CSF Flashcards
It is the major fluid in the body that surrounds the brain and spinal cord.
CEREBROSPINAL FLUID
Main function of CSF
protect brain and spinal cord by
acting as a lubricant, cushion for transport of nutrients, and
metabolic waste management.
Three main reasons of why we perform CSF in lab:
- To evaluate the function of central nervous system
- It indicates presence of infections, autoimmune disorders,
diseases of the brain and spinal cord - It can also detect early signs of seizures and dementia
is the infection of the membrane
surrounding our brain and spinal cord. E.g.,
bacterial, fungal, and viral meningitis.
Meningitis
Two main sources of CSF:
Choroid plexus (70%)
Subarachnoid space (30%)
lines the choroid plexus which is responsible for the formation of blood-brain barrier.
Choroidal cells (epithelial cells)
regulates the movement of ions, molecules, and cells between the blood and brain.
Blood brain barrier
Three Layers of meninges that protect the brain and spinal cord
Dura – outermost “hard mother”
Arachnoid – middle “spiderweb-like”
Pia – innermost “gentle mother
Three Types of Epithelial Cells
Ependymal cells
Choroidal cells
Pia Arachnoid Mesothelial cells (PAM)
lines the cerebral ventricles and pleural canals of the spinal cord. Range in shape from squamous to columnar. May be ciliated which line the central cavities of the brain and spinal column. Separate the CNS interstitial fluid from the cerebrospinal fluid in the
cavities.
Ependymal cells
main source of CSF formation
Choroidal cells
lines the mesoderm of pia and arachnoid mater.
Pia Arachnoid Mesothelial cells (PAM)
CSF ionic components such as H, K, Ca, bicarbonates, and Mg are tightly regulated by a?
specific transport system.
T or F
Glucose, urea, and creatinine diffuse freely and require 2 or more hours to regulate.
T
Volume of CSF each day
500 mL of CSF
Range of 500 mL CSF/day
0.3 to 0.4 mL/min.
Range of Adult volume of CSF
90 –150 mL
? mL in the ventricles and subarachnoid space (adult volume range)
25 mL
Neonates volume range of CSF
10 – 60 mL
Total CSF volume is replaced every - hours because of proper metabolic waste management of our body.
every 5-7 hours
The process or the procedure for obtaining CSF fluid is known as
cisternal or lumbar puncture or spinal tap (through ventricular cannulas or shunts)
The most common site for lumbar puncture
intervertebral space
between your L3 and L4 for adults.
But for pediatric patients L4 and L5.
T or F
only doctors can perform lumbar puncture procedure.
T
Normal Opening pressure for Adults
90 – 180 mm of water
Normal Opening pressure for Obese Patients
250 mm of water
Normal Opening pressure for Infants and children
10 – 100 mm of water attaining
adult range by 6-8 years of age.
if there is >250 mm of water
increased intracranial pressure which imply presence of
intracranial hemorrhage, and tumors.
if there is >200 mm of water
in a relaxed patient, no more than 2.0 mL should
be withdrawn.
When collecting CSF, we are using?
manometer
indicates the pressure because if too much pressure, it can
indicate obesity, intracranial pressure will also imply presence of bacterial infection, meningitis, hemorrhage, and tumors.
Manometer
Define VDRL and Identify what type of test do they use?
Venereal Disease Research Laboratory, test for syphilis
India ink is used for?
Cryptococcus specimens
is the most sterile among the three csf tubes.
Tube 2
Contaminants in tube 2 should be avoided because if there is, it can indicate _____?
false positive results
GROSS EXAMINATION of CSF
Normal?
crystal clear or colorless
GROSS EXAMINATION
Viscosity?
watery
Present of Turbidity and Cloudiness, basaha lang
o White blood cells > 200 uL
o Red blood cells > 400 uL
o Microorganisms
o Radiographic contrast media
o Aspirated contrast media
o Aspirated epidural fat
o Proteins > 150 mg/dL (1.5 g/L)
Bloody/pink Turbidity may indicate?
RBC > 6000 uL
- Subarachnoid and intracerebral hemorrhage
- Cerebral infarct or traumatic spinal tap
- It can be traumatic or hemorrhagic
If hemorrhagic, 3 tubes will turn into what color?
all (3) tubes color is red.
If improperly collected traumatic tap, 3 tubes will turn into what color?
first tube red, second tube light red, and third tube clear/not equal.
Refers to presence of oxidized hemoglobin from lysed red blood cells.
Xanthochromia and Associated Disease
Pink CSF may indicate?
Red blood cells lysis
Orange CSF may indicate?
-Red blood cells lysis | hemoglobin breakdown
- 2-4 hours after subarachnoid hemorrhage and take as
long as 12 hours, peak 24-36 hours and disappears on 4-
8 days.
- Hypervitaminosis A (carotenoids)
Yellow CSF may indicate?
-RBC lysis | hemoglobin breakdown
- Hyperbilirubinemia: develops after 12 hours after
subarachnoid bleed, peaks 2-4 days and persists for 2-4
weeks.
- CSF protein > 150 mg/dL (1.5 g/L)
Yellow green CSF may indicate?
Hyperbilirubinemia (biliverdin)
Brown CSF may indicate?
Meningeal metastatic melanoma
(3) Clot Formation
o Traumatic taps
o Complete spinal block (Froin’s syndrome)
o Suppurative or tuberculous meningitis
(3) Presence of High Viscosity
o Metastatic mucin producing adenocarcinomas
o Cryptococcal meningitis
o Liquid nucleus pulposus
You need to test these 4 parameters to differentiate the type of meningitis present.
PROTEIN
GLUCOSE
WBC POPULATION
LACTATE
MICROSCOPIC EXAMINATION
Identify and give its CV%.
18 large squares (1 mm2 each) depth of 0.2 m
A total volume of 3.6 uL (18 x 0.2) is examined
Fusch-Rosenthal Chamber
CV = 48%
MICROSCOPIC EXAMINATION
Identify and give its CV%.
9 1mm2 square with a depth of 0.1 mm
Neubauer Hemocytometer
CV = 45%
An automated type of cytometry that Yield rapid and reliable WBC and RBC counts
UF – 100 Flow Cytometer
in UF – 100 Flow Cytometer, what tube is utilized for the cell count
tube 3
T or F
In doing cell counting of CSF, you need to do it immediately since your white blood cells may lyse.
T
T or F
Proper storage of CSF just at 37C.
F
just at room temperature. It should not be refrigerated because a lot of elements can be present during refrigeration.
What Department of the laboratory?
common fastidious organisms such as your Hemophilus and your Neisseria which are the main contributors during an infection in CSF specimens cannot live in cold temperatures.
Microbiology Department
T or F
Remain undiluted if the CSF is clear.
T
(2) If the CSF is bloody, dilute it with?
normal saline solution (nss)
glacial acetic acid
for white blood cells to be clearer.
Crystal violets
The dilution of CSF
1:10 or 1:20 dilution if the specimen is cloudy.
remember (basaha lang)
o Charge for the cells to settle.
o Large squares constant measurement 0.1
o Small squares constant measurement 0.004
Normal Leukocyte Count for Adults
0 -5 cells /uL
Normal Leukocyte Count for Neonates
0 – 30 cells/uL
No RBC should be present in CSF (take note) . Unless if present, it is a sign of???
hemorrhage
Correcting leukocytes and protein introduced by?
traumatic tap
refers to the leukocytes added to CSF by traumatic tap. Generally, this formula can be applicable during a traumatic tap.
WBC added
T or F
Differential Counting of CSF specimens is advised
F
(2) Direct smears of the centrifuged CSF sediment are also subject to significant error from?
cellular distortion and fragmentation
The stain used for CSF is the
Wright’s Stain
Recommended method for differential count of all body
fluids.
CYTOCENTRIFUGE
-Rapid, requires minimal training and allows Wright’s staining of air-dried cytospins.
-Cell yield preservation are better than with simple
centrifugation.
CYTOCENTRIFUGE
CYTOCENTRIFUGE
___-___ cells can be concentrated from ____ mL of normal
CSF.
30 – 50 cells can be concentrated from 0.5 mL of normal
CSF.
Familiarize this
Variable artifactual distortions may be seen but can be
minimized when the specimen is fresh, albumin is added to
the specimen (2 drops of bovine serum albumin) and cell
concentration is adjust to about 300 WBC/L prior to
centrifugation.
Too cumbersome
FILTRATION AND SEDIMENTATION
Adults:
Lymphocytes and monocytes Ratio
70:30 ratio (abundant)
Adults:
Neutrophils
2±5
Adults:
Histiocytes, ependymal cells, and eosinophils
RARE
Young children (basaha na largo uy)
- Monocytes – 80% (abundant)
- Lymphocytes – 20±18
- Neutrophils - 3±5
- Histiocytes - 5±4
- Ependymal cells and eosinophils – rare
(3) CSF Examination for tumor cells has the sensitivity to:
o Leukemic patients (70%) – all
o Metastatic carcinoma (20-60%)
o Primary CNS malignancies (30%)
What is present?
It has 4-5 lobes, segmented, granules are present. So, this is a sign of bacterial infection.
Presence of neutrophils
Causes of INCREASED CSF NEUTROPHILS
- Meningitis
- Other infections:
o Cerebral abscess
o Subdural empyema
o AIDS – related CMV radiculopathy - Following seizures
- Following CNS hemorrhage
- Following CNS infarct
- Reactions to repeated lumbar punctures
- Injection of foreign material (methotrexate; contrast media)
- Metastatic tumor in contact with CSF
Presence of lymphocyte and monocyte (please memorize this)
Causes of CSF LYMPHOCYTOSIS
Meningitis
Degenerative (kamo nay basa)
Other inflammatory disorders (kamo nay basa)
cytoplasm and clump chromatins are signs of these WBCs. This can be seen during multiple sclerosis or other degenerative disorders.
Reactive Lymphocytes
Causes of CSF PLASMACYTOSIS
- Acute viral infections
- Guillain-Barre syndrome
- Multiple sclerosis
- Parasitic CNS infections
- Sarcoidosis
- SSPE
- Syphilitic meningoencephalitis
- Tuberculous meningitis
Presence of eosinophils (please memorize this)
Causes of EOSINOPHILIC PLEOCYTOSIS
- Acute polyneuritis
- CNS reactions to foreign material (drugs, shunts)
- Fungal infections
- Idiopathic hypereosinophilic syndrome
- Parasitic infections
Presence of macrophage and Presence of macrophage with hemosiderin granules (please memorize this)
-Macrophages can indicate previous hemorrhage of your CSF.
(2) Nonpathologically Significant Cells
Clustered ependymal cells
Choroid plexus cells in CSF
Cells that line the cerebral ventricles and pleural canal of
your spinal cord.
Clustered ependymal cells
These cells form your blood-brain barrier. This is the main
source of the CSF formation.
Choroid plexus cells in CSF
(2) Malignant Cells
Blast cell in CSF
Medulloblastoma
Commonly found in leukemic patients. Pathologic origin
(lymphoblast, myeloblast, monoblast). Nonpathological
origin include astrocytoma, retinoblastoma, medulloblastoma
Blast cell in CSF
A malignant cell which can indicate metastatic cancers.
Medulloblastoma
CHEMICAL ANALYSIS
Proteins (basaha lang)
-Increased presence of proteins can be seen in
pathologic conditions.
-Decreased proteins can be found in fluid-leakage in CNS
-1% of the plasma level
Normal level of protein in adults:
15 – 45 mg/dL
(#) CSF protein fell rapidly from birth to 6 months of age
(118 - 40 mg/dL), plateaued 3 -10 years (32 mg/dL) and
rose slightly from 10 -16 years of age (41 mg/dL)
Classic Lowry method - __-__mg/dL
24.1 – 48 mg/dL
Trichloroacetic acid-ponceau S method - __-__mg/dL
15 – 49 mg/dL
Biuret method - __-__mg/dL
22.3 – 50.3 mg/dL
3 Types of protein that can be seen in the chemical analysis of CSF
Albumin
Pre-albumin (transthyretin)
Transferrin (beta 2 transferrin or tau)
what protein is high because of its dual synthesis by the liver and choroid plexus.
Pre-albumin (transthyretin)
what protein is present in electrophoresis, it
migrates slowly than in serum owing to cerebral
neuraminidase digestion of sialic acid residues.
Transferrin (beta 2 transferrin or tau).
2 Methodology of Proteins
Turbidimetric methods
Colorimetric methods (protein determination)
(4)Turbidimetric methods
a. Trichloroacetic acid (TCA)
b. Sulfosalicylic acid (SSA)
c. Sodium sulfate for protein precipitation
d. Benzethonium chloride or Benzalkonium chloride
Pros and cons of Turbidimetric methods
Advantages: popular, simple, rapid, and require no special
instrumentation.
Disadvantages: temperature sensitive and require large volumes (0.5 mL)
False protein elevation: using TCA methods in the presence of methotrexate
(2) Colorimetric methods (protein determination)
a. Lowry method
b. Dye binding methods
c. Modified Biuret method
the stain used in Dye binding methods of colorimetric determination of protein
Coomassie Brilliant Blue (CBB), a rapid, highly sensitive,
uses small samples (25 – 50 u/L).
(4) Electrophoretic Techniques
- CSF electrophoresis
- Electroimmunodiffusion
- Immunofixation electrophoresis
- Ig Immunoblotting
CSF electrophoresis characteristics, Basaha lang
- MS: gammaglobulin fraction
- high resolution agarose gel electrophoresis
- MS: discrete populations of IgG
- the oligoclonal bands (kappa and lambda)
- Coomassie brilliant blue (CBB) or paragon violet stains resolve oligoclonal bands in only 5 ug of IgG
- Silver staining – 20-50x sensitive than CBB and be used on unconcentrated CSF
- Isoelectric focusing
- Polyacrylamide gel
- MS – CSF IgG | albumin ratio is > 0.25
Identify what type of Electrophoretic Techniques
* More sensitive than agarose gel electrophoresis
* Does not require CSF concentration
* Provide fewer oligoclonal bands; more diffuse bands
Immunofixation electrophoresis
(8) Other CSF Proteins
Myelin Basic Protein (MBP)
Alpha-2-macroglobulin (A2M)
Beta-2-microglobulin (B2M)
C-Reactive Protein (CRP)
Fibronectin
Beta-Amyloid Protein 42 and Beta 2 Transferrin (Tau)
Protein 14-3-3
Transferrin
Identify what type in Other CSF Proteins
- Seen in MS
- Correlate with CSF leukocyte count, intrathecal IgG synthesis, and CSF | serum albumin concentration quotient.
- Surrogate marker during acute MS exacerbations
Myelin Basic Protein (MBP)
Identify what type in Other CSF Proteins
Increased in pinocytic vesicles which reflects hemorrhage or breakdown of BBB, as occurs in bacterial meningitis.
Alpha-2-macroglobulin (A2M)
Identify what type in Other CSF Proteins
- Part of HLA class I molecule
- >1.8 mg/L = leptomeningeal leukemia or lymphoma
- Marker of neuro-Bechet’s syndrome
- HIV-1 and various malignancies have been associated
Beta-2-microglobulin (B2M)
Identify what type in Other CSF Proteins
- Acute phase reactant marker can indicate
bacterial meningitis esp. during large amounts.
- Differentiating viral (aseptic) meningitis from
bacterial meningitis (increased)
C-Reactive Protein (CRP)
Identify what type in Other CSF Proteins
Cell adhesion allows leukocytes to adhere and pass
through the vascular endothelia and migrate to the
inflammatory site
Fibronectin
Identify what type in Other CSF Proteins
Increased CSF levels of microtubule-associated tau protein
and decreased beta-amyloid protein 42 and presence of neurofibrillary tangles and amyloid plaques = diagnosis of Alzheimer’s disease (AD)
Beta-Amyloid Protein 42 and Beta 2 Transferrin (Tau)
Identify what type in Other CSF Proteins
- Present in Creutzfeldt-Jacob Disease (CJ)
- 2 proteins (130 and 131) have the same amino acid
sequence as protein 14-3-3
- Determined by immunoassay
- False (+) = stroke and meningoencephalitis
Protein 14-3-3
Identify what type in Other CSF Proteins
- CSF leakage following head trauma and recurrent
meningitis makes accurate diagnosis of the leaking
fluid.
- Immunofixation electrophoresis identifies both
- Protein electrophoresis is non-invasive, rapid,
inexpensive, and requires 0.1 mL
Transferrin
(4) Increased Fibronectin indicates?
a. All = poor prognosis
b. Burkitt’s lymphoma
c. Bacterial meningitis
d. Astrocytomas
(2) decreased Fibronectin indicates?
a. Viral meningitis
b. AIDS dementia complex
two types of Transferrin?
a. Beta-1-transferrin – present in body fluids
b. Beta-2-transferin – present only in the CNS;
What type of transferrin that allows the conversion from Beta-1-transferrin by neuraminidase?
Beta-2-transferin
Glucose basaha lang
- Fasting CSF glucose: 50 – 80 mg/dL (2.8 – 4.4 mmol/L)
- CSF glucose comprises 60% of plasma values
- Normal fasting hours is 8 hrs. and results should be compared with plasma following 4 hours fast
- Normal CSF/plasma glucose ratio: 0.3 – 0.9 with
fluctuations of blood levels because of lag in CSF glucose
equilibration time. - decreased 40 mg/dL (2.2 mmol/L) or ratio < 0.3 hypoglycorrhachia
- Increase in presence of glucose or large amounts can
indicate Bacterial, tuberculous, fungal meningitis.
Lactate basaha lang more on ranges
- Children and adults: 9.0 – 26 mg/dL (1.0 – 2.9 mmol/L)
- Newborns first 2 days of life: 10 – 60 mg/dL
- Newborns 3 – 10 days old: 10 – 40 mg/dL (1.1 – 4.4 mmol/L)
- Elevated levels (increased) reflect CNS anaerobic metabolism due to tissue hypoxia.
- Viral meningitis: < 25 mg/dL
- Bacterial meningitis: > 35 mg/dL = cut off value 30-36
Lactate range for adults mg/dL
9.0 – 26 mg/dL (1.0 – 2.9 mmol/L)
Lactate range for Newborns first 2 days of life mg/dL
10 – 60 mg/dL
Lactate range for Newborns 3 – 10 days old mg/dL
10 – 40 mg/dL (1.1 – 4.4 mmol/L)
Lactate value indication for Viral meningitis
< 25 mg/dL
Lactate value indication for Bacterial meningitis
> 35 mg/dL = cut off value 30-36
Conversion factor of lactate
Conversion factor: mg/dL x 0.1110
Elevated levels in existing hepatic encephalopathy
Glutamine
synthesized from ammonia and glutamic acid serves as the means for CNS ammonia removal.
Cerebral glutamine
Normal CSF glutamine levels
8 – 18 mg/dL
MICROBIOLOGICAL EXAMINATION
Basaha lang
Normal storage is at room temperature
Gram’s stain sensitivity ranges from 60 – 90% with greatest sensitivity corresponding to higher concentrations of bacteria (10 5 colony forming units/mL).
Patients with PMNs but no organism on gram’s stain, the more sensitive acridine orange stain may be helpful.
Immunochromatographic membrane assay with streptococcus latex agglutination bacterial antigen on H. influenzae, N. meningitis, and S. pneumoniae.
T or F
Normal storage is not at room temperature (MICROBIOLOGICAL EXAMINATION)
F
Stain used for MICROBIOLOGICAL EXAMINATION
Gram’s stain
Gram’s stain sensitivity ranges from? (%)
60 – 90%
-Not routinely performed in the laboratory
-It has high sensitivity but low specificity
Immunologic studies
Rapid detection for meningitis and detects presence of
neurosyphilis but has a very high chance of producing false
positive and false negative results.
Immunologic studies
(7) But when using immunologic studies in CSF we can use:
o Coagulation techniques
o Counterimmunoelectrophoresis
o ELISA
o FTA-Abs
o Latex agglutination
o Radioimmunoassay
o VDRL
