CSF Analysis: Laboratory Examination of Cerebrospinal Fluid

CSF Analysis includes

1. Opening pressure
2. Appearance
3. Total and differential cell counts
4. Chemical examination
5. Microbiological examination
6. Special investigations

1. Opening Pressure

After attaching the manometer to the hub of the spinal needle, patient’s legs must be gently extended and neck returned to neutral position. The CSF pressure is then taken. CSF pressure is directly related to jugular and vertebral venous pressures. Patient should be relaxed since tension, straining, or breath-holding will increase the CSF pressure, while hyperventilation will lower the opening pressure.

Normal opening pressure of CSF is:

• 60-180 mm of water in adults in lateral recumbent position
• 10-100 mm of water in children less than 8 years

Causes of increased CSF pressure:

• Tense and anxious patient
• Cerebral edema
• Subarachnoid hemorrhage
• Congestive cardiac failure
• Benign intracranial hypertension (pseudotumor cerebri).
• Intracranial mass lesion (e.g. neoplasm, abscess, hemorrhage)
• Meningitis

Causes of decreased CSF pressure:

• Leakage of spinal fluid following trauma or previous lumbar puncture
• Complete spinal block (obstruction of spinal subarachnoid space due to tumor, abscess, adhesions, herniated intervertebral disk). A large difference between opening and closing pressures usually indicates presence of a partial or complete spinal block.

If opening CSF pressure is greater than 200 mm, no more than 1- 2 ml of CSF should be removed.

2. Gross Appearance of Cerebrospinal Fluid

Normal CSF is clear and colorless like distilled water, and does not clot. Abnormal CSF may appear turbid, blood-mixed, xanthochromic, or viscous. Clot formation in CSF is abnormal and indicates increased proteins.

(a)Turbid CSF:

Can be due to the presence of:

• Leukocytes  more than 200 cells/cmm
• Red cells more than 400 cells/cmm
• Microorganisms like bacteria, fungi, or amebae
• Radiographic contrast media
• Aspiration of epidural fat during LP
• Raised proteins.

(c) Xanthochromia:

This is the yellow discoloration of CSF. For its detection, CSF is centrifuged and the supernatant is compared with another tube of same size filled with distilled water. Causes of
xanthochromia include:

• Subarachnoid hemorrhage (12 hours after bleeding episode): In subarachnoid hemorrhage, bleeding occurs in subarachnoid space usually due to rupture of a cerebral aneurysm. Patient presents with severe bursting headache of sudden onset in occipital region that may be followed by loss of consciousness. Red blood cells in CSF are hemolyzed with release of oxyhemoglobin. About a week after bleeding, macrophages and other cells of leptomeninges convert oxyhemoglobin to bilirubin. This forms a yellowish discoloration of CSF supernatant. The investigation of choice, if available, for diagnosis of subarachnoid hemorrhage is CT scan to detect blood in basal cisterns. When CT scan is negative or equivocal and xanthochromia cannot be appreciated visually, spectroscopic CSF Analysis is helpful for diagnosis of subarachnoid hemorrhage. It will show absorption peaks of oxyhemoglobin and bilirubin.
• Jaundice, when serum bilirubin is more than 6.0 mg/dl
• CSF protein more than 150 mg/dl.

Froin’s syndrome is a combination of xanthochromia, excess proteins in CSF, and spontaneous formation of a coagulum in CSF on standing. It results from complete block of subarachnoid space.

3. Cell Counts in Cerebrospinal Fluid

(1) Total leukocyte count:

Cell count on CSF is done manually on undiluted sample in a counting chamber. Total leukocyte count increases in various disorders and along with differential count gives important diagnostic information. An increase in cell count in CSF is called as pleocytosis.

It is essential to do microscopic examination of all CSF samples since white blood cell (WBC) count up to 200/cmm and red cell count upto 400/cmm are associated with clear appearance of CSF.
For correct results:

Cell count should be done as soon as possible after collection of CSF since cellular disintegration occurs rapidly. Cells also adhere to the walls of the glass tubes

CSF specimen collected in tube 3 should be used.

No dilution of CSF is usually required. A diluent should be used only if CSF is cloudy and likely to have increased leukocytes.

Method:

• CSF sample must be properly mixed. If CSF is clear, it is not diluted. If CSF appears cloudy or turbid, 1:20 dilution is made using 0.05 ml of CSF and 0.95 ml of Turk solution. (Composition of Turk solution: Glacial acetic acid 4 ml, methylene blue solution 10 drops, and distilled water to make 200 ml).
• The counting chamber is covered with the coverslip provided.
• The counting chamber is filled with the fluid and allowed to stand for 2 minutes for cells to settle.
• For counting cells in CSF, Fuchs-Rosenthal counting chamber is preferred because its depth is twice that of improved Neubauer chamber. In this, cells are counted in 5 large squares (4 corner squares and one central square).
• If undiluted CSF is used, total number of cells counted in 5 squares represents total count per cmm of CSF. If CSF is diluted, the number of cells counted is multiplied by the dilution factor (i.e. 20).

2) Differential leukocyte count:

This gives information about relative proportion of various leukocytes. If CSF has only a few cells, then it is centrifuged at high speed (3000 g) for 10 minutes and a smear is made from the sediment. If CSF contains many cells, then a smear is made directly from the uncentrifuged sample.

After staining with a Romanowsky stain, smear is examined under the microscope. Simple centrifugation of CSF often causes cell breakage and distortion. Cytospin preparation with cytocentrifuge (high speed centrifugation to concentrate cells on a slide in a uniform monolayer) has been recommended as it improves the cell yield and preserves the cell morphology well.

In normal adults, differential count shows 70% lymphocytes and 30% monocytes. In young children, a higher proportion of monocytes (up to 70%) are present.

(3) Other cells:

Apart from mature blood cells, CSF may contain immature hematopoietic cells, tissue cells (ependymal cells, pia arachnoid mesothelial cells) and malignant cells. CSF examination is commonly carried out in acute lymphoblastic leukemia to detect involvement of CNS. Increased WBC count (>5/μl) with lymphoblasts is evidence of CNS involvement.

4. Chemical Examination of Cerebrospinal Fluid

Routine chemical examination of CSF consists of estimation of proteins and glucose. CSF from tube 1 is used for chemical examination.

(a) Estimation of proteins in CSF:

Normal CSF protein level in adults is 15-45 mg/dl. A rise in CSF protein is a sensitive but non-specific indicator of CNS disease. CSF proteins may be normal during early stages of meningitis. Significant elevation (more than 150 mg/dl) occurs in bacterial meningitis.

There are various methods for estimation of CSF proteins. these include:

• Turbidimetric method using trichloroacetic acid for precipitation of proteins is commonly used. In principle, trichloroacetic acid, when added to CSF, causes precipitation of proteins and a turbid solution is obtained. Amount of turbidity is compared with the turbidity of a known (standard) concentration of protein in a photoelectric colorimeter.

If a sample is contaminated with blood while doing the lumbar puncture (traumatic tap), false elevation of proteins will occur. This can be corrected by deducting 1 mg/dl of protein for every 1000 red cells per cmm. For this correction to be accurate, red cell count and proteins should be estimated in the sample from the same tube of CSF.

If facilities for estimation of CSF proteins are not available in the laboratory, then Pandy’s test for globulins can be performed. In this test, CSF is added to saturated solution of phenol. If cloudiness develops immediately, it indicates presence of increased globulins and the test is reported as positive. If no cloudiness develops, the test is reported as negative.
Along with CSF proteins, it is necessary to simultaneously measure serum proteins for proper interpretation of results.

CSF proteins are elevated in following conditions:

• Increased capillary permeability of blood-brain barrier: Meningitis
• Mechanical obstruction to circulation of CSF (causing increased fluid reabsorption due to stasis): Spinal cord tumor
• Increased local (intrathecal) immunoglobulin (IgG) production: Multiple sclerosis, neurosyphilis, subacute sclerosing panencephalitis
• Both increased capillary permeability and increased local immunoglobulin (IgG) production: Guillain- Barré syndrome
• Hemorrhage in CSF: Traumatic tap, subarachnoid hemorrhage. Marked elevation (>500 mg/dl) is noted in complete spinal block by a tumor, bacterial meningitis, and bloody CSF.

Albumin is neither synthesized nor metabolized in CNS. Therefore, increased CSF albumin/serum albumin ratio indicates increased permeability of blood-brain barrier. Immunoglobulin G (IgG) can be synthesized in CNS. Therefore, increased CSF IgG/serum IgG ratio indicates either increased permeability of blood-brain barrier or increased intrathecal synthesis of IgG. Increased CSF IgG/albumin index indicates local IgG production. 

(c) Estimation of glucose in CSF:

Normal CSF glucose is two thirds of blood glucose (CSF to blood glucose ratio is 0.6). A sample for blood glucose should be drawn 1 hour before LP for comparison with CSF glucose. After collection, CSF sample should be immediately processed for glucose estimation because falsely low result due to glycolysis may occur.
CSF glucose is measured by glucose oxidase method.
Normal range is 45-80 mg/dl. CSF glucose <40 mg/dl is abnormal.
CSF glucose is decreased due to utilization by bacteria (pyogenic or tuberculous), leucocytes, or cancer cells in CSF.

Decreased CSF glucose occurs in following conditions:

• Acute bacterial meningitis
• Tuberculous meningitis
• Fungal meningitis
• Meningeal involvement by malignant tumor (meningeal carcinomatosis)
• Hypoglycemia
• CSF glucose is normal in viral meningitis.

 5. Microbiological Examination

Microbiological tests which can be carried out on CSF sample are:

• Direct wet mount of CSF: in suspected cases of cryptococcosis, amebic meningoencephalitis, Candida infection, and trypanosomiasis
• Gram’s smear: should be done if CSF is turbid and neutrophils are increased.
• Latex agglutination tests: for detection of bacterial and cryptococcal antigens.
• Serologic tests for syphilis
• Culture for bacteria and Mycobacterium tuberculosisPolymerase chain reaction for Mycobacterium tuberculosis and viruses.
• Ziehl-Neelsen smear: if tuberculous meningitis is suspected.

(a) Direct wet mount of CSF:

One drop of CSF deposit (obtained after centrifugation) is put on a glass slide, covered with a cover glass, and examined under the microscope with reduced illumination. look for:

• motile trypanosomes
• sluggishly moving amebae (Naegleria fowleri). N. fowleri is a free-living ameba in water which enters through the nose and reaches central nervous system. It causes a fatal type of hemorrhagic meningoencephalitis.
• Candida albicans may be seen in unstained wet mount; it appears as oval budding forms and as pseudo hyphae. When cryptococcal meningitis is clinically suspected, the wet mount is examined by dark-field microscopy.
• Alternatively, a drop of India ink is added to the drop of sediment on a glass slide, a coverslip is placed, and examined under the microscope using ×40 objective. Cryptococcus neoformans appears as spherical, budding yeast forms, 2-10 μ in diameter and surrounded by a large unstained capsule Cryptococci can be demonstrated by India ink preparation in 50% cases of cryptococcal meningitis.

(b) Gram’s smear:

This should be done if CSF is purulent and neutrophils are raised. Gram’s smear is positive in 80% untreated cases and 60% partially treated cases of bacterial meningitis. Therefore, absence of bacteria on
Gram’s smear does not rule out bacterial infection. CSF is centrifuged and a smear of the deposit is made on a glass slide. (If CSF is purulent, smear is made directly without centrifugation). Smear is air-dried, stained with Gram’s method, and examined under the oil-immersion lens for bacteria. Bacteria which commonly cause meningitis are:

• Meningococci: Gram-negative diplococci located inside neutrophils.
• Pneumococci: Gram-positive diplococci surrounded by an unstained capsule.
• Hemophilus influenzae: Gram-negative coccobacilli.
• Escherichia coli: Gram-negative rods.

In all the above types of meningitis, the accompanying cells are polymorphonuclear neutrophils. Detection of typical bacteria on Gram-staining suggests the etiologic agent of meningitis. However, definitive diagnosis requires culture of CSF. 

(d) Ziehl-Neelsen staining for Mycobacterium tuberculosis:

In tuberculous meningitis, number of tubercle bacilli is usually low in CSF. Ziehl-Neelsen or AFB staining is not a very sensitive method for detection of M. tuberculosis in CSF. AFB smears are negative in about 70% of cases of tuberculous meningitis. Fluorescent auramine stains have better sensitivity than Ziehl- Neelsen stain.

(e) Latex agglutination tests:

Latex agglutination tests for bacterial antigens are available commercially and are sensitive, rapid, and simple to perform. Currently available tests can detect N. meningitidis (groups A, B, C, Y, and W135), H. influenzae (capsular type B), S. pneumoniae and S. agalactiae.

These tests are expensive and their sensitivity is similar to that of Gram’s smear.
Owing to the occurrence of false-positive results, these tests are not recommended for routine diagnosis of meningitis. These tests are particularly useful in patients who have been partially treated and in whom Gram’s stain and culture are negative. Latex agglutination tests for cryptococcal antigens are also available and have sensitivity of 90%. These tests have largely replaced India ink preparation for diagnosis of cryptococcal meningitis.

(f) Limulus lysate assay for endotoxin produced by gram-negative bacteria:

Limulus amebocyte lysate assay is a rapid, sensitive, and specific test for the presence of endotoxin. Endotoxin is produced by gram-negative bacteria like N. meningitides, H. influenzae type b, E. coli, and Pseudomonas. This test is particularly useful as a rapid test in newborns in whom these infections are common.

(g) Serologic tests for syphilis:

If fluorescent treponemal antibody with absorption (FTA-ABS) test is positive in serum, Venereal Disease Research Laboratory (VDRL) test should be done on CSF if neurosyphilis is suspected.

VDRL test is highly specific but lacks sensitivity. Therefore, a positive test rules in but does not rule out the diagnosis of neurosyphilis. Other serological tests for syphilis are not suitable for diagnosis of neurosyphilis in CSF.
Combination of positive FTA-ABS test in serum and reactive VDRL test in CSF is diagnostic of active neurosyphilis

(h) CSF culture:

Culture of CSF is important if bacteria are seen on Gram-stained smear, or leukocytes or proteins are increased. Culture remains the gold standard for diagnois of bacterial meningitis. For culture, CSF collected in tube 2 is used. Sensitivity of culture for identification of bacteria is about 90%. In incompletely treated cases, sensitivity is less.

Usually, CSF sample is inoculated on chocolate (heated blood) agar and blood agar. In newborn infants, sample is also inoculated on McConkey’s agar. In tuberculous meningitis, culture for M. tuberculosis is positive in about 56% of cases. If larger volume of CSF is used (i.e. 10 ml) for inoculation, sensitivity for detection increases. In cryptococcal meningitis, culture is positive in 95% of cases.

(h) Polymerase chain reaction (PCR):

PCR is a highly specific and sensitive tool for diagnosis of infections of CNS. It uses probes to detect genes specific for the infecting organism. The test is rapid and requires only a small amount of CSF. High cost and availability only in a few specialist laboratories are major limitations. CSF PCR is mainly useful for diagnosis of viral infections of CNS (e.g. herpes simplex, enteroviruses, herpes zoster, etc.) and of tuberculous meningitis.

 6. Special Investigations in CSF analysis

(a) CSF protein electrophoresis:

Protein electrophoresis of normal CSF differs from normal serum in:

• presence of a prominent transthyretin band
• an extra transferrin band (called β2-transferrin or tau protein).

Protein electrophoresis of CSF is used:

• For identification of oligoclonal bands,
• To determine whether fluid submitted for examination is CSF.

1. Oligoclonal bands:

Agarose gel electrophoresis of concentrated CSF is used for detection of oligoclonal bands. These are two or more discrete bands in the gamma region. Presence of oligoclonal bands on CSF protein electrophoresis that are absent on concurrently run serum protein electrophoresis is indicative of intrathecal synthesis of immunoglobulins Oligoclonal bands have been identified in majority of patients (90%) with multiple sclerosis; however, they are not specific for this disorder. They are also seen in subacute sclerosing panencephalitis,
viral CNS infections, neurosyphilis, and Guillain- Barré syndrome.

2. CSF leakage:

Occasionally, clear fluid leaking through the nose or ear after trauma or surgery is submitted for examination to determine whether it is CSF. Due to the risk of recurrent meningitis, accurate identification of such fluid as CSF is essential. Recommended test for this purpose is protein electrophoresis with immunofixation for transferrin. Protein electrophoresis of CSF shows an extra transferrin band (called as ‘tau’ protein), which is absent in other body fluids and secretions. Tau protein is an enzymatically modified transferrin that moves just behind the unaltered transferrin in β region. Identification of two isoform bands of transferrin on protein electrophoresis is a highly sensitive and specific test for identification of fluid as CSF

Other body fluids or secretions do not show the second isoform band.

(b) Measurement of albumin and immunoglobulin G (IgG):

Comparison of IgG and albumin CSF/plasma ratio can be helpful for diagnosis of multiple sclerosis (high ratio due to intrathecal synthesis of IgG).

Critical Values in CSF analysis

• Cells: more than 10/cmm
• Glucose: below 45 mg/dl
• Proteins: more than 45 mg/dl
• Detection of pathogens on Gram stain, latex agglutination tests, Ziehl Neelsen stain, or India ink preparation
• Positive culture
• Detection of blast cells or malignant cells
• Positive polymerase chain reaction for herpes simplex

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