CSF - Cerebrospinal fluid examination - from tapping to pathological diagnosis

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Cerebrospinal Fluid
Examination

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OVERVIEW
1. Physiology
2. Functions of CSF
3. Indications
4. Recommended laboratory tests
5. Specimen collection
6. Opening pressure
7. Gross examination
 Color
 Appearance (Clear/clot/cobweb/coagulum)
 Viscosity
8. Microscopic examination
 Total count
 Differential count
i. Lymphocytes
ii. Neutrophils
iii. Plasma cells
iv. Eosinophils
v. Monocytes and macrophages
vi. Tumor cells
9. Chemical examination
 Proteins
i. Total protein
ii. Albumin
iii. IgG
iv. Other CSF proteins
 Glucose
 Lactate
 F2 isoprostanes
 Enzymes
i. Adenosine Deaminase (ADA)
ii. Creatinine Kinase (CK)
iii. Lactate Dehydrogenase (LDH)
iv. Lysozyme
 Ammonia, amines and aminoacids
10. Microbiological examination
 Bacterial meningitis
 Spirochetal meningitis
 Viral meningitis
 Fungal meningitis
 Tuberculous meningitis
 Primary amebic meningoencephalitis
11. Reference values

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* Physiology
1. CSF is derived from ultrafilteration and secretion through the choroid plexus.
2. CSF resorption occurs at arachnoidal villi predominantly along superior sagittal sinus.

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* Opening pressure
1. Opening pressure can be measured by a manometer before collection of CSF
2. The pressure varies with postural changes, blood pressure, venous return and valsalva
maneuver etc.
3. Pressure should be noted in lateral decubitus position with legs and neck in neutral
position.
manometer tube with graduation from -4 cm to +34 cm and attached to three way tap
Normals
CSF opening pressure Adult – 90-180 mm of water
Children (upto 8 years) – 10-100 mm of water
Abnormals
If pressure is elevated more than 200 mm of water, no more than 2 ml should be withdrawn
as it can lead to herniation
Elevated pressure Decreased pressure
1. straining
2. congestive heart failure
3. meningitis
4. superior venacaval syndrome
5. thrombosis of venous sinuses
6. cerebral edema
7. mass lesions
8. hypoosmolality
9. Idiopathic intracranial hypertension
(pseudotumor cerebri)
1. spinal-subarachnoid block
2. dehydration
3. circulatory collapse
4. CSF leakage – like from cribriform
plate in case of head injury

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B. Appearance
Normal
Appearance Clear
Abnormals
Turbid/cloudy Leucocyte count >200 cells/mm3
RBCs >400 cells/ mm3
Microorganisms (bacteria, fungi, amebas)
Radiographic contrast material
Aspirated epidural fat
Protein level greater than 150mg/dl
Bloody RBC counts >6000 cells/mm3
Clot Traumatic tap
Complete spinal block (Froin’s syndrome)
Suppurative or tuberculous meningitis
*Not seen in patients with subarachnoid hemorrhage
Cobweb Tuberculous meningitis
Cobweb in tuberculous meningitis in CSF

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* Microscopic examination
(A) Total cell count
Methods:
1. Manual count using Neubauer’s chamber or a Fuchs-Rosenthal type chamber (most
commonly used)
2. Count with an automated cell counter (poor precision)
3. automated flow cytometry of CSF (rapid and reliable, but expensive)
Counting using a neubauer’s chamber:
1. Sample in tube 3 is used
2. No dilution of CSF is usually required. A diluent (0.05ml CSF + 0.95 ml diluent, 1:20
dilution) is used only if CSF is cloudy and likely to contain increased number of
leucocytes. Diluent mostly used is Turk solution (glacial acetic acid + methylene blue +
distilled water)
3. Put coverslip on chamber.
4. Charge it from sides, take care that no fluid goes into the drain.
5. allow to stand for two minutes, cells will settle down.
6. Cells are counted in four corner WBC counting squares, marked ‘W’ in the figure.
7. Total count (per/mm3
)= No. of cells counted x 10
No. of squares counted
Improved Neubauer’s chamber

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Counting cells in WBC counting chamber
Normals
Total count Adults - 0-5 cells/mm3
Children – 0-30 cells/mm3
RBCs – Zero / hpf
Abnormals
Increased counts 1. Meningitis and other infections of CNS
2. Intracranial hemorrhage
3. Meningeal infiltration by malignancy
4. Repeated lumbar punctures
5. Injection of foreign substances (contrast media/drugs) in
subarachnoid space.
6. Multiple sclerosis
Correction for presence of blood in CSF
Presence of blood either due to traumatic tap or subarachnoid hemorrhage artefactually
raises the total count. This needs to be corrected by the following formula -
Corrected WBC (/mm3
) = WBC counted - WBC count in blood x RBC count in CSF
RBC count in blood

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(B) Differential cell count
Methods:
1. counting chamber – poor precision, identification of different cell types difficult, not
recommended
2. Direct smears of centrifuged CSF specimen – subjected to significant error from
cellular distortion# and fragmentation, but most commonly performed
3. Using a cytocentrifuge – recommended method for all body fluids
# cellular distortion can be minimized by adding 2 drops of 22% bovine albumin to the
specimen
Normals:
Cell type Adults (%) Children (%)
Lymphocytes # 62 +/- 34 20 +/- 18
Monocytes 36 +/- 20 72 +/- 22
Neutrophils 2 +/- 5 3 +/- 5
Histiocytes Rare 5 +/- 4
Ependymal cells Rare Rare
Eosinophils Rare Rare
#Blast like lymphocytes may be seen admixed with small and large lymphocytes in CSF of
neonates
Abnormals:
1. Increased neutrophils
Meningitis
1. Bacterial meningitis # (PMN >60%)
2. Early viral meningoencephalitis (PMN <60%, changes to lymphocytic in 2-3 days)
3. Early tuberculous meningitis
4. Early mycotic meningitis
5. Amebic encephalomyelitis
Other infections
1. Cerebral abscess
2. Subdural empyema
3. AIDS related CMV radiculopathy
Following seizures
Following CNS hemorrhage
1. subarachnoid
2. Intracerebral
Following CNS infarct
Reaction to repeated lumbar punctures
Injection of foreign material in subarachnoid space (e.g. methotrexate, contrast media)
Metastatic tumor in contact with CSF
#A total neutrophil count of >1180 cells/mm3
has 99% predictive value for bacterial meningitis

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(B) Albumin
1. Albumin is around 56-76% of total proteins in CSF.
2. Normal CSF albumin (in gm/dl) : serum albumin (in gm/dl) ratio is 1:230.
3. But this yields a very difficult decimal of 0.004 to deal with.
4. Hence the permeability of Blood brain barrier is assessed by CSF albumin : serum
albumin index, where value of CSF albumin is taken in mg/dl.
5. A traumatic tap invalidates the calculation.
CSF ALBUMIN / SERUM ALBUMIN ratio = CSF ALBUMIN (g/dl)
Serum albumin (g/dl)
CSF ALBUMIN / SERUM ALBUMIN INDEX = CSF ALBUMIN (mg/dl)
Serum albumin (g/dl)
Normals:
CSF albumin: Serum albumin ratio 0.004
CSF albumin:Serum albumin index (mg/gm) <9
Slightly elevated in infants upto 6 months of
age
Reflects immaturity of blood brain barrier
Index increases gradually after age 40
Abnormals:
9-14 Slight impairment
14-30 Moderate impairment
>30 Severe impairment

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Glucose
1. CSF glucose levels should be compared with plasma levels, ideally following a 4 hour
fast, for adequate clinical interpretation.
2. CSF glucose levels normalize before protein levels and cell counts following recovery
from meningitis, hence it is a useful parameter in assessing response to treatment.
Normals:
Fasting CSF glucose levels 60% of plasma level
(50-80 mg/dl)
Normal CSF glucose:Plasma
glucose ratio
0.3-0.9
Abnormals:
Decreased CSF fasting glucose (<40mg/dl or
ratio <0.3)
a.k.a. Hypoglycorrhachia
Increased CSF fasting glucose values
Due to: increased anaerobic glycolysis in
brain tissue and leucocytes
Due to: No clinical significance
Seen in
1. Bacterial, tuberculous and fungal
meningitis
2. meningeal involvement by malignant
tumor, sarcoidosis, cysticercosis,
trichinosis, ameba, syphilis
3. intrathecal administration of
radioiodinated serum albumin
4. subarachnoid hemorrhage
5. symptomatic hypocglycemia
6. rheumatoid meningitis

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(B) Creatine Kinase (CK)
1. CK-BB comprises of nearly 90% of brain CK activity, other 10 % being contributed by
mitochondrial CK (CKmt)
2. CK-BB starts rising in CSF after about 6 hours of ischemic insult with peak levels in
about 48 hours.
3. It is also raised following a subarachnoid hemorrhage and predicts chance of
unfavourable outcome.
Abnormals:
FOLLOWING ISCHEMIC INSULT
CK-BB <5 U/L Minimal neurologic damage
CK-BB 5-20 U/L Mild to moderate CNS injury
CK-BB 21-50 U/L Correlated with death
CK-BB >50 U/L Death occurs in all patients
FOLLOWING SUBARACHNOID HEMORRHAGE
CK-BB >40 U/L Death

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(D) Fungal Meningitis
Cryptococcus is the most common fungus isolated from CSF
Microbiological Methods:
1. India ink or nigrosin stains for capsule
2. Detection of cryptococcal antigen from CSF using latex agglutination
3. Culture
Cryptococcus in CSF stained with India Ink
Findings in CSF:
Test Findings
Opening pressure Variable
Leucocyte count Variable
Differential count Mainly lymphocytes
Protein Increased
Glucose Decreased
CSF : serum glucose ratio Low
Lactic acid Mild to moderate increased

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Test Bacterial
meningitis
Viral
Meningitis
Fungal
meningitis
Tuberculous
Meningitis
Opening pressure Elevated Usually normal Variable Variable
Leucocyte count >/= 1000/mm3
<100 / mm3
Variable Variable
Differential count Mainly
neutrophils
Mainly
lymphocytes
Mainly
lymphocytes
Mainly
lymphocytes
Protein Mild-moderate
increase
Normal – mild
increase
Increased Increased
Glucose Usually <40
mg/dL
Normal Decreased Decreased
(may be <45
mg/dL)
CSF : serum
glucose ratio
Normal /
decreased
Usually normal Low Low
Lactic acid Increased Normal – mild
increase
Mild to
moderate
increased
Mild to
moderate
increased

CSF - Cerebrospinal fluid examination - from tapping to pathological diagnosis

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