Basic Concepts in Clinical Biochemistry-A Practical Guide.7z

24.6 Calculations

Plot a graph by taking bilirubin amount at x-axis vs absorbance at y-axis, and
extrapolate bilirubin concentration of the given serum sample from the graph. The
bilirubin concentration can also be calculated by applying equation:

mgof bilirubin∗in blood=dl

¼

ðÞODof testcontrol amount of standard 100

ðÞODof standardcontrol volume of sample mlðÞ 1000 ∗∗

¼xmg=dl

*Calculate total and direct bilirubin; **To convertμg to mg since amount of
bilirubin is inμg in the volume of standard added to tubes.

24.7 Clinical Significance...............................

Normal serum contains very low concentration of direct bilirubin, i.e., 0–0.2 mg/dl,
and the total bilirubin concentration ranges from 0.2 to 1.0 mg/dl. Bilirubin analysis
is a part of hepatic function test. Increased bilirubin concentration in blood causes
jaundice. The estimation of direct (conjugated) and indirect (unconjugated) bilirubin
may help in differential diagnosis of various types of jaundices–prehepatic (hemo-
lytic), hepatic, and posthepatic (obstructive).

1. In prehepatic or hemolytic jaundice, the unconjugated bilirubin is increased due
   to increased destruction of red blood cells, and the liver is unable to cope with the
   increased demand for conjugation. In pernicious and chronic hemolytic anemia,
   the serum bilirubin rarely exceeds 3 mg/dl but may also reach up to 10 mg/dl.

Reagents Blank S 1 S 2 S 3 S 4 S 5 S 6 S 7

Total Direct

TTcDDc

Bilirubin

standard (μl)

• 20 40 60 80 100 150 200 ––

Bilirubin amount

(μg)

• 2 4 6 8 10 15 20

Serum/urine (ml) ––––––– 0.1 0.1 0.1 0.1

Distilled water

(ml)

2.0 1.98 1.96 1.94 1.92 1.90 1.85 1.80 1.9 1.9 4.9 4.9

Diazo reagent

(ml)

• 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 – 0.5 –

Diazo blank (ml) 0.5 ––––––––0.5 – 0.5

Methanol (ml) 3 3 3 3 3 3 3 3 3 3 ––

Mix well and keep in dark for 30 min at room temperature, read absorbance at 540 nm

100 24 To Estimate Total and Direct Bilirubin in Serum