Diagnostic Laboratory Section

Method of Estimation

METHOD FOR CHOLESTEROL ESTIMATION 

Sample

• Serum / plasma

Reagents

• Cholesterol Reagent
• Working cholesterol standard, 200 mg%
• Precipitating reagent

Procedure

• For total Cholesterol
  • Mark three test tubes as Blank, Standard and test and proceed as follows.

	Blank	Standard	Test
Cholesterol Reagent	3.0μL	3.0μL	3.0μL
Standard	--	15 μL	--
Serum	--	--	15μL

• Mix well and keep the tubes immediately in the boiling water bath exactly for the 90 seconds (1 ½ minutes).
• Cool them immediately to room temperature under running tap water. Measure the O.D. of the tubes at 560 nm or using a yellow green filter.

For HDL Cholesterol

Step I: HDL Cholesterol separation

Pipette into centrifuge tube	Quantity
Sample	200 μl
Precipitating reagent	2000 μl

• Mix well and keep at room temoerature for 10 minutes.
• Then centrifuge at 2000 rpm for 15 minutes to obtain a clear supernatant.

Step II: HDL- Cholesterol estimation

• Mark three test tubes, Blanks, Standard and test and proceed as follows.

	Blank	Standard	Test
Cholesterol Reagent	3.0mL	3.0mL	3.0mL
Standard	-	15 μL	-
Serum	-	-	120μL

• Mix well and keep the tubes immediately in the boiling water bath exactly for 90 seconds (1 ½ minutes).
• Cool them immediately to R.T. under running tap water. Measure the O.D of all the tubes at 560 nm using a yellow green filter.

Calculations

Total cholsterol (mg/dl) = (O.D of T / O.D of S) X 200

HDL cholsterol (mg/dl) = (O.D of T / O.D of S) X 50