Absence of significant clinical benefit for a systematic routine creatine phosphokinase measurement in asymptomatic patients treated with anti-programmed death protein (ligand) 1 immune checkpoint inhibitor to screen cardiac or neuromuscular immune-related toxicities.

AIM: Despite unprecedented results of anti-programmed death protein (ligand) 1 (PD-(L)1) immune checkpoint inhibitor in the oncology's armamentarium, immune-related adverse events (irAEs) represent a therapeutic hurdle. Currently, there is no consensual recommendation on a routinely monitored biomarker to early detect irAE. Biological markers such as serum creatine phosphokinase (CPK) are commonly used to measure muscular tissue injury. The potential of routine serum CPK monitoring to predict cardiac or neuromuscular irAE in patients treated with immunotherapy remains unknown. METHODS: In this retrospective study between January 2016 and December 2018 at Gustave Roussy Cancer Campus, 1151 cancer patients treated with anti-PD-(L)1 immunotherapy were systematically monitored with serum CPK measurements before each immunotherapy cycle. We considered significant CPK increases according to Common Terminology Criteria for Adverse Events v5.0 (CTCAEV5) of grade ≥2 severity. Comparisons were performed in patients with immune-related CPK (ir-CPK) elevations symptomatic versus asymptomatic. RESULTS: Overall, 53 of 1151 (4.6%) patients showed a CPK increase. Elevations of CPK were deemed to be immunotherapy-related in 31 of 1151 (2.7%) patients. Among them, 12 of 31 (38.7%) patients experienced symptomatic cardiac or neuromuscular irAE, whereas the other 19 of 31 (61.3%) patients remained asymptomatic. In patients with symptomatic irAE, the mean ir-CPK level was higher compared with asymptomatic patients (1271 versus 771 UI/L, P value = 0.02). In the asymptomatic group, all patients experienced a spontaneous resolution of the ir-CPK increase, and none required medical intervention. CONCLUSION: Most patients with immune-related CPK increase remained asymptomatic. The CPK serum increase did not alter the clinical management of asymptomatic patients. The results of this study did not support a significant clinical interest for a systematic routine CPK monitoring in patients amenable to anti-PD-(L)1 immunotherapy.

Neurologic autoimmunity and immune checkpoint inhibitors: Autoantibody profiles and outcomes.

OBJECTIVE: To describe neural autoantibody profiles and outcomes in patients with neurologic autoimmunity associated with immune checkpoint inhibitor (ICI) cancer immunotherapy. METHODS: In this retrospective descriptive study, 63 patients with ICI-related neurologic autoimmunity were included: 39 seen at the Mayo Clinic Neurology Department (clinical cohort) and 24 whose serum/CSF was referred to the Mayo Clinic Neuroimmunology Laboratory for autoantibody testing. Serum/CSF samples were tested for neural-specific autoantibodies. Predictors of unfavorable outcome (residual adverse event severity grade ≥3) were explored (logistic regression). RESULTS: Median age at neurologic symptom onset was 65 years (range 31-86); 40% were female. Neurologic manifestations were CNS-restricted (n = 26), neuromuscular (n = 30), combined (n = 5), or isolated retinopathy (n = 2). Neural-specific autoantibodies were common in patients with CNS involvement (7/13 [54%] in the unbiased clinical cohort) and included known or unidentified neural-restricted specificities. Only 11/31 patients with CNS manifestations had neuroendocrine malignancies typically associated with paraneoplastic autoimmunity. Small-cell lung cancer (SCLC)-predictive antibodies were seen in 3 patients with non-neuroendocrine tumors (neuronal intermediate filament immunoglobulin G [IgG] and antineuronal nuclear antibody 1 with melanoma; amphiphysin IgG with non-SCLC). A median of 10 months from onset (range, 0.5-46), 14/39 in the clinical cohort (36%) had unfavorable outcomes; their characteristics were age ≥70 years, female, CNS involvement, lung cancer, higher initial severity grade, and lack of systemic autoimmunity. By multivariate analysis, only age remained independently associated with poor outcome (p = 0.01). Four of 5 patients with preexistent neurologic autoimmunity experienced irreversible worsening after ICI. CONCLUSIONS: Neural-specific autoantibodies are not uncommon in patients with ICI-related CNS neurologic autoimmunity. Outcomes mostly depend on the pre-ICI treatment characteristics and clinical phenotype.

Immune checkpoint inhibitors-induced neuromuscular toxicity: From pathogenesis to treatment.

Immune checkpoint inhibitors (ICIs) are increasingly used and are becoming the standard of care in the treatment of various tumor types. Despite the favorable results in terms of oncological outcomes, these treatments have been associated with a variety of immune-related adverse events (irAEs). Neurological irAEs are rare but potentially severe. Neuromuscular disorders represent the most common neurological irAEs following anti-PD-1, anti-PD-L1, and anti-CTLA-4 treatment, and include myositis, myasthenia gravis, and demyelinating polyradiculoneuropathy. Instrumental findings may differ from typical neuromuscular disorders occurring outside ICIs treatment. Despite initial severity, neurological irAEs often respond to immune-modulating therapies. Prompt irAEs diagnosis, ICIs discontinuation, and early treatment with corticosteroids, together with patient education and a multi-disciplinary approach, are important for optimizing clinical outcomes. Intravenous immunoglobulin, plasma exchange, and other immune-modulating treatments should be considered in more severe cases. Consideration of re-challenging with the same immunotherapy drug may be given in some cases, based on clinical picture and initial severity of irAEs.

A survey of known immune epitopes in the enteroviruses strains associated with acute flaccid myelitis.

Enteroviruses are potentially linked to the emergence of Acute Flaccid Myelitis (AFM), a rare but very serious condition that affects the nervous system. AFM has been associated with coxsackievirus A16, enterovirus A71 (EVA71) and enterovirus D68 (EVD68). Little is known about host-pathogen interactions for these viruses, and whether immune responses may have a protective or immunopathological role in disease presentations. Towards addressing this issue, we used the Immune Epitope Database to assess the known inventory of B and T cell epitopes from enteroviruses, focusing on data related to human hosts. The extent of conservation in areas that are targets of B and T cell immune responses were examined. This analysis sheds light on regions of the enterovirus polypeptide that can be probed to induce a specific or cross-reactive B or T cell the immune response to enteroviruses, with a particular focus on coxsackievirus A16, EVA71 and EVD68. In addition, these analyses reveal the current gap-of-knowledge in the T and B cell immune responses that future studies should aim to address.

Myositis and neuromuscular side-effects induced by immune checkpoint inhibitors.

AIM: To characterise clinical presentation, laboratory and histopathologic characteristics and assess the treatment and outcome of neuromuscular side-effects of checkpoint therapy. METHODS: The side-effect registry and the institutional database from ten skin cancer centres were queried for reports on myositis and neuromuscular side-effects induced by checkpoint inhibitors. In total, 38 patients treated with ipilimumab, tremelimumab, nivolumab and pembrolizumab for metastatic skin cancer were evaluated and characterised. RESULTS: Myositis was the most frequent neuromuscular adverse event. In 32% of cases, myositis was complicated by concomitant myocarditis. Furthermore, cases of isolated myocarditis, myasthenia gravis, polymyalgia rheumatica, radiculoneuropathy and asymptomatic creatine kinase elevation were reported. The onset of side-effects ranged from the first week of treatment to 115 weeks after the start of therapy. Most of the cases were severe (49% grade III-IV Common Terminology Criteria for Adverse Events), and there were two fatalities (5%) due to myositis and myositis with concomitant myocarditis. Only half of the cases (50%) completely resolved, whereas the rest was either ongoing or had sequelae. Steroids were given in 80% of the resolved cases and in 40% of the unresolved cases. CONCLUSION: Immune-mediated neuromuscular side-effects of checkpoint inhibitors greatly vary in presentation and differ from their idiopathic counterparts. These side-effects can be life threatening and may result in permanent sequelae. Occurrence of these side-effects must be taken into consideration for patient information, especially when considering adjuvant immunotherapy with anti-programmed cell-death protein 1 (PD-1) antibodies and monitoring, which should include regular surveillance of creatine kinase.

Peripheral Nerve Hyperexcitability Syndromes.

PURPOSE OF REVIEW: This article provides a review of the clinical phenotypes and evaluation of peripheral nerve hyperexcitability syndromes. These rare diagnoses include cramp-fasciculation syndrome, Isaacs syndrome, and Morvan syndrome. Recent investigations have led to an understanding of the autoimmune underpinnings of these conditions and their specific associated antibodies. As the presentation of peripheral nerve hyperexcitability syndromes includes muscle stiffness, twitches, and spasms, which are also shared with certain central nervous system and myopathic conditions, the differential diagnosis of peripheral nerve hyperexcitability syndromes is reviewed. RECENT FINDINGS: Peripheral nerve hyperexcitability syndromes share clinical and electrodiagnostic evidence of motor nerve instability; however, their clinical presentations are varied. Case reviews have helped us understand the spectrum of symptoms associated with the three peripheral nerve hyperexcitability syndromes reviewed here: cramp-fasciculation syndrome, Isaacs syndrome, and Morvan syndrome. More recently, research has focused on understanding the voltage-gated potassium channel complex antibodies as well as neoplasms associated with these conditions. SUMMARY: The diagnosis of peripheral nerve hyperexcitability syndromes requires a high index of suspicion, support from the physical examination, familiarity with the spectrum of symptoms associated with peripheral nerve hyperexcitability syndromes, and recognition of diagnostic EMG features. Voltage-gated potassium channel complex antibodies are associated with these conditions. Optimum treatment and autoimmune pathogenesis remain areas of active research.

Neuronal autoantibodies: differentiating clinically relevant and clinically irrelevant results.

The aim of this study is to compare the rates of clinically relevant and clinically irrelevant neuronal autoantibodies among patients presenting with new neurological symptoms. We reviewed 401 neurological patients who were tested for the Mayo-Clinic paraneoplastic panel from January 2014 to December 2014 at the Johns Hopkins Hospital. We divided antibody-positive patients into two groups: clinically relevant (CR), in which a recognizable autoimmune or paraneoplastic syndrome was confirmed, and clinically irrelevant (CI), in which an autoimmune/paraneoplastic etiology was initially suspected but an alternative diagnosis was eventually found. We used Fisher's exact test for categorical variables and Mann-Whitney U test for continuous variables to identify differences between the two groups. Fifty-three patients tested positive for one or more neuronal autoantibodies. There were 17 CR (65% females, mean age 56 years), 33 CI, and 3 indeterminate patients. Compared to CI patients, CR patients were more likely to present with movement disorders or stiff person syndrome, have inflammatory CSF markers, cancer or smoking history, concomitant hyponatremia, and classical onconeuronal antibodies. CI patients were more likely to have a neuromuscular presentation, a chronic course, and antibodies against synaptic antigens. By combining the most robust differentiating factors, we developed a simple scale that predicted clinical relevance with an odds ratio of 50.3 (CI 8.2-309.9, P < 0.0001) if the score was ≥ 2. Up to 62% of neuronal autoantibody-positive patients are ultimately found to have an alternative diagnosis. Several clinical and laboratory factors can differentiate CR from CI patients to aid in interpretation of positive results.

Intracellular and non-neuronal targets of voltage-gated potassium channel complex antibodies.

OBJECTIVES: Autoantibodies against the extracellular domains of the voltage-gated potassium channel (VGKC) complex proteins, leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-2 (CASPR2), are found in patients with limbic encephalitis, faciobrachial dystonic seizures, Morvan's syndrome and neuromyotonia. However, in routine testing, VGKC complex antibodies without LGI1 or CASPR2 reactivities (double-negative) are more common than LGI1 or CASPR2 specificities. Therefore, the target(s) and clinical associations of double-negative antibodies need to be determined. METHODS: Sera (n=1131) from several clinically defined cohorts were tested for IgG radioimmunoprecipitation of radioiodinated α-dendrotoxin (125I-αDTX)-labelled VGKC complexes from mammalian brain extracts. Positive samples were systematically tested for live hippocampal neuron reactivity, IgG precipitation of 125I-αDTX and 125I-αDTX-labelled Kv1 subunits, and by cell-based assays which expressed Kv1 subunits, LGI1 and CASPR2. RESULTS: VGKC complex antibodies were found in 162 of 1131 (14%) sera. 90 of these (56%) had antibodies targeting the extracellular domains of LGI1 or CASPR2. Of the remaining 72 double-negative sera, 10 (14%) immunoprecipitated 125I-αDTX itself, and 27 (38%) bound to solubilised co-expressed Kv1.1/1.2/1.6 subunits and/or Kv1.2 subunits alone, at levels proportionate to VGKC complex antibody levels (r=0.57, p=0.0017). The sera with LGI1 and CASPR2 antibodies immunoprecipitated neither preparation. None of the 27 Kv1-precipitating samples bound live hippocampal neurons or Kv1 extracellular domains, but 16 (59%) bound to permeabilised Kv1-expressing human embryonic kidney 293T cells. These intracellular Kv1 antibodies mainly associated with non-immune disease aetiologies, poor longitudinal clinical-serological correlations and a limited immunotherapy response. CONCLUSIONS: Double-negative VGKC complex antibodies are often directed against cytosolic epitopes of Kv1 subunits and occasionally against non-mammalian αDTX. These antibodies should no longer be classified as neuronal-surface antibodies. They consequently lack pathogenic potential and do not in themselves support the use of immunotherapies.

IgG4-Related Neuromyopathy Associated With Recurrent Pleural Effusion.

OBJECTIVE: Immunoglobulin G4-related disease (IgG4-RD) is an increasingly recognized idiopathic systemic disorder associated with elevated serum IgG4 level and tissue infiltration by IgG4-positive plasma cells. Multiple neurological manifestations, including peripheral neuropathy, have been described in IgG4-RD. Our objective is to discuss a case report and review of the literature, which would expand the spectrum of IgG4-RD. METHODS: We describe the clinical features and biopsy findings in a patient with IgG4-RD who presented with features suggestive of neuromyopathy in the setting of recurrent pleural effusion and weight loss. RESULTS: Electrodiagnostic findings were suggestive of an irritable myopathy and polyradiculoneuropathy with primary demyelination and secondary axonal degeneration. Pleural biopsy and laboratory studies confirmed the diagnosis. Improvement was sustained with steroid therapy. CONCLUSIONS: We describe the first case, to our knowledge, of IgG4-related neuromyopathy associated with recurrent pleural effusion. Our case expands the clinical spectrum of IgG4-RD. Neurologists should be aware of this treatable disorder and in the appropriate clinical context consider it in the differential diagnosis of neuromyopathy.

Cramp-fasciculation syndrome in patients with and without neural autoantibodies.

INTRODUCTION: We investigated the clinical, electrophysiological and neural autoantibody characteristics in cramp-fasciculation syndrome (CFS) patients. METHODS: We reviewed Mayo Clinic records from 2000 to 2011 to identify clinically defined CFS patients who underwent neural autoantibody testing. Stored sera of patients who tested positive for antibodies to voltage-gated potassium channel complex (VGKC complex) were analyzed further for leucine-rich glioma-inactivated 1 (LGI1) or contactin-associated protein-2 immunoglobulin G (CASPR2-IgG) antibodies. RESULTS: Thirty-seven patients were identified. Twelve were seropositive for neural autoantibodies. Clinical manifestations were similar in seropositive and seronegative patients, although central and autonomic neuronal hyperexcitability symptoms were more common in seropositive cases. No patients had a malignancy. Repetitive tibial nerve stimulation at 10 Hz revealed longer afterdischarges in seropositive patients. Two of 7 patients with VGKC-complex autoimmunity demonstrated LGI1 or CASPR2-IgG antibodies. Only 2 of 12 seropositive patients required immunotherapy. CONCLUSIONS: VGKC-complex autoimmunity occurs in a minority of CFS patients. Antibody positivity was associated with extramuscular manifestations, typically without malignancy. Target antigens within the VGKC complex remain unknown in most patients.

Common pathways of autoimmune inflammatory myopathies and genetic neuromuscular disorders.

It has been shown that many hereditary motor neuron diseases are caused by mutation of RNA processing enzymes. Survival of motor neuron 1 (SMN1) is well-known as a causative gene for spinal muscular atrophy (SMA) and mutations of glycyl- and tyrosyl-tRNA synthetases are identified as a cause of distal SMA and Charcot-Marie-Tooth disease. Why and how the dysfunction of these ubiquitously expressed genes involved in RNA processing can cause a specific neurological disorder is not well understood. Interestingly, SMN complex has been identified recently as a new target of autoantibodies in polymyositis (PM). Autoantibodies in systemic rheumatic diseases are clinically useful biomarkers associated with a particular diagnosis, subset of a disease, or certain clinical characteristics. Many autoantibodies produced in patients with polymyositis/dermatomyositis (PM/DM) target RNA-protein complexes such as aminoacyl tRNA synthetases. It is interesting to note these same RNA-protein complexes recognized by autoantibodies in PM/DM are also responsible for genetic neuromuscular disease. Certain RNA-protein complexes are also targets of autoantibodies in paraneoplastic neurological disorders. Thus, there are several interesting associations between RNA-processing enzymes and neuromuscular disorders. Although pathogenetic roles of autoantibodies to intracellular antigens are generally considered unlikely, understanding the mechanisms of antigen selection in a particular disease and specific neurological symptoms caused by disruption of ubiquitous RNA-processing enzyme may help identify a common path in genetic neuromuscular disorders and autoimmunity in inflammatory myopathies.

Neuromuscular complications of hepatitis A virus infection and vaccines.

The hepatitis A virus (HAV) infects millions of people worldwide every year. Case histories report on various acute neuropathy syndromes in the context of acute HAV infection, but any causal link has not been established. Epidemiological data also cast doubt on the importance of HAV as a trigger for Guillain-Barré syndrome. The virtual absence of a chronic HAV-infected state likely explains the rare occurrence of extrahepatic immune-mediated diseases, including an absence of chronic autoimmune neuromuscular disorders. Several vaccines against HAV provide effective protection against natural infection. Isolated case histories report on an unconvincing association between HAV vaccination and neuropathy. Medical and epidemiological data show insufficient evidence to support a causal relationship between HAV vaccines and neuropathy syndromes. Aluminum hydroxide, a HAV vaccine adjuvant, is considered a trigger of the macrophagic myofasciitis syndrome. This review concludes that it seems unnecessary to routinely consider HAV infection or vaccination as triggers of neuromuscular diseases.

A review: the use of rituximab in neuromuscular diseases.

Autoimmunity plays a major role in the pathogenesis of many neuromuscular disorders such as chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, polymyositis, dermatomyositis, myasthenia gravis, Lambert Eaton syndrome, and stiff person syndrome. Although most of these disorders respond favorably to the commonly used immunomodulatory agents such as steroids, intravenous gamma globulin, plasmapheresis, and chemotherapy, some are initially refractory, whereas others gradually lose responsiveness. Therefore, alternative, selective, and novel immunosuppressive agents are used to treat these cases. Among these agents, rituximab has shown promise in some of the neuromuscular disorders with minimal side effects. Rituximab is a genetically engineered antibody that depletes CD20+ B-cells and is Food and Drug Administration- approved for treatment of non-Hodgkin lymphoma, CD20+ CLL, and rheumatoid arthritis. It carries a favorable side effects profile. However, evidence of efficacy is limited to case series and large prospective randomized controlled trials are lacking. In this article, we review and discuss the available literature on rituximab in treatment of various autoimmune neuromuscular diseases.

The role of humoral autoimmunity in gastrointestinal neuromuscular diseases.

Dysfunction of the gastrointestinal neuromuscular apparatus (including interstitial cells of Cajal) is presumed to underlie a heterogeneous group of disorders collectively termed gastrointestinal neuromuscular diseases (GINMDs). There is increasing experimental and clinical evidence that some GINMDs are immune-mediated, with cell-mediated dysfunction relatively well studied. Humoral (antibody)-mediated autoimmunity is associated with several well-established acquired neuromuscular diseases and is now implicated in an increasing number of less well-characterised disorders, particularly of the central nervous system. The role of autoimmunity in GINMDs has been less studied. Whilst most work has focused on the presence of antibodies directed to nuclear antigens, particularly in the context of secondary disorders such as paraneoplastic intestinal pseudo-obstruction, the possibility that 'functional' anti-neuronal antibodies directed to membrane-bound ion channels may cause disease (channelopathy) is now also being realised. The evidence for humoral autoimmunity as an etiologic factor in primary (idiopathic) and secondary GINMDs is systematically presented using the original paradigms previously applied to established autoimmune neuromuscular disorders. The presence of anti-enteric neuronal antibodies, although repeatedly demonstrated, still requires the identification of specific neuronal autoantigens and validated evidence of pathogenicity.

Leucoencephalopathy, transverse myelopathy, and peripheral neuropathy in association with glutamic acid decarboxylase-65 (GAD) antibodies in children with cancer.

Neurologic toxicity may occur as a direct effect of cancer and its therapy or indirectly because of a dysfunctional immune system. The authors report the development of axonal neuropathy, myelopathy, and leucoencephalopathy associated with glutamic acid decarboxylase-65 (GAD) antibodies in 4 children with progressive cancer who were heavily pretreated. Three patients with refractory leukemia and 1 with Ewing sarcoma developed paraplegia with sensory level and dorsal column dysfunction. Three developed leucoencephalopathy and 1 died of neurologic disease. All had high serum titers of GAD antibodies during the progressive phase of the illness, and the antibody levels returned to normal with the stability of the neurologic disease. Three survivors are showing gradual recovery. This syndrome of central and peripheral nervous system toxicity may have resulted from chemotherapy toxicity or from immune dysfunction, as suggested by the high GAD antibody titers.

[The use of intravenous immunglobulin in the treatment of autoimmune neuromuscular diseases]. / Az intravénás immunglobulin alkalmazása autoimmun neuromuscularis betegségekben.

Intravenous immunglobulin given in autoimmune neuromuscular disorders modulates the immune system by complex actions, including, 1. the modification of the expression and function of Fc receptors, 2. interference with the activation of the complement and the cytokine network, 3. neutralisation of antiidiotypic antibodies, 4. effects on the activation, differentiation and effector functions of the T and B cells. Controlled trials have shown that intravenous immunglobulin is effective as first-line therapy in patients with Guillain-Barré syndrome and multifocal motor neuropathy. In case of steroid resistance or coexisting diabetes mellitus, intravenous immunglobulin can be the first line therapy in chronic inflammatory demyelinating polyneuropathy as well. As an alternative therapy it can be a second-line choice in dermatomyositis, myasthenia gravis, Lambert-Eaton myasthenic syndrome, and stiff person syndrome. While it has a remarkably good safety record for long-term administration the following side effects have been observed: headache, skin rash, thromboembolic events and renal tubular necrosis. In some disorders, the appropriate dose and frequency of infusions that maintain a satisfactory therapeutic response is well defined on the basis of data of evidence-based medicine, whereas in others it still remains to be defined. For the analysis of pharmacoeconomical aspects and the mechanism(s) of response differences in the same disease categories, further studies are necessary.

Current therapies for neuromuscular manifestations of paraneoplastic syndromes.

The neuromuscular manifestations of paraneoplastic disorders result in diverse syndromes that may also occur in patients without cancer. In general, treatment of these disorders is the same whether or not there is an underlying malignancy. However, when the disorder is believed to be paraneoplastic, the main concern should be prompt detection and treatment of the tumor, as this has been shown to offer the best chance for neurologic stabilization or improvement. The paraneoplastic neuromuscular disorders can be divided into two main categories: those that are directly mediated by antibodies and those that are believed to result from other immune-mediated mechanisms, including cytotoxic T-cell responses with or without association with specific antibodies. For disorders in which the antibodies are pathogenic, therapy is aimed at removing the antibodies. For the other disorders, adjuvant therapies are for the most part empiric and include a variety of immunosuppressant and immunomodulatory agents.

Human antiganglioside autoantibodies: validation of ELISA.

Gangliosides have a hydrophilic sugar chain that contains antigenic determinants and a hydrophobic ceramide. In humans, gangliosides elicit a T-cell independent IgM response; antiganglioside IgM autoantibodies may be pentameric or polymeric. A correlation between specific neuropathies and antiganglioside autoantibodies has been confirmed. Although many neurologists attempt to lower titers of antiganglioside autoantibodies, oncologists are developing strategies to augment production of IgM antibodies that will remove immunosuppressive gangliosides from the circulation of patients and target gangliosides and kill tumor cells. Antiganglioside IgM antibodies can cause leakage of the blood-nerve barrier in a concentration-dependent and complement-independent manner, bind to neuronal gangliosides to create a neuromuscular block and serve as a marker of axonal damage in neuropathies such as multiple sclerosis. They are also a promising biomarker of early prostate cancer. There is a need to validate the protocol for enzyme-linked immunosorbent assay (ELISA) of antiganglioside IgM autoantibodies. This validation must consider the purity of gangliosides from different commercial sources, the coating of gangliosides onto a solid matrix in a manner that maximizes exposure of oligosaccharide epitopes to IgM paratopes, techniques to minimize background noise and eliminate nonspecific antibody binding, and carefully defined positive and negative controls. The validated protocol also must include a simple formula to estimate titers for several replicas. Finally, antibody titers must be converted to natural logs for statistical appraisal.