CHOLESTEROL ( CHOD / PAP method )

 

Clinical Significance :-

                       Cholesterol is the main if it found in blood bile and brain tissues. It is the main lipid associated with arteriosclerotic  vascular diseases.  It is required for the formation of steroid  and cellular membranes. The liver metabolized the cholesterol and it  is transported in the blood stream by lipoproteins . Increased level are found in  hypercholesterolemia, hyperlipidaemia  and hypothyroidism and uncontrolled diabetes ,  nephrotic syndrome and cirrhosis.  Decrease level are found in  malabsorption , hyperthyroidism, malnutrition , anemia and liver disease.

Principle :-

           Cholesterol  estersase  hydrolyses esterified cholesterol to free cholesterol to  free cholesterol  . The free cholesterol  is oxidised to form hydrogen peroxide which further  react with phenol and 4- aminoantipyrine  by the catalytic  action of peroxidase to form red colour quinonemine dye complex . intensity  of the colour formed is directly proportional to the amount of cholesterol of present in the sample.

 Material required :-

• Clean and dry glassware.                                     
• Laboratory glass Pipettes or  micro Pipettes and tips. 
• Colorimeter.

 Normal Range :-

                    Serum (Suspicious) : 220 mg/dl and above 

                          (Elevated)   : 260 mg/dl and above 

Procedure :-

                       Pipette into clean and dry test tubes labeled as Blank ( B ) , Standard ( S ), and Test ( T ) :

Addition           Sequence	B	S	T
Reagent	1ml	1ml	1ml
Standard	-	10 ul	-
Sample	-	-	10ul

 Mix well  and  incubate  at  37 ‘ C for 10 mins. Measure absorbance of the standard ( Abs . S ) and Test ( Abs . T ) against Reagent blank at 520 nm.

 

   Calculation  :-

                                              Abs . T

                    Cholesterol ( mg/dl ) =    ..............  * 200

                                              Abs . S   

Preparation and reagent and stability :-

                                        All Reagent are ready to use and are stable till the expiry date mentioned on the label , when stored at 2' to 8' C.

Quality Control :-

                  For accuracy it is necessary to run known controls with every assay.

Limitation & Precautions :-

 

1. Storage conditions as mentioned on the kit to be adhered.
2. Do not freeze or expose the reagent to high temperature and protect from direct as it may affect the performance of the kit.
3. Use clean glassware free from dust .

 General System Parameters:-

          Reaction type           : End point 

          Wave length             : 505 nm ( 490 - 530 nm )

          Temperature             : 37'C

          Incubation              : 10 minutes 

          Reagent Volume          : 1.0 ml

          Sample Volume           : 10 ul 

          Standard concentration  : 200 mg/dl

          Zero setting            : Reagent Blank

          Light Path              : 1 cm