(Courtesy: Tulip Group of Companies)
For the determination of HDL cholesterol in serum or
plasma (for in vitro diagnostic use only).
Lipoproteins are the proteins which mainly transport fats in
the bloodstream. They can be grouped into chylomicrons,
very low density lipoproteins (VLDL), low density
lipoproteins (LDL) and high density lipoproteins (HDL).
Chylomicrons and VLDL transport mainly triglycerides,
though VLDLs also transport some amount of cholesterol.
LDL carries cholesterol to the peripheral tissues where it
can be deposited and increase the risk of arteriosclerotic
heart and peripherial vascular disease. Hence, high levels
of LDL are atherogenic. HDL transports cholesterol from
the peripherial tissues to the liver for excretion, hence,
HDL has a protective effect. The measurement of total
and HDL cholesterol and triglycerides provide valuable
information for the risk assessment of coronary heart
When the serum is reacted with the polyethylene glycol
contained in the precipitating reagent, all the VLDL and
LDL are precipitated. The HDL remains in the supernatant
and is then assayed as a sample for cholesterol using the
cholesterol (CHOD/PAP) reagent.
It is recommended that each laboratory establish its
own normal range representing its patient population.
L3 : Pricipitating reagent 2.5 mL
S : HDL cholesterol standard (25 mg/dL) 5 mL
Contents are stable at 2–8°C till the expiry mentioned on
Working reagent: Pour the contents of 1 bottle of L2 (Enzyme
reagent 2) into 1 bottle of L1 (Enzyme reagent 1). This working
reagent is stable for at least 8 weeks when stored at 2–8°C.
Upon storage the working reagent may develop a slight pink
color however this does not affect the performance of the
Alternatively for flexibility as much of working reagent
may be made as and when desired by mixing together
4 parts of L1 (Enzyme reagent 1) and 1 part of L2 (Enzyme
reagent 2). Alternatively 0.8 mL of L1 and 0.2 mL of L2 may
also be used instead of 1 mL of the working reagent directly
Serum, EDTA plasma. Cholesterol and HDL cholesterol
are reported to be stable in serum for 7 days when stored
at 2–8°C. The sample should preferably be of 12 to 14 hours
Wavelength/filter : 505 nm (Hg 546 nm)/green
Pipette into a clean dry test tube :
Precipitating reagent (L3) 0.1 mL
Mix well and incubate at RT for 5 minutes. Centrifuge at
2500-3000 rpm to obtain a clear supernatant.
Pipette into clean dry test tubes labeled as blank (B),
Mix well and incubate at 37°C for 5 minutes or at RT
(25°C) for 15 minutes. Measure the absorbance of the
standard (Abs. S), and test sample (Abs. T) against the
* If only total cholesterol is to be determined use only
0.01 mL of DW/cholesterol Std/sample directly in the
HDL cholesterol in mg/dL = _______ × 25 × 2 Abs. S
(Where 2 is the dilution factor due to the deproteinization step)
Calculation of LDL Cholesterol (mg/dL)
= Total cholesterol _ (_____________ (
Friedewald’s formula is reliable provided that:
1. No chylomicrons are present, i.e. it is a fasting sample.
2. Triglyceride values are below 400 mg/dL.
3. Type III hyperlipoproteinemia is absent.
This procedure is linear upto 150 mg/dL of HDL cholesterol.
If values exceed this limit, dilute the serum with normal
saline (NaCL 0.9%) and repeat the assay. Calculate the
value using the proper dilution factor.
The supernatant should be clear. If it is hazy or cloudy,
the sample should be diluted 1 + 1 with normal saline
(NaCL 0.9%) and the precipitation step should be repeated
Anticoagulants such as fluoride, oxalates and hemolyzed
Reaction : End Point Interval : -
Zero setting : Reagent blank Reagent
: 37°C/RT Standard : 25 mg/dL × 2
Read time : - Linearity : 150 mg/dL
Coronary heart disease (CHD) risk factor can be calculated
using total lipid profile, as suggested by Castelli, et al. The
risk factor gives a most accurate and definite assessment of
The factors are calculated by the ratio of total cholesterol
to HDL—cholesterol and by the ratio of LDL—cholesterol
(Low density lipoproteins—cholesterol) to HDL—
Risk Ratio: Total/ Ratio: LDL/
HDL__Cholesterol HDL__Cholesterol
2 × Average 9.55 7.05 6.25 5.03
3 × Average 23.99 11.04 7.99 6.14
HDL Cholesterol ppt Set (PEG Precipitation Method)
(Courtesy: Tulip Group of Companies)
For the determination of HDL cholesterol in serum or
plasma (for in vitro diagnostic use only).
Lipoproteins are the proteins which mainly transport fats in
the bloodstream. They can be grouped into chylomicrons,
very low density lipoproteins VLDL, low density lipoproteins
(LDL) and high density lipoproteins (HDL). Chylomicrons
and VLDL transport mainly triglycerides, though VLDLs
also transport some amount of cholesterol. LDL carries
cholesterol to the peripheral tissues where it can be deposited
and increase the risk of arteriosclerotic heart and peripheral
vascular disease. Hence, high levels of LDL are atherogenic.
HDL transports cholesterol from the peripheral tissues to
the liver for excretion, hence HDL has a protective effect. The
provide valuable information for the risk assessment of
When the serum is reacted with the polyethylene glycol
contained in the precipitating reagent, all the VLDL and
LDL are precipitated. The HDL remains in the supernatant
and is then assayed as a sample for cholesterol using the
cholesterol (CHOD/PAP) reagent.
favourable risk level indicator
It is recommended that each laboratory establish its
own normal range representing its patient population.
L1: Precipitating reagent 10 mL
S: HDL cholesterol standard (25 mg/dL) 5 mL
Contents are stable at 2–8°C till the expiry mentioned on
After the precipitation step cholesterol reagent is
required additionally for conducting the cholesterol assay.
Serum, EDTA plasma. HDL cholesterol is reported to
be stable in serum for 7 days when stored at 2–8°C. The
sample should preferably be of 12 to 14 hours fasting.
Precipitation of VLDL and LDL:
Pipette into a clean dry test tube
Precipitating reagent (L1) 0.1 mL
Mix well and incubate at RT for 5 minutes. Centrifuge at
2500–3000 rpm to obtain a clear supernatant.
Procedure for the Cholesterol Assay
Wavelength/filter : 505 nm (Hg 546 nm)/green
Pipette into clean dry test tubes labeled as blank (B),
Mix well and incubate at 37°C for 5 mm. or at RT (25°C)
for 15 minutes. Measure the absorbance of the standard
(Abs S), and test sample (Abs T) against the blank, within
HDL cholesterol in mg/dL = _______ × 25 × 2 Abs S
(Where 2 is the dilution factor due to the deproteinization
Calculation of LDL cholesterol (mg/dL):
Triglycerides = (Total cholesterol) _ _______________
Freidewald’s formula is reliable provided that:
1. No chylomicrons are present, i.e. it is a fasting sample.
2. Triglyceride values are below 400 mg/dL.
3. Type III hyperlipoproteinemia is absent.
normal saline (NaCL 0.9%) and repeat the assay. Calculate
the value using the proper dilution factor.
The supernatant should be clear. If it is hazy or cloudy,
the sample should be diluted 1 + 1 with normal saline
(NaCL 0.9%) and the precipitation step should be repeated
Anticoagulants such as fluoride, oxalates and hemolyzed
Reaction : End point Interval :
: 37°C / RT Standard : 25 mg/dL × 2
Delay time : — React slope : Increasing
Read time : — Linearity : 150 mg/dL
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