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Laboratory Diagnosis work up of a patient with Suspected Autoimmune Encephalitis

Presenter Dr Santosh Dash Chair Persons Dr Anita Mahadevan

Dr Netravathi M

NIMHANS, BANGALORE,INDIA

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Autoimmune encephalitis

Definition The term autoimmune encephalitis is used to

describe a group of disorders:- Characterized by symptoms of the CNS (limbic,

extra-limbic, basal ganglia, autonomic structures or more wide-spread) due to autoantibodies against neuronal surface or synaptic proteins, which are likely to mediate the disease.

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First paper

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• Associate with autoantibodies to neuronal surface or synaptic proteins

Autoimmune encephalitis

(AIE)

• Associate with autoantibodies to intracellular neuronal antigens (e.g., Hu, Yo, Ri)

Paraneoplastic neurological syndromes

(PNS)

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TARGET SITES

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Prevalence

A recent population based survey has shown that autoimmune encephalitis are more common than previously believed, accounting for 21% of unselected encephalitis cases in the U.K. (Granerod et al., Lancet Infect Dis 2010;10(12):835–44

There are few studies from India which are retrospective studies but exact prevalence in india is not known.(Chandra SR et al :AIAN 2014;17:166-70, Pandit KA et al : AIIN 2013 ;Oct 577-584)

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When to suspect a case of encephalitis as having autoimmune cause ?

How to confirm the diagnosis

What investigations to do for exclusion of other mimickers .

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When to suspect ?

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Criteria for Diagnosis

Acute or subacute (< 12 wks) onset of symptoms

Evidence of CNS inflammation (at least

one of):

CSF,MRI,PET,Biopsy

Exclusion of other causes

Angella vincent :JNNP-2012;83:638-645

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SPECIAL CLINICAL FEATURES OF

INDIVIDUAL ANTIBODY

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NMDA

Age- frequently 2– 40 years, 80% Female

Clinical features

Behavioural disturbance,Psychosis

catatonia

Seizures

Movement disorders including orolingual dyskinesias and

stereotypic movement.

Dysautonomia. Barry H : BJ Psych Bull ,2015

Feb 19-23

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LGI1

Age- 30–80 years (median 60 years), 65% male Clinical Features

Faciobrachial dystonic seizures (FBDS)

limbic encephalitis

seizures including myoclonus

Rapidly progressive dementia like CJD

Sleep disorder (RBD) Dalmau J : Lancet Neurol 2008;7:327–340

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Caspr2

45–80 years (median 60 years), 85% male.

Clinical features

Peripheral nerve hyperexcitability or neuromyotonia

(Isaacs’ syndrome)

Morvan’s syndrome

Limbic encephalitis

Sleep disorders. Graus F :J Neurol 2010;257:509–517

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AMPAR Encephalitis

40–90 years (median 60 years),

90% female.

Limbic encephalitis and atypical psychosis.

Associated Autoimmune disease present.Lancaster E:

Neurology 2011;77:179–189

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Anti-GABA-B receptor encephalitis

25–75 years (median 60 year),50% female.

Limbic encephalitis with prominent seizures in

up to 80% of patients.

Antibodies directed against the B1 subunit of the

GABA receptor Ramanathan S :J Clin Neurosci 2014;21:722–730

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Glycine R

Age group 5–69 years (median 49 years)

Progressive encephalomyelitis with

rigidity(PREM)

stiff person syndrome

Vincent A:Brain2004;127:701–712

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GAD

It can be presented with

Stiff-person syndrome.

Refractory seizures.

Cerebellar syndrome.

Associated with both paraneoplastic and

nonparaneoplastic. Simabukoro MM :Epileptic Disord. 2015 Mar;17(1):9

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Salient features

Most frequently, these antibodies are directed against the voltage-gated potassium channel (VGKC) complex and the N-methyl, D-aspartate (NMDA) receptor.

The diseases are typically immunotherapy-responsive.

They are only associated with cancer in a minority of patients.

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After clinical examination following

investigations to be carried out to

Confirm the diagnosis

Exclude other mimickers

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To confirm the diagnosis

Blood TestAutoantibody

CSF STUDY

EEGMRI Brain

Functional Imaging

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Auto antibody test

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Available Assays

Different assays are available for the diagnosis of antibodies, both for CSF and serum:

Tissue-based assays (TBA; in-house or commercially

available)

Cell-based assay (CBA; in-house or com- mercially

available)

ELISA

Primary cultures of neurons

Immunoprecipitation

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1.Tissue-based assays

In the TBA, rat or mouse brains are stained with CSF or serum of patients with an indirect immunhistochemistry or immunofluorescence technique.

Anti- neuronal antibodies attach to their receptor or synaptic antigen in the rodent brain, resulting in a neuropil staining pattern in the hippocampus.

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The TBA provides an excellent screening method, which detects most of the currently known neuronal cell surface auto antibodies (with some limitations for the GlyR- and D2R-antibodies).

Also can reveal new autoantibodies.

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LGI1

Caspr2

Control

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2. Cell-based assay

In the CBA, cells (e.g., HEK293 cells) are transfected with the respective surface receptor or synaptic antigen and stained with CSF or serum of the patients with an indirect immunofluorescence technique.

Autoantibodies against the specifically expressed receptor result in a membrane staining of the cells.

It is expressed as either end-point titers or relative fluorescence units.

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Most laboratories use the CBA for the diagnosis of neuronal cell surface autoantibodies, which is a highly sensitive and specific assay

But the disadvantage that new autoantibodies are not detected.

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To reach a maximum sensitivity and

specificity, a combination of TBA as

screening method and CBA as confirmatory

test should be considered.

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3.Primary cultures of neurons

Primary cultures of hippocampal neurons are

stained with CSF or serum of patients with an

indirect immunofluorescence technique and the

autoantibodies are visualized as surface staining

of neurons.

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4.Immunoprecipitation

In the IP, autoantibodies that are present in

serum of patients bind to a specific antigen, the

antigen–antibody complex is precipitated out of

solution and measured.

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Staining of hippocampal neurons and IP is mainly used in research but may be helpful in selected individual cases

For example in samples which shows positive in TBA but negative in CBA there to characterize and ascertain that the patient’s autoantibodies recognize a yet to be identified cell surface antigen).

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Most commonly used assay

CELL BASED ASSAY

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Methodology

Cell based indirect immunofluorescence antibody (IFA) assay for the detection of NMDAR IgG antibodies was approved by the US FDA.

For this test, human embryonic kidney (HEK-293) cells transfected with the NR1 subunit of NMDAR, as well as non-transfected cells grown on Biochips are used as substrates. (Euroimmun AG, Lubeck, Germany).

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30µl of CSF/serum 1:10

was taken

Incubation for 30 min at room temperature

Rinsed with a flush of PBS-

Tween

IgG was labeled using Fluorescein-

conjugated goat anti-human IgG

antibody

Mount with slides and seen

under microscope

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Samples were classified as positive or negative

based on the intensity of surface

immunofluorescence of transfected cells in direct

comparison with non-transfected cells and control

sample.

It is based on the manufacturer's recommendations

for reading and interpretation.

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Lab procedure

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EUROIMMUN Kit Box

NMDA CASPR

AMPA 1 LGI

AMPA 2 GABA B

MOSAIC BIOCHIP

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Video of lab test

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What to test ?

Only serum Only CSF or Both

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Answer

Both should be tested (CSF and serum ) because

One can be positive and other is negative

Both can be negative but still can be

autoimmune encephalitis.

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Evidence

At the time of diagnosis of this disease

autoantibodies are always present in CSF, the

serum can be negative in up to 14% of patients,

suggesting that serum examination alone may

be insufficient to exclude AIE

Titulaer MJ :Lancet Neurol 2013;12:157-65

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CSF findings

Abnormal CSF findings in 79% of patients

CSF revealed lymphocytic pleocytosis in more than

90% of cases.

Intrathecal protein increase in 33%.

Oligoclonal bands in approximately 25%.

Glucose is mostly normal.

DALAMU j : Lancet Neurology 2008 7.1091-1098

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In our patients most of the patients have normal

CSF finding with some having mildly elevated

protein and cells.

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Antibody titer

There is a relation between antibody titers, relapses, and outcome.

It has been shown that high autoantibody titers were associated with a poorer outcome or the presence of a teratoma in NMDA disease.

A rapid decrease of CSF autoantibody titers within the first month of disease associated with a better prognosis.

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Fev 2014

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False Positive

Anti-NMDAR antibodies have been described in patients Multiple sclerosis, Seronegative NMO Creutzfeldt–Jakob disease

Antibodies against VGKC complex have also been described in patients

Amyotrophic lateral sclerosis Bickerstaff encephalitis Miller–Fisher syndrome Influenza A

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EEG

EEG is considered a sensitive test (90%), but

poorly specific (30-35%).

In early disease EEG monitoring may show

evidence of seizure activity.

Dalamu J ;Lancet Neurol. 2008

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Most common abnormality is diffuse non-

specific slowing in theta and Delta range.

PLEDs

Non-convulsive status epilepticus is also

reported.

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Epileptiform activity is less common than slow

waves but may include electrographic seizures

in approximately 60% when continuous

monitoring is undertaken.

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Interestingly all the EEG changes

disappear on treatment

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It is characterized by rhythmic delta activity at 1−3 Hz

with superimposed bursts of rhythmic 20–30 Hz beta

frequency activity “riding” on each delta wave.

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Significance of Delta brush

It resemblance to waveforms seen in premature infants.

The presence of extreme delta brush was associated with a more prolonged hospitalization (mean 128.3 ± 47.5 vs 43.2 ± 39.0 days, p = 0.008) .

increased days of cEEG monitoring (mean 27.6 ± 42.3 vs 6.2 ± 5.6 days, p = 0.012)

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19 year old with NMDA encephalitis Delta Brush

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Gentleman with VGKC encephalitis Delta Waves

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Patient with NMDA encepahlitis Epileptiform Discharges

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MRI Brain Imaging

At presentation about 50 % of the patients have abnormal MRI findings.

Most commonly increased signal on fluid-attenuated inversion recovery (FLAIR) or T2 sequences medial temporal lobes ( 40%)

Abnormalities have been reported in other areas such as corpus callosum or brainstem, insular region .

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There may be T2 signal changes in periventricular signal, particularly in the trigone area also described.

Faint or transient contrast enhancement may occur in the cerebral or cerebellar cortex and overlaying meninges.

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Severe disease courses can result in predominantly hippocampal or mild global atrophy.(Dalamu et al 2007).

Interestingly, brain atrophy was partially reversible and accompanied by clinical recovery in two patients with follow-up for 5 and 7 years (Iizuka et al., 2010)

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1st case

1.Pt R 17/F presented with acute onset behavioral

symptoms f/b stereotyped movements, mutism

and catatonic state. Her CSF routine test was

normal but NMDA was strongly positive.

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Normal MRI

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NMDA positivity

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2nd case

2. pt S 61/M presented with one month history

of seizures and recent memory loss. Seizures

were both myoclonic jerks and facio-brachial

dystonic seizures. VGKC positive

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VGKC positive

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3rd case

Pt R 19/F presented with h/o psychiatric

symptoms f/b mutism. She was on treatment by

psychiatrist for 10 month later shifted to

neurology side. O/E she had stereotype

movements. NMDA positive

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55 year old lady with VGKC Encephaltiis

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NMDA encephalitis showing diffuse cerebral atrophy

Can be Reversible

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SPECT Imaging

SPECT revealed Hypoperfusion of the frontal, parietal and medial temporal lobes, as well as thalamus, and cerebellum which was responsible for psychaitric symptoms.

Hyperperfusion in the motor strip and left temporal lobe, which are areas related to some of the patient's symptoms, including seizures, orolingual dyskinesia, and Wernicke aphasia. 

Brain Behav. 2011 Nov; 1(2): 70–722.

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PET scan

18F FDG PET/CT is more sensitive than MRI.

FDG–PET can reveal pathological changes even when MRI and CT scans are normal.

However findings can be variable depending on which phase of illness is ongoing at the time of the scan.

In the acute phase FDG-PET generally shows cerebral hypermetabolism anteriorly, with relative diffuse posterior hypometabolism. Vitaliani et al.,

200505

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New biomarker

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Mimickers

Infection( viral encephalitis HSV).

Systemic inflammatory disease.

Demyelinating disease.

Toxic-metabolic disorders.

Paraneoplastic

Neurodegenerative disorders.

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Tumor screening

Potential screening imaging modalities include

Ultrasonography of Abdomen and pelvis

Testicular ultrasound

CT chest ,abdomen and pelvis

MRI abdomen and pelvis

Mammography

PET scanning of whole body.

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Paraneoplastic Antibody screen

Following paraneoplstic tests can be done in serum.

anti-Hu

anti-Yo

anti-CV2 (also called anti-CMRP-5)

anti-Ma2

anti-Ri

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Florance et al. recommended periodic ultrasound and MRI of the abdomen and pelvis for at least two years following diagnosis is required.

Tumor surveillance for males was not recommended as the number of cases has been too small.

FLORANCE NR :Anna Neurology 66:11-18

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Blood test

FBC

LFT , renal and thyroid function.

Serological panel for Autoimmune disorders:

ESR, CRP, complement levels, ANA, dsDNA, ANCA,

ACE, SS-A, SS-B, RF, cardiolipin.

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Anti-thyroid antibodies (TPO) for Hashimotos

encephalopathy.

Serum Na to look for Hyponatraemia(60%)

CSF can be tested for Viral infection (HSV PCR)

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22 patients with new onset psychiatric symptoms who did not respond to conventional treatment .

They were analyzed using EEG, MRI,CSF, screening for malignancy, Vasculitic work-up and autoantibody tests.

2014;17:166-70

90 3 had systemic malignancy, 10 had chronic

infection, 1 with vasculitis, 1 had Hashimoto encephalopathy and 2 with non-convulsive status. 

Conclusion: All patients who present with new onset neuropsychiatric symptoms need to be evaluated for sub-acute infections, inflammation, autoimmune encephalitis and paraneoplastic syndrome.

A repeated 20 minute EEG is a very effective screening tool to detect organicity.

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Total 15 patients of autoimmune encephalitis.

The most common onset was sub acute (64%)

and four (29%) patients presented as SE.

Predominant clinical presentations was seizure

(100%).

AIAN 2013 Oct-Dec

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CSF was done in 10 patients; it was normal in

60%.

Brain MRI was done in all patients, in six (40%)

it was normal, six (40%) showed T2W and

FLAIR hyperintensities in bilateral limbic areas.

NMDA receptor antibody in seven (50%), VGKC

antibody in five (36%), two having anti GAD.

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Third study

Total 22 patients with suspected AIE were

studied over a period of 3½ years and 16

patients had positive autoimmune antibodies.

Cognitive impairment and seizures were the

predominant symptoms present in nearly all

(100%) patients followed by psychiatric

disturbances (87.5%). Netravathi M et al. Neurology India 2015 (In

Press)

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EEG

EEG was abnormal in 81.25%.

Diffuse slowing of the background activity were

the predominant EEG changes.

Epileptiform discharges were seen in 3 (18.75%)

patients with anti-NMDA encephalitis and two of

them showed evidence of extreme delta brush.

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CSF

CSF examination was available in 14 patients and

was normal in 10 (71.4%) patients.

One patient with anti-NMDA encephalitis had

lymphocytic pleocytosis with normal protein, sugar.

Three patients in each of the three subtypes of AIE

had mildly elevated protein with normal cell count

and sugars.

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MRI

MRI was abnormal in 53.3% patients.

Abnormalities were seen in all patients with voltage-

gated potassium channels (VGKC); 60% of patients

with NMDA.

Imaging was normal in 26.7% of the patients.

PET-CT was done in 4 patients (2-VGKC, 2-NMDA)

and none of them had any evidence of internal

malignancy.

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Take home Messages

It is very important to know the clinical features of AIE

to clinically diagnose a case.

Serum and CSF both to be used for diagnosis of AIE

because each having its own limitations.

Tumor work up should be carried out in all cases as it

affect prognosis.

Exclusion of other disease is important as its having

own therapeutic implication.

98 THANK YOU