LA Testing/EMSL Analytical, Inc. Allergens & Endotoxins: An Overview of Two Factors Associated with Allergies & Asthma. Gregory S. Saenz Ph.D. LA Testing/EMSL Analytical, Inc. South Pasadena, CA

Asthma Risk Factors Genetics i.e. Family History Sensitization to Indoor Allergens Atopy: Genetic tendency to develop a classic allergic disease, i.e. allergic reactions in susceptible individuals. Allergy: A hypersensitive immune response triggered by an allergen. Hypersensitivity: A reaction to an antigen producing a greater response than the initial exposure.

Allergens Endotoxins What are allergens? Sources of indoor biological allergens Well characterized indoor allergens Objectives of allergen testing Sampling for allergen testing Laboratory analysis of allergens Interpretation of results What are endotoxins Sources for endotoxins Objectives of endotoxin testing Sampling for endotoxins: air and/or dust Laboratory analysis Interpretation of results

What are allergens? Allergens are antigens that elicit symptoms of allergic disease in a previously sensitized individual; an antigen is a substance that is specifically recognized by the individual’s immune system,with subsequent development of specific antibody and/or cell-mediated immunity. Allergens are principally proteins.

Sources of Allergens Dust mites Cockroaches Cats Dogs Rats & Mice Fungi

Dust mite allergens Dermatophagoides pteronyssinus Der p 1, Der p 2, and others European Dust Mite Dermatophagoides farinae Der f 1, Der f 2, and others American Dust Mite Der p 1 and Der p 2 are the two major and most important allergens of dust mites, and thus, are the ones recommended for routine testing.

Dust Mites Geography: Temperate & humid regions. Habitats: Upholstered furniture, bedding & mattresses, and carpets. Frequency: Found in over 80% of U.S. homes (Arbes et al. 2003a) Moisture: Humidity is a limiting factor, need between 55%-73% RH. Exposure: Thought to occur through fecal pellets (~20µm) & dust particulate.

Cockroach allergens Blatella germanica Bla g 1, Bla g 2 German cockroach Periplaneta americana Per a 1 These are the two most common species and the major allergens isolated to date.

Cockroaches Geography: Temperate & humid regions. Habitats: Commonly, where food is stored and handled, i.e. especially kitchens, and where sanitary practices are inadequate. Also in bathrooms or near water. Frequency: Found in 13% of U.S. kitchens (Cohn et al. 2005). Especially prevalent in high-rise buildings, urban areas, pre-1940 construction, and low household income levels (<$20,000). Moisture: Are better able to survive lower humidity because they can actively search for water. Exposure: Cast skin, whole bodies, egg shells, fecal pellets, and saliva. Also associated with dust particulate.

Cat allergens Felis domesticus Fel d 1 Cats are in over 25% of American homes. About 2% of the US population are allergic to cats. The most abundant cat allergen is Fel d 1. Homes with cats had levels of Fel d 1 exceeding 10µ/g dust. Homes without cats had less than 1 µ/g dust.

Dog allergens Canis familiaris Can f 1 Dogs are in over 40% of American homes. Most homes without a dog had less than 10 µg/g dust of Can f 1. Homes with dogs had significantly higher Can f 1 at 120 µg/g dust.

Pets Exposure: Skin & fur (i.e. dander: dried skin scales & hair), and saliva. In some settings, they are the dominant indoor allergens. Allergens can remain airborne for longer periods of time, because they are found to be carried on smaller particulates (2-3 µm) (Chapman & Wood, 2001). Also found in dust. Allergens are easily transported to areas without pets (e.g. workplace) and clothing can be a major source of allergens.

Rat & Mouse Allergens Mus musculus Mus m 1 Rattus norvegicus Rat n 1 House Mouse and the Norway Rat. Rat & Mouse Urinary Protein Allergens. Health effects are currently under study.

Rodents Habitats: Live in close association with people. Commensal organisms. Frequency: In a study of inner-city homes, mouse allergens were present in at least one room in 95% of the sample (Phipatanakul, 2000a). Exposure: Urinary proteins, found in bedding and dust particulate. Easily airborne and dispersed with dust.

Fungal allergens Alternaria alternata Alt a 1 Aspergillus fumigatus Asp f 1 Penicillium notatum Pen n 1 Cladosporium herbarum Cla h 1 Can produce allergenic proteins as well as toxic compounds that can irritate and exacerbate asthma symptoms.

Fungi Habitats: In the indoor environment, especially associated with water damaged material, or in areas with high humidity. With sufficient moisture present, they are found on nearly every kind of building and/or household material. Exposure: Spores and hyphal fragments that can either be airborne or in dust. Release of fungal material (2-40µm) may be through air movement, mechanical disturbance, or by natural discharge mechanisms. Some may be seasonal, but many are consistently present in the indoor environment.

Objectives of Allergen Testing To identify and evaluate potential environmental allergen exposure. Unless authorized, do not provide procedures to remove or eliminate any issues found. Contact an allergist. To assess the effectiveness of allergen avoidance measures.

Sampling for Indoor Allergens Dust collector and a vacuum cleaner or a high volume pump.

Sampling for Indoor Allergens A composite sample or single samples from specific area may be taken. Room Selection: The best single predictor for overall allergen exposure is the living room floor (NSLAH), next would be the bedroom floor. For meaningful results, follow other studies. For certain allergens, kitchens and bathrooms may be better. Suggested sampling area is one square meter (m2). However, estimates are appropriate, when sampling along baseboards, nooks & crannies (e.g. kitchens). A minimum of 100 mg dust is required for analysis.

Laboratory Analysis of Allergens ELISA: Involves a pair of high-affinity monoclonal antibodies (Mab), where one Mab is used for allergen capture and a second biotinylated or enzyme labeled Mab is used for allergen detection

Interpretation of Results Guidelines for mite allergen levels (Der p 1): <2µg/g dust = Low: Not sufficient to cause symptoms 2-10 µg/g dust = Moderate: Risk for sensitization and bronchial hyperactivity >10 µg/g dust = High: Risk for acute allergic asthma attack

Interpretation of Results Suggested threshold level for cockroach: 2 U/g dust of Bla g 1 and/or Bla g 2 >2 U/g dust = risk for sensitization Measurement is in Units per gram.

Interpretation of Results Guidelines for cat allergen levels (Fel d 1): <1µg/g dust = Low: Not sufficient to cause symptoms 1-8 µg/g dust = Moderate: Risk for sensitization and bronchial hyperactivity >8 µg/g dust = High: Risk for acute allergic asthma attack

Interpretation of Results There are no established risk factors for Dog, Rodent, and Fungal Allergens. For dog allergens (Can f 1), suggested risk for sensitization at >10 µg/g dust.

Endotoxin Pyrogenic (fever-causing), cell wall component (lipopolysaccharide) of Gram-negative bacteria. Heat-stable

Known Endotoxin Areas Elevated airborne concentrations are prevalent in sewage treatment plants Swine operations Cotton textile mills Poultry houses Water damaged buildings or in the presence of a contaminated humidifier

Health Effects Produces airway inflammation (chest tightness & wheezing) Associated with increased severity of child and adult asthma Exposure during childhood may reduce allergic responses later in life Cause of Humidifier Fever May be associated with Building-Related Symptoms

Objectives of Endotoxin Testing Used to confirm exposure in agricultural and industrial environments, as well as in buildings with humidifiers and recent water damage. Caveat: Threshhold Limit Values have not yet been established due to lack of standard methods for sampling and analysis, but much research is being done to correct this.

Sampling for Endotoxins Filter Specifications: 0.45µm Polycarbonate Filter. Other filter types have been shown to bind endotoxins, thus negatively effecting the recovery (Milton & Johnson, 1995). Always take a Blank and a Baseline/Background sample.

Sampling for Airborne Endotoxins 0.45µm Endotoxin-Free Polycarbonate Filter Flow rate 2 liters/minute Duration: At least 8 hours. Some studies have sampled for up to a week (Douwes et al., 1995). While other researchers have sampled for 24 hours at a high flow rate (1132 lpm; Mueller-Anneling et al., 2004). Dales et al. (2006) suggested that a five-day air sample was a stable estimate of average exposure.

Sampling for Endotoxins in Dust Literature contains various sampling devices, but one factor has been established, Mixed-Cellulose Ester filters bind or inactive endotoxins. See allergen sampling protocol. Kitchens and bathrooms may provide stable endotoxin in dust estimates over time (Abraham et al., 2005).

Laboratory Analysis of Endotoxins Keep samples cold (e.g. 4ºC) and ship to laboratory overnight. Kinetic Chromogenic Limulus amoebocyte lysate (LAL) assay (EU – endotoxin units) Comparative analysis based upon biological activity to a reference standard Endotoxin. The result is NOT a concentration of a single chemical substance.

Results Outdoor Endotoxin levels generally <3.0 EU/m3). Mueller-Anneling et al. (2004) did an outdoor Endotoxin & PM10 study of the Southern California area. Several significant findings, of which, inland downwind sites from Los Angeles had the highest endotoxin and PM10 levels. Endotoxin levels <5.5 EU/m3.

Results Airborne Endotoxin levels in homes and in indoor work environments can be greater than the outside, especially if contaminated humidifiers are present, or after a water-damage event. ACGIH suggests levels >100X background is indicative of a problem, but EU levels in slight excess of the background can be associated with exacerbated asthma symptoms. Agriculture & industrial exposure levels of >45 EU/m3 have been associated with decreased lung function.

Further Information The Department of Housing and Urban Development, Healthy Homes Initiative is an excellent resource for all of your allergen and endotoxin needs. <www.hud.gov/offices/lead/hhi> For further education on allergens & endotoxins, the American Academy of Allergy, Asthma, & Immunology annual meeting in San Diego, February 23-27, 2007. <www.aaaai.org> References for this presentation have been provided on an additional sheet.

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