Perks plus: the new hematology analyzers
William Check, PhD
Steven Kroft, MD, conducted a search for a new hematology analyzer
in the past year. His short list of mandatory features included substantially
decreasing the manual review rate for differentials while automatically
quantitating nucleated red blood cells, reticulocytes, and platelets.
"Pretty much all the high-end instruments we looked at filled these
requirements," says Dr. Kroft, assistant professor of pathology in
the Division of Hematopathology and Immunology at the University of
Texas Southwestern Medical School in Dallas.
Dr. Kroft found that, beyond satisfying his basic criteria, several
instruments offered additional novel and desirable applications.
Some are FDA approved, such as early identification of anemias and
measuring the immature reticulocyte fraction. But the more exciting
advances are still investigational—enumerating immature granulocytes,
estimating stem cell numbers by quantitating surrogate cells, counting
reticulated platelets, and handling non-traditional specimens, particularly
bone marrow. Moreover, some instruments have the potential to offer
further novel and advantageous features, such as immunological assays,
with only software modifications. This enhancement was one factor
in breaking the tie between the top two instruments on Dr. Kroft’s
Also important was the ability to be incorporated into what Dr.
Kroft calls "a hematology island of automation"—analyzers
integrated with an automatic slidemaker/stainer under a central
controlling computer that coordinates the movement of samples. In
this system, which Dr. Kroft’s laboratory is gradually implementing,
he says, "If we needed to repeat a CBC for some reason, no one would
have to handle that tube. It would be done automatically."
Ed Wong, MD, director of hematology in the Department of Laboratory
Medicine at Children’s National Medical Center in Washington, DC,
also recently purchased a new hematology analyzer. "The more modern
instrumentation available today has improved on original designs,"
Dr. Wong says, "and we wanted to take advantage of that to see if
we could improve patient care." Many contemporary instruments "offer
a lot in improved white cell, red cell, and platelet analysis,"
Dr. Wong is satisfied with the instrument he chose, the Roche
Sysmex XE 2100. "We now have an automated machine that can do everything
we want in terms of reticulocyte and platelet counts," he says.
It also satisfies the needs of his medical center’s bone marrow
transplantation and hematology-oncology programs.
A major technological advance—incorporating a laser into
the instrument’s standard operation-has eased automated hematology
analyzers from the shadow of unfulfilled promise into the bright
light of accomplishment. (An argon ion laser is used in all instruments
except the Sysmex, which contains a semiconductor diode laser.)
"More of the current analyzers are moving in that direction," says
Dr. Kroft, who chose the Abbott Cell-Dyn 4000. "They are essentially
little automated flow cytometers for the hematology laboratory."
But instruments differ in the degree to which they "exploit that
technology to do traditional flow cytometric assays," he notes.
One of the factors in his choice of the Cell-Dyn 4000 was its greater
ability to do flow cytometric assays, particularly CD4/8 subsets,
offering considerable labor savings. "Anything that is labor-intensive
and high volume is fair game for this type of analysis," Dr. Kroft
Carol Briggs, head of hematology evaluation and development in
the Department of Haematology at University College London Hospital,
sees, too, the trend that Dr. Kroft describes. "Basically all vendors
are moving towards using more flow cytometric methods on hematology
analyzers, combining the research side with the routine side," she
says. "It would be lovely to be able to measure CD4 and CD8 counts
on HIV patients straight through on your blood sample," she says,
"and I think that will be coming." Another upcoming application
will be measurement of immature platelets, which, she says, are
getting more important. "As research techniques become more established,
people want them done routinely and with one tube of blood and reduced
number of staff and quickest turnaround," she says. A hematology
analyzer with a fully exploited laser could meet those goals. She
sees CD4 and CD8 counts as an obvious first step; the Cell-Dyn 4000
is the only analyzer now that measures them, though not at the same
time as the CBC. "A separate tube of reagent is needed," she says,
"and the test has to be preselected."
Bruce Davis, MD, associate director of clinical research at the Maine Medical
Center Research Institute, says that several high-end hematology instruments
have a limited capability to perform many of the same measurements that are
on multi-parameter flow cytometric instruments, "given that they use a combination
of laser excitation and fluorescence." The crucial question determining the
extent to which tests will move from flow cytometry to hematology is, "What
would be the volume, and is the cost of development justified by clinical utility,"
says Dr. Davis, who is also medical director of flow cytometry at Genzyme Genetics
in Santa Fe, NM. "Vendors must balance cost of development against return on
Going from the long-term prospect of doing flow cytometric
assays on a hematology instrument to the more mundane hope of reducing
manual differentials is like driving away from a Ferrari showroom
in your Volvo station wagon. For anyone running a hematology laboratory,
however, a truly automated differential would be as welcome as cost-effective,
reliable daily transportation.
"One of our big issues was that we were bottlenecking at slide
review, which was keeping our technologists very busy and increasing
our turnaround times," says Dr. Kroft. "We have a very high smear
review rate at Parkland [Hospital], in part because we have many
neonates and extremely sick patients." A major issue for Dr. Kroft
in selecting a new analyzer was to maximize the sensitivity and
specificity of flagging of abnormal blood specimens, which the Cell-Dyn
Dr. Wong says that the manual differential rate at his hospital
ranged between 50 and 60 percent. "We think we can get it down to
35 percent or so with our new instrument," he says. Because the
Sysmex XE 2100 has been online for only a few months, however, he
doesn’t know what the actual figure will be.
New analyzers reduce the rate of manual differential review partly
by quantitating nucleated red blood cells automatically, which makes
"an enormous difference," Briggs says. "Potentially, this feature
completely negates the need to do manual counts," she says. NRBCs,
which are included in and distort the white blood cell count, are
present in infants and in persons with hematologic disorders, such
as sickle cell disease and thalassemias, as well as in some persons
with cancer and infections. Older machines flagged specimens that
had NRBCs, and a technologist then had to stain those samples and
count NRBCs manually. Says Dr. Wong: "Newer machines can quantitate
NRBCs and correct the WBC count. We have a great need for this feature
in our NICU."
Dr. Kroft has a similar need. "In Parkland Hospital, there are
babies around every corner," he says. Technologists were reviewing
smears on every CBC from neonates because neonates usually have
significant numbers of reticulated RBCs, as well as RBCs with strange
morphology. Dr. Kroft calls manual review "a big time sink for our
technologists." He looked for instruments that could report out
WBCs without having to correct manually for NRBCs. At the time of
his evaluation, only two manufacturers had a quantifiable NRBC assay
that was FDA approved—Cell-Dyn 4000 and Sysmex XE 2100—so
he focused on those two.
Much the same could be said of Palmetto Health Richland in Columbia,
SC. "With our previous instrument, almost all of the samples from
babies would give the reject code on WBCs and platelets," says laboratory
supervisor Christy Knight, BS, MT(ASCP). Overcall problems were
also common on sickle cell patients. When they got a Cell-Dyn 4000,
Knight says, their slide reviews dropped by 50 percent.
Decreases in manual review derive also from the more-accurate
automated counting of reticulocytes and platelets, which, like NRBC
counting, is based on fluorescent nucleic acid dyes and laser excitation.
Reticulocyte counts, which are a fairly sensitive indicator of
RBC production in bone marrow, are most commonly ordered when investigating
anemias or if patients are taking certain drugs, says Bernard Fernandes,
MD, head of hematopathology and the blood transfusion service at
Mt. Sinai Hospital, Toronto. Reticulocyte counts also reflect recovery
of bone marrow after bone marrow transplants or chemotherapy. "Manual
counts have a very high degree of imprecision," says Dr. Fernandes.
"I would say the manual count is of very limited value."
Previous generations of hematology analyzers based only on impedance
technology were unable to perform reticulocyte counts, and the counts
were therefore performed manually. They have been done on freestanding
flow cytometers, which give accurate and precise results but are
time-consuming. "With this generation of CBC instruments, we can
get a reticulocyte count at the same time and on the same specimen
as the rest of the CBC," says Dr. Fernandes, who uses a Sysmex XE
Automated reticulocyte counting is important for several reasons,
says Stefanie McFadden, MT (ASCP), SH, core laboratory manager at
Grant/ Riverside Hospital in Columbus, Ohio. "Probably precision
is the overriding issue for me," says McFadden, whose laboratory
uses a Beckman Coulter GEN*S. In her laboratory, she found coefficients
of variation ranging from 11.1 percent to 5.3 percent for automated
reticulocyte counting, depending on the reticulocyte concentration
(from ~0.3 percent to ~5 percent of RBCs). Precision with the manual
method ranged from 46 percent to 27 percent, she found.
Says Knight at Palmetto Health Richland, "We do automated reticulocyte
counting and we love it." They had been doing manual reticulocyte
counting because they were not able to get automated reticulocyte
counting to work on their previous instrument. "We have a lot of
babies who have siderocytes," Knight says. "Our previous instrument
used a staining method that stained siderocytes as well, so we couldn’t
do babies or patients with sickle cell disease." Since getting a
Cell-Dyn 4000, she estimates that the laboratory has done two manual
reticulocyte counts in the past year out of a volume of 75 to 100
samples per week.
At North Shore Long Island Jewish Health System in New Hyde Park,
NY, reticulocyte counts are done on an ABX Pentra, says Teddie Owen,
MT, supervisor of the Faculty Practice Laboratory. The instrument
breaks down the count by hematocrit and by stages of maturity according
to DNA content. Owen says that the laboratory reports out the CBC,
then goes into another program to do the reticulocyte count, since
"we can’t run reticulocytes at the same time as the CBC."
Platelet counting has followed the same pattern as reticulocytes:
initial impedance methods, then improved accuracy and precision
with optical counting in the newest analyzers. Precision at the
low end of platelet counts is particularly important, Dr. Kroft
says. "Low-end accuracy allows us to make more informed decisions
about when to transfuse platelets," he says, citing "a significant
literature" proving that optical platelet counts are typically more
accurate in the low range.
University College London Hospital uses a threshold for platelet
transfusion of 10 (10,000/µL or 10x109/L).
"Our worry with the impedance method is not knowing when the platelet
count is really 10," Briggs says. As with reticulocytes, the impedance
method might count RBC fragments or WBC microparticles as platelets,
giving a count of 20 when it is really 10. Equally problematic,
since platelets are measured by size, is that large platelets get
excluded, leading to a falsely low count.
An accurate method is to stain with monoclonal antibodies against
CD61 and CD41 and count on a flow cytometer. Using this value as
the reference, Briggs and her coworkers found that there were problems
with impedance counts at the low end and that all instruments that
did optical counts were significantly better.
Says Dr. Kroft, "From reading the literature and comparing methods
for automated optical platelet counting, it was apparent to me that
several manufacturers now provide it on their hematology analyzers
and all perform in excellent fashion." He favored the Cell-Dyn 4000
because it performs dual impedance and optical platelet counts automatically
on every specimen. With the Sysmex XE 2100, for both NRBCs and platelets
a qualitative screen is first done. If a flag is thrown, the sample
is reflexed to generate a numeric NRBC or platelet count.
"We are currently in the process of validating that we can report
out low platelet counts with confidence," Dr. Kroft says. Their
lower limit before doing manual review has been 50,000/µL. With
the new instrument, Dr. Kroft is comfortable dropping that to 30,000.
"We will push it further, aiming for 10,000," he says.
Owen, of North Shore Long Island Jewish Health System, says she considers
being able to handle the extreme low end of platelet and WBC counts a crucial
requirement for a hematology instrument. "Some of our patients are very sick
and many have blood cancers," she says. "Once we run our controls, that is probably
the last normal we see for the day."
Beyond doing a truly automated differential count and accurately
quantitating NRBCs, reticulocytes, and platelets, high-end analyzers
can help diagnose anemia and reflect the hematopoietic status of
the bone marrow generally. One useful value is to measure directly
the hemoglobin content of reticulocytes (CHr), a patented feature
of the Bayer Advia 120.
"For several years analyzers have reported mean corpuscular hemoglobin
[MCH], the hemoglobin content of RBCs," says Carlo Brugnara, MD,
director of the hematology laboratory at Children’s Hospital, Boston,
and associate professor of pathology at Harvard Medical School.
In an analogous way, CHr gives the hemoglobin content of reticulocytes
in picograms per cell, calculated from direct measurements of volume
and hemoglobin concentration of individual reticulocytes. Because
reticulocytes reflect the functional state of the bone marrow in
the preceding four to five days, CHr gives a much closer reflection
than MCH of the real-time balance between iron and erythropoiesis.
(Lifespan of RBCs is 120 days.)
Dr. Brugnara has evaluated CHr in a variety of conditions, including
anemia of children and after therapy with recombinant human erythropoietin,
or rhEpo. "There is no gold standard for iron deficiency in erythropoiesis,"
Dr. Brugnara says. Proving iron deficiency requires treating with
IV iron and showing that patients respond with an increase in hemoglobin
and CHr. In a 1997 paper, nephrologist Steven Fishbane, MD, demonstrated
these responses following IV iron therapy in dialysis patients who
had low CHr. "We have done similar studies in classical iron deficiency
patients," Dr. Brugnara says. One advantage of using CHr is that
it responds within two to four days of initiating treatment, whereas
other parameters tend to improve much more slowly, with hemoglobin
taking four to five weeks to increase.
A particularly damaging form of iron deficiency occurs in children
ages three months to 15-18 months, when development of the brain
is crucially dependent on the availability of iron. "Even with moderate
iron deficiency, not severe enough to cause anemia, permanent and
irreversible alterations in cognitive brain function can occur,"
Dr. Brugnara says. He reported in 1999 that CHr can identify patients
who are iron deficient even in the absence of anemia. "We are now
trying further to validate this parameter and show early iron therapy
can reverse these changes," he says.
A third application of CHr is to evaluate patients receiving rhEpo.
"When you use Epo, it drives the bone marrow to such a high rate
of production that the iron that is available in normal subjects
is not enough," Dr. Brugnara says. This creates a transient state
called functional iron deficiency, which cannot be detected except
with an assay that shows what is happening in bone marrow at that
time. Several years ago Dr. Brugnara gave normal patients a short
course of Epo; all developed functional iron deficiency and, for
10 days to two weeks, produced reticulocytes and RBCs functionally
indistinguishable from those seen in patients with classical iron
deficiency. Those with more iron—ferritin values ≥
100 ng/mL—made more RBCs with the same dosage of Epo. "If
you use enough IV iron to saturate the system, that allows the patient
to get the maximum benefit from Epo," Dr. Brugnara suggests. With
Epo, an expensive drug, being given to most of the approximately
200,000 persons in the U.S. on dialysis, maximizing its benefit
Briggs has evaluated a parameter on the Sysmex XE 2100 called
RET-Y, a measure of reticulocyte size obtained from the mean value
of the forward scatter of reticulocytes. "We found that RET-Y correlated
very well with CHr," she says, "although we didn’t go to the clinical
Says Dr. Brugnara: "We have shown that measurement of reticulocyte
volume is not as sensitive as CHr. In patients taking Epo it changes
for a variety of reasons." Most important, he says, RET-Y can’t
provide the hemoglobin content per cell.
In theory, immature reticulocyte fraction, or IRF, which can be
done on any analyzer that does automated reticulocyte counting,
could be useful for evaluating bone marrow erythropoietic activity,
Dr. Kroft notes. Reticulocyte number tells how many immature RBCs
are circulating, but it doesn’t take into account their different
half-lives. "With IRF," Dr. Kroft says, "for the first time we can
get a sense of how fast or early the reticulocytes are getting pushed
out of the marrow, the true rate of production of RBCs."
Says Dr. Davis, "At the moment, IRF is probably one of the most
underutilized yet clinically beneficial parameters that high-end
hematology instruments provide." Hundreds of papers demonstrate
its utility, he adds. "If there is a question of whether RBC production
is ongoing or adequate, IRF is the most efficacious parameter to
look at. Probably its greatest utility is providing assistance in
both diagnosis and therapeutic monitoring of the anemic patient."
One major branch-point in the differential diagnosis of anemia
is between decreased RBC production and RBC destruction. "If production
is decreased, IRF will be low," Dr. Davis says. "If production is
appropriate, and the problem lies outside the bone marrow compartment,
IRF is increased." Recovery of bone marrow following Epo therapy,
chemotherapy, or bone marrow transplantation is reflected within
a few days by increased IRF.
Immature granulocyte number can be measured on contemporary analyzers,
but this parameter is not yet approved in the United States. Dr.
Fernandes, in Toronto, is evaluating its utility. "Counting immature
granulocytes could be useful in many clinical situations," he says.
It is increased in infections, many hematologic disorders, diseases
of the bone marrow, and inflammatory diseases.
Briggs notes that hematology analyzers don’t differentiate immature granulocytes
into their three stages, which a slide count does. "It doesn’t matter in a patient
with infection or inflammation," she says, "but for hematologic disorders—leukemia
or myelodysplastic syndrome—it does."
Measuring stem cell production on a hematology analyzer
is a promising but problematic proposition. Dr. Wong is using a
new parameter on the Sysmex XE 2100 that measures a subpopulation,
called hematopoietic progenitor cells, or HPC, that resembles or
correlates with CD34-positive stem cells. "I think hematopoietic
progenitor cells is a misnomer," he says. "Further work is necessary
to clarify what these cells’ characteristics are." Nonetheless,
he finds them useful in timing apheresis collections, because quantitation
of true CD34-positive stem cells requires a flow cytometer with
trained technical staff. Measuring HPC allows him to determine within
90 seconds whether to go ahead with stem cell collection.
Michael Watts, PhD, director of the stem cell laboratory in the
Haematology Department at University College London, evaluated HPC.
To mobilize stem cells from bone marrow into the circulation, patients
are given growth factors and chemotherapy. Apheresis could be timed
by doing WBC counts. However, there is a problem of "poor mobilizers,"
patients with extensive prior therapy or whose marrow has not yet
recovered from prior treatment.
Direct measurement of CD34-positive cells in peripheral blood
on a flow cytometer has been used to time apheresis. However, the
concentration of CD34-positive cells in many patients’ blood is
around 0.1 percent, a range in which the precision of a flow cytometer
drops substantially. "Our interest in the HPC test was to find something
that would reduce the number of flow cytometer measurements and
improve harvest timing beyond simply WBC recovery," says Dr. Watts.
He and his coworkers showed good reproducibility for HPC even
at very low levels. Correlation between HPC and CD34 number was
"poor," they found, consistent with the idea that the two tests
measure different subpopulations. "But that doesn’t matter since
we are only looking for the recovery phase of bone marrow," Dr.
Watts says. Most important, adequate apheresis yield predicted by
HPC was only slightly less accurate than by CD34 counts.
An "optimal strategy" would be to proceed with apheresis for HPC
counts > 10x106/L (typically >
0.1 percent), with the option of doing flow cytometry for lower
counts. This approach has the potential to reduce flow cytometer
runs by 90 percent. For now they continue to do both tests for validation.
Rare cases occur in which relying on HPC alone would result in collections
with inadequate numbers of stem cells.
Dr. Davis calls the HPC count "an imperfect surrogate" and says
it "does not necessarily give the count you want." Stimulation with
growth factors and chemotherapy drugs drives many types of cells
in the bone marrow to be produced at a higher rate, he notes. He
favors the use of IRF as a surrogate to stem cell counting, based
on several publications by Spanish and Australian groups. "IRF provides
a parameter that parallels CD34 stem cells and several instruments
already can measure it," Dr. Davis says. Dr. Kroft agrees that IRF
needs to be evaluated further. "It holds promise because it is so
easy to do," he says.
Dr. Davis is helping a working group of the International Society
for Laboratory Hematology to develop another assay, reticulated
platelets, for evaluation of thrombopoiesis. "Reticulated platelets,
simply put, are the platelet analogy to the reticulated component
of the RBC series," Dr. Davis says. Like reticulocytes, younger
platelets—those most recently produced and released by bone
marrow—have more RNA, so they are a fairly direct measurement
of bone marrow platelet production.
"Measuring reticulated platelets turns out to be a fairly informative
way to assess thrombopoiesis without doing more invasive techniques
such as bone marrow biopsies and aspirations, which are themselves
not always informative," Dr. Davis says.
Reticulated platelet measurement has been reported to discriminate between
thrombocytopenias due to platelet consumption, such as hypersplenism, and those
due to low platelet production. In principle, reticulated platelets could be
measured on any analyzer that can do a fluorescent reticulocyte count, although
no instrument offers that assay now.
All of the applications described so far use peripheral
blood samples. But a few pathologists have assessed whether hematology
analyzers can handle non-traditional specimens, particularly bone
marrow. Harold Schumacher, MD, emeritus professor of pathology and
director of the hematopathology fellowship program at the University
of Cincinnati Medical Center, ran remainder material from about
300 bone marrow biopsies through a Cell-Dyn 4000 and compared the
differential counts obtained with his hand counts. "I calculated
the relation of the myeloid cellular elements to erythroid elements
and determined abnormal cells, particularly blast cells from acute
leukemia," he says. Abnormal cells are identified by position on
the instrument’s multi-angle polarized scatter separation histograms,
with lobularity on one axis and size on the other.
Performance of the instrument was not hampered by the fact that
it was analyzing bone marrow, Dr. Schumacher found. Some people
expected the presence of marrow fat to clog up the aperture. "But
I never had problems with aperture occlusion," Dr. Schumacher says.
In spiking experiments with leukemic blast cells from culture, the
instrument proved accurate down to at least 3.6 percent blasts.
A good hematopathologist can detect down to around five percent
blasts on a slide, and a flow cytometer is sensitive down to one
to two percent, Dr. Schumacher estimates. The next logical step
would be to look at bone marrows from leukemic patients.
Maria Pedemonte, MD, medical director of Bioreference/Genpath
Laboratories in Elmwood Park, NJ, is running bone marrow samples
on a Bayer Advia 120. "My assumption," Dr. Pedemonte says, "was
that if I ran peripheral blood or bone marrow on the analyzer, by
combining the laser histogram and physical properties of the cells
with myeloperoxidase staining, I would get a good idea of lymphoid
versus myeloid and monocytic lineage." This was the case, she found,
with some leukemias having characteristic histogram patterns. For
instance, acute promyelocytic leukemia gives a very bright region
in the side scatter of the myeloperoxidase-positive channel. Like
Dr. Schumacher, she found no clogging from fat particles.
Dr. Pedemonte finds this method to be complementary to flow cytometry,
reducing the number of antibodies needed in some cases by about
one-third. "The most important thing is that this method is not
diagnostic," she says. "I use it as a screening tool in conjunction
with morphology, flow cytometry, and cytogenetics."
Some of today’s analyzers can also perform immunological assays,
potentially moving some tests from the flow cytometer to the hematology
laboratory. Says Dr. Kroft, "That is one of the things that excited
us" about the Cell-Dyn 4000, the development of immunologically
based assays for a hematology platform. One example is that it can
do a platelet count with CD61 monoclonal antibody in an almost completely
automated fashion. "It incubates a blood sample with antibody to
CD61 antigen, then enumerates platelets by fluorescence measurement,"
Dr. Kroft says. This kind of assay done on the flow cytometer is
essentially the gold standard for platelet counting. On the hematology
analyzer, it can function as a backup method to confirm a platelet
count that is very low or has abnormal platelets, such as very large
or hypogranular platelets, as in myelodysplastic syndrome. Dr. Kroft
has not yet developed the assay in his laboratory, but it is available
from the manufacturer and FDA approved so he anticipates adopting
"That is a nice feature of the Cell-Dyn 4000," Dr. Kroft says.
"It has the ability to do a variety of flow-based assays in up to
three colors." One approved assay of this type is enumeration of
T cell subsets (CD4 and CD8 cells) in HIV-infected patients. Because
this is a relatively high-volume test, it could make economic sense
to move it onto a hematology platform.
Looking further into the future, Dr. Davis sees hematology analyzers
being used to measure functional states of cells, rather than just
counting cells as particles. He is working on measuring expression
of a protein, CD64, the high-affinity Fc receptor, on neutrophils.
"I and others have a lot of evidence that neutrophils upregulate
CD64 in the acute inflammatory response," he says. "So measuring
CD64 levels on neutrophils could provide a good indicator of sepsis
or infection." Since this method is essentially done with a monoclonal
antibody and fluorescence detection, it could go on any current
high-end hematology instrument.
"If there is any area where laboratory medicine has not moved
forward in the last 10 to 20 years," Dr. Davis says, "it is providing
new diagnostics for infection or sepsis. We still count neutrophils
or look for indication of left shift or sedimentation rate as indirect
measures of inflammation." He predicts that the hematology platform
will bring activation markers onto a blood cell counter: "Its design
is ideally suited for that."
William Check is a medical writer in Wilmette, Ill.