Transcript Document

Workshop 1: A Practical Approach
to Aeroallergen Identification
How to Set Up a Sampling Station
Estelle Levetin, PhD
Learning Objectives and
Disclosure Information
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Upon completion of this workshop, participants
should be able to:
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Set up a sampling station to collect airborne pollen and fungal
spore
Recognize the most common types of pollen found in the
atmosphere
Recognize the most common types of fungal spores found in
the atmosphere
No conflicts to disclose
Aerobiological Sampling
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Sampling plan or objective
Choosing samplers: Rotorod, Burkard Spore Trap,
Lanzoni, Krammer-Collins, Allergenco
Location
One day head or 7 day head for Burkard
Preparing the samples
Slide Analysis and Identification
Data Analysis
Sampling Objective
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Pollen only or both pollen and spores
Sampling frequency
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7 days a week
5 days a week
3 days a week
Time commitment
Rotorod Samplers
Rotorod Samplers
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Models most often used by allergists have retracting
rods for intermittent operation
Standard is 10% sampling time
Head rotates at 2400 rpm
Leading edge of rod coated with grease
Pollen and spores impacted on greased surface
Efficient for pollen and spores >10 mm
Rotorod Samplers
Older Model Rotorod Sampler
Rotorod Analysis
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Collector rods placed in a special
adapter for microscopic
examination
Rods stained with Calberla’s
pollen stain
Entire surface of each rod
counted unless pollen/spore load
very high (then a subset of the
surface is analyzed)
Atmospheric concentrations
determined
Rotorod Calculations
C=N/V
C is concentration, N is the total number of pollen or spores counted
on both rods, V is the volume of air sampled by the rods
V = Rod area (m2) x D x p x RPM x t
Rod area = width of rod (1.52 mm = 0.00152m) x length of the rod (23
mm = 0.023m) x 2 (both rods), D is the diameter of the Rotorod head
(8.5 cm = 0.085m), RPM is 2400, t is minutes sampled per day
With a 5% sampling time (72 min) V = 3.226 m3
Concentration = N/3.226 m3
With a 10% sampling time (144 min) V = 6.452 m3
Concentration = N/6.452 m3
Hirst Spore Trap
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Burkard Spore Trap
Lanzoni Spore Trap
Kramer-Collins Spore
Trap
Allergenco
Burkard Spore Trap
Lanzoni VPPS Sampler
Allergenco – Samplair MK-3
Advantages of Burkard Spore Trap
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High efficiency down to
less than 5 mm
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Time discrimination
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Allows for greater
accuracy for small fungal
spores
Permits analysis for
diurnal rhythms
Permanent slides for
future reference
Location
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Roof of a building - ideal 3 to 6 stories above
ground (30 to 60 ft)
Not close to overhanging vegetation
Air flow not obstructed by nearby buildings or
other structural features
Burkard 7-day sampler head
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Standard is the 7-day sampling head
Sampler drum mounted on 7-day clock
Drum moves by orifice at 2 mm per hr
Melenex tape mounted on drum and greased
(Lubriseal, High Vacuum Grease, other)
Air is brought in at 10 l/min and impacts on greased
Melenex tape
Drum changed each week
Seven Day Sampling Head
Processing the 7-day drum
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Melenex tape removed from drum
Tape cut into seven 24 hour segments each 48 mm
long
Segments mounted on microscope slides in 10%
gelvatol (polyvinyl alcohol) and dried
Glycerin-jelly mounting medium added and a 50 mm
cover slip
Mounting medium contains pollen stain - either basic
fuchsin or phenosafarin
Melenex tape on cutting board
One-day sampling head
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Alternate head is the 24 hour head
Standard glass microscope slide is greased
and placed on the head
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Alternatively Melenex tape can be fixed on the
slide and greased
Slide is changed daily, carrier realigned
Mounting medium with stain and coverslip
are added
24 hour sampling head
Outdoor air sample from Tulsa
Analysis
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Microscopy - 400X for pollen; 1000X for
fungal spores
Different methods of microscopic analysis
are used to obtain
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Average daily concentration - Single longitudinal
traverse
Hourly or bihourly concentrations which can then
be averaged to obtain a daily average - 12
transverse traverses
Burkard Counting Methods
The Single Longitudinal Traverse Method
The Twelve Transverse Traverse Method
Comparison of methods
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Single Longitudinal
Traverse
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Quicker
Produces average daily
concentration
Good for routine
monitoring
3 or 4 longitudinal
traverses can increase
accuracy
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12 Transverse
Traverses
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Takes longer
Can determine diurnal
rhythm of airborne
allergens
All traverses can be
averaged to determine
average daily
concentration
Conversion to Concentrations
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Microscope counts are entered into a database such
as Excel
Formulas added to convert counts into
concentrations
Information needed
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Field diameter of objective lens - Variable
Flow rate (10 liters/minute) and exposure time (normally 24
hrs) for a total volume of air sampled of 14.4 m3
Calculating Concentrations for Single
Longitudinal Traverse
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C = Concentration - pollen grains/m3
N = number of pollen counted on traverse
W = Width of entire sample - 14 mm
F = field diameter of objective lens - 0.48 mm
V = total volume of air sampled- 14.4 m3
C = N x W/F x 1/V
C = N x 14mm/0.48mm x 1/14.4m3
C = N x 2.025
Example of an Excel Spreadsheet with 15 Days
of Pollen Data
Cushing
Daily Pollen concentrations
Date
Cupress Ulmus
Ambrosia Artemisia Cheno/AmCompositae
Cyperaceae
Poaceae
1-Oct-01
0
0
181
4
4
34
0
13
2-Oct-01
0
0
170
8
2
42
0
17
3-Oct-01
0
2
284
13
6
48
0
27
4-Oct-01
0
0
269
2
6
21
0
36
5-Oct-01
6
6
231
48
8
19
0
8
6-Oct-01
0
0
19
0
0
19
0
2
7-Oct-01
0
0
57
4
2
4
0
13
8-Oct-01
0
0
164
0
8
27
0
17
9-Oct-01
0
0
189
0
0
6
2
8
10-Oct-01
2
0
80
8
6
2
0
8
11-Oct-01
2
0
27
2
2
2
0
6
12-Oct-01
4
0
50
4
0
17
0
2
13-Oct-01
19
0
29
2
6
21
0
13
14-Oct-01
2
0
36
2
2
6
0
4
15-Oct-01
95
0
63
6
4
15
0
4
Identification
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AAAAI and ACAAI Aeroallergen courses
Other aerobiology courses
Reference slides
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NAB/AAAAI Pollen Slide Library
Reference slides from local specimens
Consult a botanist at a local university
Identification Manuals
Identification Manuals
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Grant Smith. 2000. Sampling and Identifying Allergenic
Pollens and Molds, AAAAI, Milwaukee
R.O. Kapp, How to Know Pollen and Spores - originally
published in 1950s - new edition
Richard Weber. 1998. Pollen Identification Ann Allergy
Asthma Immunol 80:141–7.
Lewis WH, Vinay P, Zenger VE. 1983. Airborne and
Allergenic Pollen of North America. Johns Hopkins
University Press, Baltimore, MD.
Aeroallergen Photo Library, Steve Kagan, http://allernet.net/
Essential Reference
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Grant Smith’s Sampling
and Identifying
Allergenic Pollen and
Molds
Sample Pages
How the data can be used
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Average daily concentrations can be graphed
to look at the seasonal and yearly pollen
levels
Develop regional pollen calendar
Data can be compared with patient
symptoms, peak flow readings, office visits,
emergency room visits
Prepare for peak seasons - staffing, etc
Average Daily Pollen Concentration in Tulsa Atmosphere - 2009
4000
3500
Pollen grains/m 3
3000
2500
2000
1500
1000
500
0
J
F
M
A
M
J
J
A
S
O
N
D
Airborne Ambrosia pollen in Tulsa Fall 1999
700
Pollen grains/m 3
600
500
400
300
200
100
0
8/15
8/29
9/12
9/26
10/10
10/24
Multiple Years of Data
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Data from several years can be averaged to
produce a graph of the pollen season
Smoothing techniques such as 5 day running
mean can be used to generate a smoother
curve and better estimate of the typical peak
period
31
-O
ct
24
-O
ct
17
-O
ct
10
-O
ct
3O
ct
26
-S
ep
19
-S
ep
12
-S
ep
5Se
p
29
-A
ug
22
-A
ug
15
-A
ug
Pollen grains/m 3
Airborne Ambrosia pollen in Tulsa: 20 year
mean
600
500
400
300
200
100
0
Five day running mean of airborne Ambrosia
pollen in Tulsa: 20 year mean
Peak on or about Sept 10
500
450
400
350
300
250
200
150
100
50
0
g
Au
17
g
Au
24
g
Au
31
p
Se
7
p
Se
14
p
Se
21
p
Se
28
ct
O
5
ct
O
12
ct
O
19
ct
O
26
Conclusion
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Air sampling allows the allergist to get a first
hand understanding of the local
aeroallergens, their concentration, and
season occurrence
Several years of sampling will allow for the
development of a pollen calendar which can
benefit the physician and his or her patients
Additional references
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Gregory, P. H. 1973. The Microbiology of the Atmosphere, 2nd
ed., Halstead Press, NY.
Lacey, J and J. Venette. 1995. Outdoor Air Sampling Techniques.
in Bioaerosols Handbook, C.S. Cox and C.M.Wathes, ed., Lewis
Publishers, Boca Raton, FL.
Levetin E. and Horner WE. Fungal Aerobiology: Exposure and
Measurement, in “Fungal Allergy and Pathogenicity”, ed by
Brittenbach, Crameri, Lehrer. Krager, Basel. 2002; 81: 10-27.
Weber, R (ed). 2003. Immunology and Allergy Clinics of North
America. Vol 23 (3) Aerobiology