CE UPDATE—INSTRUMENTATION
III
Margaret Chapman, MT(ASCP)SH
Hematology Analyzers
Offer New Technology
and User-Friendliness
ABSTRACT During the last quarter century, blood-cell
analysis has progressed from the use of labor-intensive
manual procedures to the use of highly automated
instruments capable of measuring many disease-associated
parameters. Modern hematology analyzers incorporate
nontraditional flow cytometry, robotics, and expert system
technologies. This article reviews the principles, applications,
and user-friendliness of these new systems.
This is the third article in a 4-part continuing education series on instrumentation. On
completion of this article the reader will be able to describe the analytical principles
used in hematology analyzers and discuss the scope and use of various hematology
analyzers currently available.
Reprint requests
to Ms Chapman,
2 DaVinci, Lake
Oswego, OR 97035;
or e-mail:
Nampachmgt@
aol.com
Hematology instruments range from handheld
photo-optical devices to sophisticated, highly
automated analyzers. Complexity varies with the
types of tests performed, the degree of automation, and the required computer connectivity.
Laboratory professionals typically select instruments on the basis of patient population and
scope of laboratory service, current and anticipated. Other considerations include operator
qualifications, service, reliability, precision, accuracy, regulatory compliance capability, and budgetary constraints. Manufacturers, in turn,
carefully research these requirements as they target specific analyzers to various laboratories.
Applications and Laboratory Impact
Small hematology analyzers, typically priced at
<$50,000, are found in small clinics and physician
offices. They are also used in hospital and commercial laboratories as backups for larger analyzers. Their limited test menu consists of a CBC
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with directly measured WBC and RBC counts,
hemoglobin level, mean cell volume (MCV), and
platelet count. The systems also calculate mean
corpuscular hemoglobin (MCH) mass, mean corpuscular hemoglobin concentration (MCHC),
and red cell distribution width (RDW) from the
measured RBC count, hemoglobin level, and
MCV. Some may also offer a partial or 5-part
automated differential WBC count.
The strengths of these small analyzers lie in
their reliability, high-quality results, simple operation, speed (especially in point-of-care testing),
low operating expense, and small sample requirement (some require only 12 µL). Throughput,
however, usually does not exceed 30 tests per hour
because the instruments lack cap-piercing and bar
code capability. They must be hand-fed, thus
introducing the necessity of extra safety precautions such as using shields and biohazard wipes.
Midsize hematology systems are used in large
clinics, small hospitals, and large hospitals or reference laboratories. They cost from $50,000 to
$100,000 and offer the same core test menu as
their smaller counterparts. An automated 5-part
differential WBC count is standard, and test
throughput ranges from 50 to 100 tests per hour.
Some midsize analyzers offer reticulocyte counts
and, by flow cytometry, the CD4/CD8 count, a
ratio of “cluster differentiation” (CD) glycoproteins useful in checking for organ rejection after
transplantation and evaluating the relative condition of patients with HIV. Midsized instruments
interface with a host computer, read bar codes,
and pierce caps for hands-off operation. Many
come with quality control software.
A large hematology analyzer costs from
$100,000 to $350,000. If included as part of an
automated laboratory transport system, the system
Principle of Operation
The principles on which hematology instrument
operations are based vary widely, especially in
large instruments. The following describe methods frequently used in hematology testing.
Determination of Hemoglobin Levels
Most instruments measure hemoglobin concentration by a modified cyanmethemoglobin procedure, in which segmented whole blood is added to
potassium cyanide and potassium ferricyanide.
The ferricyanide converts the hemoglobin iron
from the ferrous state (Fe++) to the ferric state
(Fe+++), thus forming methemoglobin that combines with potassium cyanide to form the stable
cyanmethemoglobin. The color intensity, measured
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Scientific Communications
Designed for people who do not have a laboratory
background, small hematology analyzers are easy
to learn and operate. Midsize and large systems,
though designed to operate in a more complex
environment, are also user-friendly, with automated startup, shutdown, and quality control
analysis. Maintenance procedures are simple, but
troubleshooting may be complex. Sophisticated
software displays error messages that localize the
problem and direct the operator to the trouble
area. Onboard tutorials and help files also aid in
teaching and troubleshooting.
Hematology instrument operations consist of
computation, CBC analysis, and other analyses
such as differential WBC counts. On many midsize and large analyzers, the onboard computer
does computation and analysis, which, in newer
analyzers, looks and functions like a personal
computer. The computer controls all hardware
and software operations and contains a database
of stored patient data and quality control results.
A working knowledge of personal computers is
4
Instrument Complexity
and User-Friendliness
helpful as many systems now function in a
menu-driven Windows environment. The main
menu of the instrument is typically segmented
into the patient run and results environment,
the quality control analysis and results environment, and the hardware and software utilities
section with troubleshooting logs and computer
setup information.
Modern hematology analyzers use a combination of methods to do differential WBC counts
and other tests. Using an impedance-based
method, the analysis area of the instrument
counts RBCs, WBCs, and platelets as they pass
through an aperture. The improvements in
valves, tubing, and apertures, along with automated maintenance procedures, have greatly
reduced the likelihood of problems with pinched,
plugged, or leaky tubes. The use of flow cells and
lasers has increased the sensitivity and specificity
for separating WBC populations. With these
improvements, however, comes more fluctuation,
instability, and difficulty in troubleshooting. Test Your
Trained service personnel must usually resolve Knowledge!
Look for the CE
problems with the flow cell and laser.
On each workshift, at least 2 levels (high and Update exam on
Instrumentation (002)
low) of control should be run for each directly in the April issue of
measured CBC parameter. Traditionally, a nor- Laboratory Medicine.
mal-level control is also tested. Operators should Participants will earn
understand quality control procedures as well as 4 CMLE credit hours.
the linearity and other limitations of the instrument. They should also do validation procedures
when tolerance limits are exceeded and, after
replacement of a major component or every 6
months, check the calibration to verify accuracy
for directly measured parameters.
Section
costs even more. Built for large hospital or reference laboratories running up to thousands of
tests per day, these instruments, which automate
every aspect of hematology testing, do >100
tests per hour. They interface with a laboratory
information system and some can network with
one another.
Some large instruments have “expert” systems,
computer-based programs to automate the
labor-intensive application of complex review
criteria. Expert systems can be programmed to
initiate reflex testing, rerun specimens, or make
and stain slides according to user-defined rules.
Other analyzers, instead of incorporating expert
systems, link to an external system capable of
rule-based analysis.
Hematology profiles from both midsize and
large systems now include new tests, such as reticulocyte counts and flow cytometric CD4/CD8
counts, and a method for quantifying CD64 is
being developed. Enhancements in flow cytometry make it possible for these new instruments to
also count immature (blast) cells and nucleated
RBCs (NRBCs).
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Fig 1. Hydrodynamic
focusing. Cells are
hydrodynamically
focused through a
flow cell. Courtesy
Beckman Coulter,
Miami.
count.2 The MCH and MCHC, however, are calculated from the direct measurements with the
following formulas:
MCH = hemoglobin level 3 10/RBC count
MCHC = hemoglobin level 3 100/hematocrit
Aperture
Sheath
Fluid
Some analyzers calculate additional parameters
to screen for various anemias.
Differential WBC Measurements
in a separate cuvette, is read spectrophotometrically at 540 nm and is proportional to the concentration of hemoglobin. All forms of hemoglobin
(except sulfhemoglobin) are measured by this
method.1 Some instruments use a modified cyanmethemoglobin procedure, and others use
cyanide-free colorimetric determinations.
Directly Measured CBC Parameters
Although methods for directly determining RBC,
WBC, and platelet counts differ, the 50-year-old
impedance procedure continues to be the most
frequently used, either alone or in combination
with others. In this method, cells suspended in
isotonic saline solution are forced through the
center of an aperture in single file. Each cell causes
a momentary decrease in electrical current that, in
turn, creates a pulse, the size (amplitude) of which
is proportional to the size of the responsible cell.
The number of pulses generated is proportional to
the number of cells (Fig 1).
Their impedance directly determines the RBC,
WBC, and platelet counts. The MCV is measured
by electronic cell counters, usually by dividing the
summation of the cell volumes by the RBC
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Many small hematology instruments use the
impedance method for partial or 5-part differential WBC separation because it is inexpensive
and generally reliable.3 Manufacturers of higherend analyzers, however, continually work to find
better ways to quantify differential WBC counts.
They have reduced the need to manually verify
abnormal or flagged patient results by combining different methods to maximize sensitivity
and specificity. This has improved the accuracy
in counting low-incidence cells such as NRBCs
and various immature cells, thus expanding the
list of tests done by hematology analyzers. The
development of other tests such as the reticulocyte, CD4, CD8, and CD64 counts will obscure
the line between the immunology and hematology testing environments.
Current hematology instruments combine
laser technology, impedance, radio frequency,
direct current, optimized temperatures and volumes, and staining in various ways to maximize
sensitivity and specificity for the automated differential WBC count. For example, Beckman Coulter
(Miami) instruments use VCS technology, in
which volume (V), conductivity (C), and laser
light scattering (S) are simultaneously measured
on each cell that passes through the flow cell. The
V measures cell volume and number by impedance (Fig 2), the C measures the nuclear size and
density of each cell (Fig 3) with high-frequency
current, and the scattering (S) from a laser source
(Fig 4) measures internal structure, granularity,
and surface characteristics of cells as well as provides information on the shape and structure of
individual cells.4,5
The latest generation of Beckman Coulter analyzers uses “IntelliKinetics Management” to ensure
consistency in reagent reaction temperature,
exposure time, and delivery volumes. Enhancements in instrument electronics such as improved
Scientific Communications
Fig 4. Laser light scatter. A laser light source measures
internal structure, granularity, and surface characteristics of the cells. Courtesy Beckman Coulter, Miami.
4
signal-to-noise ratio combine with IntelliKinetics to
provide better data signals for instrument algorithms
to analyze, increasing the sensitivity for flagging
abnormal results. Analysis occurs under controlled
conditions.6,7 These analyzers count reticulocytes
and can do semiautomated CD4/CD8 counts.
Hematology instruments produced by Abbott
Diagnostics (Cell-Dyn, Abbott Park, IL) use both
impedance and laser optical technology.8 Placed
in single file by hydrodynamic focusing and laminar flow, cells stream through a flow cell for
counting and analysis. WBCs are counted and
classified by laser light-scattering data, using a
multi-angle polarized scatter and separation
technique. The angle of scattering is a function of
cell size, refractive index, nuclear–cytoplasmic
ratio, nuclear shape, and granularity. The most
recent Cell-Dyn instrument uses optical scatter
and fluorescence technology with an argon laser
to count NRBCs and to separate differential
WBC parameters. The latest Cel-Dyn offers reticulocyte, CD4/CD8, and CD64 counts.
Sysmex (Long Grove, IL) hematology analyzers
simultaneously measure both cell size and intracellular information by using the radio frequency/direct current (RF/DC) method. This
method uses direct current combined with radio
frequency current to count and classify cell types.
Biologic tissues have frequency characteristics that
vary in their electroconductivity. Choosing the
Fig 3. Conductivity measurement. A cell’s nuclear size
and density is measured by conductivity as it passes
through the flow cell. Courtesy Beckman Coulter, Miami.
Section
Fig 2. Volume measurement. A cell’s volume and
number is measured through impedance as it passes
through the flow cell. Courtesy Beckman Coulter, Miami.
appropriate frequency allows for detection of certain WBC features. Another channel detects
immature WBCs where a specific reagent lyses the
cytoplasm of normal WBCs, but leaves immature
WBCs intact to be counted.
The most recent Sysmex analyzers use semiconductor laser technology to perform automated
WBC differential counts. Low- and wide-angle
light scatter is measured in a special optical detector unit. The total WBC count and the basophil
count are determined in the optical WBC/BASO
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channel from a separate specimen dilution by the
use of a specific lyse reagent. An Adaptive Cluster
Analysis (ACAS) algorithm identifies the major
WBC populations: lymphocytes, monocytes,
eosinophils, neutrophils, and basophils.8,9
Instruments manufactured by Technicon
(Bayer Diagnostics, Tarrytown, NY) use peroxidase staining and nuclear density to perform the
WBC and differential analysis. The systems use a
combination of laser light-scatter and peroxidase-based cytochemistry and a second analytic
basophil-nuclear lobularity channel, where cells
undergo cytoplasmic stripping to bare nuclei.
Information from these 2 channels integrates
into a series of distribution cytograms, including
1 based on peroxidase cytochemistry and 1
based on data from the basophil-nuclear lobularity channel.10
The hematology instruments of ABX Diagnostics (Garden Grove, CA) offer a 5-part automated
differential WBC count by combining impedance,
light transmission, cytochemistry, and fluoro-flow
cytometry into their Double Hydrodynamic
Sequential System. This approach combines a primary focused flow to measure impedance and a
second focused flow to detect light. Upper-end
ABX analyzers count reticulocytes, and offer userselected or automatic rerun of CBC, 5-part differential WBC count, and reticulocyte count.
Conclusion
The hematology analyzers of today will continue
to expand traditional boundaries of hematology
testing by using state-of-the-art technology to
improve and automate the hematology profile.
With the wide range of analyzers available, highquality patient results, good sensitivity and specificity, reliability, service, safety, ease of operation
and training, and cost effectiveness should continue to be important considerations in selecting a
hematology system.l
References
1. Brown B. Hematology Principles and Procedures. 5th ed.
Philadelphia, PA: Lea and Febiger; 1988:80.
2. Williams WJ. Hematology. 4th ed. New York, NY: McGrawHill; 1990.
3. Turgeon M. Clinical Hematology Theory and Procedures.
Baltimore, MD: Williams & Wilkins; 1985: 311-315.
4. Coulter GenS [system operator’s guide]. Hialeah, FL: Coulter Corporation; 1996.
5. Garrity P, Walters G. Concepts in New Age Hematology.
Deerfield, IL: Baxter Diagnostics.
6. Coulter STKS [operator’s/reference manual]. Hialeah, FL:
Coulter Corporation; 1991.
7. Lee P, Kessler C. IntelliKinetics technology on the Coulter
GENS hematology analyzer. Technological Innovations in Laboratory Hematology. April 1996.
8. Cell-Dyn 3000 [system operator’s manual (Rev D)].
Mountain View, CA: Abbott Laboratories.
9. Yamane T, Katsuhiro T, Kensuke O, et al. Determination of
hematopoietic stem cells in peripheral blood by automated
hematology analyzer SE-9000. Sysmex Journal.
10. Swaim W. Laboratory and clinical evaluation of white
blood cell differential counts. Comparison of the Coulter VCS,
Technical H-1, and 800-cell manual method. Am J Clin Pathol.
1991;95:381-388.
Suggested Reading
Aller R, Sheridan B. Automated CBC and 5-part differential
hematology instruments. CAP Today. December 1998;12.
Koepke J, Lotspeich-Steininger S-M. Clinical Hematology Principles, Procedures, Correlations. Philadelphia, PA: JB Lippincott; 1992.
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