A Practical Approach To Aeroallergen Identification

ACAAI Annual Meeting Nov.
7 - 11 2013, Baltimore
Workshop 1: A Practical Approach to Aeroallergen Identification
How to Set Up a Sampling Station
Estelle Levetin, PhD
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, Allergenco One day head or 7-day head for Burkard or Lanzoni Location Preparing the samples Slide Analysis and Identification Data Analysis
ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore
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 Calberlas 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 Allergenco
Burkard and Lanzoni Spore Traps
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 Permits analysis for diurnal rhythms
Time discrimination
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 Traverse

12 Transverse Traverses

Quicker Produces average daily concentration Good for routine monitoring 3 or 4 longitudinal traverses can increase accuracy
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
Example of an Excel Spreadsheet with 15 Days of Pollen Data

Cushing Daily Pollen concentrations Date Cupress Ulmus Ambrosia Artemisia Cheno/AmCompositaCyperacea Poaceae 0 0 181 4 4 34 0 13 1-Oct-01 0 0 170 8 2 42 0 17 2-Oct-01 0 2 284 13 6 48 0 27 3-Oct-01 0 0 269 2 6 21 0 36 4-Oct-01 6 6 231 48 8 19 0 8 5-Oct-01 0 0 19 0 0 19 0 2 6-Oct-01 0 0 57 4 2 4 0 13 7-Oct-01 0 0 164 0 8 27 0 17 8-Oct-01 0 0 189 0 0 6 2 8 9-Oct-01 2 0 80 8 6 2 0 8 10-Oct-01 2 0 27 2 2 2 0 6 11-Oct-01 4 0 50 4 0 17 0 2 12-Oct-01 19 0 29 2 6 21 0 13 13-Oct-01 2 0 36 2 2 6 0 4 14-Oct-01 95 0 63 6 4 15 0 4 15-Oct-01
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
10
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 80:1417. 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/ Lacey M and West J. 2006. The Air Spora: A manual for catching and identifying airborne biological particles. Springer.
Essential Reference
Grant Smiths Sampling and Identifying Allergenic Pollen and Molds
Sample Pages
11
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
12
Average Daily Pollen Concentration in the Tulsa Atmosphere - 2011

4000 3500
Pollen grains/cubic meter of air
3000 2500 2000 1500 1000 500 0 J J F F M M A A M M J J J J A A S S O O N N D D
Airborne Ambrosia pollen in Tulsa Fall 1999

700 600 Pollen grains/m3 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
13
Airborne Ambrosia pollen in Tulsa: 20 year mean

600 500
Pollen grains/m 3
400
300
200
100
0
5Se p 22 -A ug 12 -S ep 15 -A ug 29 -A ug 19 -S ep 26 -S ep 3O ct 17 -O ct 24 -O ct 10 -O ct 31 -O ct
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
31 -A ug 7Se p 24 -A ug 14 -S ep 17 -A ug 21 -S ep 28 -S ep 5O ct 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
14
Introduction to the Fungi
15
Fungi are eukaryotic organisms that are neither plant nor animal
Fungi include molds
Mushrooms
16
Puffballs
Bracket Fungi
Fungi can be unicellular such as yeast
17
Fungi usually have a thread like body made up of hyphae
Hyphae make up the mycelium
Hyphae also make up the structure of fruiting bodies such as mushrooms
18
Fungal Life Styles

Fungi are absorptive heterotrophs As absorptive heterotrophs they exist as

Pathogens Mutualistic symbionts Saprobes
Common Human Pathogen
19
Apple-Cedar Rust A Destructive Plant Pathogen
Lichens are symbiotic organisms composed of an alga and a fungus
The Majority of Fungi Are Saprobes
20
Most of the common airborne fungi are saprobes naturally occurring on leaf surfaces, decaying plant material, or in soil
Fungi reproduce by spores
Spores can result from sexual or asexual reproduction
Mycelium
Sexual Spores Asexual Spores
Mycelium
21
Spore Release Mechanisms
PASSIVE: Frequently related to wind speed and turbulence include members of the Dry Air Spora which peak in the afternoon ACTIVE: Generally require moisture common mechanism for ascospores and basidiospores

Basidiospores most abundant in predawn hours Ascospores most abundant during or following rain; however, a number of ascospores only require high humidity and are abundant in predawn hours
Types of Asexual Spores

Sporangium Conidia on hyphae
Sporangiospore or just spore
Conidium
Conidiophore Sporangiophore
Rhizopus Sporangium
22
Chains of Conidia
Fungal Classification
Mycologist recognize 8 phyla in the Kingdom Fungi based phylogenetic analysis of DNA Three of these phyla contain important allergens

Zygomycota Ascomycota Basidiomycota
ZYGOMYCOTA -- Zygospores
ASCOMYCOTA -- Ascospores
BASIDIOMYCOTA -- Basidiospores ASEXUAL (ANAMORPHIC) STAGE Conidia
23
Asexual /Anamorphic Fungi
Most are members of the Ascomycota with a small percent members of the Basidiomycota Formerly called

Deuteromycetes Imperfect Fungi (Fungi Imperfecti) Mitosporic Fungi Mold Spores
Also called

24
Members of the Zygomycota produce asexual spores in a sporangium
The zygospore (zygosporangium) is the characteristic sexual structure of the Zygomycota
Characteristic sexual structure of the Ascomycota is the ascus containing 8 ascospores
25
Ascomycota
Many members of the Ascomycota develop asci within a fruiting body Asexual spores are called conidia Fruiting bodies often called ascocarps Ascocarps can be

Flask-shaped Cup-shaped Other
Cup-shaped fruiting body
Morels are also cup-shaped fruiting body
26
Asci with ascospores from a morel
Abundant Airborne Ascospores Following Rain
Airborne Ascospores Still in Group of 8
27
Majority of Asexual Fungi Are Ascomycota
Basidiomycota

The most conspicuous fungi in the environment Basidiospores are typically produced in a large fruiting body such as

Mushrooms Bracket Fungi Puffballs
Characteristic spores are basidiospores and four basidiospores are produced externally on basidia
28
Basidia line the pores of bracket fungi and the gills of mushrooms
Single basidium with 4 basidiospores
Basidiospores are small and single-celled often with an asymmetric attachment peg
29
Rusts and Smuts
Basidiomycota also includes two groups of fungi that lack fruiting bodies Rust fungi and smut fungi Important pathogens on both native and cultivated plants
Stem Rust of Wheat
Loose Smut of Wheat
30
Common Fungal Spores

Estelle Levetin, PhD
31
Fungal Spore Characteristics

Spore size Spore shape Number of cells Attachment Scars Wall characteristics Spore color
Spore Size, Shape, and Septation

SIZE: 2m to 100 mm SHAPE: Globose, elliptical, fusiform, asymmetric, lemon-shaped, barrel-shaped, curved SEPTATION: Non-septate (one cell), single septum, transverse septa, transverse and longitudinal septa, random septa, pseudoseptate
Other Characteristics
ATTACHMENTS: Attachment scars, attachment pegs APPENDAGES WALL CHARACTERISTICS: Smooth, granular, reticulate, spines, warts, wall thickness COLOR: Hyaline (colorless) to deeply pigmented
32
Globose
Barrel-shaped
Non-septate
Random septa
Lemon-shaped
Club-shaped
Transverse septa
Attachment scars
Elliptical
Curved
Transverse and Longitudinal septa
Ornaments: spines
Asymmetric and germ pore
Cylindrical
Pseudoseptate
Appendages
Spore color
Asexual Spores in the Ascomycota

Also known as Deuteromycetes, Fungi Imperfecti (imperfect fungi), or Mitospores
33
Asexual Spores
Typically the most abundant spores in the atmosphere Asexual stage of ascomycetes Conidia often formed on specialized hyphae called conidiophores Look for attachment scars where the spores were attached to the conidiophore
Cladosporium
Cladosporium
Note the septum
34
Several species of Cladosporium are common in the atmosphere
Note the prominent attachment scars on Cladosporium conidia
Alternaria
35
Alternaria
Curvularia
Nigrospora Curvularia
Drechslera
36
Drechslera-type spores
Several genera of fungi have similar cylindrical spores

Drechslera Bipolaris Exserohilum Helminthosporium
Drechslera-type spores
Pithomyces
37
Note the colorless attachment at the base of Pithomyces spores
Epicoccum
Penicillium species
Produce distinctive conidiophores (spore bearing structures) Spores are usually spherical to oval and form in chains
38
Aspergillus species
Produce distinctive conidiophores (spore bearing structures) Spores are usually spherical to oval and form in chains
Penicillium-Aspergillus type spores
Fusarium
39
Botrytis
Oidium
Nigrospora
Nigrospora Culture
Air Sample
40
Periconia
Cercospora
Polythrincium
Peronospora
41
Stemphylium
Torula
Tetraploa
42
Spegazzinia
Stachybotrys
Phylum Ascomycota: Sexual Stage
43
Ascospores are produced in an ascus. Eight ascospores are found in each ascus without any attachment scars
Ascospores are sometimes found in groups of eight in air samples
Chaetomium ascospores
44
Leptosphaeria ascospores
Pleospora ascospores
Diatrypella ascospores
45
Sporomiella ascospores
Venturia ascospores
Many ascospores on a rainy day
46
Phylum Basidiomycota
Basidiospore
Basidium
Ganoderma basidiospores
47
Other basidiospores that are easy to recognize
Coprinus
Agrocybe - type
Coprinus and Ganoderma basidiospores
Psathyrella
Psathyrella velutina
Russula
Stropharia
48
Lycoperdon
Calvatia
Pisolithus
Scleroderma
Mixed Basidiospores
Other Spores in the Basidiomycota

Rusts and Smuts
49
Rust spores: Puccinia
Puccinia uredospores
Puccinia teliospores
Smut Spores
Smut Spores
50
Other Fungal-like Spores
Myxomycetes Slime Molds
Myxomycete (slime mold) spores
Other slime mold spores
51

A Practical Approach To Aeroallergen Identification

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ACAAI Annual Meeting Nov.

Workshop 1: A Practical Approach to Aeroallergen Identification

How to Set Up a Sampling Station

Estelle Levetin, PhD

Learning Objectives and Disclosure Information

Upon completion of this workshop, participants should be able to:

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

Pollen only or both pollen and spores Sampling frequency

7 days a week 5 days a week 3 days a week

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

Older Model Rotorod Sampler

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

V = Rod area (m2) x D x x RPM x t

Hirst Spore Trap

Burkard Spore Trap Lanzoni Spore Trap Allergenco

Burkard and Lanzoni Spore Traps

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

Allergenco Samplair MK-3

Advantages of Burkard Spore Trap

High efficiency down to less than 5 m

Permanent slides for future reference

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

Telescoping mast elevates sampler above local vegetation.

Burkard 7-day sampler head

Seven Day Sampling Head

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

Processing the 7-day drum

Melenex tape on cutting board

One-day sampling head

Alternatively Melenex tape can be fixed on the slide and greased

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

24 hour sampling head

Outdoor air sample from Tulsa

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

Burkard Counting Methods

The Single Longitudinal Traverse Method

The Twelve Transverse Traverse Method

Single Longitudinal Traverse

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

Calculating Concentrations for Single Longitudinal Traverse

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

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

Grant Smiths Sampling and Identifying Allergenic Pollen and Molds

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

How the data can be used

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

Average Daily Pollen Concentration in the Tulsa Atmosphere - 2011

3000 2500 2000 1500 1000 500 0 J J F F M M A A M M J J J J A A S S O O N N D D

Airborne Ambrosia pollen in Tulsa Fall 1999

Multiple Years of Data

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

Airborne Ambrosia pollen in Tulsa: 20 year mean

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

Introduction to the Fungi

Learning Objectives and Disclosure Information

Upon completion of this workshop, participants should be able to:

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

Fungi include molds

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore

Fungi can be unicellular such as yeast

ACAAI Annual Meeting Nov. 7 - 11 2013, Baltimore