HDL-CHOLESTEROL STABILITY AND PREPARATION OF REAGENTS

R1. Macro assays: Contents ready for use undiluted. Stable

(HDL) up to the expiry date specified when stored at +15 to
+25C.
PRECIPITANT
MANUAL R1. Rx Monza and Semi-Micro assays: Predilute the
RX MONZA precipitating reagent in the ratio 4 + 1 with redistilled
water (dilute the contents of 80 ml bottle with 20 ml
INTENDED USE redistilled water). Stable up to the expiry date specified
For the quantitative in vitro determination of HDL-Cholesterol when stored at +15 to +25C.
in serum and plasma. This product is suitable for manual use
and on the Rx Monza analyser.
MATERIALS PROVIDED
HDL-Cholesterol Precipitant
Supplementary pack for Cholesterol, CHOD-PAP method.

Cat. No. MATERIALS REQUIRED BUT NOT PROVIDED

CH 203 R1. Precipitant 4 x 80 ml Randox Lipid Controls:-
Level 1 LE 2661 or LE 2668
GTIN: 05055273201161 Level 2 LE 2662 or LE 2669
Level 3 LE 2663 or LE 2670
CLINICAL SIGNIFICANCE(1) Randox Aqueous Cholesterol Standard, Cat. No. ST 1590.
High-density lipoproteins (HDL) are one of the major classes Reagent Solution for Cholesterol CHOD-PAP Assay,
of plasma lipoproteins. They are composed of a number of Cat. Nos. CH 200, 201 and 202.
heterogeneous particles, including cholesterol and vary with
respect to size and content of lipid and apolipoprotein. HDL PROCEDURE NOTES
serve to remove cholesterol from the peripheral cells to the Only clear supernatants on centrifugation must be used. In the
liver, where the cholesterol is converted to bile acids and case of incomplete sedimentation (turbid supernatant) caused
excreted into the intestine. by elevated triglyceride concentrations, the sample should be
diluted 1 + 1 with 0.9% NaCl solution and the precipitating
An inverse relationship between HDL-cholesterol (HDL-C) step repeated. The result should then be multiplied by 2.
levels in serum and the incidence/prevalence of coronary heart
disease (CHD) has been demonstrated in a number of LDL Cholesterol: The values obtained using this assay are
epidemiological studies. The importance of HDL-C as a risk reliable provided that no chylomicrons are present in the
factor for CHD is now recognised (1). sample, the triglyceride concentration does not exceed 400
mg/dl, and the sample does not show signs of any type III
Accurate measurement of HDL-C is of vital importance when hyperlipoproteinaemia.(2)
assessing patient risk from CHD. In this diagnostic test kit a
method for direct measurement of HDL-C, without sample PROCEDURE (3)
pretreatment, is presented. Direct measurement gives
improved accuracy and reproducibility when compared to FOR USE ON RX MONZA
precipitation methods. 1. Precipitation
PRINCIPLE Pipette into centrifuge tubes:
Low density lipoproteins (LDL and VLDL) and chylomicron
fractions are precipitated quantitatively by the addition of
Sample/ Standard 200 l
phosphotungstic acid in the presence of magnesium ions. After
centrifugation, the cholesterol concentration in the HDL (high Diluted Precipitant (R1) 500 l
density lipoprotein) fraction, which remains in the
supernatant, is determined. Mix and allow to sit for 10 minutes at room temperature.
Then centrifuge for 10 minutes at 4,000 rpm, or
SAMPLE 2 minutes at 12,000 rpm.
Serum, heparinized plasma or EDTA plasma.
Separate off the clear supernatant immediately and determine
REAGENT COMPOSITION the cholesterol content by the CHOD-PAP method. The
supernatant may be stored up to five days at +2 to +25C.
Contents Initial Concentrations of Solution

R1. Phosphotungstic Acid 0.55 mmol/l

Magnesium Chloride 25 mmol/l

SAFETY PRECAUTIONS AND WARNINGS

For in vitro diagnostic use only. Do not pipette by mouth.
Exercise the normal precautions required for handling
laboratory reagents.

Health and Safety Data Sheets are available on request.

The reagents must be used only for the purpose

intended by suitably qualified laboratory personnel,
under appropriate laboratory conditions.

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 MANUAL/RX MONZA CH203

2. Cholesterol CHOD-PAP Assay CALCULATION

Pre-incubate the cholesterol reagent at +37C for at least 10 1. HDL Cholesterol
minutes before use. When using a factor:
Using fresh ddH2O perform a new Gain Calibration in cuvette
mode. Select HDL in the Run Test screen and carry out a MACRO SEMI-MICRO
water blank as instructed.
Wavelength mmol/l mg/dl mmol/l mg/dl
Pipette into a cuvette:
500 nm 4.65 180 5.43 210
Reagent Blank S0 Standard S1 Sample Hg 546 nm 7.09 274 8.27 320
ddH2O 40 l - -
Standard Supernatant - 40 l -
Sample Supernatant - - 40 l When using a standard:
CHOL Reagent 700 l 700 l 700 l Concentration of HDL Cholesterol in supernatant.

Mix, incubate for 10 minute at +37C. Mix well and insert into ASample
the Rx Monza flowcell holder and press Read. = x Conc. of Standard
AStandard
CALIBRATION
Recommended using Randox Aqueous Cholesterol Standard. 2. LDL Cholesterol(2)
Calibration is recommended with a change in reagent lot or as In mmol/l:
indicated by quality control procedures.
LDL Total Triglycerides HDL
FOR MANUAL USE Cholesterol = Cholesterol - 2.2 - Cholesterol
1. Precipitation
In mg/dl:
Pipette into centrifuge tubes:
LDL Total Triglycerides HDL
Macro Semi Micro Cholesterol = Cholesterol - 5 - Cholesterol

Sample/Standard 500 l 200 l QUALITY CONTROL

Precipitant (R1) 1000 l -- Randox Lipid Controls, Level 1, Level 2 and Level 3 are
Diluted Precipitant (R1) --- 500 l recommended for daily quality control. Two levels of controls
should be assayed at least once a day. Values obtained should fall
Mix and allow to sit for 10 minutes at room temperature. within a specified range. If these values fall outside the range and
Then centrifuge for 10 minutes at 4,000 rpm, or repetition excludes error, the following steps should be taken:
2 minutes at 12,000 rpm. 1. Check instrument settings and light source.
2. Check cleanliness of all equipment in use.
Separate off the clear supernatant within two hours and 3. Check water, contaminants i.e. bacterial growth may
determine the cholesterol content by the CHOD-PAP contribute to inaccurate results.
method. The supernatant may be stored up to five days at +2 4. Check reaction temperature.
to +25C. 5. Check expiry date of kit and contents.
6. Contact Randox Laboratories Technical Services,
2. Cholesterol CHOD- PAP Assay Northern Ireland +44 (0) 28 9445 1070.

Wavelength: 500 nm, Hg 546 nm INTERFERENCES

Cuvette: 1 cm light path The assay is unaffected by icteric samples
(bilirubin < 30 mg/dl), rheumatoid factors <1000 IU/ml,
Temperature: +20 to +25C or +37C
haemolytic samples (Hb < 500 mg/dl) and lipaemic samples
Measurement: against reagent blank
(triglyceride < 1200 mg/dl). Lipaemic samples with a
triglyceride concentration >1200 mg/dl should be diluted 1+9
Only one reagent blank per series is required.
with 0.9% (w/v) NaCl before assay. The corresponding result
should be multiplied by 10.
Pipette into test tubes:
EXPECTED VALUES (4, 5,) (NCEP GUIDELINES)
Reagent Blank Standard Sample
mg/dl mmol/l
Distilled Water 100 l -- --
Supernatant -- -- 100 l
Standard Supernatant -- 100 l --  40 1.04 Low
Reagent 1000 l 1000 l 1000 l
60 1.55 High
Mix, incubate for 10 minutes at +20 to +25C or 5 minutes at
As HDL cholesterol is affected by a number of factors such as
+37C. Measure the absorbance of the sample (Asample) and
smoking, exercise, hormones, age and sex, each laboratory
standard (Astandard) against the reagent blank within 60 minutes.
should establish its own reference ranges.

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 MANUAL/RX MONZA CH203

SPECIFIC PERFORMANCE CHARACTERISTICS

The following performance data were obtained using an Rx
Monza analyser running at a temperature of 25C.

LINEARITY
The test is linear up to a cholesterol concentration of 23.7
mmol/l (915 mg/dl). Dilute samples with a cholesterol
concentration greater than this 1+2 with 0.9% NaCl. Multiply
the result by 3.

SENSITIVITY
The minimum detectable concentration of HDL-Cholesterol
with an acceptable level of precision was determined as 0.071
mmol/l (2.75 mg/dl).

PRECISION

Intra Assay
Level 2 Level 3
Mean (mg/dl) 40.3 61.5
SD 4.23 5.4
CV(%) 10.5 8.75
n 20 20

Inter Assay
Level 2 Level 3
Mean (mg/dl) 38.2 62.9
SD 3.89 7.37
CV(%) 10.2 11.7
n 20 20

REFERENCES
1. National Institutes of Health Consensus Development
Conference Statement : Triglyceride, High Density
Lipoprotein and Coronary Heart Disease. Washington
D.C. Feb 26-28, 1992.
2. Friedewald, W.T. et al. Clin. Chem. 1972; 18: 499.
3. Lopes-Virella, M.F. et al. Clin. Chem. 1977; 23: 882.
4. Third Report of the National Cholesterol Education
Programme (NCEP) Expert Panel on Detection, Evaluation
and treatment of High Blood Cholesterol in Adults (Adult
Treatment Panel III). JAMA Publication, Vol 285, No. 19,
P2486 - 2497; 2001.
5. Jacobs, D. et al. In Laboratory and Test Handbook; Jacobs,
D.S; Kasten, B.L., De Mott, W.R., Wolfson, W.L., Eds; Lexi
- Comp Inc: Hudson (Cleveland), 1990; P. 219.

Revised 28 Apr 16 bi
Rev. 003

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