clmc 1 lecture: cerebro spinal fluid
DESCRIPTION
what you need to know when examining Cerebro-Spinal Fluid. (video not included)TRANSCRIPT
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• Specimen is collected in sterile tubes labeled 1,2 and 3 in the order in which they are withdrawn • Tube 1 – for chemical and serologic test; least affected by blood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
!!!!!!!!!!Number!of!cells!counted!x!dilution!Number!of!squares!counted!x!vol!of!1!square = !"##$/!"!
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
!"#!(!""#") = WBC! Blood !!!"#!(!"#)RBC!(Blood) !
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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!!")
(!"#!!"#$%&'!(!"!" )/(!"#$%!!"#$%&'!(!!")
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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