Name: Phenol Red Thread-based Sampling Procedure for Untargeted Tear Fluid Lipidomics in Biomarker Discovery (Video) | JoVE Journal
Uploaded: 2025-12-12T14:25:00.000+00:00
Duration: 8 min 16 s
Description: Learn a standardized phenol red thread method for reproducible tear lipidomic profiling in biomarker discovery. This protocol reduces operator variability and enables LC-MS/MS analysis of over 1,300 lipid species.

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5 minutes

This work was supported by the InnoHK initiative and the Hong Kong Special Administrative Region Government and the Research Centre for SHARP Vision at The Hong Kong Polytechnic University. The authors also gratefully acknowledge technical support from the University Research Facility in Chemical and Environmental Analysis (UCEA) and the University Research Facility in Life Sciences (ULS) of The Hong Kong Polytechnic University.

The study was approved by the Institutional Review Board (IRB) of The Hong Kong Polytechnic University. The subjects provided written informed consent before participation in the study. The reagents and the equipment used are listed in the Table of Materials.
1. Phenol Red Thread (PRT)-based collection of human tear fluid
<ol>
	<li>Wear gloves and disinfect the workstation to prevent sample contamination.</li>
	<li>Make sure the PRT package is sealed and not expired before opening it.</li>
	<li>Hold the thread by the end opposite the bent hook. Always avoid touching the bent hook half of the PRT.</li>
	<li>Ask the subject to look at the superior nasal direction.</li>
	<li>Pull down the subject's lower eyelid gently and insert the PRT with the bent hook positioned in the lower temporal palpebral conjunctiva near the outer canthus of the subject's eye (Figure 2).</li>
</ol>
<img alt="Tear duct fluorescein test diagram; eye highlighting tear drainage pathway in medical examination." class="xfigimg" src="/files/ftp_upload/69474/69474fig2.jpg" data-altupdatedat="1773047343000" data-alt="Figure 2"> 
Figure 2: Position of the phenol red thread during tear collection. <a href="https://www.jove.com/files/ftp_upload/69474/69474fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="6">
	<li>Ask the subject to close their eyes gently and tilt his/her head slightly forward to prevent the PRT from touching his/her facial skin. 
	NOTE: Tear absorbed would gradually induce a red dye front, indicating the sampled region of PRT due to the pH-sensitive phenol red. To ensure enough tear fluid for lipidomic analysis, collecting tear volume equivalent to 50 mm of PRT is preferred, typically completed within 2 min.</li>
	<li>Pull down the subject's lower eyelid gently and pull out the sampled PRT. Avoid touching the sampled region.</li>
	<li>Record the length of the sampled region of the PRT and transfer the PRT into a new sample tube with the aid of sterile forceps. 
	NOTE: If the sampled PRT length is &lt;50 mm, kindly ask the subject to rest for 1-3 min and repeat the collection with the same eye. Transfer each sampled PRT to a separate sample tube.</li>
	<li>Label and cap the sample tubes properly.</li>
	<li>Clean the gloves and forceps thoroughly with methanol and clean paper wipes to remove any tear residuals or contaminants, and then let them dry.</li>
	<li>Repeat steps 1.2-1.10 for the other eye.</li>
	<li>Seal the sample tubes with parafilm and immediately store the sample at -20°C for temporary storage or -80°C for long-term storage. Avoid unnecessary freeze-and-thaw cycles and protect the sampled PRT from light.</li>
</ol>
2. Processing of sampled PRT
<ol>
	<li>Pre-wash the micro-scissors with methanol before processing the PRTs.</li>
	<li>Discard the bent hook end (i.e., first 3 mm) of the PRTs (Figure 3).</li>
</ol>
<img alt="Chromatography diagram showing thread segments with marked cutting regions for sample collection." class="xfigimg" src="/files/ftp_upload/69474/69474fig3.jpg" data-altupdatedat="1773047343000" data-alt="Figure 3"> 
Figure 3: Physical appearance of the phenol red thread (PRT). (A) Unused PRT with bent hook end. (B) Used PRT with the indicated section cut and collected for lipid extraction. <a href="https://www.jove.com/files/ftp_upload/69474/69474fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Cut the sampled region of the PRT (i.e., region above the phenol red dye front) into 2 mm pieces and transfer these pieces to a clean 1.5 mL organic solvent-tolerant microcentrifuge tube.</li>
</ol>
3. MTBE/methanol biphasic separation for lipid extraction from sampled PRT
CAUTION: MTBE and methanol are flammable and volatile. Use in a well-ventilated area or fume hood. Avoid open flames and sparks. Wear gloves and a lab coat. Wash hands after handling.
<ol>
	<li>Place the samples on ice.</li>
	<li>Add 232 µL of ice-cold methanol (MS grade) to each sample and vortex for 15 s.</li>
	<li>Sonicate the samples in a pre-cooled ultrasonic cleanser for 15 min.</li>
	<li>Add 774 µL of MTBE (HPLC grade) and 194 µL of deionized water to each sample and vortex for 15 s. The resultant volume ratio of MTBE:methanol:water in the sample should be 4:1.2:1 (v/v/v).</li>
	<li>Sonicate the samples again in a pre-cooled ultrasonic cleanser for 15 min.</li>
	<li>Incubate the sample in the thermomixer for 8 h at 4 °C, with shaking at 1,200 rpm.</li>
	<li>Let the mixture sit at room temperature for 10 min.</li>
	<li>Centrifuge the resultant mixtures at 10,000 × g for 10 min at 4 °C.</li>
	<li>After centrifugation, the mixtures are separated into two phases (Figure 4). 
	NOTE: Upon completion of phase separation, inspect the sample for two distinct layers: the upper organic phase (MTBE-rich) should appear transparent with a slight milky opacity, while the lower aqueous phase exhibits a yellow coloration attributable to the presence of phenol red indicator. Proceed only if these phase characteristics are observed, as shown in Figure 4.</li>
</ol>
<img alt="Organic-aqueous separation diagram; liquid-liquid extraction in centrifuge tube demonstrating phase distinction." class="xfigimg" src="/files/ftp_upload/69474/69474fig4.jpg" data-altupdatedat="1773047343000" data-alt="Figure 4"> 
Figure 4: Phase separation of lipid extraction using MTBE-methanol-water: upper organic phase and lower aqueous phase. <a href="https://www.jove.com/files/ftp_upload/69474/69474fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="10">
	<li>Collect 700 µL of the upper organic fraction containing lipids carefully and transfer to a pre-chilled new 1.5 mL microcentrifuge tube.</li>
	<li>Discard the lower aqueous phase containing residual MTBE and methanol as hazardous chemical waste. Collect waste in labeled, sealed containers and dispose of it according to institutional hazardous waste protocols and local regulations. Do not pour solvents down the drain.</li>
	<li>Dry the lipid extracts using a refrigerated SpeedVac concentrator for 4 h at 4 °C. 
	NOTE: To ensure complete drying, periodically check the sample dryness starting at 2 h. Extend the drying time as necessary until samples are fully dried.</li>
	<li>Store the dried lipid extracts at -20 °C for short-term storage or -80 °C for long-term storage.</li>
</ol>
4. Reconstitution of extracted tear lipid for LC- MS/MS analysis
<ol>
	<li>Reconstitute the dried lipid extracts with 50 µL of the ice-cold methanol (MS grade)/chloroform (HPLC grade) mix (1:1, v/v) as the solvent (1 µL of solvent per 1 mm of PRT used) and follow by sonication in a pre-cooled ultrasonic cleanser for 15 min.</li>
	<li>Centrifuge the reconstituted lipid extracts at 14,000 × g and 4 °C for 10 min.</li>
	<li>Carefully transfer 40 µL of the supernatant to an autosampler glass vial with a glass insert installed.</li>
	<li>Tightly cap the vial with a non-slit septum open top cap and ready for LC-MS/MS analysis.</li>
</ol>
5. Sample acquisition by LC-MS/MS
<ol>
	<li>For Liquid Chromatography, set the column chamber and sampler temperature at 50 °C and 4 °C, respectively.</li>
	<li>For each injection, load 5 µL of the sample onto a reverse-phase LC column (C18; 1.7 µm, 100 mm × 2.1 mm) and fractionate the lipids at a flow rate of 0.3 mL/min in a 24 min separation gradient.
	<ol>
		<li>Use mobile phase A comprising a mixture of 60:40 acetonitrile (MS grade):water (v/v) with 5 mM ammonium formate (MS grade) and 0.1% (v/v) formic acid (MS grade) and mobile phase B containing 90:10 isopropanol (MS grade):acetonitrile (v/v) with 5 mM ammonium formate and 0.1% (v/v) formic acid.</li>
		<li>Use the following gradient: 0-2 min: 40% B; 2-2.5 min: 40% B; 2.5-3 min: 58% B; 3-18 min: 99% B; 18-20 min: 99% B; 20-20.1 min: 40% B; 20.1-24 min: 40% B.</li>
	</ol>
	</li>
	<li>For the Orbitrap Mass Spectrometer, set the spray voltage to +3.5 kV for positive mode or -2.5 kV for negative mode.</li>
	<li>Adjust the sheath gas flow rate to 45 Arb and the auxiliary gas flow rate to 10 Arb.</li>
	<li>Set the sweep gas flow rate to 2 Arb.</li>
	<li>Set the ion transfer tube temperature to 300 °C and the vaporizer temperature to 320 °C.</li>
	<li>Use data-dependent acquisition (DDA) mode.</li>
	<li>For MS1, set the resolution to 120,000, scan range to 100-2000 m/z, standard AGC target, auto injection time, dynamic exclusion to 6 s, and intensity threshold at 5.0e4.</li>
	<li>For MS2, set the resolution to 15,000, auto scan range, standard AGC target, dynamic injection time, isolation window at 1.6 m/z, collision energy type as higher-energy collisional dissociation (HCD), and normalized collision energy (NCE) to stepped values of 15, 30, and 40.</li>
	<li>Analyze raw data with LipidSearch software or other compatible platforms.</li>
</ol>

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<li>Lam, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipidomic+analysis+of+human+tear+fluid+reveals+structure-specific+lipid+alterations+in+dry+eye+syndrome%5BTitle%5D)+AND+%22J+Lipid+Res%22%5BJournal%5D)">Lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome.</a> J Lipid Res. 55 (2), 299-306 (2014).</li>
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<li>Bertram, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+Schirmer+strip+wetness+on+volume+absorbed%2C+volume+recovered%2C+and+total+protein+content+in+canine+tears%5BTitle%5D)+AND+%22Vet+Ophthalmol%22%5BJournal%5D)">Influence of Schirmer strip wetness on volume absorbed, volume recovered, and total protein content in canine tears.</a> Vet Ophthalmol. 24 (4), 425-428 (2021).</li>
<li>Zhan, X., Li, J., Guo, Y., Golubnitschaja, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+spectrometry+analysis+of+human+tear+fluid+biomarkers+specific+for+ocular+and+systemic+diseases+in+the+context+of+3p+medicine%5BTitle%5D)+AND+%22EPMA+J%22%5BJournal%5D)">Mass spectrometry analysis of human tear fluid biomarkers specific for ocular and systemic diseases in the context of 3p medicine.</a> EPMA J. 12 (4), 449-475 (2021).</li>
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<li>Kecskeméti, G., Tóth-Molnár, E., Janáky, T., Szabó, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+extensive+study+of+phenol+red+thread+as+a+novel+non-invasive+tear+sampling+technique+for+proteomics+studies%3A+Comparison+with+two+commonly+used+methods%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">An extensive study of phenol red thread as a novel non-invasive tear sampling technique for proteomics studies: Comparison with two commonly used methods.</a> Int J Mol Sci. 23 (15), 8647(2022).</li>
<li>Barmada, A., Shippy, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantifying+sample+collection+and+processing+impacts+on+fiber-based+tear+fluid+chemical+analysis%5BTitle%5D)+AND+%22Transl+Vis+Sci+Technol%22%5BJournal%5D)">Quantifying sample collection and processing impacts on fiber-based tear fluid chemical analysis.</a> Transl Vis Sci Technol. 9 (10), 23(2020).</li>
<li>Hao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Validation+of+the+phenol+red+thread+test+in+a+Chinese+population%5BTitle%5D)+AND+%22BMC+Ophthalmol%22%5BJournal%5D)">Validation of the phenol red thread test in a Chinese population.</a> BMC Ophthalmol. 23 (1), 498(2023).</li>
<li>Matyash, V., Liebisch, G., Kurzchalia, T. V., Shevchenko, A., Schwudke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipid+extraction+by+methyl-tert-butyl+ether+for+high-throughput+lipidomics%5BTitle%5D)+AND+%22J+Lipid+Res%22%5BJournal%5D)">Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics.</a> J Lipid Res. 49 (5), 1137-1146 (2008).</li>
<li>Ulmer, C. Z., Jones, C. M., Yost, R. A., Garrett, T. J., Bowden, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+Folch%2C+Bligh-Dyer%2C+and+Matyash+sample-to-extraction+solvent+ratios+for+human+plasma-based+lipidomics+studies%5BTitle%5D)+AND+%22Anal+Chim+Acta%22%5BJournal%5D)">Optimization of Folch, Bligh-Dyer, and Matyash sample-to-extraction solvent ratios for human plasma-based lipidomics studies.</a> Anal Chim Acta. 1037, 351-357 (2018).</li>
<li>Lam, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extensive+characterization+of+human+tear+fluid+collected+using+different+techniques+unravels+the+presence+of+novel+lipid+amphiphiles%5BTitle%5D)+AND+%22J+Lipid+Res%22%5BJournal%5D)">Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles.</a> J Lipid Res. 55 (2), 289-298 (2014).</li>
<li>Quah, J. H., Tong, L., Barbier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patient+acceptability+of+tear+collection+in+the+primary+healthcare+setting%5BTitle%5D)+AND+%22Optom+Vis+Sci%22%5BJournal%5D)">Patient acceptability of tear collection in the primary healthcare setting.</a> Optom Vis Sci. 91 (4), 452-458 (2014).</li>
<li>Saini, R. K., Prasad, P., Shang, X., Keum, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+lipid+extraction+methods-a+review%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Advances in lipid extraction methods-a review.</a> Int J Mol Sci. 22 (24), 13643(2021).</li>
<li>Chen, J., Nichols, K. K., Wilson, L., Barnes, S., Nichols, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Untargeted+lipidomic+analysis+of+human+tears%3A+A+new+approach+for+quantification+of+o-acyl-omega+hydroxy+fatty+acids%5BTitle%5D)+AND+%22Ocul+Surf%22%5BJournal%5D)">Untargeted lipidomic analysis of human tears: A new approach for quantification of o-acyl-omega hydroxy fatty acids.</a> Ocul Surf. 17 (2), 347-355 (2019).</li>
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</ol>

The authors have no conflicts of interest to declare.

Advantages of PRT-based tear sampling and lipid extraction 
The PRT-based tear lipidomics workflow presented here provides a minimally invasive and practical sampling method for comprehensive tear lipid profiling. Compared to Schirmer strips, which require prolonged contact and larger sample volumes, PRT sampling is rapid, can be completed within 2 min, well-tolerated by diverse populations, including those with reduced tear volume or heightened ocular sensitivity, and yields tear samples compatible...

Tear film is the outermost barrier that protects the ocular surface from pathogens and maintains ocular homeostasis1,2, while the lipid layer in tear film is the key component for preventing tear evaporation and maintaining tear film stability3,4. Dysregulation of the tear lipid composition has been linked to various eye diseases, including dry eye disease (DED), meibomian gland dysfunction (MGD), and allergic conjunctivitis5,6,7,8. As a result, detailed characterization of tear lipidomics has become a critical and trending research direction for a more comprehensive understanding of the molecular pathologies underlying these ocular conditions9.
While the tear lipidomic approach holds significant clinical importance, a critical challenge in tear lipidomics is establishing a standardized, reproducible collection and analysis workflow. Current tear sampling methods, Schirmer's strips and microcapillary tube, each present distinct limitations. Schirmer's strips have a large contact area with the ocular surface that often causes ocular irritation and reflex tearing9,10. In comparison, microcapillary tube collection is highly dependent on operator technique and can yield inconsistent results when performed by different personnel11. Additionally, improper use of these sampling tools may also carry a risk of ocular surface injury. These limitations could compromise the accuracy and reproducibility of downstream data12,13. Furthermore, tear collection methods that require complex handling procedures may limit their accessibility and standardization across different laboratories and clinical settings. Therefore, there is a pressing need to establish a non-invasive, robust, and reproducible tear collection and processing workflow that can preserve the native tear lipid profile.
Recent studies have highlighted the utility of phenol red thread (PRT) for downstream LC-MS/MS workflows analyses in proteomic and metabolomic applications14,15. PRT offers inherent advantages as a tear collection tool. Its thin structure, minimally invasive nature, and straightforward sampling procedure could minimize technical complexity and reduce operator-dependent variability, while being well-tolerated by most subjects14,16. However, a standardized workflow specifically for PRT-based tear lipidomic analysis has not yet been established.
This protocol introduces an integrated workflow for reproducible tear lipidomics: a user-friendly PRT-based tear collection method adapted for lipid analysis, an optimized methyl tert-butyl ether (MTBE)/methanol lipid extraction protocol with defined solvent-to-sample ratios and storage controls17,18, and a modified lipid identification method using high-resolution LC-MS/MS. This standardized approach enables a consistent tear lipidomic profiling for downstream biomarker discovery and disease characterization. Also, it is particularly well-suited for subjects with low tear volume or reduced tolerance for foreign body sensation (Figure 1).
<img alt="Phenol red thread method; diagram of human tear lipid analysis via LC-MS/MS and lipid identification." class="xfigimg" src="/files/ftp_upload/69474/69474fig1.jpg" data-altupdatedat="1773047343000" data-alt="Figure 1"> 
Figure 1: Schematic overview of the Phenol Red Thread (PRT)-based workflow for human tear lipidomics. Tear fluid is collected from subjects utilizing the PRT approach, which enables consistent sample acquisition with minimal discomfort. Sample processing involves sequential MTBE/methanol biphasic extraction, SpeedVac-mediated concentration, and reconstitution of tear lipid extracts for liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lipidomic profiles are subsequently acquired via LC-MS/MS and subjected to annotation and identification using LipidSearch software. <a href="https://www.jove.com/files/ftp_upload/69474/69474fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Acetonitrile (ACN), LC-MS grade</td><td>RCI Labscan</td><td>LM1005</td><td></td></tr><tr><td>ACQUITY UPLC CSH C18 Column</td><td>Waters Corporation</td><td>186005297</td><td>130&amp;Aring;, 1.7 &amp;micro;m, 2.1 mm X 100 mm</td></tr><tr><td>Ammonium formate, LC-MS grade</td><td>Sigma-Aldrich</td><td>55674</td><td></td></tr><tr><td>Autosampler Glass Vial</td><td>Well Rich Scientific</td><td>2ML-9-V1002</td><td>2mL Clear Glass 12*32mm Flat Base 9-425 Screw Thread Vial with Label (WITH writing pad )</td></tr><tr><td>Blue 9-425 Open Top Ribbed Screw Cap&amp;nbsp;</td><td>Well Rich Scientific</td><td>9-SP1003</td><td>with 9mm,White PTFE/Red Silicone Septa 1mm</td></tr><tr><td>Chloroform, HPLC grade</td><td>Duksan Reagents</td><td>1271</td><td></td></tr><tr><td>Eppendorf Safe-Lock Tubes, 1.5 mL</td><td>Eppendorf</td><td>30120086</td><td></td></tr><tr><td>Formic Acid (FA),&amp;nbsp; LC-MS grade</td><td>ThermoFisher Scientific</td><td>A117-50</td><td></td></tr><tr><td>Glass micro-insert</td><td>Well Rich Scientific</td><td>2ML-N2002</td><td>250 &amp;micro;l insert, clear glass, conical base with polyspring, size: 5.8*28.5mm</td></tr><tr><td>Isopropanol, LC-MS grade</td><td>RCI Labscan</td><td>LM1162</td><td></td></tr><tr><td>LipidSearch Software</td><td>ThermoFisher Scientific</td><td>OPTON-30879</td><td>Version 5</td></tr><tr><td>Methanol,&amp;nbsp; LC-MS grade</td><td>RCI Labscan</td><td>LM1115</td><td></td></tr><tr><td>Phenol Red Thread&amp;nbsp; (PRT)</td><td>Tianjin Jingming New Technology Development Co., Ltd</td><td>20192160086</td><td></td></tr><tr><td>Refrigerated Centrifuge</td><td>ThermoFisher Scientific</td><td>75007200</td><td></td></tr><tr><td>Refrigerated CentriVap Centrifugal Concentrator and CentriVap Cold Traps</td><td>Labconco</td><td>16108335</td><td></td></tr><tr><td>Screw Cap Micro Tubes</td><td>ThermoFisher Scientific</td><td>3488</td><td></td></tr><tr><td>tert-Butyl methyl ether (MTBE), HPLC grade</td><td>Duksan Reagents</td><td>1070</td><td></td></tr><tr><td>Thermo Scientific Dionex UltiMate 3000 HPLC</td><td>ThermoFisher Scientific</td><td>ULTIM3000RSLCNANO</td><td></td></tr><tr><td>Thermo Scientific Orbitrap IQ-X Tribrid MS</td><td>ThermoFisher Scientific</td><td>FSN05-10001</td><td></td></tr><tr><td>ThermoMixer C</td><td>Eppendorf</td><td>5382000015</td><td></td></tr><tr><td>Ultrasonic Cleanser</td><td>Crest Ultrasonic</td><td>P500D-45</td><td></td></tr><tr><td>Vortex Mixer</td><td>Benchmark</td><td>BV1003</td><td></td></tr></tbody></table>

phenol red thread-based sampling procedure for untargeted tear fluid lipidomics in biomarker discovery

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

Tear lipids are crucial for tear film stability and ocular surface health. Changes in tear composition could be associated with ocular and systemic diseases such as meibomian gland dysfunction and dyslipidemia. Profiling tear lipids may help biomarker discovery for disease diagnosis and management. However, common tear sampling methods present distinct limitations: Schirmer's strips frequently cause ocular irritation and discomfort due to their large contact area with the eye surface, while microcapillary tube collection could yield low reproducibility due to operator variability, especially when performed by different personnel. These limitations might compromise the accuracy and consistency of lipidomic data. This study introduces a minimally invasive phenol red thread (PRT)-based sampling method optimized for tear lipidomics. The thin structure of PRT minimizes the risk of ocular irritation and allows rapid and gentle tear collection. This user-friendly and easy-to-perform method is suitable for subjects with reduced tear volume or lower tolerance for foreign body sensation, and it enables more reproducible sample collection by reducing operator-dependent variability. Tear lipids collected by PRT were extracted using an optimized methanol/methyl tert-butyl ether (MTBE) phase separation protocol and analyzed by high-resolution LC-Orbitrap-IQX MS/MS with LipidSearch software. The workflow identified more than 700 unique tear lipid species, each characterized by specific fatty-acid-derived product ions. These results indicate that PRT-based sampling provides robust lipid recovery for tear lipidomic analysis. This minimally invasive and reproducible approach offers a practical platform for clinical and experimental tear lipid research. Ultimately, this could also facilitate biomarker discovery and disease monitoring.

Composite tear samples were collected from 16 healthy volunteers at three independent visits spaced two weeks apart (R1, oldest batch; R2, R3 collected sequentially two weeks after each). Using LipidSearch with stringent criteria (i.e., Signal-to-Noise Ratio ≥100, grade C or above, and ion intensity ≥30,000), we identified 773, 890, and 1,025 unique lipid species in R1-R3, respectively (Figure 5A). Positive ion mode identified 1,302 lipid species with 26.0% Grade A (i.e., both lipid class an...

This protocol describes a unique clinical protocol for collecting human tear fluid samples using phenol red threads and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based workflow to discover the tear lipidomic profile. The simple methyl tert-butyl ether (MTBE)/methanol biphasic separation method enables rapid tear lipid extraction with high recovery for tear biomarker discovery.

Tear lipids are crucial for tear film stability and ocular surface health. Changes in tear composition could be associated with ocular and systemic ...

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.

phenol-red-thread-based-sampling-procedure-for-untargeted-tear-fluid

Research

JoVE Journal

Medicine

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

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