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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
92
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
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