Laboratory

Urea: programming in semi- auto biochemistry analyzer

Urea

Urea (NH2CONH2) can be measured by various methods such as Berthelot, GLDH, Nessler’s reagent, etc.

It forms in the liver via the urea cycle. It is also the end product of protein metabolism. An increase of urea in the body causes an increase in blood plasma osmolality. This increases the flow of water from tissues and plasma. So, there will be an increment in urine outflow. Urea is one of the parameters for the renal function test for knowing the condition of our kidneys.

Everything that I state below will be based on a semi-auto biochemistry analyzer and can differ from one manufacturer to another.

Normal range  (approximate value) of urea

  • Globally:- 17 – 43 mg/dl
  • Women (< 50 years):- 15 – 40 mg/dl
  • Women (> 50 years):- 21 – 43 mg/dl
  • Men (< 50 years):-  19 – 44 mg/dl
  • Men (> 50 years):-  18 – 55 mg/dl
  • urine:- 20 – 36 g/l (24 hours urine collection)

Handling process

  • Wear an apron and surgical gloves before carrying out the measurement.
  • Look for the expiry date of the reagents during purchase and measurement time. Suppliers tend to give you reagent kits with a low expiry interval.
  • Once you open the reagent bottles, the lifespan will decrease. So, look in your reagent kit literature for that.
  • Bring the reagents and samples to room temperature before you can carry out any measurement.
  • Always store reagents in the refrigerator when not in use.

Working principle (Urease/ GLDH method)

1.  Principle reaction

Urea undergoes hydrolysis in the presence of urease to produce ammonia and CO2. The ammonia produced combines with 2 – oxoglutarate and NADH in the presence of GLDH to yield glutamate and NAD. The absorbance of light by NADH helps to determine the concentration of urea in the given sample.

2. Sample preparation

If you have two reagents besides the calibrator, you can make a working solution to it. The combination of these two reagents will be given in your literature. Some other companies can have a single ready to use reagent.

Once you mix the working solution to the sample test, you can directly feed into the machine.  But, always remember that sipping volume < sample volume + reagent volume. If you set the sipping volume to 500 μl, then the total sample to feed into the machine should be more than 500 μl.

If the concentration of urea is very high (out of range/ non-linear curve), then you can dilute it with 0.9% NaCl or distilled water in a ratio of 1:y. For urine sample y= 50. Then when you get the result, multiply it with the dilution factor (1+y).

Programming for urea in a semi-auto biochemistry analyzer (GLDH method)

urea, GLDH, semi-auto biochemistry analyzer

 

Working principle (Berthelot method)

1. Principle reaction

According to the Berthelot method, the urease enzyme causes the breakdown of urea to ammonia and carbon dioxide. The ammonia further reacts with hypochlorite and phenolic chromogen to give a green-colored complex

2. Sample preparation

For accurex:- You need to prepare an enzyme solution by mixing enzyme and diluent. The ratio of their composition will be given in your reagent bottles. Then add 0.01 ml of sample/ calibrator to 1 ml of enzyme solution. For making a reagent blank, you do not need a sample/calibrator. Now, incubate the mixture at 370C for 3 mins. Again add 1 ml of chromogen solution to the mixture. Incubate it at 370C for 5 mins. Then feed the solution into the machine.

For tulip:- Mix 1 ml of buffer reagent, 0.1 ml of enzyme reagent, and 0.01 ml of sample/calibrator together. For making reagent blank, replace sample/calibrator with distilled water. Now, incubate the mixture at 370C  for 5 mins. Then add 0.2 ml of chromogen reagent. Incubator it at 370C for 5 mins. Finally, feed the solution into the machine.

If the concentration of urea is very high (out of range/ non-linear curve), then you can dilute it with 0.9% NaCl or distilled water in a ratio of 1:y. For urine sample y= 50. Then when you get the result, multiply it with the dilution factor (1+y).

Programming urea in a semi-auto biochemistry analyzer (Berthelot method)

urea, berthelot method,semi-auto biochemistry analyzer

Related Articles

Leave a Reply

Your email address will not be published. Required fields are marked *

Back to top button