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Page 4 of 16 Bhatia et al. Hepatoma Res 2018;4:9 I http://dx.doi.org/10.20517/2394-5079.2018.04

Assessment of hepatobiliary function
At the end of 10th week, 99m Tc-mebrofenin hepatobiliary functional test was performed to assess
hepatocellular function, biliary obstruction and to quantify hepatic extraction fraction (HEF). 185-200 MBq
of Tc- sodium pertechnetate prepared in normal saline was mixed with mebrofenin according to the
99m
instructions provided by the manufacturer (BRIT, India). The scintillator counter was calibrated at 140 KeV
99m
with a window setting of ± 20% using Tc as a radioactive source. Mice were then positioned over the
scintillation counter immediately after the intravenous administration of Tc-mebrofenin with liver and
99m
mediastinum in the field of view. Liver activity and blood pool activity was monitored as a function of time
and then used to measure the percentage of activity retained by the hepatic tissue (hepatic retention). The
time required for maximum uptake of mebrofenin (T ) as well as the time at which the activity reduces to
peak
its half (T ) was also calculated for the hepatic and cardiac tissues.
1/2 peak
Assessment of hematological parameters
Collection of whole blood samples
Blood samples from mice of different groups were collected through an ocular vein in sterilized eppendorf
containing anticoagulant at the end of treatment period. Blood samples were mixed properly and processed
for the estimation of various hematological parameters.

Hemoglobin
The hemoglobin (Hb) content was estimated in whole blood by cyanmethemoglobin method as given
[26]
by Dacie and Lewis . The estimation is based on the oxidation of hemoglobin to cyanmethemoglobin
in presence of potassium ferricyanide. The intensity of red colored complex thus formed was measured
spectrophotometrically at 540 nm against a Drabkin's solution and expressed as g/dL.

Red blood cells
Total red blood cell (RBC) counts were measured in non-coagulated whole blood as per the method described
by Dacie and Lewis . Hayem's fluid is an isotonic solution consisting of sodium sulphate, sodium chloride
[26]
and mercuric chloride. The measurement is based on the dilution of blood samples with Hayem's fluid and
then counting of the RBCs in four corners and one central square of a Neubauer's chamber. The RBC counts
were further expressed as counts × 10 /mm .
3
6
Total leucocyte count
The counting of total leucocytes (TLC) was performed in whole blood according to the method of Dacie
and Lewis using Turk's fluid. The glacial acetic acid in this fluid causes the destruction of RBCs while
[26]
the gentian violet helps in the staining of white blood cells (WBCs) nuclei makes them visible under the
microscope. Counting of cells was done in four corner WBC squares and expressed as counts × 10 /mm .
3
3
Platelet count
The counting of blood platelets was done in a hemocytometer using 1% ammonium oxalate as a platelet
diluting fluid . In this, oxalate induces the complete hemolysis of RBCs and preservation of platelets. The
[26]
number of platelets were counted in whole blood in all the central RBC squares under a microscope and
expressed as counts × 10 /mm .
5
3
Differential leucocyte count
Differential leucocyte counting was performed to compute the presence and number of different type of
leucocytes in blood according to the method of Dacie and Lewis . The percentage (%) counts of neutrophils
[26]
and lymphocytes were determined by observing a blood smear under a microscope.