Electronic Impedance

The impedance principle of cell counting is based on the detection and measurement of
changes in electrical resistance produced by cells as they traverse a small aperture. Cells
suspended in an electrically conductive diluent such as saline are pulled through an aperture
(orifice) in a glass tube. In the counting chamber, or transducer assembly, low-frequency
electrical current is applied between an external electrode (suspended in the cell dilution) and
an internal electrode (housed inside the aperture tube). Electrical resistance between the two
electrodes, or impedance in the current, occurs as the cells pass through the sensing aperture,
causing voltage pulses that are measurable. Oscilloscope screens on some instruments display
the pulses that are generated by the cells as they interrupt the current. The number of pulses is
proportional to the number of cells counted. The size of the voltage pulse is directly proportional
to the size (volume) of the cell, which allows discrimination and counting of cells of specific sizes
through the use of threshold circuits. Pulses are collected and sorted (channelized) according to
their amplitude by pulse height analyzers.
The data are plotted on a frequency distribution graph, or size distribution histogram, with
relative number on the y-axis and size (channel number equivalent to a specific size) on the xaxis.
The histogram produced depicts the volume distribution of the cells counted. Size thresholds
separate the cell populations on the histogram, and the count is the cells enumerated between
the lower and upper set thresholds for each population. Size distribution histograms may be
used for the evaluation of one cell population or subgroups within a population. The use of
proprietary lytic reagents to control shrinkage and lysis of specific cell types, as in the older
Coulter S-Plus IV, STKR, and Sysmex E-5000 models, allows separation and quantitation of
white blood cells (WBCs) into three populations (lymphocytes, mononuclear cells, and
granulocytes) for the three-part differential on one size distribution histogram.
Several factors may affect size or volume measurements in impedance or volume displacement
instruments. Aperture diameter is crucial, and the red blood cell (RBC)/platelet aperture is
smaller than the WBC aperture to increase platelet counting sensitivity. On earlier systems,
protein buildup occurred, decreasing the diameter of the orifice, slowing the flow of cells, and
increasing their relative electrical resistance. Protein buildup results in lower cell counts, which
result in falsely elevated cell volumes. Impedance instruments once required frequent manual
aperture cleaning, but current instruments incorporate burn circuits or other internal cleaning
systems to prevent or slow protein buildup. Carryover of cells from one sample to the next also
is minimized by these internal cleaning systems. Coincident passage of more than one cell at a
time through the orifice causes artificially large pulses, which results in falsely increased cell
volumes and falsely decreased cell counts. This count reduction, or coincident passage loss, is
statistically predictable (and mathematically correctable) because of its direct relationship to cell
concentration and the size or effective volume of the aperture. Coincidence correction typically
is completed by the analyzer computer before final printout of cell counts from the instrument.
Other factors affecting pulse height include orientation of the cell in the center of the aperture
and deformability of the RBC, which may be altered by decreased hemoglobin (Hb) content.
Recirculation of cells back into the sensing zone creates erroneous pulses and falsely elevates
cell counts. A backwash or sweepflow mechanism has been added to prevent recirculation of
cells back into the sensing zone, and anomalously shaped pulses are edited out electronically.
The use of hydrodynamic focusing avoids many of the potential problems inherent in a rigid
aperture system. The sample stream is surrounded by a sheath fluid as it passes through the
central axis of the aperture.

Laminar flow allows the central sample stream to narrow sufficiently to separate and align the
cells into single file for passage through the sensing zone.The outer sheath fluid minimizes
protein buildup and plugs, eliminates recirculation of cells back into the sensing zone with
generation of spurious pulses, and reduces pulse height irregularity because off-center cell
passage is prevented and better resolution of the blood cells is obtained. Coincident passage
loss also is reduced because blood cells line up one after another in the direction of the flow.
Laminar flow and hydrodynamic focusing are discussed further in Chapter 33.
Radiofrequency
Low-voltage DC impedance, as described previously, may be used in conjunction with RF
resistance, or resistance to a highvoltage electromagnetic current flowing between both
electrodes simultaneously. Although the total volume of the cell is proportional to the change in
DC, the cell interior density (e.g., nuclear volume) is proportional to pulse size or change in the
RF signal. Conductivity, as measured by this high-frequency electromagnetic probe, is
attenuated by nucleus-to-cytoplasm ratio, nuclear density, and cytoplasmic granulation. DC and
RF voltage changes may be detected simultaneously and separated by two different pulse
processing circuitsTwo different cell properties, such as impedance and conductivity, can be
plotted against each other to create a twodimensional distribution cytogram or scatterplot .Such
plots display the cell populations as clusters, with the number of dots in each cluster
representing the concentration of that cell type. Computer cluster analysis can determine
absolute counts for specific cell populations. The use of multiple methods by a given instrument
for the determination of at least two cell properties allows the separation of WBCs into a fivepart differential (neutrophils, lymphocytes, monocytes, eosinophils,and basophils). DC and RF
detection are two methods used by the Sysmex analyzers to perform WBC differentials.
Optical Scatter
Optical scatter may be used as the primary methodology or in combination with other methods.
In optical scatter systems (flow cytometers), a hydrodynamically focused sample stream is
directed through a quartz flow cell past a focused light source. The light source is generally a
tungstenhalogen lamp or a helium-neon laser (light amplification by stimulated emission of
radiation). Laser light, termed monochromatic light because it is emitted at a single wavelength,
differs from brightfield light in its intensity, its coherence (i.e., it travels in phase), and its low
divergence or spread. These characteristics allow for the detection of interference in the laser
beam and enable enumeration and differentiation of cell types.
Optical scatter may be used to study RBCs, WBCs, and platelets. As the cells pass through the
sensing zone and interrupt the beam, light is scattered in all directions. Light scatter results from
the interaction between the processes of absorption, diffraction (bending around corners or the
surface of a cell), refraction (bending because of a change in speed), and reflection (backward
scatter of rays caused by an obstruction).
The detection of scattered rays and their conversion into electrical signals is accomplished by
photodetectors (photodiodes and photomultiplier tubes) at specific angles. Lenses fitted with
blocker bars to prevent nonscattered light from entering the detector are used to collect the
scattered light.
A series of filters and mirrors separate the varying wavelengths and present them to the
photodetectors. Photodiodes convert light photons to electronic signals proportional in
magnitude to the amount of light collected. Photomultiplier tubes are used to collect the weaker

signals produced at a 90-degree angle and multiply the photoelectrons into stronger, useful
signals. Analogue-todigital converters change the electronic pulses to digital signals for
computer analysis.
Forward-angle light scatter (0 degrees) correlates with cell volume or size, primarily because of
diffraction of light. Orthogonal light scatter (90 degrees), or side scatter, result from refraction
and reflection of light from larger structures inside the cell and correlates with degree of internal
complexity.
Forward low-angle scatter (2 to 3 degrees) and forward high-angle scatter (5 to 15 degrees)
also correlate with cell volume and refractive index or with internal complexity.
Differential scatter is the combination of this low-angle and high-angle forward light scatter and
is primarily used on Siemens systems for cellular analysis. The angles of light scatter measured
by the different flow cytometers are manufacturer and method specific.
Scatter properties at different angles may be plotted against each other to generate twodimensional cytograms or scatterplots, as on the Abbott CELL-DYN instruments. Optical scatter
may also be plotted against absorption, as on the Siemens systems, or against volume, as on
the larger Coulter systems. Computer cluster analysis of the cytograms may yield quantitative
and qualitative information.
RESEARCHER: TAGUMASI, NERREN L.
REFERENCE:
Rodak, B. F., Fritsma, G. A., & Keohane, E. M. (2012). Hematology: Clinical Principles and Applications
4th
Edition. Saunder Elsevier Inc. pp..598 -601