AMERICAN
JOURNAL
OF
CLINICAL
PATHOLOGY
Editorial
W h e r e
Identification
D i d
of
T h i s
Sample
C o m e
Mix-ups
In this issue of the American Journal of Clinical Pathology,
Shibata reports on the "Identification of mismatched fixed
specimens with a commercially available kit based on the polymerase chain reaction.'" Although Shibata has previously published an isolated case report of a mislabeled specimen diagnosed by DNA testing,2 this publication marks the first series
(16) on the important issue of specimen misidentification and
its resolution by the new "DNA fingerprint" technologies.
With literally thousands of biopsies performed annually in
the United States in every hospital, it is inevitable that examples of sample mix-ups will come to light. Moreover, clinical
laboratory samples may also have been switched and can now
be tested for identity. Although this is most easily performed on
blood samples, we have even demonstrated the ability to verify
the identity of some urine samples obtained for drug testing.
Sample identity errors occur in three primary contexts: prelaboratory labeling errors; laboratory labeling errors, particularly during the placement of specimens in cassettes and during
histologic slide preparation; and inclusion of fragments from
other specimens, so-called floaters, particularly during histologic slide preparation. The typical case involves a diagnosis of
cancer by the pathologist in a patient without other signs of
cancer. This represents an area of great potential liability for
the hospital and professional staff.3
Attorneys, patients, and pathologists have sought the help of
the Armed Forces DNA Identification Laboratory (AFDIL)
more for clarification of alleged sample mix-ups than for criminalistic testing, parentage testing, remains identification
testing, determination of human origin, or any other single
purpose. It has also been the experience of commercial laboratories performing polymerase chain reaction (PCR)-based
DNA testing that many requests relate to alleged sample
mix-ups.
It is foreseeable that hospitals may wish to store a drop of
blood collected by the blood bank during routine blood crossmatching, perhaps as" a mere air-dried blood stain, from all
patients on whom a biopsy is performed. Currently, some medical examiners store a drop of blood in case later questions of
identification arise.
The PCR has revolutionized DNA testing and, indeed, even
biologic science. Truly, PCR is nothing more than a simple
method of copying a small region of DNA; a millionfold amplification of a target DNA is common. It is easy, quick, and
inexpensive to perform. It is highly sensitive and specific. Moreover, PCR-based DNA tests can yield meaningful results when
the DNA has largely been broken down and degraded.
Invention of PCR is credited to Kary Mullis, who had been
working at the Cetus Corporation (later merged with PerkinElmer); however, the patent rights, other than for identity testing, were later sold to Roche. The PCR technique was first
The opinions expressed are those of the author and do not necessarily reflect those of the Department of Defense or the Department of the
Army.
592
by
F r o m ?
DNA
Testing
published in 1985.4 The original concept was demonstrated
using typing of the /3 hemoglobin, but the first commercial
application was in human lymphocyte antigen (HLA) DQ-a
testing. Thefirstdescription of the utility of HLA DQ-a testing
for forensic purposes was described in 1986.5 Since then, there
have been many publications on the use of PCR dot/blots for
identification.6,7
Significantly, the first case in which DNA testing was introduced into court in the United States was in the 1986 case of
Pennsylvania v. Pestinikas* That case involved the HLA DQ-a
dot/blot DNA testing by Ed Blake of Forensic Science Associates (Richmond, CA). The operators of a rest home in Pennsylvania were charged with the negligent homicide of an elderly
gentleman who had died of apparent starvation. The autopsy
results were questioned, resulting in an exhumation. In response, the original autopsy physician then alleged that the
internal organs had been switched with those of another body.
The body had been embalmed and buried for approximately 1
year. The DNA was very degraded (average fragment size was
100 base pairs). The 82-base pair HLA fragment amplified
from the internal organs matched that of other body tissues.
The defendants were convicted of negligent homicide, but acquitted of the charge of tampering with the body.
It is significant that PCR-based testing can be performed,
although not always successfully, on specimens that have been
formalin-fixed, paraffin-embedded, and stained on a glass
slide. In the case of questionable results, actual DNA sequencing of the amplified DNA fragments can be performed to confirm or clarify test results. Classic DNA testing performed by
restriction fragment length polymorphisms (RFLP), also
known as the Southern Blot technique, cannot be performed
on fixed paraffin-embedded tissue specimens.
Performance of the DNA testing using the commercially
available kit (AmpliType, Perkin-Elmer, Norwalk, CT) is simple and straightforward. The test kit has proven to be a robust
assay that is not difficult to perform properly, and the manufacturer's manual is understandable and easily followed. No major equipment is needed, except a thermal cycler ($4000 to
$ 12,000). Kits are sold in lots of 50 for $825. We calculate costs
as less than $35 per test, including kit costs, labor, and controls.
The testing can be performed by one technician in less than
1 day.
Since the introduction of the commercial kit in February
1990, more than 1 million tests have been produced and distributed. More than 200 forensic scientists have been trained to
perform HLA DQ-a typing. More than 75 crime laboratories
and more than 100 nonforensic laboratories have purchased
DQ-a kits to date. HLA DQ-a test evidence has been used in
more than 200 court cases. With the exception of less than a
handful of cases (none on the basis of inadequacy of the technology), this technology has gained widespread courtroom acceptance (Personal communication, Kristen Garvin, PerkinElmer, October 1, 1993).
The commercial kit involves a "reverse dot/blot analysis," in
which amplified sample DNA fragments are probed by a com-
AMERICAN JOURNAL OF CLINICAL PATHOLOGY
593
Editorial
plementary fragment of DNA bound to membrane strips. The
probes are known as allele specific oligonucleotide or sequence
specific oligonucleotide probes, which are of sufficient length
to be specific yet short enough to require an exact sequence
match. As labels are attached to the sample DNA during the
amplification process, a colorimetric reaction indicates probe
hybridization. Accordingly, blue spots on the strip indicate one
or another specific alternative HLA sequence. The process is
analogous to an enzyme-linked immunosorbent assay with antibody bound to a solid phase.
Testing for human identification is less demanding than for
some other purposes, such as infectious disease testing where
only a few copies of target DNA may be present. However,
laboratories must be alert to problems arising from cross-contamination, as the PCR technique is exquisitely sensitive.
Recently, the College of American Pathologist's Molecular
Pathology Resource Committee issued a checklist for accreditation of molecular pathology laboratory testing, including the
performance of PCR-based DNA testing and specifically as applied to the area of identification. Furthermore, the College of
American Pathologists, as well as others, offers proficiency testing surveys that include HLA DQ-a testing.
The major drawback to HLA DQ-a testing is its limited discriminatory power, at least compared with classic RFLP testing. It yields a discriminatory power of approximately 1 in 20.
However, as Shibata points out, this discriminatory power is
adequate for the determination of most clinical sample mixups. Moreover, the manufacturer has just begun sales of a second dot/blot strip with other discriminating probes, commonly
known as the "Polymarker kit," which when used in conjunction with the HLA DQ-a strip will yield an average discriminatory power of more than 1 in 2000. These kits are similar in
cost ($995 per 50 kits).
In the future, DNA testing will become a large part of the
clinical testing performed in the pathology laboratory. Genetic
testing is no longer a matter of rare inherited diseases, but of
such diseases as hypertension and atherosclerosis. With the pro-
gression of the Human Genome Project, the molecular structure of the human organism is becoming far better known and
this is bound to affect the practice of medicine and the face of
health care. DNA testing in the future will not only be useful
for genetic testing of blood samples, but for tumor diagnosis,
prognosis, monitoring, and determination of chemotherapeutic resistance of tumors, as well as for diagnosis of infectious
agents.
VICTOR WALTER WEEDN, MD,
JD
Lieutenant Colonel. US Army, Armed Forces
Institute of Pathology
Program Manager
Department of Defense DNA Registry
REFERENCES
1. Shibata D. Identification of mismatchedfixedspecimens with a
commercially available kit based on the polymerase chain reaction. Am J Clin Pathol 1993; 100:666-670.
2. Shibata D, Namiki T, Higuchi R. Identification of a mislabeled
fixed specimen by DNA analysis. Am J Surg Pathol
1990;14:1076-78.
3. Mazer v. Lipschutz, 327 F.2d 42 (3d Cir. 1963).
4. Mullis KB, Faloona F. Specific synthesis of DNA in vitro via a
polymerase-catalyzed chain reaction. Methods Enzymol
1985;155:335-350.
5. Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. Analysis
of enzymatically amplified beta-globin and HLA DQ alpha
DNA with allele specific oligonucleotide probes. Nature
1986;324:163-166.
6. Blake E, Mihalovich J, Higuchi R, Walsh S, Erlich H. Polymerase
chain reaction (PCR) amplification and human leukocyte antigen (HLA)-DQ-alpha oligonucleotide typing on biological evidence samples: Casework experience. J Forensic Sci
1992;37:700-726.
7. Comey CT, Budowle B. Validation studies on the analysis of the
HLA DQ-alpha Locus using the polymerase chain reaction. J
Forensic Sci 1991;36:1633-1648.
8. Commonwealth of Pennsylvania v. Pestinikas, court of Common
Pleas, Lackawanna County, #CR1019A-D/CR1020A-E, Dec.
28, 1988.
Vol. 100- No. 6