understood that one skilled in the art can readily identify different thresholds accordingly. The threshold value can also be varied to adjust the accuracy of diagnosis, trading off type I (false positive) vs type II (false negative) errors. The present methods can more efficiently detect skin pathologies, transplantation complications (e.g., rejection, infection, vascular failure, inflammation) comparing to traditional methods (e.g., clinical assessment and histological studies). The present methods can detect transplantation complications at an earlier time comparing to traditional methods. The present methods can detect transplantation complications within 4 hours, 8 hours, 12 hours. 16 hours, 20 hours, 24 hours. 36 hours. 48 hours, 60 hours, 72 hours, or 96 hours after operation. Comparing to traditional methods, the methods described herein can detect transplantation complications at least 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, or 96 hours sooner. Significant differences in graft temperature can be detected as early as post operative day (POD) 1 and 2. By comparison clinical and histological assessment may not be able to detect signs of transplantation complications until POD 3.

[0086] Comparing to a syngeneic transplant, an allogeneic transplant can lead to a lower temperature in the test area. Comparing to a syngeneic transplant, an allogeneic transplant can lead to a temperature drop of more than more than 0. 1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C, 1°C, 1.1 °C, 1.2°C, 1.3°C, 1.4°C, 1.5°C,

[0087] 1.6°C, 1.7°C, 1.8°C, 1.9°C, 2°C, 2.1°C, 2.2°C, 2.3°C, 2.4°C, 2.5°C, 2.6°C, 2.7°C,

[0088] 2.8°C, 2.9°C. 3°C, 3.1°C, 3.2°C, 3.3°C, 3.4°C, 3.5°C, 3.6°C, 3.7°C, 3.8°C, 3.9°C, 4°C, 4.1°C. 4.2°C, 4.3°C, 4.4°C. 4.5°C, 4.6°C, 4.7°C. 4.8°C, 4.9°C, 5°C, 5.5°C, 6°C. 6.5°C, 7°C, 7.5°C, 8°C, 8.5°C, 9°C, 9.5°C, or 10°C. Comparing to a syngeneic transplant, an allogeneic transplant can lead to a temperature drop of more than 1°C within 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, or 96 hours after operation. Without wishing to be bound by theory, an allogeneic transplant may lead to a lower temperature in the test area by causing vascular dysfunction and / or ischemia. For example, rejection may cause clotting in the capillaries, leading to ischemia and lower temperature in the test area. The present methods can detect skin pathologies, transplantation complications, or the risk thereof by detecting vascular dysfunctions in the test area. The present methods can detect skin pathologies, transplantation complications, or the risk thereof by detecting local ischemia in the test area.

[0089] The subject’s skin can be pigmented (e.g., Fitzpatrick skin type IV -V). The subject’s skin can be non-pigmented (Fitzpatrick skin type I-II). The present methods can effectively detect early signs of transplantation complications (e.g., rejection, infection, vascular failure, inflammation) irrespective of whether the subject’s skin is pigmented.

[0090] The transplantation can be an organ transplantation. The transplantation can be a transplantation of the heart, kidneys, liver, lungs, pancreas, intestine, and thymus. The transplantation can be a tissue transplantation. The transplantation can include the transplantation of bones, tendons (both referred to as musculoskeletal grafts), cornea, skin, heart valves, nen es and veins. The transplantation can include Vascularized Composite Allotransplantations (VC As), auto-transplantations, free flap transfers, and sentinel skin flaps. The transplantation can include skin transplants (e.g., full thickness skin grafts, partial thickness or split skin grafts). The transplantation can be a cell transplantation. The transplantation can be a transplantation of stem cells, bone marrow cells, or immune cells (e.g., CAR-T cells).

[0091] In some cases, the intended transplantation (e.g., kidney transplantation) does not involve a skin tissue, but a skin tissue (e g., a sentinel flap) is transplanted to assess the risk of rejection for the intended transplantation. A sentinel flap can be transplanted before the intended transplantation. A sentinel flap can be transplanted along with the intended transplantation.

[0092] The present methods can determine the presence or risk of skin pathologies and / or transplantation complications based solely on temperature data. In addition to temperature data, the present methods include obtaining other types of data from the subject. The present methods can include obtaining blood oxygen data and / or heart rate data from the subject. The present methods can detennine the presence or risk of skin pathologies and / or transplantation complications based on temperature, blood oxygen, tissue oxygen, and / or heart rate. Without wishing to be bound by theory, skin pathologies and / or transplantation complications may lead to increased blood oxygen, decreased tissue oxygen concentration and / or increased heart rate. The present methods can include obtaining other physiological parameters including blood pressure, respiratory rate, ECG, and / or blood analysis.

[0093] The methods based on IR-gun measurements can more accurately detect skin pathologies and / or transplantation complications (e.g., rejection, infection, vascular failure, inflammation) comparing to the methods based on FLIR measurements. In some cases, AUC analysis reflects a higher sensitivity and specificity of IR-gun measurements than FLIR measurements with an AUC of 83.54% in the pigmented group and 74.32% in the non-pigmented group using combined IR-gun temperature data from POD 1-2 (FIG. 2B). Comparing to the methods based on FLIR measurements, the methods based on IR-gun measurements can lead to an AUC that is higher by 5%, 10%, 15%, 20%, or 25%. Comparing to the methods based on FLIR measurements, the methods based on IR-gun measurements can lead to a specificity that is higher by 5%. 10%. 15%. 20%. or 25%.

[0094] The present methods can more accurately detect skin pathologies and / or transplantation complications (e.g., rejection, infection, vascular failure, inflammation) comparing to traditional methods (e.g., clinical assessment and histological analysis). As shown in FIG. 3C, for the experimental groups combined it was observed that the daily AUC for temperature assessment (72.22 to 91.22) was consistently higher than the AUC for clinical scoring (54.46 to 73.08) from POD 1 until POD 4. Further, the AUC of the rejection groups for temperature assessment and clinical scores are high and align well for POD 5 to POD 7 (76.29 to 100 and 78.57 to 100, respectively), with both techniques predicting rejection with very high confidence and accuracy. Comparing to the traditional methods, the methods described herein can lead to an AUC that is higher by 5%, 10%, 15%, 20%, or 25%. Comparing to traditional methods, the methods described herein can lead to a specificity that is higher by 5%, 10%, 15%, 20%, or 25%. The present methods can achieve higher sensitivity and specificity compared to traditional diagnostic methods (e.g., clinical assessment).

[0095] Treatment or Additional Diagnostic Procedures

[0096] The disclosure provides methods of treating a subject based on the detection of skin pathologies, transplantation complications, or the risk thereof. The present methods can further include treating the subj ect with: a. immunosuppressants, which can be chosen or adjusted at the discretion of the clinician, and can be introduced locally or systemically, by injection, ingestion, inhalation or through other means; b. removal of the graft to avoid adverse outcomes for the recipient; and / or c. additional diagnostic procedures (e.g., biopsies and histology assessments). The present methods can determine a risk of developing complications. Based on the risk of developing complications, the subject can be treated with an immunosuppressive agent. In the last several decades, the available number of immunosuppressive agents has increased greatly. Agents can be used as induction therapy (at the time of transplant), as maintenance therapy (to prevent rejection of the allograft), or for treatment of acute rej ection.

[0097] Recommendations for immunosuppressive medications are necessarily complex, because combinations of multiple classes of immunosuppressive drugs are used and because the choice among different regimens are determined by the tradeoffs between benefits and hann. Typically, a greater degree of immunosuppression may reduce the risk of rejection, but may also increase the risk of infection and cancer. Standard post transplantation therapy consists of a calcineurin inhibitor, antiproliferative agents, and corticosteroids. The calcineurin inhibitors, cyclosporine and tacrolimus, are a mainstay of maintenance immunosuppression. They inhibit IL -2 transcription, and suppress effector T-cells. Both have similar side effects, such as new-onset diabetes after transplantation, hyperlipidemias, hypertension, osteopenia and a decreased glomerular filtration rate. They both also cause significant nephrotoxicity.

[0098] The present methods can determine a risk of developing transplantation complications (e.g., rejection, infection, vascular failure, inflammation). Based on the risk of developing transplantation complications, the graft can be removed to avoid adverse outcomes. The same patient can then receive a different graft, if indicated.

[0099] In some cases, removal of the transplanted organ / tissue / cell is recommended when the temperature difference between the temperature of the test area and the control temperature exceeds a threshold value. The threshold value can be 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C, 1°C, 1.1 °C, 1.2°C, 1.3°C,

[0100] 1.4°C, I.5°C, 1.6°C, 1.7°C, 1.8°C, 1.9°C, 2°C, 2.1°C, 2.2°C, 2.3°C, 2.4°C, 2.5°C,

[0101] 2.6°C, 2.7°C. 2.8°C, 2.9°C, 3°C, 3.1°C, 3.2°C, 3.3°C, 3.4°C, 3.5°C, 3.6°C, 3.7°C,

[0102] 3.8°C, 3.9°C. 4°C, 4.1°C, 4.2°C. 4.3°C, 4.4°C, 4.5°C. 4.6°C, 4.7°C, 4.8°C, 4.9°C,

[0103] 5°C, 5.5°C, 6°C, 6.5°C, 7°C, 7.5°C, 8°C, 8.5°C, 9°C, 9.5°C, or 10°C. The threshold value can be higher than 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C, 1°C. 1.1 °C, 1.2°C, 1.3°C, 1.4°C, 1.5°C, 1.6°C, 1.7°C, 1.8°C, 1.9°C, 2°C, 2.1°C, 2.2°C. 2.3°C, 2.4°C, 2.5°C. 2.6°C, 2.7°C, 2.8°C, 2.9°C, 3°C, 3.1°C, 3.2°C, 3.3°C, 3.4°C, 3.5°C, 3.6°C, 3.7°C, 3.8°C, 3.9°C, 4°C, 4.1°C, 4.2°C, 4.3°C, 4.4°C,

[0104] 4.5°C, 4.6°C. 4.7°C, 4.8°C, 4.9°C. 5°C, 5.5°C, 6°C. 6.5°C, 7°C, 7.5°C, 8°C, 8.5°C, 9°C, 9.5°C, or 10°C. The threshold value can be lower than 0. 1°C, 0.2°C, 0.3°C,

[0105] 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C, 1°C, 1.1 °C, 1.2°C, 1.3°C, 1.4°C, 1.5°C,

[0106] 1.6°C, 1.7°C, 1.8°C, 1.9°C, 2°C, 2.1°C, 2.2°C, 2.3°C, 2.4°C, 2.5°C, 2.6°C, 2.7°C,

[0107] 2.8°C, 2.9°C. 3°C, 3.1°C, 3.2°C, 3.3°C, 3.4°C, 3.5°C, 3.6°C, 3.7°C, 3.8°C, 3.9°C,

[0108] 4°C, 4.1°C. 4.2°C, 4.3°C, 4.4°C. 4.5°C, 4.6°C, 4.7°C. 4.8°C, 4.9°C, 5°C. 5.5°C, 6°C.

[0109] 6.5°C, 7°C, 7.5°C, 8°C, 8.5°C, 9°C, 9.5°C, or 10°C. The threshold value can be 1 -

[0110] 2°C, 1.2-2°C, or 1.2-1.6°C. The threshold value can be 0.5-3°C, 1-3°C, 1-2°C, 1-

[0111] 1.6°C, or 1.4-1.6°C. The threshold value can be 1.4-1.6°C. The threshold value can be

[0112] 1.45°C. It should be noted that this threshold is dependent on the device type and specifics used, location of sensing, and other operational factors. The threshold value can also be varied to adjust the accuracy of diagnosis, trading off type I (false positive) vs type II (false negative) errors. In some cases, removal of the transplanted organ / tissue / cell is recommended when the test area temperature is lower than the control temperature by more than 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C, 1°C, 1.1 °C, 1.2°C, 1.3°C, 1.4°C, 1.5°C, 1.6°C, 1.7°C, 1.8°C, 1.9°C,

[0113] 2°C, 2.1°C, 2.2°C, 2.3°C, 2.4°C, 2.5°C, 2.6°C, 2.7°C, 2.8°C, 2.9°C, 3°C, 3.1°C,

[0114] 3.2°C, 3.3°C, 3.4°C, 3.5°C, 3.6°C, 3.7°C, 3.8°C, 3.9°C, 4°C, 4.1°C, 4.2°C, 4.3°C, 4.4°C, 4.5°C. 4.6°C, 4.7°C, 4.8°C. 4.9°C, 5°C, 5.5°C. 6°C, 6.5°C, 7°C, 7.5°C, 8°C, 8.5°C, 9°C, 9.5°C, or 10°C. In some cases, removal of the transplanted organ / tissue / cell is recommended when the test area temperature is low er than the control temperature by more than 0.5-10°C, 0.5-6°C, 0.5-3°C, 1-3°C, 1-2°C, 1-1.6°C, or 1.4-1.6°C. In some cases, removal of the transplanted organ / tissue / cell is recommended when the test area temperature is lower than the control temperature by more than 0.2-0.4°C. In some cases, removal of the transplanted organ / tissue / cell is recommended when the test area temperature is lower than the control temperature by more than 1.4-1.6°C. In some cases, removal of the transplanted organ / tissue / cell is recommended when the test area temperature is lower than the control temperature by more than 4-6°C.

[0115] The present methods can determine a risk of developing complications. Based on the risk of developing complications, the subject can be treated by further diagnostics as shown in FIG. 4A. As shown in FIG. 1A, use of thermal assessment can be combined with other data to make a risk assessment of the chance of early rejection based upon which further diagnostics can be recommended or, alternatively, to refrain from diagnostics. Given the high negative predictive values of thermal assessment alone, avoiding further invasive diagnostics can be a key contribution of adding this assessment to routine of care. For example, if the risk of developing complications is high, the subject is further subjected to biopsies, surgical explorations. CT scans, and / or Doppler ultrasound analysis. In some cases, if the risk of developing complications is low, the subject is not subjected to additional treatment or diagnostic procedures. In this way, the methods described herein prevents waste of medical resources and reduces the discomfort of the patient. Since it is easier to confirm the presence of a pathological condition with lower temperatures, it can be used as a referral for biopsies which can be used as a differential diagnostic method. It could reduce the need for invasive and risky diagnostic procedures such as serial biopsies, which can result in bleeding or inflammation. Due to the facile nature of the methods described herein, they may be useful for both clinical as well as at- home monitoring for grafts post-transplant (FIG. 4B). As shown in FIG. 4B, thermal assessment can be used in longitudinal timeline of transplantation and postoperative care. After transplantation, temperature can be measured daily or, if otherwise indicated, in the hospital as well as in the patient’s home as little expertise is required to obtain the measurements. By use of cloud communication these data can be transferred and tracked by in-hospital physicians, or in-device notifications to contact caretakers can be incorporated. As shown in FIG. 4C, the methods described herein can be used in inflammatory skin diseases or flap vascular failure of auto- and allotransplants. The FLIR image on the top shows a pigmented pig (Yucatan) with highlighted in white the inguinal ligament and in red the thermally identified saphenous artery. Manual palpation and visual assessment were insufficient for identification of the pedicle; however, using FLIR imaging, the vascular pedicle was successfully identified prior to procurement of the bilateral saphenous flaps.

[0116] In addition to transplantation complications, the methods described herein can be used for diagnosis and monitoring of several pathologies related to inhibited skin perfusion such as cellulitis, chronic vasculopathy, bum wounds, vessel patency in peripheral arterial disease, and surgical flaps. Intra-operatively, detection of signs of early graft dysfunction might also benefit from thermal assessment as a diagnostic tool (FIG. 4C). Furthermore, the use of sentinel skin transplants may be used as a rejection detection tool in solid organ transplantation. The non-contact nature of the methods described herein is highly suitable for immediate clinical translation, as a supportive approach to enhance prediction of rejection. A potential clinical plan would involve several key steps: first, measure temperature profiles in autologous skin, VC A, and free flap transfer transplants to establish standard temperature benchmarks for all patients, ideally involving a cohort of different pigmentation levels. This would effectively be a control group for non-rejection graft monitoring. The next phase would involve testing temperature profiles in allogenic VC A patients and sentinel flap clinical trials (currently ongoing) to further validate its effectiveness. Thermal assessment could revolutionize the benefits of sentinel flaps in solid organ transplant patients, regardless of their skin pigmentation levels. Moreover, for application to research, thermal assessment has been mentioned as a technique to increase standardization and reproducibility in bum wound models and the effect of treatment in these models.

[0117] EXAMPLES

[0118] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0119] EXAMPLE 1: Detection of transplantation complications or the risk thereof

[0120] Pigmented and non-pigmented allogeneic and non-pigmented syngeneic control transplant recipients underwent daily thermal assessment using an infrared (IR) gun and a forward-looking IR (FLTR) imaging of VCAs and surrounding healthy skin using a rodent partial hindlimb transplant model. Daily clinical assessment was performed, and biopsies were taken on POD 1, 3, and 7.

[0121] The following animals were used in the following examples. 60 rats (male, 250±50 grams) were used for all experiments, of which 42 were inbred Lewis rats, 11 Brown Norway rats, and 7 Buffalo rats (Charles River Laboratories, Wilmington, MA). The animals received humane care in accordance with the National Research Council guidelines and the experimental protocols were approved by the IACUC of Massachusetts General Hospital (Boston, MA).

[0122] Study design

[0123] Partial hindlimb transplants were performed in three different surgical groups (FIG. 1A): (1) non-pigmented syngeneic control (no rejection) group (n=12) in which Lewis rats were donors: (2) pigmented allogeneic (rejection) group (n=l 1) in which Brown Norway rats were donors; (3) non-pigmented allogeneic (rejection) group (n=7) in which Buffalo rats were donors. In all groups, Lewis rats were recipients. Buffalo and Lewis rats are considered albino animals therefore would be considered Fitzpatrick skin type I. Brown Norway rats have a non- Agouti brown coat meaning they are solid-colored. No equivalent scale to the Fitzpatrick skin types exists for rats, how ever, Brown Norway rats are considered to be closest to a Fitzpatrick skin ty pe IV -V. Use of pigmented animal models with similar immunological compatibility allows for cross-pigmentation measurements on the same timeline providing positive and negative control groups.

[0124] VCA transplantation

[0125] After induction using isofl urane (5%) inhalation with 100% O2, general anesthesia was sustained with inhaled isoflurane (1-3%) and anesthesia depth was confirmed with a toe pinch test. Partial hindlimbs were procured as described earlier. Briefly, grafts include the knee joint with 10 mm distal femur and 10 mm proximal femur and tibia, along with thigh muscle groups with the inguinal fat pad and calf muscles as well as the surrounding skin paddle. Femoral vessels were skeletonized and ligated 5 minutes after IV administration of lOOIU / mL / kg heparin in the penile dorsal vein. The femoral artery was cannulated with a 24G angio catheter and secured with a 6 / 0 nylon suture. The femoral vein was cut after ligation. Immediately after procurement, a pressure-controlled manual flush with 3 mL (200IU) of heparin saline at room temperature was performed. Next, the VCA was transplanted into a Lewis rat. Recipient vessels w ere prepared on the contralateral side in a similar fashion to the donor. Vessels were ligated distally and prepared for anastomosis. A longitudinal incision in the flank was made with subsequent tunneling to the groin area for VCA insertion. Femoral arteries and veins w ere anastomosed using a self-developed adjusted cuffing technique to allow' for application to partial hindlimb transplant. Skin on the donor VCA was excised to create an oval flap in the flank which was secured with interrupted 5-0 sutures. Inguinal fat pad and groin skin incision were similarly closed with interrupted 5-0 vicryl sutures.

[0126] Postoperative assessments using IR technologies

[0127] Postoperatively, temperature measurements were taken daily as displayed in FIG. 1A using a temperature IR gun (Digisense, Cat. N° 20250-07) and FLIR thermal images (FLIR ONE®1Pro - iOS). The FLIR thermal images are shown in FIG. 1C. Both devices were held at approximately 20 cm distance to the region of interest. Gun measurements were taken of the center and periphery of the flap (transplanted tissue), control measurements were taken of the skin immediately dorsal to the flap. FLIR images were taken of the entire flank area. For analysis, the mean temperature of the flap area and the mean of an area immediately dorsal of the flap w as taken in a blinded fashion. Diurnal variations in body temperature were accounted for by control measurements of healthy skin in the same animal, ensuring the reliability of the results by reducing environmental influences on the temperature.

[0128] Average temerpature differences between VCA (test area) and surrounding skin (control) are shown in FIG. ID (IR gun) and FIG. IE (FLIR camera) as mean and error with 95% Confidence Interval (CI). The temperature differences show (i) a significant difference from POD1 onwards comparing both pigmented (*) (n=8) and non-pigmented (°) (n=7) allogeneic groups to syngeneic control (n=12). Apart from POD7, no significant difference is found between the two rejection groups (+) (ii) Temperature assessment using FLIR shows a similar trend despite the loss of sensitivity7. * / ° p < 0.0332; ** / °° / ++p < 0.0021; *** / oo° p < 0.0002; **** / ooo°p< 0.0001.

[0129] As shown in FIG. 2A, temperature differences between graft and native skin of POD 1 and 2 were combined for AUG analysis. As shown in FIG. 2A(i), temperatures measured by IR gun showed significant difference between allogeneic and syngeneic groups regardless of pigmentation (n=8). As shown in FIG. 2A(ii), while not significant, temperature assessment using FLIR shows a similar trend (n=l 1 ). As shown in FIG. 2B, for the same time points, AUC analysis shows high specificity / sensitivity of temperature using the IR Gun in detecting rejection in both the (i) pigmented (83.54%) group and (ii) non-pigmented (74.32%) group. Similarly, as shown in FIG. 2C, temperature assessment using FLIR shows an AUC of (i.) 61.9% in the pigmented and of (ii.) 60. 12% in the non-pigmented rejection group. **** / oooop < 0.0001.

[0130] Postoperative assessments using clinical assessment

[0131] Postoperatively, daily flap images (e.g., cell phone pictures) were taken for blinded clinical assessment by six blinded clinicians using a clinical VC A rejection score. The daily flap images are shown in FIG. IB. Briefly, grade 0 constitutes no difference between graft and native skin. Grade 1 shows mild erythema, grade 2 moderate erythema with beginning of scaling and scabbing, grade 3 severe erythema and scabbing with areas of epidermolysis, and grade 4 constituting full-thickness graft epidermolysis with areas of necrosis.

[0132] As shown in FIG. 3A(i), representative clinical and temperature images show' similar results on POD1. As shown in FIG. 3A(ii), on POD3, a drop in temperature of the VC A compared to surrounding skin is seen. Clinically, erythema and epidermolysis, while subtle can be observed to be more pronounced in the non- pigmented group. As shown in FIG. 3A(iii), by POD7, rejection is pronounced in both rejection groups showing epidermolysis, necrosis and oozing of lymph fluid. Temperature drop of the VCA is more pronounced, regardless of the pigmentation of the flap.

[0133] Histology’

[0134] On postoperative day (POD) 1, 3 and 7 skin and muscle biopsies were taken (FIG. 1). On POD 7 additional muscle biopsies were taken. Biopsies were fixed in formalin and processed for histopathological examination. Slides were stained with hematoxylin and eosin (H&E). A blinded evaluation by a pathologist was performed for all biopsy samples and using the Banff criteria score to assess acute cell-mediated rejection. Briefly, grade 0 is considered no rejection, grade I mild (mild perivascular infiltration, no involvement of epidermis), grade II moderate (moderate perivascular infiltration, possible mild epidermal involvement), grade III severe (dense inflammation and epidermal involvement) and grade IV necrotizing acute rejection (frank necrosis of the epidermis and other skin structures). For the skin samples, a mean Banff score was calculated for comparison. Muscle tissues were evaluated and scored using the histology injury scoring system (HISS) for hypoxia-induced muscle injury.

[0135] Light microscopy images of histological analysis are shown in FIG. 3B, at xlO (scale bar 250 pm) with H&E staining. As shown in FIG. 3B(i), histological analysis showed normal features on POD1. As shown in FIG. 3B(ii), at POD3. focal epidermal necrosis was observed in both rejection groups with epidermal thickening (#), infiltration (*), microthrombi (±) and apoptotic bodies (f). As shown in FIG. 3B(iii), by POD7, full-thickness skin necrosis ( ) with severe loss of architecture (§) and thrombi (±) was seen in the rejection groups.

[0136] As shown in FIG. 3C, analysis of clinical assessment scores and histology grading shows rejection was identifiable at a slightly earlier time in the nonpigmented group. Conversely, blinded microscopic Banff evaluation show s no significant differences between groups and shows more severe rejection than clinical assessment suggests. In FIG. 3C(i) and FIG. 3C(iv), the part above the dotted black line indicates moderate to severe rejection.

[0137] As shown in FIG. 3D, Association between temperature assessment, clinical rejection score and histological Banff score (daily ROC curve based AUC of individual data points for each type of assessment versus POD curve) show that temperature assessment has an earlier association with rejection than both other scores (dotted line at 75%). * p < 0.0332; ** / °° p < 0.0021; *** p < 0.0002.

[0138] Statistical analysis

[0139] Temperature data is analyzed using a linear mixed effects model with the ty pe of transplant (3 levels; pigmented allogeneic, non-pigmented allogeneic, nonpigmented syngeneic) and POD (8 levels; POD 0-7) as fixed effects while also accounting for their interaction. Locations on the flap (3-4 per subject) and subjects (7-12 per condition) were treated as random variables for the temperature gun data. For FLIR data, average temperature for the whole flap is used for analysis, thereby only subject is treated as the random variable. Multiple comparisons were performed using Tukey’s corrected multiple comparisons test with 8 families (one for each time point). The appropriateness of the model was confirmed with a residual plot that showed no correlation of the residuals with the predicted values, and the nonnality assumption was confirmed with a QQ plot that showed high coincidence between the predicted and actual residual values (FIGS. 5A-5B).

[0140] Discriminative performance of thermal assessment for detecting graft rejection in the early PODs (POD 1 and 2) was evaluated using two separate methods. Firstly, a linear mixed effects model with type of transplant (3 levels as described above) and only early PODs (2 levels; POD 1 and POD 2) as the fixed effects were used while accounting for their interaction. For the discriminatory analysis, post-hoc analysis using multiple comparisons with Tukey’s correction was performed under the assumption of one family for the entire transplant ty pe. Secondly, a binary classification system is applied, and corresponding receiver operating characteristic (ROC) curves were generated, that independently compared two pairings: pigmented rejection with the non-rejection group, and the non-pigmented rejection group with the non-rejection group. The binary7classifiers also utilized temperature values from POD 1 and 2 for each pairing type. Furthermore, the effectiveness of each pairing was compared for each individual POD.

[0141] Clinical rejection score differences between groups were analyzed using a mixed-effects model with multiple comparisons. The time-series plots are represented as mean with 95 Confidence Interval (CI), bar charts are represented as mean with Standard Deviations (SD). All statistical analyses were perfonned using Prism 9 for Mac OSX (GraphPad Software, La Jolla, CA). p-Values less than 0.0332 were considered to be significant.

[0142] Results

[0143] As shown in FIGS. 1A-1E, temperature evaluation allows diagnosing rej ection as early as day 1 post-transplant. As shown in FIGS. 2A-2C, infrared gun is more accurate in detecting rejection compared to FLIR. As shown in FIGS. 3A-3D, clinical diagnosis of rejection is not possible before day 3, and is delayed in pigmented skin grafts.

[0144] Overall, clinical and histological assessments indicated signs of rejection on postoperative day (POD) 3. In contrast, thermal assessment using the IR gun detected significant differences as early as POD 1, notably a decrease in temperature, when compared to syngeneic control transplants. This demonstrates the capability of thermal assessments to identity' early signs of rejection before clinical symptoms become apparent.

[0145] The findings suggest that thermal assessments can serve as a non-contact, objective adjunct tool for early detection of graft rejection, with consideration of skin pigmentation. This approach may reduce the need for invasive biopsies, thereby improving patient comfort and reducing potential complications associated with current diagnostic methods.

[0146] Transplants in all three groups were successful until end of study as defined by visual assessment using the vascular patency test.

[0147] EXAMPLE 2: Data analysis

[0148] Postoperative thermal trend analysis indicates rejection can be detected as early as day 1

[0149] Representations of daily clinical images are shown in FIG. IB and corresponding FLIR images are shown in FIG. 1C, which readily reveal visual indicators of graft rejection in a pigment-agnostic manner as early as POD1. Temperature difference between VC A and surrounding skin using the IR-gun (FIG. ID) was assessed using a mixed effects model as described in the methods section and found significant effect of both the fixed effects and their interaction (p<0.0001). The standard deviation for the random effects (subject x location) is 0.51. The model was also found to have highly effective matching, indicating that the mixed effects model was the appropriate choice for analysis (pO.OOOl). Furthermore, post-hoc analysis to compare means for each POD shows a significant difference between the pigmented (p<0.0001) and non-pigmented (p=0.0068) rejection groups compared to the nonrejection group from POD 1 onwards. While the level of significance fluctuates and shows a decrease on POD3 in both groups, it remains significant until the end of study. FLIR temperature assessment (FIG. IE) shows a similar trend in mixed effects analysis (fixed effects and interaction significant with p<0.05, matching effective at p .OOOl, SD of random effect: 1.17) as well as post-hoc multiple comparisons, even though statistically significant differences are not observ ed until POD 6.

[0150] Infrared gun shows superior sensitivity and specificity compared to FLIR

[0151] FIG. 2A shows that thermal assessment indicated significant differences between rejection and non-rejection groups as early as POD 1 and 2, however only in the case of IR gun the average temperature difference reached statistical significance. Fitting of the mixed effects model on the data from the IR gun showed a statistically significant effect of the fixed effects, i.e. POD and type of transplant (p<0.005), however, no effect of interaction between POD and transplant type was found (p>0.05), allowing for grouping POD 1 and 2 data for post-hoc comparison. Tukey’s corrected multiple comparison for temperatures showed statistically significant difference between each of the rejection groups with the non-rejection group (p<0.0001). Neither the mixed effects model, nor the post-hoc comparison for the data from the FLIR measurements reached statistical significance (p>0.05). Correspondingly, AUC analysis reflects a higher sensitivity and specificity of IR-gun measurements than FLIR measurements with an AUC of 83.54% in the pigmented group and 74.32% in the non-pigmented group using combined IR-gun temperature data from POD 1-2 (FIG. 2B). AUC analysis of all other PODs is shown in FIGS. 6A-6B and 7A-7B. Similar to the daily thermal trend analysis, daily AUC curves show some fluctuation. To minimize data dependence on daily fluctuations in FIG. 2 a temporal component was integrated by using the average of POD 1 and 2. As shown in FIGS. 6A-6B, daily AUC curves of IR gun measurements show significant differences from POD1 onw ards. Daily average temperature differences measured by IR gun show significant differences between rejection and non-rejection groups regardless of pigmentation, which increases as the rejection worsens. For the same time points, AUC analysis shows high specificity / sensitivity of temperature using the IR Gun in detecting rejection in both the pigmented and non-pigmented group from POD 1 onwards. As shown in FIGS. 7A-7B, daily AUC curves of FLIR camera measurements show a similar trend but significant differences from POD 6 onwards. Daily AUC curves of thermal assessment using FLIR show a similar trend as the IR gun. However, significance is not reached until POD 6.

[0152] Clinical assessment does not diagnose rejection before day 3

[0153] Representations of daily clinical images are show n in FIG. 3A, and corresponding histological images in FIG. 3B. In both rejection groups at POD 1, the mean clinical assessment score was 0.25 (± 0.21) (FIG. 3C-ii), indicating minimal observable changes at this early stage. In the non-pigmented group the mean score was 0.35 (± 0.03), while the pigmented group w as only scored at a mean of 0.15 (± 0.33). At POD 3, the mean score increased to 1.65 (± 0.14), suggesting grafts show mild to moderate erythema with some showing the beginning of scaling and scabbing. Similarly, the non-pigmented group was scored lower at 1.22 (± 0.28). By POD 7, the mean score of both rejection groups further increased to 3.47 (± 0.04), reflecting pronounced clinical signs of severe erythema with areas of epidermolysis and necrosis or crust, consistent with graft rejection. This far into the rejection process, mean scores between the rejection groups were more similar with 3.57 (± 0.27) in the non- pigmented group and 3.2 in the pigmented group. The mean day on which rejection was clinically diagnosed was at 2.71 ± 0.44) and 2.96 (± 0.35) in the non-pigmented and pigmented grafts respectively, highlighting slightly earlier diagnosis in the non- pigmented group compared to the pigmented group (FIG. 3C-iii). In the non-rejection group, grafts showed normal postoperative recovery signs which could be confused with early stages of rejection, however, none of the grafts showed high clinical rejection scores, as expected.

[0154] Histological assessment does not detect rejection before day 3

[0155] Histology at POD 1, 3, and 7 is shown in FIG. 3C-iv and its analysis in FIG. 3C-vi. In both experimental groups, on POD 1, no pathological findings related to rejection were detected in skin tissue. Muscle tissue showed mild to moderate ischemic changes as displayed in FIGS. 8A-8C. At POD 3, skin samples showed focal epidermal necrosis resulting in a Banff score of III in both experimental groups. Muscle tissue showed moderate edema and inflammation. By POD 7. a Banff score of IV was found in both experimental groups based on severe ischemic changes with early necrosis of muscle tissue and full-thickness skin necrosis, indicative of advanced histological rejection, as shown in more detail in FIGS. 9A-9C. In the non-rejection group, no pathological signs were found in skin nor muscle tissue. No significant differences were found between the non-pigmented and pigmented rejection groups.

[0156] Comparison between thermal, clinical, and histological assessment

[0157] As shown in FIG. 3C, for the experimental groups combined it was observed that the daily AUC for temperature assessment (72.22 to 91.22) was consistently higher than the AUC for clinical scoring (54.46 to 73.08) from POD 1 until POD 4. This aligns well with our hypothesis that temperature-based assessment can provide an early measurement of the comorbidities associated with rejection. Further, the AUC of the rejection groups for temperature assessment and clinical scores are high and align well for POD 5 to POD 7 (76.29 to 100 and 78.57 to 100, respectively), with both techniques predicting rejection with very high confidence and accuracy.

[0158] EXAMPLE 3: Detection of transplantation complications or the risk thereof

[0159] All animals were purchased from Sinclair BioResources. Female 4-5-month- old Yucatan pigs (15.2-17.8 kg) were used for the neck flap surgeries (n = 2). Female 3-5-month-old Yucatan pigs (13.2-24. 1 kg) w ere used for the autologous surgeries (n = 2). The average overall w eight of the experimental pigs was 15.4 ± 2.31 kg. All animals were acclimatized for at least five days before surgery’. Research was conducted according to the principles outlined in the Guide for Laboratory Animal Facilities and Care prepared by the National Academy of Sciences, National Research Council. The Institutional Animal Care and Use Committee (IACUC) approved the research protocols and living quarters for the animals. Before surgery, all animals are held nil per os for 12 h. The pigs were anesthetized using ketamine (10 mg / kg) and xylazine (2 mg / kg), given intramuscularly (IM), and maintained under isoflurane following intubation. All procedures were performed under sterile conditions with continuous monitoring of the animal’s vitals throughout the surgery. All pigs underwent pain and infection management with 0. 12 mg / kg Buprenorphine Extended- Release (Bup ER) given subcutaneously (SQ), 4 mg / kg Carprofen SQ, and 40 mg / kg Cefazolin IV or 5 mg / kg Excede IM.

[0160] Neck flap isolation with induced ischemia

[0161] Following intubation, the pigs were placed in a lateral decubitus position to access the right anterolateral neck. A horizontal elliptical preoperative marking was made on the side of the neck. The apex of the flap was placed midline. Procurement of the flap was performed by careful dissection of the skin, subcutaneous tissue, and superficial strap muscles. The sternocleidomastoid muscle was divided, and dissection continued medially until the main perforating vessels branching from the right internal carotid artery (RICA) and the right external jugular vein (REJV) were identified (Supplementary Material SI). These vessels were carefully isolated. After the flap was elevated and islanded on its pedicle, Acland clamps were applied to the tw o main perforating vessels, achieving complete ischemia. Ischemia was maintained for 20 min for each flap. During this time, the flap was returned to its anatomical position. After the ischemia period, the clamps were released, and the vessels were inspected for any damage that may have occurred secondary to clamping. Once the vessels were deemed stable, with no evidence of injury. the flap was placed back into the lateral neck defect. The deep dermal layer was then closed with an interrupted, inverted 3-0 Vicryl suture, and the skin was reapproximated using a 4-0 Vicryl running subcuticular suture.

[0162] Autologous VRAM flap surgery

[0163] The VRAM flap is designed in a lenticular shape measuring roughly 15 x 6 cm and is procured from the left lower abdominal quadrant, capturing the nipples. The skin is incised, and electrocautery dissection is performed through the superficial ventral abdominal muscles down to the rectus fascia. Dissection continues down into the pelvis, and the rectus muscle is separated from the peritoneum and elevated with the flap. The external iliac vessels are identified and dissected distal to the bifurcation of the internal iliac vessels, where the left external iliac artery (LEIA) and vein (LEIV) are divided with surgical clips or silk ties. The flap, containing the pedicle, is then removed from the abdominal cavity and intravascularly flushed with 50-100 ml of normal saline containing heparin sulfate (1,000 IU / L). The warm ischemia time was between 50 and 90 min for each flap.

[0164] The abdominal defect created from the VRAM flap procurement was closed using size 1 (4 metric) DURAMESHTM Mesh Suture (Chicago, IL). The parietal peritoneum opening was closed in a lateral to medial fashion using the suturable mesh. Continuous over and over sutures were placed within the Scarpa fascia in a caudal to cranial fashion to successfully re-approximate the tissue. Complete closure of the abdominal skin was achieved using a continuous nonabsorbable 4-0 Vicryl running subcuticular suture. The abdominal wound was then covered with Tegaderm and left open to air starting on postoperative day (POD) 1.

[0165] Following abdominal flap procurement and wound closure, the animals were placed in a lateral decubitus position to access the right anterolateral neck. After placement of a contraleral (left) central line catheter, a horizontal lenticular shape was outlined on the right neck. A composite soft tissue defect was created by excising skin, subcutaneous tissue, and superficial strap muscles, just smaller than the size of the flap. Dissection was continued through the sternocleidomastoid muscle, brachiocephalicus, and stemocephalicus muscle until the RICA and REJV were identified. After the removal of the neck tissue and identification of the RICA and REJV, the vessels were carefully isolated and dissected for 5 cm, then proximally clamped in preparation for microvascular anastomosis.

[0166] Microsurgical anastomosis

[0167] The VRAM flap pedicle was placed into the neck tissue defect and all vessels were prepared for anastomosis by visual inspection, excision of adventitia, and irngation with heparinized saline. Using at least 3.5X magnifying surgical loupes, the LEIA and LEIV are hand-sewn, end-to-end, to the RICA and REJV, respectively, using 9-0 nylon interrupted sutures. Proximal clamps are then removed to analyze the quality of the anastomoses and once it is deemed satisfactory, the flap is inset, and a fenestrated Penrose drain is placed. The deep dermal layer is then closed with an interrupted, inverted 3-0 Vicryl suture, and the skin is reapproximated using a 4-0 Vicr l running subcuticular suture. Postoperative care

[0168] The neck of each pig was wrapped with two layers of McKesson tubular elastic retainer net (size 7), to protect the wound. Food and water were reinstituted immediately after each animal recovered from surgery'. All flaps were monitored daily via physical examination (palpation, tactile warmth assessment, and visual inspection). The integrity of the anastomosis of each flap was assessed by visual inspection of flap color as well as the presence of bleeding following daily punch biopsies. The animals were also followed for any fluid collections (seromas / hematomas) around the flap, which were drained percutaneously if clinically significant.

[0169] The oxygen and temperature sensor

[0170] The oxygen and temperature sensor includes an electronic reader and oxygenresponsive material. The sensor operates through the principle of phosphorescent quenching, where the phosphorescence lifetime of a sensor molecule is inversely proportional to the partial pressure of oxygen (pO2) via collisional interactions. For example, at high oxygen concentrations, the lifetime of the phosphorescence is short, meanwhile the phosphorescence lifetime increases in the setting of decreasing pO2. Phosphorescence lifetime can be calibrated and related to pO2 via the Stem-Volmer equation.

[0171] The oxygen sensing material uses a highly sensitive, ultrabright metalloporphyrin molecule (18) embedded within a polypropylmetharcylate (PPMA) polymer material. This porphyrin-PPMA material is packaged within a film composed of an atmosphere-facing layer comprised of Bioclusive polyurethane dressing material and a skin-face layer comprised of breathable silicone impregnated with titanium dioxide. The material was developed to be moisture and humidity-insensitive and has been previously tested in both animal and human studies. The atmosphere-facing layer serves to insulate the porphyrin-PPMA material from atmospheric oxygen, while the skin-facing layer isolates the sensor from the skin and improves the amount of collected light via backscattering. Prior testing of this material has found it does not leach porphyrin into the skin at levels detectable via inductive plasma coupled mass spectroscopy analysis. The electronic phosphorescence lifetime reader was designed to be standalone, battery-powered, and self-contained for mobile detection and recording of transcutaneous partial oxygen pressure. The reader is comprised of both a reader head and body, where the head is made to adhere to the sensor material and is connected to the body by a ribbon cable. The reader’s head excites the sensor material via two 385 nm LEDs and the subsequent phosphorescence emission is collected with a photodiode. A thermistor is additionally positioned on the sensor head to measure skin temperature. The body of the sensor is based on a Particle Photon Arduino microcontroller mounted on a custom circuit board. To record oxygen and temperature data, an Adafruit secure digital (SD) card reader was added to the circuit board. The sensor runs custom firmware written in C that orchestrates phosphorescence lifetime measurements through a sinusoidal modulation / modulo operator method and records the oxygen partial pressure and temperature data on the SD card.

[0172] Sensor materials and readers were calibrated using a custom developed calibration device and protocol. A programmable gas mixer was used to precisely deliver known air / nitrogen gas ratios over time, and a heating element was used to automatically change the temperature experienced by the sensor and material. This allowed the sensors to be calibrated over a large range of oxygen partial pressures and temperatures, which was found to be critical for the successful operation of the device. Analysis and calibration of the sensor were accomplished using a custom Python program that calculated the phosphorescence lifetime, translated the values to pO2, and exported the data into CSV files. In this study, the sensors were placed either on the graft site or on an abdominal (inguinal) control site at predetermined sequential time points following surgery. The abdominal control location was selected as the skin is relatively thin, allowing for high oxygen transport for routine oxygen detection and measurement.

[0173] Transcutaneous temperature

[0174] The oxygen and temperature sensor was used at daily intervals following the neck flap and autologous transplant procedures. The temperature measurements of the abdominal skin control sites were observed to be relatively consistent across all animals with a mean surface temperature of 33.4°C (FIG. 10B). In contrast, the temperatures of both the autologous and the neck flaps were observed to rise in the postoperative period (FIG. 10A); this increase was found to be statistically significant across all animals using a partial Bayesian linear mixed effects analysis comparing graft temperature and day post-surgery (p = 0.015). Temperature was also found to be predicted by the ty pe of surgical procedure (neck flap vs. autologous transplant, p = 0.037). The difference in temperature between grafts and their respective controls (FIG. 10C) was not observed to be statistically significant.

[0175] EXAMPLE 4: Detection of transplantation complications or the risk thereof

[0176] Allogeneic (allograft) and syngeneic (autograft) control transplant recipients underwent daily thermal assessment using a transcutaneous sensor (covering a circular area with a diameter of ~1.5 centimeter) using a pig transplant model. Control temperature was measured from a patch of healthy skin. Daily clinical assessment was performed, and biopsies were taken for histological analysis.

[0177] The temperatures of the graft sites are shown in FIG. 11. As shown in FIG.

[0178] 11, autografts showed consistent temperatures post-surgery. Allograft pigs, which did not receive immunosuppression, showed rapid rejection of the graft with a decrease in temerpature occurring beginning on Day 5. Linear mixed effects model showed statistically significant relationship of histological rejection with allograft status (allo vs auto) (p = 0.002). Linear mixed effects model showed statistically significant relationship of histological rejection with interaction of allograft status Temperature (p = 0.004).

[0179] OTHER EMBODIMENTS

[0180] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for noninvasively determining the risk of developing transplantation complications in a test area in a subject, comprising:(a) measuring a first temperature at a first time point in a first region of said subject using a temperature measurement device that does not substantially apply any one of thermal energy, positive pressure, and negative pressure to said first region of the subject, wherein the first region is within the test area.

2. The method of claim 1, further comprising:(b) providing a control temperature;(c) determining, based on the difference between the first temperature and the control temperature, the risk of developing of transplantation complications.

3. A method for treating transplantation complications at a test area in a subject, comprising:(a) measuring a first temperature at a first time point in a first region of said subject using a temperature measurement device that does not substantially apply any one of thermal energy, positive pressure, and negative pressure to said first region of the subject, wherein the first region is within the test area.

4. The method of claim 1, further comprising:(b) providing a control temperature;(c) determining, based on the difference between the first temperature and the control temperature, the risk of developing of transplantation complications; and(d) applying an immunosuppressive agent to the subject if the risk of developing transplantation complications is high.

5. The method of claim 2 or claim 4, wherein step (b) comprises measuring a control temperature at a control region of said subject using the temperature measurement device, wherein the control region is outside the test area.

6. The method of claim 2 or claim 4, wherein step (b) comprises providing a historical control temperature.

7. The method of any one of claims 1-6, further comprising: measuring a second temperature at a second time point in the first region, and determining, based on temporal temperature changes in the first region, the risk of developing of transplantation complications.

8. The method of any one of claim 7, further comprising: measuring a third temperatures at a third time point in the first region, and determining, based on temporal temperature changes in the first region, the risk of developing of transplantation complications.

9. The method of any one of claims 1-8, wherein the temperature measurement device comprises an infrared sensor or a near-infrared sensor.

10. The method of any one of claims 1-9, wherein the temperature measurement device comprises an infrared gun or a forward-looking infrared (FLIR) camera.

11. The method of any one of claims 1-10, wherein the temperature measurement device comprises a thermistor, a resistance temperature detector, a thermocouple, and / or a semiconductor-based temperature sensor.

12. The method of any one of claims 1-1 1, wherein the test area is an area on the subject’s skin.

13. The method of any one of claims 1-12, wherein the subject’s skin is Fitzpatrick skin type IV-VI.

14. The method of any one of claims 1-13, wherein the first temperature is an average temperature of the test area.

15. The method of any one of claims 1-14, wherein step (a) comprises measuring a peripheral temperature and a center temperature of the test area.

16. The method of claim 15, wherein step (a) comprises calculating the average temperature of the test area by taking the average of the peripheral temperature and center temperature.

17. The method of claim 2 or claim 4, wherein step (c) comprises determining that the risk of developing of transplantation complications is high when the temperature difference is at least 1-5 °C.

18. The method of claim 2 or claim 4, wherein step (c) comprises determining that the risk of developing of transplantation complications is high when the temperature difference is at least 0.2-0.4 °C.

19. The method of any one of claims 1-18, further comprising measuring a blood oxygen level and / or a tissue oxygen level.

20. The method of any one of claims 1-19, wherein the first temperature is obtained within 48 hours after transplantation.

21. The method of claim 20, wherein the first temperature is obtained within 24 hours after transplantation.

22. The method of any one of claims 1-21, wherein the method comprises determining the risk of developing of transplantation complications within 72 hours after transplantation.

23. The method of any one of claims 1-22. wherein the transplantation comprises a skin graft (e.g., full thickness skin graft, partial thickness or split skin graft).

24. The method of any one of claims 1-23, wherein the transplantation comprises vascularized composite allotransplantation (VC A).

25. The method of any one of claims 1-22, wherein the transplantation comprises solid organ transplantation.

26. The method of any one of claims 1-25. further comprising obtaining a biopsy from the subject to assess transplantation complications via histological analysis.

27. The method of claim 26, wherein the biopsy is only obtained after the risk of developing of transplantation complications is determined to be high based on temperature analysis.

28. The method of any one of claims 1-27, further comprising using surgical exploration, CT scan, and / or doppler ultrasound analysis to assess transplantation complications.

29. The method of any one of claims 1 -28, wherein the temperature measurement device does not contact the subject’s skin.

30. The method of any one of claims 1-29, wherein the method comprises training a machine learning model using a set of training data and determining the risk of developing of transplantation complications using testing data.

31. The method of claim 30, wherein the training data comprises temperatures at multiple locations within the test area.

32. The method of claim 30, wherein the training data comprises temperatures at multiple time points.

33. The method of claim 4, wherein the immunosuppressive agent is selected from cyclosporine, a steroid such as methylprednisolone, antithymocute globulin,alemtuzumab, clazakizumab. imlifidase, carfilzomib, tocilizumab, mTOR inhibitors, leflunomide, plasmapheresis with IVIG, rituximab, bortezomib. eculizumab, azathioprine, FK-506, or 15 -deoxy spergualin.

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