Sysmex Journal International Vol.9 No.

1 (1999)

Principles of Measurement in Hematology Analyzers

Manufactured by Sysmex Corporation

Keiji FUJIMOTO

Scientific Division, Sysmex Corporation, 4-4-4 Takatsukadai, Nishi-ku, Kobe 651-2271, Japan.

This article describes the measurement principles found in hematology analyzers manufactured by Sysmex Corporation and currently
available on the market. To aid in understanding, a brief historical background of the analytical principles involved is presented. This is
followed by general descriptions of particle behavior in an electric field and coincidence. The details of the measurement principles of
individual parameters, RBC, WBC, PLT, reticulocytes, immature WBC and NRBC are presented together with the relevant reagent
reactions.
As technology advances, so does the availability of new analyzers to satisfy customer needs.
Sysmex Corporation will continue to develop innovative and user friendly products of high clinical utility.
(Sysmex J Int 9 : 31 − 44, 1999)

Automated Hematology Analyzer, RBC, WBC

FOREWORD
In this article the principles of measurement used in the
Sysmex hematology analyzers available on the market
are described. Because of the wide application of mea-
surement principles in Sysmex products, a general expla-
nation of the measurement principles will be presented
first. Then each measurement parameter such as RBC,
WBC, PLT, immature WBC, NRBC, reticulocyte and
hemoglobin, is explained.
UNDERSTANDING
THE BASIC KNOWLEDGE OF
THE MEASUREMENTS
In this section, basic but important knowledge for under-
standing the measurement principles in biological cell
analysis is described.
Cell counting by electric capacitance method
Around 1960, when Sysmex first tried to develop a
hematology analyzer, the founder of the Coulter
Company (now Beckman-Coulter Co.), Mr. Wallace H.
Coulter1), took out a technical patent for the electric resis-
tance method he had developed. Due to the risk of
infringement of this patent, Sysmex could not apply this
method to its product.
To avoid this technical patent, Sysmex developed the
capacitance method rather than the resistance method.
As shown in Fig. 1, when a particle is located in the
detection area, a change in electric capacitance occurs
and this is in proportion to the volume of the particle.
Applying this method, we can count the total number of
cells as they pass through the detection area (a small
aperture). A detailed description of this method is given
in the article by Tatsumi2) and Okada3), in this issue.
This method has not been directly applied in our recent
products alone. The technology is, however, still used in
some of our products as a part of the high energy electric
impedance method for the purpose of detection and dif-
ferentiation of WBCs, which will be described in a later
section in this article.
Cell counting by electric impedance method
(Note : The word “Impedance” used here has almost the same meaning
as “Resistance”. Theoretically, impedance contains resistance, capaci-
tance and reactance. In electric impedance method, electric resistance
does major contribution. As then in this article, electric impedance
method and electric resistance method are almost the same.)
This is the most popular method applied in hematology
analyzers manufactured not only by Sysmex but also by
other companies. In this method, biological cells such as
WBC, RBC and PLT are regarded as completely non-

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 Sysmex Journal International Vol.9 No.1 (1999)

Fig. 1 The principle of measurement by electric capacitance method

(a) shows the cross section of aperture. Two electrodes are located across the aperture. The aperture is then regarded
as a condenser. The electric capacitance is the change from (b) no cell present to (c) a cell located in the aperture.
The total change of capacitance is proportional to the volume of the cell.

Fig. 2 The principle of measurement by electric impedance method
When a cell suspended in electrolyte solution passes through the
aperture, the change of electric impedance is detected as a pulse. The
total pulse number corresponds to total cell count and each pulse
height is proportional to the corresponding cell volume.
Fig. 3 Coincidence phenomenon
When more than two cells are located in the aperture (or sensing
zone), the coincidence phenomenon is observed. Cell 1 and Cell 2
are detected as a single large pulse, therefore one of the cells is not
counted (coincidence loss). The degree of coincidence loss depends
on the concentration of cells.

conductive resistivity particles. Coincidence

When a blood cell passes through an aperture (sensing
zone) suspended in electrolyte solution, the change of
electric impedance is detected (Fig. 2). The change of
impedance is in proportion to the volume of the cell
detected and can thus separate, for example, RBC from
PLT depending on the degree of impedance change. In
the case of WBC counting, after hemolysis of RBCs, the
same procedure is applied.
This method has been employed in major hematology
analyzers for many years. Two major problems, how-
ever, have remained in the quest for more precise and
accurate measurements. These are coincidence and non-
identification.
In the electric impedance method, as a particle passes
through the aperture it changes the resistivity between the
immersed elecotrodes located on either side in a manner
analogous to the electric capacitance method.
Ideally, if the particles go through the sensing zone one
by one, we can count the total number detected in the
sensing zone. However, simultaneous occupancy of the
sensing zone by more than one particle occurs. This phe-
nomenon is called “coincidence” (Fig. 3) and the result-
ing count error is known as the coincidence error4, 5) . The
magnitude of the coincidence error increases with the
concentration of cells suspended. For major hematology
analyzers, by using the measurement results from several

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 Sysmex Journal International Vol.9 No.1 (1999)

samples of different concentration, the coincidence cor-

rection formula can be established. This correction for-
mula may be integrated into the analyzer’s computer and
the coincidence corrected result reported (Fig. 4).
Two simple models of coincidence are illustrated in Fig. 5
and will help to analyze the coincidence phenomena and
other problems described below.
In order to minimize coincidence physically, the hydro-
dynamic focusing method has been developed and
assembled in some analyzers.

Hydrodynamic focusing method

Fig. 4 Coincidence correction formula
The dotted line represents the actual measurement results.
The higher concentrations of suspended cells produce higher
coincidence loss. This actual measurement curve depends on
the type of instrument. The solid line is the count corrected
by the coincidence correction formula.
The coincidence phenomenon is simplified into two mod-
els, namely horizontal interaction and vertical interaction
(Fig. 5).
When the vertical interaction occurs, a large single signal
is observed even although two cells pass through the
sensing zone simultaneously (Fig. 5b). The count result
itself may be corrected by the coincidence correction for-
mula described previously. However, from the single
large pulse generated, it is not possible to decide if this
arises from one large cell or two small cells. In case of
horizontal interaction, sometimes an M-shaped signal
occurs as a result of two cells located near each passing
horizontally through the sensing zone (Fig. 5a).

Fig. 5 Simplified representation of the two types of coincidence

In the case of horizontal interaction (a), one wide M-shaped pulse is produced; in the case of vertical
interaction (b), one large pulse is observed. From the observed pulse, inversely, we can not identify the
existence of two cells in the aperture.

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 Sysmex Journal International Vol.9 No.1 (1999)

In 1969, Thom, et al. (Photo 1) using a large scale model

investigated the effect of axial and non-axial particle
flow through the electrical field both at the entrance and
sides of the sensing zone. In this study single particles
passing close to the wall of the sensing zone also pro-
duced M-shaped pulses similar to that illustrated in Fig.
5a. This M-shaped signal was produced by only one par-
ticle and was completely independent of the coincidence
phenomenon. It was due to the passage of the cell close
to the wall of the sensing zone where high current density
exists6,7) (Fig. 6). This observation indicated the impossi-
bility of determining the cause of the M-shaped pulse,
non-axial passage through the sensing zone or horizontal
interaction of a couple of cells. While the count result
may be valid because of coincidence correction, there is
no way to correct the measurement of cell volume. Photo 1 Prof. Dr. Reinhard Thom and the author
Thom, by using an hydrodynamic focusing method (Fig. 7), This photo was taken in his home in July, 1998. He
attempted to resolve this problem8). invented the hydrodynamic focusing method as applied
In hydrodynamic focusing a steady flow of diluent, the to electric impedance detection in the Free University
sheath is drawn through the aperture and the cell suspen- of Berlin.
sion is injected into this moving body of liquid in a fine
stream close to the aperture entrance.
Using this principle the vertical interaction coincidence
phenomenon described above is dramatically reduced.
In a hydrodynamically focused system, no particle there-
fore goes near the wall or the entrance angle of the sens-
ing zone (aperture), where high current density exists. As
a result all M-shaped signals detected are due to the hori-
zontal interaction coincidence phenomenon.
The horizontal interaction coincidence phenomenon may
be further decreased by the choice of a suitable dilution
ratio of measurement sample.
As described above, there is much merit in the applica-
tion of hydrodynamic focusing for cell analysis.
However it took time to develop this method for com- Fig. 6 The effect of inhomogeneity of the electrical field
mercial hematology analyzers because of the need for within the sensing zone6)
precision components and a highly sophisticated fluid When a cell passes axially though the sensing zone, a
circuit design. Gaussian style pulse is observed. When a cell passes
In 1980, TOA Medical Electronics Co., Ltd. (the corpo- through the sensing zone non-axially the signal produced
rate name was changed to Sysmex Co.) in Japan released is M-shaped.
a fully automated hematology analyzer, the E Series,
was the first fully automated hydrodynamically focused
electric impedance system worldwide. This measurement
technology is incorporated in other recent systems such
as NE Series9-11), SE Series and XE-2100TM.
One of the major practical merits of this method for
hematology laboratories is the dramatic improvement in
the resolution of the cell volume distribution curves. Fig. 8
shows the comparison of distribution curves from a mix-
ture of three different monosized latex particles with and
without hydrodynamic focusing. A much clearer volume
distribution histogram is obtained by the hydrodynami-
cally focused analyzer.
Hydrodynamic focusing when applied to RBC measure-
ments produces a clear disrimination between PLT and
RBC. It enables subtle change in the RBC volume distri-
bution curves in patients with iron deficiency under
going treatment to be recognized. It permits recognition Fig. 7 Schematic diagram of hydrodynamic focusing
of multiple RBC populations in, for example, patients method
with iron deficiency who have received blood transfu- The cell stream is sheathed by cell free reagent. As a
sion. result, all cells are directed through the centre of the sens-
A major disadvantage of the electric impedance method ing zone (axial flow). As a result the vertical interaction
is the difficulty in distinguishing large platelets from coincidence can be ignored (Fig. 5b).

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 Sysmex Journal International Vol.9 No.1 (1999)

Fig. 8 A comparison of resolving power with and without hydrodynamic focusing method
Mixed latex particles of 4.3 fL, 12 fL and 19 fL are measured. The distribution curve by E Series with
the hydrodynamic focusing method (the histogram on the left) is superior to the non-focused system.

the signal height of this cell corresponds to a larger ellip-

Fig. 9 Actual measurement volume of a spherical cell
By the electric impedance method, a spherical cell produces an ellip-
tical shadow in the electric field as indicated by the dashed line. The
volume of this spherical cell is overestimated by 30 to 50 % or alter-
natively the shape factor is 1.3 to 1.5.
soid cell. This is illustrated by the dashed line in Fig. 9.
This is an example of the “shape factor” phenomenon.
When a fresh or non-stabilized RBC passes through the
sensing zone, its shape is transformed into an ellipsoid
creating an ellipsoid electronic shadow. Thus the volume
of the electronic shadow conforms very closely to the
physical volume of the cell. If, on the other hand, a latex
particle is passed through the sensing zone it retains its
spherical shape but produces a fusiform electronic
shadow equivalent to 1.3-1.5 times of the actual volume
of the sphere. Particles with other shapes produce even
more complex electronic shadows. Experimentally, the
shape factor of spherical cells such as latex particles
compared with fresh RBC is 1.3-1.5. This means that a
latex particle having the same theoretical volume as a
fresh RBC produces a signal which is 1.3-1.5 times larger.
Understanding this phenomenon and the use of shape
factor will help the calibration of the distribution curves
using latex particles.

extremely microcytic or fragmented RBC even using
hydrodynamic focusing method. This capability is better
with light scattering methods as will be described in a
later section. The electric impedance method, however,
can estimate cell volume more precisely than the light
scattering method. In both methods, morphological
abnormality of RBCs, for example spherocytosis, or
elliptocytosis is not detected unless the MCH or MCHC
is out of normal range. Morphological abnormality is
best detect by image analysis technology.
Shape factor
Thom, et al. challenged the accurate measurement of the
volume of latex particles by analyzer using an electric
impedance instrument even with hydrodynamic focusing.
During this investigation, it was found that different sig-
nal heights were produced by different shaped cells with
the same theoretical volume6, 7, 12).
Since the system was hydrodynamically focused, this
phenomenon cannot be explained by vertical interaction
coincidence and/or non-axial particle flow.
Using a large scale model, these workers demonstrated
that when a spherical cell goes through the sensing zone,
RF/DC method
During the early evolution of technology for hematology
analyzers, the measurement parameters expanded from
only WBC and RBC to PLT, and then to WBC 3 part dif-
ferential (lymphocyte, neutrophil, and other cells).
Sysmex paralleled this development with many then
“state of the art” analyzers.
To progress further, however, and develop the WBC 5
part differential count required additional technologies.
In the electric resistance method, detection pulses corre-
spond to cell volume. As described above, Sysmex
developed the electric capacitance method for cell count-
ing originally for the purpose of avoiding the Coulter
patent. Electric capacitance signals depend not only on
the volume of cells but also on their internal contents
such as granules and nuclei. So long as the integrity of
WBC could be maintained, measurement of electric
resistance and capacitance simultaneously would result in
cell volume and internal information for the different
cells. This suggested that it might be possible to achieve
further differentiation of WBC using this combination of
t e c h n o l o g i e s13). This became a reality when Sysmex
developed the WBC 5 part differential count using such a
combination.

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 Sysmex Journal International Vol.9 No.1 (1999)

Fig.10 The principle of measurement of automated reticulocyte analyzer R-1000

The R-1000 analyzes reticulocytes by flow cytometry, using an argon laser as the light source. Whole blood sam-
ples are stained with a fluorescent dye and passed through a laser beam in the sheathed flow cell. The fluorescently
labeled cells are irradiated using an argon laser beam and the forward scatter and the side fluorescence emitted
from each cell in the sheath flow are detected. While the former is used as an indicator of relative cell size, the lat-
ter is used as an indicator of RNA content. Combining these two parameters, reticulocyte count and reticulocyte
ratio are determined.

D << 2λ / π Rayleigh theories

2λ / π < D < 10λ / π Mie theories

The Mie theory provides a solution to the way in which

Fig. 11 An example of scattergram produced by the R-1000
Vertical axis indicates cell volume and horizontal axis indi-
cates the intensity of fluorescence. The scattergram is divided
into PLT area, mature RBC area and reticulocyte area.
Cell counting by the light scattering method
When light reaches the surface of any material, scatter-
ing occurs. The detailed theory of the light scattering
mechanism is very complex. Basically, there are two
major light scattering theories depending upon the rela-
tive relationship between the wave length (λ) of incident
light and the diameter of objective (D);
light is spread as its component fields act on a small
sphere suspended in some background medium and
applies particularly to the case of blood cells.
More details of the light scattering method can be found
in the article by Groner, et al. in this issue. The light
scattering method only as applied in Sysmex products
will be discribed here.
In Sysmex products, we have applied the light scattering
method by using an argon ion laser (488 nm wave length
and 7.5 mW output power) in the fully automated reticu-
locyte analyzer, the R-1000 16). Cell size is determined
using forward light scatter and simultaneously RNA con-
tent by fluorescence after automated staining with the
fluorochrome Auramine O. At the time of its introduc-
tion, only the argon laser possessed sufficient power and
a suitable wavelength for the small amount of RNA in
each reticulocyte17). From the cell volume and fluores-
cence, a scattergram was produced from which it was
possible to discriminate and count mature RBC, reticulo-
cytes and PLT (Figs 10 and 11).

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 Sysmex Journal International Vol.9 No.1 (1999)
In general, the light scattering method is constructed by
complex formulae with many parameters of size, shape,
and surface condition of the cell, which are not simple
linear functions between the scattered light intensity and
the cell volume. This means, for example, that twice the
light intensity does not imply twice the volume. This is
inconvenient for the precise detection of cell volume, the
phenomenon however works well in the discrimination
between large PLT and small RBC which have the same
volume but different surface characteristics.
The argon gas laser has a life span of several thousand
hours. The price of the argon laser is around US$ 2,000 -
3,000 and partly accounts for the high production cost of
the analyzer.
Sysmex has now studied the application of an alternative
laser light source. A semi-conductor laser (excitation
wave length 650 nm, power output 2 mW) developed by
TOSHIBA ELECTRIC Co. was used in studies on cell
analysis. Building on experience obtained during devel-
opment of the R-1000, we achieved a new application of
this semi-conductor laser for WBC differential, resulting
in the development of a fully automated hematology ana-
lyzer, the SF-300018).
THE MEASUREMENT PRINCIPLES
IN EACH PARAMETER
In this section, the measurement principle for each para-
meter generated in Sysmex hematology analyzers is
described.
Red blood cells (RBC) and platelets (PLT)
Currently, RBC and PLT are counted in a single mea-
surement channel and unit with the same sample prepara-
tion procedure.
Electric impedance method
As described above, in this method, all cells are assumed
to be non-conducting particles. The change of imped-
ance is detected when cells suspended in electrolyte solu-
tion pass through the sensing zone, normally 70-100 µm
diameter and depth (Fig. 2). In whole blood, there are
approximately 5 × 1012/L. To avoid or minimize the
coincidence phenomenon, whole blood is prediluted
25,000 to 50,000 times with electrolyte solution, called
the diluent.
Predilution may be performed manually or automatically
in the instrument. The composition of the diluent is care-
fully designed to maintain the suspended cells in good
condition interms of shape and size by controlling the
osmotic pressure, pH, etc. and the addition of some
preservatives.
As stated previously, M-shaped signals are produced by
non-axial cell flow in the sensing zone and can result in
misleading measurements unless corrected. Some recent
Sysmex products such as KX-21TM, K-4500TM and SF-
3 0 0 0 TM do not have hydrodynamic focusing but can
detect and eliminate such M-shaped signals automatically
by means of editing circuitry. Coincidence loss is also
automatically corrected. The frequency and exact shapes
of these M-shaped signals depends on the deformability
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of the cells. Fresh blood cells and stabilized blood cells
such as control materials exhibit very different degrees of
deformability.
Electric impedance method
-hydrodynamic focusing method
In high end analyzers (XE-2100, SE Series, NE Series),
the presence of hydrodynamic focusing method removes
the need for editing circuitry (Fig. 7). This method mini-
mizes coincidence and the occurrence of M-shaped sig-
nals producing very reliable distribution curves and count
results.
Accurate focusing of the sheathed flow in the sensing
zone requires precision component and sophisticated
fluid control technology. Any small air babbles produced
near the sensing zone will disturb accurate focusing and
produce electric noise.
We add a special surfactant in the sheath reagent, which
produces few bubbles and any formed are rapidly
removed from the sensing zone.
Light scattering method
Basically, Sysmex Corporation relies upon electric
impedance as the primary measurement method because
it can estimate the volume of each cell exactly.
However, in some abnormal samples, such as those with
large PLTs and small RBCs or fragments of them, the
impedance method can have difficulty in discriminating
between RBCs and PLTs.
In such cases this discrimination is much better with the
light scattering method. In the XE-2100, however, both
impedance and scattered light methods co-exist. The
PLT counts by impedance are presented as the primary
results. In the above-mentioned abnormal samples, in
which the PLT counts by the two measurement methods
fail to agree, a computer algorithm is applied and the
reported result is switched to that produced by scattered
light19).
WBC counts and differential
Electric impedance method
WBC can be categorized into lymphocytes, neutrophils,
eosinophils, basophils and monocytes in normal samples.
Early hematology analyzers were unable to differentiate
the different WBC types and therefore only a total count
was provided.
The population ratio between WBC and RBC is around
1 : 1,000 even in normal samples, which means that RBC
interference during estimation of WBCs may occur. To
increase accuracy and precision of WBC count it is nec-
essary to hemolyze the RBCs.
The development of lysing reagents for RBCs but not
for WBC was difficult. In the early developmental
stages of hematology analyzers, saponin was used as a
lysing reagent for RBCs, but this also affected WBCs
which were stripped of their cytoplasm, and their
nuclei shrunk more or less uniformly. As a result of
many investigations, more sophisticated lysing reagent
were developed, by which the WBC cytoplasm was
still stripped and the nuclei shrunk but the degree of
shrinkage depended on the cell type. The differing
degrees of shrinkage made possible the WBC 3 part
 Sysmex Journal International Vol.9 No.1 (1999)

differential, separating lymphocytes, neutrophils and

other cells (Fig. 12, Photo 2). This method is still
used in the K Series instruments. In some semi-auto-
mated analyzers, a WBC 2-part differential count (or
lymphocytes and other WBC) is provided.

Electric impedance method -RF/DC method

During developmental work on lysing reagents, we pro-
duced a highly sophisticated reagent which hemolyzes
RBCs but not WBCs (Photo 3).
WBCs, although of different sizes, comprise a nucleus,
cytoplasm and frequently granules. The nature of these
components varies with the type of cell. These character-
istics can be used for differentiating the types of white
cell : therefore, it becomes possible to differentiate Fig. 12 An example of WBC 3 part differential
WBCs by detecting volume and internal information. All RBCs are hemolysed. The WBCs are de-nucleated and
As mentioned above, by applying both of the electric their nuclei are shrunk. The degree of shrinkage of the nuclei
impedance (DC) method and electric capacitance (RF) is dependent upon the type of WBC. A WBC 3 part differential
method, we can estimate the cell volume and its internal then becomes possible (left to right are the lymphocytes, the so-
content (nucleus, granules etc.). Even if two cell types called mixed cells comprising mainly monocytes and finally the
have the same volume, they can be differentiated if they neutrophils).
have different internal contents (Fig. 13).
No reagents are available which can maintain the original
shape of WBCs over a prolonged period and at the same
time hemolyze the RBCs completely. In fully automated
hematology analyzers, sample preparation procedures
such as dilution, mixing, reaction with reagents, tempera-
ture and timing are perfectly standardised by computer
controlled sequences. Thus in a short, fixed time, which
is sufficient for the lysing reagents to act, measurement
data with good precision is obtained by fully automated
analyzers. However, with manual sample preparation
using those reagents, as is necessary in semi-automated
instruments, it is difficult to achieve good 3- and 5- part
differentiation.
Basophil and eosinophil absolute numbers are much
lower than other WBC types in normal samples. In some
analyzers, independent channels for those cells are Before reaction : a single WBC and three RBCs
assembled and use the impedance method of measure- are clearly observed
ment (Fig. 14).

Light scattering method

The impedance method detects volume and internal con-
tent of cells. The optical method achieves 5 part differ-
entiation by using light scatter and fluorescence with
staining of organelles, granules and nuclei.
Fig. 15 illustrates the principle of measurement of the
WBC differential in the SF-3000 by means of a semi-
conductor laser. The scattered light from the surface of
the cell provides information on its size. Some incident
light penetrates the inside of cell and produces scattered
light from cytoplasmic granules and the nucleus.
The information gleaned from light scatter depends on
the angle of scatter. Generally, lower angle scattered
light contains information on cell size and higher angle After reaction : the WBC has been de-nucleated
scattered light contains information on the internal com- and three RBCs have disappeared
position of the cell.
The simultaneous observation of such different angles of
scattered light can provide a scattergram producing inde- Photo 2 Morphological change of a WBC and some RBCs
pendent parameters (Fig. 16). before and after reaction with lysing reagent
The detection devices for the different scattered light
angles are assembled on the same electronic board,
which permits a simple and reliable optical design. In

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 Sysmex Journal International Vol.9 No.1 (1999)

a : neutrophil b : lymphocyte

c : monocyte

d : eosinophil e : basophil

Photo 3 Morphological appearance of WBCs in the lysing reagent for the 5 part differential

Radio frequency method

Direct current method

Fig. 13 Schematic model of RF (Radio Frequency) and DC (Direct Current) detection method
Simultaneous application of RF and DC in the sensing zone, produces information on cell size
and internal composition as the cell goes through the sensing zone.

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In the eosinophil detection channel, a specific alka- In the basophil detection channel, a specific acidic
line lysing reagent hemolyses RBCs and all WBCs lysing reagent hemolyses RBCs and all WBCs except
except eosinophils. Such residual eosinophils are basophils. Such residual basophils are detected by
detected by the electric impedance method. the electric impedance method.

Fig. 14 The measurement principle for eosinophil and basophil detection

In the SE-9000, two independent channels are constructed to detect those cells.

Low angle scattered light detection model Low angle scattered light detection principle

High angle scattered light detection model High angle scattered light detection principle

Fig. 15 The principle of measurement of WBC differentiation in SF-3000 using the

light scattering method
When hydrodynamically focused cells pass through the flow cell, a focused semi-conduc-
tor laser light beam illuminates the cells. Each cell produces scattered light of low angle
and high angle. Low angle scattered light provides cell size information, and high angle
scattered light contains information on the internal composition of the cell.
For more clear discrimination of eosinophils from other granulocytic cells, a specific
stain dyes eosinophils granules clearly separating them from other cell types.

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DIFF WBC/BASO

Fig. 16 Example of the scattergram produced by the SF-3000

Side scattered light intensity Side scattered light intensity

Fig. 17 Example of the scattergram produced by the XE-2100 (WBC 4 part differental)

Side scattered light intensity

Fig. 18 Example of the scattergram produced by the XE-2100 (basophil detection)

the SF-3000, for the purpose of clear discrimination of
eosinophils from other granulocytic cells, a special stain
exclusively dyes eosinophils and changes their scattering
characteristics. In the standard scattergram the location
of basophils coincides with that of the neutrophils. The
SF-3000 therefore first performs a WBC 4 part differenti-
ation (lymphocytes, monocytes, eosinophils and neu-
trophils) and then in the next sequence uses a specific
lysing reagent to count basophils.
The XE-2100 uses almost the same method. In XE-2100,
however, we detect side scatter intensity (90° scatter)
rather than high angle scattered light for information on
cell content. At the same time, all cells are stained by a
fluorescence dye and the side fluorescence intensity pro-
duced by RNA/DNA derived from organelle and nuclei
is measured. This information provides 4 part differenti-
ation (Fig. 17). Basophils are counted using low angle
forward scatter and side scatter (90° scatter) (Fig. 18)
after lysis of other WBCs.

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Immature WBC
All automated hematology analyzers perform as
“Screening” instruments. This means that instruments
identify samples as “Normal” and “Abnormal” (morpho-
logical and/or population abnormalities). In the case of
“Normal” samples, the operator can directly report the
instrument results, but in the case of “Abnormal” sam-
ples, the operator should re-check and/or verify those
samples using other procedures.
The ideal instrument has very low false positive and false
negative rates. The confident detection of immature
WBC, not normally present in the peripheral blood is
very important in this respect.
Sysmex has achieved this capability in the SE-9000 by
using the combination of capacitance (RF) and imped-
ance (DC) methods in conjunction with special reagents.
Immature WBCs contain less lipid than mature cells. In
the presence of special lysing reagents (polyoxyethylene-
series non-ionic surfactant and sulfur-containing amino
acid) mature cells are disrupted, their granules are eluted
and only the nuclei remain. Immature cells of the granu-
locytic series behave in a different way. There is cell
membrane damage, but before the intracellular compo-
nents are eluted the lysing reagent enters the cell and
binds to the membrane and the granules thus fixing the
cell. In this process the sulfur-containing amino acid in
the lysing reagent acts as a protector for the cell against
the action of the surfactant. Mature WBCs, on the other
hand are lysed by the super-surfactant in the same way as
RBCs (Fig. 19).
Since different immature cells also have differing charac-
teristics, such as nuclear size and granule content depend-
ing on their degree of maturity, we have realized that
suitable selection and concentration of amino acid offers
the possibility for qualitative differentiation of immature
WBCs (Fig. 20).
This detection method is present in the SE-9000 and the
XE-2100 and contributes to their superior sensitivity for
abnormal cells. Recently, some laboratories using the SE-
9000 found that this channel could be used for the detec-
tion of hemopoietic progenitor cells in patients on stem
cell mobilising regimes, thus indicating the optimum
time for apheresis. This offers an excellent alternative to
the expensive and time-consuming flow cytometric assay
of CD34 + cells. Sysmex provides special computer soft-
ware in SE-9000 for this application.

Fig. 19 Mechanism of lysis of mature WBCs

The difference in composition of mature and immature granulocytes is shown. A specific lysing reagent
containing a polyoxyethylene series nonionic surfactant and a sulfur-containing amino acid lyses mature
WBCs. Immature cells of the granulocyte series are protected by the amino acid against lysing.

Fig. 20 Further qualitative differentiation of immature cells of the granulocyte series in the IMI channel
The effect of the lysing reagent differs with each type of immature granulocte allowing qualitative differentiation.
Mature WBCs are, however, completely lysed and shrunken being located in blue colored cluster in the scattergram.

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 Sysmex Journal International Vol.9 No.1 (1999)

Nucleated red blood cell (NRBC)
The detection of NRBC has clinical significance.
In almost hematology analyzers, some or all of nucleated
RBCs have been recognized as lymphocytes due to their
similar nuclear size. This results in overestimation of
the total WBC count. Hitherto it has been difficult to
count NRBC by automated methods. During the devel-
opment of WBC differential from 3 part to 5 part, the
detection capability of NRBC have been has improved.
Today, most sophisticated hematology analyzers have a
flagging system to indicate the presence of NRBC in a
sample. This flagging system, however, can not produce
an NRBC count.
Sysmex has established NRBC quantitative estimation in
addition to qualitative flagging in XE-2100. In this ana-
lyzer, first, complete hemolysis of RBCs is produced by a
specific lysing reagent, which simultaneously denucle-
ates, shrinks and slightly stains the nuclei of NRBCs. At
the same time, this lysing reagent maintains the shape
of the WBCs and stains their intracytoplasmic organelles
and nuclei.
The different degrees of staining between NRBCs and
WBCs can be detected by semi-conductor laser, the fluo-
rescence intensity of the WBCs being much stronger than
for NRBCs. This permits easy differentiation and quanti-
tative estimation (Figs 21 and 22).
Reticulocyte counting
Until recently, reticulocyte counting has been performed
by visual microscopy of supravitally stained preparations.
A fully automated method was first realized in 1988 by
Sysmex, the R-1000, which has proven to be a popular
method worldwide. This capability is adopted in the XE-
2100 and also as an optional attachment (RAM-1) for the
SE-9000.
In these devices, RBCs are stained by fluorescence dye
and activated by an argon or a semi-conductor laser.
Fluorescence mainly from DNA and RNA in WBCs,

Fig. 21 The method principle for NRBC detection in the XE-2100 - the mechanism of lysis-
A new reagent has been developed for NRBC detection in the XE-2100. The RBCs are completely lysed. The WBCs,
however, retain their original shape. The NRBC are de-nucleated and shrunken. The specific dye contained in the reagent
stains the cytoplasm and nucleus of the WBCs quite strongly while the staining of the NRBC is comparatively weak.

Fig. 22 The principle for NRBC detection in the XE-2100 - the detection method-
The reaction with the new reagent is explained in Fig. 21. The cell stream is focused hydrodynamically in the flow cell.
Individual cells are activated in the flow cell by a semi-conductor laser and produce forward scatter and fluorescence. The
different staining characteristics between WBCs and NRBCs achieves clear discrimination of those cells and makes NRBC
counting possible.

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 Sysmex Journal International Vol.9 No.1 (1999)
reticulocytes and PLTs is detected by scattering of light.
Fluorescence intensity (RNA/DNA concentration) and
scattered light (cell size) are expressed in a scattergram,
in which mature RBCs, reticulocytes, PLTs and WBCs
are discriminated (Fig. 11).
The fluorescent dye in the instruments stains RNA and
DNA simultaneously. The concentration of DNA in
WBC exceeds that of RNA in reticulocytes, therefore the
fluorescence intensity of the WBC far exceeds that of the
reticulocytes.
Large PLTs and RBCs including reticulocytes are dis-
criminated by curve threshold set by a computer algo-
rithm rather than straight line. Moreover, as described
above, in the light scattering method, the discrimination
capability between large PLTs and RBCs is superior to
the electric impedance method. With normal samples,
discrimination and enumeration of reticulocyte are done
perfectly.
In some abnormal cases with poor discrimination, the
instrument gives a flag as an alarm and suggests re-
checking by an alternative method.
In these analyzers, reticulocytes are differentiated into
three categories, the most immature (youngest) reticulo-
cyte, moderately mature reticulocytes and most mature
reticulocyte. This classification provides sensitive para-
meters to indicate bone marrow activity.
Hemoglobin measurement
Hemoglobin concentration measurement is regarded as
one of the most important parameters produced by hema-
tology analyzers. In Sysmex hematology analyzers,
RBCs are first hemolysed, and the hemoglobin released
is converted to a single stable form such as oxyhemoglo-
bin, cyanmethemoglobin or SLS hemoglobin.
Those stabilized hemoglobins are measured by spec-
trophotometric methods. The details are described in a
separate article in this issue.
CONCLUSION
This article has described the measurement principles
employed in Sysmex hematology analyzers. Since the
time we released our first analyzer to the market, we have
continued to develop innovative technologies for our
products in order to produce reliable measurement
results. We are, however, not satisfied that even our
newest products are the perfect or ultimate machine. We
in order promise to continue basic investigation and
development in order to provide improved and/or innova-
tive products.
The rate of development of technical innovations contin-
ues unabated. We, Sysmex Corporation would like to
continue to provide customer oriented products by listen-
ing to customer opinions.
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References
1) Coulter WH : Means for counting particles suspended in a fluid.
USP No. 2656508.
2) Tatsumi N, et al. : Principle of blood cell counter -development of
electric impedance method. Sysmex J Int, 9 (1) : 8-20, 1999.
3) Okada T : Development and problem of automated hematology
analyzer. Sysmex J Int, 9 (1) : 52-57, 1999.
4) Bader, et al. : Theory of coincidence counts and simple practical
methods of coincidence count correction for optical and resistive
pulse particle counters. Rev Sci Instrum, 42 (10) : 1407, 1972.
5) Helleman PW : Investigation on the fundamental assumptions
underlying coincidence loss theories. Chapter 8 of the Coulter
Electronic Particle Counter, De Bilt Holland, 1972.
6) Thom R, et al. :Die elektronische Volumenbestimmung von
Blutkörperchen und ihre Fehlerquellen. Z Ges Exp Med, 151 :
331-349, 1969.
7) Kachel V : Sizing of cells by the electrical resistance pulse tech-
nique. Chapter 7 of Cell Analysis 1, Plenum Press, New York,
1982.
8) Thom R, Kachel V : Fortschritte fuer die elektronishe Grössenbes-
timmung von Blutkörperchen. Blut, 21 : 48-50, 1970.
9) Tatsumi N, et al. : Evaluation of a new blood cell counter with
sheath flow system. Cytometry, 6 : 395-400, 1985.
10) Rowan RM : Blood cell volume analysis. pp.16-17, Albert Clark
and Company Ltd., London, 1983.
11) Okada T : Performance of automated hematology analyzers
E Series. Sysmex J, 6 (3) : 148-157, 1 9 8 3 .
12) Thom R : Einfluß von Reagenzien und Materialien auf hämatolo-
gische Meßergebnisse und Probleme der Standardisierung mit
Kalibriermaterial. Lab Med, 9 : 98-108, 1985
13) Fujimoto K : A basic theory in clinical laboratory -white blood
cell differentiation by hematology analyzers-. Kensa to gijutsu,
7 (12) : 1472-1476, 1989.
14) Mullaney PF, Dean PN : The small angle light scattering of bio-
logical cells. Biophysical J, 10:764-772, 1970.
15) Groner W : Optical technology in blood cell counting. Sysmex J
Int, 9 (1) : 21-30, 1999.
16) Kojima K, et al. : An automated optoelectronic reticulocyte counter.
Am J Clin Pathol, 92 : 57-61, 1989.
17) Takami T, et al. : Outline of automated reticulocyte analyzer R-
1000, Sysmex J, 11 (Suppl.II) : 18-26, 1988.
18) TOA Medical Electronics Co., Ltd. : Evaluation of the automated
hematology analyzer SF-3000. Sysmex J Int, 6 : 4-15, 1996.
19) Inoue H : Overview of automated hematology analyzer XE-2100.
Sysmex J Int, 9 (1) : 58-64, 1999.
20) Hamaguchi Y, et al. : A reagent for three part differenticial in
WBCs. Sysmex J, 8 (11) : 12-22, 1985. (in Japanese)
21) Yamane T, et al. : Determination of hematopoietic stem cells in
peripheral blood by automated hematology analyzer, SE-9000.
Sysmex J Int, 7 : 57-62, 1997.
22) TOA Medical Electronics Co., Ltd. : Introduction of the hematopoi-
etic progenitor cell (HPC) screening module. Sysmex J, 20 (2) :
142-149, 1997.
23) Chang CC, Kass L : Clinical significance of immature reticulocyte
fraction determined by automated reticulocyte counting. Am J Clin
Pathol, 108 : 69-73, 1997.
24) Watanabe K, et al. : Reticulocyte maturity as an indicator for esti-
mating qualitative abnormality of erythropoiesis. J Clin Pathol,
47 : 736-739, 1994.
25) Oshiro I, et al. : New method for hemoglobin determination by using
sodium lauryl sulfate (SLS). Clin Biochem, 15 (1) : 83-88, 1982.
26) Hamaguchi Y : Overview of the principle of Sysmex's hemoglobino-
metry. Sysmex J Int, 9 (1) : 45-51, 1999.