HEMOCYTOMETRY

The Hemocytometer
The hemocytometer is a thick glass slide used to count erythrocyte, leukocyte and platelet in the
blood. It may also count cells in other body fluids. The hemocytometer should meet the specification of
the National Institute of Standards and Technology. When viewed from the top, it has two raised
platform surrounded by depressions (Moats) on those sides which form an H. On both sides of the
platforms is raised ridge in which the cover glass is placed. The central platform are exactly 0.1 mm and
over than the ridge and each contains one ruled area composed of square, one square is divided into nine
large square, 1mm x 1mm. These four-corner square used for WBC count are divided each into 16 small
squares. The central large square is divided into 25 small square 0.2 x 0.2mm each of which are
subdivided into 16 smaller square 0.05 mm x 0.05mm. The corner and central squares of the 25 small
square are used for RBC count. Other used 10 square in platelet count the large Center Square is used
though the number of square. Some advocate using 25 squares, other 10 and 5.
Calculation of cell counts:
In all cells, the general formula for calculating cell count is:
Ct = Cc x D x DF
A
Where : Ct = Total cell count
Cc = No. of cell counted
D = depth factor (mm) which is the distance between the ruled platform and
cover glass, 0.1 mm in improved Neubauer
Df = dilution factor which is the ratio of the blood and dilution fluid used.
A = total area of the square where the cells are counted.
The volume ruled area in 0.9 mm3 ( ). The volume of one large square is 0.1 mm3 (ul ).
The volume of each area of 25 square is (0.2 x 0.2 x 0.1 mm3 ) A clean thick cover glass , ground to a
perfect plane, which meet the NIST specification is positioned so that it cover both ruled areas. With it
in place, the depth of the fluid ( depth factor ) in the improved Neubauer. Hemocytometer is 0.1 mm.
The Ruled Areas / Counting Chamber
To view the ruled areas, the hemocytometer is placed on the microscope stage, using the low
power (10 x) objective with one ruled area positioned over the high source. The course adjustment knob
is used to carefully move the hemocytometer closer . the objective is almost touching the cover glass.
Looking to the eyepiece or ocular, the course adjustment is then used to lower the hemocytometer until
the ruled area is in focus. The fine adjustment knob is then used to achieve a sharp focus. The etched
lines of the ruled area are more easily viewed when the amount of light is minimised. The white cell
counting areas are located by moving carefully the stage or the hemocytometer. The red cell counting
areas are located by first locating the central square. The high power (40 x) objective is carefully rotated
into place and is used in locating the small squares. For sharper focus used the adjustment knob. With
microscope a slight rotation with fine adjustment, sharper focus is with only. When moving the
hemocytometer, the low power objective should be used. The oil immersion objective is never used.
Counting Pattern :
A left to right, right to left counting pattern is used to insure no duplication. The count begins
to the upper left corner and proceeds t in a serpentive manner. Boundary lines divide square, which may

be single, double, or triple. When triple line is present, the outer line is considered as a boundary .All
cell within the square, cell which touch the left boundary of the square, and cell which touch the top
boundary of the square are counted. Cell which touches the right and lower boundary of the square
should not be counted in that square eyes though they may lie within the square. The cells on the
designated squares should be counted on both sides of the squares. The result for each side should be
recorded. The counts for the both sides are then totalled and the average is calculated.
Blood Diluting Pipettes
Before blood cells may be counted microscopically, blood must be diluted. This is because
cellular element in the blood is so concentrated. Blood diluting pipettes used to dilute blood and other
body fluid. Blood and diluting fluid are mixed within the pipet. The Thoma style pipettes are used
mostly for erythrocyte, leukocyte, and platelet count. However they may also be used for counting cell
in spinal, sinovial and other body fluids and for counting sperms. A blood diluting pipettes have three
basic parts: 1.) A long calibrated stem into which the sample and diluting fluid are aspirated. 2.) The
bulb in which the contents are mixed and 3.) A short stem in which a suction or pipettes filler is
attached. Pipettes used must meet the specification National Institute of standards and Technology
( NI ) and ST must certified by NIST to have a + 1 % accuracy.
The red cell diluting pipette is used to dilute blood for a red cell or erythrocyte count. The
0.5 on the long stem is a mark to which blood specimen is drawn. The 101 mark on the short stem is the
mark to which diluting fluid mixed with blood specimen is drawn. The red bead identifies the rbc
pipette and also mixer the content.
The white blood cell diluting pipette is used to dilute blood for leukocyte count. It hold a
smaller volume and makes a lower dilution. Blood is also drawn in 0.5 mark. The diluting fluid mixed
with blood is drawn to the 11 mark. The white bead identifies the WBC pipette.
The important steps using a pipette are: filling the pipette , mixing the content, and
dispensing the diluted sample. Pipettes must be clean and dry for accurate dilution. The beads rolls
freely when the pipette is completely dry. Cell counts provide valuable information to the physician. It is
important that the cell count be an accurate as possible. Blood dilution must be performed carefully and
precisely. Anticoagulated venous blood must be mixed before sampling by 20 - 30 gentle inversion
avoiding shaking.
Filling the Pipette
The sample is drawn carefully and slowly to the 0.5 mark by aspiration on capillary action
after allowing pipetting aid and placing tip of pipette into the sample. The excess sample is then wiped
from the outside of the pipette tip with soft tissue, without touching the tip thereby withdrawing some of
the sample. Diluting fluid is then aspirated to the 11 or 101 mark while slowly rotating the pipette. The
sample and diluting fluid must be drawn exactly to the 0.5 mark and 11 / 101 mark respectively to
accurate dilutions.

Mixing Fluids :
After place index finger over pipette tip carefully removed pipette filler in a horizontal
position. For hand mixing, Hold pipette with a thumb and index / middle finger on each pipette tip.

Then rotate it in a figure eight motion for 2 to 3 minutes. For automatic shaking, carefully insert pipette
into shaker and set time at least 2 minutes.
Dispensing Fluids
Immediately dispense fluid after shaking to avoid settling of cells. With the index finger
over the tip of the short stem control the flow and the rest of the finger holding the pipette vertically,
discard four to five drops of fluid unto piece of gauze. This drops consist mostly of diluting fluid. The
next drop is then charge to a counting chamber. Place clean cover glass to cover both ruled area of
clean hemacytometer by touching the tip of the pipette to where cover glass and raised platform melt.
Let one - half to one drop of fluid flow by capillary action. The fluid should flow into the chamber in a
smooth unbroken stream. It should not be allowed to over flow into the depression or moats. After filling
stand the hamocytometer for two minutes to allow cell to settle.