CSF Examination

Muhammad Asif Zeb lecturer Hematology IPMS-KMU

ContentsIntroductionFunction Sample CollectionCSF Lab ExaminationChemical AnalysisSerological ExaminationOther TechniquesReferences

GENERAL INFORMATION:

Both the brain and spinal cord are covered by three protective membranes referred to as the meninges. The outermost layer is called the dura mater and is composed of tough connective tissue. The middle layer is the arachnoid named for it spider web like appearance. The delicate innermost layer which is in direct contact with the brain and spinal cord is called the pia mater. An inflammation of the meninges is referred to as meningitis.

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). CSF is produced in the ventricles of the brain by a collection of rich vascular protrusions called the choroid plexus. Excess CSF is continuously reabsorbed by arachnoid villi and returned to the venous system thus maintaining a consistent amount of fluid

FUNCTIONS OF CSFTHE TWO CHIEF FUNCTIONS OF CSF (SPINAL) FLUID ARE:1. To protect and cushion the brain and the spinal cord against possible injury.

2. To deliver nutrients from the blood to the Central Nervous System and removeswastes

Normal CSFThin, colourless, clear fluid

CSF is the 3rd major fluid of the bodyAdult volume 90-150 mLNeonate volume 10-60 mL

0-5 WBC’s /mm3 (neonates 0-30/ mm3 ) (Lymphocytes & monocytes)

Occasional ependymal or choroid plexus cellsProtein 15-45mg/dlGlucose 50-80mg/dlChloride 113-130 mEq/LSterile

Lumber puncture or collection of CSf

The procedure used to obtain cerebrospinal fluid is referred to as Lumbar Puncture or a Spinal Tap.

PurposeThe purpose of CSF analysis is to diagnose medical Disorders that affect the CNS.

Viral and Bacterial Infections.Tumors of Nervous System.Syphilis, a Sexually transmitted disease.Bleeding around the brain and spinal cord.Multiple sclerosis,a disease that affects the myelin coating of the nerve fibers.Guilain-Barre syndrome,an inflammation of the nerves.

Collection Technique

full flexion

Level of entry

Spinal needle

Spinal needle

Under strict aseptic precautions.,

Collecting the CSF

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Collection: 3-10 ml is collected

Tube 1Tube 1 Chemistry & Chemistry & immunologyimmunology

Tube 2Tube 2 MicrobiologyMicrobiology

Tube 3Tube 3 Cytology and Cytology and HematologyHematology

AnalysisPhysical examinationChemical examinationMicroscopic examination

Appearance of CSFclear and colourless (normal); bright red - This indicates the presence of blood. The appearance of fresh blood in all three tubes supports the diagnosis of a subarachnoid hemorrhage. xanthochromic (yellow) - jaundice

turbid - This indicates the presence of white cells and is suggestive of a CNS infection.

Presence of clotNo clotIf present indicate high protein contentCondition With patient traumatic tapComplete spinal blockage Tuberculosis meningitisBacterial infection

Amount of CSFHow much collected

Microscopic examinationCell counts

Noramal less than 5/ul TLC count RBC count Smear preparation for Differential count to see the morphology

of WBC,sUsually few lymphocytes are present

How to count TLC and RBC???

Clinical significance of Increase TLC

Neutrophili leukocytosis =Neisseria meningitis, Hemophilus influenza and E. coliLymphocytic/monocytic leukocytosis=T.b, Viral infection, fungus or with syphilisMalignancy= Leukemia,

Leukocyte-differential

Most patients with bacterial infections=neutrophils while fungal, tubercular and viral infection = lymphocytes.

Leukocyte-differential (Contd..)

other conditions.LeukemiaPlasma cell leukemiaPresence of tumor in brain.Metastasis to CSF

Gram stain and cultureA Gram’s stain should be perform on all CSF.Cultures are also set up in order to retrieve and identify organisms present in the CSF.

If the physician suspects a viral, mycobacterial, or rickettsia organism,

it is important that special culture techniques be followed for isolation of these organisms.

Chemical analysis

CSF PROTEIN DETERMINATIONCSF Glucose DETERMINATION

ProteinNormal CSF protein concentration is 15 - 45 mg/dl. It is derived from the plasma.

Increased levels are associated with:- infection- altered capillary permeability- decreased absorption- local biosynthesis of gamma globulin

Sulfosalicylic acid testTake 3 ml of 3% sulfosalicylic acid in a tube add 1 ml of supernatant clear CSF in it.Cloudiness indicate the presence of protein

Principle (Biuret Method): Any compound(proteins) containing three or more peptide bonds reacts with alkaline copper tartrate reagent to form a blue to purple coloured

substance. The intensity of colour produced is

proportional to the number of peptide bonds reacting and, therefore, to the amount of proteins.

Biuret MethodProcedure: To 2 ml CSF add 2 ml 10%trichloracetic acid, mix well and allow to stand for 5 min. Centrifuge at high speed and discard the supernatant. Mark this tube containing precipitate as test. Take another test tube and mark it blank. To both tubes add 1 ml 15% NaOH. Shake the “test” tube to dissolve them precipitate. Add 0.5 ml 5% Copper sulphate and 4 ml distilled water. Mix thoroughly and centrifuge at high speed. Transfer the supernatant to corresponding clean tubes marked. Read absorbance of the “test” against “blank” in a colorimeter at 550 nm.

CSF GLUCOSE DETERMINATION

• Normal CSF glucose is: 60 to 70 % of the patient's serum glucose concentration.

It is generally decreased in bacterial meningitis, as well as in meningitis associated with a fungus, tuberculosis or syphilis.

Chemical analysis (Contd..)

Decreased protein levels are less commonly encountered.Common reasons are removal of large amounts of CSF and leakage of CSF caused by trauma.Glucose concentrations can be determined using the same methodologies as used for blood.The most common finding is a low glucose concentration associated with most types of CNS infections.If patient is hyperglycemic, then the CSF glucose concentrations will like wise be elevated and an infection may be masked.If the patient is hypoglycemic the physician may be misled by a low CSF glucose.

Chemical analysis (Contd..)

Lactate determination is considered to be of some use by physicians in differentiating bacterial, tuberculous and fungal meningitis from viral meningitis.The lactate concentration in viral meningitis is generally normal or low in comparison to that in bacterial, tuberculous, or fungal meningitis.

Serologic examinationSerologic examination of CSF is typically involved with diagnosis of neurosyphilis.A fluorescent treponemalantibody absorption (FTA-ABS) test for CSF has also been developed.The VENERAL DISEASE RESEARCH LAB. (VDRL) Test is also used.TPHA test or TPPA test for syphlus

Other techniquesIt is important to recognize other techniques that have been used and may still be used for CSF.

Immunoelectrophorsis (CIE), latex agglutination tests for bacterial antigens, enzyme linked immunosorbent assay (ELISA) and limulus of gram-negative bacterial).

The nucleic acid probes using PCR are the newest of the techniques being investigated for indentification of specific infectious agents.

Thank You