CHAPTER VI

CEREBROSPINAL BLUD EXAMINATION

shiefly dinlyeate from the blood (a plaona thra filtrate), and is

relatively sinnilar to serum but differs in its concentration of the
major constituents,

The major constituents are:

The protein . which is extremely low with no fihrinoeon
The glucose which is
approximately two thirds that of
the blood augar.
The chloride sistinea which is
about 25% higher than the plasma
chloride,
Another difference of cerebrospival fluid from plasma is that
many of the crystalloids in the CSF are of different concentrations.
For
example sodium and chloride owof high levels while potassium,
calcium, bicarbonate, phosphate, sulphate and glucose are of lower
levels.

THE PHYSIOLOGY AND SOURCES or CEREBROSPINAL

FLUID:

Theecrebrospinal fluid is formed in the highly vascular

choroid plexuses (tufts of capullary blood vessels) in the ventricles
of the brain by filtration (secretion and diffusion) from the blood
plasma. A small portion is secreted by the ependvinal colls lining
the ventriele. This fluid then enters the subarachnoid space through
the foramina of Luselika and Magendie, circulating upward over
the cerebral hemispheres as well as downward over the spinal cord,
and entering venous blon (blood stream) throuh arachnoid villi
of the dural sinuses.

LOCATION OF THE FLUID

1. Internally it fills the ventricles of the brain (ventricular

fluid), the cisternae, (cisternal fluid) and the canal of the
spinal cord.
2. Externally it fills the space between the pia and arachnoid
membranes surrounding the brain and spinal cord (spinal
fluid).

221
 PeRicAROiaLFLUid

Normal Vol: 10-2C m

1
Pericardrd) efuaor: presme

cadh

yehem

e Maumasid do

*SLE

* DeA
FUNCTIONS 0F THE FLUID
1.
protection of the brain from injury by acting a8
fluid buffer
(cushion ).
It acts as & mediumm of exchange for the transter of dis
lyzable material
cord
the blood nream and the spioal

33. 11 equalizes the pressure between the brain and the

cord.

1.
It nerves as an exeretory channel in the elinination of
products of nervous motabollem.

COLLECTION OF THE FLUID

The spinal fluid in collected

by it physician by making 01 spinal
puncture. Aside from all clean and steriled equipment and 01*
terialsfor use, precautions are also be taken: nuch na avoidance
of blood in the collection, addting anticoagulant if noomcarr. and
immediate performance of the tests to he done.
TYPES OF PUNCTURE:

1 Lumbar puncture
It is relatively safe and simple procedure, but it should ant

be done unless there are definite indicationa. Puncture in

between the third and the fourth vertebrae of the Imbar
region.
Indications:
A) Diagnosis

1) To obtain spinal fluid for study

2) To estimato intracranial pressuro
3) To test for spinal block
4) To introduce air or a lipoidal substance.

B) Therapeutic
1) To introduce serum, penicillin, streptomycin, Of onenthe.

tic substance.
2) To remove blood or irritative exudates.

0909
Contraindications:

B) Presence of
generally increased Intracranial
2. Cisternal
puncture, (cistera magna
This is
somewhat more dangervus than a lumbar
ture and is usually
done ouly under the following conditionss
a)Blocked spinal canal
b) Deformity of the vertebrae
c) Infections of the tissues of the back
3. Ventricular puncture
This is done
in infants for last resort, although frequeutly done
whose fontanelles are still opon, bit rarely su
adults except in connection with ventriculosraphe.
AMOUNT TO BE COLLECTED:

1. At lease 8 to 10 ce. of fluid io necewsary for # complete

examination
2. It should be collected
in 3 sterile, chemically clean test tubve,
numbered 1, 2, and 3.
3. The first drops are place in tube bas and may contain some
blood from the puncture.
This fluid should not be used, unless it is necessary for
bacteriological examination (smear and culture).
b) Presence of blood effects all tests except for chlorides
4. Collect 7 cc. in tube 2 for serological, bacteriological, and
chemical tests (Hematologie-differential count and bie
chemical tests)
5. Collect about 2 cp. in tube 3 for cell count and qualitative
protein tests (colloidal gold test).
6. When the fluid obtained in xanthoobromic (canary yellow),
it is advisable to add # trace of lithium oxalate lo tulies 2
and 3 to prevent clotting.

7. The cell count and examination for bocteria and sugar musl
be dowe at once, while the remaining tosts can be delayed
several hours if the specimen is kept in the refrigerator.
 Venous blood should be drawn at the same time as the spinal
fluid if chemical tests are to be dome
especially for chloride
and sugar,

COMPOSITION OF NORMAL SPINAL FLUID:

1. Glucose
45-100 mg./100 ml.
2. Urea
8-28 mg,/100 ml.
8. Sodium 117.137 mEg/L
4. Potassium 2.33-4.59 mEg/L
5. Phosphorus, luorganic 1.2-2.1 mg./100 ml.

0.07-2.8 mg./100 ml.

7, Chlorides 113-127 mEg/L

(710-390 mg./100 ml.)

8. Acid-base balance
a) PC02
b) HC03 22.9 mEg/L

15.45 mg./100 mi.

9. Total protein
a) Lumbar 20-40 mg /100 ml.

b) Cisternal 15-25 mg./100 mil

e Ventricular 5-10 mng./100 ml.

10. Fibrinogen

11. Electrophoretic separation of lumbar

fluid mean walnes:

a) Prealbumin 46 = 13%
49.5 ‡ 65%

globulin 6.7 + 2.09

c) Alpha
globulin 8.3 + 2.1%
d Alpha 2
e) Beta and tetraglubin 18.5 % 4.8%

Gamma globulin 112 + 2.7%

12. Calcium Clumbar) 2.32 mEq./L

13. Magnesium
2.20 mEa./L

224
 14.
15. Creatinine
16. Glutamic
17.
Lactic oxalacetic

Cells
dehydrogenase
18. Phosphohexose transminase 04-1.5
mg./100 ml.
isomerase
MACROSCOPICOR 042
0.19 units
8.50
1.
PHYSICAL Bodansky units
Amount.
a) The EXAMINATIO
per normal
amount is
pound of
about
b) 100 to body roughly
There is 150 cc.weight. estimated
Also
of
the increased everyday to
roughly
aboutbe
throughmeninges amount
due to inacuteproduction estimated 1
CC.

meabilitythe
to he

capillaries
of the increased and
chronie
There is
and
also anchoroid plexus. transudation
probably congestion
infections
and due increased
to of
plasma
increased
to
increase the production
amount in
2. to
Pressure: permeability of the
of acute
and
a
Normal inflammatory chronie
70 and pressure
capillaries. exudate
200 mm.for the
average of 100 of waterhorizontalposition
3.
Color: to 150 (0-8 mm. varies between
mm• of
a)
mercury)withthe
Normally
water,
the fluid
is
as clear and
b)
Bright red due colorless Ds distilled
while inserting to fresh blood
from s vessel
natant fluid is the needle; upon punctured
c) Dull red
clear. centrifugation the super.
or brown
skull or in some { depending on the
intracranial age
of the lesion)
and chronie

bogins, so that a hemorrhatc After 48 hours

of at
least this
after the yellow
centrifugation. or red color of the lip ne ad
is recog.
fluid

d) Yellow (xanthochromio) may be

due to
resulting from disintegration of RBC blood pigments
within the suba.
rachnoid space or to altered permeability of the
which under normal conditions are excluded.
blood plasma

225
 inflammatory
0) Greenish or grayish due to pus cells in severe

reactions and in acute meningitis.

4. Transparency:
Normal spinal fluid 13 clear. Fewer than 200 white cells/cu.
of the fluid
mm. does not give rise to macroscopic clouding
Haziness is produced by 200-500 white cells/cu. mm, and
over 500 white cells/cu. mm, cause turbidity.
In acute meningitis, the fluid may exhibit varying degrees
of cloudiness, from slight turbidity to the capacity of
pure. pus.
b) In the loss acute stage of epidemic meningitis, it is sOmE
times quite clear.
It is usually clear in tuberculosis and syphilitic meningitis,
tubes, paliomyelitis and encephalitis.
5. Coagulation:
a) Normal spinal fluid does not coagulate.
b) Abnormal:
1 The fluid clots when there is an increase in proteina
including fibrinogen
2) Numerous small clots (coagulate) occur in paresis.
3) A "cobweh" or pine tree or "weblike" clot dedicate
coagulum is typical of tuberculous meningitis, it forms
on the surface of the fluid and extends down the middle
of the tube.

a) Twelve or more hours may he required for its for.

mation.

b) The absence of pellicle does not exclude tuberculous

meningitis.
c) In purulent meningitis, large clot are seen.
4) Heavy coagulation and sediment occur in acute sup-
purative meningitis.
5) Complete coagulation with xanthochromia without
hemorrhage occurs in Froin's syndrome (spinal suba-
rachnoid block}

Test for Fibrinogen (Screening)

a) Add 1/3 volume 10% NaOH to a few ec. of spinal fluid
and shake.

296
 b) Observe fine fibrin flocculation that ultimately condenves
on the surface.

Sediment:
Normally mo sediment. Often present in meningitis.
Reaction.
The normal reaction is slightly alkaline pH 7.30- 7.45.
Specific Gravity:
The normal specifie gravity is 1.006-1.008.

Queckenstedt Test
The test is to confirm the presence of any subarachnoid block.
Normally, if both jugular veins are manually compressed CSF
pressure riges rapidly to over 300 mm. CSF rapidly returns to
normal when compression ceases. In sinus thrombosis. subarachnoid
block at the foramen magnum, or a mass lesion at the spinal
canal, the rise of CSF may be decreased or delayed. For positive
result delayed or decreased rise of CSF pressure.

CHEMICAL EXAMINATION

I. PROTEINS GLOBULIN (QUALITATIVE TESTS):

General Considerations:

a) The protein of chief interest is globulin.

b) The test for globulin is value less when applied to fluid
containing blood, owing to the presence of serum globulins.
If the fluid is cloudy, it should be centrifuged and the
clear supernatant fluid used for the test.
d) Globulin is increased in
meningitis and latent syphilis.
1. Nonne - Apelt Test for Globulin:
Principle:
A
50% saturated solution of ammonium sulfate precipitates
globulin.

Reagent:
Merck' Purified neutral ammonium

85 gma.

227
 Distilled Water
Boil and filter as BOOD 100 cc.
as at 1s cooled

Procedure:

ml. of
in
eaturated solution of ammonium auliate
a test tube.

b) Add 1 ml.
of spinal fluid and mix
c)
by inverting the tube.
Allow the tube to stand for three minutes.
d) Fluid that contains a
normal amount of globulin will remain
clear or become slightly opalescent. if an excess of globulin
1s present, a
cloudy precipitate will form,
2. Rogs-Jones Test

Principle:
Same as Nonne-Apelt.

Procedure:
a) Place 2 Cc. of ammonium sulfate to the bottom of the tube
b) Add 1 Cc. of
spinal fluid using medicine dropper (overlay).
c) A. clear-cut, thin grayish-white rings appears at the zone
of contract for positive reaction. Under normal condition,
the ring will appear after 5 minutes,
d) Observe for 3 minutes then mix the solution.

e) Interpretations.
+ ring in 3 minutes and no trace after mixing.

ring in 3 minutes and faint opalescent after mixing.

ring in 3
minutes and definite cloud after mixing.
++++ - ring in 3 minutes and heavy cloud after
mixing,

For Albumin Test:

a Shake the content of the tube used in the globulin test

and filter.

b) Acidify with 1 drop of 10% acetic acid and boil.

c) A slight cloudiness is normal.

228
 Principle:
The reagent precipitates albumin und globulin.
Reagent:
Phenol crystals
Distilled water

Procedure

Place e. of saturated aqueous solution of phesol in

small test inhe.
b) Add 1 Barge drop of spinal fuid.
o) A bloih white eland immediately Torm 14 BOon
the drop nises with the reagent in increased globulin.
d Normal fluids may show * faint trace but this should
he reported on negative

Noguchi's Teet Qualitative Detection of Proteins):

Place 0.2 mi. of spinal fmid into a teat tuhe.
b) Add 1 ml. of a 10% solution of butyric acid in normal
salt solution.
e) Heat to boiling.
d) Add 02 ml. normal sodiuna hydroxide.
Allow to stand for about 10 minutes
Precipitation ie positive reaction. however in believe cants

precipitates are delayed for an hour or more Faint

opalestence is normal

Total Proteins Quantitative Test

The protein 1* precipitated from the spinal fluid with sed

tumautate, dimalved and presipitated. The nitrogen lo detenained
 portion after being put into solution with the aid
of sodium hydroxide.

Coveral Considerations:
a Presence of blood gives false high values.
b) Presence of bacteria gives unreliable resulia.
31
Delay of analysis increases the value detained unless flurd
is kept sterile and tightly corked.
Quantitative Test for Total Protein:
1. Direct Teet
a)Prepare a test tube (label and tube unknown and the
other blank).
b) Place 1 ml. of water to
tube marked blank and 1 ml.
of spinal fluid to tube marked unknown.
d)
Read the value by spectrophotometer method then cal.
culate as follows.
Gm. of protein X 1,000 20
mags. protein
per 100 ml.
20 spinal fluid
2. Sicard-Cantelouble Teat
This technique uses a special graduated 21 cm. long tube
having a 7 mm. diameter. The graduation in the bottom is
0.2 cc. and the topmost is 4 cc.

Procedure:
a) Place 4 co. of spinal fluid into a test tube.
b) Heat to 600 or 809C.
e) Add 12 drops of 33% trichloracetic acid.
d) Allow to stand for 5 minutes, then invert the tuhe 3
few times.

Let stand for 24 hours and read the quantity of

the sediment.

Interpretations:
Ist graduation equal to 0.22 on. protein/L.
And graduation equal to 0.44 m. protein/L
3rd graduation equal to 0.56 g. protein/L.

230
 4th graduation equal to 0.71 em. protein/L
5th graduation equal to 0.85 gunk. protein /L
The normal value of protein does not exceed 0.30 pib
protein/L..

Il. SUGAR:

General Considerations:
8 The test for engar must he performed within one-half
hour after withdruwal of the fluid, hecance glucose
gradually decomposes on standing,
b) Blood for glueose determination must be drawn al the
same time.

0) If possible, the spinal fluid and blood for this test shoold
drawn before breakfust
d) The glucose in spinal fluid ia normally 60% of that
in blood.

Qualitative Test:
1. Benedict's Test

8) Place 0.5 cc. of Benedict's qualitative reagent to sent

tube,

b) Add 4.5 ce. of distilled water.

o) Heat to boiling,
d) Add 1 cc. of spinal fluid.
e) Boil again for 1 to 2 minutea
Allow to cool.
Interpretations:
Normal sugar in CSF - turhid greenish yellow
Absence of sugar (pathologieal) hote. no change of color.
Excess of protein but no sugar --deep purplish-violet or

pinkish-violet color.

Qualitative Test:
Nelson-Somogyi Test
Blank (1 tube)

231
 0)
Paper
Standards2.0(3ml. of distilled
tuber)
Paper 2.0 ml. water auto
of nugor tube.
0)
Pipet2..00 inl working
of
standard(005
working Mo/ml.) intosugar
Unknowns standard 10.20
1)
11
Pipet 2.0 ml. tube
per
mg./ml.) intosugar
of
test.)
To all spinal fluid
tubea: inta
each tube.
a) Add
2.0 ml.
b) Place of
allsaline
tubes into copper.
After 8 boiling water Mix.
bath to minutes,place bothfor8
d) Add cool.
Do not tubes into minutes. Time.
2.0 mi.
of the disturb room
the temperature water
ternate
acid. phosphomolybdie
method: Add precipitate.
Mix. 3.0 of
This acid. Mix. (The
(e) and alternate
stabilizes the a-
e To
(1).
stabilize the color, color, phosphomolobye
eliminating
water both steps
again for 2 place the tubes into
At the
end of 2 minutes.
Time. the
boiling
water bath to minutes,
cool. return. to the room
g) Dilute
10 the
25 temperature
mix thoroughly ml. mark with distilled
by
Pour into cuvetteinversion.
water.
h) Stopperand
and read
millimicrons. against the blank at
420
Calculations.
O.D. (unknown) X Mg. standard
- Mg. % glucose
O.D. (standard)
NOTE:

The standards are made so that 2.0 ml. volume will contain
the following mg. contribution:
a) (0.05 mg./ml.) 0.1 mg. equivalent to 50 mg. %
b) (0.10 mg./ml.) 0.2 mg. equivalent to 100 mg. %

929
 c) (0.02 mg./ml.) 0.4 mg, equivalem to 200 mg.
Calculate the glocose, using the standard that reads closest
10

the unknown.

CHLORIDES:

General Considerations:

a) Blood for Chloride determination should be drawn at the

same time the spinal fluid is withdrawn.

b) The chlorides in spinal fluid is normally 25% higher than

that in the blood.

Test - Quantitative:
1. Schales and Schales
Procedure the same as in the Chemistry procedure.

IV. ALBUMIN-GLOBULIN RATIO TEST - LANGE'S

COLLOIDAL GOLD TEST

Principle:
The changes of the colloidal gold are the result
in color
of differences in the aggregation of colloidal particles due
to varying quantities of gamma globulin in the spinal fluid.

General Considerations
a) Spinal fluid containing blood cannot be used, because
variable reactions and false positive reaction is mostly
to occur.

b) If the spinal fluid cell count is above normal, the

fluid should be centrifuged and the supernatant fluid
used for the test.

Procedure:
a) Arrange a series of 12 chemically clean, dry, test tubes
in a rack with an opaque glass back.
b) Place 0.9 cc. of freshly prepared 04% NaCL solution
in the first test tube and 0.5 cc. in each of the next 10
tubes.

Place the 0.85 cc. of a 1% N&CL solution in the 12th

tube.

233
Reading and

The
tubes are scored by the
Unchange}deep red appearance of the (huid in the tubo.
Red to blue

Lilae to purple
Deep blue
Pale blue
(partial precipitate)
(complete)
NOTE.

A trace of
blood in the CSF
may give rise to a
meningitis curve,
while bacterial contamination makes the readl unreliable.
PREPARATION OF COLLOIDAL GOLD SOLUTION:
General Considerations:

a) Use
chemically clean glasswares.
b) Clean again with
aqua regia (1 volume HNO3 to 3 volume
and rinse thoroughly first with
single distilled water
then with double distilled water.
¢) All chemicals should be Merck's "Blue Label",
d) The solutions should be made in double distilled water
and the sodium citrate solution made fresh each time.

Borowskaja's Modification of Lange's Test

Procedure:

a) Add 10 cc. of A 1% solution of acid yellow gold chloride

t0 950 cc. of double distilled water in a 2 liter beaker.
b) Heat to 90 degrees centigrade and add 50 cc. of a 1%
solution of sodium citrate.

Boil, stirring constantly until a dark-red color appeara, then

watch carefully to the daylight until a cherry red color
appears without any evidence to blue. The longer the
solutin boils, the more sensitive it becomes.

235
 b) Finally, add 1 cc. of the
mastic solution to each tube.
Mix well and set
aside at
hours, or room temperature for 12 to 24
in the incubator for six to twelve hours.
Tubes
in which the reaction is complcte will show
precipitate with clear a heavy
supernatant fluid,
Colloidal Benzoin Test

This test
is similar to many respects to the mastic test. It
specific for
not
is
neurosyphilis,
same results as the more
but does give practically the
complicated colloidal gold test.
Reagents:
Benzoin Solution
Sumatra benzoin resin
Absolute alcohol 10 cc.

Filter the clear supernatant liquid after 48 hours. This stock

solution is prepared everytime you use it.
Stock Solution
Add 3 co. of stock solution drop by drop with constant
shaking
to 20 of double distilled water. Heat 369C in a water
both with constant shaking.
Salt Solution

Make a 0.01% solution of sodinm chloride in a double distilled

water.

CHEMICAL TESTS FOR TUBERCULOSIS MENINGITIS:

1. Tryptophane Test

Reagents:
a) Concentrated hydrochlorie acid
b A 2% solution of formalin (1:20 dilution of formalin.
water)
A 0.06% solution of sodium nitrite.

Procedure:
a Place 2 or 3 cc. of spinal fluid in a large test tube.

b) Add 15 10 78 00 of concentrated hydrochloric acid.

 Add 2 or 3 drops of 2% solution of formalin.
d) Shake the tube and allow the
mixture to stand for + to
5 minutes.
Add carefully (over lay) to form a
2 cc. of 0.06% solution of sodim supernatant
layer 1 to
nitrite.
Allow the mixture to stand for two to three minutes.
g) Positive reaction is indicated by the presence of violet
ring
at the zone of contact of two layers.
Negative reaction if brown ring or absence of a colored
ring.

NOTE.

A purple ring is given by bloody, purulent or xanthochronie

fluids. The test is positive in a very large percentage of tuber.
culous meningitie. but it is not diagnostic. It is usually nega-
tive in syphilitie meningitis, poliomyelitis and brain tumor.

2. Levingon's Test

Reagents:
a) A 1% solution of mercuric chloride.
b) A 3% solution of sulfonalycylie acid.
Procedure:
a) Place 1 mm. of CSF to each of two.

b) Add 1 cc. of 2% aqueous solution of sulfosalycylic acid

to test tube 2.

e)Stopper the tubes and allow to stand at room tem-

perature for 24 hours.

d) Measure the heights of the sediments.
Normal CSF has less than 2 mum. sediments.
Abnormal D increase proteing

Sediment in mercuric chloride is light and featherly

and forms slowly.

Sediment in malfosalyeylic acid compact and heavy

and forms rapidly.
In tubercutous meningitis, sediment in mercuric chloride
1s 2 mon. or more and 2 or more times that in the
acid tube.

238
Method for Moderate White Cell Count

Diluting Fluid:
Crystal violet
0.2 gan.
Glacial acetie acid
10.0 ml.
Distilled water
to 100 ml.
Procedure:

Mix specimen thoroughly. If not cloudy or bloody,

very
draw CSF to mark 1 in a white cell counting
then draw diluting fluid to mark 11, producing pipet and
& dilution
of 1:10.

b) Mix, discard 1/3, and

place 1 drop on each side of the
blood counting chamber, in the method for
lenkocyte
counting chamber. and add the results
of all 8 squares
counted.

Calculation:

Divide the sum by 8 to obtain

the number/single large square.
100 to obtain the number
a 1 mm3 of cells 2n
The first 10 converts the number found in 0.01
mm?,
to the
dilution found in 1.0 mm?, and the second 10 reptesens
numborfactor.
If the total cell count is low or moderate a rough estimate of
the differential count
can
be obtained by classifying the cells
seen in the counting chamber.
Method for High White Cell
Count:

The white cell count

as outlined for white purulent cerebrospinal fluid is done
on a

cells on the peripheral blood.

Method for Counting Mixture of White and Red Cells:
To find the true white cell
count when the cerehrospinal fluid
is bloody, perform red and white cell counts
on the
well as on the patients blood
cerebrospinal fluid specimen. Multiply
86

of the red cell count of the ratio

the fluid to the red cell count of
the blood
by the blood leukoyte count of the spinal fluid.

240
Examples
RBC of blood 5 million and of spinal fuld 25:000 WBC of
blood 12,000 and of spinal fluid 70/mm
85,000 X 12,000
5,000,000

10 white cells/mm° represents the true CSF white

cell count.

Normal blood will add 1 leukocyte for each 700 red cells

Significance of Total White Call Count:

The normal spinal fluid in essentially free of cells, containing

the
from 0.5 cells/mm', chiefly small lymphocytes Infants at
age of a few weeks may have 08 many a6 30 lymphocytes/mm* in
the CSF.

Variations in Diseases:

The total eell count as usually normal in multiple

epilepsy, brain tumor, meningismus, and cerebral arteriosclerosis,
and 50% of
elevated in syphilis (29-100), viral meningitio,
It. is

incephalitis, and reaches highest in pyogenic meningitis.

are not increased
Sometimes the cell count and the protein
3 corresponding sucrears
together. The celis are increased withoutprotein is increased with-
inprotein in aseptic meningitis, and the
or blockage, syndrome and in
out an increase of cells in Froin,
Guillain-Barre syndrome.

confirmed by stained
An increased cell count must always be
3

smear and a differential count.

Preparation of Smear for Differentio! Count:

described.
Various method of cell concentration have heen
Centrifugation is the easiest hut least satisfactory, as cells may be
the
damaged. Sedimentation techniques are probably hest, hut
is not
necessary equipment
commercially available. Sedimentation
to the
of cells directly onto the slide. appears to be superior

241