About the Measure
	
Domain:

	Respiratory	
Measure:

	Blood, Hair, and Saliva Assays for Tobacco Smoke Exposure

Definition:

	To assess smoking and environmental tobacco smoke (ETS) exposure by measuring cotinine, a metabolite of nicotine. Blood (serum), hair, or saliva specimen may be used for the bioassay.

About the Protocol

Description of Protocol:

	A biospecimen is collected from the respondent to measure cotinine, a marker of either smoking or of environmental tobacco smoke exposure. The most common methods are via a blood (serum), urine, hair, or saliva sample obtained from the respondent. Note: urine protocol is found in the PhenX Toolkit:

 https://www.phenxtoolkit.org/index.php?pageLink=browse.protocols&id=90500   

Cotinine in a serum sample may be quantified by isotope dilution-high performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (ID HPLC-APCI MS/MS) and measured in saliva by a High Sensitivity Salivary Cotinine Quantitative EIA Kit.

Hair cotinine is a useful biomarker of longer term smoke exposure and a small amount of hair may represent smoke exposure over a period of 2 to 3 months. Hair nicotine is measured by isotope dilution gas chromatography mass spectrometry.
	
Protocol text:

	Blood (serum) - a trained nurse or phlebotomist collects blood via standard venipuncture procedures in a sterile 3-mL standard, non-anticoagulated Vacutainer® tube. The blood may be centrifuged 30-60 minutes after clotting. 1.5 mL is the minimum amount of serum needed and 2 mL is preferred. Aliquot the serum into polypropylene cryovials and then freeze at -20°C until analysis.

Serum cotinine is measured by an isotope dilution-high performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (ID HPLC-APCI MS/MS). The detection limit was .015 ng/mL and the below the limit of detection value was .011ng/mL. See source references for details.

Hair - a trained interviewer or technician uses scissors to cut a small amount of hair (approximately 30 milligrams) close to the rear of the person’s scalp and approximately 3 centimeters in length. Each hair sample is stored in a clean envelope with the cut end of the hair identified. Store envelopes in a refrigerator until analysis.

The hair is weighed, spiked (i.e. a known quantity of analyte is added) to 3.3 ng/mg, and liquid-liquid extraction is performed by shaking the sample for 60 minutes. The hair is added to a centrifuge tube and washed with dichloromethane to remove nicotine/cotinine that has adhered to the surface.

After the hair is dry, complete the extraction process described in the Kim et al. 2008 manuscript. Finally, perform isotope dilution gas chromatography mass spectrometry on the hair sample to identify the hair nicotine/cotinine concentration in ng/mg.

Saliva - the respondent should insert the oral swab under his/her tongue, keep in place for 1 to 2 minutes, and then insert the swab into the collection tube. 0.5 mL of saliva is sufficient to perform cotinine analysis on the sample. Refrigerate sample within 30 minutes after collection, then freeze within 4 hours at -20° C until analysis. Note: oral swabs should not be used for children under the age of 6. Sorbettes® and cotton rope are alternatives for young children.

Saliva samples may be analyzed via kits such as the High Sensitivity Salivary Cotinine Quantitative EIA Kit available through Salimetrics®. Batches of frozen samples may also be shipped directly to a laboratory such as Salimetrics® for analysis.

Vacutainer® is a registered trademark of Becton, Dickinson and Company.
Salimetrics® is a registered trademark of Salimetrics LLC.
Sorbettes® isa registered trademark of Salimetrics LLC.
	
Participant:
	All ages (for serum and hair test), ages 6 + (for saliva test)	
Source: 

	Blood (Serum)
National Center for Health Statistics. National Health and Nutrition Examination Survey. NHANES 2005-2006 Laboratory Procedure Manual for Cotinine. Retrieved May 11, 2009, from http://www.cdc.gov/nchs/data/nhanes/nhanes_05_06/cot_d_met_cotinine.pdf

National Center for Health Statistics. National Health and Nutrition Examination Survey. NHANES 2005-2006 Laboratory Procedures Manuals. Retrieved April 27, 2009, from
http://www.cdc.gov/nchs/about/major/nhanes/nhanes2005-2006/lab05_06.htm

Hair
Kim, S.R., Wipfli, H., Avila-Tang, E., Samet, J, Breysse, P. Method validation for measurement of hair nicotine level in nonsmokers. 2009. Biomed. Chromatogr. 23: 273–279

Saliva
Salimetrics LLC. High Sensitivity Salivary Cotinine Quantitative EIA Kit.
Retrieved May 4, 2009, at http://www.salimetrics.com/

Salimetrics LLC. Saliva Collection and Handling Advice.
Retrieved May 4, 2009, at http://www.salimetrics.com/
	
Personnel and Training Required: 

	Venipuncture is necessary to obtain blood for serum analysis. Venipuncture requires a trained nurse or phlebotomist. Hair or saliva samples can be self-collected by children aged 10 or older with adequate instructions.

A laboratory technician trained to process and analyze biological specimens is necessary to analyze serum, hair, or saliva samples.	
Equipment Needs: 

	Standard venipuncture, hair and saliva collection supplies that have been sterilized are necessary. Laboratory supplies and instruments are needed to measure cotinine in the biological fluids. Biological samples may be shipped to laboratories that specialize in these types of analysis.
	
General References: 

	Bernowitz, N.L. (1999). Biomarkers of Environmental Tobacco Smoke Exposure.
Environ Health Perspect. 107(Suppl 2):3 49-355.

Kuo, H.W., Yang, J.S., Chiu, M.C. (2002). Determination of urinary and salivary cotinine using gas and liquid chromatography and enzyme-linked immunosorbent assay. J Chromatogr B Analyt Technol Biomed Life Sci. 768(2):297-303.

Dhar, Preeti. (2004). Measuring tobacco smoke exposure: quantifying nicotine/cotinine concentration in biological samples by colorimetry, chromatography and immunoassay methods. J Pharm Biomed Anal, 35(2004), 155-168.

Benowitz, Neal L. (1996). Cotinine as a biomarker of environmental tobacco smoke exposure. Epidemiol Rev, 18(2), 188-204.