Purpose of the Test
To determine the level of direct (conjugated) bilirubin in serum samples.
Principle
Direct bilirubin reacts with diazotized sulfanilic acid to form a blue colored azobilirubin complex. The intensity of the color formed is directly proportional to the concentration of direct bilirubin in the sample.
Equipments
1. Semi-autoanalyzer
2. Cuvettes
3. Micropipettes
4. Timer
5. Spectrophotometer
6. Total Bilirubin Reagent (Contains sulfanilic acid and hydrochloric acid)
Sample Type
Serum preferred for the Test
Procedure
1. in 3 clean dry tubes add reagents and sample as mentioned in the Table below
| Sample Blank | Sample | |
|---|---|---|
| Direct Bilirubin RGt | 1000 μl | 1000 μl |
| Activator / Substrate | – | 50 μl / 1 drop |
| Sample | 100 μl | 100 μl |
Procedure
2. Mix Well and incubate for 10 mins at room Tempreature or 5 min @ 37c’ (protected from light.)
3. using the colorimeter or spectrophotometer read the absorbance of Sample against Sample blank at filter wavelenght 540 nm
4. then do the calculations based on the following formula
Total Bilirubin = (Abs.Test – Abs. Sample Blank) X (Factor)
Reference range: Adult: 0.0 – 0.3 mg/dL Newborn: 1 – 2 mg/dL
Normal Range
Quality Control:
1. Run two levels of control each day of testing.
2. Control results should fall within established range.
Warning :
1. Hemolysis causes falsely Increased results.
2. Photodegradation can falsely lower results.
M. Sulieman
mohammad@mlsgaate.com
Check reagent expiration. Inspect instrument for errors. Run controls before testing samples.
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