Comparison of collection methods for tear fluid biomarkers

1. Introduction

The main medical context in which tear fluid biomarkers are studied is ophthalmology, particularly the anterior segment of the eye (62.4%)[1]. Endocrinology, including metabolic diseases and diabetes, is also studied, and papers on neurological diseases have increased since the 2000s[1]. In breast cancer, tear fluid is regarded as one of the biofluids used for biomarker discovery[2].

Most of the proteins detected in plasma (118) were also detected in tear samples, and tear samples additionally showed 34 unique proteins as well as over 400 microRNAs[3].

Many collection methods are used in tear fluid biomarker research, and no single standardized method has been established yet[1, 4].

2. Main collection methods and reported use[1]

Across the 1,484 articles reviewed in tear fluid biomarker research, the reported use of collection methods was as follows:

  • Glass capillary tubes: 45%
    The tip is placed at the tear meniscus and tear fluid is drawn from the forniceal reservoir of the conjunctival sac into the tube by capillary action.
  • Schirmer strips: 25%
    A filter paper strip is placed in the lower conjunctival sac and absorbs tear fluid[4].
  • Ocular surface wash: 3.7%
    A fixed volume of saline solution is applied to the ocular surface and the diluted tear fluid is then collected with a glass capillary or a micropipette.
  • Sponges: 2.6%
    An absorbent material such as a cellulose or polyurethane mini sponge or a porous polyester rod is placed in the lower tear meniscus for a set period of time.
  • Swabs: 1.5%
    The conjunctiva is swabbed, collecting tear fluid along with conjunctival cells.
  • Conjunctival impression cytology: 1.1%
    A membrane or filter paper is applied to the ocular surface to collect its outermost cell layers, and some tear fluid is obtained along with the cells.
  • Contact lenses: 0.3%
    Tear fluid is collected simply by removing the worn lens from the ocular surface.
  • Other: 8%
    Rarely reported methods, including micropipettes, filter paper discs, polyester wicks, and polyester rods, are grouped together.

3. Detailed comparison of collection methods

This table summarizes the characteristics of each collection method as reported in the literature. For comparison, the characteristics of SMTube are shown for the same items (highlighted column).

The table scrolls horizontally; the first column stays in place.

CriterionGlass Capillary TubesSchirmer StripsSpongesOcular Surface WashSwabsConjunctival Impression CytologyContact LensesSMTube
Collection volume / QuantificationDraws 5–10 µL from the forniceal reservoir of the conjunctival sac by capillary action[1]. The collected tear fluid volume itself is recorded[4]The wetting length (mm) is recorded but does not correspond directly to a specific volume[1]. Volumes vary between manufacturers[4]Volume can be estimated by weighing the sponge before and after collection, but retention within the material markedly reduces recoverable components[1]Dilutes the collected tear fluid, preventing accurate measurement of the original volume[1]Collects an undetermined volume and may retain analytes within the material, complicating precise quantification[1]Not specifically designed for tear collection; some tear fluid is obtained along with the cells[1]Protein deposition on the lens and absorption may reduce or deplete specific tear fluid components[1]—
Ocular surface contact and irritationMinimal contact with the ocular surface, thereby avoiding conjunctival irritation[1, 4]Irritation of the ocular surface after removal of the strip, particularly in DED patients[1]Placed in the lower tear meniscus for a set time; mechanical contact with the ocular surface[1]Flushing the ocular surface with a fixed volume of saline; a gentle way of collection[1]Collected by swabbing the conjunctiva[1]A membrane or filter paper is applied to the ocular surface[1]Associated with low irritation and can be worn for a long time, but even normal, uncomplicated lens wear can trigger inflammation[1]Touched to the lower tear meniscus only, without being inserted into the conjunctival sac[5]
Reflex tearingPerformed with care to avoid touching the ocular surface epithelium, to prevent reflex tearing[1]Can stimulate reflex tearing[4]Mechanical contact may induce reflex tearing[1]—The mechanical swabbing action may stimulate reflex tearing[1]——Strip meniscometry was reported to induce no reflex tearing[6]
Cellular contaminationAvoids collection of epithelial cells[1, 4]Collects conjunctival epithelial cells[1, 4]——Collects tear fluid along with conjunctival cells[1]Collects the outermost layers of the ocular surface[1]——
Time and techniqueSlow to perform and requires well-trained, skilled hands; difficult in some participant groups[1, 4]Placed in the lower conjunctival sac for about 5 minutes[4]. Inexpensive and easy to learn[1]Generally easy and quick to perform[1]After instillation the participant closes the eye and rolls the eyeball several times before collection with a glass capillary or micropipette[1]A simple and inexpensive process[1]—Collected simply by removing the lens from the ocular surface; user-independent[1]Contact for 5 seconds per eye, without anesthesia[5]

* "—" indicates not described in the cited literature.

4. Collection methods by research area

How the collection method affects results

Reflex tearing increases the tear fluid flow rate and dilutes tear fluid components, so topical anesthetics are sometimes used to ensure that only basal tear fluid is collected; the impact of anesthetic eye drops on tear fluid composition, however, requires further research[1].

The level of proteins extracted from Schirmer strips is higher, and differs in content, compared with glass capillary tubes[1]. Fifty-four unique proteins were identified from Schirmer strips versus 13 from capillary tubes, with secreted (serum) proteins predominating in capillary samples and a combination of cellular and serum proteins in Schirmer strip samples[7]. In a comparison of samples collected from the same participants with 2- or 3-µL Microcap tubes (capillaries) and with Schirmer strips, the mean total protein of the Schirmer strip samples was reported to be ten times higher than that of the capillary samples[8]. In a within-donor comparison, Schirmer strips also recover ocular surface-derived cellular material, which broadens the detected proteome[9]. In contrast, the total protein content per 1 µL was comparable between the two methods, and the bands of the surfactant proteins (SP-A to SP-D) were more intense in capillary-collected tears[10].

Consequently, results obtained using different collection methods are not directly comparable[4, 11].

Dry eye disease, meibomian gland dysfunction, and other eye diseases

The profiling of inflammatory proteins in tear fluid is advancing in dry eye disease (DED)[12]. Increased expression of S100A6, S100A9, S100A8, S100A4, GSTP1, and annexin A1 has been reported in the tear fluid of dry eye patients, and most of these proteins are known to participate in inflammatory processes[12]. S100A8 and S100A9 have also been reported to correlate with meibomian gland dysfunction severity and redness[13].

Additionally, changes in BDNF in normal tension glaucoma (NTG) and markers like MMP-9 in keratoconus have been investigated[13].

Collection method: In the tear proteome study of dry eye and meibomian gland dysfunction described above, tear fluid was collected with sponges (discovery phase and validation 1) and with glass capillary tubes (validation 2)[12].

Neurodegenerative diseases (AD, PD, MS)

Changes in tear protein profiles have been reported in Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS)[14, 15].

Collection method: The studies covered by systematic reviews in this field used Schirmer strips and capillary tubes[14, 15], as well as sponges[15]. In two Parkinson's disease studies, basal tears (with topical anesthesia) and reflex tears (without anesthesia) were collected with Schirmer strips[14, 16].

Collection issues

In this field, the collection method and storage conditions affect the reliability of the results[14]. It is important to avoid inducing reflex tearing during sampling[14, 15]. Performing a Schirmer test without anesthesia yields mainly reflex tears[16]. Currently, there is only one published tear-collection protocol specific to AD[15].

Diabetes / Diabetic retinopathy

In a systematic review and meta-analysis of ocular complications of diabetes (19 studies, 1,413 participants), the tear fluid concentration of TNF-α was significantly higher in the group with complications than in controls or in diabetes without complications, and VEGF showed a similar trend[17]. IL-6 and IL-8 were also increased in the complications group, while lactoferrin, lysozyme, and IL-1RA were lower, with lactoferrin reaching significance[17].

Collection method: In the studies included in the meta-analysis, tear fluid was collected with methods such as Schirmer strips or glass capillary tubes[17].

Collection issues

In the same meta-analysis, a subgroup analysis by collection method (glass capillary versus Schirmer strip) showed differences in the heterogeneity values for some markers, suggesting that the collection method can introduce heterogeneity into the results[17].

Cancer (e.g., breast cancer)

A proteomic analysis of tear fluid from breast cancer patients reported increased expression of S100A8 (7.8-fold), S100A9 (10.2-fold), and galectin-3 binding protein (3.0-fold). On ELISA validation, S100A8 and S100A9 were significantly higher in breast cancer patients, whereas galectin-3 binding protein was higher in the control group[18].

Tear fluid is regarded as one of the biofluids used for biomarker discovery in breast cancer, alongside blood (serum/plasma), urine, saliva, and nipple aspiration fluid[2].

Collection method: In this proteomic analysis of tear fluid from breast cancer patients, tear fluid was collected with Schirmer strips[18].

Extracellular vesicles

Tear-derived extracellular vesicles (EVs, such as exosomes) are being investigated as a source of biomarkers for ocular and neurodegenerative diseases[19].

In a study using a system that isolates exosomes from about 10 µL of tears within 5 minutes, 904 proteins were identified, and CALML5, KRT6A, and S100P were shown to classify dry eye disease. In addition, 484 microRNAs were investigated, and miR-145-5p, miR-214-3p, miR-218-5p, and miR-9-5p were reported to be dysregulated during the development of diabetic retinopathy[20].

Collection method: The tear extracellular vesicle studies covered by the review used Schirmer strips and ocular surface wash[19]. In the exosome isolation study described above, tear fluid was collected with Schirmer strips[20].

Collection issues

There is currently no standardized protocol for tear sample handling, from collection to analysis[19]. The first challenge is identified as the method of tear collection[19].

5. State of standardization

There is currently no standardized recommendation for collection methods[1, 4].

The TEAR-RG guidelines standardize reporting, not the collection method itself[21]. A global practice survey has also been conducted to assess current methods[22].

Manufacturers and researchers are expected to align with standards proposed by networks such as the Tear Research Network (TRN) in the future[11].

References

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