How to perform CSF Examination- Detailed procedure for Physical, chemical,cell counts

What is CSF?

Cerebrospinal fluid (CSF) is an ultrafiltrate of plasma contained within the ventricles of the brain and the subarachnoid spaces of the cranium and spine. It performs vital functions, including providing nourishment, waste removal, and protection to the brain . CSF can be tested for the diagnosis of a variety of neurological diseases, usually obtained by a procedure called lumbar puncture

Intended use: useful in the diagnosis of bacterial, fungal, mycobacterial, and viral central nervous system (CNS) infections and, in certain settings, for help in the diagnosis of subarachnoid hemorrhage (SAH), CNS malignancies, demyelinating diseases, and Guillain-Barré syndrome.

Sample collection: sample collection CSF is collected in a series of sequential tubes. The subsequent tubes collected will not be contaminated with peripheral blood .

TEST PROCEDURE:

Routine CSF consists of:

Physical examination of CSF

Normal cerebrospinal fluid (CSF) is clear and colorless: Includes volume, appearance, clot formation, and color of the specimen. A cloudy colorless CSF sample is associated with an increase of nucleated cells, such as WBCs or malignant cells. A clear, but yellow CSF could be due to a previous subarachnoid hemorrhage (SAH), often called a brain bleed, or an abnormal chemical constituent, such as bilirubin.

Red or bloody CSF is associated with red blood cells.

Xanthochromia: refers to pale pink,orange to yellow color in the supernatant of centrifuged CSF .Yellow, pink, and light orange are commonly associated with red blood cell degradation products.

To detect xanthochromia supernatant fluid should be compared with distilled water.

visible xanthochromia can be due to following

1.Oxyhemoglobin from artifactual red cell lysis in needle or detergent contamination in collecting tube.

2.bilirubin in jaundiced person

3.CSF protein over 150mg/dl present in traumatic tap, pathological sates like soinal block,polyneuritis,meningitis

5.hypercarotenemia,melanin (mets of melanoma) ,rifampicin

Differential diagnosis of bloody CSF

1. Normally, CSF is collected in a sequential series of three or four tubes. In traumatic tap, the hemorrhagic fluid usually clears between the first and third collected tubes as blood contamination will decrease or clear as the tubes are collected., but remains relatively uniform in subarachnoid hemorrhage

2..Evidence of eythrophagocytosis or hemosiderin laden macrophages indicate a subarachnoid bleed in absence of prior traumatic tap.

 3. D-dimer by latex agglutination immunoassay test for cross-linked fibrin derivative D dimer is specific for fibrin degradation and negative in traumatic taps, The D-dimer is positive in patients with subarachnoid hemorrhage.

4. Blood clots in a bloody CSF indicate a traumatic tap. Blood present from a subarachnoid hemorrhage will not clot as it has been subjected to the body’s fibrinolytic system. 

Traumatic Tap	Subarachnoid Hemorrhage
Blood in decreasing amounts	Blood in equal amounts (sequential tubes )
Clot formation	No clot formation
Colorless supernatant	Xanthochromia
Hemosiderin and hematoidin crystals not present	Hemosiderin and hematoidin crystals present
Negative D-dimer	Positive D-dimer

Clot formation is noted in an uncentrifuged specimen. May be present in traumatic taps, complete spinal block, or suppurative or tuberculous meningitis. clots interfere with cell count accuracy by trapping inflammatory cells.

Viscous CSF: encountered in patients with metastatic mucin producing adenocarcinoma, cryptococcal meningitis, liquid nucleus pulposus.

Chemical examination of CSF:

Includes the estimation of glucose and micro proteins . If a single bulb of the specimen is sent then microscopic examination is done first and then the remaining fluid is centrifuged at 2000 rpm for 10 minutes , of which the supernatant is used to estimate glucose and proteins and the sediment is stained with Leishmans for the differential count.

CSF proteins under normal conditions contains very little proteins, (15-45mg/dl) which is below 1% of the normal serum protein level. In case of inflammation of the meninges under toxic conditions or development of tumors, the barrier between the blood and brain become more permeable and increased amounts ofprotein enter the subarachnoid space and appear in the CSF.

Reference Range- 15-45 mg/dl

Infants have significantly higher CSF protein levels than older children and adults.

Glucose level in CSF is about 20 mg/dl less than that in blood, which means 40-85 mg/dl of CSF, but the amount, may vary with blood sugar levels. The glucose level in spinal fluid is reduced in bacterial meningitis, which may be due to increased number of leukocytes and pathogenic organisms, and both contribute to increased glycolysis. Decreased glucose level is not seen in viral meningitis, primary brain tumor, or vascular accidents. It is low in metastatic tumor and insulin shock, and elevated in diabetic coma.

Reference Range- 40-85 mg/dl

Lactate: Lactate measurement has been used as an adjunctive test in differentiating viral meningitis from bacterial, fungal, and tubercular in which routine parameters give equivocal results .Reference level for adults 9-26mg/dl. Persistently raised lactate levels in CSF are associated with poor prognosis in patient with head trauma

ADA(Adenosine deaminase): because ADA is abundant in T lymphocytes, which are increased in tuberculosis its measurement is recommended in the diagnosis of meningeal tuberculosis

Type of Meningitis	Glucose	Lactate	Protein
Bacterial	Marked decrease	>35 mg/dl	Marked increase
Viral	Normal	Normal	Moderate increase
Fungal	Normal to decreased	>25 mg/dl	Moderate to marked increase
Tubercular	Decreased	>25 mg/dl	Moderate to marked increase

Microscopic examination of CSF:

Includes the leukocyte count and red blood cell count of the whole (uncentrifuged) specimen. Inaddition, the sediment of the centrifuged specimen is taken for the study of the stained smear, for the direct examination of abnormal cells and differential count. Hematology analysis is typically performed on the last tube collected to ensure that any peripheral blood that may have contaminated the sample during the lumbar puncture has cleared .

Hematologic analysis of CSF samples should be performed within one hour after the fluid has been obtained

I. Total leukocyte count or total nucleated cell count: Normal CSF is virtually free of cells, although a many as 5 cells/microlitre is considered as normal for adults and older childrens , it is upto 30 cells /microlitre in neonates and these are small lymphocytes. The presence of neutrophils or monocytes is never normal.

Increased leukocyte count suggests the possibility of infective meningitis.

Procedure for Estimation of Total Leukocyte Count

1) Gently mix the specimen.

2)Examine a drop of the fluid placed on a slide with cover slip ,it will give estimate for dilution. Normal CSF has very few cells and can be counted on the chamber directly without dilution. When few cells are present, the entire ruled area of both sides of the chamber should be counted (a total of 18 large squares).

3)If more than 90 cells are seen on undiluted sample, dilution is required For dilution : Add 20 µl of CSF diluting fluid in the labeled tubes to the tubes add 380 µl of well-mixed specimen, if u see less than 10 cells after dilution make a smaller dilution.

4) Mix well and charge both sides of two Neubauer chambers.

5) Let it stand for 5 minutes and then observe under high power

objective.

6) Count the number of WBC’s & RBC’s in four WBC squares (take a mean of both sides)

7) Calculate as follows:

WBC/RBC count/microlitre= (Number of cells counted/4) x 10

8)In cases where RBC’s interfere in counting of WBC’s, 380 µl of

WBC diluting fluid is added to 20 µl of sample. Mix well and

keep for 10-15 mins. Charge the chamber and count 4 WBC

squares. Calculate as follows:

WBC count/microlitre= (Number of cells counted/4) x 200

Differential Leukocyte Count:

For smear examination for estimation of differential count:

1) Process the sample using cytospin at 2000 rpm for 1min, Excessive red blood cells can obscure the nucleated cells, especially when concentrated. If this occurs, a small dilution of the CSF with 2% acetic acid can be made before cytocentrifugation of the sample. The acetic acid will lyse the red cells leaving the nucleated cells intact.

2) Smear formed on slide are left to dry

3) Stain the slide with Leishman stain and observe under oil immersion

Type of Meningitis	Predominant Cells
Bacterial	Neutrophils
Viral	Lymphocytes
Fungal	Lymphocytes, monocytes, eosinophils
Tubercular	Lymphocytes, monocytes,

CSF cytology for diagnosing malignant spread, primary CNS lymphoma, parasitic infestation primary amoebic meningoencephalitis. Malignancies involving the CNS are most often metastatic malignancies, such as breast and lung carcinomas or leukemia.

Read further on https://www.aafp.org/pubs/afp/issues/2003/0915/p1103.html