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>
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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.

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

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia 
(impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort.
One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey 
et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, 
the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an 
expedient and effective delivery method for clinical gene transfer application.
Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Wharton's duct 
(Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at 
the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the 
guidelines of the Canadian Council on Animal Care.
For this experiment, a trypan blue solution is infused into the gland through the opening of the Wharton's duct using a 
insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated 
into the gland successfully.

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton&#039;s Duct

A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands.

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds

We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. Collecting site-specific biochemical markers is important to understand the relationship between levels in serum and surgical wound, determine any associations between mediator release, pain, analgesic use and other outcomes of interest, and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry. This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. We have measured wound exudate and serum mediators at the same time intervals as patient's pain scores and analgesics consumption for up to 48 hours post-cesarean delivery. Using this methodology we have been able to detect various biochemical mediators including nerve growth factor (NGF), prostaglandin E2 (PG-E2) substance P, IL-1&#x03B2;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&#x03B3;, G-CSF, GM-CSF, MCP-1 and MIP-1&#x03B2;. Studies applying this human surgical wound bioassay have found no correlations between wound and serum cytokine concentrations or their time-release profile (J Pain. 2008; 9(7):650-7).1 We also documented the utility of the technique to identify drug-mediated changes in wound cytokine content (Anesth Analg 2010; 111:1452-9).2

This article provides a detailed and visual description of a methodology for collecting and measuring biochemical inflammatory and nociceptive mediators at the surgical wound site following cesarean delivery. This human bioassay has been used to determine correlations between wound and serum cytokine concentrations and drug-mediated changes in wound cytokines, chemokines and neuropetides.

We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. ...

Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.

Frailty syndrome is commonly seen in the aged and reflects multi-system physiological change. However, with reduced functional reserve and resilience frailty is also known to be common in the HIV infected population. This study outlined an easily administered screening test to identify HIV patients with frailty. When significant components of frailty are identified, clinicians will be able to focus on amelioration of the problem and promote reversion to the pre-frail state.

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Clinical Assessment of Spatiotemporal Gait Parameters in Patients and Older Adults

Spatial and temporal characteristics of human walking are frequently evaluated to identify possible gait impairments, mainly in orthopedic and neurological patients1-4, but also in healthy older adults5,6. The quantitative gait analysis described in this protocol is performed with a recently-introduced photoelectric system (see&#160;Materials table) which has the potential to be used in the clinic because it is portable, easy to set up (no subject preparation is required before a test), and does not require maintenance and sensor calibration. The photoelectric system consists of series of high-density floor-based photoelectric cells with light-emitting and light-receiving diodes that are placed parallel to each other to create a corridor, and are oriented perpendicular to the line of progression7. The system simply detects interruptions in light signal, for instance due to the presence of feet within the recording area. Temporal gait parameters and 1D spatial coordinates of consecutive steps are subsequently calculated to provide common gait parameters such as step length, single limb support and walking velocity8, whose validity against a criterion instrument has recently been demonstrated7,9. The measurement procedures are very straightforward; a single patient can be tested in less than 5 min&#160;and a comprehensive report can be generated in less than 1&#160;min.

This protocol is used to evaluate spatial and temporal gait variables of neurological/orthopedic patients and older persons by means of a recently-introduced floor-based photocell system.

Spatial and temporal characteristics of human walking are frequently evaluated to identify possible gait impairments, mainly in orthopedic and ...

A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The capillaroscopy technique can provide insight into a patient&#8217;s microvascular health, leading to a variety of potentially valuable dermatologic, ophthalmologic, rheumatologic and cardiovascular clinical applications. In addition, tumor growth may be dependent on angiogenesis, which can be quantitated by measuring microvessel density within the tumor. However, there is currently little to no standardization of techniques, and only one publication to date reports the reliability of a currently available, complex computer based algorithms for quantitating capillaroscopy data.1 This paper describes a new, simpler, reliable, standardized capillary counting algorithm for quantitating nailfold capillaroscopy data. A simple, reproducible computerized capillaroscopy algorithm such as this would facilitate more widespread use of the technique among researchers and clinicians. Many researchers currently analyze capillaroscopy images by hand, promoting user fatigue and subjectivity of the results. This paper describes a novel, easy-to-use automated image processing algorithm in addition to a reproducible, semi-automated counting algorithm. This algorithm enables analysis of images in minutes while reducing subjectivity; only a minimal amount of training time (in our experience, less than 1 hr) is needed to learn the technique.

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy-to-learn methodology for directly visualizing capillaries in the microcirculation. However, only one publication to date describes the reliability of a complex software program available for quantitating capillaroscopy data. Here, we present a simple, reliable protocol for quantitating capillaries using a standardized algorithm.

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The ...

Murine Drinking Models in the Development of Pharmacotherapies for Alcoholism: Drinking in the Dark and Two-bottle Choice

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments are limited to three FDA-approved medications that are largely ineffective even when combined with psychosocial intervention, as is evident by the high relapse rate. As such, the search for more novel treatments represents an important public health goal. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds: two-bottle choice (TBC) and drinking in the dark (DID). The former allows mice to voluntary drink in moderation while the latter induces mice to voluntary consume a large amount of alcohol in a short period that mimics binge drinking. The simple and high throughput nature of both of these paradigms allow for rapid screening of pharmacological agents or for identifying strains of mice that exhibit certain voluntary drinking behavior.

Alcohol Use Disorder (AUD) is a major national health problem and the development of more effective treatments is&#160;required to offset the needs of this patient population. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds.

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments ...

Collecting Hair Samples for Hair Cortisol Analysis in African Americans

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, including humans. The advantage of hair cortisol concentration is that it reflects a retrospective representation of hypothalamic-pituitary-adrenal (HPA) axis function over time, much like hemoglobin A1C represents glycemic control. However, obtaining hair samples can be challenging, due to the cultural beliefs and hair care practices of minority participants. For example, African Americans may be reluctant to provide samples. Additionally, few researchers are trained to collect hair samples from African Americans. The purpose of this paper is to present a culturally informed protocol to help researchers obtain hair samples from African Americans. To illustrate the representative results of this protocol implementation, de-identified data from African Americans that participated in a community-based study on chronic stress are provided. Hair practice preferences are assessed. The participants are made comfortable by showing pictures of hair samples prior to cutting their hair. The single strain twist and gently pull method is used to collect approximately 30 - 50 strands of hair from the posterior vertex region of the scalp. This protocol will significantly improve collection of hair samples from African Americans.

Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, ...

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In these cases, questions remain regarding disease pathogenesis and effective interventions. An optimized technique that allows functional assessment of the salivary glands is invaluable for investigating exocrine gland biology, dysfunction, and therapeutics. Here, we present a step by step approach to performing pilocarpine stimulated saliva secretion, including tracheostomy and the dissection of the three major murine salivary glands. We also detail the appropriate murine head and neck anatomy accessed during these techniques. This approach is scalable, allowing for multiple mice to be processed simultaneously, thus improving the efficiency of the work flow. We aim to improve the reproducibility of these methods, each of which has further applications within the field. In addition to saliva collection, we discuss metrics for quantifying and normalizing functional capacity of these tissues. Representative data are included from submandibular glands with depressed salivary gland function 2 weeks following fractionated radiation (4 doses of 6.85 Gy).

We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands.

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In ...