Blood cell counters

• Definition: A blood cell counter is a device that measures the number

and size of red blood cells, white blood cells, platelets, and
hemoglobin in your blood. The results of a blood cell count are used
to determine the risk of disease
• A complete blood count (CBC) is a blood test that measures amounts
and sizes of your red blood cells, hemoglobin, white blood cells and
platelets.
Types of blood cell counters
• There are several types of blood cell counters, including:
• Hematology analyzers: Automated counters that use electrical
impedance and light scatter to measure blood cell concentrations.
They are often used in healthcare settings to perform complete blood
counts (CBCs).
• Coulter counters: Use impedance to measure changes in electrical
resistance as cells pass through a small aperture
• Flow cytometers: Analyze individual cells in a fluid stream to provide
information about cell size, complexity, and fluorescence.
• Manual cell counting chambers: Glass slides or specialized chambers
that allow you to count cells under a microscope manually.
• Automated cell imaging systems: Use digital imaging and computer
vision algorithms to count and analyze cells automatically.
• Handheld cell counters: Portable devices for quick cell counting in
field settings
Coulter counter
• 1. scheme of coulter counter, 2. Coulter counter instrument
Coulter Principle
• During the Coulter Principle measurement, as a particle passes through the sensing zone
when the liquid is drawn from the container, a volume of the electrolyte equivalent to the
immersed volume of the particle is displaced from the sensing zone. This causes a short-
term change in the resistance across the aperture. This resistance change can be
measured either as a voltage or current pulse. By measuring the number of pulses and
their amplitudes, one can obtain information about the number of particles and the
volume of each individual particle.
• The number of pulses detected during measurement is the number of particles measured,
and the amplitude of the pulse is proportional to the volume of the particle. Because this
is a single-particle measurement process, it yields the highest resolution that any particle
characterization technique can achieve. The particle diameter can be determined at the
resolution of voltage or current measurement which can be very accurately using current
electronics technology. The distribution amplitude can be determined to the accuracy of a
single particle.
Hematology analyzer
Principle of Automated Haematology
Analyser
• The automated blood cell counting process is very fast and can process up to 60-80 blood samples
in an hour. There are three detector blocks in an automated haematology analyser. RBCs and
platelets are counted in the same block whereas, WBCs are counted in a separate block. All the
red cells in the blood directed towards the WBC counting block are lysed first using the
stromatolyser solution. This solution is composed of an organic quarternary ammonium salt (8.5
g/L) and sodium chloride (0.6 g/L). After lysis, only the nuclei of the WBCs along with a thin rim of
cytoplasm remains, which are counted and plotted in the WBC histogram.
• The size of the WBCs after lysis corresponds to the size of their nuclei; hence neutrophil is the
largest after lysis even though originally monocyte is the largest WBC. Cells are counted by
passing a dilute solution of the blood through an aperture across which an electrical current is
flowing. The passage of cells through the current changes the impedance between the terminals
(Coulter principle). The sizing and counting of blood cells is based on this measurable change in
the electrical impedance.
• Inside an automated haematology analyser, the EDTA anticoagulated blood is divided into three
channels and enters WBC detector block, RBC detector block and haemoglobin detector block.
Flow cytometers
Principle of Flow Cytometry
• The basic principle of flow cytometry is the passage of cells in single file in front of a laser so
they can be detected, counted and sorted. Cell components are fluorescently labelled and
then excited by the laser to emit light at varying wavelengths
• The fluorescence can then be measured to determine the amount and type of cells present in
a sample. Up to thousands of particles per second can be analysed as they pass through the
liquid stream. A beam of laser light is directed at a hydrodynamically-focused stream of fluid
that carries the cells. Several detectors are carefully placed around the stream, at the point
where the fluid passes through the light beam. One of these detectors is in line with the light
beam and is used to measure Forward Scatter or FSC. Another detector is placed
perpendicular to the stream and is used to measure Side Scatter (SSC). Since fluorescent labels
are used to detect the different cells or components, fluorescent detectors are also in place.
The suspended particles or cells, which may range in size from 0.2 to 150μm, pass through the
beam of light and scatter the light beams. The fluorescently labelled cell components are
excited by the laser and emit light at a longer wavelength than the light source.
Manual cell counting chambers
Hemocytometer Principle
• The hemocytometer, an essential tool in cell counting and measuring cell density, offers more than
just carefully etched lines delimiting nine 1 x 1 mm (1mm2) sectors. To provide even greater
accuracy, each sector is further divided into 16 or 25 smaller squares of equal size, depending on
the specific version of the hemocytometer being used. These smaller squares facilitate precise
counting by creating a grid pattern that ensures consistent distribution of cells for analysis.
• During the counting process, a specially designed slide is positioned on top of the chamber, leaving
a narrow 0.1mm gap between the surface of the chamber and the bottom of the slide. This
deliberate spacing serves a crucial purpose: it establishes a known volume for analysis. By
considering the dimensions of the chamber and the gap, a precise volume of 0.1 mm3 (equivalent
to 1E-4 mL) is obtained.
• When cells are introduced into the chamber, they naturally occupy the available space within the
gap. Consequently, any visible cells within each of the 1mm2 sectors are representative of the cell
population present in the 1E-4 mL volume. This ingenious design enables researchers and scientists
to extrapolate cell density at a small scale, providing a quantifiable measure of cells per milliliter
(cells/mL).
Handheld cell counters
The Scepter 3.0 Handheld
Automated Cell Counter principle
• The use of a hemocytometer can be time consuming, susceptible to subjective
judgements by the operator and some cell types, such as those that form clusters, are
particularly difficult to count using this method. Cell counting equipment is available
offering alternative cell quantification methods including the Scepter™ Cell Counter.
The Muse® Cell Analyzer enables flow cytometry-based assessment of cell count and
viability. The Scepter™ Cell Counter is a portable, handheld cell counter that measures
volume using the Coulter Principle. It can quantify cells based on size and will
discriminate larger cells from smaller debris, unlike vision-based techniques, which
rely on object recognition software and cannot reliably detect small cells. The
Scepter™ cell counter detects every cell and displays the population as a histogram of
cell size distributions. From the histogram, count all the cells or use the gating function
to count a chosen subpopulation. By monitoring changes in your histogram, you can
gain insight into the health and quality of your cell culture from one experiment to the
next.
Automated cell imaging systems
Definition & Core Principles
• Live-cell imaging enables scientists to observe and document the behavior of living
cells over time, providing a dynamic perspective on cellular processes as they unfold
naturally. This method elevates the study of cells beyond traditional microscopy’s
capabilities.
• Live-cell imaging integrates advanced microscopy techniques, such as fluorescence
and confocal microscopy, with the capacity to maintain cells in a life-supporting state
and elevates the study of cells beyond traditional microscopy’s capabilities. This
combination allows for the real-time visualization of cellular processes, ensuring
observations are as close to the natural state of the cells as possible.
• In addition to combining microscopy techniques, using fluorescent proteins and dyes
to highlight specific components helps unveil the intricacies of molecules and
structures within the cellular landscape, offering unprecedented insights into cellular
dynamics.