Abstract
Objectives:
laboratory tests for work-up of hereditary and acquired neuropathies of peripheral nerves are frequently uncritically utilized. This overview focuses on the most common laboratory tests and investigations needed for diagnosing PNPs by the general neurologist.
Method:
Literature search.
Results:
laboratory tests recommended for the work-up of hereditary and acquired neuropathies should be chosen according to the individual and family history, clinical presentation, and electrophysiological findings. Laboratory tests should be selected specifically according to the suspected type of neuropathy to avoid unnecessary tests and expenses. Work-up should include as few samples as necessary for uncovering the etiology and should consider the sensitivity/specificity of the tests applied.. Basic screening tests for neuropathies should include a blood cell count, thyroid, renal and liver function tests, blood glucose levels, HbA1c, vitamin-B12, and immunofixation. Other laboratory investigations should be carried out only if a specific phenotype is present or if unexpected changes of the disease course occur. In these cases referral to a neuromuscular center is recommended.
Conclusions:
Laboratory tests are helpful for the diagnosis of acquired and hereditary neuropathies but these tests should be ordered according to the history, clinical presentation and findings on electrophysiological investigations. If basic laboratory parameters fail to uncover the etiology, patients should be referred to a center specialized in neuromuscular disorders.
LIST OF ABBREVIATIONS
Acute motor axonal neuropathy Bickerstaff brainstem encephalitis Blood sedimentation rate Chronic ataxic neuropathy, ophthalmoplegia, IgM paraprotein, cold agglutinins and antidisialosyl antibodies Contactin-associated protein Chronic idiopathic axonal polyneuropathy Circulating immune complexes Chronic inflammatory demyelinating polyneuropathy Contactin-1 C-reactive protein Cerebrospinal fluid Distal acquired demyelinating and sensory neuropathy Electromyography Extractable nuclear antigen Guillain Barre syndrome Glomerular filtration rate Myelin-associated glycoprotein Miller-Fisher syndrome Multifocal motor neuropathy Nerve conduction studies Next generation sequencing Oral glucose tolerance test Pharyngeal-cervical-brachial variant of GBS Polyneuropathy PNP, organomegaly, endocrinopathy, monoclonal protein, skin changes Rheuma factor Anti-Sjögren’s-syndrome-related antigen-A Anti-Sjögren’s-syndrome-related antigen-B Trans-glutamine-2 antigen Varicella zoster virus
INTRODUCTION
Polyneuropathies (PNPs) are among the most common neurological disorders, with a prevalence of 2–3% in the general population [1]. The prevalence increases with age amounting to 8% for patients > 55 years of age and 24% for patients > 65 years of age [2]. Besides the clinical examination and nerve conduction studies (NCSs) and electromyography (EMG), laboratory investigations are an important part of the diagnostic procedure. However, in 11–31% of the PNPs, the underlying cause remains unclear [3], why they are called chronic idiopathic axonal PNP (CIAP). However, also causes of demyelinating PNPs may remain unclear. The prevalence of CIAP increases with age. In patients with a PNP aged 70–79 years, 32% are diagnosed with a CIAP whereas only 12% are diagnosed with a CIAP in patients aged 40–49 years [4]. In clinical practice a standard laboratory test battery is often used for all PNPs. Mostly, this test battery consists of a wide range of investigations, rather than specific investigations selected for a particular patient. Ideally, laboratory testing should be adopted to the PNP phenotype and the disease progression. A subacute symmetric motor PNP demands a different approach than a chronic symmetrical sensory PNP. Recent studies showed that even in distal symmetric PNPs recommended tests are underutilized while other expensive unnecessary tests are widely used [5, 6].
This paper discusses the most important and most common laboratory tests used in the diagnostic work-up of PNPs and addresses the relevance and diagnostic yield of these tests in relation to the different PNP phenotypes. Such a phenotype-based selection of laboratory tests should guide general neurologists to choose the optimal laboratory test in the evaluation of PNPs. Electrophysiological classifications should be included in the consideration of diagnostic tests. This overview focuses on the common laboratory tests and investigations needed for diagnosing PNPs by the general neurologist.
METABOLIC TESTS
Glucose, HbA1c, oral glucose-tolerance test (oGGT)
Diabetes mellitus is the most common cause of a PNP in Western Europe [7]. Diabetic PNP typically causes chronic distal symmetrical sensory PNP or a distal symmetrical small-fiber neuropathy. In 11% of the undiagnosed patients with such a phenotype, an increased blood-glucose is found [8]. If the HbA1c value is > 5.6 an oGGT should be carried out. An abnormal oGTT can be found in 11–21% of these patients [8]. Although in patients with newly diagnosed diabetes, a distal symmetric PNP was more common that in the general population, this was not the case for patients that had increased glucose-intolerance [9]. However, there is a strong association between metabolic syndrome, obesity and dyslipidemia with distal symmetric PNP [10].
RENAL FUNCTION
In patients who undergo dialysis due to chronic renal failure, 60–100% develop a PNP [11]. These PNPs are axonal and primarily sensory but patients may also develop motor symptoms with disease progression. PNPs develop particularly in patients suffering from severe kidney-dysfunction (GFR < 12 ml/min), while mild kidney-dysfunction does usually not cause PNP [11].
THYROID FUNCTION
In patients with thyroid dysfunction (either hypo- or hyperthyroidism), 19–42% had symptoms of a distal symmetric PNP, which could be confirmed by NCSs in 17–24% of the patients (axonal) [12]. On the other hand, in patients with distal symmetric PNP, thyroid dysfunction is found in only 0–4% [13].
BLOOD CELL COUNT, CRP/BSR, AND LIVER FUNCTION PARAMETERS
Although these measures are routinely checked in clinical practice during the diagnostic work-up of a PNP, they are non-specific. These measures merely give a global indication for systemic disease that may underlie the PNP, for example auto-immune diseases (see below). CRP and BSR may be elevated in patients with infectious neuropathy or vasculitis.
VITAMINS
Vitamin-B12, methyl-malonate and homocysteine
Vitamin-B12 deficiency can cause a distal symmetric PNP, with mainly (as in funicular myelosis) proprioceptive dysfunction. About 4% of the distal symmetric PNPs are caused by vitamin-B12 deficiency [8, 12]. Important to note is that a normal blood level of vitamin-B12 does not exclude a clinical relevant, relative vitamin-B12 deficiency. In these cases increased methyl-malonate and homocysteine levels can help diagnosing the relative vitamin-B12 deficiency [14]. However, methyl-malonate and homocysteine can be also increased in hypothyroidism, kidney-dysfunction or hypovolemia. Homocysteine is also increased in vitamin-B6 deficiency and in patients with heterozygote homocysteinemia [2]. In these cases, the clinical presentation determines the relevance and interpretation of abnormal laboratory findings.
Determination of vitamin-B1, folic acid, vitamin-B6, or vitamin-D should not be carried out routinely as neuropathies due to deficiency or intoxication, as in the case of vitamin-B6, are rare [15].
PARAPROTEIN
A paraprotein is found in about 1% of the general population and 3–4% in the population > 50y of age, but the majority is non-symptomatic [16]. However, paraproteins are more frequently found in patients with undiagnosed PNP than in the general population [17], IgM paraproteins are strongly associated with PNP, while a causal relation between IgG or IgA paraproteins and PNP remains unclear, unless in the context of PNP, organomegaly, endocrinopathy, monoclonal protein, skin changes (POEMS) syndrome or AL amyloidosis [18]. IgM paraproteinemic neuropathies typically present as chronic, ataxic, sensory neuropathies with absent or minimal clinical weakness although NCSs show marked prolonged distal motor latencies (distal acquired demyelinating and sensory (DADS) neuropathy). Approximately 50% of these patients have antibodies against the myelin-associated glycoprotein (MAG). MAG antibodies may be also elevated in patients with psoriatic neuropathy. In patients with chronic, ataxic neuropathy with ophthalmoplegia, M-protein, cold agglutinins and disialosyl antibodies (CANOMAD) antibodies against GQ1b, GD1b, GD3, or GT1b are frequently elevated (Table 1). When paraprotein testing is ordered, serum protein immunofixation electrophoresis is preferable over serum protein electrophoresis as it is more sensitive to detect mild gammopathies [19]. Once a monoclonal gammopathy is detected an oncological work-up is mandatory due to the risk of an occult malignancy [20, 21].
MMN: multifocal motor neuropathy, AMAN: acute motor axonal neuropathy, AMSAN: acute motor and sensory axonal neuropathy, PCB: pharyngeo-cervico brachial subtype, BBE: brainstem Bickerstaff encephalitis, CANOMAD: chronic ataxic neuropathy, ophthalmoplegia, IgM paraprotein, cold agglutinins and antidisialosyl antibodies.
COLLAGENOSIS-, IMMUNE-NEUROPATHY-, AND VASCULITIS-ASSOCIATED ANTIBODIES
PNPs occur in 10–80% of patients with auto-immune disease, such as systemic lupus erythematosus, rheumatoid arteritis, sclerodermia, Wegener-granulomatosis, Churg-Strauss syndrome, Sjögren syndrome, polyarteriitis nodosa und microscopic polyangiitis [22]. As a presenting symptom, the phenotype is mostly that of a mononeuritis multiplex, promoting work-up for these differentials. Distal symmetric or, less common, autonomic PNPs, were also described, but seem to be rare and occur mostly in chronic cases when the diagnosis has already been established. A sensory PNP with profound ataxia can be the presenting symptom of Sjögren syndrome [23].
ANA, p- and c-ANCA, rheuma-factor (RF), SSA, SSB, C3, C4, C3d, CIC, anti-dsDNA, ENA
ANA, p- and c-ANCA and RF are useful laboratory tests to screen for an undiagnosed auto-immune disorder. If they are abnormal, further analysis can follow measuring C3, C4 and C3d, as well as the circulating immune-complexes (CIC). In case ANA are increased, further investigations should include ENA, SSA, SSB, anti-dsDNA, and anti-CCP antibodies to further assess the relevance of the findings.
Angiotensin-converting enzyme (ACE) and soluble interleukine-2-receptor (sIL2)
In 6–18% of patients with sarcoidosis, a neuropathy is the presenting symptom; mostly a small-fiber-neuropathy or a mononeuritis multiplex [24]. In case of clinical suspicion of a sarcoidosis, ACE and sIL2 should be determined [25]. If serum levels are normal, the test should be repeated in the cerebrospinal fluid (CSF).
Endomysium IgA and trans-glutamine-2 (TG2)
Whether coeliac disease causes PNP or not is still under debate [26]. In a study of 28.232 patients with coeliac disease based on gut-biopsies, only 0,7% had neuropathy. When testing for coeliac disease is considered, antibodies against endomysium (IgG and IgA) and against trans-glutamine-2 (TG2, IgA) are more specific than anti-gliadine-antibodies, which are neither specific, nor sensitive. Endomysium IgA or TG2 antibodies should be determined if celiac disease associated neuropathy is suspected but celiac disease is not yet confirmed [27]. Celiac disease-associated neuropathy is a small or large fiber neuropathy and most frequently of the axonal type.
Neuropathy specific antibodies
Anti-ganglioside antibodies
Ganglioside antibodies can be found in acute and chronic neuropathies [28]. The various antibodies are usually associated with very specific phenotypes (Table 1). Acute neuropathies are associated with IgG-antibodies; chronic neuropathies with IgM-antibodies. It should be noted that low levels of these antibodies can also be found in the general population. Specific anti-ganglioside antibodies should be determined only in case of clinical and electrophysiological evidence for a neuropathy as listed in Table 1.
Nodal and paranodal antibodies
In recent years, nodal and paranodal antibodies were found in patients with acute and subacute immune-mediated neuropathies. Mostly antibodies against neurofascin-isoforms (NF-155 and NF-186) or contactin-1 (CNTN1) were reported. Also a few patients with antibodies against CASPR-1 were detected [29]. In case series antibodies against CNTN1 were found in 2,2–8,7% of the patients, against NF-155 in 2,0–8,0% and against NF-186 in < 2% of patients with immune-mediated neuropathies [30]. Antibodies against CNTN1 are of the IgG4 isotype and the significance of IgM isotype antibodies is currently unknown [31]. Patients, positive for anti-NF-155 antibodies, often present with an onset at early age, show tremor and ataxia, and do not respond well to immunoglobulins. Recently, IgM-NF155 antibodies have been found in 2–4% of CIDP patients [32]. Because of reduced availability and high costs of these tests, they should be carried out only in case of an undermined immune-neuropathy.
Paraneoplastic antibodies
Paraneoplastic neuropathies are mostly associated with anti-Hu and anti-CV2/CRMP5 antibodies. Anti-Ma2, anti-Yo and anti-amphiphysin antibodies were only described in single cases [33]. The prototypic phenotype of an anti-Hu or anti-CV2/CRMP5 mediated neuropathy is a subacute, sensory, multifocal or asymmetric PNP. The upper extremities, face and trunk can be involved at an early stage of the disease and these neuropathies are frequently painful or ataxic [34]. Paraneoplastic antibodies should be ordered if there is subacute sensory neuropathy and if an occult neoplasm is suspected, if there was no complete remission of a neoplasm, or if neuropathy started time-linked to the development of the malignoma.
VIROLOGICAL AND BACTERIOLOGICAL EXAMINATIONS
Infectious neuropathies are rare in the Western world. However, some infectious diseases should be excluded if the patient history fits. The most frequent infectious neuropathy is acute ganglionitis due to varicella-zoster virus (VZV) [35], why VZV antibodies should be determined in case of acute or subacute, painful proximal neuropathies. Hepatitis-C and associated cryoglobulinemia can cause a vasculitis presenting as mononeuritis multiplex or as painful pure sensory or sensorimotor neuropathy [36]. HIV-infections can cause a distal symmetric neuropathy which is mostly sensory [37]. Infections with B.-burgdorferi-sensu-lato can cause a neuropathy as part of neuroborreliosis. If serum IgG- and IgM-antibodies are normal, neuroborreliosis becomes unlikely, except within the first 6 weeks after the infection [38]. When elevated, neuroborreliosis should be confirmed by CSF investigations showing pleocytosis, intrathecal immunoglobulin-synthesis (IgM, IgA, and IgG) and borrelia-specific antibodies (IgG or IgM). Cytokine CXCL13 can be elevated in neuroborreliosis but is non-specific as it is also increased in HIV-infection, neurosyphilis, and cryptococcal meningitis [39]. The clinical presentation of borreliosis-associated neuropathy is that of a radiculoneuritis (Bannwarth syndrome) or cranial nerve palsy. Particularly the facial nerve (80%) can be involved [40]. In the advanced stage of neuroborreliosis, axonal PNP or neuritis can develop in association with acrodermatitis chronica atrophicans.
Other infectious causes of neuropathies are rare in the Western world but should be considered in travelers, foreign workers, migrants or immigrants (e.g. ZIKA, diphtheria, leishmaniosis, chikungunya, leptospirosis, dengue) [41].
In Guillain-Barre syndrome (GBS) the proof of a preceding Campylobacter jejuni or mycoplasma infection can be helpful for determining the prognosis. Specificity and sensitivity of tests for detecting viral, bacterial, protozoal, funghal, or parasitological causes are usually high but some of these agents may be missed if not considered and if only non-specific tests were applied.
CEREBROSPINAL FLUID (CSF)
CSF investigations are helpful for the investigation of suspected acute or chronic immune-mediated neuropathies and for infectious neuropathies. In GBS patients, 64% show increased CSF protein. This number increases with duration of the disease: 49% on the first day, 88% after 2 weeks [42]. The cell count is typically normal (85%) and has to be below 50/μL [42]. In CIDP, CSF protein is typically increased > 100 mg/dl [43]. The diagnosis should be challenged if CSF protein values are normal or only marginally increased [44]. Recently, new reference values for CSF protein have been proposed [45] and their application to CIDP increased the specificity of elevated CSF protein in the diagnosis of CIDP from 39% to 57% –64% [46].
GENETICS
A hereditary neuropathy should be considered when a typical disease course, a typical phenotype with characteristic NCS findings, a positive family history, or other specific laboratory features (e.g. very-long chain fatty acids in adrenomyeloneuropathy) are evident. However, a negative family history does not exclude a hereditary neuropathy. In these cases foot deformity, distal calf atrophy, absent pain and unclear symptom onset, and ulcerations and mutilation suggest a hereditary cause. The most common genetic neuropathies are those caused by mutations in the PMP22, MPZ, GJB1 or MFN2 genes [47]. An overview is beyond the scope of this paper as many more genes are known today being responsible for various other hereditary neuropathies. The reader is here referred to the OMIM-database (ref_link) or other genetic databases. If the clinical and electrophysiological phenotype under investigation is very specific, like hereditary neuropathy with liability for pressure palsies (HNPP) or a uniformly demyelinating sensory-motor neuropathy, monogene testing can be considered (PMP22 deletion in HNPP, PMP22 duplication in CMT1A). If a hereditary small fiber neuropathy is suspected monongenic testing for mutations in TTR, SCNA9, SCNA10, or SCNA11 should be carried out. However, for less specific phenotypes gene panels or next generation sequencing (NGS) approaches are more appropriate. If acute intermittent porphyria is suspected (severe abdominal pain, nausea, vomiting, tachycardia, hypertension, convulsions, and neuropathy that may lead to respiratory insufficiency), determination of porphobilinogen in urine (increased) is recommended before genetic testing [48].
NERVE BIOPSY
Nerve biopsy today is required only in rare conditions. It should be considered in case of suspected neuropathy due to non-systemic vasculitis, sarcoidosis, amyloidosis, CIDP (if suspected but diagnostic criteria are not met), lymphoma, or leprosy. Nerve biopsy should be indicated and carried out only in centers specialised in neuromuscular disorders.
DISCUSSION AND CONCLUSIONS
Laboratory tests are helpful for the diagnosis of acquired and hereditary PNPs (Table 2). These tests should be ordered in relation to the history, clinical presentation and findings on NCSs and EMG in a stepwise manner (Table 3). The first step is usually carried out by the general neurologist and he should apply only those tests recommended in this overview in case of an appropriate clinical and NCS presentation. Laboratory tests should be selected specifically according to the suspected type of neuropathy to avoid unnecessary tests and expenses. Work-up should include as few samples as necessary for detecting the underlying cause. Work-up should also consider the sensitivity/specificity of the tests applied. If tests produce many false positive or false negative results, cost-effectiveness will decline. Uncritical ordering of many tests as screening, because of missing diligence or incompetence, should be avoided. The risk of taking too few samples is that abnormal results may be missed. However, if a stepwise approach is adopted and the patient is referred in a second step to a center specialised in neuromuscular disorders, cost-effectiveness will increase, If the first step yielded negative results, and negative tests are not repeated, costs will go down and the risk of missing abnormal findings will be minimized. The risk of ordering too many investigations is that costs are high and that many negative results are generated. In case the general neurologist is confronted with a neuropathy of undetermined cause and if first step laboratory tests yield negative or inconclusive results, patients should be referred to a center specialized in neuromuscular disorders.
Proposed laboratory battery for the most common polyneuropathy (chronic sensory-predominant axonal neuropathy without weakness) in patients aged > 60y)
Relevant laboratory tests (what to do) for the work-up of neuropathies in relation to the clinical presentation
A: chronic, predominantly sensory, distal, symmetric, axonal (painless or minimal pain), B. chronic, demyelinating, motor, sensory, or sensory-motor, symmetric, C: chronic, motor, predominantly axonal, D: chronic, painful, sensori-(motor), distal, E: mononeuritis multiplex, F: small fiber neuropathy, G: acute, demyelinating, proximal, H: acute or subacute, axonal, NCS: nerve conduction studies, oGTT: oral glucose tolerance test, GFR: glomerular filtration rate, *: if normal determine methyl-malonate and homocysteine, #: if celiac disease is suspected but not yet confirmed, &: TTR-gene, §: used as a screening test.
CONFLICT OF INTEREST
The authors have no conflict of interest to report.
FUNDING
No funding was received.
AUTHOR CONTRIBUTIONS
All authors contributed equally. All authors have read the journal’s position on issues involved in ethical publication