Serum Bilirubin Test With Jendrassik & Grof’s Method

Jendrassik and Grof’s Method is one of the most common methods for the estimation of Serum or Bilirubin in Serum. If you don’t know about this substance then know that hemoglobin is responsible for the formation of this substance and when it becomes Bilirubin complex, it gets transported to the liver. Since this is formed in the liver, certain reactions take place in it. Most of the bilirubin in the liver gets converted into the conjugated form. Now this conjugated form will not remain here and will excrete itself into the bile. This then gets stored in the gall bladder and then it is received by the small intestine. So, this a little about bilirubin. Let us now move to the study of the serum bilirubin test with Jendrassik and Grof’s method. You are going to learn its principle, reagents, and procedure in this article.

Principle and Reagent of Jendrassik and Grof’s Method:

Every test has a certain principle and so does this one. In fact, the principle of this test is much simpler and easier to perform than many others. If you have read the above paragraph well then you can easily understand the principle mentioned below. So start reading it.

In the above paragraph, I talked about Director conjugated bilirubin, this is responsible for the whole reaction. It reacts with sulfanilic acid which results in the yield of Azo-bilirubin. This compound has the tendency to be absorbed in 546 nm. Now wasn’t this the simplest principle you ever read? A simple reaction of Total Bilirubin present in the serum and its reaction with diazotized sulfanilic acid. This yields a compound called azo-bilirubin and we are done with the principle of this easy test. Let us now talk about the reagent of the test.

The reagent for the test are already prepared but you need to know their names and composition. So following are the reagent that we are going to use in this test.

Reagent 1 is Direct Bilirubin Reagent which consists of Hydrochloric Acid and Sulfanilic Acid.
Reagent 2 is Direct Diazo Reagent which also consists of Hydrochloric Acid and Sulfanilic Acid but in different quantity.
Reagent 3 is Total Bilirubin Reagent which is Sodium Nitrite.
Reagent 4 is Total Diazo Reagent which will be Sodium Nitrite but just 1 mol.

All of this reagent will remain stable at a temperature of 2-8 degrees. The second condition for their stability is until their expiry date. Once you have a good understanding of the principle, reagents, and their quantities, we are good to move on with our test of estimation of bilirubin in serum with Jendrassic and Grof’s Method.

Procedure of Bilirubin Test:

Here is a step by step procedure to perform Serum Bilirubin Test. Make sure that you follow each and every step as it is written. The only precaution for this test will be to take the measurements of all the chemicals correctly. Once you have all the reagents and have them measured correctly, we are good to proceed with the test. Let us perform it now.

Take two reagents Total Bilirubin and Total Diazo Bilirubin. Mix with them with serum sample and then put them in the incubator. It must be kept there for at least 10 minutes at room temperature. Now read the absorbance at 546 nm.
Repeat the same procedure for direct bilirubin and take the direct reagents in this case.

Formulas for Finding Results: Given below are the formulas to find the results.
For Direct Bilirubin = (Abs of TD – Abs of BD x 26.31 (Factor).
For Total Bilirubin = (Abs of T_t– Abs of B_t)x 26.31 (Factor).

Precautions for Serum Billirubin Test with Jendrassik & Grof’s Method.

Here is a list of precautions for performing this test.

Storage conditions as mentioned on the kit to adhere.
Use clean glassware and micro tips while pipetting.
Do not freeze or expose the reagents to a higher temperature as it may affect the performance of the kit.
Avoid contamination of the reagent during the assay process.
Sequence of reagents addition should be followed strictly as per the procedure
If final volume is not sufficient to fill the cuvette, double all specified volumes. This does not affect the calculations.

After taking all these precautions, your test will be a successful one.

Final Words:

So, this was all from this test. If you have any query or feedback about the article, you can leave it in the comments section. Keep visiting our website All Med Tests for more.