2.
Flows through the subarachnoid space
CHAPTER 10: located between the arachnoid and pia
mater
CEREBROSPIN 3. Reabsorbed back into the blood
capillaries in the arachnoid
AL FLUID granulations/villi at a rate equal to its
production
BLOOD-BRAIN BARRIER
CEREBROSPINAL FLUID used to represent the control and
filtration of blood components to
First recognized by Cotugno (1764) the CSF and then to the brain
Functions: Composed of capillary endothelium
held together by tight junction and
To supply nutrients to the nervous tissue choroid plexuses
Removes metabolic wastes Allows essential metabolites to pass
from the blood to the CNS
Produces a mechanical barrier to cushion Blocks most molecules >500
the brain and spinal cord against trauma daltons
The brain and spinal cord is lined by SPECIMEN COLLECTION AND
meninges which has 3 layers: HANDLING
• Dura mater o Methods of Collection
outermost layer that lines the skull and Lumbar Puncture
vertebral canal routine method for CSF collection
• Arachnoid mater between 3rd, 4th or 5th lumbar
vertebrae
middle layer, a filamentous inner patient is in fetal position or lateral
membrane decubitus position
• Pia mater Other methods:
innermost layer, a thin membrane lining Ventricular Puncture
the surfaces of the brain and spinal cord used in infants with open fontanels
Cisternal Puncture
1. Produced in the choroid plexuses of the collected in the sub-occipital region
two lumbar ventricles and the third and Lateral Cervical puncture
fourth ventricles
ORDER OF PRESERVATI
COLLECTIO ON
N
Tube Chemistry Frozen formation proteins meningitis
1 and Serology and
Tube Microbiology Room clotting
2 Temperature factors
Tube Hematology/ Refrigerated Xanthochr Hemoglob Old
3 Cell Count omic in hemorrhage/lys
Bilirubin ed cell from
Carotene traumatic tap
Normal Values for CSF
Protein RBC;
Color: colorless Melanin degradation/ele
Viscosity: same with water vated serum
Clarity: crystal clear bilirubin levels;
Specific gravity: 1.006-1.008 Increased
serum levels;
pH: 7.30-7.45
Disorders
Pressure: 50-200 mL H20
affecting BBB;
APPEAR CAUSE MAJOR Melanin;
ANCE SIGNIFICANC melanosarcoma
E Oily Radiograp
Slightly WBCs Meningitis hic
Hazy contrast
Cloudy/ WBCs Meningitis, media
Turbid/ microorga disorders
Milky nism, affecting BBB,
Xanthochromia
increased production of
proteins or IgG within the termed used to describe CSF
lipid CNS supernatant that is pink, orange or
concentrat yellow
ion most common cause:
Clotted Increased Froin's disease Pink = very slight amount of
proteins oxyhemoglobin
and Orange = heavy hemolysis
clotting Yellow = conversion of
factors oxyhemoglobin to unconjugated
Bloody RBCs Non- bilirubin
pathologic:
traumatic tap Chemical Examination
Pathologic: o CEREBROSPINAL PROTEIN
hemorrhage
Pellicle Increased Tubercular most frequently performed chemical test
on CSF
Normal value: 15-45 mg/dL Rapid CSF production
* values are higher in infants and older Recent puncture
persons
Water intoxication
Proteins normally found in CSF:
Methods:
Albumin
1. Qualitative Tests
PrealbuminHaptoglobin and
Tests Reagent Positive
Ceruloplasmin
Nonne-Apelt Ammonium sulfate Cloudy p
Transferrin
Rose Jones Ammonium sulfate White
IgG with small amounts of IgA
Proteins not normally found in CSF: Pandy's Phenol Blueish
IgM, fibrinogen and beta lipoprotein Nogochi 10% butyric acid Precip
Colloidal Gold Test Colloidal gold
Elevated Results solution
Meningitis
Hemorrhage 2. Quantitative Tests
Turbidimetric
Primary CNS tumors
precipitation of protein using:
Multiple sclerosis Trichloroacetic acid (TCA) - reagent
of choice, precipitates both albumin
Guillain -Barre syndrome
and globulin
Neurosyphilis Sulfosalicylic Acid (SSA) -
precipitates albumin unless comine
Polyneuritis with sodium sulfate.
Myxedema Dye-Binding Technique
protein error of indicators
Cushing disese
advantages:
Connective tissue disease Requires smaller sample size
Less interference from
Polyneuritis external sources
Stains used: Coomassie Brilliant
Diabetes
Blue G250 and Ponceau S
Uremia Nephelometry
Decreased Results uses benzalkonium chloride
Protein Fractions
CSF leakage/trauma
To accurately determine whether IgG provide a valuable measure of the
is increased because it is being effectiveness of current and future
produced within the CNS or is treatments
elevated as the result of a defect in Radioimmuno diffusion
the blood—brain barrier, com -
parisons between serum and CSF CSF GLUCOSE
levels of albumin and IgG must be Normal value: 60-70% of plasma
made. glucose
CSF / Serum Albumin Index blood glucose should be drawn 2
to evaluate the integrity of the hours prior to spinal tap
bloodbrain barrier specimens should be tested
CSF/serum albumin index = CSF immediately because glycolysis
albumin (mg/dL) / Serum albumin occur rapidly in CSF
(g/dL) Clinical Significance
CSF lgG Index
measures IgG synthesis within the Increased:
CNS Result of plasma glucose
lgG index = CSF lgG elevations
(rng/dL)/serum lgG (g/dL) / CSF a DM, encephalitis and conditions
bumin
associated with intracranial
(nWdL)/serum albumin (g/dL)
pressure
Electrophoresis Decreased:
to detect oligoclonal bands Aids in determining the
Multiple Sclerosis causative agent of meningitis
presence of two or more Alterations in the
oligoclonal bands in the mechanisms of glucose
CSF that are not present in transport across BBB
the serum part when Increased glucose use by
accompanied by an brain cells
increased IgG index CSF GLUTAMINE
Other disorders with oligoclonal bands Normal value:
but not in serum: Encephalitis, chemical test frequently
neurosyphilis, Guillain-Barre syndrome and performed in CSF but not in
neoplastic disorders blood
Myelin Basic Protein used an indirect measure of CSF
presence in the CSF indicates recent ammonia
destruction of the myelin sheath that
As CSF ammonia increase --->
protects the axons of the neurons
alphaketoglutarate decreases =
(demyelination)
Coma
used to monitor course of multiple
sclerosis
Elevated: Liver disorder that - Any cell count should be done
results in increased blood immediately
ammonia and CSF ammonia
- WBCs and RBCs begin to lyse
-
Coma of unknown within Ihr.
-
Reye's syndrome
- 40 % of leukocytes disintegrate
*Disturbance in consciousness
occurs when levels are after 2 hrs
>35mg/dL WBC COUNT
CSF LACTATE Clarity Dilution
Normal value: 10-24 mg/dL
Slightly hazy 1:10
aids in diagnosing and managing
meningitis cases Hazy 1:20
sensitive method for evaluating Slightly cloudy 1:100
the effectiveness of antibiotic Slightly bloody 1:200
therapy or Cloudy
used to monitor severe head Bloody or 1:10000
injuries Turbid
• LD ISOENZYMES
- help diagnose meningitis by RBC Count
confirming the needed presence of PMN - done only when there is traumatic tap
and lymphocytes and correction for leukocytes or proteins is
needed
Increased LD Condition
Isoenzymes - RBC count = Total cell count — WBC count
LDI and LD2 Brain tissue
destruction Total Cell Count
LD2 and LD3 Viral meningitis cells arc counted in the 4 corner
LD4 and LD5 Bacterial meningitis squares and the center square of
the hemocytometer
Pasteur pipet is used to load the
• Microscopic Examination hemocytometer
o Counting Chambers used:
CELL COUNT
1. Improved Neubauer counting
chamber
- Specimens that contain up to
# of cells x Dilution / # of cells
200 WBCs or 400 RBCs/uL may
counted x Vol. of I square =
appear clear so it is necessary to
cells/uL2.
examine all specimens
microscopically.
2. Fuchs-Rosenthal counting albumin produces adequate cell
chamber yield
Addition of albumin increases
• Quality Control of CSF and other cell yield and decreases cellular
Body Fluid Cell Counts distortion frequently seen on
All diluents should be checked cytocentrifuged specimens
biweekly for contamination by o Centrifugation
examining them in a counting
specimen is centrifuged for 5 to
chamber under 400x magnification.
10 minutes
Contaminated diluents should be
supernatant fluid is removed and
discarded and new solutions be
stored for additional tests
prepared.
slides made from the suspended
The speed of the cytocentrifuge
sediment are allowed to air dry
should be checked monthly with a
and are stained with Wright's
tachometer.
stain
The timing of cytocentrifuge should
• CSF Cellular Constituents
be checked with a stopwatch.
Normal:
If non-disposable counting chambers
are used, they must be soaked in a Lymphocytes
bactericidal solution for at least 15 Monocytes
minutes and then thoroughly rinsed Neutrophils
with water and cleaned with
isopropyl alcohol after each use. Abnormal:
Immature WBCs, eosinophils,
• DIFFERENTIAL COUNT plasma cells, macrophages,
increased tissue cells and
specimen should be concentrated
malignant cells
and performed on a stained smear
Neutrophils
100 cells should be counted,
classified and reported in percentage contains cytoplasmic vacuole
following cytocentrifugation
If <100 cells = report only the
numbers of the cell types seen Increased ff:
• Methods of Specimen Concentration - CNS hemorrhage, repeated
o Sedimentation lumbar punctures and injection
o Filtration of medications or radiographic
o Cytocentrifugation dye
Pyknotic nuclei: indicate
As little as 0. 1mL of CSF
degenerating cells; may resemble
combined with 1 drop of 30%
nucleated RBCs but usually have pneumoencephalography and in
multiple nuclei fluid obtained from ventricular
Lymphocytes taps or during neurosurgery
- Reactive lymphocytes appear in clusters and can be
containing increased dark blue distinguished from malignant
cytoplasm and clumped cells by their uniform appearance
chromatin are frequently present Choroidal cells
during viral infection in
- seen singularly and in clumps
conjunction with normal cells
- Increased lymphocytes are seen - nucleoli are usually absent, and
in cases of both asymptomatic nuclei ependymal, choroid plexus,
HIV infection and AIDS lymphoma, and have a uniform
- A moderately elevated WBC appearance
count (less than 50 WBCs/u L) Ependymal Cells
with increased normal and - have less defined cell membranes
reactive lymphocytes and plasma and are frequently seen in clusters
cells may indicate multiple - nucleoli are often present
sclerosis or other degenerative Spindle-shaped cells
neurologic disorders.
- usually seen in clusters and seen
Eosinophils
with systemic malignancies
- Increased eosinophils are seen
in the CSF in association with • Malignant Cells of Hematologic
parasitic infections, fungal Origin
infections and introduction of Lymphoblasts, myeloblasts, and
foreign material, including monoblasts in the CSF are frequently
medications and shunts into the seen as a serious complication of
CNS acute leukemias.
Macrophages Lymphoma cells
- remove cellular debris and - resemble large and small
foreign objects such as RBCs lymphocytes and usually appear in
- appear within 2 to 4 hours after clusters of large, small, or mixed
RBCs enter the CSF and are cells based on the classification of
frequently seen following the lymphoma
repeated taps.
- nuclei may appear cleaved, and
- finding of increased
prominent nucleoli are present
macrophages indicates a previous
hemorrhage • Malignant Cells of Nonhematologic
Nonpathologically Significant Origin
Cells - primarily from lung, breast, renal, and
- most frequently seen after gastrointestinal malignancies
diagnostic procedures such as
- Cells from primary CNS tumors Latex Agglutination Test and ELISA
include astrocytomas, retinoblastomas, - detection of Streptococcus group B, H.
and medulloblastomas influenzae Type B, S. pneumoniae, N.
- usually appear in clusters and must be meningitidis A, B, C, Y, W135, M.
distinguished from normal clusters of tuberculosis, Coccidioides immitis, and E.
cells coli K1 antigens
o Bacterial Antigen Test (BAT)
Microbiologic Examination
should be used in combination
with results from the
Gram Stain hematology and clinical
chemistry laboratories for
routinely performed on CSF in all
diagnosing meningitis
cases of meningitis
o Reverse Latex Agglutination
all smears and cultures should be
performed on concentrated detects C. neoformans antigen in
specimens serum and CSF
CSF should be centrifuged at 1500 g more sensitive than indian ink
for 15 minutes False (+): Interference of
Organisms most frequently encountered: rheumatoid factor
Gram (+) o Lateral Flow Assay (LAF)
S. pneumoniae provide a rapid method for
S. agalactiae and L. monocytogenes detecting C.
(newborns) neoformans
Gram (-) o Limulus Lysate Test
H. influenzae, E. coli and N diagnosis of meningitis caused
meningitides by gram (-) organisms by
detecting endotoxins found in
Blood Culture their cell walls
done because the causative agent will be uses blood cells of the horseshoe
in both blood and CSF crab termed as 'amebocytes"
Acid Fast/ Fluorescent Antibody Stains which contains copper complex
for Tubercular meningitis responsible for the blue color
Indian Ink coagulates the amebocyte lysate
within 1 hour at 37 degrees
detects the presence of Cryptococcus
celcius
neoformans
Serologic Examination
o VDRL - recommended for CSF by CDC
to diagnose syphilis due to its ability to
detect active cases of syphilis
o RPR - not recommended for use on CSF
because it is less sensitive and less specific
than the VRDL
o FTA-ABS - used with care to avoid
contamination with blood because it
remains positive in the serum of
treated cases of syphilis
