This publication was made possible by the Clinical and Translational Science Collaborative of Cleveland; KL2TR000440 from the National Center for Advancing Translational Sciences (NCATS) component of the National Institutes of Health; and NIH Roadmap for Medical Research. Dr. Jodi Ford's work in this area has been funded by the National Institutes of Health National Institute on Drug Abuse (1R21DA034960) and The Ohio State University Institute for Population Research seed grant funding from the National Institute of Child Health and Development (2P2CHD058484). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Dr. Laudenslager's work in this area has been funded by a contract from the Patient-Centered Outcomes Research Institute (CE-1304-6208), a grant from the Administration for Children and Families (90YR0058), and a grant from the Developmental Psychobiology Research Group Endowment Fund of the Department of Psychiatry, University of Colorado Denver Anschutz Medical Campus.

The study is approved by the University Hospitals Cleveland Medical Center Institutional Review Board, and the protocol follows the guidelines of participant protection and data safety as indicated by the review committee.
1. Prepare Participant for Hair Sample and Hair Sample Collection Questions
<ol>
	<li>Explain to participant that the hair sample collected is less hair than that is lost normally in brushing each day from the back of the head (posterior vertex). Tell the participant that the site for the hair sample is hidden by surrounding hair and, thus, is not visible after collection. Inform the participant that the sample will be used to measure a hormone called cortisol present in the hair10,11.</li>
	<li>Take out the sample photo and show the participant the amount of hair that will be collected (30 - 50 strands). 
	NOTE: See Figure 1 for photo of a hair sample.</li>
	<li>Ask the participant the hair care practice12 questions listed in Table 1.</li>
</ol>
2. Collect the Hair Sample
<ol>
	<li>Tell the participant that the sample is collected in a private area behind a portable partition, curtain, or closed door. Assure participants that, if they wear a wig or hairpiece, their hair is not cut in an area where others may see. Measure hair length from scalp to end by pulling the hair gently and taut.</li>
	<li>If the hair is short (less than 3 cm), follow the short hair protocol in step 3 (although if the hair is less than 1 cm long, the sample cannot be collected, and, ideally, the hair should be at least 3 cm long). For hair longer than 6 cm, follow the long hair protocol in step 4. Otherwise, continue to the next step.</li>
	<li>Ensure that materials are ready by placing the hair cutting supplies (a sheet of aluminum foil, business envelope, salon grade scissors, large and small tooth parting comb, alcohol preps, painters tape, permanent marker, non-latex gloves [optional], and hair clips)10 on a surface next to the participant. Clean the scissors and comb with the alcohol prep pad before collecting the sample. Cut out an 8 x 10&#34; sheet of aluminum foil to use for the sample.</li>
	<li>Put on non-latex gloves. Take the comb and part the hair horizontally along the posterior vertex of the scalp between the tips of the ears.</li>
	<li>Take a clip and move the hair from the top of the part, then place another clip at the bottom to expose a 5 x 10 cm rectangle between the two clips of loose hair (about the size of a deck of playing cards).</li>
	<li>Ask the participant if they prefer use of a wide tooth or narrow tooth comb through the 2 - 3&#34; rectangle of hair. Ask the participant how she or he would like to have the remaining hair in the comb discarded. Give the hair in the comb to the participant, or, if (and only if) the participant states that it is okay to discard the loose hair, discard the remaining hair from the comb into a trash can.</li>
	<li>Grasp approximately 30 - 50 strands of hair to the right of the 2 - 3&#34; rectangle and gently pull and twist the hair away from the scalp in a rolling motion between the fingers.</li>
	<li>Ensure that the sample is collected as close to the scalp as possible, but do not cut the scalp. Use salon-grade scissors and make a clean straight cut.</li>
	<li>Grasp the hair sample and ensure that the root end is aligned carefully along the cut.</li>
	<li>Attach the hair to the sheet of aluminum foil by taping with painter&#39;s tape. Do not cover the scalp end, and do not use painter&#39;s tape on hair that is shorter than or equal 3 cm. Place the loose cut hair into the center of the foil and fold into a square, ensuring that the hair does not fall out of the foil.</li>
	<li>Take the marker and label the scalp end on the aluminum foil. Use premade bar-coded labels to attach to the foil and envelope.</li>
	<li>Fold the aluminum foil without folding the hair on the scalp end to prevent bed head (crumpled and bent hair), place it in the business size envelope, and seal.</li>
	<li>Label the outside of the envelope with the participant&#39;s unique study identification number, date, and the name of the primary investigator, or use a bar code label if available.</li>
	<li>Store the sample in a dry area at room temperature.</li>
</ol>
3. Sample Collection for Short Hair (1 - 3 cm)
<ol>
	<li>Follow steps 2.3 and 2.4.</li>
	<li>After parting, ask the participant to hold the parted hair close to the scalp. Cut the hair along the part, while holding the hair tightly with index finger and thumb10.</li>
	<li>Place loose hairs in foil and fold it securely. Do not tape the hair to the foil.</li>
	<li>Follow steps 2.11 through 2.14.</li>
</ol>
4. Sample Collection for Long Hair (longer than 6 cm)
<ol>
	<li>Part the hair left to right at the posterior vertex.</li>
	<li>Use the hair clips to hold back the extra hair.</li>
	<li>Create a twist of hair and hold tightly with the index finger and thumb so as not to drop the sample10.</li>
	<li>Make a clean straight cut as close to the scalp as possible. Collect 30 - 50 strands of hair.</li>
	<li>If the participant has thin hair, then cut 2 - 3 small areas (1 cm apart) across the posterior vertex to conceal the site of the cut.</li>
	<li>Follow steps 2.10 through 2.14.</li>
</ol>
5. Sample Processing
<ol>
	<li>Store the foil packets containing the hair sample at room temperature indefinitely until analysis. 
	Note: Hair cortisol is extremely stable in hair and can be stored for years. In fact, cortisol has been measured in hair samples from mummified remains11.</li>
	<li>Analyze hair cortisol as previously published12. Samples can be shipped overnight at ambient temperature.</li>
</ol>

<ol>
	<li>Sauve, B. K. G., Walsh, G., Tokmakejian, S., Van Uum, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cortisol+in+human+hair+as+a+biomarker+of+systemic+exposure.">Measurement of cortisol in human hair as a biomarker of systemic exposure.</a> Clin Invest Med. 30 (5), 183-191 (2007).</li><li>Wright, K. D., Hickman, R., Laudenslager, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+cortisol+analysis:+a+promising+biomarker+of+HPA+activation+in+older+adults.">Hair cortisol analysis: a promising biomarker of HPA activation in older adults.</a> Gerontologist. 55, 140-145 (2015).</li><li>Awad, G. H., 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=Beauty+and+body+image+concerns+among+African+American+college+women.">Beauty and body image concerns among African American college women.</a> J Black Psychol. 41 (6), 540-564 (2015).</li><li>Capodilupo, C. M., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+and+race+matter:+the+importance+of+considering+intersections+in+Black+women's+body+image.">Gender and race matter: the importance of considering intersections in Black women's body image.</a> J Couns Psychol. 61 (1), 37-49 (2014).</li><li>Miracle, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Belmont+Report:+the+triple+crown+of+research+ethics.">The Belmont Report: the triple crown of research ethics.</a> Dimens Crit Care Nurs. 35 (4), 223-228 (2016).</li><li>Fishman, B. M., Bobo, L., Kosub, K., Womeodu, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultural+issues+in+serving+minority+populations:+emphasis+on+Mexican+Americans+and+African+Americans.">Cultural issues in serving minority populations: emphasis on Mexican Americans and African Americans.</a> Am J Med Sci. 306 (3), 160-166 (1993).</li><li>Webb, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Louisiana+voodoo+and+superstitions+related+to+health.">Louisiana voodoo and superstitions related to health.</a> HSMHA Health Rep. 86 (4), 291-301 (1971).</li><li>Ford, J. L., Boch, S. J., McCarthy, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+hair+collection+for+cortisol+measurement+in+population+research+on+adolescent+health.">Feasibility of hair collection for cortisol measurement in population research on adolescent health.</a> Nurs Res. 65 (3), 249-255 (2016).</li><li>Wosu, A. C., 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=Hair+cortisol+in+relation+to+sociodemographic+and+lifestyle+characteristics+in+a+multiethnic+US+sample.">Hair cortisol in relation to sociodemographic and lifestyle characteristics in a multiethnic US sample.</a> Ann Epidemiol. 25 (2), 90-95 (2015).</li><li>Webb, E., 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=Assessing+individual+systemic+stress+through+cortisol+analysis+of+archaeological+hair.">Assessing individual systemic stress through cortisol analysis of archaeological hair.</a> J Archaeolog Sci. 37 (4), 807-812 (2010).</li><li>Russell, E., 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=Toward+standardization+of+hair+cortisol+measurement:+results+of+the+first+international+interlaboratory+round+robin.">Toward standardization of hair cortisol measurement: results of the first international interlaboratory round robin.</a> Ther Drug Monit. 37 (1), 71-75 (2015).</li><li>Bosley, R. E., Daveluy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+to+natural+hair+care+practices+in+black+patients.">A primer to natural hair care practices in black patients.</a> Cutis. 95 (2), 78-80 (2015).</li><li>Loussouarn, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=African+hair+growth+parameters.">African hair growth parameters.</a> Brit J Derm. 145 (2), 294-297 (2001).</li><li>Hoffman, M. C., Karban, L. V., Goodteacher, A., Laudenslager, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+processing+and+shampooing+impact+cortisol+measured+in+human+hair.">Chemical processing and shampooing impact cortisol measured in human hair.</a> Clin Invest Med. 37 (4), 252-257 (2014).</li><li>Ramirez, J., Elmofty, M., Castillo, E., DeRouen, M., Shariff-Marco, S., Allen, L., 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=Evaluation+of+cortisol+and+telomere+length+measurements+in+ethnically+diverse+women+with+breast+cancer+using+culturally+sensitive+methods.">Evaluation of cortisol and telomere length measurements in ethnically diverse women with breast cancer using culturally sensitive methods.</a> J Community Genet. 8, 75-86 (2017).</li></ol>

The Behavioral Immunology and Endocrinology Laboratory is designated as a Laboratory of Excellence by Salimetrics, LLC.

Essential steps in the hair collection protocol include providing an example of how much hair will be taken, showing the sample site (posterior vortex), use of the single strain twist and gently pull method, and securing the sample with painter&#39;s tape away from the scalp end. By gently twisting and pulling the sample, the curly hair is lengthened and the scalp end alignment is maintained. This reduces laboratory time needed to align the scalp ends, measure, and cut the sample. Although thinning shears have been used ...

Collecting samples of saliva, blood, or urine for cortisol analysis has unique procedural challenges and greater participant burden, as the procedure requires multiple saliva samples, venipuncture samples, or 24-hour urine collection. Obtaining cortisol from hair samples is a simple and acceptable alternative to using saliva, serum, or urine samples1,2. Hair cortisol provides a retrospective representation of hypothalamic-pituitary-adrenal (HPA) axis function over time. Approximately 3 cm of hair collected from the scalp is equal to average cortisol release for the past 3 months1. However, researchers encounter unique challenges when attempting to collect hair samples across diverse populations. Such challenges include religious or spiritual beliefs including exposing hair (e.g., Muslim women), hair as sacred (American Indian groups), or cultural norms that connect hair to standards of beauty and self-image.
In the African American culture, particularly among women, scalp hair is a very personal and important aspect of their body image3,4. Further, historical experiences (e.g., the Tuskegee experiments) continue to foster mistrust in the research process5. Some African American folklore and cultural beliefs also suggest that the hair sample could be used in rituals that could cause harm6,7. Researchers should be aware of potential challenges that they may face in studies when African American participants are asked to provide hair samples.
In addition to issues surrounding body image, the specific techniques for collecting and handling hair samples from African Americans may differ from that of European Americans6,7. For example, African Americans may have straight, thin, curly, kinky, coarse, or a combination of these hair types, each requiring different techniques to collect and secure adequate hair samples for processing. Recent studies on samples of African American teens8, as well as multi-ethnic samples9, showed that collection of hair for cortisol level measurement was acceptable among African American participants. At present, there are no visual training procedures designed to provide researchers with the skill and cultural knowledge to collect hair samples in African Americans. The purpose of this paper is to present a culturally informed protocol to help researchers obtain adequate hair samples from African American participants. This method was developed with careful consideration of physical, psychosocial, and cultural characteristics relevant to collection of hair samples from African American adults. To date, there are no visual methods of collecting these samples, and this protocol will be the first guide that details proper hair sample collection from African American adults. To illustrate the representative results of the protocol implementation, de-identified data are presented from African Americans that participated in a community-based study on chronic stress that were aged 65 and older. Both male and female research assistants from a variety of racial backgrounds contacted potential participants from a clinic generated list and flyers. The primary investigator, who is an African American woman, spoke at community centers and senior subsidized housing apartments to older adult African Americans about the study, including a description of the hair collection methods.

<table><tbody><tr><td>&amp;nbsp;Salon grade scissors</td><td>Cricket</td><td>S-3 550</td><td>Carded Professional Hair Shear, 5.5 inch</td></tr><tr><td>Large tooth comb</td><td>Goody</td><td>No catalog number__Goody Stylista Rake Comb&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Link to Company website&amp;nbsp; http://www.goody.com/Products/Styling_Tools/Goody_Stylista_Rake_Comb?colId=d11c04a1-1dd3-4848-a313-046fc6c109ec&amp;amp;c_id=35a36c88-d359-4104-a53c-dfbfd8452780</td></tr><tr><td>Small tooth comb</td><td>Goody</td><td>No catalog number_ Goody Start.Style.Finish. Link to company website http://www.goody.com/Products/Styling_Tools/Goody_StartStyleFinish_Combs?colId=d11c04a1-1dd3-4848-a313-046fc6c109ec&amp;amp;c_id=35a36c88-d359-4104-a53c-dfbfd8452780</td><td></td></tr><tr><td>Alcohol preps</td><td>Kendall Webcol Alcohol Preps</td><td>No catalog number. Kendall Webcol Alcohol Preps</td><td>https://www.buyemp.com/product/kendall-webcol-alcohol-preps</td></tr><tr><td>Painter&#039;s tape</td><td>3M Scotch</td><td>ScotchBlue ORIGINAL Painter&amp;rsquo;s Tape</td><td>https://www.scotchblue.com/3M/en_US/scotchblue/products/painters-tape/</td></tr><tr><td>Permanent marker</td><td>Sharpie</td><td>Black Sharpie Fine Point</td><td>http://www.sharpie.com/sharpie/fine/SHFineBlackFine.html</td></tr><tr><td>Non-latex gloves</td><td>Prot&amp;eacute;g&amp;eacute;</td><td>93997</td><td>Protege&amp;reg;Nitrile Powder-Free Examination Gloves http://aureliagloves.com/product/protege/</td></tr><tr><td>Hair clips&amp;nbsp;</td><td>Goody</td><td>Goody Start.Style.Finish. Curl Clips</td><td>http://www.goody.com/Products/Styling_Tools/Goody_StartStyleFinish_Curl_Clips</td></tr><tr><td>Envelope</td><td>Office Depo</td><td>330808</td><td>Office Depot&amp;reg; Brand Clasp Envelopes, 9&quot; x 12&quot;, Brown, Box Of 100</td></tr><tr><td>Foil sheets&amp;nbsp;</td><td>Choice&amp;nbsp;</td><td>1229105</td><td>Box of 500 Sheets Food Service Interfolded Pop-up foil sheets&amp;nbsp;</td></tr><tr><td>Pre-weighed 2 ml cryovial</td><td>Wheaton, Millville, NJ, USA</td><td>W985902</td><td></td></tr><tr><td>100% isopropanol</td><td>Spectrum</td><td>I1275 | 67-63-0</td><td></td></tr><tr><td>Liquid nitrogen&amp;nbsp;</td><td>Ohio State Unversity Stores</td><td>Gas Cylinder Warehouse</td><td>http://osustores.osu.edu/prodservices/liquidnitrogen.aspx</td></tr><tr><td>Stainless steel ball bearing&amp;nbsp;</td><td>McMasterCarr</td><td>9291K42</td><td>https://www.mcmaster.com/#standard-stainless-steel-balls/=1acqby0</td></tr><tr><td>Specially milled aluminum cassettes</td><td>FisherScientific&amp;nbsp;</td><td>Retsch 20.745.0001</td><td></td></tr><tr><td>HPLC grade methanol</td><td>FisherScientific&amp;nbsp;</td><td>A452N1-19</td><td>https://www.fishersci.com/shop/products/methanol-hplc-fisher-chemical-6/p-216129</td></tr><tr><td>Microcentrifuge tube</td><td>Eppendorf North America</td><td>22363352</td><td>https://online-shop.eppendorf.us/US-en/Laboratory-Consumables-44512/Tubes-44515/Eppendorf-Safe-Lock-Tubes-PF-8863.html?_ga=2.36544352.1776616592.1511283212-1254533852.1511283212</td></tr><tr><td>Cortisol ELISA Kit</td><td>Salimetrics LLC, State College, PA, USA</td><td>1-3002 (5PK 1-3002-5)</td><td>https://www.salimetrics.com/assay-kit/cortisol-salivary-elisa-eia-kit</td></tr><tr><td>Centtrifuge for the micorcentrifuge tube</td><td>Beckman Coulter</td><td>Microfuge 20 series</td><td>http://centrifugebybeckman.com/?page_id=727/?pi_ad_id=creative?utm_term=microfuge</td></tr></tbody></table>

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.

In our study, twenty-one African Americans (mean age = 75; SD = 6.9; 85% female) consented to participate. Four participants (19%) refused to provide a hair sample and two (9.5%) could not provide a sample because their hair was too short. A total of 15 hair samples were obtained and processed (Table 2). The length of the hair samples that were processed ranged from 1 cm to 4.8 cm. Technical problems included samples were poorly aligned at the scalp end, tangled, or curly...

Watch this Scientific Journal Video about Collecting Hair Samples for Hair Cortisol Analysis in African Americans at JoVE.com

Collecting Hair Samples for Hair Cortisol Analysis in African Americans | Medicine | Video

Collecting Hair Samples for Hair Cortisol Analysis in 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, ...

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17.9K Views.  The Ohio State University. Cortisol concentratie haaranalyse biedt een alternatief voor traditionele maatregelen van cortisol; echter, om te verzamelen haar monsters van Afro-Amerikanen, wetenschappers moeten cultureel geïnformeerd en bevoegde. Het doel van dit protocol is om aan te tonen van een cultureel geïnformeerd techniek om te verzamelen haar monsters voor cortisol analyse van Afro-Amerikanen.

Video: Collecting Hair Samples for Hair Cortisol Analysis in African Americans

A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the biological output produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis in humans. While studies have explored how modifications to the TSST influence stress responsivity, few studies have created a modified TSST appropriate for vulnerable youth that elicits a significant cortisol stress response. Thus, the current study sought to modify or adjust the TSST in a culturally sensitive way for a vulnerable sample of 14 year-old adolescents. The present study took place within the context of a longitudinal birth cohort study of Mexican American families in California called the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS). The CHAMACOS sample was optimal to test the effectiveness of a modified culturally appropriate TSST, as it is comprised of Mexican American youth, who are often excluded from research. These youths also have experienced significant early life adversity. Example modifications included timed prompts, alternative math tasks, use of same-ethnicity peers as confederates, debriefing immediately after the conclusion of the TSST, and using an unknown youth examiner to administer the debrief. Saliva samples were collected at baseline (after a resting phase), and then again at 15, 30, and 45 min post-TSST onset to assess cortisol concentration. A pilot study of 50 participants (50% female) have been analyzed for cortisol reaction to the TSST. Results confirmed that this modified version of the TSST was successful at eliciting a significant cortisol reaction, with a wide range of variability likely due to individual differences. Goals for modifications and ethnicity considerations are discussed. This study provides the foundation for future research to utilize a modified TSST with vulnerable youth.

Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the ...

Developmental Biology

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. ...

Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial species, the koala (Phascolarctos cinereus), faces prolonged exposure to anthropogenic-induced stressors and assessment of chronic stress in wild populations is urgently warranted. One of the most effective ways to measure chronic stress is through analyzing the glucocorticoid hormone cortisol in hair or fur, as it supports physiological and behavioral responses. This laboratory validation study aims to test current techniques to validate an optimal hormone extraction method to be used as a non-invasive measure of cortisol in koala fur. It is recognized that using non-invasive techniques to measure stress hormones is preferred over traditional, invasive techniques due to their ideal practical and ethical standpoints. Additionally, it is comparatively easier to acquire fur from koalas than it is to acquire samples of their blood. This study used samples of koala fur acquired from the Adelaide Koala and Wildlife Hospital to run a number of hormone extraction techniques in an attempt to validate an optimal cortisol extraction method. Results showed that 100% methanol provided the most optimal solvent extraction compared to 100% ethanol or 100% isopropanol based on parallelism results. In conclusion, this method of cortisol extraction from koala fur provided a reliable non-invasive assay that could be used to study chronic stress in koalas.

Cortisol Measurement in Koala Phascolarctos cinereus Fur

We present a protocol to determine the optimal extraction solvent to measure cortisol from koala fur. The solvents used in this protocol are methanol, ethanol and isopropanol. Determining an optimal extraction solvent will aid in reliably measuring fur to determine the impact of chronic stress on koalas.

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial ...

Biochemistry

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) and quantify any differences in fin cortisol levels among three main sturgeon species. Fins were harvested from 19 sacrificed sturgeons including seven beluga (Huso huso), seven Siberian (Acipenser baerii), and five sevruga (A. stellatus). The sturgeons were raised in Iranian farms for 2 years (2017-2018), and cortisol extraction analysis was conducted in South Korea (January-February 2019). Jawbones from five H. huso were also used for cortisol extraction. Data were analyzed using the general linear model (GLM) procedure in the SAS environment. The intra- and inter-assay coefficients of variation were 14.15 and 7.70, respectively. Briefly, the cortisol extraction technique involved washing the samples (300 &#177; 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80&#160;rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 &#176;C) for 7 days, further drying the samples using a bead beater at 50 Hz for 32 min and grinding them into powder, applying 1.5 mL methanol to the dried powder (75 &#177; 5 mg), and slow rotation (40 rpm) for 18 h at room temperature with continuous mixing. Following extraction, samples were centrifuged (9,500 x g for 10 min), and 1 mL supernatant was transferred into a new microcentrifuge tube (1.5 mL), incubated at 38 &#176;C to evaporate the methanol, and analyzed via enzyme-linked immunosorbent assay (ELISA). No differences were observed in fin cortisol levels among species or in fin and jawbone cortisol levels between washing solvents. The results of this study demonstrate that the sturgeon jawbone matrix is a promising alternative stress indicator to solid matrices.

In this study, we present a protocol for cortisol extraction from the fin and jawbone of sturgeon species. Fin and jawbone cortisol levels were further examined by comparing two washing solvents followed by ELISA assays. This study piloted the feasibility of jawbone cortisol as a novel stress indicator.

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) ...

Environment

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.

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 ...

Providing Meaningful Environmental Enrichment and Measuring Saliva Cortisol in Pigs Housed on Slatted Flooring

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a substantial pressure coming from customers, legislators and other stakeholders, to make management changes to improve the well-being of these animals. Several studies have demonstrated that providing environmental enrichment to pigs allows them to express their natural behavior, such as rooting and exploring, as well as nesting prior to farrowing, and is associated with reduced stress and improved production and welfare. However, many considerations should be taken into account when providing environmental enrichment, such as the type of floor, drainage and sewage systems, the pigs&#39; stage in life, the material, as well as its hanging method, height and location within the pen. The objectives of this paper are (1) to give methodologic information on how to provide a relatively simple and practical meaningful environmental enrichment for pigs which are housed on slatted floors during the different stages of their life, and (2) to demonstrate how to collect saliva samples for the measurement of cortisol concentrations, as a biomarker for acute stress. Protocols include information regarding the use of jute, cotton ropes, straw in racks, as well as chewable silicone sticks devices as environmental enrichment in pens of farrowing and lactation, weaners and finishers. In addition, the use of cotton rope for a non-invasive saliva samples collection for cortisol concentrations analysis is detailed. The protocols provided are relevant for professionals aiming to improve and monitor animal welfare, in both research and industrial swine farming.

This protocol demonstrates how to provide practical meaningful environmental enrichment for pigs which are housed on slatted flooring during the different stages of their lives, and how to collect saliva samples in a non-invasive manner for the measurement of cortisol concentrations, as a biomarker for acute stress.

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a ...

Behavior

Human Circadian Phenotyping and Diurnal Performance Testing in the Real World

In our continuously developing &#39;around the clock&#39; society, there is a need to increase our understanding of how changes in biology, physiology and psychology influence our health and performance. Embedded within this challenge, is the increasing need to account for individual differences in sleep and circadian rhythms, as well as to explore the impact of time of day on performance in the real world. There are a number of ways to measure sleep and circadian rhythms from subjective questionnaire-based methods to objective sleep/wake monitoring, actigraphy and analysis of biological samples. This paper proposes a protocol that combines multiple techniques to categorize individuals into Early, Intermediate or Late circadian phenotype groups (ECPs/ICPs/LCPs) and recommends how to conduct diurnal performance testing in the field. Representative results show large differences in rest-activity patterns derived from actigraphy, circadian phase (dim light melatonin onset and peak time of cortisol awakening response) between circadian phenotypes. In addition, significant differences in diurnal performance rhythms between ECPs and LCPs emphasizes the need to account for circadian phenotype. In summary, despite the difficulties in controlling influencing factors, this protocol allows a real-world assessment of the impact of circadian phenotype on performance. This paper presents a simple method to assess circadian phenotype in the field and supports the need to consider time of day when designing performance studies.

Here, we present a method to investigate diurnal rhythms in performance following accurate categorization of participants into circadian phenotype groups based on the Munich ChronoType Questionnaire, gold standard circadian phase biomarkers and actigraphic measures.

In our continuously developing &#39;around the clock&#39; society, there is a need to increase our understanding of how changes in biology, physiology and ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Collecting Hair Samples for Hair Cortisol Analysis in African Americans

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