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