METHOD FOR MEASURING AVERAGE CORTISOL AND GLUCOSE CONCENTRATIONS, USING EARWAX.

Abstract

A method for measuring glucose and cortisol levels in earwax, wherein the measured cortisol and glucose levels are interpreted as average cortisol and glucose levels, and a medical device that provides effective, reliable, safe 5 and hygienic self-extraction of earwax.

Description

METHOD FOR MEASURING AVERAGE CORTISOL AND GLUCOSE CONCENTRATIONS USING EARWAX BACKGROUND OF THE INVENTION A. FIELD OF THE INVENTION The present invention relates to a method for measuring cortisol and glucose concentrations, and, in particular, to a method for measuring average cortisol and glucose levels from earwax. B. DESCRIPTION OF THE STATE OF THE ART 1st Problem Lack of a reliable and harmless method to measure the average concentration of glucose levels. Chronic diseases account for the majority of deaths (71%) worldwide, with diabetes being the fourth leading cause (WHO, 2018). Furthermore, according to the same WHO report, 650 million adults suffer from another chronic condition: obesity. Even more striking is the figure indicating that 39% of adults over 18 are overweight. Unfortunately, this additional chronic condition is also showing an upward trend. In fact, the global prevalence of obesity nearly tripled between 1975 and 2016 (NCD-RisC et al., 2017). These two epidemic diseases are closely related. Almost 90% of patients with the most common type of diabetes, or type 2 diabetes, are overweight (Wu et al., 2014). Moreover, these diseases commonly share the same metabolic alteration: a chronic elevation of glucose levels. In fact, this finding is necessary for diagnosing diabetes. Furthermore, individuals with elevated glucose levels, but not in the diabetic range, are up to 4.5 times more likely to be obese (Meigs et al., 1998). Current short-term glucose measurement methods, such as serum glucose, have significant limitations in assessing average glucose concentration. This is because glucose levels fluctuate considerably throughout the day. Furthermore, diurnal alterations, caused by stressful events (Dagogo-Jack, 2010), can significantly impact glucose levels. RQtij nn / nznz / E / YiAi or smoking (Frati et al., 1996), high blood pressure (Modan et al., 1985), Body Mass Index (BMI) (Hiller et al., 1988) and physical activity (Alien et al., 2009) can also affect their levels. Several glucose measurements, such as fasting and postprandial glucose concentrations, have been standardized to provide a more accurate glycemic level. However, undergoing these laboratory tests can be quite demanding for patients, and they do not accurately reflect mean glucose levels, which are the level necessary for long-term glycemic control in diabetic patients. In fact, these levels are usually below average, as seen in fasting serum glucose (FSG), or below that value, as in postprandial serum glucose (PSG) (Peter et al., 2006). Glycated hemoglobin (HbA1c) is a protein subunit of red blood cells, called hemoglobin, that shows positive correlations with both fasting and postprandial glycemic indices. It is commonly used as a long-term average glucose index (Monnier et al., 2006; Bonora et al., 2001; Rohlfing et al., 2002), which is why it is considered the gold standard for reflecting average glucose concentration. However, compared to diabetic patients, healthy individuals show weak associations between postprandial and fasting HbA1c levels (van 't Riet et al., 2010).In fact, a very large study showed correlations of 0.46 between fasting glucose (FSG) and HbA1c, and 0.33 when polyglycemic control (PSG) was associated with HbA1c in the general population, while in the diabetic population these results were 0.71 and 0.79 for the same associations (van 't Riet et al., 2010). These results undermine the ability of HbA1c to act as a screening test (Dagogo-Jack, 2010). On the other hand, fasting glucose levels show a stronger association with HbA1c than postprandial glucose levels with the same protein in healthy individuals and in diabetic patients with poor glycemic control (Monnier et al., 2006). This means that HbA1c could be within a normal range in diabetic patients who frequently indulge in dietary indiscretions. This certainly diminishes the ability of HbA1c to closely monitor average glucose levels in these patients. Therefore, a truly accurate method. RQtij nn / nznz / E / YiAi to reflect the average concentration of glucose levels must weigh postprandial and fasting glycemic levels in a balanced way. HbA1c is a protein measured to identify the average plasma glucose concentration over the past three months, but this is more heavily influenced (75%) by plasma glucose concentrations from the past month (Leow, 2016; Mortensen & Válund, 1988; Tañara & Shima, 1993). Therefore, HbA1c does not provide accurate information over shorter periods, which are necessary for tighter glycemic control, such as when hypoglycemic drugs are prescribed (Goldstein et al., 2004; Kim et al., 2012; Koenig et al., 1976). It also has some additional limitations. For one, it is not a completely accurate method, given that some common variables, such as those associated with aging, also affect HbA1c levels (Dagogo-Jack, 2010). Furthermore, some common disorders, such as anemia (Sundaram et al., 2007) and various hemoglobinopathies (up to 7%) (Weatherall, 2011), can also affect its levels. Even long working hours have been associated with higher HbA1c levels (Azami et al., 2018). Moreover, it is an expensive and often unavailable laboratory test (Sacks, 2011). Ultimately, it is an indirect approximation of average glucose levels, since it is a protein, rather than the sugar itself, that is measured directly.Due to all the reasons mentioned above, some authors even doubt its actual validity as a diagnostic test for diabetes mellitus and glucose intolerance (Dagogo-Jack, 2010). More recently, glycated albumin has been used as an index during intermediate periods of 2 to 4 weeks. However, sociodemographic variables, such as age or BMI (Miyashita et al., 2007), or disorders that affect albumin metabolism, such as thyroid dysfunction, nephrotic syndrome, or liver cirrhosis (KJ Kim & Lee, 2012), make glycated albumin a rather erratic measure in clinical practice (Huh et al., 2014). Furthermore, it has not yet been validated as a diagnostic method. It is also important to note that all the above samples, whether measuring glycemic levels, HbA1c, or glycosylated albumin, are obtained from samples Blood glucose tests are expensive because they require skilled workers, such as nurses, to collect the blood. Furthermore, they can be associated with side effects, such as bleeding and / or local infections, which are even more frequent and complicated in patients with metabolic disorders, such as diabetes. Nevertheless, and regardless of all the aforementioned disadvantages, blood glucose levels remain the most frequently requested laboratory test in Primary Health Care Centers in several countries (Salinas et al., 2014; Zunic, 2012), also representing the third highest laboratory cost for health systems (Zunic, 2012). HbA1c is also one of the most requested laboratory tests, and it is believed that it is still under-requested (Salinas et al., 2012).Therefore, there is an unquestionable need to develop not only a more beneficial sample in terms of being able to obtain a direct measurement of glucose concentration over different periods of time, but also a method that is more economical and harmless. 2d0Problem Lack of a reliable and practical method for measuring the average concentration of cortisol levels Depression is another chronic and epidemic disorder. Its clinical diagnosis is considered highly unreliable (Lieblich et al., 2015). This may explain the inherent heterogeneity of this syndrome. A significant effort has been made to develop an accurate biomarker that can improve the consistency of this diagnosis. Measuring cortisol levels has been the most popular biomarker, as it is the most frequently observed neurobiological alteration in this syndrome (Pariente, 2009). However, due to the reactive profile of its secretion, the results for this hormone have been highly inconsistent. In fact, it not only has its own very pronounced circadian rhythm (Bhagwagar, 2003; Bhagwagar et al., 2005), but also several common variables, such as food intake (Gibson & Checkley, 1999), nicotine (Steptoe & Ussher, 2006), physical exercise (Hill et al., 2008) and stress levels (Kirschbaum et al.(1993; Sharpley, 2012) can affect their short-term levels. This means that most current biological samples, such as the... RQtij nn / nznz / E / YiAi plasma or serum, are not the most appropriate to reflect the average concentration of cortisol, which is the level needed to describe the alteration of this hormone that is related to the different types of depressive disorders. Not long ago, hair samples began to be used to measure average cortisol levels (Dettenborn et al., 2012). This biological sample has been found to provide an index of average cortisol concentration, as it accumulates the hormone without being affected by confounding variables, typical of short-term measurements (Short et al., 2016a). However, it also has several limitations. Most of its validation studies have been conducted by comparing capillary cortisol concentration (HCC) with a single or aggregated daytime cortisol samples, without taking into account nighttime cortisol levels. This may explain why, until now, most correlation coefficients between hair and the aggregation of one or more short-term saliva samples have been quite modest (D'Anna-Hernandez et al., 2011; Sauvé et al., 2007; van Holland et al., 2012; Xie et al., 2011).In fact, few capillary validation studies have been properly conducted. The ideal study should correlate HCC with continuous cortisol levels or with cortisol levels measured throughout the day and night. Indeed, studies that have associated HCC with 24-hour urine collection have shown variable results. While Sauvé et al. (2007) found only a moderate correlation between HCC and 24-hour urine cortisol collection, Short et al. (2016b) found no significant association. Ultimately, it may also be possible that hair is not as good a reflection of average cortisol concentration levels as previously thought. For example, it is not entirely clear whether sweat glands, which are indeed affected by acute influences, also contribute some of the cortisol that accumulates within the hair (Sharpley, 2012). However, it is clear that sebaceous glands, which undoubtedly deliver cortisol into the hair, are nerved and therefore influenced by fine networks of nerve fibers (Okumura, 1967). Furthermore, acute influences can also affect the hair growth cycle. There is accumulating evidence indicating that neurohormones and neurotransmitters released during the stress response can also play a role. RQtij nn / nznz / E / YiAi significantly influence the hair growth cycle (Paus et al., 2006, 1997; Botchkarev, 2003). Furthermore, the spheroids deposited within the hair follicle, and thus within the future hair-free segment, also depend on local metabolic variables that reflect the hair growth state (Terao & Katayama, 2016). This would explain why the hair protocol suggests cutting this keratinized tissue from the posterior vertex of the scalp, as less hair growth variability has been observed in this area (Pragst & Balikova, 2006). This may mean that, although acute cortisol confounding variables may not affect HCC, these local covariates could. It may be possible to argue, then, that hair, instead of accurately reflecting long-term systemic cortisol levels, provides an index of long-term local cortisol levels.Hair sampling also faces several practical problems that hinder its widespread clinical use. The area with the least variability in hair growth, the posterior vertex of the scalp, is simultaneously the region most affected when people begin to lose their hair. In fact, this type of baldness (type IV) affects up to 40% of men and 10% of women over 40 years of age (Hamilton, 1951). Furthermore, this figure does not include the large percentage of people who cannot provide a sample simply because they do not have the minimum required hair (at least one centimeter [cm], representing the retrospective cortisol concentration of the past month). Indeed, a recent study showed that up to 30% of participants were unable or unwilling to provide this sample for various reasons, including aesthetic ones (Fischer et al., 2016).It is important to note that precisely cutting 1 mm of hair may be an impossible task. However, being able to discriminate the average cortisol level between weeks can be extremely important for clinicians. In fact, the antidepressant effect typically begins to show after 3, rather than 4, weeks of treatment (Tanum & Malt, 1996). This means that the actual antidepressant effect, in terms of long-term cortisol level changes, may not be accurately described using hair samples. Ultimately, unlike non-keratinized tissue, its analysis is very slow. In fact, while... RQtij nn / nznz / E / YiAi While analyzing a saliva sample can take 4 hours and 20 minutes, analyzing a hair sample takes over 30 hours, meaning almost eight times more working hours. Therefore, it is a highly inefficient process. This may explain why its cost can be up to 44.3% higher than analyzing a short-term cortisol sample, such as saliva (Bristow, 2017). All of the aforementioned variables definitely hinder its widespread clinical use. Several covariates can also affect cortisol levels in hair. Gender, for example, can affect HCC. Several studies have shown that men have higher HCC than women (Garcia-Leon et al., 2018; Vanaelst et al., 2012). Although the “wash-out” effect, caused by external factors such as UV radiation or the use of cleansing products, has been ruled out below the 0.45 cm closest to the hair root (Dettenborn et al., 2010), it is unknown whether an additional wash-out effect, or, in other words, an effect when the sample has just been removed from the scalp, also reduces cortisol levels. Furthermore, hair studies do not agree on a single type of cortisol extraction, even though significant variability in cortisol levels has been linked to this step in hair cortisol analysis.In fact, up to 3.5 times more cortisol has been extracted when the sample has been pulverized, rather than cut into small pieces (Davenport et al., 2006). Ultimately, it is unclear whether some cosmetic treatments, such as hair dye, also have an effect on HCC (Manenschijn et al., 2011; Sauvé et al., 2007). 3rd Problem: Lack of an efficient and safe device for obtaining cerumen samples for analysis. Unfortunately, to date, no self-cleaning device is as safe and effective as the traditional clinical method for collecting earwax samples from the outer ear. This means that, regardless of the potential usefulness of earwax sampling for measuring long-term average glucose and cortisol levels, its widespread clinical use seems unrealistic. Clinical earwax collection would be prohibitively expensive, as only qualified physicians can perform it safely. RQtij nn / nznz / E / YiAi Furthermore, although cleaning the outer ears is not medically recommended, millions of people practice this dangerous habit daily. In fact, cotton swabs, the most common method for self-cleaning the ears (Khan et al., 2017), are also the main risk factor for several external ear diseases, such as impacted earwax and bleeding (Ahmed et al., 2014; Nussinovitch et al., 2004). Therefore, considering their potentially serious side effects, their effectiveness should not be the sole reason for understanding their popularity. Other hypotheses have also been proposed. It has been suggested that these devices can have an addictive effect. Indeed, stimulation of the sensory fibers surrounding the external auditory canal can elicit various pleasurable visceral stimuli. However, chronic use can trigger a vicious cycle, described as an itch-scratch cycle that tends to perpetuate itself over time. Thus, increased use of these devices could not only explain an increase in itching but also their subsequent abuse (Mochizuki et al., 2014; Pata et al., 2003).Therefore, simply warning about their potential side effects may prove insufficient to curb their enormous demand, given their commercial success and high expectations for continued rapid growth in major global markets. One brand alone, for example, reported sales of $189.3 million in the US in 2005 and $204.8 million in 2014. Furthermore, a recent market study revealed that their sales have shown growth rates of 20% in the United States, 32% in China, and 26% in Europe between 2011 and 2017, and are projected to grow at rates of 20%, 24%, and 19%, respectively, over the next five years (Hexa Reports, 2017). Finally, and perhaps most importantly, they are not a standard extraction method, as their effectiveness depends on the energy applied by each user, rather than the energy supplied by the device itself.Therefore, it is necessary to develop a reliable, efficient, and safe alternative to these popular and risky devices that allows for the extraction of earwax for analysis. Unfortunately, so far, this has not been possible. Currently, several ear cleaning products on the market have shown no effectiveness. RQtij ηη / ηζηζ / E / γίΛΐ effect or practically minimal. In fact, a comprehensive systematic review showed that, although some ceruminolytic solutions, such as those containing mineral oils, may have some benefit, it is unclear which ones specifically provide it and to what extent. Furthermore, to date, no device (mechanical or electrical) is as effective as clinical extraction performed by a specialist using a syringe, such as the Reiner-Alexander syringe (Clegg et al., 2010). 1st Solution Earwax reflects the average concentration of glucose and cortisol. Few biological samples can provide an average of glucose and cortisol levels. Adipose tissue may be one of them, due to its known properties for accumulating substances (Szymczak & Milewicz, 1998). However, taking an adipose tissue biopsy from patients seems extremely impractical, as it is not only a risky procedure but also much more expensive than taking blood samples. However, another, more readily available biological sample could provide these levels. Earwax is an oily secretion that is also composed primarily of lipids (Inaba et al., 1987). It is secreted by the apocrine and sebaceous glands within the ear canal (Montagna, 1955).This secretion could provide an accurate average of glucose and cortisol levels since it would not be affected by local and acute influences, such as those caused by nerve fibers or local metabolic variables that affect their concentration. In fact, unlike the sebaceous glands of the hair follicle, the apocrine and sebaceous glands of the ear have been shown to be non-nerved (Bende, 1981). Bees also produce their own wax. The role of beehives also suggests that earwax may offer additional advantages over blood samples. On the one hand, bees are able to store (accumulate) their sugar (honey) in their hives (Fratini et al., 2016), and on the other hand, due to its bacteriostatic properties, it is not consumed by microorganisms (Ghanem, 2011). In fact, this property is also shared with human earwax (Stoeckelhuber et al., 2006). Therefore, this suggests that earwax may not only be able to accumulate glucose and cortisol levels over long periods but may also be protected from epidermal flora. This implies that earwax could be collected from patients' homes, since unlike blood samples, no special storage or transport conditions would be needed. Cortisol and glucose are two highly reactive substances. Measuring their chronic levels is crucial because they are altered in epidemic disorders. However, current biological samples can only measure their levels over short periods, or, even with samples capable of accumulating them over longer periods, their widespread clinical use is impractical and costly. Earwax could be a practical, viable, and reliable biological sample for determining these substances over extended periods. However, it is unknown whether this secretion accumulates these reactive substances over long periods. Therefore, the applicant developed an analytical method for detecting glucose and cortisol levels using earwax samples. A systematic review evaluated the method of the present invention, and a pilot study assessed the efficiency and reliability of the earwax analysis, along with determining whether glucose and cortisol could be detected in this novel sample. Finally, the effectiveness of various types of sponges for removing artificial earwax from pigskin was also tested. Results: Cortisol levels have not been previously measured in earwax. Cortisol and glucose are detected in this oily secretion. The time required to analyze cortisol in earwax was much shorter than the time required to analyze the same substance using hair. A cellulose sponge with specific abrasive and absorbent properties was the most efficient at removing earwax from a piece of pigskin. Conclusion: Earwax may be the most accurate and efficient sample for measuring long-term cortisol and glucose levels. A cellulose sponge may be an effective, economical, and safe material for its collection. 2nd Solution Design of a safe and effective device for obtaining earwax «Qh / nn / nznz / E / YiAi In view of the aforementioned problems, the applicant developed a medical device that provides reliable, effective, safe, and hygienic self-extraction of earwax. Furthermore, the self-extraction earwax device of the present invention is capable of providing a suitable sample for the analysis method for the detection of glucose and cortisol using this new biological sample. SUMMARY OF THE INVENTION Therefore, a primary objective of the present invention is to provide a method for measuring glucose and cortisol levels in earwax. Another main objective of the present invention is to provide a new medical device that provides reliable, effective, safe, and hygienic self-extraction of earwax. These and other objectives and advantages of the method for measuring glucose and cortisol levels in earwax and the medical device of the present invention will become evident to those persons of ordinary skill in the art, from the following detailed description of embodiments of the invention, which will be made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a left side view of a first embodiment of the medical device of the present invention showing the cross-section of the tip including the sponge. Figure 2 is a perspective view of the tip of the medical device of the present invention without the sponge. Figure 3 is a top view of the tip of the medical device of the present invention without the sponge. Figure 4 is a left side view of a second embodiment of the medical device of the present invention showing the cross-section of the tip including the sponge. RQtij nn / nznz / E / YiAi Figure 5 is a graph showing the results of the association between the baseline-EGC sample and the FSG sample that was taken one month later (Follow-up). RQtij nn / nznz / E / YiAi Figure 6 is a graph showing Basal-HbA1c and FSG. Figure 7 is a graph showing EGC-Follow-up and PSG-Follow-up. Figure 8 is a graph showing HbAlc Monitoring and PSG Monitoring. Figure 9 is a graph showing the results of the association between the baseline EGC sample and the average glycemic levels. Figure 10 is a graph showing the results of the association between BasalHbA1c and mean glycemic levels. Figure 11 is a graph showing the results of the association between Follow-up-EGC and mean glycemic levels. Figure 12 is a graph showing the results of the association between HbA1c monitoring and average glycemic levels. Figure 13 is a graph showing the results of the association between HCC and cortisol concentration in cerumen (ECC). DETAILED DESCRIPTION OF THE INVENTION The method for measuring long-term glucose and cortisol levels in earwax of the present invention will be described according to a preferred embodiment thereof, wherein, in its most general embodiment, the method of the present invention comprises: Collect earwax samples by any suitable means. The minimum amount of earwax required to measure average cortisol and glucose levels was 0.8 mg; Prepare earwax samples for measuring cortisol and glucose levels according to the means or methods of measurement; Measure cortisol and glucose using any known means or method in which cortisol and glucose levels are interpreted as the average levels of the same substances. Earwax samples can be extracted by traditional means, for example, using a Reiner-Alexander syringe or using any suitable extraction device. Sample preparation can be carried out in different ways depending on the cortisol and glucose measurement method to be used. Samples may consist of pure, dried earwax. Glucose and cortisol analysis can be performed using various methods, such as immunohistochemistry or ELISA. In a first specific embodiment of the present invention, the external earwax is obtained by using a Reiner-Alexander syringe. In this specific implementation, sample preparation and measurement of cortisol and glucose levels are carried out as follows: Sample preparation Cortisol extraction a) Drying the earwax samples using N2 vapor at room temperature, until all the water evaporates from the sample. This step can also be done using lyophilization; b) Weigh the dry wax samples to normalize the amount of cortisol in dry weight. Normalization means that the measured weight is adjusted to a common scale to allow for comparison of the data; c) Homogenize the dried samples with 1 ml of phosphate-buffered saline (PBS) to obtain a cerumen solution in PBS. The amount of phosphate-buffered saline (PBS) can be 10 volumes by weight of cerumen, for example, for 100 g of cerumen if 1000 µl of phosphate-buffered saline (PBS) is used. Alternatively, any hydrophilic solvent can be used, such as physiological saline. RQtij ηη / ηζηζ / Ε / γίΛΐ d) Divide the solution obtained in step c) into a first portion of solution, a portion of a second solution and add each portion of solution to a respective tube; e) Adding 0.5 ml of diethyl ether to the first portion of solution to obtain a cerumen solution in PBS mixed with 0.5 mg of diethyl ether; since the ratio between both substances is 1:1. However, other suitable families of substances can also be used. f) Shake the tube containing the solution obtained in step e) for a period of time of at least one minute to mix the solution obtained in step f) and add 0.5 mg of diethyl ether after resuspending, the ratio with PBS being 1:1; g) Cool the mixed solution obtained in step g) to a temperature of -18 to 21°C, preferably -20°C, for a period of at least two hours to ensure that the liquid portion is frozen and does not contaminate it with the organic fraction. This step allows the extraction of compounds that are specifically soluble in diethyl ether, such as cortisol. This is because while the diethyl ether fraction remains liquid at -20°C, the phosphate fraction freezes. h) Extract from the cooled solution the compounds that are specifically solubilized in diethyl ether; i) Dry the remaining fraction of the liquid solution using the N2 displacement method. However, other methods such as evaporation can also be used; j) Store the dry fraction obtained in step i) at -80°C for later use; k) Adding 300 pg of cortisol to the second portion of solution in order to obtain a cerumen solution in PBS mixed with cortisol; I) Adding 0.5 ml of diethyl ether to the solution obtained in step k) in order to quantify the amount of purified cortisol as a way of evaluating the efficiency of the extraction method; m) Shake the tube containing the solution obtained in step I) for a period of time of at least one minute to mix the solution obtained in step RQtij nn / nznz / E / YiAi m) and add 0.5 mg of diethyl ether after resuspending, the ratio with PBS being 1:1; n) Cool the mixed solution obtained in step m) to a temperature of -18 to 21° C, preferably -20° C for a period of time of at least two hours in order to ensure that the liquid part is frozen, and does not contaminate the organic fraction; o) Extract from the cooled solution the compounds that are specifically solubilized in diethyl ether; p) Dry the remaining fraction of the liquid solution again using the N2 displacement method; q) Store the dry fraction obtained in step i) at -80° C for later use; r) In a 3rd tube -without the presence of homogenized solution- 0.5 ml of 300 pg / ml of purified cortisol solution was dissolved in PBS at pH 7. This step was performed as a way to obtain the efficiency of the cortisol extraction protocol from earwax. s) Carry out the same procedure used for the first portion of solution and for the second portion of solution to extract cortisol from it. Quantification of cortisol ELISA techniques are used, according to the manufacturer's instructions (Enzo Life Sciences, Farmingdale, NY) to quantify the amount of cortisol concentration in earwax samples, in which the quantification of cortisol is carried out as follows: a) Reconstitute the extracted samples using a buffer assay provided by the manufacturer, which allows for the quantification of cortisol using competitive colorimetric ELISA techniques. Add the buffer to the extracted samples to obtain a solution and let the solution stand for 20 minutes. 20 minutes was sufficient to rehydrate the solution to a more easily resuspended buffer. However, other time ranges can also be used. Afterward, shake the solution for 1 minute to homogenize it. For the purpose of this application, we will understand a buffer to be a stable solution because it maintains its pH within a certain range, regardless of whether a base or acid is added. b) Using a standardized curve for cortisol levels - microplate reader (NovoStar) - to measure the total amount of cortisol in the sample; c) Normalization of the quantified amount per gram of dry earwax using fluorometric techniques in which the fluorometer is excited within a range of 530-570 nm and read within an emission range of 590-600 nm. Since several variables, such as age, sex, different medical conditions, and stress levels, could affect cortisol levels in earwax, we used cholesterol, as it is not affected by the aforementioned variables, thus avoiding confounding the cortisol results found by the effect of the aforementioned covariates. Quantification of glucose A fraction of the previously dissolved earwax solution in PBS, used to measure cortisol levels, was used to measure glucose levels. Glucose quantification was performed as follows: a) Measure glucose levels using the SERA-PAK PLUS kit (Bayer Healthcare) for glucose levels in earwax solution, following the manufacturer's instructions. Glucose absorptions are quantified in triplicate at 505 nm. The glucose concentration (mg / dl) is obtained using the average absorptions, and the total amount of glucose in the dissolved solution is calculated according to the initial weight of the samples after a normalization process. In a second specific embodiment of the invention, earwax is obtained by means of the extraction device of the present invention comprising: A handle (1) having a first end (2) and a second end (3), said second end (3) having coupling means which in a preferred embodiment of the invention may comprise a thread (4); A detachable head (tip) comprising a base (5) and a longitudinally extendable, elongated sponge support (6) directly dependent on an upper part of the base, wherein the lower part of the base has a housing that RQtij ηη / ηζηζ / Ε / γίΛΐ includes an internal threaded pattern (7) to receive the thread (4) of the handle (1), and in which the sponge of the support (6) has a star-shaped cross-section; An elongated sponge (8) having a longitudinal housing located in the center (not shown) having a star-shaped cross-section to receive the sponge support (6) from the base (5). The handle (1) and base (5) may include any suitable coupling means for attaching the handle, such as a pressure joint (9). The sponge (8) can preferably be made of cellulose and is glued to the sponge support (6) using a non-allergenic glue. As described above, the sponge holder (6) has a star-shaped cross-section, which enhances earwax removal while rubbing the sponge (8) inside the ear. However, its cross-section can be any suitable shape. The base (5) is wider than the handle (1) and acts as a safety brake that makes it difficult to insert the tip into the ear canal. The handle (1) is characterized by having a rotationally symmetrical shape, which allows the user to rub the sponge inside the ear by rotating it within the external auditory canal. The sponge (8) is packaged and sealed under humid conditions to maintain its softness. The humectant used is magnesium chloride (MgCl₂), which acts as an antimicrobial agent to prevent the growth of microorganisms during its storage life. Magnesium chloride not only prevents the growth of microorganisms on the sponge but also supports earwax removal. Furthermore, it has also been used to treat dermatitis (Zhai et al., 1999), which is the most common side effect of using cotton swabs (Ahmed et al., 2014). Other known antimicrobial agents may also be used. Earwax is obtained by inserting the tip with the sponge (8) into the ear and rotating the sponge (8) inside the ear canal for about 30 to 60 seconds. In the second embodiment of the invention, sample preparation is carried out by adding 500 ml of a PBS buffer solution to a 5 ml tube. The sponge is detached from its plastic support and inserted into the tube. After the sponge absorbs all the solution, it is squeezed and reabsorbed repeatedly for a period of 2 minutes or as required, and then removed from the tube. The resulting solution is then dried by displacement with N2, and the contents are resuspended in 500 ml of distilled water. The solution in the tube is stored at 4°C until further use; however, it can be cooled within another suitable temperature range. It will be obvious to a person skilled in the art that other quantities of PBS can be used. The weight-to-volume ratio is typically 1:2, so 500 ml was used. In this second specific embodiment, cortisol levels are measured using ELISA techniques, and glucose levels are measured using the SERA-PAK PLUS Kit (Bayer Healthcare) for glucose levels in earwax solution, following the manufacturer's instructions. Other suitable and known methods, such as liquid chromatography-tandem mass spectrometry (LC-MS / MS), may be used. Pilot study Earwax removal The volunteers' ears were cleaned using a Reiner Alexander syringe because, to date, it is the only viable and safe method for effectively removing earwax from the external ears (Clegg et al., 2010). This syringe is the traditional method used by medical specialists (otolaryngologists) to remove impacted earwax. Before cleaning both ears, the external auditory canal was examined using an otoscope to rule out any external ear pathology, such as impacted earwax or a perforated eardrum. Briefly, the Reiner-Alexander syringe slowly injects water at 37 degrees Celsius into the external auditory canal. The process of injecting the warm water creates a sensation of gentle pressure in the ear, which helps to loosen the earwax. The expelled water and the volume of earwax removed were collected in a kidney dish, weighed using a digital scale, and stored at 4 degrees Celsius. rq^j nn / nznz / E / YiAi Cortisol and glucose analysis using earwax samples Cortisol was extracted according to the first specific embodiment of the invention. The quantification of cortisol and glucose was also performed according to the first specific embodiment of the invention. The following pilot study was conducted in a group of healthy participants to standardize the cortisol and glucose extraction protocol for earwax samples. Glucose and cortisol results using cerumen samples (Table 11.1) Table 11.1: Pilot study of cortisol and glucose analysis using human wax samples from five volunteers (Volunteers 0-4). «ai? / nn / nznz / E / Yi Sample pg. Total cortisol in sample Dry cortisol secretion (pg / mg) Glucose measurement (mg / dl) mg total glucose in sample Dry glucose secretion (mg / mg) Volunteer 0 7251.7 127.0 16.3 815.0 14.3 Volunteer 1 4917.6............... ϊ n 8 38.5 1925 0 43(8 Volunteer 2 3450.8 862.7 3.6 180.0 45.0 Volunteer 3 3978.3 180.8 5.4 270.0 12.3 Volunteer 4 3279.8 489.5 4.2 210.0 31.3 VOLUNTEER 23564.6 O 1 +15000 pg· VOLUNTARY CORTISOL 1 + 300 pg. CORTISOL 5199.3............... — — — VOLUNTARY 2 + 300 pg. CORTISOL 3765.3 VOLUNTARY 3 + 300 pg. CORTISOL 4249.1 The effectiveness of the cortisol extraction and quantification protocol in cerumen samples compared to plasma samples As explained previously, the cortisol extraction efficiency was monitored by dissolving 0.5 mL of 300 pg / mL purified cortisol solution in PBS at pH 7, following the same extraction protocol used with earwax. The results showed an average cortisol concentration of 281.48 ± 5.16 pg / mL, corresponding to the average cortisol concentration of the three tubes (289.2 pg / mL, 273.42 pg / mL, and 281.6 pg / mL). This yields an extraction efficiency of 93.8 x 1.72%, indicating high efficacy of the cortisol extraction protocol in earwax samples. The efficiency of the extraction procedure was also evaluated by adding 300 pg of purified cortisol to the homogenized solution of dissolved earwax prior to the addition of diethyl ether (see Methods). This was done to determine whether the 15 components of earwax interfere with cortisol purification. The results are shown in Table 2, demonstrating a mean recovery rate of 99.3% of purified cortisol from cerumen wax homogenates, confirming the high efficiency of the cerumen protocol for measuring cortisol levels. Table II.2: Recovery rate of cortisol levels in cerumen samples RQtij nn / nznz / E / YiAi Dry weight of extracted cerumen (pg / mg) Recovery rate (%) of 300 pg of added cortisol Volunteer 0 127.0 108.7% Volunteer 1 44.0 94.0% Volunteer 2 4.0 105.0% Volunteer 3 22.0 90.0% Volunteer 4 6.7 99.0% The measurement procedure was also evaluated using three serum samples (participants 0-2) from a previous project, which served as a control group (Table 3). The cerumen protocol for measuring cortisol levels was confirmed to be highly reliable because when cortisol levels were measured again using the same five serum samples, but using the current cerumen protocol, the results were virtually identical (compare columns 3 and 4 in Table 3). Table II.3: Serum cortisol levels of three participants (control group) RQfr / nn / nznz / E / Yi Sample Pg. Total cortisol in the sample Original serum levels Cortisol levels (ng / ml) Same serum samples, but using the current cerumen protocol to measure cortisol levels (ng / ml) Participant 0 73364.9 70.5 71.3 Participant 1 153712.6 147.8 139.1 Participant 2 79258.5 76.2 65.4 Finally, after observing Tables 11.4 and 11.5, it can be seen that the cortisol and glucose analyses of earwax after using the Reiner-Alexander syringe were faster in measuring cortisol levels in the hair. Table 11.4: Time required to analyze cortisol and glucose using cerumen samples after using the Reiner-Alexander syringe. RQfr / nn / nznz / E / Yii PROCEDURE Quantification of analysis time Cortisol Glucose Sample centrifugation Sample drying with N2 before extraction 00:00 08:30 00:00 08:30 Sample extraction with organic solvent 02:10 02:10 Sample drying after extraction 00:40 00:40 Quantification protocol 04:00 01:00 TOTAL TIME 15:20 12:20 Table 11.5: Time and costs associated with capillary cortisol analysis and saliva samples. Biological sample Cortisol Cortisol cspiler^ Cost (per unit)8 Cost (Euro) 27.3 64.51 Hour (hours) Treatment Hour (hours) Techniques Hour 0 0:26 Incubation 0 24:00 Rotary evaporator Hour 0 3:00 Total Processing time 0 27:26 Analysis time (hours) Technical time (hours) 0:05 0:06 Centrifugation 0:25 0:25 Robot time 4:00 4:00 Total, analysis 4:30 4:31 Total time (hours) Processing + Analysis 4:30 31:57 φ: These values ​​were obtained courtesy of Bristow, M. BIOMARKER ANALYSIS LABORATORY QUOTATION AT ANGLIA RUSKIN ENTERPRISE (2017), Cambridge. Root? / nn / nznz / E / Yi It can also be seen in Table II.4 that when the Reiner-Alexander syringe is used, cerumen samples must be dried prior to glucose and cortisol level analysis, which significantly increases the total time required for sample analysis. However, the device of the present invention removes earwax without the injection of any solution. Conclusion The results of this pilot study showed that glucose and cortisol levels are detectable in human earwax samples. The time required to analyze cortisol in earwax was significantly less than the time required to analyze capillary cortisol. It was also found that the most suitable sponge for removing artificial earwax is made of cellulose. Validation study Method The participants recruited were predominantly volunteer staff and students from the Catholic University of the North (UCN) in Coquimbo, Chile, and its surrounding area. All participants were evaluated by the same clinical investigator. The sample comprised thirty-seven healthy participants; 20 were women, the mean age was 29.9 years and the average BMI was 25.6 kg / m2 All participants were recruited during the Southern Hemisphere winter (between July 6 and August 3, 2018). It has been previously demonstrated that the seasons affect the triglyceride composition of earwax (Cipriani et al., 1990). Individuals of Asian descent and those with intellectual disabilities were excluded due to the different characteristics of their earwax in terms of composition and quantity, respectively (Cipriani et al., 1990; Crandell & Roeser, 1993). Participants reported no current or past-month history of medical conditions, including ear pathologies such as impacted earwax, perforated eardrum, otitis, or metabolic disorders such as diabetes and glucose or lactose intolerance. Selected participants had not taken any medication for at least the previous month.Subjects were also excluded if they reported any illicit substance use or having been exposed to any type of severe stressor, as defined by DMS-III (Pichot, 1986) during the previous month. The validation study consisted of two interviews conducted one month apart: one during the baseline visit (Day 1) and another during follow-up (Day 30). During the baseline assessment, participants underwent a comprehensive clinical interview to rule out any medical conditions, such as ear pathologies, metabolic diseases, or psychiatric disorders. Sociodemographic data were also recorded during this assessment. Once participants were enrolled in the study, their ears were cleaned using the Reiner-Alexander syringe, which remains the safest and most practical clinical method for effectively removing earwax (Clegg et al., 2010). This syringe is the traditional method used by specialist physicians to remove impacted earwax.Participants were instructed to avoid using cotton swabs or any other ear cleaning method during the follow-up period. This allowed the research assistant to collect a standardized earwax sample thirty days later (the follow-up assessment). rq^j nn / nznz / E / YiAi The comparable amount of earwax between the right and left ear (Cipriani et al., 1986) allowed for the design of a prospective case-control study, rather than a cross-sectional study with prospective characteristics. Therefore, during the follow-up assessment, the left ear was cleaned using the Reiner-Alexander syringe [controls] and the right ear using the extraction device of the present invention [Trears]. Self-assessment of certain environmental factors during the month prior to the start of the study, such as the frequency and severity of typical daily environmental annoyances, was assessed using the Daily Annoyance Scale (Kanner & Coyne, 1981), and more unexpected environmental factors, such as significant life events, were assessed using the Recent Life Change Questionnaire (RLCQ; Miller & Rahe, 1997).Participants also assessed their perceived stress levels over the past month using the Perceived Stress Scale (PSS; Cohen, 1994). All psychometric tools were validated in Spanish. Finally, a standardized satisfaction survey was administered to evaluate participants' experience using the device of the present invention. This evaluation was conducted using the 5-point Likert scale technique (Spector, 1985). Some categorical and continuous variables, such as participants' knowledge and frequency of use of cotton swabs, were also recorded in this survey. Anthropometric variables, such as weight, height, body mass index (BMI), and waist circumference, were also recorded during this visit. General results: Results of the sociodemographic, anthropometric and self-administered questionnaire. rq^j ηη / ηζηζ / Ε / γίΛΐ Results: Table III. 1: Sociodemographic and anthropometric variables. Root? / nn / nznz / E / Yii Variable Results N: Female 20; (%) (54.1) Age (Years), 29.9, Mean (SD) (1.4) Marital status: single (yes), 32; N (%) (86.5) Postgraduate studies 16; N, (%) (43.2) Ethnic origin Mixed race, 36, n (%) (96.3) Asian race 0, n (%) (0) Alcohol (yes), 6 10, n (%) (27.0) Units' 1.3; mean, (SD) (0.5) Tobacco (yes), 9, n (%) (24.3) Contraceptive pill (yes), 9, n (%) (52.9) Intellectual developmental disorder 0; Mean (SD) (0) Medical or psychiatric comorbidity, 0, n (%) (0) Hair washing frequency 4.9, (week) (0.3) Cosmetic treatment / 1 1, n (%) Yes (2.7) Medications8, 0, n (%) (0) S: at least one unit in the past week &: any medication, including psychotropic drugs and steroids. 5: One unit of alcohol is measured as 10ml or 8g of pure alcohol. This is equivalent to a single 25ml measure of whiskey (Alcohol by Volume [ABV] 40%), or a third of a pint of beer (ABV 5-6%), or half a standard glass (175ml) of red wine (ABV 12%). Ω: dyeing, bleaching, permanent straightening, or perming YOU Table III.2: Anthropometric Results Variable Q1 Median Average (SD) Q3 Height (cm) Sample 160 167 166.7, 173 Average, , x complete (1.4) Women ------ .........Ϊ60 161Ό (1-8) Men 168 173 172.7, 176 (1-3) Weight (kg) Sample 62 72 72.5, 78 Average complete (2.5) (SD) Women 57.5 65.5 64.6, 72 (2.0) Men 72 75 81.8, 95 (3.9) BMI (Kg / m2), Sample 23.3 24.9 25.6, 26.7 Average . x complete (0.6) Women 22 8......... .........24.6 24.2. ..........25.5 (0.6) Men 24.1 25.4 27.2, 31.2 (1-1) Waist circumference (cm), r (2.4) Average Women 70.5 78 78.8, 87 (SD) (2.3) Men 88 93 94.4, 102 (3.4) BMI: Body Mass Index. Q1: First quartile, Q3: Third quartile Root? / nn / nznz / E / Yi RQfr / nn / nznz / E / Yi Table III.3: Results of the self-administered questionnaires Perceived Stress Scale (PSS) Questionnaire, Mean (SD) Life Events Score (RLCQ), Mean (SD) History of Major Life Events (RLCQ) (last month), N (%) Number of Troubles (last month), Mean (SD) Troubles Severity Index, Mean (SD) Subjects with a higher number (>25) of troubles (last month), N (%) Subjects who have trouble coping with their troubles (last month) N (%) RLCQ; Recent Life Change Questionnaire, PSS: Perceived Stress Scale Results 22.6, JUL..................................... 141.2, (20.8) 10, (27.0) 16.7, (1.7) 22.9, (2.8) 9; (24.3) 1, (2.7) Overall, Tables III.1, III.2, and III.3 show that the participants comprised a fairly homogeneous young sample, consisting mainly of 5 women (54.1%). They were also a healthy group in terms of anthropometric variables. However, this group of participants was exposed to a greater number and more severe discomforts and life events than other samples of healthy Chilean controls (Herane-Vives et al., 2018). This may explain why their perception of stress was higher than that of other samples of healthy Latinos (Cohen, 1994). Evaluation of the extraction device of the present invention fTrears©) Background: To date, no self-cleaning device is as effective and safe as the traditional clinical method for removing earwax. If such a device existed, it could be used for the safe collection of earwax samples for analysis. In this study, we evaluated the reliability, effectiveness, and user experience of the first medical device designed for the safe self-collection of earwax samples for analysis, called Trears. Methods: The external ears of 37 healthy participants without otological pathology, including impacted cerumen, were cleaned during a baseline visit using a traditional clinical method (the Reiner-Alexander syringe). The effectiveness of both methods was assessed by comparing the weights of 37 earwax samples from the right ear obtained using Trears with the weight of the same number of samples obtained from the left ear using the Reiner-Alexander syringe one month later, with both samples representing the retrospective month of cerumen secretion. The reliability of both methods was compared using Coefficients of Variation (CV). Participants also evaluated the self-cleaning experience of Trears using a standardized satisfaction survey. Results: The reliability of both methods was not significantly different (p>0.05). Trears was significantly more effective than using the Reiner-Alexander syringe to remove earwax (p<0.01). Trears tips with 50% (105.1 ml) moisture were more effective at removing earwax than Trears with 12.5% ​​(30 ml) moisture (p<0.05). Participants found using Trears safer and more comfortable than using cotton swabs. No participants reported adverse events from using Trears. Conclusion: Trears can be an equally reliable and safe, but more efficient and therefore more economical method than the clinical method for removing cerumen from the healthy ear for analysis. Introduction The results of the previous pilot study confirmed that an abrasive and absorbent cellulose sponge was highly effective in removing artificial earwax from an animal's skin (Herane-Vives & Benohr, 2018). However, its effectiveness had not yet been tested in the human ear. In this prospective case-control study design, the reliability, effectiveness, and safety of the extraction device of the present invention (Trears), which incorporates this sponge, will be evaluated. RQhj ηη / ηζηζ / Ε / γίΛΐ Earwax samples The clinical research assistant was explicitly trained in the use of the Reiner-Alexander syringe by an otolaryngologist on May 30, 2018. Before cleaning both ears, the external auditory canal was examined using an otoscope to rule out the presence of any external ear pathology, such as impacted cerumen or a perforated eardrum. Briefly, the Reiner-Alexander syringe slowly injects water at 37 degrees Celsius into the external auditory canal. The process of injecting warm water with the syringe creates a sensation of gentle pressure in the ear, which dislodges earwax. The expelled water and the volume of earwax removed were collected in a kidney dish, weighed using a digital analytical scale, and stored at 4 degrees Celsius. During the follow-up visit, participants self-cleaned their right ear using Trears, according to the manufacturer's instructions (Diagram 1). Diagram 1: Trears instructions for use. Ear Cleaning Device / í\ Warning « This product should not be used on children under 12 years of age ® This product should not be used on individuals with ear tubes * Do not use if you experience pain, discomfort, hearing loss, ear pressure, or bleeding from the ears « Trears™ should not be used in the following situations without the supervision of a physician: - Individuals suffering from impacted earwax Individuals who suffer from hearing loss or deafness due to excessive earwax production Individuals suffering from Meniere's syndrome, or any form of dizziness, including vertigo - Individuals who suffer from hearing difficulties or other ear problems - Individuals with a current ear infection - Individuals with ear tubes - Individuals who have undergone mastoid surgery • Individuals with current or previously perforated eardrums - Individuals with any external malformation of the ear / T\ Caution Please do not reuse or wash the disposable tips. The cleaning tips are for single use only (one per ear canal). Reusing the tips may cause infection or damage to the ear. If desired, you can clean the Trears™ device with warm water and dry it thoroughly before storing. ® Earwax is produced near the entrance to the ear canal, known as the external auditory canal. Do not strike or force Trears™ deeper into the ear than the safety brake allows. Forcing Trears™ beyond the limit set by the safety brake can damage the eardrum and auditory system, which may result in permanent hearing loss and / or tinnitus / pain / dizziness. "Impacted earwax should be removed by a health professional." ® Practice safe use of this product by following the step-by-step instructions. Improper use of Trears™ could cause serious injury. Read all instructions carefully before use. rq^j nn / nznz / E / YiAi Read the instructions carefully before using TREARS™ TREARS Instructions for use Please follow these instructions for safe and effective ear cleaning 5 _____________________________________________________________________________________________________ Preparation 1. Wash your hands. 2. Remove the Trears™ device and a sealed disposable tip from the box and place it on a clean surface. Warning: Do not use or reuse cleaning tips if they were not properly sealed. 3. Remove a cleaning tip from its packaging and insert it into the Trears™ device by turning it clockwise until it clicks. Cleaning 4. Carefully and gently insert Trears™ (with a disposable tip) into the ear. Do not force the cleaning tip into the ear canal. Caution: The device has a safety brake; do not insert the device into the ear canal beyond what the brake allows. 5. Clean the ear by rubbing the cleaning tip all over the inside walls of the ear for about 30 to 60 seconds. You will be able to safely clean your ear thanks to the Trears™ safety brake. rq^j nn / nznz / E / YiAi Ear cleaning device Instructions for use Please follow these instructions for safe and effective ear cleaning 5 _____________________________________________________________________________________________________ Provision 6. Remove the Trears™ cleaning tip by turning it counterclockwise. 7. Dispose of the previously used cleaning tip in any household waste container. The tip is biodegradable. Warning: Do not flush down the toilet. 8. Repeat the same procedure on the other ear. 9. Store the Trears™ device and its sealed cleaning tips in a dry place. Disposable pointed sponges were pre-moistened to varying levels of moisture using a mineral oil solution containing magnesium chloride (MgCl2). The magnesium oil used in the study was a 31% magnesium chloride solution, which is also used for massage, skin regeneration, and skin care. Due to its high magnesium content, the solution has a smooth, nourishing, and fluid texture. It does not contain natural oil but has a silky, oil-like feel. Each milliliter of magnesium oil contained approximately 103 mg of elemental magnesium. Four earwax samples were labeled, weighed, and stored at 4 degrees Celsius. All earwax samples were dried, labeled, weighed, and stored at 4 degrees Celsius. Statistical analysis The normality of the data distribution was assessed using statistical and graphical tests such as histograms. All samples were non-normally distributed (all p < 0.05). Therefore, we used an adapted version of the Coefficient of Variation (CV) to assess the reliability of both cerumen sampling methods because, while there is ample evidence for comparing normal samples, the same is not true for well-documented non-parametric data. Thus, CV comparisons were performed using the sample squared relative dispersion vector (SSRD) (supplementary appendix). However, equality between the mean SSRD of each sample is equivalent to equality with its respective CV.Linear regression analysis was used to determine the association between the volume of earwax extracted by Trears and different biological variables, or between the same volume and some of the questions in the user satisfaction survey. The significance level was set at p-0.05 (two-tailed). Supplementary appendix: Let X = (X1;...,Xn) be a sample of a random variable and let CVX2= Dxlos unbiased estimators of the mean and standard deviation, and the estimator of the coefficient of variation of the variable, respectively. „ ,. . _ ¿nCXi-X)2nCXi-X)2^ We define Dx;= ( / ' ,ΛV (nl)X2' ' (nl)X2J as the vector of the relative squared dispersion of the sample (VSSRD) and we note that if Dx is the average of VSSRD, then: CVX2= Dx Therefore, the coefficient of variation is non-negative if: X=(X1.....Xn) and Y=(Y1.....Ym) are samples of two random variables, then: CVX— CVy, only if Dx— Dy. It is possible to use tests for the significant difference between the means of Dx and DY to test the significant difference between the coefficients of variation of X and Y. Detailed results from the sociodemographic, anthropometric, and self-administered questionnaires can be found in Tables III.1, III.2, and III.3, respectively. Most participants found Trears to be very convenient, effective, and safe. They also described its use as more effective, safer, and as pleasant as using cotton swabs. Although only 14.3% would be willing to purchase this product, the majority said they might consider that option (60.7%) (Table V.1). Although the amount of baseline earwax samples extracted from the left and right ears did not differ, Trears extracted significantly more earwax than the Reiner-Alexander syringe (p<0.001) (Table IV.2). Both ears showed a significant increase in cerumen production after cleaning with the Reiner-Alexander syringe (both p<0.05). The amount of left earwax sample extracted was also significantly greater than the baseline right earwax sample (p<0.05). The results for the CVs were: Right-baseline: 51%, Left-baseline: 59%, Right-Follow-up: 71%, and Left-Follow-up: 59%. Comparisons of the CVs with the two sampling methods were not significantly different (all p>0.05); this result was reinforced by observing a similarity between the samples in terms of the relative deviation of the earwax weight of each participant. No side effects were reported from the use of either extraction method. While the thickness of the Trears tips showed no difference with respect to the amount of wax extracted, those sponges with 50% moisture or 105.1 mMgCl2 (Table IV.3) extracted more earwax than those with 12.5% ​​moisture (p<0.05) (Table IV.4). No biological or psychological variable varied the amount of cerumen secreted (all p>0.05) (Table IV.5).Although the different variations of Trears did not change participants' evaluations of their experience of use, subjects under a greater number or more severe stressors considered its effectiveness to be weaker and worse than that of cotton swabs (Table IV.6). Tables: rq^j nn / nznz / E / YiAi Tables IV.1: Trears Satisfaction Survey RQfr / nn / nznz / E / Yii Xs^fefefesfe Q1 Mediase Ofoossfefe as 1 2 w ¿Coo-s, dessrsoo ¡ss is sfefe vks*4 efe S3¡r0 ¡OSOfer SUS osdoo·1 S-fefeV feofefeíxfe δ ««Λ Si” 3C« á . 4.® I s feross 'Oef'CSÍ, 8- ro.y «ssct, ¡fe 4 , « S «Sí 2 .;Qvs san segoro xmíeeres ¿«se fue ei aso de de Sussssr»,. 5«tiy ssgoro 4 4 4.3 ó 4 iCsrossj fes fe®í®fst»s“ de· Sig^fe? 0 Sí fío 31 0, WO MS ¿Os* ^s» fessoeosío m í>U«p®s de Csxi T«KÍÍÍ5 : . ... 3ρΰε& A$gm«s ; Kars w Csssouos» éoffea 1 i 3 fe ; :¡ ;2®δ; {3.8; fe X ÍX?> ) Í31-2 égysóWdO. ai Ρίί3*ΤΜ$£$ίί5 6 ? ¿Crees <$se sí «so de >««»»* SÓSfeSO qos S! OS fe? OSSOOOS ¿S SlgOofes? 5 «sJmrwJSawesx» en >ses»so®rsfe; 5- «rtreres&oefí» deacwsf» 3 3 fefe U.45 4.5 ;feSSS SOS fe OSO 'fes yí'SSf <.. fes >· .osos d« SlgOdÓO fe exO'fefesfesfeOíóe fes ¿feásoer ¡3», fe extremsásnsente If «ssorfes < ex'tmner&mens» S O'SS.SSÜOJ OS . fe ext '•s'-^soso-oos? .fe o xSOOO SS 8 4 4 s 8 i Coso® ¿«fe-fe fe si doeo®o«^V >·'ΐ<.Χ $.\?<^ 5 4 04^ 4 W óCsse o^n¿$ ¿ss «fe ,· Os «SOSO; SO ^O3Vs'fe. fe áe«?mi»S» S feS feS^Si 4. Table IV.2: Cerumen removal comparisons between the use of the ReinerAlexander syringe and the Trears device. Root? / nn / nznz / E / Yi Left Ear Right Ear Method Reiner-Alexander Syringe (mg) Reiner-Alexander Syringe Evaluation 01 Median Average (d) 03 Median Average (sd) 03 P-value Baseline (day 0) 6.2 8.5 10.7 (1.1) 15.7 5.1 7.9 9.2 (0.7) 13.7 0.07 Method Reiner-Alexander Syringe (mg) Trials (mg) Evaluation 01 Median Average Trial 03 01 Median Average Average (sd) 03 P-value Follow-up (day 30) 12.0 173 19.1 (1.8) 19.6 79.8 124.7 155.8 (18-2) 200.3 <0.001* the p-value utíítzsoúo íe prueba t repetiría ψ: se obtener el valor p Vvlicoxon rriatched-paí?'3 íjgned-ranxs test *: =i vaior p fue íígnir kati vo en e: nive· D.D5 RQfr / nn / nznz / E / Yii Table IV.3: Trears Moisture % Volume of MgCl2 Average SD 12.5 30 0 25 56.6 5.2 30 63.7 2.5 50 105.1 5.6 Table IV.4: Analysis of the linear regression model between Trears variants and the cerumen obtained using Trears. Trears Variants Amount of cerumen obtained by Trears B Confidence Interval P-value Trears sponge moisture (%) 25 39.6 -105.9; 185.2 0.58 30 26.7 -63.7; 117.2 0.55 50 147.1 71.1; 223.2 <0.01* Trears tip thickness (mm) 4.5 19.4 -67.6; 106.5 0.65 *:p significant at p<0.05 Table IV.5: Linear regression model between the analyses between the extracted ears using Trears and some biological and psychological variables RQfr / nn / nznz / B / Yii Variables B p-value Confidence Interval Age 0.6 0.78 -3.8; 5.0 Gender 53.8 0.14 -19.0; 126.7 Alcohol (unit / ? -2.3 0.73 -16.0; 11.4 Tobacco -26.0 0.54 -112.9; 60.8 BMI 4.3 0.37 -5.41; 14.0 Waist circumference 0.8 0.53 -1.8; 3.4 Contraceptive pill 31.8 0.18 -16.5; 80.2 PSS -1.0 0.72 -6.9; 4.9 Number of complaints -0.2 0.91 -3.8; 3.4 Severity of complaints -0.6 0.58 -2.7; 1.6 RLCQ -0.1 0.51 -3.4; 0.2 Severe RLCQ'1' -17.6 0.43 -62.7; 27.6 φ: One unit of alcohol is measured as 10 ml or 8 g of pure alcohol. This is equivalent to a single 25 ml measure of whiskey (Alcohol by Volume [ABV] 40%), or one-third of a pint of beer (ABV 5–6%), or half a standard glass (175 ml) of red wine (ABV 12%). Ψ: Recent Life Events Questionnaire Table IV. 6: Linear regression models between some results of the Jígg^ satisfaction survey. And variables «dcdógkats or variants of Tregs P Questions of the Jggggj satisfaction survey How would you describe your experience with the user? How would you describe it? Do you think the effectiveness of is more effective than Trgg^? cotton swabs for cleaning your ears? Do you think the use of JM& is more common than cotton swabs? ----- Variables Volume of wax obtained per (mg) &, 5.7 p- case CS 41.5 510 &. j.3.5 T □ hO p- $.80 Cl 43.8; 34.3 & 18.7 p- AL . J... 0.29 C -253: 45.7 i 25 Gs ¢.72 1.¾ 2.0 G.4 $.6$ í -1.2; i -31 10 03 1.9 -0.2 Q.S7 -2Λ i 30 -0.7 S.1S -1-8; 0.4 -5.7 2.17 ; -1.¾ : -18 0.4 i 0.Q4* -0.1 -0.3 3.70 -2.1; 1.4 50 di M7 -1G; OS 034 -14; ΐ -37 $.5 3.23 -1,¾ 0.5 -0.1 0.99 -1.5: 1.7 ss : Tip thickness Máme?© of nt-ciessiss -0.4 ClBQ 550 $.21 3.17 -4.7; Ξ = Pl -5 . 338 : -1.1; : -32 $3 i 0Ό2 ϊ -9Λ; Γ-3.5 : -5.9 : $.65 0.02* 3.08; G.SS -7.2; -0.7 0.12 <0.9 0.70 9.51 -0.52: 0.77 -3.9; 2.C «3 Gravedad de !ss -w ü.7S -S.2; £.2 -73 0.03 ; -15.1; i -5.8 * 0..S $..G4- 11.1; 0.1 -14 0.58 -6.4; 3.6 1 1 r-íúrneyo iv^sles se íes »tfSF:tOE vitales 15.2 <ίώ 1 £.43 ¡133 74. S 36.2 -03; 0.4 18.5 -0.3 0.53 ; -73.2; ; - ί 42 0 : 28 4 0.20 -0.7; Í -0.6 $.1 i $.LS 6.53' 74.2: 15.3 -ΪΊ; -$.4 -7.5 -0.2 0.69 0.57 -484; 31.4 -6.9; 03. rq^j ηη / ηζηζ / Ε / γίΛΐ Esíres -aS: 43 £3 ¿Ccn qué -G.4; 3.G •áe 3.35 i · -03 Ϊ 03 í RQfr / nn / nznz / E / YiA Discusión We found that cerumen production increased significantly after baseline cleaning with the Reiner-Alexander syringe in both ears. However, Trears removed more earwax than the clinical method at the follow-up visit. Regardless of the sampling method used and the amount of cerumen obtained, the volume obtained relative to the sample average did not vary significantly among participants. The majority of participants (10) found the use of Trears to be comfortable, effective, and safe. They also considered its use to be more effective, safe, and as comfortable as using cotton swabs. It is noteworthy that, regardless of the extraction method used, the amount of earwax, instead of decreasing, increased significantly during the 15-month follow-up period. This could be explained by the ceruminous glands increasing their earwax production as a compensatory mechanism after the reduced earwax production resulting from the baseline cleaning of both ears. This difference can also be understood in light of the specific characteristics of our study protocol. We instructed participants to avoid cleaning their ears during the 20-month follow-up period in order to extract a standardized earwax sample representing the retrospective month of earwax production. This possibility was reinforced by the observation that 35.7% of the study participants are very frequent users of cotton swabs, and another 28.6% use them at least sporadically. Warm water injection using the Reiner-Alexander syringe extracted slightly more earwax (2.25 ± 0.18 mg / week) than the extraction method used by Cipriani et al. (1986), which combined the effect of an unspecified mechanical extraction method and a 3:1 v / v alcohol / ether solution (2.02 ± 0.22 mg / week). We also confirmed the results of Cipriani et al. from 1986 regarding the lack of significant variation in the volume of earwax produced by both ears after comparing baseline weights. However, the baseline earwax volume did not reflect the amount of earwax secreted one month later, as Trears extracted more than eight times more earwax than the syringe from the ears of healthy, primarily mixed-race individuals. The use of cotton swabs is expected to continue increasing. Therefore, developing a safe and standardized alternative for self-collecting earwax is a real necessity. Unfortunately, all current self-cleaning products or devices show little to no effect compared to various clinical methods. However, Trears proved to be not only as effective as the traditional clinical method for removing earwax from healthy ears, but also capable of extracting more earwax. The material of the Trears tips, made of abrasive and absorbent cellulose sponge, had already shown promise in removing artificial liquid earwax from a piece of animal skin. Furthermore, the Trears tips incorporated a mineral oil that had already demonstrated some earwax removal effectiveness.We also confirmed that a 50% mineral oil concentration significantly increased the amount of earwax extracted by Trears, compared to the same devices using less moist tips (12.5%). Therefore, we should produce Trears with that moisture percentage. It is thus possible to speculate that the additive effect of the Trears tip material plus the mineral oil could explain our results. Trears proved to be as reliable as the most popular clinical extraction method. This is because the variability in earwax weight (distance relative to the mean) was similar between the clinical and self-extraction methods (Inter-Reliability) (p-0.20). This result indicates that the new device is indeed a consistent method, since the comparator, or reference device, also proved to be consistent in sampling cerumen from healthy ears when the VRSDs obtained using the same clinical method (Intra-Reliability) were compared with each other (p-0.85). Measurement of the average glucose concentration from earwax Background: An increase in the long-term average glucose concentration is associated with epidemic and chronic diseases. Currently, no single test is accurate, affordable, convenient, and safe for chronically measuring glucose levels. Earwax may meet these criteria. The applicant associated fasting and baseline glucose levels with earwax samples and glycated hemoglobin (HbA1c). Methods: 37 healthy participants provided two right earwax samples and two serum samples, collected one month apart. Baseline samples were obtained after an 8-hour fast, and follow-up samples after a standard caloric intake. While baseline earwax glucose concentration (Baseline-EGC) represented the average of previous fasting and postprandial glucose levels over an unspecified time, follow-up EGC (Follow-EGC) represented the same average, but only from the previous month. Both HbA1c samples represented the retrospective average glucose concentration over a period of one to three months. Average plasma glucose (glycemic) was calculated as the average of baseline and follow-up levels. Baseline and follow-up levels were compared for each sample. The effect of multiple co-occurring factors was investigated in these samples.Fasting serum glucose (FSG) was correlated with its respective baseline CGE and HbA1c samples. The same correlation was performed between postprandial serum glucose (PSG) and its respective follow-up CGE and HbA1c samples. The average glycemic level was correlated with all the aforementioned HbA1c and CGE samples. Different glycemic levels were predicted using the baseline and follow-up CGE and HbA1c samples. Results: All follow-up sample concentrations were higher than their respective baseline concentrations. Cerumen samples were not affected by any covariate. While all associations between EGC and glycemic levels showed high positive correlations (all the RQhj nn / nznz / E / YiAi (R>0.60; p<0.001), the associations of HbA1c with different glycemic levels showed only moderate or weak correlations (all R<0.50; 0.10>p>0.01). Baseline ECG predicted the greatest increase in mean glycemic levels (p<0.001). Conclusion: Earwax is more accurate than HbA1c in reflecting glycemic levels. Earwax is more stable than a single blood sugar sample and than HbA1c for measuring long-term glucose concentration. Follow-up earwax results suggest that earwax is a better sample than HbA1c for predicting long-term (one-month) average glycemic levels. Introduction Although glucose levels have been measured in earwax samples in other studies (Masuda et al., 1978; Shichjo & Masuda, 1979; Herane-Vives & Benohr, 2018), including in diabetic patients (Khasanov & Popova, 1984), it was unknown whether the glucose concentration (GC) found in those earlier studies accurately reflected glycemic levels. Therefore, in this study, we measured GC, glycemic, and HbA1c levels during fasting and after a standardized meal in a sample of healthy participants. Baseline and follow-up levels were compared between the two samples. The effect of several covariates on glucose levels was also investigated. Fasting serum glucose [FSG] was correlated with their respective baseline EGC and HbAic levels, and the same correlation was made between postprandial serum glucose [PSG] and their respective follow-up EGC and HbAic levels.The average glucose level correlated with all the aforementioned HbA1c and CGE samples. Different glycemic levels were predicted using baseline samples and follow-up CGE and HbA1c. We hypothesized that: 1) All follow-up concentrations would be higher than their respective baseline concentrations; 2) Earwax glucose levels would represent a more stable level than blood glucose and HbA1c in order to reflect the average systemic glucose concentration; 3) All associations between EGC and different blood glucose levels would be stronger than the associations between RQtij nn / nznz / E / YiAi HbA1c and the same glycemic measures; and 4) the baseline EGC would predict the greatest increase in the average glycemic levels. RQtoj ηη / ηζηζ / Ε / γίΛΐ Methods: Healthy participants provided two samples of right earwax and two serum samples, collected one month apart. Baseline measurements were taken after an 8-hour fast, and follow-up samples were taken after the ingestion of a standardized meal. Although the retrospective time period of glucose accumulation in the earwax of the baseline sample was unknown, the follow-up sample covered the last month of accumulation. Both HbA1c samples represented the retrospective average glucose concentration over a period of one to three months. The average glucose level was calculated as the average of baseline and follow-up glucose levels. Baseline and follow-up levels were compared between each sample. The effect of several covariates was investigated in these biological samples.Fasting serum glucose (FSG) correlated with its respective baseline CGE and HbA1c levels, as did postprandial serum glucose (PSG) with its respective follow-up CGE and HbA1c levels. The mean glycemic level correlated with all the aforementioned HbA1c and CGE measurements. Earwax samples: The baseline right earwax sample was taken after an 8-hour fast. The follow-up right earwax sample was collected two hours after the start of ingestion of a standardized 236 ml liquid meal of Ensure Avance®. All samples were labeled and stored at 4 degrees Celsius. Earwax samples using the Reiner-Alexander syringe. Earwax samples obtained by ear irrigation were dried using the nitrogen displacement method. Specifically, each 50 ml sample was divided into four tubes, each fitted with a cannula connected to a nitrogen gas tank. A constant temperature of 25°C was maintained using a temperature-controlled water bath. When the nitrogen flow was opened, it displaced the evaporating water, allowing the sample to dry. Once the baseline samples, as well as the remaining sample obtained one month later, were dried, the weights of the dried earwax samples were determined by subtracting the weight of the empty tube (previously weighed) from the weight of the tube containing the dried sample. Finally, 125 ml of PBS was added to each tube, the contents of each tube were resuspended, and the samples were combined into a single 5 ml tube containing the total resuspended sample in 500 ml of PBS. This tube was stored at 4°C until use. Earwax samples with Trears Earwax samples obtained using the TREARS® instrument were processed by washing the sponge with 500 mL of PBS for 2 minutes. However, the N2 drying time was much shorter because the TREARS extraction mechanism does not inject any liquid solution, unlike the Reiner-Alexander syringe. Specifically, 500 mL of PBS buffer solution was added to a 5 mL tube. The sponge was detached from its plastic holder and inserted into the tube. After the sponge absorbed all the solution, it was repeatedly squeezed and reabsorbed for 2 minutes before being removed from the tube. The resulting solution was then dried by N2 displacement, and the contents were resuspended in 500 mL of distilled water. The solution in the tube was stored at 4°C until analysis. Serum samples Fasting blood samples were drawn using a 3 mL syringe from the antecubital vein and a blood collection tube without anticoagulant. All serum samples were collected in the morning. A functional blood glucose (FBG) sample was collected in the morning of the baseline visit. Participants were instructed to refrain from eating or drinking for eight hours prior to this assessment. A follow-up FBG sample was collected during the subsequent visit. These follow-up samples were taken two hours after the start of ingestion of a standardized 236 mL liquid meal of Ensure Avance®. RQtoj nn / nznz / E / YiAi containing 1.5 kcal / ml, given by 24.3% protein, 44.8% carbohydrate, 28.8% fat, 1% fiber, and 1.1% Beta-Hydroxy-Beta-Methyl. The average serum glucose concentration was estimated from the average between fasting and postprandial serum glucose levels. Serum glucose analysis: Blood samples obtained under fasting and postprandial conditions were stored at 4°C for 24 hours to allow coagulation and serum separation. They were then centrifuged at 1000 x g for 20 minutes at 4°C. Subsequently, the serum was separated from the pellet using a 1 ml syringe and collected in 2 ml labeled plastic tubes. Once obtained, the serum samples were stored at -20°C until further analysis. Quantification of glucose from cerumen and serum samples: The amount of glucose was quantified using enzymatic oxidation assays and labeling of oxidized glucose in 96-well plates, according to the instructions provided by the supplier (BioVision Inc., Milpitas, CA, USA). A standard curve of 0, 2, 4, 6, 8, and 10 nmol of glucose standard per well was used. Fifty milliliters of standards, aqueous fraction, and a 1:25 dilution of serum were added to the remaining wells along with 50 mL of glucose mixture, containing glucose assay buffer, 2 mL of glucose probe, and 2 mL of enzymatic glucose mixture. The mixture was incubated for 30 min at 37°C, protected from light, immediately after measuring the absorbance at 570 nm using a microplate reader (NovoStar). The absorbance of the standard curve was fitted to a line equation and the glucose content was calculated by interpolation within the fitted curve. Quantification of glycosylated hemoglobin from serum samples:The amount of glycosylated hemoglobin (HbA1c) was quantified using the sandwich ELISA method, according to the instructions provided by its supplier (Abbexa Ltd., Cambridge, UK), using a standard curve of 0.30125, 6.25, 12.5, 25, 50, 100, and 200 ng / mL of HbA1c standard. Standards and 100 mL of undiluted sera were added to the wells of an HbA1c-coated plate and incubated for 90 minutes at 37°C with shaking. After discarding the contents and washing twice with the wash solution, 100 mL of a biotin-conjugated HbA1c detection antibody was added. They were then incubated for 60 minutes at 37°C with shaking. After discarding the contents and washing 3 times with wash buffer, 100 ml of a Strepatavidin-Rabinite Peroxidase (HRP) conjugated solution was added to each well and incubated for 30 minutes at 37°C with shaking.After discarding the contents and washing five times with wash buffer, 90 ml of TMB substrate were added to each well and incubated in the dark at 37°C for 20 minutes. Finally, 50 ml of reaction solution were added to each well, and the absorbance at 450 nm was measured using a microplate reader (NovoStar). The absorbance of the standard curve was fitted to a straight line, and the sample absorbances were interpolated on this curve. Statistical analysis Data were checked for normality using the Kolmogorov-Smirnov test and graphical methods. All values ​​were normally distributed (all p > 0.05). Therefore, repeated t-tests were used to compare baseline and follow-up glucose levels using different samples. Linear regression analysis was used to predict different glycemic levels using CGE and HbA1c samples and their association with various biological and psychological variables. Pearson correlations were used to determine the associations between baseline and follow-up CGE and different glycemic levels, or between baseline and follow-up HbA1c and different glycemic levels. Cohen's criteria were used for correlations: weak (0.1–0.3), moderate (0.3–0.5), and strong (0.5–1.0) (J. Cohen, 2013). The time required to analyze each specimen was also recorded.The significance level was set at p-0.05 (two-tailed). Results Detailed results from the sociodemographic, anthropometric, and self-administered questionnaires can be found in Tables III.1, III.2, and III.3, respectively. The use of Trears significantly decreased the time required to measure glucose in earwax compared to the time required when using the Reiner-Alexander syringe (compare Tables III.4 and V.1). All follow-up concentrations were higher than their respective baseline concentrations (Table V.2). Earwax samples proved to be more stable than HbA1c or glucose levels, as their glucose concentration was not affected by any covariate (Table V.3). Age had a direct effect on the baseline HbA1c sample. Furthermore, while a greater number of years of education increased follow-up HbA1c and PSG levels, smoking decreased FSG and PSG levels (Table V.3).While all associations between EGC and glycemic levels showed high positive correlations (all R>0.60; p<0.001), associations of HbA1c with different glycemic levels showed moderate or weak correlations (all R<0.50; 0.10 <p<0.01) (Figuras 5, 6, 7 y 8). La muestra de seguimiento de HbA-ic mostró una correlación más fuerte con FSG (R-0,48, p<0,001) que la asociación entre la basal de HbAic y FSG (R-0,43, p<0,001) (Figuras 6 y 8). Ambas asociaciones de EGC fueron más fuertes que las observadas cuando HbAic se asoció con los mismos niveles de glucosa sérica. Sin embargo, la muestra de EGC de seguimiento mostró una asociación más fuerte con PSG (R-0,90, p<0,001) (Figura 7), que la Basal-EGC con niveles FSG (R-71 p<0.001) (Figura 5).The average glycemic level also showed a stronger association with earwax, rather than with HbA1c samples (see Figures 9, 10 and 11, 12) and, among them, the postprandial cerumen sample also showed a stronger association with the average glycemic level (R-0.84, p<0.001) (Figure 11). rq^j ηη / ηζηζ / Ε / γίΛΐ Table V.1: Time required to analyze different specimens PROCEDURE Quantification Time Cortisol in ear wash Cortisol using TEARS Glucose in ear wash Glucose using TEARS Cortisol in serum HbAlc in serum Glucose in serum Sample centrifugation 00:00:00 00:00:00 00:00:00 00:00:00 00:20:00 00:20:00 00:20:00 Sample drying with N2 prior to extraction 08:30:00 00:47:00 08:30:00 00:47:00 00:00:00 00:00:00 00:00:00 Sample extraction with organic solvent 02:10:00 02:10:00 02:10:00 02:10:00 00:00:00 00:00:00 00:00:00 Post-extraction sample drying 00:40:00 00:40:00 00:40:00 00:40:00 00:00:00 00:00:00 00:00:00 Quantification protocol 04:00:00 04:00:00 01:00:00 01:00:00 04:00:00 04:00:00 01:00:00 TOTAL TIME 15:20:00 07:37:00 12:20:00 04:37:00 04:20:00 04:20:00 01:20:00 RQfr / nn / nznz / E / Yi Table V.2: Comparadones is the time required by i» basic samples and se segoímler. t® ds giscosa, HbAXc and EGC. Been Right Right Pife Time of 1» morning.. 841: i month sO.65 00:50 00::44 Teacher 8asal-£GC inmai / l; S»SMS-EG£ úimol / l} Q1 MetSs'i Mean .......................ί.&Λ.......... Q3 Q1 Median i Mean .......................Lfeál.......... Q3 p-tíir Results 60.5 755 176.7: 1461 ¢2..5 81.5 81.9: 92.7 ; (2.91 ii.5 <&sr Maestra Basal-HbAíC hg / b Follow-up- HhA:c hg / b Crl Median : Mean £8 01 Median i Mean Q3 Restiftados 43.5 654 ; 654: 4.3 31.2 524 74.6 7.5 4.5: 51 -5. Samples FSG i|WO; / l; FSG ínmol / O Q1 Median : Mean Q3 01 Median : Mean : (ti Q3 p-fef Highlights 3.2 4.5 :43; íG.3 i 5.2 4.9 54 15.4: i Í3.25 6.6 -sO.GVV «s: the wlor e was obtained utiiartóo 1 repeated t test. *: ei value p ive signiíkativs is the 0.05 level. HbA.c femoglofea jlirasuada. í-SG: Fasting serum glycolysis P5G: Postprandial serum glucose. Tab V3: Regression models- of! e^re see» cwssbés * months»es of kissing and following h will be of the ofe, turned and HbAk. 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Consult figures 5, 6, 7, 8, 9, 10, 11 and 12 Raí? / nn / nznz / E / Yi RQfr / ηη / ηζηζ / Ε / γΐΛ Táia V.4: Mees íes de «giBiási lina I éare los. test levels, monitoring and medical monitoring of ECG and HbAk a® f SG, KG and ei level giucémks pemeb Samples of cersímeny Mustias of Glkma BG PSG ^el glüc&nko pío; neda f Λ vf ' P-®, G BíS^ESC (nsiíífll 12.4 74; 17.4 9.5 mr 1.3' 17.8 132 <aoor 65:1 δ S 8.4 taz 54; 113 12.1; 17,0 12Λ 16:15.1 Etó-HbAkWI Q j <0.01 2.5: 15.8 m : <0,01 34:20,1 11.7 <0Ό1Λ 4.0:194 SepñÉñts-HbAkM 5.8 Q $7 <4; 12.1 12,0 : <o.cr 4.119,5 15 sor 2.2:10.7 EGC Easss: ΰή«= Onwssihi, -S Skis gusai So^^si ΐκ» Soase HbA-¿ Sjsík hBüsgo&g. Tht ¡isas yes £GC G^aemk ®d HbAk ss??s:al®ss site sssn iOiR 'tel' ______________________________________________________________________________________ Discussion All follow-up concentrations, using glucose, HbA1c, and EGC samples, were found to be significantly stronger than their respective baseline concentrations, confirming that the study was conducted appropriately. Earwax was a more stable sample than HbA1c and glucose samples, as its glucose levels were not affected by any covariate. While all associations between EGC and glucose levels showed high positive correlation coefficients (all R > 0.60; p < 0.001), the associations of HbA1c between both glucose levels showed only moderate or weak correlations (all R < 0.50; p < 0.10). <p<0.01). tanto las medidas de seguimiento egc como hbaic, mostraron que la correlación más fuerte con psg fue encontrada entre el y (r-0,90, p<0,001 r-0,48, p<0.01, respectivamente). el mayor aumento los niveles glucémicos se predijo basal en hbaic (r-13,2, p<0,001; r11,7, p<0,01, respectivamente).Previous findings corroborated that the strength of the association between HbA1c and postprandial or fasting glucose levels is modest in healthy individuals (van 't Riet et al., 2010). This may be explained by the moderate sample size, or because HbA1c typically shows a stronger association in individuals with elevated glucose levels, such as those observed in diabetic patients (van 't Riet et al., 2010). It is important to note that the time period for which the baseline earwax glucose (EGG) is measured is not entirely comparable with the follow-up sample, as they represent different periods of glucose accumulation in the earwax. In fact, the study design only allowed us to standardize the amount of earwax secreted from the follow-up samples after conducting a baseline ear cleaning. It is possible that some previous episodes of intense physical activity or stressful events prior to enrollment in this study may have momentarily increased the baseline earwax glucose levels that were accumulated in that sample. In this sense, the baseline-EGG sample may represent the long-term accumulation of several fasting episodes and postprandial glycemic levels.However, the fact that the greatest increase in average glucose levels was preceded by the baseline HbA1c measurement, as well as by baseline EGC, suggests that EGC better represents the average glucose concentration, rather than its fasting or postprandial concentration. It is well known that HbA1c is primarily influenced by fasting glucose (FSG), rather than polysomnography (PSG), because people spend more time fasting than eating during a 24-hour day (Monnier et al., 2006). In fact, baseline EGC may also be influenced by fasting glucose levels, as this glucose concentration was lower than in the earwax sample taken one month later (p<0.01). Earwax was undoubtedly a better indicator of serum glucose levels than HbA1c. This is not only because all correlations between CGE and glycemic levels were much stronger than the correlation coefficients observed between HbA1c and blood sugar levels, but also because the correlation between follow-up CGE and PSG showed a stronger association (R=0.90; p<0.001) than the relationship between baseline CGE and FSG (R=0.71; p<0.001). Therefore, a baseline CGE that exclusively covers a fasting period may exhibit an even stronger association than the correlation observed between follow-up CGE and different glycemic levels. Previous results indicating that HbA1c levels are affected by age were corroborated. HbA1c was also affected by the level of education. It is very likely that the type of work performed can explain this. It has been shown that jobs requiring a high level of education are also associated with an increase in working hours (Uehata, 1991), which, in turn, are also associated with an increase in HbA1c (Azami et al., 2018). We also verified previous results indicating that smoking decreases FSG and PSG. Conversely, earwax was also a more stable sample, as its glucose levels were not affected by any covariate. Our results suggest that EGC should begin to be measured in diabetic and obese patients. Some earlier studies have used the area under the curve, rather than the mean between fasting and postprandial glucose levels, to estimate average glucose concentration (Avignon et al., 1997). However, the mean between fasting and postprandial glucose levels has also proven to be a very accurate index. In fact, Svendson and colleagues found that average glucose levels derived from approximately 2 to 300 measurements in each of 18 patients with type 1 diabetes correlated almost perfectly (R-0.96) with HbA1c (Aaby Svendsen et al., 1982). Ozmen et al. found that average plasma glucose levels derived from fasting and postprandial plasma glucose levels also correlate strongly with HbA1c in patients with type 2 diabetes (Ozmen et al., 2006).Recently, the average of postprandial and fasting glucose levels has also been used in women with gestational diabetes mellitus (Koren et al., 2016). Therefore, this index may correlate even better with average glucose levels in healthy individuals, since their 24-hour blood glucose levels vary much less than in diabetic patients (Praet et al., 2006). Interindividual differences related to participants' ability to absorb different food components can also affect their glucose levels (Freckmann et al., 2007). This is why some studies use the glucose tolerance test after ingestion of 75 g of glucose, instead of postprandial levels after a standardized meal (Ensure) containing various nutrients, such as proteins, lipids, and glucose, which have different absorption rates, thus varying the final PSG level. However, we used a widely used test: the delivery of a liquid meal that is easily absorbed. Furthermore, participants with food allergies, such as lactose intolerance, were excluded. Regarding the differences in blood glucose levels between plasma and serum, some studies report that plasma glucose is higher than serum glucose, while other studies find no difference. However, serum glucose measurement is not recommended for diagnosing diabetes (American Diabetes Association, 2010). We did not use FSG or PSG levels for any type of diagnosis; we simply recruited a sample of healthy participants to investigate their glucose levels using different biological samples. In conclusion, earwax is more accurate than glycemic and HbA1c samples in reflecting average glucose levels, as it was a more stable sample and its glucose concentration better predicted the average glucose concentration, rather than its fasting or postprandial levels. Measurement of cortisol levels using earwax samples Background: The diagnosis of depression is considered unreliable. This may be explained by the great heterogeneity of this syndrome. An accurate biomarker could improve the consistency of this diagnosis. Cortisol levels have been commonly measured in depression, reflecting the frequency of this neurobiological disorder. However, cortisol results using short-term samples have been highly divergent, given the reactive secretion profile of this hormone. Therefore, these short-term samples are inappropriate for reflecting average cortisol concentrations, given the large number of acute influences that can affect cortisol levels in these samples. Hair cortisol concentration (HCC) has been documented as accurately reflecting long-term cortisol levels, as it accumulates the hormone over extended periods. However, its widespread use seems unrealistic.Furthermore, it is not entirely clear whether some acute influences do affect HCC. Cortisol concentration (ECC) may be a more convenient and accurate measure to reflect long-term average cortisol concentration. RQhj nn / nznz / E / YiAi Methods: The ears of 37 healthy participants were cleaned during a baseline visit. One month later, the ECC was analyzed from the participants' right ear. During that follow-up visit, participants also provided 1 cm of hair, representing the retrospective month of capillary cortisol accumulation. ECC and HCC were compared and correlated. Results: The ECC was significantly higher than the HCC (P<0.001). ECC and HCC showed a significant moderate positive association (R-0.39; p-0.03). While men had a higher HCC than women (p<0.001), ECC was not affected by gender. Conclusion: The ECC can be another sample that accurately reflects the average cortisol concentration. Compared to hair, earwax accumulates higher cortisol concentrations. Earwax was also a more stable sample, as its cortisol levels were not affected by any covariate. Ultimately, the time required to analyze the ECC was significantly less than the time required to analyze the HCC. Objectives and hypotheses: Although we recently found that this hormone is detectable in this secretion (Herane-Vives & Benohr, 2018), we did not know if the level found represented the average systemic concentration of the hormone. Therefore, in this study, we correlated earwax cortisol concentration (ECC) and hair cortisol (HCC) in a sample of 37 healthy participants. We hypothesized that: 1) The time required to analyze ECC would be less than the time required to analyze HCC, 2) ECC would be higher than HCC, 3) ECC and HCC would be positively correlated with each other, and 4) Common short-term cortisol covariates for hair would not affect ECC. Methods: We compared and cortisol levels obtained from the right follow-up earwax sample and a 1-cm hair sample obtained during the same visit. Both samples represented the retrospective month of cortisol accumulation. The effect of several covariates was investigated in these samples. The time required to analyze the ECC was also recorded. Earwax sample with Trears: (see diagram 1 in the instructions) Cortisol analysis of earwax: Purification of cortisol from earwax: After obtaining earwax samples using the TEARS® device, the samples were resuspended in 500 mL of PBS and homogenized using a 1 mL syringe. Then, 500 mL of diethyl ether was added, and each sample was vortexed for 1 minute and then incubated at -20°C for 2 hours. After this time, the liquid fraction of each sample (organic fraction) was transferred to a new, properly labeled 5 mL tube and dried using the N2 displacement method described above. Once dry, the samples were resuspended in 500 mL of PBS, and cortisol was quantified. Separately, the aqueous fraction remaining after diethyl ether extraction was used to quantify glucose levels in the earwax. Quantification of cortisol from cerumen samples: The amount of cortisol was quantified using ELISA, according to the instructions provided by the supplier (Enzo Life Sciences, Farmingdale, NY, USA). Cortisol levels were quantified using competitive colorimetric ELISA techniques, employing a standard curve of 0, 156, 313, 625, 1250, 2500, 5000, and 10000 pg / mL of standard cortisol. One hundred milliliters of standard solutions, organic sample fractions, and serum dilutions were added to the wells of a plate coated with an anti-mouse antibody. In addition to the aforementioned solutions, 50 µL of a blue conjugate containing cortisol covalently bound to alkaline phosphatase and 50 µL of a mouse monoclonal antibody against cortisol were added to all wells.Once the antibody was added, the wells were incubated for 2 hours with shaking to allow the cortisol present in the samples / standards to compete with the cortisol in the antibody-conjugated sample. This conjugated antibody remained bound to the well through its interaction with the antibody. The second antibody adhered to the walls of the wells. After 2 hours, the wells were thoroughly washed, and 200 ml of para-nitrophenyl phosphate (pNpp) were added to each well and incubated for 1 hour without shaking. This allowed the para-nitrophenyl phosphate to be transformed by an alkaline phosphatase-mediated enzymatic reaction into para-nitrophenol, producing a color inversely proportional to the amount of cortisol present. Finally, 50 ml of a solution to stop the enzymatic reaction was added to each well.The plate is read at 405 nm using a microplate reader (NovoStar). The absorbance of the standard curve is fitted to a 4-point logistic curve, and the sample absorbances are interpolated on this curve, yielding the sample concentration in pg / mL. To calculate the pg / mg of cerumen, the concentration is multiplied by the sample volume (500 µL) and divided by the weight of the dry sample. To calculate the serum concentration, the concentration is multiplied by the dilution factor. Hair samples. A physician trained in sample collection cut the hair of all participants. The presence and frequency of any confounding variables and / or procedures that could affect hair cortisol levels were measured, including cosmetic treatments (dyeing, bleaching, permanent straightening) and hair washing frequency. Hair samples were taken from the vertex at the back of the head and cut with clean scissors as close to the scalp as possible. For this study, four strands of hair were required from different locations at the back vertex, each approximately 1 centimeter thick. In the laboratory, 1 cm of hair was cut from each strand, measured from the end to the scalp surface, representing approximately one month of hair growth equivalent to the retrospective cortisol production level of the past month.The total weight of the four 1 cm segments from each hair strand is approximately equivalent to 25–50 mg of hair. Once collected, the hair samples were stored at room temperature in the dark in a sealed container. Capillary cortisol analysis. Prior to analysis, hair samples were washed in 1 mL of isopropanol to remove external contaminants. The isopropanol was then removed from the vial, and the hair was dried at room temperature for 48 hours. Once completely dry, five ceramic beads were added to each tube, and the hair samples were ground to a powder using the MPbio Fast Prep machine (MP Biomedicals, LLC). To extract cortisol, 1.75 mL of methanol was added to each sample, and the samples were incubated for 20 hours with continuous rotation. Hair, methanol, and ceramic beads were decanted into a polypropylene tube (Sarstedt AG & Co, Germany) that separated the ceramic beads from the rest of the mixture. The tube was centrifuged at 3000 RCF to separate the ground hair from the methanol, and 1.25 mL of the pure methanol supernatant was decanted into a 2 mL polypropylene cryovial. The methanol was removed using a vacuum centrifuge (Sean Speed ​​40, Labgene), and the tubes were frozen at -80°C until needed for the cortisol ELISA. Cortisol levels were determined using a commercially available competitive ELISA (Salimetrics LLC, USA). Samples were thawed and reconstituted with 0.125 mL of cortisol assay diluent and tested according to the manufacturer's protocol. Results are expressed as picograms of cortisol per milligram of hair.All hair samples were analyzed at the Biomarker Laboratory of Anglia Ruskin, Cambridge, UK (www.anqlia.ac.uk) (Albermann & Musshoff, 2012). www.anqlia.ac.uk. Statistical analysis The normality of the data distribution was assessed using the Kolmogorov-Smirnov test and graphical methods, such as histograms. Both ECC and HCC values ​​were normally distributed. Therefore, we used a paired-samples t-test to compare ECC and HCC. Pearson correlations were used to determine the association between HCC and ECC. Cohen's criteria were used for correlations (J. Cohen, 2013). Linear regression analysis was used to determine the association between cortisol concentration and various biological and psychological variables. The time required to analyze the ECC was also recorded. The significance level was set at p = 0.05 (two-tailed). Results Detailed results from the sociodemographic, anthropometric, and self-administered questionnaires can be found in Tables III.1, III.2, and III.3, respectively. The time required to analyze ECC using both Trears and rcm nn / nznz / E / YiAi with the Reiner-Alexander syringe was compared (compare Tables II.4 and VI.1). The time required to analyze ECC using Trears was more than four times shorter than the time required to analyze HCC (Table VI.1). ECC was significantly higher than HCC (Table VI.2). While men had higher HCC compared to women (P<0.001) (Table VI.3), gender did not affect ECC. ECC showed a moderate positive correlation with HCC (R=0.39, p=0.03) (Figure 13). RQfr / nn / nznz / E / Yi Table VI.1: Time required to analyze the ECC Quantification time (hours) Cortisol Process using TEARS Hair Process* Sample Centrifugation 00:00 Techniques 0:26 Sample Drying with N2 before Extraction 00:47 Incubation 24:00 Sample Extraction with Organic Solvent 02:10 Rotary Evaporator 3:00 Sample Drying after Extraction 00:40 Total Processing Time 27:26 Quantification Protocol 04:00 Technical Time (Hours) 0:06 Centrifugation 0:25 Robot Time 4:00 Total Analysis 4:31 FOTAL TIME 07:3731:57 &: These values ​​were obtained courtesy of Bristow, M. BIOMÁRKÉR ANÁLYSIS LABORATORY QUOTATION ATANGLIA RUSKIN ENTERPRISE (2017), Cambridge. RQfr / ηη / ηζηζ / Ε / γΐΛ Table VI.2: Comparisons between HCC and ECC -- (pg / mg) (pg / mg) P-value Q1 Median Average Q3 Q1 Median Average Q3 79.8 124.7 137.8; 200.3 7.6 9.7 9.7; 10.9 <0.001* | (20.8) (0.7) ECC: Cortisol concentration in cerumen; HCC: Cortisol concentration in hair *: P-value significant at 0.05 Q1: First quartile, Q3: Third quartile T&b VL3: Linear regression model between covariates and HCC and between covariates and ECC Variables ECC ípíMW HCC <pa. mq) p-vaíue a p p-vakie edad 48 0.78 -3.8 0.1 048 -at 9.-2: género 53.8 0,14 -19,0; 125,7 3.7 <0.301' % 5.3 afcotoá (unidad}ο -23 0,73 -18.q; 11.4 o.so -0.4: 3,6 tabaco -26.0 0,54 -112.5: 80,8 3.0 q.as -5..¾ bmi íkq otvi 4.3 0,37 -5.4; 14.0 0,3 0.12 -ai; a? cicunterefícia de cintura (cmj 08 0.s3 -1.8 3.4 07 0,17 <-0.2 02 pastüea antkonceptwb -8..6 0.82 -87,8; 70,8 <9.1 0.98 mí lavado peto 13.1 o.:9 -7.9; 38.1 as 0j5 -az.: 1,5 tratamiento cosmético si.4 0.47 147.9; 310,7 -6.3 0.87 7.5 p55 -133 0,72 -6.9; <-0.1 85 quinera -02 0.93 -3.8; <-0,1 0,50 -az; ai gravedad eas modestias -<16 0.58 -2 7: 1.8 -sj; número rlcq -ai 0,5' -0.4; 0.2 0.88 < "g: ' severidad -17.5 043 <-ai -1.7; 1.7RQfr / ηη / ηζηζ / Ε / γΐΛ PS5: fscsS ds tsrs i en psri bfe. RiCQ; cuesianang fe «ventas fe vida --fe-?.:¿iss unidad fe fefe; if you want to talk about it.. ES» «©¿vate 3 isa cedida ¿asa fe 25 «si de stisfc? (Akchot «5 ííSiüisen [Wj ACSS), □ üs teres fe sss pinte de serves ¡ASV :5-6¾) ® medium sepe ggándsr (275 rfe da· vina tasa j&sv 3¾ gs swaradon as? estafe da gcagrsfe g fe gBstgrafe.__________________________________ See Figure 13. Discussion: ECC using Trears was significantly more efficient than using the Reiner-Alexander syringe. Performance was even better when comparing the time required to analyze HCC. Earwax concentrated significantly more cortisol than hair. Earwax was also a more stable sample than hair for reflecting cortisol levels, as its concentration was not affected by any covariate. We corroborated the results of previous studies indicating that men have higher HCC levels compared to women (Garcia-Leon et al., 2018; Vanaelst et al., 2012). Hair proved to be another biological sample capable of accumulating cortisol over long periods, showing a positive correlation with a specimen already widely validated for measuring these levels. This property was reinforced by observing that this novel biological sample was not affected by any acute influences. The ECC (Electron Cortisol Concentration) may show a stronger correlation with 24-hour urine collection or continuous cortisol levels, as some evidence suggests that the HCC is affected by acute influences, such as sweat (Sharpley, 2012) and nerve fibers (Okumura, 1967). Earwax accumulates a higher concentration of cortisol than hair. Cortisol is secreted indirectly into the hair duct through a multi-compartment model that is not fully understood. On the other hand, cortisol is secreted directly into the external auditory canal through a simple uni-compartment model, explained by the presence of ceruminous glands. A potential limitation relates to ECC / HCC comparisons. Although this study used the same sample of participants, its results were not strictly comparable because these specimens were analyzed in two different laboratories. Clark et al. (1998), for example, showed a significant bias ratio of up to 1.2 among five different immunoassays in controls subjected to a standard corticotropin test (Jeremy Cohen et al., 2006). However, we found an ECC / HCC ratio of up to 14.3.Aside from the fact that this fraction is extremely large, both laboratories used ELISA techniques. Therefore, it is extremely unlikely that the difference could have been explained by chance. Future studies should correlate the ECC with cortisol levels in nails, another specimen that has shown the potential to accumulate cortisol long-term (Izawa et al., 2015). Ultimately, the ECC could begin to be measured in depressed patients. Conclusion: Trears may be a more economical, convenient, and effective method for self-collection of external earwax samples in healthy individuals. This device can also replace the use of cotton swabs, which pose a high risk. Earwax samples reflect average cortisol and glucose levels. Common acute influences do not affect glucose and cortisol levels in this sample.

Claims

1. A method for measuring glucose and cortisol levels in earwax comprising the steps of: Extracting earwax samples by any suitable means; Preparing earwax samples for the measurement of cortisol and glucose levels according to measurement methods; Measuring cortisol and glucose using any known means or method wherein the cortisol and glucose levels are interpreted as the average levels of the same substances.

2. A method for measuring glucose and cortisol levels as claimed in claim 1, wherein: Earwax extraction is performed using a Reiner Alexander syringe from the ears; Sample preparation is carried out by: a) Drying the earwax samples until all water has evaporated from the sample. This step can also be carried out by lyophilization; b) Weighing the earwax samples to normalize the amount of cortisol on a dry weight basis. Normalization means that the measured weight is adjusted to a common scale to allow for data comparison; c) Homogenizing the dried samples with 1 ml of phosphate-buffered saline (PBS) to obtain an earwax solution in PBS. The amount of phosphate-buffered saline (PBS) can be 10 volumes by weight of earwax; for example, 1000 µL of phosphate-buffered saline (PBS) is used for 100 g of earwax. Furthermore,Any hydrophilic solvent may be used, such as physiological saline; d) Divide the solution obtained in step c) into a first portion of solution, a second portion of solution, and add each portion of solution to a respective tube; RQfr / nn / nznz / E / Yii e) Add a solvent to the first portion of solution in a 1:1 ratio between the PBS and the first portion of solution in order to obtain a cerumen solution in PBS mixed with solvent; f) Shake the tube containing the solution obtained in step e) for a period of at least one minute to mix the solution obtained in step f) and add 0.5 mg of diethyl ether after resuspending, the ratio with PBS being 1:1; g) Cool the mixed solution obtained in step g) to a temperature of -18 to -21°C,preferably -20°C for a period of at least two hours to ensure that the liquid portion is frozen and does not contaminate the organic fraction. This step allows the extraction of compounds that are specifically soluble in diethyl ether, such as cortisol. This is because while the diethyl ether fraction remains liquid at -20°C, the phosphate fraction freezes; h) Extract from the cooled solution the compounds that are specifically soluble in diethyl ether; i) Dry the remaining fraction of the liquid solution; j) Store the dried fraction obtained in step i) at -80°C for later use; k) Add 300 pg of cortisol to the second portion of solution to obtain a cerumen solution in PBS mixed with cortisol; l) Add 0,5 ml of solvent to the solution obtained in step k) in order to quantify the amount of purified cortisol; m) Shake the tube containing the solution obtained in step i) for a period of at least one minute to mix the solution obtained in step m) and add 0.5 mg of diethyl ether after resuspending, the ratio being 1:1 with PBS; n) Cool the mixed solution obtained in step m) to a temperature of -18 to -21°C, preferably -20°C for a period of at least two hours in order to ensure that the liquid part is frozen, and does not contaminate the organic fraction; RQfr / nn / nznz / E / Yi o) Extract from the cooled solution the compounds that are specifically solubilized in diethyl ether; p) Dry the remaining fraction of the liquid solution; q) Store the dried fraction obtained in step i) at a temperature between -20 and -90°C for later use; r) Dissolution of 0,5 ml of 300 pg / ml purified cortisol solution in PBS at a pH between 6.8 and 7.

2. This step was performed as an attempt to obtain the efficiency of the cortisol extraction protocol from earwax. s) Carry out the same procedure used for the first portion of solution and for the second portion of solution to extract cortisol from it. The measurement of cortisol is carried out by: a) Reconstituting the extracted samples using a buffer assay provided by the manufacturer, which allows the quantification of cortisol, using competitive colorimetric ELISA techniques, adding the buffer to the extracted samples to obtain a solution,a) Allowing it to settle. b) Using a standardized curve for cortisol levels – microplate reader (NovoStar) – to measure the total amount of cortisol in the sample; c) Normalization of the quantified amount per dry gram of earwax using fluorometric techniques in which the fluorometer is excited within a range of 530–570 nm and read within an emission range of 590–600 nm. Since several variables, such as age, sex, different medical conditions, and stress levels, could affect cortisol levels in earwax, cholesterol levels, which are not affected by the aforementioned variables, were used to avoid confounding the cortisol results with the aforementioned covariates. Glucose measurement was performed using the SERA-PAK PLUS Kit (Bayer Healthcare) for glucose levels in the dissolved earwax solution, following the manufacturer's instructions.where the glucose absorptions are quantified in triplicate at 505 nm and the glucose concentration (mg / dl) is obtained using their absorption averages, and the total amount of glucose in the dissolved solution is calculated according to the initial weight of the sample.

3. A method for measuring glucose and cortisol levels as stated in claim 2, wherein in step a) the earwax is dried by means of N2 vapor at room temperature.

4. A method for measuring glucose and cortisol levels as stated in claim 2, wherein in step a) the earwax is dried using lyophilization.

5. A method for measuring glucose and cortisol levels as stated in claim 2, wherein in step e) the solvent comprises diethyl ether.

6. A method for measuring glucose and cortisol levels as stated in claim 2, wherein in step i) the remaining fraction of the liquid solution is dried using N2 steam at room temperature.

7. A method for measuring glucose and cortisol levels as stated in claim 2, wherein in step I) the solvent comprises diethyl ether.

8. A method for measuring glucose and cortisol levels as stated in claim 2, wherein in step p) the remaining fraction of the liquid solution is dried by N2 vapor at room temperature.

9. A method for measuring glucose and cortisol levels as stated in claim 2, wherein in step a) concerning the measurement of cortisol, the solution is allowed to stand for a period of time of 20 minutes.

10. A method for measuring glucose and cortisol levels as claimed in claim 2, wherein in step a) concerning the cortisol measurement, the solution is stirred for a period of 1 minute. RQfr / nn / nznz / E / Yii 11. A method for measuring glucose and cortisol levels as claimed in claim 1, wherein: Earwax removal is carried out by means of an extraction device comprising: A handle having a first and second end, said second end having coupling means; A removable sponge head (tip) comprising a base and an elongated member extending longitudinally directly dependent on an upper portion of the base, wherein the lower portion of the base has coupling means for receiving the coupling means of the handle, and wherein the elongated member has a star-shaped cross-section; An elongated sponge having a centrally located longitudinal housing for receiving the elongated member of the base; wherein the earwax is obtained by inserting the tip with the sponge into the ear and rotating the sponge within the ear canal;Sample preparation is carried out by: adding a PBS buffer solution to a tube; separating the sponge from its plastic holder and inserting it into the tube; after the sponge has absorbed all the solution, it is repeatedly squeezed and reabsorbed; squeezing the sponge and removing it from the tube. Drying the resulting solution; resuspending the resulting contents in ultrapure (distilled) water; and storing the resulting solution until use; and Cortisol levels are measured using ELISA techniques and glucose levels are measured using a SERA-PAK PLUS kit (Bayer Healthcare) for glucose levels of the dissolved earwax solution, following the manufacturer's instructions.

12. A method for measuring glucose and cortisol levels as stated in claim 11, wherein in the sample preparation the ratio between the weight of the sponge and the volume of PBS is 1:

2.

13. A method for measuring glucose and cortisol levels as stated in claim 11, wherein in the sample preparation the resulting solution is dried by N2 displacement.

14. A method for measuring glucose and cortisol levels as stated in claim 11, wherein in the preparation of the sample the resulting solution is stored at 4°C.

15. A method for measuring glucose and cortisol levels as stated in claim 1, wherein the extraction device has the following additional feature: The handle coupling means comprise a thread; and The base coupling means comprise a housing including an internal threaded pattern for receiving the handle thread.

16. A method for measuring glucose and cortisol levels as claimed in claim 1, wherein the extraction device has the following additional feature: The elongated member has a star-shaped cross-section, which improves the extraction of earwax while rubbing the sponge inside the ear; The longitudinal housing located in the center has a star-shaped cross-section to receive the elongated member at the base; 17. A method for measuring glucose and cortisol levels as stated in claim 1, wherein the extraction device has the following additional feature: The sponge is made of cellulose.

18. A method for measuring glucose and cortisol levels as stated in claim 1, wherein the extraction device has the following additional feature: The sponge is attached to the elongated member using a non-allergenic glue.

19. A method for measuring glucose and cortisol levels as stated in claim 1, wherein the extraction device has the following additional feature: Its base is wider than the handle, acting as a safety brake that makes it difficult to insert the tip into the ear canal.

20. A method for measuring glucose and cortisol levels as claimed in claim 1, wherein the extraction device has the following additional feature: The sponge is packaged and sealed under humid conditions to maintain its moisture using a humectant comprising Magnesium Chloride (MgCl2).

21. A method for measuring glucose and cortisol levels as stated in claim 1, wherein the extraction device has the following additional feature: The earwax is obtained by inserting the tip with the sponge into the ear and rotating the sponge inside the ear canal for about 30 to 60 seconds.

22. A device for earwax removal comprising: A handle having a first and second end, said second end having coupling means; A removable sponge head (tip) comprising a base and an elongated member extending longitudinally directly dependent on an upper portion of the base, wherein the lower portion of the base has coupling means for receiving the coupling means of the handle, and wherein the elongated member has a star-shaped cross-section; An elongated sponge having a centrally located longitudinal housing for receiving the elongated member of the base; wherein earwax is cleaned from an ear by inserting the tip with the sponge into the ear and rotating the sponge within the ear canal.

23. A device for earwax removal according to claim 22, wherein: The handle coupling means comprise a thread; and The base coupling means comprise a housing including an internal threaded pattern to receive the handle thread.

24. A device for earwax removal according to claim 22, wherein: The elongated member has a star-shaped cross-section, which improves the removal of earwax while rubbing the sponge inside the ear; The longitudinal housing located in the center has a star-shaped cross-section to receive the elongated member at the base; 25. A device for earwax removal according to claim 22, wherein the sponge is made of cellulose.

26. A device for earwax removal according to claim 22, wherein the sponge is glued to the elongated member using a non-allergenic glue.

27. A device for earwax removal according to claim 22, wherein the base is wider than the handle, acting as a safety brake that prevents the tip from being inserted into the ear canal.

28. A device for earwax removal according to claim 22, wherein the sponge is packaged and sealed under moist conditions to keep it soft with a moistened moisture.

29. A device for earwax removal according to claim 22, wherein the sponge is packaged and sealed under moist conditions to maintain its softness using a humectant comprising Magnesium Chloride (MgCl2).

30. A device for earwax removal according to claim 22, wherein the earwax is obtained by inserting the tip with the sponge into the ear and rotating the sponge inside the ear canal for about 30 to 60 seconds.