Total Erythrocyte count (TRBC)

Blood pipette (Sahli’s) 50 micro liter and 20 micro liter
2)      Hemocytometer /Neubauer chamber
3)      Microscope with minimum 10x and 40x objective.
Reagent : -
                     Red Cell Diluting Fluid
 The Red cell diluting fluid is isotonic to Red cells, i.e it prevents hemolysis. The flowing are the formula of dacies fluid……………
 Tri sodium citrate                                                    -  3.013 g
Commercial formal dehyde 37% formalin              - 1.0 ml
Distilled water                                                         - 100 ml

(Dissolved thorough with a stirrer and fluid is ready to use)

Procedure :-
1)      A test tube is taken and marked as the patient identity number
2)      4.0 ml of RBC diluting fluid is taken in to the tube
3)      20 micro liter of well mixed anti coagulated blood specimen is drawn the sahli’s  pipette
4)      The pipette is hold horigently and out side is wiped carefully to check the perfect amount of blood present in the pipette.
5)      The blood specimen is added in to the RBC diluting fluid . the solution is well mixed by swinging it is left undisturbed for 2 minutes.
6)      The hemocytometer is placed the flat surface of the work bench. Then the cover slip is placed on the counting chamber.
7)      The well mixed diluting Blood solution is taken in the pipette  and allowed to seep in to the counting chamber by capillary action . it is made sure that three is no air bubble and there is no overfilling beyond the ruled area.
8)      The counting chamber is left on the bench for 3 minute to allow the cells to settle.
9)      The counting chamber is placed on the stage of the microscope . first switched to low power objective and then switched carefully to high dry objective (40x) . the vision of the field is set  in the middle smaller squares of the RBC chamber ( 16 middle small squares)
10)  The number of RBC is counted seen on the 5 small square of which each square measure 0.2 x 0.2 = 0.04 square mm/mm2
11)  A total of all the cells counted in five square are calculated . the same procedure is applied to the other side counting chamber is made finally. The calculation is made.

Calculation : -  
                                                       No of RBC counted x Dilution
  No of RBC (Per micro l) =          --------------------------------------
                                                        Area counted x Depth of fluid
Note : - Area x Depth of fluid  =  Volume of fluid in the counting chamber.

Now , Dilution = 201
Area counted   =  0.2x0.2x5
                        = 0.04x5
                        = 0.2 square / mm2
Depth of fluid   =                    0.1 mm
                                                 Dilution
Hence,                     -------------------------------------
                                  Area counted x Depth of fluid
                                                      
                                                201
                            =     ---------------------
                                           0.2 x 0.1
                            =              10,050
                                             Dilution
Factor       =  ----------------------------------------------
                       Area counted  X   Depth of fluid
                   =   10.000
So, the formula can be rewrite as No of RBC (per micro liter)
No of Cells counted in 5 square X 10.000
              5.00000  cell/mm3
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