CEREBROSPINAL

FLUID (CSF)

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 CEREBROSPINAL FLUID (CSF)
• Major fluid of the body that circulates in the Central
Nervous System (CNS)
• First recognized by Contugno in 1764
• Three meninges that lines the brain and spinal cord
a) Dura Mater (“hard mother”) – outermost layer; lines
the skull and vertebral canal
b) Arachnoid Mater (“spider-web like”) – middle layer;
filamentous inner membrane
• CSF flows in the SUBARACHNOID SPACE
c) Pia Mater (“gentle mother”) – innermost layer; thin
membrane lining the surfaces of brain and spinal cord
 CEREBROSPINAL FLUID (CSF)
• Produced by the choroid plexuses, capillary networks
found in the 2 lateral ventricles and the third and fourth
ventricles
– The endothelial cells of the choroid plexuses have BLOOD-BRAIN
BARRIER (very-tight fitting junctures that prevent the passage of
many molecules)
– BBB protect the brain harmful chemicals in the blood and prevent
the passage of helpful substances including antibodies and
medications
• CSF is reabsorbed back into blood capillaries through the
arachnoid villi/granulations
– Arachnoid villi/granulations – ONE-WAY VALVE ; responds to
pressure within the CNS and prevents reflux of the fluid
 CSF
• CSF is produces from selective filtration under
hydrostatic pressure and active transport
secretion. Thus, the chemical composition of the
CSF DOES NOT resemble an ultrafiltrate of plasma
• 20mL of CSF is produced per hour
• Total CSF Volume in ADULTS
– 90-150mL
• Total CSF Volume in NEONATES
– 10-60mL
 CSF Collection and Handling
• CSF COLLECTION
– Ventricular puncture
• Done in infants with open fontanels
– Cisternal puncture
• Dangerous ; done in sub-occipital region
• Performed if there is (1) blockage of spinal canal, (2) vertebrae
deformity, (3) infection of the back
– Spinal tap (lumbar puncture – L3, L4, L5)
• The volume of CSF removed is dependent on the
age (adult vs neonate) and opening pressure of the
CSF
– ↑Opening pressure – CSF withdrawn slowly, collect
SMALL AMOUNTS only.
 CSF Collection and Handling
Specimens are collected in three sterile tubes
– Tube 1 – CHEMISTRY and SEROLOGY
– Tube 2 – MICROBIOLOGY
– Tube 3 – HEMATOLOGY
– Tube 4 – May be used for microbiology to exclude skin
contamination or for additional serologic tests
If only one tube can be collected, it must be tested
first by MICROBIOLOGY
 CSF Collection and Handling
• CSF tests are performed in a STAT basis
• If not possible,
– Hematology(Tube 3) is REFIGERATED
– Microbiology (Tube 2) remains at ROOM TEMP.
– Chemistry and Serology (Tube 1) must be FROZEN
 CSF Appearance
• Normal: COLORLESS and CRYSTAL CLEAR
• Abnormal Variations
1. Cloudy / Turbid / Hazy / Milky
o Increased protein and lipids
o Infection due to increased WBC (>200 cells/ul)
 Centrifugation of CSF must be done in CAPPED TUBES
2. Oily
o Radiographic contrast media
 CSF Appearance
3. Xanthochromic – CSF supernatant is
pink/orange/yellow
CAUSES
o Presence of RBC degradation products – most common
cause
 PINK – slight amount of oxyhemoglobin
 ORANGE – increased hemolysis
 YELLOW – oxyhemoglobin that is converted to unconjugated
bilirubin
o Elevated serum (unconjugated) bilirubin
o Presence of carotene pigment
o Markedly increased serum protein (>150mg/dL)
o Other pigments: melanin, rifampin, etc.
 CSF Appearance
4. Bloody (approximately 6000 RBCs/uL)
CAUSES
a. Intracranial hemorrhage
b. Traumatic tap
 REMEMBER: RBCs begin to lyse within 1
hour; 40% of leukocytes disintegrate after 2
hours
 INTRACRANIAL TRAUMATIC TAP
HEMORRHAGE

1. Distribution of blood EVEN UNEVEN

Tube 1 = Tube 2 = Tube 3 Tube 1 > Tube 2 > Tube 3>

2. Clot Formation NEGATIVE POSITIVE

(CSF has no fibrinogen) (Leakage of plasma
fibrinogen)
3. Appearance of XANTHROCHROMIC Clear supernatant
supernatant (NOTE: recent hemorrhage
produces clear supernatant
and protein contamination
produces xanthrochromia)
4. Presence of POSITIVE NEGATIVE
ERYTHROPHAGES

5. D-dimer POSITIVE NEGATIVE

 (+) CLOT but NOT BLOODY CSF
• Due to damage to blood brain barrier caused
by:
1. Meningitis
• Web-like clots (PELLICLE formation) observed after
12-24 hours of refrigeration – indicates TUBULAR
MENINGITIS
2. Blockage of CSF circulation
3. Froin syndrome
 Calculation of CSF Cell Counts
The standard Neubauer calculation formula used for
blood cell counts is also applied to CSF cell counts to
determine the number of cells per microliter.
 CSF CELL COUNT
A. Manual Cell Count – uses a hemocytometer
 Before performing CSF cell counts,
o CHECK CLARITY FIRST
o CLEAR = no need for dilution
o NOT CLEAR = dilute with normal saline for TOTAL CELL COUNT
= dilute with 3% glacial acetic acid to lyse the red
cells and stain with methylene blue to
differentiate neutrophils from monocytes
o Cells are counted in the FOUR CORNER SQUARES and
CENTER SQUARE on both sides of the hemocytometer
for total cell count and WBC count
 CSF Dilutions
CLARITY DILUTION AMOUNT OF AMOUNT OF
SAMPLE DILUENT
1. Slightly Hazy 1:10 30 uL 270 uL

2. Hazy 1:20 30 uL 570 uL

3. Slightly 1:100 30 uL 2,970 uL

Cloudy
4. Slightly 1:200 30 uL 5,970 uL
Bloody
5. Cloudy / 1:10,000 0.1 mL of 1:100 9.9 mL
Bloody / Turbid dilution
 CSF CELL COUNT
• RBC Count : NOT routinely done (performed if
TRAUMATIC TAP HAS OCCURRED)
• RBC value will be used for WBC and TOTAL PROTEIN
concentration CORRECTION
• If peripheral blood RBC and WBC count is normal,
the shortcut formula is:
– SUBTRACT 1 WBC for every 700 RBC
– SUBTRACT 8mg/dL TP for every 10,000 RBC/uL

– Other books: SUBTRACT 1mg/dL TP for every 1,200 RBC

 CSF Cell Count
• WBC COUNT
– Routinely performed in the clinical laboratory
– Diluent
• 3% acetic acid and methylene blue
– Reference value:
• ADULT : 0-5 WBC/ uL
• NEONATES : 0-30 WBC/ uL
 CSF Cell Count
• DIFFERENTIAL CELL COUNT
– Performed on a stained smear (Wright’s stain)
– Specimen must be concentrated before preparing the
smear
– Concentration can be performed through
• Sedimentation
• Filtration
• CENTRIFUGATION – most common ; 5-10 minutes
- remove supernatant but do not discard
• CYTOCENTRIFUGATION – most recommended method in all
body fluid cell count
– NOTE: Save the supernatant for additional tests
 CSF Cell Count
• CYTOCENTRIFUGATION
– 0.1mL CSF is added in a conical chamber
– Cells are forced in a monolayer
– 30% ALBUMIN is added to
• Increase cell yield
• Decrease cellular distortion (cytoplasmic vacuoles,
nuclear clefting, prominent nucleoli, indistinct nuclear
and cytoplasmic borders, and cellular clumping)
– QC: Check daily for bacterial contamination using
0.2 mL saline and 2 drops of 30% albumin
 Quality Control of CSF and other
Body Fluid Cell Counts
• BIWEEKLY basis
– All diluents must be checked for contamination
– Examination of diluent must be done in a counting
chamber under 400x magnification
• MONTHLY basis
– SPEED of cytocentrifuge must be checked in a
TACHOMETER and the timing should be checked with a
STOPWATCH
• For nondisposable counting chambers
– Soak in bactericidal solution for at least 15 minutes.
Rinse with water and clean with isopropyl alcohol after
each use
 CSF Cell Count
• DIFFERENTIAL CELL COUNT – done primarily to
identify type of organism causing meningitis
• CSF Normal Cells: Lymphocytes and
monocytes
• NOTE: Occasional neutrophils are also observed in
CSF using improved concentration methods
– ADULT: Lymphocyte (70%) ; Monocyte (30%)
– CHILDREN: 80% Monocytes
 CSF Cell Count
• DIFFERENTIAL CELL COUNT
–PLEOCYTOSIS – increased number of normal
CSF cells
1. LYMPHOCYTES and MONOCYTES
o Increased in cases of FUNGAL, TUBERCULAR,
VIRAL, or PARASITIC meningitis and multiple
sclerosis (demyelinating disease
o Reactive lymphocytes with dark blue cytoplasm
– VIRAL INFECTION
 CNS Cell Count
2. NEUTROPHILS
– Predominant in cases of BACTERIAL meningitis, but can
also be seen in early stages (1-2 days) of VIRAL,
FUNGAL, TUBERCULAR, AND PARASITIC MENINGITIS
– Increased neutrophils due to meningitis may contain
ingested bacteria
– OTHER CAUSES: CNS hemorrhage, repeated lumbar
punctures, injections of medication or radiographic dye
– Neutrophil with PYKNOTIC NUCLEI indicates
degenerating cells and may resemble nucleated RBCs
 CNS Cell Count
3. MACROPHAGES
– Appear within 2-4 hours after RBCs enter the CSF
and are frequently seen following repeated taps
– Indicates a PREVIOUS HEMORRHAGE
– ERYTHROPHAGES – macrophages with ingested red
cells
– Degradation of phagocytosed RBCs lead to formation
of
• Dark-blue or black IRON-CONTAINING hemosiderin
granules
• Yellow IRON-FREE hematoidin crystals
 CSF Cell Count

4. EOSINOPHILS – seen in PARASITIC and

FUNGAL infections primarily Coccidiodes
immitis
5. NUCLEATED RBCs (metarubricytes) – seen in
TRAUMATIC TAP ; bone marrow
contamination
NON-PATHOLOGIC CELLS in CSF
• Most frequently seen after diagnostic procedures (e.g.
pneumoencephalography) and in fluid obtained from
ventricular taps or duing neurosurgery
1. Choroidal cells – from the epithelial lining of the
choroid plexus; seen singularly and in clumps;
NUCLEOLI are usually ABSENT and nuclei have a
uniform appearance.
2. Ependymal cells – from the lining of the ventricles
and neutral canal; have less defined cell membranes
and are frequently seen in clusters; NUCLEOLI are
often PRESENT.
3. Spindle-shaped cells – represent lining cells from
arachnoid; usually seen in clusters and may be seen
with systemic malignancies
 ABNORMAL CELLS IN CSF
MALIGNANT CELLS OF HEMATOLOGIC ORIGIN
– Lymphoblasts, Myeloblasts, Monoblasts = ACUTE
LEUKEMIA
– Lymphoma cells = dissemination from the lymphoid
tissue
MALIGNANT CELLS OF NONHEMATOLOGIC ORIGIN
– Metastatic carcinoma cells = lung, breast, renal, and
gastrointestinal malignancies
– Astrocytomas, Retinoblastomas, Medulloblastomas =
Primary CNS Tumors
 CSF Cell Count
B. Automated Cell Count
o Advantages
Increased precision
Standardization
Faster turn around time
o CSF COUNT ANALYZERS
ADVIA 2120i
Sysmex XE-5000
Iris iQ200 with Body Fluids Module
Beckman Coulter LH780
UniCel DxH800
 Major Constituents of CSF
• Because filtration of molecules is selective and
controlled by blood-brain barrier, CSF does NOT
resemble an ultrafiltrate of plasma
1. PROTEIN – lower than plasma due to absence of
HMW molecules like fibrinogen and IgM
2. GLUCOSE – 60-70% of plasma glucose
3. SODIUM, CHLORIDE, and MAGNESIUM – higher in
CSF than plasma
4. POTASSIUM and CALCIUM – lower in CSF than
plasma
 CHEMICAL TESTS FOR CSF
1. CSF PROTEIN
– Most frequently performed chemical test on CSF
– REFERENCE VALUE: 15-45 mg/dL
• NOTE: Reference value is higher among infants and
people over age 40
– INCREASED PROTEIN in cases of
1. Multiple sclerosis
2. Meningitis
3. Intracranial hemorrhage
 CHEMICAL TESTS FOR CSF
1. CSF PROTEIN
– Normal CSF Protein constituents
• ALBUMIN – Major protein
• PREALBUMIN (Transthyretin) – Second most prevalent
• ALPHA-GLOBULINS – Ceruloplasmin & Haptoglobin
• BETA-GLOBULIN – Transferrin
– “TAU” – separate carbohydrate deficient transferrin
fraction seen in CSF and not in serum
• GAMMA-GLOBULINS – IgG (major) with some IgA
– CHON not found in CSF: IgM, beta-lipoprotein,
fibrinogen
QUALITATIVE TEST FOR CSF TP
1. PANDY’S TEST
– Reagent: Phenol
– (+) Result: Faint Blue CLOUD
2. ROSS JONES
3. NONNE-APELT
} 3% ammonium sulfate
– (+) Result: White ring of precipitate
 QUANTITATIVE TEST FOR CSF TP
1. TURBIDIMETRIC
a. Trichloroacetic Acid (TCA) – PREFERRED
precipitating agent because it precipitates
both albumin and globulin
b. Sulfosalicylic Acid (SSA) – precipitates
albumin only (REMEDY: Add
Sodium/ammonium sulfate to precipitate
globulin)
 QUANTITATIVE TEST FOR CSF TP

2. DYE BINDING METHOD

–Uses Coomasie Brilliant Blue Dye
–Original Color: RED DYE + PROTEIN =
BLUE COLOR (Spectro)
 QUANTITATIVE TEST FOR CSF TP
3. IMMUNOFIXATION
ELECTROPHORESIS (IFE) and
ISOELECTRIC FOCUSING (IEF)
–Uses silver stain to visualize the bands
–Method of choice when determining
whether a fluid is actually CSF
 CSF PROTEIN
4. CSF ELECTROPHORESIS
–Needed in the diagnosis of neurologic
disorders associated with abnormal CSF
particularly multiple sclerosis
–PRIMARY PURPOSE: detect oligoclonal
bands (increased IgG)
–NOTE: Serum electrophoresis must be
simultaneously done with CSF
Electrophoresis
 OLIGOCLONAL BANDS
OLIGOCLONAL BANDS
SERUM CSF
•Leukemia POSITIVE POSITIVE
•Lymphoma
•Viral Infections (HIV Infection)
oMultiple Sclerosis NEGATIVE POSITIVE
oEncephalitis
oNeurosyphilis
oGuillian-Barre syndrome
oNeoplastic disorders

NOTE: In multiple sclerosis, the

oligoclonal banding REMAINS
upon remission but disappears
in other conditions
 CSF Protein
• To determine whether increased IgG is due to
synthesis within CSF or due to damage in the blood-
brain barrier, serum and CSF levels of albumin are
compared. Tests include:
– CSF/serum albumin index = evaluates integrity of
the blood-brain barrier
– CSF IgG index = measures IgG synthesis within
the CNS
 CSF PROTEIN

• An index value of <9 represents an intact blood-brain

barrier

• Comparison of CSF/serum albumin index with the

CSF/serum IgG index
• Values >0.70 indicate IgG production within the CNS
MYELIN BASIC PROTEIN (MBP)
• Presence in the CSF indicates recent
destruction of the myelin sheath that
protects the axons of the neurons
(demyelination)
• Also provide a valuable measure of the
effectiveness of current and future
treatments.
 CHEMICAL TESTS FOR CSF
2. CSF GLUCOSE
– Glucose enters the CSF via selective transport
– REFERENCE VALUE: 60-70% of blood glucose
(Approx. 65 mg/dL)
– A blood glucose test must be run for comparison
• BLOOD GLUCOSE should be drawn 2 hours before the
spinal tap to allow time for equilibration between
blood and fluid.
 CHEMICAL TESTS FOR CSF
2. CSF GLUCOSE
– INCREASED – not significant
– DECREASED – determines cause of meningitis
• Bacterial meningitis – markedly decreased CSF
glucose and increased neutrophils
• Tubercular meningitis – decreased CSF glucose and
increased lymphocytes
• Viral meningitis – NORMAL CSF glucose, increased
lymphocytes
 CHEMICAL TESTS FOR CSF
3. CSF LACTATE
– Valuable aid in diagnosing and managing
meningitis cases
– REFERENCE VALUE: 10-24 mg/dL
– INCREASED CSF Lactate is primarily due to oxygen
deprivation
oMeningitis
oHead injuries
oHydrocephalus
oIntracranial hemorrhage (RBS contains high amount
of lactate)
 CHEMICAL TESTS FOR CSF
3. CSF LACTATE
–Inversely proportional to CSF glucose
• Increased CSF Lactate: FUNGAL, TUBERCULAR,
and highest in BACTERIAL MENINGITIS
(>35mg/dL)
• Normal CSF Lactate: VIRAL MENINGITIS
–RBCs contain high concentrations of lactate,
and falsely elevated results may be obtained
on xanthochromic or hemolyzed fluid
 CHEMICAL TESTS FOR CSF
4. CSF GLUTAMINE
– NORMAL VALUE: 8-18 mg/dL
– By-product of ammonia and α-ketoglutarate
– Removes toxic ammonia
– Concentration is directly proportional to serum ammonia
– PREFERRED over direct measurement of ammonia since it is
more stable than ammonia
– Testing is done for patients with COMA OF UNKNOWN
ORIGIN (depletion of α-ketoglutarate)
– INCREASED in cases of Reye’s Syndrome (childhood
complication following ASPIRIN intake by patients with viral
infection)
 CHEMICAL TESTS FOR CSF
5. CSF LACTATE DEHYDROGENASE (LD) –
with 5 isoenzymes
–SERUM: LD 2> 1 > 3 > 4 > 5
• Myocardial infarction: (LD1 > LD2)
–CSF: LD 1 > 2 > 3 > 4 > 5 (L1 is predominant)
• Neurological abnormality: (LD2 > LD1)
MICROBIOLOGICAL TESTS FOR CSF
• Identifies the causative agent in meningitis
• Preliminary tests
– Gram staining
– Acid-fast staining
– India ink preparation
– Latex agglutination tests
• Confirmatory test
– CSF culture
• NOTE: Both culture and staining methods are
performed on CSF precipitate
MICROBIOLOGICAL TESTS FOR CSF
1. GRAM STAINING
– Routinely performed on CSF from all suspected cases of
meningitis
– Performed on concentrated specimens
• CSF is centrifuged at 1500 g for 15 minutes
– Organisms most frequently encountered include
Streptococcus pneumoniae
Haemophilus influenzae
Escherichia coli
Neisseria meningitidis
Streptococcus agalactiae
}
Listeria monocytogenes encountered in newborns
MICROBIOLOGICAL TESTS FOR CSF
2. ACID-FAST STAINING
– not routinely performed unless tubercular
meningitis is suspected
3. INDIA INK STAINING
– Detects the presence of thickly encapsulated
Cryptococcus neoformans
– Gram stain reaction of Cryptococcus neoformans
= classic starburst pattern
MICROBIOLOGICAL TESTS FOR CSF
4. LATEX AGGLUTINATION TEST
– Detects presence of C. neoformans antigen in serum
and CSF
– More sensitive method than India Ink preparation
– Presence of rheumatoid factor causes false-positive
reactions
5. LATERAL FLOW ASSAY (LFA)
– Rapid method for detecting C. neoformans
– Utilizes a reagent strip coated with monoclonal
antibodies that react with the cryptococcal
polysaccharide capsule
MICROBIOLOGICAL TESTS FOR CSF
6. ELISA
– Detect the following organisms and antigens
 Streptococcus group B
 H. influenzae Type B
 S. pneumoniae
 N. meningitidis A, B, C, Y, W135
 Mycobacterium tuberculosis
 Coccidiodes immitis
 E. coli K1 antigens
 MISCELLANEOUS
Naegleria fowleri
– An opportunistic parasite found in ponds, small lakes,
and even chlorinated swimming pools
– The Naegleria enters the nasal passages and migrates
along the olfactory nerves to invade the brain
– Motile trophozoites can be observed microscopically by
examining a wet preparation of CSF
– Nonmotile trophozoites may be seen on cytocentrifuged
stained smears accompanied by increased WBCs and no
bacteria
 SEROLOGIC TESTING
• Diagnosis of NEUROSYPHILIS
• Venereal Disease Research Laboratory (VDRL) –
CDC recommended method for diagnosis of
neurosyphilis
• Other tests:
o Fluorescent treponemal antibody-absorption (FTA-ABS)
– avoid serum contamination (serum remains positive
even after treatment)
o Rapid plasma reagin (RPR) test – not recommended
(less sensitive than VDRL)
 MENINGITIS
1. BACTERIAL MENINGITIS
– Causative agent (encapsulated)
• Birth to 1 month – Group B streptococcus
(S. agalactiae) (neonatal meningitis)
• 1 month to 5 y/o – H. influenzae
• 5 y/o to 29 y/o – N. meningitidis
• > 29 y/o – S. pneumoniae
• Newborn, elderly, immunosuppressed –
Listeria monocytogenes
MICROBIOLOGICAL TESTS FOR CSF
1. BACTERIAL MENINGITIDIS
– Lab Tests
• Increased: CSF, WBC, neutrophils, LD4 & LD5, protein,
lactate (>35 mg/dL)
• Decreased: glucose
• LIMULUS LYSATE TEST – POSITIVE
– Test for bacterial endotoxin (for gram-negative bacteria only)
– REAGENT – blood of horseshoe crab (Limulus polyphemus)
(blue color due to COPPER)
– Amoebocytes in crab release CHON (lysate) in the presence of
endotoxin)
– (+) Result: CLUMPING
MICROBIOLOGICAL TESTS FOR CSF
2. VIRAL MENINGITIS
–Causative agent : ENTEROVIRUS,
Echovirus, Coxsackie and Polio virus,
Arbovirus
–Increased: WBC count (lymphocytes),
LD2 and LD3, protein
–Normal: Glucose, lactate
MICROBIOLOGICAL TESTS FOR CSF
3. FUNGAL MENINGITIS
–Causative agent: Cryptococcus neoformans
(encapsulated)
–Increased: WBC count (lymphocytes and
monocytes), protein, lactate (> 25 mg/dL)
–Decreased: Glucose
–Laboratory test: India Ink stain / negative
stain / indirect stain
• CAPSULE – unstained
• BACKGROUND - black
MICROBIOLOGICAL TESTS FOR CSF
4. TUBERCULAR MENINGITIS
– Causative Agent: Mycobacterium tuberculosis
– Increased: WBC count (lymphocytes and monocytes),
protein, lactate (> 25 mg/dL)
– Decreased: Glucose
– Laboratory Test: AFB (+) Red staining bacteria
– (+) PELLICLE formation (web-like clot) upon refrigeration
for 12-24 hours
 PELLICLE FORMATION IN CSF
• Enhanced by refrigeration of the sample
• MACROSCOPICALLY, it appears as small fine
clots seen after refrigeration of CSF for a
period of 12-24 hours
• MICROSCOPICALLY, it consists of white
blood cells against a fibrinous background and
must be examined for bacteria through gram
stain and culture
 PELLICLE FORMATION IN CSF
• NORMAL: no clots due to absence of
fibrinogen
• VARIATIONS:
– Small clots – paresis (incomplete paralysis)
– Large clots – purulent meningitis
– Web-like clots – TB meningitis
– Clotting en masse – blockage in CSF circulation