workshop 1: a practical approach to aeroallergen identification estelle levetin, phd how to set up a...
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Workshop 1: A Practical Approach to Aeroallergen Identification
Estelle Levetin, PhD
How to Set Up a Sampling Station
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Learning Objectives and Disclosure Information Upon completion of this workshop, participants
should be able to: 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
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Aerobiological Sampling
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
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Sampling Objective
Pollen only or both pollen and spores Sampling frequency
7 days a week 5 days a week 3 days a week
Time commitment
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Rotorod Samplers
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Rotorod Samplers
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 m
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Rotorod Samplers
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Older Model Rotorod Sampler
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Rotorod Analysis
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
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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 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
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Hirst Spore Trap
Burkard Spore Trap Lanzoni Spore Trap Kramer-Collins Spore
Trap Allergenco
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Burkard Spore Trap
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Lanzoni VPPS Sampler
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Allergenco – Samplair MK-3
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Advantages of Burkard Spore Trap
High efficiency down to less than 5 m Allows for greater
accuracy for small fungal spores
Time discrimination Permits analysis for
diurnal rhythms Permanent slides for
future reference
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Location
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
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Burkard 7-day sampler head
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
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Seven Day Sampling Head
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Processing the 7-day drum
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
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Melenex tape on cutting board
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One-day sampling head
Alternate head is the 24 hour head Standard glass microscope slide is greased
and placed on the head 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
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24 hour sampling head
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Outdoor air sample from Tulsa
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Analysis
Microscopy - 400X for pollen; 1000X for fungal spores
Different methods of microscopic analysis are used to obtain Average daily concentration - Single longitudinal
traverse Hourly or bihourly concentrations which can then
be averaged to obtain a daily average - 12 transverse traverses
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The Single Longitudinal Traverse Method
The Twelve Transverse Traverse Method
Burkard Counting Methods
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Comparison of methods
Single Longitudinal Traverse Quicker Produces average daily
concentration Good for routine
monitoring 3 or 4 longitudinal
traverses can increase accuracy
12 Transverse Traverses Takes longer Can determine diurnal
rhythm of airborne allergens
All traverses can be averaged to determine average daily concentration
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Conversion to Concentrations Microscope counts are entered into a database such
as Excel Formulas added to convert counts into
concentrations Information needed
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
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Calculating Concentrations for Single Longitudinal Traverse 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/VC = N x 14mm/0.48mm x 1/14.4m3
C = N x 2.025
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Cushing Daily Pollen concentrationsDate Cupress Ulmus Ambrosia Artemisia Cheno/AmCompositaeCyperaceaePoaceae
1-Oct-01 0 0 181 4 4 34 0 132-Oct-01 0 0 170 8 2 42 0 173-Oct-01 0 2 284 13 6 48 0 274-Oct-01 0 0 269 2 6 21 0 365-Oct-01 6 6 231 48 8 19 0 86-Oct-01 0 0 19 0 0 19 0 27-Oct-01 0 0 57 4 2 4 0 138-Oct-01 0 0 164 0 8 27 0 179-Oct-01 0 0 189 0 0 6 2 8
10-Oct-01 2 0 80 8 6 2 0 811-Oct-01 2 0 27 2 2 2 0 612-Oct-01 4 0 50 4 0 17 0 213-Oct-01 19 0 29 2 6 21 0 1314-Oct-01 2 0 36 2 2 6 0 415-Oct-01 95 0 63 6 4 15 0 4
Example of an Excel Spreadsheet with 15 Days of Pollen Data
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Identification
AAAAI and ACAAI Aeroallergen courses Other aerobiology courses Reference slides
NAB/AAAAI Pollen Slide Library Reference slides from local specimens Consult a botanist at a local university
Identification Manuals
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Identification Manuals
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/
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Essential Reference
Grant Smith’s Sampling and Identifying Allergenic Pollen and Molds
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Sample Pages
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How the data can be used
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
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Average Daily Pollen Concentration in Tulsa Atmosphere - 2009
0
500
1000
1500
2000
2500
3000
3500
4000
J F M A M J J A S O N D
Pol
len
grai
ns/m
3
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Airborne Ambrosia pollen in Tulsa Fall 1999
0
100
200
300
400
500
600
700
8/15 8/29 9/12 9/26 10/10 10/24
Po
llen
gra
ins
/m3
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Multiple Years of Data
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
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Airborne Ambrosia pollen in Tulsa: 20 year mean
0
100
200
300
400
500
600
15-A
ug
22-A
ug
29-A
ug
5-Sep
12-S
ep
19-S
ep
26-S
ep
3-Oct
10-O
ct
17-O
ct
24-O
ct
31-O
ct
Po
llen
gra
ins/
m3
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Five day running mean of airborne Ambrosia pollen in Tulsa: 20 year mean Peak on or about Sept 10
0
50
100
150
200
250
300
350
400
450
500
17-A
ug
24-A
ug
31-A
ug
7-Sep
14-S
ep
21-S
ep
28-S
ep
5-Oct
12-O
ct
19-O
ct
26-O
ct
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Conclusion
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
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Additional references
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