NEW INSIGHTS INTO THE TEAR FERNING TEST FOR DRY EYE Remigio López-Solís1, Leonidas Traipe2, Allister Gibbons2, Daniela Salinas1,2 1 ICBM (Biología Celular y Molecular), Facultad de Medicina, Universidad de Chile 2 Fundación Oftalmológica Los Andes No finantial relationships between the authors and any company or person exist in regard to the present study

Background Objectives Fern-like crystalloids are formed when a sessile drop of tear fluid is dried onto a glass surface: tear ferning Reduced ferning (Rolando’s scores III and IV) usually occurs among Dry Eye patients The mechanism of this alteration is poorly understood. The process of ferning formation has not been analyzed systematically Objectives General: To contribute to the characterization of normal human tear fluid by the analysis of ferning images Specific: To investigate the mode of progression of a normal Tear Ferning as well as its eventual alterations in tear samples from Dry Eye patients.

Methods Subjects: Healthy adult volunteers of both sexes, 22-50 y.o. (n=30) who were not displaying blepharitis, allergy, Dry-Eye or had not experienced ocular surgery were included. Those subjects were not consumers of medication, alcohol or cigarrettes and were not contact-lens wearers. Patients with Dry Eye (n=10, AAO and OSDI criteria) secondary to various rheumatologic or dermatologic diseases, were also included. In all cases, informed consents were obtained. Tear Collection: Absorption by positioning polyurethane minisponges on the outer third of the margin of the lower lid for 3 min (see Figure). Details in Cornea 25 (3), 312-318 (2006). Ferning test: A tear aliquot (1 microliter) was placed on the surface of a glass slide and positioned under a dark-field microscope. Video images (230/sec) were taken using a Canon G10 camera and set on-line with a PC screen. Image capture and processing were performed using conventional software.

Tear fluid collection and ferning assay 1. Absorption with polyurethane minisponges; 2 + 3. Transference of tear fluid to Eppendorf tubes by centrifugation; 4. One µL of tear on glass surface; 5. Ferning progression and image capture

Tear Ferning from a healthy subject III II I Transition band Three regions are identified: I: Hyaline or amorphous II: Major ferns III: Minor ferns A fourth component (Transition band) is usually present between regions I and II

Early Mid TEMPORO-SPATIAL SEQUENCE IN NORMAL TEAR FERNING 0 5 6 (min) (16ºC; <60% RH) 0 5 6 (min) Early (slow) Mid 8 8.15 8.30 (min)

Late Final Contact inhibition Cont. TEMPORO-SPATIAL SEQUENCE IN NORMAL TEAR FERNING (16ºC; <60% RH) 8.40 9.00 (min) Late (fast) Final Contact inhibition 9.15 9.30 (min)

EFFECT OF TEMPERATURE AND HUMIDITY ON FERNING PRODUCTION Long ferns at Region II 16°C; RH < 60% Control drying (9 min) 6°C ; RH < 60% Slow drying (> 17 min) 6°C; >80% RH No ferning (> 30 min)

Lipids? TEAR LIPIDS IN FERNING ORGANIZATION Intense spontaneous browning of an outer region (close to Transition Band) in an aged ferning suggests its oxidable lipidic character Differential distribution of a lipid dye during tear ferning SUDAN III Lipids? SUDAN BLACK BSA solution TEAR 1 mg/mL BSA, bovine serum albumin served as control. Dyes were mixed with tear fluid at a 1/0.1 v/v ratio just before ferning test

Size of fern-like tear crystalloids (region III) depends on the rate of drying One-µL aliquot of tear from a healthy subject was dried under standard laboratory conditions (drying time: 9.5 min) In parallel, one-µL aliquot from the same tear sample was dried under mild vacuum (drying time: 6 min). Note the significantly smaller fern-like crystalloids in region III

ALTERED FERNINGS IN TEAR SAMPLES FROM DRY EYE PATIENTS Altered regions II and III In both examples, Region II is absent. Both appearances comprising small fern-like crystalloids in region III would suggest a high-drying rate (accelerated evaporation). However, a significant outer lipidic layer is also present.

Conclusions Ferning formation is highly dependent on the environmental conditions. Temperature affects positively drying rate. Relative Humidity affects negatively ferning formation. In a normal ferning some basic regions (I, II, III and transition band) can be identified. These are formed asynchronously: I is the first one and precedes II and this latter one precedes III. II and III are formed rapidly at the end of the process. Tear lipids become mostly located at Region I during ferning formation. II and III are contact-inhibited during their formation in a normal ferning. Thus, a slow drying would allow a more developed region II (long ferns) while a fast drying results in a high number of small ferns at region III. The regional structure of a ferning becomes markedly altered in different ways when the assay is performed on tear samples from Dry Eye patients. Small ferns at region III, which denotates accelerated evaporation, and absence of region II, which is a “fern-organizer structure”, are frequent observations.