CHOLESTEROL (CHOL) REAGENT COMPOSITION

Enzymatic Endpoint Method Contents Concentrations in the Test

MANUAL
RX MONZA
R1. Reagent
INTENDED USE Pipes Buffer 80 mmol/l, pH 6.8
For the quantitative in vitro determination of Cholesterol in serum 4-Aminoantipyrine 0.25 mmol/l
and plasma. This product is suitable for manual use and on the RX Phenol 6 mmol/l
monza analyser. Peroxidase ≥ 0.5 U/ml
(E.C.1.11.1.7, Horse Radish, +25C)
Cat. No. Cholesterol esterase ≥ 0.15 U/ml
CH 200 R1. Reagent 6 x 30 ml (E.C.3.1.1.13. Pseudomonas, 37C)
6 x 30 ml CAL. Standard 1 x 5.5 ml Cholesterol oxidase ≥ 0.10 U/ml
GTIN: 05055273201130 (E.C.1.1.3.6. Nocardia, 37C)
CH 201 R1. Reagent 6 x 100 ml
6 x 100 ml CAL. Standard 1 x 5.5 ml SAFETY PRECAUTIONS AND WARNINGS
GTIN: 05055273201147 For in vitro diagnostic use only. Do not pipette by mouth.
Exercise the normal precautions required for handling laboratory
CH 202 R1. Reagent 8 x 250 ml reagents.
8 x 250 ml CAL. Standard 1 x 5.5 ml
GTIN: 05055273201154 This kit contains components classified as hazardous in
accordance with the Regulation (EC) No.1272/2008:
CLINICAL SIGNIFICANCE (1,2,3)
Cholesterol measurements are used in the diagnosis and treatments Cal. Standard
of lipid lipoprotein metabolism disorders. Lipids play an important
role in the body; they serve as hormones or hormone precursors,
aid in digestion, provide energy, storage and metabolic fuels, act as
functional and structural components in biomembranes and form
insulation to allow nerve conduction and prevent heat loss. WARNING
Flammable liquid and vapour
In clinical chemistry, over the last decade however, lipids have Causes serious eye irritation.
become associated with lipoprotein metabolism and atherosclerosis.
The Abell Kendell method, reported by Abell et al (1952) involved Precautions:
extraction of cholesterol by organic solvents and subsequent alkaline Keep away from heat.
hydrolysis of the cholesterol esters. This reaction is highly specific
but the reagents involved are corrosive and the method IF ON SKIN (or hair): Take off immediately all contaminated
cumbersome, rendering it impractical for routine laboratory use. clothing. Rinse skin with water/shower.
IF IN EYES: Rinse cautiously with water for several minutes.
The use of cholesterol oxidase following specimen saponification as
Remove contact lenses, if present and easy to do. Continue
described by Richmond (1973) provided the first step toward a
totally enzymatic procedure. In 1974 Allain et al. and Roeschlaw et rinsing.
al. published the first fully enzymatic procedure for cholesterol
determinations replacing chemical saponification with enzymatic Disposal of all waste material should be in accordance with
saponification. local/national guidelines.
Safety data sheet available on request.
ASSAY PRINCIPLE (4)
The cholesterol is determined after enzymatic hydrolysis and The reagents must be used only for the purpose
oxidation. The indicator quinoneimine is formed from hydrogen intended by suitably qualified laboratory personnel,
peroxide and 4-aminoantipyrine in the presence of phenol and under appropriate laboratory conditions.
peroxidase.
Cholesterol STABILITY AND PREPARATION OF REAGENT
Cholesterol ester + H2O Cholesterol + Fatty acids (R1) Reagent
esterase
Contents ready for use. The reagent is stable up to the expiry
Cholesterol
date, when stored at +2 to +8°C, in the absence of
Cholesterol + O2 Cholestene-3-one + H2O2 contamination, protected from light.
oxidase

peroxidase
(CAL.) Standard
2H2O2 + phenol + 4-Aminoantipyrine quinoneimine + 4H2O
Contents ready for use. Stable up to the expiry date when
SPECIMEN COLLECTION AND PREPARATION (5) stored at +2 to +8°C.
Serum: may be used.
Plasma: EDTA (1 mg/ml) or heparin (up to 75 U/ml) may be used. MATERIALS PROVIDED
Do not use citrate, oxalate or fluoride. Reagent
Plasma and Serum samples may be stored for up to 4 days at +4°C. Standard

MATERIALS REQUIRED BUT NOT PROVIDED

Randox Assayed Multisera Level 2 (Cat. No. HN 1530) and
Level 3 (Cat. No. HE 1532)
Randox Calibration Serum Level 3 (Cat. No. CAL 2351)

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 MANUAL/RX MONZA CH200

PROCEDURE QUALITY CONTROL

Using fresh ddH2O perform a new Gain Calibration in cuvette Randox Assayed Multisera, Level 2 and Level 3 are recommended
mode. Select CHOL in the Run Test screen and carry out a for daily quality control. Two levels of controls should be assayed
water blank as instructed. at least once a day. Values obtained should fall within a specified
range. If these values fall outside the range and repetition excludes
Pipette into a cuvette: error, the following steps should be taken:
1. Check instrument settings and light source.
Reagent Blank S0 (µl) Standard S1(µl) Sample(µl) 2. Check cleanliness of all equipment in use.
3. Check water. Contaminants, i.e. bacterial growth, may
ddH2O 5 - - contribute to inaccurate results.
Standard - 5 - 4. Check reaction temperature.
Sample - - 5 5. Check expiry date of kit and contents.
Reagent 500 500 500
6. Contact Randox Laboratories Technical Services, Northern
Ireland +44 (0) 28 9445 1070.
Mix, incubate for 10 min at +20 to +25°C or 5 minutes at
+37°C. Insert into the RX monza flowcell holder and press
Read within 60 minutes. INTERFERENCE
Haemoglobin values up to 200 mg/dl, bilirubin up to 507 µmol/l
CALIBRATION FOR RX MONZA and triglycerides up to 4.64 mmol/l do not interfere with the
Recommended using CAL standard in kit or Randox Calibration test.
Serum Level 3. Calibration is recommended with a change in
reagent lot or as indicated by quality control procedures. NORMAL VALUES IN SERUM/PLASMA (6)
Risk levels
FOR MANUAL USE
Value Interpretation
Wavelength: 500 nm, Hg 546 nm
Cuvette: 1 cm light path < 5.17 mmol/l (200 mg/dl) Desirable blood cholesterol
Temperature: +20 to +25°C, +37°C 5.17 - 6.18 mmol/l (200-239 mg/dl) Borderline-high
Measurement: against reagent blank blood cholesterol
≥ 6.20 mmol/l (240 mg/dl) High blood cholesterol
Pipette into cuvette:
It is recommended that each laboratory establish its own
Reagent blank (µl) Standard (µl) Sample (µl) reference range to reflect the age, sex, diet and geographical
location of the population.
Distilled H2O 10 --- ---
Standard --- 10 --- SPECIFIC PERFORMANCE CHARACTERISTICS
Sample --- --- 10 The following performance data were obtained using an RX
Reagent 1000 1000 1000 monza analyser running at temperature of +25°C.

Mix, incubate for 10 min. at +20 to +25C or 5 minutes at LINEARITY

+37C. Measure the absorbance of the sample (A sample) against The test is linear up to a cholesterol concentration of 656 mg/dl
the reagent blank within 60 minutes. (17.0 mmol/l). Dilute samples with a cholesterol concentration
greater than this 1 + 2 with 0.9 % NaCl. Multiply the result by 3.
MANUAL CALCULATION
SENSITIVITY
1. Using a standard The minimum detectable concentration of Cholesterol with an
Conc. of cholesterol in sample = acceptable level of precision was determined as 13.7 mg/dl (0.354
Asample mmol/l).
x conc. of standard
Astandard PRECISION
Intra Assay
2. Using a Factor Level 2 Level 3
Mean (mg/dl) 3.65 7.18
Wavelength mmol/l mg/dl SD 0.100 0.088
CV(%) 2.74 1.23
Hg 546 nm 21.7 x A 840 x A n 20 20
500 nm 14.3 x A 553 x A
Inter Assay
Level 2 Level 3
STANDARDISATION Mean (mg/dl) 3.65 7.18
Randox Calibration Serum Level 3 is traceable to Cholesterol SD 0.143 0.333
reference material NIST 909b and NIST 1952a. CV(%) 3.92 4.64
n 20 20

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 MANUAL/RX MONZA CH200

CORRELATION
This method (Y) was compared with another commercially
available method (X) and the following linear regression equation
obtained:

Y = 0.963 X - 0.157
and a correlation coefficient of r = 0.9943

44 patient samples were analysed spanning the range 0.42 to

15.85 mmol/l.

REFERENCES
1. Abell, L.L, Levey, B.B., Brodie B.B., et al, J. Biol Chem 195 :
357, 1952.
2. Richmond, N., Clin Chem 19 : 1350 - 1356, 1973.
3. Roeschlau, P., Bernt, E. and Gruber, J.W., Clin Chem Clin
Biochem. 12 : 403, 1974.
4. Trinder, P., Ann clin Biochem 6 : 24, 1969.
5. Clinical Laboratory Diagnostics. 1st Edition (1998) p169;
Lothar Thomas ed. TH-Books Verlagsgesellschaft mbH,
Frankfurt/Main, Germany.
6. 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.

Revised 12 Dec 17 ml
Rev. 004

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