Objective assessment of the drainage function of the lacrimal drainage system based on the dye disappearance test

Introduction

Modern dacryology possesses methods for sufficiently accurate diagnosis of the anatomical state of the lacrimal drainage system. For this purpose, radiological methods are used, having preliminarily filled the lumen of the lacrimal passages with a radiopaque contrast agent. The spatial resolution of currently available clinical instruments allows reliable assessment of objects with a linear size of less than 0.5 mm, which is sufficient for the current development of lacrimal surgery (1).

At the same time, the analysis of the actual drainage function of the lacrimal drainage system remains a complex diagnostic problem to this day. All available methods are based on detecting the disappearance rate of a certain substance from the ocular surface after its instillation. Known tests include the dye disappearance test, where a colored solution is instilled into the conjunctival cavity (a separate variation involves instilling a substance with a characteristic taste) (2), as well as lacrimal scintigraphy, where a radiopharmaceutical is instilled and its kinetics are observed using a γ-camera (3). The first method is most commonly used in clinical practice because it is simple to implement, reproducible, and economically feasible, although it is not without drawbacks. The main disadvantage is the difficulty in interpreting the obtained data, which is associated with a certain subjectivity in assessing the volume of dye remaining on the ocular surface, as well as the non-standard volume of dye instilled into the conjunctival sac.

Syringing (irrigation) of the lacrimal drainage system is considered the gold standard clinical test for assessing anatomical lacrimal obstruction. This procedure involves flushing the lacrimal passages with saline to determine patency. While highly valuable for confirming anatomical obstruction, syringing has limitations: it is not a quantitative test, it cannot assess the degree of partial obstruction, and when the lacrimal pathway is patent, it does not provide reliable information about functional tear drainage capacity (functional epiphora).

All this explains the feasibility of developing new methods for assessing the drainage function of the lacrimal drainage system that would combine the advantages of known methods while eliminating the subjectivity in interpreting the obtained data.

The objective of this study was to develop an objective method for assessing the drainage function of the lacrimal drainage system based on the dye disappearance test. We present this article in accordance with the STARD reporting checklist (available at https://qims.amegroups.com/article/view/10.21037/qims-2025-757/rc).

Methods

The study was conducted at the Krasnov Research Eye Institute. All patients were recruited from the outpatient clinic of this institution. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of the Krasnov Research Institute of Eye Diseases (No. 2020-31) and informed consent was obtained from all participants.

The study included 70 patients (140 observations), comprising 60 cases without anatomical lacrimal drainage impairment, 50 cases with dacryostenosis, and 30 cases with lacrimal duct obstruction. The mean age of patients was 58±16 years (range, 23–90 years). The diagnosis was established based on clinical data, and in cases of dacryostenosis or lacrimal duct obstruction, it was confirmed by computed tomography with contrast dacrycocystography (CT-DCG). Patients with dacryostenosis and complete obstruction presented with epiphora symptoms, while those without lacrimal drainage impairment did not report epiphora.

A priori power analysis based on preliminary data (expected effect size d=0.8, α=0.05, power =80%) indicated that a minimum of 64 observations per group comparison would be required. Given the three-group design and paired-eye data, we enrolled 70 patients (140 eyes), which provided sufficient power for the primary outcome (fluorescein retention at 5 minutes). Post-hoc power analysis confirmed 92% power to detect intergroup differences (P<0.001).

All patients underwent the dye disappearance test as follows. The tear film was stained with fluorescein using fluorescein sodium ophthalmic strips United States Pharmacopeia (USP) (Contacare Ophthalmic and Diagnostics, Vadorara, India). Fluorescein sodium ophthalmic strips (10% w/v) were moistened with 20 µL of sterile saline immediately before use, delivering approximately 5–7 µL of a 0.5–1.0% fluorescein solution to the conjunctival sac—consistent with clinical standards and described previously in other protocols. To minimize fluorescence quenching, patients were instructed to blink gently three times to ensure uniform tear film distribution, and all imaging was performed within the linear response range of the camera sensor, as verified by preliminary calibration. Ocular surface photography was performed under standardized environmental conditions: room temperature maintained at 22–24 ℃ and humidity at 40–60%. This controlled for potential effects of external factors on tear film evaporation and ensured test consistency. After 1 minute, the ocular surface was photographed under cobalt-blue light with a Wratten yellow filter on the slit lamp objective; similar photographs were taken at 3 and 5 minutes. Imaging was performed using a Dixion S390 photo-slit lamp (Mediworks, Shanghai, China) and corresponding software. An example set of eye images submitted for analysis is shown in the Figure 1.

Figure 1 Example of eye images after dye instillation. The upper row shows the right eye of a patient with normal drainage function of the lacrimal system, while the lower row presents the left eye of the same patient, a case of dacryostenosis. The images were taken at 1, 3, and 5 minutes after dye instillation. The number of green pixels is indicated, based on which the fluorescein retention rates were calculated.

Using ImageJ (National Institute of Health, USA), areas corresponding to the “green” color in the HSB (hue, saturation, brightness) color scheme (hue: 40–130; saturation: 60–255; brightness: 30–255) were selected on the images. Although the images appear monochromatic under cobalt-blue excitation, background structures (e.g., eyelashes, conjunctival vessels, corneal reflections) can contribute non-specific signal in grayscale. Using the HSB color space allows selective isolation of the true fluorescein emission (green band) based on hue, while excluding artifacts using saturation and brightness thresholds, thereby improving segmentation specificity. The total number of green pixels on the first (1 minute), second (3 minutes), and third (5 minutes) images was calculated. The fluorescein retention was then computed as the proportion of green pixels on the second and third images relative to the number of green pixels on the first image, which was taken as 100%. Similarly, the fluorescein retention was calculated for the time interval between 1 and 5 minutes.

The obtained fluorescein retention values were compared among patients in three groups (without anatomical lacrimal drainage impairment, with dacryostenosis, and with lacrimal duct obstruction), as well as separately between two groups (without impairment and with impaired lacrimal drainage).

Statistical analysis was performed using parametric methods, as all values followed a normal distribution. The analysis was conducted using IBM SPSS Statistics 26 (IBM, USA). The distribution was assessed using the Shapiro-Wilk test, differences between groups were evaluated using t-tests for two-group comparisons and analysis of variance (ANOVA) for three-group comparisons. Our primary hypothesis was that digital quantification of dye retention would significantly differ between eyes with and without anatomical lacrimal obstruction. The secondary hypothesis was that combining retention values from multiple time points would improve diagnostic accuracy. Because both eyes of each patient were included, we accounted for inter-ocular correlation using a linear mixed-effects model with patient ID as a random effect for group comparisons. For receiver operating characteristic (ROC) and logistic regression analyses, we used generalized estimating equations (GEE) with an exchangeable correlation structure. The logistic regression model was developed to create a multivariate diagnostic tool leveraging the most informative time intervals (1–3 and 1–5 minutes). Differences were considered significant at P<0.005. ROC analysis was performed to assess sensitivity and specificity, with the optimal threshold defined as the point on the ROC curve with the maximum Youden index.

Results

Descriptive statistics for fluorescein retention are presented in Table 1.

Statistical analysis revealed that fluorescein retention at 3 minutes did not differ significantly (P=0.278 for three-group comparison and P=0.115 for two-group comparison). In contrast, fluorescein retention at 5 minutes showed statistically significant differences (P<0.001 for both three-group and two-group comparisons), as did the clearance between 3 and 5 minutes (P<0.001 for both comparisons). The threshold values for the parameters analyzed are shown in Table 2.

A logistic regression model was trained using a combination of fluorescein retention parameters at 1–3 and 1–5 minutes. The model coefficients were: intercept b₀=–5.0; b₁=3.0; b₂=4.0. The sensitivity of this model for detecting lacrimal drainage pathology was 88%, specificity was 93%, and area under the curve (AUC) was 0.95.

Discussion

The dye disappearance test for assessing the drainage function of the lacrimal system has been used for a long time, but its first formal description dates back to 1961 (4). The study involved instilling one drop of a 0.5% fluorescein solution into the conjunctival sac. After an exposure time of 1–5 minutes, a moistened cotton wick was placed under the anterior end of the inferior nasal concha using a probe. The test was considered positive if the cotton wick became stained. Studies on the diagnostic value of this method, known as the Jones test, showed that it was positive in 78% of subjects without lacrimal drainage pathology (5). The authors noted that in some cases, dye partially remained on the ocular surface by the end of the test. A formal assessment of dye retention on the ocular surface was proposed, using a scoring system. When no reduction in tear film staining intensity was observed during the test, the result was scored as 4 points, whereas complete disappearance of staining was scored as 0 points. Intermediate states were scored as 1, 2, or 3 points, depending on the staining intensity (6). The authors reported a 95% positive result rate in subjects without lacrimal drainage impairment, confirming the diagnostic advantage of this method over the classic Jones test. In subsequent years, this scoring system became widely used under the name “fluorescein dye disappearance test” (2,7), while in Russian literature, the term “canalicular color test” (proposed by JM West in 1918 for a different diagnostic procedure) is traditionally used, although scoring is typically not employed (8). The simplicity of this method likely contributed to its widespread adoption, but it still does not allow direct quantitative assessment of the lacrimal drainage function.

The concept of tear clearance was actively developed in the 1980s. Various automatic and semi-automatic methods were proposed to determine clearance values, initially based on direct continuous fluorophotometry: a technique involving the measurement of light intensity emitted by fluorescein dissolved in the tear film (9,10). However, the results of this test are significantly influenced not only by tear drainage through the lacrimal system but also by tear secretion. These methods are complex and labor-intensive. Later, methods for assessing tear clearance by analyzing tear meniscus morphometric parameters using optical coherence tomography were proposed, providing some insight into tear production but limited clinical information about lacrimal drainage function (11,12). The addition of polymethylmethacrylate particles to the tear film improved diagnostic value by enabling the observation of particle movement in the tear film (13).

In the 1950s, a colorimetric method for assessing tear clearance was proposed (14), and its diagnostic value was later thoroughly investigated (2). A drop of fluorescein solution is instilled into the conjunctival sac. After 3 minutes, the degree of fluorescein retention on the ocular surface is assessed. A Schirmer test strip is placed under the eyelid and left for 5 minutes with the eyes closed. Ten minutes after fluorescein instillation, the test strips are reinserted for another 5 minutes. The degree of strip staining is then compared to reference samples stained with known fluorescein concentrations. This method can be implemented in practice but is time-consuming and requires a constant supply of reference strips, making it cumbersome and limiting its popularity.

Our proposed method combines the simplicity of the “canalicular color test” with the objectivity of fluorophotometry. It involves sequential assessment of the number of green pixels corresponding to fluorescein dissolved in the tear film, followed by calculation of the difference expressed as a ratio. As the results show, fluorescein retention values computed using this method significantly differ between normal and anatomical impaired lacrimal drainage, with the 5-minute assessment demonstrating slightly higher diagnostic value than the 3-minute assessment. This supports the recommendation of this method for clinical practice. These temporal patterns likely reflect the biphasic drainage dynamics in lacrimal system pathophysiology. During the initial phase (0–3 minutes), residual pump function of the lacrimal sac—mediated by orbicularis oculi contraction during blinking—may facilitate fluorescein clearance even in partial obstruction. This mechanism becomes overwhelmed as the sac reaches saturation capacity (typically by 3–5 minutes), revealing the true drainage impairment. The delayed diagnostic signal (≥5 minutes) thus represents exhaustion of this physiological reserve. To verify this hypothesis, it would be valuable to conduct further studies in patients with atonic epiphora or complete punctal obstruction where the lacrimal sac’s suction function is known to be absent.

The proposed method has several limitations. False-positive results (e.g., clearance >85% without pathology) may occur due to uneven fluorescein distribution, particularly in patients with unstable tear films. False negatives (clearance <55% with confirmed obstruction) might result from tear hypersecretion masking drainage impairment. To minimize errors, we recommend: conducting tests after patient acclimatization to stabilize tear production, excluding patients with acute conjunctivitis or severe dry eye, implementing automated region-of-interest (ROI) selection in ImageJ to exclude eyelid and lash artifacts. It should be noted that our study assessed anatomical lacrimal drainage impairment confirmed by CT-DCG, rather than functional impairment. While anatomical obstruction typically correlates with functional impairment, there can be cases of functional epiphora without anatomical obstruction, which would require lacrimal scintigraphy for proper diagnosis. It should also be noted that retention values exceeding 100% were observed in some obstructed eyes. This likely reflects increased fluorescein concentration due to tear film evaporation in the absence of effective drainage, or transient pooling in the inferior fornix. Our method does not aim to quantify absolute fluorescein concentration but to provide a reproducible relative index that distinguishes pathological from normal drainage. Since this phenomenon occurs predominantly in eyes with impaired outflow, it does not compromise the method’s diagnostic utility for differentiating “normal” from “pathological” drainage function.

Compared to syringing, our method provides a quantitative assessment of drainage function rather than a binary patent/obstructed result. While syringing remains essential for confirming anatomical patency, our method offers additional information about the functional capacity of the drainage system, which may be particularly valuable in cases of partial obstruction where syringing may show patency despite functional impairment.

The method’s simplicity and objectivity make it suitable not only for integration with existing ocular surface analyzers but also for development of dedicated software. A mobile application utilizing machine learning algorithms could: automatically detect stained areas in smartphone images captured via slit-lamp adapters, provide real-time clearance percentage calculations, compare results against normative database values. Such an application would reduce reliance on expensive equipment and facilitate primary care screening.

This standardized approach holds particular value for research applications by: enabling multi-center data comparison without inter-observer variability, establishing quantitative inclusion criteria for clinical trials, objectively monitoring post-intervention dynamics (e.g., post-dacryocystorhinostomy).

Conclusions

Our study presents a novel quantitative approach to assess lacrimal drainage function using digital analysis of fluorescein retention patterns. The method demonstrates significant diagnostic value, with clearance measurements at 5 minutes showing superior discriminative capacity (AUC 0.95) compared to the 3-minute evaluation, effectively differentiating between normal drainage, partial obstruction, and complete obstruction cases. The technique combines the practicality of traditional dye tests with objective quantification through standardized image analysis, overcoming the subjectivity limitations of conventional methods while maintaining clinical feasibility.

These findings suggest that temporal analysis of fluorescein retention provides valuable insights into both functional and anatomical aspects of lacrimal drainage. The method’s ability to detect progressive changes in clearance rates makes it particularly suitable for monitoring disease progression and treatment outcomes in clinical practice, while its standardized approach offers potential for multicenter research applications. Future refinements should focus on automating the analysis pipeline and establishing normative values across different patient populations to further enhance diagnostic accuracy.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://qims.amegroups.com/article/view/10.21037/qims-2025-757/rc

Data Sharing Statement: Available at https://qims.amegroups.com/article/view/10.21037/qims-2025-757/dss

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://qims.amegroups.com/article/view/10.21037/qims-2025-757/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of the Krasnov Research Institute of Eye Diseases (No. 2020-31) and informed consent was obtained from all participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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