Method Article

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

DOI:

10.3791/67530

⸱

June 13th, 2025

 ,  ,  ,  ,  ,  ,  , 
1Human Performance Clinical Research Laboratory, Department of Health and Exercise Science, Colorado State University, 2Anschutz Health and Wellness Center, University of Colorado Anschutz Medical Campus

* These authors contributed equally

In This Article

Summary

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

Abstract

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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.

Introduction

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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.

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Protocol

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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.

  1. Setup for through-the-wall sampling (Figure 1)
    1. 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.
  2. Attach the extension line for through-the-wall sampling.
    1. 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.
    2. 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.
    3. 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.
    4. 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.
    5. 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.
    6. 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.
    7. Move to the participant'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.
    8. Remove the cap on the extension line and attach the line to the IV extension set (connected to the participant).
    9. Once the extension line is connected to the IV, return to the antechamber.
      1. 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.
      2. 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 °C/98.6 °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.
    10. 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.
    11. Once the extension line is successfully established, return to the participant'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.
    12. 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.

2. Sampling blood through the extension line

  1. Drawing a blood sample.
    1. 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.
      1. 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.
    2. 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 "waste" syringe, which consists of the saline in the extension line and the IV extension set.
    3. 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.
    4. 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.
    5. 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.
    6. Remove the 5 mL waste syringe and replace it with a warm 10 mL saline flush.
    7. 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.
    8. Push up to 10 mL of warmed saline, ensuring no blood remains in the extension line.
    9. Twist the OFF tab on the stopcock to close the line to both syringes.
      1. 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.
      2. 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.
    10. Replace all syringes to prepare for the next draw.
      NOTE: Do not leave stopcock ports without syringes attached, as this will compromise sterility.

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Results

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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...

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Discussion

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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...

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Disclosures

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The authors have nothing to diclose.

Acknowledgements

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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 & 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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
9-in Standard-Bore IV Extension Set with 1 mL Priming Volume, Removable SmartSite Needle-Free Connector, Slide Clamp and Spin Male Luer LockMedlineIME22059EH
0.9% Sodium Chloride injection USP 10 mL in a 10 mL flush syringe ZRExcelsior MedicalEMZE010001
Alcohol prep padMedlineMDS090737
BD Insyte Autoguard Winged 20 G catheterMedline381534
BD Plastipak 3 mL syringeFischer Scientific309657
CoFlexNLMedline5200CP
IV extension set 192 in, smallbore tubingICU MedicalB2202It is necessary to create an account with ICU medical to order supplies
Non-woven gauze spongesMedlineNON25223
TegadermMedline1624W
Ultra(TM) 3-way stockcockw/swivel male luer lockMedlineMX2311L

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Tags

Through The Wall SamplingCircadian RhythmsIndwelling CatheterSerial Blood SamplingPlasma MelatoninPolysomnography RecordingCardiometabolic HealthSleep Architecture

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