Data from patients with a clinical diagnosis of an AAN who were diagnosed and treated in our outpatient clinic for autonomic disorders between July 2012 and June 2019 were retrospectively evaluated. Inclusion criteria for the AAN group were based on current literature concerning the occurrence, course, symptoms and diagnosis of AAN. Patients were assigned to the diagnosis of AAN if the following criteria were fulfilled:

1.

Patients experiencing initial symptoms between the ages of 35 and 60 years, oriented at the mean values of symptom occurrence by Nakane et al. [1] and Suarez et al. [2] Typical causes and course of AAN: autoimmune or neoplastic diseases [1] and/or a typical clinical course (at least 1): previous event:, e.g., viral infection [1, 2], onset: subacute or gradual [1], orthostatic hypotension as the first autonomic symptom [1, 3] and/or a monophasic course without complete recovery [2]

2.

Typical clinical presentation (at least 5 typical autonomic symptoms or at least 3 autonomic symptoms combined with 1 extra-autonomic symptom)

Autonomic complaints [1, 2]: hypo/anhidrosis/heat intolerance, pupillary dysfunction, circulatory disorder, bladder disorder, sicca syndrome, dry skin, gastrointestinal symptoms and sexual dysfunction.

Extra-autonomic symptoms: sensory dysfunction, especially dysesthesia and neuropathic pain [2, 3] and central nervous system involvement –, e.g., psychiatric symptoms [1].

Typical examination results (at least 2): pathological skin biopsy [6], elevated gACHR-Ab [1] or N-type calcium channel antibodies [4], cardiovagal and adrenergic dysfunction on tilt table testing [2], low norepinephrine levels when supine [1], sudomotor dysfunction [2] in the sympathetic skin response and/or pinprick disease [3]—abnormalities in mechanical testing (QST).

The following exclusion criteria were defined to rule out Lambert Eaton syndrome: at least one typical examination result (typical electrophysiology or detection of PQ—calcium channel antibodies [7]) and one typical clinical sign (proximal muscle weakness, abnormal cranial nerves/oculobulbar symptoms and areflexia [8]) or two typical examination results.

Patients were excluded if pure autonomic failure (PAF) was suspected. PAF had to be considered if there were at least two clinical signs: olfactory complaints [9], advanced average age of more than 60 years [9], injuries of themselves or others while sleeping [9], hyperreflexia or Babinski’s sign [10].

Patients were classified as seropositive if they had antibodies against gACHR [1], N-type calcium channel [4] or other antibodies associated with autoimmune diseases affecting the autonomic nervous system [against gangliosides, potassium channel and glutamate-decarboxylase-65 (GAD-65)].

In the clinical routine, each patient was examined by an experienced physician as follows: detailed history, physical examination, blood sampling, QST, skin biopsy and tilt table testing with continuous blood pressure measurement.

The patient’s history included the experienced symptoms, the course of the disease, secondary diagnoses and demographic data such as age and sex. For analysis, the anamnestic data were processed in a qualitative manner and classified into subgroups based on complaints of the eyes, gastrointestinal tract, sweating, mouth and skin, bladder function, circulatory function, and neuropathic pain. Relevant subgroups for secondary diagnoses were other autoimmune diseases, neoplasia, Ehlers–Danlos-syndromes, systemic lupus erythematosus, Sjögren’s syndrome, and stiff person syndrome. Further criteria were the first autonomic symptom and the date of first diagnosis.

The physical examination included sensitivity, cranial nerves, pupillomotor function, temperature sensation and reflexes, as well as skin condition. In clinical routine, the blood sample (serum gel tube) taken was tested for antibodies against the following structures: N-type calcium channel (Radioreceptor assay, RRA), g-ACHR, (RRA), gangliosides (ELISA), GAD-65 (enzyme immunoassay), PQ-type calcium channel (RRA) and potassium channel (radioimmunoassay). These antibody samples were analysed in an external laboratory (SYNLAB, Leverkusen, Germany).

QST was used to examine small fibre function. It was performed in all patients under standardized conditions as described previously [11] on the dorsum of one hand and foot (mostly the left).

Skin biopsies were obtained from the right lateral lower leg (10 cm above the lateral malleolus) under local anaesthesia with 0.5 ml prilocaine and epinephrine (Xylonest, Aspen Germany GmbH, Munich, Germany). Intraepithelial nerve fibre density (PGP9.5 immunofluorescence), axon branching and focal swellings (> 1.5 µm), rarefication of the subepidermal plexus and innervation of imaged sweat glands were examined as described previously [12]. The IENFD was considered reduced when below the published normative values for the respective sex and age group [13]. Differences of autonomic symptom burden were compared between AAN patients with and without nerve fibre damage in the skin biopsies concerning autonomic symptoms, norepinephrine levels and tilt table diagnosis.

Two out of the following three criteria must be fulfilled for the diagnosis of a definite SFN [5]: (1) At least two clinical signs of SFN (e.g., reduced temperature sensitivity, or pinprick hypoalgesia, allodynia, hyperalgesia), (2) abnormal thresholds in QST and/or (3) reduced intraepidermal nerve fibre density (IENFD) in a punch biopsy taken from the distal leg.

Statistics

IBM SPSS version 25/27, STATISTICA 7.1 (Statsoft, Inc.) and Excel for Mac were used for statistical analysis. Qualitative results of anamnesis and physical examination were evaluated by frequency counts. The chi square test was used to investigate possible differences between seropositive and seronegative patients and in AAN patients with and without small fibre nerve damage regarding nominal values, and the Mann–Whitney U test was used to assess metric values.

QST values were compared with healthy controls (age: 55.0 ± 5.7 years; mean ± SD) that were matched for age and sex derived from the internal clinical database. After log- and z-transform, QST data were analysed using a two-way mixed ANOVA comparing groups (AAN vs. controls as ‘between factor’) and body regions (hand vs. foot as ‘within factor’). Differences in QST values depending on the serostatus were investigated with multiple ANOVAs based on z score QST data. Differences in IENFD were assessed using a Mann–Whitney U test.

Correlations of IENFD and QST values were assessed using correlation coefficients with IENFD as the determining factor. Missing data were removed pairwise. Finally, the symptomatic pattern, the presence of antibodies and the results of QST and skin biopsies of AAN patients were also presented descriptively.