Cerebrospinal Fluid

Presented by: Farhad Jahanfar

Contents

Chemical examination

Physical examination

Routine examination of csf

Specimen collection

Introduction

Cytoligical examination

Microbiological examination

First recognized by Cotugno in 1764

Both the brain and spinal cord are covered by three protective membranes referred to as the meninges

Between the arachnoid layer and the pia mater is a space called the subarachnoid space. It contains a clear, colorless fluid referred to as Cerebrospinal Fluid (CSF).

Supply nutrients to the nervous tissue,remove metabolic wastes and produce a mechanical barrier

CSF

CSF

CSF is produced in the choroid plexuses of the twolumbar ventricles and the third and fourth venticles.

choriod plexuses are capillary networks that formthe CSF from plasma by mechanisms of selective filtration under hydrostatic pressure and active transport secretion.

CSF

In adults, approximately 20 mL of fluid is produced every hours.( 500 ml per Day)

the total volume of CSF circulating throughout the adult Central Nervous System approximately 90 - 150 ml.

blood-brain barrier

Blood-csf barrier

CSF

CSF

Cerebrospinal fluid (CSF) analysis may be used to help diagnose a wide variety of diseases affecting the central

nervous system.

Infectious diseases such as meningitis and encephalitis

Bleeding (hemorrhaging)

Tumors

inflammation , autoimmune disorders, Guillain-Barré syndromeOr sarcoidose and multiple sclerosis

How is it used?

Specimen Collection and Handling(LP(

CSF is routinely collected by lumbar puncture between the third, fourth, or fifth lumbar vertebrae

Tube 1 for chemical and serologic tests because these tests are least affected by blood or bacteria

introduced as a result of the tap procedure;

Tube 2 is usually designated for the microbiology laboratory

Tube 3 is used for the cell count, because it is the least likely to contain cells introduced by the spinal tap

• Hematology tubes are refrigerated.

• Microbiology tubes remain at room temperature.

• Chemistry and serology tubes are frozen.

tests are performed on a STAT basis

CSF physical characteristics

.

Pressure

Increased CSF pressure: tumors, infection, abnormal accumulation of

CSF within the brain (hydrocephalus) , or bleeding.

Viscosity same consistency as water

thicker" may be seen in people with certain types of cancers or meningitis.

Appearance clear, cloudy or turbid, milky,xanthochromic, and hemolyzed/bloody.

90~180mmH2O Adults, 10~100mmH2O in children

WBC>200RBC>400

RBC>600

Xanthochromia is a term used to describe CSF supernatantthat is pink, orange, or yellow

pink (very slight amount of oxyhemoglobin( orange (heavy hemolysis(

yellow (conversion of oxyhemoglobin to unconjugated bilirubin(

Other causes of xanthochromia include elevated serumbilirubin, presence of the pigment carotene, markedlyincreased protein concentrations.

Traumatic Collection (Tap(

Grossly bloody CSF can be an indication of intracranial hemorrhage,but it may also be due to the puncture of a blood vessel during the spinal tap procedure.

1.Uneven Distribution of Blood

2.Clot Formation3.Xanthochromic Supernatant

Three visual examinations:

CSF glutamine

CSF LDH

Serological test

CSF glucose

CSF protein CSF lactic acid

CSF chemical tests

CSF Glucose

normal is about 2/3 the concentration of blood glucose (60% to 70%)

For an accurate evaluation of CSF Glucose,a blood glucose test must be run for comparison(2 hours prior (

Specimens should be tested immediately because glycolysis occurs rapidly in the CSF

CSF Glucose

CSF Glucose , WBC(neutrophils) :bacterial meningitis

CSF Glucose ,WBC(lymphocytes) : tubercular meningitis

CSF Glucose(Normal) ,WBC(lymphocytes) :viral meningitis

CSF Protein only a small amount is normally present in CSF

Normal values for total CSF protein are usually listed as

15 to 45 mg/dL higher values are found in infant

CSF IgG increased multiple sclerosis, herpes encephalitis, connective tissue diseases

and older persons.

the CSF contains protein fractions similar tothose found in serum

CSF

damage to the integrity of the BBB

Intrathecal synthesisprotein

CSF Protein

CSF/serum albumin index=CSF albumin (mg/dL(Serum albumin (g/dL(

IgG index =CSF IgG (mg/dL)/serum IgG (g/dL(

CSF albumin (mg/dL)/serum albumin (g/dL(

An index value less than 9 represents an intact BBB

values greater than 0.70 are indicative of IgG production within the CNS

Bacterial

Myelin basic protein(MBP(

Myelin protein:myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein.

seen when the covering of nerves (myelin) breaks down, such as with multiple sclerosis.

Measurement of the amount of MBP in the CSF can be used to monitor the course of MS.

CNS trauma, tumor, and viral encephalitis,peripheral neoropathy,vasculitis. Guillain-Barré syndrome

CSF protein electrophoresis

oligoclonal bands

The bands are located in the gamma region of the protein electrophoresis, indicating immunoglobulin production.

presence of two or more oligoclonal bands in the CSF that are not present in the serum can be a valuable tool in the diagnosis MS.

Transthyretin(Pre-Albumin(

β2 Transferrin(tau)

α2 Macroglubolin(AMG(

CSF normally contain very low levels of AMG

there are several components to the blood-CSF barrier, with a few large pinocytic vesiclcs transporting proteins up to 1500A into the CSF.

Polyneuropathies:Increase of AMG

The level of AMG alone, or its relationship to levels of albumin and IgG, may therefore be helpful in differential diagnosis of

Neurological disorders.

In patients with bacterial meningitis, CSF AMG levels are on average 15 times higher than is the case with nonbacterial meningitis

CSF levels of AMG normally increase with age

AMG

Dye-binding methods such as Coomassie brilliant blue or Ponceau S are used.

Methodology

Turbidimetry

(Trichloroacetic acid&sulfosalicylic acid precipitation)

urinary protein reagent strips

CSF Lactate

In bacterial,tubercular, and fungal meningitis, the elevation of CSF lactate to levels greater than 25 mg/dL occurs.

Levels greater than 35 mg/dL are frequently seen with bacterial meningitis, whereas in viral meningitis, lactate levels remain lower than 25 mg/dL

falsely elevated results may be obtained on xanthochromic or hemolyzed fluid

CSF Glutamine

Glutamine is produced from ammonia and -ketoglutarate by

.the brain cells

The normal concentration of glutamine in the CSF is 8 to 18 mg/dL.

may be increased with liver disease, hepatic encephalopathy or Reye syndrome

Some disturbance of consciousness is almost

always seen when glutamine levels are more than 35 mg/dL(coma(

used to differentiate between bacterial and viral meningitis; the level is usually increased with bacterial meningitis and not with viral meningitis; may also be elevated with leukemia or stroke.

CSF LDH

Isoenzymes LD1 and LD2 are found in brain tissue.

Isoenzymes LD2 and LD3 are found in lymphocytes.

Isoenzymes LD4 and LD5 are found in neutrophils

CSF LDH

LD5 All

Bacterial meningitis Viral meningitis Metastatic tumors Primary neoplasm

LD4,LD5 LD1,LD2,LD3

CPK: Raised CPK is found in: Brain infract Brain tumors polineoropathy MS

CK-BB < 5 Slight injury

Mild injury 5<CK-BB <20

21>CK-BB< 50 Death

CRP: It is markedly increased with bacterial meningitis.

it is often used to distinguish between bacterial and viral meningitis

Tubercular meningitis L/15< : ADA

Tumor markers — Carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and hCG may be increased in metastatic tumors.

PCR has been especially useful in the diagnosis

of viral meningitis

Serologic test: VDRL,FTA-ABS

Microscopic Examination

The cell count that is routinely performed on CSF specimens is the leukocyte (WBC) count.

Any cell count should be performed immediately(1 Hours)

Normal adult CSF contains :0 -5 WBCs/uL 0-30 WBC/ul : newborns

Children: 0-20 WBC/ul

0 :RBC

Calculation of CSF Cell Counts

Number of cells counted × dilution

Number of squares counted(9) × volume of 1 square(0.1(

cells/uL=

WBC Count

Dilutions for total cellcounts are made with normal saline

Lysis of RBCs must be obtained prior to performing the WBC count on either diluted or undiluted specimens

3% glacial acetic acid(Marcano(

Number of cells counted × dilution

Number of squares counted(4) × volume of 1 square(0.1(

WBC/ul=

Corrections for ContaminationCorrections for Contamination

WBC (added(=WBC(blood) ×RBC (CSF(

RBC (blood(

The specimen is centrifuged for 5 to 10 minutes

Differential Count

the suspended sediment are allowed to air dry and are stained with Wright’s stain

When the differential count is performed, 100 cells should be counted, classified, and reported in terms of percentage

:The WBC count seen in normal adult CSF isapproximately 70 %lymphocytesand 30 %monocytes

Increase Lymphocytes: Viral & Fungal meningitisMultiple sclerosis

Increased neutrophils: Bacterial Meningitis

cerebral abscess CNS hemorrhage

CNS infarct

77% of patients with bacterial meningitis will have a WBC count higher than 1,000 /mm,3

99% will have more than 100 per mm3. Having less than 100 WBCs/ mm3 is viral meningitis.

CSF gram stain

direct observation of microorganisms under a microscope

hemophilus influenza Neisseria meningitidis

Microbiological examination

A CSF culture is used to detect infectious organisms in the CSF(blood agar,chocolate agar)

Acid-fast staining should be done if tuberculosis is clinically suspected

Wright-gimsa staining: for Differential Count

Pathogens• Strep. pneumoniae

• Strep. agalactiae (Group B(• Listeria monocytogenes

• Aerobic Gram negative bacilli• Staphylococcus species

Haemophilus influenzae Neisseria meningitidis Streptococcus pneumoniae

5% Sheep blood agar

Chocolate agar