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Tissue floaters and contaminants in the histology laboratory


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Anatomic pathology diagnosis is often based on morphologic features. In recent years, an appropriate increased attention to patient safety has led to an emphasis on improving maintenance of patient identity. Decreasing or eliminating cross-contamination from one specimen to another is an example of a patient identity issue for which process improvement can be initiated. To quantify the presence of cross-contamination from histology water baths and the slide stainers. We assessed for the presence of contaminants in water baths at cutting stations and in linear stainer stain baths. We assessed the potential for tissue discohesion and carryover in tissue samples and we assessed the potential for carryover onto blank slides sent through the stainer. In the 13 water baths examined (totalling 195 L of water), only one fragment of tissue was identified. The stain baths, however, contained abundant tissue contaminants, ranging in size from 2 to 3 cells to hundreds of cells. The first sets of xylenes and alcohols were the most heavily contaminated. Cross-contamination to blank slides occurred at a rate of 8%, with the highest frequency in the late afternoon. Cross-contamination can present a significant challenge in the histology laboratory. Although the histotechnologists' water baths are not heavily contaminated, the stainer baths do contain contaminating tissue fragments. Cross-contamination does occur onto blank slides in the experimental setting.
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Arch Pathol Lab Med—Vol 133, June 2009 Contaminants in Histology—Platt et al 973
Tissue Floaters and Contaminants in the
Histology Laboratory
Eric Platt, BS; Paul Sommer; Linda McDonald, MT, ASCP; Ana Bennett, MD; Jennifer Hunt, MD
Context.—Anatomic pathology diagnosis is often based
on morphologic features. In recent years, an appropriate
increased attention to patient safety has led to an emphasis
on improving maintenance of patient identity. Decreasing
or eliminating cross-contamination from one specimen to
another is an example of a patient identity issue for which
process improvement can be initiated.
Objective.—To quantify the presence of cross-contami-
nation from histology water baths and the slide stainers.
Design.—We assessed for the presence of contaminants
in water baths at cutting stations and in linear stainer stain
baths. We assessed the potential for tissue discohesion and
carryover in tissue samples and we assessed the potential
for carryover onto blank slides sent through the stainer.
Results.—In the 13 water baths examined (totalling 195
L of water), only one fragment of tissue was identified. The
stain baths, however, contained abundant tissue contami-
nants, ranging in size from 2 to 3 cells to hundreds of cells.
The first sets of xylenes and alcohols were the most heavily
contaminated. Cross-contamination to blank slides oc-
curred at a rate of 8%, with the highest frequency in the
late afternoon.
Conclusions.—Cross-contamination can present a signif-
icant challenge in the histology laboratory. Although the
histotechnologists’ water baths are not heavily contami-
nated, the stainer baths do contain contaminating tissue
fragments. Cross-contamination does occur onto blank
slides in the experimental setting.
(Arch Pathol Lab Med. 2009;133:973–978)
The production of histologic sections for diagnosis is a
fundamental process that is critical for all diagnostic
work in anatomic pathology (AP). Most clinical AP labo-
ratories function in a similar fashion, from grossing of
specimens in a designated space (gross room), to process-
ing of tissues in processors, to embedding of tissues in
paraffin wax, to cutting of sections on a microtome, to
staining of glass slides for final review by a pathologist. It
is critical that the pathologist be confident that the diag-
nostic material represented on that final hematoxylin-eo-
sin (H&E)–stained slide truly represents the patient’s di-
agnostic material. And, most laboratories have established
checks and balances within their processes to ensure the
integrity of the tissue and the identity of the tissue
throughout the flow of specimens.
As every diagnostic pathologist realizes, however, this
process is subject to errors and to possible mishaps at es-
sentially every step, ranging from those that even occur
before the laboratory (specimen identity issues) to those
that occur within the laboratory. Frank specimen mix-ups
have been the subject of reports in the literature but are
also the subject of reports in the lay press, especially when
Accepted for publication September 12, 2008.
From the Department of Anatomic Pathology, The Cleveland Clinic,
Cleveland, Ohio.
The authors have no relevant financial interest in the products or
companies described in this article.
Presented as a platform presentation at the annual meeting of the
United States and Canadian Academy of Pathology, Denver, Colo,
March 2008.
Reprints: Jennifer Hunt, MD, Department of Anatomic Pathology,
L25, The Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH (e-mail:
bad outcomes occur. Molecular approaches to resolving
identity in tissue specimens have been reported to provide
excellent results in most cases.
One area that is a constant issue for the diagnostic pa-
thologist is the possibility of tissue floaters and contami-
nants that are transferred to the glass slides during tissue
processing. A retrospective review
estimated that up to
3% of diagnostic slides in AP have tissue contaminants.
These contaminants can occur at any step in the process-
ing of tissues, although certain steps have been identified
as having high potential for contamination. High-risk
steps include the transfer of tissue to the glass slides in
the water bath and traditional H&E staining procedures,
which rely on dipping and dunking slides into sequential
staining baths.
These tissue floaters are particularly trou-
blesome because they are often only on the glass slide and
not in the block. This makes the molecular assessment
quite difficult due to the floaters small nature and the fact
that the DNA may be altered by the staining process.
Therefore, approaches to minimize the possibility for con-
tamination during the sectioning and staining process
would be highly desirable in the AP laboratory.
This study sought to quantify and assess the risk for
tissue floaters and contaminants in the 2 areas that are the
source for most oaters: the water bath and the traditional
linear H&E stainer.
General Methods
For the purposes of this study, standard formalin fixation in
10% neutral buffered formalin and paraffin embedding of tissues
was performed on all samples. The tissue processing was per-
formed using standard techniques on traditional processors (Lei-
974 Arch Pathol Lab Med—Vol 133, June 2009 Contaminants in Histology—Platt et al
Table 1. Staining Solutions and Contaminants in
Staining Baths*
Bath No. Solution
No. of Contaminants per Slide
(Entire Bath Contents)
1 Xylene 49
2 Xylene 33
3 Xylene 73
4 Xylene 41
5 Xylene 14
6 100% Alcohol 8
7 100% Alcohol 126
8 95% Alcohol 194
9 95% Alcohol 101
10 Water 1
11 Hematoxylin 5
12 Hematoxylin 2
13 Hematoxylin 2
14 Hematoxylin 0
15 Water 0
16 Define solution 4
17 Water 0
18 Bluing solution 20
19 Water 0
20 70% Alcohol 9
21 Eosin 1
22 100% Alcohol 2
23 100% Alcohol 4
24 100% Alcohol 6
25 Xylene 0
26 Xylene 1
27 Xylene 0
* This table demonstrates the solution in each of the staining baths
on the linear stainer (see Bath No. and Solution columns). When the
entire bath contents were analyzed for contaminants, different numbers
of tissue fragments were seen in the lineup (see No. of Contaminants
per Slide column). All stain solutions were purchased from Surgipath
Medical Industries, Inc, Richmond, Ill.
ca Microsystems, Bannockburn, Ill; Sakura Finetek USA, Inc, Tor-
rance, Calif). Typical microtome (Leica; Surgipath Medical In-
dustries, Inc, Richmond, Ill; Microm International GmbH, Wall-
dorf, Germany) and water bath setups (Leica; Baxter
International, Inc, Deerfield, Ill) were used, again according to
standard histology procedures. Slides were stained using a tra-
ditional linear stainer (also known as a ‘‘dip and dunk’’ stainer)
(Leica). The linear stainer is a semiautomated machine. The slides
are racked, with a maximum of 20 slides per rack, and dipped
sequentially into each bath for the appropriate amount of time.
They are then coverslipped on a separate automated coverslipper,
and finally they are labeled by hand. Fluids that were collected
and analyzed were processed on the ThinPrep machine (Hologic
[formerly Cytyc], Marlborough, Mass). For the comparison stain-
ing, and particularly for assessment of contaminants, slides were
stained in a continuous workflow, discrete slide stainer (Sym-
phony, Ventana Medical Systems, Inc, Tucson, Ariz).
Water Bath Contamination Assessment
The first experiment was designed to assess the number and
type of contaminants in the water bath that histotechnologists
use to float sections as they are cutting. The water in the bath
was harvested in its entirety, approximately 1.5 L, and was bro-
ken down into aliquots of 50 mL of water. Each aliquot was spun
down separately, using a centrifuge. The supernatant was dis-
carded and the pellets were all recombined in ThinPrep solution
and a ThinPrep slide was prepared for histologic review. This
was performed at the end of the histotechnologists cutting day,
which usually represented an 8-hour shift. The histotechnologists
were asked to save the water from their baths if they did need
to change it during the course of their day, and the same pro-
cedure was followed in that situation.
Linear Staining Bath Contamination
The second experiment was designed to assess contaminants
in the staining solutions of the traditional dip and dunk linear
staining setup. There are 27 staining baths in the linear staining
setup. They consist of the series of xylenes, alcohols, water, and
specific stains shown in Table 1. Each bath contains about 250
mL of fluid. In our laboratory, the average number of blocks cut
per day is 1092, and the average number of H&E slide stains that
are processed is approximately 1637. The stains are generally
changed once a day.
Initial Assessment of Staining Baths. Initially, a pilot ex-
periment was performed to assess overall potential for contami-
nants. In this experiment, 20 mL of uid was extracted from each
water bath at the end of 3 separate days, before the staining so-
lutions were changed for the next day’s work. The fluid was spun
down and ThinPrep slides were prepared and stained with the
Symphony stainer. The slides were assessed for the presence, size,
and type of tissue contaminants. Contaminants were defined as
fragments that were more than 2 cells in size and contained at
least one nucleus. Single keratinocytes were specifically excluded.
Full Assessment of Staining Baths. The second experiment
to assess for contaminants in the staining baths involved taking
the entire contents of each stain bath on 1 day. At the end of the
work day (approximately 5
), each bath was harvested. This
was split into 50-mL aliquots, which were individually spun
down and pelleted. The pellets were combined and ThinPrep
slides were made. Slides were stained on the Symphony stainer
and were assessed microscopically for the exact number, size, and
type of tissue contaminants. Contaminants were defined as frag-
ments that were more than 2 cells in size and contained at least
one nucleus. Single keratinocytes were specifically excluded.
Cross-Contamination From Slide Pickup
The final experiment was designed to determine whether con-
taminants in the stainer baths could be carried over to other
slides during the process of staining. We also wanted to under-
stand whether the contaminants in the water baths could be pick-
ed up on slides and be the source for tissue floaters. To assess
this slide pickup during the staining process, we did 3 separate
Contaminants Picked Up on Slides Prepared With Tissue
Sections. The first experiment was to assess slides that had tis-
sue on them for the presence of tissue floaters picked up during
the staining process. Ten source tissues of different types were
obtained from residual tissues from the gross room. These tis-
sues included colon cancer, endometrial curettings, lung paren-
chyma, bone marrow with bone spicules, fibrous tissue, breast
cancer, fibrous breast tissue, skin, thyroid, and gastrointestinal
stromal tumor. The source tissues were cut into 3 different-sized
fragments: approximately 2 mm in diameter, 4 mm in diameter,
and 6 mm in diameter. These sets of differently sized fragments
were embedded into 3 paraffin blocks, such that one block con-
tained small fragments, one contained medium-sized fragments,
and one contained large-sized fragments. Forty slides were cut
from each block, using meticulous techniques; the histotechnol-
ogist was asked to change the water bath frequently and to min-
imize the potential for cross-contamination. These slides were
stained, with 20 from each group stained in the linear stainer
and 20 stained in the Symphony stainer. The slides were then
assessed for external tissue contaminants and floaters, for lifting
and discohesion of the known tissue fragments, and for move-
ment of these discohesive fragments from one area of the slide
to another.
Contaminants Picked Up on Blank Slides Run Alone and
Alternating With Tissue Sections. We then assessed the pos-
sibility of cross-contamination occurring in the staining baths,
from adherence to slides as they are passed through the linear
stainer. To do this, we sent 40 routine histology blank slides
through the stainer at the end of an average load day, with no
slides that contained tissues. For comparison, 40 slides were also
sent through the Symphony stainer. Then, 200 charged slides
Arch Pathol Lab Med—Vol 133, June 2009 Contaminants in Histology—Platt et al 975
Figure 1. Contaminants in the staining baths. A, This image shows a
representative area on a slide prepared by spinning down the contents
of an entire stain bath. Note the density of contaminating tissue frag-
ments, the large sizes of these fragments, and the fact that the mor-
phology is quite well preserved (hematoxylin-eosin, original magnifi-
10). B, This image shows a higher power view of one of the
contaminating fragments from a stain bath. The fragment is morpho-
logically interpretable and sizable (hematoxylin-eosin, original mag-
were sent through the stainer in alternating positions with slides
that had routine tissue on them. This latter experiment was per-
formed at hourly intervals throughout the course of an average
load day. All of the slides were then assessed microscopically for
the presence of any tissue contaminants. Contaminants were de-
fined as fragments that were more than 2 cells in size and con-
tained at least one nucleus. Single keratinocytes were specifically
Water Bath Contamination Assessment
Thirteen water baths were analyzed, representing 13
different histotechnologists. The average number of blocks
cut per histotechnologist on the routine cutting rotations
is 15 to 20 per hour, or approximately 130 blocks on an
average 8-hour shift. Of all the uid spun down and an-
alyzed microscopically, only one tissue fragment was
identified. This was a large fragment with approximately
100 cells, and it consisted of lymph node tissue. There
were many acellular contaminants in the water baths.
These included keratin, fragments of paraffin, and minute
specks of India ink.
Linear Staining Bath Contamination
Initial Assessment of Staining Baths. The initial as-
sessment of the staining baths demonstrated tissue floaters
and contaminants throughout the staining baths (Table 1).
However, the highest density of contaminants was found
in the first xylenes and alcohols. In these baths early in
the staining lineup, the slides had between 12 and 30 dif-
ferent contaminating fragments on them. The contami-
nants ranged in size from 4 cells to more than 50 cells.
The largest fragments were up to approximately 0.5 mm
in diameter on the glass slide (Figure 1). Some of the con-
taminating fragments were morphologically malignant.
Importantly, the contaminating fragments were extremely
well preserved and could be easily recognized histologi-
cally. Abundant debris was also seen in the background
of the slides. This included keratin, unrecognizable foreign
debris, and minute specks of India ink.
Full Assessment of Staining Baths. The day that was
selected for the assessment was a moderately busy day in
the histology laboratory, with approximately 1192 blocks
processed and approximately 1734 H&E slides stained.
The slides demonstrated contaminants that were again
present in each staining bath but were concentrated in the
early baths. Quantitation of the exact number of tissue
contaminants was performed in this experiment (Figure
2; Table 1). The average number of contaminants per stain-
ing bath was 25.6 (median, 4; range, 0–194). The maximum
number of contaminants was seen in the first set of alco-
hols (stain baths 6–9 in the stain lineup). These baths con-
tained 8, 126, 194, and 101 contaminating fragments, re-
spectively. In this complete sampling of the baths, a large
variety of tissue types was seen, ranging from epithelial
fragments, to lymphoid fragments, to stromal fragments.
The size of the fragments was also quite variable. Many
of the contaminants were around 10 to 30 cells. However,
some were also quite large, measuring 0.5 to 1.0 mm on
the glass slides. Again, in this experiment, some of the
fragments showed morphologically malignant features.
Cross-Contamination From Slide Pickup
Contaminants Picked Up on Slides Prepared With Tis-
sue Sections. In the experiment assessing whether con-
taminants were picked up on prepared tissue sections, we
found several interesting things. First, in tissue sections
containing our known source tissues, we frequently saw
displacement of fragments of the tissue across the slide.
This was especially true of the more friable tissue types,
such as the colon cancer. These displaced fragments were
located at some distance from the source tissue on the
glass slides. This appeared to be due to discohesion or
lifting of the tissue fragments during staining. Second, the
linear stainer had significantly more of these displaced
tissue fragments than the Symphony stainer (45% of slides
vs 22%, P.007). Finally, 2 of the slides from the linear
stainer (3% of the slides stained) had foreign tissue frag-
ment floaters. In other words, these slides had tissue con-
taminants that did not match the type of tissues contained
in the known source tissue block. None of the slides
stained on the Symphony had foreign contaminants.
Contaminants Picked Up on Blank Slides Run Alone
and Alternating With Tissue Sections. We found that
the 40 blank slides run through the linear stainer and the
976 Arch Pathol Lab Med—Vol 133, June 2009 Contaminants in Histology—Platt et al
Figure 2. Graph of number of contaminating fragments from the stainer baths when the entire bath was evaluated. The x-axis has the stain bath
identity and the y-axis has the overall number of contaminants on the ThinPrep slide that was prepared from the contents.
40 run through the Symphony stainer did not pick up
contaminants. When the blank slides were alternated with
tissue sections, and this was performed hourly, we did
find 16 of the 200 blank charged slides harbored tissue
floaters (Figure 3; Table 2). The number of contaminant
fragments per slide ranged from 1 to 3 contaminants. The
tissue contaminants were relatively small, with an average
size of 13.9 cells (range, 4–50 cells). These contaminants
were identified at various time points during the second
half of the day. However, no contaminants were seen be-
tween 7
and 11
. The contaminants could be easily
morphologically identified as specific cell types. The cell
types include adipose and fibrous tissue, stromal tissue,
skeletal muscle, and epithelial tissue (Figure 4).
One of the most serious issues faced in AP is misiden-
tification of tissues, which includes mislabeled specimens,
block identification problems, and tissue contaminants.
fact, patient identification errors in surgical pathology are
the most rapidly growing category of malpractice claims
involving pathologists.
Most laboratories have elaborate
identification processes to avoid specimen mix-ups, in-
cluding regulations for how tissue samples and requisi-
tions are matched up and checked during grossing, for
how cross-checking of identity is done at each step, and
for how the blocks and samples are labeled for accuracy.
However, it is often difficult to eliminate all risk of tissue
contaminants, especially given the hands-on nature of
grossing, processing, embedding, sectioning, and staining
of tissue sections. Floaters and contaminants can occur at
every step, including pickups at the gross bench during
prosection, inside the processor, and at every step in the
histology process.
In one study, the percentage of diagnostic slides with
tissue floaters or contaminants was estimated to be almost
3% through retrospective review.
Up to 30% of these
floaters consisted of abnormal or frankly malignant tis-
sues. Many times these contaminants can be resolved at
the histologic level, particularly when the tissue contam-
inant is derived from a completely different organ system.
And, it has been shown that molecular analysis, using a
DNA fingerprinting assay similar to those used in forensic
analyses, can resolve most potential tissue floaters.
However, in approximately 1% of cases, the tissue contam-
inant cannot be resolved, either histologically or through
the use of molecular technology.
Because of the diagnostic issues that contaminants can
cause, the AP laboratory has a responsibility to reduce
potential for error in every way possible. Process improve-
ment and quality assurance initiatives should focus par-
ticularly on reducing the potential for false positives that
can arise secondary to tissue floaters or contaminants.
This study was aimed at further classifying the types of
floaters and contaminants that occur during histology
processing in the AP laboratory. In particular, we closely
examined the potential for contaminants during the cut-
ting of slides from the water bath and during the staining
of slides on a traditional linear dip and dunk type of H&E
stainer, which are considered to be the areas of most prob-
able tissue floater contamination.
Arch Pathol Lab Med—Vol 133, June 2009 Contaminants in Histology—Platt et al 977
Figure 3. Graph of the number of contaminants that were picked up on blank charged slides as they were sent through the linear stainer. The
x-axis has the time of day and the y-axis has the percent of slides with contaminants.
Table 2. Number and Type of Contaminants Picked
Up on Blank Charged Slides Sent Through the Linear
Staining Setup at Various Time Points
No. of
Percentage of
Slides With
No. of
Slides With
Our results demonstrate that contaminants are present
in the water bath for the microtome, although in very low
concentrations. We postulated that because the tissue sec-
tions floated in the water bath are completely maintained
within a thin sheet of paraffin, fragmentation and break-
ing off of tissue fragments that can cause floaters is less
prevalent at this point in processing. That being said, there
is still a very real potential for tissue floaters and contam-
inants from water bath contaminants. Further study of dif-
ferent time points in the day, different volumes of slides
being cut, and during a series of different days may be
useful in determining the overall risk of oaters from the
water bath.
In contrast, the potential for tissue contamination dur-
ing the staining procedure may be much higher, because
tissue is deparaffinized during the first steps in making
an H&E stain. As the slides are dipped up and down into
the staining baths, the deparaffinized tissue can fragment
and small discohesive pieces can break free and lift from
the slide. We demonstrated this by using tissue blocks
with known tissue types within the blocks in serial sec-
tions. We noted frequent lifting and discohesion on these
slides, especially when the native tissues were either fri-
able (colon cancer), fragmented (endometrial curettings),
or naturally more difficult to adhere to a slide (boney frag-
Knowing that discohesion occurs during the staining
process, we were not surprised to also find high levels of
contamination in the staining baths for the linear stainer.
The contaminants were most abundant in the early baths
of the staining lineup, particularly in the first set of xy-
lenes and first set of alcohols. The latter baths (100% al-
cohol 2 and 95% alcohol 2) had a very high average
number of contaminants, compared with all the other
baths (107.3 fragments vs 11.4 fragments, averaged). How-
ever, tissue contaminants were found sporadically all the
way through the staining lineup. The contaminating frag-
ments ranged in size from a few cells all the way up to
fragments of 0.5 to 1 mm. These larger fragments may
978 Arch Pathol Lab Med—Vol 133, June 2009 Contaminants in Histology—Platt et al
Figure 4. Contaminants seen on glass slides that were sent through
the linear stainer. These images show representative contaminants that
were picked up onto a charged glass slide that was sent through the
linear stainer with other tissue-containing slides (hematoxylin-eosin,
original magnifications
40 [A] and
60 [B]).
contain hundreds of cells. Most of the contaminant frag-
ments were very well preserved morphologically and
some contained frankly malignant cells. It is highly likely
that the contamination of the baths is dependent on the
volume of slides being stained and the time point during
the day that the samples are taken. Additionally, the re-
sults may be sporadic and different baths could be at risk
depending on tissue type. Further experiments are needed
to fully explore the reason for the variable presence of
specific contaminants in the different baths.
Lastly, we wanted to determine whether contaminants
in the staining baths could adhere to slides that were pass-
ing through the staining lineup. Through a set of experi-
ments using blank slides run either with or without other
tissue slides, we found that cell fragments could indeed
make their way onto blank slides and adhere. Up to 25%
of the blank slides that were run through the lineup might
contain contaminants, with this maximum number being
reached at 3
on the study day. The highest number of
contaminants on a single blank slide passed through the
baths was 3. The fragments again ranged in size from 4
cells to 1 large fragment that contained approximately 100
cells. These results indicate that there is a risk for carry-
over from the stain baths to slides that are being passed
through the linear stainer.
There are several things that a laboratory can do to limit
the risk of tissue contaminants and floaters from the water
bath and a traditional linear stainer. First, meticulous
cleaning of the microtome water bath and frequent clear-
ing or changing of the water will almost certainly alleviate
the rare contaminants that may pose a risk for carryover
to subsequent sections being mounted. Second, because
the stainer baths are a potential reservoir of tissue contam-
inants, changing the staining fluids may alleviate some of
the potential for carryover from this source. And, as higher
numbers of the contaminating fragments are localized to
the first xylenes and alcohols, frequently changing these
baths in particular may be useful. Finally, these experi-
ments were performed on one specific linear stainer in-
strument. It is not known if they are transferrable to other
machines that use similar technology. It is, however, un-
likely that there is the same risk of cross-contamination on
a newer instrument that uses a discrete slide staining pro-
cess (Symphony, Ventana). In this continuous workflow in-
strument, the stain aliquots are used only once per slide
and the potential for slide-to-slide or floater cross-contam-
ination is theoretically nonexistent. Additional studies are
needed to fully understand the spectrum of carryover and
cross-contamination that occurs in routine histology pro-
cessing and the impact that these can have on workflow.
We gratefully acknowledge Ventana Medical Systems, Inc, for
the placement of the Symphony instrument in our laboratory for
the purposes of performing this study.
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... This rate of allogeneic contamination would be compatible with a reported 8% rate of contamination during histologic slide preparation. 6 We further evaluated how the detection of contamination affected reporting in 262 cases with greater than 5% contamination (Figure 7). A total of 147 cases (147 of 7571; 1.9%) were reported without modification. ...
... In the anatomic pathology laboratory, contaminating tissue may be present in the paraffin block or on the glass slide. 6 In the molecular laboratory, contamination can occur during the complex process of multiplexed library preparation either during manual or automated pipetting, or it can be a result of reagent impurity, such as the adapter contamination issue in our laboratory. Some massively parallel sequencing technologies are prone to index switching and cross-library contamination. ...
Context.— The presence of allogeneic contamination impacts clinical reporting in cancer next-generation sequencing specimens. Although consensus guidelines recommend the identification of contaminating DNA as a part of quality control, implementation of contamination assessment methods in clinical molecular diagnostic laboratories has not been reported in the literature. Objective.— To develop and implement a method to assess allogeneic contamination in clinical cancer next-generation sequencing specimens. Design.— We describe a method to detect contamination based on the evaluation of single-nucleotide polymorphic sites from tumor-only specimens. We validate this method and apply it to a large cohort of cancer sequencing specimens. Results.— Identification of specimen contamination is validated via in silico and in vitro mixtures, and reference range and reproducibility are established in a panel of normal specimens. The algorithm accurately detects an episode of systemic contamination due to reagent impurity. We prospectively apply this algorithm across 7571 clinical cancer specimens from a targeted next-generation sequencing panel, in which 262 specimens (3.5%) are predicted to be affected by greater than 5% contamination. Conclusions.— Allogeneic contamination can be inferred from intrinsic cancer next-generation sequencing data without paired normal sequencing. The adoption of this approach can be useful as a quality control measure for laboratories performing clinical next-generation sequencing.
... Retrospective and prospective case reviews have shown that floaters occur in 0.01%-1.2% of histologic slides and represent a potential source of diagnostic error. 1,2 When the tissue is from an entirely different organ, it is easy to deem the abnormal histological finding as a tissue floater. Interestingly, it can be challenging to identify a floater when it is from the same anatomical location and often requires additional steps, including deoxyribonucleic acid analysis, to be taken to prove it is a contaminant. ...
... Studies have shown friable native tissues are more likely to have discohesion and lead to tissue floaters. 1 Those tissue fragments that are concerning for floaters will likely occur along the edge of a slide or may be close to the actual patient specimen but not in direct contact. This is seen on our patient's slide in which the floater is not in direct contact with the patient's specimen (Figure 4). ...
Full-text available
A tissue floater or extraneous cross-contamination tissue on a microscopic slide is rare; however, it is a potential cause of diagnostic error. Occasionally, on collecting and processing of specimens, cross-contamination of tissue occurs leading to pathologic findings that are inconsistent with endoscopic findings. If the extraneous tissue is neoplastic, it can lead to a false-positive diagnosis. We present a case of discordant pathological and endoscopic diagnosis of invasive squamous carcinoma of the esophagus.
... 3 In a study assessing the prevalence of contaminants originating from the water bath and slide stainer, floaters were identified on 25% of blank slides when screening for contaminants of any size and type, but on only 3% of tissue sections when using different, more restrictive identification criteria. 5 A proposed explanation from the study authors was that different laboratories differ in ''cleanliness'' for different processing steps. 2 We suggest that taken together, prior efforts to produce generalizable, reproducible, and practical information on microscopic slide contamination highlight that the measured prevalence of contaminants is highly dependent on the identification method and criteria used, and that the lack of standardization in nomenclature and investigation complicates safety event measurement. ...
Context.—: Tissue contaminants on histology slides represent a serious risk of diagnostic error. Despite their pervasive presence, published peer-reviewed criteria defining contaminants are lacking. The absence of a standardized diagnostic workup algorithm for contaminants contributes to variation in management, including investigation and reporting by pathologists. Objective.—: To study the frequency and type of tissue contaminants on microscopic slides using standardized criteria. Using these data, we propose a taxonomy and algorithm for pathologists on "floater" management, including identification, workup, and reporting, with an eye on patient safety. Design.—: A retrospective study arm of 1574 histologic glass slides as well as a prospective study arm of 50 slide contamination events was performed. Using these data we propose a structured classification taxonomy and guidelines for the workup and resolution of tissue contamination events. Results.—: In the retrospective arm of the study, we identified reasonably sized benign tissue contaminants on 52 of 1574 slides (3.3%). We found size to be an important parameter for evaluation, among other visual features including location on the slide, folding, ink, and tissue of origin. The prospective arm of the study suggested that overall, pathologists tend to use similar features when determining management of potentially actionable contaminants. We also report successfully used case-based ancillary testing strategies, including fluorescence in situ hybridization analysis of chromosomes and DNA fingerprinting. Conclusions.—: Tissue contamination events are underreported and represent a patient safety risk. Use of a reproducible classification taxonomy and a standardized algorithm for contaminant workup, management, and reporting may aid pathologists in understanding and reducing risk.
... This is but one flawed process step, with others publishing their own documentation of contamination errors in additional processes at microtome water baths and linear batch staining platforms that affect slide production in histology. [2][3][4] The process flow map of tissue pathology is illustrated in the upper level of boxes in FIGURE 1 . No process of tissue pathology is contamination free. ...
I am pleased to write this editorial on a topic that has been of concern to me for quite some time. If you are a medical director, responsible for the quality and safety of your laboratory operations, or if you have been the unfortunate pathologist associated with a contaminant-related sentinel event and, possibly worse, a medical-legal claim, you will understand. If not, you may want to read on. In this issue of the American Journal of Clinical Pathology, Carll and Pytel¹ designed an experiment demonstrating tissue contamination of cassettes transported in formalin-filled containers. To provide a lay analogy, they have shown that if you take a communal bath with no provision to drain, filter, or cleanse the tub, then the expectation is that whatever was in the tub from its current or prior occupants will potentially contaminate you. Most people would reply, no thanks. This is but one flawed process step, with others publishing their own documentation of contamination errors in additional processes at microtome water baths and linear batch staining platforms that affect slide production in histology.
... Floaters generally indicate contamination subsequent to the point of tissue embedding and have been described as occurring at the cutting bench as well as during tissue staining, whether performed manually or with automated linear stainers. 3,4 Floaters often have morphologic features that belie their nature as contaminants; these features include an abnormal intensity of staining, tissue section folding or shearing, and disjointed location on the glass slide relative to the reference (noncontaminant) tissue section FIGURE 1 . When suspicion of contamination arises, floaters can be conclusively eliminated by recutting the tissue from the paraffin block. ...
Objectives: Tissue carryovers are contaminants of surgical pathology cases in which extraneous tissue is incorporated into tissue blocks. Carryovers occur most frequently at the grossing or embedding stations, but little is published about them. We sought to analyze their transmission during transit to the histology lab. Methods: Cassettes of friable donor tissue were mixed with cassettes of spongy recipient tissue in formalin-filled containers and agitated by shipment via pneumatic tube. The tissue cassettes were processed, embedded as blocks, and cut as usual. Liquid samples were prepared from the submission containers as well as from workstation submission containers and histology tissue processor waste. Results: A high rate of contamination (14.9%) was observed under these artificial conditions. Friable donor tissue, including urothelium and colorectal adenocarcinoma, were promiscuous contaminants, as were placental villi. Fluid from submission containers showed viable tumor cells and fragments, which were also present in workstation submission containers and in tissue processor waste fluid. Conclusions: This study implicates liquid transport media as a possible avenue of contamination during submission and transportation of tissue cassettes for histologic processing. Attention should be given to the friability of submitted tissue and physical agitation of the cassettes in transit. Such contaminants may be present in the fluid in tissue submission bins and in tissue processor fluid.
... This dataset is structured to mimic retrieval tasks in clinical practice. The patch-to-scan classification may appear trivial but has relevant clinical applications such as "floater detection" where we need to find the origin of a foreign tissue [26,18,25]. To generate the training and test datasets, Babaie et al. [1] slide a window with no overlapping over each WSI to crop patches of size 1000×1000 and ignored background patches by analyzing both homogeneity and gradient change of each patch. ...
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The Kimia Path24 dataset has been introduced as a classification and retrieval dataset for digital pathology. Although it provides multi-class data, the color information has been neglected in the process of extracting patches. The staining information plays a major role in the recognition of tissue patterns. To address this drawback, we introduce the color version of Kimia Path24 by recreating sample patches from all 24 scans to propose Kimia Path24C. We run extensive experiments to determine the best configuration for selected patches. To provide preliminary results for setting a benchmark for the new dataset, we utilize VGG16, InceptionV3 and DenseNet-121 model as feature extractors. Then, we use these feature vectors to retrieve test patches. The accuracy of image retrieval using DenseNet was 95.92% while the highest accuracy using InceptionV3 and VGG16 reached 92.45% and 92%, respectively. We also experimented with "deep barcodes" and established that with a small loss in accuracy (e.g., 93.43% for binarized features for DenseNet instead of 95.92% when the features themselves are used), the search operations can be significantly accelerated.
... Common causes of this problem appear to be because of the pickup of floaters in the water bath during sectioning, 2 or during staining of slides when deparaffinized tissue can easily fragment. 3 The dilemma pathologists often face is whether such a tissue floater truly belongs to the case in question, or if instead it represents a true contaminant from another patient's sample in which case it should be ignored. This poses a major conundrum when the tissue floater contains malignant cells, and is unlikely to be derived from an obviously different organ or tumor. ...
Context.— Pathologists may encounter extraneous pieces of tissue (tissue floaters) on glass slides because of specimen cross-contamination. Troubleshooting this problem, including performing molecular tests for tissue identification if available, is time consuming and often does not satisfactorily resolve the problem. Objective.— To demonstrate the feasibility of using an image search tool to resolve the tissue floater conundrum. Design.— A glass slide was produced containing 2 separate hematoxylin and eosin (H&E)-stained tissue floaters. This fabricated slide was digitized along with the 2 slides containing the original tumors used to create these floaters. These slides were then embedded into a dataset of 2325 whole slide images comprising a wide variety of H&E stained diagnostic entities. Digital slides were broken up into patches and the patch features converted into barcodes for indexing and easy retrieval. A deep learning-based image search tool was employed to extract features from patches via barcodes, hence enabling image matching to each tissue floater. Results.— There was a very high likelihood of finding a correct tumor match for the queried tissue floater when searching the digital database. Search results repeatedly yielded a correct match within the top 3 retrieved images. The retrieval accuracy improved when greater proportions of the floater were selected. The time to run a search was completed within several milliseconds. Conclusions.— Using an image search tool offers pathologists an additional method to rapidly resolve the tissue floater conundrum, especially for those laboratories that have transitioned to going fully digital for primary diagnosis.
... Floaters and carry-overs may result in nearmissed events, thus affecting final histological diagnosis as demonstrated in a study. 18,19 Most of the artefacts during staining are due to altered staining intensity as a result of low-quality stains, impurities attributed to insufficient filtration of staining solutions, and lack of potential of hydrogen (pH) and temperature monitoring 17,19 (Figure-3). Majority of technicians never consulted equipment manual for handling operating errors; whereas the technical persons of the relevant companies supplying the equipment lacked the sufficient knowledge and competency to handle trouble-shootings. ...
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Abstract Objective: To evaluate pre-microscopic errors in anatomical pathology. Methods: The cross-sectional descriptive study was conducted at the Department of Pathology of a tertiary care hospital in Lahore, Pakistan, from September, 2016, to January, 2017, and comprised surgical pathology specimens. Errors were noted across the pre-microscopic process. Defects per million opportunities were calculated to determine sigma metric value in every step, from requisition to slide preparation. Root cause analysis was applied to the process of histology preparation to identify the root cause of each previously identified problem using Eindhoven classification. All errors were recorded on a pre-designed proforma. Results: There were 2420 specimens. While errors were encountered in all phases of the pre-microscopic process, but the (G6: n=1085, 44.83%), followed by requisition (R3: n=893, 36.9%) and cover slipping (C1: n=776, 32.06%). Conclusion: Development of standard procedures and protocols with staff training is likely to help in controlling the errors. Keywords: Anatomical pathology, Eindhoven, Errors, Root cause analysis, six sigma metrics.
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Oxyntic gastric heterotopia (GH) in the colon is not common. Its presence in a colon tubular adenoma is even rare. A 73-year-old woman with a history of resected colon carcinoma underwent periodical colonoscopies for the removal of tubular adenomas for 12 years. In the last colonoscopy, a sessile, non-ulcerated polyp, centrally depressed, with a smooth surface, measuring 20 mm, located at 50 cm from the anal verge was excised. A histological study identified a tubular adenoma with focal low-grade dysplasia and ectopic gastric oxyntic epithelium. The GH, composed of parietal and chief cells, and was found incidentally. Oxyntic GH in a tubular adenoma is extraordinarily rare. To the best of our knowledge, there is only one previously published case. The main possible difficulties and∕or errors in the diagnosis include a tissue floater or a cross-contaminant. Precise diagnosis of oxyntic GH is basic for appropriate management. Diagnosis relies on histopathological examination. The immunohistochemical study for mucin 6 (MUC6) can confirm the nature of the epithelium. Oxyntic GH has the potential to produce serious complications including tumor development. However, GH is considered a benign disease and adenocarcinoma rarely occurs in the heterotopic mucosa. The optimal treatment of oxyntic GH associated with a tubular adenoma is endoscopic complete polypectomy.
Tissue contaminants in anatomical pathology are not uncommon. While issues related to the presence of extraneous tissue on glass slides are often easily resolved, this is not always the case and several factors may contribute to diagnostic difficulty. Because of this, familiarity with the different steps involved in handling specimens in the anatomical pathology laboratory is essential when troubleshooting possible cross-contaminants. Most commonly, the specimen constituting the source of cross-contamination is handled before the actual contaminated case; however, this is not always so. In this article, we review the steps involved in processing pathology specimens as they pertain to cross-contamination; share an approach covering how to troubleshoot and prevent tissue contaminants in a systematic and practical manner; present some examples from our own experiences; and compare our experience to what is reported in the literature. The information included in this article will be of use to all members of the anatomical pathology team including medical laboratory technologists, laboratory managers and supervisors, pathologist assistants, trainees in pathology, and pathologists.
The finding of possibly contaminant tissues or cells in surgical or cytology case material can be a challenging problem in diagnostic anatomical pathology samples. The reported rates of occurrence have ranged from 0 to 8.8% (including prospective and retrospective cases). A diagnostically dissimilar tissue fragment, whether contiguous with other tissue or among other fragments within a paraffin section, and which is not incompatible with the case tissue, often requires a rigorous investigation to confirm or deny its relevance to the case. Fluorescence in situ hybridization using dual red and green DNA probes to regions of the X and Y chromosomes, respectively, were used in one case where the potential contaminant was suspected to have originated from a male patient. The putative contaminant tissue fragment was confirmed as male, with cells having one X and one Y chromosome, unlike the other tissue fragments on the slide with two X chromosomes. In a second case, DNA polymorphisms were used to compare allelic patterns that were informative not only in proving the extraneous tissue as a contaminant, but in addition, could be used to trace the latter to its original tissue source. The molecular tools of fluorescence in situ hybridization in sex-mismatched cases and of DNA microsatellite probes that are applicable to paraffin sections can provide definitive identifiers of tissues and individual cells. They are important adjuncts to histology for the anatomical pathologist when faced with the diagnostic problems of tissue contamination encountered in routine practice.
To develop a multi-institutional reference database of extraneous tissue (contaminants) in surgical pathology. In 1994, participants in the College of American Pathologists Q-Probes quality improvement program performed prospective and retrospective evaluations of extraneous tissue found in surgical pathology microscopic sections for a period of 4 weeks or until 1000 slides were reviewed in each participating laboratory. Two hundred seventy-five surgical pathology laboratories institutions, predominantly from North America. Extraneous tissue contamination rate for slides in prospective and retrospective reviews; staffing and practice procedures; location of extraneous tissue on slides; type of extraneous tissue (normal, abnormal, nonneoplastic, neoplasm, microorganisms, etc); class of extraneous tissue (slide or block contaminants); source of extraneous tissue (different or same case); origin of extraneous tissue (pathology laboratory, physician's office or operating room); and degree of diagnostic difficulty caused by extraneous tissue. Three hundred twenty-one thousand seven hundred fifty-seven slides were reviewed in the prospective study and 57083 slides in the retrospective study. There was an overall extraneous tissue rate of 0.6% of slides (2074/321757) in the prospective study and 2.9% of slides (1653/57083) in the retrospective study. Of those slides with extraneous tissue, the extraneous tissue was located near diagnostic tissue sections in 59.5% of the slides reviewed prospectively and in 25.3% of slides reviewed retrospectively; deeper sections were performed to evaluate extraneous tissue in 12.2% of prospective cases and in 3.1% of retrospective cases. Of the laboratories, 98% had written guidelines for changing solution in tissue processors, and 64.9% had guidelines for maintaining water baths free of extraneous tissue. A total of 98.9% used lens paper, filter bags, or sponges for processing fragmented and small specimens. Written protocols for documentation of extraneous tissue in surgical pathology reports were established in 6.1% of laboratories, for removal of extraneous tissue from blocks in 5.7%, and for removal of extraneous tissue from microscopic slides in 4.7%. In 24% of laboratories no comment or record was kept to document extraneous tissue. Extraneous tissue consisted of neoplasm in 12.7% of the prospectively reviewed slides and in 6.0% of the retrospectively reviewed slides. For the prospective study, 59.4% of extraneous tissue was classified as slide contaminants, and 28.4% was found to be contaminants within the paraffin block; for the retrospective study, 72.9% was classified as slide contaminants and 15.9% as block contaminants. For the prospective study, 63.2% of extraneous tissue was presumed to be from a different case, and in the retrospective study, 48.5% was presumed to be from a different case. Over 90% of extraneous tissue was thought to originate from the pathology laboratory. The degree of diagnostic difficulty caused by extraneous tissue was judged to be severe in 0.4% of slides in the prospective study and 0.1% of slides in the retrospective study. In the prospective study, it could not be determined whether the tissue in the diagnostic sections was extraneous in 0.6% of slides, and in the retrospective study, it could not be determined whether tissue in the diagnostic sections was extraneous in 0.1%. This study has documented the frequency, type, origin, source, and diagnostic difficulty of extraneous tissue and presents benchmarks of extraneous tissue experienced in the general practice of surgical pathology.
A recurring problem in surgical pathology practice is specimen mix-up and floater contamination. While many cases can be resolved histologically, a significant number remain unclear and may have serious clinical and medicolegal implications. To design a microdissection and genotyping assay to identify contaminating floater tissues in paraffin-embedded tissues that is optimized for small samples, and to use the assay to resolve a series of clinical cases with floater tissues. Twenty-one cases of possible tissue floater contamination in paraffin-embedded tissue blocks were included. Using 4 unstained, 4-microm-thick histologic sections, multiple sites were microdissected under direct visualization either by hand or by laser capture microdissection. Nonneoplastic and neoplastic tissues were sampled. Polymerase chain reaction was performed for a panel of 10 polymorphic microsatellite markers at 1p34, 3p26, 5q21, 9p21, 10q23, and 17p13. Allele size and content were analyzed semiquantitatively by fluorescent capillary electrophoresis, and the genotypes for the tissues in the paraffin-embedded tissue blocks were compared for identity. Tissue identification was successful in all cases, despite small tissue sample size and fixation effects. Comparative analysis of neoplastic tissue floaters and the presumptive source tumor was performed when possible to control for possible allelic loss or microsatellite instability. Microdissection and genotyping are effective and reliable means to objectively resolve problems of possible floater contamination. Even minute tissue samples provide sufficient DNA template for polymerase chain reaction microsatellite analysis. Because of the potential clinical implications of floaters, we recommend that all suspected floaters that would change a diagnosis from benign to malignant be subjected to genotyping assay to confirm the identity of the floater tissue.
A total of 272 surgical pathology claims reported to The Doctors Company from 1998 through 2003 were reviewed. They were analyzed and repetitive patterns involving both specimen type and category of diagnostic error were identified. These patterns were then compared with those uncovered in a prior review of 218 surgical pathology claims reported from 1995 through 1997 to identify trends and see if new patterns of diagnostic error had emerged.
A 62-year-old woman presented with recurring right upper quadrant pain and underwent a routine laparoscopic cholecystectomy. A large gallstone was found impacted in the fundus of the gall bladder. Interestingly, aside from noting mild inflammation of the gall bladder wall, microscopic examination of the specimen identified 2 fragments of benign thyroid tissue. Given the routine nature of the surgical procedure and lack of abnormality detected during the operation, the attending pathologist suspected extraneous tissue contamination ("floater") of the pathology specimen and submitted the block and slides to Molecular Pathology. The thyroid tissue-containing fragments and gallbladder wall were independently isolated and subjected to genetic fingerprinting using a standard forensic DNA identification panel. All fragments showed the identical fingerprint, strongly suggesting that they belonged to the same patient. The results indicated that the thyroid tissue was from an ectopic rest adjacent to the gall bladder, which has been reported only very rarely in the previous literature and illustrates the unusual use of molecular genetic testing to confirm the presence of ectopic tissue versus contamination.
This review summarizes our experience using blinded review as a method to measure quality in surgical pathology. It details the specifics about how the review is performed, the weaknesses in the method, and then summarizes our results so far. These results suggest that error rates correlate with the individual pathologist, the type of specimen, the type of diagnosis, subspecialization, and the number of pathologists who review a case. Errors do not correlate with workload. This method is relatively unbiased and effective at identifying significant errors in real-life clinical practice. The drawback to this method is the amount of work involved. Blinded review, performed so that errors can be corrected in a timely manner, and eventually integrated into an interlaboratory review process, represents a realistic and fair method to provide quantitative quality assurance information.