CHEMISTRY REAGENT KITS
           ------->CHOLESTEROL (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 + O     CO  cholestenone3. One+HO

2 H2O2.4AAP.DHBS POD quindye+4H2O

Composition+

Final concentration in the reactive medium

1.ENZYME  
Phosphate buffer 100mmol/L
Cholesterol Esterase 300 U/L
Cholesterol Oxidase 100U/L
Hydroxybenzoicacid 20 mmol/L
4-Aminophenazone 0.5mMol/L
Peroxidase >  200 U/L
Surface active agents 10g/L
Stabilizers 1g/L
2.BUFFER  
Potassium Phospha 100mMol/L
3.  STANDARD  
Cholesterol (STD)   200 mGs/dL (5.18mMol/L)
REAGENTS SUPPLIED
Cat. No. A 141
1.Enzyme Powder      10mL 10 vials
2.Buffer                       10mL 10 Bottles
3.Standard (200 mGs/dL)4mL 1 Bottle
Cat. No. A140  
1.Enzyme Powder      10mL 3 vials
2.Buffer                       10mL 3 Bottles
3.Standard (200 mGs/dL)4mL 1 Bottle

Preparation of Working Reagent
Dissolve on enzyme vial (Reagent No. 1) with 10mL buffer (Reagent No. 2) A uniform solution may take place after5 minutes which is ready to use.

STABILITY
At 2-8°C for 18 months. The reconstituted reagent is stable for atleast 25 days at 2-8°C away from light.

SYSTEM PARAMETERS

Reaction

End-Point

Direction of reaction

increasing

Temperature 37°C
Wave Length                      510 nM (500-546 nM)
Standard Concentration

200 mg/dL

Absorbance Range 0-2°A
Cuvette Path Length 1 cm
Reaction Time 10mins.
Linearity 700 mg/dL
Max. Limit of Blank Reagent <     0.100
Final ColourStability  30mins.
Reagent Volume 100µL
Sample Volume 10µL

SAMPLE
Sample can be serum of plasma which has no sign of haemolysis. Common anticoagulants have no interference on this assay. Cholesterol is affected by food intaeke. Therefore, keep the patient fasting for

MANUAL METHOD
1.
   Pipette into 3 Test Tubes Blank mL Sample Standrd mL
   Cholesterol Reagent No. 1 1.00 1.00 1.00
   Distilled water 0.02 - -
   Standard Reagent No. 3 - - -
   Sample or HDL Supernatant - 0.02 -

2.Mix well. Incubate at 37°Cfor 10 minutes or 15 minutes at 30°C + 5°C.
3.After incubation add 2mL distilled water for 3 mL cuvette size read at 510 nM (500-546    nM) or GREEN filter against Blank. Final colour is stable upto 30 minutes away from bright    light.
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METHOD FOR HDL CHOLESTEROL
   Pipette into 3 test tubes Blank mL Sample Standrd mL
   Reagent 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 II Pipette into 3 Test Tubes
1.  Cholesterol Reagent No. 1.......................mL
2.  HDL Reagent.......................................mL
3.  Supematentfrom STEP I. (given below)........mL
4.  Cholesterol Std (200 mGs/dL)...................mL

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. Add 2ml distilled water for 3 mL cuvette size. Final colour is stable upto 30 minutes away from bright light. Note : Standard 200 mGs/dL volume is only 0.02 mL where as sample volume is 0.1 mL.

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.

METHOD FOR HDL CHOLESTEROL
STEP I
Serum     :     0.2 mL
HDL Reagent : 0.05 mL
Mix well, wait for 10 mins. and centrifuge. Separate clear
supernatent and estimate cholesterol level of the supermatent
asgiveninSTEPII.

RESULTS
Compute Total Cholesterol in mGs/dL =
                                              A O. D. Test

EXPECTEDVALUES
Total Cholesterol:
150 to 200 mGs/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 RESULTS
Compute    HDL Cholesterol in mGs/dL = O. D. Test HDL - O.D. HDL Blank
x50
O.D. HDL Std. - O.D. HDL Blank

EXPECTED VALUES
HDL Cholesterol
Men : 30 to 70 mGs/dL (0.78 to 1.55 nMol/L)
Women:40to60mGs/dL(1.03to1.81nMol.L

LIMITATIONS
This assay is linear up to 700 mg/dL 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 AgNO3’ HgCl2’ Teepol or similar substances, and false result may be obtained by the contamination of H2O2’ 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.

BIBLIGRAPHY
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.

4.  GROVE T.T.Clin,Chem.(1979),25,560. McDaniel.R.C.,and Devine,J.E.Ann,Clin.Lab              Scl.10.155(1980)

5.  N.TieTZ Ed. Fundamentals of Clin. Chem. WEYWMAN. AEET.alAm.J.Med 56:13(1974).

6.   ZSIGMOND. E.K. et. al. Anesth Analg(Cleve)51.220(1972).