Feature

Hematology Analyzer: From

Workhorse to Thoroughbred
Ellen Sullivan
DOI: 10.1309/TMQ6T4CBCG408141

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 Feature
ematology analyzers are the workhorses of the
H
clinical laboratory. High-end, high-volume
analyzers deliver reliable red blood cell counts,
platelet counts, and 5-part differentials of
white blood cells, identifying lymphocytes,
monocytes, neutrophils, eosinophils, and ba-
sophils. Nucleated red blood cell counts and
immature granulocytes are emerging as sixth and seventh param-
eters. While electrical impedance still has a firm foothold in de-
termining the overall number and size of cells, flow cytometry
techniques have proven their worth in differentiating white
blood cells and identifying abnormal cells.
The increasing sophistication of flow techniques on the
analyzer raises some interesting questions. What will be the di-
viding line between the hematology analyzer and the flow cy-
tometer? Will new methods push some tests back to the
hematology laboratory? Will that drive up costs for routine tests?
Finally, are pathologists and laboratory professionals using the
advanced technology appropriately, not just to increase
efficiency, but also to improve medical-decision making?
Evolution of the Analyzer
The first automated cell counters came out in the 1950s
based on Coulter’s electrical impedance principle in which cells
pulled through an aperture break an electric circuit, indicating
both the presence of a cell and the size of the cell. “Those were
the ‘prehistoric’ analyzers that just did counts and indices—mean
corpuscular volume, mean corpuscular hemoglobin, mean cor-
puscular hemoglobin concentration,” said Kathleen Finnegan,
MS, MT(ASCP)SH, Clinical Assistant Professor and Chair of
the Clinical Laboratory Sciences Program at Stony Brook Univer-
sity, State University of New York. “If you’ve ever counted cells,
you know it’s very monotonous, and 2 technologists could never
do it the same. So it has taken out all of that variability.”
In the 1970s, automated platelet counters, 7-parameter
complete blood count (CBC) analyzers, and 3-part differential
leukocyte counters (for lymphocytes, monocytes, and granulo-
cytes) entered the market. For the first time, manual differentials
were not the only way to analyze white blood cells. In the
1980s, a single instrument could produce a 10-parameter CBC.
The 1990s brought further advancements in leukocyte differen-
tials with the use of flow-cell techniques based on either electri-
cal impedance or light scatter properties.
Manufacturers often seek to separate their instruments from
the pack by focusing on the particular package of technologies they
use to differentiate white blood cells or to count platelets, but
pathologists and laboratory professionals say it is hard to tell the
difference between most analyzers. “They all use similar technol-
ogy,” said Finnegan, who taught the 2005 ASCP continuing med-
ical laboratory education (CMLE) course, “Meeting Morphology
Challenges: When Automated Analyzers Need Your Help.” “It’s
just that they add bells and whistles to tweak it differently.”
For instance, one analyzer may determine leukocyte differ-
entials by inserting a fluorescent dye into the cell nucleus and
measuring how strongly it fluoresces. One may alter the perme-
ability of a cell and see how quickly it absorbs a dye. Another
may measure enzyme activity in a cell placed in a particular sub-
strate. Then there is the Volume Conductivity and Scatter
(VCS) method that analyzes cells in their “near-native” state.
“The new technologies are great,” said William G. Finn,
MD, FASCP, Associate Professor of Pathology, Director of
Hematopathology, and Associate Director of Clinical Pathology
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Laboratories at the University of Michigan. “They’re evolving
toward flow cell-based technologies, where cells are interrogated
cell by cell by optical systems that can then measure lots of
things we never used to measure. The problem is that manufac-
turers appropriately want to take their own proprietary step to
have their own identity. So they become very good at one thing,
and maybe not as good at another.”
Bruce H. Davis, MD, of the Maine Medical Center Re-
search Institute in Scarborough, ME, said all of the analyzers on
the market are generally reliable. “The differences are really
minor in terms of bells and whistles that may appeal to one per-
son or another,” he said. “The decision typically comes down to
cost or someone is in a buying group so the decision has already
been made.”
Ralph Taylor, Beckman Coulter’s Vice President of Product
Management for Cellular Analysis Clinical Business, said money
did not used to be an issue. “Now hematology is becoming a
very competitive market, and sometimes pricing (rather than best
available technology) does influence the acquisition of analyzers.”
The newest, high-volume analyzers can run from a single
instrument in the neighborhood of $75,000 to a multiple-in-
strument and automation system in excess of $200,000 dollars,
depending on the configuration. A complete laboratory automa-
tion system (that includes hematology, chemistry and immuno-
chemistry analyzers with automated input, output, and
refrigerated stockyards) can cost in the millions of dollars.
Application of Flow Principles
Barb Connell, MS, MT(ASCP)SH, Diagnostic Market
Manager for Sysmex America, agrees that analyzers are compara-
ble in certain areas, namely, total white and red blood cell
counts, hemoglobin counts, and platelet counts. “Yes, we count
those normal, routine type samples pretty much equally for the
most part,” she said. “But, are analyzers pretty much the same
across the board? No, absolutely not.” Some analyzers are prima-
rily based on impedance principles, some use flow principles to
perform laser light scatter, and others use fluorescent flow cy-
tometry, she said.
“We’re using some fluorescent dyes that stain the unique
characteristics of the cells to really separate them further,” Con-
nell said. “By doing that, we’ve been able to add additional pa-
rameters to our differential and RBCs (red blood cell counts),
including a nucleated RBC count that’s very sensitive on the low
end, and quantitative immature granulocyte counts.” New pa-
rameters for 2006 include Reticulated Hemoglobin Equivalent
(RET-He), which is used to monitor erythropoiesis and the im-
mature platelet fraction (IPF), she said.
Taylor said the leaps in technology are starting to slow as
the whole hematological platforms mature. Nonetheless, there
are still plenty of refinements happening. “Almost standard now
is a CBC with a nucleated red blood count,” he said. “That’s
one of the newest changes. Also, the precision and accuracy of
low platelet counts has improved considerably for most hematol-
ogy analyzers.”
Manufacturers also are improving linearity and reportable
range across systems. “Often you can be linear over a certain set
of counts but your reportable range can be higher,” said Taylor.
“The range is getting wide because it stops people having to do
dilutions to bring it back into the linear range. It’s part of the
effort to take out manual intervention.”
Another increasingly standard feature on the high-end ana-
lyzers is counting cells from body fluids, Taylor said. “The ability
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to perform a white count and red count on body fluid samples is
a labor-intensive activity in the lab,” he said. “It’s usually done
manually on a hemocytometer, and is time-consuming and re-
quires a skilled member of the laboratory staff to perform the
count, and labs are looking to reduce manually performed tests.”
The next big step in hematology is the extended differen-
tial, Taylor said. “While today’s analyzers flag for suspect blast
cells, suspect immature granulocytes and atypical lymphocytes,
now the next step will be to count those parameters instead of
just flagging. You will find a lot of analyzers today report them
in some shape or form on an RUO parameter—research use
only. But it’s not too far away. Most of the major companies are
working on it. That’s where their focus is going.”
Linda Sandhaus, MD, FASCP, Director, Core Laboratory
for Hematology, University Hospitals of Cleveland, Cleveland,
said today’s analyzers deliver good quantitative but not qualita-
tive information. “They’re good for counting particles and
telling you what general category of particle it is: a red cell, a
platelet, leukocyte,” she said. “They’re less reliable in what I
would call the qualitative determinations. So they can tell you
it’s a granulocyte, but they’re not going to be as precise in telling
you what stage of granulocyte maturation it is.”
Davis, who is also president of Trillium Diagnostics of
Scarborough, ME, said the next generation of analyzers will
better assess cell maturation, and that generation may be only
3 to 5 years away.
“My sense is that currently all the manufacturers have pretty
well refined the technology surrounding the Coulter
(impedance) principle and tweaked their software to the point
where they can extract about as much information as they can,”
Davis said. “What we’re going to see in the coming years, and
we’ve seen some of it to date, is the attempt to introduce some
new, novel technologies using particularly some cell functional as
well as cell surface protein expression that indicates the stage of
maturation or even the cell function.”
Where the Analyzer Ends and the
Cytometer Begins
Already some analyzers have started to integrate what many
people might consider standard flow cytometry assays, said Davis.
Antigen markers such as CD4 and CD8 are beginning to appear
on some analyzers. Connell says Sysmex’s instrument is “as close
to a flow cytometer as you can get without actually using antigen
markers to actually tag those cells. We’ve maybe reached our limit
with what we can do currently on the analyzer.”
Connell sees analyzers continuing to develop the technol-
ogy of flow cytometry, and flow cytometry becoming simpler.
“Eventually, there won’t be a difference between the flow
cytometer and the hematology analyzer, but it’s going to take
someone to see that advantage and continue to make advance-
ments in hematology,” she said
Davis, too, sees the eventual integration of what may have
been considered standard tests that went to flow cytometry com-
ing back to hematology. “For example, I will not be surprised if
some of the instruments will be able to do fetal red cell counting
to replace the Kleihauer Betke manual technique,” he said.
“That’s already done by flow cytometry in some labs, but I think
bringing it back into the hematology lab will gain wider accept-
ance and will probably finally bring that assay from being a hor-
rible assay from a precision perspective to something that’s a
little more in line with what we expect for diagnostic assays in
the 21st century.”
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Feature
The line between hematology analyzers and flow cytometers
will probably continue to shift for the foreseeable future as tech-
nologies or methodologies evolve, Davis predicted. “The reticu-
locyte count is a good example,” he said. “First it was manual,
then it was done on a flow cytometer, then it swung back to the
automated hematology instrument once the methodologies were
made in an automated way.”
Patricia K. Kotylo, MD, FASCP, of Pathology Associates,
Indianapolis, IN, thinks it would be possible to adapt some of
the simpler tests to the hematology analyzer. “Routine T-cell
subsets, maybe eventually a very obvious, forthright chronic or
acute leukemia where all the cells are uniform with a very clear
phenotypic profile you could do as well,” she said. “If they can
accurately identify where cells of interest are using scatter charac-
teristics, you could adapt something to a CBC analyzer. Some
more problematic cases that have mixed cell populations or re-
ally small populations of cells with unusual or more aberrant
phenotypic profiles might be more difficult.”
Now hematology is becoming a very
competitive market, and sometimes pricing
(rather than best available technology) does
influence the acquisition of analyzers.
Finnegan does not look forward to the day hematology
analyzers are serving as flow cytometers. “We don’t need that
level,” she said. “We’re not going to be flowing every patient.
A CBC costs about $1.50 to $2. To flow a patient is more like
$50 to $60. I don’t know that the doctors are looking for that
sophistication. Hematology is a routine test. There should be
routine things that you look at. If a patient does get flagged
and it is abnormal, then we have other tests, but a basic hospi-
tal and your doctor’s office are not going to want to do all that
other stuff.”
Davis, however, said the more advanced tests would be
run separately, so they would not increase the cost of routine
CBCs. “I don’t envision that a complicated acute leukemia
workup or large panels that are used in flow cytometry for
diagnostic workups will rapidly come back to the hematology
lab,” he said.
Kotylo, who taught “Clinical Applications of Flow Cytome-
try” in the 2006 ASCP’s CLME course, “Diagnostic
Hematopathology” said there’s no getting around the fact that
flow cytometry is expensive, but there are ways to reduce costs
by combining reagents in different ways. Another factor may
slow the transition of flow tests to the hematology analyzer, and
that is the loss of revenue. “Some people don’t want to lose flow
business because they feel that it’s already been hit with the de-
creased reimbursement,” she said.
Reliability and reproducibility of flow results are other im-
portant considerations for the hematology laboratory. The im-
pedance-based analyzers “are workhorses for high-volume labs
that are reporting many CBCs in a busy hospital setting,” said
Kotylo. “They have to be reliable. They have to be fast. You have
to make sure it works and is cost-effective. We know that their
accuracy and reproducibility are its strength.”
As flow cytometry applications continue to emerge, she
added, they will have to be proven and established. “Because of
the type of technology that flow is, you have to have good quality
May 2006 䊏 Volume 37 Number 5 䊏 LABMEDICINE 275
 Feature
control and standardization of your instrument and your
reagents,” Kotylo said. “If you don’t do that, you can get some
erroneous results. You have to have someone who is trained,
knows what they are doing, knows what they are looking at. It’s
pretty sophisticated. Your best techs are the ones working with
this technology.”
At Trillium, Davis is working on an assay based on CD64
expression on neutrophils to detect infection or sepsis that can
be integrated into a high-end hematology analyzer.
“Within 3 to 5 years we’re going to see a new set of param-
eters that’s going to somewhat revolutionize lab hematology or at
least wake us up out of the doldrums which in my mind has
been going on for 10 or 15 years with little change,” he said.
“Those instruments that already have an ability to measure fluo-
rescence are in a much better situation in terms of being
amenable to add-ons or assays adapted for that. Not to say that
somebody couldn’t get clever with just using light scatter princi-
ples, but my bias is that fluorescence affords a better degree of
sensitivity and specificity.”
Middleware, Rules, and Automation
While the forecasters look to the future, instrument manu-
facturers today still have to angle for an advantage over their
competitors. In addition to highlighting differences in technol-
ogy, companies distinguish their products by adding data man-
agement software that gives the instruments the ability to
auto-verify normal cells based on each laboratory’s set of rules,
significantly reducing review rates and leaving medical technolo-
gists more time to focus on abnormal cells. (See the upcoming
July 2006 issue of LABMEDICINE for more on middleware
and other new software applications for the laboratory.)
“At the level of the analyzer, it is difficult to distinguish
the advantages of different products,” said Bruce A. Fried-
man, professor of pathology at the University of Michigan
Medical School and Director of Pathology Data Systems at
the UM Medical Center in Ann Arbor. “So to a certain ex-
tent, by producing a (middleware) device that will perform
certain key functions after a result has been generated, they
Instrument manufacturers are also
promoting automation systems to high-volume
laboratories to help with staff shortages.
have a way of distinguishing their product in the market.
First and foremost, (the in vitro diagnostic companies) are
into the middleware market as a way of protecting their key
business, but they feel they have to be in information man-
agement to survive in the market.”
Maria C. Grana, BS, MT(ASCP)SH, Technical Manager in
charge of Hematology, Blood Bank, and Flow Cytometry at
Quest Diagnostics in Miami, says computer software has im-
proved significantly from one generation of analyzers to the
next. “They have updated the computer 100% and they have
much better discrimination for manual differentials,” she said.
“The ability to reduce the microscopic review rate is very impor-
tant. If you have an instrument that is accurate, then you can
actually look at the ones you need to look at—the ones with
problems—which is a better use of our technologists. And these
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instruments can do that. That’s what I think labs are looking for:
ease of use, efficiency, and reduced microscopic review rate.”
Finn is concerned that some pathologists and laboratory
professionals may focus too much on getting the best technology
rather than optimizing the technology to improve medical deci-
sion-making. “You can buy the fanciest analyzer in the world,
but if you are repeating every abnormal result, you have instantly
negated the power of your technology,” he said. “That assumes
that abnormal results are innately more likely to be incorrect
than normal results, which isn’t true actually. I’ve heard of labo-
ratories auto-validating only normal results, and there’s no logic
to that whatsoever.”
Finnegan said that every laboratory has to determine the
criteria they will use for what gets reviewed and what gets
scanned and generates a manual differential. In her laboratory,
there’s no doubt that the overall number of manual differentials
has decreased. “And that has taken out the drudge,” she said. “So
you can spend time with an abnormal differential and not feel
that you’re rushing through it because you’ve got 17 million
other slides that you’ve got to do behind it.”
Middleware allows laboratories to set up rules for auto-vali-
dation and suspect flags based on sample location or patient
population. For example, if a laboratory handles a lot of samples
from cancer patients, the system can be set up to auto-verify ab-
normal red cells on those samples.
The important thing is not just to auto-validate normal
samples, but to reduce the number of false positive flagging, said
Taylor. “The manual differential is the most technically demand-
ing in the lab,” he said. “It’s the most labor intensive. The bottom
line is reducing the amount of time someone goes to the micro-
scope. They only want to go to the microscope when it is abnor-
mal, and not when it’s normal and you flagged it incorrectly.”
Instrument manufacturers are also promoting automation
systems to high-volume laboratories to help with staff shortages.
With an automation system, a technologist places the samples
on the automation line. The system then routes tubes to the an-
alyzer, from the analyzer to an outlet for additional testing, or
into a temperature-controlled “stockyard” where they may be
recalled quickly for add-on tests. Automated slidemakers and
stainers further reduce hands-on staff time. Middleware can be
considered a component of an automation system, but it can
also be purchased separately.
“Automation will continue to grow in the hematology labo-
ratory as the number of technologists continues to decrease,”
said Sysmex’s Connell. “You need to have sophisticated systems
where you can put your samples on and go do other work and
only be available to look at those truly abnormal samples.”
Beckman Coulter’s Taylor agreed. “I think the state of the art
of hematology analyzers is moving in the direction of less interac-
tion with the hematology sample,” he said. “The ability to auto-
mate for walk-away systems to improve productivity is a big area.”
Most automation systems are customized for each laboratory
setup. In some cases, standardized configurations are available.
Grana, who is a member of the ASCP Board of Registry
Hematology Examination Committee, said her hematology lab-
oratory at Quest Diagnostics has a proprietary laboratory infor-
mation system with flagging algorithms for specimen
management and reflexing. “This optimizes the technologists’
time as well,” she said.
Finn cautioned against automating for the sake of automating.
“I heard about a lab that undertook a major robotic deployment
project and built in an up-front automation lab, high-cost, high-
tech, sounds like a wonderful bit of progress, and they repeated
labmedicine.com
 Feature

Automated Blood Cell Counting

Specimen: Whole blood
Method Description

Most automated blood cell counters measure or calculate

the following parameters: hemoglobin content of RBCs, hemat-
ocrit, RBC count, mean corpuscular volume (MCV) of RBCs,
mean corpuscular hemoglobin (MCH), mean corpuscular hemo-
globin concentration (MCHC), platelet count, mean platelet
volume, and WBC count with differential.
Hemoglobin is measured directly from the whole blood speci-
men using a method based on cyanomethemoglobin formation.
Enumeration of the RBCs, the WBCs, and the platelets can
be achieved by several methods. Many cell counters employ an
electrical impedance method. This method relies on the change
in conductance evoked by cells passing through a small aperture.
The aperture size differs for RBC, WBC, and platelets. The
change in conductance results in an electric impulse that can be
detected and recorded. This method also allows for the measure-
ment of the cell volume. Analysis of the WBCs requires the lysis
of the erythrocytes. After performing red cell lysis, the different
populations of the WBCs are identified by flow cytometry
within the cell counter.

Reprinted from: Laposata M. Laboratory Medicine: Clinical

Pathology in the Practice of Medicine. ASCP©2002.

every CBC that isn’t normal,” he said. “You have just negated that
investment by one silly lapse in medical decision-making.”
Optimizing the Automated Analyzer
Finn said there is too much emphasis on how good the
analyzers are getting, and not enough on optimizing the use of
automated and manual technologies. Part of the problem is
that pathologists in charge of hematology laboratories trained
as residents in anatomic pathology and did not learn about
laboratory medicine.
“There is not a culture of training residents to be medical
decision-makers in the clinical laboratory,” he said. “That’s
where you have to start. A lot of pathologists are performing
validation functions when they should be performing interpre-
tive functions. The concepts aren’t firm with them. There are 2
functions in the laboratory: One is to stand by the numbers
you release, and the other is to interpret the results you release.”
The next step is to practice evidence-based medicine, says
Finn. “If you look at the last 10,000 cases that you released and
see no evidence that they could not have been auto-verified with
the exact same results, then you should be auto-verifying,” he
said. “If you look at the last 10,000 blood smears that a patholo-
gist reviewed and there was no medical value added to that, then
you shouldn’t be doing that. At the same time, if you released
10,000 results that could have had medical value added, you
should have been reviewing those. We are very poor on
evidence-based practice.”
Another challenge is to help the technologists understand
the information coming off the hematology analyzer. Sandhaus,
who teaches ASCP CMLE courses on preventing errors in the
hematology laboratory and on diagnostic hematopathology, said
that technologists’ understanding is variable.
“Most of the techs in my lab really don’t have that much of
an understanding of the technology of the instrument,” she said.
“Also, their understanding of the graphic displays is very limited,
so as part of our continuing education in the lab, we try to em-
phasize correlating the graphic displays with their morphologic
findings, so they can get more out of that information.”
Even the CBC batteries have become overly complex, said
Sandhaus. “There’s just a ton of data that’s produced on every
CBC analysis,” she said. “We’ve got all these numbers that are
produced, all these indices, graphic displays, and interpretive
comments. All of that information in some way needs to be in-
tegrated into that laboratory’s algorithm for which results they’re
going to review and which ones they’re going to auto-validate. If
you make that process more complex, you’re going to be slowing
down your lab considerably. You have to weigh the benefits of
more information against the additional complexity that it intro-
duces into your whole operation.”
To be fair, Finn said, it would take a biophysicist to un-
derstand everything the analyzers are doing to cells. “We can
let the manufacturers be the biophysicists,” he added. “But we
are all smart enough to run the instruments right if we get the
right medical attitude to doing it. Medical decision-making
really has to take hold in the application of high-throughput
laboratory hematology. The first step is to have a medical di-
rector who is devoted to the practice of laboratory medicine
and is not just a spectator.”
Finn said medical directors should use the technology to free
up technologists to do more interpretive work of cells that are in-
terrogated by methods other than microscopes, to teach them how
to read the flow-based histogram data and the distributions on the
cells that have been manipulated with different reagents.
“The potential is basically accumulating behind this wave of
technology,” he said. “The technology is advancing rapidly, and

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 Feature
A
Flow Cytometry
Specimen: Whole blood, any body fluid, dispersed bone
marrow aspirate, disrupted tissue
Method Description

Flow cytometry is a method in which a single cell can be

characterized by size, shape, and biochemical or antigenic
composition.
The first flow cytometry figure illustrates the basic princi-
ples of flow cytometry. The cells flow in a single stream
through a cuvette, where they are exposed to a beam of
intense light. A single cell scatters the light in all directions.
Forward scatter, resulting from diffraction, correlates with cell
volume. Side scatter (right angle) is a result of refraction and
approximates internal cellular granularity. Populations of cells
such as neutrophils and lymphocytes, which differ by size
and/or granularity, can be identified using forward and side
scatter data.
The second flow cytometry figure depicts an example of
the use of fluorescence to detect different cell populations.
Monoclonal antibodies used in identifying cytoplasmic and
cell surface antigens most commonly are labeled with fluores-
cent compounds. Fluorescent compounds, like fluorescein or
R-phycoerythrin, have different emission spectra, allowing for B
the identification of cells based on the color of the emitted
light. A cell suspension is incubated with 2 monoclonal anti-
bodies, each labeled with a different fluorochrome. As cells
with bound antibodies pass through the cuvette, laser light at
488 nm excites the fluorescent compounds causing them to
fluoresce at specific wavelengths. A system of lenses and filters
detects the emitted light and translates it into an electric signal
that can be analyzed by the computer. The side scatter and
forward scatter and the intensity of emitted light at specific
wavelengths characterize each cell type. The data collected
from thousands of events are gathered, analyzed, and plotted
on a histogram. Flow cytometry is highly useful in the diagno-
sis of leukemias and lymphomas. The use of different antibody
markers allows for the precise identification of cells.

Reprinted from: Laposata M. Laboratory Medicine: Clinical

Pathology in the Practice of Medicine. American Society for Clini-
cal Pathology, Chicago: ©2002.

we should be grateful for that and encouraged by that, but be-
hind this wave, there is accumulating so much potential for the
appropriate utilization of the technology, and I just don’t see the
mindset of clinical pathology evolving as quickly as the technol-
ogy, and that’s unfortunate. The rate-limiting steps are really the
attitudes toward medical practice in the clinical laboratory. People
have to start leading and defining how we practice medicine with
tools that include automated platforms. There are smart ways to
use smart technology and we’re not always using them.”
That’s not to say that hematology laboratories should not
embrace the technological advances. “It doesn’t have to be
either/or,” Finn said. “You can get new technology and improve
your attitude about medical practice.”
In the end, said Kotylo, emerging technology is going to
come no matter what. “You can’t keep hanging on to the way it
was,” she said. “I think what you’ll see in the future is that flow
will be less isolated, and it’s going to be more a part of morphol-
ogy and molecular diagnostics.” LM

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