# Conjunctivitis and Total IgE in Lacrimal Fluid: Lacrytest Screening

Authors: Susana Monzón; Elena Arrondo; Joan Bartra; Ferran Torres; María Basagaña; M. del Mar San Miguel; Rosario Alonso; Anna Cisteró-Bahima
Journal: Journal of Allergy
 (2009)

---

## Abstract

Total tear IgE has been considered to play an important role in allergic conjunctivitis, and measurement has been considered useful for diagnosis. The aim of this study was to ascertain whether Lacrytest®, a new commercialised method to detect IgE levels in lacrimal fluid, could constitute a screening test for the diagnosis of allergic conjunctivitis. This was a cross-sectional study. Patients with seasonal and perennial allergic conjunctivitis, vernal keratoconjunctivitis and a control group were included. Clinical history, ophthalmic examination, skin prick test and conjunctival provocation test were obtained. Lacrytest® was later performed in all groups. Fifty-four patients were enrolled: thirty with IgE-mediated conjunctivitis and, nine with vernal keratoconjunctivitis and fifteen controls. Lacrytest® was negative in all controls, positive in 20% of the IgE-mediated conjunctivitis group and in 88.9% of the vernal keratoconjunctivitis group. Global statistically-significant differences were found among the three groups (P=.003). Sensitivity of the test in the IgE-mediated conjunctivitis group was 20%, specificity 100%, positive predictive value 100%, and negative predictive value 38.46%, while in VKC sensitivity was 88.88%, specificity 100%, positive predictive value 100%, and negative predictive value 93.75%. Our data confirm that this test is not useful for screening allergic conjunctivitis. Lacrytest®, while not providing any useful information to an allergist, could be helpful for ophthalmologists to confirm an IgE-mediated or VKC conjunctivitis.


---

## Body

## 1. Introduction

Allergic
conjunctivitis constitutes a group of diseases affecting the ocular surface;
however, different kinds of conjunctival disorders are grouped under this
umbrella term for this single clinical entity. Seasonal and perennial allergic
conjunctivitis (SAC and PAC) can be defined as recurrent and bilateral
conjunctival inflammation with exacerbations in different seasons of the year
caused by direct exposure of the ocular surface to airborne allergens. Both are
mainly dependent on classical type I hypersensitivity in which patients have
positive skin prick tests and specific IgE in serum to airborne allergens.
Itching is the major symptom in this type of conjunctivitis. Ocular findings
are scant or even absent and are not related to symptom intensity [1].Vernal
keratoconjunctivitis, a chronic severe inflammatory disease of the conjunctiva
usually recurring bilaterally and seasonally (spring and summer), occurs predominantly
in male children and young adults with a personal or family history of atopy.
Itching is the most significant symptom in these patients, although cobblestone
papillae, excess mucus, and intense photophobia may be observed. Corneal
involvement may occur and result in permanent vision damage. The pathogenesis
is more complex than that of SAC and PAC, and a leading role of an inflammatory
network not confined to the classical IgE-mast cell immediate hypersensitivity
paradigm, but characterised mainly by Th2-type inflammation with mast cells, basophils,
eosinophils, and polyclonal IgE activation, has been suggested. SPT and serum specific IgE antibody test are often not positive,
although total serum IgE levels are high. Eosinophils are present in both tears
and conjunctival scrapings [2].A
new lacrimal test based on total IgE determination has been commercialised to
diagnose allergic conjunctivitis. Total tear IgE has been considered to play an
important role in allergic conjunctivitis and it has been shown that the
measurement of tear IgE concentrations can aid the diagnosis of this condition [3–5].
Lacrytest (ADIATEC S.A, Nantes,
France) is a
rapid immunoassay for total IgE determination in tears. This assay indicates,
in a qualitative manner, the presence of total class E immunoglobulin in tears
with levels above the normal value (<2 KU/L, 3 ng/mL) [3]. In order
to investigate whether Lacrytest could be a screening tool to diagnose
allergic conjunctivitis, we analysed the results of the test in patients with
allergic conjunctivitis and compared them with a control group in a cross-sectional
study.


## 2. Methods

### 2.1. Patients and Study Design

Patients
were systematically enrolled from October 2004 to April 2005. The study
included two centres: Institute Universitari Dexeus of Barcelona
(Allergy Department) and Mutua of
Terrassa (Ophthalmology Department). Patients were preselected according to a
clear history of allergic conjunctivitis. A clinical history was taken and an
ophthalmic examination and finally a skin prick test (SPT) to airborne
allergens and a conjunctival allergen provocation test (CPT) were performed if
the SPT was positive. Antihistamines were prohibited for three days before skin
testing and conjunctival challenge. Selected patients gave their written
informed consent.Patients
were divided into three groups depending on their diagnosis. The vernal
keratoconjunctivitis (VKC) group was based on clinical history and ophthalmic
examination (giant papillae or superficial keratitis). SPTs were not considered because are often not positive [2].Seasonal and perennial allergic conjunctivitis
(IgE-mediated allergic conjunctivitis) were diagnosed by clinical history,
positive SPT to pneumoallergens and a positive conjunctival-specific
challenge test. Ophthalmic examination was not a basis to diagnose
them because they are acute
forms of conjunctivitis and some patients could not have ocular symptoms and
signs of active allergic conjunctivitis at the moment of the visit.The control group comprised patients
with no symptoms of allergy (atopic dermatitis, rhinitis, or asthma) or
conjunctivitis in their clinical history, and with normal ophthalmic
examination and negative SPT.After
the division into three groups and with or without signs of active allergic
conjunctivitis in that moment, Lacrytest was performed in one eye for the
control and vernal keratoconjunctivitis groups and in both eyes for the IgE-mediated
allergic conjunctivitis group: in one eye immediately after the conjunctival-specific
challenge test and in the other in which the allergen was not instilled to
compare the results between eyes.


### 2.2. Skin Prick Test

All
patients underwent SPT performed according to standard procedure [6] with Dermatophagoides pteronyssinus, Dermatophagoides farinae, Dermatophagoides microceras, cat and dog
epithelium, moulds (Aspergillus
fumigatus, Alternaria alternata), pollens (Phleum pratense, Cynodon
dactilon, Phragmites comunis, Olea europaea, Platanus acerifolia, Cupressus
arizonica, Pinus radiata, Parietaria judaica, Artemisia vulgaris, Chenopodium album, Plantago lanceolata), latex, and cockroach (Blatella germanica). These allergens were supplied by ALK-ABELLÓ (Madrid, Spain).
Histamine and isotonic saline were used as positive and negative controls,
respectively. Mean wheal diameters of 3 mm were considered a positive reaction.


### 2.3. Conjunctival Allergen-Specific Provocation Test (CPT)

This test was performed with the
allergen suspected of being one of the aetiologic causes of IgE-mediated
conjunctivitis in each patient, chosen on the basis of clinical history
correlation and SPT results. The CPT was conducted according to
Möller et al. [7].  Each patient
was skin prick tested on the volar surface of the forearm with four ten-fold serial dilutions of the
specific allergen extract; the CPT was
started with the dilution before the one that was positive in the SPT (mean
wheal diameter 3 mm) and one drop was instilled into the conjunctival sac. The
dilutions instilled were increased every twenty minutes until the test proved
positive. Criteria for a positive test were congestion of the conjunctival
mucosa, itching, and eye watering.


### 2.4. Lacrytest

Lacrytest
is an assay for total IgE determination in tears. A strip is placed in the
lower conjunctival fornix and, when wet with tears, is removed. Total IgE reacts
with a gold-labelled antibody and is immobilised with the uptake of anti-IgE
antibody. Signal intensity is dependent on the total IgE level. For normal
value, below 2.5 KU/L, no line was obtained. The positive results could be
divided among the three groups on a semiqualitative scale: for low total IgE
levels, 2.5 to 10 KU/L, intensity of the signal in the IgE reactive field was lower
than that of the control line; for medium total IgE levels, 10 to 40 KU/L,
intensity was closer to that of the control line; and for high total IgE levels>40 KU/L, intensity was stronger than the control. The test was performed
according to the manufacturer’s instructions.


### 2.5. Statistical Analysis

A descriptive analysis of
demographic characteristics and clinical symptoms was carried out. The
following statistical tests were used for the inferential analysis: Fisher’s
exact test for qualitative variables, Mann-Whitney U or Kruskall-Wallis tests
(for 2 or 3 groups, resp.) for semiquantitative ordinal measurements,
and the MacNemar test for binary paired data. Sensitivity, specificity, and
positive predictive value were calculated with standard formulae [8].
The analysis was performed using SPSS v10.0 (SPSS,
Inc., Chicago IL), and the CIA software for
calculation of the 95% confidence intervals [9]. The level of
significance was established at P=.05 (two-sided).


## 2.1. Patients and Study Design

Patients
were systematically enrolled from October 2004 to April 2005. The study
included two centres: Institute Universitari Dexeus of Barcelona
(Allergy Department) and Mutua of
Terrassa (Ophthalmology Department). Patients were preselected according to a
clear history of allergic conjunctivitis. A clinical history was taken and an
ophthalmic examination and finally a skin prick test (SPT) to airborne
allergens and a conjunctival allergen provocation test (CPT) were performed if
the SPT was positive. Antihistamines were prohibited for three days before skin
testing and conjunctival challenge. Selected patients gave their written
informed consent.Patients
were divided into three groups depending on their diagnosis. The vernal
keratoconjunctivitis (VKC) group was based on clinical history and ophthalmic
examination (giant papillae or superficial keratitis). SPTs were not considered because are often not positive [2].Seasonal and perennial allergic conjunctivitis
(IgE-mediated allergic conjunctivitis) were diagnosed by clinical history,
positive SPT to pneumoallergens and a positive conjunctival-specific
challenge test. Ophthalmic examination was not a basis to diagnose
them because they are acute
forms of conjunctivitis and some patients could not have ocular symptoms and
signs of active allergic conjunctivitis at the moment of the visit.The control group comprised patients
with no symptoms of allergy (atopic dermatitis, rhinitis, or asthma) or
conjunctivitis in their clinical history, and with normal ophthalmic
examination and negative SPT.After
the division into three groups and with or without signs of active allergic
conjunctivitis in that moment, Lacrytest was performed in one eye for the
control and vernal keratoconjunctivitis groups and in both eyes for the IgE-mediated
allergic conjunctivitis group: in one eye immediately after the conjunctival-specific
challenge test and in the other in which the allergen was not instilled to
compare the results between eyes.


## 2.2. Skin Prick Test

All
patients underwent SPT performed according to standard procedure [6] with Dermatophagoides pteronyssinus, Dermatophagoides farinae, Dermatophagoides microceras, cat and dog
epithelium, moulds (Aspergillus
fumigatus, Alternaria alternata), pollens (Phleum pratense, Cynodon
dactilon, Phragmites comunis, Olea europaea, Platanus acerifolia, Cupressus
arizonica, Pinus radiata, Parietaria judaica, Artemisia vulgaris, Chenopodium album, Plantago lanceolata), latex, and cockroach (Blatella germanica). These allergens were supplied by ALK-ABELLÓ (Madrid, Spain).
Histamine and isotonic saline were used as positive and negative controls,
respectively. Mean wheal diameters of 3 mm were considered a positive reaction.


## 2.3. Conjunctival Allergen-Specific Provocation Test (CPT)

This test was performed with the
allergen suspected of being one of the aetiologic causes of IgE-mediated
conjunctivitis in each patient, chosen on the basis of clinical history
correlation and SPT results. The CPT was conducted according to
Möller et al. [7].  Each patient
was skin prick tested on the volar surface of the forearm with four ten-fold serial dilutions of the
specific allergen extract; the CPT was
started with the dilution before the one that was positive in the SPT (mean
wheal diameter 3 mm) and one drop was instilled into the conjunctival sac. The
dilutions instilled were increased every twenty minutes until the test proved
positive. Criteria for a positive test were congestion of the conjunctival
mucosa, itching, and eye watering.


## 2.4. Lacrytest

Lacrytest
is an assay for total IgE determination in tears. A strip is placed in the
lower conjunctival fornix and, when wet with tears, is removed. Total IgE reacts
with a gold-labelled antibody and is immobilised with the uptake of anti-IgE
antibody. Signal intensity is dependent on the total IgE level. For normal
value, below 2.5 KU/L, no line was obtained. The positive results could be
divided among the three groups on a semiqualitative scale: for low total IgE
levels, 2.5 to 10 KU/L, intensity of the signal in the IgE reactive field was lower
than that of the control line; for medium total IgE levels, 10 to 40 KU/L,
intensity was closer to that of the control line; and for high total IgE levels>40 KU/L, intensity was stronger than the control. The test was performed
according to the manufacturer’s instructions.


## 2.5. Statistical Analysis

A descriptive analysis of
demographic characteristics and clinical symptoms was carried out. The
following statistical tests were used for the inferential analysis: Fisher’s
exact test for qualitative variables, Mann-Whitney U or Kruskall-Wallis tests
(for 2 or 3 groups, resp.) for semiquantitative ordinal measurements,
and the MacNemar test for binary paired data. Sensitivity, specificity, and
positive predictive value were calculated with standard formulae [8].
The analysis was performed using SPSS v10.0 (SPSS,
Inc., Chicago IL), and the CIA software for
calculation of the 95% confidence intervals [9]. The level of
significance was established at P=.05 (two-sided).


## 3. Results

Fifty-four
patients (30 males, 24 females) were included. Mean (standard deviation) age
was 30.6 (20.2) years (range: 5–76). The main demographic characteristics and
different clinical conjunctivitis groups are summarised in Table1.Table 1
Demographic characteristics
and groups of study.
PatientGroupSexAgeSPTPatientGroupSexAgeSPT11F14+312M8−21M37+322M5−31F52+332M65−41F12+342M9−51F39+352F25−61F28+362F76−71F49+372F74−81M30+382F74−91F24+392F14−101M21+402M34−111F25+412F52−121F14+422F43−131F15+432F36−141F43+442M25−151M33+452F17−161M24+463F8−171F39+473M9−181M33+483M10+191M36+493F7+201M28+503F6+211M27+513F8−221F33+523M9−231F18+533F9−241F70+543M8−251M55+261M13+271F59+281M56+291M39+301M56+1- IgE-mediated conjunctivitis
group. 2- Control group. 3- VKC. M: male. F: female. +: Positive SPT. −:
Negative SPT.The
Lacrytest was positive in only 6 patients with IgE-mediated conjunctivitis
(20%) and 8 patients with vernal keratoconjunctivitis (88.9%) and was negative in
all controls (100%). Differences were significant when the three groups were
compared (P=.003); pair-wise comparisons of VKC versus the other groups were
statistically significant (P<.001 for both comparisons), but not between
IgE-mediated conjunctivitis and control groups (P=.157). (Figure 1).Figure 1
Lacrytest global results.
Number of subjects is shown on the top of each column.If
the result of the ocular test in its positive semiqualitative scale (negative,
low, medium, and high) is analysed, the results among the three groups were also
significant (P<.001); pair-wise comparisons of control versus VKC and VKC versus
IgE-mediated conjunctivitis were statistically significant (P<.001 for both
comparisons), but not between control versus IgE-mediated conjunctivitis groups
(P=.066) (Figure 2).Figure 2
Lacrytest qualitative results. Number of subjects is shown on the
top of each column.Results
of the lacrimal test used in IgE-mediated conjunctivitis improved after the
conjunctival provocation test had been applied to the contralateral eye in each
subject. All 6 previously positive results remained positive, but 14 new
positives were observed among the 24 previously negative (58.3%, McNemar testP<.001) (Figure 3).Figure 3
Lacrytest results in IgE-mediated
conjunctivitis group.Regarding
the Lacrytest results (Table2), in the IgE-mediated conjunctivitis group
sensitivity was 20%, specificity 100%, positive predictive value 100%, and negative predictive value 38.46%, while in
VKC sensitivity was 88.88%, specificity 100%, positive predictive value 100%,
and negative predictive value 93.75% (Table 3). If results after the CPT were
analysed in the IgE-mediated conjunctivitis group (Table 2), sensitivity was
66.66%, specificity 100%, positive predictive value 100%, and negative predictive
value 60%.Table 2
Sensitivity and
specificity of the Lacrytest (IgE-mediated conjunctivitis before and after CPTversus control group).
IgE-mediated conjunctivitis before
CPTControlTotalIgE-mediated conjunctivitis after CPTControlTotalTEST +606TEST +20020TEST−241539TEST−101525Total301545Total301545Sensitivity: 20%Sensitivity: 66.66%Specificity: 100%Specificity: 100%Positive predictive value: 100%Positive predictive value: 100%Negative predictive value: 38.46%Negative predictive value: 60%Table 3
Sensitivity and
specificity of Lacrytest (VKCversus control group).
VKCControlTotalTEST +808TEST−11516Total91524Sensitivity: 88.88%Specificity: 100%Positive predictive value: 100%Negative predictive value: 93.75%


## 4. Discussion

The
eye is one of the major targets of allergic disorders, either alone or
accompanied by other allergic affection [10]. The ocular component may be the most
prominent and sometimes disabling feature of the allergy. The acute forms (seasonal
and perennial allergic conjunctivitis) are commonly seen in an Allergy department,
and over 50% of patients also have a history of rhinitis, whereas the more
chronic forms [11] (vernal keratoconjunctivitis, atopic
keratoconjunctivitis, and giant papillary conjunctivitis) can be seen more often
in an Ophthalmology department since patients report only ocular symptoms. The acute
forms are mediated by a type I hypersensitivity response and the chronic forms
have a more complex immunological basis and a chronic inflammatory component [2].Based
on the finding of high total IgE levels in allergic conjunctivitis tears, a new
diagnostic method has been commercialised (Lacrytest. ADIATEC S.A., Nantes, France),
which qualitatively indicates the presence of total class E immunoglobulins in
tears with levels above normal values. Positive levels of the test indicate an
allergic conjunctivitis and negative levels rule out this aetiology.We
decided to evaluate the lacrimal test because it was rapid and easy to perform
and a prospective study was designed to ascertain its reliability. Our clinical
data showed the Lacrytest to be positive in only 6 of 30 patients (20%) with
IgE-mediated conjunctival allergy, confirmed by SPT and CPT. This is a very low
value compared with other studies in which the IgE-mediated conjunctivitis
group showed the most pronounced local IgE production [12, 13]; however
the test showed an increased positive value after conjunctival specific
provocation (20 of 30 patients; 66.7%). Tear IgE levels are known to have a
significant influence on intensity of the inflammatory process and the
correlation between CPT and lacrimal IgE has been demonstrated [14].
During allergic reactions, the local synthesis of IgE is elevated and total
tear IgE increases because of the
lacrimal IgE that have filtered through the blood-tear barrier [15];
thus, it is logical that the results proved more positive after exposure to the
allergen. In any event, these findings merit further studies to confirm and
explain this result.In
the vernal keratoconjunctivitis group, the Lacrytest was positive in 8 of 9
patients (88.9%). The results obtained in VKC concur with those in the literature.
Locally-produced IgE levels have been shown to be the largest contributor to
the severity of allergic conjunctivitis [16], particularly in chronic
forms, and local IgE production in VKC is increased compared with controls [13, 14] although no clinical differences were observed in atopic
keratoconjunctivitis (AKC) [17].The
Lacrytest has very high specificity and positive predictive value (100%); thus,
a positive test could point to an allergic aetiology, but the low number of positive
cases found in the group of IgE-mediated allergy (sensitivity 20%, negative
predictive value 38.46%) indicates that this test cannot be the only parameter to
achieve an aetiological diagnosis, as occurs with serum IgE. This test is not
useful for allergists because we have other more sensitive and specific methods
to diagnose IgE-mediated allergic conjunctivitis; however, it could be helpful
to ophthalmologists to confirm an IgE-mediated reaction or VKC if the result is
positive. Negative results should be sent to the allergist for the allergic aetiology
to be definitively confirmed or ruled out.



---

*Source: 518903-2009-04-12.xml*