PATOLOGI KLINIK 3.

CEREBROSPINAL FLUID EXAMINATION

ASISTEN 2012

INTRODUCTION
Cerebrospinal fluid (CSF) is a bodily fluid that can be

found in the entire cerebral/spine cavity.

Cerebrospinal fluid has a total volume of around 90150 ml in adult that resides mostly in the subarachnoid
(around 125ml)
Cerebrospinal fluid is also found in the neonates as
much as 10-60 ml
Suprisingly, in every 5-7 hours total CSF volume is
replaced.

INTRODUCTION
CSF is formed in the ventricular choroid plexus by a

combined process of active secretion and

ultrafiltration from plasma.
CSF leaves the ventricular system through the
medial and lateral foramina, flowing over the brain
and spinal cord surface within subarachnoid space .
CSF resorption occurs at arachnoid vili.
CSF formation rate in adult is about 500ml/day or
20ml/hour

FUNCTION OF CSF
Provides physical support
Protective effect againts sudden change in
acute venous(respiratory and postural) and
arterial blood pressure impact pressure
Provides excretory waste function
Transported hypothalamus-releasing factor
to the cells of median eminence
Mantains central nervous system ionic
homeostasis

SPECIMEN COLLECTION
CSF may be obtained by :
Lumbar puncture
Cisterna puncture
Lateral cervical puncture
Ventricular cannulas/shunt
but the most common used is lumbar
puncture

Meningeal
infection

Demyelinating
disease
INDICATION FOR
LUMBAR PUNCTURE

Subarachnoid
hemorrhage

Primary or
metastatic
malignancy

Indication of Lumbar Puncture: 4 major disease category (American College of Physician,

1986)

The most important role of CSF examination is in diagnosis

of bacterial, fungal, mycobacterial, and amoebic
meningitis.

CONTRAINDICATION OF LUMBAR
PUNCTURE
Intracranial lesion : lumbar puncture will
higher the risk of transtentorial hernia
Local infection in lumbar area
Coagulopathy
Vertebral lesion

LUMBAR PUNCTURE
Lumbar puncture is usually done in L3-L4 or L4-

L5
Lumbar puncture is performed with patient in the
lateral recumbent position or sitting upright. The
lateral recumbent position is preferred because it
allows accurate measurement of the opening
pressure.
The patient is instructed to remain in the fetal
position with the neck, back, and limbs held in
flexion
Correct patient positioning is an important
determinant of success in obtaining CSF.

COMPLICATION
The most common complication is

headache. Hydration and use of a 20 or 25

gauge needle have been recommended as
preventive measures.
Rare complication is extradural,subdural, or
subarachnoid spinal hematoma with
paraplegia, in patient with bleeding
tendency due to thrombocytopenia or
anticoagulant.

CSF EXAMINATION
1. Gross Examination
2. Microscopic Examination
- Total Cell Count
- The Differential Count
3. Chemical Examination
- Globulin
- Glucose Test
- Protein

GROSS EXAMINATION
CSF appearance

Normal = crystal clear (appearance and viscosity comparable to water)

Cloudy or turbid CSF = may be caused by ;

- leukocyte (>200cells/microL)
- erythrocyte (>400 cells/microL)
- microorganism (bacteria, fungi,amoeba)
- aspiration of epidural fat during lumbar puncture
- contrast media/ radiographic contrast media

Viscous CSF
May be encountered in patient with metastatic mucin producing adenocarcinoma, cryptococcal
meningitis due to capsular polysaccharide, or liquid nucleus pulposus resulting from needle
injury to annulus fibrosus.

GROSS EXAMINATION

Clot formation
may occur due to an increase of fibrinogen ( associated with condition such as traumatic tap,
subarachnoid block, suppurative meningitis, and tuberculous meningitis

Pellicle -> very fine clots

may be detected by observing the surface of CSF after 12 to 24 hours refrigerated

Pink-red CSF
Indicates the presence of blood and is grossly bloody when RBC count exceeds 6000microL. It
may originate from subarachnoidal hemorrhage, intracerebral hemorrhage, or cerebral infarct, or
from traumatic spinal tap

Microscopic Examination
1. Total Cell Count
Procedure
.
Total cell count are performed on undiluted
CSF sample in manual counting chamber ( Fuch-Rosenthal chamber or Improved
Neubauer chamber) without staining
.
Total Cells are counted in 18 large square ( 1 mm3 each with depth 0.2 mm ) in FuchRosenthal chamber
.
or in nine 1mm2 squares of 0.1 depth in Improved Neubauer hemocytometer.

Normal range for leukocyte CSF

Adult
= 0-5 cells (lymphocyte and monocyte)/microL
Neonates = 0-30 mononuclear cells/ microL
.

The leukocyte count should be made as a routine test and must be made within the first
half hour after withdrawal of CSF sample.

No RBCs should be present in normal CSF. If numerous, pathologic process is probable such
as trauma, malignancy, infarct, hemorrhage.

2. The Differential Count

if total cell count is high (>500 cells/ cu.mm.) :
Count directly from spinal fluid

If total cell count is low :

Centrifuge the spinal fluid in an open bottom centrifuge tube with a rubber stopper.
Discard the supernatant fluid
Smear the cells on glass slide
Stain with aqueous solution or Wright or Methylene blue method

Interpretation
Adult
: 0-10 leukocytes per cubic milimeter (all small lymphocytes)
<1 year
: 30 lymphocytes
1-4 year
: 20 lymphocytes
5 year- puberty
: 10 leukocytes
In adult normal CSF contain small number of lymphocytes and monocytes in
approximate 70 : 30 ratio
Small number of neutrophils (PMNs) may also be seen in normal CSF

Pleocytosis (increase cell count) :

- Due to an irritative or inflammatory lesion of brain, spinal cord, or
meninges.
- More common in cerebral tumour than in chronic encephalitis
- Most of the cells have their origin in meninges
- Acute process will tend to have a predominance of segmented
granulocytes ( especially pyogenic infection)
- Chronic infection and non bacterial acute reactions (toxic) usually
have predominance lymphocytes.

Moderate increase in cells (10-100/cubic milimeter) with lymphocyte predominating,

is
found in :

Early tuberculous meningitis

All form of neurosyphillis

Anterior poliomyelitis

Encephalitis lethargica

Multiple sclerosis, etc.

Increase in cells to 1000/cubic milimeter with lymphocyte predominating, is found in :

Late tuberculous meningitis

Poliomyelitis

Acute syphillic meningitis

Great increase in cells (>1000/cubic milimeter) with segmented granulocytes

predominating
is found in :

Pyogenic meningitides

Influenza meningitidis

Brain abscess

When any infectious process gives rise toan aseptic inflammation of leptomeninges

CHEMICAL EXAMINATION
1. Globulin
A globulin test should be made on all body fluids. Any
amount of blood will be interfered the test
a) Pandys Test
To 1 ml of a saturated aqueous solution of phenol (5-10%)
in small test-tubes, add 1 drop of spinal fluid. A bluish
white ring is immediately formed if an excess of globulin
is present. This test is more sensitive than Ross-Jones Test
b) Ross-Jones Modification of Nonnecs Apelt Test
Overlay 1 ml of spinal fluis on 2 ml pf a saturated solution
of ammonium sulphate. A white or gray ring at point
contact indicates an excess of globulin.

2. Glucose Test (GOD-PAP Method)

Objective : To determine glucose concentration in CSF
Principle : Glucose concentration is determine after enzymatic
oxidation by glucose oxidase. The colorimetric indicator is
quinoneimine, which is generated from 4-aminoantipyrine
and phenol by hydrogen peroxidase under the catalytic
action of peroxidase( Trinders reaction). The reaction as
follow;

Glucose + O2

GOD( Glucose Oxidase)

Gluconic acid + H2O2

POD (Peroxidase)
2H2O2 + 4 aminoantipyrine + Phenol
Quinoneimine+ 4H2O

REAGENTS
Phosphate buffer Ph 7.5 250 mmol/L
Phenol 5 mmol/L
4-Aminoantipyrine
0.5 mmol/L
Glucose Oxidase (GOD)
10 kU/L
Peroxidase (POD) 1 kU/L
Standard :
100 mg/dl (5,55 mmol/L
STORAGE INSTRUCTION & REAGENTS STABILITY
The reagent is stable up to the end if the indicated expired date, if stored at 2-8oC
, protected from light and avoided from contamination. Do not freeze the reagent!
The standards is stable up to the end of the indicated expired date, if stored at 2-25oC
Note : It has to be mentioned, that the measurement is not influenced by occasionally
occuring color changes, as long as the absorbance of the reagent is <0.3 at 546
nm
WARNING AND PRECAUTION
1. The reagent contains sodium azide (0.95 g/L) as preservative. Do not swallow. Avoid
contact with skin and mucous membrane
2. Take the necessary precaution for the use of laboratory reagents.
ADDITION REAGENT
Trichloroacetic acid solution 300 mmol/L for deproteination, stable at +15p C up to
+25p C.

PROCEDURE
Sample or Standard
Distilled water
Reagent

Blank

Sample or Standard

10 microL
1000 microL

10 microL
1000 microL

Mix, incubate 20 minutes, at 20-25oC, Read the absorbance againts

the blank within 60
CALCULATION

Glucose ( mg/Dl) = absorbance Sample X Concentration

Standard/Cal (mg/dL)
absorbance std/cal
CONVERSION FACTOR
Glucose (mg/dL) x 0,05551 = Glucose (mmol/L)
NORMAL RANGE
Fasting
35-50 mg/dL

INTERPRETATION
Decrease
Bacterial, tuberculous, and fungal meningitis
Meningeal involvement by malignant tumor
Sarcoidosis, cysticercosis, trichinosis, ameba
Acute syphillitic meningitis
Intrathecal administration of radioionated serum albumin
Subarachnoid hemorrhage
Symptoatic hypoglycemia
Rheumatoid meningitis
Increase
No clinical significance
Traumatic tap may also cause a spurious increase in CSF glucose

3. Protein
Principle
Protein forms a purple coloured complex with cupric ions in alkaline solution. The
reaction takes its name from the simple compound biuret which reacts in the same
way.
The intensity of the purple colour is measured at 540 nm/ yellow green filter and
compared with a standard of known protein concentration .
Test Principle :
The principle of this total protein assay is by biuret reaction.In alkaline solution,
cupric
ions react with all compounds with two amide or peptide bonds linked either
directly or
through an intermediate carbon atom to form a violet colored complex. The
intensity of
this colored complex is directly proportional to the protein concentration in the
sample.
Reagent 1
Reagent 2
Sodium Hydroxide
Sodium Hydroxide
Potassium sodium Tartrate
Potassium Sodium Tartrate
Potassium Iodide
Copper Sulphate

Procedure
Pipette into
test tube

Blank

Std/Cal

Sample

Sample, Std/Cal

20

20

Dist Water

20

Reagent

1000

1000

1000

Mix, incubate for 5 min, at 20-25/77 OC

Read absorbance againts reagent blank within 60 min

INTERPRETATION
Referrence range
Adult : 15-45 mg/dL
Elevated
Increase permeability of the blood brain barrier
Decreased resorption af arachnoid villi
Mechanical obstruction of CSF due to spinal block above the
puncture site
Increase intrathecal immunoglobulin (Ig) synthesize
Low
Young children between 6 month-2 years of age
Condition associated with increase CSF turn over :
Removal of large CSF volume
CSF leaks induce by trauma or lumbar puncture
Increased intracranial pressure
Hyperthyroidism

CEREBROSPINAL FLUID FINDINGS IN

MENINGITIS

CEREBROSPINAL FLUID
FINDINGS
Brain Tumor
Appearance : Clear and colorless, or xantochromia (pale pink to
yellow color in supernatant of centrifugated CSF)
Spinal fluid present : may be elevated
Protein level : may be increased
Glucose level : normal
Cell Count : Normal or slightly elevated lymphocyte
Subdural Hematoma
CSF findings vary depending on whether the hematoma is recent
or several days old. In recent subdural (within 7 days of injury),
CSF is usually bloody or xanthochromic. In late cases CSF is clear.
The classic CSF triad : elevated protein level, xanthochromic,
relatively normal cell count present in 50% patient