Cerebrospinal fluid biology and physiology examination.ppt

Csf analysis

1. .

2. Central Nervous System: Overview
Brain
Spinal cord

3. Spinal Cord Regions
Cervical
Thoracic
Lumbar
Sacral

4. Protecting the Brain
Hair, skin, cranium
Venous sinus blood
Meninges
Dura mater
Arachnoid membrane
Pia mater
Cerebrospinal fluid

5. Circulation of CSF
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Due to pulsation of blood in choroid plexus
Due to pulsation of ependymal cells
Lateral ventricles interventricular
foramen of Monroe third ventricle
mesencephalic aqueduct (aqueduct of
Sylvius) fourth ventricle
foramen of Luscka Magendi spinal
cord central canal; also, out the lateral
apertures to the subarachnoid space to the
venous system absorbed by the
arachnoid villi projections.

7. The Basics……Physiology
500 ml of CSF – Produced Daily = (0.3ml-0.4ml/min)
Total volume ( adult) varies from 90 – 150 ml , 25ml in ventricles and
remainder in subarchanoid space.
Neonates volume is 10-60 ml .
Total volume of CSF is replaced in every 5 – 7 hrs.
70% of CSF is derived by ultrafiltration and secretion through the
choroid plexsus and the remainder through ventricle ependymal lining
and cerebral subarachnoid space.

8. CEREBROSPINAL FLUID
The Basic Components very different from plasma
pH is lower ( 7.28-7.32)
Electrolytes- Osmolality (280 – 300 mOsm/L)
Low in proteins which include- Transthyretin, transferin
Prealbumin, Albumin, Alpha1 , Alpha2, Beta, Gamma globulin.
Na, K, Cl, Ca, Mg, Fe, P, Zn, HCO
3,
Co2 and glucose are lower
NH3, Glutamine, Creatinine , Urea, Urate
-

9. Blood Brain Barrier
Extensive capillaries & sinuses
Astrocyte foot processes: secrete paracrines
Tight junctions: limit permeability
Protects brain: from hormones & circulating chemicals
Many glucose transporters

11. Blood Brain Barrier
The BBB consists of two morphologically distinct components :
 Capillary endothelia
 Choroid plexus ependymal cells connected by tight junctions
overlies fenestrated capillaries.
 +,K+,Ca+,Mg2+ tightly regulated-H
 Glucose, urea, and creatinine diffuse freely.
Proteins pass by passive diffusion
THUS BBB MAINTIANS EQUILIBRIUM.

12. Functions of the CSF
a)It provides physical support (Buoyancy) since 1500gm brain weighs about 50gm
when suspended in CSF.
b) It confers a protective effect against sudden changes in acute venous and arterial
blood pressure or impact .
c) It provides an excretory waste function since the brain has no lymphatic system.
d) It is the pathway whereby hypothalamus releasing factors are transported to the
cells of the medial eminence
e) it maintains central nervous system ionic homeostasis.

14. Specimen Collection
Lumbar puncture (LP) is the insertion of a
needle into the subarachnoid space for diagnostic
or therapeutic purposes.
This allows access to the cerebrospinal fluid (CSF)
in which the brain and spinal cord float.
Lumbar region is most often used as it allows the
needle to be inserted below the end of the spinal
cord.

15. Specimen Collection Sites
Cerbrospinal fluid may be obtained by
- Lumbar puncture
- Cistern puncture
- Lateral cervical puncture
- through ventricular cannulas and shunts.

17. Indication of lumber puncture
Diagnostic and therapeutic
 Infection - Suspicion of meningitis
 Trauma / Aneurysm - Suspicion of subarachnoid hemorrhage
Suspicion of central nervous system diseases
Degenerative/Demyelinating such as Guillain-Barré syndrome

malignancies /carcinomatous meningitis
Therapeutic relief of pseudotumor cerebri
Therapeutics' – Meningeal Chemotherapy (Leukemias) etc.

18. Absolute contraindications to lumbar puncture
Unequal pressures between the supratentorial and
infratentorial compartments, and or raised intracranial
pressure usually inferred by characteristic findings on the
brain CT scan:
Midline shift
Loss of suprachiasmatic and basilar cisterns
Loss of the superior cerebellar cistern
Loss of the quadrigeminal plate cistern
Posterior fossa mass
Infected skin over the needle entry site

19. Relative contraindications to lumbar puncture
Increased intracranial pressure (ICP)

Coagulopathy
Brain abscess

20. Diseases Detected BY CSF Examination
High Sensitivity , High Specificity
Bacterial, Tuberculous and Fungal meningitis
High Sensitivity, Moderate Specificity
 Viral meningitis, CNS syphilis, Sub Arachnoid hemorrhage, Multiple
sclerosis Paraspinal Abscess, Infectious polyneuritis,
Moderate sensitivity, High Specificity.
 Meningeal Malignancy /Metastasis
Moderate sensitivity , Moderate Specificity
Intracranial Hemorrhage, Subdural hematoma ,Viral encephalitis

21. Specimen Collection
CSF specimen is usually divided in into three serially collected
sterile tubes:
Tube 1 is for chemistry and immunology studies,
Tube 2 is for microbiological examination
Tube 3 is for cell studies /cytology
If traumatic tap ( Hemorrhagic ) discard tube 1 and 2 , use tube 3
Tube one should never be used for microbiological purpose.
Glass tubes should never be used for cell counts
Differentials should be carried out within one hr of collection
Refrigeration should be avoided for culture of H Influenza and N
Meningitides

22. Opening Pressure changes
A manometer should be attached prior to fluid removal to record opening
pressure.
The normal opening adult pressure is 90-180 mm of water in lateral
decubitus position Neonates:10-100 mm of water
Physiological Changes:
CSF pressure varies with postural changes, blood pressure, venous
return, Valsava maneuver, and factors that alter cerebral flow.
Slightly higher in patients sitting up and also alters in respiration.
Pressures maybe as high as 250mm of water are seen in obese individuals

23. Elevated opening pressure
Tensed/ stressed individual
Hypo-osmolality
Cerebral edema
Meningitis
Superior vena cava syndrome
Thrombosis of venous sinuses
Mass lesions
Congestive heart failure
Conditions inhibiting CSF absorption
OPENING PRESSURE ABNORMALITY MAYBE THE ONLY
ABNORMALITY WHICH IS SEEN IN CASES OF CRYPTOCOCCAL
MENINGITIS, PSEUDO TUMOR CEREBRI.

24. Decreased CSF Pressure
Decreased opening pressure
Spinal-subarachnoid block
CSF leakage
Dehydration
Circulatory collapse

25. Gross Examination – color/appearance
Upto < 20ml of CSF can be removed.
Normal CSF is clear (Like water) and colorless and has viscosity
similar to water.
Turbidity or cloudiness begins with Leukocyte counts >200cells/μl or
Red cells counts >400cells/ μl, Protein level >150mg/dl
other causes:
- Grossly bloody fluids ( >6000cells/ μl)
- Microorganism (bacterial, fungi, ameobas)
- Radiographic contrast material
- Aspirated epidural fat
-
Cells counts of >50cells/dl can be detected by expert observer when the test
tube with fluid is put against sunlight in 90’ angle , it gives a “sparkling or a
snowy effect” as suspended particles scatter light known as TYNDALL ‘S
EFFECT

26. Gross examination……….cntd
Clot formation is seen in traumatic taps , complete spinal block
(froin’s syndrome) suppurative and tuberculous meningitis. It
is NOT seen in subarachnoid hemorrhage.
Fine surface pedicles may be observed after refrigeration for
12 to 24 hrs. Clots interfere with cell counts accuracy by
trapping cells.
Viscous CSF - metastatic mucinous adenocarcinomas,
crypotococcal meningitis, or liquid nucleus pulposus rupture.
Pink red color usually indicates presence of blood , grossly
bloody CSF

27. Xanthochromia
Xanthochromia refers to pale pink to yellow color in supernatant of the
centrifuged CSF, due to the lysis of the RBCs and hemoglobin break down.
Pale pink to orange xanthochromia is usually detected in case of Subarachnoid
hemorrhage 2 to 4hrs back with a peak intensity in 12hrs and disappearing in 24
to 48hrs time.
Pink/ Yellow – RBC, Hb, Bilirubin (bilirhachia), Protein; RBC Lysis
Orange- RBC, Hb ,Caroteinoids;
Brown –Melanin.
Other significant causes causing xanthochromia
 CSF protein levels >150mg or traumatic taps (>100,000 RBC’s/μl) or
 pathological states as in Complete spinal block, polyneuritis and meningitis
 Rifampin therapy

28. Microscopic Examination
CSF counts peformed on manual counting chamber.
Cell counts in CSF samples are usually too low for accurate
precision by automated machines.
Counted in Neubauer’s chamber or Fuch’s Rosenthal
Chamber.
All 9 large squares of the Neubauer’s chamber are used.
NORMAL COUNTS:
Adult CSF Leukocyte count – 0-5 cells/ cu. mm
Neonate CSF Leukocyte count – 0- 30 cells/ cu. Mm
NO RBC’s are present in CSF.

29. Causes of Eosinophilia
Fungal Infections
 Parasitic infestations
 Idiopathic hyper Eosinophilic syndrome
Acute polyneuritis

30. Protein Analysis
Estimate protein (for protein precipitation by denaturation)
Trichloacetic acid
Sulphosalicylic acid method
Sodium sulphate.
Calorimetric methods
Lowry method
Dye binding method using Coomassie Brilliant Blue or
Ponceau S Modified biuret method.
Generally accepted Protein levels 15 – 45 mg/dl

31. Increased CSF Protein
Traumatic spinal puncture
Increase blood-CSF permeability
Arachnoiditis
Meningitis
Hemorrhage(subarachnoid, intracerebral)
Endocrine :
Decreased endocrine function
Milk alkali syndrome
Diabetic neuropathies,
Metabolic disorders: Uremia, dehydration, hereditary neuropathy, myelopathies
Drug toxicity
Ethanol, Phenothiazines, Phenytoin
CSF circulation defects
Mechanical obstruction (tumor, abscess, herniated disk), Loculated CSF effusion
Increased IgG synthesis
Neurosyphilis, Multiple Sclerosis, Subacute sclerosing panencephalitis
Increased IgG synthesis and blood – CSF permeability
Guillain- Barre’ Syndrome, collagen vascular diseases, Polyradiculopathy

32. Electrophoresis and CSF
On CSF Electrophoresis normal bands seen are – transthyretin and an
extra band of transferrin.
 CSF Rhinorrhoea or otorrhoea due to trauma can be assessed by protein
electrophoresis
CSF oligoclonal bands (2 or more discrete bands in gamma region) seen
in, Viral CNS infections, Neurosyphilis, Cryptoccocal infections, Multiple
sclerosis, Sub Acute sclerosing panencephalitis, Guillain Barre’ syndrome,
transverse myelitis, meningeal carcinomatosis, Gliobalstoma multiforme.
Alpha 2 macroglobulin : Polyneuropathies, Subdural haemorrhage
Beta 2 microglobulin : Leptomeningeal leukemia, Lymphoma, HIV
infections

33. Glucose
50-80 mg/dl
CSF/plasma glucose ratio:
0.3-0.9
40% of plasma glucose
High CSF glucose
Traumatic tap
Non meningeal syphilis
Epidemic encephalitis
Diabetic coma
Diabetes mellitus
Low CSF glucose
Bacterial meningitis
Tuberculous meningitis
Viral meningitis
Cysticercosis
Trichinosis
Ameobic meningitis
SAH
Sarcoidosis
Rheumatoid meningitis

34. Ammonia/amines
Glutamine: 5-20 mg/dl
>35 mg/dl: hepatic encephalopathy
Electrolytes
Chloride 113-130 mEq/l
Decreased in TB meningitis
Tumor markers
Carcinoembryonic Antigen
Beta HCG
Beta glucoronidase
Lysozyme(muramidase)
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35. Enzymes
Lactate
Dehydrogenase
10% serum level
Total LD level 40 u/l ( adult)
70 u/l (neonate)
LD1, LD2, LD3: TB
meningitis
LD4, LD5: bacterial
meningitis
LD1,LD2: viral meningitis
LD3,LD4,LD5: SAH
LD5: metastases
CPK-BB raised in
 trauma, hypoxia, shock,
stroke, seizures, meningitis,,
malignancy .
Prognostic marker for
CNS injury
<5 U/L: Normal
6-20U/L: mild
21-50U/L: moderate
>50U/L: severe
ADA (adenosine deaminase)
> 15 u/l Higher ADA level are in tubercular meningitis.
< 15 u/l non tubercular meningitis. .

36. Microbiological examination
second aliquot collected in a sterile container
Stored in incubator
Gross
Cobweb (veil clot): ZN stain
Turbid
Viscous
Wet Mount
Unstained
Gram’s staining / ZN staining
India ink preparation
Lactophenol cotton blue preparation
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37. Causes of meningitis
Bacterial
Meningococcus
Pneumococcus
Haemophilus
Coliforms (neonates)
Streptococci Gr B (neonates)
Staphylococci (iatrogenic)
Staph. epidermidis (shunts)
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38. Gram Stain
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Differential staining: Gram negative cocci in
pairs (diplococci) intracellular, Neisseria
Meningitidis

39. Gram Stain
Hemophilus influenzae: Gram negative
coccobacilli
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40. Z-N stain
Tuberculous meningitis ZN Stain
Fluorescent stain: auramine phenol/ auramine
rhodamine stain [more sensitive]
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41. Protozoa causing meningitis
Trophozoites
CystsCysts
AcanthamoebaAcanthamoeba
NaegleriaNaegleria
HartmanellaHartmanella

42. Protozoa causing meningitis
Toxoplasma
Trypanosomes

43. Fungal Meningitis
Blastomycosis
Histoplasma capsulatum
Cryptococcus neoformans
Coccidioidomycosis
Candida albicans
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44. India ink preparation
Cryptococcus : Capsulated budding yeasts seen as
bright round objects against a dark background
(Negative staining)
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45. Lactophenol cotton blue preparation
Candida
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BlastomycosisBlastomycosis

46. Lactophenol cotton blue preparation
Histoplasma
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47. Viral Meningitis
Coxsackie
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EchovirusEchovirus
Enterovirus: Coxsackie, Echovirus, PoliovirusEnterovirus: Coxsackie, Echovirus, Poliovirus
Mumps virusMumps virus
Herpes simplex, Varicella zosterHerpes simplex, Varicella zoster
MeaslesMeasles
AdenovirusAdenovirus
ArbovirusArbovirus

48. Cytology for malignant cells
Metastases to the leptomeninges
Primary tumors
Hematolymphoid malignancies
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49. DiagnosisDiagnosisGrossGrossOpeninOpenin
g g
pressurpressur
e(mm e(mm
water)water)
Protein Protein
(mg/dl)(mg/dl)
Fasting Fasting
glucose glucose
(mg/dl)(mg/dl)
CellCell
CytologyCytology
(/cumm)(/cumm)
Other Other
findingsfindings
Ac. Ac.
Pyogenic Pyogenic
meningitismeningitis
Purulent, Purulent,
coarse coarse
clot/turbidclot/turbid
II 50-50-
15001500
0-450-45 25-25-
10000, 10000,
PMNPMN
Gm Gm
stain, stain,
Culture, Culture,
PCRPCR
TB TB
meningitismeningitis
Pale Pale
yellow, yellow,
hazy, hazy,
cobwebcobweb
II 45-50045-50010-4510-4525-1000, 25-1000,
LL
AFB, AFB,
Culture, Culture,
PCRPCR
Viral Viral
meningitismeningitis
Clear/ Clear/
turbidturbid
N/IN/I 20-20-
200+200+
NN <500,L<500,LSerology Serology
viral viral
antibodiantibodi
eses
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