Finger immersion test of vasomotor sympathetic function. Hand is immersed for 30 min in a heated water bath. Digital pictures are taken (from left to right) at baseline and after 5, 15 and 30 min of immersion. Pictures in (a) reflect changes in subject with small fiber peripheral neuropathy. Pictures in (b) are from normal control
Methods of Testing
Quantitative sensory testing: The younger a child is, the more difficult it can be to accomplish a reliable or quantitative sensory examination. When cooperation and focused attention allow quantitative testing of clinical sensory thresholds (thermal thresholds for small fiber function), normal results are helpful. However, it has been demonstrated that abnormal thermal thresholds can be feigned; therefore, abnormal quantitative thermal thresholds are less reliable . The frequency of false negative results for subjective pinprick thresholds in patients with objective evidence of SFN has been reported at 19%, higher than one might anticipate in adults . Common sense suggests that reliability would be even less in children. Computer-assisted sensory examinations are used in adult patients but the same caveats apply with respect to false positive results, particularly with younger patients who tend to be more distractable.
Nerve Conduction Studies: Sensory nerve conduction studies assess the function of large myelinated sensory nerve fibers. Some disorders may affect all types of nerve fibers; however, if the abnormality is limited to small nerve fibers, standard sensory nerve conduction studies will be normal.
Contact Heat Evoked Potential (CHEP): This technique applies cycles of heating and cooling using a heat foil thermode placed over glabrus skin on an extremity while recording the evoked potential over a CZ scalp electrode. This evoked potential is mediated by A delta fibers and demonstrates increased latency and decreased amplitude in subjects with documented small fiber neuropathy as compared to normal adult controls . This causes no tissue injury and is considered non-invasive but does involve a minor degree of discomfort. There are no published reports using this technique in children.
Microneurography: A technique has been developed to record impulse traffic within peripheral nerves using specialized coated tungsten electrodes. The burst frequencies from single unmyelinated afferent and efferent fibers tend to be relatively uniform within an individual over time. However, there are wide variations between individuals which make it difficult to establish reference ranges. The technique is time-consuming and requires a skilled evaluator. Movement interferes with the recording which is likely why this has not been applied to children. At present, this technique functions more as a research tool than a clinical diagnostic test .
While autonomic testing is relatively new, experience is being gained with the use of these techniques in children and adolescents. Normative data is being systematically gathered and it will be very helpful when this process is complete with results published.
Adrenergic: Short duration Head Up Tilt tests (HUT) are performed to assess the adrenergic support of heart rate and blood pressure in response to orthostatic stress. Protocols for autonomic tilt testing are simpler and shorter, and do not use infusions of vasoactive drugs, in contrast to cardiac tilt testing. A preceding interval of 10–30 minutes of supine rest is required followed by passive elevation to a 70° HUT on a motorized tilt table. Heart rate, blood pressure and clinical well-being are monitored for 10 minutes. Other parameters such as end-tidal CO2, oxygen saturation, respiratory rate and regional cerebral perfusion are also monitored in our laboratory. With the data obtained, other causes of dizziness, such as occult hyperventilation or isolated cerebral hypoperfusion, can be identified. Adolescents frequently present with dizziness and data have been gathered on healthy controls to demonstrate that the range of normal heart rate responses to HUT is broader in adolescents than adults . Normative data from larger groups of adolescents are being collected at present and will be useful for clinical correlation. Adrenergic function can also be assessed from evaluation of blood pressure responses during a Valsalva maneuver. The rebound increase of blood pressure during phase II of the Valsalva maneuver and the phase IV overshoot of blood pressure both rely on adrenergic support (Fig. 19.2).
Heart rate (upper tracing) and blood pressure responses (lower two traces) for a 7 year old girl performing Valsalva maneuver. Timing of Valsalva pressure indicated by solid bar above top graph. Cartoon below physiologic tracing identifies components of the blood pressure response to Valsalva maneuver
Cardiovagal: The heart rate response to deep breathing (HRDB) is a quantification of the variation in heart rate that occurs during normal breathing— “sinus arrhythmia”. It is easy to perform, requiring children to use visual or auditory cues to pace deep breathing at 6 cycles per minute while their heart rate is recorded. The mean is calculated from the maximal excursions of heart rate during the largest five consecutive responses. Normal values have been collected from 9 years of age and older with an inverse relationship between age and HRDB [10, 11]. Valsalva Ratio (VR) is vagally mediated and is the ratio of the highest heart rate occurring during a 15 second Valsalva maneuver (performed on a mouthpiece with an air leak) to the lowest heart rate occurring within 30 seconds of completion. Normal values in adults are between 1.5 and 2.9 and several studies [10, 11] have documented very similar findings 6–17 year olds. It is difficult to get younger children to sustain pressure on the mouthpiece long enough to accomplish this measurement. Cardiovagal responses can be obtained by other maneuvers including squat to stand, assuming a squat or forcefully coughing; however, reference ranges have not been adequately established for these responses in either adults or children.
Electrochemical Skin Impedance: A device that uses reverse iontophoresis and chronoamperometry to assess sweating on the palms and soles has been demonstrated to correlate with epidermal nerve fiber density. The Sudoscan device uses stainless steel plates against which palms and soles are placed for a 3 minute scan with the subject in a standing position . This is a recently developed device so, despite the relative simplicity of use, there are no reports describing the use of this technique in children.
Heart rate variability: Measures of heart rate variability (HRV) are obtained by monitoring the electrocardiogram (ECG) for intervals of time between 5 minutes and 24 hours, analyzing sequential R-R intervals. Heart rate variability is created by the naturally opposing influences of sympathetic and parasympathetic tone on the sinoatrial node (cardiac pacemaker). Physiologic state and age affect the degree of variability [13, 14]. While cardiologists report variables relating to time domain and frequency parameters, a scatter plot of successive R-R intervals (Poincaré plot) creates an immediate visual image of HRV (Fig. 19.3). Decreased variability appears as a cigar-shaped pattern with robust heart rate variability appearing in the shape of an ice-cream cone.
Poincaré plots of heart rate variability. Normal (middle graph): “ice cream cone” shaped plot with labels indicating higher heart rates in circle to left, lower heart rates in circle to right, “line of identity” in red. ROHHAD (Rapid onset Obesity, Hypoventilation, Hypothalamic Dysfunction and Autonomic Dysregulation) (right graph): narrow plot, note decreased heart rate variability at lower heart rates. CCHS (Congenital Central Hypoventilation Syndrome) (left graph): narrow “cigar” shaped plot indicating decreased heart rate variability
Pupillometry: Basal size of the pupils and velocity of change in size reflect a balance between sympathetic and parasympathetic tone. Normal values have been collected . The ambient light, medications and presence of any ocular disease must be taken into consideration when testing pupillary function. In addition, any stimuli that might trigger sudden anxiety or fear should be avoided as alterations in the sympathetic/parasympathetic balance will affect pupil function.
Sudomotor: Production of sweat in response to heat stress or emotional stress is a sympathetically-mediated autonomic function that is mediated by acetylcholine as the neurotransmitter. Function at the level of the peripheral axon reflex can be measured with quantitative sudomotor axon reflex testing (QSART/QSWEAT). A plastic capsule is used to focally place acetylcholine onto a patch of skin. A small electric current is used to iontophorese the acetylcholine through the skin where it stimulates the sweat glands. A retrograde impulse through the small nerves innervating the sweat gland reflexively stimulates an adjacent sweat gland and the amount of reflex sweating generated onto an adjacent patch of skin is measured. Four capsules are placed along the limbs in a length-dependent fashion (foot, distal leg, proximal thigh and distal forearm) and stimulated simultaneously . This technique has been used in toddlers and young children successfully; however, normative data are sparse under 10 years of age . A different technique can be used to evaluate the entire central to peripheral sudomotor apparatus: thermoregulatory sweat testing (TST). An individual who is well-hydrated and who is not taking any antihistamine or other medication that suppresses sweating is placed in a controlled hot and humid environment. It takes 45–60 minutes in such a setting to increase core temperature by 1°C or to 38.0°C (whichever is higher). All individuals without disease will sweat under these conditions. A moisture sensitive powder is placed over the ventral surface of cheeks, trunk and extremities. A color-coded response is evident, indicating where the individual subject was able to sweat . Lesions from the hypothalamus through the spinal cord and out to focal/generalized nerve distributions can interfere with sweating. The disadvantage of the TST is that it requires that a special chamber or environment be fabricated to warm the patient. The advantage is that this procedure is non-invasive and can be repeated over time to follow the course of a disease (Fig. 19.4). Also, the entire ventral surface of the body is being studied and focal and/or strictly length dependent abnormalities can be outlined (Fig. 19.5). In 2011, a decade’s worth of experience obtaining TSTs in children at Mayo Clinic Rochester was reviewed. Children between 2 and 18 years of age were examined with valid, clinically useful results obtained in 94% of studies .
TST (Thermoregulatory Sweat Test) performed serially in a 6 year old boy with small fiber peripheral neuropathy to demonstrate ongoing clinical improvement
TST (thermoregulatory sweat test). (a) Demonstrates involvement of only the distal foot/toes that was not detected by standard QSART testing which assesses proximal foot as most distal site. (b) Demonstrates length dependent anhidrosis involving both lower extremities. (c) A cartoon of the sweat pattern on the entire body, for the subject in (b), demonstrating trunk and upper extremity involvement as well
Sympathetic Skin Response (SSR): Surface electrodes placed over the palm and sole of the feet are used to record sympathetic skin responses (SSR) . A number of types of sensory stimuli can be used to provoke this response in sympathetic nerves: auditory, tactile, electrical. A novel use of SSR was described by Pereon and colleagues when they used auditory signals of varying intensity to determine the hearing threshold for a child post cochlear implant . SSR is non-invasive but habituates quickly to repeated sensory stimuli. It is usually judged on its presence or absence.
Vasomotor: Vasoreactivity has not been studied extensively with techniques that are clinically applicable. One simple test that has been developed is to look for wrinkling or “pruning” of the skin in response to timed water immersion or application of a topical anesthetic under an occlusive dressing. Development of pruny skin is a sympathetically mediated vasoconstrictive phenomenon  (Fig. 19.1). Skin changes have been subjectively graded.
Standard Nerve Histology: Peripheral nerve biopsies are a “gold standard” for evaluating the constituent elements of nerves. However, biopsies are invasive, require general anesthesia in children and leave a small functional sensory or motor deficit. Sural sensory nerves are traditionally biopsied just proximal to the lateral malleolus in diffuse or generalized processes and the ensuing clinical sensory deficit is minimal and generally well-tolerated. Nevertheless, the invasive nature of this procedure, and the difficulties inherent in repeating such a biopsy to assess response to treatment, have led to development of less invasive testing methods.
Intraepidermal Nerve Fiber Density (IEFND): Small nerve fibers can be seen (and quantified) in the epidermis and in sweat glands. In adults, 3 mm full thickness skin biopsies are obtained under local anesthesia with a skin punch biopsy tool. Samples are usually taken at five sites along the limbs. Normal values for epidermal nerve fiber density, nerve fiber length and nerve branching density have been obtained from 18 to 90 years of age . McArthur and colleagues obtained 3 mm skin punch biopsies under local anesthesia for 98 patients, eight of whom were between 13 and 19 years of age (mean 18 years). The IENFD at the level of the thigh and distal leg were both higher than in other controls aged 20–79 years of age. However, the thigh/distal leg ratio was the same as in all other controls up to 60 years of age . While this type of biopsy is less invasive than a standard nerve biopsy, it is still invasive and more difficult to justify performing without clear normal values for young children and adolescents, especially at multiple sites.
Sweat Gland Nerve Fiber Density: Sweat glands are incidentally obtained in most of the skin punch biopsies done to determine IEFND . Normative values are available for adults but not yet for children and adolescents.
Corneal Confocal Microscopy (CCM): A non-invasive method for evaluating nerve integrity, corneal confocal microscopy, has been used to quantify corneal nerve fiber density, corneal nerve fiber length and corneal nerve branch density in various medical conditions prone to small fiber neuropathy such as diabetes mellitus. Chen and colleagues evaluated a cohort of 63 patients with type I diabetes mellitus between 14 and 85 years of age and an equivalent number of age-matched controls. Motor and sensory nerve conduction studies, IENFD and CCM were performed on all subjects. While IEFND has been shown to detect early small nerve fiber injury when nerve conduction studies and quantitative sensory testing are still normal, CCM was shown in this study to have a slightly better sensitivity and specificity. CCM is non-invasive and can be scored in an automated manner that provides rapid results. Obtaining corneal tomographic images does not require undue cooperation from children and adolescents so it is a promising biomarker of early diabetic neuropathy [22, 23].
MR Neurography (MRN) with Dorsal Root Ganglion Imaging: An MRN protocol has been developed that allows imaging of bilateral dorsal root ganglia. This has been used to evaluate patients with Sjögren syndrome when other quantitative sensory physiologic tests were unrevealing . The imaging demonstrated enlargement of the dorsal root ganglia in affected individuals that was reversed after immunotherapy. The limitation for this procedure in children is the time required for them to be motionless during imaging, as in other magnetic resonance imaging studies; therefore, it would likely require anesthesia in younger children.
Ultrasound: 25 adults with small fiber peripheral neuropathy confirmed by IEFND and age/BMI-matched controls were studied by ultrasound. Cross-sectional areas of superficial peroneal sensory and sural sensory nerves was increased in the subjects with SFPN as compared to controls. This technique does not provide much information regarding the specific etiology but may serve as a non-invasive, low cost initial screening approach .
If the phenotypic presentation and/or family history strongly suggest a Mendelian disorder and a gene test (or affordable gene test panel) is available to confirm that diagnosis, proceeding directly to genetic testing may be a minimally-invasive and cost efficient approach. There are circumstances when physiologic testing, in conjunction with the clinical findings, will significantly narrow the differential diagnosis. For example, findings on nerve conduction studies and/or pupillometry can focus the diagnostic possibilities to the spectrum of inherited peripheral neuropathies .