BODY FLUID ANALYSIS

Chapter One

Cerebrospinal fluid analysis

 Acknowledgements
 Addisa Ababa University
 Jimma University
 Hawassa University
 Haramaya University
 University of Gondar
 American Society for Clinical Pathology
 Center for Disease Control and Prevention-Ethiopia
 Chapter outline
 Introduction to Cerebrospinal fluid
 Routine laboratory assays
 Collection of sample
 Gross appearance
 Cell counts
 Chemical analysis
 Morphological Examination
 Microbiological Examination
 Serological Examination
CEREBRAL SPINAL FLUIDS
 Learning Objectives
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.
Learning Objectives (continued)

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.
 CSF Formation and Physiology

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
hour.
 Formation and Physiology

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
production
Cerebrospinal fluid (CSF)
 Fluid in the space called sub-arachnoid space between
the arachnoid mater and pia mater
 Protects the underlying tissues of the central nervous
system (CNS)
 Serve as mechanical buffer to
 prevent trauma,
 regulate the volume of intracranial pressure
 circulate nutrients
 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
 Cerebrospinal fluid analysis

 Collection of CSF sample

 Routine Laboratory assays of CSF
 Gross appearance
 RBC &WBC counts
 Morphological Examination
 Microbiological Examination
 Serological Examination
 CSF cont’d
 Maximum volume of CSF
 Adults 150 mL
 Neonates 60 mL
 Rate of formation in adult is 450-750 mL per day or 20 ml
per hour
 reabsorbed at the same rate to maintain constant
volume
 Collection by lumbar puncture done by experienced
medical personnel
 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.
 Collecting CSF specimen
 Collected in three
sequentially labeled tubes
 Tube 1 Chemical
and immunologic tests
 Tube 2
Microbiology
 Tube 3 Location of CSF
Hematology (gross
examination, total
WBC & Diff)
 This is the list likely
to contain cells
introduced by the
puncture procedure
 Report the appearance of the c.s.f.

 As soon as the c.s.f. reaches the laboratory, note its

appearance.
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.
 Report the appearance of the c.s.f.

 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.
 Report the appearance of the c.s.f.

 Following a subarachnoid haemorrhage, the fluid may

appear xanthrochromic, i.e. yellow-red (seen after
centrifuging).
 Clots in c.s.f. Indicates a high protein concentration
 with increased fibrinogen, as can occur with
 pyogenic meningitis or when there is spinal
constriction.
 Clinical Significance

 Diagnosis of meningitis of bacterial, fungal,

mycobacterial and amoebic origin or differential
diagnosis of other infectious diseases
 subarachnoid hemorrhage or intracerebral
hemorrhage
 Principle of the test

 CSF specimen examined visually and microscopically

and total number of cells can be counted and identified

Specimen:
 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-
8oC.
 All specimens should be handled as biologically
hazardous
 Uptake and Utilization of
Glucose
 Glucose is major energy substrate for brain as
well as a major carbon source for many
molecules.
 Brain uses 20-25% of total oxygen and 15% of
cardiac output is directed to CNS.
 Glucose Utilization
 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.
 Brain Utilization of Glucose
 Glycolysis--conversion to lactic acid
 Hexokinase has high activity in brain
 Serves to trap glucose and maintain concentration
gradient for diffusion
 Brain Utilization of Glucose
 2-deoxyglucose is also taken up by brain and
phosphorylated by hexokinase, but then
becomes trapped
 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.
 Brain Utilization of Glucose
 Pentose shunt
 Provides source of D-ribose for synthesis of
DNA and RNA
 Produces NADPH required for lipid syntheses
 Most active during development
 Utilization of Amino Acids
 Concept: Amino acids serve many functions in
CNS
 Peptide and Protein synthesis
 Precursors for transmitters
 Neurotransmitters
 Proteins in the CNS
 Concept: Neurons must produce those proteins
essential for their special functions:
 conduction of action potentials
 synaptic transmission
 axoplasmic transport
 establishment of specific connections
Specific Neural/GlialPeptides/Proteins
 Structual-cytoskeletal
 Cell Surface proteins play a role during
development in directing neural connections
 Contractile proteins
 function in axoplasmic movement
 Neurotubular protein
 Glial proteins (glial fibrillary protein)
 Specific Neural/Glial Peptides/Proteins
 Enzymes for transmitter synthesis and
degradation
 Transmitter receptors
 Membrane transporters
 Ion Channels
 Growth Factors
 Synaptic Vesicle
MICROSCOPIC OBSERVATIONS
OF CEREBRAL SPINAL FLUID
 Neutrophils and Bands
 Morphologically identical to neutrophils and
bands in blood
 Occasionally granulation disappears and
pseudo-hypersegmentation is observed.
 Lymphocytes
 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
 Macrophages

 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.
 Hematoidin Crystals
 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.
 Immature Granulocytes
 Metamyelocytes, myelocytes, and
promyelocytes may be found in fluids, though
they are rarely seen.
 They are morphologically identical to those in
the blood
 May be due to bone marrow contamination in
CSF
 Blasts
 Morphologically similar to blasts found in the
blood
 There may be some clover-leaf shaped nuclei
due to cytocentrifugal distortion.
 May be found in all fluids
 Seen in association with leukemias,
lymphomas
 Bone marrow contamination of CSF
 Nucleated Red Blood Cells
 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
WBCs
 NRBC’s are commonly due to peripheral blood
or bone marrow contamination of CSF
 Abnormal Lymphocytes
 Plasmacytoid lymphs: Identical in morphology
to plasmacytoid lymphs in blood
 Found in all fluids.
 Mott cells: Plasma cells with numerous clear
cytoplasmic vacuoles containing
immunoglobulins
 Reactive Macrophages
 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
lymphocytes
 Malignant Cells
 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
 Microorganisms
 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
laboratory
 Bloody CSF
 When the CSF is pinkish red, this usually
indicates the presence of blood, which may
have resulted from:
 Sub arachnoid hemorrhage
 Intra cerebral hemorrhage
 Infarct
 traumatic tap
Order of Draw of Lumbar Puncture
 1st - Chemistry
 2nd - Microbiology
 3rd - Hematology
 Physical Examination
 Color – Xanthochromia
 Hyperbilirubinemia
 Increased Protein
 Turbidity
 Increased White Blood Cells (Pleocytosis)
 CSF Supernatant
 A traumatic tap shows progressively
decreasing RBC in serial samples
 Generally, in subarachnoid hemorrhage, the
RBC would be consistent from one tube to the
next
 CSF Supernatant
 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
 Cell Count
 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
 Cell Count
• Viral infections are associated with an increase
in mononuclear cells
• An increase in mononuclear cells may also be
seen with:
– cerebral abscess
– acute leukemia
– Lymphoma
– intracranial vein thrombosis
– cerebral tumor
– multiple sclerosis
 Cell Count cont’d
 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
bacterial meningitis
 Cell Count cont’d
Method
 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 leucocytes.
 CSF Cell Count procedures
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.
 CSF Cell Count procedures
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
 Improved Neubauer ruled chamber
Modified Fuchs-Rosenthal ruled
chamber
Calculation of CSF Cell Counts
 Chemistry Tests

 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
barrier,
 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.
.
Measurement of c.s.f total protein
 Use the supernatant fluid from centrifuged
c.s.f. or uncentrifuged c.s.f. when the sample
appears clear.
 Total protein can be measured in c.s.f. using a
colorimetric technique or a visual comparative
technique,
 Normal Total c.s.f. protein is normally 0.15–0.40
g/l (15–40 mg%).
Measurement of c.s.f. total protein
 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.
 Clinical Significance of Elevated Protein
Values
 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
 Increased CSF Protein >80mg/dL
 Diabetes Mellitus
 Brain tumor
 Meningioma
 Acoustic neuroma
 Ependymoma
 Encapsulated brain abscess
 Spinal cord tumor
 Multiple Sclerosis
 Acute purulent Meningitis
Increased CSF Protein >80mg/dL

Granulomatous Meningitis
Carcinomatous Meningitis
Syphilis (protein may be normal if longstanding)
Guillain-Barre Syndrome (Infectious polyneuritis)
Cushing's Disease
Connective tissue disease
Uremia
Myxedema
Cerebral hemorrhage
 Cerebrospinal Fluid Glucose

 Glucose enters the CSF by selective transport across

the bloodbrain
 barrier, a normal value 60% to 70% that of the plasma
glucose.
 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
blood glucose.
 Specimens should be tested immediately because
glycolysis occurs rapidly in the CSF.
 Clinical Significance of Elevated glucose
Values
The diagnostic significance of CSF glucose is confined
to the finding of values that are decreased in relation to
plasma values.
Elevated CSF glucose values are always a result of
plasma elevations.
Low CSF glucose values can be of considerable
diagnostic value in determining the causative agents
in meningitis.
The finding of a markedly decreased CSF glucose
accompanied by an increased WBC count and a large
percentage of neutrophils is indicative of bacterial
meningitis.
 Measurement of c.s.f. glucose

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
Benedict’s reagent.
 Measurement of c.s.f. glucose

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
mg%).
Raised c.s.f. glucose: Occurs when the blood glucose
level is raised (hyperglycaemia) and sometimes with
encephalitis.
Low c.s.f. glucose: The c.s.f. glucose concentration is
reduced in most forms of meningitis, except viral
meningitis.
In pyogenic bacterial meningitis it is markedly
reduced and may even be undetectable.
 Glucose
 Low glucose levels, as compared to plasma
levels, are seen in:
 bacterial meningitis
 cryptococcal meningitis
 malignant involvement of the meninges and
sarcoidosis
 Glucose levels are usually normal in viral
infections of the CNS
 CSF Normal Adult Lab Ranges
 Normal CSF Levels:
Protein (10 - 45 mg/dL)
Glucose (40 - 70 mg/dL)
 Physical Appearance
Clear/colorless
RBC <5/mL
WBC <5/mL
 Lactate
 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
 CSF India ink preparation

 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 encapsulated
yeasts.
 India ink preparation
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
 Wet preparation to detect amoebae

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.
 Wet preparation to detect amoebae

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
cont’d
 Exercise
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
for:
6 The presence of which of the following cells is
increased when a CNS shunt malfunctions?
 Summary
 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
CSF
 References:

 Urinalysis and body fluids / Susan King Strasinger, 5th ed. 2008
 District laboratory practice in tropical countries. 2nd ed. Part I. Monica
Cheesbrough, 2005
 Text book of urinalysis and body fluids. Doris LR, Ann EN, 1983
 Urinalysis and body fluids: A color text and atlas. Karen MR, Jean JL.
1995
 Clinical chemistry: Principles, procedures, correlation. 3rd ed. Michael
L. Bishop et al. 1996
 Tietz Text book of clinical chemistry. 3rd ed. Carl AB, Edward RA,
1999
 Clinical chemistry: Theory, analysis, correlation 4th ed. Lawrence AK.
2003
 ASCP Document
 Urinalysis lecture note . Mistire W. , Dawite Y.

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