Transcript Immunoblot assay for determining the incidence allergens specific

Sensitization to pollen , animal dander ,
house dust mites and moulds among
patients with allergic rhinitis in warm
dry climates
Abbas H. Alsaeed M Med Sci, PhD, CLC, MRCpath
Assistant Professor and Consultant Hematologist
King Saud University, College of Medical Sciences
Dept. Of Clinical Laboratory Sciences
1
Variations in Defining Allergic Rhinitis
1.
2.
3.
Allergic rhinitis was defined as the selfreported presence, in the previous year, of
usual nasal blockage and discharge apart
from colds or the flu, provoked by allergens,
with or without conjunctivitis (Ng & Tan, 1994)
Rhinitis was defined as sneezing or a runny
or blocked nose not due to a cold or the flu
(Sly, 1999)
Individuals with rhinitis were defined as those
suffering with three or more symptoms of
rhinitis either throughout the year (perennial)
or for part of the year (seasonal) (Frosh et al
1999)
2
Background
 Pollutants
have a direct effect on the nose by
causing cellular damage and stimulating the
release of inflammatory mediators (Bascom,
1991).
 Prevalence rates of rhinitis symptoms among
office workers reported in the western
countries were around 30%, but could be as
high as 60% in some offices (Finnegan 1984;
Viegi et al 1991)
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ANIMAL EPITHELIA DANDERS
1-Cats are considered to be the most highly
allergenic animal species for most people.
2-Dog, responsible for allergens, usually
specific to epithelial proteins.
3-Horse is reported as a source of a very
potent allergen. Many patients develop
severe allergy, even an indirect contact to
horse is enough to release symptoms.
4
HOUSE DUST MITES (HDM)
Dust mites are microscopic organisms,
belong to the kingdom animalia, phylum
arthropoda and class arachnia.
The organism’s genus and species name
are Dermatophagoides pteronyssinus and
Derm. farinae.
HDM is widely suspected to be an important
factor in the pathogenesis of allergic
disease .
5
MOULD
Mould, e.g. alternaria alternata is a common
and cosmopolitan species occurring on
many plants and other substrates including
soils, food and textiles.
It is frequently found on condensed window
frames and it considered an outdoor mould
and appears when weather is warm.
A. alternata is considered one of the most
allergenic mould in the world (Agarwal et al
1982).
6
Symptoms of Allergic Rhinitis
1- Sensitive to specific allergens, e.g. dust
2- Pruritus of the nose, eyes palate, ears
3- Sneezing more than two at a time
4- Watery rhinorrhoea
5- Coexistant asthma or eczema
6- Seasonal symptomas
7- Family history of allergies.
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Classification Allergic Rhinitis
 Seasonal Allergic
Rhinitis (Hayfever)
(SAR)
 Perennial Allergic
Rhinitis (PAR)
 Occupational
Allergens
 Pollen
from a variety of
trees, grasses, and
weeds.
 HDM,
Mold, Animal
epithelial dander, Food
(rare)
 Chemicals
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Perennial Allergic Rhinitis (PAR)

Patients with PAR are more likely than those
with SAR to have asthma (Kubetin 2001).

Positive family history of allergic rhinitis.

Personal history of collateral allergy such as
eczematous dermatitis, urticaria, and/or
asthma.
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Non-allergic Triggers
Cold air
2. Smoke and perfumes
3. Strong odors
4. Spicy/hot food
5. Alcohol
6. Pregnancy / hormones
7. Decongestant nasal sprays
8. Medications, e.g. antihypertensive agents
(Druce 1998).
1.
10
Symptoms of non-allergic rhinitis
1.
2.
3.
4.
History of negative allergen specific IgE
test
Sensitive to temperature changes, smoke,
perfume or environmental irritants
Adult onset of symptoms
Nasal crusting or drying.
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Common Comorbidity
Perennial
Seasonal
Sinusitis
50%
23%
Asthma
22%
16%
Otitis Media
14%
14%

20-15% of allergic rhinitis patients had asthma
 75-80% of asthma patients had allergic rhinitis
(Kirn 2001).
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AIMS
1- To investigate
sensitization to pollen,
animal dander, HDM and mould in adult
patients with allergic rhinitis in warm dry
climates using an immunoblot assay.
2- To estimate the prevalence of rhinitis
symptoms in warm climate.
3- To study the relationship between rhinitis
symptoms and allergens.
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Subjects
 Thirty–eight
adult patients (25 males and 13
females) with diagnosed allergic rhinitis

without a previous history of treatment
attending the outpatient ENT clinic, from
April 2002 to March 2005 on General
Hospital, Saudi Arabia.
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Investigations
History: present illness, past medical history,
family history, environmental history, and
impact on quality of life.
2. Physical exam: nose, eyes, ears, lung, skin.
3. Nasal exam with rhinoscope / flexible
nasendoscope.
4. Nasal smear for eosinophils.
5. Nasal mucociliary clearance assessment:
saccharin test, and ciliary best frequency
measurement.
6. Nasal airway assessment.
7. Allergy tests: Allergen specific IgE
1.
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Allergy Test (Methods)
METHOD
TEST
Blood Test
allergen immunoblot assay
(RIDA allergen screen)
Blood serum is
combined with allergen
in trough
Radio-allergosorbent allergy Blood serum is
testing (RAST)
combined with allergen
in test tube.
Skin Test
Provocation testing
Direct exposure (e.g.
via inhalation or
ingestion to a likely
allergen
Prick testing
Percutaneous
Scratch testing
Percutaneous
Intradermal testing
Intradermal injection
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METHOD
 Allergens are coated to the surface of
nitrocellulose membranes lying in trough.
 Patient
serum pipette into the reaction
trough.
 Allergy
screen detection antibody were
added.
 Streptavidin
conjugate with alkaline
phosphatase that binds to biotin in the
test fields were added.
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Continue

Substrate a specific enzyme color reaction
of alkaline phosphatase were added.
 The reaction stopped by rinsing with water
and analysis the results on the test strips
(RIDA allergy screen 2002).
 Negative control had not been coated with
allergen,
biotin-labeled
bovine
serum
albumin had been applied as positive control.
 The color intensity on the test fields is
directly reflect to specific IgE antibody in the
serum of the patients.
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Figure (1) Illustrates the results appearance on
the surface of nitrocellulose membrane
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Results

Specific IgE antibodies were detected in 65.8%
of all the subjects .
 Males demonstrated a higher rate than females
(76% vs. 46.2%) or a M/F ratio of ~ 2:1.
Parameters Number
Allergen
Allergen
Absent (%) Present (%)
P value
Patients
38
13 (34.2%)
25 (65.8%)
< 0.005
Male
25
06 (24.0%)
19 (76.0%)
< 0.005
Female
13
07 (53.8%)
06 (46.2%
0. 5
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Continue Results
 The
sensitization rate was highest for
pollen while results for the other allergen
groups are as follows :
 pollen (68.3%), animal dander (23.9%),
HDM (6.0%), and moulds (1.8%).
 Among allergenic pollen mixed grasses
were the most common cause
of
sensitization (11.1%) while alder, birch,
rye and oak have the same, albeit lower
sensitization rates (9.4%).
 These plants are commonly cultivated for
greening purposes in urban areas .
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Continue results
 Sensitization
rate to cat and dog dander are
(9.4% and 8.5% respectively),
 while dander from horse, guinea pig and
golden hamster caused
lower rates of
sensitization (3.4% - 1.7%).
 Dermatophagoides
pteronyssinus
and
Dermatophagoides farinae
are the most
prevalent indoor sensitizers (4.3% and 1.7%,
respectively).
 Sensitization
to mould is relatively rare
( 1.8%).
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 Animal
dander (23.9%) and HDM (6.0%)
altogether pose a significant although less
important source of exposure,
 with
only rare cases involving mould
(1.72%), leaving pollen as the leading
cause of sensitization,
 attributable in part to the all too common
utilization of plants in urban areas (like
Riyadh) which are purposely cultivated for
greening purposes and in part to existing
dry climatic conditions
23
Table (2) Distribution of allergen antibodies in rhinitis
patients.
Allergen
(antigen system)
Pollens (Aeroallergens)
Alder
Birch
Hazel
Grasses
Rye
Mugwort
Plantain
Oak
Allergen
Antibodies No.
Allergen
Antibodies
Antibodies (%) System (%)
68.3
11
11
10
13
11
8
5
11
9.4
9.4
8.5
11.1
9.4
6.8
4.3
9.4
Animal epithelia dander
Cat
Horse
Dog
Guinea pig
Golden Hamster
Rabbit
23.9
11
4
10
1
2
-
9.4
3.4
8.5
0.9
1.7
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Allergen
(antigen system)
Allergen
Antibodies No.
Allergen
Antibodies (%)
House dust Mites
Derm. pteronyssinus
2
1.7
Derm. farinae
5
4.3
Mould
Alternaria alternate
Pencillium notatum
Cladosporium hebarum
Antibodies
System (%)
6.0
1.8
-
-
Aspergilus fumigatus
1
1
0.9
0.9
Total
117
100
100
25
Figure ( ) Diagram showing the overlapping of
sensitization to combination of allergens (%)
among patients with allergic rhinitis
26
 Allergic
patients are not only allergic to
one sole type of allergen since many of
them will display sensitization to a
combination of allergens .
 The largest subset of patients display a
combination of sensitization to both pollen
and dander
27
Table (3) Patients with one or multiple allergen
antibodies developed
No. of allergen
Antibodies developed
No. of patients
and percentage
Sensitized patients
(Percentage)
1
06 (15.8%)
24 %
2
01 (2.6%)
4%
3
03 (7.9%)
12 %
4
02 (5.3%)
8%
5
03 (7.9%)
12 %
6
03 (7.9%)
12 %
7
01 (2.6%)
4%
8
04 (10.5%)
16 %
9
01 (2.6%)
4%
11
01 (2.6%)
4%
Total
65.1
100
28
In most studies the prevalence of seasonal allergic
rhinitis is significantly higher than perennial
allergic rhinitis , and range from 4.5 % to 38.3 %
(Charpin et al 1993; Dotterud et al 1994) .
In Finland the prevalence of allergic rhinitis is
around 14% (Pekkanen et al, 1997).
in Australia 27% ( Woolcock et al 2001), while in
India 20-30% of the general population suffers
from allergic rhinitis (Anonymous, 2000).
In the United States allergic rhinitis seems to be
exceedingly common where 9-40% of the
population may have some form of allergic
rhinitis at anytime of the year
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Figure (2) Relative prevalence of allergens & seasons:
the allergen clender
12
% Cases No.
10
8
6
4
2
0
Winter
Spring
Pollens
Dander
Summer
HDM
Autumn
Moulds
30
In a related study in Ankara, Turkey, grass pollen were
found to be the major allergens in a study by
Sener et all (2003).
In Kuwait, higher sensitization rates were seen among
pollen belonging to plants which were imported
from other countries for shading purposes or for
binding sand, like Chenopedium and Bermuda
grass (Dowalsan et al, 2000).
In most European and North American countries
allergens with the highest sensitization rates
among patients with nasal symptoms were also
pollen/aeroallergens followed by house dust mites
and cat dander
31
 HDM
were reported to have the highest
rate of sensitization among patients with
allergic rhinitis in Thailand (Pumhirum et al,
1997), Singapore (Chow et al, 1999) and
in Taiwan especially among asthmatic
patients (Tsai et al, 1998).
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Report of Prevalence Rates in Allergic Rhinitis
(variation in methodology)
Prevalence Age
Country
Year
24.0%
62.5%
51.0%
27.6%
41.5%
22.0%
15.9%
26.3%
UK
Turkey
Turkey
Singapore
Singapore
Denmark
Italy
Bangkok
1991
1994
1996
1994
1994
2000
2002
2002
16-65
H. School
H. school
6-7
12-15
7-17
20-44
U student
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Prevalence of Rhinitis in UK
Prevalence Age
Author
Year
35.1%
6-7
Lau et al.
1998
44.0%
13-14
Leung et al.
1997
29.8%
12-18
Leung et al.
1997
49.5%
Workers
Tong et al.
2001
34


Discussion
This study clearly suggests that pollen
constitute the most important group of
sensitizing allergens in patients with allergic
rhinitis in this region with as many as 68.3%
of all positive cases sensitized to at least
one pollen.
In this study IgE - mediated sensitization to
fungal allergen was rare in allergic rhinitis
patients which means that people in a
certain locality had less exposure to mould
allergens
35


Pet ownership is not popular in Saudi Arabia
and detailed studies on animal
dander
sensitivity are lacking. Pet ownership
is
undoubtedly
a more common practice in
other parts of the non-Moslem world, such as
in Europe and America, where more and more
people are choosing a solitary existence,
accompanied only by a pet animal at home.
Allergen calendars could be a helpful guide in
determining probable provocative factors
among allergic rhinitis patients presenting with
a history of sensitization . Spring heralds an
epidemiologic peak in the number of cases
yearly .
36
Conclusions
1.
2.
3.
4.
5.
6.
The results of this study show that even in warm climates
pollen , animal dander and HDM allergens may be important
sensitizing allergens .
Pollens of local horticultural plants are the main sensitizing
allergens among patients with allergic rhinitis in the locality
studied, which may play a role in the pathogenesis of allergic
respiratory diseases
and
should therefore
be considered
important factors in the management of patients affected in regions
presenting with a generally warm dry climate most of the year .
The practice of greening the country seems to contribute to
increased rates of allergic sensitization of persons prone to
allergic rhinitis.
Subtle changes in social practices, like pet ownership, increasing
mobilisation and introduction of new potentially allergen.
The results of this study are mostly consistent with the results of
other studies made in other parts of the world, with some variations
due to differences in environmental conditions and social practices .
Local environmental and genetic factors are probably involved
in the pathogenesis of the disease .
37
Treatment
Allergen
avoidance
* Identification of the
responsible allergens
* Health education on allergen
avoidance
Medical treatment * Antihistamines.
* Topical corticosteroids.
* Anticholinergics.
* Decongestants
Immunotherapy-injection
Sublingual
38
Treatment
Symptoms
Symptomatic treatments
Nasal obstruction
due to nonallergic
rhinitis
* Azelastine HCL nasal
spray
* Oral decongestant
Non-purulent chronic * Increase water intake.
post nasal drip
* Nasal saline irrigation.
* Humidity to bedroom.
Bilateral anterior
* Intranasal cortisteroid.
rhinorrhea
* Atrovent spray.
* Nasal saline
39