Between August and November 2004, clinical laboratories
that use Vacutainer SST, Vacutainer SSTII, and Microtainer test tubes
from BD Diagnostics received bulletins from the company, alerting them
to an immunoassay interference related to the tubes that had been documented
when a particular set of assays was performed on certain platforms from
"Through the application of Six Sigma methods, BD scientists and engineers
have been able to identify an interference between a constituent of our
product and a limited number of assays," says John Boselli, vice president
of quality for BD Diagnostics Preanalytical Systems. BD’s studies have
confirmed, he says, that other assays on the affected instrument systems
do not exhibit these interferences. "BD has also learned that there are
other instrument platforms that appear not to exhibit any immunoassay
interferences associated with our products," Boselli adds. (Related article:
"Instruments, platforms, and assays affected
by the tube-related interference").
Alan Remaley MD, PhD, and his colleagues at the National Institutes of
Health were among the first labs to identify the interference in February
2004, when an NIH endocrinologist noticed that total T3 results
were discordant in comparison with other thyroid test results the laboratory
generated. "We study a lot of patients with thyroid disorders, and in
addition to other thyroid function tests, we perform total T3
tests on all of our patients, even though generally total T3
is not a widely ordered assay. I think that’s probably why this came to
our attention first," explains Dr. Remaley, director of the immunoassay
laboratory in the Department of Laboratory Medicine at the NIH.
Kenneth Blick, PhD, professor in the Department of Pathology and director
of chemistry at the University of Oklahoma Health Sciences Center, Oklahoma
City, says his lab identified a similar type of immunoassay interference
with its folic acid and cortisol assays at about the same time. "One of
our technologists had picked up discrepancies with cortisol, just from
evaluations we were performing in the lab," Dr. Blick says. Cortisol results
generated between March and May 2004 at his institution appeared to be
20 percent to 30 percent higher than those performed earlier in 2004.
Dr. Remaley and his colleagues witnessed a similar bias with the NIH’s
total T3 assay, discovering upon initial review that 25 percent
to 30 percent of all of the total T3 results generated by
his lab between March and May were above the upper limit of the reference
range. "Because we are a research institution, this wouldn’t necessarily
raise a red flag, but upon further review, we discovered that most of
those patients were euthyroid," he says.
Further, the lab noticed discrepancies between the results of total T3
assays performed on two different instrument platforms, says Glen
Hortin, MD, PhD, director of clinical chemistry, Department of Laboratory
Medicine, NIH. "We have two instruments in our laboratory on which we
can perform T3 testing," Dr. Hortin says. "One was relatively
severely affected, but we saw no effect on the other analyzer." This led
the NIH researchers to suspect that the interference was out of the ordinary.
As more laboratories began to notice interferences with particular immunoassay
tests—chiefly endocrine assays—they turned to their manufacturers
for solutions. While these manufacturers worked with BD, of Franklin Lakes,
NJ, to solve the problem, Drs. Remaley and Hortin joined forces with Raffick
Bowen, PhD, clinical chemistry fellow in the NIH Department of Laboratory
Medicine, and other NIH colleagues to study its cause and scope. Their
findings appear in an article published online before print in Clinical
Chemistry on Dec. 2 (www.clinchem.org/cgi/content/abstract/clinchem.2004.043349v1).
Using three types of serum test tubes—plastic Vacuette tubes from
Greiner Bio-One (Longwood, Fla.), BD Vacutainer glass tubes, and BD Vacutainer
plastic SST tubes—the researchers analyzed serum samples from apparently
healthy volunteers, 15 males and 34 females, who ranged in age from 30-60
years. They performed 17 immunoassays (n = 49), 30 clinical chemistry
tests (n = 20), and 33 immunology assays (n = 15) to determine which assays
and tubes showed clinically significant interferences, and examined tube
effects by adding pooled serum to different tube types.
The researchers discovered that when measured by the Immulite 2000 but
not by the AxSym analyzer, total T3 results were significantly
higher for samples collected in the SST tubes (mean value: 2.81 nmol/L)
than either the glass (mean value: 2.15 nmol/L) or Vacuette (mean value:
2.24 nmol/L) tubes. The effect was large enough to substantially shift
the distribution of patient values, increasing the percentage of values
above the reference interval from 11.3 percent to 35.8 percent. Results
from several other tests statistically differed among tube types, but
the differences were not considered to be clinically significant.
Importantly, they found interference not only from SST and SSTII tubes,
but also from plastic plain red-top and tiger-top tubes. "Other tube types
are giving falsely elevated results with the T3 and possibly
other immunoassays," Dr. Bowen says. That the plain red-top and tiger-top
tubes are implicated also is no surprise, he adds, because they have internal
tube coatings (i.e. silicone).
The NIH investigators also confirmed BD’s observations about the cause
of the interference. "We don’t understand the mechanism of the interference
fully, but we do now know that it is a tube additive," Dr. Remaley says.
The immunoassay interference observed with the total
T3 assay was the tip of the iceberg, Dr. Remaley says. "There
were many tests that were affected, and some even far worse than total
T3." For example, vitamin B12 showed a positive
bias approaching 40 percent.
At the University of Oklahoma Health Sciences Center, the 30 percent
increase seen in total T3 was a great concern. "If somebody
had had a level that was toward the upper end of the reference range,"
Dr. Blick says, "this could have pushed them over the cutoff and could
have caused a diagnosis of hyperthyroidism."
However, says Laurence Demers, PhD, distinguished professor of pathology
and laboratory medicine at Penn State University, M.S. Hershey (Pa.) Medical
Center, clinicians expect variability when ordering total T3
results, mainly because the assay has always had a great deal of variability
associated with it. Thus, a 25 percent to 30 percent positive bias may
not be clinically significant, Dr. Demers says: "The recovery on the analytical
systems that measure total T3 is not that good, so it doesn’t
take much to upset the equilibrium that exists between plucking out an
antigen with an antibody and having it interfered with by some factor
that’s in the sample." In addition, for total T3 , he says,
"the variability from sample to sample in patients can run as high as
25 percent, which is about the same as the percentage of bias that’s been
reported in conjunction with the tube-related interference."
Since total T3 is a confirmatory test that’s usually ordered
for patients in whom hyperthyroidism is suspected and the T4
result is borderline elevated, total T3 generally is not a
major test in the thyroid workup, Dr. Demers notes. He says, "Although
an interference such as that described in the Clinical Chemistry
paper could cause a decision zone to be affected, it’s a minor change
that’s generally not quite enough to cause a clinically significant change
in patient management."
But other affected assays could be more problematic. The one that most
concerns D. Robert Dufour, MD, professor of pathology at George Washington
University, Washington, DC, in terms of the assay giving false-positive
results, is the hepatitis B surface antigen. Under certain circumstances,
the affected hepatitis B assay is subject to false-positives, and though
the test manufacturer recommends that all positive hepatitis B surface
antigens be confirmed by a neutralization assay, most clinical laboratories
don’t perform this confirmatory test. "In our laboratory," Dr. Dufour
says, "we do the neutralization assay with weakly positive samples, but
not in all of our positives. So there is the potential that some patients
were misclassified as being hepatitis B positive based on that test, particularly
since it’s probably the most widely used test for hepatitis B surface
Another affected assay is a tumor marker for breast cancer, the BR test
from Bayer Diagnostics of Tarrytown, NY, which has been demonstrated to
have a positive bias of about 20 percent when using the affected test
tubes. "With a 20 percent positive bias, a value of 35, which is the cutoff,
would become a value of 42," says Martin Fleisher, PhD, chairman of the
Department of Clinical Laboratories at Memorial Sloan-Kettering Cancer
Center, New York. "It doesn’t sound like a big deal," he says, "but protocols
are written so that patients will be treated in a certain way if a value
is above or below a certain cutpoint. If that cutpoint is at 35, but then
spuriously becomes a 42 because of an immunoassay interference, obviously
the patient is being treated in a fashion that would be outside of the
limits of the protocol."
Dr. Blick points out that not all immunoassays are "up for grabs." It’s
the small-molecule competitive assays that appear to be most affected.
"The sandwich, or noncompetitive, assays are okay," he says.
Since no one knows exactly when the interference began
to appear, reference laboratories with affected systems are now offering
to go back as far as October 2003 to re-test potentially affected samples.
Clinical laboratories across the nation are having to make difficult decisions
about whether to re-test samples.
"We went back and re-collected specimens and tested everything," Dr.
Fleisher says. "At the time the technical bulletins were released, BD
did not specifically indicate that the tests not listed were unaffected.
We could not take the chance."
Because the interference varied from lot-to-lot, with some test tubes
demonstrating biases and others returning accurate results, without performing
some type of analysis labs were unable to determine whether the lots of
tubes they were using were affected. At the same time, a similar variability
was observed in selected reagent lots used in the affected assays. Fortunately,
Memorial Sloan-Kettering’s lots of tubes appeared to be okay. "If we would
have had a bad lot, however, it would have affected us tremendously because
we have thousands of patients and numerous protocols that are being monitored
all of the time, and small changes in test results are examined frequently,"
Dr. Fleisher says.
The University of Oklahoma Health Sciences Center had recently changed
to a new type of specimen collection protocol, so only cortisol and folic
acid were affected. The affected tests were sent to a reference laboratory
that ran a platform not affected by the interference. "We don’t have a
phlebotomy team, so all of the nurses and the house staff collect samples
all over the center, throughout three hospitals, and we have stocks of
tubes everywhere," Dr. Blick says. Tubes come to the laboratory via a
pneumatic tube system. "For us, it was just an impossible task to go around
and replace all of the tubes or instruct everybody to collect cortisol
and total T3 in glass tubes. Nevertheless, this was the solution
BD was promoting at the time," he says.
BD began distributing a new test tube in October 2004, which it is referring
to as the "adjusted product," and the company expects that once it reaches
all of the affected laboratories, the new tubes will go a long way in
remedying the situation. "In the spirit of trying to restore platform
performance, to make sure we can drive the best possible clinical outcomes,
we decided to take the positive action to adjust our products so that
we could improve the performance of these systems. And we’ve achieved
that," Boselli says.
Ana Stankovic, MD, PhD, MSPH, worldwide medical director of BD Diagnostics
Preanalytical Systems, says: "Going forward, if customers are experiencing
elevated results, the best strategy is to purchase the adjusted product.
But if labs are unable to get the new tubes, they can continue to use
BD’s plain serum tubes until the new tubes become available."
Regardless of what company’s test tube a laboratory
uses to collect specimens, labs need to begin considering the test tube
as a factor that can affect laboratory analyses, Dr. Hortin says. "Using
mass spectrometry, Dr. Steve Drake in our laboratory looked at some of
the components that are shed by test tubes, and he saw a variety of polymers
in the samples that are used as surface coatings for tubes," he says (Clin
Chem. 2004;50:2398-2401). Dr. Hortin now believes collection tubes
are much more complex devices than laboratorians have commonly appreciated
in the past. And he says immunoassay interferences related to tubes are
not a problem unique to BD. "It’s a potential problem with all tubes,"
"Test tubes are not totally inert materials that act as containers. They
have substances in them that are serving as clot activators and surfactants
that are affecting how the components interact with the surface wall.
In addition, there are lubricants that go in the stopper to determine
how well the vacuum is contained, and substances that can leach out of
the gel in the gel tubes."
The lab community can take steps to reduce the probability that the interference
problem will recur. First, Dr. Demers says, if manufacturers work on eliminating
other sources of variability in immunoassays, overall bias can be reduced.
"It would also be helpful if manufacturers would use state-of-the-art
test tubes when establishing ranges for their assays, or perform their
studies in a number of different tubes."
Proficiency testing could also help ensure that tube-related immunoassay
interferences are minimized. "When CAP or other proficiency testing providers
send out challenges," Dr. Fleisher says, "they could ask labs to tell
them what kinds of tubes they are using. Tubes are part of the test system,
and it’s really the same thing as asking what type of reagents or instruments
are being used to perform a test."
Labs might also consider adding new types of statistical analyses to
their protocols. "One of the devices we used in our analysis of the problem
was a moving average," says NIH’s Dr. Bowen. "We were actually able to
track our data from the past three years, and we noticed a shift in the
actual mean of our total T3 results, so labs can probably
monitor shifts in their results by using a moving average of their patient
data, assuming that patient distribution doesn’t change," he says.
Adds Dr. Remaley: "It’s an insensitive technique, but it was sensitive
enough to pick up this problem. Proficiency tests are incapable of detecting
this because you don’t draw the sample in those tubes, so I think the
only way to do this currently is to look at your own results." Diagnostic
companies will have to do their part in helping eliminate these interferences,
Dr. Blick’s suggestion would be what he’s going to do in his own laboratory:
"We’re going to take our control material and add it to any future new
lots of BD collection tubes and mix it in such a way that the sample comes
into contact with the lubricant on the cap, clot activators, etc." Then
they will centrifuge to expose it to the serum separator gel, and run
the assay on that processed control material to look for changes.
For the moment, labs must rely on manufacturers to provide collection
tubes that will enable accurate testing. Drs. Blick and Bowen have tested
BD’s adjusted product, and both say the new tubes seem to be considerably
improved. To help prevent future interference issues, Boselli says, BD
has formed a cross-functional instrument company liaison group. The group’s
focus is to establish close working relationships with instrument company
partners at all levels of product development and post-market surveillance.
Still, a larger, perhaps more troubling, quality control worry remains.
"Those of us in the laboratories have always felt," Dr. Fleisher says,
"that once we have validated a test on a particular instrument using particular
reagents, we were home safe—we knew everything about the test and
the problems that existed. But now we have to worry about not only in
what tube the sample is being collected, but under what conditions it
is being collected."
This situation has uncovered "a weakness in our approach and a chink
in our armor," Dr. Remaley sums up, "revealing that our current practices
are imperfect for detecting this type of problem."
Sue Parham is a writer in Edgewater, Md. To read more about the affected
products, read the technical bulletins released by BD on the following
Web site: www.bd.com/vacutainer/techbulletins.
BD is encouraging laboratorians who are seeking additional information
or support to contact the company at Vacutainer_TechServices@bd.com
or at 800-631-0174.