specialist testing of csf - acb
TRANSCRIPT
PowerPoint Presentationassociated with:
antibodies
interconnected cavities known
midline
cord iihptcresources.weebly.com
CSF formation
• Within each ventricle is a region of choroid plexus, a
network of ependymal cells
through the capillary walls of the choroid plexus in the
lateral ventricles
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
puncture:
Useful in the differential diagnosis of the following conditions:
• Meningitis – fever, headache, stiff neck
• Encephalitis – confusion, reduced GCS
en.wkipedia.org
range
Significance
Cell
count
Red
cells
• Intracranial bleed
• Traumatic LP
White
cells
<5 per mm3 Samples with increased numbers of white cells undergo a differential cell count to
distinguish the different types
eg MS
Culture & gram
stain
Identifies if bacteria are present and differentiates them into 2 large groups; gram positive
and gram negative
Glucose >60% of
• 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
cerebral artery into the
cerebral aneurysm
headache, classically occipital
~95% of cases within 48hrs
en.wikipedia.org
en.wikipedia.org
Xanthochromia
www.circ.ahajournals.org
converted to
bilirubin
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 oxyhaemoglobin absorbance (NOA)
www.birmingham.ac.uk
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
(3) It becomes apparent that the sample was not protected
from light during transport to the referral lab. How does this
affect your interpretation?
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
(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
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
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
packs (dark tubes, envelopes), transport options & the risks
associated with them
known as plaques
CNS are disrupted
• Symptoms include blurred/double
vision, muscle weakness,
balance and co-ordination,
criteria for diagnosis
anatomical locations:
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
MS
CSF & serum with extra bands in CSF
Type 4 pattern – identical pattern of
bands in CSF & serum, systemic IgG
synthesis, not MS
pattern of bands in CSF & serum,
consistent with monoclonal paraprotein
Case 2
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
C1 S1 C2 S2 C3 S3 C4 S4 C5 S5
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
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
• Check internal QC
• 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
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
Case 3
decreased visual acuity.
results and pituitary MRI suggested a non-functioning
13mm pituitary tumour.
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?
1 2 3 4 5
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
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
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
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
Lane 5 – Neat CSF control sample
The transferrin band in the fluid sample has not migrated
as either tetrasialotransferrin or asialotransferrin (compare
to control).
?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
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.
1 2 3 4 5
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?
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.
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
Positive control 9949.04 9971.06 Target 0.81-
1.51
Patient sample 7688.7 7702.5
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?
<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
Positive control 9949.04 9971.06 9960.05 16.60 Target 0.81-
1.51
Patient sample 7688.7 7702.5 7695.6 12.83
Case 4
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
Any questions?
antibodies
interconnected cavities known
midline
cord iihptcresources.weebly.com
CSF formation
• Within each ventricle is a region of choroid plexus, a
network of ependymal cells
through the capillary walls of the choroid plexus in the
lateral ventricles
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
puncture:
Useful in the differential diagnosis of the following conditions:
• Meningitis – fever, headache, stiff neck
• Encephalitis – confusion, reduced GCS
en.wkipedia.org
range
Significance
Cell
count
Red
cells
• Intracranial bleed
• Traumatic LP
White
cells
<5 per mm3 Samples with increased numbers of white cells undergo a differential cell count to
distinguish the different types
eg MS
Culture & gram
stain
Identifies if bacteria are present and differentiates them into 2 large groups; gram positive
and gram negative
Glucose >60% of
• 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
cerebral artery into the
cerebral aneurysm
headache, classically occipital
~95% of cases within 48hrs
en.wikipedia.org
en.wikipedia.org
Xanthochromia
www.circ.ahajournals.org
converted to
bilirubin
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 oxyhaemoglobin absorbance (NOA)
www.birmingham.ac.uk
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
(3) It becomes apparent that the sample was not protected
from light during transport to the referral lab. How does this
affect your interpretation?
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
(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
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
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
packs (dark tubes, envelopes), transport options & the risks
associated with them
known as plaques
CNS are disrupted
• Symptoms include blurred/double
vision, muscle weakness,
balance and co-ordination,
criteria for diagnosis
anatomical locations:
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
MS
CSF & serum with extra bands in CSF
Type 4 pattern – identical pattern of
bands in CSF & serum, systemic IgG
synthesis, not MS
pattern of bands in CSF & serum,
consistent with monoclonal paraprotein
Case 2
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
C1 S1 C2 S2 C3 S3 C4 S4 C5 S5
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
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
• Check internal QC
• 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
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
Case 3
decreased visual acuity.
results and pituitary MRI suggested a non-functioning
13mm pituitary tumour.
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?
1 2 3 4 5
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
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
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
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
Lane 5 – Neat CSF control sample
The transferrin band in the fluid sample has not migrated
as either tetrasialotransferrin or asialotransferrin (compare
to control).
?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
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.
1 2 3 4 5
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?
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.
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
Positive control 9949.04 9971.06 Target 0.81-
1.51
Patient sample 7688.7 7702.5
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?
<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
Positive control 9949.04 9971.06 9960.05 16.60 Target 0.81-
1.51
Patient sample 7688.7 7702.5 7695.6 12.83
Case 4
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
Any questions?