Specialist Testing of CSF

Katherine Birch

katherine.birch@thewaltoncentre.nhs.uk
Outline
1) Introduction to CSF
2) Common CSF tests
3) Less-common CSF tests & the conditions they are
associated with:
• Xanthochromia & subarachnoid haemorrhage
• Oligoclonal bands & multiple sclerosis
• Beta-2-transferrin & ?CSF leak
4) Myasthenia gravis & serum acetylcholine receptor
antibodies
5) Case examples and discussion
Basic anatomy of the brain

The human brain has 4

interconnected cavities known
as ventricles:

• 2 lateral ventricles, left & right

• The third ventricle in the
midline
• The fourth ventricle, the
central canal of the spinal
cord iihptcresources.weebly.com
CSF formation
• Within each ventricle is a region of choroid plexus, a
network of ependymal cells
• CSF is predominantly formed by plasma ultrafiltration
through the capillary walls of the choroid plexus in the
lateral ventricles
• Fluid shifts across other vascular beds within the CNS
are also involved
• Solutes enter CSF by active transport and diffusion
• In adults, the total volume of CSF is approx 135 mL
produced at a rate of 500 mL/day so the fluid is typically
exchanged every 6 hours
 CSF flow

www.slideshare.net
CSF collection
CSF collection is usually by lumbar
puncture:
• Potentially uncomfortable
• Post LP headache
• Risk of bleeding and infection
en.wkipedia.org

Useful in the differential diagnosis of the following conditions:

• Meningitis – fever, headache, stiff neck
• Encephalitis – confusion, reduced GCS
• De-myelination – reduced mobility
• Acute onset of headache
• Neuropathy – impaired sensation & movement
• Dementia – decreased ability to think & remember
 Common CSF tests
Test Reference Significance
range
Red <10 per mm3 Increased in:
cells • Intracranial bleed
Cell • Traumatic LP
count
White <5 per mm3 Samples with increased numbers of white cells undergo a differential cell count to
cells distinguish the different types
• Increased lymphocytes - viral meningitis, TB meningitis, chronic inflammatory diseases
eg MS
• Increased polymorphs/granulocytes – bacterial meningitis, brain abscess

Culture & gram Identifies if bacteria are present and differentiates them into 2 large groups; gram positive
stain and gram negative

Glucose >60% of Decreased in:

simultaneously • Bacterial meningitis
determined • Hypoxia
plasma • Hypoglycaemia
concentration • SAH
• Meningeal carcinoma
Total protein 0.2-0.4 g/L Increased in
• Blood brain barrier breakdown – infections (eg acute bacterial meningitis), malignancy
• Cell damage within the CNS - toxic damage, trauma
• Local synthesis within the CNS - MS
Lactate 1.1-2.4 g/L Increased in:
• Cerebral hypoxia
• Bacterial meningitis
• Inherited metabolic disorders
 Subarachnoid Haemorrhage

• SAH = blood escape from a

cerebral artery into the
subarachnoid space
• Most commonly due to rupture of a
cerebral aneurysm en.wikipedia.org

• Typically present with severe, acute

headache, classically occipital
• Primary method of diagnosis is high
contrast CT scan which will detect
~95% of cases within 48hrs
en.wikipedia.org
Xanthochromia
(2) Oxidation of
haem ring
produces biliverdin (3) Biliverdin is
converted to
bilirubin

(1) Following
haemorrhage
www.circ.ahajournals.org
erythrocytes lyse
and liberate
oxyhaemoglobin

Oxyhaemoglobin may be produced following a traumatic tap when blood is

artefactually introduced to the subarachnoid space at the puncture site during LP.

Steps (2) and (3) are entirely dependent on enzymes present in macrophages and
other cells of the leptomeninges therefore bilirubin can only form in vivo.
Spectrophotometric
analysis of CSF
Revised national guidelines Cruickshank et al. Ann Clin
Biochem 2008; 45: 238-44

• Specimens should be taken >12hrs but <14days after

onset of symptoms
• Least blood stained fraction (usually last & ideally at
least the 4th)
• Protect from light
• Centrifuge
• Using a cuvette with a 1cm path length, perform a zero
order scan between 350-600nm on an undiluted
specimen
Xanthochromia interpretation

1) Draw a baseline which forms a

tangent to the scan between
350-400nm and again between
430-530nm. Baseline should
never cut the scan.

2) Measure the absorbance

above the baseline at:
i. 476nm; the net bilirubin
absorbance (NBA)
ii. The absorption maximum
between 410-418nm; the net www.birmingham.ac.uk
oxyhaemoglobin absorbance
(NOA)
 Xanthochromia interpretation

Ann Clin Biochem 2008; 45: 238-44

Case 1
• A 51 year old male patient presents to A&E with a 5hr
history of severe headache and vomiting.
• A CT scan is performed which shows no evidence of
haemorrhage.
• When the patient is 12hrs post onset of symptoms an LP
is performed and the CSF is sent to the lab for
xanthochromia analysis.
• The on-site lab do not have a spectrophotometer so the
sample is centrifuged and stored in the fridge until it is
collected by a courier and transported to a referral lab.
Case 1
(1) The referral lab scan the sample and get the following
trace. What are the NBA and the NOA?
Case 1
(2) How would you interpret the result?

(3) It becomes apparent that the sample was not protected

from light during transport to the referral lab. How does this
affect your interpretation?

(4) What factors would you consider when deciding

whether it would be appropriate to offer the xanthochromia
test on-site in the lab at the originating hospital?
Case 1 - answers
(1) The referral lab scan the sample and get the following
trace. What are the NBA and the NOA?

NOA = 0.067 – 0.019 = 0.048

NBA not detected
Case 1 - answers
(2) How would you interpret the result? NBA<0.007 and
NOA<0.1, no evidence to support SAH

(3) It becomes apparent that the sample was not protected

from light during transport to the referral lab. How does this
affect your interpretation? Bilirubin degrades in light (decay
rate of 0.005 AU/hr in daylight), could be a false negative
result, interpret with caution. Would you still make the
numeric results available to the clinician? There are many
issues associated repeating an LP. Need to report the
incident and perform a root cause analysis to determine
why the sample was not protected from light.
 Case 1 - answers
(4) What factors would you consider when deciding whether
it would be appropriate to offer the xanthochromia test on-
site in the lab at the originating hospital?
Clinical need – number of patients, required turnaround time,
past incidents
Lab facilities – spectrophotometer (appropriately sized quartz
cuvettes), software packages, staff capacity to take on test
especially out-of-hours, experience & expertise of staff,
maintaining competency, availability for senior advice out-of-
hours
Other options – provide wards with xanthochromia collection
packs (dark tubes, envelopes), transport options & the risks
associated with them
 Multiple sclerosis
• MS is an inflammatory
demyelinating condition
• Damaged areas of myelin are
known as plaques
• Nerve impulses to and from the
CNS are disrupted
• Symptoms include blurred/double
vision, muscle weakness,
stiffness and spasms, loss of
balance and co-ordination,
bladder & bowel problems
sickkids.ca
Diagnosis of MS
NICE guidance 2014 [CG186] refer to the 2010 Mcdonald
criteria for diagnosis
• Core requirement is the demonstration of multiple CNS
lesions developed at different times and in different
anatomical locations:
– History of attacks
– MRI pattern of lesions
– Evoked potentials
– CSF oligoclonal bands
• Exclude alternative diagnoses
Associationlymesansfrontieres.com
Oligoclonal bands
• In normal CSF, immunoglobulins are not produced within
the CNS but only come from blood
• In MS, B lymphocytes are induced to migrate from the
blood into the brain where they differentiate into plasma
cells which secrete immunoglobulins intrathecally (local
synthesis within the CNS only)
• Intrathecal immunoglobulin synthesis can be detected
using isoelectric focusing of paired CSF and serum.
Proteins are separated electrophoretically using an
agarose gel with a pH gradient. Proteins will migrate until
they reach their pI point (pH at which the protein has net
zero charge).
 Oligoclonal bands
Type 1 pattern – normal, no IgG bands
in CSF or serum
CS

Type 2 pattern – oligoclonal IgG bands

present in CSF only, consistent with
CS
MS
Type 3 pattern – matching bands in
CSF & serum with extra bands in CSF

CS
Type 4 pattern – identical pattern of
bands in CSF & serum, systemic IgG
synthesis, not MS
CS
Type 5 pattern – identical ladder
pattern of bands in CSF & serum,
consistent with monoclonal paraprotein
CS

J Neurol Neurosurg Psychiatry 1994; 57: 897-902

 Case 2
1. Interpret the following isoelectric focusing results.

C1 S1 C2 S2 C3 S3 C4 S4 C5 S5

2. Sample 4 was an EQA sample. When the EQA report is

issued your lab interpretation is out of consensus with other
participants. How do you investigate?
 Case 2 - answers
1. Interpret the following isoelectric focusing results.

Patient 1 – Type 2 pattern

Patient 2 – Type 1 pattern
Patient 3 – Type 1 pattern
Patient 4 – Type 4 pattern
Patient 5 – Type 1 pattern

C1 S1 C2 S2 C3 S3 C4 S4 C5 S5
 Case 2 - answers
2. Sample 4 was an EQA sample. When the EQA report is
issued your lab interpretation is out of consensus with
other participants. How do you investigate?
• Check for transcription errors & sample labelling
• Double check interpretation with multiple staff members
• Repeat the analysis
• Repeat the analysis with fresh EQA samples
• Check internal QC
• Check reagents, kits, gels etc in date & stored correctly
• Repeat previous EQA samples
• Ask EQA scheme organisers for advice
• If a causative error is identified, put plans in place to ensure
the error can’t happen again
Beta-2-transferrin
• Carbohydrate free form of transferrin (aka asialo-
transferrin)
• Not found in blood, mucus or tears, specific marker for CSF
• Can identify CSF leakage from the nose (rhinorrhoea) or
the ear (otorrhoea) or other fluids
• Beta trace protein is now a well-established alternative
• Beta trace protein is a prostaglandin D2 synthase
synthesized in the CNS by glial cells & the choroid plexus
so concentrations are much higher in CSF than serum
• Beta trace protein measurement can be automated using
nephelometry/turbidimetry.
Beta-2-transferrin
Electrophoretic separation followed by immunochemical
detection using an anti-transferrin antibody
Case 3
• A 43yr old female presented with headaches and
decreased visual acuity.
• Visual field defect was confirmed and serum endocrine
results and pituitary MRI suggested a non-functioning
13mm pituitary tumour.
• The patient underwent trans-sphenoidal surgery for the
macroadenoma. In the weeks following surgery a clear
nasal discharge started to appear
Case 3
1. A sample of the discharge was collected to identify
whether or not the fluid was CSF. Unfortunately due to a
misunderstanding at the lab the sample was not
centrifuged and was not referred for beta-2-transferrin
testing for several days. The following results were
generated. How would you report the results?
Lane 1 – Patient fluid sample neat
Lane 2 – Patient fluid sample diluted 1 in 5
Lane 3 – Patient fluid sample diluted 1 in 10
Lane 4 – Patient serum sample
1 2 3 4 5
Lane 5 – Neat CSF control sample
 Case 3
2. A second sample of the discharge was collected and was
handled appropriately. The following results were generated.
How would you report the results?
Lane 1 – Patient fluid sample neat
Lane 2 – Patient fluid sample diluted 1 in 5
Lane 3 – Patient fluid sample diluted 1 in 10 1 2 3 4 5

Lane 4 – Patient serum sample

Lane 5 – Neat CSF control sample

3. The patient also reported being thirsty and complained of

polyuria. What would you be suspicious of and how would you
confirm the diagnosis?
Case 3 - answers
1. Lane 1 – Patient fluid sample neat
Lane 2 – Patient fluid sample diluted 1 in 5
Lane 3 – Patient fluid sample diluted 1 in 10
1 2 3 4 5 Lane 4 – Patient serum sample
Lane 5 – Neat CSF control sample

The transferrin band in the fluid sample has not migrated

as either tetrasialotransferrin or asialotransferrin (compare
to control).

Report “Equivocal result due to transferrin degradation

?due to bacterial contamination. Please send repeat fresh,
centrifuged specimen.”
 Case 3 - answers
2. Lane 1 – Patient fluid sample neat
Lane 2 – Patient fluid sample diluted 1 in 5
Lane 3 – Patient fluid sample diluted 1 in 10
Lane 4 – Patient serum sample 1 2 3 4 5

Lane 5 – Neat CSF control sample

Report “Beta 2 transferrin detected in fluid sample, fluid is

consistent with CSF”
Implications for patient; infection risk, may require further surgery
and a CSF drain
The serum sample shows tetrasialotransferrin only. There are very
rare genetic variants where asialotransferrin is present in the blood
(& also some alcoholics). This can cause a false positive result for
beta 2 transferrin in the fluid. By sending a paired serum sample, a
false positive result has been ruled out in this case.
 Case 3 - answers
3. The patient also reported being thirsty and complained of
polyuria. What would you be suspicious of and how would
you confirm the diagnosis?

Following pituitary surgery, patients are at risk of developing

transient cranial diabetes insipidus. Check serum sodium and
osmolality and 24hr urine volume and osmolality. If polyuria
and an inappropriately dilute urine are confirmed, proceed to
a water deprivation test.
Myasthenia Gravis
• Characterised by fluctuating,
fatigable weakness of muscles
under voluntary control

• Pathogenesis involves auto-

antibodies against the nicotinic
acetylcholine receptor in the
post-synaptic neuromuscular
junction
 Case 4
A 61yr old male presents with double vision, eye droop & muscle
weakness in his hands & fingers. A request for serum acetylcholine
receptor antibodies (AChR Ab) was sent.
The lab uses a sandwich radioimmunoassay:
i. 125I labelled acetylcholine receptor is used as the antigen

ii. If autoantibodies to the receptor are present in the serum

they will bind the labelled receptor
iii. This complex is then precipitated using a second antibody
to human IgG
iv. The amount of radioactivity (in counts per minute, cpm) in
the sediment is directly proportional to the concentration of
acetylcholine receptor autoantibodies in the sample
v. Calibrators, controls and samples are all treated the same
and run in duplicate. 2 tubes are prepared without any
calibrator, control or sample to get a total activity.
 Case 4 - results
The mean activity from the total activity tubes was 59990 cpm
Sample Activity 1 Activity 2 Mean Percentage AChR Ab
(cpm) (cpm) activity binding B/T concentration
(cpm) (%) (nmol/L)
Calibrator A 679.9 544.9 0

Calibrator B 1984.5 2105.1 0.2

Calibrator C 4054.2 4457.7 0.5

Calibrator D 8756.6 9282.2 1.2

Calibrator E 13741.2 13865.9 3

Calibrator F 17836.3 17811.7 8

Positive control 9949.04 9971.06 Target 0.81-

1.51
Cut-off control 3767.5 3779.8 Target 0.3-0.45

Patient sample 7688.7 7702.5

Case 4
1. Calculate the mean activity from the duplicates for each
calibrator, control and the patient samples

2. Calculate the percentage binding for the calibrators,

controls and the patient sample as follows:
% B/T = (Mean activity/Total activity) x 100

3. Plot a standard curve on semi-logarithmic graph paper

Case 4
4. Use the standard curve to calculate the concentration of
AChR Ab in the controls. Are the results within target?

5. Use the standard curve to calculate the concentration of

the AChR Ab in the patient sample. What is the
interpretation?
AChR Ab concentration (nmol/L) Interpretation
<0.25 Negative
0.25-0.4 Equivocal
>0.4 Positive
 Case 4 - answers
The mean activity from the total activity tubes was 59990 cpm
Sample Activity 1 Activity 2 Mean Percentage AChR Ab
(cpm) (cpm) activity binding B/T concentration
(cpm) (%) (nmol/L)
Calibrator A 679.9 544.9 612.4 1.02 0

Calibrator B 1984.5 2105.1 2044.8 3.41 0.2

Calibrator C 4054.2 4457.7 4255.8 7.09 0.5

Calibrator D 8756.6 9282.2 9019.4 15.03 1.2

Calibrator E 13741.2 13865.9 13803.6 23.01 3

Calibrator F 17836.3 17811.7 17823.9 29.71 8

Positive control 9949.04 9971.06 9960.05 16.60 Target 0.81-

1.51
Cut-off control 3767.5 3779.8 3773.65 6.29 Target 0.3-0.45

Patient sample 7688.7 7702.5 7695.6 12.83

Case 4
4. Positive control 1.4 nmol/L (within target)
Cut-off control 0.35 nmol/L (within target)

5. AChR Ab concentration (nmol/L) Interpretation

<0.25 Negative
0.25-0.4 Equivocal
>0.4 Positive

Patient AChR Ab concentration = 0.85 nmol/L (positive)

Any questions?

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