Fk Unswagati Cerebro Spinal Fluid Analysis

7/30/2019 Fk Unswagati Cerebro Spinal Fluid Analysis

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CEREBRO SPINAL FLUID

ANALYSISDEPARTMENT OF CLINICAL PATHOLOGY

MEDICAL SCHOOL- UNIVERSITAS PADJADJARAN/

RUMAH SAKIT DR. HASAN SADIKIN

BANDUNG


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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 andlubricates the central nervous system (CNS)


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Cerebrospinal fluid (CSF)

INCREASED AMOUNT :

acute and chronic congestion of the

meningens due to increasedtransudation; acute and chronic

infections

PRESSURE : 100-150 mm of water


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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


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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


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(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


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(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


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(PRE-ANALYTICAL STEPS)

The specimen should be divided into

three sterile tubes, labeled 1,2, and 3:

1. chemistry and immunological exami-

nation

2. microbiological examination

3. cell count and differential count


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(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 forseveral hours in the refrigerator


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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


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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 superna-

tant should be clear


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Microscopic examination:

LEUKOCYTE CELL COUNT

DIFFERENTIAL COUNTINGInterpretation:

Normal only mononuclear (MN) cells

(lymphocytes or an occasional monocyte)

are found


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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 glucoseexamination


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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


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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 theCSF


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NONNE-APELT TEST..

Positive result : a clear-cut, grayish-white 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 :


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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


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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


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NONNE-APELT TEST

++ = a flocculate precipitate in a slightly

cloudy fluid

+++ = a heavy flocculate precipitate in aclear fluid


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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


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CSF EXAMINATION :

GLUCOSE

DEPARTMENT OF CLINICAL PATHOLOGY



BANDUNG


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CSF GLUCOSE

Must be performed within 1.5 hrs after with-

drawl

Blood glucose: must be performed in thesame time

If possible, the blood and CSF are drawn

before breakfast

Normally : CSF glucose 60% blood glucose

level


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CSF GLUCOSE

Method : chemical & enzymatic

Chemicalmethods are no longer used,

because of lack of specificity, except ortho-

toluidine method

ENZYMATIC method :

Glucose oxidase (less specific than

hexokinase) Hexokinase (generally accepted reference

method)


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CSF GLUCOSE

GLUCOSE OXIDASE-PAP :

glucoseH2O

-D-glucose + O2 gluconolactone

oxidase O2peroxidase

gluconic acid + H2O2 H2O + OnOn + phenylamine-phenazone colorchanges

+ H2

O Measured by photometer in specific wavelength


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CSF GLUCOSE

HEXOKINASE :

hexokinase

Glucose + ATP glucose 6-phosphate + ADP

Mg++

G6PD

Glucose 6-phosphate + NADP 6 phosphoglucono-

lactone + NADPH + H+

More expensive, but better in specificity and precision


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CSF EXAMINATION :TOTAL PROTEIN

DEPARTMENT OF CLINICAL PATHOLOGY



BANDUNG


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PRINCIPLE OF THE TEST :

Protein in the presence of copper ions form

a violet blue color complex in alkaline solu-

tion (Biuret method)

The reagents used are same with reagents in

serum/plasma protein measurement


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REAGENTS :

R1 : Sodium hydroxide

Potassium sodium tartrate

R2 : Sodium hydroxidePotassium sodium tartrate

Potassium iodide

Copper sulphate


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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 programmedfirst, then get the fixed FACTOR to calculate

the amount of protein


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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


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Laboratory Activity

PATOLOGI KLINIK


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CEREBROSPINAL FLUID

Lumbar puncture between 3rd, 4th & 5th

lumbar vertebra

No special preparation

Freshly collected (30 minutes)

Chemistry

& serologymicrobiology

Hematology

& microbiology

1 2 3


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MACROSCOPIC

Color

Turbidity

ReactionSpecific Gravity

Coagulation

CELL COUNTING

RBC

WBC

ROUTINE ANALYSIS

CHEMISTRY

Glucose

Enzim (LDH, CK)

Protein

Qualitatitive: Nonne

& Pandy test


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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


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PANDY TEST

Add 1cc of phenol

solution to a tubeAdd 1 large drop of

spinal fluid

Normal:

Negative

Positive reaction:

bluish-white cloud

forming immediately

around the drop.

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Fk Unswagati Cerebro Spinal Fluid Analysis