Creatinine ( Alkaline Picrate Method )

 Clinical significance :-

                          Creatinine is a catabolic product of creatinine phosphate which is used by the skeletal muscle. The daily production depends on muscular mass and it is excreted out of the body entirely by the Kidneys. Elevated level are found in renal dysfunction , reduced renal blood flow (shock,  dehydration,  congestive heart failure )  diabetes. Decreased level are found in Muscular dystrophy.

Principle :-

             Picric acid in an alkaline medium react with creatinine  to form Orange coloured Complex with the alkaline picrate. Intensity of the colour formed is directly proportional to the amount of creatine and present in the sample.

 Material required :-

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

 Normal Value :- 

                 Males :  0.6 - 1.2   mg/dl

                 Female:  0.5 - 1.1   mg/dl

Sample :-

           Serum .

Procedure : - 

                      Deproteinization of specimen :

                      Pipette into a clean dry test tube

Picric Acid	2.0 ml
Sample	0.2 ml

Mix well and centrifuge at 2500 – 3000 rpm for 10 min. to obtain a clear supernatant.

Addition Sequence	B	S	T
Supernatant	-	-	1.1 ml
Picric Acid ( L – 1)	1.0 ml	1.0 ml	-
Distilled Water	0.1 ml	-	-
Creatinine Standard	-	0.1 ml	-
Buffer Reagent (L – 2 )	0.1 ml	0.1 ml	0.1 ml

 Mix well  and  keep the test tube  at  R.T. for exactly 20  mins. Measure absorbance of the standard ( Abs . S ) and Test ( Abs . T ) against Reagent blank .

 

   Calculation  :-

                                              Abs . T

                   Uric Acid ( mg/dl ) =    ..............  * 2.0

                                              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 .
4. Before the assay bring all reagents to room temperature.

 General System Parameters:-

          Reaction type           : End point 

          Wave length             : 520 nm 

          Temperature             : R.T.

          Incubation              : 20 minutes 

          Reagent Volume          : 1.10 ml

          Sample Volume           : 200 ul 

          Standard concentration  : 2 mg/dl

          Zero setting            : Reagent Blank

          Light Path              : 1.0 cm