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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.
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