Name: a protocol of manual tests to measure sensation and pain in humans
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The authors acknowledge the following funding sources: the Duquesne University Faculty Development Fund grants awarded to Kimberly Szucs, PhD and Alex Kranjec, PhD and to Benedict Kolber, PhD and Matthew Kostek, PhD. We also acknowledge Rachel Sweetnich for experimental assistance and funding from the Duquesne University Pain Undergraduate Research Experience program awarded to Sweetnich (Mentors: Szucs and Kostek).

All tests with human subjects should be approved by the Institutional Review Board at the individual institution. All testing described for the current study was approved by the Duquesne University Institutional Review Board for human subject research. Training for and descriptions of each measure are as follows:
1. Cutaneous Mechanical Sensitivity Assay13
NOTE: Enrolled participants are asked to sit in a chair, with support provided for the extremity to be...

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We have demonstrated cost-effective and simple qualitative and quantitative sensory tests that can be used to assess mechanical sensation, thermal sensation and pain, and pressure pain in human subjects. The value of these assays is their ease of implementation and low amount of necessary training time. Each experimenter received a minimal amount of training (one trial observation and one trial implementation). Thus, multiple technicians could be trained in one day. The results suggest strong inter-experimenter and withi...

Chronic pain conditions are a worldwide clinical problem. More than 1.5 billion people worldwide suffer from chronic pain, and approximately 5% of the global population suffers from neuropathic pain, with incidence rates increasing with age1. In America, it is estimated that pain affects more people than diabetes, heart disease, and cancer, combined2. While awareness of this problem is increasing, treatments are not always successful, can be expensive, and may have serious side effects, including addiction. Research on treatments is ongoing, but as pain varies greatly between individuals, pain measurement for research or diagnosis can be problematic. In particular, the reliance on qualitative approaches, such as the Visual Analog Scale (VAS), for determining treatment efficacy has been problematic because of the subjective and personal nature of pain3. As more research laboratories and smaller clinics around the world answer questions about and treat pain, measures that are accurate, consistent, portable, quantitative, and affordable are in great demand.
A key distinction in pain measurement is acute versus chronic pain. Acute pain is a normal response to injury, infection, or another noxious stimulus. Acute pain normally resolves with treatment and time, and the pain location is usually site-specific. Chronic pain, however, can be related to an initial bout of acute pain, or it can be idiopathic. Chronic pain may relate to the site of injury, but it is often widespread throughout the body4. Chronic pain can last for weeks, months, and even years, causing substantial physical, psychological, and monetary burdens on patients and their families, employers, and societies. The ability to identify and quantify pain is critical for correct diagnosis, evaluation of ongoing treatment, and development of new analgesic treatments. Quantitative and qualitative sensory testing are thus critical for diagnosis and treatment.
Several methods can be used to examine peripheral sensation and pain: nerve conduction velocity (NCV), somatosensory evoked potentials (SEP), skin biopsies, and quantitative sensory testing (QST). Clinicians also routinely use bedside neurologic sensory testing, but this testing is not calibrated and does not use a standardized set of instructions5. Exams of NCV and SEP can be informative, but compared to QST, they require highly specialized equipment, typically only examine large nerve fibers, only measure loss of function, and do not test the entire somatosensory system6,7. Skin biopsies are used to assess nerve fiber density, but compared to QST, they are invasive and require tissue processing and microscopy time, which could take several days to accomplish8. Furthermore, the biopsy only examines a small, specific area of the somatosensory system and does not test nerve function. QST measurements overcome most of the limitations of other testing methods. Recently, standardized normative data for QSTs have been made available, which further add to their utility to assess pain and neural sensation9-11. We therefore focus the current protocol on QST measures for chronic pain.
New technologies have made the assessment of pain and physical sensation (e.g., pressure and heat) precise and reliable within well-equipped laboratories that have established internal protocols12. Many of these technologies, however, are not easily portable and are cost-prohibitive for new or small research laboratories and medical clinics. Additionally, protocols for technology use are not standardized across laboratories13, which can affect reliability. Therefore, the goal of this manuscript is to demonstrate effective and reliable pain and sensory measures that can be conducted with equipment that is available in most clinics or research laboratories. The rationale for the development of the current protocol is that while many people suffer from chronic pain conditions, and accurate assessment of pain is needed for diagnosis and treatment, there are no published protocols with visual demonstrations of assays.
An example of a nearly fully automated device for testing acute pain is the Neuro Sensory Analyzer, which can reliably assess thermal pain sensation, as demonstrated by Angst et al. following a cutaneous burn in human subjects14. The unit is modular, and additional sensory testing devices can be added. In their study, Angst et al. also demonstrate pressure sensory testing with the use of Punctuated Pressure Probes, which were custom built. While these probes should offer more consistent results, few laboratories or clinics have them.
The current protocol demonstrates QST measures for chronic pain: von Frey filaments for cutaneous sensory testing, a radiant (&#34;Hargreaves&#34; method) and contact heat technique, and pressure algometry for deep tissue pain. These QST measurements are not unique. Rather, they are the most common and generally accepted measurements for human sensory testing in medical clinics, hospitals, and research laboratories13,15,16. Mechanical and thermal stimulation are used to examine cutaneous and deep sensation. These measures, furthermore, include the evaluation of both small and large fiber sensitivity for normal sensation and pain. To assess deep tissue pain (muscle), pressure algometry is used, which is the most frequently applied technique for the quantification of pain in soft tissue such as muscles17,18. Both A-delta and C fibers mediate pain induced by pressure stimulation19. Stimulation of both fibers is an advantage and a disadvantage, in that it examines multiple pathways, making it a good overall measure, but it is also less specific. To examine touch sensitivity, mechanical stimulation of skin with von Frey filaments is used because they are one of the most commonly used sensory devices in pain and medical neural clinics. Von Frey filaments stimulate A-beta fibers,20 but are not specific as both low threshold mechanoreceptors and nociceptors can be activated21. The use of these filaments has been criticized, mainly because of potential variability of the application procedure (degree of filament indention or accidental movement of the hand) and concerns that the mechanical filament characteristics may change over time22,23. This protocol addresses these issues by providing detailed instructions with a script and calibration of filaments.
For thermal pain, radiant heat using the &#34;Hargreaves&#34; method (visible light and ramping temperature) and a heat block to examine contact heat are used. Contact and radiant heat activate thermal receptors differently and can even confound one another. It has been shown that dynamic contact can inhibit thermal nociception24. This is similar to the concept of thermal referral, in which touch contributes to normal temperature perception25-27. Therefore, one measure of thermal sensation and two measures of thermal pain are included. First, radiant heat is used to determine the threshold for temperature change detection (starting from room temperature). Second, the radiant heat source is used to determine the threshold for heat pain. The detection of warm thermal change (non-nociceptive) is mediated in part by transient receptor potential (TRP) channels on C fibers, while heat pain is mediated by TRPV1/V2 and other higher-threshold channels on C and A-delta fibers28-30. At threshold determination, rapid skin heating activates first A-delta fibers, corresponding to the &#34;first pain,&#34; followed by a C fiber-mediated &#34;second pain,&#34; described as &#34;throbbing, burning, or swelling&#34;31. Heating gives a preferential activation of C fibers and is the best evaluation of second pain32. In the contact heat assay, a constant nociceptive temperature is applied to determine the qualitative intensity and affective aspects of pain.
Another variable considered in developing the QST protocol is anatomical location. For acute or location-specific pain, the anatomical site of the pain is typically used for testing. Because the protocol was designed with chronic pain conditions in mind, we take a more global approach. The protocol assesses sensation on the forearm and leg instead of the hand, as it has been shown that heat pain thresholds are significantly higher on the hand than on the forearm33 and that thermal nociception can be perceived on the hand, although less frequently and less intensely than on the forearm24. While the protocol was designed for the majority of chronic pain conditions, we caution users that some chronic pain conditions affect specific anatomical regions, and this should be taken into account when modifying the protocol for a specific patient population.
While these QST measures are the most commonly used and are accepted as some of the most reliable, they are inexpensive and common enough that most clinics and research laboratories might already have access to them, can afford them, and can transport them. This QST protocol is useful to any laboratory or clinic where measures are needed for humans with chronic pain. To date, there are currently no published visual reports demonstrating a protocol for the use and reliability of these measures. Based upon this protocol demonstration and tips on improving reliability, a laboratory or clinic could easily examine their own test-retest reliability. Because many clinics will need to utilize several technicians to measure all patients, inter-rater reliability data would be useful in selecting a protocol. We include a small set of data that suggests that the protocol has good reliability, but each clinic and laboratory is strongly advised to use this as an example, as each clinic and each patient population with chronic pain is unique.
Notes on injury risk for sensory and pain testing:
Risk of injury related to cutaneous mechanical testing is extremely rare and unlikely. Mechanical testing is safe and widely used. Risks to the individual are minimal because 1) this is not a painful or noxious stimulus; 2) subjects are instructed that they may stop any procedure at any time, with no adverse consequences; and 3) the level of sensation experienced by subjects is well below their tolerance level and threshold for pain.
Risk of injury related to thermal pain testing is minimal. Thermal testing is safe and widely used. While thermal testing does produce pain, risks to the individual are minimal because 1) the pain is transient in nature and generally subsides immediately after the procedure; 2) subjects are instructed that they may stop any procedure at any time, with no adverse consequences; and 3) the level of pain experienced by subjects is below their tolerance level. With Hargreaves thermal stimulation, there is a very slight risk of receiving a burn, but this is minimized by the following: 1) the positive lockout of stimulus parameters above 50 &#176;C; 2) the built-in shutdown system in the stimulator that prevents the delivery of prolonged or high-intensity stimuli (20 sec); and 3) the electronic thermometer that measures the temperature at the glass surface before and during each use (see below in the instrument section). Pain threshold trials will proceed only if the temperature detected at the 20 sec cutoff is &#8804;50 &#176;C.
Risk of injury related to pressure pain testing is minimal. Pressure testing is safe and widely used. While pressure testing does produce pain, risks to the individual are minimal because 1) the pain is transient in nature and generally subsides immediately after the procedure; 2) subjects are instructed that they may stop any procedure at any time, with no adverse consequences; 3) the level of pain experienced by subjects is below their tolerance level; and 4) the pain applied is never more than that subject&#39;s pain threshold, which is well below any pressure that could cause damage. A rare side effect of pressure testing is bruising at the stimulus site. In this situation, a subject should not be retested at the bruised site. The chance for bruising can be minimized by study exclusion of individuals that bruise easily or are taking blood thinners.
During the enrollment period, participants are given a full description of all sensory and pain measures that will be used. With initial consent, all participants are allowed to experience all sensory and pain measures before full enrollment. All sensory and pain assays are based on well-established assays used in both healthy human participants and in chronic pain patients34. All assays involve either innocuous (non-painful stimuli) or acute noxious stimuli (painful stimuli) that do not damage tissue. The time between different tests is &#62;5 min, to allow the subject to rest and to reduce the potential for sensory fatigue or sensitization. The sequential order of tests is the same during each testing session. Specific sites of testing are limited to the T1 dermatome on the left and right forearms and L3/S2 dermatome on the left and right calves. All sites for testing are marked with a marker, and individual sites are spread out to avoid overlapping receptive field activation (Figure 1). See the Materials and Equipment Table for the full materials list. For retest reliability studies, individual subjects were tested by two experimenters in a single day.

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="86">
			<td height="86" style="height:87px;width:224px;">Pressure Algometer / Force Dial</td>
			<td style="width:144px;">Wagner Instruments</td>
			<td style="width:111px;">FDK 20</td>
			<td style="width:427px;">The pressure algometer quantifies pressure pain threshold.&#160; It has a rubber tip attachment that is applied to the marked skin site by the investigator.&#160; The dial records the pressure and is reset after each measurement.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:224px;">von Frey cutaneous stimulators</td>
			<td style="width:144px;">Touch Test</td>
			<td style="width:111px;">NC1275-01 through -08</td>
			<td style="width:427px;">These von Frey filaments are commonly used to examine sensitivity in research and clincial settings.&#160; Our set of 8 filaments covers a range of sensitivites.&#160; The individual filaments are 1.65 mN, 2.36 mN, 2.44 mN, 2.83 mN, 3.22 mN, 3.61 mN, 3.84 mN, 4.08 mN.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:224px;">&#34;Hargreaves&#34; apparatus, testing platform</td>
			<td style="width:144px;">Custom</td>
			<td style="width:111px;">n/a</td>
			<td style="width:427px;">One complete base and four supporting columns are used to form a platform for a sheet of safety glass through which the heat source directs heat to the subjects arm or leg that is resting on the glass.&#160; The heat lamp is placed beneath the glass.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">0.64cm Pyrex safety glass</td>
			<td style="width:144px;">DuPont</td>
			<td style="width:111px;">n/a</td>
			<td style="width:427px;">Safety glass is important to avoid injury in the unlikely event of a fracture in the glass surface.</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;width:224px;">Electronic thermometer / thermocouple 53 IIB</td>
			<td style="width:144px;">Fluke</td>
			<td style="width:111px;">3821062</td>
			<td style="width:427px;">The thermocouple is used for thermal testing.&#160; The thermocouple is placed on the glass underneath the subject&#39;s arm or leg and measures the temperature at the glass level.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">IITC Plantar Analgesia Meter&#160;</td>
			<td style="width:144px;">Life Science Inc. Woodland Hills, CA</td>
			<td style="width:111px;">390</td>
			<td style="width:427px;">This is the heat source and timer for Hargreaves testing.&#160; The unit&#39;s heat source has an &#8220;idle state&#8221; that allows exact placement of the heat source.&#160; The heat source is radiant light and the light beam is focused to the top of the glass to creates a 4X6mm intense spot on the arm or leg.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:224px;">Examiner script</td>
			<td style="width:144px;">Custom</td>
			<td style="width:111px;">n/a</td>
			<td style="width:427px;">A written script for the examiner is used for every testing session.&#160; Because pain and sensitivity can be affected by envrionmental stresses, we attempt to maintain as much consistency as possible between subjects.&#160; The examiner reads directly from the script every time a measure is made to ensure&#160; verbal consistency.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Markers for testing site</td>
			<td>Sharpie</td>
			<td>n/a</td>
			<td style="width:427px;">Washable markers may be preferable for situations where multiple days of testing is not necessary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Constant heat stimulus block</td>
			<td>Benchmark Scientific</td>
			<td>BR10-00</td>
			<td>This block is digitally controlled. The surface of the block is 2x3cm.</td>
		</tr>
	</tbody>
</table>

a protocol of manual tests to measure sensation and pain in humans

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study demonstrates a quantitative sensory testing protocol using techniques to measure tactile sensation and pain threshold for pressure and heat using portable and easily accessed equipment. These techniques and equipment are ideal for new laboratories and clinics where cost is a concern or a limiting factor. We demonstrate measurement techniques for the following: cutaneous mechanical sensitivity on the arms and legs (von-Frey filaments), radiant and contact heat sensitivity (with both threshold and qualitative assessments using the Visual Analog Scale (VAS)), and mechanical pressure sensitivity (algometer, with both threshold and the VAS). The techniques and equipment described and demonstrated here can be easily purchased, stored, and transported by most clinics and research laboratories around the world. A limitation of this approach is a lack of automation or computer control. Thus, these processes can be more labor intensive in terms of personnel training and data recording than the more sophisticated equipment. We provide a set of reliability data for the demonstrated techniques. From our description, a new laboratory should be able to set up and run these tests and to develop their own internal reliability data.

&#160;Here, we describe the implementation of cost-effective qualitative and quantitative assays to measure innocuous sensation and pain in human participants using the VAS (Figure 2). The visual representation is important, because accurate and precise results of these examinations are dependent upon correct and consistent protocol execution by the technician. Additionally, it is valuable to know if multiple technicians performing the technique as described can collect r...

Watch this Scientific Journal Video about A Protocol of Manual Tests to Measure Sensation and Pain in Humans at JoVE.com

A Protocol of Manual Tests to Measure Sensation and Pain in Humans

The goal of this procedure is to demonstrate a battery of quantitative techniques for sensory and pain measurement in humans. The equipment and techniques described are commonly found in pain clinics or are easy to obtain.

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study ...

The overall goal of this data collection procedure is to measure pressure and heat pain thresholds as well as tactile sensation in humans. These sensory measures include both quantitative measures of sensation as well as qualitative measures that help asses individuals sensory and emotional experience during testing. This method can help answer key questions in the Study of Chronic Pain Conditions by providing concrete data points to evaluate the efficacy of various therapies in the context of pain.The main advantages of these techniques are that they're easy to implement and offer reliability between experimentaters. The equipment is easily purchased, stored, and transported by most clinics and research laboratories around the world. Individuals will struggle with these methods because small changes in the time of stimulus application, placement of stimulus application, and/or application pressure can introduce significant variability in the data set.Application procedures of these tests are seemingly subjective, which can lead to inconsistent results. Visual demonstration is therefore critical to ensure accuracy and precision across multiple tests, multiple technicians, and multiple patients. Escort the subject into the testing room and have them sit on an exam table that provides arm support.Give the filament to the subject and let them gently bend it against the skin of their hand. During each trial, ask the subject to look away from their forearm. Start by applying the smallest filament at 0.078 millinewtons to the subject's forearm until it boes and ask if they feel the filament.Complete four positive trials where the filament is applied to the forearm, and one randomized negative trial where the filament is not applied, but the subject is still asked if they feel the filament. Finally, score the trials on the data form, such that if a subject detects greater than three of the positive trials, and zero of the negative trials for a filament, record that filament as the subject's mechanical sensory threshold. For a single filament, if the subject detects less than two of the positive trials, and or greater than zero of the negative trials, then start another round of five trials with the next biggest filament until the sensory threshold is reached.Begin by showing the radiant heat device to the subject and allowing them to feel the stimulus with their hand. Next, ask the subject to rest their forearm on the room temperature glass plate, which is covered with a rubber insulating sheet except for the small opening for stimulus presentation. Use a mirror to position the light source under a marked area on the subjects forearm.Then, for each trial, ask the subject to depress the stop button when they feel the temperature change. To achieve the withdraw threshold, set the device to ramp up the temperature so that the stimulus temperature reaches 47 degrees Celsius at 10 seconds. Begin by showing the constant heat block to the subject and allowing them to feel the stimulus with their hand.Next, apply the stimulus for three seconds to the marked location on the left forearm. Finally, immediately following the stimulus, ask the subject to evaluate the quality and unpleasantness of the pain using a standard zero to 10 visual analog scale. Before starting the experimental trial, allow the subject to handle the algometer, and apply the stimulus to themselves under careful supervision.Complete a pain threshold trial by placing the probe on the subject's forearm and gradually applying pressure until the stimulus transitions from innocuous pressure to painful pressure. Ask the subject to say stop when they want the stimulus removed from their forearm. Complete two trials to the forearm in two distinct sights on the right and left arm to avoid damage to a single area.Then, remove the algometer from the subject. Record the greatest applied pressure as the pressure pain threshold for the trial. Finally, to complete a constant pressure trial, apply a painful stimulus for three seconds and ask the subject to evaluate the quality and unpleasantness of the pain on two standard, zero to 10 visual analogue scales.Inform the subject that on the quality scale, zero is represented as no pain, and 10 is represented as the worst pain imaginable. On the unpleasantness scale, zero is represented as not unpleasant, and 10 is represented as the most unpleasant sensation imaginable. This intra-class correlation coefficient analysis examines the reliability between two examiners, male and female.Inter-experiment reliability was strong for mechanical, thermal, and pressure testing. Such that average inter-experiment reliability measures were all statistically significant except for the mechanical sensitivity test. In addition, all visual analogue scale results for constant heat and constant pressure, showed significant correlations between the male and female experimenters.Once mastered, these techniques can be completed in 30 minutes if performed properly. While attempting these procedures, it's important to remember to remain as consistent as possible when applying stimuli to the subject's testing areas. This is especially important for application of the Von Frey filament for cutaneous mechanical sensitivity, and the algometer for pressure sensitivity.Following these procedures, other methods that evaluate cold hypersensitivity and mechanical pain, can be performed in order to answer additional questions related to other sensory modalities that are altered in the context of pain. Quantitative sensory testing is regularly applied in pre-clinical, and clinical testing. When combined with traditional qualitative measures, the entire procedure provides a valuable snapshot of an individuals sensory and emotional experience to noxious stimuli.After watching this video, you should have a good understanding of how to measure quantitative and qualitative sensation and pain in humans. This includes tactile sensation and pain threshold for pressure and heat, as well as VAS qualitative assessments. Don't forget that working with noxious stimuli in human subjects can be hazardous, and precautions should always be taken when performing this procedure.

Research

JoVE Journal

Behavior

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Frame-by-Frame Video Analysis of Idiosyncratic Reach-to-Grasp Movements in Humans

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Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats

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