Manual Cell Counting (by microscope)

Since blood cells are counted per unit volume (per liter), it is vital that the volume of
blood, in which the cells are counted, corresponds to a known quantity. This makes the
Neubauer counting chamber a useful method.

A special objective slide on

which two 3 x 3 mm long
counting grids were engraved
comprises the Neubauer
counting chamber.

Neubauer counting chamber from above with cover

slip
Notice the two counting grids which are recognized
as crosses.

The counting grid is composed of

9 big squares, measuring 1 x 1
mm . From these squares, the
central square contains 25
medium sized squares each
measuring 0.2 x 0.2 mm. These
are further divided into 16 small
squares each measuring 0.05 x
0.05 mm. The large central
square is also called the
erythrocyte grid. The squares
highlighted in red correspond to
80 small squares, that are used to
establish the erythrocyte and
platelet counts. The large
squares marked in blue are used
to establish the leukocyte count.

Schematic representation of the

Neubauer counting chamber's counting grid:
blue = area of the leukocyte count
red = areas of the erythrocyte and platelet counts
 During the chamber count, all cells
that lie on the left and the lower
border or that just touch it are
counted. None of the cells on the
upper and the right border or those
that just touch it will be counted.
This procedure is also known as
the L-form rule
Filled circles = count
Empty circles = do not count

Schematic representation of a medium-sized

square
(0.2 x 0.2 mm) of the Neubauer counting
chamber's erythrocyte grid.

On both sides of the counting

grid, an edge can be found. A
cover slip is placed on top of it.
If the cover slip is properly
placed, a distance of 0.1 mm
Schematic representation of the Neubauer (chamber depth) will exist
counting chamber between the counting grid and
side view the cover slip. The volume that is
blue = chamber depth over the squares of the counting
net corresponds to 0.1µl in a
large square (1mm x 1mm x 0.1
mm = 0.1mm3 = 0.1µl) and to
0.00025µl in a small square
(0.05 mm x 0.05 mm x 0.1 mm =
0.00025mm3 = 0.00025µl).
Photographic representation of the Neubauer
counting chamber
side view
Notice the short distance of 0.1 mm between
object slide and the cover slip

Manual Erythrocyte Counting

In a manual erythrocyte count, 10 µl of EDTA blood (collected with an Eppendorf

pipette) is diluted in 1990 µl of isotonic erythrocyte dilution solution. This results in a
dilution of 1:200. This suspension must be well-mixed and be immediately placed into
the counting chamber. After approximately 3 minutes, the erythrocytes will have
settled, and one may begin counting the erythrocytes in 80 small squares.

The calculation of the erythrocyte count is achieved by following the formula below
using these factors:

I. the number of erythrocytes (Ec) counted in the small squares,

II. the dilution of the cell solution,
III. the number of counted small squares,
IV. the volume above one small square, and
V. the conversion factor which is necessary in order to arrive at the volume of one
liter. Since we are dealing with one µl, this is equal to 106 (1 µl = 1 x 106 L or 1
L = 1 x 106 µl).

number of
counted erythrocytes(I) x dilution(II) erythrocytes erythrocytes x 106(V)
= =
number of counted squares(III) µl L
x volume above one small square(IV)

Example with 420 counted erythrocytes:

420 Ec x 200
84,000 Ec 4,200,000 Ec 4,200,000 Ec x 106
80 x 0.00025 µ = = = = 4.2 x 1012 Ec/L
0.02 µl µl L
l

Minimum number of erythrocytes to be counted = 400

In 80 small squares, around 400 erythrocytes are counted for normal erythrocytes
values. This yields a coefficient of variation (variability) of ±5%. This constitutes the
highest acceptable amount of random error (accuracy). If, however, the erythrocytes are
decreased in one patient (anemia, except for the thalassemias), scattering increases and
the accuracy, therefore, decreases. In polycythemia on the other hand, the accuracy
increases with an increasing erythrocyte count. In this latter caes, a higher dilution must
be used. In low erythrocyte counts on the other hand, the number of counted squares
must be increased or the dilution (e.g. 1:100) must be decreased in order to bring the
accuracy into an acceptable range.

Manual Leukocyte Counting

IIn the mnual leukocyte count, 50 µl of EDTA-blood (collected with an Eppendorf

pipette) is mixed together with 950 µl dilution solution (e.g. Türk's solution, collected
with an Eppendorf pipette). This constitutes a dilution of 1:20. The erythrocytes will be
lysed, and the leukocyte nucleus will be stained. The counting chamber is immediately
filled after mixing. After 2 minutes, one may begin counting the leukocytes in the 4
large squares.

Calculation of the leukocyte count is achieved by following the formula below using
these factors:
 I. the number of leukocytes (Lc) counted in the big squares,
II. the dilution of the cell solution,
III. the number of counted big squares,
IV. the volume above a big square, and
V. the conversion factor which is necessary in order to come to the volume of one
liter. Since we are moving in the µl area, this is equal to 106 (1 µl = 1 x 106 L or
1 L = 1 x 106 µl).

number of counted leukocytes(I) x dilution(II)

leukocytes leukocytes x 106(V)
number of counted squares(III) = =
µl L
x volume above one big square(IV)

Example with 150 counted leukocytes:

150 Lc x 20 3,000 Lc 7,500 Lc 7,500 Lc x 106

= = = = 7.5 x 109 Lc/L
4 x 0.1 µl 0.4 µl µl L

Minimum number of leukocytes to be counted = 100

Since the leukocyte count already shows greater physiologic variations, a coefficient of
variation of ±10% is acceptable. At least 100 leukocytes must be counted to achieve
this. As previously mentioned, accuracy decreases with decreasing leukocyte counts and
increases with increasing values. In low leukocyte counts, either the number of counted
squares must be increased or the dilution decreased (e.g. 1:10) in order to bring the
accuracy into the acceptable range.

Since normoblasts are not recognized as such in this procedure, a large number of
normoblasts can invalidate results. This is also true for mechanical leukocyte counts.
The number of normoblasts can only be accurately identified in a blood film and is
expressed as the number of normoblasts per 100 leukocytes. If there are more than 5
normoblasts per 100 leukocytes, a correction must be used.

The normoblast correction is achieved by using the formula below:

number of counted leukocytes x 100

number of normoblasts per 100 leukocytes + 100
Example with 12 normoblasts per 100 leukocytes with a leukocyte count of 5.6 x 109/L:
5.6 x 109/L x 100 5.6 x 109/L x 100 5.6
= = x 109/L = 5.0 x 109/L
12 + 100 112 1.12

Manual Platelet Counting

In a platelet count, 50 µl EDTA blood (collected with an Eppendorf pipette) is mixed in

950 µl dilution solution (collected with an Eppendorf pipette). This results in a dilution
of 1:20. The mixture must stand approximately 5 minutes so that the erythrocytes are
completely lysed. Then the suspension is mixed and put into the counting chamber. The
chamber is left in a moist environment for 20-30 minutes so the platelets can settle
without the chamber drying. Like the erythrocyte count, 80 small squares are counted.
Calculation of the platelet count is achieved by using the formula below using these
factors:

I. the number of platelets (Tc) counted in the small squares,

II. the dilution of the cell solution,
III. the number of counted squares,
IV. the volume above a square, and
V. the conversion factor which is necessary in order to come to the volume of one
liter. Since we are moving in the µl area, this is equal to 106 (1 µl = 1 x 106 L or
1 L = 1 x 106 µl).

number of counted platelets(I) x dilution(II)

platelets platelets x 106(V)
number of counted squares(III) = =
µl L
x volume above one small square(IV)

Example with 230 counted platelets:

230 Tc x 200
4,600 Tc 230,000 Tc 230,000 Tc x 106
80 x 0.00025 µ = = = = 230 x 109 Tc/L
0.02 µl µl L
l

Minimum number of platelets to be counted = 100

A coefficient of variation of ±10% is also acceptable for platelets. At least 100

platelets must be counted. In the case of low platelet counts, the number of counted
squares must be increased or the dilution must be decreased (e.g. 1:10) in order to bring
the accuracy into an acceptable range. One can also count a second chamber.

Manual leukocyte differential

To manually classify leukocytes, a blood film is stained with May-Gruenwald-Giemsa.

The different types of leukocytes from the film are counted under a microscope. Since
the leukocytes are not evenly distributed in the film and the same cell may not be
counted more than once, the preparation should be systematically screened.

Pull film Push film

At least 100 leukocytes should be counted and classified. Ideally, 2 x 100 cells (in two
blood films) should be counted. It is nearly impossible to count more than 100
leukocytes in severe leukopenias. On the other hand, in the case of very high leukocyte
counts, 400 leukocytes should be counted. Percentages achieved in this way are
converted to absolute values via the leukocyte count (e.g. 20% lymphocytes with a
leukocyte count of 6.0 x 109/L corresponds to an absolute lymphocyte count of 0.2 x 6.0
= 1.2 x 109/L).

Precision must be discussed again. Since leukocytes such as eosinophils and basophils
only constitute a small part of the total number of leukocytes, the accuracy of their
counts is rather small when only 100 leukocytes are counted. This is especially
important when the leukocyte count is very high (e.g. 1 eosinophil per 100 leukocytes in
a leukocyte count of 60.0 x 109/L already corresponds to 60.0 x 107/L). To what degree
the leukocyte differential values can vary, independent of the number of differentiated
cells, can be determined from the Rümke table (see below).

$ If the actual percentage of a patient's basophils are 5%, for example, the value found
by counting 100 leukocytes may be between 2 and 11%. Only by counting 10,000 cells
(performed accurately only by automated counters), has the obtained value a precision
of ±10%. If the percentage of a cell type is 50% a precision of ±10% is achieved with
500 counted leukocytes.$

Rümke table
% = percentage of a type of leukocyte to the total number
shaded dark red = percentage of identified leukocytes
(Table modified from the CD-ROM "Das interaktive Handbuch der
Hämatologie")

Manual Reticulocyte Counting

Reticulocytes are stained without fixation (supravital staining) with either new
methylene blue or brilliant cresyl blue and applied to an ordinary glass slide. A special
eyepiece is used which reduces the field of vision to a small square for counting.
Around 1000 erythrocytes (500 in each of the two films) are counted, and the
reticulocytes which are recognizable due to their reticular markings are counted. The
number of reticulocytes per erythrocytes is expressed as a percent.
The lower the number of reticulocytes, the more inaccurate the value. When 1000
erythrocytes are counted, a coefficient of variation of ±10% is achieved for a
reticulocyte percentage of 1-2% (= normal range).

Created on 6/3/2000. Last changed on 8/3/2000. Address all remarks to Dr. med. U. Woermann