Download Diapositiva 1 - ASCRS/ASOA 2010

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


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
Transition band
Three regions are identified:
I
II
III
I: Hyaline or amorphous
II: Major ferns
III: Minor ferns
A fourth component (Transition
band) is usually present
between regions I and II
TEMPORO-SPATIAL SEQUENCE IN NORMAL TEAR FERNING
(16ºC; <60% RH)
0
5
6 (min)
Early
(slow)
Mid
8
8.15
8.30 (min)
Cont.
TEMPORO-SPATIAL SEQUENCE IN NORMAL TEAR FERNING
(16ºC; <60% RH)
8.40
9.00
(min)
Late
(fast)
Final
9.15
9.30
(min)
Contact
inhibition
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)
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.