AUTOMATED BLOOD CELL
ANALYSIS
Objectives
To explain the different principles of automated blood cell
counting and analysis: Electronic impedance,
Radiofrequency, and Optical scatter;
To describe how the general principles are implemented on
different instruments: Beckman Coulter instrumentation,
Sysmex instrumentation, Abbott instrumentation, and
Siemen’s Healthcare Diagnostics instrumentation
To identify the parameters directly measured on the four
analyzers discussed;
To identify sources of error in automated cell counting and
determine appropriate corrective action.
INTRODUCTION
Automated blood cell analyzers typically provide eight (8)
standard hematology parameters (CBC), plus a three-part,
five-part, or six-part differential leukocyte count in less than 1
minute on 200 mL or less of whole blood.
Automation allows more efficient workload management and
more timely diagnosis and treatment of disease.
GENERAL PRINCIPLES OF
AUTOMATED BLOOD CELL ANALYSIS
Most automated blood cell analyzers rely on two basic
principles of operation:
- Electronic Impedance (resistance)
- Optical scatter
Electronic Impedance
The principle of cell counting is based on the detection and
measurement of changes of electrical resistance produced by
cells as they traverse a small aperture.
A voltage pulse of short duration is produced for each cell that
passes through the aperture.
The magnitude of voltage is proportional to the cell volume or size,
and the number of voltage pulses is proportional to the frequency of
particles passing through the aperture.
RBCs and WBCs are counted in duplicate or triplicate. Each of the
duplicated counts must agree within a standardized range of
deviation from each other.
The MCV is often determined directly from the voltage-pulse
heights from the RBC count or histogram curve.
The Hb of the sample is obtained spectrophotometrically from the
WBC dilution.
Platelets are counted in duplicate or triplicate in the RBC aperture
bath. Particles ranging from 2 to 20 fL [1 femtoliter (fL)= 10−15
L= 1 cubic micrometer (mm3)] in the RBC bath are sorted as
platelets and plotted as a platelet histogram.
RBC indices are computed parameters commonly obtained from
automated cell counters.
(a) The HCT is computed from the RBC count and the MCV and
calculated from the following formula:
HCT%= [RBC × MCV] ÷ 10
The MCH is computed from the MCV and the MCHC and
calculated from the following formula (note that 1 μg = 1 picogram
[pg]):
MCH pg= [MCV × MCHC] ÷ 100
The MCHC is computed from the Hb and HCT and calculated from
the following formula:
MCHC%= [Hb ÷ HCT] × 100
Factors that may affect volume
measurements in impedance or volume
displacement instruments:
Aperture diameter
Protein buildup
Carryover over of cells from one specimen to the next
Coincident passage of more than one cell at a time through
the orifice
Orientation of the cell in the center of the aperture and
deformability of the RBC, which may be altered by decreased
hemoglobin content
Recirculation of cells back into the sensing zone
Radiofrequency
While electronic impedance or low-voltage DC impedance is
used for the measurement of the total cell volume, which is
proportional to the change in DC, the radiofrequency or the
high-frequency electromagnetic current is used for
measurement of the cell interior density, which is proportional
to pulse height 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.
Optical Scatter
Also known as Flow cytometers.
In this system, a hydrodynamically focused sample stream is
directed through a quartz flow cell past a focused light source.
The light source is generally a tungsten-halogen 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 bright-field light in its
intensity, its coherence (i.e., it travels in phase), and its low
divergence or spread.
Principle:
As the cells pass through the sensing zone and interrupt the
beam, light is scattered in all directions. Light scatter result
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.
Analog-to-digital converters change the electronic pulses to
digital signals for computer analysis.
Forward-angle light scatter (0 degrees) correlates with cell
volume, primarily because of diffraction of light.
Orthogonal light scatter (90 degrees), or side scatter, results
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 two-dimensional 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
Beckman Coulter systems.
Computer cluster analysis of the cytograms may yield
quantitative and qualitative information.
Principal Instruments
Beckman Coulter Instrumentation
Sysmex Instrumentation
Abbott Instrumentation
Siemens Healthcare Diagnostics Instrumentation
Overview
These Hematology analyzers have some common basic
components:
- Hydraulics system
- Pneumatics
- Electrical system
Beckman Coulter Instrumentation
Characteristics:
Has two measurement channels in the hydraulic system for
determining the hemogram data.
The RBC and WBC counts and hemoglobin are considered to
be measured directly.
The aspirated whole-blood sample is divided into two
aliquots, and each is mixed with an isotonic diluent.
The first dilution is delivered to the RBC aperture chamber,
and the second is delivered to the WBC aperture chamber.
In the RBC chamber, RBCs and platelets are counted and
discriminated by electrical impedance as the cells are pulled
through each of three sensing apertures.
Particles 2 to 20 fL are counted as platelets, and particles
greater than 36 fL are counted as RBCs.
Particles between ~35 and 90 fL are considered lymphocytes,
particles between 90 and 160 fL are considered
mononuclears, and particles between 160 and 450 fL are
considered granulocytes.
When cell populations overlap or a distinct separation of
populations does not exist, a region alarm (R flag) may be
triggered that indicates the area of interference on the
volume-distribution histogram.
An R1 flag represents excess signals at the lower threshold
region of the WBC histogram and a questionable WBC count.
This interference is visualized as a high takeoff of the curve
and may indicate the presence of nucleated RBCs, clumped
platelets, unlysed RBCs, or electronic noise.
Potential Errors from Cell Counting
Instrument
Instrumental errors (Errors in a single Parameter Machine)
Errors caused by the nature of the specimen
Instrumental Errors
Negative error: - excessive lysing of RBCs
Positive error: - aperture plugs – most common
- Extraneous electrical pulses from improperly
grounded equipment
- Bubbles caused by too vigorous shaking
Either Positive or Negative: - Improper settings of aperture
current
Errors caused by the nature of the
specimen
Giant platelets may be counted as RBCs or WBCs
Fragments of leukocytes cytoplasm, such as may be present
during leukemia therapy, may be counted as platelets or
RBCs.
Increased numbers of schistocytes may make accurate
erythrocyte and platelet count impossible
Agglutination of erythrocytes, leukocytes or platelets will
cause false negative results for each of the respective cell
counts
Agglutinated red cells or platelets may also cause false
positive leukocyte counts
Platelet satellitism will result in falsely low platelet count
Some abnormal RBCs tend to resist lysis, which mat result in
high WBC counts.
Ex. Sickle cells, extremely hypochromic cells and target cells.
The problem can be solved by delaying 2 to 3 minutes
between the addition of the lysing reagent and counting.
(Steininger)
