Session 1202: Basic Aeroallergen Course Setting up a Sampling Station Estelle Levetin, PhD
Disclosure No conflicts to disclose
Aerobiological Sampling Sampling plan or objective Samplers: Rotorod, Burkard Spore Trap Location One day head or 7 day head for Burkard Preparing the samples Slide Analysis Identification Data
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
Rotorod Samplers
Rotorod Samplers Models typically used have retracting rods Head rotates at 2400 rpm, leading edge of rod coated with silicon grease Pollen and spores impacted on greased surface Generally operated at 10% sampling time Efficient for pollen and spores >10 m m
Rotorod Samplers
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
Rotorod Calculations C = N / V C is concentration, N is the number of pollen or spores counted on both rods, V is the volume of air sampled by the rods V = Rod area (m 2 ) x D x p x RPM x t Rod area = width of rod (1.52* mm = 0.00152* m) x length of the rod (23 mm = 0.023 m) x 2 (both rods), D is the diameter of the Rotorod head (8.5 cm = 0.085 m), RPM is 2400, t is minutes sampled per day With a 5% sampling time (72 min) V = 3.226 Concentration = N/3.226 pollen grains/m 3 *NOTE: Width of rods may vary slightly
Burkard Spore Trap Advantages High efficiency down to less than 5 m m Allows for greater accuracy for small fungal spores such as basidiospores and small ascospores Time discrimination Permits analysis for diurnal rhythms Permanent slides for future reference
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
Telescoping mast elevates sampler above local vegetation. Parapet around roof requires platform to elevate the orifice above the wall
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
Seven Day Sampling Head
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
Melenex tape on cutting board
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
24 hour sampling head
Outdoor air sample from Tulsa
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
Burkard Counting Methods The Single Longitudinal Traverse Method The Twelve Transverse Traverse Method
Comparison of methods Single Longitudinal 12 Transverse Traverse Traverses Quicker Takes longer Produces average Can determine diurnal daily concentration rhythm of airborne allergens Good for routine monitoring All traverses can be averaged to determine 3 or 4 longitudinal average daily traverses can increase concentration accuracy
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 m 3
Calculating Concentrations for Single Longitudinal Traverse C = Concentration - pollen grains/m 3 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 m 3 C = N x W/F x 1/V C = N x 14mm/0.48mm x 1/14.4m 3 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/AmComposita Cyperacea 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 AAAAI and ACAAI Aeroallergen courses Other aerobiology courses such as the New Orleans Aeroallergen Course Reference slides NAB/AAAAI Pollen Slide Library Reference slides from local specimens Consult a botanist at a local university Identification Manuals
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. Lacey, Maureen and J. West. 2006. The Air Spora: A Manual for Catching and Identifying Airborne Biological Particles, Springer. 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 Grant Smith’s Sampling and Identifying Allergenic Pollen and Molds
Sample Pages
http://allernet.net/
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
Average Daily concentration of Airborne Pollen in Tulsa - 2008 3000 2500 2000 Pollen grains/m 3 1500 1000 500 0 J F M A M J J A S O N D
Airborne Ambrosia pollen in Tulsa Fall 1999 700 600 Pollen grains/m 3 500 400 300 200 100 0 8/15 8/29 9/12 9/26 10/10 10/24
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
Mean airborne Ambrosia pollen in Tulsa: 1987-2007 600 500 400 300 200 100 0 g g g p p p p t t t t t c c c c c u u u e e e e O O O O O A A A S S S S - - - - - - - - - - - - 3 0 7 4 1 5 2 9 5 2 9 6 1 1 2 3 1 2 2 1 1 2
Five day running mean of airborne Ambrosia pollen in Tulsa Peak on or about Sept 10 700 600 Pollen grains/m 3 500 400 300 200 100 0 8/15 8/29 9/12 9/26 10/10 10/24
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
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
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