CHOLESTEROL & HDL CHOLESTEROL

(CHOD- PAP Method) Step 1 - For HDL Cholesterol:

For the determination of Cholesterol and Pipette into a centrifuge tube :
HDL Cholesterol in serum. Serum / Plasma 200 µl
(For In vitro Diagnostic Use Only) HDL Reagent (A2) 300 µl
SUMMARY: Mix well allow to stand at R.T. for 5 minutes. Centrifuge at 3000 rpm for 10 minutes to get a
Cholesterol is the main lipid found In blood, bile and brain tissues. It Is the main lipid associated with clear supernatant.
arteriosclerotic vascular diseases. It Is required for the formation of steroids and cellular If the supernatant is not clear (high TGL Level) dilute the sample 1:1 with normal Saline, (result
membranes. x 2)
The liver metabolises the cholesterol and It is transported In the blood stream by lipoproteins. Step 2 - For Total and HDL Cholesterol:
Pipette into 4 test tubes labeled Blank (B), Standard (S), Total Cholesterol (Tc) and HDL
PRINCIPLE: Cholesterol (TH) as shown below :
Cholesterol Esterase
Cholesterol esters + H2O Cholesterol + Fatty acids Addition Sequence B(µl) S(µl) TC(µl) TH(µl)
Cholesterol Oxidase Cholesterol Reagent (A1) 1000 1000 1000 1000
Cholesterol + O2 Cholestenone+ H2O2
Standard (S) - 10 - -
Peroxidase
H202 + 4 Aminoantipyrine+ Phenol Red Quinoneimine dye + H2O Specimen - - 10 -
Supernatant (from Step 1) - - - 100
Intensity of the colour formed is directly proportional to the amount of cholesterol present in the Mix well and Incubate for 5 minutes at 37°C (or) 10 minutes at R.T. Read the absorbances of
sample. Standard (S), Total Cholesterol (Tc) and HDL Cholesterol (TH ) against Blank (B) at 505 nm or
with green filter (500 - 540 nm).
CONTENTS:
Pack size Cholesterol Reagent(A1) HDL Reagent(A2) Standard (s) CALCULATIONS:
Abs.Tc
2x25ml 2x25ml 5ml 1ml 1. Cholesterol in mg/dl= -------------------------- × 200
Abs.S
1x60ml 1x60ml 5ml 1ml
Abs.TH
2x50ml 2x50ml 10ml 2ml 2. HDL Cholesterol= -------------------- × 50
Abs.S
STORAGE/STABILITY:
Contents are stable at 2 - 8°C till the expiry mentioned on the labels. NORMAL REFERENCE VALUES:
Total Cholesterol : 130 - 250 mg/dl
REAGENT PREPARATION:
Reagents are ready to use for the given procedure. HDL Cholesterol (Male) : 30 - 70 mg/dl
(Female) : 35 - 90 mg/dl
SAMPLE : It is recommended that laboratories establish their own normal range representing its patient
Serum, EDTA Plasma. Cholesterol is reported to be stable in the sample for 7 days when stored population
at 2 - 8°C. The sample should preferably of 12 to 14 hours fasting.
LINEARITY:
GENERAL SYSTEM PARAMETER: This procedure is linear upto 1000 mg/dl. If values exceed this limit, dilute the serum with
Reaction End Point Sample Vol. 10µl normal saline (NaCI 0.9%) and repeat the assay. Calculate the value using the proper dilution
Wavelength 505nm (500-540nm) Reagent Vol. 1000µl factor.
The linearity of HDL Cholesterol is 150 mg/dl.
Blank Reagent blank Standard 200 mg/dl
NOTE:
Incubation 37°C 5 min / R.T.10 min React. Slope Increasing
Anticoagulants such as fluorides and oxalates result in false low values. The test is not
Delay Time 5 Sec Linearity 1000 mg/dl influenced by Hb values upto 20 mg/ dl and bilirubin upto 10 mg/dl.
Read Time 5 Sec Units mg/dl REFERENCES:
Trinder, P., (1969) Ann. Clin. Biochem, 6:24
ASSAY PROCEDURE:
Allain, C.C., etal, (1974) Clin. Chem, 20:470
Wavelength / filter : 505 nm (Hg 546 nm) / Green Flegg, H.M., (1972) Ann. Clin. Biochem. 10:79
Temperature : 37°C / R.T.
Light Path : 1 cm