3. Addisa Ababa University
University of Gondar
American Society for Clinical Pathology
Center for Disease Control and Prevention-Ethiopia
6. Upon completion of this chapter the student will be able to:
1.Describe the formation of CSF from blood.
2. Describe the appearance of normal CSF.
3. Define xanthochromia and state its significance.
4. Differentiate between CSF specimens caused by
intracranial hemorrhage and a traumatic tap.
5 Differentiate between CSF specimens caused by
intracranial hemorrhage and a traumatic tap.
7. 8. Given the laboratory observations of a bloody CSF,
evaluate the supernatant and propose the type of
pathological condition associated with a clear
supernatant versus a xanthochromic supernatant.
9. Compare the difference of pathological conditions
associated with the types of cells observed in a CSF.
10. List the normal range of glucose, protein, and cell
count for a CSF.
11. Evaluate abnormal laboratory results with a
pathological condition related to CSF.
12. Discuss appropriate collection requirements for CSF
following a lumbar puncture.
8. First recognized by Cotugno in 1764, cerebrospinal fluid
(CSF) is a major fluid of the body.
CSF provides a physiologic system to supply nutrients to
the nervous tissue, remove metabolic wastes, and
produce a mechanical barrier to cushion the brain and
spinal cord against trauma.
CSF is produced in the choroid plexuses of the two
lumbar ventricles and the third and fourth venticles. In
adults, approximately 20 mL of fluid is produced every
9. The fluid flows through the subarachnoid space
located between the arachnoid and pia mater.
To maintain a volume of 90 to 150 mL in adults
and 10 to 60 mL in neonates.
the circulating fluid is reabsorbed back into the
blood capillaries in the arachnoid
granulations/villae at a rate equal to its
11. Fluid in the space called sub-arachnoid space between
the arachnoid mater and pia mater
Protects the underlying tissues of the central nervous
Serve as mechanical buffer to
regulate the volume of intracranial pressure
remove metabolic waste products from the CNS
Act as lubricant
Has composition similar to plasma except that it has
less protein, less glucose and more chloride ion
13. Maximum volume of CSF
Adults 150 mL
Neonates 60 mL
Rate of formation in adult is 450-750 mL per day or 20 ml
reabsorbed at the same rate to maintain constant
Collection by lumbar puncture done by experienced
About 1-2ml of CSF is collected for examination
lumbar puncture is made from the space between the
4th and 5th lumbar vertebrae under sterile conditions.
14. Location of CSF
Collected in three
sequentially labeled tubes
Tube 1 Chemical and
Tube 2 Microbiology
Tube 3 Hematology
total WBC & Diff)
This is the list likely
to contain cells
introduced by the
15. As soon as the c.s.f. reaches the laboratory, note its
Report whether the fluid:
– is clear, slightly turbid, cloudy or definitely purulent
(looking like pus),
– contains blood,
– contains clots.
Normal c.s.f. Appears clear and colourless.
16. Purulent or cloudy c.s.f.
Indicates presence of pus cells, suggestive of acute
pyogenic bacterial meningitis.
Blood in c.s.f.
This may be due to a traumatic (bloody) lumbar
puncture or less commonly to haemorrhage in the
central nervous system. When due to a traumatic
lumbar puncture, sample No. 1 will usually contain
more blood than sample No. 2.
17. Following a subarachnoid haemorrhage, the fluid may
appear xanthrochromic, i.e. yellow-red (seen after
Clots in c.s.f. Indicates a high protein concentration
with increased fibrinogen, as can occur with
pyogenic meningitis or when there is spinal
19. Diagnosis of meningitis of bacterial, fungal,
mycobacterial and amoebic origin or differential
diagnosis of other infectious diseases
subarachnoid hemorrhage or intracerebral
20. CSF specimen examined visually and microscopically
and total number of cells can be counted and identified
the third tube in the sequentially collected tubes
must be counted within 1 hour of collection (cells
disintegrate rapidly). If delay is unavoidable store 2-
All specimens should be handled as biologically
21. Glucose is major energy substrate for brain as
well as a major carbon source for many
Brain uses 20-25% of total oxygen and 15% of
cardiac output is directed to CNS.
22. When body glucose supply is decreased, other organs
decrease glucose utilization to maintain adequate
supply of glucose to brain.
Other organs can readily switch to oxidation of another
substrate for energy production.
Under certain conditions, such as chronic starvation,
the brain can oxidize other substances but maintains a
minimal obligatory requirement for glucose.
23. Glycolysis--conversion to lactic acid
Hexokinase has high activity in brain
Serves to trap glucose and maintain concentration
gradient for diffusion
24. 2-deoxyglucose is also taken up by brain and
phosphorylated by hexokinase, but then
Marker to correlate changes in neural activity with
changes in glucose utilization.
Enolase, an enzyme in glycolytic pathway,
exists in nerve cells in unique isoform (neuron
specific enolase, NSE)
Used as a specific marker for neurons.
25. Pentose shunt
Provides source of D-ribose for synthesis of
DNA and RNA
Produces NADPH required for lipid syntheses
Most active during development
26. Concept: Amino acids serve many functions in
Peptide and Protein synthesis
Precursors for transmitters
27. Concept: Neurons must produce those proteins
essential for their special functions:
conduction of action potentials
establishment of specific connections
Cell Surface proteins play a role during
development in directing neural connections
function in axoplasmic movement
Glial proteins (glial fibrillary protein)
29. Enzymes for transmitter synthesis and
31. Morphologically identical to neutrophils and
bands in blood
Occasionally granulation disappears and
pseudo-hypersegmentation is observed.
32. Almost identical morphology to lymphocytes
in the blood
Due to "flattening-out" of the lymphs during
cytocentrifugation, nucleoli may be visible.
Found in all fluid
33. Leukophages:Macrophagescontaining phagocytized
WBC. WBCs are often pyknotic and easily confused
with NRBC's. Found in all fluids.
Erythrophages: Macrophages containing phagocytized
RBC or RBC fragments. May contain several RBC.
Found in all fluids.
Siderophages: Macrophages containing phagocytized
particles of hemosiderin, which stain a blue-black color.
34. These are bright-yellow diamond-shaped
crystals of hemosiderin
intracellular or extracellular on the slide.
They are iron-negative on the Prussian blue
stain and therefore
Can be noted on the patient report without
performing an iron stain.
35. Metamyelocytes, myelocytes, and
promyelocytes may be found in fluids, though
they are rarely seen.
They are morphologically identical to those in
May be due to bone marrow contamination in
36. Morphologically similar to blasts found in the
There may be some clover-leaf shaped nuclei
due to cytocentrifugal distortion.
May be found in all fluids
Seen in association with leukemias,
Bone marrow contamination of CSF
37. NRBC are rarely seen body fluids. If observed,
they should be reported as the number of
NRBC per number of WBC counted
They must be differentiated from pyknotic
NRBC’s are commonly due to peripheral blood
or bone marrow contamination of CSF
38. Plasmacytoid lymphs: Identical in morphology
to plasmacytoid lymphs in blood
Found in all fluids.
Mott cells: Plasma cells with numerous clear
cytoplasmic vacuoles containing
39. These are most common in CSF from small
children with subarachnoid hemorrhage but
may be found in all body fluids
May be very difficult to distinguish
morphologically from large atypical
40. Malignant cells may be shed from solid tissue
(non-hematopoietic) neoplasms into CSF or
body cavity fluid submitted for cell counts
Fluid will be turbid or bloody
Malignant cells are usually seen in clusters of 3-
5 or more, but may occur singly
41. Intracellular bacteria or yeast can be observed
in acute bacterial or fungal infections
It is important to coordinate your findings with
those of the Microbiology Section of the
42. When the CSF is pinkish red, this usually
indicates the presence of blood, which may
have resulted from:
Sub arachnoid hemorrhage
Intra cerebral hemorrhage
44. Color – Xanthochromia
Increased White Blood Cells (Pleocytosis)
45. A traumatic tap shows progressively
decreasing RBC in serial samples
Generally, in subarachnoid hemorrhage, the
RBC would be consistent from one tube to the
46. After the CSF is centrifuged, the supernatant
fluid is clear in a traumatic tap, but it is
xanthochromic in a subarachnoid hemorrhage
Xanthochromia of the CSF refers to a pink,
orange, or yellow color of the supernatant after
the CSF has been centrifuged
47. The white cell count is increased when there is
inflammation of the central nervous system,
particularly the meninges
Bacterial infections are usually associated with
the presence of neutrophils in the CSF
48. • Viral infections are associated with an increase
in mononuclear cells
• An increase in mononuclear cells may also be
– cerebral abscess
– acute leukemia
– intracranial vein thrombosis
– cerebral tumor
– multiple sclerosis
49. A white cell count with an indication whether
the cells are pus cells or lymphocytes, is
required when the c.s.f. appears slightly cloudy
or clear or when the
Gram smear does not indicate pyogenic
To identify whether white cells in the c.s.f. are
polymorphonuclear neutrophils (pus cells) or
lymphocytes, dilute the c.s.f. in a fluid which stains the
cells. Istonic 0.1% toluidine blue is recommended
because it stains lymphocytes and the nuclei of pus
cells blue. C. neoformans yeast cells stain pink. Red
cells remain unstained. The motility of trypanosomes is
not affected by the dye.
When toluidine blue is unavailable, isotonic methylene
blue can be used which will also stain the nuclei of
51. 1 Mix 1 drop of the c.s.f. (sample No. 2 uncentrifuged
c.s.f.) with 1 drop of toluidine blue diluting fluid,
2 Assemble a modified Fuchs-Rosenthal ruled
counting chamber, making sure the chamber
and cover glass are completely clean.
When unavailable, an improved Neubauer (preferably
Bright-Line) chamber can be used.
A Fuchs-Rosenthal chamber is recommended because it
has twice the depth (0.2 mm) and is more suitable for
counting WBCs in c.s.f.
52. 3 Using a fine bore Pasteur pipette or capillary
tube, carefully fill the counting chamber with the
well-mixed diluted c.s.f. The fluid must not overflow into
the channels on each side of the chamber.
4 Wait 2 minutes for the cells to settle.
5 Count the cells microscopically. Focus the cells and
rulings using the 10 objective Count the cells in 5 of
the large squares
55. Because CSF is formed by filtration of the plasma,
chemicals in the CSF are that are found in the plasma.
chemical composition is controlled by the blood-brain
normal values for CSF chemicals are not the same as
the plasma values.
Abnormal values result from alterations in the
permeability of the blood-brain barrier or increased
production or metabolism by the neural cells in
response to a pathologic condition.
56. Use the supernatant fluid from centrifuged
c.s.f. or uncentrifuged c.s.f. when the sample
Total protein can be measured in c.s.f. using a
colorimetric technique or a visual comparative
Normal Total c.s.f. protein is normally 0.15–0.40
g/l (15–40 mg%).
57. When the total protein exceeds 2.0 g/l (200
mg%), the fibrinogen level is usually increased
sufficiently to cause the c.s.f. to clot. This may
occur in severe pyogenic meningitis, spinal
block, or following haemorrhage.
58. The causes of elevated CSF protein include damage to
the blood-brain barrier, production of immunoglobulins
within the CNS, decreased clearance of normal protein
from the fluid, and degeneration of neural tissue.
Meningitis and hemorrhage conditions that damage the
blood-brain barrier are the most common causes of
elevated CSF protein.
Many other neurologic disorders can elevate the CSF
protein, and finding an abnormal result on clear fluid
with a low cell count is not unusual
61. Glucose enters the CSF by selective transport across
barrier, a normal value 60% to 70% that of the plasma
If the plasma glucose is 100 mg/dL, then a normal CSF
glucose would be approximately 65 mg/dL. For an
accurate evaluation of CSF glucose, a blood glucose
test must be run for comparisonCSF glucose is
analyzed using the same procedures employed for
Specimens should be tested immediately because
glycolysis occurs rapidly in the CSF.
62. The diagnostic significance of CSF glucose is confined
to the finding of values that are decreased in relation to
Elevated CSF glucose values are always a result of
Low CSF glucose values can be of considerable
diagnostic value in determining the causative agents
The finding of a markedly decreased CSF glucose
accompanied by an increased WBC count and a large
percentage of neutrophils is indicative of bacterial
63. Glucose must be measured within 20 minutes of the
c.s.f. being withdrawn otherwise a falsely low result
will be obtained due to glycolysis.
Use the supernatant fluid from centrifuged c.s.f. or
uncentrifuged c.s.f. if the sample appears clear.
Glucose can be measured in c.s.f. using a colorimetric
technique or a simpler semiquantitative technique using
64. Normal c.s.f. glucose: This is about half to two thirds
that found in blood i.e. 2.5–4.0 mmol/1 (45–72
Raised c.s.f. glucose: Occurs when the blood glucose
level is raised (hyperglycaemia) and sometimes with
Low c.s.f. glucose: The c.s.f. glucose concentration is
reduced in most forms of meningitis, except viral
In pyogenic bacterial meningitis it is markedly
reduced and may even be undetectable.
65. Low glucose levels, as compared to plasma
levels, are seen in:
malignant involvement of the meninges and
Glucose levels are usually normal in viral
infections of the CNS
67. In bacterial and cryptococcal infection, an increased
CSF lactate is found earlier than a reduced glucose
In viral meningitis, lactate levels remain normal, even
when neutrophils are present in the CSF
Raised levels may also occur with severe cerebral
hypoxia or genetic lactic acidosis
68. When cryptococcal meningitis is clinically suspected,
e.g. patient with HIV disease, or when yeast cells are
detected when performing a cell count or examining a
Gram smear, examine an India ink preparation or a
wet preparation by dark-field microscopy for
69. 1 Centrifuge the c.s.f. for 5–10 minutes. Remove
the supernatant fluid and mix the sediment.
2 Transfer a drop of the sediment to a slide, cover
with a cover glass and examine by dark-field
Microscopy or add a drop of India ink , use nigrosin 200
g/l (20% w/v solution.
3 Examine the preparation using the 40 objective Look for
oval or round cells, some showing budding, irregular in
size, measuring 2–10 m in diameter and surrounded by
a large unstained capsule
70. Examine a wet preparation for motile amoebae
when primary amoebic meningoencephalitis is clinically
suspected (rare condition caused by N. fowleri) or the
c.s.f. contains pus cells with raised protein and low
glucose, but no bacteria are seen in the Gram smear.
Red cells may also be present.
71. 1 Transfer a drop of uncentrifuged purulent c.s.f. or a
drop of sediment from a centrifuged specimen to a
slide and cover with a cover glass.
2 Examine the preparation using the 10 and 40
objectives, with the condenser closed sufficiently
to give good contrast. Look for small, clear,
motile, elongated forms among the pus cells. Use
the 40 objective
74. 1 A CSF WBC count is diluted with:
2 A total CSF cell count on a clear fluid should be:
3 The purpose of adding albumin to CSF before
cytocentrifugation is to:
4 The primary concern when pleocytosis of neutrophils
and lymphocytes is found in the CSF is:
5 Neutrophils with pyknotic nuclei may be mistaken
6 The presence of which of the following cells is
increased when a CNS shunt malfunctions?
75. You should now be able to discuss the
formation and collection procedure for cerebral
spinal fluid, normal and abnormal findings in
CSF and methods of analysis and evaluation of
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