Workshop 1: A Practical Approach to Aeroallergen Identification

Estelle Levetin, PhD

How to Set Up a Sampling Station

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

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

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 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

Rotorod Samplers

Older Model Rotorod Sampler

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 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

Hirst Spore Trap

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

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

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

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

The Single Longitudinal Traverse Method

The Twelve Transverse Traverse Method

Burkard Counting Methods

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

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

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

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

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

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/

Essential Reference

Grant Smith’s Sampling and Identifying Allergenic Pollen and Molds

Sample Pages

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 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

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

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

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

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

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