Name: Through-the-Wall Blood Sampling Method to Minimize Sleep Disruption in Clinical Settings (Video) | JoVE Journal
Uploaded: 2025-06-13T14:00:00.000+00:00
Duration: 6 min 39 s
Description: Learn a through-the-wall blood sampling method to minimize sleep disruption in clinical research. This technique enables continuous 24-hour collection of metabolic markers without disturbing circadian rhythms.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This research was supported in part by T32HL149646 to GSM and R01DK125653 and R01HL168081 to JLB. We would also like to thank Stephanie Laing RN, BSN, a Professional Research Nurse with the Colorado Clinical &#38; Translational Sciences Institute (CCTSI) and the University of Colorado Anschutz Medical Campus Clinical Translational Research Center for reviewing the methods and providing insights and edits to the final version of this manuscript.

The methods described in this paper are used in protocols currently approved by the Institutional Review Board of Colorado State University and the Colorado Multiple Institutional Review Board. Representative data were derived from such studies for which written informed consent was obtained before participation. All supplies and equipment used are listed in the Table of Materials. Additional method considerations are presented in the discussion.
1. Setup and attachment of the extension line
NOTE: Prior to any clinical procedure, appropriate hand hygiene must be performed using a solution with a minimum of 70% isopropyl alcohol. Personal protective equipment (PPE), including but not limited to gloves, lab coats, and closed-toe shoes, must be worn at all times.
<ol>
	<li>Setup for through-the-wall sampling (Figure 1)
	<ol>
		<li>Outside of the participant room, set up the blood draw station with the following supplies: disposable absorbent pad, 3-way stopcock valve, 120 in extension line, 10 mL saline flush syringe, warmed 10 mL saline flush syringe in warming device, 5 mL syringe for waste, 3 mL syringe for blood collection (size may vary depending on blood draw volume), and a biohazard waste container. All supplies are listed in the Table of Materials.</li>
	</ol>
	</li>
	<li>Attach the extension line for through-the-wall sampling.
	<ol>
		<li>On the disposable absorbent pad, attach the 3-way stopcock to the female Luer lock side of the extension line. 
		NOTE: Ensure that the exposed end of the stopcock does not touch any surfaces (including gloved hands). If contaminated, replace the stopcock to ensure sterility.</li>
		<li>Place the 10 mL saline flush and 3 mL sample syringe (with the seal broken) on the stopcock. 
		NOTE: Once the stopcock is attached to the extension line, the 2 remaining stopcock attachment sites are identical; therefore, the saline flush and sample syringes may be placed at either remaining stopcock attachment. Breaking the seal refers to pulling back on the syringe plunger to release before the syringe is attached to the participant, ensuring that the acute negative pressure from the seal break does not result in damage to the blood vessel.</li>
		<li>Twist the OFF tab of the stopcock so that the extension line is closed to the 3 mL sample syringe. This will open the stopcock to the 10 mL saline flush.</li>
		<li>Push saline through the extension line. As saline reaches the end of the line, carefully remove the cap and let the saline drip into a cup and/or onto the absorbent pad to ensure the line is completely filled with saline. Reattach the cap to the end of the now primed extension line.</li>
		<li>Twist the OFF tab of the stopcock so that the line is closed to both syringes. Leave the syringes attached to the stopcock. 
		NOTE: The maximum volume in the line is 3.4 mL.</li>
		<li>Feed the capped end of the extension line through the wall port to the participant room. Ensure that the cap remains in place to keep the line sterile.</li>
		<li>Move to the participant&#39;s room to attach the extension line to the participant. Clean the distal end of the extension line and the attachment site of the IV extension set (connected to the participant) with a sterile alcohol prep pad.</li>
		<li>Remove the cap on the extension line and attach the line to the IV extension set (connected to the participant).</li>
		<li>Once the extension line is connected to the IV, return to the antechamber.
		<ol>
			<li>Remove and discard the used saline flush from the stopcock and attach a warmed 10 mL saline flush. Twist the OFF tab of the stopcock to close the line to the sample syringe. This will open the stopcock to the new 10 mL saline flush.</li>
			<li>Push saline into the extension line to ensure no leaks and the IV is still patent. Twist the OFF tab of the stopcock so that the line is closed to both syringes. 
			NOTE: Saline flush must not exceed human body temperature (37 &#176;C/98.6 &#176;F). Verify saline temperature using a thermometer placed in the warming device. A minimum of 4.4 mL of saline is needed to flush the entire line as the volume in the extension line is 3.4 mL +, and the volume in the IV extension set is 1 mL.</li>
		</ol>
		</li>
		<li>Continue to slowly push the saline flush while twisting the OFF tab of the stopcock to close the line to both syringes. This will maintain pressure in the extension line and minimize the backflow of blood.</li>
		<li>Once the extension line is successfully established, return to the participant&#39;s room to wrap the IV extension set with a self-adherent, elastic bandage to ensure that it does not get pulled during sleep. Secure the extension line to the bed frame with tape.</li>
		<li>Discuss with the participant their sleeping preferences (i.e., side sleeper, back sleeper, etc.) and provide excess tubing to allow freedom of movement during sleep. Ask the participant to test the range of motion. 
		NOTE: It is important to remind the participant that they must request staff assistance to disconnect the extension line to use the restroom.</li>
	</ol>
	</li>
</ol>
2. Sampling blood through the extension line
<ol>
	<li>Drawing a blood sample.
	<ol>
		<li>Prior to any blood draws, ensure that the extension line is still primed with saline and confirm that the OFF tab of the stopcock is twisted such that the line is closed to both syringes.
		<ol>
			<li>If blood can be seen in the extension line, flush a minimum of 4.4 mL of warmed saline to clear the extension line and IV extension set of any blood.</li>
		</ol>
		</li>
		<li>Attach one 5 mL syringe (waste syringe) and one 3 mL syringe (sample syringe) to the stopcock. Twist the OFF tab of the stopcock to open the extension line to the 5 mL syringe (waste syringe). 
		NOTE: Before collecting a blood sample, it is necessary to draw and discard a &#34;waste&#34; syringe, which consists of the saline in the extension line and the IV extension set.</li>
		<li>Draw with the 5 mL syringe until whole blood begins to enter the syringe (following at least 4.4 mL of waste). 
		NOTE: The color of the liquid in the waste syringe will begin as clear, followed by pink, and finally dark red once the extension line is filled with whole blood.</li>
		<li>Once whole blood is visible in the 5 mL syringe, twist the OFF tab of the stopcock to close the line to the 5 mL waste syringe.</li>
		<li>Draw the desired amount of blood with the 3 mL syringe. Twist the OFF tab of the stopcock to close the extension line to both syringes. 
		NOTE: Blood must not remain in the extension line for longer than 5 min to reduce the risk of clotting. If the blood draw is slow, multiple smaller sampling syringes may be used to reduce pressure on the vein. Set a timer to determine the duration of the blood draw. At 5 min, flush the line to prevent clotting. A new draw may be attempted, or the draw may be skipped. While some protocols incorporate blood-sparing techniques, such as reinfusing the waste syringe to minimize blood loss, this protocol does not implement such measures.</li>
		<li>Remove the 5 mL waste syringe and replace it with a warm 10 mL saline flush.</li>
		<li>Twist the OFF tab on the stopcock to close the line to the empty 3 mL syringe. This will open the line to the saline flush.</li>
		<li>Push up to 10 mL of warmed saline, ensuring no blood remains in the extension line.</li>
		<li>Twist the OFF tab on the stopcock to close the line to both syringes.
		<ol>
			<li>Continue to slowly push the saline flush while twisting the OFF tab of the stopcock to close the line to both syringes. This will maintain pressure in the extension line and minimize the backflow of blood.</li>
			<li>Use any remaining saline in the syringe to flush through the stopcock into the empty 3 mL syringe. Clear the stopcock further of the saline by removing the saline syringe and pulling back on the 3 mL syringe to remove any remaining liquid in the stopcock.</li>
		</ol>
		</li>
		<li>Replace all syringes to prepare for the next draw. 
		NOTE: Do not leave stopcock ports without syringes attached, as this will compromise sterility.</li>
	</ol>
	</li>
</ol>

<ol><li>Cox, K. H., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circadian+clock+genes+and+the+transcriptional+architecture+of+the+clock+mechanism%5BTitle%5D)+AND+%22J+Mol+Endocrinol%22%5BJournal%5D)">Circadian clock genes and the transcriptional architecture of the clock mechanism.</a> J Mol Endocrinol. 63 (4), R93-R102 (2019).</li>
<li>Broussard, J. L., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBroussard%2C+JL+%22Biological+Timekeeping%3A+Clocks%2C+Rhythms+and+Behaviour%22">Biological Timekeeping: Clocks, Rhythms and Behaviour</a>. , Springer. India. (2017).</li>
<li>Chellappa, S. L., Vujovic, N., Williams, J. S., Scheer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+circadian+disruption+on+cardiovascular+function+and+disease%5BTitle%5D)+AND+%22Trends+Endocrinol+Metab%22%5BJournal%5D)">Impact of circadian disruption on cardiovascular function and disease.</a> Trends Endocrinol Metab. 30 (10), 767-779 (2019).</li>
<li>Broussard, J. L., Van Cauter, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disturbances+of+sleep+and+circadian+rhythms%3A+novel+risk+factors+for+obesity%5BTitle%5D)+AND+%22Curr+Opin+Endocrinol+Diabetes+Obes%22%5BJournal%5D)">Disturbances of sleep and circadian rhythms: novel risk factors for obesity.</a> Curr Opin Endocrinol Diabetes Obes. 23 (5), 353-359 (2016).</li>
<li>Hanlon, E., Van Cauter, E. V., Tasali, E., Broussard, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHanlon%2C+E+%22Endocrine+Physiology+in+Relation+to+Sleep+and+Sleep+Disturbances.+Principles+and+Pract+Sleep+Medicine%22">Endocrine Physiology in Relation to Sleep and Sleep Disturbances. Principles and Pract Sleep Medicine</a>. , Elsevier Saunders, MO. (2021).</li>
<li>Prasad, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+smartphone+intervention+to+promote+time+restricted+eating+reduces+body+weight+and+blood+pressure+in+adults+with+overweight+and+obesity%3A+a+pilot+study%5BTitle%5D)+AND+%22Nutrients%22%5BJournal%5D)">A smartphone intervention to promote time restricted eating reduces body weight and blood pressure in adults with overweight and obesity: a pilot study.</a> Nutrients. 13 (7), 2148(2021).</li>
<li>Procopio, S. B., Esser, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clockwork+conditioning%3A+aligning+the+skeletal+muscle+clock+with+time-of-day+exercise+for+cardiometabolic+health%5BTitle%5D)+AND+%22J+Mol+Cell+Cardiol%22%5BJournal%5D)">Clockwork conditioning: aligning the skeletal muscle clock with time-of-day exercise for cardiometabolic health.</a> J Mol Cell Cardiol. 198, 36-44 (2025).</li>
<li>Holtman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+circadian+timing+following+a+one-week+in-home+sleep+extension+manipulation+in+habitually+short-sleeping+adolescents%5BTitle%5D)+AND+%22Sleep%22%5BJournal%5D)">Changes in circadian timing following a one-week in-home sleep extension manipulation in habitually short-sleeping adolescents.</a> Sleep. 48 (2), zsae273(2024).</li>
<li>Chellappa, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Daytime+eating+prevents+internal+circadian+misalignment+and+glucose+intolerance+in+night+work%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">Daytime eating prevents internal circadian misalignment and glucose intolerance in night work.</a> Sci Adv. 7, eabg9910(2021).</li>
<li>Skene, D. J., Arendt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+circadian+rhythms%3A+physiological+and+therapeutic+relevance+of+light+and+melatonin%5BTitle%5D)+AND+%22Ann+Clin+Biochem%22%5BJournal%5D)">Human circadian rhythms: physiological and therapeutic relevance of light and melatonin.</a> Ann Clin Biochem. 43 (Pt 5), 344-353 (2006).</li>
<li>Powell, D. J., Schlotz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Daily+life+stress+and+the+cortisol+awakening+response%3A+testing+the+anticipation+hypothesis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Daily life stress and the cortisol awakening response: testing the anticipation hypothesis.</a> PLoS One. 7 (12), e52067(2012).</li>
<li>Wright, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+sleep+deprivation+and+circadian+misalignment+on+cortisol%2C+inflammatory+markers%2C+and+cytokine+balance%5BTitle%5D)+AND+%22Brain+Behav+Immun%22%5BJournal%5D)">Influence of sleep deprivation and circadian misalignment on cortisol, inflammatory markers, and cytokine balance.</a> Brain Behav Immun. 47, 24-34 (2015).</li>
<li>Ikegami, K., Refetoff, S., Van Cauter, E., Yoshimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interconnection+between+circadian+clocks+and+thyroid+function%5BTitle%5D)+AND+%22Nat+Rev+Endocrinol%22%5BJournal%5D)">Interconnection between circadian clocks and thyroid function.</a> Nat Rev Endocrinol. 15 (10), 590-600 (2019).</li>
<li>Kessler, L., Nedeltcheva, A., Imperial, J., Penev, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+serum+TSH+and+free+T4+during+human+sleep+restriction%5BTitle%5D)+AND+%22Sleep%22%5BJournal%5D)">Changes in serum TSH and free T4 during human sleep restriction.</a> Sleep. 33 (8), 1115-1118 (2010).</li>
<li>Van Cauter, E., Plat, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiology+of+growth+hormone+secretion+during+sleep%5BTitle%5D)+AND+%22J+Pediatr%22%5BJournal%5D)">Physiology of growth hormone secretion during sleep.</a> J Pediatr. 128 (5 Pt 2), S32-S37 (1996).</li>
<li>Morselli, L., Leproult, R., Balbo, M., Spiegel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+sleep+duration+in+the+regulation+of+glucose+metabolism+and+appetite%5BTitle%5D)+AND+%22Best+Pract+Res+Clin+Endocrinol+Metab%22%5BJournal%5D)">Role of sleep duration in the regulation of glucose metabolism and appetite.</a> Best Pract Res Clin Endocrinol Metab. 24 (5), 687-702 (2010).</li>
<li>Kapcala, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Twenty-four-hour+prolactin+secretory+patterns+in+women+with+galactorrhea%2C+normal+menses%2C+normal+random+prolactin+levels+and+abnormal+sellar+tomograms%5BTitle%5D)+AND+%22J+Endocrinol+Invest%22%5BJournal%5D)">Twenty-four-hour prolactin secretory patterns in women with galactorrhea, normal menses, normal random prolactin levels and abnormal sellar tomograms.</a> J Endocrinol Invest. 7 (5), 455-460 (1984).</li>
<li>Sassin, J. F., Frantz, A. G., Kapen, S., Weitzman, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+nocturnal+rise+of+human+prolactin+is+dependent+on+sleep%5BTitle%5D)+AND+%22J+Clin+Endocrinol+Metab%22%5BJournal%5D)">The nocturnal rise of human prolactin is dependent on sleep.</a> J Clin Endocrinol Metab. 37 (3), 436-440 (1973).</li>
<li>Chaput, J. -P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+insufficient+sleep+and+circadian+misalignment+in+obesity%5BTitle%5D)+AND+%22Nat+Rev+Endocrinol%22%5BJournal%5D)">The role of insufficient sleep and circadian misalignment in obesity.</a> Nat Rev Endocrinol. 19 (2), 82-97 (2023).</li>
<li>Broussard, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sleep+restriction+increases+free+fatty+acids+in+healthy+men%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">Sleep restriction increases free fatty acids in healthy men.</a> Diabetologia. 58 (4), 791-798 (2015).</li>
<li>Vaughn, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endocannabinoid+signalling%3A+has+it+got+rhythm%5BTitle%5D)+AND+%22Br+J+Pharmacol%22%5BJournal%5D)">Endocannabinoid signalling: has it got rhythm.</a> Br J Pharmacol. 160 (3), 530-543 (2010).</li>
<li>Lindgren, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+islet+and+incretin+hormone+responses+in+morning+versus+afternoon+after+standardized+meal+in+healthy+men%5BTitle%5D)+AND+%22J+Clin+Endocrinol+Metab%22%5BJournal%5D)">Differential islet and incretin hormone responses in morning versus afternoon after standardized meal in healthy men.</a> J Clin Endocrinol Metab. 94 (8), 2887-2892 (2009).</li>
<li>Wesselius, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quality+and+quantity+of+sleep+and+factors+associated+with+sleep+disturbance+in+hospitalized+patients%5BTitle%5D)+AND+%22JAMA+Intern+Med%22%5BJournal%5D)">Quality and quantity of sleep and factors associated with sleep disturbance in hospitalized patients.</a> JAMA Intern Med. 178 (9), 1201-1208 (2018).</li>
<li>Markwald, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+insufficient+sleep+on+total+daily+energy+expenditure%2C+food+intake%2C+and+weight+gain%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Impact of insufficient sleep on total daily energy expenditure, food intake, and weight gain.</a> Proc Natl Acad Sci U S A. 110 (14), 5695-5700 (2013).</li>
<li>McHill, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+circadian+misalignment+on+energy+metabolism+during+simulated+nightshift+work%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Impact of circadian misalignment on energy metabolism during simulated nightshift work.</a> Proc Natl Acad Sci U S A. 111 (48), 17302-17307 (2014).</li>
<li>Depner, C. M., Melanson, E. L., McHill, A. W., Wright, K. P. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mistimed+food+intake+and+sleep+alters+24-hour+time-of-day+patterns+of+the+human+plasma+proteome%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Mistimed food intake and sleep alters 24-hour time-of-day patterns of the human plasma proteome.</a> Proc Natl Acad Sci U S A. 115 (23), E5390-E5399 (2018).</li>
<li>Mullington, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sleep+loss+reduces+diurnal+rhythm+amplitude+of+leptin+in+healthy+men%5BTitle%5D)+AND+%22J+Neuroendocrinol%22%5BJournal%5D)">Sleep loss reduces diurnal rhythm amplitude of leptin in healthy men.</a> J Neuroendocrinol. 15 (9), 851-854 (2003).</li>
<li>Ona, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+technique+for+improved+blood+sampling+during+sleep+studies%5BTitle%5D)+AND+%22Psychophysiology%22%5BJournal%5D)">A technique for improved blood sampling during sleep studies.</a> Psychophysiology. 31 (6), 609-610 (1994).</li>
<li>Schlierf, G., Dorow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diurnal+patterns+of+triglycerides%2C+free+fatty+acids%2C+blood+sugar%2C+and+insulin+during+carbohydrate-induction+in+man+and+their+modification+by+nocturnal+suppression+of+lipolysis%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Diurnal patterns of triglycerides, free fatty acids, blood sugar, and insulin during carbohydrate-induction in man and their modification by nocturnal suppression of lipolysis.</a> J Clin Invest. 52 (3), 732-740 (1973).</li>
<li>Schlierf, G., Reinheimer, W., Stossberg, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diurnal+patterns+of+plasma+triglycerides+and+free+fatty+acids+in+normal+subjects+and+in+patients+with+endogenous+%28type+IV%29+hyperlipoproteinemia%5BTitle%5D)+AND+%22Nutr+Metab%22%5BJournal%5D)">Diurnal patterns of plasma triglycerides and free fatty acids in normal subjects and in patients with endogenous (type IV) hyperlipoproteinemia.</a> Nutr Metab. 13 (2), 80-91 (1971).</li>
<li>Swanson, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((turnover+markers+after+sleep+restriction+and+circadian+disruption%3A+a+mechanism+for+sleep-related+bone+loss+in+humans%5BTitle%5D)+AND+%22J+Clin+Endocrinol+Metab%22%5BJournal%5D)">turnover markers after sleep restriction and circadian disruption: a mechanism for sleep-related bone loss in humans.</a> J Clin Endocrinol Metab. 102 (10), 3722-3730 (2017).</li>
<li>Van Cauter, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+glucose+regulation+and+insulin+secretion+by+circadian+rhythmicity+and+sleep%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Modulation of glucose regulation and insulin secretion by circadian rhythmicity and sleep.</a> J Clin Invest. 88 (3), 934-942 (1991).</li>
<li>Rynders, C. A., Morton, S. J., Bessesen, D. H., Wright, K. P. Jr, Broussard, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circadian+rhythm+of+substrate+oxidation+and+hormonal+regulators+of+energy+balance%5BTitle%5D)+AND+%22Obesity+%28Silver+Spring%29%22%5BJournal%5D)">Circadian rhythm of substrate oxidation and hormonal regulators of energy balance.</a> Obesity (Silver Spring). 28 (Suppl 1), S104-S113 (2020).</li>
<li>Thosar, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circadian+rhythm+of+vascular+function+in+midlife+adults%5BTitle%5D)+AND+%22Arterioscler+Thromb+Vasc+Biol%22%5BJournal%5D)">Circadian rhythm of vascular function in midlife adults.</a> Arterioscler Thromb Vasc Biol. 39 (6), 1203-1211 (2019).</li>
<li>Thosar, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morning+impairment+in+vascular+function+is+unrelated+to+overnight+sleep+or+the+inactivity+that+accompanies+sleep%5BTitle%5D)+AND+%22Am+J+Physiol+Regul+Integr+Comp+Physiol%22%5BJournal%5D)">Morning impairment in vascular function is unrelated to overnight sleep or the inactivity that accompanies sleep.</a> Am J Physiol Regul Integr Comp Physiol. 315 (5), R986-R993 (2018).</li>
<li>Dimsdale, J. E., Coy, T., Ziegler, M. G., Ancoli-Israel, S., Clausen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+sleep+apnea+on+plasma+and+urinary+catecholamines%5BTitle%5D)+AND+%22Sleep%22%5BJournal%5D)">The effect of sleep apnea on plasma and urinary catecholamines.</a> Sleep. 18 (5), 377-381 (1995).</li>
<li>Klerman, E. B., Gershengorn, H. B., Duffy, J. F., Kronauer, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparisons+of+the+variability+of+three+markers+of+the+human+circadian+pacemaker%5BTitle%5D)+AND+%22J+Biol+Rhythms%22%5BJournal%5D)">Comparisons of the variability of three markers of the human circadian pacemaker.</a> J Biol Rhythms. 17 (2), 181-193 (2002).</li>
<li>Benloucif, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measuring+melatonin+in+humans%5BTitle%5D)+AND+%22J+Clin+Sleep+Med%22%5BJournal%5D)">Measuring melatonin in humans.</a> J Clin Sleep Med. 4 (1), 66-69 (2008).</li>
<li>Lammers, W. J., Badia, P., Hughes, R., Harsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temperature%2C+time-of-night+of+testing%2C+and+responsiveness+to+stimuli+presented+while+sleeping%5BTitle%5D)+AND+%22Psychophysiology%22%5BJournal%5D)">Temperature, time-of-night of testing, and responsiveness to stimuli presented while sleeping.</a> Psychophysiology. 28 (4), 463-467 (1991).</li>
<li>Alikhan, R., Forster, R., Cohen, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heparin+for+the+prevention+of+venous+thromboembolism+in+acutely+ill+medical+patients+%28excluding+stroke+and+myocardial+infarction%29%5BTitle%5D)+AND+%22Cochrane+Database+Syst+Rev%22%5BJournal%5D)">Heparin for the prevention of venous thromboembolism in acutely ill medical patients (excluding stroke and myocardial infarction).</a> Cochrane Database Syst Rev. 2014 (5), CD003747(2014).</li>
<li>Roth, B., Ekelund, M., Fan, B. G., Ekstrom, U., Nilsson-Ehle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+heparin+and+low+molecular+weight+heparin+on+lipid+transport+during+parenteral+feeding+in+the+rat%5BTitle%5D)+AND+%22Acta+Anaesthesiol+Scand%22%5BJournal%5D)">Effects of heparin and low molecular weight heparin on lipid transport during parenteral feeding in the rat.</a> Acta Anaesthesiol Scand. 40 (1), 102-111 (1996).</li>
<li>Weintraub, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+intravenous+heparin+administration+in+humans+causes+a+decrease+in+serum+lipolytic+activity+and+accumulation+of+chylomicrons+in+circulation%5BTitle%5D)+AND+%22J+Lipid+Res%22%5BJournal%5D)">Continuous intravenous heparin administration in humans causes a decrease in serum lipolytic activity and accumulation of chylomicrons in circulation.</a> J Lipid Res. 35 (2), 229-238 (1994).</li>
<li>Schallom, M. E., Prentice, D., Sona, C., Micek, S. T., Skrupky, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heparin+or+0.9%25+sodium+chloride+to+maintain+central+venous+catheter+patency%3A+a+randomized+trial%5BTitle%5D)+AND+%22Crit+Care+Med%22%5BJournal%5D)">Heparin or 0.9% sodium chloride to maintain central venous catheter patency: a randomized trial.</a> Crit Care Med. 40 (6), 1820-1826 (2012).</li>
<li>Rahman, S. A., Wright, K. P. Jr, Lockley, S. W., Czeisler, C. A., Gronfier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterizing+the+temporal+dynamics+of+melatonin+and+cortisol+changes+in+response+to+nocturnal+light+exposure%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Characterizing the temporal dynamics of melatonin and cortisol changes in response to nocturnal light exposure.</a> Sci Rep. 9 (1), 19720(2019).</li>
</ol>

The authors have nothing to diclose.

The through-the-wall blood sampling method presented here is widely used in sleep and circadian research to collect blood during sleep with minimal participant disturbance. The procedures described -- including preparation, setup, and use of the extension line -- have broad applications in both clinical and research settings. By employing this technique, researchers can successfully obtain blood samples over a 24-h period, including during scheduled sleep. Furthermore, polysomnography (PSG) confirms that under ideal cond...

Diurnal and circadian rhythms are biological cycles involved in the regulation of physiologic, metabolic, and behavioral processes over 24 h. Diurnal rhythms are biological patterns that follow a day/night cycle and are driven by environmental or behavioral cues. In contrast, circadian rhythms are near 24-h cycles in biology driven by an intrinsic network of molecular clocks, independent of external cues (note: a detailed explanation of circadian clocks is beyond the scope of this article; interested readers are directed to Cox et al.1 for further information and Broussard et al.2 for a discussion of clinical circadian protocols). Both diurnal and circadian rhythms play critical roles in health, with disruptions associated with increased risks for developing cardiovascular disease and type 2 diabetes3,4. This understanding is rooted in decades of research involving time series measurements in controlled laboratory conditions. Results from such studies have helped distinguish between endogenous circadian influences and external environmental or behavioral effects on biological processes and their roles in maintaining physiological homeostasis2,5. This body of work laid the foundation for more recent translational research investigating the impact of sleep and circadian timing of behaviors on health-related outcomes6,7,8,9.
Hormones and metabolic factors exhibit distinct 24-h oscillations. Melatonin, for example, rises during the biological night and remains low during the biological day10. In contrast, the cortisol rhythm is characterized by suppression during the early part of the biological night, followed by a surge before awakening11, which is altered during sleep deprivation12. Thyroid-stimulating hormone (TSH) rises in the biological evening and is suppressed during sleep13. The TSH rhythm is disrupted during sleep deprivation, leading to increased TSH levels during the biological night14. Rhythmic patterns of several other circulating factors are similarly impacted by sleep-wake cycles. For example, growth hormone (GH) secretion is closely tied to slow-wave sleep (SWS), with a pronounced release shortly after sleep onset, especially in men15. Furthermore, GH secretion in conjunction with increased glucagon during sleep helps accommodate the prolonged overnight fast and leads to reduced glucose utilization to maintain stable blood glucose levels and prevent hypoglycemia16. Similar in pattern to GH, prolactin rises sharply after sleep onset and peaks midway through the sleep period, with similar rises occurring during daytime naps17,18. Additional circulating factors that exhibit 24-h rhythms include adrenocorticotropic hormone, luteinizing hormone, glucose, and insulin (for a more comprehensive summary, see Hanlon et al.5).
Many circulating factors involved in hunger, appetite regulation, and energy balance also exhibit fluctuations over the 24-h period. Ghrelin, which stimulates hunger, peaks before meals and declines during the nocturnal sleep period, while leptin, a satiety hormone, rises at night, likely suppressing hunger during the overnight fast19. Other metabolic signals, such as free fatty acids, endocannabinoids, and satiety peptides like GLP-1 and PYY, exhibit variations that are influenced by both sleep and eating patterns19,20,21,22.
Taken together, the oscillations outlined above illustrate the complex interplay among sleep, circadian processes, and physiological homeostasis which can only be fully appreciated by profiling the 24-hour circulating patterns of relevant physiological variables.
Elucidation of 24-h rhythms of circulating factors is essential for understanding daily fluctuations in physiological function, as well as how sleep and circadian disruption impact health. However, traditional blood sampling methods can interfere with the very processes that drive and regulate these rhythms23, highlighting the need for minimally invasive approaches that allow for accurate insights into 24-h profiles.
Therefore, the goal of this methods article is to describe in sufficient detail the steps involved in reliably obtaining frequent blood samples over 24 h from human research participants using a through-the-wall blood sampling technique. The described technique, employed by many sleep and circadian laboratories20,24,25,26,27,28,29,30,31,32,33,34,35,36, addresses technical challenges involved in collecting blood samples during sleep. In the following sections, the technique itself is described with a supplemental video demonstration. In addition, representative data from a clinical study utilizing the method are presented. Finally, the article closes with a discussion of the limitations, pitfalls, and important considerations of using the method in clinical research.

<table><tbody><tr><td>9-in Standard-Bore IV Extension Set with 1 mL Priming Volume, Removable SmartSite Needle-Free Connector, Slide Clamp and Spin Male Luer Lock</td><td>Medline</td><td>IME22059EH</td><td></td></tr><tr><td>0.9% Sodium Chloride injection USP 10 mL in a 10 mL flush syringe ZR</td><td>Excelsior Medical</td><td>EMZE010001</td><td></td></tr><tr><td>Alcohol prep pad</td><td>Medline</td><td>MDS090737</td><td></td></tr><tr><td>BD Insyte Autoguard Winged 20 G catheter</td><td>Medline</td><td>381534</td><td></td></tr><tr><td>BD Plastipak 3 mL syringe</td><td>Fischer Scientific</td><td>309657</td><td></td></tr><tr><td>CoFlexNL</td><td>Medline</td><td>5200CP</td><td></td></tr><tr><td>IV extension set 192 in, smallbore tubing</td><td>ICU Medical</td><td>B2202</td><td>It is necessary to create an account with ICU medical to order supplies</td></tr><tr><td>Non-woven gauze sponges</td><td>Medline</td><td>NON25223</td><td></td></tr><tr><td>Tegaderm</td><td>Medline</td><td>1624W</td><td></td></tr><tr><td>Ultra(TM) 3-way stockcockw/swivel male luer lock</td><td>Medline</td><td>MX2311L</td><td></td></tr></tbody></table>

through-the-wall blood sampling method to minimize sleep disruption in clinical settings

Through-the-Wall Blood Sampling Method to Minimize Sleep Disruption in Clinical Settings

Physiological rhythms, including diurnal and circadian rhythms, play a critical role in health, with disturbances increasingly linked to cardiovascular disease and type 2 diabetes. These rhythms are closely tied to sleep-wake cycles, as changes in sleep timing or duration can alter diurnal and circadian rhythms, thereby disrupting physiological homeostasis. Additional factors, such as the timing of behaviors like food intake and/or physical activity, may modulate 24-h rhythms in manners that support health or promote chronic disease risk. 
Despite the growing recognition of the importance of biological rhythms, obtaining serial blood samples required to elucidate patterns in circulating factors remains a significant challenge in clinical research. Routine blood draw methods can be disruptive to sleep, potentially altering sleep and biological rhythms and subsequently influencing variables of interest. This underscores the need for minimally invasive approaches that allow for the assessment of 24-h biological processes without disturbing sleep or biological rhythms. 
To address these challenges, many clinical and research groups employ a technique known as a "through-the-wall" blood sampling method. This approach uses extended tubing connected to an indwelling catheter, threaded through a wall or partition to a location outside the participant's room. By enabling blood collection without having to enter the room, this method minimizes disruptions, thereby preserving the integrity of the 24-h profiles of circulating variables of interest and minimally disturbing sleep. 
Here, we provide a detailed description of the through-the-wall blood sampling method and demonstrate its feasibility and efficacy in collecting blood samples during scheduled sleep opportunities. Representative data from a clinical study are presented, and additional considerations, including the impact of a difficult blood draw on sleep architecture, are discussed.

24-h Melatonin using through-the-wall blood sampling during sleep 
The method described above allows for the assessment of 24-h patterns of circulating factors that would otherwise be missed by a single time-point sample. Melatonin, a hormone frequently measured by through-the-wall blood sampling, serves as the most widely accepted surrogate for assessing the rhythm of the central circadian clock, which resides in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus. Because direct access to...

Here, we present a through-the-wall, minimally invasive method to sample blood during sleep in clinical settings.

Physiological rhythms, including diurnal and circadian rhythms, play a critical role in health, with disturbances increasingly linked to cardiovascular ...

We study how sleep and circadian rhythms impair cardiometabolic health. Ultimately, we would like to find strategies to reduce the rest of diseases associated with sleep and circadian disruption. To study things that change in the body over 24 hours, we need to have a window into the system during the entire period.This can be tough because we don't want to disturb sleep in circadian rhythms by going into a participant's room while they're sleeping to get a blood sampling. To get around that, we use a through the wall blood sampling protocol. We address the gap by using a long IV line that we place through the wall from which we can sample blood from outside the participant room.This allows us to sample blood with minimal disruption to sleep and circadian rhythms. Our findings will allow researchers to access metabolic health markers such as glucose and insulin across 24 hours without disturbing sleep, which can help clarify the influence of sleep and circadian rhythms on disease risk. To begin, attach the extension line for through the wall sampling by connecting the three-way stop to the female lure lock end of the extension line.After breaking the seals, place the 10 milliliter saline flush syringe and the three milliliter sample syringe onto the stop cock. Twist the off tab of the stop cock to close the extension line to the three milliliter syringe and open it to the 10 milliliter saline flush. Push saline through the extension line until it reaches the end.Carefully remove the cap and allow the saline to drip into a cup or onto the absorbent pad. Twist the off tab to close the extension line to both syringes, keeping them attached to the stop cock. Feed the capped end of the extension line through the wall port into the participant room, ensuring the cap remains in place to maintain sterility.In the participant's room, clean the distal end of the extension line and the attachment side of the IV extension set. Using a sterile alcohol prep pad, remove the cap from the extension line and attach it to the IV extension set connected to the participant. Now return to the anti chamber.Remove and discard the used saline flush from the stop cock and attach a warmed 10 milliliter saline flush. Twist the off tab to close the line to the sample syringe and open it to the new saline flush. Push saline through the extension line to verify the absence of leaks and confirm that the IV line remains patent.Slowly continue pushing the saline flush while simultaneously turning the off tab to fully seal the system. Once the extension line is confirmed to be functioning, return to the participant's room to wrap the IV extension set with a self adherent elastic bandage to prevent dislodgement during sleep. Tape the extension line securely to the bed frame.Attach afive milliliter waste syringe ensuring that the extension line is saline primed and connect a three milliliter sample syringe to the stop cock. Twist the off tab to open the line to the five milliliter waste syringe. Using the five milliliter syringe, collect at least 4.4 milliliters of waste.Then continue drawing until whole blood appears. Now twist the off tab of the stop to close the line to the five milliliter waste syringe. Use the three milliliter syringe to draw the desired amount of blood and twist the off tab to close the extension line to both syringes.Remove the five milliliter waste syringe and replace it with a warm 10 milliliter saline flush. Twist the off tab to close the line to the three milliliter sample syringe. Opening it to the saline flush.Push up to 10 milliliters of warm saline into the extension line, ensuring no blood remains in the line. Remove the blood filled sample syringe for further processing and replace it with an empty three milliliter syringe. Twist the off tab to close the line to both syringes.Slowly continue to push the saline flush while turning the off tab to maintain pressure in the extension line and minimize blood back flow. Use any remaining saline to flush through the stopcock into the empty three milliliter syringe. Then remove the saline syringe and pull back on the three milliliter syringe to extract any remaining liquid from the stop cock.Replace all syringes to prepare for the next blood draw. The 24 hour plasma melatonin concentration profile of a participant was measured under dim light conditions. The findings suggest that the melatonin levels follow a distinct rise and fall pattern consistent with a normal circadian rhythm, confirming that the through the wall blood sampling method accurately captures hormonal changes during the biological night.In a recent study, the success rate for through the wall blood sampling was 67%corresponding to 304 out of 454 total blood draw attempts, unsuccessful blood draws accounted for 33%attempts with 94%of these failures attributed to positional issues such as the participant lying on or occluding the extension line. During an easy blood draw, polysomnography recordings showed stable electroencephalogram, electrogram, electromyogram, and electrocardiogram signals with no evidence of arousal or sleep stage change. A difficult blood draw was associated with disrupted electroencephalogram, electrogram, and electromyogram activity along with an elevated heart rate indicative of arousal.

through-wall-blood-sampling-method-to-minimize-sleep-disruption

Research

JoVE Journal

Medicine

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements.  Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Here, we present a protocol to improve the quality of data from home sleep testing by providing a method to enhance instructions through a structured participant visit. This protocol includes the implementation of a step-by-step educational manual with photos to ensure proper placement of equipment.

Multi-Modal Home Sleep Monitoring in Older Adults

Sleep loss and circadian misalignment contribute to numerous operational accidents and incidents. The effectiveness of countermeasures and work scheduling designs aimed at mitigating fatigue can be challenging to evaluate in operational environments. This manuscript summarizes an approach for collecting sleep, circadian, fatigue, and performance data in complex operational environments.

Collecting Sleep, Circadian, Fatigue, and Performance Data in Complex Operational Environments

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.

Human Circadian Phenotyping and Diurnal Performance Testing in the Real World

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

The present protocol describes a simple and efficient method for the real-time and dynamic collection of rat heart blood using the microdialysis technique.

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Luteinizing hormone (LH) pulsatility is a hallmark of reproductive function. We describe a protocol for remote activation of specific neuronal populations linked to serial automated blood collection. This technique allows timed hormonal modulation, multiplexing, and minimizing manipulation effects on LH levels in conscious freely moving, and undisturbed animals.

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice

Here we present a combination of effective rat restriction and subclavian vein puncture methods that enable rapid, safe, and repeated blood collection in rats without anesthesia.

Subclavian Vein Blood Sampling in Conscious Rats

Learn a through-the-wall blood sampling method to minimize sleep disruption in clinical research. This technique enables continuous 24-hour collection of metabolic markers without disturbing circadian rhythms.

Through-the-Wall Blood Sampling Method to Minimize Sleep Disruption in Clinical Settings

Explore More Videos