Chapter Six
Cerebrospinal Fluid Analysis (CSF Analysis)
•Formation and Physiological Role
•CSF is a fluid surrounding brain and spinal cord
•CSF provides a physiologic system to supply
nutrients to the nervous tissue, remove
metabolic wastes, and produce a mechanical
barrier to cushion the brain and spinal cord
against trauma
Formation:
•The brain and spinal cord are lined by the meninges, which consists of
three layers: the dura matter, arachnoid, and pia matter. [ Fig.7.1]
•The outer layer is the dura mater that lines the skull and vertebral
canal.
•The arachnoid is a filamentous (spiderlike) inner membrane.
•The pia mater is a thin membrane lining the surfaces of the brain and
spinal cord.
•CSF is produced in the choroid plexuses and is present in the brain
and in the subarachnoid space around both the brain and the spinal
cord.
The choriod plexuses are capillary networks that form the CSF from
plasma by mechanisms of selective filtration under hydrostatic
pressure and active
transport secretion.
•Therefore, the chemical composition of the CSF does not resemble
an ultra filtrate of plasma.
•Capillary walls throughout the body are lined with endothelial cells
that are loosely connected to allow passage of soluble nutrients and
wastes between the plasma and tissues.
•In the choroid plexuses, the endothelial cells have very tight-fitting
junctures that prevent the passage of many molecules.
•This tight-fitting structure of the endothelial cells in the choroid
plexuses is termed the blood-brain barrier (BBB).
•Maintaining the integrity of the blood-brain barrier is essential to
protect the brain from chemicals and other substances circulating in
the blood that could harm the brain tissue.
In contrast, the junctures also prevent the passage of helpful
substances including antibodies and medications
•Disruption of the blood-brain barrier by diseases such
as meningitis allows leukocytes, proteins, and additional
chemicals to enter the CSF
Collection and Preservation
CSF is routinely collected by lumbar puncture (LP) which is also called spinal tap
between the third, fourth, or fifth lumbar vertebrae.
CSF is collected by physicians
Specimens are collected in three sterile tubes, which are labeled 1, 2, and 3 in
the order in which they are withdrawn
Tube 1 is used for chemical and serologic tests because these tests are least
affected by blood or bacteria introduced as a result of the tap procedure;
Tube 2 is usually designated for the microbiology laboratory;
Tube 3 is used for the cell count, because it is the least likely to contain cells
introduced by the spinal tap procedure
Considering the discomfort to the patient and the possible complications that
can occur during specimen collection, laboratory personnel should handle CSF
specimens carefully.
•Ideally, tests are performed on a STAT basis. If this is not possible,
specimens are maintained in the following manner:
•Hematology tubes are refrigerated.
•Microbiology tubes remain at room temperature.
• Chemistry and serology tubes are frozen.
•Traumatic Collection (Tap)
•Grossly bloody CSF can be an indication of intracranial hemorrhage,
but it may also be due to the puncture of a blood vessel during the
spinal tap procedure.
•Three visual examinations of the collected specimens can usually
determine whether the blood is the result of hemorrhage or a
traumatic tap
Uneven Distribution of Blood:
Blood from a cerebral hemorrhage will be evenly distributed
throughout the three CSF specimen tubes, whereas a traumatic
tap will have the heaviest concentration of blood in tube 1, with
gradually diminishing amounts in tubes 2 and 3.
Clot Formation:
Fluid collected from a traumatic tap may form clots owing to
the introduction of plasma fibrinogen into the specimen.
Bloody CSF caused by intracranial hemorrhage does not contain
enough fibrinogen to clot.
Xanthochromic Supernatant
Xanthochromia is a term used to describe CSF supernatant
that is pink, orange, or yellow.
A variety of factors can cause the appearance of
xanthochromia, with the most common being the presence of
RBC degradation products.
RBCs must usually remain in the CSF for approximately 2 hours
before noticeable hemolysis begins; therefore, a xanthochromic
supernatant would be the result of blood that has been present
longer than that introduced by the traumatic tap.
Other causes of xanthochromia include elevated serum
bilirubin, presence of the pigment carotene, markedly increased
protein concentrations, and melanoma pigment.
Examination
Physical Examination
•Appearance
•Normal CSF is crystal clear
•The major terminology used to describe CSF appearance
includes crystal clear, cloudy or turbid, milky,
xanthochromic, and hemolyzed/bloody
•Clot Formation
•Fluid collected from a traumatic tap may form clots owing to
the introduction of plasma fibrinogen into the specimen.
•Bloody CSF caused by intracranial hemorrhage does not
contain enough fibrinogen to clot.
Microscopic Examination
•Total Cell Count
•The cell count that is routinely performed on CSF specimens is the
leukocyte (WBC) count.
•Any cell count should be performed immediately, because WBCs
(particularly granulocytes) and RBCs begin to lyse within 1 hour, with
40% of the leukocytes disintegrating after 2 hours.
•Methodology: An improved Neubauer counting chamber is routinely
used for performing CSF cell counts. Automated hematology analyzers are
not recommended. Clear specimens may be counted undiluted
•Normal value:
•Adult CSF contains 0 to 5 WBCs/µL.
•The number is higher in children, and as many as 30 mononuclear
cells/µL can be considered normal in newborns
•Differential Cell Count
•The differential count should be performed on a stained
smear and not from the cells in the counting chamber.
•The specimen is centrifuged for 5 to 10 minutes,
supernatant fluid is removed and saved for additional tests,
and slides made from the suspended sediment are allowed to
air dry and are stained with Wright’s stain.
•When the differential count is performed, 100 cells should be
counted, classified, and reported in terms of percentage.
•If the cell count is low and finding 100 cells is not possible,
report only the numbers of the cell types seen
•The cells found in normal CSF are primarily lymphocytes
and monocytes
Chemistry Tests
•Routine chemistry tests include Total protein, Glucose,
lactate and Glutamine
•Methodology: Uses the same method applied for
determination of the analytes in blood
•Protein
1. Normal concentration is 15 to 45 mg/dL.
2. Elevated values are most frequently seen in patients
with meningitis, hemorrhage, and multiple sclerosis.
•Glucose
1. Normal value is 60% to 70% of the plasma
concentration.
2. Decreased levels are seen in patients with
bacterial, tubercular, and fungal meningitis.
•Lactate
1. Levels >35 mg/dL are seen in patients with bacterial
meningitis.
2. Levels >25 mg/dL are found in patients with tubercular and
fungal meningitis.
3. Lower levels are seen in patients with viral meningitis
•Destruction of tissue within the CNS owing to oxygen deprivation
(hypoxia) causes the production of increased CSF lactic acid levels.
•Therefore, elevated CSF lactate is not limited to meningitis and can
result from any condition that decreases the flow of oxygen to the
tissues
•Glutamine-is produced from NH3 and α-ketoglutarate to remove
ammonia which is toxic metabolic waste.
1. Normal concentration is 8 to 18 mg/dL.
2. Levels >35 mg/dL are associated with some disturbance of
consciousness.
Microbiology Tests
Gram stain, acid-fast stain, India ink preparation, and latex
agglutination tests are used for preliminary diagnosis of meningitis
Culture is used for confirmation of meningitis
•Gram stain
•The CSF should be centrifuged and slides and cultures
should be prepared from the sediment
•Even when concentrated specimens are used, at least a
10% chance exists that Gram stains and cultures will be
negative.
•Thus, blood cultures should be taken, because the
causative organism is often present in both the CSF and the
blood
•Organisms most frequently encountered include
Streptococcus pneumoniae (gram-positive cocci),
Haemophilus influenza (pleomorphic gram-negative rods),
Escherichia coli (gram negative rods), and Neisseria
meningitidis (gram-negative cocci).
•The gram-positive cocci, Streptococcus agalactiae and the
gram-positive rods Listeria monocytogenes may be
encountered in newborns
•Acid Fast
•Acid-fast or fluorescent antibody stains are not routinely performed
on specimens unless tubercular meningitis is suspected.
•Considering the length of time required to culture mycobacteria, a
positive report from this smear is extremely valuable
•Indian Ink
•Specimens from possible cases of fungal meningitis are Gram
stained and often have an India ink preparation performed on them to
detect the presence of thickly encapsulated Cryptococcus
neoformans
•As one of the more frequently occurring complications of AIDS,
cryptococcal meningitis is now commonly encountered in the clinical
laboratory.
•India ink have a sensitivity of about 25%, increasing to 53% with
multiple lumbar punctures.
•Detection of cryptococcal antigen from sera or CSF using latex
agglutination has higher sensitivity, ranging from 60-95%.
•Latex Agglutination
•Latex agglutination and enzyme-linked immunosorbent
assay (ELISA) methods provide a rapid means for
detecting and identifying microorganisms in CSF. Test kits
are available to detect Streptococcus, influenzae , S.
pneumoniae, N. meningitides and E. coli antigens.
•The bacterial antigen test (BAT) does not appear to be as
sensitive to detection of N. meningitidis as it is to the other
organisms.
•The BAT should be used in combination with results from
the hematology and clinical chemistry laboratories for
diagnosing meningitis.
•The Gram stain is still the recommended method for
detection of organisms.
Serologic Testing
•In addition to the serologic procedures performed for
identification of microorganisms, serologic testing of the CSF
is performed to detect the presence of neurosyphilis
•Although many different serologic tests for syphilis are
available when testing blood, the procedure recommended by
the Centers for Disease Control and Prevention to diagnose
neurosyphilis is the Venereal Disease Research Laboratories
(VDRL), even though it is not as sensitive as the fluorescent
treponemal antibody-absorption (FTA-ABS) test for syphilis.
•If the FTA-ABS is used, care must be taken to prevent
contamination with blood, because the FTA-ABS remains
positive in the serum of treated cases of syphilis
•The rapid plasma reagin (RPR) test is not recommended for
use on CSF, because it is less sensitive and specific than the
VDRL.