We acknowledge the support from Ruhr-University Bochum for our research on neuromuscular ultrasound.

All examinations for this work were performed in compliance with institutional guidelines of the Ruhr-University Bochum, Germany.
1. Experimental preparations
<ol>
	<li>Patient preparation
	<ol>
		<li>Check the patient inclusion criteria: examine adult patients diagnosed with polyneuropathy, suspicious of inflammatory origin.</li>
		<li>Check the patient exclusion criteria: do not examine patients with open wounds or infections in the regions to be examined.</li>
	</ol>
	</li>
	<li>Instrumental checkpoints
	<ol>
		<li>Check the integrity of the ultrasound machine and all the materials used (see Table of Materials).</li>
		<li>Enter patient name and details in the ultrasound machine before starting the ultrasound examination (depending on the machine).</li>
		<li>Choose an appropriate ultrasound probe (preferred 14-18 MHz) (see Table of Materials) and preset for the neuromuscular ultrasound.</li>
		<li>During the whole examination, adjust the depth and focus on obtaining optimal image quality.</li>
		<li>Whenever possible, examine the complete course of each nerve in a cross-sectional view. 
		NOTE: The nerves recommended for examination are: median, ulnar, radial nerve, cervical roots, brachial plexus, and vagal nerve, as well as tibial, fibular, and sural nerve (Figure 3). The examination of each of these nerves is shown in the next section and the video. The entire ultrasound examination according to the following protocol will take ~30-45 min.</li>
	</ol>
	</li>
</ol>
2. Ultrasound examination
<ol>
	<li>Start examining the arm nerves with the patient sitting in a neutral position with the arm resting supinated on a surface, e.g., the leg.</li>
	<li>Place some ultrasound gel over the transducer probe, the wrist, the forearm, the elbow, and the upper arm.</li>
	<li>For examination of the median nerve, start by performing a transverse scan at the wrist level.</li>
	<li>Move proximally to follow the anatomical course of the median nerve to the upper arm.</li>
	<li>Measure the CSA of the median nerve at the following sites: at the entrance to the carpal tunnel (retinaculum flexorum); at the forearm (10-15 cm proximal to retinaculum flexorum; at the elbow (crook of the elbow); at the upper arm next to the brachial artery (at the middle of the distance between the medial epicondyle and axillary fossa).</li>
	<li>For examination of the ulnar nerve, start by performing a transverse scan at the level of the wrist ulnar to the median nerve.</li>
	<li>Move proximally to follow the anatomical course of the ulnar nerve along the sulcus to the upper arm. 
	NOTE: Moving toward the upper arm, let the patient raise the arm bent at the elbow to examine the sulcus and upper arm.</li>
	<li>Measure the CSA of the ulnar nerve at the following sites: at the entrance to Guyon&#39;s canal; at the forearm (10-15 cm proximal to Guyon&#39;s canal); at the elbow (between the medial epicondyle and olecranon); at the upper arm (at the middle of the distance between the medial epicondyle and axillary fossa).</li>
	<li>To examine the radial nerve, let the patient hold the arm in front of the stomach bent in the elbow and scan the radial nerve directly next to the humerus.</li>
	<li>Use color duplex mode to avoid confusion with the accompanying artery and vein. 
	NOTE: Color duplex mode shows blood flow in arteria profunda brachii, and might show low flow in the corresponding vein, while no flow occurs in the radial nerve. Additionally, the vein can be compressed by exerting external pressure, and the nerve cannot.</li>
	<li>Measure the CSA of the radial nerve at the following site: radial nerve in the spiral groove.</li>
	<li>Carry on with the examination of the vagal nerve, cervical nerve roots, and the brachial plexus.</li>
	<li>Place ultrasound gel at the middle of the neck.</li>
	<li>To examine the vagal nerve, perform a transverse scan at the middle of the neck and find the carotid artery. 
	NOTE: The vagal nerve can be found directly next to the carotid artery and jugular vein.</li>
	<li>Measure the CSA of the vagal nerve at the following site: at carotid sheath at the level of the carotid bifurcation.</li>
	<li>For the examination of cervical nerve roots, C5, C6, C7 move the probe dorsal and a little up and down. 
	NOTE: The cervical nerve roots appear between the anterior and posterior tubercle of the transverse process. C7 can be recognized by the absence of the anterior tubercle from its transverse process, while both anterior and posterior tubercles are found with the other cervical nerve roots.</li>
	<li>Measure either the CSA or the diameter of the cervical nerve roots at the most proximal location possible, where the nerve root exits over the transverse process: C5; C6; C7.</li>
	<li>To examine the brachial plexus, follow the anatomical course of the cervical nerve roots distally and find them perform trunks and cords.</li>
	<li>Measure the CSA of the plexus at the following sites: Intrascalene space (between anterior and medial scalene muscle); Supraclavicular space (next to A. subclavia).</li>
	<li>Carry on with the examination of the leg nerves.</li>
	<li>Let the patient lie down to one side with the legs slightly bent. Place some ultrasound gel over the transducer probe, the popliteal fossa, the fibula, the malleolus, and the lower leg.</li>
	<li>For examination of the fibular nerve, feel the fibular head, place the transducer directly behind it, and then follow the course of the nerve to the popliteal fossa.</li>
	<li>Measure the CSA of the fibular nerve at the following sites: just proximal to the &#64257;bular head; in the popliteal fossa.</li>
	<li>To examine the tibial nerve in the popliteal fossa, find the fibular nerve and the popliteal artery in the popliteal fossa. 
	NOTE: The tibial nerve can be found just above the popliteal artery in most cases.</li>
	<li>Measure the CSA of the tibial nerve at the following site: in the popliteal fossa.</li>
	<li>For examination of the tibial nerve at the ankle, place the probe directly behind the medial malleolus. 
	NOTE: The tibial nerve can be found just next to the posterior tibial artery in most cases.</li>
	<li>Measure the CSA of the tibial nerve at the following site: at the level of the medial ankle.</li>
	<li>For examination of the sural nerve, place the probe at the lateral ankle. 
	NOTE: The sural nerve can be found next to a superficial vein in most cases.</li>
	<li>Follow the anatomical course of the sural nerve proximally to the lower leg.</li>
	<li>Measure the CSA of the sural nerve at the following site: between the lateral and medial head of the gastrocnemius muscle.</li>
	<li>Perform all measurements on both sides.</li>
	<li>Save the results of all the measurements (depending on the ultrasound machine) and end the examination. 
	NOTE: Figure 3 gives an overview of all measuring sites for CSA.</li>
</ol>

The authors declare no conflicts of interest related to this manuscript.

Nerve ultrasound is a helpful additional diagnostic tool in polyneuropathies. It can give information on the possible causes of polyneuropathy depending on the extent and pattern of nerve enlargement. Moreover, CSA alterations in the longitudinal disease course of patients with CIDP were described to correlate to clinical disease course and treatment response33,34,35,36.
<p class="jove_cont...

Next to clinical examination, evaluating any large-fiber polyneuropathy includes an electrophysiological examination to characterize the motor or sensory system&#39;s involvement and differentiate axonal from demyelinating damage1. In axonal polyneuropathy, toxic and diabetic neuropathy are the leading causes, while in demyelinating polyneuropathies, hereditary or inflammatory neuropathies such as CIDP should be considered2,3,4. Commonly used diagnostic criteria for CIDP are the European Federation of Neurological Societies/Peripheral Nerve Society (EFNS/PNS) criteria established in 2005 and revised in 2010 and 20215. These define clinical and electrophysiological criteria to diagnose CIDP and describe additional criteria such as nerve biopsy to detect demyelination or inflammation. However, in some cases, despite a thorough diagnostic workup, the cause of neuropathy remains ambiguous. In these cases, nerve ultrasound offers a complementary method to examine the nerves not functionally but morphologically6. Several studies proved the use of nerve ultrasound as an additional tool in diagnosing CIDP, so that the 2021 revised EFNS/PNS criteria implemented nerve ultrasound in the guideline5. The advantage of nerve ultrasound compared to other imaging methods such as magnet resonance neurography (MRN) is that it can be used directly by the treating neurologists as a bedside tool; it is relatively cost-efficient. It can be used repeatedly, as it is noninvasive and not painful.
Typical characteristics of CIDP observed in nerve ultrasound are cross-sectional-area (CSA) increase7,8, also found in hereditary polyneuropathies. In CIDP, this affects individual nerve segments heterogeneously7,9.
A variety of examination protocols have been published10,11,12,13,14,15&#160;trying to clarify normal CSA values and determine the adequate anatomical positions of ultrasound examination. Some of these positions are similar in most examination protocols. However, a widely accepted protocol to standardize the examination process and simplify the interpretation of the measurements does not exist.
This article demonstrates the nerve ultrasound examination using a standardized protocol for polyneuropathies, presents various reference values for the CSA, and shows typical pathological findings in patients with inflammatory neuropathies.
Technical requirements for nerve ultrasound 
The neuromuscular ultrasound is performed in B-mode (Brightness mode, two-dimensional image with gray levels) using the compound imaging of the corresponding sonographic device6,16. Compound imaging enables electronic control of the piezoelectric elements in the sonic probe (transducer) to illuminate the target structure from different angles17. The ultrasound waves are reflected in several directions due to the histological structure of the peripheral nerves. As a result of the sound coming from different angles, a more significant part of the otherwise lost reflections gets back to the sound probe (receiver) and can generate images. For neuromuscular ultrasound, a high-resolution ultrasound probe with 18 MHz linear array transducer, for deeper nerves, an additional 12 MHz linear array probe (e.g., to display tibial and fibular nerve in the popliteal fossa) is used6,16. Transducers with lower frequencies result in reduced spatial and lateral resolution so that the differentiation of the nerve boundaries from the surrounding structures is less precise. The optimal settings can be kept constant using a preset for neuromuscular imaging provided by the manufacturer. During the examination, the image depth and the focus position must be adjusted to the structure to be examined and constantly adapted to the position of the nerve. The B-image gain and the depth-dependent gain can be adjusted for image optimization with uniform brightness. Blood vessels are often close to neural structures and are often used as landmarks to make the measurements at the same position. To depict their anatomical interaction and distinguish between nerves and vessels, it is also necessary to display the flow velocity and direction using pulsed Doppler and color-coded duplex sonography16,18. The pulse repetition frequency must be adapted to the expected low flow velocities in the&#160;blood vessels of the extremities, or the power Doppler must be selected for color-coding16.
Nerves reflect the ultrasound waves differently from different angles of incidence so that the sonographic image varies in echogenicity (anisotropy)16,19. The best image is achieved from an orthograde angle since the ultrasonic waves are reflected most strongly by the nerves in this angle. For avoiding artificial anisotropy or nerve deformity, the probe must therefore be held in a neutral position during the examination without applying additional pressure perpendicular to the nerves (Figure 1). The cross-sectional area (CSA) is measured within the thin, hyperechoic epineurium (Figure 2) to avoid alterations of the epinerval tissue in the measurement19. More details on technical ultrasound can be found in References6,16,17,18,19,20,21.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Affiniti 70</td>
			<td>Philips GmbH</td>
			<td>n/a</td>
			<td>with preset for neuromuscular ultrasound</td>
		</tr>
		<tr>
			<td>L18-5 linear array transducer</td>
			<td>Philips GmbH</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>C + V Pharma Depot GmbH</td>
			<td>n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

nerve ultrasound protocol to detect dysimmune neuropathies

Nerve ultrasound is increasingly used in the differential diagnosis of polyneuropathy as a complementary tool to nerve conduction studies. Morphological alterations of the peripheral nerves, such as increasing the cross-sectional area (CSA), have been described in various immune-mediated polyneuropathies. The most prominent morphological changes in nerve ultrasound have been described for the chronic inflammatory demyelinating polyneuropathy (CIDP)-spectrum disease. CIDP may be distinguished from hereditary and other polyneuropathies by measuring the extent and pattern of nerve swellings (CSA increase). Typical findings in demyelinating inflammatory neuropathies are multifocal nerve swellings with inhomogeneous fascicular structure, while CSA increase in demyelinating hereditary neuropathies occurs in a more generalized and homogenous manner. In other non-inflammatory axonal neuropathies, nerves can appear with normal or slight CSA increases, especially in typical entrapment sites. This article presents technical requirements for nerve ultrasound, an examination procedure using a standardized examination protocol, current reference values for the CSA, and typical sonographic pathological findings in patients with inflammatory neuropathies.

Each ultrasound laboratory should establish its CSA reference values by collecting data from the healthy local population, as specific ultrasound machines and examiner or population-dependent variables can lead to slightly different results in each laboratory. However, to indicate which CSA values can be considered normal, data from two leading German nerve ultrasound groups and a recent meta-analysis of all published reference values so far13,14,<sup ...

Watch this Scientific Journal Video about Nerve Ultrasound Protocol to Detect Dysimmune Neuropathies at JoVE.com

Nerve Ultrasound Protocol to Detect Dysimmune Neuropathies

This article presents a protocol for nerve ultrasound in polyneuropathies to aid the diagnosis of inflammatory neuropathies.

Nerve ultrasound is increasingly used in the differential diagnosis of polyneuropathy as a complementary tool to nerve conduction studies. Morphological ...

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2.4K Views.  Ruhr-University Bochum. This article presents a protocol for nerve ultrasound in polyneuropathies to aid the diagnosis of inflammatory neuropathies.

Video: Nerve Ultrasound Protocol to Detect Dysimmune Neuropathies

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Quantification of Optic Nerve Cross Sectional Area on MRI: A Novel Protocol using Fiji Software

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