BIOCHEMICAL ANALYSIS OF CEREBRO-SPINAL FLUID

1. UNIVERSITY OF BUEA
7/22/2019
1
Faculty of Health Sciences
Department of MLS
Chemical Pathology: CPY 602,
BIOCHEMICAL ANALYSISOF CEREBRO-SPINALFLUID
Presented By
Lamngwa Benard Nfor HS18P036

2. OUTLINE
Introduction
Formation of CSF/anatomy
Circulation of CSF
Functions of CSF
Blood brain barrier
Biochemical composition of CSF
CSF analysis and pathophysiology
Laboratory investigations
Management of some CSF related infections
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3. OBJECTIVES
After this presentation, the students should be able to:
Describe the formation and location of cerebrospinal fluid
(CSF)
Describe the appearance and state the composition of
normal CSF
Understand the formation and functions of CSF
Understand some pathology associated to CSF
Understand biochemical investigations of CSF and
interpretation of such results
Have an idea of management of some related disease
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4. INTRODUCTION
•The cerebrospinal fluid (CSF) is a dynamic,
metabolically active fluid surrounding the brain and
spinal cord and has many important functions.
•It is very valuable as a diagnostic aid in the
evaluation of inflammatory conditions, infections
involving the brain, spinal cord, and
subarachnoid haemorrhage.
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5. FORMATION OF CSF/ANATOMY
•Both the brain and spinal cord are covered by three
protective membranes referred to as the meninges. The
outermost layer is called the dura mater and is composed
of tough connective tissue.
•The middle layer is the arachnoid named for it spider web
like appearance.
•The delicate innermost layer which is in direct contact with
the brain and spinal cord is called the pia mater. An
inflammation of the meninges is referred to as meningitis.
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6. FORMATION OF CSF ANATOMY CONT.
•Between the arachnoid layer and the pia mater is a space
called the subarachnoid space. It contains a clear, colorless
fluid referred to as Cerebrospinal Fluid (CSF).
•CSF is produced in the ventricles of the brain by a collection
of rich vascular protrusions called the choroid plexus.
•Excess CSF is continuously reabsorbed by arachnoid villi
and returned to the venous system thus maintaining a
consistent amount of fluid under an intracranial pressure
between 50 - 180 mmHg.
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7. fddf
•vdfd
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8. fddf
•vdfd
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9. FORMATION OF CSF /ANATOMY CONT.
•Generally, the total volume of CSF circulating throughout
the adult Central Nervous System (Brain and Spinal Cord)
is approximately 90 - 150 ml.
•In newborns this volume is 10 -60 ml.
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10. CIRCULATION OF CSF
•CSF flows from the lateral ventricles through the third and
fourth ventricles and into the subarachnoid space.
• From the fourth ventricle, the CSF either continues to the
central canal of the spinal cord or leaves for the
subarachnoid space by passing through the medial aperture
(foramen of Magendie) and paired lateral apertures
(foramina of Luschka) in the roof and lateral recesses of the
fourth ventricle, respectively.
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11. CIRCULATION OF CSF
•CSF flows from the lateral ventricles through the third and
fourth ventricles and into the subarachnoid space.
• From the fourth ventricle, the CSF either continues to the
central canal of the spinal cord or leaves for the
subarachnoid space by passing through the medial aperture
(foramen of Magendie) and paired lateral apertures
(foramina of Luschka) in the roof and lateral recesses of the
fourth ventricle, respectively.
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12. FUNCTIONS OF CSF
•Protection : CSF protects the brain from damage by
buffering the brain. It acts as a cushion
•Buoyancy: The actual mass of the human brain is about
1400 grams;
however, the net weight of the brain suspended in the CSF
is equivalent to a mass of 25 grams. which allows
the brain to maintain its density without being impaired
by its own weight.
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13. FUNCTIONS OF CSF cont.
•Chemical stability/Homeostasis: CSF maintain the
distribution of necessary substance and waste product
between CNS and Blood stream
•Prevention of brain ischemia: made by decreasing the amount
of CSF in the limited space inside the skull. This decreases
total pressure . transport of biomolecules to the brain
•Clearance of catabolites (CO2, lactate)
•Maintenance of constant intracranial pressure
•Clearin of waste: Removes waste from the brain through the
bllod for elimination via kidneys.
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14. BLOOD BRAIN BARRIER
•. A protective barrier separates the brain from circulating
blood and regulates the distribution of substances between
the blood and the CSF.
• If this protective barrier disrupts, it may result in a change
in the normal level or type of constituents of CSF.
•The blood–brain barrier restricts the passage of pathogens,
the diffusion of solutes in the blood, and large or hydrophilic
molecules into the cerebrospinal fluid (CSF), while allowing
the diffusion of hydrophobic molecules (O2, CO2,
hormones) and small polar molecules.
•Cells of the barrier actively transport metabolic products
such as glucose across the barrier using specific transport
proteins.7/22/2019 14

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16. BIOCHEMICAL COMPOSITION OF CSF
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17. Collection of CSF
•The patient is placed in a lateral position with the knees
bent in full flexion up to the chest(fetal position) (sit and
bend position also possible)
•Introduction of 1% lidocain into the subcutaneous space
•Insertion of a spinal needle into the subarachnoid space at
the L3-L4 or L4-L5 intercostal spaces (inserted until the
second “give”- subarachnoidal space).
•Removal of the stylet of the needle in order to collect the
fluid
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18. fddf
•vdfd
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19. 7/22/2019 19

20. Collection of CSF
• Approximately 10-12 ml can be taken from an adult and
less than 3 ml from a neonate. Care must be taken not to
contaminate the sample with blood.
•The fluid is collected into 3 tubes via free passive flow
through the needle. Up to four tubes of CSF can be
collected aseptically.
•Each tube should contain 2-4 mL of fluid. The tubes should
be labelled with a number indicating the order in which
they were collected. Each tube goes to a different section of
the laboratory for testing
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21. Collection of CSF
• Tube #1 Chemistry: Contains debris from the puncture and
occasionally blood in a "traumatic tap".
Since it is the most likely to be contaminated with microbes, tissue
fluid and blood cells which could yield misleading results. It
is best used for chemistry and immunological
determinations.
• Tube #2 Microbiology. May contain some blood cell contaminants
but is suitable for microbiological studies.
• Tube #3 Haematology. Has the least cellular or debris
contamination and therefore is used for cell counts, white cell
differentials and the examination of abnormal cells e.g. tumor
cells
• Tube #4, Molecular & Reference Lab7/22/2019 21

22. CSF ANALYSIS AND PATHOPHYSIOLOLOGY
• CSF is investigated to aid in the diagnosis of SAH
(subarachnoid hemorrhage), Meningitis, Demyelinating
disorders, CNS malignancy, Head injury with CSF leak and
other less common tests, the majority of the investigations
involve cytology and microbiology and chemistry.
•Meningitis
•Can be bacterial ( pyogenic or tuberculous), viral or fungal
( Cryptococcus). Apart from direct organism identification,
biochemistry and cell counts can help distinguish between
the types of meningitis.
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23. CSF ANALYSIS AND PATHOPHYSIOLOGY cont.
• In classical pyogenic meningitis ( eg Strep
Cryptococcal) CSF protein levels are high > 0.8 g/L,
glucose is much lower than 60% of blood value, high
counts of PMNL’ s ( polymorphonuclear lymphocytes)
occur and the CSF is turbid.
•TBM (tuberculous meningitis) can be differentiated by a
mixed PMNL/lymphocyte high cell count and a low CSF
chloride.
•Viral meningitis often has a lymphocytosis with a normal
glucose, normal protein levels and a clear CSF.
•Fungal infections present with CSF lymphocytosis, a high
protein count and normal glucose
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24. fddf
•vdfd
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25. CSF ANALYSIS AND PATHOPHYSIOLOGY cont.
• CSF GLUCOSE
•Glucose enters the CSF from the plasma by 2 mechanisms,
diffusion and active transport. Glucose is the primary
source of energy for the brain. Brain is 5 % body mass but
uses 20% glucose.
•The level of CSF glucose is influenced by the concentration
and duration of the plasma glucose level.
•CSF glucose levels are roughly two-thirds of plasma
glucose levels
•CSF glucose can only be interpreted reliably with a blood
sample taken within 15 min before or after an LP.
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26. CSF ANALYSIS AND PATHOPHSIOLOGY cont.
 CSF CHLORIDE
The diagnosis of tuberculous meningitis is notoriously
difficult to make. Chloride (less than 110mmol/1) is used to
distinguish tuberculous from viral and bacterial meningitis.
A CSF chloride of less than 100mmol/l is thought to be
virtually diagnostic of TBM.
CSF PROTEIN. Spinal fluid is an ultrafiltrate of plasma
that lacks high molecular weight proteins such as beta
lipoprotein, alpha-2 macroglobulin, IgM, etc.
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27. CSF ANALYSIS AND PATHOPHYSIOLOGY cont.
 Measurement of CSF protein is also used to
distinguish septic from aseptic meningitis.
Protein concentrations >1 g/L are often viewed as
diagnostic for bacterial, fungal, or tuberculous meningitis,
reflecting the presence of the pathologic organism
Unfortunately, the increase of CSF protein is a sequela in
many CNS disease processes, usually in association with
increased permeability of the bloodbrain barrier, vasogenic
brain edema, hypercellularity, and release of brain-specific
proteins during cell death
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28. CSF ANALYSIS AND PATHOPHYSIOLOGY cont.
 SUBARACHNOID HAEMORRHAGE
In subarachnoid haemorrhage red cells are a diagnostic
feature in the CSF, so careful LP is essential.
Red cells that have been in the CSF for longer than 4
hours cause a yellow staining of the CSF called
xanthochromia and the presence of this in fresh CSF
confirms bleeding into the subarachnoid space from a
source other than contamination during the LP. Keep in
mind that there are other causes of
xanthochromia. Xanthochromia refers to a yellow, orange
or pink colour7/22/2019 28

29. CSF ANALYSIS AND PATHOPHYSIOLOGY
cont.
HEAD INJURY
• In patients with rhinorrhoea or otorrhoea, post head
injury or spontaneously, it is important to ascertain
whether CSF is present in the fluid, which would
confirm a CSF leak. This is done by identifying beta-
2-transferrin in the fluid leak sample, using
electrophoresis and immunofixation
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30. CSF ANALYSIS AND PATHOLOGY cont.
• Protein index:
• This assesses the amount of intrathecal protein synthesis that may
occur in an inflammatory disease and assesses the permeability of
the blood-brain barrier in relation to increased intrathecal synthesis
(eg IgG).
• Albumin is used as a reference protein.
• A normal CSF Albumin/Serum Albumin ratio is less than 9. CSF
IgG index = CSF IgG/Serum IgG ÷ CSF Albumin/Serum Albumin
Usually the CSF IgG index will be 0.3-0.8. If > 0.8, this indicates
increased intrathecal synthesis such as may be seen in multiple
sclerosis..
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31. LABORATORY INVESTIGATIONS
• Physical examination
•Normal CSF is:
•Colorless
•Clear
•Free of clots
•Free of blood
•If CSF is cloudy (turbid) ,
perform microscopic
examination:
•Usually due to leucocytes
•may be due to micro-
organisms
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32. LABORATORY INVESTIGATIONS
• Physical examination
•Normal CSF is:
•Colorless
•Clear
•Free of clots
•Free of blood
•If CSF is cloudy (turbid) ,
perform microscopic
examination:
•Usually due to leucocytes
•may be due to micro-
organisms
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33. Blood & Hemoglobin pigments in CSF
Traumatic tap
•bright red color
• RBCS in decreasing
number as the fluid is
sampled
Subarachnoid hemorrhage
(SAH)
•Xanthochromia
(hemoglobin breakdown
pigments) = RBCs lysis &
metabolism previously
occurred (at least 2 hr
earlier)
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34. Examination of CSF
(Biochemical analysis of CSF)
•Tests of interest:
•Glucose
•Protein
Total
Specific:
Albumin
Immunoglobulin
Others (e.g. myelin basic protein; MBP)
 Lactate
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35. Glucose
• Glucose enters CSF via facilitative transporter (GLUT)
• CSF glucose is ~ 2/3 that of plasma 50 - 80 mg/dl
• A plasma sample must be obtained ~ 2-4 hr before CSF
sample
• In hypoglycemia, CSF glucose may be very low and In
hyperglycemia glucose is raised.
• Measure CSF Glucose immediately or preserve the
specimen with and antiglycolytic
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36. Glucose
•↑ CSF glucose conc. (hyperglycorrhachia) :
•Not clinically informative
•Provides only confirmation of hyperglycemia
•↓CSF [glucose] (hypoglycorrhachia):
1)Disorder in carrier-mediated transport e.g. TB meningitis,
2)Active metabolism of glucose by cells or organisms:
•e.g. acute purulent, amebic, & fungal meningitis
3)Increased metabolism by the CNS
4)Glucose is measured by the use of a Spectrophotometer
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37. Proteins
•The Proteins mostly found in the CSF is albumin
•Source of CSF proteins:
• 80% from plasma by ultrafiltration
• 20% from intrathecal synthesis
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38. proteins cont.
•Increase CSF total protein
Must be compared to the serum protein conc.
Useful nonspecific indicator of pathological states:
•Lysis of contaminant blood (traumatic tap)
•↑ Permeability of the epithelial membrane due to:
•Bacterial or fungal infection, cerebral hemorrhage
•↑ production by CNS tissue in, e.g Multiple sclerosis
(MS)
•Obstruction e.g. in tumors and abscess
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39. Proteins cont.
•Total protein is measured spectrophotometrically
•The Pandys test is also used for the qualitative
detection of total protein in CSF
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40. Albumin
• Albumin is produced solely in the liver
• Its presence in CSF must occur through BBB
• Measured by the protein electrophoresis method
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41. Immunoglobulin
• CSF IgG can arise:
• from plasma cells within CSF
• from the blood through BBB
•Increase IgG conc. and normal Alb conc. of CSF
suggests local production of IgG, e.g.,
• Multiple sclerosis (MS)
• Subacute sclerosing panencephalitis (SSPE)
• IgG can be measured by electrophoretic method
• Also measured by immunoassays.
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42. Management of CSF related infections
Meningitis
Bacterial meningitis is a medical emergency
Empirical selection of therapy is usually necessary, and
treatment should be started at the first suspicion of bacterial
meningitis.
A single dose of benzylpenicillin can be given if the person is
outside hospital, but cefotaxime is the preferred treatment in
hospital
Chloramphenicol is an option for those who have an allergy to
both penicillin and cephalosporins.
Treatment is given for 5 days for meningococcus, and 10 days
for Haemophilus influenzae or pneumococcus.
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43. Management of CSF related infections
Meningitis
Amphotericin is active against all common fungi that cause
systemic infection (Candida, Aspergillus, Mucor and
Cryptococcus species). Cryptococcus is incriminated in
cryptococcal meningitis.
Tuberculosis is usually treated with a multidrug regimen because
of the rapid development of resistance. Rifamycins (rifabutin,
rifampicin), Isoniazid, Pyrazinamide and Ethambutol are first line
regimen.
Other drugs can be used as second-line treatments in multidrug-
resistant tuberculosis. These include cycloserine, capreomycin,
amikacin, ciprofloxacin, moxifloxacin, azithromycin,
clarithromycin and p-aminosalicylic acid7/22/2019 43

44. Management of CSF related infections
Acute brain injury
Mannitol is occasionally used to reduce ischaemic cerebral
damage, for example after neurosurgery or in acute traumatic
brain injury.
Fluid loss via the kidney should be replaced with
intravenous crystalloid to avoid dehydration.
Dexamethasone is often used to reduce oedema
around malignant tumours in the brain and those
compressing the spinal cord
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45. REFERENCES
1. Cerebrospinal fluid analysis in infectious diseases of the nervous system: when to ask, what to ask, what
to expect, Luís dos Ramos Machado1,3, José Antonio Livramento2,3, Liliana Scaff Vianna4
2. Scott LA, Tintinalli JE, Brewer KL, Salgado CD. Lumbar punctures for
3. suspected meningitis in adults. Infect Dis Clin Pract 2008;16:298-302.
4. 2. Van de Beek D, de Gans J, Tunkel AR, Wijdicks EFM. Communityacquired bacterial
meningitis. N Engl J Med 2006;354:44-53Dalia Mohamed Moemen
5. Cerebrospinal fluid, Assistant lecturer of Medical Microbiology and Immunology,
Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura
Universit
6. Clinical Utility of Biochemical Analysis of Cerebrospinal Fluid Mark A. Watson and
Mitchell G. ScottRoutine c erebrospinal fluid ( CSF ) a nalysis
7. F. Deisenhammer,1 A. Bartos,2 R. Egg,1 N. E. Gilhus,3 G. Giovannoni,4 S. Rauer,5
Sellebjerg,6 H. Tumani
8. CSF ANALYSIS IN METABOLIC DISORDERS© CSF Neurotransmitter Request
Form (.pdf)7/22/2019 45

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