CEREBROSPINAL FLUID

ANALYSIS
I Nyoman Wande
Clinical Pathology Department

Medical Facultyof Udayana University

05/25/16

Overview:
Production of CSF
Indications of LP
Contraindications of LP
Routine of CSF analysis
CSF analysis in several disease

05/25/16

PRODUCE OF CSF
Produced at
choroid plexus of ventricel walls (70%) ,
ependymal cells of ventricel &
cerebral subarachnoid

500 mL of CSF is formed/day, although

only 90-150 ml is present in the system at any

time.
Reabsorption of CSF occurs at the arachnoid
villi
05/25/16

05/25/16

COMPOSITION OF CSF
Protein (<<) : 15-45 mg/dL
Glucose (<) : 60-80% of blood glucose
K+, Ca++, Phosphat, Sulphat (<)
Na+, Cl-, :10-15% higher than blood

by active secretion of choroid

plexus
05/25/16

FUNCTION OF CSF
MECHANICAL FUNCTION
Shock absorben prevent to shaky
Lubbricant, between brain-skull; medullaspinalis- columna
vertebrae

CONTROL TO VOLUME (PRODUCTION)

As the filler of space due to atrophic region
or decrease volume production if space occupying lession
happened

TRANSPORT FUNCTION
To clean/wash the brain from metabolite & toxic agent and
than excreted
Transport food to cells of CSF

Tabel. Diseases detected by laboratory examination

of CSF
Sensitivity/specificity

Diseases

High sensitivity, high

specificity

Bacterial, tuberculous, and

fungal meningitis

High sensitivity, moderate Viral meningitis, subarachnoid

specificity
hemorrhage, multiple sclerosis,
CNS syphilis, infectious
polyneuritis, paraspinal abcess
Moderate sensitivity, high
specifisity

Meningeal malignancy

Moderate sensitivity,
moderate specificity

Intracranial hemorrhage, viral

encephalitis, subdural
hematoma

05/25/16

INDICATION FOR LUMBAL

PUNCTURE

Diagnosis of CNS infection

Diagnosis of subarachnoid hemorrhage
Diagnosis of meningeal malignancy
Diagnosis of demyelinating disease
Diagnosis of CNS vasculitis
Intrathecal treatment of meningeal malignancy
Intrathecal treatment of fungal meningitis

ABSOLUTE CONTAINDICATION OF
LUMBAR PUNCTURE
Increased intracranial pressure
Skin infection in the lumbal region

Specimen Collection
Lumbar puncture (LP) is the insertion of a needle into

the subarachnoid space (the area under the membrane

that surrounds the brain and spinal cord) of the lumbar
(lower back) region for diagnostic or therapeutic purposes.
This allows access to the cerebrospinal fluid (CSF) in
which the brain and spinal cord float.
Although the subarachnoid space can be accessed from
other levels, the lumbar region is most often used as it
allows the needle to be inserted below the end of the
spinal cord.

05/25/16

11

05/25/16

05/25/16

13

Position & site of LP

SEATED POSITION

LYING POSITION

CSF STORAGE
CSF should be analysed immediately (less than 1 h)
after collection.
If storage is required for later investigation this can be
done at 48C (short term) or at 20C (long term).
Only protein components and RNA (after appropriate
preparation) can be analysed from stored CSF (Good
Practice Point).

05/25/16

15

CSF ANALYSIS
Macroscopic
Microscopic
Microbiology
Chemistries
Serology
PCR
05/25/16

16

Recommended Laboratory Test

on CSF

Routine

Opening CSF Pressure

Total cell count
Differential cell count (stained smear)
Glucose (CSF/plasma ratio)
Protein

Useful in certain circumstances

Cultures (bacteria, fungi, viruses, Mycobacterium tuberculosis)
Stains (Grams stain, acid-fast stain)
Fungal and bacterial antigens
Polymerase chain reaction (TB, viruses)
Cytology
Protein electrophoresis
VDRL test for syphilis
Fibrin-derivative D-dimer

CSF Macroscopic
1. Turbidity / cloudy
2. pH
3. Specific Gravity (SG)
4. Color
5. Sediment
6. Pellicle
7. Pressure
05/25/16

18

CSF color and appearance

Normal CSF is crystal clear, with the
appearance and viscosity of water.
Abnormal CSF may appear hazy, cloudy,
smoky, or bloody
Clotting of CSF is abnormal and indicates
increased protein or fibrinogen levels

05/25/16

19

TURBIDITY / CLOUDY :
Opalescent
Turbid
1. Hemorrhage
2. Present of inflamation cells, bacteria, or fungi
Cells (400-600/cmm pleocytosis)
4. Pleocytosis in clear CSF
Meningitis TBC
Tabes dorsalis
Poliomyelitis
Meningitis luetica

pH : normal : alkalis
SG : 1.003 1.008
COLOR : clear & colorless
1.

Xanthochromia:

2.

subarachnoid hemorrhage,
traumatic LP,
delayed processing of sample,
jaundice (especially bilirubin > 4 mg/dL),
protein > 150 mg/dL,
presence of carotene pigment
Red : artificial bleeding, subarachnoid bleeding

3.

Brown : long time bleeding + hemolysis

4.

Gray : leukocyte (purulent inflamation)

5.

Green

: meningitis meningococcen

PELLICEL
Normally : formed if CSF allow 24 hours at
room temperature
Meningitis
purulenta
Pellicel fast formed
minute - 1 hour, big
form, coarsely

Meningitis TBC
Pellicel formed after
12-24 hours, form like
spider nest

CSF Microscopic
Examination of cells: Total cell count,
differential cell count

05/25/16

23

CELL COUNT
1. Fuchs-Rosenthal chamber (4X4X0,2cmm)
2. Improved Neubauer chamber (3X3X0,1cmm)
3. Dilution: 10/9X (turk reagent)
4. Formula
Fuchs-Rosenthal
Improved Neubauer

= 1/3N cell/cmm
= 5/4N cell/cmm

5. Normally = 0-5/cmm
Child < 5 y.o: N < 20/cmm)
05/25/16

24

DIFFERENTIAL CELLS COUNT

1. Differentiated to PMN & MN
PMN > MN : purulent, acute
MN > PMN : serous, chronis / reconvalesence

2. Diff. Count do in smear of CSF, with

Wright staining / methylene blue
3. Count of 100 cells,
4. Result report in % of
PMN = %,
MN = %)
05/25/16

25

MiCROBIOLOGY EXAMINATION
1.
2.
3.

05/25/16

Direct smear
Culture & Sensitivity test
Viral isolation

26

05/25/16

27

CHEMISTRIES ANALYSIS
1. Protein Qualitative
None Apelt (Rose-Jones)
Pandy

2. Protein Quantitative
SSA method
Biuret method

3. Glucosa
GOD-PAP
Hexokinase

05/25/16

28

CHEMICAL ANALYSIS
qualitative protein
None Apelt (Rose-Jones)
To know elevation of globulin concentration
Principle of the test :
globulin sedimented in saturated (NH4)2SO4 solution
In to reaction tube 7mm + 1ml saturated (NH4)2SO4 sol.
And than add 0,5 ml CSF
Allow for 3 minute
+ WHITE RING appearing at the juncture of the liquid

05/25/16

29

INTERPRETTION OF NONE APELT TEST

- : No appearing of WR
+ : WR slightly, mixing WR less
+2 : WR clear, mixing opalescent
+3 : WR more clear, mixing turbid
+4 : WR very clear, mixing very turbid

05/25/16

30

CHEMICAL ANALYSIS
qualitative protein
PANDY TEST
To know elevation of globulin and albumin
concentration
Principle of the test:
protein sedimented in saturated phenol
solution in water
To a watch glass + 1 ml Pandy reagen,
Add 1 drop of CSF
cloudiness againts a black bacground indicates
05/25/16 increased amount of globulin

31

INTERPRETATION OF PANDY TEST

- : clear & colorless
+ : Opalescent ~ 50-100 mg% of protein
+2 : Cloudy ~ 100-300 mg% of protein
+3 : Very cloudy ~ 300-500 mg% of protein
+4 : Cloudy like milk and formed
sedimentation ~ > 500 mg% of protein

05/25/16

32

CSF SEROLOGY
Serology test for sifilis (VDRL)
CSF immunoglobulins
Specific antibodies (measles, rubella, HSV, HIV,CMV,
Toxoplasma)

05/25/16

33

Average and Range of

Cerebrospinal Fluid Protein

05/25/16

34

CSF Parameters in some

Neurological Disease

05/25/16

35

Typical Cerebrospinal Fluid Finding

in Various Types of Meningitis

05/25/16

36

Conclusion
CSF analysis High sens and high spec
for the diagnosis meningitis: bacterial,
tuberculous, and fungal.
Routine CSF analysis:
Opening CSF Pressure
cell count
Differential cell count (stained smear)
Glucose (CSF/plasma ratio)
Protein
05/25/16

37

Learning Task

Trigger scenario:

A boy 4 years old came to emergency department with chief

complained seizure dan fever. History of previous illness, she was
suffering from productive cough and rhinitis since six days a go.

Learning task:

Mention the kind of CSF test should be done!

Described the location of CSF puncture in this patient!

Mention and described the macroscopic examination of CSF should be

done?

Described of qualitative protein analysis of CSF.

What are the CSF findings in bacterial meningitis?

Mention the differential diagnosis of this patient!

Self assessment:
1. How is CSF product ?
2. What is the indication and contraindication of lumbar
puncture ?
3. How to interprete the result of each test ?
4. How to differentiate the red colour of CSF due to the
artificial bleeding and the subarachnoidal bleeding ?
5. Procedure collection of CSF?

05/25/16

39

05/25/16

40