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 mm

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 (m2) 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/m3

*NOTE: Width of rods may vary slightly

Burkard Spore Trap

Advantages

High efficiency down to less than 5 mm 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

Parapet around roof

requires platform to elevate

the orifice above the wall

Telescoping mast

elevates sampler above

local vegetation.

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/V

C = N x 14mm/0.48mm x 1/14.4m3

C = N x 2.025

Cushing Daily Pollen concentrations

Date Cupress Ulmus Ambrosia Artemisia Cheno/AmCompositaeCyperaceaePoaceae

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

Example of an Excel Spreadsheet with

15 Days of Pollen Data

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

0

500

1000

1500

2000

2500

3000

J F M A M J J A S O N D

Po

llen

gra

ins/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

Mean airborne Ambrosia pollen in

Tulsa: 1987-2007

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-O

ct

10-O

ct

17-O

ct

24-O

ct

31-O

ct

Five day running mean of airborne

Ambrosia pollen in Tulsa

0

100

200

300

400

500

600

700

8/15 8/29 9/12 9/26 10/10 10/24

Po

lle

n g

rain

s/m

3

Peak on or about Sept 10

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