The role of the fern test in the treatment of rhinitis

Abstract

Background:
The fern test is a method for assessing the characteristics of the nasal section in the treatment of patients with mucous dysfunction of the airway.
Objective: The aim of this study was to investigate the role of the fern test in patients with rhinitis and to assess the classification of each type of rhinitis (types I-IV) in clinical practice.

Methods:
A cross-sectional study, which included consecutive patients from a third level Rhinology Unit, worked with 182 patients with rhinitis and 30 healthy subjects as control. The patients were subdivided according to their type of rhinitis: allergic rhinitis (59), infectious rhinitis (32), polyps (31), NARES (Non-allergic rhinitis with eosinophilia syndrome) (30) and NARNE (non-allergic rhinitis with neutrophils) (30).

Results:
The control subjects had only type I or II rhinitis, whereas patients with rhinitis usually showed type III or IV. Allergic rhinitis and nasal polyps had the most serious deterioration according to the fern test (type IV).

Conclusions:
The fern test is effective for assessing mucus alterations in patients with rhinitis and it could be included as a new parameter in the study of rhinitis as a potential biomarker of the function of damaged epithelial cells.

Full text

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184 Rev Alerg Mex. 2019;66(2):184-191
éergia M
Revista
Al xico
Colegio
Mexicano de
Inmunología
Clínica A.C.
Original article
The role of the fern test in the treatment of rhinitis
El papel de la prueba de “helecho” en el tratamiento
de la rinitis
Matteo Gelardi,1 Giuseppe Porro,1 Nicola Quaranta,1 Brigida Sterlicchio,1 Michela Silvestri,2 Giorgio Ciprandi3
1University of Bari Aldo Moro, Department of Basic Medical Scien-
ce, Neuroscience and Sensory Organs, Bari, Italy
2Istituto Giannina Gaslini, Genoa, Italy
3Ospedale Policlinico San Martino, Genoa, Italy
How to cite this article: Gelardi M, Porro G, Quaranta N, Sterlicchio B, Silvestri M, Ciprandi G.The role of the fern
test in the treatment of rhinitis. Rev Alerg Mex. 2019;66(2):184-191
ORCID
Matteo Gelardi, 0000-0003-4406-0008; Giuseppe Porro, 0000-0003-2860-7608; Nicola Quaranta,
0000-0001-6214-2336; Brigida Sterlicchio, 0000-0002-6606-4811; Michela Silvestri, 0000-0003-4804-2421;
Giorgio Ciprandi, 0000-0001-7016-8421
Abstract
Background: The fern test is a method for assessing the characteristics of the nasal section in the
treatment of patients with mucous dysfunction of the airway.
Objective: The aim of this study was to investigate the role of the fern test in patients with rhinitis
and to assess the classifi cation of each type of rhinitis (types I-IV) in clinical practice.
Methods: A cross-sectional study, which included consecutive patients from a third level Rhinology
Unit, worked with 182 patients with rhinitis and 30 healthy subjects as control. The patients were
subdivided according to their type of rhinitis: allergic rhinitis (59), infectious rhinitis (32), polyps
(31), NARES (Non-allergic rhinitis with eosinophilia syndrome) (30) and NARNE (non-allergic
rhinitis with neutrophils) (30).
Results: The control subjects had only type I or II rhinitis, whereas patients with rhinitis usually
showed type III or IV. Allergic rhinitis and nasal polyps had the most serious deterioration according
to the fern test (type IV).
Conclusions: The fern test is effective for assessing mucus alterations in patients with rhinitis and
it could be included as a new parameter in the study of rhinitis as a potential biomarker of the
function of damaged epithelial cells.
Keywords: Rhinitis; Fern test; Infl ammation of the mucosa
Correspondence: Giorgio Ciprandi.
gio.cip@libero.it
Received: 2018-08-28
Accepted: 2019-01-24
DOI: 10.29262/ram.v66i2.544

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Gelardi M et al. Role of the fern test in the treatment of rhinitis
Background
Mucus is a complex, viscoelastic, and adherent se-
cretion that is produced and secreted by specialized
goblet cells and mucous cells in the columnar epi-
thelia that line the lumen of all the mucosa that are
exposed to the external environment.1,2 In particular,
mucus covers the inner linings of organs of the re-
spiratory tract, the gastrointestinal tract, the repro-
ductive tract, and the ocular surface. The concept of
mucus is very old as its existence was already known
from the time of the Ancient Greeks: phlegm was
one of 4 humors. Mucus is widely produced in both
kingdoms, plant and animal.3
From a physiological point of view, mucus car-
ries out many protective functions for the underlying
epithelia, such as mucosal lubrication, which is use-
ful for material transport and cellular hydration of
epithelial cells, mainly in the respiratory tract, eyes
and mouth, which are exposed directly to the drying
evaporative effects of air, and it provides a barrier
against noxious agents and exposure to pathogens
by trapping them.4 It also works as a cleansing trans-
port, in which the external particles that are trapped
in the mucus layer can be eliminated from organ
cavities by cilia-facilitated expulsion of the mucus
layer5 and provides a selectively permeable gel layer
for the diffusion, exchange and absorption of gases
(eyes and lungs) and nutrients (gastrointestinal tract)
with the underlying epithelium.6
The rheology of nasal mucus deserves prop-
er attention in clinical practice.7,8 From a chemical
point of view, mucus should be seen as an integrat-
ed structure of biopolymers. The physical charac-
teristics of mucus are complex, for example; it is a
non-Newtonian fl uid, and its physical characteris-
tics range from a viscous liquid to an elastic solid.
Mucus can be measured considering its consistency
by viscosity (resistance to fl ow) and elasticity (stiff-
ness). It must be noted that impaired mucus rheolo-
gy may have signifi cance on its functions, including
lubrication, barrier, and defense against infective
agents and noxious substances.8
Resumen
Antecedentes: La prueba de “helecho” es un método que sirve para evaluar las características
de la secreción nasal en el tratamiento de pacientes con disfunción de la mocosa de la vía aérea.
Objetivo: El objetivo del presente estudio fue investigar el papel de la prueba de helecho en
pacientes con rinitis y evaluar la clasifi cación de cada tipo de rinitis (tipos I a IV) en la práctica
clínica.
Métodos: Estudio transversal en el que se incluyeron pacientes consecutivos de una unidad de
rinología de tercer nivel. Se incluyeron 182 pacientes con rinitis y 30 sujetos sanos como controles.
Los pacientes se subdividieron según el tipo de rinitis: alérgica (59), infecciosa (32), pólipos (31),
rinitis eosinofílica no alérgica (30) y rinitis no alérgica con neutrófi los (30).
Resultados: Los sujetos control solo presentaron rinitis tipo I o II, mientras que los pacientes con
rinitis generalmente mostraban tipo III o IV. La rinitis alérgica y los pólipos nasales tuvieron el
deterioro más grave según la prueba de helecho (tipo IV).
Conclusiones: La prueba de helecho es efectiva para evaluar las alteraciones del moco en
pacientes con rinitis y podría incluirse como un nuevo parámetro en el estudio de la rinitis como
biomarcador potencial de la función de las células epiteliales dañadas.
Palabras clave: Rinitis; Prueba de "helecho"; Infl amación de la mucosa
Abbreviations and acronyms
AR, allergic rhinitis
MGG, May-Grünwald-Giemsa
NARES, non-allergic rhinitis with eosinophilia
NARNE, non-allergic rhinitis with neutrop hils

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186 Rev Alerg Mex. 2019;66(2):184-191
Gelardi M et al. Role of the fern test in the treatment of rhinitis
Mucus is a thixotropic gel that is able to proper-
ly respond to shear stress. This ability depends on its
composition. The components of mucus are mucins,
DNA fragments, lipids, ions, proteins, cells, cellu-
lar debris, and water.9,10 In this regard, mucins are
the most important component of the mucus as it is
formed by cross-linked, bundled, and entangled mu-
cin fi bers which are produced and secreted by both
goblet cells and sero-mucinous glands.
The bio-rheological properties of the nasal mu-
cus are the cohesive forces, the load-bearing capaci-
ty, the spinnability, the thixotropy, the creep, and the
adhesiveness. This complexity may easily explain
how disorders could signifi cantly affect the compo-
sition and function of nasal mucus. Infections, in-
fl ammatory disorders (including allergic and non-al-
lergic infi ltrates), polyps, drugs, and primary mucus
defects may signifi cantly alter the composition and
function of mucus.
The properties of mucus may be assessed by
using sophisticated tests, but a simple way could be
represented by the nasal cytology. Long time ago,
Papanicolaou proved that some cervical mucus that
was spread on a slide and left to dry was able to
crystallize with a characteristic shape, like an “ar-
borization”: a fern-like reaction.11 He brilliantly con-
ceived that this phenomenon could depend on the
ovulation time as a refl ection of the estrogenic activ-
ity that was able to affect the characteristics of the
mucus. Further on, it was proven, by inducing the
mucus-fern phenomenon,12,13 that the alterations of
mucus, depending on the cycle of sexual hormones,
occurred also in other mucus-secreting surfaces of
the body; including the nose, salivary glands, and
lacrimal glands. Abou-Shabanah and Plotz proved
that the fern reaction depended on electrolyte, pro-
tein and/or saccharide concentrations.14 So, the fern
test was used to assess the characteristics of mucus
in several disorders. Rolando et al. defi ned a scoring
system to assess the fern test15 and reported fern im-
pairment in different disorders at an ocular level.16,17
These outcomes were confi rmed by further studies
that were always conducted at an ocular level,18,19 so
the fern test is commonly used in clinical practice in
an ophthalmological setting. However, only another
study considered the fern phenomenon in the nose:
it compared nasal and cervical mucus. The authors
concluded that mucus is an ancestral biological
component, but they did not speculate about nasal
disorders.20 Thus, we aimed to investigate the role
of the fern test in patients with rhinitis in clinical
practice.
Methods
Globally, 182 consecutive patients (102 males and
80 females, mean age: 43.15 ± 19.03 years) with
rhinitis were visited at the Rhinology Unit because
of their intensive treatment and they were included
in this study.
The inclusion criteria were:
• Adult men and women.
• Documented diagnosis of rhinitis, including al-
lergic rhinitis, infectious rhinitis, non-allergic
rhinitis (NARES or NARNE), or nasal polyps.
• Presence of nasal symptoms.
The exclusion criteria were:
• Concomitant comorbidities and chronic illness-
es that could interfere in the interpretation of the
fi ndings.
• Current treatments able to interfere in the inter-
pretation of the fi ndings. In addition, 30 healthy
subjects were included in the study.
The Review Board approved the procedure
and a written informed consent was given by all
participants.
A nasal endoscopy was carried out with a 3.4
mm diameter fl exible fi beroscope (Vision-Sciences®
ENT-2000) to assess the nasal cavities.
Nasal cytology involves: sampling, processing
and microscope reading. The sampling requires the
collection of cells from the surface of nasal mucosa,
which is done by a sterile disposable curette. Sam-
ples should be collected from the middle portion of
the inferior turbinate. The procedure is performed
under anterior rhinoscopy, with an appropriate light
source, and the procedure is completely painless. The
obtained sample is smeared immediately on a glass
slide and it is air-dried. The sample staining is carried
out by using the common May-Grünwald-Giemsa
(MGG) staining because of its ability to correctly
identify the infl ammatory nasal cells. The traditional
MGG staining procedure requires about 30 minutes.
The stained sample is read at optical microscopy,
with a 1000x objective with oil immersion. Fifty is

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Gelardi M et al. Role of the fern test in the treatment of rhinitis
the minimum number of fi elds in order to identify
a suffi cient number of cells. The count of each cell
type was expressed by a semi-quantitative grading
as it was described previously.21 The detection of eo-
sinophils, mast cells, bacteria or fungal ifae clearly
identifi es a pathological condition. In nasal cytolo-
gy, bacterial infectious rhinitis is usually character-
ized by the presence of many neutrophils, with intra
and extracellular bacteria that can be easily identi-
fi ed at optical microscopy. Lymphocytes and mac-
rophage can accompany the neutrophilic infi ltrate.22
Allergic rhinitis (AR) is always characterized by the
presence of infl ammatory cells (eosinophils, mast
cells, neutrophils, and lymphocytes) in the nose.
Non-allergic infl ammatory rhinitis is defi ned by the
presence of infl ammatory cells in the nasal muco-
sa and by the absence of allergic sensitization. The
best known and fi rst described non-allergic rhinitis
was NARES: the presence of eosinophils is not only
predominant but also massively present with an ex-
pression that is even higher than in seasonal rhinitis.
Another important type of infl ammatory non-aller-
gic rhinitis is NARNE; which is characterized by a
massive presence of neutrophils without concomi-
tant bacterial colonization.23 Nasal polyps may show
an infl ammatory pattern, as previously reported.24
For the fern test, nasal specimens were taken,
as well as the traditional nasal cytology. The collect-
ed smears were put on microscope slides and left to
dry at room temperature for 10 minutes. They were
examined under a phase contrast microscope at 100×
and 400× magnifi cation and they were classifi ed from
type I to type IV according to the Rolando scoring.25
In type I (normal pattern), the fern pattern is uniform
with closely branching arborization. In type II, the
ferns are well distinguished but with less branching.
In type III, there are scattered ferns with rare branch-
ing, and, in type IV, no fern pattern is seen.
Statistical analysis
Descriptive statistics were performed and reported
in terms of absolute frequencies or percentages for
qualitative data and in terms of medians with fi rst
and third quartiles (1q-3q) or means with standard
deviation for quantitative data. The comparison of
frequency distributions was made by means of the
chi-square test or the Fisher’s exact test in case of
expected frequencies < 5. All tests were two-tailed,
and p-values < 0.05 have been considered statisti-
cally signifi cant. The analyses were performed using
the GraphPad Prism software, GraphPad Software
Inc, CA, USA
Results
Table 1 reports demographic and clinical character-
istics of control and patients with different types of
Table 1. Demographic and clinical characteristics of controls and patients with rhinitis
Controls
(30)
Patients with
rhinitis
(182)
Allergic
rhinitis
(59)
Infectious
rhinitis
(32)
Nasal polyps
(31)
NARES
(30)
NARNE
(30)
Age
(mean ± SD) 37.63 ± 17.87 43.15 ± 19.03 38.29 ± 18.46 35.75 ± 20.36 56.39 ± 11.57 48.93 ± 16.38 41.13 ± 19.84
Sex
(male/female) 15/15 102/80 3/27 16/16 19/12 17/13 16/14
n%n%n%n%n%n%n%
Allergy 0 0 89 48.90 59 66.29 10 11.24 12 13.48 5 5.62 3 3.37
ASA
sensitivity 0 0 20 10.98 1 5.00 3 15.00 10 50.00 4 20.00 2 10.00
Asthma 0 0 31 17.03 10 32.26 5 16.13 10 32.26 4 12.90 2 6.45
Smoke 0 0 59 32.40 11 18.64 14 23.73 11 18.64 12 20.34 11 18.64
Current 48 26.40 9 81.82 12 85.71 8 72.73 10 83.33 9 81.82
Past 11 6.04 2 18.18 2 14.29 3 27.27 2 16.67 2 18.18
NP = nasal polyps, NARES = non-allergic rhinitis with eosinophils, NARNE = non-allergic rhinitis with neutrophils, SD = standard deviation,
ASA = acetylsalicylic acid

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Gelardi M et al. Role of the fern test in the treatment of rhinitis
rhinitis. Allergic rhinitis was the most frequently di-
agnosed type of rhinitis (59 out of 182); all the other
types of rhinitis i.e. infectious, polyposis, NARNE,
NARES were found almost equally among patients.
Allergy was a common comorbidity, ASA sensi-
tivity was more frequently present in patients with
nasal polyps, asthma was more commonly reported
in patients with allergic rhinitis and nasal polyps, and
smoking was reported in about 1/3 of patients.
Considering nasal specimens, we found that
type II was the most common one in control subjects
(76.67 %), whereas type III (59.34 %), followed by
type IV (28.02 %), was the most frequently observed
among patients with rhinitis (fi gure 1 and table 2). It
is worthy of note that no control subject had a type
III or type IV fern pattern. The frequency of differ-
ent fern patterns was signifi cantly different among
patients with different types of rhinitis and control
subjects (p = 0.0066).
In type I fern patterns, only 12.5 % of nasal
specimens were collected from patients with rhini-
tis (i.e. infectious); all the other were isolated from
control subjects (87.5 %) (fi gure 2). More than half
of the nasal specimens characterized by type II fern
pattern were collected from control subjects, where-
as the remaining specimens were recovered from pa-
tients with allergic rhinitis, infectious rhinitis, NA-
RES or NARNE (fi gure 2). Allergic rhinitis was the
most frequent type of rhinitis (37.96 %), followed
by infectious (19.44 %) associated to a type III fern
pattern. Differently, considering type IV, about 30 %
and 25 % of this pattern was found in patients with
nasal polyps or in patients with allergic rhinitis, re-
spectively (fi gure 2).
We also observed that the type of rhinitis that
was most commonly found in the most “pathologic”
fern patterns, i.e. type III or type IV, was allergic rhi-
nitis, which affected about 1/3 of the patients whose
specimens had type III or type IV fern patterns. The
other 2/3 were almost equally distributed among the
other types of rhinitis (fi gure 3).
In order to verify possible differences in fern
patterns between control subjects and patients with
rhinitis, we analyzed each type of rhinitis separate-
ly. Patients with infectious rhinitis, with NARNE
or with allergic rhinitis had a type III fern pattern
more frequently, whereas control subjects had a type
II pattern more frequently (p < 0.0001, each com-
parison), (data not shown). In patients with NARES
or nasal polyps, type III and type IV were the most
frequent patterns whereas, in control subjects, type
II was predominant (p < 0.0001, each comparison).
No statistically signifi cant difference in fern
patterns was found in patients with different de-
Figure 1. The four grades
of nasal ferning taken at
100x magnifi cation, classi-
fi ed from type I to type IV
according to the Rolando
scoring.25 Panel A, type I
(normal pattern): the fern-
ing pattern is uniform with
closely branching arboriza-
tion. Panel B, type II: the
ferns are well distinguished
but with less branching.
Panel C, type III: there are
scattered ferns with rare
branching. Panel D, type
IV: no ferning pattern is
seen. Light microscope at
400× magnifi cation.

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Gelardi M et al. Role of the fern test in the treatment of rhinitis
grees of nasal eosinophilia or neutrophilia (data
not shown).
Discussion
The defi nition of rhinitis assumes the concept of an
infl ammatory reaction; the characteristics of nasal
infl ammation, mainly regarding the peculiar cellular
infi ltrate, allow the differentiation of several types of
rhinitis. However, a common denominator in infl am-
matory disorders is the presence of impaired mucus.
Altered mucus is per se a pathogenic factor that in-
duces a vicious circle that involves a deterioration of
infl ammatory disorders.
Nasal cytology is commonly used in clinical
practice to defi ne the type of rhinitis, to quantify
the cellular infi ltrate, and to monitor infl ammatory
change over time.21 Traditionally, the nasal cytolo-
gy assessment considers cellular presence and mor-
phology, bacteria, and biofi lm. However, mucus is a
component that is neglected in the treatment of nasal
disorders. A possible explanation is the complexity
of diagnostic tests able to assess the characteristics
of mucus.
In this regard, the fern test could be a very
simple and useful method to investigate mucus in
patients with rhinitis. The current study, conducted
in a real-world setting, such as a Rhinology Unit,
proved that rhinitis is associated with impaired fern
patterns. In particular, healthy subjects have only
type I or II fern patterns and never III or IV. On the
Table 2. Frequency of different ferning Types within each rhinitis group
Study groups Disease
Ferning type
I II III IV
n%n%n%n%
Patients with
rhinitis
1 0.55 22 12..09 108 59.34 51 28.02
Allergic rhinitis 0 0 5 8.47 41 69.49 13 22.03
Infectious rhinitis 1 3.13 5 15.63 21 65.63 5 15.63
NARES 0 0 5 16.67 12 40 13 43.33
NARNE 0 0 7 23.33 18 60 5 16.67
nasal polyps 0 0 0 0 16 51.61 15 48.39
Control subjects 7 23.33 23 76.67 0 0 0 0
NARES = non-allergic rhinitis with eosinophils, NARNE = non-allergic rhinitis with neutrophils.
Figure 2. Frequency of
different rhinitis within
each ferning type.
100
90
80
70
60
50
40
30
20
10
0
Control subjects
Polyposis
NARNE
NARES
Infectious rhinitis
Allergic rhinitis
87.5
12.5 11.11
11.11
11
15.56
51.11
14.81
16.67
11
19.44
37.96 25.49
9.8
9.8
29.41
25
I II III IV
Ferning (type)
Percentage

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190 Rev Alerg Mex. 2019;66(2):184-191
Gelardi M et al. Role of the fern test in the treatment of rhinitis
other hand, patients with rhinitis frequently present
type III or IV fern patterns. Allergic rhinitis and na-
sal polyps are most frequently associated with type
IV fern patterns. This fi nding could depend on more
intense infl ammation, even though a clear-cut rela-
tionship between cellular infi ltrate and fern pattern
was not reported.
The most relevant outcome of the present study
is the feasibility of the fern test in the treatment of
patients with rhinitis. The fern test may be consid-
ered a reliable tool for assessing the characteristics
of the mucus, and its fi ndings have clinical rele-
vance. It is conceivable that impaired fern patterns,
such as type III and IV, could be the expression of
damaged epithelial cells, mainly concerning mu-
ciparous cells. In fact, patients with rhinitis usually
present altered fern patterns. In other words, type I
(and type II) fern patterns could mean the wellness
of the epithelium.
However, the present study has some limita-
tions, including the cross-sectional design, the lack
of mediator’s assessment, and the lack of assessment
of clinical symptoms. Therefore, other studies should
be designed to fulfi ll these unmet needs. In particu-
lar, it is necessary to correlate the severity of nasal
disorders with the type of fern patterns. If a strong re-
lationship between the type of fern pattern and severe
symptoms and/or infl ammatory aspects exists, this
test could be fruitful in phenotyping patients with
rhinitis. Therefore, the fern test could be considered
a potential biomarker that is useful for defi ning epi-
thelial damage in nasal disorders. This issue should
be properly investigated in further studies.
In conclusion, the fern test could be considered
a fruitful method to assess mucus alterations in pa-
tients with rhinitis and it could be included as a new
parameter in the treatment of rhinitis as a potential
biomarker of damaged epithelial cell function.
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