Mass cytometry-based analysis of the composition of the immune system in peripheral blood and cerebrospinal fluid in narcolepsy

Narcolepsy is a chronic sleep disorder affecting 0.06% of the population in
European countries. Narcolepsy is characterized by the variable expression of
four symptoms referred as the *narcoleptic tetrade*: Excessive daytime
sleepiness (EDS) affecting 100% of the patients, cataplexy presenting in 70% ,
hypnagogic hallucinations in 15-66% and sleep paralysis in 17-80%. Only
cataplexy is specific for narcolepsy whereas hallucinations and sleep paralysis
are also common in healthy subjects. Three biological markers are tightly
associated with the disease: a) sleep onset REM sleep (SOREM), i.e. sleep of
narcoleptic patients will start with REM-sleep within 15 minutes compared to
about 60 minutes in healthy controls; b) specific HLA-subtypes, most notably
HLA-DQB1*0602 found in up to 90% across all ethnic groups as compared to 12-38%
in the normal population; c) a reduction or absence of hypocretins(=orexin) in
the cerebrospinal fluid (CSF). There is a low prevalence of familial cases:
1-2% risk of developing narcolepsy in a first-degree relative suggesting the
existence of other genetic factors. Since the loss of hypocretin was described
in narcolepsy, considerable effort in clarifying the pathogenesis of this
disorder and in identifying targets for potential therapies has been made.
However, important issues such as the pathophysiology of the disease remain
unclear.

Achieving more detailed information about the possible auto-immune process that
underlies narcolepsy by:
(1) Assessing differences in immune composition between peripheral blood
samples of healthy controls (n=30) and type 1 narcolepsy patients (n=30) using
mass cytometry and an antibody panel consisting of 36 cell surface markers.
(2) Comparing the specific immune composition findings in peripheral blood in
(1) with the immune composition in cerebrospinal fluid (CSF), in only type 1
narcolepsy patients using flow cytometry.

In both narcolepsy patients and HLA-matched healthy controls blood will be
drawn. In patients undergoing a lumbar puncture for clinical purposes
additional CSF will be collected. Only basic data will be collected (age, sex,
etnicity, length, weight, vaccination status, disease history and month of
birth, date of collection, sleepiness (Epworth Sleepiness Scale)). All patients
will be given the opportunity to reconsider and withdraw their participation
from the study without disclosing their motivation.

Peripheral blood samples (and cerebrospinal fluid in patients) are collected
from patients and healthy controls after obtaining informed consent. Peripheral
blood mononuclear cells will be isolated from heparin anticoagulated blood
using Ficolll-PaqueTM density-gradient centrifugation and cryopreserved at
-80°C. After thawing them, they are stained with antibodies targeting x immune
cell surface molecules and mass cytometry data acquisition is performed, as
previously described (Bendall et al., 2011). For data analysis, SPADE analyses
(Qiu et al., 2011), the Barnes-Hut implementation of t-SNE (van der Maaten,
2014) are performed. Subsequently, phenotypically distinct subsets are
identified with ACCENSE (Shekhar et al., 2014). Immune cells in cerebrospinal
fluid will be analyzed using fluorescence-activated cell sorting.

The drawing of blood may cause pain and/or bruising; fainting may occur during
or shortly after the intervention. If so, the subject will be instructed to lie
down immediately.

Note, that we only collect CSF from narcolepsy type 1 patients in whom a lumbar
puncture is already performed as part of the clinical workup for patients. The
drawing of CSF (which will be performed only in narcolepsy patients who already
undergo a lumbar puncture as part of the clinical workup) may cause pain.
Subjects are already in a horizontal position, which decreases the risk of
fainting. A common side effect is headache due to the dural punction (10-30%).
In rare cases, a postpunctional hypoliquorrhoe syndrome develops for which it
is necessary to provide a second puncture and inject a small amount of the
subject*s blood (blood patch) to close the dural gap.

It is extremely unlikely that the laboratory tests in blood and CSF will
provide unexpected results with medical implications. If this may be the case,
patients will be invited to the outpatient clinical to discuss the findings and
an appropriate clinical workup will follow. Again, such an event is extremely
unlikely.

All reasonable safeguards to prevent unauthorized people getting access to
uncoded medical or personal information will be undertaken. All data collected
are stored under the subject*s code instead of the name. All accounts are
password protected. Every precaution has been taken to assure that computer
confidentiality is maintained. The name, initials, first letters of name or
surname, and the date of birth are not entered in the database.