- Email: [email protected]
Pollen Identification
Review article Supported by a grant from Zeneca Pharmaceuticals
Pollen identification Richard W Weber, MD*
Learning Objectives: The purpose of this review is to introduce the reader to a systematic approach to categorizing pollen types. This will enable the reader to recognize pollen characteristics of the most common botanical sources and determine the relevant contributors to the local aeroallergen burden. Data Sources: Allergenic plant and pollen atlases for pollen characteristics, allergy texts for procedures and overviews, and relevant reviews from the English medical literature (1985 to present). Results: By applying a visual gestalt utilizing grain number, size, shape, surface structures, and internal detail, one is able to identify pollen source to the appropriate botanical taxonomic level. Identification may be possible only to the family or order, but most frequently to the genus, and occasionally to the species. Conclusions: Outdoor sampling allows the identification of aeroallergen burden in a locale. In conjunction with field work, the relevant sources can be identified as well as time of pollination. This allows the physician to correlate symptoms with exposure and relevant sensitization. Ann Allergy Asthma Immunol 1998;80:141–7.
INTRODUCTION The purpose of this review is to provide a basis for identification of the majority of aeroallergenic pollens. Through the consideration of several variables discussed below, the observer can develop a system of pattern recognition, or gestalt, to assist in characterizing the airborne burden in a specific locale. This should be combined with a familiarity with what plants are actually growing in the area of concern. This may be best achieved by contacting a botanist who has knowledge of the local flora. Such a person may be working for a community or state government, or perhaps a university extension. Additionally, there are a number of texts that catalogue regional plants, and the interested party will * From Department of Medicine, Allergy/ Clinical Immunology Division, National Jewish Medical and Research Center, Denver, Colorado. Received for publication August 11, 1997. Accepted for publication in revised form December 9, 1997.
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find these helpful resources.1–3 Field trips at varying times of the year will familiarize one with the times of anthesis for the local plants. Knowing the local flora will assist in identification of the particles found on the sampler; this in turn will allow interpretation of patient’s symptom chronology and the relevance of in vivo or in vitro sensitivity. This review will use numerous descriptive phrases that are not common usage. These terms are defined throughout the text. For ease of referral, these phrases are placed in bold face where they are defined. BACKGROUND Higher plants use two methods of pollen dispersal: wind or vector. While a variety of animals may transport pollen, insect vectors are by far the most prevalent. Plant pollinated through the agency of an insect carrier are called entomophilous. Only about ten percent of higher plants utilize wind pollination, these are called anemophilous, and are the principle cause of
pollinosis. Plants that typically are both insect-pollinated and wind-pollinated are amphiphilous. Some primarily entomophilous plants, such as linden (Tilia), produce a large enough pollen load that appreciable surplus amounts become airborne. SAMPLE PREPARATION Principles of aeroallergen sampling have been reviewed previously by Burge.4 The reader is referred to that article for discussion of techniques and tabulation. Advantages of various samplers will not be discussed here other than to state than older gravimetric techniques such as the Durham sampler are no longer adequate, and volumetric sampling is strongly recommended. Once the sample is obtained, it is necessary to prepare it for microscopic examination. Pollens may be stained with numerous agents, but the standard preparation is with Caberla’s solution, which utilizes basic fuchsin. This stain permanently stains the pollen outer wall (exine), while not staining the great majority of mold spores; it imparts a pink to red color to the pollen grains, which persists in permanent mounts. The formulation of Caberla’s solution is as follows5: glycerin 5.0cc 95% ethyl alcohol 10.0cc distilled water 15.0cc saturated basic fuchsin 2 drops (or ⬃50mg powder) If the solution is staining too intensely, it may be diluted with the dyefree solution.
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MORPHOLOGY IN POLLEN GRAIN IDENTIFICATION
Figure 1. Composite cross-sectional detail of pollen grain wall (not all features found on individual grains). Surface projections not drawn to scale for ease of illustration. See text for definitions.
STRUCTURE OF POLLEN GRAINS The pollen wall consists of three layers, the outer two comprising together the exine, and the inner layer the intine. The exine is resistant to degradation, and withstands acid-heat exposure (a process called acetolysis). The intine is cellulose, and is susceptible to such treatment. Beneath the intine lies the cytoplasm of the grain, or the protoplast, enveloped by the cell membrane, the plasmalemma (Fig 1).1 The exine has two layers: the innermost featureless, but impervious, nexine, and the outer sexine, which is penetrated by numerous micropores, as well as larger apertures: pores and fur-
rows. These apertures are areas of thinned exine, and act as points of egress for the pollen tube during successful pollination. The apertures and micropores probably also act as avenues for the elution of allergenic proteins, which may have enzymatic and signal recognition functions in instigating the fertilization process. The sexine may have separate sublayers: an outer roof, or tectum, a foot layer abutting on the nexine, and a central segment comprised of columns, or baculae, separating the two. Additionally, the outer surface of the sexine may have a variety of projections and surface markings which are discussed below.
Figure 2. Photomicrographs showing representative shapes and clusters of pollen grains (not all same scale). A. spherical monoporate monad (grass, Graminae); B. wedge shape (bulrush, Scirpus); C. rhomboid tetrad (broadleaf cattail, Typha latifolia); D. split exine and extruded intine and stellate protoplast (cedar, Juniperus); E. square shape (ash, Fraxinus); and F. grain with two air bladders (pine, Pinus).
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Number of Grains The majority of pollens captured on samplers are single grains, or monads. Representative pollen types are shown in Figures 2 and 3. Some, however, are multiple, such as the 16-grain mimosa (Acacia) polyad, or the 4-grain tetrad of broadleaf cattail, Typha latifolia (Fig 2C). The latter is a “rhomboid” tetrad, while wood rush, Luzula, and members of the heath family (Erica) have a tetrahedral configuration. Occasionally, pollens will clump together, giving the appearance of a polyad, but with a little pressure on the coverslip, these will usually separate easily. Size Pollen grains vary greatly in size, from 2 to 5m to about 250m, although most wind-pollinated grains are between 20 to 60m. Nettle, Urtica, and the mulberries, Morus and Broussonetia, all produce pollens in the 10 to 15m range. While corn, Zea, and some conifers, Abies and Pseudotsuga, may be over 100m. Certain entomophilous pollens such as pumpkin, Cucurbita, may be as large as 250m. Shape Pollen grains are generally elliptical or spherical, but other distinct shapes exist. Pine, fir, and spruce pollens (Pinus, Abies, and Picea, respectively) have two air bladders, or vesicles, one either side of the grain. This gives the pollen grain a “Mickey Mouse cap” appearance when viewed in the equatorial plane (Fig 2F). These bladders may be either clear, taking on the fuchsin stain, or be darkly pigmented. Footballshaped or triangular are not uncommon, or even wedge-shaped, like a kernel of corn (sedge and bulrush, Carex and Scirpus, Fig 2B). Pores in the grain may act as points on the surface where the exine angles, giving the pollen a triangular, square, or polygonal shape, depending on the numbers of pores (ash, Fraxinus, Fig 2E). An exception is linden, which has three pores occupying the flat sides of a somewhat triangular shaped grain, rather than the
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Figure 3. Photomicrographs showing representative pollen grain surface features (not all same scale). A. three-furrowed, tricolpate (maple, Acer); B. spined, echinate, tricolporate (sunflower, Helianthus); C. periporate chenopod-amaranth; and D. triporate grain with distinctive aperture features of annulus, aspis, and oncus (birch, Betula).
corners. In the case of sages (Artemisia), the exine invaginates somewhat at the pores, bulging between, giving the grain a distinctive scalloped appearance. The degree of elongation of elliptical pollen grains can be used in identification. Kapp suggested using the ratio of polar to equatorial diameters, the P/E index.6 The pole of a grain is defined as either the location of a single pore or midpoint of a single furrow, or the end where furrows converge (tricolpate or tricolporate grains). The following descriptive phrases are used depending on the P/E index: ⬎2.0 perprolate (very elongated) 1.3–2.0 prolate (slightly elongated) 0.75–1.3 subspheroidal 0.50 – 0.75 oblate (slightly flattened) ⬍0.5 peroblate (very flattened) Surface Structures The majority of pollen grains can be identified by surface structures in the
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sexine, the most distinctive being apertures: pores and furrows (Fig 4). Additionally, projections off the grain surface may be characteristic, as may be ridge patterns on the pollen surface. These structures are the primary means of identification to the taxonomic level of genus, and can sometimes delineate species. Pores. Pollens with pores are called porate. Pores are primarily circular, but sometimes elliptical, and may be single or multiple. The appropriate prefix specifies whether there is a single pore, two, three, or numerous pores (mono-, di-, tri-, or periporate, respectively). Members of the grass family invariably are monoporate, as are several grass allies (Fig 2A). Triporate grains are perhaps the next most common. A few species have two pores, such as the mulberries and wood nettle (Parietaria), and occasionally aberrant grains of species normally having 3 pores (nettle and birch, Urtica and Betula) will only possess two. Weeds of the closely related chenopod and amaranth families have multiple pores, generally between 20 and 80 per grain. Under the microscope, these pollens
look like golf balls (Fig 3C). In distinction, plantains (Plantago) have fewer pores, usually 6 to 11. Sweet gum, Liquidambar, has 12 to 20 pores, which are bulging with dark granules. Grains with ⱖ4 pores oriented along the grain’s equator (crown-like) are called stephanoporate (Fig 3). For some periporate pollens such as walnut, Juglans, the pores are entirely on the equator and one hemisphere. There are several modifications of the exine around pores which may be helpful in classification (Fig 5). The pore sometimes possesses a cap or plug, the operculum. An aspis is a thickening of the exine around a pore, like a shield. An annulus, or ring, around the pore may either be a thickening, or a thinning of the exine. A thickening of the intine under a pore is called an oncus. A band may arch between pores, and is called an arcus; although it may appear to be within the inner portion of the grain, it is thickened sexine, forming a ridge along the surface of the grain. Birch is a triporate
Figure 4. Types of pollen apertures (structures depicted in dotted lines are situated on far side of pollen grain). A. inaperturate; B. monocolpate; C. tricolpate, equatorial plane; D. tricolpate, polar view; E. tricolporate; F. periporate; G. monporate; H. triporate, equatorial plane; I. triporate, polar view; and J. stephanoporate.
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pollen with a very distinctive annulus, aspis, and oncus (Fig 3D). Alder, Alnus, which is pentaporate, likewise has distinctive aspides and very prominent arci. While some species of birch may have arci, these are much less apparent than with alder grains.7,8 Furrows (colpi). These are slits or boat-shaped areas of thin exine (Fig 4). Grains with furrows are called colpate. The furrows may be quite long, stretching from pole to pole, or be quite short and unapparent. The same prefixes are used as with pores to designate number and orientation. Pollens which possess both furrows and pores are called colporate. The pores in such cases are located within the furrows, which occasionally may be quite short, barely extending beyond the pore, as seen with the ragweeds, Ambrosia spp. Maples and oaks (Acer and Quercus) are colpate but may show bulging furrows which appear colporate in polar view (Fig 3A). Sculpturing. Surface texturing into ridges or depressions may occasionally identify one species from another, although such detail is frequently difficult to discern under normal conditions of light microscopy. A pollen with a smooth surface, such as with the grasses, is called psilate. Patterns may be reticulate (net-like), striate (roughly parallel ridges), or rugulate (irregular pattern). Most maples have a distinctly striated surface; boxelder, however, has a more irregular pattern. Cottonwoods, Populus, have a granular, irregular surface texture. The roof of the exine may have prominent projections of various shapes: baculate, or rod-like; clavate, club-like or tennis-racket shaped; echinate, spiny; gemmate, door knob shaped; scabrate, rough or flecked; and verrucate, warty or bumpy (Fig 1).6 These projections may be very distinctive, and readily apparent, or may be very subtle and hard to see, requiring careful observation under high resolution with oil immersion. Many composite weeds (including the ragweeds, Ambrosia) have obvious spines (Fig 3B). Those of the ragweeds are short and broad-based, while those of
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Figure 5. Composite cross-sectional detail of pore-associated structures (not all features found on individual grains). See text for definitions.
primarily entomophilous species such as goldenrod and sunflower (Solidago and Helianthus, respectively) have longer spines. Sages are baculate, but the rod-like projections are less easy to appreciate. Bald cypress (Taxodium) and redwood (Sequoia) pollens resemble those of the cedars and junipers (see below) with the exception of a single irregular projection, an “exit papilla.” Interior Structures While the protoplast of many pollens is nondescript, internal characteristics of certain pollens are helpful for identification. The presence of a thick intine helps identify cottonwoods. The intine may be particularly dense, as seen with Cypress family members (cedars and junipers, Juniperus), with an irregular stellate appearance of the protoplast. Juniperus species, particularly, have a thin exine, which frequently splits open on the microscope slide, extruding the clear round intine and protoplast portion of the grain, leaving the stained exine appearing like a “Pacman” (Fig 2D). Docks and sorrels, Rumex, have prominent internal starch granules, giving the interior a “popcorn” appearance. Grasses also have starch granules, but these are much less apparent. SUMMARY OF MAJOR AIRBORNE POLLEN GROUP CHARACTERISTICS The following section summarizes pollen attributes of the major groupings of
aeroallergenic plants. It is not meant to be exhaustive, but rather highlights the average appearance and significant characteristics within each group. Pollen characteristics have been derived from several sources.6 –12 Further botanical taxonomic information on these plants may be found in several sources.1,13,14 Pinaceae Members of the pine family (pine, spruce, and fir; Pinus, Picea, and Abies, respectively) generally have two air bladders, are inaperturate, and range in size from 40 to 100m. Larches and Douglas fir (Larix and Pseudotsuga) do not have vesicles. Hemlocks (Tsuga spp.) may have two bladders, which are either fully formed or rudimentary, or have none. Cupressaceae Cedars, junipers, and arbor vitae (Juniperus and Thuja spp.) are psilate and inaperturate, and smaller than pines, ranging from 20 to 35m. A markedly thickened intine is up to 6.5m. Taxodiaceae Bald cypress and redwoods (Taxodium and Sequoia spp.) are up to 36m, with a thickened intine. They characteristically have a single exit papilla. Graminae (Poaceae) Grasses are all very similar in appearance: psilate, spheroidal, from 20 to 60m. There is much overlap in size, and with the exception of corn (90 to 110m), this cannot separate genus or
ANNALS OF ALLERGY, ASTHMA, & IMMUNOLOGY
species. Grains are monoporate, with a prominent operculum. Compositae (Asteraceae) Members are tricolporate, with pores and furrows more or less prominent. Ragweeds (Ambrosia) have short furrows with indistinct pores, while sages have more apparent apertures. Short, broad-based spines on ragweeds and marshelders (Iva), longer spines on goldenrod, sunflower, daisy (Solidago, Helianthus, Aster, respectively). Sages (Artemisia) without spines, scalloping. Size is between 15 to 30m. Chenopodiales Members of the closely related amaranth and chenopod families have very similar pollens which are periporate, ranging from 20 to 80 pores per grain. Pore size and number show genus and species differences, but the overlap is sufficient to make identification below the level of botanical order very difficult. Grain size is between 20 and 35m. Plantaginaceae Plantains (Plantago) are periporate, but with significantly fewer pores than the chenopods or amaranths. Usual pore number is between 6 to 11. Size is between 20 and 30m. Betulaceae The birch family has tri- to pentaporate grains, with distinct pore-associated findings such as annuli, aspidae, and arci. Birch (Betula) usually has three pores, while alder has 4 or 5. Size is between 20 and 35m. Alder (Alnus) with distinct oncus and arci. Fagaceae The beech family has tricolpate or tricolporate members, ranging from 10 to 15m (chestnut, Castanea) to 25 to 40m (oak and beech, Quercus and Fagus). Shapes are from prolate to triangular in polar view.
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Aceraceae The majority of Acer species are tricolpate, with large furrows, and a striate surface. Boxelder (Acer negundo) is tricolpate with a rugulate texture. Vine maple (Acer circinatum) is tricolporate. Grains range from 25 to 40m in size. Juglandaceae Walnuts and butternuts (Juglans) are periporate, with 10 to 15 pores on the equator and one hemisphere (heteropolar). Pores are aspidate with indistinct onci. Hickory and pecans (Carya) are usually triporate with pores evenly spaced close to the equatorial plane. Large oblate grains ranging from 35 to 55m. Oleaceae Grains of ash and olive (Fraxinus and Olea) are tri- to tetracolpate, with short furrows. Surfaces are finely or coarsely reticulate, respectively. Size varies from 20 to 30m. Salicaceae Cottonwood and aspen (Populus) have inaperturate grains, while willows (Salix) are tricolpate. Willows have a reticulate surface and are between 10 and 20m. Populus species have a granular texture, and are larger, between 20 and 35m. Ulmaceae Elms (Ulmus) are stephanoporate with usually five pores, although ranging from 4 to 7. Surface is wavy and convoluted. Grains are oblate with equatorial diameter 25 to 35m. Hackberry (Celtis) pollens have 3 to 10 pores, with a thick undulating, granular sexine, 25 to 40m. REFERENCES 1. Lewis WH, Vinay P, Zenger VE. Airborne and allergenic pollen of North America. Baltimore: Johns Hopkins University Press, 1983. 2. Whitson TD, Burrill LC, Dewey SA, et
3.
4. 5.
6. 7. 8. 9. 10.
11. 12.
13. 14.
al. Weeds of the west. Jackson, WY: University of Wyoming, 1991. United States Department of Agriculture Forest Service. Range plant handbook. Dover Publications, New York, 1988. Burge HA. Monitoring for airborne allergens. Ann Allergy 1992;69:9 –18. Sheldon JM, Lovell RG, Mathews KP. Standard technic for atmospheric pollen testing by gravity method. In: A manual of clinical allergy. Philadelphia: WB Saunders Co, 1953: 363– 6. Kapp RO. How to know pollen and spores. Dubuque, Iowa: Wm C Brown, 1969. Brown GT. Pollen-slide studies. Springfield, Illinois: Charles C Thomas, 1949:47–53. Wodehouse RP. Pollen grains. New York: Hafner, 1935. Ogden EC. Manual for sampling airborne pollen. New York: Hafner, 1974. Bassett IJ, Crompton CW, Parmelee JA. An atlas of airborne pollen grains and common fungus spores of Canada. Hull, Quebec: Printing and Publishing, Supply and Services Canada, 1978. Smith EC. Sampling and identifying allergenic pollens and molds. San Antonio, TX: Blewstone Press, 1984. Smith EC. Sampling and identifying allergenic pollens and molds. volume II. San Antonio, TX: Blewstone Press, 1986. Weber RW, Nelson HS. Pollen allergens and their interrelationships. Clin Rev Allergy 1985;3:291–318. Solomon WR, Weber RW, Dolen WK. Common allergenic pollen and fungi. In: Bieeman CW, Pearlman DS, Shapiro GG, Busse WW, eds. Allergy, asthma, and immunology from infancy to adulthood, 3rd edition. Philadelphia: WB Saunders Company, 1996: 93–114.
Requests for reprints should be addressed to: Richard W Weber, MD Department of Medicine National Jewish Medical & Research Center 1400 Jackson St, Rm B103A Denver, CO 80206
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CME Examination No. 008-002 Questions 1–20: Weber RW. Ann Allergy Asthma Immunol 1998;80:140 – 6 CME Test Questions 1. The pigment used to stain pollen grains in Caberla’s solution is a. toluene blue. b. basic fuchsin. c. phenosafranin. d. eosin. e. hematoxylin. 2. The two outer layers of the pollen wall are called the a. ectomorph and endomorph. b. intine and plasmalemma. c. sexine and nexine. d. baculum and protoplast. e. aspis and oncus. 3. Plants that are both wind- and vector-pollinated are called a. amphiphilous. b. aerobic. c. entomophilous. d. anemophilous. e. anaerobic. 4. Apertures that are elongated are a. pores. b. colpi. c. operculae. d. papillae. e. vesicles. 5. Grasses are invariably a. diporate. b. tetracolpate. c. monocolpate. d. monoporate. e. monocolporate. 6. Members of the Coniferales order which have pollens with air bladders include a. larch, ginkgo, and Australian pine. b. juniper, cedar, and hemlock. c. yew, bald cypress, and joint pine (Ephedra). d. myrtle, heath, and Norfolk Island pine. e. pine, spruce, and fir. 7. A distinguishing feature of docks and sorrel (Rumex spp.) grains is a. exit papilla.
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8.
9.
10.
11.
12.
b. thin exine that frequently splits and extrudes inner portion of grain. c. prominent starch granules. d. large single furrow. e. more than 60 pores. Members of the Chenopodiales order (amaranths and chenopods) are periporate with what range of pore number? a. 4 to 11 b. 3 to 15 c. 12 to 20 d. more than 60 e. 20 to 80 “Stephanoporate” refers to what attribute? a. arrow-headed surface ornamentation b. pores found within furrows c. crown-like string of pores around the grain equator d. ⬎20 pores e. ⱖ4 furrows aligned around grain equator The sexine of the pollen grain a. may be composed of a tectum, series of columns, and foot layer. b. is the origin of the pollen tube. c. is the outer portion of the intine. d. contains the pollen’s nuclear material. e. is susceptible to acetolysis. Pollen grains that have no pores or furrows are called a. psilate. b. operculate. c. oblate. d. percolate. e. inaperturate. Plantain pollen may be differentiated from amaranth pollen by a. being pericolpate rather than periporate. b. having bulging, granular pores. c. being triporate.
13.
14.
15.
16.
17.
18.
d. having fewer pores, 6 to 11 rather than ⬎20. e. having pores limited to one hemisphere. Grains being very elongated are a. called perprolate. b. have a P/E index of ⬍0.5. c. called peroblate. d. called echinate. e. called obstinate. Bald cypress or redwood pollens may be distinguished from cedar pollen by a. having two air bladders. b. being monoporate. c. having single exit papillae. d. possessing a single vesicle. e. being tetrads. A triporate grain with prominent annulus, aspis, and oncus is characteristic of which of the following pollens? a. broad-leafed cattail b. mountain cedar c. aspen d. plantain e. birch What is an annulus? a. a shield-like thickening extending away from a pore b. a ring-like thickening or thinning around a pore c. a string of pores around the equator of a grain d. an aperture filled with debris e. arrow-shaped surface ornamentation A pollen grain with surface patterns assuming a net-like appearance is called a. reticulate. b. rugulate. c. psilate. d. pronate. e. obfuscate. Walnut pollen may be distinguished from sweetgum pollen by a. walnut has bulging pores.
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b. sweetgum has 40 to 60 pores compared to ⬍20 for walnut. c. walnut is heteropolar with pores in one hemisphere. d. walnut is tricolporate. e. there are no distinguishing characteristics. 19. Surface ornamentation that is warty or bumpy is called a. gemmate.
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b. scabrate. c. clavate. d. echinate. e. verrucate. 20. Cottonwood and grass pollens may be distinguished from each other by all but which of the following criteria? a. In certain locales cottonwood pollinates earlier than grass.
b. While both are subspheroidal, cottonwood is inaperture while grasses are monoporate. c. Cottonwood has a rugulate to granular texture while grasses are psilate. d. Cottonwood is triangular while grass is perprolate. e. Grass has an operculum.
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Instructions for Category I CME Credit Certification. As an organization accredited for continuing medical education, the American College of Allergy, Asthma, & Immunology (ACAAI) certifies that when the CME material is used as directed it meets the criteria for two hours’ credit in Category I of the American College of Allergy, Asthma, & Immunology CME Award and the Physician’s Recognition Award of the American Medical Association. Instructions. Category I credit can be earned by reading the text material, taking the CME examination and recording the answers on the perforated answer sheet entitled, “Continuing
Medical Education,” which can be found after the examination. Please record your ACAAI identification number and the quiz identification number in the spaces and scanning targets provided on the answer sheet. Your ACAAI identification number can be found on your ACAAI membership card, nonmembers of the College will be assigned an ACAAI identification number and this should be left blank on the answer sheet. The quiz identification number can be found at the beginning of the CME examination. Use a No. 2 or soft lead pencil for marking the answer sheet. You may
erase but do so completely in order to prevent computer reading errors. Your ACAAI identification number and quiz identification number will be used to record your credit hours earned on the CME transcript system. No records of individual performance will be maintained. Tear out the perforated answer sheet and print your name and address in the spaces provided. Return it within one month after the Annals is received to the American College of Allergy, Asthma, & Immunology, 85 West Algonquin Rd, Suite 550, Arlington Heights, IL 60005. Answers will be published in the next issue of the Annals of Allergy, Asthma, & Immunology.
Answers to CME examination—Annals of Allergy, Asthma, & Immunology, January 1998 (Identification No 008 – 001) Vaswani SL and Creticos PS. Metered dose inhaler: past, present, and future. Ann Allergy Asthma Immunol 1998;80:11–23. 1. e 7. e 13. e 19. e 25. b 2. c 8. a 14. d 20. c 26. c 3. b 9. e 15. e 21. b 27. e 4. a 10. b 16. c 22. e 5. b 11. b 17. e 23. c 6. b 12. c 18. a 24. c
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Pollen identification Richard W Weber, MD*
Learning Objectives: The purpose of this review is to introduce the reader to a systematic approach to categorizing pollen types. This will enable the reader to recognize pollen characteristics of the most common botanical sources and determine the relevant contributors to the local aeroallergen burden. Data Sources: Allergenic plant and pollen atlases for pollen characteristics, allergy texts for procedures and overviews, and relevant reviews from the English medical literature (1985 to present). Results: By applying a visual gestalt utilizing grain number, size, shape, surface structures, and internal detail, one is able to identify pollen source to the appropriate botanical taxonomic level. Identification may be possible only to the family or order, but most frequently to the genus, and occasionally to the species. Conclusions: Outdoor sampling allows the identification of aeroallergen burden in a locale. In conjunction with field work, the relevant sources can be identified as well as time of pollination. This allows the physician to correlate symptoms with exposure and relevant sensitization. Ann Allergy Asthma Immunol 1998;80:141–7.
INTRODUCTION The purpose of this review is to provide a basis for identification of the majority of aeroallergenic pollens. Through the consideration of several variables discussed below, the observer can develop a system of pattern recognition, or gestalt, to assist in characterizing the airborne burden in a specific locale. This should be combined with a familiarity with what plants are actually growing in the area of concern. This may be best achieved by contacting a botanist who has knowledge of the local flora. Such a person may be working for a community or state government, or perhaps a university extension. Additionally, there are a number of texts that catalogue regional plants, and the interested party will * From Department of Medicine, Allergy/ Clinical Immunology Division, National Jewish Medical and Research Center, Denver, Colorado. Received for publication August 11, 1997. Accepted for publication in revised form December 9, 1997.
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find these helpful resources.1–3 Field trips at varying times of the year will familiarize one with the times of anthesis for the local plants. Knowing the local flora will assist in identification of the particles found on the sampler; this in turn will allow interpretation of patient’s symptom chronology and the relevance of in vivo or in vitro sensitivity. This review will use numerous descriptive phrases that are not common usage. These terms are defined throughout the text. For ease of referral, these phrases are placed in bold face where they are defined. BACKGROUND Higher plants use two methods of pollen dispersal: wind or vector. While a variety of animals may transport pollen, insect vectors are by far the most prevalent. Plant pollinated through the agency of an insect carrier are called entomophilous. Only about ten percent of higher plants utilize wind pollination, these are called anemophilous, and are the principle cause of
pollinosis. Plants that typically are both insect-pollinated and wind-pollinated are amphiphilous. Some primarily entomophilous plants, such as linden (Tilia), produce a large enough pollen load that appreciable surplus amounts become airborne. SAMPLE PREPARATION Principles of aeroallergen sampling have been reviewed previously by Burge.4 The reader is referred to that article for discussion of techniques and tabulation. Advantages of various samplers will not be discussed here other than to state than older gravimetric techniques such as the Durham sampler are no longer adequate, and volumetric sampling is strongly recommended. Once the sample is obtained, it is necessary to prepare it for microscopic examination. Pollens may be stained with numerous agents, but the standard preparation is with Caberla’s solution, which utilizes basic fuchsin. This stain permanently stains the pollen outer wall (exine), while not staining the great majority of mold spores; it imparts a pink to red color to the pollen grains, which persists in permanent mounts. The formulation of Caberla’s solution is as follows5: glycerin 5.0cc 95% ethyl alcohol 10.0cc distilled water 15.0cc saturated basic fuchsin 2 drops (or ⬃50mg powder) If the solution is staining too intensely, it may be diluted with the dyefree solution.
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MORPHOLOGY IN POLLEN GRAIN IDENTIFICATION
Figure 1. Composite cross-sectional detail of pollen grain wall (not all features found on individual grains). Surface projections not drawn to scale for ease of illustration. See text for definitions.
STRUCTURE OF POLLEN GRAINS The pollen wall consists of three layers, the outer two comprising together the exine, and the inner layer the intine. The exine is resistant to degradation, and withstands acid-heat exposure (a process called acetolysis). The intine is cellulose, and is susceptible to such treatment. Beneath the intine lies the cytoplasm of the grain, or the protoplast, enveloped by the cell membrane, the plasmalemma (Fig 1).1 The exine has two layers: the innermost featureless, but impervious, nexine, and the outer sexine, which is penetrated by numerous micropores, as well as larger apertures: pores and fur-
rows. These apertures are areas of thinned exine, and act as points of egress for the pollen tube during successful pollination. The apertures and micropores probably also act as avenues for the elution of allergenic proteins, which may have enzymatic and signal recognition functions in instigating the fertilization process. The sexine may have separate sublayers: an outer roof, or tectum, a foot layer abutting on the nexine, and a central segment comprised of columns, or baculae, separating the two. Additionally, the outer surface of the sexine may have a variety of projections and surface markings which are discussed below.
Figure 2. Photomicrographs showing representative shapes and clusters of pollen grains (not all same scale). A. spherical monoporate monad (grass, Graminae); B. wedge shape (bulrush, Scirpus); C. rhomboid tetrad (broadleaf cattail, Typha latifolia); D. split exine and extruded intine and stellate protoplast (cedar, Juniperus); E. square shape (ash, Fraxinus); and F. grain with two air bladders (pine, Pinus).
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Number of Grains The majority of pollens captured on samplers are single grains, or monads. Representative pollen types are shown in Figures 2 and 3. Some, however, are multiple, such as the 16-grain mimosa (Acacia) polyad, or the 4-grain tetrad of broadleaf cattail, Typha latifolia (Fig 2C). The latter is a “rhomboid” tetrad, while wood rush, Luzula, and members of the heath family (Erica) have a tetrahedral configuration. Occasionally, pollens will clump together, giving the appearance of a polyad, but with a little pressure on the coverslip, these will usually separate easily. Size Pollen grains vary greatly in size, from 2 to 5m to about 250m, although most wind-pollinated grains are between 20 to 60m. Nettle, Urtica, and the mulberries, Morus and Broussonetia, all produce pollens in the 10 to 15m range. While corn, Zea, and some conifers, Abies and Pseudotsuga, may be over 100m. Certain entomophilous pollens such as pumpkin, Cucurbita, may be as large as 250m. Shape Pollen grains are generally elliptical or spherical, but other distinct shapes exist. Pine, fir, and spruce pollens (Pinus, Abies, and Picea, respectively) have two air bladders, or vesicles, one either side of the grain. This gives the pollen grain a “Mickey Mouse cap” appearance when viewed in the equatorial plane (Fig 2F). These bladders may be either clear, taking on the fuchsin stain, or be darkly pigmented. Footballshaped or triangular are not uncommon, or even wedge-shaped, like a kernel of corn (sedge and bulrush, Carex and Scirpus, Fig 2B). Pores in the grain may act as points on the surface where the exine angles, giving the pollen a triangular, square, or polygonal shape, depending on the numbers of pores (ash, Fraxinus, Fig 2E). An exception is linden, which has three pores occupying the flat sides of a somewhat triangular shaped grain, rather than the
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Figure 3. Photomicrographs showing representative pollen grain surface features (not all same scale). A. three-furrowed, tricolpate (maple, Acer); B. spined, echinate, tricolporate (sunflower, Helianthus); C. periporate chenopod-amaranth; and D. triporate grain with distinctive aperture features of annulus, aspis, and oncus (birch, Betula).
corners. In the case of sages (Artemisia), the exine invaginates somewhat at the pores, bulging between, giving the grain a distinctive scalloped appearance. The degree of elongation of elliptical pollen grains can be used in identification. Kapp suggested using the ratio of polar to equatorial diameters, the P/E index.6 The pole of a grain is defined as either the location of a single pore or midpoint of a single furrow, or the end where furrows converge (tricolpate or tricolporate grains). The following descriptive phrases are used depending on the P/E index: ⬎2.0 perprolate (very elongated) 1.3–2.0 prolate (slightly elongated) 0.75–1.3 subspheroidal 0.50 – 0.75 oblate (slightly flattened) ⬍0.5 peroblate (very flattened) Surface Structures The majority of pollen grains can be identified by surface structures in the
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sexine, the most distinctive being apertures: pores and furrows (Fig 4). Additionally, projections off the grain surface may be characteristic, as may be ridge patterns on the pollen surface. These structures are the primary means of identification to the taxonomic level of genus, and can sometimes delineate species. Pores. Pollens with pores are called porate. Pores are primarily circular, but sometimes elliptical, and may be single or multiple. The appropriate prefix specifies whether there is a single pore, two, three, or numerous pores (mono-, di-, tri-, or periporate, respectively). Members of the grass family invariably are monoporate, as are several grass allies (Fig 2A). Triporate grains are perhaps the next most common. A few species have two pores, such as the mulberries and wood nettle (Parietaria), and occasionally aberrant grains of species normally having 3 pores (nettle and birch, Urtica and Betula) will only possess two. Weeds of the closely related chenopod and amaranth families have multiple pores, generally between 20 and 80 per grain. Under the microscope, these pollens
look like golf balls (Fig 3C). In distinction, plantains (Plantago) have fewer pores, usually 6 to 11. Sweet gum, Liquidambar, has 12 to 20 pores, which are bulging with dark granules. Grains with ⱖ4 pores oriented along the grain’s equator (crown-like) are called stephanoporate (Fig 3). For some periporate pollens such as walnut, Juglans, the pores are entirely on the equator and one hemisphere. There are several modifications of the exine around pores which may be helpful in classification (Fig 5). The pore sometimes possesses a cap or plug, the operculum. An aspis is a thickening of the exine around a pore, like a shield. An annulus, or ring, around the pore may either be a thickening, or a thinning of the exine. A thickening of the intine under a pore is called an oncus. A band may arch between pores, and is called an arcus; although it may appear to be within the inner portion of the grain, it is thickened sexine, forming a ridge along the surface of the grain. Birch is a triporate
Figure 4. Types of pollen apertures (structures depicted in dotted lines are situated on far side of pollen grain). A. inaperturate; B. monocolpate; C. tricolpate, equatorial plane; D. tricolpate, polar view; E. tricolporate; F. periporate; G. monporate; H. triporate, equatorial plane; I. triporate, polar view; and J. stephanoporate.
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pollen with a very distinctive annulus, aspis, and oncus (Fig 3D). Alder, Alnus, which is pentaporate, likewise has distinctive aspides and very prominent arci. While some species of birch may have arci, these are much less apparent than with alder grains.7,8 Furrows (colpi). These are slits or boat-shaped areas of thin exine (Fig 4). Grains with furrows are called colpate. The furrows may be quite long, stretching from pole to pole, or be quite short and unapparent. The same prefixes are used as with pores to designate number and orientation. Pollens which possess both furrows and pores are called colporate. The pores in such cases are located within the furrows, which occasionally may be quite short, barely extending beyond the pore, as seen with the ragweeds, Ambrosia spp. Maples and oaks (Acer and Quercus) are colpate but may show bulging furrows which appear colporate in polar view (Fig 3A). Sculpturing. Surface texturing into ridges or depressions may occasionally identify one species from another, although such detail is frequently difficult to discern under normal conditions of light microscopy. A pollen with a smooth surface, such as with the grasses, is called psilate. Patterns may be reticulate (net-like), striate (roughly parallel ridges), or rugulate (irregular pattern). Most maples have a distinctly striated surface; boxelder, however, has a more irregular pattern. Cottonwoods, Populus, have a granular, irregular surface texture. The roof of the exine may have prominent projections of various shapes: baculate, or rod-like; clavate, club-like or tennis-racket shaped; echinate, spiny; gemmate, door knob shaped; scabrate, rough or flecked; and verrucate, warty or bumpy (Fig 1).6 These projections may be very distinctive, and readily apparent, or may be very subtle and hard to see, requiring careful observation under high resolution with oil immersion. Many composite weeds (including the ragweeds, Ambrosia) have obvious spines (Fig 3B). Those of the ragweeds are short and broad-based, while those of
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Figure 5. Composite cross-sectional detail of pore-associated structures (not all features found on individual grains). See text for definitions.
primarily entomophilous species such as goldenrod and sunflower (Solidago and Helianthus, respectively) have longer spines. Sages are baculate, but the rod-like projections are less easy to appreciate. Bald cypress (Taxodium) and redwood (Sequoia) pollens resemble those of the cedars and junipers (see below) with the exception of a single irregular projection, an “exit papilla.” Interior Structures While the protoplast of many pollens is nondescript, internal characteristics of certain pollens are helpful for identification. The presence of a thick intine helps identify cottonwoods. The intine may be particularly dense, as seen with Cypress family members (cedars and junipers, Juniperus), with an irregular stellate appearance of the protoplast. Juniperus species, particularly, have a thin exine, which frequently splits open on the microscope slide, extruding the clear round intine and protoplast portion of the grain, leaving the stained exine appearing like a “Pacman” (Fig 2D). Docks and sorrels, Rumex, have prominent internal starch granules, giving the interior a “popcorn” appearance. Grasses also have starch granules, but these are much less apparent. SUMMARY OF MAJOR AIRBORNE POLLEN GROUP CHARACTERISTICS The following section summarizes pollen attributes of the major groupings of
aeroallergenic plants. It is not meant to be exhaustive, but rather highlights the average appearance and significant characteristics within each group. Pollen characteristics have been derived from several sources.6 –12 Further botanical taxonomic information on these plants may be found in several sources.1,13,14 Pinaceae Members of the pine family (pine, spruce, and fir; Pinus, Picea, and Abies, respectively) generally have two air bladders, are inaperturate, and range in size from 40 to 100m. Larches and Douglas fir (Larix and Pseudotsuga) do not have vesicles. Hemlocks (Tsuga spp.) may have two bladders, which are either fully formed or rudimentary, or have none. Cupressaceae Cedars, junipers, and arbor vitae (Juniperus and Thuja spp.) are psilate and inaperturate, and smaller than pines, ranging from 20 to 35m. A markedly thickened intine is up to 6.5m. Taxodiaceae Bald cypress and redwoods (Taxodium and Sequoia spp.) are up to 36m, with a thickened intine. They characteristically have a single exit papilla. Graminae (Poaceae) Grasses are all very similar in appearance: psilate, spheroidal, from 20 to 60m. There is much overlap in size, and with the exception of corn (90 to 110m), this cannot separate genus or
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species. Grains are monoporate, with a prominent operculum. Compositae (Asteraceae) Members are tricolporate, with pores and furrows more or less prominent. Ragweeds (Ambrosia) have short furrows with indistinct pores, while sages have more apparent apertures. Short, broad-based spines on ragweeds and marshelders (Iva), longer spines on goldenrod, sunflower, daisy (Solidago, Helianthus, Aster, respectively). Sages (Artemisia) without spines, scalloping. Size is between 15 to 30m. Chenopodiales Members of the closely related amaranth and chenopod families have very similar pollens which are periporate, ranging from 20 to 80 pores per grain. Pore size and number show genus and species differences, but the overlap is sufficient to make identification below the level of botanical order very difficult. Grain size is between 20 and 35m. Plantaginaceae Plantains (Plantago) are periporate, but with significantly fewer pores than the chenopods or amaranths. Usual pore number is between 6 to 11. Size is between 20 and 30m. Betulaceae The birch family has tri- to pentaporate grains, with distinct pore-associated findings such as annuli, aspidae, and arci. Birch (Betula) usually has three pores, while alder has 4 or 5. Size is between 20 and 35m. Alder (Alnus) with distinct oncus and arci. Fagaceae The beech family has tricolpate or tricolporate members, ranging from 10 to 15m (chestnut, Castanea) to 25 to 40m (oak and beech, Quercus and Fagus). Shapes are from prolate to triangular in polar view.
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Aceraceae The majority of Acer species are tricolpate, with large furrows, and a striate surface. Boxelder (Acer negundo) is tricolpate with a rugulate texture. Vine maple (Acer circinatum) is tricolporate. Grains range from 25 to 40m in size. Juglandaceae Walnuts and butternuts (Juglans) are periporate, with 10 to 15 pores on the equator and one hemisphere (heteropolar). Pores are aspidate with indistinct onci. Hickory and pecans (Carya) are usually triporate with pores evenly spaced close to the equatorial plane. Large oblate grains ranging from 35 to 55m. Oleaceae Grains of ash and olive (Fraxinus and Olea) are tri- to tetracolpate, with short furrows. Surfaces are finely or coarsely reticulate, respectively. Size varies from 20 to 30m. Salicaceae Cottonwood and aspen (Populus) have inaperturate grains, while willows (Salix) are tricolpate. Willows have a reticulate surface and are between 10 and 20m. Populus species have a granular texture, and are larger, between 20 and 35m. Ulmaceae Elms (Ulmus) are stephanoporate with usually five pores, although ranging from 4 to 7. Surface is wavy and convoluted. Grains are oblate with equatorial diameter 25 to 35m. Hackberry (Celtis) pollens have 3 to 10 pores, with a thick undulating, granular sexine, 25 to 40m. REFERENCES 1. Lewis WH, Vinay P, Zenger VE. Airborne and allergenic pollen of North America. Baltimore: Johns Hopkins University Press, 1983. 2. Whitson TD, Burrill LC, Dewey SA, et
3.
4. 5.
6. 7. 8. 9. 10.
11. 12.
13. 14.
al. Weeds of the west. Jackson, WY: University of Wyoming, 1991. United States Department of Agriculture Forest Service. Range plant handbook. Dover Publications, New York, 1988. Burge HA. Monitoring for airborne allergens. Ann Allergy 1992;69:9 –18. Sheldon JM, Lovell RG, Mathews KP. Standard technic for atmospheric pollen testing by gravity method. In: A manual of clinical allergy. Philadelphia: WB Saunders Co, 1953: 363– 6. Kapp RO. How to know pollen and spores. Dubuque, Iowa: Wm C Brown, 1969. Brown GT. Pollen-slide studies. Springfield, Illinois: Charles C Thomas, 1949:47–53. Wodehouse RP. Pollen grains. New York: Hafner, 1935. Ogden EC. Manual for sampling airborne pollen. New York: Hafner, 1974. Bassett IJ, Crompton CW, Parmelee JA. An atlas of airborne pollen grains and common fungus spores of Canada. Hull, Quebec: Printing and Publishing, Supply and Services Canada, 1978. Smith EC. Sampling and identifying allergenic pollens and molds. San Antonio, TX: Blewstone Press, 1984. Smith EC. Sampling and identifying allergenic pollens and molds. volume II. San Antonio, TX: Blewstone Press, 1986. Weber RW, Nelson HS. Pollen allergens and their interrelationships. Clin Rev Allergy 1985;3:291–318. Solomon WR, Weber RW, Dolen WK. Common allergenic pollen and fungi. In: Bieeman CW, Pearlman DS, Shapiro GG, Busse WW, eds. Allergy, asthma, and immunology from infancy to adulthood, 3rd edition. Philadelphia: WB Saunders Company, 1996: 93–114.
Requests for reprints should be addressed to: Richard W Weber, MD Department of Medicine National Jewish Medical & Research Center 1400 Jackson St, Rm B103A Denver, CO 80206
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CME Examination No. 008-002 Questions 1–20: Weber RW. Ann Allergy Asthma Immunol 1998;80:140 – 6 CME Test Questions 1. The pigment used to stain pollen grains in Caberla’s solution is a. toluene blue. b. basic fuchsin. c. phenosafranin. d. eosin. e. hematoxylin. 2. The two outer layers of the pollen wall are called the a. ectomorph and endomorph. b. intine and plasmalemma. c. sexine and nexine. d. baculum and protoplast. e. aspis and oncus. 3. Plants that are both wind- and vector-pollinated are called a. amphiphilous. b. aerobic. c. entomophilous. d. anemophilous. e. anaerobic. 4. Apertures that are elongated are a. pores. b. colpi. c. operculae. d. papillae. e. vesicles. 5. Grasses are invariably a. diporate. b. tetracolpate. c. monocolpate. d. monoporate. e. monocolporate. 6. Members of the Coniferales order which have pollens with air bladders include a. larch, ginkgo, and Australian pine. b. juniper, cedar, and hemlock. c. yew, bald cypress, and joint pine (Ephedra). d. myrtle, heath, and Norfolk Island pine. e. pine, spruce, and fir. 7. A distinguishing feature of docks and sorrel (Rumex spp.) grains is a. exit papilla.
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8.
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b. thin exine that frequently splits and extrudes inner portion of grain. c. prominent starch granules. d. large single furrow. e. more than 60 pores. Members of the Chenopodiales order (amaranths and chenopods) are periporate with what range of pore number? a. 4 to 11 b. 3 to 15 c. 12 to 20 d. more than 60 e. 20 to 80 “Stephanoporate” refers to what attribute? a. arrow-headed surface ornamentation b. pores found within furrows c. crown-like string of pores around the grain equator d. ⬎20 pores e. ⱖ4 furrows aligned around grain equator The sexine of the pollen grain a. may be composed of a tectum, series of columns, and foot layer. b. is the origin of the pollen tube. c. is the outer portion of the intine. d. contains the pollen’s nuclear material. e. is susceptible to acetolysis. Pollen grains that have no pores or furrows are called a. psilate. b. operculate. c. oblate. d. percolate. e. inaperturate. Plantain pollen may be differentiated from amaranth pollen by a. being pericolpate rather than periporate. b. having bulging, granular pores. c. being triporate.
13.
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d. having fewer pores, 6 to 11 rather than ⬎20. e. having pores limited to one hemisphere. Grains being very elongated are a. called perprolate. b. have a P/E index of ⬍0.5. c. called peroblate. d. called echinate. e. called obstinate. Bald cypress or redwood pollens may be distinguished from cedar pollen by a. having two air bladders. b. being monoporate. c. having single exit papillae. d. possessing a single vesicle. e. being tetrads. A triporate grain with prominent annulus, aspis, and oncus is characteristic of which of the following pollens? a. broad-leafed cattail b. mountain cedar c. aspen d. plantain e. birch What is an annulus? a. a shield-like thickening extending away from a pore b. a ring-like thickening or thinning around a pore c. a string of pores around the equator of a grain d. an aperture filled with debris e. arrow-shaped surface ornamentation A pollen grain with surface patterns assuming a net-like appearance is called a. reticulate. b. rugulate. c. psilate. d. pronate. e. obfuscate. Walnut pollen may be distinguished from sweetgum pollen by a. walnut has bulging pores.
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b. sweetgum has 40 to 60 pores compared to ⬍20 for walnut. c. walnut is heteropolar with pores in one hemisphere. d. walnut is tricolporate. e. there are no distinguishing characteristics. 19. Surface ornamentation that is warty or bumpy is called a. gemmate.
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b. scabrate. c. clavate. d. echinate. e. verrucate. 20. Cottonwood and grass pollens may be distinguished from each other by all but which of the following criteria? a. In certain locales cottonwood pollinates earlier than grass.
b. While both are subspheroidal, cottonwood is inaperture while grasses are monoporate. c. Cottonwood has a rugulate to granular texture while grasses are psilate. d. Cottonwood is triangular while grass is perprolate. e. Grass has an operculum.
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Instructions for Category I CME Credit Certification. As an organization accredited for continuing medical education, the American College of Allergy, Asthma, & Immunology (ACAAI) certifies that when the CME material is used as directed it meets the criteria for two hours’ credit in Category I of the American College of Allergy, Asthma, & Immunology CME Award and the Physician’s Recognition Award of the American Medical Association. Instructions. Category I credit can be earned by reading the text material, taking the CME examination and recording the answers on the perforated answer sheet entitled, “Continuing
Medical Education,” which can be found after the examination. Please record your ACAAI identification number and the quiz identification number in the spaces and scanning targets provided on the answer sheet. Your ACAAI identification number can be found on your ACAAI membership card, nonmembers of the College will be assigned an ACAAI identification number and this should be left blank on the answer sheet. The quiz identification number can be found at the beginning of the CME examination. Use a No. 2 or soft lead pencil for marking the answer sheet. You may
erase but do so completely in order to prevent computer reading errors. Your ACAAI identification number and quiz identification number will be used to record your credit hours earned on the CME transcript system. No records of individual performance will be maintained. Tear out the perforated answer sheet and print your name and address in the spaces provided. Return it within one month after the Annals is received to the American College of Allergy, Asthma, & Immunology, 85 West Algonquin Rd, Suite 550, Arlington Heights, IL 60005. Answers will be published in the next issue of the Annals of Allergy, Asthma, & Immunology.
Answers to CME examination—Annals of Allergy, Asthma, & Immunology, January 1998 (Identification No 008 – 001) Vaswani SL and Creticos PS. Metered dose inhaler: past, present, and future. Ann Allergy Asthma Immunol 1998;80:11–23. 1. e 7. e 13. e 19. e 25. b 2. c 8. a 14. d 20. c 26. c 3. b 9. e 15. e 21. b 27. e 4. a 10. b 16. c 22. e 5. b 11. b 17. e 23. c 6. b 12. c 18. a 24. c
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