Blood cell count principles

BLOOD CELL COUNT PRINCIPLES

AUTOMATIC METHOD
Complete blood count performed by an automated analyzer that counts the numbers and
types of different cells within the blood. It aspirates a very small amount of the sample
through the narrow tubing. Within this tubing, there are sensors that count the number of
cells going through it, and can identify the type of cell; this is called flow-cytometry. For
detection light detectors are used as well as the measurement of electrical impedance. One
way the instrument can tell what type of blood cell is present is by size. Other instruments
measure different characteristics of the cells to categorize them.

MANUAL METHOD
This measurement is made with a microscope and a specially ruled chamber
(hemocytometer) using diluted blood.

Figure no. 1. The hemocytometer lateral and top view

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Figure no. 2. The grid pattern of the Trk chamber

The hemocytometer consists of a thick glass microscope slide with a rectangular indentation
that creates a chamber. This chamber is engraved with a grid of perpendicular lines. The
device is carefully crafted so that the area bounded by the lines is known, and the depth of
the chamber is also known. It is therefore possible to count the number of cells or particles in
a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall.
Several types of hemocytometers are used; we use Trk counting chamber, engraved with
two straight and one "H" shaped deep moats, which enclose the cover glass mounting
supports and separate the two engraved areas (see Figure no. 1). Each counting surface has
an area of 9 mm2. The depth between the cover glass and the surface of the slide is
1/10 mm. The grid of the counting chamber consists of perpendicular lines situated at
1/5 mm and 1/20 mm respectively (see Figure no. 2). The perpendicular lines delimit squares
and rectangles where the blood cells are counted:
small squares with the area of 1/400 mm2 for red blood cell count,
big squares with the area of 1/25 mm2 for white blood cell count,
rectangles with the area of 1/100 mm2 for platelet count.
Diluting pipettes (Potains pipettes) are capillary (very thin) pipettes with a mixing chamber. In
the mixing chamber there is a mixing bead, colored in red for the red blood cell pipette and in
white for the white blood cell pipette. On the capillary part of the pipette are marks for 0.5 and
1 l, and above the mixing chamber is a mark for 101 l on the red cell pipette and for 11 l
on the white cell pipette (see Figure no. 3). A rubber tube with a mouthpiece is attached to
the top-end of the pipette.

Figure no. 3. Diluting (Potain) pipettes for red (left) and white (right) blood
cell count

White blood cell count

11

WHITE BLOOD CELL COUNT

MATERIALS
hemocytometer,
diluting pipette for white blood cells,
diluting fluid (Trk's solution: glacial acetic acid 0.5 ml, gentian violet 1% 1.5 ml,
distilled water ad 150 ml),
light microscope,
for blood sample collection: cotton balls, alcohol, sterile needles, rubber gloves.

PROCEDURE
Cleanse the hemocytometer and cover glass with a piece of cotton saturated with alcohol
and let air dry. Prepare the hemocytometer by placing the cover glass on its mounting
supports.
To avoid risk of infection, wash your hands with soap and water before and
after doing any blood tests! When manipulating blood samples from another
person, use disposable rubber gloves! Dispose of the used needles in special
containers!
Cleanse the tip of the finger with a piece of cotton saturated with alcohol. Let the finger air
dry. Using a sterile, disposable needle, quickly make a single puncture in the top of the
cleansed finger (hold firmly between the thumb and forefinger) deep enough so that blood
flows freely from the wound. Wipe off the first drop of blood with a piece of cotton; when a
second drop has accumulated, proceed with the filling of the pipette. Do not fill the pipettes
with blood until sufficient blood has welled up on the fingertip since these pipettes have a
very small bore and blood clots extremely easily in them. The finger must not be squeezed.
Place the pipette tip just within the drop of blood. Suck up a continuous column of blood in
the tube to the 0.5 mark on the pipette by using the mouthpiece. Wipe the excess blood from
the tip of the pipette. Immerse the pipette tip in the white blood cell diluting fluid (Trk's
solution); and while holding the pipette vertically, suck the diluting fluid exactly to the 11
mark. Dilution should be done very quickly and precisely to prevent clotting of the blood and
to insure accuracy. Thus a 20-fold dilution is obtained (the volume of the mixing chamber is
11 l1 l=10 l, containing 0.5 l of blood).
Close the ends of the pipette with your thumb and middle finger and shake the pipette for 3
minutes.
Throw away the first two drops from the pipette (they come from the capillary part of the
pipette and contain only diluting fluid) and with the third drop fill the hemocytometer: let the
drop fall near the cover glass and the chamber will fill due to capillarity. The excess fluid will
flow in the moats.
Place the hemocytometer in a light microscope and examine the sample using low-power
objective (10 x) and weak light. Count the white blood cells in 25 big squares considering the
cells inside the squares and from two sides.

RESULTS
Report the number of white blood cells as cells/mm3. First the average number of white blood
cells in a big square must be calculated: N (total number of found white blood cells)/25

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(number of squares). This must be reported to the volume corresponding to a big square:
area*height of the chamber. Last the results must be corrected with the dilution of the blood.

NWRC

N
N

NSq * Volume * Dilution NSq * Area * Height * Dilution

NWBC

N
N

* 25 * 10 * 20 N * 200
1 1 1
25
25 * * *
25 10 20

DATA INTERPRETATION:
Normal range:
adults: 6000-8000/mm3
newborn, young children: 14000-20000/mm3
Higher values leukocytosis:
physiological: after effort, after meals, women: menstruation, pregnancy, childbed
pathological: infection, inflammation, poisoning
Lower values leucopenia: anaphylactic shock, viral infections, X-ray exposure.

Eosinophil count

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EOSINOPHIL COUNT
MATERIALS
hemocytometer,
diluting pipette for white blood cells,
diluting fluid (eosin solution: eosin 2% 5 ml, acetone 5 ml, distilled water 90 ml),
light microscope,
for blood sample collection: cotton balls, alcohol, sterile needles, rubber gloves.

PROCEDURE
Prepare the hemocytometer and disinfect finger as described above.
To avoid risk of infection, wash your hands with soap and water before and
after doing any blood tests! When manipulating blood samples from another
person, use disposable rubber gloves! Dispose of the used needles in special
containers!
Make a finger puncture and suck blood in the pipette to the 1 mark as described above. Fill
the pipette with eosin solution to the 11 mark, obtaining a 10-fold dilution. Shake the pipette
for 3 minutes and then fill the hemocytometer with the third drop of fluid.
Place the hemocytometer in a light microscope and examine the sample using low-power
objective and weak light. Identify the eosinophils colored in bright red. Count the eosinophils
on the whole surface of the grid (9 mm2).

RESULTS
Report the number of red blood cells as cells/mm3. The total number of found eosinophils
must be reported to the volume corresponding to the grid: area*height of the chamber. Last
the results must be corrected with the dilution of the blood.

NEo

N
N

Volume * Dilution Area * Height * Dilution

NEo

N
N
N
* 10 * 10 * 100
1 1
9
9
9* *
10 10

DATA INTERPRETATION
Normal range: 100-200/mm3
Higher values eosinophilia:
allergies,
infection with parasites.
Lower values eosinopenia: burns, shock, administration of steroid hormones.