CEREBRO SPINAL FLUID ANALYSIS

DEPARTMENT OF CLINICAL PATHOLOGY MEDICAL SCHOOL- UNIVERSITAS PADJADJARAN/ RUMAH SAKIT DR. HASAN SADIKIN BANDUNG

Cerebrospinal fluid (CSF)

Produced 500 mL/ day Produced by ultra filtration from blood plasma and secreted by cells of the choroids plexus water, circulates nutrients, and cushions and lubricates the central nervous system (CNS)

Cerebrospinal fluid (CSF) INCREASED AMOUNT : acute and chronic congestion of the meningens due to increased transudation; acute and chronic infections PRESSURE : 100-150 mm of water

Sample collection of CSF:

(PRE-ANALYTICAL STEPS)
CSF is collected by lumbar puncture (between VL3-4 or VL4-5) It is done only by definite indications:

1. DIAGNOSTIC or
2. THERAPEUTIC

Sample collection of CSF: (PRE-ANALYTICAL STEPS)

DIAGNOSTIC : 1.To study the CSF 2.To estimate intracranial pressure 3.To test the spinal block 4.To introduce air or lipoidal substance

Sample collection of CSF: (PRE-ANALYTICAL STEPS)..

THERAPEUTIC:

1. To introduce penicillin, streptomycin, or an anesthetic 2. To remove blood or irritative substance

CONTRAINDICATIONS :
1. Subtentorial tumors 2. Greatly increased intracranial pressure

Sample collection of CSF:

(PRE-ANALYTICAL STEPS).

Freshly collected (within 30 minutes)

The amount of CSF to be collected : 8-10 ml The first drop may contain blood from the puncture and should not be used

Sample collection of CSF: (PRE-ANALYTICAL STEPS)

The specimen should be divided into three sterile tubes, labeled 1,2, and 3: 1. chemistry and immunological examination 2. microbiological examination

3. cell count and differential count

Sample collection of CSF: (PRE-ANALYTICAL STEPS)..

When xanthochromic CSF is obtained, add a trace of Lithium oxalate to prevent clotting The cell count, bacteria and glucose examinations must be done at once, whilst the others can be delayed for several hours in the refrigerator

ROUTINE ANALYSIS: (ANALYTICAL STEPS)

Macroscopic examination:
Color, Normal: clear and colorless Turbidity, Normal: clear Reaction, Normal : alkaline Specific Gravity, Normal : 1.003-1.008 Coagulation, Normal: does not coagulate

Macroscopic examination:..

If the blood in the specimen is due to a traumatic puncture, the CSF in the third tube should be clearer than those in tube 1 or 2, after centrifugation, the supernatant should be clear

Microscopic examination:
LEUKOCYTE CELL COUNT DIFFERENTIAL COUNTING Interpretation: Normal only mononuclear (MN) cells (lymphocytes or an occasional monocyte) are found

CHEMICAL EXAMINATION :

PROTEIN : Nonne test, Pandy`s test, and Total protein. The main interest protein is globulin Interfered by blood in CSF CSF GLUCOSE :should be done at the same time with serum glucose examination

NONNE APELT TEST

Detection Globulin and albumin :

Is due to the blood interfering (Falsely high)

If the CSF is cloudy centrifuged the clear supernatant can be used for the test

NONNE-APELT TEST.

GLOBULIN

1cc CSF + 1cc ammonium sulfate (saturated)

Procedure : place the tip of pipette containing amm.sulfate to the bottom of the test tube which already contains with CSF. Let the amm.sulfate solution layers underneath the CSF

NONNE-APELT TEST..

Positive result : a clear-cut, grayishwhite ring appears at the contact zone between two fluids Observe for 3 minutes. If a ring is formed, then shake the tube to mix the fluids. Result should be report as follows :

NONNE-APELT TEST.

+ = a ring appears within 3 min, only visible against dark background, leaving no trace on mixing

++ = a faint, opalescence after mixing +++ = a definite cloud after mixing

++++ = a heavy cloud after mixing

NONNE-APELT TEST

ALBUMIN Shake the tube used in globulin test Add 1 drop of 10% acetic acid, boil Report as follows: Negative = a slight cloudiness + = a definite cloudiness, with a fine precipitate

NONNE-APELT TEST

++ = a flocculate precipitate in a slightly cloudy fluid +++ = a heavy flocculate precipitate in a clear fluid

PANDYS TEST 1cc phenol (saturated aqueous solution) + 1 large drop of CSF Normal CSF may show a faint trace, and should be report as NEGATIVE
POSITIVE : a bluish-white cloud forming immediately around the drop of CSF :abnormal amount of globulin

CSF EXAMINATION : GLUCOSE

DEPARTMENT OF CLINICAL PATHOLOGY MEDICAL SCHOOL- UNIVERSITAS PADJADJARAN/ RUMAH SAKIT DR. HASAN SADIKIN BANDUNG

CSF GLUCOSE
Must be performed within 1.5 hrs after withdrawl Blood glucose: must be performed in the same time If possible, the blood and CSF are drawn before breakfast Normally : CSF glucose 60% blood glucose level

CSF GLUCOSE Method : chemical & enzymatic Chemical methods are no longer used, because of lack of specificity, except orthotoluidine method

ENZYMATIC method : Glucose oxidase (less specific than hexokinase) Hexokinase (generally accepted reference method)

CSF GLUCOSE
GLUCOSE OXIDASE-PAP :
glucose H2O

-D-glucose + O2

gluconolactone
oxidase peroxidase O2

gluconic acid + H2O2 H 2O + O n On + phenylamine-phenazone color

changes

+ H 2O

Measured by photometer in specific wavelength

CSF GLUCOSE
HEXOKINASE :
hexokinase

Glucose + ATP
Mg++

glucose 6-phosphate + ADP

G6PD

Glucose 6-phosphate + NADP lactone + NADPH + H+

6 phosphoglucono-

More expensive, but better in specificity and precision

CSF EXAMINATION : TOTAL PROTEIN

DEPARTMENT OF CLINICAL PATHOLOGY MEDICAL SCHOOL- UNIVERSITAS PADJADJARAN/ RUMAH SAKIT DR. HASAN SADIKIN BANDUNG

PRINCIPLE OF THE TEST :

Protein in the presence of copper ions form a violet blue color complex in alkaline solution (Biuret method) The reagents used are same with reagents in serum/plasma protein measurement

REAGENTS :

R1 : Sodium hydroxide Potassium sodium tartrate R2 : Sodium hydroxide Potassium sodium tartrate Potassium iodide Copper sulphate

The convention procedure, from serum/plasma to CSF

The crucial things to do are : 1. The standard solution must be diluted 100x 2. The amount of reagent and sample : inversion from serum/plasma measurement (i.e. 20 L reagent + 1000 L CSF) The photometer should be programmed first, then get the fixed FACTOR to calculate the amount of protein

INTERFERING FACTORS :

Ascorbic acid > 30 mg/dl Bilirubin > 40 mg/dl Hemoglobin> 500 mg/dl Triglyceride > 1000 mg/dl IV polydextrans : falsely too high levels

Laboratory Activity

PATOLOGI KLINIK

CEREBROSPINAL FLUID
Lumbar puncture between 3rd, 4th & 5th lumbar vertebra No special preparation Freshly collected (30 minutes)

Chemistry & serology

microbiology

Hematology & microbiology

ROUTINE ANALYSIS
MACROSCOPIC Color Turbidity Reaction Specific Gravity Coagulation CELL COUNTING CHEMISTRY

Glucose
RBC WBC Enzim (LDH, CK) Protein Qualitatitive: Nonne & Pandy test

NONNE TEST

Pipette 1 cc of spinal fluid into a small test tube

Gently add 1 cc of ammonium sulfate solution, to make 2 layers of solution

Positive reaction: a clear cut, thin, grayish-white ring appears at the zone of contact of the two fluids within a few second. Normal : negative

PANDY TEST

Add 1cc of phenol solution to a tube

Add 1 large drop of spinal fluid

Normal: Negative

Positive reaction: bluish-white cloud forming immediately around the drop.