Inflammatory cytokines play a critical role in the pathogenesis of allergic conjunctivitis and represent important targets for tear-based diagnostics. Th2-type cytokines, particularly interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13), are central mediators in allergic inflammatory responses. Specifically, studies have shown that Th2 pathway-associated cytokines are important in the pathogenesis of Behçet’s disease. Literature indicates that IL-4 levels are elevated in the tears of patients with VKC, AKC, and SAC [7]. IL-5 levels are increased in ocular allergy patients with proliferative lesions (P < 0.05), mainly promoting the activation, proliferation, and differentiation of eosinophils [8]. IL-13 is elevated in the tears of AKC patients (median: 197.1 pg/mL), significantly higher than healthy controls (median: 90.4 pg/mL, P < 0.001), VKC patients (median: 60.0 pg/mL, P < 0.05), and SAC patients (median: 80.9 pg/mL, P < 0.001), but its level is not associated with severe complications in AKC [9]. Among all allergic conjunctivitis patients and controls, tear IL-13 and periostin levels are weakly but significantly positively correlated (r = 0.3329, P < 0.05) [9]. However, tear periostin is elevated in AKC patients (median: 444.0 ng/mL), and its level is significantly associated with severe complications. In the large papillae group, periostin is markedly higher (median: 678.8 ng/mL) than in the no/mild papillae group (median: 23.6 ng/mL, P < 0.001), and is also significantly higher in the corneal damage-positive group (median: 543.7 ng/mL) compared to the negative group (median: 77.0 ng/mL, P < 0.01) [9]. Therefore, tear periostin is an effective biomarker for assessing AKC disease severity [9]. In addition to Th2-type cytokines, other inflammatory cytokines also play important roles in the pathogenesis of allergic conjunctivitis. Tumor necrosis factor-α (TNF-α) is significantly elevated in the tears of VKC patients [10]. Studies indicate that interleukin-6 (IL-6) and its soluble receptor (sIL-6R) are key in allergic conjunctivitis. In VKC and giant papillary conjunctivitis (GPC) patients, tear sIL-6R levels are significantly increased, with net optical density values over eight times higher than healthy controls, and this elevation is statistically significant (VKC group P < 0.01; GPC group P < 0.01) [11]-[14]. In chronic inflammatory conditions such as dry eye, sIL-6R is also characteristically upregulated, as seen in the tears of patients with Sjögren’s syndrome (2.38 ± 0.98 ng/mL), which is significantly higher than normal controls (0.16 ± 0.34 ng/mL, P < 0.01) [11]. These findings suggest that sIL-6R is a potential quantitative indicator of ocular surface inflammatory activity. Interleukin-1β (IL-1β) is elevated in all types of allergic conjunctivitis and is involved in the initiation and amplification of the inflammatory cascade [12].

Chemokines play a central role in the recruitment and activation of inflammatory cells in allergic conjunctivitis. CCL17/thymus and activation-regulated chemokine (TARC) and CCL24/eotaxin-2 have recently attracted significant research attention. Simultaneous detection of CCL17/TARC, CCL24/eotaxin-2, and IL-16 levels in tear fluid helps differentiate between acute and chronic allergic inflammation [13]. Studies have demonstrated that the levels of CCL17/TARC, CCL24/eotaxin-2, and IL-16 in the tears of patients with AKC and VKC are significantly higher than those in patients with AC. Notably, CCL24/eotaxin-2 shows a distinct and significant increase in VKC patients’ tears, with its net optical density intensity more than eight times higher than that of healthy controls, a difference that is statistically significant (P < 0.05) [14]. Antibody array analysis further confirmed that both eotaxin-1 (CCL11) and eotaxin-2 (CCL24) are markedly upregulated in the tears of VKC and GPC patients [14]. Monocyte chemoattractant protein-1 (MCP-1/CCL2) is elevated across all types of allergic conjunctivitis and is involved in the recruitment of monocytes/macrophages [15]. Additionally, research from other mucosal tissues also provides supporting evidence for the ocular surface chemokine network. Research indicates that in an HTLV-I-infected salivary gland epithelial cell (SGECs) model, the expression of RANTES/CCL5 is significantly upregulated in a time-dependent manner. Compared with the baseline at 0 hours without co-culture, the concentration of RANTES/CCL5 in the supernatant after 96 hours of co-culture is significantly increased (P < 0.05) [16]. Since mucosal epithelial cells (including those of the ocular surface, respiratory tract, and salivary glands) often share similar pattern recognition receptors and inflammatory signaling pathways (such as NF-κB) when responding to pathogens or inflammatory stimuli, this finding suggests that in the inflammatory environment of allergic conjunctivitis, activated ocular surface epithelial cells may also serve as an important source of RANTES/CCL5.

In patients with seasonal allergic conjunctivitis (SAC), total tear IgE levels are significantly higher than those in healthy controls (p < 0.01) and show a significant positive correlation with total serum IgE levels (r = 0.44, p < 0.01) [17]. More importantly, tear IgE levels can more directly reflect the allergic status of the ocular surface. Improvement in symptoms and therapeutic response in SAC patients are significantly associated with decreased tear IgE concentrations (correlation coefficient r = −0.71, P < 0.001); the tear IgE level in patients with recurrence (278.5 ± 40.2 IU/mL) is significantly higher than in those without recurrence (125.3 ± 22.7 IU/mL), independent samples t-test t(56) = 9.34, P < 0.001 [18]. Recent studies have found that point-of-care testing (POCT) for tear IgE can effectively distinguish between type I and type IV allergic conjunctivitis, thereby providing guidance for personalized treatment [19]. Secretory immunoglobulin A (sIgA) plays an important role in maintaining ocular surface immune homeostasis. Studies have shown that the total sIgA level in tears of VKC patients (28.7 ± 6.8 µg/mL) is significantly lower than that of healthy controls (45.2 ± 5.1 µg/mL), with an independent samples t-test result of t(38) = 8.45, P < 0.001 [20]. The role of specific IgA antibodies in allergic conjunctivitis requires further investigation [21].

Matrix metalloproteinase-9 (MMP-9) plays a crucial role in corneal wound healing and inflammatory responses. Elevated levels of MMP-9 are observed in the tear fluid of patients with allergic conjunctivitis. Secretory phospholipase A2 Group IIa (sPLA2-IIa) is also upregulated in the tears of allergic conjunctivitis patients, potentially contributing to tear film instability by degrading the lipid layer and thereby exacerbating ocular surface inflammation [22]. Eosinophil cationic protein (ECP) is a specific marker for eosinophil activation. ECP levels are elevated in the tears of all types of allergic conjunctivitis patients, with the highest concentrations found in those with AKC and VKC [23]. ECP levels are significantly correlated with clinical severity; the mean ECP concentration in the severe group (38.74 ± 5.51 ng/mL) is significantly higher than in the moderate (35.42 ± 6.39 ng/mL) and mild groups (31.13 ± 8.74 ng/mL). One-way ANOVA indicates that the differences between groups are statistically significant (F = 9.413, P < 0.001), and post hoc pairwise comparisons (LSD-t test) show significant differences between the severe and moderate groups as well as between the severe and mild groups (P < 0.05), making ECP an important indicator of disease activity [24].

Neurotransmitters play a crucial role in both the itching symptoms and the maintenance of chronic inflammation in allergic conjunctivitis. Substance P levels are elevated in the tears of patients with SAC and VKC, contributing to neurogenic inflammatory responses [25]. Nerve growth factor (NGF) is increased in the tears of patients with AKC and VKC and may be involved in sustaining chronic neuropathic pain [26]. Vasoactive intestinal peptide (VIP) levels rise significantly in tears following allergen challenge, which may be associated with the exacerbation of allergic symptoms [27]. Additionally, thymic stromal lymphopoietin (TSLP) derived from epithelial cells plays a key role in initiating and amplifying Th2-type immune responses. Studies show that TSLP levels in the tears of VKC patients are significantly elevated and correlate with disease severity [28]. IL-13, which acts in synergy with TSLP, is not only a core cytokine in the Th2 pathway but also plays an important role in the dialogue between epithelial and immune cells, serving as a potential biomarker for disease subtyping and treatment response evaluation [9][28].

Enzyme-linked immunosorbent assay (ELISA) is a classical method for detecting tear fluid biomarkers, distinguished by its high specificity and good reproducibility. However, ELISA suffers from drawbacks such as low throughput, large sample requirements, and lengthy detection times, limiting its widespread adoption in routine clinical practice [29]. Although radioimmunoassay (RIA) offers high sensitivity, it poses risks of radioactive contamination and is now used less frequently.

The development of multiplex bead assay technology has greatly enhanced the efficiency and throughput of tear fluid analysis. This technique enables the simultaneous detection of dozens of biomarkers within a single sample, significantly reducing the required sample volume. Studies utilizing multiplex bead assays have measured cytokines such as IFN-γ, TNF-α, IL-2, IL-4, IL-5, and IL-10, and found that IL-10 levels are decreased in the tears of allergic patients, while the ratios of TNF-α/IFN-γ, IL-5/IFN-γ, and IL-5/IL-10 are significantly increased [12]. Membrane-based antibody array technology represents another multiplex detection approach. Using this method to assess 16 inflammatory mediators, researchers observed enhanced signals for IL-2, IL-4, IL-5, and IFN-γ in open-eye samples from allergic patients, and elevated levels of IL-1α and TNF-α in closed-eye samples [30]. Liquid-phase chip technology has also demonstrated advantages in the detection of tear cytokines [31].

The development of proteomics technologies has provided powerful tools for the discovery of tear fluid biomarkers. iTRAQ quantitative proteomics analysis revealed that levels of hemoglobin, transferrin, heme-binding protein, mammaglobin B, and secretoglobin 1D1 were significantly elevated in the tears of VKC patients compared to normal controls [32]. Liquid chromatography-mass spectrometry (LC-MS) offers higher precision and accuracy, enabling the identification of 890 proteins in tear samples as small as 4 - 10 μL, with high reproducibility [7]. Data-independent acquisition (DIA) mass spectrometry represents a significant advancement in recent years. This technique allows for the detection of all detectable proteins in a sample within a single analysis, providing more comprehensive data for biomarker discovery. Studies have demonstrated that DIA mass spectrometry has great potential in tear proteomics, with the promise of identifying additional disease-specific biomarkers [33]. Preliminary results from DIA-MS-based tear proteomics have already been achieved in AC research [34].

The development of point-of-care testing (POCT) technologies has provided significant support for the clinical translation of tear fluid diagnostics. The i-ImmunDx™ tear IgE detection platform can complete testing within 15 minutes using only a minimal tear sample, greatly enhancing clinical convenience [18]. This platform demonstrated strong diagnostic performance in distinguishing between type I and type IV allergic conjunctivitis, with an area under the ROC curve of 0.896 [19]. This rapid and user-friendly testing method assists clinicians in promptly assessing the ocular surface inflammatory status of patients and guiding treatment decisions [35][36].

Tear fluid analysis demonstrates significant value in the differential diagnosis of allergic conjunctivitis subtypes. Distinct types of allergic conjunctivitis are associated with unique profiles of tear fluid biomarkers. In patients with VKC, levels of IL-5, RANTES, and eotaxin-1 are significantly higher than those observed in SAC patients, facilitating differential diagnosis [16]. Tear fluid in AKC patients is characterized by elevated IL-4, whereas GPC is primarily associated with increased IL-8 [37]. The combined detection of multiple biomarkers enables more accurate disease classification and provides a basis for personalized treatment [38].

The levels of tear fluid biomarkers are closely associated with the severity of allergic conjunctivitis. Previous studies have confirmed that MMP-9 levels are elevated in the tears of contact lens wearers and are significantly correlated with corneal involvement and the formation of giant papillae, reflecting the extent of structural damage to ocular tissues [21][39]. One key study demonstrated that during contact lens wear, the concentration of tear MMP-9 (median: 4.6 ng/mL; range: 0.0 - 41.7 ng/mL) was significantly higher than the baseline level prior to lens wear (median: 0.0 ng/mL; range: 0.0 - 0.0 ng/mL), P < 0.001 [21][39]. Changes in the levels of HA, TSLP, ECP, and other markers in the tears of pediatric AC patients can also reflect disease severity [40].

Tear fluid analysis provides objective indicators for monitoring the therapeutic efficacy of allergic conjunctivitis. Studies have shown that levels of multiple biomarkers in patient tears decrease significantly following effective treatment. In AKC patients treated with tacrolimus eye drops, tear ECP levels decreased markedly from 2680.22 ± 2342.7 ng/ml before treatment to 195.71 ± 164.46 ng/ml [41] (P < 0.05). In AC patients treated with emedastine eye drops for seven days, tear IL-16 levels significantly decreased (P < 0.05), corresponding with improvements in clinical symptoms [13]. Dynamic changes in tear cytokines may serve as sensitive indicators of therapeutic response [42].

Tear fluid analysis can assist in guiding personalized treatment for allergic conjunctivitis. By measuring tear IgE levels, it is possible to differentiate between IgE-mediated type I hypersensitivity reactions and non-IgE-mediated type IV hypersensitivity reactions, thus providing a basis for selecting appropriate anti-allergic medications [19]. For patients exhibiting elevated levels of specific cytokines in tear fluid, targeted therapies may be considered. For instance, patients with increased IL-4 and IL-13 levels may benefit from anti-IL-4/IL-13 receptor antibody therapy [43]. In pediatric AC patients, the expression of non-coding RNAs such as miR-19b and miR-146a in tears may also serve as potential therapeutic targets [44].

Although tear fluid analysis demonstrates significant potential in the diagnosis and treatment of allergic conjunctivitis, several challenges remain. The first is the issue of standardization, as there is a lack of unified protocols for sample collection methods, storage conditions, and detection procedures, which affects the comparability and reproducibility of results [45]. The second challenge is cost control; high-throughput detection techniques such as proteomics analysis are expensive, limiting their adoption in routine clinical practice. Additionally, barriers to clinical translation exist, as the transition from research findings to clinical application requires rigorous validation, including large-scale clinical studies and assessment of diagnostic efficacy [46].

The future development of tear fluid detection technologies will primarily focus on the following aspects: First, establishing standardized testing procedures and quality control systems to enhance the comparability and reproducibility of results [38]. Second, developing more convenient, rapid, and cost-effective detection technologies to promote the widespread clinical application of POCT technologies [47]. Third, integrating multi-omics approaches, including proteomics, metabolomics, and transcriptomics, to construct a more comprehensive biomarker network [48]. For example, metabolomics can systematically analyze changes in small molecule metabolites in tears (such as lipids, amino acids, and energy metabolism products), revealing real-time metabolic reprogramming and oxidative stress states in ocular surface cells under allergic inflammatory conditions, and identifying more dynamic functional biomarkers distinct from proteins. Transcriptomics, on the other hand, can reveal molecular subtyping of the disease, activation of key signaling pathways, and potential new therapeutic targets at the gene regulation level by analyzing the expression profiles of extracellular RNA in conjunctival epithelial cells or tears. Finally, employing artificial intelligence and machine learning techniques to develop diagnostic models based on multiple biomarkers, thereby improving diagnostic accuracy and efficiency [49].