OLFU-QC MEDICAL TECHNOLOGY 2022

CLINICAL CHEMISTRY | CCHM321LAB

LECTURE: HDL CHOLESTEROL| 1st SEMESTER |MIDTERM

HDL PRECIPITATION BLOOD TEST PREPARATION light .

HDL CHOLESTEROL
METHOD PRECIPITATING (PHOSPHOTUNGSTIC ACID)
SUMMARY AND PRINCIPLE
1. The determination of serum HDL Cholesterol is a
useful tool in identifying patients at risk of
developing coronary heart disease. Low HDL
cholesterol levels are associated with an increased
risk of coronary artery disease.
2. HDL Cholesterol precipitating reagent is for use in
conjunction with cholesterol reagent for enzymatic
determination of HDL Cholesterol in serum or
plasma.
3. Compared to the conventional Ultra centrifugation
method, the precipitation method is simple and
time saving particularly when combined with single
step enzymatic cholesterol reagent.
Phosphotungstate/Mg2+ precipitates Chylomicrons,
LDL and VLDL fractions. High density lipoprotein
(HDL) fraction remains unaffected in supernatant.
Cholesterol content of HDL fraction is assayed using
Cholesterol reagent.
PREPARATION, STORAGE & STABILITY
• HDL-cholesterol Precipitating Reagent is ready to
use as supplied. The kit should be stored at 2-8C
and is stable till the expiry date indicated on the
label.
CALCULATION:
mg Abs.of sample
HDL Cholesterol ( dL ) = x 100
Abs.of standard
REFERENCE VALUES FOR NORMAL PEOPLE
HDL cholesterol - 30 to 70mg/dL
Question 2.
How is LDL-C measured?
• LDL-C is most often measured indirectly, using a
calculation based on other blood lipid analytes.
Historically, the Friedewald calculation2 has been
the most common approach. This equation,
developed in the 1970s, incorporates total
cholesterol, HDL cholesterol (HDL-C), and
triglyceride concentrations:
LDL - C(mg /dL) = total cholesterol - HDL - C -
(triglycerides / 5)
➢ where "triglycerides/5" is used to represent very
low-density lipoprotein-C (VLDL - C) .

PROCEDURE FOR HDL SEPARATION: (SUPERNATENT)

• Take 500 serum/plasma and add 500 HDL
precipitating reagent to it. Mix well and centrifuge
at 4000 rpm for 10 mins to obtain clear
supernatant. Decant supernatant in clean container
and label it for further use.

NOTE:
Cholesterol reagent is not provided in this kit but
required for the estimation of Cholesterol contents in
supernatant.

COMPONENT OF REAGENT
COMPONENT CONCENTRATION
Phosohototungstic Acid 2.4 mmol/l
Magnesium Chloride 40 mmol/l
Stabilizers and inactive
ingredients

ASSAY PROCEDURE
Blank Standard Test
Reagent 1000 μl 1000 μl 1000 μl
Standard NA 50 μl NA
Sample NA NA 50 μl
(supernatant)
Mix the reagent and sample/standard in the above-
mentioned ratio and incubate for 5 mins at 37C or 10
mins at R.T.
Aspirate reaction mixture into flow cell and measure
the absorbance.
The final colour is stable for 2 hours if not exposed to

CORONEL, BARBIE H. | BSMLS 3-YA-4 1