About 80-85% of the total bilirubin originates from the degradation of hemoglobin in liver Kupffer cells, spleen, and bone marrow. This unconjugated (primary, indirect, or water-soluble) bilirubin, which binds with the plasma albumin, is soluble in lipids and toxic. In contrast to the unconjugated, conjugated or secondary, or direct bilirubin is soluble in water and is excreted by the kidney. It occupies the remaining 20- 15% of the total bilirubin.
Bilirubin Normal Range
The range shown below is the approximate range for a normal person. Thus, it can slightly vary from one laboratory to another.
- Direct:- upto 0.2 mg/dl
- Total:- upto 1.0 mg/dl
If the range of bilirubin in the serum of the person goes out of range, then he/she is recommended to visit the doctor for further investigation.
Causes and Symptoms of High Bilirubin
The breakdown of red blood cells is the cause of the release of bilirubin. Then it goes to the liver. After that the liver mixes it with the bile juice produced by the gall bladder. Finally, the bile juice carries it to the digestive system. Finally, it gets excreted out through the body.
Thus any defect to this system can be the cause of high bilirubin in the blood. Some of them are as follows.
- Hemolytic Anemia:- It is the condition in which the red blood cells break down at a higher rate.
- Liver dysfunction such as Hepatitis, Gilbert’s Syndrome, etc.
- Diseases that cause an abnormality in the bile functioning such as Gallstones, Bile Duct Inflammation, etc
An increase in the level of bilirubin normally leads to jaundice. The other symptoms are as follows.
- Fatigue and weakness
- Nausea and vomiting
- Pain in the chest, abdomen, etc.
- Dark urine
Process of Bilirubin Test
1. Principle Reaction
When bilirubin and the diazonium salt of sulfanilic acid reacts, an azobilirubin is formed. The intensity of color due to azobilirubin is directly proportional to the concentration of bilirubin present in the serum. But, when you are measuring the total bilirubin, you need to add an accelerator, dimethylsulphoxide (DMSO). The DMSO helps to solubilize the free bilirubin present in the serum.
2. Sample Preparation
The table shown below is valid for the reagents of Analyticon, Tulip, Accurex, and Diasys Companies. So, If you have a reagent of any other company then there can be a minor variation in the procedure. Also, it is valid for the programming of both the total and direct bilirubin reagents.
First of all, make a working solution out of the given two reagents. The ratio of their composition will be given in the literature. But, if you have only one reagent besides the calibrator, then that will act as a working solution.
While preparing the sample solution or sample blank, mix the sample with the reagent. Then, incubate the solution at room temperature for 10 mins. Then, feed the solution in a semi-auto biochemistry analyzer.
The unique feature of this reagent is that it requires a sample blank. Since every patient sample is unique to one another, so for every test, you need to make both, sample test, and sample blank. For making a blank solution, you need only a bilirubin reagent, which is generally the first reagent.
Always remember that volume of the total sample preparation should be greater than the set value of sippling volume in the instrument (500 μl in our case). Else, the machine will suck up the air and give you the fault reading.
Even though it is an end-point method, you may not get a standard/calibrator for the calibration process. Instead of that, you may need to put the factor value. This factor will be available in your literature.
If the concentration of bilirubin in the sample is very high (out of range/ non-linear curve) then dilute the solution with 0.9% NaCl or distilled water at the ratio of 1:y. Finally, when you get the result, multiply it with the dilution factor (1+ y).
3. Programming of Bilirubin Reagent in a Semi-auto Biochemistry Analyzer
4. Handling of Bilirubin Reagents
- 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.