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 37°C. 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 37°C for 10 minutes or 15 minutes at 30°C±5°C. 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
-
ALLAIN, C.C.POON, L.S.CHAN, G.S.G. RICHMOND W..F.U.P.C. Clin. Chem (1974) 20,470.
- BURSTEIN.M. SCHOLNICK. H.R., MORFIN. R.J. of lipids Res. (1970) 11.58.
- LOPES VIRELLAM, STONEP, ELLISS., CORWELL J.A. Clin. Chem. 1977,23,882.
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