CHEMISTRY REAGENT KITS
                         ------->CHOLESTEROL TOTAL & HDL
                                                         (CE- CO-PAP, Enzymatic, End-Point)
                                             
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CLINICAL SIGNIFICANCE
Total Cholesterol: Increased levels are associated with atherosclerosis, nephrosis, diabetes mellitus, myxoedema, obstructive jaundice. Decreased levels are observed in cases of hyperthyroidism, certain anaemias, malabsorption and wasting syndrome. HDL Cholesterol: Decreased levels are associated with increased risk of developing coronary artery diseases and other atherosclerotic diseases.

PRINCIPLE
In blood, Cholesterol is associated with two kinds of lipoproteins: high density lipoproteins (HDL) and low density lippoteins (LDL and VLDL). Recent studies have shown that the level of Cholesterol bound to HDL is a better indicator of cardiovascular risk than the total cholesterol level.

cholesterol esters   CE    cholesterol + fatty acids
cholesterol + O2    CO   cholestenone3. One+H2O2

2H202-4AAP.+DHBS    POD  quindye +

4H20

COMPOSITION
Final concentration in the reactive medium
1.  ENZYME

 

Cholesterol Esterase 160U/L
Cholesterol Oxidase 100U/L
Hydroxybenzoic acid 20 mmol/L
4-Aminophenazone 0.5 mMol/L
Peroxidase > 20U/L
Surface active agents 10g/L
Stabilizers 1 g/L

2.  BUFFER

 
Potassium Phosphate 100 mMol/L

3.  STANDARD

 
Cholesterol (STD)   (5.18 mMol/L)

4. HDL Reagent

 
Sodium Phosphotungstate 20 mMol/L
MgCI2 10 mMol/L

REAGENTS SUPPLIED
Cat.No.A140

1.  Enzyme Powder 10mL
    3 vials
2.  Buffer 10mL 3 Bottles
3.Standard (200 mGs/dL) 4mL 1 Bottle
4.HDL Reagent 3mL          1 Bottle.

Cat.No.

1.  Enzyme Powder 10mL 10 vials
2.  Buffer 10mL 10 Bottles
3.Standard (200 mGs/dL) 4mL 1 Bottle
4.HDL Reagent 3mL          1 Bottle.

Preparation of Working Reagent
Dissolve one enzyme vial (Reagent No. 1) with 10 mL buffer (Reagent No. 2) A uniform solution may take place after 5 minutes which is ready to use.

STORAGE & STABILITY
At 2-8°C for 18 months. The reconstituted reagent is stable for atleast 25 days at 2-8°C away from light.
Store HDL Reagent at RT

SYSTEM PARAMETERS

Reaction

        End-point

Direction of reaction         increasing
Temperature         37°C
Wave Length         51 OnM (500-
        546 nM)
Standard Concentration

        200 mg/dL

Absorbance Range         0-2°A
Cuvette Path Length         1 cm
Reaction Time

        10 mins.

Linearity

        700 mg/dL

Max. Limit of Blank Reagent         < 0.100
Final Colour Stability         30 mins
ReagentVolume         1000 pi.
Sample Volume

        10pL

SAMPLES
Sample can be serum or plasma which has no sign of haemolysis. Common anticoagulants have no interference on this assay. Cholesterol is affected by food intake. Therefore, keep the patient fasting for atlease 8 hrs. prior to sample collection.

METHOD FOR TOTAL CHOLESTEROL
Pipette into 3 Test tubes Blank mL Standard mL Test mL
Working Reagent No. 1 1.00 1.00 1.00
Sample  - - 0.01
Standard - 0.01 -

Mix well. Incubate for 10 minutes at 37C. Read the absorbance of sample and standard at 510 nM ( 20) against Blank Reagent and sample / standard volume can be altered proportionately.

METHOD FOR HDL CHOLESTEROL
STEP I
Serum                                                0.2 mL
HDL Reagent                                    0.1 mL
Mix well, wait for 10 mins. and centrifuge at > 3000 RPM. Separate clear supernatent and estimate cholesterol level of the supermatent as given in STEP II.

Pipette into 3 Test tubes Blank mL Standard mL Test mL
1   Working Cholesterol Reagent No. 1     1.00 1.00 1.00
2   HDLReagent 0.1 0.1- -
3   SupernatantfromSTEPI - - 0.1
4   Cholesterol Std (200 mGs/dL) - 0.02 -

Mix well. Incubate at 37C for 10 minutes or 15 minutes at 30C5C. After incubation read at 510 nM (500-546nM) or GREEN filter against HDL blank. Final colour is stable upto 30 minutes away from bright light.

NOTE : Standard 200 mGs/dL volume is only 0.02 mL (20 |iL) where as sample volume is 0.1mL(100nL).

RESULTS

                                                                                 ΔO.D. Test
Compute Total Cholesterol In mGs/dL = -----------------------   x  200
                                                                                 ΔO.D. STD

                                                                       O. D. Test HDL - O.D. HDL Blank O.D.
Compute HDL Cholesterol in mGs/dL=------------------------------------------------X60
                                                                                HDL Std.   -   O.D. HDL Blank

EXPECTED.VALUES

Total Cholesterol:

150  to  200m Gs/dL
(3.88 to 6.47 mMol/L)

As with all diagnostic methods, the final diagnosis should not be made on the result of a single test as well as laboratory diagnosis must be confirmed with clinical manifestations.

HDL Cholesterol
                   Men     :      30 to 70    mGs/dL
                                       (0.78to1.55nMol/L)

                Women :       40 to 60    mGs/dL
                                       (1.03to1.81nMol.L)

WARNING
This reagent system is for in vitro Diagnostic use only. This reagent system is containing preservatives and components that have not established for safety if contacted on broken skin or eye or taken orally. In case of such incidents wash off with plenty of water.

LIMITATIONS
This assay is linear up to 700 mg/dL Total Cholesterol. For values higher than this limit dilute the sample with 0.9% normal saline and multiply the results by dilution factor i.e. by 2 for 1:1 dilution. The enzyme system is inactivated by contamination of AgN03. HgCI2.Teepol or similar substances, and false result may be obtained by the contamination of H2Oz Hypochlorite etc.

QUALITY CONTROL
To ensure adequate quality control, each kit should be tested against a standard control sera. It should be realised that the use of quality control material checks both instrument function, temperature control, cleanliness of glass wares and accuracy of pipetting. It is appropriate to establish each laboratory's accuracy constant and interpret values accordingly. Similarly laboratory findings should be established by clinical manifestations.

BIBLIOGRAPHY

    1. ALLAIN,   C.C.POON,   L.S.CHAN,   G.S.G.   RICHMOND W..F.U.P.C. Clin. Chem (1974) 20,470.
    2. BURSTEIN.M.   SCHOLNICK.   H.R.,   MORFIN.   R.J.   of lipids Res. (1970) 11.58.
    3. LOPES   VIRELLAM,   STONEP,   ELLISS.,   CORWELL J.A. Clin. Chem. 1977,23,882.