Name: collecting hair samples for hair cortisol analysis in african americans
Uploaded: 2018-06-10T14:00:00
Description: Watch this Scientific Journal Video about Collecting Hair Samples for Hair Cortisol Analysis in African Americans at JoVE.com

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 ...

<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>

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.

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Salon grade scissors</td>
			<td style="width:232px;">Cricket</td>
			<td style="width:312px;">S-3 550</td>
			<td style="width:479px;">Carded Professional Hair Shear, 5.5 inch</td>
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			<td height="42" style="height:42px;width:216px;">Large tooth comb</td>
			<td style="width:232px;">Goody</td>
			<td style="width:312px;">No catalog number__Goody Stylista Rake Comb&#160;&#160;&#160;</td>
			<td>Link to Company website&#160; http://www.goody.com/Products/Styling_Tools/Goody_Stylista_Rake_Comb?colId=d11c04a1-1dd3-4848-a313-046fc6c109ec&#38;c_id=35a36c88-d359-4104-a53c-dfbfd8452780</td>
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			<td height="21" style="height:21px;width:216px;">Small tooth comb</td>
			<td style="width:232px;">Goody</td>
			<td colspan="2">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&#38;c_id=35a36c88-d359-4104-a53c-dfbfd8452780</td>
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			<td height="42" style="height:42px;width:216px;">Alcohol preps</td>
			<td style="width:232px;">Kendall Webcol Alcohol Preps</td>
			<td style="width:312px;">No catalog number. Kendall Webcol Alcohol Preps</td>
			<td>https://www.buyemp.com/product/kendall-webcol-alcohol-preps</td>
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			<td height="21" style="height:21px;width:216px;">Painter&#39;s tape</td>
			<td style="width:232px;">3M Scotch</td>
			<td>ScotchBlue ORIGINAL Painter&#8217;s Tape</td>
			<td>https://www.scotchblue.com/3M/en_US/scotchblue/products/painters-tape/</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Permanent marker</td>
			<td style="width:232px;">Sharpie</td>
			<td style="width:312px;">Black Sharpie Fine Point</td>
			<td>http://www.sharpie.com/sharpie/fine/SHFineBlackFine.html</td>
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			<td height="42" style="height:42px;width:216px;">Non-latex gloves</td>
			<td style="width:232px;">Prot&#233;g&#233;</td>
			<td style="width:312px;">93997</td>
			<td style="width:479px;">Protege&#174;Nitrile Powder-Free Examination Gloves http://aureliagloves.com/product/protege/</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hair clips&#160;</td>
			<td style="width:232px;">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 height="21">
			<td height="21" style="height:21px;width:216px;">Envelope</td>
			<td style="width:232px;">Office Depo</td>
			<td>330808</td>
			<td>Office Depot&#174; Brand Clasp Envelopes, 9&#34; x 12&#34;, Brown, Box Of 100</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Foil sheets&#160;</td>
			<td style="width:232px;">Choice&#160;</td>
			<td>1229105</td>
			<td>Box of 500 Sheets Food Service Interfolded Pop-up foil sheets&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pre-weighed 2 ml cryovial</td>
			<td>Wheaton, Millville, NJ, USA</td>
			<td>W985902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% isopropanol</td>
			<td style="width:232px;">Spectrum</td>
			<td style="width:312px;">I1275 | 67-63-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid nitrogen&#160;</td>
			<td style="width:232px;">Ohio State Unversity Stores</td>
			<td>Gas Cylinder Warehouse</td>
			<td>http://osustores.osu.edu/prodservices/liquidnitrogen.aspx</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel ball bearing&#160;</td>
			<td>McMasterCarr</td>
			<td>9291K42</td>
			<td>https://www.mcmaster.com/#standard-stainless-steel-balls/=1acqby0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Specially milled aluminum cassettes</td>
			<td style="width:232px;">FisherScientific&#160;</td>
			<td style="width:312px;">Retsch 20.745.0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HPLC grade methanol</td>
			<td style="width:232px;">FisherScientific&#160;</td>
			<td style="width:312px;">A452N1-19</td>
			<td>https://www.fishersci.com/shop/products/methanol-hplc-fisher-chemical-6/p-216129</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tube</td>
			<td style="width:232px;">Eppendorf North America</td>
			<td style="width:312px;">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 height="21">
			<td height="21" style="height:21px;">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 height="42">
			<td height="42" style="height:42px;width:216px;">Centtrifuge for the micorcentrifuge tube</td>
			<td style="width:232px;">Beckman Coulter</td>
			<td style="width:312px;">Microfuge 20 series</td>
			<td>http://centrifugebybeckman.com/?page_id=727/?pi_ad_id=creative?utm_term=microfuge</td>
		</tr>
	</tbody>
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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

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, ...

This method can help researchers acquire adequate hair samples in a culturally sensitive manner. The main advantages of this technique are its culturally informed approach and the twist and roll maneuver that is used to secure the samples. Implementation of this technique will help increase the participation of African American subjects in studies about the influence of longterm cortisol exposure and health outcomes.Our protocol takes into account a careful consideration of physical, psychosocial, and cultural characteristics relevant to the collection of hair samples from the African American adults. Visual demonstration of this method is critical for appreciation of the importance of hair in the African American community and to provide the necessary steps for improving the participants experience. Before collecting the sample, explain to the participant that the sample collected is less hair than is lost in normal everyday brushing from the back of the head.Inform the participant how the site for the sampling is hidden by the surrounding hair and therefore not visible after collection, and that the sample will be used to measure a hormone called cortisol that is present in the hair. Show the participant a sample photo to illustrate the amount of hair that will be collected. Then, ask the participant the hair care practice questions listed in the table.Next, escort the participant to a private area. Don a pair of non-latex gloves, and clean a pair of salon grade scissors and a large and small tooth parting comb with an alcohol prep pad. Gently pull the hair in the sample taut to allow measurement from the scalp to the ends of the strands.Use the comb to part the hair horizontally along the posterior vertex of the scalp between the tips of the ears, and use a clip to secure the hair from the top of the part. Then, place another clip at the bottom of the part to expose a five by 10 centimeter rectangle between the two clips of loose hair, and ask the participant if they prefer a wide tooth or a narrow tooth comb. After combing the hair rectangle, ask the participant how they would like to have the remaining hair in the comb discarded.And, grasp approximately 30 to 50 strands of hair to the right of the five by 10 centimeter rectangle. Gently pull and twist the hair away from the scalp in a rolling motion between the fingers, and use the scissors to make a clean, straight cut with the root ends aligned carefully along the cut. Use painter's tape to secure the hair sample in the center of an eight by 10 inch piece of aluminum foil, and use a marker to label the scalp end of the sample.Fold the foil into a square so the hair cannot fall out of the foil, but without bending the hair. Place the foil into a business envelope. After sealing, use a premade barcode label to label the envelope with the participant's unique study identification number, and store the sample in a dry area at room temperature.If you do not have a premade barcode label, you can write the participant's unique id number, date, and hair sample on an envelope. The hair cortisol can then be analyzed according to standard protocols. 21 African Americans consented to participate in this representative study.Four participants ultimately refused to provide a hair sample, and two could not provide a sample because their hair was too short. A total of 15 usable hair samples with lengths ranging from one to 4.8 centimeters were obtained and processed from the other study participants. Technical problems included samples that were poorly aligned at the scalp end, too small of a sample, curly, or with painter's tape being over the scalp end.Once mastered, this technique can be completed in 15 minutes if performed properly. While attempting this procedure, it's important to remember to thank the participants and offer to give the participants any unused hair. Although thinning shears have been used in previous studies, shears are not the preferred tool for cutting hair because their use results in uneven scalp end alignment and an increased time for preparing the hair samples for analysis.This technique paved the way for researchers in the hair cortisol analysis field in obtaining culturally informed hair samples from African American study participants and in quantifying the potential influences of longterm cortisol exposure on health in African Americans. After watching this video, you should have a good understanding of how to approach and to collect adequate hair samples for cortisol concentration analysis from African American adults. Don't forget that working with salon grade scissors can be extremely sharp, so take care not to handle the blade ends of the scissors and not to press the scissors directly against the participant's scalp.

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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. ...

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 ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is ...

Capturing Oral Submucous Fibrosis Tissue Samples for Spatial-Omics Analysis

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

Author Spotlight: Unlocking the Mysteries of Oral Potential Malignancies 

Laser capture microdissection of oral submucous fibrosis tissues allows for precise extraction of cells from histological regions of interest for the analysis of multi-omics data with morphological and spatial information.

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina ...

Analyzing Tibetan Medicinal Plant APB Using 2D-HPLC-MS with Molecular Networking

2D-HPLC-MS Technology Combined with Molecular Network for the Identification of Components in Tibetan Medicine Aconitum pendulum

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate chemical composition of the Tibetan medicinal plant APB. Through the implementation of 2D-HPLC, enhanced separation of complex mixtures was achieved, enabling the isolation of individual compounds for subsequent analysis. The molecular network approach further aided in elucidating structural relationships among these compounds, contributing to the determination of potential bioactive molecules. This integrated strategy efficiently identified a wide array of chemical components present within the plant. The findings revealed a diverse spectrum of chemical constituents within APB, including alkaloids, among others. This research not only advances understanding of the phytochemical profile of this traditional Tibetan medicine but also provides valuable insights into its potential therapeutic properties. The integration of 2D-HPLC-MS and molecular network proves to be a powerful tool for systematically exploring and identifying complex chemical compositions in herbal medicines, paving the way for further research and development in the field of natural product discovery.

Author Spotlight: Integrating 2D-HPLC-MS and Molecular Networking in Natural Medicine Analysis

This study utilizes two-dimensional high-performance liquid chromatography-mass spectrometry (2D-HPLC-MS) technology in conjunction with molecular networking to unravel the intricate chemical composition of the Tibetan medicinal plant Aconitum pendulum Busch (APB). The article provides a detailed protocol for the systematic exploration and identification of complex chemical components of herbal medicines.

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate ...