Laboratory Diagnosis in Neurology, 1 Ed.

Non-Pathogen-Related Inflammatory Disorders: Autoimmune Diseases

B. Wildemann

Multiple Sclerosis

MS is a chronic encephalomyelitis and as such produces characteristic CSF abnormalities which, in addition to clinical features and demonstration of inflammatory lesions by MRI, have diagnostic significance (Andersson et al., 1994; Reiber, 1998; Reiber et al., 2009).

CSF Analysis

MS-specific CSF changes (Table 10.11) relate to the cytology and, with much higher sensitivity and specificity, the protein analysis.

Pathological CSF findings are to be expected in more than 95% of patients with MS, irrespective of clinical course and disease activity (relapsing-remitting, secondary chronic progressive, primary chronic progressive). If the cytological and/or protein findings and the clinical and MRI features are compatible with MS, repeated CSF analysis over the course of the disease is not necessary.

Cytological Findings

CSF cell count and cell morphology show changes in about 50–60% of MS patients. Moderate pleocytosis of 5–30 cells/μL—less often over 30 cells/μL—is characteristic. Cell counts higher than 50/μL are exceptional in MS and in terms of the differential diagnosis suggest other inflammatory diseases of the nervous system. Cytomorphological changes include lymphomonocytic pleocytosis with a mild to moderate increase in activated lymphocytes and plasma cells. T lymphocytes of the CD 4-positive helper cell type dominate and express various activation markers and adhesion molecules; there is also an increased percentage of B cells.

Protein Findings

Total protein and albumin quotient (QAlb). Total protein levels are normal or slightly increased up to a maximum of 800–900 mg/L. The albumin quotient (QAlb) is normal or in some cases indicates mild blood–CSF barrier dysfunction (<15 × 10−3).

Table 10.11 Characteristic CSF findings associated with multiple sclerosis (see also Fig. 19.1)

CSF parameter


Diagnostic sensitivity, %

Cell count, cells/μL







> 30





Activated lymphocytes, mild ↑ (2–5%)



Plasma cells, mild ↑ (2–5%)


Barrier function, QAlb, × 10−3

< 8





Total protein (mg/L)

< 450



up to 800–900


Intrathecal immunoglobulin fractions*)









Oligoclonal IgG bands






MRZ reaction







Varicella zoster





Glucose, lactate



*) (Reiber et al., 2009)

Immunoglobulins. The most common and most characteristic laboratory finding in MS is a humoral immune response. Dominant and persistent intrathecal IgG synthesis is the most sensitive parameter of MS-associated chronic CNS inflammation. It is demonstrated quantitatively in the quotient diagram and, much more sensitive, qualitatively by the detection of oligoclonal IgG fractions by isoelectric focusing (IEF) of parallel CSF and serum samples—since, unlike quantitative IgG analysis, IEF recognizes even minor local IgG production (Chap. 5, “Relative Sensitivities of Immunodetection Methods”).

• Local IgG synthesis: Locally synthesized IgG as measured in the quotient diagram, is found in 82% of patients with clinically definite MS (Tables 5.6 and 5.7). The intrathecal fraction of IgG in the total CSF IgG correlates with the B-cell response detected cytologically. It varies between individuals and may temporarily drop after corticosteroid administration.

• Local IgM/IgA synthesis: Additional synthesis of IgM or IgA occurs in about 41% or 14% of MS patients, respectively (Tables 5.6 and 5.7). An isolated IgA or IgM response in the CSF is reason to doubt the MS diagnosis; rather, it suggests a pathogen-induced inflammatory disease (see below).

• Oligoclonal IgG: Intrathecal IgG synthesis resulting from CNS-specific inflammation in patients with clinically definite MS is indicated by the detection of oligoclonal IgG bands (OCB) of type 2 (OCB in CSF only) and type 3 (OCB in CSF and, in addition, identical OCB in CSF and serum) (see Figs. 4.134.14, and 4.15). Identification of OCB has a sensitivity between 96% (≥ 4 OCB) and 98% (2–3 OCB). The OCB pattern varies between individuals but is relatively constant within individuals during the course of the disease and is unaffected by treatment.

The specificity of OCB is comparatively low. With variable sensitivities and different distribution patterns, oligoclonal IgG is also detected in the CSF and serum in association with numerous infectious and autoimmune inflammatory disorders, and sometimes also in noninflammatory diseases of the nervous system.

Immunoreactivity in MS patients. Unlike with infections of the nervous system, the immune reactivities comprised within the OCB of MS patients are not specifically directed against pathogen antigens; rather, they seem to result from a nonspecifically triggered humoral immune response within the CNS. In addition to antibody reactivities directed against viral antigens (see MRZ reaction, below), antibodies against various myelin proteins and other oligodendrocyte—or astrocyte—specific proteins are present in the CSF of 30% of MS patients, although they are not necessarily found among the OCB; they include antibodies to myelin basic protein (MBP), myelin-associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), cyclic nucleotide phosphohydrolase (CNPase), oligodendrocyte-specific protein (OSP), α/β-crystallin, and transaldolase (Correale et al., 2002). The immune reactivities in the CSF described so far probably account only for a fraction of the OCB (< 5%). It is a particular challenge to interpret the specificities in oligoclonal IgG. The predominance of B memory cells migrating into perivascular tissues and CSF (Meinl et al., 2006; Archelos et al., 2008) supports the observation that the intrathecal antibodies in the brain and CSF are high-affinity antibodies which have undergone antigen-driven maturation (Robinson-Agramonte et al., 2007). The detection of somatic hypermutations in the gene loci coding for hypervariable regions of Ig heavy chain genes (Correale et al., 2002) and of ectopic lymphoid follicles in the meninges (Franciotta et al., 2008) further supports this view of true antigen-related B cell development. This makes other current concepts, such as the molecular mimicry model, less likely. However, there is no evidence that myelin-specific antigens belong to targets of the intrathecal B cell repertoire (Owens et al., 2009) or that particular micro-organisms are causative. The recently expanded evidence of an altered immune reactivity to EBV as a nonspecific activator of B cells in patients with MS has, however, reinforced the idea that indirect or bystander activation contributes to the immune response within the CNS (Franciotta et al., 2008). With regard to this discussion, it is of note that the B cell repertoire is different in CSF and aqueous humor of the eye in the individual MS patient (Reiber et al., 2010).

MRZ Reaction

The intrathecal IgG fraction contains antibodies that are directed—polyspecifically and without causal significance—against various viral antigens and other pathogen-associated antigens (Chap. 19, “Evaluation”). Especially common is the detection of antibodies against measles virus (78%), rubella virus (65%), and varicella zoster virus (55%) (“MRZ reaction”), intrathecal synthesis of which is determined by calculating the relevant pathogen-specific antibody indices (AIs) (Chap. 5, “Antibody Detection”). Less often, antibodies against herpes simplex virus (25%), Borrelia species (25%), Chlamydiaspecies (30%), or Toxoplasma gondii (10%)—but also anti dsDNA antibodies (19%)—are present in the IgG fraction which is produced locally in the CSF (Reiber et al., 1998; Correale et al., 2002; Graef et al., 1994).

A positive MRZ reaction in the form of a single (M, R, Z), double (MR, MZ, RZ), or triple combination (MRZ) is detected in more than 90% of MS patients. It reflects with high specificity the presence of a chronic autoimmune-type inflammatory process, since positive antibody indices for several pathogens of the MRZ group do not occur in infections, and this test (unlike OCB, which appear in acute diseases as well) implies an immune response that has already been in existence for some time. If only one antibody index is positive, a monospecific infection of the nervous system should be ruled out.

Other CSF Parameters

Myelin basic protein (MBP), an etiologically nonspecific marker of myelin damage, may be quantitatively detected in CSF. It is not detectable in normal CSF. In MS, MBP concentrations in CSF rise during an acute exacerbation and decline rapidly when the disease activity subsides. In chronic MS, MBP levels are usually not elevated (Whitaker, 1998). In very active MS, MBP is potentially suited as a surrogate marker during therapy.

Pitfalls in Interpreting CSF Findings in Cases of Suspected MS

A suspected diagnosis of MS must be reconsidered if of the following are found:

image Cell count > 40–50 cells/μL

image Pure barrier dysfunction and/or

image Absence of oligoclonal IgG synthesis

image Prominent local IgA synthesis

image Prominent local IgM synthesis

Important differential diagnoses:

Isolated pleocytosis without humoral immune response:

image Neurosarcoidosis

image Neural manifestations with systemic vasculitis and connective tissue disease

Isolated barrier dysfunction:

image Space-occupying processes

Isolated IgA synthesis with or without pleocytosis:

image Tuberculous meningitis and tuberculoma

image Multifocal septic encephalitis

image Brain abscess

image Whipple's disease

image Leprosy

image Adenoleukodystrophy

Isolated IgM synthesis with or without pleocytosis:

image Neuroborreliosis

image Mumps meningoencephalitis

image Neural manifestations of non-Hodgkin lymphoma

Diagnostic and Prognostic Implications of CSF Analysis in Monosymptomatic Clinical Episodes

Early diagnosis of MS. Intrathecal IgG synthesis, which can be qualitatively detected with extremely high sensitivity, is very important in the early diagnosis of MS. According to the McDonald diagnostic criteria established in 2001 and revised in 2005 (McDonald et al., 2001; Polman et al., 2005) and the recommendations of the Multiple Sclerosis Therapy Consensus Group in Germany (MS Therapie Konsensus Gruppe, 2000, 2002), a diagnosis of MS is definite if, after a first monosymptomatic episode with typical MRI findings (at least two cranial T2 lesions), two conditions are met:

• As the most sensitive MS-specific laboratory finding, OCB are detected in the CSF.

• Cranial MRI (≥ 30 days after the first episode) reveals one new T2 lesion.

In patients with monosymptomatic optic neuritis, if more than three lesions are found on cranial MRI, and OCB or a positive MRZ reaction are detected in the CSF, the risk of a second disease episode within 4 years (and, hence, clinically definite MS) is increased. A similar relation has also been shown for monosymptomatic myelitis and brainstem syndromes.

Predicted course of disease. There are no clear correlations between cellular and humoral changes in the CSF on the one hand and the type and severity of MS on the other. Recent studies indicate that, in patients with relapsing-remitting and secondary progressive MS, a high number of B cells and a low number of monocytes correlate with progression (Cepok et al., 2001). In line with the potential prognostic importance of the B cell response, a few studies suggest that a more rapid progression is associated with a pronounced humoral immune response, while a milder course of the disease is associated with absence of local IgG synthesis. Recent data show that in pediatric MS the presence of intrathecal IgM synthesis is associated with a slower progression (Stauch et al., 2010). The role of intrathecal IgM synthesis for the disease course in patients with adult MS is contradictory and is confounded by a bias in the methods to detect “oligoclonal IgM.”

Pediatric MS and ADEM

Paediatric MS already at first clinical manifestation shows the complete, neuroimmunological data pattern in CSF (oligoclonal IgG, intrathecal IgM, MRZ reaction) implicating that inflammatory signs do not evolve gradually in patients with MS. As pediatric and adult MS differ quantitatively but not qualitatively in neuroimmunological patterns, CSF abnormalities do not allow discrimination between “early” and “late” onset MS (Reiber et al., 2009). The absence of oligoclonal IgG in an acute disseminated encephalomyelitis (ADEM) contradicts the later emergence of MS.

Serum Analysis

Diagnostic Importance

Antinuclear antibodies. In more than 20% of MS patients, serum titers of antinuclear antibodies (ANAs) are slightly to moderately increased. Positive ANA findings are independent of the course of the disease or its duration and severity, but they are detected more frequently during active phases of the disease (Collard et al., 1997).

Myelin oligodendrocyte glycoprotein antibodies. In a recent study (Berger et al., 2003), serological detection of IgM antibodies against myelin oligodendrocyte glycoprotein (MOG) in patients with a monosymptomatic episode was predictive of the risk of having an early second episode. The prognostic importance of MOG antibodies for the progression to clinically definite MS is currently unclear, since the results have not been confirmed by subsequent studies (Lampasona et al., 2004; Lim et al., 2005; Rauer et al., 2006; Kuhle et al., 2007).

Other markers. Numerous studies have tested the value of various cytokines and adhesion molecules as surrogate markers for both the activity and course of the disease.

In the absence of sufficient evidence, and given their lack of specificity, determination of these serum variables is currently not recommended in routine diagnostics.

Treatment Monitoring

Neutralizing antibodies. Some MS patients treated with β-interferons as immunoprophylactic agents develop neutralizing antibodies (NAB) against the three available preparations of interferon-beta (Betaferon, containing interferonbeta-1 b, and Avonex and Rebif, both containing interferonbeta-1 a). These antibodies may be accompanied by a loss of response to treatment, but often are present only transiently. If the therapeutic effect wears off, both clinically and in the MRI, determination of these antibodies may be helpful. However, there is a problem in the lack of standardized test methods for the detection and determination of their neutralizing potential. A current initiative aims to establish reference laboratories for NAB testing in several European countries.

Mx proteins. β-Interferons induce specific and stable expression of Mx proteins in leukocytes. When the effect wears off, the serum levels of Mx proteins decrease (Kracke et al., 2000). Mx proteins are therefore a valuable surrogate marker for interferon activity, and their determination may be helpful when failure of β-interferon treatment is suspected.

Acute Demyelinating Encephalomyelitis and MS Variants

CSF Analysis

Multiple sclerosis must be distinguished from various other inflammatory, often fulminant demyelinating diseases of the CNS. These include:

• Acute demyelinating encephalomyelitis.

• Acute MS (Marburg type).

• Balo's concentric sclerosis.

• Acute hemorrhagic leukoencephalitis (Hurst's disease).

• Neuromyelitis optica (Devic's syndrome).

Special clinical features. Distinct clinical features include:

• Frequent parainfectious occurrence of acute demyelinating encephalomyelitis and acute hemorrhagic leukoencephalitis, which can be characterized radiologically by coincidental hemorrhages in addition to multifocal demyelination.

• In neuromyelitis optica, simultaneous occurrence of monophasic or relapsing myelitis and optic neuritis without associated clinical or radiological cerebral involvement.

Special features of CSF variables. The CSF variables show changes typical of MS, with the following special features:

• Acute demyelinating encephalomyelitis, acute MS (Marburg type), Balo's concentric sclerosis, and acute hemorrhagic leukoencephalitis may show more pronounced lymphomonocytic pleocytosis (> 50 cells/μL)

• Neuromyelitis optica shows more pronounced pleocytosis (> 50 cells/μL); cytomorphology shows predominance of neutrophilic granulocytes, and, occasionally, a few eosinophilic granulocytes

• Any of these disease entities may show a more pronounced barrier dysfunction (Qalb > 10 × 10−3) and a higher increase in total protein (> 900 mg/L)

• Acute demyelinating encephalomyelitis, acute hemorrhagic leukoencephalitis, and neuromyelitis optica more rarely show production of OCB, and OCB are more likely only to be transiently detectable

• Neuromyelitis optica and acute demyelinating encephalomyelitis show no MRZ reaction.

Serum Analysis

Routine serum markers are of no significant importance in the diagnosis of MS-related idiopathic demyelinating diseases. Neuromyelitis optica, presumably a mainly humorally mediated disease, can occur in connection with other antibody-associated autoimmune diseases. Recently, a distinct autoantibody (NMO-IgG) has been detected in patients with neuromyelitis optica; NMO-IgG has a sensitivity of 58–76% and a specificity of up to 99%, and selectively targets the aquaporin-4 water channel located in astrocytic foot processes (Lennon et al., 2004, 2005; Jarius et al., 2007; Paul et al., 2007). Determination of NMO-IgG is currently only possible in specialized laboratories.


Epidemiology and Clinical Features

Definition. Sarcoidosis is a multisystem disorder of unknown etiology that manifests primarily in the skin, lungs, and lymph nodes. The organs affected by this disease contain noncaseating epithelioid granulomas.

Neurosarcoidosis, i. e., involvement of the central or peripheral nervous system in sarcoidosis, is rare:

• It affects 5% of patients with systemic sarcoidosis.

• It has a prevalence at autopsy (in systemic sarcoidosis) of 15%.

• It is the first symptom in 10–30% of patients with systemic sarcoidosis.

• It is extremely rare in the general population, with an incidence of less than 0.2 per 100 000.

• It causes the following spectrum of clinical symptoms:

– Basal meningitis (cranial nerves II and VII predominantly affected).

– Focal encephalitis (hypothalamus and hypophysis predominantly affected).

– Myelopathy, spinal cord compression.

– Peripheral neuropathy.

– Myositis.

Table 10.12 Characteristic CSF findings associated with neurosarcoidosis

CSF parameter


Diagnostic sensitivity, %

Initial CSF pressure

May be ↑


Cell count, cells/μL






Eosinophilia may be detectable


CD 4/CD 8 ratio, may be ↑


Barrier function, QAlb, × 10−3



Total protein (mg/L)

Up to 800–900


Intrathecal immunoglobulin fractions, IF > 0


Up to 70

Oligoclonal IgG bands

May be detectable

Up to 70


May be ↓








Serum ACE




May be ↑



May be ↑



The diagnosis of neurosarcoidosis is difficult. It is based on demonstration of systemic sarcoidosis and the exclusion of other neurological diseases, or—in the case of isolated involvement of the nervous system—on biopsy of intracranial or extracranial lesions and histological evidence of the pathognomonic granulomas. Laboratory tests are of supplementary value for establishing the diagnosis, but laboratory variables with specificity for neurosarcoidosis do not exist.

CSF Analysis

Cytological and protein findings. CSF cytology and protein findings are pathological in up to 70% of patients with neurosarcoidosis, although the changes are nonspecific (Nowak and Widenka, 2001; Vinas and Rengachary, 2001). Normal CSF findings do not exclude neurosarcoidosis. Typical changes include (Table 10.12):

• Lymphomonocytic pleocytosis (10–200 cells/μL).

• Occasional occurrence of CSF eosinophilia.

• Increased CD 4/CD 8 lymphocyte ratio.

• Barrier dysfunction.

• Mild to moderate increase in total protein.

Intrathecal IgG synthesis and oligoclonal IgG fractions are found in up to 70% of patients. CSF glucose may be decreased, particularly in connection with basal meningitis, and the CSF opening pressure may be increased.

ACE and other markers. Angiotensin-converting enzyme (ACE) is a secretion product of epithelioid cells within the sarcoid granulomas. The diagnostic specificity of ACE is moderate, since increased levels may be present in other diseases as well. Pathological ACE levels in CSF are expected in 55% of patients with neurosarcoidosis and occur in 5% of patients with systemic sarcoidosis without clinically manifest involvement of the nervous system, and in 13% of patients with other neurological diseases (Nowak et al., 2001). Other laboratory variables that are inconsistently elevated in neurosarcoidosis are lysozyme and β2-microglobulin.

Serum Analysis

When neurosarcoidosis is suspected, serum analysis is essential to show systemic disease activity.

Routine variables. Changes in routine biochemical and hematological variables associated with sarcoidosis include:

• Increased erythrocyte sedimentation rate.

• Increased alkaline phosphatase activity.

• Hypercalcemia and hypercalciuria.

• Hypergammaglobulinemia.

• Mild eosinophilia and monocytosis in the differential blood count.

Hypercalcemia and hypercalciuria are caused by increased intestinal calcium absorption due to increased release of 1,25-dihydroxy vitamin D by epithelioid granulomas.

Angiotensin-converting enzyme. Serum ACE is elevated in 70–80% of patients with systemic sarcoidosis. Isolated neurosarcoidosis, which is extremely rare, does not cause an increase in ACE. A pathological serum ACE level in association with abnormal gallium scintigraphy is strongly suggestive of pulmonary sarcoidosis.

Table 10.13 Stiff person syndrome

Clinical variants

Associated symptoms

Classic SPS

Fluctuating rigidity of leg and trunk muscles, muscle spasms

Progressive encephalomyelitis with rigidity and myoclonus (PERM)

In addition, impaired oculomotor function, pyramidal tract symptoms, cognitive problems

Stiff limb syndrome

Rigidity of only one limb

Jerking stiff person syndrome

Brainstem contribution predominates

Supplementary Analysis

In pulmonary sarcoidosis, bronchoalveolar lavage reveals an increase in the number of lymphocytes (> 50%) and CD 4 lymphocytes, as well as an increased CD 4/CD 8 lymphocyte ratio (> 5–12).

Stiff Person Syndrome and Other Neurological Diseases Associated with GAD Antibodies

Stiff Person Syndrome

Stiff person syndrome (SPS) is a chronic encephalomyelitis of either autoimmune or, rarely, paraneoplastic etiology (Meinck et al., 1994; Meinck, 2000; Vianello et al., 2002).

SPS and its clinical variants are closely associated with numerous autoimmune diseases, the majority of which are part of the autoimmune polyendocrine syndrome (Table 10.13):

• Diabetes mellitus type 1.

• Thyroiditis.

• Pernicious anemia.

• Psoriasis.

• Connective tissue diseases (Sjögren's syndrome, scleroderma, rheumatoid arthritis, SLE).

• Vitiligo.

• Myasthenia gravis.

Less often, and usually accompanied by additional symptoms, SPS can also be a paraneoplastic condition associated with breast cancer or small-cell lung carcinoma.

Serum and CFS Analysis

GAD autoantibodies. In a high percentage of patients with SPS, various autoantibodies are detected in serum and CSF. Antibodies against glutamic acid decarboxylase (GAD) are the characteristic, and most sensitive, laboratory variable for SPS (Meinck et al., 2001; Meinck and Thompson, 2002; Vianello et al., 2002) (Chap. 7, “Antineural Antibodies”). GAD is an enzyme produced predominantly and ubiquitously in neurons and also in beta cells of the pancreas; it converts glutamate to the inhibitory transmitter γ-aminobutyric acid (GABA). High titers of GAD antibodies occur in 60–80% of SPS patients; they are almost always produced intrathecally. Antibody indices for GAD antibodies are sometimes excessive, and markedly higher than in other infectious and autoimmune inflammations of the nervous system.

GAD antibodies do not distinguish between an autoimmune and a paraneoplastic etiology of SPS. There is no correlation between the amount of antibodies in the serum and CSF and the severity or clinical course of the disease.

Pitfalls with GAD Antibodies

GAD antibodies also occur in many patients with type 1 diabetes mellitus, but the titers are usually much lower.

Other autoantibodies. In paraneoplastic SPS, the autoimmunity is rarely directed against GAD. In the majority of cases, high-titer antibodies against amphiphysin, a protein concentrated in nerve endings, are found in serum and CSF. In one patient, antibodies against gephyrin have been found.

Cytological and protein findings in the CSF. Cell count and total protein in CSF are slightly increased in a few patients. OCB are detected in about 50% of patients, indicating intrathecal IgG synthesis (Table 10.14).

Table 10.14 Characteristic laboratory findings associated with stiff person syndrome

CSF parameter


Diagnostic sensitivity, %

Cell count, cells/μL

Normal, or mild ↑





Barrier function, QAlb, × 10−3



Total protein, mg/L

Normal, or mild ↑


Oligoclonal IgG bands

May be detectable


Glucose, lactate



GAD antibodies in serum and CSF

Autoimmune SPS



Paraneoplastic SPS


GAD antibody index

Mostly positive, up to 500


Amphiphysin antibodies in serum and CSF

Paraneoplastic SPS


Gephyrin antibodies in serum and CSF

Paraneoplastic SPS

Isolated case

Table 10.15 Classification of myasthenia gravis

Clinical forms

Frequencies and special features

Generalized myasthenia gravis

• Early manifestation (≤ 40 years)

• Late manifestation (≥ 40 years)


Frequently, additional autoimmune diseases

Thymoma-associated myasthenia gravis


Ocular myasthenia gravis

Up to 20%

Initial symptom in 50–70% of patients with generalized myasthenia gravis

Other Neurological Diseases Associated with GAD Antibodies

Recent findings suggest that not only SPS but also cerebellar syndromes are associated with high titers of GAD autoantibodies in serum and CSF, and also with insulin-dependent diabetes mellitus and autoimmune polyendocrinopathy. These presumably autoimmune-mediated forms of ataxia should be included in the differential diagnosis of cerebellar diseases, since they can be treated with high-dose immunoglobulins. GAD antibodies in serum and CSF are also found in connection with refractory temporal lobe epilepsy and palatal myoclonus (Vianello et al., 2002).

Myasthenia Gravis and Other Disorders of Neuromuscular Transmission

Definition of Myasthenia Gravis

Myasthenia gravis is an autoimmune disease that causes muscle fatigue and weakness due to impaired neuromuscular transmission.

Etiology and Classification

Myasthenia gravis (MG) is caused by autoantibodies directed against nicotinic acetylcholine receptors (AchR) at the neuromuscular junctions. These antibodies interfere with the expression and function of these receptors.

Myasthenia gravis may be generalized or only ocular, and it may occur as a pure autoimmune disease or as a paraneoplastic condition associated with thymoma (Vincent et al., 2001) (Table 10.15).


Detection of anti-AchR antibodies in serum is of high diagnostic relevance. The diagnostic interpretation of AchR antibodies and other antibodies associated with MG depends on the subtypes of the disease (Table 10.15).

CSF analysis does not play a role in the laboratory diagnosis of MG.

Serum Analysis

AchR autoantibodies. AchR antibodies are detected in the serum of 85% of patients with generalized myasthenia (both autoimmune and paraneoplastic: in 80% of those with mild and 90% of those with moderate or severe generalized myasthenia) and in 50% of patients with purely ocular myasthenia (Table 10.16) (Palace et al., 2001; Vincent et al., 2001; Pascuzzi, 2002). A definite positive AchR antibody finding is diagnostic, but a negative titer does not exclude the diagnosis, since seronegative forms do occur. The AchR antibodies are polyclonal, differently composed in different individuals, and contain both pathogenic and nonpathogenic reactivities. This means that absolute titers do not correlate with the severity of the disease. However, intraindividually, a rise or fall in AchR antibody titer does reflect a change in the clinical severity.

The primary indication for AchR antibody determination is to confirm a clinically or pharmacologically suspected diagnosis of MG. Repeated determination is indicated if there are marked changes in clinical findings and/or treatment, or if surgery is scheduled. However, routine monitoring of the titer gives no useful information when the disease is stable.


Pitfalls in Interpreting AchR Antibody Findings

The quality of AchR antibody determination varies between laboratories. For this reason:

• AchR antibodies should be determined in laboratories that regularly participate in interlaboratory comparison testing.

• As a matter of principle, AchR antibody determination should always be done in the same laboratory.

• An unexpected positive finding should be followed up by repeat testing at another reference laboratory, to rule out a false-positive result.

Absence of AchR antibody. Ten percent to 20% of patients with MG—often adolescents and children—are seronegative for AchR antibodies. In up to 50% of patients with AchR antibody-seronegative MG, serum contains autoantibodies against MuSK, a muscle-specific receptor tyrosine kinase that is expressed at the neuromuscular junction (Hoch et al., 2001; Vincent and Leite, 2005) (Table 10.16). These antibodies impair the function of the MuSK protein that is essential for the formation of the neuromuscular junction. So far, determination of MuSK antibodies has only been possible in specialized laboratories. In addition, more than 60% of individuals with seronegative MG harbor IgG1 lowaffinity antibodies against AchR, as described recently (Leite et al., 2008). Assays for their detection are not yet routinely available.

Autoantibodies against musculoskeletal proteins. In addition to AchR antibodies, other autoantibodies are found in the serum of patients with thymoma-associated MG; they are directed against the musculoskeletal protein titin and the ryanodine receptor (RyR) (Romi et al., 2000) (Table 10.16):

• Titin antibodies occur with a prevalence of 74–95% in the serum of patients with thymoma-associated MG, but also in 58–78% of patients with late-onset generalized MG, and even in up to 90% of patients with MG manifestation after the age of 60 (Aarli et al., 1998: Baggi et al., 1998; Buckley et al., 2001).

• RyR antibodies are found in thymoma-associated MG (50–70%) and late-onset MG (14%) (Aarli et al., 1998; Baggi et al., 1998).

Titin antibodies and RyR antibodies therefore predict thymoma only when the disease appears before age 40.

Determination of these antibodies can be helpful when a thymoma cannot be definitely shown radiologically. However, since thymectomy is indicated in patients with generalized MG prior to age 40 irrespective of the radiological finding, the diagnostic value of musculoskeletal antibodies is limited overall. A better correlation with thymoma-associated MG has recently been described for autoantibodies against the cytokines interferon-α and interleukin-12. Both these subtypes are detected predominantly in paraneoplastic MG, and distinct increases in the titers indicate recurrence of the tumor. However, determination of cytokine antibodies has not yet become established in routine diagnostics.

Other autoantibodies. Some patients with MG produce plenty of antibodies associated with other autoimmune diseases, particularly ANAs, dsDNA antibodies, and thyroid antibodies.

Lambert-Eaton Myasthenic Syndrome


The Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease and is caused by autoantibodies against calcium channels of the presynaptic membrane, which interfere with the release of acetylcholine at the neuromuscular junction. LEMS can occur as a purely autoimmune disease, but in about 60% of patients it is paraneoplastic, usually in association with small-cell lung carcinoma (see also below, “Paraneoplastic Neurological Syndromes”).


Serum autoantibodies against the P/Q type voltage-gated calcium channels (VGCC antibodies) of cholinergic nerve endings are characteristic and confirm the diagnosis. VGCC antibodies are highly specific for LEMS but do not differentiate between the autoimmune and paraneoplastic forms of the syndrome. In contrast, serum antibodies with specificity for SOX1, an antigen which is selectively expressed in small-cell lung cancer and, within the CNS, predominantly in the Bergmann glia of the cerebellum (formerly called anti-glial nuclear antibodies, AGNA), are highly suggestive of paraneoplastic LEMS. So far, these antibodies have been found exclusively in patients with LEMS associated with small-cell lung cancer, but not in those with idiopathic LEMS. The sensitivity of SOX1 antibodies for detecting paraneoplastic LEMS is 65% (Graus et al., 2005; Sabater et al., 2008). Testing for SOX1 antibodies is confined to specialized laboratories.


Etiology and Clinical Features

Neuromyotonia develops as an autoimmune disease or, in 20% of patients, as a paraneoplastic condition associated with small-cell lung cancer or thymoma (see below, “Paraneoplastic Neurological Syndromes”). It can also occur in combination with thymoma-associated MG or neuropathy. Clinically, neuromyotonia is characterized by muscular fasciculation and spasms.



In about 40% of patients with neuromyotonia, measurable amounts of autoantibodies against voltage-gated potassium channels (VGKC antibodies) are found in the serum. These antibodies confirm the diagnosis but—like AchR antibodies in MG and VGCC antibodies in LEMS—do not differentiate between the autoimmune and paraneoplastic etiologies of the syndrome.

Polymyositis, Dermatomyositis, and Inclusion Body Myositis

Clinical Features

Dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM) are inflammatory immunogenic myopathies. The cardinal symptoms are muscle weakness, muscular atrophy, and myalgia; they develop in the three forms at different speeds and have distinct patterns of distribution (Table 10.17).

Typical extramuscular manifestations are:

• In DM:

– Skin lesions (heliotropic exanthema).

– After age 40, association with malignant tumors.

• In DM and PM:

– Cardiac involvement.

– Pharyngeal and esophageal involvement.

– Alveolitis, pulmonary fibrosis.

– Polysynovitis.

– Overlap syndromes (scleroderma, mixed connective tissue disease, Jo-1 syndrome, see below).


In addition to neurophysiological and imaging techniques, serology and muscle biopsies are of cardinal importance for the diagnostic classification.

Serum Analysis

Muscle enzymes. As a sign of muscle damage, creatine kinase (CK) and myoglobin are increased. In acute DM or PM, CK may reach up to 50 times the normal value, and values usually correlate with the activity of the disease. Rarely, acute manifestations of PM and DM with normal CK levels are seen. IBM produces less pronounced increases, with CK levels reaching up to 10 times the normal value. Transaminases, especially glutamate oxalacetate transaminase (GOT), are markedly increased in acute and subacute PM, while DM only shows a slight increase in GOT.

Myositis-associated autoantibodies. Myositis-associated antibodies directed against various non-organ-specific nuclear and cytoplasmic antigens are common in PM, less common in DM, and absent in IBM. Detection of antibodies is especially successful when extramuscular manifestations or an overlap syndrome are present in addition to myositis. Because of their low sensitivity, myositis-associated antibodies are only of minor or moderate diagnostic value, although their detection makes it easier to assign the myositis to one of the various myositis-associated syndromes (Table 10.18).

Muscle Biopsy

If myositis is suspected, muscle biopsy is the diagnostic gold standard and should be performed if possible before immunosuppressive therapy is started. Biopsy identifies PM, DM, and IBM with certainty and excludes other neuromuscular diseases (Pongratz, 2000; Mastaglia et al., 2003).

The diseases are histologically and immunohistologically characterized as follows:

• DM (Fig. 10.3):

– Perivascular and perifascicular inflammatory infiltration (perifascicular myositis).

– Perifascicular atrophy.

– Endothelial proliferation of intramuscular vessels and tubulovesicular inclusions in the vascular walls (electron microscopic finding).

– Cellular infiltrates composed predominantly of B cells and CD 4+ T cells.

– C5b-9 complement deposition.

• PM (Fig. 10.4):

– Endomysial inflammatory infiltrates (diffuse myositis).

– Absence of perifascicular atrophy.

– Absence of microangiopathy.

– Dominance of CD 8+ T cells in inflammatory infiltrates, with invasion into nonnecrotic muscle fibers.

– MHC class I expression of muscle fibers.

• IBM (Fig. 10.5) → endomysial inflammatory infiltrate with:

– Dominance of CD 8+ T cells with invasion into nonnecrotic muscle fibers.

– Rimmed vacuoles with eosinophilic cytoplasmic inclusions within muscle fibers.

– Deposition of various proteins within the cytoplasmic inclusions (ubiquitin, β-amyloid protein, β-amyloid precursor protein, apolipoprotein E, and others).

– Filamentary inclusions within the rimmed vacuoles and in the nuclei (electron microscopic finding).

Table 10.18 Myositis-associated autoantibodies and syndromes (according to Genth, 2000)


Frequency, %

Myositis-associated syndromes

Directed against aminoacyl-tRNA synthetases:

Anti-histidyl-tRNA synthetase (Jo-1)

Anti-threonyl-tRNA synthetase (Pl-7)

Anti-alanyl-tRNA synthetase (Pl-12)

Anti-isoleucine-tRNA synthetase (EJ)

Anti-glycyl-tRNA synthetase (OJ)


80–90% of these are directed against Jo-1

Jo-1 Syndrome

• Myositis

• Fibrosing alveolitis

• Polysynovitis

Anti-signal recognition particle (anti-SRP)


Mostly acute polymyositis

• Often resistant to treatment

• Poor prognosis

• Cardiac involvement (40%)



Mostly chronic (recurrent) dermatomyositis, good prognosis



Scleromyositis, dermatomyositis

• Myositis (~50%)

• Signs of systemic sclerosis

• Signs of dermatomyositis

• Good prognosis



Myositis–scleroderma overlap syndrome (30–55%), myositis (50%)




Mixed connective tissue disease (Sharp's syndrome): arthritis, Raynaud's phenomenon, limited scleroderma, myositis (~50%)

Obligatory diagnostic tests when myositis is suspected:

• CK.

• Transaminases.

• Myoglobin if CK markedly increased.

• Muscle biopsy.

Optional diagnostic test when myositis is suspected:

• Myositis-associated antibodies.

Guillain-Barré Syndrome and Other Immune-Mediated Neuropathies

Acquired immune-mediated neuropathies include Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy, paraproteinemic polyneuropathy, and cryoglobulinemic polyneuropathy. The laboratory diagnosis of neuropathies occurring in association with systemic vasculitis and connective tissue disease is discussed below (“Systemic Vasculitis and Connective Tissue Diseases”).

Table 10.19 Classification of Guillain–Barré syndrome (according to Winer, 2001)


Special features

Acute inflammatory demyelinating polyneuropathy (AIDP): classic motor-sensory GBS

Most common form in Europe and North America

Acute motor axonal neuropathy (AMAN): primary axonal GBS

Common in China and Japan

Acute motor axonal and sensory neuropathy (AMSAN): primary axonal GBS with a sensory component

Common in China and Japan

Miller-Fisher syndrome (MFS)

Ophthalmoplegia, ataxia, areflexia

Guillain-Barré Syndrome

GBS, or acute idiopathic polyneuritis, is the prototype of rapidly progressive flaccid tetraparesis and areflexia. It is divided into different subtypes according to the dominance of the underlying pathological demyelination or axonal damage (Table 10.19).


Fig. 10.3 a–f Dermatomyositis. (Courtesy of Prof. H. H. Göbel, Institute of Neuropathology, University of Mainz, Germany.)

a Perifascicular atrophy of muscle fibers. Modified trichrome staining.

b The major histocompatibility complex I (MHC I) is expressed on all muscle fibers. Immunoperoxidase reaction.

c B lymphocytes between muscle fibers. Immunoperoxidase reaction.

d T lymphocytes between muscle fibers. Immunoperoxidase reaction.

e Tubulovesicular inclusions in an endothelial cell. Electron micrograph.

f Intramuscular capillary with incomplete lining as the result of endothelial necrosis. Electron micrograph.


Fig. 10.4 a–d Polymyositis. (Courtesy of Prof. H.H. Göbel, Institute of Neuropathology, University of Mainz, Germany.)

a Muscle fibers of variable sizes, one necrotic muscle fiber undergoing phagocytosis, and interstitial cells. Modified trichrome staining.

b Expression of MHC I on all muscle fibers. Immunoperoxidase reaction.

c Aggregates of macrophages within individual necrotic muscle fibers. Immunoperoxidase reaction.

d T lymphocytes surrounding a muscle fiber, spreading through the interstitial space, and infiltrating an otherwise intact muscle fiber.

CSF Analysis

Alongside clinical criteria and neurophysiological variables, CSF analysis is the third pillar in the diagnosis of GBS.

Albuminocytological dissociation. A characteristic change in the CSF is that there is an increase in total protein while at the same time the cell count is normal or only slightly increased (Table 10.20). This finding is explained by the inflammatory process affecting primarily the nerve roots with resulting barrier dysfunction (reduced CSF flow). The pathological increase in total protein is the most sensitive parameter of CSF analysis, and in the internationally used diagnostic criteria (van der Meche et al., 2001) is regarded as supportive of the diagnosis of GBS.

Cytological findings. The cell count is normal or initially slightly increased up to a maximum of 50 cells/μL. Mild pleocytosis is found particularly in HIV-1-seropositive persons who develop GBS as an early neurological manifestation of HIV infection. Cytomorphologically, lymphocytes and monocytes predominate; only occasionally, an increased number of activated lymphocytes and a few plasma cells are found.

Protein findings. Total protein may still be normal during the 1st week after the onset of the disease; normal levels are found in about 60% of patients. Subsequently, the levels increase and may reach up to 5000 mg/L within 2–3 weeks after the first symptoms appeared. This coincides with blood–CSF barrier dysfunction with an albumin quotient (QAlb) of up to 50 × 10−3. Changes in total protein may be detectable over several months. As a sign of a systemic and often parainfectious immune reaction, oligoclonal IgG fractions may be found in serum and CSF (type 4; see Fig. 4.13). Local synthesis in quantitatively measurable amounts of one or more immunoglobulin classes, or CSF-restricted OCB, are not characteristic of GBS.


Fig. 10.5 a–d Inclusion body myositis. (Courtesy of Prof. H.H. Göbel, Institute of Neuropathology, University of Mainz, Germany.)

a Muscle fibers of various sizes, partly surrounded by endomysial connective tissue. Modified trichrome staining.

b Intense inflammatory infiltration around and between muscle fibers. Hematoxylin–eosin staining.

c Partly atrophic muscle fibers separated by endomysial connective tissue, one muscle fiber with numerous cytoplasmic bodies, and other muscle fibers with vacuoles, some of them autophagic. Modified trichrome staining.

d Numerous T8 lymphocytes between the muscle fibers. Immunoperoxidase reaction.

Table 10.20 Characteristic CSF findings associated with Guillain-Barré syndrome

CSF parameter


Diagnostic sensitivity, %

Cell count, cells/μL




Up to a maximum of 50





There may be a few activated lymphocytes and plasma cells


Barrier function, QAlb, × 10−3

Up to 50



Normal values possible during the first week


Total protein, mg/L

Up to 5000



Normal values possible during the first week


Intrathecal immunoglobulin fractions, IF > 0



Oligoclonal IgG bands

There may be identical bands in serum and CSF (type IV)


Glucose, lactate



Table 10.21 Autoantibodies against gangliosides in the serum of patients with Guillain-Barré syndrome

Antibody reactivity

Clinical syndrome

Sensitivity, %

GQ 1 b (IgG)

Miller-Fisher syndrome



GBS with ophthalmoplegia


GM1 (IgG)



GD 1 a (IgG)

• Common in patients with AMAN/AMSAN


GalNAc-GD 1 a (IgG)

• Common after infection with Campylobacter jejuni


GM2 (IgM)

GBS (common after infection with CMV)


Serum Analysis

Autoantibodies against gangliosides. Distinct ganglioside antibodies are detected in the serum of some patients with the GBS subtypes listed in Table 10.19 (Nachamkin et al., 1998; Winer, 2001; Willison and Yuki, 2002). Overall, autoantibodies against diverse gangliosides are detected in the serum of 30% of all patients with GBS during the acute phase of the disease. The highest levels are observed in the Miller-Fisher variant of the syndrome (MFS) (Table 10.21).

• GQ 1 b antibodies: In 90% of cases, Miller-Fisher syndrome is characterized by serum antibodies with specificity for the ganglioside GQ 1 b. The reactive antigens are concentrated in the paranodal regions of extramedullary sections of the oculomotor nerve and are also present in cerebellar neurons, thus implying a pathogenic role of the ganglioside-specific humoral immune response in the development of MFS-associated ophthalmoplegia and ataxia. GQ 1 b antibodies are also detected in GBS patients with ophthalmoplegia.

• Antibodies to GM1 and GM2 gangliosides: These occur frequently in GBS patients in whom the disease develops after infection with Campylobacter jejuni or cytomegalovirus. For GM1-positive neuropathies, a correlation with rapid progression and severe course without sensory involvement has been described.

• GD 1 a antibodies: Significant associations of antibodies against GD 1 a and GalNAc-GD 1 a with two GBS subtypes—acute motor axonal neuropathy (AMAN) and acute motor and sensory axonal neuropathy (AMSAN)—-have also been reported.

On the whole, GQ 1 b antibodies have high diagnostic sensitivity and specificity for Miller-Fisher syndrome. The diagnostic value of the other ganglioside antibodies is comparatively low. Although various studies suggest a prognostic importance of various immune reactivities, the current state of knowledge does not justify routine testing for ganglioside antibodies for the evaluation and treatment of GBS patients.

Pathogen detection. About 75% of GBS patients have a history of respiratory or gastrointestinal infection during the weeks preceding the first symptoms. In up to 50% of GBS patients, there is serological evidence of a recent infection; associations with Campylobacter jejuni (30%) and cytomegalovirus (15%) are most common. A definite causality has also been demonstrated for infections with Epstein-Barr virus (EBV), Mycoplasma pneumoniae, and HIV. The evidence of a recent acute infection with these pathogens is a four-fold increase or decrease of IgG and/or IgM titers in serial serum samples. Campylobacter jejuni, cytomegalovirus, and Mycoplasma carry epitopes with specificities for some of the above-mentioned antibody reactivities, suggesting that molecular mimicry plays a role in the development of these humoral immune responses.

A requirement for successful culture of Campylobacter jejuni is the preparation of stool cultures within a few days after the first symptoms appeared.

Pitfalls in Interpreting Laboratory Findings in GBS

A suspected diagnosis of GBS must be reconsidered in the following cases:

• Total protein is normal in the second CSF sample (≥ 1 week after the first symptoms).

• Cell counts are higher than 50 cells/μL.

• Pronounced local immunoglobulin synthesis is found.

The quality of ganglioside antibody determination by ELISA can vary between laboratories, since not all laboratories use recommended standardized methods.

Chronic Inflammatory Demyelinating Polyneuropathy

Clinical Features

Chronic inflammatory demyelinating polyneuropathy (CIDP) is an immunogenic disease with a chronic progressive or chronic relapsing course. Clinically, it is an ascending sensorimotor polyneuropathy with hyporeflexia or areflexia and develops over a period of more than 8 weeks (for an overview, see Kieseier et al., 2002).


According to recent findings it may be possible to distinguish subgroups, and this is reflected in the recently suggested diagnostic criteria (Table 10.22).

CSF analysis. As in GBS, there is an increase in total protein (500–5000 mg/L) due to blood–CSF barrier dysfunction QAlb ≥50 × 10−3), while the cell count is usually normal or only slightly increased. There are no other specific anomalies. According to current diagnostic criteria, an increase in CSF protein to above 450 mg/L is obligatory, and a cell count below 10 cells/μL supports a definite diagnosis of CIDP.

Table 10.22 Diagnostic criteria for chronic inflammatory demyelinating polyneuropathy (according to Saperstein et al., 2001; Koller et al., 2003)



Clinical criteria



• Main criteria

Symmetric proximal and distal weakness

• Secondary criteria

Distal weakness or impaired sensibility

Deep tendon reflexes

Hyporeflexia or areflexia


At least 2 months

Additional diagnostic criteria

Neurophysiology: 2 of 4 criteria

• Partial conduction block

• Reduced motor nerve conduction velocity

• Prolonged distal motor latency

• Prolonged F-wave latency



• Obligatory

• Supportive

Total protein: > 450 mg/L

Cell count: < 10/μL


Demyelination and remyelination

Diagnostic certainty

Definite CIDP

Clinical main criteria and neurophysiological and CSF criteria are met, perhaps biopsy (not essential)

Probable CIDP

Clinical main criteria and neurophysiological or CSF criteria are met and biopsy

Possible CIDP

Clinical main criteria and one of the 3 additional diagnostic criteria are met or clinical secondary criteria and 2 of the 3 additional diagnostic criteria are met

Serum analysis. Autoantibodies against gangliosides and myelin components are found in isolated cases, but the immune reactivities reported so far are of no diagnostic relevance. Serological tests for infections do not have a role in the diagnosis of CIDP. In large series, 16–32% of patients had a history of infection within the 6 months preceding the first symptoms of CIDP.

Nerve biopsy. CIDP can be distinguished from chronic noninflammatory neuropathies by nerve biopsy (Bouchard et al., 1999). Detection of demyelination supports the diagnosis of probable CIDP and possible CIDP (Table 10.22). Sural nerve biopsy reveals the following:

• Signs of segmental demyelination and remyelination with or without axonal degeneration.

• Endoneural edema.

• Mononuclear inflammatory infiltration.

• Proliferation of Schwann cells.

• Onion-bulb formation.

• Wallerian degeneration in older lesions.

In view of the lack of specificity and the fact that signs of inflammation are not always detectable, the diagnostic value of a nerve biopsy is debated by many authors. Nevertheless, a biopsy is an important auxiliary in the often difficult task of diagnosing CIDP.

Multifocal Motor Neuropathy

Clinical Features

Multifocal motor neuropathy (MMN) may possibly be a variant of CIDP. It is characterized by distinct clinical findings (for an overview, see Fischer et al., 2002):

• Multifocal, asymmetric paresis.

• Usually starting distally in the arms.

• Weakening or loss of deep tendon reflexes (DTR) in affected muscle groups, and preservation of DTR in nonaffected muscle groups.

• Absence of sensory deficits (clinically and neurographically).

• Muscular atrophy during the course of the disease.

• Cranial nerve VII may be involved.


Important findings for diagnosing MMN—although nowadays no longer absolutely essential—have been the detection of multifocal conduction blocks by motor neurography (in about 30% of patients) and the serological detection of IgM antibodies against GM1 ganglioside. Differential diagnosis of MMN versus spinal muscle atrophies and motor neuron diseases is of primary importance because it can be effectively treated with intravenous immunoglobulins.

CSF analysis. The only pathological marker—and present only in up to 30% of MMN patients—is a mild increase in total protein of up to 1000 mg/L.

Serum analysis. Serum IgM antibodies to GM1 are found in 30–60% of MMN patients, depending on the ELISA protocol used, and with modified technology even in up to 90% (Steck, 2000). A negative test result does therefore not exclude the presence of MMN. The GM1 antibodies are predominantly polyclonal, rarely monoclonal. In a few cases, GM2 antibodies have also been reported in MMN. Unlike in GBS, there is no evidence yet that infection-associated molecular mimicry triggers the humoral immune response to gangliosides.

Table 10.23 MGUS and malignant paraproteinemia (according to Dengler and Heidenreich, 1999)



Malignant paraproteinemia


• IgM, ~ 60%

• 10% cryoglobulin activity

• IgG, ~ 30%

• IgA, ~ 10%

• Mostly IgG or IgA

• Less often IgM

Light chain type

κ (80–90%)

Predominantly λ

Bone marrow

Free of inflltration

Inflltration by malignant B-cell clones

Serum immunoglobulins

0.75–1.5 g/100 mL

IgG and IgA, > 3 g/100 mL IgM, > 1.5 g/100 mL

Additional findings

• Polyneuropathy

• Over the years, transition to malignant paraproteinemia in ~35% of cases

• Polyneuropathy

• Anemia

• Bence Jones paraproteinuria

• Amyloidosis

Associated malignant tumor

• IgM

• IgG or IgA

• IgG and IgA

Waldenström's disease

Multiple myeloma

• POEMS syndrome

• Castleman's tumor

Paraproteinemic Polyneuropathy

Etiology and Classification

Paraproteinemic polyneuropathy develops in the context of clonal B-cell proliferation and synthesis of monoclonal immunoglobulin fractions of the classes IgM, IgG, and/or IgA. The paraproteins (M proteins) are either formed in isolation (monoclonal gammopathy of undetermined significance, MGUS) or in association with a malignant hematological disease (malignant paraproteinemia) (see Chap. 7, “Autoantibodies”). The characteristics of the two forms are listed in Table 10.23.


Serum analysis. Paraproteins are detected in serum by immunoelectrophoresis or immunofixation, or in urine as Bence Jones proteins. The immune reactivities of paraproteins and the associated neuropathies are summarized in Table 10.24. In about 50% of patients, IgM paraproteins have a specificity for myelin-associated glycoprotein (MAG), as detected by ELISA or immunoblot. The immunoreactive epitope is not only expressed on MAG but also on acidic glycolipids—sulfate-3-glucuronyl paragloboside (SGPG) and sulfate-3-glucuronyl-neolactose paragloboside (SGLPG)—as well as the myelin glycoproteins P0 and PMP-22, thus causing cross-reactivities against ganglioside antigens. In only about 5% of patients with IgM MGUS, the paraprotein shows reactivity with sulfatides and even less often with disialosyl gangliosides (GD 1 b, GD 3, GT1 b, GQ 1 b). The immune reactivities of IgG and IgA paraproteins in patients with MGUS are largely unknown (Steck, 2000; Willison and Yuki, 2002).

Nerve biopsy. Typical histological, immunohistochemical, and electron microscopic findings in sural nerve biopsies in MAG-reactive neuropathies include:

• Segmental demyelination.

• Concomitant axonal degeneration.

• Dilatation of myelin lamellae.

• IgM and complement deposition onto myelin lamellae.

Cryoglobulinemic polyneuropathy


Cryoglobulins are immunoglobulin fractions that precipitate or gel at cold temperature (Chap. 7, “Autoantibodies”). They are divided into three types (Table 10.25). Cryoglobulins are formed essentially or, often, secondarily in relation to:

• Hepatitis B (types I and II).

• Hepatitis C (types I and II).


• Malignant paraproteinemia (types I and II).

– Multiple myeloma.

– Waldenström's disease.

– POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes).

• Autoimmune diseases (type III).

The associated neuropathies develop via vasculitis. They are sensory or sensorimotor, often painful, asymmetric of the multiplex type, and axonal.


For the detection of cryoglobulins, blood must be collected using a warmed-up syringe and centrifuged at 37°C. Separate serum samples are stored at 37°C, 20°C, and 4°C and checked after 4, 24, 72, and 96 hours for cold precipitation or gelling. The cryoprecipitate is then characterized by immunoelectrophoresis and/or immunofixation using mono—or polyvalent antisera.


Table 10.25 Cryoglobulins (according to Kuhl et al., 2001)

Type I

Type II

Type III

• Monoclonal

– IgM (predominantly)

– IgG

– IgA

• Immunoglobulin fragments (Bence Jones protein)

• 25%

• Several classes

• Monoclonal components

– IgM, rarely IgG (monoclonal)

– IgG (polyclonal)

• 25%

• Polyclonal

• Complexes of IgM against autologous IgG, rarely IgM-IgG-IgA complexes

• 50%

Systemic Vasculitis and Connective Tissue Diseases

Classification and Clinical Features

Systemic vasculitis and connective tissue diseases are systemic disorders characterized by vascular inflammation induced by immune reactions. An exception is the rarely occurring primary angiitis of the CNS (PACNS), which affects exclusively the cerebral vessels and is not associated with extracerebral manifestations. The various forms of systemic vasculitis are divided into different syndromes according to the caliber of the vessels predominantly affected and to the various immunoserological, virological, and immunohistochemical findings (Moore and Richardson, 1998; Gross, 1999) (Table 10.26). Neuropsychiatric syndromes occur especially in necrotizing vasculitis of the medium-sized and small vessels, less often with vasculitis of the large vessels. Among the connective tissue diseases, involvement of the nervous system is most common in systemic lupus erythematosus (SLE), less often in rheumatoid arthritis, scleroderma, Sjögren's syndrome, and mixed connective tissue disease (MCTD, Sharp's syndrome). Neurological deficits may be the first symptom of these diseases and develop as a result of vasculitis of neural vessels. An exception is CNS involvement in SLE (CNS SLE), which is caused by noninflammatory small-vessel vasculopathy or thrombophilic diathesis, but rarely by cerebral vasculitis (< 10%).

Table 10.26 Neurological complications associated with systemic vasculitis and connective tissue diseases

Systemic vasculitis/connective tissue diseases

Complications, % Peripheral nervous system

Central nervous system

Medium-sized vessels


Polyarteritis nodosa



Primary angiitis of the CNS



Small vessels


Churg-Strauss syndrome



Microscopic polyangiitis



Wegener's granulomatosis



Behçet's disease



Essential cryoglobulinemic vasculitis



Hypersensitivity vasculitis



Large vessels



Giant-cell (temporal) arteritis



Takayasu's arteritis


Systemic lupus erythematosus



Rheumatoid arthritis



Sjögren's syndrome






Sharp's syndrome




The definitive diagnosis of neuromanifestations associated with systemic vasculitis or connective tissue disease may create considerable difficulties, especially when neurological deficits are the first symptom. Laboratory diagnosis is helpful, but not always specific and should always be interpreted in the context of clinical and histological data, and perhaps angiographic findings. Particularly in the case of cerebral symptoms, the differential diagnosis should include secondary effects of immunosuppressive treatment (infections, malignant non-Hodgkin lymphoma). Laboratory findings associated with systemic vasculitis and connective tissue diseases are summarized in Tables 10.28 and 10.29.

CSF Analysis

CSF analysis is of little value. The routine markers are normal or slightly increased in a nonspecific way. The following have diagnostic significance:

• In Behçet's disease and CNS SLE: signs of meningitis with mononuclear and/or granulocytic pleocytosis.


– Intrathecal IgG production may be present.

– OCB may be present.

– A positive MRZ reaction may be present.

– Presence of antineural antibodies (see below).

– Increased or intrathecally produced levels of IL-6 and prolactin.

• In Churg-Strauss syndrome: eosinophilia.

In view of the often long-term immunosuppressive treatment of systemic vasculitis and connective tissue diseases, CSF analysis plays a decisive role in excluding complicating infections. Differentiating between infections and the above disease activities is facilitated by determining procalcitonin concentrations in serum and CSF, for procalcitonin is clearly increased in bacterial infections. However, a moderate procalcitonin increase may also occur in vasculitis or connective tissue disease relapses with marked disease activity.

Serum Analysis

The systemic character of these diseases is of primary diagnostic importance. Laboratory findings reveal a set of inflammatory symptoms:

• Markedly increased ESR and CRP level.

• Anemia.

• Leukocytosis and thrombocytosis.

Laboratory parameters relevant for individual syndromes include:

• In SLE and Sjögren's syndrome: leukopenia, thrombopenia, anemia.

• In Churg-Strauss syndrome: eosinophilia.

Table 10.27 Immune complex-mediated forms of systemic vasculitis and connective tissue disease

Systemic vasculitis

Connective tissue disease

Essential cryoglobulinemic vasculitis

Systemic lupus erythematosus

Hypersensitivity vasculitis

Rheumatoid arthritis

Polyarteritis nodosa (associated with infection)

Sharp's syndrome

• In immune-complex-mediated diseases (Table 10.27):

– Decrease in complement (C 3, C 4, CH50).

– Detection of factor VIII-associated antigen and soluble thrombomodulin as indicators of vasculitis-associated endothelial damage.

Systemic Necrotizing Vasculitis

Typical laboratory findings in systemic vasculitis are summarized in Table 10.28.

Anti-neutrophil cytoplasmic antibodies. Anti-neutrophil cytoplasmic antibodies (ANCAs) are of high diagnostic value for systemic necrotizing vasculitis, which predominantly affects small vessels. ANCAs are detected by indirect immunofluorescence using fixed human neutrophilic granulocytes (Chap. 7, “Autoantibodies”). Depending on the fluorescence pattern, two subtypes can be accurately distinguished by means of ELISA: cANCAs yield cytoplasmic immunofluorescence and react with the enzyme proteinase 3 as the specific target antigen (PR3-cANCA), and pANCAs produce perinuclear immunofluorescence and are specific for the enzyme myeloperoxidase (MPO-pANCA) (see Fig. 7.3 a, b). PR3-cANCAs are present in about 90% of patients with Wegener's granulomatosis and MPO-pANCAs in about 90% of patients with microscopic polyangiitis. PR3-cANCAs or MPO-pANCAs are found in about 30% of patients with Churg-Strauss syndrome.

Other autoantibodies. Antibodies against endothelial cells (AECAs) are detected in the majority of patients with Takayasu's arteritis. Cryoglobulins, i. e., complexes of monoclonal and/or polyclonal immunoglobulins that precipitate in the cold, are characteristic of cryoglobulinemic vasculitis (Chap. 7, “Autoantibodies”).

Pathogen detection. Cryoglobulinemic vasculitis and in some cases also polyarteritis nodosa are associated with infections; they are frequently caused by hepatitis C virus (HCV) and hepatitis B virus (HBV). Direct and indirect detection of the viruses is therefore diagnostically important. An association with infections or drugs is often found also in hypersensitivity vasculitis.

Connective Tissue Diseases

Typical laboratory findings in connective tissue diseases are summarized in Table 10.29.

Antinuclear antibodies. In almost all connective tissue diseases, antinuclear antibodies (ANAs) are present. They are detected by indirect immunofluorescence tests using cultured cells and may be directed against a multitude of nuclear target antigens (Chap. 7, “Autoantibodies”). Among these ANAs are autoantibodies against defined nuclear constituents; they are highly specific for individual syndromes and in many cases facilitate the prognostic assessment (Table 10.30; for details, see specialist textbooks). The fluorescence pattern in the immunofluorescence test (homogeneous, granular, nucleolar, centromeric) gives only a rough orientation with regard to ANA specificity; fine differentiation is then by ANA characterization using immunoblot and/or ELISA.



Other autoantibodies. Additional autoantibodies include:

• IgM rheumatoid factors: IgM antibodies against the Fc region of IgG are characteristic of rheumatoid arthritis and type II essential cryoglobulinemia.

• Phospholipid antibodies: These are directed against cardiolipin and other negatively charged phospholipids. They are detected by ELISA in the presence of β2-glycoprotein 1 as a cofactor. Phospholipid antibodies occur in 15−70% of patients with SLE and in 95−100% of patients with primary antiphospholipid syndrome (APS), and also in various other rheumatic and nonrheumatic diseases.

• Lupus anticoagulant: these antibodies are directed against phospholipid−protein complexes on the cell membrane and are associated with an increased tendency to thrombosis. In the presence of lupus anticoagulant, the clotting time in a phospholipid−dependent coagulation test (e. g., aPTT) is prolonged. Lupus anticoagulant occurs in association with primary APS, SLE, and other autoimmune diseases.

• Autoantibodies against ribosomal P protein: These are specific for phosphoproteins of the ribosomal subunit and are present in 10−20% of patients with SLE.

• Antineural antibodies against surface antigens of neuroblastoma cell lines: These antibodies frequently occur in the serum and CSF of patients with CNS SLE. Only specialized laboratories can determine them, and their diagnostic value is unclear.

• Antibodies against α-fodrin: According to recent studies, antibodies against α-fodrin are found in many patients with primary and secondary Sjögren's syndrome, in whom they correlate with the activity of the disease (Watanabe et al., 1999). Their determination is only possible in specialized laboratories.

• Antibodies against aquaporin-4 (NMO-IgG, AQP4-Ab): These antibodies are detectable in approximately 50% of patients with SLE or Sjögren's syndrome and concomitant NMO spectrum disorders (NMO-SD), which include optic neuritis, longitudinally extensive transverse myelitis (LETM), and classic neuromyelitis optica (NMO), and rare cases of brainstem encephalitis, but are negative in SLE and Sjögren's syndrome patients without NMO-SD. So far, it is unclear whether NMO-SD in patients with SLE/Sjögren's syndrome represents an entity of its own or reflects neurological disease activity of the collagen vascular disorder (Pittock SJ et al, 2008).

Clinical-immunological correlations in CNS SLE. In the case of CNS involvement in SLE, uncovering the etiology of neuropsychiatric symptoms is facilitated by clinical-immunological correlations (West et al., 1995). Focal SLE-associated symptoms develop predominantly as a result of embolic or thrombotic stroke syndromes. Correlated findings are ischemic lesions on cranial MRI and high serum titers of phospholipid antibodies. Symptoms that suggest diffuse CNS damage (headache, seizures, psychosis, coma) develop primarily as a result of noninflammatory vasculopathy and correlate with normal findings or multifocal small hyperintensities on cranial MRI. Laboratory findings may reveal antineural autoantibodies in CSF (not in serum). SLE psychosis coincides frequently with serological detection of autoantibodies against ribosomal P protein.


Urine analysis. Of the connective tissue diseases, renal involvement is seen primarily in SLE. When SLE is suspected on the basis of clinical and laboratory findings, in addition to urinary sediment evaluation, it is recommended that creatinine clearance and proteinuria should be determined in the 24-hour urine even if retention values are normal.

Stepwise Diagnosis

Laboratory diagnosis of neuropsychiatric manifestations in rare forms of systemic vasculitis and connective tissue diseases should be undertaken one step at a time in order to avoid ordering unnecessary and expensive laboratory tests (Table 10.31).


Paraneoplastic Neurological Syndromes


Paraneoplastic neurological syndromes are rare tumor-associated diseases of the nervous system, affecting less than 1% of cancer patients (Table 10.32). They are caused by autoimmune reactions against onconeural antigens, i. e., neural epitopes expressed ectopically in tumor tissues. The resulting dysfunction causes distinct unifocal or multifocal syndromes depending on the preferential or overlapping involvement of different anatomical structures. Paraneoplastic neurological syndromes precede detection of the inducing tumor by months or years. They are therefore of extraordinary diagnostic value for occult cancers (for an overview, see Voltz, 2002; Darnell and Posner, 2003).


Laboratory parameters of cardinal diagnostic importance for paraneoplastic neurological syndromes are high-titer antibodies against various neural proteins (Tables 7.2 and 10.34). These antibodies can be detected in serum and CSF as a sign of an onconeural immune response.

CSF Analysis

Cell counts and total protein in the CSF are pathologically changed in about 50% of patients with paraneoplastic neurological syndromes (Table 10.33). A normal CSF finding does not exclude these diseases.

Table 10.32 Paraneoplastic syndromes of the peripheral and central nervous systems

Peripheral nervous system

Central nervous system

• Myasthenia gravis (MG)

• Lambert-Eaton myasthenic syndrome (LEMS)

• Neuromyotonia

• Subacute sensory neuronopathy (SSN)

• Autonomic neuropathy

• Paraneoplastic retinopathy

• Optic neuritis

• Paraneoplastic encephalomyelitis (PEM)

• Limbic encephalitis

• Rhombencephalitis

• Paraneoplastic cerebellar degeneration (PCD)

• Paraneoplastic opsoclonus-myoclonus syndrome (OMS)

• Motor neuron disease (MND)

• Stiff person syndrome (SPS)

Table 10.33 Characteristic CSF findings associated with paraneoplastic syndromes of the nervous system

CSF parameter


Diagnostic sensitivity, %

Cell count/μL





Transformed lymphocytes →

Plasma cells →


Barrier function, QAlb, × 10−3

< 8–10


Total protein, mg/L



Intrathecal immunoglobulin fractions, IF > 0



Oligoclonal IgG bands



Typical changes include mild lymphomonocytic pleocytosis of 30–40 cells/μL, slightly elevated total protein, and quantitative or qualitative detection of intrathecal IgG production.

Pleocytosis is usually detected early on but disappears within weeks or months after the onset of neurological symptoms. Locally synthesized IgG may persist. Diagnostically relevant antineural antibodies (Tables 7.2 and 10.34), if detected, are usually produced intrathecally in paraneoplastic syndromes of the CNS, but not in paraneoplastic disorders affecting peripheral nerve structures (Stich et al., 2007). An elevated antibody index and/or bands with specificity for onconeural antigens within CSF-restricted OCB has been shown for the antigens Hu, Yo, Ri, CRMP/CV2, amphiphysin, and Ma2 (Vega et al., 1994; Stich et al., 2007; Jarius et al., 2008a). In contrast to this, abnormal CSF findings including OCB are uncommon in limbic encephalitis associated with VGKC antibodies (Jarius et al., 2008 b).

Serum Analysis

Antibodies. The various associated antibody reactivities (see Chap. 7, “Antineural Antibodies”) and the most commonly associated malignant tumors are listed in Table 10.34.

Target antigens. In peripheral paraneoplastic conditions the humoral immune response is directed against cellular membrane proteins of known function (usually ion channels), while in paraneoplastic conditions of the CNS it is directed against cytoplasmic or nuclear proteins of as yet unknown function. Recently, antibodies against VGKC—which so far have only been described in the context of neuromyotonia—have also been found in patients with limbic encephalitis (Vincent et al., 2004). Furthermore, antibodies targeting conformational epitopes of the NMDA receptor (NMDA-R) of hippocampal neurons define a newly identified variant of limbic encephalitis in young women, which is associated with ovarian teratoma (Dalmau et al., 2007). The expression of onconeural CNS antigens in the associated malignant tumor probably contributes to the breaking down of immune tolerance. Target antigens are listed in Table 10.35.

Diagnostic implications. Serological detection of antibodies against the various onconeural proteins occurring in association with paraneoplastic syndromes of the CNS confirms the paraneoplastic etiology of neurological disease almost without exception. Only antibody responses against ion channels (VGKC, NMDA-R) in central paraneoplastic conditions such as limbic encephalitis are not invariably associated with an underlying tumor. The paraneoplastic syndrome type also has a high predictive value with regard to the type and organ specificity of the underlying neoplasm.

Pitfalls in Diagnosing Paraneoplastic Syndromes

In interpreting the clinical findings and the serological results, it should be borne in mind that various syndromes also occur as nonparaneoplastic autoimmune diseases, and that the antibodies associated with the syndrome do not always distinguish between a paraneoplastic and a purely autoimmune etiology.

Detection. Antineural antibodies are identified by immunohistochemical detection of typical intracellular cell structures in neural cells through the use of brain sections of various species (Fig. 10.6) in combination with characterization of the molecular weight of the target antigens by Western blot (Chap. 7, “Antineural Antibodies”). Diagnosis on the basis of immunohistochemistry alone is insufficient. The diagnostic procedure can be supplemented by Western blot or ELISA using recombinant antigens, if available (see Chap. 7, “Antineural Antibodies”). Antibodies against ion channels and acetylcholine receptors are identified by immune precipitation of labeled protein preparations with specificity for the respective channel or receptor (Chap. 7, “Autoantibodies”). Detection of a specific antibody should prompt an intense search for a tumor, and if results are negative the search should be repeated at regular intervals.


Table 10.35 Target antigens of antineuronal antibodies


Target antigen



P/Q type VGCC

Ion channel



Acetylcholine receptor


Ion channel

Sarcomere protein of striated muscles



Ion channel



Ca2+-binding intracellular protein, plays a role in signal transduction in the retina


HuD, HuC, Hel-N1, HuR

Family of neuron-specific RNA binding proteins, characterized by RNA recognition motives*




Neuron-specific RNA binding proteins*


CDR34, CDR62–1, CDR62–2, CZF

Leucine zipper proteins*





Ma1 antigen

Family of CNS/testis-specific proteins*


Ma2 antigen

Family of CNS/testis-specific proteins*


PCA-2 antigen



mGluR1 antigen

Metabotropic glutamate receptor



Concentrated in nerve endings, plays a role in endocytosis of synaptic vesicles



Transcription factor: plays a role in the development of the nervous system; in the adult brain predominantly expressed in the Bergmann glia of the cerebellum


N-methyl-D-aspartate receptor

Ion channel

Nova, neuro-oncological ventral antigen; CDR, complementary determining region; CZF, cerebellar zinc finger. (For other abbreviations see Table 10.34.)

* Proteins with largely unknown functions.


Fig. 10.6a, b Antineural antibodies. (Courtesy of Dr. U. Wurster, Department of Neurology, Medical School Hannover, Germany.)

a Hu-positive neurons in a section of monkey cerebellum. Neurons show nuclear staining but no staining of the nucleolus. Immunoperoxidase reaction. × 100.

b Yo-positive Purkinje cells in a section of human cerebellum. In addition to the granular cytoplasm, the dendrites are also stained. Immunoperoxidase reaction. × 400.

Clinical–Immunological Correlations

The association between certain syndromes and various antibodies is diverse (Table 10.36). Some immune responses are closely associated with just one syndrome, e. g., VGCC antibodies with LEMS. Other immune responses are highly specific for more than one clinical syndrome or, in particular, for multifocal syndromes. Typical examples are anti-Hu antibodies, which are present in all syndromes that may be part of the paraneoplastic encephalomyelitis/subacute sensory neuronopathy (PEM/SSN) syndrome. Finally, several immune responses may be associated with one specific form of syndrome. A characteristic example is paraneoplastic cerebellar degeneration (PCD), which can occur with anti-Yo, anti-Ri, anti-Tr, anti-Hu, and anti-GluR1 antibodies. Here, the respective antibody subtype is highly specific for the underlying malignant tumor. It is also possible for several syndromes to coincide, for example, LEMS and the anti-Hu syndromes (PEM or PCD).

Pitfalls in Diagnosing and Interpreting Laboratory Findings in Paraneoplastic Neurological Syndromes

Onconeural antibodies occur in only about 50% of patients with paraneoplastic neurological syndromes. Hence, a negative serodiagnosis does not exclude their presence. Several of the syndromes listed in Table 10.32 also occur as nonparaneoplastic autoimmune diseases:

• Myasthenia gravis.

• Lambert-Eaton myasthenic syndrome.

• Neuromyotonia.

• Opsoclonus-myoclonus syndrome (parainfectious).

• Stiff person syndrome.

• Sensory neuronopathy (Sjögren's syndrome).

Various antibody specificities are of high diagnostic importance for distinct clinical syndromes, but cannot provide a basis for differentiating between paraneoplastic and purely autoimmune etiologies:

• Antibodies against acetylcholine receptors (myasthenia gravis).

• Antibodies against nicotinic ganglionic acetylcholine receptors (autonomic neuropathy).

• Antibodies against voltage-gated calcium channels (Lambert-Eaton syndrome).

• Antibodies against voltage-gated potassium channels (neuromyotonia, limbic encephalitis).

• Antibodies against glutamate decarboxylase (stiff person syndrome).

In some paraneoplastic or purely autoimmune syndromes, distinct antibody specificities may suggest a paraneoplastic origin:

• Antibodies against titin, ryanodine receptor, interleukin-α, interleukin-12 (myasthenia gravis, see above, “Myasthenia Gravis”).

• Antibodies against amphiphysin (stiff person syndrome).

• Antibodies against SOX1 (LEMS).

In rare cases, antibody-positive patients do not develop a malignant tumor, and various antineural antibodies (anti-Hu) may be detected at low titers in some cancer patients who do not have a paraneoplastic neurological syndrome.

Table 10.36 Clinical–immunological correlations associated with paraneoplastic syndromes of the nervous system

Immune response


One immune response—one syndrome






Limbic encephalitis

One immune response—several syndromes


PEM/SSN, limbic encephalitis, rhombencephalitis, PCD, POM


PEM/SSN, limbic encephalitis, rhombencephalitis, PCD




Neuromyotonia, limbic encephalitis

Several immune responses—one syndrome

Anti-Yo, anti-Ri, anti-Tr, anti-Hu, anti-mGluR1


Anti-Hu, anti-Ma2, anti-VGKC, anti-NMDA-R

Limbic encephalitis

Several immune responses—overlapping syndromes

Anti-Hu and anti-VGCC

PEM and LEMS, or PCD and LEMS


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Further Reading

Berlit P (ed). Klinische Neurologie. 3rd ed. Heidelberg: Springer Verlag; 2010

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