Development of a new grading scale for tear ferning
Introduction
The chemical analysis of tear film composition is difficult due to the small volumes available, and to the transparent and dynamic nature of tears [1]. Clinicians and scientists recognise that biochemical analysis of osmolarity and other key components in a tear sample is the way forward, but the small volumes involved make biochemical analysis particularly challenging [2], [3]. Techniques available are limited by the need for expensive equipment that is difficult to use under normal clinical conditions [4]. A simple, clinical tear film test, that is quick and inexpensive to perform, and can indicate the biochemical properties of the tear film, would be very useful.
One potential and clinically suitable test involves drying a tear sample on a glass microscope slide to produce a crystallisation pattern in the form of a fern [5], [6], [7]. This phenomenon occurs with many body fluids and follows a characteristic formation process. The first discovery of tear crystallisation was reported by Fourcroy and Vauquelin in 1791 [8], but remained unnoted until 1946, when observed by Papanicolaou while studying cervical mucus [9]. Ferning patterns have been used to test different body fluids, such as vaginal and cervical mucus as an indicator of the menstrual cycle [10], oestrogen activity and ovulation [11], [12], [13], [14] and early pregnancy [13], [15]. Ferning has also been used to test saliva [16], to consider the observation of salivary ferning as a new technique for determining the fertile period [17], and to correlate salivary ferning and the fertile period [18], and using of salivary ferning in ovulation detection in family planning [19].
Crystallisation begins with the formation of a nucleus, consisting of a regularly arranged number of ions. The nucleus is formed by aggregation when the solute evaporates and dissolved ions are concentrated until super-saturation of the tear film is reached [7]. The nucleation process begins at the peripheral edges of the drop, where the solution is thinnest and super-saturation is reached rapidly [7]. Each nucleus has the ability to grow into a large crystal unit with the addition of more ions, and, so long as the sample solute is able to diffuse into areas with a lower solute concentration area, normal crystals can form. This requires a slow growth rate, low solution viscosity and low impurity levels to permit free solute diffusion.
The absence of these conditions can lead to dendritic crystal growth [20]. In this situation the stems grow longer and branch at regular intervals along the main stem. The reason for this regularity is not understood [7], but it is known that fern-like dendritic growth can be promoted by increasing the evaporation rate of the drop, by reducing atmospheric humidity, by increasing the drying temperature, or when impurities are present in low concentration, which acts as additional nuclei for crystal deposition [7].
Since tears are a complex solution, with many organic and non-organic components, the tear fern pattern produced by drying a sample depends on the composition of the tear sample [4], [7]. This variation in pattern has been suggested as a simple test for tear film quality at a gross biochemical level. This phenomenon gives tear ferning the potential, and the features, to be used as a diagnostic test in the clinic [5], [21]. Previous studies have demonstrated it to show good repeatability [22], sensitivity and specificity [21], [23], [24].
Different scales for grading tear ferning patterns have been proposed [6], [21], [25] with the Rolando scale being adopted as the main method used in previous published work in this area. However, the Rolando scale was not originally developed to produce a repeatable, standardised grading instrument, rather it arose from Rolando's observation that the Type I and II patterns were found in the majority of normal eyes, while Types III and IV were found in the majority of keratoconjuctivitis sicca (KCS) eyes [6].
The main difficulty with using the Rolando scale lies with this gross categorisation of ferning patterns, restricting sensitivity – the variance around Types I and II is particularly large – and not all types of tear ferning patterns are represented by the scale [22]. If the tear ferning test is to become part of routine clinical examination of the tear film, it is important to have a grading scale that has been developed to meet the needs of the clinician, and to address the four fundamental design requirements of a grading scale [26].
The aim of this paper is to report on the development of an improved subjective grading scale for clinicians, and the comparison of the new subjective scale with the Rolando scale.
Section snippets
Methods
A digital image library was compiled from tear ferning patterns produced using a standardised protocol, all images were observed under digital microscope (Leica DMRA2) with 10× magnification, and all images were saved in JPEG file format [22]. In total, 560 images of tear ferning patterns were produced from tear samples collected from 157 subjects, and all images were graded to 0.25 increments of the Rolando scale, for increased sensitivity [26]. Sixty images were selected by the authors,
Grading of image library using the Rolando Scale
The median grades for each Type were calculated (Table 5), indicating non-linearity across the scale, i.e. small difference between Types I and II, but large between Types III and IV. The variance around each grade also differed.
The non-parametric equivalent of ANOVA (Kruskal–Wallis test) was used to compare the scores for the 60 images using the Rolando scale and a statistically significant difference was found between the grades (p < 0.001; Fig. 2). Post hoc testing indicated that homogeneous
Discussion
This series of studies has led to the development of a new tear ferning grading scale, which has improved discrimination and repeatability over the previous Rolando grading scale. The final 5-point grading scale demonstrated good linearity in grading score across the ferning image library, and significant differences were found between the mean scores of the 5 scales. Reproducibility between sessions was also better with the new scale compared to Rolando's scale, indicating improved reliability.
Disclosure
None of the authors has any proprietary interest in this manuscript.
References (36)
- et al.
Spatial location studies on the chemical composition of human tear ferns
Ophthalmic Physiol Opt
(2000) - et al.
Ocular ferning test. A qualitative test for mucus deficiency
Ophthalmology
(1982) Tear crystallization test: two centuries of history
Ocul Surf
(2004)A general survey of the vaginal smear and its use in research and diagnosis
Am J Obstet Gynecol
(1946)- et al.
A biochemical study of the cervical and nasal mucus fern phenomenon
Am J Obstet Gynecol
(1957) - et al.
Efficacy of methods for determining ovulation in a natural family planning program
Fertil Steril
(1999) Grading scales for contact lens complications
Ophthalmic Physiol Opt
(1998)Tear osmolarity: a new gold standard?
Adv Exp Med Biol
(1984)- et al.
Performance of tear osmolarity compared to previous diagnostic tests for dry eye diseases
Curr Eye Res
(2010) - et al.
Factors responsible for tear ferning
Cornea
(1991)
Tear mucus ferning test in normal and keratoconjunctivitis sicca eyes
Chibret Int J Ophthalmol
The basis of tear ferning
Clin Exp Optom
Observation on the crystallization of the cervical mucus
Acta Obstet Gynecol Scand
A simple test for the determination of ovulation, estrogen activity, and early pregnancy using the cervical mucus secretion
Am J Obstet Gynecol
Some problems related to ovarian function, in Pincus G (ed)
Recent Prog Horm Res
The fern test. A critical analysis
Obstet Gynecol
Scanning electron microscopy of cervical mucus crystallization
Obstet Gynecol
Tear and saliva ferning tests in Sjogren's syndrome (SS)
Clin Rheumatol
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