JMD 2001, Vol. 3, No. 1
Copyright © 2001 American Society for Investigative Pathology & Association for Molecular Pathology
Molecular Approaches to Identification of Tissue Contamination in Surgical Pathology Sections
Maria J. Worsham*,
Sandra R. Wolman
and
Richard J. Zarbo*
From the Department of Pathology,
*
Henry Ford Hospital, Detroit, Michigan; and the Uniformed Services University of the Health Sciences,
F. Edward Hebert School of Medicine, Bethesda, Maryland
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Abstract
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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.
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Introduction
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The finding of potentially contaminating tissue in a surgical or
cytology case can be a vexing diagnostic problem in anatomical
pathology. The ambient rate of this occurrence demonstrates
considerable variation, dependent on the method of detection. In a 1994
College of American Pathologists Q-Probes study of data from 275
laboratories, an overall extraneous tissue frequency of 0.6% (range,
0–1.8%) was detected based on prospective review at the time of case
sign-out. However, the frequency rose to 2.9% (range, 0–8.8%) when
slides were reviewed retrospectively with the specific intent to find
contaminants.1
On occasion, however, an apparently inconsistent tissue fragment that
is not incompatible with the case tissue may be encountered within the
section. Frequently, the source of contamination can be traced within
the laboratory itself or, less commonly, to the physician’s office or
operating room. But in 4 to 7% of cases the origin of the extraneous
tissue is uncertain.1
In the Q-Probes study, roughly 30%
of the extraneous tissues encountered prospectively were abnormal or
neoplastic, 10% presented some degree of diagnostic difficulty, and in
0.6% it could not be determined whether the tissue was truly alien.
Such findings raise the possibility of disease with serious medical
consequences, requiring the clinician to subject the patient to
additional diagnostic studies, possibly necessitating an additional
tissue biopsy, or to initiate close clinical surveillance. The
pathologist must use every available means to pursue the origin of such
tissue fragments in hopes of determining their contaminant status.
Although visual assessment may yield an educated guess, molecular
approaches to selected cases can result in definitive exclusion of
tissue identity. In this paper we present two cases where molecular
approaches were used to decipher the origins of diagnostically
problematic tissue contaminants encountered in routine surgical
pathology practice.
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Materials and Methods
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Patient 1
Molecular Approach: Fluorescence in Situ
Hybridization (FISH)
A routine hematoxylin and eosin section reviewed in Surgical
Pathology included several fragments of gastric mucosa and submucosa
with mild chronic inflammatory infiltrate and a smaller fragment
showing poorly differentiated adenocarcinoma. The specimen (Figure 1A
, Case A) was appropriately labeled as an endoscopic gastric biopsy
from an elderly male with an endoscopic appearance of diffuse gastric
mucosal erythema and pebbling, but no mass or ulcer. However, several
years earlier, this individual had had a gastric adenocarcinoma
in situ in a fundic polyp that was treated by local
excision/polypectomy. In the same tray of slides for a pathologist’s
review was a gastric biopsy from a female whose endoscopic examination
was highly suggestive of malignant ulcer. This specimen (Figure 1B)
was
also appropriately labeled (Case B) and showed a poorly differentiated
adenocarcinoma that was histologically similar to that seen in the
smaller fragment of Case A.
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Figure 1. A: A routine H&E section of an endoscopic gastric biopsy
from an elderly male with diffuse gastric mucosal erythema and
pebbling, labeled Case A. B: A routine H&E section of a
gastric biopsy from a female whose endoscopic examination was highly
suggestive of malignant ulcer
(contaminant), labeled
Case B. C: FISH on tissue section derived from the gastric
biopsy illustrated in A. Note the presence of one red signal
(X chromosome) and one
green signal (Y
chromosome) in gastric mucosal cells.
D: FISH on the tissue section derived from the gastric
biopsy shown in B
(contaminant). Note the
presence of two red signals (representing X
chromosomes) and absence of green signals.
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The original slides and tissue blocks from both cases were available
for study. Additional sections were cut for examination by fluorescence
in situ hybridization (FISH). Hybridization was performed on
5-µm sections from both cases according to previously
published procedures.2, 3
Before hybridization, the region
corresponding to the smaller questionable fragment in the Case A
section was circled with a diamond pencil. Dual probes for the X and Y
chromosomes were used (DXZ1 for the centromeric region of the X and
DYZ3 for the distal heterochromatic region of the Y (Oncor, Inc.,
Gaithersburg, MD). The X probe was labeled with digoxigenin and
a rhodamine (red) fluorophore and the Y probe with avidin and a
fluorescein (green) fluorophore.
Patient 2
Molecular Approach: DNA Polymorphisms-Microsatellite Markers
Three needle core biopsy samples of the prostate examined at
several levels of a hematoxylin and eosin (H&E)-stained section were
composed of benign prostatic tissue (Case C, Figure 2A
). Included on only one of the three levels was a much smaller fragment
infiltrated with prostatic adenocarcinoma (Figure 2B)
. No residual
tissue from this small piece was evident in the tissue block Case C for
retrieval or recuts. However, the malignant area was noted to be
morphologically similar to that observed in another prostate needle
biopsy case processed simultaneously (Case D). The Case D sections
(Figure 2B)
showed near total replacement by prostatic adenocarcinoma,
with Gleason score 8 (patterns 3 + 5) showing glandular perineural
invasion. Both surgical samples were needle biopsies of the prostate
from elderly males, and neither clinical nor serological
prostate-specific antigen findings were helpful.
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Figure 2. A: H&E section from Case C composed of benign prostatic
epithelium. B: H&E section from Case D showing moderately to
poorly differentiated prostatic adenocarcinoma, with Gleason pattern 3
+ 5 (Gleason score =
8), and with perineural invasion. C:
Autoradiograph of microsatellite analysis of DNA from Case C and Case D
(putative source of
contaminant). Lane 1: DNA-1
microdissected from the H&E-stained tissue section area of alleged
contaminant labeled Case D present on the slide from Case C. Lane
2: DNA-1' microdissected from tissue sections of a paraffin block
of prostate adenocarcinoma from which Case D was suspected to
originate. Lane 3: DNA-2 microdissected from the H&E-stained
tissue section area of normal prostatic epithelium from Case C.
Lane 4: DNA-2' microdissected from tissue sections
originating from a paraffin block of Case C.
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The fragments from the original H&E-stained slide (Case C) were
microdissected individually and placed in separate sterile tubes. The
smaller fragment (prostatic adenocarcinoma, probable contaminant) was
labeled DNA-1, and a larger fragment (benign prostatic epithelium)
labeled DNA-2. DNA was extracted using a lysis buffer with Tween and 5
µl of proteinase K, with a 2-hour incubation at 55°C
followed by boiling for 15 minutes. DNA (3–6 µml) was amplified by
polymerase chain reaction (PCR) using a polymorphic VNTR marker D1S80,
and incorporating radioactive 32dCTP. The
reaction products were run on a 6% denaturing polyacrylamide gel and
subsequently exposed to X-ray film.
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Results
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In Patient 1, cells of the gastric mucosa in larger fragments of
Case A (Figure 1A)
contained one red signal (one copy of the X
chromosome) and one green signal (Figure 1C)
. The section from Case B
(Figure 1B)
demonstrated 2 red signals by FISH in glandular epithelial
nuclei. The fragment within the circled area showed only red signals
(Figure 1D)
, indicating two copies of the X chromosome per cell and
absence of the Y chromosome. The chromosomal discrepancy confirmed the
fragment as a contaminant. On the basis of both the morphological
similarities and sex chromosome correspondence, the fragment was
considered to be a contaminant of Case A, most likely originating from
Case B.
In Patient 2, the bands of DNA-1 and DNA-2 migrated differently,
indicating that the two samples were genetically distinct (Figure 2C)
.
Additional DNA samples were extracted from separate sections recut from
different paraffin blocks of the original benign prostatic epithelium
Case C, and labeled DNA-2' and from the original potentially
contaminating tissue (Case D), and labeled DNA-1'. The DNA alleles
extracted from Case C (DNA-2') showed a pattern identical to that of
the larger, benign fragment (DNA-2), and those of Case D, DNA-1' were
identical to the bands seen in the DNA-1 sample from the stained
original fragment that contained the focus of adenocarcinoma. Thus, it
was concluded that the small fragment seen in one section from Case C
represented a contaminant from Case D.
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Discussion
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Surgical specimen mix-ups, even in the most stringently controlled
clinical laboratory setting, are often inevitable. Potential tissue
mismatches can be resolved in several ways. The immunostaining method
of blood group antigens A, B, and O determinants in tissue sections can
identify patients’ tissues correctly.4
Recently,
the use of DNA-based PCR techniques performed on DNA isolated from
paraffin-embedded tissues can correctly determine whether tissue
samples have been interchanged, and can correctly assign specimens to a
patient. Kit-based PCR assays, which amplify and distinguish different
genotypes at the highly polymorphic human leukocyte antigen
locus4, 5
can be applied routinely to fixed-tissue
specimens to confirm the identity of cases where there is
potential tissue contamination. Short tandem repeat sequences or
microsatellites that vary in their repeat number between individuals
are well suited to PCR of minute tissue sections and are an effective
method to confirm surgical specimen mix-ups.6
In this study we have used relatively simple molecular approaches
employing FISH and microsatellite marker analyses to confirm the
contaminant status of suspect tissue fragments in surgical pathology
slides. These approaches are useful both for gender-mismatched tissues,
where simple evaluation of X and Y chromosomes is sufficient for
confirmation of contamination, and for same-gender contaminants which
require the additional steps of microdissection and DNA extraction,
followed by microsatellite marker analysis. The latter, which reveals
distinct DNA fingerprints, can lead to positive identification of the
source of contamination (depending on availability of source tissue and
DNA and on the extent of fingerprinting), whereas the former simply
excludes portions of tissue or certain cells from the diagnostic
evaluation of an individual case without definitive identification of
the source of contamination.
The assessment of DNA fingerprint patterns is limited in certain
respects. The suspected contaminant usually presents as a very small
area, which may be lost in subsequent cuts from the block and,
therefore, lost to additional analyses. The potential number of blocks
from which the contaminant may have originated may be numerous,
necessitating laborious, costly, and extensive microsatellite analyses
and DNA extractions. In Patient 2, although the allelic pattern of
marker DS180 was informative, permitting the recognition of the
extraneous tissue as different, the finding of identity with a single
PCR marker is not unequivocal. Positive identification may require the
use of several markers to exclude a tissue fragment as a possible
contaminant, adding to the labor and expense.
The issue of patient sample misidentification and tissue contamination
is an important one. The Q-Probes quality improvement databases,
derived from many institutions, provide a glimpse of the magnitude of
this problem encountered in surgical pathology laboratories in the
1990s. The overall extent of specimen identification deficiencies
approaches 50% in the poorest performing (10th percentile)
laboratories.7
Types of deficiencies that could lead to
the incorrect assignment of patient tissues include processing
specimens with (i) no label on container, (ii) no requisition slip,
(iii) no patient identification on either container or requisition
slip, (iv) patient name on container or requisition slip does not match
that on master patient index, (v) wrong patient name on both container
and requisition slip, and (vi) incorrect patient name keyed by remote
order entry. Many of these identification deficiencies in the
pre-analytic aspect of surgical pathology testing would be unknown to
the pathologist examining the tissues, assuming the error had not been
detected in the process of accessioning. The laboratory should have in
place specific quality control criteria for specimen rejection from the
accessioning process to prevent such errors from entering the system.
Laboratory users guides should specify that either the clinician or
his/her designee must rectify these types of deficiencies when detected
before the specimen is processed. Such errors may also be caught after
the fact by the clinicians who recognize an inconsistent or unexpected
result or a diagnosis returned on a patient who had had no prior
biopsy. Often, most of the latter error types originate in the clinical
setting, where the tissue sample is placed in an unlabeled container
and subsequently mislabeled by ancillary personnel.
In prospective slide evaluation, the situation that approximates actual
case sign-out by the pathologist, the frequency of contaminant tissue
is quite high, approaching 2.9%.1
Although most
contaminant tissues are loosely referred to as "floaters," in
truth, contaminants derived from the water bath during slide
preparation pose less of a problem than tissue contaminants that are
present within the paraffin block.1
The origin of a
floater is more readily detected as it usually is present only once,
implying that it floated onto the slide, usually from a contaminated
water bath from sections previously cut by the same microtome. The
tissue contaminant present within the block is more difficult to
assess, but when present on subsequent tissue sections, tissues are
available for molecular probing. Furthermore, preservation of the
morphological context allows the geographic localization of even minute
contaminants during microscopic fluorescence examination and subsequent
verification with conventional stains by comparing pre- and
post-sectioned slide levels.
From the Q-Probes data, it appears that the two most common
conditions after normal extraneous tissue are tissue that is
neoplastic or tissue that is non-neoplastic but otherwise abnormal.
Although in over 70% of cases, the source of contamination is a
different case, even the 15% of same-case between- or within-specimen
contaminants may pose serious problems.1
A common example
of this is the alleged knife blade-induced focus of vascular invasion,
which is a subject of lively debate. In this study, we have shown how
FISH in one case, and molecular genetic techniques in another, can be
used to clarify potential cases of tissue contamination arising in
routine surgical pathology practice.
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Footnotes
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Address reprint requests to Maria J. Worsham, Ph.D., Diplomate ABMG, Department of Pathology, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202. E-mail: mworsha1{at}hfhs.org
Supported by National Institutes of Health grant RO1 CA 70923
and American Cancer Society grant RPG-96–093.
Accepted for publication November 22, 2000.
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References
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-
Gephardt GN, Zarbo RJ: Extraneous tissue in surgical pathology: a College of American Pathologists Q-Probes study of 275 laboratories. Arch Pathol Lab Med 1996, 120:1009-1014[Medline]
-
Worsham MJ, Wolman SR, Carey TE, Zarbo RJ, Benninger MS, Van Dyke DL: Common clonal origin of synchronous primary head and neck squamous cell carcinomas: analysis by tumor karyotypes and fluorescence in situ hybridization. Hum Pathol 1995, 26:251-261[Medline]
-
Worsham MJ, Wolman SR, Carey TE, Zarbo RJ, Benninger MS, Van Dyke DL: Chromosomal aberrations identified in culture of squamous carcinomas are confirmed by fluorescence in situ hybridization. Mol Pathol 1999, 52:42-46[Abstract]
-
Ota M, Fukushima H, Akamatsu T, Nakayama J, Katusyam T, Hasekura H: Availability of immunostaining methods for identification of mixed-up tissue specimens. Am J Clin Pathol 1989, 92:665-669[Medline]
-
Shibata D: Identification of mismatched fixed specimens with a commercially available kit based on the polymerase chain reaction. Am J Clin Pathol 1993, 100:666-670[Medline]
-
Tsongalis GJ, Berman MM: Application of forensic identity testing in a clinical setting: specimen identification. Pathology 1997, 18:385-389
-
Nahkleh RE, Baker PB, Zarbo RJ: Autopsy result utilization: a College of American Pathologists Q-probes study of 256 laboratories. Arch Pathol Lab Med 1999, 123:290-295[Medline]
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Abstract
Introduction
