Laboratory diagnosis of CSF

1. CSF EXAMINATION
Dr Abdul Hafeez Kandhro
Senior Lecturer
B.Sc, M.Sc; Medical Technology,
M.Phil Biochemistry
Ph.D. Medical Technology
(Mahidol University, Bangkok , Thailand)

2. What is Cerebrospinal fluid (CSF) ?
• Cerebrospinal
fluid (CSF) is a
clear, colorless
liquid found in
the brain and
spinal cord.

3. CSF Composition
• Daily about 1500 ml of CSF is formed
• Volume 130 - 150 ml (the whole volume of CSF is replaced several times
a day)
• Colour Colorless
• Appearance Clear
• Pressure 60 - 150 mm of H2O (about 8-15 mm of Hg) in Spine and 200 -
250 mm of H2O in Sitting position.
• pH 7.35 (7.3 - 7.4)
• Specific gravity 1.007 (1.003 to 1.008)
• Protein 15 - 45 mg/dl
• Sugar 40 - 80 mg/dl
• Chloride 720 - 750 mg/dl
• Sodium 144 154 mEq/L
• Potassium 2.0 - 3.5 mEq/L
• Creatinine 0.5 - 1.2 mg/dl
• Urea 5 - 15 mg/dl
• Uric acid 0.5 - 4.5 mg/dl
• Cholesterol 0.2 - 0.6 mg/dl
• Cells : 0-5 lymphocytes/cumm (Neutrophils absent).

4. CSF Functions
• The brain and spinal cord make up central
nervous system (CNS). The CNS controls and
coordinates everything including, muscle
movement, organ function, and even complex
thinking and planning.
• CSF helps protect this system by acting like a
cushion against sudden impact or injury to the
brain or spinal cord.
• It contains glucose, electrolytes, amino acids, and
other small molecules found in plasma, but has
very little protein and few cells.

5. CSF Functions
• Since the brain lacks a true lymphatic system,
excess protein in the brain tissue spaces are
carried through the Perivascular spaces and into
the Subarachnoid spaces by the Cerebrospinal
fluid.
• By passing through the Arachnoid villi, the CSF
carries the protein back into the venous blood
stream. This route via the Perivascular spaces may
also be utilized to flush out cellular debris in the
brain following an infection and other metabolic
wastes.

6. CSF Functions
• CSF also removes waste products from the brain
and helps CNS work properly.

7. How CSF is formed
• CSF is produce by an active process where Sodium
ions are transported across the Epithelial cells and
pushed outside of the Choroid plexus.
• The positive Sodium ions then attract negative
Chloride ions.
• This changes the osmotic gradient and the CSF
with the higher ion concentration draws water
across the Choroid plexus membrane (osmosis).

8. • Glucose, Bicarbonate and Sodium ions are then
transported out of the blood capillaries by other
processes.
• This brings the composition of CSF similar to that
of plasma, although the quantities of chloride
ions, potassium ions and glucose are lower in the
CSF.
• Fluid secreted from the Choroid plexus of the
lateral passes through the first and third ventricles
and into the fourth ventricle.
How CSF is formed

9. • By exiting the fourth ventricle through the two
lateral foramina and the midline foramen, the
Cerebrospinal fluid enters the Cisterna magna.
• This then drains into the Subarachnoid space
which surrounds the entire brain and Spinal cord.
• Eventually CSF flows through the Arachnoidal villi
and is emptied into the several venous sinuses of
the Cerebrum. It is then returned into the venous
circulation
How CSF is formed

10. • Pressure:
• Increased:
– Brain Tumor.
– Meningitis, Enccephalitis.
– Neurosyphilis.
– Subarachnoid and Intracerebral hemorrhage.
– Hypertensive Encephalopathy.
– Venous sinus Thrombophlebitis.
– Hydrocephalus.
– Benign raised Intracranial tension.
– Uremia
– Emphysema.
• Decreased:
– Repeat L.P. soon after the brain tumor.
– Subarachnoid spinal block.
– Subdural hematoma.
Significant Features of CSF

11. • Appearance :
• Turbid: Pyogenic meningitis.
• Cobweb: T.B. meningitis.
• Blood tinged:
– Subarachnoid hemorrhage.
– Trauma to spinal blood vessels.
– Bleeding diathesis.
• Xanthochromia (Yellow tinting of the C.S.F.).
– Following hemorrhage in the C.S.F.
– High protein content of the C.S.F (Guillain Barre syndrome,
spinal block, neurofibroma, etc.).
– Jaundice.
– Tumors near the cauda equina, around the ventricles and
acoustic neuroma.
Significant Features of CSF

12. • Proteins :
• Increased:
– Meningitis.
– Encephalitis including Poliomyelitis.
– Disseminated sclerosis.
– Guillain Barre Syndrome.
– Neurosyphilis.
– Spinal cord compression.
– Intra- cranial tumor.
– Cerebral arteriousclerosis.
• Decreased: ( Not known)
• Glucose :
• Increased:
– Diabetes mellitus.
– Following I.V. glucose administration.
– Encephalitis.
• Decreased: Meningitis.
Significant Features of CSF
Chlorides :
Increased : (Not known)
Decreased :
Purulent and Tuberculous
meningitis.
Systemic hypochloremia.

13. • Cells :
• Polymorphonuclear leucocytosis :
– Pyogenic meningitis.
– Acute syphilitic meningitis.
– Actute poliomyelities (early stage).
– Epidural abscess.
• Lymphocytosis :
– Meningitis - tuberculous, viral, syphilitic & late stage of pyogenic.
– Encephalitis.
– Poliomyelitis- later stages.
– Disseminated sclerosis.
– Cerebral tumor.
– Cortical venuous thrombophlebitis.
• Eosinophil : Cerebral or spinal cysticercosis.
• Malignant Cells: Cerebral or spinal malignancy.
Significant Features of CSF

14. • Diagnostic :
– Absolute :
• Meningitis
• Subarachnoid Haemorrhage
– Relative :
• Neurosyphilis
• Unexplained Coma
• Guillain barre Syndrome.
• Multiple Sclerosis
– Radiological
• Myelography
• Pneumoencephalography
• Therapeutic :
– To Introduce Drugs :
• Methothexate 0.25 mg/kg biweekly in leukemia.
• Gentamicin 10-20 mg in Gram Negative meningitis.
• Crystalline Penicillin 10,000 20,000units in pyogenic meningitis.
– To reduce raised intro-cranial tension in Hypertensive Encephalopathy.
– To administer spinal anesthesia.
Indications for CSF

15. • Position : The patient is placed on his side at the
edge of the bed with the knee drawn up and the
head flexed. It can also be done with the patient
sitting and bending forward.
• Site : In the 3rd lumber space. This space lies in
the plane which joints the highest points on the
iliac crest. The skin over the back from the lower
thoracic vertebrae to the coccyx is sterilized with
cetavlon, ether, Iodine and spirit. The part is
draped.
• Local anesthesia : The skin to be punctured is
infiltrated with 5 ml of 2% lignocaine. Infiltration
is done upto ligamentum flava.
Collection of CSF

16. • Puncture : A lumber puncture needle with a stilette is
introduced after 2-3 minutes into the anesthetized
space, with the cutting edge of the bevel in the
direction parallel to the fibers of the ligamentum
flava. The needle is introduced through the resistance
of supraspinous ligament.
• The interspious ligament is then easily negotiated. At
about 4-7 cm, the firmer resistance of ligamentum
flavum popping sensation as the dura is breached.
• The stilette is then withdrawn and the fluid is
collected slowly in 4-5 sterile test tubes for
biochemical, cytological and serological assay. The
first few drops are collected separately and may be
used for bacteriological culture.
Collection of CSF

18. • Laboratory investigations of Cerebrospinal fluid are
consist of mainly the primary examination;
• A) Physical Examination,
• B) Microscopic Examination and
• C) Chemical Examination.
• CSF must be examined immediately within one hour,
after the specimen is collected.
• The blood cells and the offending organisms sought
for diagnosis rapidly lyse on standing while the
bacteria multiply with delay in analysis resulting in
changes in the chemical composition (e.g. glucose).
Lab Diagnosis of CSF

19. • A. Physical Examination
• Physical examination ( mainly
appearance and clot formation)
is done with an Uncentrifuged
specimen.
• Initial clot formation is noted
before the centrifugation while
one of the tubes can be left for
observing 24-hour clotting, or
leave the supernatant to
observe clotting after
processing the sediment.
Lab Diagnosis of CSF

20. • A. Physical Examination
• Appearance :
• Examine the appearance of CSF and record
accurately as:
[Clear, Cloudy, Blood-stained, or Xanthochromic]
• Determine the specific gravity of CSF with the help of
a hand Refractometer or by the weight method.
• Normal CSF is crystal clear and colourless.
• When a pathologic condition is present, there may be
a change in colour; Blood, pus, bacteria, and their
products may be found. Pus formation due to
infection leads to cloudy CSF which is most often
associated with an increase in this number of pus
cells or leukocytes.
Lab Diagnosis of CSF

21. • A. Physical Examination
• Clot formation :
• Normal spinal fluid does not coagulate or clot.
• Clot formation is due to the presence of fibrinogen
which converts to fibrin to produce the clot.
• Fibrinogen is absent in normal CSF but increases
when the blood-brain barrier is disturbed.
• The type of clot depends on certain diseases and is
associated with increased protein concentration.
• A delicate, fine clot is typical of tuberculous
meningitis; a large clot is associated with purulent
meningitis; and a complete and spontaneous clot is
found in case of spinal constriction.
Lab Diagnosis of CSF

22. • B. MICROSCOPIC EXAMINATION
• Microscopic examination of the CSF includes;
• the Leukocyte count of the uncentrifuged whole
specimen,
• Examination of the wet mount is used for
detection of Trypanosoma and Cryptococcus by
using India Ink preparation.
• The sediment of the centrifuged specimen is
taken for the study of the stained smear; Direct
examination of the specimen for infectious agents
& for the differential count.
Lab Diagnosis of CSF

23. • B. MICROSCOPIC EXAMINATION
• Total Leukocyte Count :
• Normal CSF is virtually free of cells, although as
many as 5 WBC/mm3 is considered as normal and
these are small mononuclear cells (lymphocytes).
• The presence of neutrophils and/or monocytes
are the indication of abnormality. Increased
leukocyte count suggests the possibility of
infective meningitis.
• A cloudy specimen may have a high leukocyte
count (>500 WBC/cmm).
Lab Diagnosis of CSF

24. • B. MICROSCOPIC EXAMINATION
• Differential Leukocyte Count :
• The Differential Leukocyte Count provides information regarding the
relative distribution of various white blood cells - lymphocytes,
neutrophils and others. The basic procedure is the same as describe
under hematology.
• Preparation of a thin smear of uncentrifuged, well mixed CSF
specimen on a microscopic slide for differential leukocyte count (as like
hematology), if the specimen is cloudy and Total Leukocyte count of CSF
is >500 WBC/cmm.
• If <200 WBC/cmm Centrifuge the specimen in a centrifuge tube at 2500
rpm for 10 minutes.
• Transfer the supernatant to another tube for chemical analysis. Save the
last drop of the specimen.
• Tap the sediment to mix with the last drop of fluid in the centrifuge tube.
• Prepared a smear on a clean and dry glass slide and leave to dry.
• Stain with Leishman’s stain
• Make a differential leukocyte count under 1000X magnification (oil
immersion) of the microscope.
Lab Diagnosis of CSF

25. • B. MICROSCOPIC EXAMINATION
• Wet mount Preparation of CSF :
• Wet mount of CSF is prepare in case of suspected sleeping
sickness (Trypanosomiasis) and Cryptococcosis.
Trypanosomes are motile, flagellate, intercellular protozoa
found in the blood at early stage of infection; their
presence in CSF indicates late stage of the disease.
• Place a drop of suspended CSF sediment on a clean and dry
glass slide.
• Put a coverslip on the drop.
• Examine under the high power objective (40X). Look for
motile flagellates. In case of positive finding it may
correlated with increased lymphocyte count, Increased
globulin level and presence of mononuclear vacuolated
large cells etc.
Lab Diagnosis of CSF

26. • B. MICROSCOPIC EXAMINATION
• CSF Cytology
• A CSF sample is specially treated so that a
microscope can be used to look for abnormal
cells.
• This is often done when a central nervous system
tumor or metastatic cancer is suspected.
• The presence of certain abnormal cells, such as
tumor cells or immature blood cells, can indicate
the type of cancer present.
Lab Diagnosis of CSF

27. • C. Chemical Examination of CSF
• Chemical examination of Cerebrospinal fluid
(particularly glucose, Total Protein and Globulin) play
an important role for detect the nature of meningitis.
• Glucose level in spinal fluid is about 40 - 80 mg/dl.
• Glucose Reduced in bacterial meningitis which may
be due to increase number of leukocytes and
pathogenic organisms and both contribute to
increased glycolysis.
• Also low in metastatic tumour and insulin shock
• Elevated Glucose in diabetic coma, in some
pathogenic states such as brain tumour (70 - 100
mg/dl) and also in case of diabetes mellitus.
Lab Diagnosis of CSF

28. • C. Chemical Examination of CSF
• Total Protein concentration of Cerebrospinal fluid
is about 15 - 45 mg/dl, which is below 1% of the
normal serum protein level.
• Incase of inflammation of the meninges under
toxic conditions or development of tumours, the
barriers between the blood and brain become
more permeable and increased amounts of
protein enter the subarachnoid space and appear
in the CSF.
Lab Diagnosis of CSF

29. • C. Chemical Examination of CSF
• Globulin is the protein of chief interest in the
study of CSF. This is because immunoglobulins
develop within the CSF in case of infection. Blood
tap gives erroneous results.
• Pandy test and Sulphosalicylic acid turbidometry
tests are convenient semiquantitative procedures
for globulin and total protein respectively.
Lab Diagnosis of CSF

30. • C. Chemical Examination of CSF
• LACTATE DEHYDROGENASE. This enzyme is
elevated in bacterial and fungal meningitis,
malignancy, and subarachnoid hemorrhage.
Lab Diagnosis of CSF

31. • Culture and Sensitivity test of CSF :
• The first tube is always used for bacterial culture
while the other tubes are used for microscopic
examination of the whole specimen (leukocyte
count) or examination of the smear of the
sediment.
• The sediment (more concentrated) should be
used for Bacterial and Fungal culture.
• The common pathogenic bacteria responsible for
meningeal problems are: Neisseria meningitidis,
Haemophilus influenzae, Streptococcus
pneumonia and Mycobacterium tuberculosis.
Lab Diagnosis of CSF

32. • Serological Tests
• Syphilis
• This involves testing for antibodies that indicate
neurosyphilis.
• The fluorescent treponemal antibody-absorption
(FTA-ABS) test is often used and is positive in
persons with active and treated syphilis.
• The test is used in conjunction with the VDRL test
for nontreponemal antibodies, which is positive in
most persons with active syphilis, but negative in
treated cases.
Lab Diagnosis of CSF

33. • Serological Tests
• Tests to detect antibodies produced by the
immune system against specific disease-causing
microbes may be helpful in infections where
culture and molecular testing are insensitive (e.g.,
West Nile virus, Lyme disease that infects the
nervous system, etc.).
• Testing of CSF to detect proteins or antigens
released by certain microbes, including the fungi
Cryptococcus neoformans/gattii or Histoplasma
capsulatum, may also be performed depending on
exposure risk and signs and symptoms
Lab Diagnosis of CSF

34. • Molecular testing of the CSF
• By polymerase chain reaction (PCR) assays can be
performed to detect nucleic acid from various
pathogens that may be present in the sample.
• This method detects bacterial, viral, fungal or
parasitic genetic material (DNA, RNA) and is
particularly helpful if the microbe does not grow
in routine culture or if the patient has been on
antibiotics.
Lab Diagnosis of CSF