WBC Histogram

Interpretations of 3-Part Differentiation

Sysmex Xtra Online | July 2011

Basis for automated cell counts is the concentration analysis of corpuscular peripheral
blood components per defined volume. The performance spectrum in the class of
automated compact systems (for example, Sysmex KX-21 or Sysmex K-4500) also
includes a pre-differentiation of white blood cells, in addition to the eight parameters
of the CBC. Based on this trimodal pre-differentiation, a lot of information is provided
for the interpretation of results and indications of abnormalities. Deviations from
normal ranges are detected – such as, for example, an increased or reduced white
blood cell count.

Such abnormal values require more precise clarifications, and, respectively, a micro-
scopic differentiation to detect and identify haematologic diseases. Initial information
about the reason for an abnormal white blood cell count is provided by a classification
of white blood cells into three separate subpopulations which is called a pre-differenti-
ation and which immediately provides important information to the examiner. These
are results on the differential blood count, especially regarding the percentage of
lymphatic and myeloid white blood cell in the total white blood cells count. With
a careful interpretation of all parameters, pre-differentiation allows the detection of
normal blood samples and the results can be transmitted without delay to the treating
physician. Samples are considered ‘normal’ when their analysis results are within the
reference ranges and if the ordering physician did not request further haematologic
examination. Accordingly, pre-differentiation will bring about a reduction of the number
of differential blood counts so that pathological and suspicious blood counts can be
concentrated on. Without a specific problem at issue, it is entirely sufficient to separate
white blood cells into lymphocytes and neutrophils. Any suspicion of a viral or inflam-
matory disease can thus also be confirmed or disproved.

Reagent effect on cells

Various white blood cells forms are fixed to a defined size by means of a special lyse
reagent. The lyse reagent effect on white blood cells causes the cells to shrink to a
defined volume according to their cell type – to be thus presented as a separate
population in the histogram. After this special lysis treatment, cells will be shown
in a histogram according to their size (Fig. 1).
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WBC Histogram Interpretations of 3-Part Differentiation 2

Before adding lyse reagent

Cell diameter in µm
Neutrophils Neutrophils 10 – 15
Basophils Basophils 9 – 14
Eosinophils
Eosinophils 11 – 16
Monocytes
Lymphocytes Monocytes 12 – 20
Lymphocytes 7 – 12
µm

After adding lyse reagent

Lymphocytes Cell volume in fl
Monocytes Lymphocytes 30 – 80
Basophils Neutrophils Basophils 60 – 120
Eosinophils Eosinophils 70 – 130
Monocytes 80 – 140
Neutrophils 120 – 250
fl

Fig. 1 Normal haematologic result

Monocytes present the largest population in the peripheral blood with a diameter of
approx. 20 μm and are shrunk the most. The effect on eosinophils and basophils is also
relatively major, so that these three populations find themselves in one mixed popula-
tion. The lyse reagent’s influence is most minor for lymphocytes and it is also minor
for neutrophils. The x-axis of the WBC histogram indicates cell size in femtolitres.
This cell size in the WBC histogram does not show the actual volume of the white
blood cells presented since the measuring signals following lysis treatment are no
longer in relation to the volume of native white blood cells.

Histogram interpretation
All measured pulses are shown in the volume distribution curve. Red blood cells are
lysed in the separate WBC channel so as not to interfere with the white blood cells.
Platelets are separated from the white blood cells by means of the lower discriminator
(threshold value LD = lower discriminator) so that only white blood cells are actually
shown. The volume distribution curve should be located within the two discriminators
(LD + UD) and not only begin at the base line but also end there. If a deviation from the
base line occurs, the analyser provides a corresponding warning message to indicate
required follow-up actions. The various deviations are explained in the following text.
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WBC Histogram Interpretations of 3-Part Differentiation 3

Deviation of the histogram on the lower discriminator

In case of a deviation of the base line on the lower discriminator, the analyser
generates a ‘WL’ warning (WL = WBC lower discriminator Fig. 2). There are various
causes for this:
n Platelets aggregates (clotted sample,
EDTA incompatibility)
n Lyse-resistant red blood cells
n Erythroblasts
n Cryoagglutinates
n Giant Platelets

Fig. 2 ‘WL’ warning due to interference on Fig. 3 erythroblasts and one lymphocyte
lower discriminator

In case of a ‘WL’ warning, the WBC count value should be checked since it might have
a false increase due to the effect of one of the above-mentioned interference factors.
The most frequent cause for such an interference are platelets aggregates. These
aggregates affect the WBC result because the analyser cannot differentiate between
aggregates and white blood cells. In this case, however, the platelets histogram also
suggests the presence of aggregates which can also be seen in the manual differential
blood count. To obtain a correct result, it is necessary to take another blood sample.

The presence of erythroblasts can also

measured WBC lead to a ‘WL’ warning. The nucleated
value x 100
Correct WBC value = erythroblasts (Fig. 3) are also counted
100 + number of
erythroblasts* as white blood cells. Accordingly, the
WBC value of these samples must be
* Number of erythroblasts counted to 100 WBC corrected using the formula you can
find to the left.
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WBC Histogram Interpretations of 3-Part Differentiation 4

White blood cells values may be distorted

WBC WL* 49.4x10 /L 9
due to lyse-resistant red blood cells in
LYM % WL -.–
MXD % WL -.–
premature infants and in patients with
NEUT % WL -.– higher osmotic resistance. The lyse rea-
gent’s effect on the red blood cells mem-
brane – which is very firm in this case –
will not be sufficient so that the white
blood cells value is interfered with. The
Fig. 4 ‘slide curve’ typical ‘slide curve’ in the white blood
cells histogram is characteristic for this
(Fig. 4). In this case, the sample must be diluted to increase the effect of the lyse rea-
gent on the red blood cells. A falsified white blood cells count due to giant platelets
is extremely rare but should be taken into account for the sake of completeness.

Deviation of the histogram on the upper discriminator

With a deviation on the upper discriminator (UD) (Fig. 5), the system generates the
warning ‘WU’ (WU = WBC upper discriminator). In this case, the linearity limit of the
measurement is mostly exceeded. Due to the greatly increased cell count (WBC > 100 x
10³ μL), not all cells can be shown in the histogram; the WBC count is perhaps higher
yet. The sample should be diluted at a ratio of 1:5 to obtain a reliable count.

Fig. 5 ‘WU’ warning on upper discriminator Fig. 6 ‘T2’ alarm. ‘T1’ was found, but not ‘T2’

T1 and T2 flags
Aside from the two delimiting discriminators, so-called ‘valley discriminators’ are addi-
tionally used. These discriminators seperate the white blood cells into the three popu-
lations. The two valley discriminators ‘T1’ or ‘T2’ are flexible in certain areas; i. e. they
can largely adjust to the sample. With certain abnormalities of the sample, a reliable
separation of the populations through valley discriminators will not be possible. In
this case, the equipment generates flag ‘T1’ or ‘T2’ (Fig. 6). When a ‘T1’ or ‘T2’ flag is set,
the pre-differentiation values should not be used because the populations have not
been precisely separated from each other. Here, clarification is recommended through
manual differential blood count. However, the total WBC count is correct if no ‘WL’
or ‘WU’ warning has been generated.
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WBC Histogram Interpretations of 3-Part Differentiation 5

F1, F2 and F3 flags

Contrary to the ‘T1’ and ‘T2’ flag, the corresponding valley was found in this case;
however, the valleys are conspicuously far away from the base line. It is accordingly
possible that the fractions are mixed; i. e. fraction 1 and fraction 2, or fraction 2 and 3
merge into each other over large areas. Even in such a case, the total number of white
blood cells is counted correctly, provided that no additional ‘WL’ or ‘WU’ warning is
given; however, the pre-differentiation should be verified by manual differential
count.

F1 F2 F3

Fig. 7 ‘F1’, ‘F2’, ‘F3’ abnormal differential

Summary
The above described warning messages enable the user to detect positive samples
and to react with follow-up actions because of the warnings. Morphological changes –
mostly preliminary stages or abnormalities of the myeloid and lymphatic cell series –
require manual differentiation. Pre-differentiation information will reduce the rate of
microscopic differentiation to clinically relevant cases. Working time can thus be saved
without loss of quality.

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