Dynamics of tear fluid desiccation on a glass surface: a contribution to tear quality assessment
© Traipe-Castro et al.; licensee BioMed Central Ltd. 2014
Received: 19 May 2014
Accepted: 20 May 2014
Published: 4 June 2014
Fern-like crystalloids form when a microvolume of tear is allowed to dry out at ambient conditions on a glass surface. Presence of crystalloids in tear “microdesiccates” is used to evaluate patients with Dry-Eye disease. This study aims to examine morphologically the desiccation process of normal tear fluid and to identify changes associated with accelerated tear evaporation. Tear microdesiccates from healthy (Non-Dry Eye) and Dry Eye subjects were produced at ambient conditions. Microdesiccate formation was monitored continuously by dark-field video microscopy. Additionally, accelerated desiccation of tear samples from healthy subjects was conducted under controlled experimental conditions. Particular morphological domains of tear microdesiccates and their progressive appearance during desiccation were compared.
In normal tear microdesiccates, four distinctive morphological domains (zones I, II, III and transition band) were recognized. Stepwise formation of those domains is now described. Experimentally accelerated desiccation resulted in marked changes in some of those zones, particularly involving either disappearance or size reduction of fern-like crystalloids of zones II and III. Tear microdesiccates from Dry Eye subjects may also display those differences and be the expression of a more synchronous formation of microdesiccate domains.
Morphological characteristics of tear microdesiccates can provide insights into the relative rate of tear evaporation.
Fern-like microcrystalloids are formed when some biological fluids are placed as sessile microdrops on glass surfaces and allowed to dry out at ambient conditions [1–3]. In the case of tear fluid, failure in the formation of fern-like structures has been closely associated with Dry Eye disease [4–6]. Up-to date, the Tear Ferning test using the Rolando’s scoring system has proven useful as a laboratory aid in Dry Eye assessment [4, 7–9]. Accordingly, abundance of fern-like tear crystalloids is suggestive of normality (scores I and II) whereas reduced tear ferning (scores III and IV) is usually observed among Dry Eye patients [5, 6, 10]. Based on our experience with over six hundreds tear ferning assays, desiccation of a tear sessile drop collected from a healthy subject on a glass surface results in circular tear “microdesiccates” comprising four discrete morphological domains or zones, namely, an outer structured hyaline zone I, a band of regularly and centripetally oriented crystalloids that are distinctive of zone II, a central zone III comprising typical randomly distributed fern-like structures differing to each other in robustness, length and branching, and, finally, a transition band, which is a noticeable structure located between zones I and II that seems to serve to anchor the zone II ferns . All these structural or morphological components of a tear “microdesiccate”, besides the fern-like crystalloid structures, may also be the expression of a normal tear composition and, probably, a normal tear film.
A number of reports have shown that tear protein profiles may exhibit major alterations when the tear fluid subjected to desiccation has been sampled from Dry Eye patients [12, 13]. On the other hand, changes in lipid composition or mucin composition of tear fluid resulting from dysfunctional Meibomium glands or from goblet cell deficiency, respectively, have been associated with Dry Eye [14–18]. Thus, the reduced ability to organize tear ferns can be associated with changes of diverse origin in the tear composition. At least some of those biochemical changes may affect the ability of the tear fluid to retain water as it might occur in patients expressing the evaporative subtype of Dry Eye [19, 20]. Accordingly, it would be expected that the formation of fern-like structures during desiccation of microvolumes of tear fluid on glass surfaces may be also influenced by or be an expression of the rate of tear evaporation. The present study was aimed at characterizing the progression of desiccation of tear fluid from healthy subjects (absence of Dry Eye) on a glass surface using a morphological approach. The study also aimed to evaluate the effect of various experimental conditions that enhance evaporation of tear samples collected from single healthy subjects both on the dynamics of desiccation and the morphological features of the resulting tear microdesiccates. Finally, the study characterized morphologically a differing progression of desiccation of tear fluid collected from both eyes of a mild/moderate Dry Eye case.
Rate of tear desiccation
Tear microdesiccates from healthy subjects display a similar four-domain design
Temporal appearance of the morphological zones occurring in a normal tear microdesiccate
Effect of experimentally accelerated tear desiccation on the four-domain morphology of tear microdesiccates
Temporal progression of the formation of tear microdesiccates from cases of patients with Dry Eye
In this study we have described the dynamics of formation of a domain-based structure produced by desiccation of a sessile tear drop on a glass flat surface. Overall, our observations indicate that each fern-like or leaf-like crystalloid in the tear microdesiccate is formed from an origin site and that its orderly lineal growth and branching end when the growth frontlines of the crystalloid get in contact with other forming or already formed crystalloid.
Spontaneous desiccation of a microvolume of tear fluid collected from healthy subjects gives rise to tear microdesiccates consisting of four domains named zones I, II, III and transition band . We have now shown that those four domains of a normal tear desiccate are formed asynchronously. The outermost hyaline zone I of the tear microdesiccate is the first domain to form at an early stage of desiccation. This structure represents a “pinning” stage of the desiccation process by which the sessile drop of the tear fluid becomes firmly bound to the glass surface thus preventing a decrease in the contact surface between the tear and the glass. Later, during a second stage of the desiccation process, water loss occurs at the water/air interface while the tear drop would be undergoing successive changes of shape [25, 26]. During this period, only some degree of disordered movement can be hardly seen by dark-field microscopy in the fraction of tear that remains in a liquid or semiliquid state. At the onset of the third stage, from specific points usually located at roughly regular distances close to the hyaline zone, fern-like or leaf-like crystalloids of zone II start to grow unidirectionally towards the center of the circular area of desiccation. While zone II crystalloids are still experiencing its unidirectional centripetal growth, disperse crystalloids start to appear asynchronously near the center of the desiccation area (zone III). Each of these nascent zone III crystalloids emerges from a single site, is usually multibranched and becomes larger, less regular in structure and more robust than zone II crystalloids. The highly diverse extents of these zone III crystalloids, and the ones of their branches, seem to depend stochastically on the contact inhibition phenomenon. Formation of zone III crystalloids would represent a fourth and final stage of the tear desiccation process. Contact between zone II and zone III crystalloids inhibits their growth and demarcates the end of the organization of a tear microdesiccate. Overall, formation of zones II and III are late, separate and partly overlapped events during desiccation of normal tear. Their formation in the final stages of desiccation occurs much faster than the visible events of the previous stages of tear desiccation. Anyhow, formation of a tear microdesiccate from any normal tear fluid sample seems to occur in an orderly sequence of events.
Formation of tear crystalloids can reflect the presence of definite components in the tear fluid, mostly proteins, mucins and salts [27, 28]. In support of that view and regardless of significance for desiccation that those tear components may have, in this study we have given some evidence showing that tear microdesiccates from any single subject are essentially identical whereas tear desiccates produced from tear samples provided by different subjects can vary markedly. However, the morphological characteristics of tear desiccates produced from a single tear sample are dependent not only upon the composition of the tear fluid but also on the conditions under which desiccation takes place . In this study we showed that a single sample of normal tear may produce morphologically different microdesiccates under different drying conditions. Thus, under drying conditions that enhance tear water evaporation, such as reduced air pressure, reduced partial water pressure or higher ambient temperature, crystalloids would be originated from a higher number of origins. Because the growth of single crystalloids seems to occur continuously until they make contact with other forming or already formed crystalloids, then a faster desiccation will result in smaller crystalloids. Altogether, these experimental observations suggesting a link between morphological features of tear microdesiccates and relative rate of tear evaporation may provide an additional source of valuable information on tear evaporation from single eyes that may help to assess and diagnose ocular surface disorders [4, 7–9, 12, 18, 23, 30–36]. In our view, the abundance of small crystalloids instead of the vigorous zone III crystalloids in a tear microdesiccate would represent the morphologic expression of a rapid evaporation of the tear water. In this regard, images of honeycomb structures occurring in tear ferning tests of Dry Eye patients, as reported by other laboratories , would be the expression of abundant groups of small crystalloids whose growth from a high number of origin sites was rapidly halted by their contact with similar growing neighbor groups of crystalloids.
Spontaneous desiccation of a microvolume of tear fluid collected from healthy subjects on a flat glass surface gives rise to tear microdesiccates consisting of well-defined morphological domains. Major fern-like crystalloids are a major feature of some of those domains. Formation of the microdesiccate domains occurs in an orderly sequence of asynchronic events. Under experimental conditions of accelerated desiccation major crystalloids are replaced by smaller crystalloids and asynchrony is lost. The same phenomenon does occur when tear microdesiccates are prepared from tear samples collected from some Dry Eye patients. The observed link between morphological features of tear microdesiccates and relative rate of tear evaporation may provide an additional source of valuable information on tear evaporation from single eyes.
All nine healthy subjects (age range 20–59 years old) included in this study fulfilled the following criteria: a) Normal visual parameters, b) Schirmer I test over 10 mm at 5 min , c) Fluorescein break-up time over 10 seconds , d) OSDI questionnaire scoring below 15 , e) Tear ferning test score I or II [5, 6], f) No previous eye surgery and g) No medication during the last three months . In addition, three patients (age range 20–55 years old) diagnosed with Dry Eye according to the DEWS guidelines , who had not experienced eye surgery and who were regularly attending the Fundación Oftalmológica Los Andes Ophthalmology Clinic in Santiago, Chile, were invited to be volunteer tear donors. Each one of the diagnostic tests was performed by single trained personnel. The study was conducted between March 2011 and June 2013 in accordance with the tenets of the Helsinki Declaration of 1975 and the guidelines of both the Ethics Committee of the Faculty of Medicine, University of Chile and the Ethics Committee of Fondecyt-Chile (Fondo de Desarrollo Científico y Tecnológico-Chile).
Tear fluid was collected using polyurethane minisponges, as reported elsewhere . Samples were taken always around 9–11 AM to control for eventual circadian variations. For each eye a single 3-minute tear sample was taken. The amount of sample was determined by gravimetry. Desiccation assays were conducted immediately after tear collection.
Excepting specific experiments (see Results section), one-microliter aliquots of fresh samples of tear fluid were taken with a P2-Gilson micropipette fitted with an ultrafine tip and placed sharply on a point of a microscope slide that was positioned horizontally. Tear aliquots were allowed to dry spontaneously at ambient conditions of temperature (range 15-25°C), relative humidity (range 40-45%) and altitude (520 m above sea level). Micrographs of the dry samples were taken under a dark-field microscope (Zeiss Axiostar Plus, objective lens = 2.5X, ocular lens = 10X) fitted with a Canon Powershot G10 14.7 megapixel digital camera. Fern images were classified as types I through IV according to Rolando’s criteria [5, 6].
Progression of tear desiccation
Desiccation of one-microliter aliquots of tear fluid was performed under the microscope as described above, except that to this purpose the digital camera was adjusted for capturing high resolution videos. Video-images (30 frames per second) were taken during 1 second at time-intervals of 5 seconds. The endpoint of desiccation was defined as the one in which growth of tear crystalloids was fully halted. Time for full desiccation of each tear sample was scored. In order to prevent any artifactual effect on tear desiccation produced by heat transfer, the lamp of the microscope was turned on only when video-images were being recorded.
Absorbing polyurethane mini-sponges (PeleTim®) and microcentrifuge tubes for tear collection were obtained from VOCO, Cuxhaven, Germany and from Axygen Scientific, California, USA, respectively. Sterile Schirmer tear test strips and fluorescein strips were acquired from Alcon Laboratories, Santiago, Chile. Microscope slides were purchased from Isolab Laborgeräte, Wertheim am Main, Germany.
Written informed consent was obtained from the patient for the publication of this report and any accompanying images. Confidentiality of patient information was protected.
This study was partially supported by Grant 1110325 from Fondo Nacional de Ciencia y Tecnología (Fondecyt), Chile.
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