Phenol Red Thread-based Sampling Procedure for Untargeted Tear Fluid Lipidomics in Biomarker Discovery

We established a more standardized, easy-to-operate phenol red thread-based method to help achieve reproducible tear lipidomic profiles for future tear biomarker discovery. The current challenge is that Schirmer strips irritate while capillaries show operator variabilities in tear collection. This hinders experimental reportability and inter-study comparisons.

To begin, wear gloves and disinfect the workstation thoroughly to prevent any contamination of the sample. Hold the phenol red thread by the end opposite the bent hook, making sure not to touch the bent hook region. Ask the subject to direct their gaze towards the superior nasal direction.

Then pull down the subject's lower eyelid gently, and insert the phenol red thread with the bent hook placed in the lower temporal palpebral conjunctiva near the outer corner of the eye. Now, ask the subject to close their eyes gently and tilt their head slightly forward to prevent the phenol red thread from contacting the skin on the face. Pull down the subject's lower eyelid again, and gently remove the phenol red thread, avoiding contact with the sampled region.

Record the length of the sampled red region. And using sterile forceps, transfer the PRT into a new sample tube. Now label and cap sample tube properly.

If the sampled length is less than 50 millimeters, ask the subject to rest for one to three minutes and then repeat the tear fluid collection using the same eye. Place each newly sampled PRT into a separate sample tube. Using methanol and clean paper wipes, clean the gloves and forceps thoroughly to eliminate any tear residues or contaminants.

Then allow them to air dry completely. Seal all sample tubes with parafilm and immediately store them at minus 20 degrees Celsius for temporary storage or minus 80 degrees Celsius for long-term storage. Pre-wash the micro scissors and forceps with methanol before processing the phenol red threads.

Cut off the bent hook end approximately the first three millimeters from each phenol red thread. Cut the sampled region up to the phenol red dye front into two millimeter pieces and collect these segments with a clean 1.5 milliliter organic solvent-tolerant microcentrifuge tube. Discard the bent hook end and unwanted region of PRT.

Place the samples on ice to maintain a low temperature before extraction. Now, add 232 microliters of ice cold mass spectrometry grade methanol to each sample and vortex for 15 seconds to mix thoroughly. Sonicate the samples in a pre-cooled ultrasonic cleanser for 15 minutes.

Then add 774 microliters of HPLC grade methyl tert-butyl ether and 194 microliters of deionized water to each sample. Vortex again for 15 seconds to mix the solvents thoroughly. Sonicate the samples again in a pre-cooled ultrasonic cleanser for another 15 minutes.

Post-sonication, incubate the samples in a ThermoMixer set to four degrees Celsius with shaking at 1, 200 revolutions per minute for eight hours. Then let the mixture sit at room temperature undisturbed for 10 minutes. Centrifuge the mixtures at 10, 000 G for 10 minutes at four degrees Celsius.

After centrifugation, ensure the mixtures are visibly separated into two distinct liquid phases. Carefully collect 700 microliters of the upper organic phase containing lipids and transfer it into a pre-chilled new 1.5 milliliter microcentrifuge tube. Discard the lower aqueous phase, which contains residual methyl tert-butyl ether and methanol as hazardous chemical waste.

Dry the lipid extracts using a refrigerated SpeedVac concentrator set at four degrees Celsius for four hours. Then store the dried lipid extracts at minus 20 degrees Celsius for short-term storage or minus 80 degrees Celsius for long-term storage. Prepare the methanol-chloroform solvent mixture by mixing methanol with chloroform in a one-to-one volume ratio.

Reconstitute the dried lipid extracts using 50 microliters of the ice cold methanol-chloroform solvent mixture. Sonicate the reconstituted solution in a pre-cooled ultrasonic cleanser for 15 minutes. Now, centrifuge the reconstituted lipid extracts at 14, 000 G and four degrees Celsius for 10 minutes.

Carefully transfer 40 microliters of the resulting supernatant into an autosampler glass vial equipped with a glass insert. Tightly cap the vial with a non-slit septum open top cap to prepare it for liquid chromatography tandem mass spectrometry analysis. Using stringent criteria, 773, 890, and 1, 025 unique lipid species were identified in replicate one, replicate two, and replicate three sample groups respectively.

Positive ion mode identified 1, 302 lipid species with 26%grade A, 13.2%grade B, and 60.8%grade C identifications. Negative ion mode identified 257 lipid species with about 8.2%grade A, 22.9%grade B, and 68.9%grade C identifications. Despite storage-related variations, 394 unique lipid species were consistently identified across all three sample sets.

Inter-subject coefficient of variation across three visits was 21.5%in positive mode and 31.3%in negative mode. This protocol addresses the absence of a standardized workflow for reproducible tear lipidomic analysis and can enhance the inter-study comparability. Our PRT-based method minimizes irritation and enables rapid tear collection and reduces operator variability.

Our future research will validate tear lipidomic biomarkers in large cohorts and integrate multiomics for personalized diagnostics.