A Novel Earwax Method to Measure Acute and Chronic Glucose Levels

Diabetes is the fourth cause of death globally. To date, there is not a practical, as well as an accurate sample for reflecting chronic glucose levels. We measured earwax glucose in 37 controls. Participants provided standard serum, glycated hemoglobin (HbA1c) and earwax samples at two time-points, one month apart. The specimens measured baseline fasting glucose, a follow-up postprandial glucose level and a between sample chronic glucose, calculated using the average level on the two occasions. The baseline earwax sample was obtained using a clinical method and the follow-up using a novel self-sampling earwax device. The earwax analytic time was significantly faster using the novel device, in comparison to the clinical use of the syringe. Earwax accurately reflected glucose at both assessments with stronger correlations than HbA1c. Follow-up postprandial concentrations were more significant than their respective fasting baseline concentrations, reflecting differences in fasting and postprandial glycemia and more efficient standardization at follow up. Earwax demonstrated to be more predictable than HbA1c in reflecting systemic fasting, postprandial and long-term glucose levels, and to be less influenced by confounders. Earwax glucose measurements were approximately 60% more predictable than HbA1c in reflecting glycemia over a month. The self-sampling device provided a sample that might accurately reflect chronic glycemia.

inside the external ear canal. The process of syringing creates a sensation of mild pressure in the ear as the warm water from the syringe flushes the wax out. The expelled water and the extracted earwax secretion were collected in a kidney basin. During the follow-up visit, participants self-clean their right ears using the earwax self-sampling device, according to the manufacturer instructions (www.trears.com). The four labelled earwax samples were stored at 4 °C until they were analysed.

Earwax Analysis using the Reiner-Alexander Syringe
The extracted solution of water plus earwax secretion was stored in a 50 ml cryovial. Earwax samples were then resuspended with 500 µl Phosphate-Buffered Saline (PBS) to homogenise the sample. Then, 500 µl of diethyl ether was added to each sample and wobbled for one minute using a vortex. The resulting solution was stored at -20°C for 2 hours. Glucose levels were then analysed from the hydrophilic fraction. The earwax solution was dried using the displacement method of N2 at 25 degree Celsius. Following this, 125 µl of PPBS was added to resuspend each solution. The resulting solution containing earwax samples was stored at 4 degrees Celsius until analysed.

Earwax Analysis using the earwax self-sampling device
The earwax self-sampling device tips were cleaned using 500 µl of PBS for 2 minutes. Then, the same previous steps that were done for analysing earwax sample using the metallic syringe were followed when the novel device was also used.

Blood Samples:
All blood samples, baseline and follow up, for every participant, were taken in the morning. Blood samples were taken from the cephalic vein using a 3cc syringe without using any anticoagulants.
Participants had been previously instructed to avoid eating or drinking anything 8 hours before the baseline assessment. Another blood sample was taken during the morning of the follow-up visit. The follow-up samples were taken 2 hours after consuming a standardised liquid meal, 236 ml of Ensure Avance®. This meal contains 1.5 kcal/ml, given by 24.3% protein, 44.8% carbohydrate 28.8% fat, 1% fibre and 1.1% Beta-hydroxy-beta-methyl butyrate. FSG and HbA1c levels were analysed from baseline samples. HbA1c levels and PSG were analysed from the follow-up samples. The chronic glucose level over the preceding one-month period was calculated using the mean between the baseline and the follow-up sample of glycaemia.

Serum Glucose Analysis:
Blood samples were coagulated after being stored at 4°C for 24-h. It allows the separation of the serum fraction from the clotting factors, such as fibrinogen. Following the separation, serum samples were centrifugated (1000 x g) for 20 min at 4°C. The resulting pellet was separated from the solution.
The solution was recollected in labelled tubes of 2 ml. These tubes were stored -20°C until their content was analysed.

Glucose Analysis using Serum and Earwax Samples
Glucose levels were measured using enzymatic oxidation testing and the labelling of oxidized glucose using 96-well plates, according to manufacturer instructions (BioVision Inc., Milpitas, CA, USA). Diluted standard assay with 1:25 of serum were added to the wells including 50 µl of a mixed portion of glucose which contains 2 µl of probed glucose and 2 µl of an enzymatic mix for glucose.
The resulting solution was incubated for 30 min at 37°C and protected from direct light. Absorbance was read at 570 nm using a microplate reader (NovoStar).

Haemoglobin analysis
Total haemoglobin was measured using the standard colorimetric method of cyanmethemoglobin, according to the manufacturer instructions (Valtek Laboratory, Santiago, Chile). Briefly, 1 µl of blood were incubated for 10 minutes with 250 µl of Drabkin solution at room temperature. Sample absorbances were measured at 540 nm using a microplate reader (NovoStar).

Glycated Haemoglobin Analysis:
HbA1c was measured using a sandwich ELISA technique, according to the manufacturer's instructions (Abbexa Ltd., Cambridge, UK). 100 µl of the diluted standards were added into the standard wells. An antibody, specific to HbA1c, is already precoated onto the 96 well plates. A competitive inhibition reaction was initiated between biotin labelled HbA1c and unlabelled HbA1c with the pre-coated antibody specific to HbA1c. The unbound conjugates were washed away 3 times.
The wells that contained HbA1c, underwent a colour change, from a blue coloured product yellow, after adding acidic stop solution. The intensity of the yellow colour was inversely proportional to the HbA1c amount bound on the plate. The optical density absorbance was measured spectrophotometrically at 450 nm in a microplate reader, and the concentration of HbA1c was calculated. % HbA1c was calculated using the National Glycohemoglobin Standardization Program (NGSP) units which express the percentage of HbA1c over the total amount of haemoglobin.