This ppt is mainly for how to approach a suspected case of autoimmune encephalitis

1. Laboratory Diagnosis work up
of a patient with Suspected
Autoimmune Encephalitis
 Presenter Dr Santosh Dash
 Chair Persons Dr Anita Mahadevan
Dr Netravathi M
1
NIMHANS, BANGALORE,INDIA

2. 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.
2

3. First paper
3

4. • 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)
4

5. 5
TARGET SITES

6. 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)
6

7. 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 .
7

8. 8
When to suspect ?

9. Criteria for Diagnosis
9
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

10. SPECIAL CLINICAL FEATURES OF
INDIVIDUAL ANTIBODY
10

11. NMDA
 Age- frequently 2– 40 years, 80% Female
Clinical features
 Behavioural disturbance,Psychosis
 catatonia
 Seizures
 Movement disorders including orolingual dyskinesias
and stereotypic movement.
 Dysautonomia.
11
Barry H : BJ Psych Bull ,2015
Feb 19-23

12. 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)
12
Dalmau J : Lancet
Neurol 2008;7:327–340

13. 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.
13
Graus F :
J Neurol 2010;257:509–517

14. AMPAR Encephalitis
 40–90 years (median 60 years),
 90% female.
 Limbic encephalitis and atypical psychosis.
 Associated Autoimmune disease present.
14
Lancaster E:
Neurology 2011;77:179–
189

15. 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
15
Ramanathan S :J Clin
Neurosci 2014;21:722–730

16. Glycine R
 Age group 5–69 years (median 49 years)
 Progressive encephalomyelitis with
rigidity(PREM)
 stiff person syndrome
16
Vincent A:
Brain2004;127:701–712

17. GAD
It can be presented with
 Stiff-person syndrome.
 Refractory seizures.
 Cerebellar syndrome.
 Associated with both paraneoplastic and
nonparaneoplastic.
17
Simabukoro MM :Epileptic
Disord. 2015 Mar;17(1):9

18. 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.
18

19.  After clinical examination following
investigations to be carried out to
Confirm the diagnosis
Exclude other
mimickers
19

20. To confirm the diagnosis
Blood Test
Autoantibody
CSF STUDY
EEGMRI Brain
Functional
Imaging
20

21. Auto antibody test
21

22. 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
22

23. 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.
23

24.  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.
24

25. 25
LGI1
Caspr2
Control

26. 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.
26

27.  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.
27

28.  To reach a maximum sensitivity and
specificity, a combination of TBA as
screening method and CBA as confirmatory
test should be considered.
28

29. 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.
29

30. 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.
30

31.  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).
31

32. Most commonly used assay
CELL BASEDASSAY
32

33. 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).
33

34. 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
34

35.  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.
35

36. Lab procedure
36

37. 37

38. EUROIMMUN Kit Box
38

39. NMDA CASPR
AMPA 1 LGI
AMPA 2 GABA
B
MOSAIC BIOCHIP

40. Video of lab test
40

41. 41

42. What to test ?
Only serum Only CSF or Both
42

43. 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.
43

44. 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
44
Titulaer MJ :Lancet
Neurol 2013;12:157-65

45. 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 7.109
1098
45

46. 46

47. 47

48.  In our patients most of the patients have normal
CSF finding with some having mildly elevated
protein and cells.
48

49. 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.
49

50. 50
Fev 2014

51. 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
51

52. 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
52

53.  Most common abnormality is diffuse non-
specific slowing in theta and Delta range.
 PLEDs
 Non-convulsive status epilepticus is also
reported.
53

54.  Epileptiform activity is less common than slow
waves but may include electrographic seizures
in approximately 60% when continuous
monitoring is undertaken.
54

55. Interestingly all the EEG changes
disappear on treatment
55

56. 56

57.  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.
57

58. 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)
58

59. 19 year old with NMDA encephalitis
Delta Brush59

60. Gentleman with VGKC encephalitis
Delta Waves
60

61. Patient with NMDA encepahlitis
Epileptiform Discharges61

62. 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 .
62

63.  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.
63

64.  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)
64

65. 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.
65

66. Normal MRI
66

67. 67

68. 68

69. NMDA positivity
69

70. 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
70

71. 71

72. 72

73. 73

74. VGKC positive
74

75. 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
75

76. 76

77. 77

78. 55 year old lady with VGKC Encephaltiis
78

79. NMDA encephalitis showing diffuse
cerebral atrophy79
Can be
Reversible

80. 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.
80

81. 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
81

82. New biomarker
82

83. Mimickers
 Infection( viral encephalitis HSV).
 Systemic inflammatory disease.
 Demyelinating disease.
 Toxic-metabolic disorders.
 Paraneoplastic
 Neurodegenerative disorders.
83

84. 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.
84

85. 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
85

86.  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
86

87. 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.
87

88.  Anti-thyroid antibodies (TPO) for Hashimotos
encephalopathy.
 Serum Na to look for Hyponatraemia(60%)
 CSF can be tested for Viral infection (HSV PCR)
88

89. 89
 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. 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.

91. 91
 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. 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.

93. 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)
93

94. 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.
94

95. 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.
95

96. 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.
96

97. 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.
97

98. THANKYOU98