Tissue floaters and contaminants in the histology laboratory

Abstract

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.

Full text

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:
huntj2@ccf.org).
bad outcomes occur. Molecular approaches to resolving
identity in tissue specimens have been reported to provide
excellent results in most cases.
1,2
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
3
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.
3
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 floaters: the water bath and the traditional
linear H&E stainer.
MATERIALS AND METHODS
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 fluid 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
PM
), 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
experiments.
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-
cation
⫻
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-
nification
⫻
20).
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
excluded.
RESULTS
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 fluid 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
AM
and 11
AM
. 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).
COMMENT
One of the most serious issues faced in AP is misiden-
tification of tissues, which includes mislabeled specimens,
block identification problems, and tissue contaminants.
3
In
fact, patient identification errors in surgical pathology are
the most rapidly growing category of malpractice claims
involving pathologists.
4
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.
3
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.
1,2,5
However, in approximately 1% of cases, the tissue contam-
inant cannot be resolved, either histologically or through
the use of molecular technology.
2,3
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.
6
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
Time
No. of
Contaminant
Fragments
Percentage of
Slides With
Contaminants
No. of
Slides With
Contaminants
7
AM
000
8
AM
000
9
AM
000
10
AM
000
11
AM
151
12
PM
3153
1
PM
151
2
PM
3153
3
PM
8255
4
PM
4102
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 floaters 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-
ments).
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
PM
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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