1. Dr. Saha presented on the evaluation of CSF and discussed its history, production, circulation, absorption, and functions. 2. The presentation covered CSF specimen collection via lumbar puncture and laboratory tests including cell counts, chemistries, and cytology. 3. Increased proteins, cells, or ratios between CSF and serum components can indicate conditions like meningitis, tumors, or increased blood-brain barrier permeability.

1. EVALUATION OF CSF
PRESENTER- DR S SAHA PGT MD ( Path)
MODERATOR- DR P BHATTACHARYAY
Assistant Professor
DATE- 05.10.2012
DEPARTMENT OF PATHOLOGY
RGKMC&H

2. CONTENT

3. HISTORY RELATED CSF
Presence of fluid in brain known from ancient time.
Hippocrates (460-375BC)commented on water surrounding
brain.
Discovery of CSF was attributed to Emanuel
Swedenberg(1688-1722)
Heinrich Irenaus Quincke first discovered lumbar puncture
needle.

4. INTRODUCTION
Amount adult- 60-150ml , neonate-10-60ml.
Daily production -500 ml. ( @ 0.3-0.4 ml/min)
Total volume replaced in every 5-7 hours.
Conc. greater than plasma: Na+, Cl-, Mg++ &
Glutamine.
Conc. less than plasma: Glucose, K, Ca, Fe,
Uric acid, Zn, Thyroxin.

5. SECRETION & ABSORPTION
70% of CSF secreted – choroid plexus.
Remaining CSF- Ventricular ependymal lining cell
& cerebral subarachnoid space.
CSF pass through medial & lateral foramina,
flowing over brain & spinal cord.
CSF absorption occur - arachnoid villi.

6. CONTD

7. FUNCTION OF CSF
Physical support to brain.
Protective effect against sudden changes in acute
venous & arterial blood pressure.
Excretory function.
Maintain ionic homeostasis.
Pathway for transport of certain factor in CNS.

8. HYDROCEPHALUS
Abnormal accumulation of cerebrospinal fluid (CSF) in
the ventricles, or cavities, of the brain.
Increased intracranial pressure inside the skull and
progressive enlargement of the head, convulsion, tunnel
vision and mental disability.
 2 types –communicating & noncommunicating.

9. SPECIMEN COLLECTION
CSF is collected by lumbar puncture needle.
20-22 G; Pediatric 25-27 G.
Flexible.
12 cms in length.
Parts: Needle proper & Stilette.
Lumbar, cisternal, lateral cervical puncture
method.
L3-L4 (adult) L4-L5( children)

10. CONTD…..
Indications: meningeal infection.
subarachnoid haemorrhage.
primary or metastatic malignancy
de-myelinating disease.
Normal CSF pressure-  90-180 mm water in adults.
10-100 mm water in infants &
children.
Pressure > 250mm water- Intracranial hypertension
due to ICH.
Tumors.
Meningitis .

11. CONTD…
Decreased pressure :- Spinal- subarachnoid block.
Dehydration.
Circulatory collapse.
CSF leakage.
Significant drop :- Herniation or spinal block above puncture
site.
Upto 20 ml CSF can be normally removed.
 Glass tubes should be avoided .
Specimen divided serially into 3 sterile tubes :-
Tube 1 : Chemical examination & immunologic study.
Tube 2 : Microbiologic examination.
Tube 3: Total &differential count + cytology .

12. LUMBAR PUNCTURE -PROCEDURE

13. PROCEDURE OF COLLECTION

14. DISEASE DETECTION BY LABORATORY
EXAMINATION OF CSF
 ↑ sensitivity; ↑specificity: Bacterial, TB & fungal
meningitis.
 ↑sensitivity; ↔specificity: Viral meningitis, SAH,
MS, CNS syphilis, Paraspinal abscess, Infectious
polyneuritis.
 ↔sensitivity;↑specificity: Meningeal malignancy.
 ↔sensitivity;↔specificity: Intracranial hemorrhage,
Viral encephalitis, Subdural hematoma .

15. RECOMMENDED CSF LABORATORY TESTS
Routine : Opening CSF pressure.
Total cell count ( WBC & RBC ).
Differential cell count.
Glucose ( CSF/plasma ratio ).
Total protein.
Useful under certain conditions :
Cultures.
Gram stain , acid fast stain.
Fungal & bacterial antigens.
Enzymes (LDH,ADA,CK-BB).
Cytologic examination.
Electrophoresis, PCR.
Protein (crp,tau,b-amyloid).
VDRL –syphilis.

16. DEPARTMENT OF PATHOLOGY

17. GROSS EXAMINATION
Normally- crystal clear & colourless ; viscosity like water.
May be turbid , cloudy frankly purulent or pigmented.
Turbidity – WBC > 200 cells/µl,RBC >4OO cells/µl
micro organisms, radiographic contrast,
aspirated epidural fat, protein > 150 mg/dl.
Clot formation – traumatic taps, complete spinal blocks,
suppurative & T.B meningitis.
Viscosity increased – metastatic mucin producing
adenocarcinomas, cryptococcal
meningitis.
Pink- red CSF – traumatic tap, sub arachnoid haemorrhage,
ICH, cerebral infarct.

18. XANTHOCHROMIA
Pale-pink to yellow colour to the supernatant of the
centrifuged CSF.
After centrifugation, compare with a tube of
distilled water.
Due to RBC lysis & Hb breakdown.
Orange: OxyHb.
Yellow: Bilirubin.
Yellow green: Biliverdin.
Brown: Meningeal metastatic melanoma.

19. D/ D OF BLOODY CSF
In traumatic tap, hemorrhagic fluid clears
between1st & 3rd tubes; remains uniform in SAH.
Xanthochromia, Erythrophagocytosis &
Hemosiderin-laden macrophages indicate a sub-
arachnoid bleed in absence of prior trauma.
Commercial latex agglutination immunoassay: For
fibrin derivative D-Dimer, is specific for fibrin
degradation, is negative in traumatic tap

20. MICROSCOPIC EXAMINATION
Cell counts are performed on undiluted CSF in a manual
counting chamber.
Automated counting of WBC & RBC ( Talstad-1984) – poor
precision.
Recently- automated flow cytometry of CSF using UF-100 flow
cytometer –yields rapid, reliable count; Van Acker,2001.
The normal leucocyte count in adults- 0-5 cells/µl.
in neonates- 0-30 cells/µl.
No RBC should be present in the CSF.

21. DIFFERENTIAL CELL COUNT
DC performed in a counting chamber has poor
precision.
Direct smear of centrifuged specimen- cellular
distortion.
Filtration & sedimentation are other methods.
Traumatic puncture may result in the presence of
bone marrow cells, cartilage cells, squamous cells,
ganglion cells, and soft tissue elements.
In addition, ependymal and choroid plexus cells
may rarely be seen. Moreover, blast-like primitive
cell clusters, most likely of germinal matrix origin,
are sometimes found in premature infants with
intraventricular hemorrhage .

22. CHOROID PLEXUS CELLS IN CSF.
CLUSTER OF BLAST LIKE CELLS IN CSF OF PREMATURE
NEWBORN.

23. CONTD
Among acellular component –
Corpora amylacea- spherical proteinaceous
structure, seen commonly brain in elderly,
occasionally found in CSF.
Powder crystal- By starch granules from powder
use in gloves, crystal may be mistaken with spore
of cryptoccous.

24. MANUAL COUNTER

25. CSF REFERENCE VALUES FOR DIFFERENTIAL
CYTOCENTRIFUGE COUNTS
Cell type Adult (%) Neonate (%)
Lymphocyte 62 34 20 18
Monocyte 36 20 72 22
Neutrophil 2 5 3 5
Histiocyte Rare 5 4
Ependymal cell Rare Rare
Eosinophil Rare Rare

26. CAUSE OF CSF NEUTROPHILIA
Meningitis
Bacterial meningitis
Early viral meningoencephalitis
Early tuberculous meningitis
Early mycotic meningitis
Amebic encephalomyelitis
Other infections
Cerebral abscess
Subdural empyema
AIDS-related CMV radiculopathy
Following seizures
Following CNS hemorrhage
Subarachnoid
Intracerebral
Following CNS infarct
Reaction to repeated lumbar punctures
Injection of foreign material in subarachnoid space .
metastatic tumor in contact with CSF

27. CSF NEUTROPHIL
Shortcut to csf neutrophil.lnk

28. CAUSE OF CSF LYMPHOCYTOSIS
Meningitis: Viral, Tb, Fungal, Syphilis, Leptospira,
Early bacterial.
Degenerative disorders: MS, GBS, Drug abuse
encephalopathy.
Handl Syndrome: Headache+ Nero deficit+
Lymphocytosis.
Sarcoidosis.
Polyneuritis.
CND periarteritis.

29. CSF CYTOLOGY (LYMPHOCYTE TO MONOCYTE DISTRIBUTION
RATIO 70:30).

30. PLASMA CELL IN CSF
Plasma cells, not normally present in CSF, may appear in a
variety of inflammatory conditions along with large and small
lymphocytes and in association with malignant brain tumors.
Multiple myeloma may also rarely involve the meninges.
Causes of CSF plasmacytosis :-
 Acute viral infections
Guillain–Barré syndrome
Multiple sclerosis
Parasitic CNS infestations
Sarcoidosis
Subacute sclerosing panencephalitis
Syphilitic meningoencephalitis
Tuberculous meningitis

31. CONTD….

32. CAUSES OF EOSINOPHIL IN CSF
Generally rare. Most commonly found in parasitic infections.
Most common causes of CSF eosinophilia are :-
 Acute polyneuritis
CNS reaction to foreign material (drugs, shunts)
Fungal infections
Idiopathic eosinophilic meningitis
Idiopathic hypereosinophilic syndrome
Parasitic infections
Infrequently associated with :-
 Bacterial meningitis
Leukemia/lymphoma
Myeloproliferative disorders
Neurosarcoidosis
Primary brain tumors.

33. CONTD….

34. MONOCYTE & MACROPHAGE IN CSF
 Increased CSF monocytes
lack diagnostic specificity,
usually part of a mixed cell
reaction, seen in
tuberculous and fungal
meningitis.
 Macrophages with
phagocytosed erythrocytes
(erythrophages) appear
from 12-48 hours following a
subarachnoid hemorrhage
or traumatic tap.

35. OTHER CELLS IN CSF
Cerebrospinal fluid examination for tumor cells has moderate
sensitivity and high specificity (97-98%).
Sensitivity depends on the type of tumor.Leukemic patients
has the highest sensitivity (about 70%), followed by
metastatic carcinoma (20-60%) and primary CNS
malignancies (30%).
Leukemic involvement of the meninges is more frequent in
ALL than in AML.
A leukocyte count over 5 cells/μL with unequivocal
lymphoblasts in cytocentrifuged preparations is commonly
accepted as evidence of CSF involvement.
High grade NHL like large cell immunoblastic,lymphoblastic &
burkitt’s lymphoma may involve meninges.

36. BLAST CELL IN CSF

37. LYMPHOMA CELL IN CSF

38. DEPARTMENT OF BIO- CHEMISTRY

39. PROTEIN MEASUREMENT
Protein Plasma: CSF
80% Of CSF protein
derived from blood, in Prealbumin 14
concentration of less Albumin 236
than 1% of plasma Transferrin 142
level. Ceruloplasmin 366
IgG 802
Normal value-15-45
IgA 1346
mg/dl. (adult)
90mg/dl (term infant) Fibrinogen 4940
115 mg/dl(preterm
infant)

40. CONDITION ASSOCIATED WITH
INCREASED CSF PROTEIN
Traumatic spinal puncture
Increased blood–CSF permeability
Arachnoiditis (e.g., following methotrexate therapy)
Meningitis (bacterial, viral, fungal, tuberculous)
Hemorrhage (subarachnoid, intracerebral)
Endocrine/metabolic disorders
Milk–alkali syndrome with hypercalcemia
Diabetic neuropathy
Hereditary neuropathies and myelopathies
Decreased endocrine function (thyroid, parathyroid)
Other disorders (uremia, dehydration)
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–Barré syndrome
Collagen vascular diseases (e.g., lupus, periarteritis)
Chronic inflammatory demyelinating polyradiculopathy

41. METHODOLOGY
Turbidimetric method – based on TCA or
sulphosalicylic acid & sodium sulphate for protein
precipitation. This method is simple, rapid, require
no special instrumentation.
Colorimetric methods – Lowry method, dye binding
method using CBB, & modified biuret method.
CBB method is rapid, highly sensitive, use in small
sample size.
Immunologic method measure specific protein,
require 25-30µl of CSF, simple to perform.

42. CSF SERUM ALBUMIN RATIO
Assess permeability of BBB.
Normal ratio- 1:230.
CSF/ serum albumin index- CSF albumin/ serum
albumin.
<9= Intact barrier.
9-14= Slight impairment of barrier.
14-30= Moderate impairment of barrier.
>30= Severe impairment of barrier.

43. CSF SERUM IMMUNOGLOBULIN RATIO
CSF/ serum IgG ratio = CSF IgG (mg/dl)
serum IgG (g/dl)
Normal ratio is 1:390.
CSF IgG index = CSF IgG ( mg/dl) x serum albumin (g/dl)
serum IgG ( g/dl) x CSF albumin (mg/dl)
It can increase in intrathhecal IgG synthesis or increased IgG
crossover. Normal level- 3.0-8.7.
% of CSF IgG increases in multiple sclerosis.
Increased CSF IgM & kappa light chains- marker of MS.

44. OTHER CSF PROTEIN
 Proteins Major diseases
 Alpha-2-macroglobulin Subdural hemorrhage,
bacterial meningitis.
 Beta-amyloid and tau proteins Alzheimer's disease.
 Beta-2-microglobulin Leukemia/lymphoma &
Bechet’s syndrome.
 C-reactive protein Bacterial and viral Meningitis.
 Fibronectin Lymphoblastic leukemia,AIDS
 Methhemoglobin Mild subarachnoid/subdural
 haemorrhage.

 Myelin basic protein Multiple sclerosis, other
 tumors.
 Protein 14-3-3 Creutzfeldt–Jakob disease
 Transferrin CSF leakage (otorrhea)

45. CSF GLUCOSE
Derived from blood glucose.
Fasting CSF glucose- 50-80 mg/dl .
Normal CSF/ plasma glucose level – 0.3-0.9.
CSF values below 40 mg/dl or ratios below 0.3 are
considered to be abnormal.
Hypoglycorrhachia is a characteristic finding of
bacterial, tuberculous, and fungal meningitis.
Decreased CSF glucose – due to increased anaerobic
glycolysis in brain tissue by leukocytes and impaired
transport into the CSF.

46. CONTD
CSF glucose levels normalize before protein levels
and cell counts during recovery from meningitis ,
useful parameter in assessing response to
treatment.
Increased CSF glucose is of no clinical
significance, reflecting increased blood glucose
levels within 2 hours of lumbar puncture. A
traumatic tap may also cause a spurious increase
in CSF glucose.

47. CSF LACTATE
CSF & blood lactate level are largely independent.
Normal level Newborn-10-60 mg/dl.
Older child & adult-9-26 mg/dl.
Lactate measurement - helps in differentiating viral
meningitis from bacterial, mycoplasma, fungal, and
tuberculous meningitis
In viral meningitis - lactate levels are usually below
25 mg/dL and almost always less than 35 mg/dL,
whereas bacterial meningitis has levels above 35
mg/dl.
Persistently elevated ventricular CSF lactate levels
are associated with a poor prognosis in patients
with severe head injury.

48. CSF F-2 ISOPROSTANES
F-2 isoprostane level are inceased in patients with
alzheimer disease.

49. CSF ENZYMES
Adenosine deaminase (ADA)
Since ADA is particularly abundant in T lymphocytes, which
are increased in tuberculosis – useful in the diagnosis of
pleural, peritoneal, and meningeal tuberculosis.
More recently, ADA levels greater than 15 U/L were found to
be a strong indication of tuberculous meningitis since
nontuberculous meningitis consistently had levels less than
15 U/L.
Creatine kinase (CK)
Increased CSF CK activity has been reported in numerous
CNS disorders including hydrocephalus, cerebral infarction,
various primary brain tumors, and subarachnoid hemorrhage.
Since the CK-BB isoenzyme comprises about 90% of brain CK
activit, so CK isoenzyme measurements are more specific for
CNS disorders than total CK.

50. CONTD…
Lactate dehydrogenase-
LDH-1, LDH-2 isoenzyme is very high in brain.
CSF –LDH level is high – CNS leukaemia, lymhoma,
metastatic carcinoma, bacterial meningitis, SAH.
Lysozyme –
Since the enzyme is particularly rich in neutrophil and
macrophage,its activity is very low in normal CSF. However,
CSF lysozyme activity is significantly increased in patients
with both bacterial and tuberculous meningitis.
Ammonia, Amines, and Amino Acids –
CSF ammonia levels vary from 30-50% of the blood values.
Elevated levels are generally proportional to the degree of
existing hepatic encephalopathy.

51. CSF ELECTROLYTE
Osmolality 280–300 mOsm/L
Sodium 135–150 mEq/L
Potassium 2.6–3.0 mEq/L
Chloride 115–130 mEq/L
Carbon dioxide 20–25 mEq/L
Calcium 2.0–2.8 mEq/L
Magnesium 2.4–3.0 mEq/L
Lactate 10–22 mg/dL
pH Lumbar fluid 7.28–7.32

52. CSF TUMOR MARKERS
Carcinoembryonic antigen (CEA)-
Metastatic carcinoma of leptomeninges.
Human chorionic gonadotropin (HCG)-
Choriocarcinoma and malignant germ cell tumors with a
trophoblastic component.
Alpha-fetoprotein-
Increased in germ cell tumors with yolk sac elements.
Elevation of CSF ferritin is a sensitive indicator of CNS
malignancy but has very low specificity since it is also
increased in patients with inflammatory disorders.

53. DEPARTMENT OF MICROBIOLOGY

54. BACTERIAL MENINGITIS
A thorough and prompt microbiologic examination of CSF –
useful for a definitive diagnosis.
Bacterial Meningitis-
The most common agents of bacterial meningitis are-
group B streptococcus (neonates)
Escherichia coli (newborn to 1 month)
Neisseria meningitidis (3 months and older)
Streptococcus pneumoniae (3 months and older)
Haemophilus influenzae (3 months to 18 years)
Listeria monocytogenes (neonates, elderly, alcoholics, and
immunosuppressed)
 Cerebrospinal fluid shunts, head trauma, and neurosurgery
place patients at risk for CNS infections from
Staphylococcus species, aerobic Gram-negative bacilli, and
Propionibacterium species.

55. CSF GRAM STAIN SHOW GRAM
NEGATIVE DIPLOCOCCI

56. CSF GRAM STAIN SHOW GRAM
POSITIVE ROD

57. CONTD….
The Gram stain remains an accurate, rapid method to
diagnose CNS infections.
All specimens should be concentrated by centrifugation before
Gram stain and culture.
Recent tools used -
 Binax NOW® Streptococcus pneumoniae antigen test-an
immunochromatographic membrane assay.
Latex agglutination bacterial antigen tests (BAT) -detect H.
influenzae, N. meningitidis, S. pneumoniae, and beta-
hemolytic group-B streptococcus.
The limulus lysate assay - very sensitive test for the
presence of endotoxin, a product of most Gram-negative
bacteria. It is particularly useful as a rapid test in the
newborn where early diagnosis and treatment are critical.
Polymerase chain reaction .

58. SPIROCHETAL MENINGITIS.
The diagnosis of CNS infection in patients with
syphilis relies primarily on CSF parameters and
serologic testing.
Abnormalities in CSF protein and cell counts are
common in syphilitic meningitis, although they are
nonspecific.
The standard nontreponemal test performed on CSF
is the VDRL . If there are few erythrocytes
contaminating the CSF, the VDRL specificity is high
but sensitivity is low.
Treponemal tests, such as the treponemal antibody
absorption (FTA-ABS), are both sensitive and
specific for syphilis.

59. VIRAL MENINGITIS
Enteroviruses (echoviruses, Coxsackieviruses, polioviruses)
are responsible for up to 80% of meningitis cases, with a
seasonal peak in late summer.
Most patients present with a CSF pleocytosis,neutrophils may
be observed early in the infection, patients soon develop a
predominance of lymphocytes.
Reverse transcriptase polymerase chain reaction (RT-PCR) is
significantly more sensitive than cell culture – evolving as the
‘gold standard’ for the diagnosis of viral meningitis.
PCR amplification of HSV-2 DNA in CSF may be useful in the
early diagnosis of HSV encephalitis.
False negatives might occur in very early infections and
bloody taps.
Serum and CSF serologies for HSV antibody may be useful,
when PCR becomes negative ( after 2 weeks.)

60. HIV MENINGITIS
A wide variety of CSF abnormalities are
lymphocytic pleocytosis, elevated IgG indexes, and
oligoclonal bands.
Identifying opportunistic infections is the most
important indication for examining the CSF.
Serious fungal infections may exist in the presence
of little or no CSF parameter abnormalities

61. FUNGAL MENINGITIS
Cryptococcus is the most frequently isolated fungal pathogen
from CSF.
India ink or nigrosin stains show cryptococcus capsular halos.
Detection of cryptococcal antigen from sera or CSF using
latex agglutination has high sensitivity, ranging from 60-95%
False negatives due to a prozone effect, low concentration of
polysaccharide, Early disease, intraparenchymal infection,
infection with nonencapsulated Cryptococcus neoformans
variants may occur.
Conversely, sera or CSF from patients with rheumatoid factor
or Trichosporon beigelii infections may be falsely positive.
If clinical suspicion for dimorphic or filamentous fungi is high,
large volumes of CSF (approximately 15-20 mL) are optimal
for culture to improve recovery of fungal organisms.

63. TUBERCULAR MENINGITIS
Abnormal CSF with elevated protein and lymphocytic predominance
are the hallmark features of tuberculous meningitis.
The sensitivity of CSF acid-fast stains for the diagnosis of
tuberculous meningitis is highly variable.
PCR nucleic acid amplification for detecting Mycobacterium
tuberculosis DNA-specific sequences - yields rapid and accurate
diagnosis of tuberculous meningitis.
DOT enzyme linked immunosorbent assay (DOT ELISA) has been
standardized to detect tuberculosis antigens and antibodies against
M. tuberculosis in CSF.
Other tests,e.g-ligase chain reaction amplification is reportedly a
rapid method for the early diagnosis of tuberculous meningitis.
Moreover, adenosine deaminase (ADA) levels are significantly higher
in tuberculous meningitis than in other types of meningitis and CNS
disorders.
Indeed, a level greater than 15 U/L is a strong indicator of
tuberculous meningitis ( Choi, 2002 ).

64. PRIMARY AMEBIC
MENINGOENCEPHALITIS
This rare disease is caused by the free-living ameba Naegleria
fowleri or Acanthamoeba species.
Naegleria is more likely to cause an acute inflammatory
response with a neutrophilic pleocytosis, decreased glucose
level, an elevated protein concentration, and the presence of
erythrocytes.
Gram stain is always negative.
Acanthamoeba more often produces a granulomatous
meningitis.
Motile Naegleria trophozoites may be visualized by light or
phase-contrast microscopy in direct wet mounts, allowing
rapid diagnosis.
Can also be identified on Wright's or Giemsa-stained
cytospins, but must be distinguished from macrophages.
 Acridine orange stain is useful to differentiate ameba (brick
red) from leukocytes (bright green).

65. LUMBAR CSF FINDINGS IN
MENINGITIS
TEST BACTERIAL VIRAL FUNGAL TUBERCULOUS
PRESSURE INCREASE NORMAL VARIABLE VARIABLE
COUNT >1000 <100 VARIABLE VARIABLE
DIFFERENTI NEUTROHIL LYMPHOCYTE LYMPHOCYTE LYMPHOCYTE
AL COUNT
PROTEIN Mild↑ NORMAL ↑ ↑
GLUCOSE <40mg% NORMAL ↓ ↓
LACTATE Mild↑ N-Mild↑ Mild-Mod ↑ Mild-Mod ↑

66. REFERENCE
 Henry’s Clinical Diagnosis & Management by Laboratory
Methods, 21st edition, 426-427, Year 2007.
 Todd-Sanford Clinical Diagnosis by laboratory methods, 15th
Edition, 1254-1265, Year 1969.
 Medical lab manual for tropical countries, Vol II, 1st edition,
160-173, Year 1984.
 www.google.com
 images.google.com
 images.yahoo.com
 www.answers.com
 www.wikipedia.org

67. THANK YOU