Csf analysis presentation

Ceribrospinal Fluid Analysis is a very important investigations in all laboratories. You can asses this ppt for your routine laboratory work.

1. Cerebrospinal Fluid Analysis
BY
BIJO AUGUSTINE

2. Anatomy and Physiology
Cerebrospinal fluid (CSF) is present within
the subarachnoid space surrounding the
brain in the skull and the spinal cord in
the spinal column.
Total volumes:
–Adults:140 - 170 mL
–Children:10 - 60 mL

3. Functions of CSF
It’s main function is to protect the brain and
the spinal cord from injury by acting as a
fluid cushion.
It is the medium through which nutrients
and the waste products are transported
between brain/spinal cord and the blood.

4. Formation and composition of
CSF
 CSF is derived by ultra filtration of plasma
and by secretion through the choroid plexus
located in the ventricles of the brain.
 Reabsorbtion of CSF occurs at the
arachnoid villi which projects in the venous
sinuses in the duramater.
 CSF is produce at the rate of 500 mL/day.

5. Formation andComposition of
CSF
Blood brain barrier maintains the relative
homeostasis of CNS environment by
tightly regulating the concentration of
substances by specific transport systems for
H+, K+, Ca2+, Mg2+, HCO3-.
 Glucose, urea and creatinine diffuse freely
between blood and the CSF.

6. Proteins cross freely by passive diffusion
along the concentration gradient and is also
influenced by molecular weight.
Formation and Composition of
CSF

8. Composition of Normal CSF
·
Protein -15 - 45 mg/dL
Glucose -50 - 80 mg/dL
Urea - 6.0 - 16 mg/dL
Uric acid - 0.5 - 3.0 mg/dL
Creatinine - 0.6 - 1.2 mg/dL
Cholesterol - 0.2 - 0.6 mg/dL
Ammonia -10 – 35 μg/dL

9. Composition of Normal CSF
Sodium -135 – 150 mEq/L
Potassium - 2.6 – 3.0 mEq/L
Chloride -115 – 130 mEq/L
Magnesium - 2.4 – 3.0 mEq/L
Cells - 0 – 5 Lymph/μL

10. Clinical Application of CSF Examination
In the diagnosis of
b.Bacterial, viral or fungal meningitis.
c.Encephalitis.
d.Malignant infiltrates like in acute leukemia,
lymphoma.
e.Subarachnoid hemorrhage.
f.Spinal canal blockage leading to elevated
intracranial tension.
g.Sub acute sclerosing pan encephalitis (SSPE)

11. Characteristics of normal CSF
•Color - Colorless
•PH - 7.28 – 7.32
•Appearance - Clear
•Sp. Gravity- 1.003 – 1.004
•No clot formation on standing
•Total solids - 0.85 – 1.70 g/dL
•PO2 - 40 – 44 mmHg

12. Collection and Processing
•Lumbar puncture, cisternal puncture, lateral
cervical puncture, shunts & cannulas
•Opening pressure = 90 - 180 mm H
2
O (+/-)
•Approximately 15 - 20 cc fluid collected
•Process within 1 hour without refrigeration - STAT
•Three tube set-up:
–Tube 1:Chemistry and Immunology (Frozen)
–Tube 2:Microbiology (Room temperature)
–Tube 3:Cell count, differential, cytology (Refrigerated)

15. Diagnosis by CSF
•High sensitivity, high specificity
–Bacterial, TB, and fungal meningitis
•High sensitivity, moderate specificity
–Viral meningitis, SAH, CNS syphilis, abscess
•Moderate sensitivity, high specificity
–Meningeal malignancy
•Moderate sensitivity, moderate specificity
–Intracranial hemorrhage, viral encephalitis,
subdural hematoma

16. Routine Lab Tests
•Required
•Opening CSF pressure
•Macroscopic Examination
•Total cell count and differential (stained)
•Glucose (CSF/plasma ratio)
•Protein
•Optional
•Cultures, gram stain, antigens, cytology
•Protein electrophoresis, VDRL, D-dimers

17. Gross Examination
•Normal CSF is clear, colorless
•Viscosity equal to water
•Clot seen in traumatic tap, not SAH
•Viscous CSF with increased protein exudate
•Turbidity:
–WBC > 200 cells/uL
–RBC > 400 cells/uL
–Microorganisms, increased protein

18. Clot/Coagulation formation
Allow the specimen of CSF to stand over over
night and examine the sample for fibrin clot,
which is formed if the sample contains
fibrinogen. Also note the nature of the clot.
- Delicate clot, which resembles a cobweb, is
characteristically seen in tubercular meningitis
due to marked increased in CSF proteins. The
clot may have entrapped tubercle bacilli,
which could be demonstrated microscopically
by staining for acid-fast bacilli.

19. Clot/Coagulation formation
Corase clot is formed in pyogenic meningitis,
traumatic tap and in case of complete spinal
block.
pH Determination.
pH can be measured by using pH paper or
using pH meter.

20. Xanthochromia
•Pink, orange, or yellow discoloration
•RBC lysis or hemoglobin breakdown
•May be seen within hours of LP
•Peak intensity at 24 - 36 hours
•RBC > 6000/μL (SAH, ICH, infarct, traumatic)
•Oxyhemoglobin, bilirubin, increased protein
•Carotinoids, melanin, rifampin therapy

21. Differential Dx of Bloody CSF
•Traumatic tap - blood clears between tubes
•Xanthochromia - pink tinge, RBCs
•SAH - blood does not clear or clot

22. Microscopic Exam of CSF
Total WBC Count
Normal CSF contains 0-8 lymph and no
RBCs.
Procedure
Glass slides
Counting chamber
Cover slip of thickness with size of 22 23 mm
˟
CSF diluting fluid – 1% Toludine blue or 1 %
violet – stains the WBC without lysing the RBC,
thus enabling to count both RBC and WBC in the

23. •Same chamber. The stain is mixed with the
CSF in the ratio 1:9 dil.
•Dilute acetic acid – 0.1 gm of crystal violet
is added to 1 ml glacial acetic acid is made
up to 50 ml by adding distilled water. Few
drops of phenol is also added to this. As this
fluid lyses the red cells it is useful in case of
blood tinged CSF. In such case the RBC
count estimated separately using undiluted
CSF sample.

24. Procedure
Dilution – if CSF is clear there is no need
for dilution and both RBS and WBC can be
counted simultaneously in the same
chamber.
If CSF is cloudy then make a dilution of
1:10 or 1:20.One can also pipette out 900μL
of CSF diluting fluid in the tube and 100μL
of CSF to it.

25. Counting of cells
•Charge the counting chamber properly
without any air bubbles.
•Wait for 5 minutes before counting, to
allow the cells in CSF to settle down.
•Count the cells in all 9 squares by using low
power objective.
Calculation
WBC in CSF/cumm(μL)=No.of celldep.dilu.
ˣ ˣ
Area counted

26. Important points
•Cells in CSF should be counted
immediately with out delay to prevent
degeneration of cells which will give false
low counts.
•Bloody/ traumatic tap adds approximately
1-2 WBC per 1000 RBCs. Hence in the
estimation of total leucocytes count a
deduction is made equivalent to 1 WBC for
every 700 erythrocytes counted.

27. Differential leucocytes count
•Chamber differential.
•Differential cell count
•Leishman’s stained smear or Gimsa’s
Stained smear.

28. Reference Intervals for CSF
Cell type Adults(%) Neonates(%)
Lymphocytes 62 20
Monocytes 36 72
Neutrophils 2 3
Histiocytes Rare 5
Ependymal Rare Rare
Eosinophils Rare Rare

29. Increased Neutrophils in CSF
•Meningitis (bacterial, early TB, fungal)
•Other infections
•Following seizures
•Following CNS hemorrhage
•Following CNS infarct
•Reaction to repeated LP
•Foreign materials
•Metastatic tumor

30. Increased Lymphocytes in CSF
•Meningitis (aseptic, viral, L monocytogenes)
•Parasitic infections
•Degenerative disorders
–Encephalopathy due to drugs, GBS
•Other inflammatory conditions
–Sarcoidosis, polyneuritis, periarteritis involving
the CNS

31. Plasmacytosis in CSF
•TB meningitis
•Syphilitic meningitis
•Parasitic infection
•Sarcoidosis
•Acute viral infections

32. Immature cells in CSF

33. Eosinophilic pleocytosis in CSF
•Commonly associated with
•Parasitic infections
•Fungal infections
•Reaction to foreign material
•Infrequently associated with
•Bacterial or tuberculous meningitis
•Viral, rickettsial infection, lymphoma,
sarcoidosis

34. Chemical Analysis
•Total protein non-specific marker of disease
•Turbidimetric methods based on TCA or
SSA & sodium sulfate for precipitation
•Simple, rapid, no special instrumentation
•300 different proteins have been isolated
from CSF using two-dimensional
electrophoresis and silver staining

35. Conditions Associated with
Increased CSF Total Protein
•Increased blood-CSF permeability
–Meningitis (bacterial, fungal, TB)
–Hemorrhage (SAH, ICH)
–Endocrine disorders
–Mechanical obstruction (tumor, disc, abcess)
–Neurosypilis, MS, CVD

36. Electrophoresis
•Identification of oligoclonal bands
•2 or more discrete bands in the gamma
region absent or of lesser intensity in
concurrently run patient’s serum
•Silver stain more sensitive than paragon
violet
•IFE better resolution and more specific
•Sensitivity = 83 - 94%

37. Glucose estimation in CSF
•CSF glucose is derived from blood glucose
hence, ideally CSF glucose level should be
compared with fasting plasma glucose level
for adequate clinical interpretation.
•Clinical Significance.
•CSF glucose less than 40 mg/dL or
CSF/plasma glucose less than 0.3 are
considered abnormal (normal CSF/Plasma
glucose ratio may very from 0.3 – 0.9)

38. •Increased CSF glucose is of no clinical
significance.
•Causes of decreased CSF glucose
•Meningitis-Bacterial, fungal tubercular and
syphilitic meningitis.
•Tumors involving the meninges.
•Subarachnoid hemorrhage.
•Cerebral ameobiasis.

39. Bacterial Meningitis
•0 - 1m: Group B strept & E. coli (GNR)
•1m - 5y: H. influenzae
•5 - 29y: N. meningitidis
•>29y: S. pneumoniae
•Listeria monocytogenes common in
newborns, elderly, and other
immunocompromised hosts

40. Bacterial Meningitis
•Gram’s stain sensitivity = 60 - 90%
•Depends on organism, experience,
•Culture sensitivity = 80 - 90%
•Latex agglutination becoming more widely
used due to simplicity and accuracy

41. Test AppearancePressure WBC/μL Protein mg/
dL
Glucose mg/
dL
Chloride
Normal
CSF
Clear 90 – 180
mm
0-8 lymph.15-45 50-80 115-130
mEq/L
Acute
bacterial
meningitis
Turbid Increased1000
-10000
100 – 500< 40 Decreased
Viral
meningitis
Clear Normal to
moderate
increase
5-300,
rarely
>1000
Normal to
mild
increased
Normal Normal
Tubercular
meningitis
Slightly
opaque
cobweb
formation
Increased/
decreased,
spinal
block
100-600
mixed or
lymph.
50-300 due
to spinal
block
DecreasedDecreased
Fungal
meningitis
Clear Increased40-400
mixed
50-300 DecreasedDecreased
Acute
syphilitic
Clear IncreasedAbout 500
lymph
Increased
but <100
Normal normal

42. Bacterial Meningitis

43. Neurosyphilis
•Darkfield microscopy for spirochetes
•CSF FTA-ABS 100% sensitive
•Negative test rules out diagnosis
•VDRL nearly 100% specific
•Positive test rules in neurosyphilis
•RPR unsuitable for CSF (higher FP than
VDRL)

44. Neurosyphilis

45. Viral Meningitis
•Enteroviruses (echoviruses, coxsachie,
polio viruses) account for 80% cases
•Diagnosis of exclusion, rarely use cultures
•Viral inclusions for CMV, HSV
•PCR for HSV available
•Usually requires brain biopsy

46. HIV
•Wide variety of abnormalities with or
without neurological disease
•Lymphocytic pleocytosis, elevated IgG, and
oligoclonal bands
•ID of opportunistic (fungal) infections main
reason for examining CSF

47. Fungal Meningitis
•India ink for cryptococcal capsular halos
•50% sensitivity
•LA and CF antibodies now available
•Sensitivity as high as 96%

50. Tuberculous Meningitis
•Early diagnosis extremely difficult
•Sensitivity for acid-fast stains 10%
•Large volumes of CSF recommended
•Higher levels of adenosine deaminase
•ELISA and PCR now available
•Sensitivity = 50 - 82%
•Specificity = 90 - 100%

51. Primary Amoebic
Meningoencephalitis (PAM)
•Rare disease caused by free-living amoeba
Naegleria fowleri or Acanthamoeba species
•Motile Naegleria trophozoites may be seen
with light microscope
•Acridine orange stain can differentiate
amoeba (brick red) from leukocytes (bright
green)

53. CSF Cytology
•Cytological evaluation of CSF is an
effective means for diagnosing many
disorders involving the central nervous
system. Preparatory methods for CSF
examination are discussed and normal and
reactive conditions involving lymphoma,
leukemia, meningeal carcinomatosis and the
subarachnoid spread of primary brain
tumors are evaluated by primary cytological
examination of CSF

54. Thank you