Study funded by NINR, K23NR012017-01A1 Adrenocortical Response and Neuro-psychiatric Symptoms in Alzheimer&#39;s Disease.

1. Recruitment
<ol>
	<li>Obtain appropriate Institutional Review Board approval of collection protocol with special attention to consenting subjects with impaired capacity and the collection of biological specimens9-10.</li>
	<li>Exclude participants with potential for blood leakage into saliva by assessing oral health (i.e. &#34;Did you observe bleeding gums in the past two&#160;days?&#34;)11.</li>
</ol>
2. Preparation of Saliva Collection Supplies
<ol>
	<li>Prior to scheduling home visit to consented participants, gather and assemble necessary supplies: 4 Salimetrics Children&#39;s Swabs (SCS) , 4 swab storage tubes, 4 color-coded tube caps, 5 white labels, 1 plastic zip-lock bag, saliva collection instruction sheet, 5 nitrile gloves, and storage box.</li>
	<li>Apply nitrile glove. Using gloved hand, place 4 SCS into 4 storage tubes and cap with yellow, orange, purple, and blue color caps, respectively.</li>
	<li>Place label on storage tube to indicate time of collection (on awakening- yellow cap, 30 min post awakening-orange cap, mid day before lunch-blue cap, evening before dinner-purple cap).</li>
	<li>Place the 4 capped storage tubes plus saliva collection instruction sheet and 4 nitrile gloves in zip-lock bag and label bag with subject ID and date of collection.</li>
	<li>Schedule short home visit to subjects previously consented and on day prior to saliva sampling. On arrival to home provide plastic bag of supplies and instruction sheet.</li>
</ol>
3. Saliva Collection Instruction
<ol>
	<li>Review instruction sheet (see Figure 1 for example). Emphasize the need to record start and stop time of saliva collection.</li>
	<li>Record the name, dosage, and schedule of all prescription and OTC medications taken by the participant within the last 48 hr12.</li>
	<li>Instruct participant to tilt head forward to allow saliva to pool on the floor of mouth.</li>
	<li>Instruct saliva collector (SC) to apply nitrile glove to dominant hand (only required for hand holding swab)9.</li>
	<li>Instruct SC to uncap the swab storage tube to retrieve oral swab. Do not remove the basket insert from the tube.</li>
	<li>Instruct SC to place the oral swab under the front of the tongue of the older adult to absorb about 3 drops (75 &#181;l) of pooled saliva, and to HOLD the oral swab in place for 2 min to ensure that it is saturated. Swab will become opaque when saturated.</li>
	<li>Have the SC return the saturated oral swab back into the basket of the swab storage tube and replace the cap.</li>
	<li>Have the SC immediately place tube in storage box and place in freezer. Discard glove in trash bin.</li>
	<li>Have SC record start and stop times on saliva instruction sheet.</li>
</ol>
4. Provide Additional Instructions and Answer Questions
<ol>
	<li>Instruct SC that they will be collecting saliva sample on four occasions the following day. The times of collection are on awakening, 30 min post awakening, mid morning, and evening13,14.</li>
	<li>Instruct SC to avoid sample collection within 1 hr after a major meal.</li>
	<li>Have SC instruct patient to rinse mouth with water 10 min prior to saliva collection.</li>
	<li>Discard samples visibly contaminated with blood.</li>
	<li>Instruct SC that saliva samples should remain in freezer until retrieved by study team. Confirm that study team will schedule a time and day to retrieve the frozen samples and bring new saliva collection supplies for any future collection date.</li>
</ol>
5. Saliva Handling and Processing
<ol>
	<li>Once samples are removed from subject&#39;s freezer, keep frozen during transport to lab using insulated freezer bag.</li>
	<li>On arrival to lab, replace white label on tube with sequential barcode.</li>
	<li>Confirm sequential barcode matches subject ID, date of collection, and time of collection.</li>
	<li>Return saliva sample to freezer (-20 &#176;C) to precipitate the mucins.</li>
</ol>
6. Optional: Salivary Cortisol Assay Protocol
<ol>
	<li>Ensure all salivary assay kit reagents are at room temperature.</li>
	<li>After thawing saliva samples, vortex and centrifuge at 1,500 x g for 15 min.</li>
	<li>Prepare wash buffer following kit instructions.</li>
	<li>Pipette 25 &#181;l&#160;of standards and controls into appropriate wells of plate layout. Standards and samples should be assayed in duplicate.</li>
	<li>Pipette 200 &#181;l&#160;enzyme conjugate into wells.</li>
	<li>Mix plate by placing on rotator for 5 min at 500 rpm (then incubate at room temperature for 55 min).</li>
	<li>Wash and blot plate with wash buffer 4x.</li>
	<li>Add 200 &#181;l&#160;tetramethylbenzidine (TMB) solution .</li>
	<li>Again mix the plate for 5 min at 500 rpm then incubate in the dark for an additional 25 min.</li>
	<li>Add 50 &#181;l&#160;stop solution and mix for 3 min at 500 rpm.</li>
	<li>Within 10 min of adding stop solution wipe off plate bottom. Read with a plate reader at 450 nm.</li>
	<li>Calculate cortisol concentration using the average optical density. Subtract the average optical density in nonspecific binding wells from the average optical density in zero, standard, control, and sample wells.</li>
	<li>Estimate the concentrations of the controls and unknowns using software capable of logistics.</li>
</ol>
7. Optional: Salivary Alpha Amylase Assay Protocol
<ol>
	<li>Set plate reader to incubate at 37 &#176;C, and read in center measurement kinetic mode at 1 min and 2 min.</li>
	<li>Use 405 nm filter and no reference filter.</li>
	<li>Dilute saliva samples with the alpha-amylase diluent provided.</li>
	<li>Prepare 1:200 dilution by first preparing a 1:10 dilution of the saliva by pipetting 10 &#181;l&#160;of saliva into 90 &#181;l&#160;alpha-amylase diluent. Then, further dilute by pipetting 10 &#181;l&#160;of the 1:10 dilution into 190 &#181;l&#160;alpha-amylase diluent (1:20).</li>
	<li>Heat alpha-amylase substrate solution to 37 &#176;C using a preheated microtiter plate incubator.</li>
	<li>Add 8 &#181;l&#160;of diluted saliva samples to individual wells.</li>
	<li>Simultaneously add 320 &#181;l&#160;of preheated (37 &#176;C) alpha-amylase substrate solution.</li>
	<li>For reading kinetically in 37 &#176;C plate reader, immediately place plate in reader and start reader.</li>
	<li>Subtract 1 min readings from the 3 min reading and multiply by the conversion factor.</li>
	<li>Calculate amylase activity as &#916;Abs./min x TV x DF /MMA x SV x LP = U/ml. Where &#916;Abs./min = Absorbance difference per minute ; TV = Total assay volume (0.328 ml) ; DF = Dilution factor ;MMA = Millimolar absorptivity of 2-chloro-p-nitrophenol (12.9) ;SV = Sample volume (0.008 ml) ; LP = Light path = 0.97(specific to plate received with kit).</li>
</ol>

<ol>
	<li>Granger, D. A., Fortunato, C. F., Beltzer, E. K., Virag, M., Bright, M. A., Out, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focus+on+methodology:+Salivary+bioscience+and+research+on+adolescence:+An+integrated+perspective.">Focus on methodology: Salivary bioscience and research on adolescence: An integrated perspective.</a> Journal of Adolescence. 3 (10), 1081-1095 (2012).</li><li>Savla, J. T., Granger, D. A., Roberto, K. A., Davey, A., Blieszner, R., Gwazdauskas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortisol+and+alpha+amylase+reactivity+to+care-related+stressors+in+spouses+of+persons+with+mild+cognitive+impairment.">Cortisol and alpha amylase reactivity to care-related stressors in spouses of persons with mild cognitive impairment.</a> Psychology and Aging. , (2013).</li><li>Hodgson, N. A., Freedman, V., Granger, D., Erno, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biobehavioral+correlates+of+relocation+in+the+frail+elderly:+salivary+cortisol,+affect,+and+cognition.">Biobehavioral correlates of relocation in the frail elderly: salivary cortisol, affect, and cognition.</a> Journal of the American Geriatrics Society. 52 (11), 1856-1862 (2004).</li><li>Woods, D. L., Kim, H., Yefimova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morning+cortisol+in+relation+to+behavioral+symptoms+of+nursing+home+residents+with+dementia.">Morning cortisol in relation to behavioral symptoms of nursing home residents with dementia.</a> Biological Reseach in Nursing. (2), 13-132 (2011).</li><li>Granger, D. A., Johnson, S. B., Gellman, M. D., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+Biomarkers.">Salivary Biomarkers.</a> Encyclopedia of Behavioral Medicine. , 1005-1009 (2012).</li><li>Nater, U. M., Rohleder, N., Schlotz, W., Ehlert, U., Kirschbaum, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+the+diurnal+course+of+salivary+alpha-amylase).">Determinants of the diurnal course of salivary alpha-amylase).</a> Psychoneuroendocrinology. 32, 392-401 (2007).</li><li>Navazech, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+collecting+saliva.">Methods for collecting saliva.</a> Annals of the NY Academy of Sciences. 694, 72-77 (1993).</li><li>Dickerson, S. S., Kemeny, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+stressors+and+cortisol+responses:+A+theoretical+integration+and+synthesis+of+laboratory+research.">Acute stressors and cortisol responses: A theoretical integration and synthesis of laboratory research.</a> Psychology Bulletin. 130, 355-291 (2004).</li><li>Scannapieco, F. A., Papandonatos, G. D., Dunford, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+between+oral+conditions+and+respiratory+disease+in+a+national+sample+survey+population.">Associations between oral conditions and respiratory disease in a national sample survey population.</a> Annals of Periodontology. 3, 251-256 (1998).</li><li>Reibel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+and+oral+diseases.+Update+on+the+evidence,+with+recommendations.">Tobacco and oral diseases. Update on the evidence, with recommendations.</a> Medical Principles and Practice. 1, 22-32 (2003).</li><li>Kivlighan, K. T., Granger, D. A., Schwartz, E. B., Nelson, V., Curran, M., Shirtcliff, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+blood+leakage+into+the+oral+mucosa+and+its+effects+on+the+measurement+of+cortisol,+dehydroepiandrosterone,+and+testosterone+in+saliva.">Quantifying blood leakage into the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva.</a> Hormones and Behavior. 46, 39-46 (2004).</li><li>Granger, D. A., Hibel, L. C., Fortunato, C. K., Kapelewski, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medication+effects+on+salivary+cortisol:+Tactics+and+strategy+to+minimize+impact+in+behavioral+and+developmental+science.">Medication effects on salivary cortisol: Tactics and strategy to minimize impact in behavioral and developmental science.</a> Psychoneuroendocrinology. 34, 1437-1448 (2009).</li><li>Nater, U. M., Rohleder, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+alpha-amylase+as+a+noninvasive+biomarker+for+the+sympathetic+nervous+system:+Current+state+of+research.">Salivary alpha-amylase as a noninvasive biomarker for the sympathetic nervous system: Current state of research.</a> Psychoneuroendocrinology. 34 (4), 486-496 (2009).</li><li>Hellhammer, J., Fries, E., Schweisthal, O. W., Scholtz, W., Stone, A. A., Hagemann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Several+measurements+are+necessary+to+reliably+assess+the+cortisol+rise+after+awakening:+State-+and+trait-components.">Several measurements are necessary to reliably assess the cortisol rise after awakening: State- and trait-components.</a> Psychoneuroendocrinology. 32, 80-86 (2007).</li><li>Pruessner, J., Kirschbaum, C., Meinlschmid, G., Hellhammer, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+formulas+for+computation+of+the+area+under+the+curve+represent+measures+of+total+hormone+concentration+versus+time-dependent+change.">Two formulas for computation of the area under the curve represent measures of total hormone concentration versus time-dependent change.</a> Psychoneuroendocrinology. 28, 916-931 (2003).</li><li>Twisk, J. W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Applied multilevel analysis: A practical guide. , (2006).</li><li>Hox, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Multilevel analysis: Techniques and applications. , (2002).</li><li>Adam, E. K., Hoyt, L. T., Granger, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diurnal+alpha-amylase+patterns+in+adolescents:+associations+with+puberty+and+momentary+mood+states.">Diurnal alpha-amylase patterns in adolescents: associations with puberty and momentary mood states.</a> Biological Psychology. 88, 170-173 (2011).</li><li>Dorn, L. D., Lucke, J. F., Loucks, T. L., Berga, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+cortisol+reflects+serum+cortisol:+analysis+of+circadian+profiles.">Salivary cortisol reflects serum cortisol: analysis of circadian profiles.</a> Annals in Clinical Biochemistry. 44, 281-284 (2007).</li><li>Donzella, B., Talge, N. M., Smith, T. L., Gunnar, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+spear+or+not+to+spear:+Comparison+of+saliva+collection+methods.">To spear or not to spear: Comparison of saliva collection methods.</a> Developmental Psychobiology. 50 (7), 714-717 (2007).</li><li>Beltzer, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+flow+and+alpha-amylase:+Collection+technique,+duration,+and+oral+fluid+type.">Salivary flow and alpha-amylase: Collection technique, duration, and oral fluid type.</a> Physiology and Behavior. 101 (2), 289-296 (2010).</li><li>Granger, D. A., Johnson, S. B., Szanton, S. L., Out, D., Schumann, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporating+salivary+biomarkers+into+nursing+research:+an+overview+and+review+of+best+practices.">Incorporating salivary biomarkers into nursing research: an overview and review of best practices.</a> Biological Research in Nursing. 14 (4), 347-356 (2012).</li><li>Shultheiss, O. C., Stanton, S. J., Harmon-Jones, E., Beer, J. 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In the interest of full disclosure, DAG is founder and serves as the Chief Scientific and Strategy Advisor at Salimetrics LLC (State College, PA) and SalivaBio LLC (Baltimore, MD). These relationships are managed by the policies of the committee on conflict of interest at the Johns Hopkins University School of Medicine, and Arizona State University&#39;s Office of Research Adherence and Integrity.

To advance our understanding of the meaning of individual differences in biological sensitivity and susceptibility in frail older adults investigators will need to engage caregivers in the collection of biological specimens. These collection protocols need to be minimally invasive, easy to accomplish, and yield quality specimens. Since one major challenge is ensuring that caregivers collect the samples at the correct time family caregivers designated as saliva collectors should be screened for ability to understand writt...

The theoretical perspective guiding lifespan developmental science emphasizes the need to integrate biological, behavioral, social, and contextual factors into the study of health and behavior1. Contemporary theoretical models champion the notion that individual differences in biological sensitivity and susceptibility to contextual factors may moderate risk versus resilience, and can forecast morbidity and mortality in vulnerable populations. Considerable research attention has focused on these issues early in life development1, but only recently have investigators been able to test the core ideas in studies with medically and cognitively frail older adults2-4. One reason for this trend is the development of procedures to noninvasively measure differences in neurobiological activity including the hypothalamic-pituitary-adrenal (HPA) axis and autonomic nervous system (ANS) within and across individuals via oral fluids (saliva)5,6.
Salivary measures can allow for the collection of normative longitudinal data, to compare changes between different treatment groups, to compare effectiveness of interventions, or can be extended to include measurement of coregulation or activity between multiple individuals. The data obtained from these measures can shed light on stress-linked biological processes in health conditions common in older adults including cancer, heart disease or neurocognitive disorders7. Saliva has received growing attention as a biospecimen due to the perceptions of sample collection as an easy to learn, inexpensive, minimally invasive, and acceptable8. Nonetheless, the conventional methods of collection requiring participants to drool through a straw into a small tube have been shown to be too complicated or undesirable for older, frail participants. New methods are required that can standardize the preanalytical phase of collection and processing to deliver the most accurate and meaningful results. The field of salivary bioscience is advancing rapidly and the purpose of this presentation is to provide an update of developments and a practical roadmap for investigators interested in integrating these measurement approaches into research on aging. In this protocol we demonstrate (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) the procedures for measuring HPA axis and ANS activity via salivary cortisol and salivary alpha amylase (sAA), and (3) representative individual differences in salivary cortisol and sAA data.

<table border="0" cellpadding="0" cellspacing="0" width="650">
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		<col />
		<col />
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	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Salimetrics Children Swab (SCS)</td>
			<td style="width:95px;">Salimetrics</td>
			<td style="width:140px;">5001.06</td>
			<td style="width:223px;">store at RT 50/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Swab storage tube (2 ml)</td>
			<td style="width:95px;">Sarstedt</td>
			<td style="width:140px;">51.1534.004</td>
			<td style="width:223px;">100/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Barcode lables</td>
			<td style="width:95px;">CISBR</td>
			<td style="width:140px;">n/a</td>
			<td>contact CISBR to order</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Color coded polypropylene vial caps (for SST)</td>
			<td style="width:95px;">Sarstedt</td>
			<td>(see below)</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">White</td>
			<td style="width:95px;">Sarstedt</td>
			<td>65.793.517</td>
			<td style="width:223px;">1,000/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blue</td>
			<td style="width:95px;">Sarstedt</td>
			<td>65.793.513</td>
			<td style="width:223px;">1,000/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yellow</td>
			<td style="width:95px;">Sarstedt</td>
			<td>65.793.514</td>
			<td style="width:223px;">1,000/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Green</td>
			<td style="width:95px;">Sarstedt</td>
			<td>65.793.515</td>
			<td style="width:223px;">1,000/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orange</td>
			<td style="width:95px;">Sarstedt</td>
			<td>65.793.516</td>
			<td style="width:223px;">1,000/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Red</td>
			<td style="width:95px;">Sarstedt</td>
			<td>65.793.518</td>
			<td style="width:223px;">1,000/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Black</td>
			<td style="width:95px;">Sarstedt</td>
			<td>65.793.519</td>
			<td style="width:223px;">1,000/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Violet</td>
			<td style="width:95px;">Sarstedt</td>
			<td>65.793.520</td>
			<td style="width:223px;">1,000/pack</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mixed Colors</td>
			<td style="width:95px;">Sarstedt</td>
			<td>65.793.999</td>
			<td style="width:223px;">1,000/pack</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Salivary Cortisol Enzyme Immuno Assay Kit</td>
			<td style="width:95px;">Salimetrics</td>
			<td style="width:140px;">1.3102</td>
			<td style="width:223px;">Diagnostic Kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Salivary Alpha Amylase Enzyme Immuno Assay Kit</td>
			<td style="width:95px;">Salimetrics</td>
			<td style="width:140px;">1.1902</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Nitrile gloves</td>
			<td style="width:95px;">VWR</td>
			<td style="width:140px;">(see below)</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">X-Small</td>
			<td style="width:95px;">VWR</td>
			<td style="width:140px;">82026-423</td>
			<td style="width:223px;">100/box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Small</td>
			<td style="width:95px;">VWR</td>
			<td style="width:140px;">82026-424</td>
			<td style="width:223px;">100/box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Medium</td>
			<td style="width:95px;">VWR</td>
			<td style="width:140px;">82026-426</td>
			<td style="width:223px;">100/box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Large</td>
			<td style="width:95px;">VWR</td>
			<td style="width:140px;">82026-428</td>
			<td style="width:223px;">100/box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">X-Large</td>
			<td style="width:95px;">VWR</td>
			<td style="width:140px;">82026-43</td>
			<td style="width:223px;">100/box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulated Shipping Container</td>
			<td>ThermoFisher</td>
			<td>Varies based on size</td>
			<td><a href="https://us.vwr.com/store/catalog/product.jsp?catalog_number=14100-420">Visit VWR to select size and order</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">4 in Sample Storage Box (7 x 7)</td>
			<td style="width:95px;">Sarstedt</td>
			<td style="width:140px;">95.064.949</td>
			<td style="width:223px;">1 box holds 49 SSTs</td>
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</table>

collecting saliva and measuring salivary cortisol and alpha-amylase in frail community residing older adults via family caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

The protocol is meant to provide one example of how salivary biomeasures can be collected in the community setting. The procedures are designed to measure the diurnal profile of salivary alpha amylase and salivary cortisol concentrations (Figure 2).
Analytic Strategy
Cortisol results have been shown to be positively skewed. Therefore it is suggested to use log transformation to the data to yield a normal distribution. Several strategies are available t...

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Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

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11.3K Views.  Johns Hopkins University School of Nursing. We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.