The document provides a comprehensive overview of cerebrospinal fluid (CSF), including its anatomy, functions, and the procedures involved in its collection and analysis. It outlines the role of CSF in diagnosing central nervous system disorders and describes normal and abnormal characteristics of CSF findings that indicate various medical conditions. Methods for chemical, microscopic, and serological examinations, as well as newer diagnostic techniques using PCR, are also discussed.

1. CSF Examination
Muhammad Asif Zeb
lecturer Hematology
IPMS-KMU

2. Contents
Introduction
Function
Sample Collection
CSF Lab Examination
Chemical Analysis
Serological Examination
Other Techniques
References

3. GENERAL
INFORMATION:
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.

4. 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

5. FUNCTIONS OF CSF
THE TWO CHIEF FUNCTIONS OF CSF (SPINAL)
FLUID ARE:
1. To protect and cushion the brain and the spinal
cord against possible injury.
2. To deliver nutrients from the blood to the Central
Nervous System and removes
wastes

6. Normal CSF
Thin, colourless, clear fluid
CSF is the 3rd
major fluid of the body
Adult volume 90-150 mL
Neonate volume 10-60 mL
0-5 WBC’s /mm3
(neonates 0-30/ mm3
)
(Lymphocytes & monocytes)
Occasional ependymal or choroid plexus cells
Protein 15-45mg/dl
Glucose 50-80mg/dl
Chloride 113-130 mEq/L
Sterile

7. Lumber puncture or collection
of CSf
The procedure used to obtain
cerebrospinal fluid is referred to as
Lumbar Puncture or a Spinal Tap.

8. Purpose
The purpose of CSF analysis is to diagnose
medical Disorders that affect the CNS.
Viral and Bacterial Infections.
Tumors of Nervous System.
Syphilis, a Sexually transmitted disease.
Bleeding around the brain and spinal cord.
Multiple sclerosis,a disease that affects the myelin
coating of the nerve fibers.
Guilain-Barre syndrome,an inflammation of the
nerves.

9. Collection Technique

10. full flexion

12. Level of entry

13. Spinal needle

14. Spinal needle

15. Under strict aseptic precautions.,

18. Collecting the CSF

19. 19
Collection: 3-10 ml is collected
Tube 1Tube 1 Chemistry &Chemistry &
immunologyimmunology
Tube 2Tube 2 MicrobiologyMicrobiology
Tube 3Tube 3 Cytology andCytology and
HematologyHematology

20. Analysis
Physical examination
Chemical examination
Microscopic examination

21. Appearance of CSF
clear and colourless (normal);
bright red - This indicates the presence of blood.
The appearance of fresh blood in all three tubes
supports the diagnosis of a subarachnoid
hemorrhage.
xanthochromic (yellow) - jaundice
turbid - This indicates the presence of white cells
and is suggestive of a CNS infection.

22. Presence of clot
No clot
If present indicate high protein content
Condition
With patient traumatic tap
Complete spinal blockage
Tuberculosis meningitis
Bacterial infection

23. Amount of CSF
How much collected

24. Microscopic examination
Cell counts
Noramal less than 5/ul
 TLC count
 RBC count
 Smear preparation
for Differential count to see the morphology of
WBC,s
Usually few lymphocytes are present

25. How to count TLC and
RBC???

26. Clinical significance of Increase
TLC
Neutrophili leukocytosis =
Neisseria meningitis, Hemophilus influenza
and E. coli
Lymphocytic/monocytic leukocytosis=
T.b, Viral infection, fungus or with syphilis
Malignancy=
Leukemia,

27. Leukocyte-differential
Most patients with
bacterial infections=neutrophils
while
fungal, tubercular and viral infection =
lymphocytes.

28. Leukocyte-differential (Contd..)
other conditions.
Leukemia
Plasma cell leukemia
Presence of tumor in brain.
Metastasis to CSF

29. Gram stain and culture
A Gram’s stain should be perform on all CSF
.
Cultures are also set up in order to retrieve and identify
organisms present in the CSF.
If the physician suspects a viral, mycobacterial, or rickettsia
organism,
it is important that special culture techniques be followed for
isolation of these organisms.

30. Chemical analysis
CSF PROTEIN DETERMINATION
CSF Glucose DETERMINATION

31. Protein
Normal CSF protein concentration is 15 - 45
mg/dl. It is derived from the plasma.
Increased levels are associated with:
- infection
- altered capillary permeability
- decreased absorption
- local biosynthesis of gamma globulin

32. Sulfosalicylic acid test
Take 3 ml of 3% sulfosalicylic acid in a
tube
add 1 ml of supernatant clear CSF in it.
Cloudiness indicate the presence of
protein

33. Principle (Biuret Method): Any
compound
(proteins) containing three or more peptide
bonds reacts with alkaline copper tartrate
reagent to form a blue to purple coloured
substance.
The intensity of colour produced is proportional
to the number of peptide bonds reacting and,
therefore, to the amount of proteins.

34. Biuret Method
Procedure:
To 2 ml CSF add 2 ml 10%trichloracetic acid, mix well and allow to
stand for 5 min.
Centrifuge at high speed and discard the supernatant.
Mark this tube containing precipitate as test.
Take another test tube and mark it blank.
To both tubes add 1 ml 15% NaOH. Shake the “test” tube to
dissolve them precipitate. Add 0.5 ml 5% Copper sulphate and 4 ml
distilled water.
Mix thoroughly and centrifuge at high speed.
Transfer the supernatant to corresponding clean tubes marked.
Read absorbance of the “test” against “blank” in a colorimeter at 550
nm.

35. CSF GLUCOSE DETERMINATION
• Normal CSF glucose is: 60 to 70 % of the patient's
serum glucose concentration.
It is generally decreased in bacterial meningitis, as well
as in meningitis associated with a fungus, tuberculosis
or syphilis.

36. Chemical analysis
(Contd..)
Decreased protein levels are less commonly
encountered.
Common reasons are removal of large amounts of
CSF and leakage of CSF caused by trauma.
Glucose concentrations can be determined using the
same methodologies as used for blood.
The most common finding is a low glucose
concentration associated with most types of CNS
infections.
If patient is hyperglycemic, then the CSF glucose
concentrations will like wise be elevated and an
infection may be masked.
If the patient is hypoglycemic the physician may be
misled by a low CSF glucose.

37. Chemical analysis (Contd..)
Lactate determination is considered to be of
some use by physicians in differentiating
bacterial, tuberculous and fungal meningitis
from viral meningitis.
The lactate concentration in viral meningitis is
generally normal or low in comparison to that
in bacterial, tuberculous, or fungal meningitis.

39. Serologic examination
Serologic examination of CSF is
typically involved with diagnosis of
neurosyphilis.
A fluorescent treponemalantibody
absorption (FTA-ABS) test for CSF has
also been developed.
The VENERAL DISEASE RESEARCH
LAB. (VDRL) Test is also used.
TPHA test or TPPA test for syphlus

40. Other techniques
It is important to recognize other techniques that have
been used and may still be used for CSF.
Immunoelectrophorsis (CIE), latex agglutination tests
for bacterial antigens, enzyme linked immunosorbent
assay (ELISA) and limulus of gram-negative bacterial).
The nucleic acid probes using PCR are the newest of
the techniques being investigated for indentification of
specific infectious agents.

41. Thank You