Diagnosis of SAH

1
Diagnosis of SAH

• The laboratory perspective
• Dr Marie Parsons
• Principal Clinical Biochemist

2
Some statistics

• Demonstration of blood on CT- will, in
  experienced hands be positive in 98 of patients
  with SAH presenting within the first 12h after an
  event.
• This positivity falls to 50 in patients
  presenting after 1 week
• There is a need for a technique for detecting
  those CT negative patients presenting with a
  history suggestive of SAH who actually have had
  an event, and to eliminate the possibility in the
  remainder without the need for angiography.

3
What happens to the red blood cells following
haemorrhage into the CSF ?

• Lysis and phagocytosis
• Liberated oxyhaemoglobin is converted in vivo
  in a time dependant manner into bilirubin and
  sometimes methaemoglobin.
• The bilirubin is what can convey the yellow/
  xanthochromic appearance to the CSF.
• Of the 3 pigments only bilirubin arises solely
  from in vivo conversion.
• CSF bilirubin will also be increased when CSF
  total protein or serum bilirubin is increased
• Oxyhaemoglobin and methaemoglobin may be produced
  both in vitro and in vivo.

4
How do we detect bilirubin in CSF?

• Do we hold it up to the light and look for the
  yellow colour of the CSF ?
• NO Evidence clearly indicates visual
  inspection is not a reliable method
• Do we analyse the CSF for bilirubin using
  chemical methods available on our analyser?
• NO Chemical methods have not been adequately
  validated in CSF.
• Do we perform scanning spectrophotometry ?
• YES

5
Principles of spectrophotometery
Beer Lamberts Law
Absorbance concentration x abs coefficient of
substance x light path length
6
Scanning spectrophotometry

• CSF centrifuged gt2000rpm for 5 mins
• ASAP after receipt.
• Perform a spectrophotometric scan
• between 350 and 600nm in a cuvette
• with a 1cm path length.
• The scan should be inspected and the
• presence of the following pigments
• recorded.
• OxyHb - Peak absorbance 410-418nm
• Bilirubin- Broad peak 450- 480nm or a
• shoulder adjacent to the OxyHb peak.
• MetHb Rarest pigment 403 410nm

7
How are the scans analysed ?

• A predicted baseline is drawn which forms a
  tangent to the scan between 350 and 400nm and
  between 430 and 530.
• The absorbance of the scan at 476nm above this
  predicted baseline is measured, this is the NBA-
  Net Bilirubin Absorbance
• The absorbance of any oxyhaemoglobin above this
  predicted baseline is also measured, this is the
  NOA-Net OxyHb Absorbance

8
Normal CSF with no bilirubin
9
OxyHb with zero NBA
10
OxyHb with increased NBA
11
Usefulness of CSF spectrophotometry

• CSF spectrophotometry is of particular value in
  the investigation of CSF with an increased
  erythrocyte count as there is no other reliable
  way of distinguishing between SAH and a traumatic
  puncture
• It is also of value in the investigation of CSF
  with a normal red cell count from a patient
  presenting several days after an event by which
  time the cells may no longer be present.

12
Specimen requirements
13
Where can it go wrong (1) ?

• Not protecting the sample for spectrophotometery
  from light

Bilirubin is a light labile substance, it decays
at a rate of 0.005AU/h. Samples not protected
from exposure to light may give false negative
results.
14
Where can it go wrong (2) ?

• Insufficient sample provided. A minimum of 1ml is
  required (15-20 drops).
• Insufficient sample volume means that instead
• of the light passing cleanly through the sample,
  the sample
• level may be below that of the light path making
• measurement impossible or the light may hit the
• sample meniscus scattering the light so that Beer
  
• Lamberts law no longer applies and an incorrect
  result will
• be achieved.

15
Where can it go wrong (3) ?

• Failure to provide a serum sample for total
  protein and bilirubin measurement
• serum bilirubin level is gt20µmol the adjusted
• net bilirubin is calculated using an equation
• requires the serum bilirubin and total
• protein levels. Unless this calculation is done
  we
• are unable to tell if the CSF bilirubin is raised
  due
• to SAH or due to the raised serum bilirubin level
  crossing
• the blood brain barrier.

16
Patient MM
17
Patient CS
18
Patient EW
19
Patient LB
20
Protocol for sample collection in ? SAH

• Protocol approved by the Procedural Documents
  Approval Committee Dec 2009
• Produced by Clinical Biochemistry in conjunction
  with Dept of Microbiology and the Emergency
  Assessment Unit.
• Document Number 289 in the e-library

21
Information provided by the protocol

• The roles and responsibilities of those involved
  in sample collection and analysis
• Specimen requirements
• Clinical Information required by the laboratory
  to accurately interpret the scan results.
• Availability of the analytical service
• If CNS infection is in the differential diagnosis
  that the LP MUST be carried out immediately and
  the sample sent to microbiology. In these cases
  the sample if taken lt12h post onset of headache
  will not be suitable for spectrophotometric
  analysis.

22
Acknowledgements

• Dr David Gannon EAU
• Dr Tony Elston Microbiology
• Dr Gillian Urwin- Microbiology
• Jackie Mann Clinical Biochemistry
• Clinical Biochemistry staff