Urinalysis and Body Fluids CRg
Unit 4
Cerebrospinal Fluid
Overview of Body Fluid Analysis
Laboratory responsibilities
Accurate & timely results
Source of information
Normal values
Reliability of results, effects of medication, etc.
Proper specimen collection and handling
Laboratory exam of body fluids
Physical characteristics
Chemical constituents
Morphologic elements
Culture for microorganisms
Ancillary studies
Cerebrospinal Fluid (CSF)
Composition and formation
CSF is the 3rd major fluid of the body
Adult volume 90-150 mL
Neonate volume 10-60 mL
Cerebrospinal Fluid (CSF)
Produced at the Choroid plexus of the 4 ventricles
by modified Ependymal cells
At rate @20 ml / hr (adults)
Med training says @ 150 ml/day is produced
CSF flows through the
Subarachnoid space
Where a volume of 90 150 ml
is maintained (adults)
Reabsorbed at the
Arachnoid villus /
granulation
to be eventually reabsorbed
into the blood
Cerebrospinal Fluid (CSF)
Blood Brain Barrier
Occurs due to tight fitting endothelial cells that
prevent filtration of larger molecules.
Controls / restricts / filters blood components
Restricts entry of large molecules, cells, etc.
Therefore CSF composition is unlike bloods
** CSF is NOT an ultrafiltrate
Cerebrospinal Fluid (CSF)
Blood Brain Barrier
Essential to protect the brain
Blocks chemicals, harmful substances
Antibodies and medications also blocked
Tests for those substances normally blocked can
indicate level of disruption by diseases: ie
meningitis and multiple sclerosis.
Cerebrospinal Fluid (CSF)
CSF functions
Supplies nutrients to nervous tissues
Removes metabolic wastes
Protects / cushions against trauma
Cerebrospinal Fluid (CSF)
Four major categories of disease
Meningeal infections
Subarachnoid hemorrhage
CNS malignancy
Demyelinating disease
Cerebrospinal Fluid (CSF)
Indications for analysis
To confirm diagnosis of meningitis
Evaluate for intracranial hemorrhage
Diagnose malignancies, leukemia
Investigate central nervous system
disorders
Cerebrospinal Fluid (CSF)
Specimen collection and handling
Routinely collected via lumbar puncture
between 3rd & 4th, or 4th & 5th lumbar
vertebrae under sterile conditions
Intracranial pressure measurement taken
before fluid is withdrawn.
Cerebrospinal Fluid (CSF)
Cerebrospinal Fluid (CSF)
Specimen collection and handling
Tube 1 chemistries and serology
Tube 2 microbiology cultures
Tube 3 hematology
Testing considered STAT
Specimen potentially infectious
Cerebrospinal Fluid (CSF)
Specimen collection and handling
If immediate processing not possible
Tube 1 (chem-sero) frozen
Tube 2 (micro) room temp
Tube 3 (hemo) refrigerated
Cerebrospinal Fluid (CSF)
Appearance
Normal - Crystal clear, colorless
Descriptive Terms hazy, cloudy, turbid, milky, bloody,
xanthrochromic
Often are quantitated slight, moderate, marked, or grossly.
Unclear specimens may contain increased lipids, proteins, cells
or bacteria. Use precautions.
Clots indicate traumatic tap
Milky increased lipids
Oily contaminated with x-ray
media
Cerebrospinal Fluid (CSF)
Appearance
Xanthrochromic Yellowing discoloration
of supernatent (may be pinkish, or orange).
Most commonly due to presence of old blood.
Other causes include increased bilirubin,
carotene, proteins, melanoma
Cerebrospinal Fluid (CSF)
Appearance
Clots indicates increased fibrinogen & usually due to traumatic tap, but may indicate
damage to blood-brain barrier. (see below)
Pellicle formation in refrigerated specimen associated with tubercular meningitis.
Pellicle formation - picture at right (pellicle in L. tube, R is normal)
Milky increased lipids
Oily contaminated with x-ray media
Traumatic collection vs
cerebral hemorrhage
Cerebral hemorrhage
Even distribution of blood in the numbered tubes
Clot formation possible
Xanthrochromic supernatent
RBCs must have been in CSF @ 2+ hours
- D-dimer, fibrin degradation product from
hemorrhage site
Microscopic presence of erythrophages, or
siderophages, Hemosiderin granules
Cerebrospinal Fluid (CSF) - procedures
All specimens should be examined microscopically hematology
Stat priority, RBC lyse in 1 hour, WBC in 2 hrs. Refrigerate if
not able to process immediately.
Electronic counters generally unusable. Manual count
No dilution usually required (use saline if needed)
Standard Neubauer hemacytometer counting chamber
Neubauer hemacytometer / counting chamber
Formula for calculations results in # cells / uL
Count and record cells from both sides of the chamber.
Average the two sides
Multiply by dilution factor (if no dilution is made, this number is 1)
Divide by number of squares counted X volume of each square
Large squares, such as # 1-9 below have volume of 0.1
Small squares in center # 5 have volume of 0.004
ave. # cells counted x dilution
# squares counted x volume of each square
Cerebrospinal Fluid (CSF)
Expected results
Normally 0 RBCs/uL regardless of age
WBCs
Adult up to 5 mononuclear WBCs/uL
Newborn up to 30 mononuclear WBCs/uL
Children (1-4) - up to 20 mononuclear /uL
Children (5+) up to 10 mononuclear / uL
Increased numbers = Pleocytosis
Cerebrospinal Fluid (CSF)
WBC counts
3% acetic acid can be used to lyse RBC
Methylene blue staining will improve
visibility
Cerebrospinal Fluid (CSF)
Correction of WBC count for traumatic tap
contamination.
Uses ratio of WBCs to RBCs in blood and compares
it to same ratio (WBC/RBC) in CSF
If patients peripheral cell counts are normal, can
subtract 1 WBC for each 700 RBCs counted in
CSF.
Great chance for considerable error, makes this
of little value.
Cerebrospinal Fluid (CSF)
QC
CSF controls
Check techniques
Check of reagents
Check of centrifuges
Decontaminate all counting chambers in
bleach water for @ 15 minutes. Rinse in
water and cleaned again with alcohol.
Cerebrospinal Fluid (CSF)
CSF Slide Differential
Wrights stained smear of concentrated
sediment.
Cytocentrifuge - places cells on filter/
membrane. Increases number of cells to
evaluate, however, risk of cell distortion
from the centrifugation process.
Use of albumin reduces cell distortion
Cerebrospinal Fluid (CSF)
Count and differentiate 100 nucleated cells.
Any cell found in peripheral blood may be seen in CSF,
other nucleated cells and malignant cells can also be found.
Entire smear should be evaluated for
abnormal cells, inclusions within cells, Clusters, Presence of
intracellular organisms
Normal differential values
Adults: 70% lymps, 30% monos.
Children / newborns: monocyte
Types of cells
Neutrophils occasionally (with normal count)
Macrophages increase following CVA
Ependymal cells, and normal lining cells can also be seen.
Cerebrospinal Fluid (CSF)
Entire smear should be evaluated for
abnormal cells
inclusions within cells
Clusters
Presence of intracellular organisms
Cerebrospinal Fluid (CSF)
Lymphocytes & monocytes / macrophages Mono / macro, segs and lymph
L lymphocytes & macrophages
Cerebrospinal Fluid (CSF)
Eosinophils
Often associated with parasitic / fungal
infections, allergic reactions including reaction to
shunts and other foreign objects.
Cerebrospinal Fluid (CSF)
Ependymal cells
Normal cell, unique to CSF
Line the ventricles, produce CSF
fluid
Large cell with distinct
round/oval nucleus, sometimes
found in sheets
Cerebrospinal Fluid (CSF)
Suspicious / unclassified or malignant cells are reported as other or
unclassified AND are sent to pathology. (as seen below)
Cytology send unstained slide to cytology / pathology
1986 CAP CM10 CSF blasts (appearance similar to peripheral blood, always
consult with hematology specialist / pathologist) ( see below right)
Cerebrospinal Fluid (CSF)
left is 1988 CAP CM 25 CSF 250x malignant cells
Remember we classify them as other or unclassified
and take the slide to the cytologist / pathologist
Right is leukemic cells found in CSF
Cerebrospinal Fluid (CSF)
Cellular inclusions ASCP 21 CSF erythrophage, with few
iron granules forming
Erythrophage
Siderophage
Hematoidin crystals (see below)
1991 CAP 30 CSF hematoidin crystal / bilirubin crystal
ASCP 6 macrophage, lymphocyte, siderophage
Cerebrospinal Fluid (CSF)
CSF Quality Control
Commercial quality control samples available
>>>>>>>>>>>>>>>>>>>>>>>>>>>
Chemistry
Blood brain barrier causes selective filtration
Abnormal values
from altered permeability
Increased production
Increased metabolism
Cerebrospinal Fluid (CSF) - protein
Normal 15 45 mg/dL .
Albumin fraction. If IgG from damaged B-B, or CNS
produced? Can electrophoresis to evaluate oligoclonal /
malignant bands.
Decreased levels not significant
Increases levels
Damaged B-B (as in meningitis or hemorrhage)
Production of immunoglobulins within CNS (MS)
Degeneration of neural tissue
Dye-binding methods preferred
Alkaline biuret
Coomassie brilliant blue - a blue color produced is
proportional to the amount of protein present (Beers Law)
Cerebrospinal Fluid MS Panel
Multiple Sclerosis
Diagnosis is difficult no one specific test
CSF Protein electrophoresis
Looking for oligoclonal bands
Myelin Basic Protein
Abnormal protein that indicates demyelination
of neuron axons
Measurement used to monitor course of disease and
effectiveness of treatment
IgG levels (both serum and CSF)
IgG index = CSF IgG mg/dl) / serum IgG (g/dl)
CSF albumin (mg/dl) / serum albumin (g/dl) NV < 0.77
Albumin (both serum and CSF)
IgG synthesis rate.
Cerebrospinal Fluid (CSF) - glucose
Selectively transported across blood-brain barrier
Normal values: 60-70% of blood glucose
STAT procedure, glycolysis reduces level quickly.
Procedure performed as for blood specimen
Decreased levels seen in bacterial & fungal meningitis
Hypoglycemia
Brain tumors
Leukemias
Damage to CNS
Cerebrospinal Fluid (CSF)
CSF Lactate
Normal values = 11-22 mg/dL
Increase as result of hypoxia
Bacterial meningitis. Head injury
CSF Glutamine
Normal 8-18 mg/dL
Increased levels associated with increases in ammonia (toxin)
CSF Enzymes
Lactate dehydrogenase (LDH or LD)
5 isoenzyme types; LD1&LD2 are in brain tissue
Creatine kinase (CPK or CK)
Isoenzyme CK3/ CK-BB from brain tissue
Following cardiac arrest, patients with CSF levels <17 mg/dL
have favorable outcome.
Differential Diagnosis of Meningitis
by Laboratory Results
Bacterial Viral Tubercular Fungal
Increased WBC count Increased WBC count Increased WBC count Increased WBC count
Neutrophils Lymphs Lymps & Monos Lymphs & Monos
Marked protein Mod. protein Mod-Marked protein Mod-Marked
protein
Marked glucose normal glucose glucose Normal to glucose
Lactate > 35 Lactate normal Lactate > 25 mg/dL Lactate > 25
mg/dL mg/dL
+ gram stains Pellicle formation + India ink with
Cryptococcus
neoformans
+ bacterial + immunological
antigen tests test for C. neo.
Cerebrospinal Fluid (CSF)- microbiology
Gram stain Extremely important for early diagnosis of bacterial meningitis
Even when well performed, 10% false negatives occur
Use of Cytospin to concentrate specimen increases sensitivity
Cultures- Aerobic & Anaerobic. Culture blood at same time
Organisms
Newborns
E. coli & group B Strep.
Children
Streptococcus pneumoniae
Hemophilus influenzae
Neisseria meningitidis
Adults -
Neisseria meningitidis
Streptococcus pneumoniae
Staph. aureus (if a shunt is present)
Mixed cells and intracellular bacteria
Immunocompromised
Cryptococcus neoformans,
Candida albicans, Coccidioides, or
any opportunistic organism
Cerebrospinal Fluid (CSF)
India-ink / nigrosin preparation
Negative stain to view the encapsulated
Cryptococcus neoformans (often AIDs /immunocompromised
complication)
Instead of stain, can also use dark field
microscopy for same effect.
These direct procedures have @ 25-50%
sensitivity
Prefer latex agglutination tests, better results
Cerebrospinal Fluid (CSF)
Serology
VDRL (Veneral Disease Research
Laboratory)
For detection of neurosyphilis
On CSF test low sensitivity, but great
specificity
FTA-Abs also used on CSF, more sensitive, but
must prevent blood contamination.
Differential Diagnosis of Meningitis
by Laboratory Results
Bacterial Viral Tubercular Fungal
Increased WBC count Increased WBC count Increased WBC count Increased WBC count
Neutrophils Lymphs Lymps & Monos Lymphs & Monos
Marked protein Mod. protein Mod-Marked protein Mod-Marked
protein
Marked glucose normal glucose glucose Normal to glucose
Lactate > 35 Lactate normal Lactate > 25 mg/dL Lactate > 25
mg/dL mg/dL
+ gram stains Pellicle formation + India ink with
Cryptococcus
neoformans
+ bacterial + immunological
antigen tests test for C. neo.
Cerebrospinal Fluid (CSF)
CSF Quality Control
Commercial quality control samples
available
END of CSF