This study aims at validating a two-step protocol for Plexin D1 antibodies (IgGs) detection and to at investigating their frequency in cryptogenic small fiber neuropathies (SFNs).

Plexin D1-IgGs are a recently discovered antibody biomarker of neuropathic pain. Intrathecal injection of Plexin D1-IgG induces mechanical and thermal hypersensitivity in animal models.
Plexin D1-IgG have been reported in patients with SFNs with a prevalence ranging from 9% to 13% with heterogeneity in cohort selection and assays employed for antibody testing. 

An in-house enzyme-linked immunosorbent assay (ELISA), using recombinant extracellular domain of Plexin D1 was developed. 56 samples from healthy controls (HCs) were used to determine positivity cut-off (>5 standard deviations). Optic deviation was normalized for serum-specific background noise. Tissue-based immunofluorescence assay (TBA) on rat dorsal root ganglia (DRG) was employed to confirm ELISA positive samples. TBA was defined as positive when surface staining of small DRG cells was observed.
Forty-one patients with cryptogenic SFN, 10 patients with defined non-autoimmune SFN and additional 7 HCs with available serum samples were retrospectively enrolled. All patients fulfilled “definite” criteria for SFN.

Plexin D1-IgG were detected in 5/41 patients with cryptogenic SFN by ELISA and confirmed by TBA in 3/41 (7%). ELISA positive, but TBA negative samples had borderline Plexin D1-IgG ELISA results.
Plexin D1-IgG were not identified in HCs or non-autoimmune SFNs (0/17).
Two Plexin D1-IgG positive patients (66%) were female and median age at onset was 32 years (median: 28-36). All patients had subacute onset of neuropathic pain in a non-length dependant distribution in 2/3 (66%). 

Plexin D1-IgG are detected in 7% of cryptogenic SFN. Clinical features of Plexin D1-IgG positive patients were suggestive of an autoimmune etiology. A two-step approach employing ELISA for screening and TBA for confirmation improves diagnostic accuracy compared to ELISA alone.