CEREBROSPINAL FLUID Joseph T. Sabido

FORMATION AND PHYSIOLOGY

CSF Physiology •  A major fluid in the

body and was first recogised by Contungo in 1764

Domenico Cotugno, (January 29 1736-October 6, 1822) was an

Italian Physician

Functions of CSF

Removes metabolic wastes Produce mechanical barrier to cushion the brain and spinal cord against trauma Provides a physiologic system to supply nutrients to the nervous tissue

ANATOMY

MENINGES •  Lines the brain and

spinal cord; 3 layers •  Dura mater - outer layer;

lines the skull and vertebral canal

•  Arachnoid - filamentous (spider – like) inner membrane

•  Pia mater – thin membrane lining surfaces of the brain and spinal cord.

•  Subarachnoid space – located between the arachnoid and the pia mater

ARACHNOID GRANULATION CELLS Act as one-way valve Respond to pressure within the Central Nervous system Prevent reflux of CSF

CSF FORMATION

CHOROID PLEXUSES •  A structure within the

ventricles of the brain where CSF is produced

•  Composed of modified ependyman cells

•  Mechanisms: •  Selective filtration

under Hydrostatic pressure

•  Active transport secretion

Sites of CSF Production

BLOOD BRAIN BARRIER •  Formed by the tight-fitting

junctures of the endothelial cells lining the choroid plexuses

•  Prevents passage of molecules

•  Disruption of Blood brain barrier by diseases such as Meningitis and Multiple Sclerosis allows Leukocytes, proteins and chemicals to enter the CSF.

•  CSF flows through the subarachnoid space and is reabsorbed back into the blood capillaries in the arachnoid granulations/ villae at a rate equal to its production • Adults

• 20 mL CSF every hour is produced • 90 – 150 mL total CSF

• Neonates • 10 – 60 mL total CSF

SPECIMEN COLLECTION AND HANDLING

SPECIMEN COLLECTION •  Collection is made by lumbar puncture between the 3rd, 4th or 5th

lumbar vertebra •  Gloves and face shield should be worn during collection and

processing •  Fluid for centrifugation must be with capped tubes

•  Specimen is collected in sterile tubes labeled 1,2 and 3 in the order in which they are withdrawn •  Tube 1 – for chemical and serologic test; least affected by blood

or bacteria •  Tube 2 – f or microbiology laboratory •  Tube 3 – for cell count; least likely to contain cells induced by the

spinal tap •  Tube 4 – (optional) for microbio lab to provide better exclusion of

skin contamination or for additional serologic tests

•  Ideally, CSF test are done on a STAT basis, if not, specimens should be kept in the ff. manner: •  Hematology tubes - refrigerated •  Microbiology tubes - room temp •  Chemistry & serology tubes - frozen

VIDEO

APPEARANCE OF CSF

•  Crystal clear - normal •  Cloudy, turbid or milky

•  Increased lipid or protein concentration •  Infection (presence of WBCS)

•  Xanthochromic (pink, orange or yellow CSF) •  Presence of RBC degradation products •  Very slight amount of oxyhemoglobin (pink) •  Heavy hemolysis (orange) •  Unconjugated bilirubin (yellow) •  Other causes

•  Elevated serum bilirubin •  Presence of pigment carotene •  Increased protein concentrations •  Melanoma pigment

•  Oily – caused by radiographic contrast media •  Bloody – caused by RBCs due to hemorrhage or

traumatic tap •  Clotted and pellicle

•  Protein – disorders affecting the blood brain barrier •  Clotting factors – introduced by traumatic tap

(clotted), tubercular meningitis (pellicle) • 

TRAUMATIC COLLECTION VS. INTRACRANIAL HAEMORRHAGE

Trauma&c  collec&on  (Tap)   Intracranial  hemorrhage  

Defini&on   Bloody  CSF  due  to  puncture  of  a  blood  vessel  during  spinal  tap  procedure.  

Bloody  CSF  due  to  bleeding  within  the  cranial  vault  which  includes  cerebral  and  subarachnoid  hemorrhage  

Uneven  distribu&on  of  blood  

Heaviest  concentra=on  of  blood  on  tube  1  with  gradually  diminishing  on  tubes  2  and  3.  

Evenly  distributed  on  all  tubes  

Clot  forma&on  

   

CloBng  present  due  to  introduc=on  of  plasma  fibrinogen  

CloBng  is  absent  

Xanthochromic  supernatant  

A  xanthochromic  supernatant  would  be  the  result  of  blood  that  has  been  present  longer  than  2  hours  aFer  trauma=c  tap.  Introduc=on  of  serum  protein  from  a  trauma=c  tap  

   

•  Very  recent  hemorrhage  produce  a  clear  supernatant  

•  Microscopic  finding  of  macrophages  containing  ingested  RBCs  (erythrophagocytosis)  or  hemosiderin  granules  

•  Detec=on  of  the  fibrin  degrada=on  product,  D-­‐dimer,  by  latex  agglu=na=on  immunoassay  indicates  forma=on  of  fibrin  at  a  hemorrhage  site.        

CELL COUNT •  Cell count routinely performed on CSF is the leukocyte

[WBC] count •  RBC counts are determined only when traumatic tap has

occurred and correction for leukocytes and protein is desired •  RBC count = total cell count – WBC count

•  Should be performed immediately because WBCs and RBCs begin to lyse within one hour with 40% of leukocytes disintegrating after 2 hours

METHODOLOGY •  Normal adult CSF = 0-5 WBCs/µL •  Number is higher in children [as many as 30

mononuclear cells/µL] •  Specimens that contain up to 200 WBCs or 400 RBCs/

µL may appear clear, which makes it necessary to examine all specimens microscopically

•  Neubauer counting chamber: routinely used for CSF cell counts

•  Electronic counters aren’t traditionally used: high background counts and poor reproducibility if low counts

Calculation of Cell Counts •  Similar to standard Neubauer calculation formula used

for blood cell counts •  Formula can be used for both diluted and undiluted

specimens

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Total Cell Count •  Clear specimens may be counted undiluted as long as

there is no overlapping of cells •  Calibrated automatic pipettes are used when dilutions

are required •  Dilutions of total cell counts are made with NSS, mixed

by inversion, and loaded into the hemocytometer using a Pasteur pipette

•  Cells are counted in the four corner squares and the center square on both sides of the hemocytometer

WBC Count •  Lysis of RBCs must be obtained prior to counting •  Diluting fluid: 3% glacial acetic acid to lyseRBCs + methylene blue

to stain WBCs, providing better differentiation between neutrophils and mononuclear cells

•  Preparation of a clear specimen that does not require dilution •  Place 4 drops of mixed specimen in a clean tube •  Rinse a Pasteur pipette with 3& glacial acetic acid •  Draw 4 drops of CSF into the pipette and allow to sit for 1 minute •  Mix the solution in the pipette, discard the first drop, and load the

hemocytometer •  Count as in the total cell count •  Multiply the counted cells by the dilution factor

Corrections for Contamination •  Corrects WBCs and protein artificially introduces into the

CSF as a result of a traumatic tap •  Determination of CSF RBC and blood WBC count is

necessary to perform correction

•  Approximate WBC CSF count can be obtained by subtracting the added WBC from the actual count

•  If peripheral blood RBC and WBC are within normal range, subtract 1 WBC for every 700 RBCs in CSF

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Quality Control •  Liquid commercial controls for spinal fluid RBC and WBC counts are

available •  In-house controls are also available •  Biweekly basis: all diluents should be checked for contamination by

examination in a counting chamber under 4x magnification •  Contaminated diluents should be discarded and new solutions must

be prepared •  Monthly basis: centrifuge speed must be checked with a tachometer

and timing should be checked by a stop watch •  Non-disposable counting chambers must be soaked in a bactericidal

solution for at least 15 minutes, thoroughly rinsed with water and cleaned with isopropyl alcohol

DIFFERENTIAL COUNT ON CEROBROSPINAL FLUID SPECIMEN

•  Performed on a stained smear •  Specimen should be concentrated prior to the preparation of the

smear •  Methods for specimen concentration •  Sedimentation – not routinely used, produce less cellular distortion •  Filtration – not routinely used, produce less cellular distortion •  Centrifugation •  Cytocentrifugation •  Specimen is centrifuged for 5 to 10 mins. •  Supernatant fluid is removed and saved for additional tests •  Suspended sediments are air dried and stained with Wright’s stain •  100 cells is counted, classified and reported in terms of percentage

Cytocentrifugation •  Fluid is added to conical chamber, while specimen is

centrifuged, cells in the fluid are forced into a monolayer within a 6-mm diameter circle on the slide.

•  Filter paper absorbs the fluid producing a more concentrated area of cells.

•  0.1 ml CSF added with 1 drop of 30% albumin produces an adequate cell yield for when processed

•  Cells from center and periphery of slide is examined because cellular characteristics may vary between areas of slide

•  Addition of albumin increases the cell yield and decreases cellular distortion

•  Cellular distortions •  Cytoplasmic vacuoles and Nuclear clefting •  Prominent nucleoli •  Indistint nuclear and cytoplasmic borders •  Cellular clumping resembling malignancy •  Daily control slide for bacteria is prepared using

0.2 ml saline and 2 drops of 30% albumin.

Number  of  WBCs  counted  in  chamber  

Number  of  cells  counted  on  cytocentrifuge  slide  

0   0-­‐40  1-­‐5   20-­‐100  6-­‐10   60-­‐150  11-­‐20   150-­‐250  20   250  

•  The table above is used for comparison of the number of WBCs counted in the chamber to the number of cells counted on cytocentrifuge slide.

•  Chamber count should be repeated if too many cells are seen on the slide. •  New slide should be prepared if not enough cells are seen on the slide.

CSF CONSTITUENTS •  Lymphocytes and monocytes commonly found in normal

CSF •  In adults, lymphocytes are predominant to monocytes

(70:30) •  In children, ratio is reversed •  Pleocytosis •  increased normal cells found in CSF •  High CSF WBC count with a majority of neutrophils is

considered indicative of bacterial meningitis •  Moderately elevated CSF WBC count with high

percentage of lmyphocytes and monocytes suggests meningitis of viral, tubercular, fungal, or parasitic origin.

Predominant Cells seen in CSF Cell  type   Major  Clinical  Significance   Microscopic  Findings  

Lymphocytes   Normal  Viral,  tubercular,  and  fungal  

meningi=s  

All  stages  of  development  may  be  found  

Neutrophils   Bacterial  meningi=s  Early  cases  of  viral,  

tubercular,  and  fungal  meningi=s  

Cerebral  hemorrhage  

Granules  may  be  less  prominent  than  in  blood  

Cells  disintegrate  rapidly  

Monocytes   Normal  Viral,  tubercular,  and  fungal  

meningi=s  Mul=ple  sclerosis  

Found  mixed  with  lymphocytes  

Macrophages   RBCs  in  spinal  fluid  Contrast  media  

May  contain  phagocy=zed  RBCs  appearing  as  empty  vacoules  or  ghost  cells,  hemosiderin  granues  

and  hematoidin  crystals  

Blast  forms   Acute  leukemia   Lymphoblasts,  myeloblasts  or  monoblasts  

Lymphoma  cells  

Disseminated  lymphomas  

Resemble  lymphoctyes  with  cleF  nuclei  

Plasma  cells   Mul=ple  sclerosis  Lymphocyte  reac=ons  

Tradi=onal  and  classic  forms  seen  

Ependymal,  choroidal,  and  spindle-­‐shaped  cells  

Diagnos=c  procedures   Seen  in  clusters  with  dis=nct  nuclei  and  dis=nct  cell  walls  

Malignant  cells  

Metasta=c  carcinomas  Primary  central  nervous  

system  carcinoma  

Seen  in  clusters  with  fusing  of  cell  borders  and  nuclei  

CSF REFERENCE RANGE VALUES FOR CYTOCENTRIFUGE COUNTS

CELL TYPE ADULTS, % NEONATES, %

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

Neutrophils •  Bacterial meningitis- neutrophils contain

phagocytized bacteria •  Increased level

•  seen in early stages (1-2 days) of viral, fungal, tubercular and parasitic meningits

•  CNS hemorrhage, repeated lumbar punctures, injection of medications or radiographic dye

•  May contain cytoplasmic vacuoles after cytocentrifugation

•  Little clinical significance •  Pyknotic nucleii •  resembles nRBC but with multiple

nucleii •  indicate degenerating cells

Lymphocytes and Monocytes •  Common in cases

of viral, tubercular, and fungal meningitis

•  Increased level •  Asymptomatic HIV

infection and AIDS

•  Reactive lymphocytes with dark blue cytoplasm and clumped chromatin present during viral infections in conjuction with normal cells

•  Moderately elevated WBC count (less than 50 WBC/ul) with increased normal and reactive lymphocytes and plasma cells may be an indicative of multiple sclerosis

Eosinophils •  Increased level •  Parasitic infections •  Fungal infections

(Coccidioides immitis) •  Introduction of foreign

material

Macrophages •  Purpose in CSF

• Remove cellular debris and foreign objects such as RBCs

•  Appear w/in 2-4 hours after RBCs enter the CSF and frequently seen in following repeated taps

•  More cytoplasm than monocytes in peripheral blood

•  Increased level •  Indicative of previous hemorrhage

•  Degradation of phagocytized RBCs result in appearance of dark blue or black iron-containing hemosiderin granules

•  Yellow hematoidin crystals represent further degradation •  Iron-free, consisting of hemoglobin and

uncjugated bilirubin

Hemosiderin-laden macrophages (siderophages) from the cerebrospinal fluid of a patient with subarachnoid hemorrhage. Hemosiderin crystals (golden-yellow) are also present.

Nonpathologically Significant Cells •  Most frequently seen in

•  Pneumoencephalography •  Fluid obtained from ventricular taps •  Neurosurgery

•  Cells often appear in clusters and distinguished from malignant cells by uniform appearance

Choroidal Cells •  From epithelial lining

of choroid plexis •  Singularly or in

clumps •  Nucleoli are usually

absent and nuclei have uniform appearance

Ependymal Cells •  From lining of

ventricles and neural canal

•  Less defined cell membranes and frequently seen in clusters

•  Nucleoli usually present

Spindle-Shaped Cells •  Represent lining cells

from arachnoid •  Usually seen in

clusters and seen with systemic malignancies

MALIGNANT CELLS OF HEMATOLOGIC ORIGIN •  Acute leukemias

•  Lymphoblasts •  Myelobasts •  Monoblasts

•  Nucleoli are more prominent than in blood smears •  Lymphoma cells

•  Indicates dissemination from lymphoid tissue •  Resemble large and small lymphocytes •  Usually appear in clusters of large, small, or mixed cells based

on the classification of the lymphoma •  Nuclei may appear cleaved and prominent nucleoli are present

Acute lymphoblastic leukemia in cerebrospinal fluid. Note uniformity of the blast cells.

Acute myeloid leukemia in cerebrospinal fluid

Burkitt’s lymphoma in cerebrospinal fluid. The cells are characterized by blue cytoplasm with vacuoles and slightly clumped chromatin pattern

CHEMISTRY TESTS

•  CSF is formed by filtration of plasma •  Normal values for CSF chemicals are

not the same as the plasma values due to: • Selective filtration process • Blood Brain barrier controls

chemical composition

•  Abnormal values result from: •  alterations of the blood brain barrier •  increased production or metabolism by

the neural cells in response to pathologic condition

•  Seldom have the same diagnostic significance as plasma abnormalities

CEREBROSPINAL PROTEIN •  Protein determination is the most frequently

performed chemical test on CSF •  Normal CSF contains very small amount of

protein •  Normal Values for Total CSF Protein:

•  15 to 45 mg/dL •  Values are method dependent •  Higher values found in infants and older

persons

•  CSF Protein fractions: •  Major CSF protein: Albumin •  Second most prevalent fraction: Prealbumin •  α-globulins: Haptoglobin and Ceruloplasmin •  Major β- globulin: Transferrin •  “Tau”- a separate carbohydrate deficient transferrin

fraction •  CSF gamma globulins:

•  Primarily IgG •  Small amount of IgA

•  IgM, Fibrinogen and β- lipoprotein not found in normal CSF

Cerebrospinal Fluid and Serum Protein Correlations

CSF    (mg/dL)  

Plasma    (mg/dL)  

Plasma/CSF  Ra&o  

Prealbumin   1.7   23.8   14  Albumin   15.5   36.00   236  

Ceruloplasmin   0.1   36.6   366  Transferrin   1.4   204   142  

IgG   1.2   987   802  IgA   0.13   175   1346  

Clinical Significance of Protein Values •  Elevated total Protein values seen in pathologic

conditions •  Abnormally low values:

•  Fluid is leaking from CNS •  Elevated CSF protein caused by:

•  Damage to the blood brain barrier •  Production of immunoglobulins within CNS •  Decreased clearance of normal protein from

the fluid •  Degeneration of neural tissue

•  Meningitis and hemorrhage conditions • Damage blood brain barrier • Most common causes of CSF

protein elevation •  Other causes of elevated CSF protein

• Neurologic conditions

Clinical Causes of Abnormal CSF Protein Values

Elevated  Results  

•  Meningi=s  •  Hemorrhage  •  Primary  CNS  tumors  •  Mul=ple  Sclerosis  •  Guillain-­‐Barré    Syndrome  •  Neurosyphilis  •  Polyneuri=s  •  Myxedema  •  Cushing  disease  •  Connec=ve  Tissue  disease  •  Polyneuri=s  •  Diabetes  •  Uremia  

Decreased  Results  

• CSF  leakage/trauma  • Recent  Puncture  • Rapid  CSF  produc=on  • Water  Intoxica=on  

Artificially-Induced Plasma proteins •  Plasma protein

•  can be artificially introduced into a specimen by a traumatic tap in the same manner as blood cells

•  Correction Calculation •  Similar to that used in cell counts •  Available for protein measurements •  If to be used, both the cell count and the protein

determination must be done on the same tube •  Normal blood hematocrit and serum protein values

•  Subtracting 1 mg/dL of protein is acceptable

Methodology •  Two most routinely used techniques for

measuring Total CSF Protein use the principles of: •  Turbidity production • Dye-binding ability

•  Nephelometry •  Automated instrumentation form of

turbidity method

Protein Fractions •  Measurement of individual Protein Fractions

•  Required in diagnosis of neurologic disorders associated with abnormal CSF protein

•  Appearance of Protein in CSF as a result of damage to integrity of Blood Brain barrier •  Contains fractions proportional to those in plasma •  Albumin present in highest concentrations

•  Higher proportion of IgG •  Shown by diseases (including Multiple Sclerosis) that

stimulate the immunocompetent cells in the CNS

•  Comparison of Serum and CSF levels of Albumin and IgG •  To accurately determine whether IgG is increased

because: •  It is being produced within the CNS or •  Elevated as the result of a defect in the blood brain

barrier •  CSF/serum albumin index

•  Evaluate integrity of the blood brain barrier •  CSF IgG index

•  Measure IgG synthesis within CNS

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•  Index value less than 9 •  Represents an intact blood brain barrier

•  Index increases relative to the amount of damage to the barrier

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•  Normal IgG index •  Vary slightly among laboratories

•  Values greater than 0.70 •  Indicative of IgG production in CNS

ELECTROPHORESIS • Method of choice when

determining if fluid is actually CSF • Appearnace of Tau – extra isoform of transferrin found only in CSF

•  Purpose of CSF Protein Electrophoresis • Detection of Oligoclonal bands

• Oligoclonal bands represent Inflammation within CNS

•  bands located in gamma region of protein electrophoresis •  indicates immunoglobulin production

•  Simultaneous Serum Electrophoresis •  Ensure that the oligoclonal bands are present as a

result of neurologic inflammation •  Leukemia, Lymphoma and Viral infections

•  may produce serum banding which can appear in CSF as a result of

•  blood brain barrier leakage •  traumatic introduction of blood into CSF

specimen •  HIV Infection

•  Banding representing both systemic and neurologic involvement

•  seen in serum and CSF

•  Multiple sclerosis •  Presence of two or more oligoclonal bands in CSF not present in

serum •  Increased IgG index

•  Other neurologic disorders that produce Oligoclonal banding not present in serum: •  Encephalitis •  Neurosyphilis •  Guillain-Barre Syndrome •  Neoplastic disorders

•  Oligoclonal banding remains positive during remission of multiple sclerosis but disappears in other disorders

•  Agarose Gel Electrophoresis followed by Coomassie brilliant blue staining •  Most frequently performed

•  Immunofixation electrophoresis (IFE) and Isoelectric focusing ( IEF) followed by Silver staining •  Better resolution

MYELIN BASIC PROTEIN •  Present in CSF in demyelinating diseases such as

Multiple sclerosis •  Normal levels in CSF is less that 4 ng/mL •  In acute exacerbations of multiple sclerosis, MBP levels

can be in excess of 8 ng/mL •  Increased in conditions such as:

•  Trauma to the head •  Hypoxia •  Myelopathy •  Intrathetical administration of chemotherapy

CSF GLUCOSE •  Glucose

• Enters the CSF by selective transport across the blood brain barrier

• Normal value: 60 % to 70 % that of the plasma glucose

•  If plasma glucose is 100 mg/dL, then CSF glucose is approximately 65 mg/dL

•  Blood Glucose Test • Must be run for comparison in order to

determine an accurate evaluation of CSF glucose

•  Blood should be drawn about 2 hours prior to the spinal tap which allows time for equilibration between the blood and fluid

• CSF glucose analyzed using the same procedures employed for blood glucose

• Specimens should be tested immediately because Glycolysis occurs rapidly in CSF

•  Elevated CSF glucose values • Result of plasma elevations

•  Decreased/Low CSF glucose values •  Primarily caused by

• alterations in the mechanisms of glucose transport across the blood brain barrier

• Increased use of glucose by the brain cells

Considerable diagnostic value in determining the causative agents of meningitis

•  Bacterial Meningitis •  Markedly decreased CSF glucose •  Increased WBC count •  Large percentage of neutrophils

•  Tubercular Meningtits •  WBCs are Lymphocytes instead of neutrophils

•  Viral Meningitis •  Normal CSF Glucose •  Increased number of Lymphocytes

Major Laboratory Results for the Differential Diagnosis of Meningitis

Bacterial   Viral   Tubercular   Fungal  Elevated  

WBC  Count  Elevated  

WBC  count  Elevated  

WBC  count  Elevated  

WBC  count  Neutrophils  present  

Lymphocytes  present  

Lymphocytes  and  

Monocytes  present  

Lymphocytes  and  

Monocytes  present  

Marked  protein  eleva=on  

Moderate  Protein  eleva=on  

Moderate  to  marked  protein  eleva=on  

Moderate  to  marked  

Bacterial   Viral   Tubercular   Fungal  Lactate  level  >  35  mg/dL  

Normal  lactate  level  

Lactate  level  >  25  mg/dL  Pellicle  

forma=on  

Lactate  level  >  25  mg/dL  

Posi=ve  India  ink  with  

Cryptococcus  neoformans  

Posi=ve  gram  stain  and  bacterial  

an=gen  test  

Posi=ve  immunologic  test  for  C.  neoformans  

Markedly  decrease  

glucose  level  

Normal  glucose  level  

Decrease  glucose  level  

Normal  to  decreased  glucose  level  

Cerebrospinal Fluid Lactate •  CSF Lactate Levels

•  Valuable aid in the diagnosis and management of meningitis cases

• Greater than 25 mg/dL • Bacterial, tubular and fungal

meningitis • Greater than 35 mg/d:

• Bacterial meningitis

•  Lower than 25 mg/dL • Viral meningitis

•  CSF lactate levels remain elevated during treatment

•  Levels fall rapidly when treatment is successful

•  Offers a sensitive method for evaluating the effectiveness of antibiotic therapy

•  Frequently used to monitor severe head injuries

•  Increased CSF Lactic Acid levels •  Caused by destruction of tissue within CNS owing to

oxygen deprivation (Hypoxia) •  Not limited to meningitis •  Can result from any condition that decreases the

oxygen flow to tissues •  Falsely elevated results

•  Obtained on Xanthochromic or hemolyzed fluid •  RBCs contain high concentrations of lactate

Cerebrospinal Fluid Glutamine •  Glutamine

•  Produced from ammonia and α-ketoglutarate by the brain cells

•  Production serves to remove the toxic metabolic waste product ammonia from the CNS

• Normal concentration in CSF: 8 to 18 mg/dL

•  Elevated values •  Found in association with Liver

disorders that result in increased blood and CSF ammonia

•  Increased glutamine synthesis is caused by excess ammonia in CNS

• Glutamine levels more than 35 mg/dL • Disturbance of consciousness is seen

•  75 % of children with Reye Syndrome

•  Determination of CSF glutamine levels •  Indirect test for the presence of excess

ammonia in CSF •  Preferred over the direct measurement

of CSF ammonia because the glutamine concentration remains more stable than the concentration of volatile ammonia in collected specimen

• Correlates with clinical symptoms much better than does the blood ammonia

•  As CSF ammonia concentration increases, supply of α-ketoglutarate decreases • Glutamine can no longer be produced to

remove toxic ammonia • Coma ensues

•  CSF Glutamine Test • Requested procedure for patients with

coma of unknown origin

SUMMARY OF CSF CHEMISTRY TESTS

Protein   1.  Normal  concentra=on  is  15  to  45  mg/dL  2.  Elevated  values  are  most  frequently  seen  in  

pa=ents  with  meningi=s,  haemorrhage  and  mul=ple  sclerosis.      

Glucose      

1.  Normal  value  is  60%  to  70%  of  the  plasma  concentra=on.  

2.  Decrease  levels  are  seen  in  pa=ents  with  bacterial,  tubercular,  and  fungal  meningi=s.      

Lactate      

1.   Levels  >  35  mg/dL  are  seen  in  pa&ents  with  bacterial  meningi&s  

2.   Levels  >  25mg/dL  are  found  in  tubercular  and  fungal  meningi&s.  

3.   Lower  levels  are  seen  in  pa&ents  with  viral  meningi&s.  

Glutamine      

1.  Normal  concentra=on  is  8  to  18  mg/dL  2.  Levels  >  35  mg/dL  are  associated  with  some  

disturbance  of  consciousness.      

MICROBIOLOGY TEST

•  The role of microbiology laboratory in the analysis of CSF •  Identification of the causative agent in the

meningitis. •  For positive identification, the microorganism

should be: •  Recovered from the fluid by growing it on the

appropriate culture medium, can take for 24 hours in cases of bacterial meningitis to 6 weeks for tubular meningitis. CSF culture is a confirmatory than a diagnostic procedure.

•  Methods of Preliminary Diagnosis in a Microbiology Lab: • Gram Stain • Acid Fast Stain •  India ink preparation • Latex agglutination test.

GRAM STAIN •  Routinely performed on CSF from all

suspected cases of meningitis. •  All smears and culture should be

performed in concentrated specimens because often only a few organisms are present in the onset.

•  CSF should be centrifuged at 1500g for 15 minutes

ACID FAST STAIN •  Used in the detection of Mycobaterium tuberculae

INDIA INK PREPARATION • Performed on CSF to detect the

presence of thickly encapsulated Cryptococcus neoformans.

• Now commonly encountered in the clinical laboratory.

LATEX AGGLUTINATION TEST •  Detects the presence of C. neoformans in

the serum and CSF provide a more sensitive method of India Ink preparation.

•  Interference of rheumatoid factor causes false positive results.

•  Done with ELISA (Enzyme Linked Immunoassay) to produce fewer false positive results and provides a rapid detection of microorganisms in the CSF.

SEROLOGIC TESTING

•  This is done to detect the presence of neurosyphilis. • Use of Penicillin reduced the number of

neurosyphilis cases. •  Other Serologic tests for Syphilis: VDRL,

FTA-ABS. •  The purpose of doing a test for Syphilis on

the CSF is to detect active cases of Syphilis within the CNS.

TEACHING CEREBROSPINAL FLUID ANALYSIS

• Many of the problems that occur in CSF analysis are: • Inadequate training of the personnel performing the tests.

• Inadequate fluid left for students to practice

• More satisfactory results with using Simulated Spinal Fluid Procedure • Provides the teaching laboratory with a specimen suitable for all types of cell analyses and glucose and protein determinations

• Advantages • absence of bicarbonate

• Bicarbonate causes bubbling with acidic diluting fluids

• Absence of calcium • Calcium prevents clot formation when blood is added

• Stability of 38 hours under refrigeration

• No distortion of cell morphology • Presence of glucose and protein