Histology labs eye new kids on the block
Special stains and immunostains
October 2003
Cover Story
William Check, PhD
“To every thing there is a season,” the Book of Ecclesiastes
tells us. After a long season of static practice, the field of surgical
pathology is entering a season of technological advancement. And
it is doing so with a vengeance.
“For the most part, we have been practicing surgical pathology in the same
ways for at least 50 years,” says Azorides Morales, MD, professor
and chairman of the Department of Pathology at the University of
Miami School of Medicine and chief of pathology services at Jackson
Memorial Hospital, Miami. “I do not mean to say that there
haven’t been advances. There is no question that a lot of
instrumentation has been developed. And hardly a day goes by when
someone doesn’t develop a new marker or a new technique. But
the basic handling of solid tissues hasn’t changed.”
Dr. Morales has helped develop an instrument that could radically alter this
situation—a continuous tissue processor based on modified
microwave technology. “This processor provides the ability
to complete surgical reports within two to three hours after surgery
and to do them in a continuous fashion,” says Dr. Morales.
About 70 percent of surgical cases in his section are processed
and signed out on the same day the specimen gets to the laboratory.
“This is truly unheard of,” Dr. Morales says.
Technological advances downstream of tissue processing are find-
ing their way into more histology laboratories, partly in response
to increasing specimen volume. “The number of specimens we
handle goes up by at least 10 percent per year,” says Brendan
Boyce, MD, head of surgical pathology at the University of Rochester
Medical Center, NY, “because more patients are being screened,
such as with colonoscopy.
“We have been automating as much as we possibly can,” Dr. Boyce says.
His section uses automated coverslippers, an automated slide labeler,
and automated processors for both special stains and immunohistochemistry.
David Hicks, MD, section head of surgical pathology at the Cleveland Clinic Foundation,
also finds increasing volume a challenge, particularly in light
of staff shortages. “We do approximately 80,000 surgical specimens
per year,” Dr. Hicks says, “so we have had to be creative
about how best to utilize staff, as well as to respond to pressures
for timeliness and quality and error reduction.
“One way to do this is to move in the direction of automation in histology,”
he continues. “In a sense, histology has lagged behind.”
He has automated most routine histochemistry special stains as well
as immunohistochemistry.
Besides increasing efficiency, automation can reduce errors, says Christopher Otis,
MD, director of surgical pathology, Baystate Medical Center, Springfield,
Mass., and associate professor of pathology at Tufts University
School of Medicine, Boston. “One of the growing concerns is
correct identification of specimens, avoiding giving a diagnosis
to the wrong person,” Dr. Otis says. “Hospitals are
becoming increasingly concerned about how we track cases as they
come through the laboratory, from the time the biopsy is obtained
and accessioned to when it is signed out by the pathologist and
the report is delivered to the clinician.” For Dr. Otis’
laboratory, which processes between 800 and 1,200 blocks daily,
this can be a challenge. To meet this challenge, he has adopted
more advanced applications of information technology, such as bar
coding and block labeling.
On the test side, immunohistochemistry is advancing as well. “Immunostains
are definitely a growing area,” Dr. Hicks says, “particularly
tumor markers. The number of antibodies on our menu is growing,
and test volume is growing as well.”
In the opinion of Allen M. Gown, MD, medical director and chief pathologist at
PhenoPath Laboratories, Seattle, “Batteries of immunostains
have largely replaced special stains.”
“In the past,” he says, “we used methods like reticulum stains,
because there was not much else we could do to further identify
the nature of certain tumors.” Special stains are still useful
for identifying organisms such as mycobacteria and fungi, Dr. Gown notes.
“We have had a fairly large expansion of reagents useful in diagnostics,”
adds Paul Swanson, MD, director of anatomic pathology at the University
of Washington Medical Center, Seattle. “Immunostains are much
more widely used, although it is not necessarily true that diagnostic
accuracy or predictive value of the technique has advanced in parallel.”
(See “Special stains and immunostains.”)
Of the many automated instruments, a continuous tissue processor
could have the greatest impact. “It will revolutionize the
practice of anatomic pathology,” Dr. Morales says. In traditional
surgical pathology, everything is handled in batches. “We
gross tissues all day long,” he says. “Then they are
placed in automated processors overnight. In the next day or two
they are handled by histotechnologists in batches. In laboratory
medicine we don’t do that any longer,” he notes. “When
specimens come to the laboratory, for the most part we place them
in analyzers one at a time. Continuous processing allows us to respond
to patients’ needs immediately.”
In surgical pathology the technology needed to expedite tissue processing has
not been available. Solid tissue converted into slides is subject
to many agents—heat, vacuum, agitation—and the process
takes 10 to 12 hours, so it is done overnight. Tissues are presented
to a pathologist at a minimum of 24 hours after material is received.
Microwave technology, introduced into pathology 20 or more years ago, accelerates to a
considerable extent the diffusion of solvents into tissues. “Unfortunately,”
Dr. Morales says, “for the most part the microwaves available
for use in histology are modified kitchen microwaves.” That
situation is changing. Dr. Morales was approached in 1996 by father
and son physicians named Essenfeld in Caracas, Venezuela, who were
working to optimize microwave exposure for solid tissues. Dr. Morales
joined forces with them, and in one year the team had developed
a manual microwave method for histology. An automated version soon
followed, and Sakura Finetek, Torrance, Calif., then designed and
built a new processor that is now in beta testing. In several years
of clinical work with the prototype, Dr. Morales says, “we
have processed close to 1 million samples and we have not spoiled
a single one.”
To design a new microwave oven specifically for histology, the team contacted
Microwave Materials Technologies, Knoxville, Tenn., which developed
a way to provide microwave energy uniformly throughout the retort
chamber where solutions are placed. “All samples receive just
about the same energy,” Dr. Morales says. In addition, they
receive very low wattage radiation. “Rather than pulsing microwave
energy, we have it on continuously at low wattage, 150 watts for
a few seconds at the most,” he explains. For the rest of the
time it is at 50 to 60 watts. “We are handling tissue in a
very gentle fashion that prevents overheating that could damage it.”
New reagents were introduced to aid this process. “We eliminated or reduced
to a significant extent the toxicity of the reagents that we use,”
Dr. Morales says. Conventional processing typically uses formalin,
alcohol, xylene, and paraffin, whereas the new process uses a mixture
of acetone, isopropyl alcohol, polyethylene glycol, and mineral
oil and paraffin. And the volume of reagents has been reduced by about 80 percent.
To accommodate the automated instrument, other steps have had to be standardized.
“Most difficult was to standardize grossing,” Dr. Morales
says. To standardize the thickness of tissue sections, his team
developed simple grossing boards that have slots with preset depths.
“If we want 1.5-mm-thick sections, we put the tissue in that
slot and slice it,” Dr. Morales says.
The processor accepts samples semicontinuously, from one to 40 samples every 15
minutes. Processing takes one hour. A histotechnologist can remove
samples every 15 minutes and proceed with embedding, microtomy,
staining, and other steps, providing virtually continuous flow.
Dr. Morales says this flow is a “tremendous advantage for the staff, not
only pathologists but also histotechnologists.” In most histology
laboratories, histotechnologists come in at odd hours of the morning,
such as 3 AM. With the new processor, everything is done during
the day. One group of histotechnologists starts at 7 AM and another
at 11 AM. “The OR for the most part is inactive on weekends,”
Dr. Morales says, “so histotechnologists only come on the
weekend for emergencies.” This makes it more attractive for
people to work there. Moreover, he says, “we don’t run
the instrument overnight, so if anything goes wrong, we can handle
the problem right away.”
With the automated processor, pathologists are working only 1.5 to two hours behind
the surgeons. “We now provide same-day diagnosis 70 percent
of the time,” Dr. Morales says. “Before, less than one
percent of our samples were diagnosed and a report provided on the
same day.” Rapid turnaround lessens patient anxiety and helps
the flow of patients through the institution. Moreover, Dr. Morales
notes, “We no longer receive calls in the laboratory asking
for a diagnosis. I can tell you this is irreversible here.”
Dr. Morales says “one of the most fascinating aspects” of the new
processing method is that it is “molecular friendly.”
“That means that if we handle the tissue appropriately before we place
it in the instrument, we can extract RNA and proteins from the paraffin
block,” he says. “With conventional overnight processors
that is basically impossible, because the chemicals used are not
molecular friendly.”
Sakura Finetek is commercializing the new processor. “It is my understanding
that it will be available by the end of 2003,” Dr. Morales
says. Large laboratories like his, which has 30,000 surgicals per
year and processes about 120,000 pieces of tissue, will find the
processor desirable, he says. The new instrument, Tissue-Tek Xpress,
handles more than 900 samples in an eight-hour shift, doing the
work of three conventional Sakura Tissue-Tek VIP processors. Dr.
Morales suggests that it may be advantageous for smaller laboratories
as well. “Do you want to wait till the next day or next week
to provide diagnosis to your patients?” he asks. “And
do you want to archive every single piece of tissue that you handle
for possible future molecular assays?”
Sakura has developed an automated embedding device that complements the automated processor.
Currently, after tissue is processed, histotechnologists remove
it from cassettes and embed it in paraffin. In the new system, tissue
will go directly from the processor into the embedding instrument,
called Tissue-Tek AutoTEC, without further handling. AutoTEC also
uses a special tissue cassette. “What we have been using from
the beginning of time can’t be sectioned,” Dr. Morales
says. With the new cassette, tissue is embedded and then sectioned
through the cassette. “This will increase efficiency tremendously,” he adds.
Existing forms of automation can help solve another problem in surgical
pathology: human errors that can lead to misdiagnosis. “Surgical
pathology is unlike most clinical pathology laboratories, where
many things are automated and mistakes can usually be backtracked
to a central point where accessioning and bar coding take place,”
Dr. Otis says. Multiple steps are inherent to histology laboratories,
starting in the clinician’s office or the clinic where specimens
are labeled by hand. After delivery, pathology accessions specimens
by hand through a computer system. “Computer systems help
in a lot of ways,” Dr. Otis says. “They standardize
through default specimen-type dictionaries how to handle tissues
from the beginning. That eliminates some error.”
Next, grossing is done by hand. “You must make sure you pick up and gross
the right specimen,” Dr. Otis says. It’s possible to
make embedding mistakes in which specimens are switched. Slide labeling
creates “a big potential for error.” A technologist
can hand-label a slide with a specimen accession number and then
put the wrong slices on it. It’s also possible to put the
wrong computer-printed labels on slides. “All this falls into
the preanalytical area, which is fraught with potential error,” Dr. Otis says.
He describes a hypothetical but plausible error in the preanalytical stage. “It
would not be uncommon to get specimens from two consecutive patients
on whom the same immunostain was ordered,” he says. Assume
that the stain ordered was p16 antigen, which helps identify high-grade
squamous intraepithelial lesion. What if the labeled slides got
tissues from the wrong patients because they picked up the wrong
tissue sections? In essence, they were crisscrossed. “Perhaps
that incident would be identified at the multiheaded microscope
conference,” Dr. Otis says, “because the morphology
did not make sense with the IHC results. Even so, that raises a
question—how many times do we fail to recognize that kind
of event? What is the denominator? No laboratory has a handle on
that. It would be naive to assume that it never happens.”
In the analytical phase, the pathologist might pick up the wrong slide, one that doesn’t
correlate with the protocol requisition slip linking the slide to
the patient. “This is the critical part of signout,”
Dr. Otis notes. “With us, each slide has at least a surgical
pathology number, the specimen type, and the patient’s name.”
Postanalytical problems can occur at transcription if the pathologist dictates
the wrong number. That would yield the correct diagnosis for the
tissue but link it to the wrong patient. Errors might not be picked
up at sign-out. “Since many of us are using computerized electronic
signouts, we won’t have the requisition at the time of signing
out to verify that what we are seeing correlates to the correct
patient,” Dr. Otis says.
“Fortunately, these things happen very infrequently, because we have set up checks
and balances throughout the system. But we can do better,” he admits.
To reduce the risk of error further, Dr. Otis’ section has started to use
bar coding for accessioning cases. Bar coding gets away from manual
input of patient information, after the initial assignment of a
bar code to each patient at entry into the hospital or when the
specimen arrives in pathology. “Our requisition forms have
bar-code labels and we are working on getting a bar code on slides,”
Dr. Otis says. If you have a reader next to each microscope, that
eliminates some potential for switching slides. Bar coding a slide
is even more useful: You slip the slide under the reader and the
computer information appears as well.
At that point, Dr. Otis says, “You are in the histology laboratory asking,
Is the right slide picking up the right tissue section and getting
the right paper label? That gets back to bar-code technology that
labels a specimen paraffin block. Under a bar-code reader, the bar
code tells what is to be done with that block, such as how many
slides to make.” The slide producer puts information on slides
with indelible ink and produces the number of slides that paraffin
block requires with the patient number and other identifiers on
it. “We are now working on bar coding blocks and slides,”
Dr. Otis says. “That will eliminate preanalytical errors of
patient and case number and block identification.”
Acknowledging the possibility of error and identifying each step where error might
occur is critical, Dr. Otis says. “In our laboratory, we handle
about 45,000 surgical specimens per year, 800 to 1,200 per day,”
he says. “It is optimistic to think that out of those 1,000
or so blocks, errors will not happen. They will, and in most cases
they can be resolved. Any laboratory that handles that amount of
material has to have steps in place to minimize misidentification.”
The same is true of smaller laboratories.
To complicate matters, histology work is being centralized because it’s
hard and possibly not cost-effective to automate small histology
laboratories. “We have already integrated two other hospitals’
histology laboratories,” Dr. Otis says, “which adds
new potential errors—lost specimens and mislabeled specimens.”
Possible safeguards include integrating computer systems and eliminating
all steps except picking up specimens and delivering them to the
central hospital laboratory. Also, Dr. Otis has set up standardized
surgical pathology reporting checklists for the whole health system.
In Dr. Otis’ view, automation in histology has lagged behind because of marketplace
issues. “Histology laboratories are a relatively small marketplace,”
he says, “not like automated clinical chemistry analyzers.
Companies have to see whether it’s worth it to invest R&D
for hardware design compared to what they will get back on their
investment.” The computerized nature of most histology laboratories
will start to make that return attractive, he predicts. There are
now several choices in bar coders and block labelers that are integrated
and interfaced with computer programs that label the number of sections
needed. “In the next few years this will all come into place,” Dr. Otis says.
One of the biggest reductions in human labor has come from
the adoption of automated stainers. “We are certainly aware
of the shortage of histotechnologists,” Dr. Gown, of PhenoPath,
says. “Automation is one way that laboratories can address
that.” Automation has made immunostains more accessible in
general, he notes. “The capability of doing special stains
by instrument has been a big advance.”
Dr. Hicks’ laboratory at the Cleveland Clinic uses the Ventana Nexxus for special
stains. It basically automates all steps, including reagents, temperatures,
and times. “We have some stains that are not yet on the machine,”
Dr. Hicks says, “and we have been working with Ventana to
adapt some of our most frequently ordered special stains to automation.”
This would be a huge help, he says, and would reduce staff exposure
to potentially toxic reagents. Some of the special stains currently
automated in Dr. Hicks’ lab are Congo red, acid-fast bacterial
dye, Gomori silver stain for fungal organisms, trichrome for collagen,
and Steiner stain for spirochetes. “The stain set we would
really love to see on the machine is the Movat stain for elastic
fibers, which is helpful for evaluating blood vessels,” he says.
In the immunohistochemistry laboratory, Dr. Hicks has Ventana Benchmark stainers, which accept
freshly cut slides and have the online capability to bake and deparaffinize
and do antigen retrieval as well. “Basically, all of our immunostains
are on the instrument, other than immunofluorescence,” Dr.
Hicks says. “It has reduced our turnaround time for most immunostains
from 24 hours to around two to six hours.” Immunostains ordered
by noon are reported out the same day. “The clinicians love
it,” he says. “Automated staining really has been a
plus for the laboratory, as the number of antibodies we do has risen.
And new antibodies are easier to work up and incorporate into the instrument.”
Automated staining instruments have two further advantages, in Dr. Hicks’ experience.
First is improved consistency and quality, since the machine controls
all parts of the staining process. For immunostains particularly,
this has been a boon, with about a 70 percent reduction in repeat
stains. Repeats are ordered if controls are not staining appropriately,
the pathologist feels that the stain is too weak or too strong,
or a specimen came off the slide (which can happen during manual
antigen retrieval but is less likely with an instrument). The laboratory
absorbs the cost of repeat stains, so reducing the number saves time and money.
Second are worker issues, from increased productivity to greater histotechnologist
job satisfaction. “We can handle higher volumes of work with
the same number of technologists,” Dr. Hicks says, “because
they are not changing reagents, running timers, and moving slides.”
And technologists now have time to look at the stains they run and
evaluate them, so they don’t have to wait until a pathologist
sees a slide to recognize problems. “Our technologists enjoy
troubleshooting and often call problems to my attention,”
Dr. Hicks says. “The more you work with them, the better they
get, which reinforces my experience that they want to know and understand
how the results of their work are used and they appreciate when
someone takes time to explain it to them.”
(Because Dr. Hicks finds that “more-informed technicians do a better job
and are more interested,” he has started a twice-monthly lecture
series for histology technologists in which staff pathologists,
house staff, or trainees talk on technical topics related to the
laboratory. “The laboratory has responded tremendously to
that,” he says, “and staff has also. We have had no
problem getting staff to give these talks.” Dr. Hicks himself
frequently gives workshops and lectures at National Society of Histotechnology
meetings, mostly about immunohistochemistry.)
Dr. Hicks is now working on a further step in automating the histology laboratory.
This includes testing a new automated routine H&E stainer and
interfacing the LIS with laboratory instruments to allow transfer
of stains ordered to the instruments’ computers. In this way,
a bar code can be generated for the stainers, eliminating the need
for redundant data entry by the technical staff and potentially
reducing errors. Dr. Hicks calls this project “a major task.”
The new routine stainer will automate baking, deparaffinization,
staining, and even coverslipping, he says. “The hope is that
this approach will free up valuable technician time and make our
staff more productive.”
DakoCytomation, too, is marketing automated histology instruments; it makes a histochemistry
stainer and an immunostainer. “Dako’s instruments are
quite good also,” Dr. Hicks says. “For us, the main
issues were the individual needs of our laboratory and a history
of collaboration with Ventana. I know a number of excellent laboratories
that are equipped with Dako instruments and are happy with them.”
“It is not the specific instrument that is important,” he emphasizes,
“but the concept of automation that adds efficiency and productivity
in histology. The goal here is not to reduce [the number of] people,
but to make them more productive.”
Dr. Swanson’s laboratory at the University of Washington Medical Center uses DakoCytomation
instruments. “We just brought in the Artisan system for histochemistry,”
he says. Like Dr. Otis, he finds that an automated instrument increases
reproducibility and allows more efficient use of technologists.
However, he is not automating immunohistochemistry. “Basically, unless
there is a clear quality or manpower issue in the laboratory, immunostaining
performed manually can still provide a superior product,”
he explains. “I have worked in three different large academic
centers in my career, and I have been blessed to work with laboratorians
with technical expertise in IHC who provide day in and day out reproducible
quality results.” Eventually, he thinks that an instrument
may be more cost-effective in terms of manpower. Still, he says,
“if there are no quality issues on the table, I don’t
see the need for automation [of IHC].” In smaller laboratories,
in settings where there are worker shortages, or when it is difficult
to devote one or more technologists to IHC, automation may be a
more urgent consideration, in Dr. Swanson’s view. “We
may yet find that automation suits our needs, but at the moment
that is not the case.”
Beyond current automation devices, Dr. Boyce, of the University of Rochester Medical
Center, sees yet another need. “What I would like to see developed
is a robotic device for handling small specimens like biopsies,”
he says. “We can get from one to 15 bottles from individual
patients, each of which has a number of pieces of tissue in it.
Someone has to take off the tissues, dictate what was received,
how many pieces of tissue, describe them, then pass them into a
teabag and into a cassette.” This process is labor-intensive.
A robot that would suck out the contents of each bottle, scan the
number of pieces of material, and count and size them would be “a
tremendous advance,” Dr. Boyce says. There would have to be
a mechanism to make sure all contents of the container have been
removed, that nothing has stuck to the bottle. The robot could feed
directly into an automated processor of the type Dr. Morales developed.
In general, Dr. Boyce says: “A great deal of what we do, I believe, could
be done by robots with technical oversight. We shouldn’t have
people doing tedious tasks, standing for hours on end taking lids
off containers and describing what they see. We have at least one
person doing this full-time essentially all day.”
Dr. Boyce’s section grosses about 40,000 specimens per year, and large reference
laboratories can do twice that many. “I would have thought
some of these large places would have developed something like this,”
he says. “I suspect it hasn’t happened because no one
has really pushed forward to make it happen. It may also be that
the cost of machines versus the number of specimens has been prohibitive
up to the last few years. But as more people get more biopsies taken
and we deal with more specimens, the time is coming when most laboratories
could benefit from robots.”
When that happens, histology will truly be enjoying the season of automation.
William Check is a medical writer in Wilmette, Ill.
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