LDL Cholesterol
Enzymatic Colorimetric
Code: HB028
1 x 30 ml + 1 x 10 ml
Store at 2-8ºC. Liquid. HDL/LDL calibrator included.
Clinical significance
Samples
The low density lipoproteins cholesterol (LDLc) particle consists
of lipoproteins that transport cholesterol to the cells. LDLc’s are
often called “bad cholesterol” because high levels are a risk
factor for coronary heart disease and are associated with
obesity, diabetes and nephrosis.
Serum: After sampling, the test should be performed without
delay. Repeated freezing and thawing should be avoided.
Stability of the sample: 7 days at 2-8°C
Principle
LDLc assay does not require any sample pre-treatment or fractionation
step and measure serum LDL-C levels directly through the use of
specially formulated surfactants. The assay consist of two steps. In the
first step no-LDL lipoproteins are eliminated under specific conditions.
CHE
Chol. Esters + H2O⎯⎯−−⎯→ Cholest. + fatty acids
CHOD
Cholesterol + O2⎯−−−−−⎯⎯→ 4-Cholestenon + H2O2
catalase
2 H2O2 −−−−−−⎯→ 2 H2O + O2
In the 2nd step, the remaining LDL cholesterol can be measured
specifically as a color development (quinone pigment) after a series of
enzymatic reactions. The intensity of the color formed is proportional to
the LDLc concentration in the sample.
CHE
Chol. Esters + H2O⎯⎯−−⎯→ Cholest. + fatty acids
CHOD
Cholesterol + O2⎯−−−−−⎯⎯→ 4-Cholestenon + H2O2
POD
2 H2O2 + TOOS + 4-AA −−−−−−⎯→ Quinonimine + 4H2O
Reagent Composition
Reagent 1
Enzymes
PIPES pH 7,0 ………………......….50 mmol/L
Cholesterol esterase (CHE)...........≥ 600 U/L
Cholesterol oxidase (CHOD).........≥ 500 U/L
Catalase .…………………...…...…≥ 600 KU/L
TOOS…………………...….....………2 mmol/L
Reagent 2
Enzymes
PIPES pH 7,0.........………….……..50 mmol/L
4-AA………...………………………...4 mmol/L
Peroxidase (POD)...……...…….……≥ 4 KU/L
Procedure
1. Wavelength 600 nm (590-700); Temperature 37°C; Cuvette 1
cm light path.
2. Adjust the instrument to zero with distilled water.
3. Pipette into a cuvette:
Blank
Calibrator
Calibrator
--5 µl
Sample
----R1
375 µl
375 µl
Mix, incubate 5 min at 37°C.
Add:
R2
125 µl
125 µl
Mix, incubate 5 min at 37°C
Read the absorbance (abs) against the blank
Sample
--5 µl
375 µl
125 µl
Calculation
LDLc (mg/dl) =
= (Abs. Sample / Abs. Stand.) x calibrator conc.
Conversion factor: mg/dl x 0,02586 = mmol/l.
Quality control
Control sera are recommended to monitor the performance of assay
procedures. If control values are found outside the defined range,
check the instrument, reagents and calibrator for problems. Each
laboratory should establish its own QC scheme and corrective
actions if controls do not meet the acceptable tolerances.
Normal and pathological human sera (HBC01, HBC02) are available.
Reference values
HDL/LDLCalibrator Lyophilized human serum…conc: see label
For in vitro diagnostic use only.
Levels of the risk:
Less than 100 mg/dl
Desirable
130-160 mg/dl
Medium
160 mg/dl and above
High
These values are for orientation purpose. Each laboratory
should establish its own reference range.
Precaution
Performance characteristics
Calibrator: Components from human origin have been tested
and found to be negative for the presence of HbsAg, HCV and
antibody to HIV(1/2). However handle cautiously as potential
infectious.
Traceability: Values are assigned according to the Cholesterol
Reference Method Laboratory Network (CRMLN).
Preparation
R1 and R2 are ready to use.
Calibrator: Dissolve the contents with 1 ml of distilled water. Cap
and mix gently to dissolve contents.
Storage and stability
All the components of the kit are stable at 2-8°C up to the date of
expiration as specified, when stored tightly closed, protected
from light and contaminations prevented during their use.
R1 and R2: Once opened is stable 4 weeks at 2-8°C
Calibrator: once reconstituted is stable 2 weeks at 2-8°C or 3
months at –20°C.
The reagents should be a clear solution. If turbidity or
precipitation has occurred, the reagent should be discarded.
Additional equipment
- Spectrophotometer or colorimeter measuring at 600 nm
- Matched cuvettes 1,0 cm light path
- General laboratory equipment
Measuring range: from 7 mg/dl (detection limit) to 1000 mg/dl
(linearity limit). If the obtained results are greater than 1000
mg/dl, dilute the sample 1:2 with saline solution, repeat the
determination, and multiply the result by factor 2.
Presicion:
Mean (mg/dl)
SD
CV (%)
Intra-assay (n=10)
71,75
108,6
177,6
0,44
1,05
1,93
0,62
0,96
1,09
Inter-assay (n=10)
98
153
207
2,44
3,39
3,63
2,5
2,21
1,75
Sensitivity: 1 mg/dl = 0,0015 Abs
Accuracy: Results obtained using CYPRESS DIAGNOSTICS
reagents did not show systematic differences when compared
with other commercial reagents.
Interferences
No interferences were observed with ascorbic acid up to 50
mg/dl, hemoglobin up to 500 mg/dl, bilirubin up to 30 mg/dl,
rheumatoid factors up to 1000 IU/ml or lipaemic samples up to
1200 mg/dl. Lipaemic samples with a triglyceride concentration
> 1200 mg/dl should be diluted 1/10 with NaCl 9 g/l and multiply
the results by 10.
Bibliography
Kaplan A et al. Lipoprotein. Clin Chem The C.V. Mosby Co. St Louis.
Okada M. et al. J. Lab. Clin. Mad., 1998; 132, 195-201
Young DS. Effects of drugs on Clinical Lab. Tests, 4th ed AACC Press 1995
Young DS. Effects of diseases on Clinical Lab. Tests, 4th ed AACC 2001
Burtis A et al. Tietz Textbook of Clinical Chemistry, 3rd ed AACC 1999
Tietz N W et al. Clinical Guide to Laboratory tests, 3rd ed AACC 1995.
Langdorp, 06. 2011.
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