DNA PRroFILING as AN ADJUNCT QuaLiry ConTROL/
QUALITY ASSURANCE IN ForENsIc ToxICOLOGY

INTRODUCTION

Acquiring accurate and authentic analytical data
on biological evidence to seek the chemical basis for
the cause of accident or death is the main objective of
forensic toxicology laboratories. For achieving that
objective, strict adherence to quality control/quality
assurance (QC/QA) procedures is essential. Such ad-
herence allows not only the correct scientific interpre-
tation, but also the judicial admissibility, of analytical
results with a high degree of confidence. This perspec-
tive is of particular importance in aircraft accident
investigations, wherein multiple types of postmortem
specimens are collected for analysis from the victims,
and depending upon the nature of an aircraft acci-
dent, victim bodies are frequently scattered, disinte-
grated, commingled, contaminated, and/or putrefied.
The complications associated with the identification
of remains and matching of tissues frequently hamper
investigation, including toxicological evaluation.
However, such complications are effectively resolved
in our laboratory by DNA profiling. Since environ-
mental and biological factors responsible for putrefac-
tion also damage DNA (Budowle et al., 1991), the
genetic material is potentially degraded in postmor-
tem samples generally obtained from aviation acci-
dent sites.

Analysis of degraded DNA by the conventional
restriction fragment length polymorphism technique
is not as effective as it is by the method based on the
polymerase chain reaction (PCR), a directed in vitro
DNA synthesis. The former is more suitable for fresh
samples containing non-degraded, high molecular
weight DNA. This technique is also relatively cum-
bersome and time consuming (Sajantila et al., 1991).
The PCR-based analysis has been successfully em-
ployed for analyzing minute amounts of degraded,
low molecular weight DNA in contaminated and
putrefied samples (Hochmeister et al., 1991; Reynolds
and Sensabaugh 1991; Sajantila et al., 1991). Thus,
the PCR analysis was adopted in the present study.

Besides the identification of victims and of post-
mortem tissues, we apply DNA profiling in resolving
other postmortem toxicology QC/QA issues, such as
accessioning/analytical errors and interpretation of
unusual analytical results. Covering the whole spec-
trum of forensic toxicology operation, three situations
—tissue mismatching/commingling, unacceptable batch
analysis, and unusual analytical finding — are hereby
exemplified to illustrate the effective applicability of
the DNA analysis in addressing those issues. In these
situations, the identifications of the samples were
determined by target-DNA amplification and DNA
hybridization using sequence-specific probes. During
this process, six independently inherited genetic mark-
ers were examined (AmpliType®, 1995). The com-
bined discriminating power of these genetic markers
statistically established the fidelity of the submitted
bio-samples in the three described situations.

SITUATIONS

Tissue mismatching/commingling

In a biohazard shipping box, five bags containing
postmortem tissues from an aircraft accident, involv-
ing two occupants, were submitted for toxicological
evaluation. The types of tissues were liver, lung,
kidney, and muscle; all samples appeared to be putre-
fied. Out of these five bags, two bags were clearly
marked: the name of one victim was written on one
bag, while the name of the other victim on the second
bag. On the other hand, doubting the exact origin of
the tissues, the remaining three bags were labeled with
the names of both victims. Samples from the five bags
were analyzed for DNA to establish the source of the
tissues so that toxicological analyses could effectively
be performed on the appropriately identified samples.

Unacceptable batch analysis

In our laboratory, the accessioning of samples for
analysis basically requires the preparation of two types
of documents: batch-review and batch-analysis. The


The concentrations of atropine found in various
tissues of the victim are presented in Table 3. Atropine
was present in the amounts of 318 ng/ml of blood and
727 ng/g of lung. It was not detected in the other
tissues (liver, spleen, and brain). The observed blood
concentration was considerably higher than the lethal
level (200 ng/ml) reported in an atropine poisoning
case (Baselt and Cravey, 1995). The selective presence
clearly demonstrated that the present case did not
follow the general poisoning trend, wherein the agent
should have been distributed throughout the body.
The possibility of samples originating from different
sources was ruled out by the DNA analytical results,
as both blood and liver specimens were genetically
identical (Table 4). Atropine’s localized presence could
be attributed to its administration by emergency medi-
cal care personnel directly into the thoracic cavity for
resuscitation and to the circulatory failure preventing
the drug’s further distribution. This aspect is further
supported by the case history that emergency medical
personnel were at the aircraft crash site. Similar selec-

tive postmortem distribution patterns of lidocaine
have also been reported in three cases following its
endotracheal intubation for cardiopulmonary resus-
citation (Moriya and Hashimoto, 1997).

In an effective forensic toxicology operation, a
QC/QA program must be properly implemented and
maintained to withstand professional, as well as judi-
cial, scrutiny of analytical findings. Although toxicol-
ogy laboratories have their standardized QC/QA
procedures to fulfill the necessary requirements, some
unique situations warrant special attention. The three
situations described herein clearly demonstrate the
practical application and effectiveness of the DNA
profiling in resolving some postmortem toxicology-
related issues. The adopted PCR technique is simple,
less time-consuming, and suitable for the analysis of
degraded DNA in the putrefied samples generally
encountered in postmortem aviation accidents. This
molecular biology approach to address the forensic
issues improves not only the degree of certainty but
also the authenticity of toxicological results.

Table 3: Atropine concentrations in various tissues.

Specimen Atropine Concentration
Blood 318 ng/ml
Lung 727 ng/g
Liver None detected
Spleen None detected
Brain None detected

Table 4: DQA1 and PM typing results of the blood and liver fluid from

Situation 3.
Genetic Markers
Sample DQA1 LDLR GYPA HBGG _ D7S8 GC
Blood 1.2; 1.2 AB AB BB AA AA
Liver fluid 1.2;1.2 AB AB BB AA AA



(QuantiblotTM, 1993). DNAs in the extracts were
immobilized on the Biodyne® B membrane (Life
Technologies Inc., Gaithersburg, MD) by vacuum
blotting using a filtration manifold system (The Con-
vertibleTM, Life Technologies, Gaithersburg, MD).
Hybridization of the biotinylated probe, which has
high affinity for the streptavidin moiety of the horse-
radish peroxidase-streptavidin enzyme conjugate
(HRP-SA), allowed for chemiluminescent detection
upon catalytic oxidation of a luminol-based reagent
by peroxidase. Chemiluminescent signals were de-
tected on HyperfilmTM-ECL (Amersham Life Science
Inc., Arlington Heights, IL) after 30-minute exposure
and development using a QX-70 medical film proces-
sor (Konica Medical Corporation, Wayne, NJ). The
intensity of the sample signals was visually compared
with that of the DNA standards processed simulta-
neously. This method selectively determines human
DNA semiquantitatively in the range of 0.15 to 10 ng.
Based on the DNA amounts, appropriate volumes of
the extracts were utilized in the subsequent DNA
amplification.

DNA amplification

In vitro DNA replication by Taq-polymerase was
carried out according to the manufacturer’s directions
for DQAI1 and polymarker (PM) amplification
(AmpliType®, 1995). The reaction mixture also con-
tained 160 g/ml BSA to minimize inhibition of
amplification by any inhibitors present in the extracts.
Target regions of six genetic loci were synthesized
from 2-10 ng of the extracted DNA ina thermal cycler
(GeneAmp PCR System 9600, Perkin Elmer Corpo-
ration, Foster City, CA) in 32 cycles. Of the six loci,
one locus was human leukocyte antigen (HLA) DQA1.
The remaining loci were PM: low density lipoprotein
receptor (LDLR), glycophorin A (GYPA), hemoglo-
bin G gammaglobin (HBGG), D7S8 (locus on chro-
mosome 7 linked with cystic fibrosis), and group
specific component (GC). Each thermal cycle con-
sisted of three steps: denaturation (95°C, 30 seconds),
primer annealing (63°C, 30 seconds), and primer
extension (72°C, 30 seconds). The last cycle was
followed by the extension step at 72°C for 10 minutes,
then cooling to 15°C for storage. To inactivate the
polymerase, 5 1] of 0.2 M ethylenediaminetetraacetic
acid (EDTA) was added to each of the amplified

samples. A 6-1] aliquot from the reaction mixture was
saved for possible product gel analysis. All amplified
samples were kept at -70°C until typed.

PCR product verification

Following the procedure for verification of PCR
amplification (AmpliType®, 1995), gel electrophore-
sis was performed on PCR products using an agarose
preparation (Agarose for the Separation of
GeneAmpTM PCR Products, Perkin Elmer Corpora-
tion, Foster City, CA) and a Horizontal Mini-Gel
Electrophoresis Unit (Sigma-Aldrich Techware, St.
Louis, MO). The 10 x 7.5 x 0.4-cm gels with imbed-
ded sample wells were cast from a solution of 4%
agarose in 0.5 X TBE (45 mM Tris, 45 mM borate, 1
mM EDTA) and 0.5 g/ml ethidium bromide (Sigma
Chemical Company, St. Louis, MO). Six-[1l of each
amplified sample in 2 yl of the buffer (20 mM Tris,
2.5 mM EDTA, 50% glycerol, 0.2% bromophenol
blue) was then loaded into individual sample well
after the gel had been submerged under a sufficient
volume of the running buffer (0.5X TBE and 0.5 [1g/
ml ethidium bromide). Along with the samples was
also loaded the GIBCO BRL 123 Base Pair DNA
ladder (Life Technologies Inc., Gaithersburg, MD) as
the molecular weight marker. At pH 8.3 and under
105-volt electrophoresis, DNA fragments migrated
from cathode to anode according to their size. Elec-
trophoresis was carried out for approximately 1.5
hours, when the bromophenol blue dye front had
traveled at least 7.5 cm from the wells for adequate
resolution of the six PCR products bands. Subse-
quently, gels were photographed under UV-illumina-
tion (U.V. P. Inc., San Gabriel, CA) using a red 23A
filter (Tiffen Manufacturing Corp., Hauppage, NY).
The intercalated ethidium bromide fluoresced in the
UV light and allowed visual identification of the
DNA bands.

Hybridization and detection

Two different probe-strips were used for the DNA
typing of the amplified samples. The DQAI strip
contains 11 probes for detecting eight alleles of the
HLA-DQAI locus (1.1, 1.2, 1.3, 2, 3, 4.1, 4.2, and
4.3), whereas 14 probes of the PM strip distinguish
the alleles of the five PM loci (LDLR, GYPA, HBGG,
D7S8, and GC). Hybridization of the amplified-


batch-review form of a particular batch contains details
of the samples, which include aliquot numbers, posi-
tive and negative blind controls, case numbers, sample
types and amounts, victim names, etc., while the
batch-analysis form entails only aliquot numbers, and
sample types and amounts. Locations of negative and
positive blind controls randomly vary from batch to
batch. The order in which the samples are listed on a
review form is the same as on the corresponding
analysis form. Analysts do not have access to the
review forms. After the completion of the batch analy-
sis, they submit the results to a reviewer. The reviewer
evaluates the findings and compares them with the
corresponding batch-review form.

In the present situation, a batch comprising a total
of seven blood samples was accessioned and submit-
ted for cyanide analysis. It entailed five aircraft acci-
dent-related case samples and blind controls (one
negative and one positive). Review of the batch analy-
sis revealed that the positive blind control was re-
ported as negative. Thus, the batch was rejected. Since
it was not certain whether the error occurred during
the accessioning or the analysis, attempts were made
to identify the source by subjecting all seven samples
for DNA analysis. Along with these were analyzed the
known positive and negative controls from which the
blind controls for the batch were initially prepared; it
was presumed that both controls originated from
different biological sources. The observed DNA pro-
files in the batch were compared with those of the
known controls, and the order of sample listing on the
batch-review form was matched with that on the
cyanide batch-analysis form.

Unusual analytical finding

Multiple specimens from an aircraft accident vic-
tim were submitted for analytical toxicology. Routine
analysis disclosed the presence of atropine at unusual
toxic concentrations in the blood and lung samples,
but the drug was not detected in the liver, spleen, and
brain samples. The presence of atropine in the bio-
logical samples could be related to three possible
scenarios: intentional or accidental atropine poison-
ing, atropine administration by emergency healthcare
providers into the thoracic cavity to modify the vas-
ovagal activity, and tissues originating from different
subjects. To verify the tissue origin, DNA analysis was
performed on two selected tissue types (blood and
liver), because atropine was present in the blood but
absent in the liver—a primary drug metabolism site.

MATERIALS AND METHODS

Materials

All reagents were of analytical grade, and the sol-
vents were of chromatographic grade. These reagents
and solvents, immunoassay and DNA analysis kits,
standards, internal standards, derivatizing agents, and
other necessary supplies were obtained from commer-
cial sources. Different immunoassay kits for drug
screens were purchased from Abbott Laboratories
(Abbott Park, IL), Roche Diagnostic Systems (Nutley,
NJ), and Diagnostic Products Corporation (Los An-
geles, CA). The DNA analysis kits (QuantiblotTM
Human DNA Quantitation Kit; AmpliType®
PM+DQAI PCR Amplification and Typing Kit) were
supplied by Perkin-Elmer Corporation (Foster City,
CA). Bovine serum albumin (BSA) of 96-99% purity
(Catalog No. A3350) was purchased from Sigma
Chemical Company (St. Louis, MO).

Routine forensic toxicology

Submitted samples were analyzed for drugs,
volatiles, carboxyhemoglobin (COHb), and cyanide
using standard laboratory procedures. For drugs, vari-
ous immunoassays and chromatographic techniques
were used. Samples were analyzed for volatiles by
headspace-gas chromatography. COHb was measured
spectrophotometrically. Blood cyanide was determined
colorimetrically using the chloramine-T/pyridine/
barbituric acid reagent.

DNA extraction

DNA from whole blood and other tissue samples
was extracted in the presence of Chelex® 100 resin
(Bio-Rad Laboratories, Richmond, CA) following the
procedure of Walsh et al. (1991). To 3-30 mg of each
of the samples, 200 pl of the 5% Chelex® resin
suspension in water was added, and the mixtures were
incubated in a water bath at 56°C for 30 minutes and
then at 90-100°C for 8 minutes. During this process,
divalent metal ions are chelated and the denatured
DNA is released into the medium. The resin inhibits
DNA degradation at high temperature in low ionic
strength medium (Singer-Sam et al., 1989).

DNA quantitation

The amount of DNA in the obtained extracts was
determined using QuantiblotTM Human DNA
Quantitation Kit. Each 5 pl-extract was processed
following the recommendations of the manufacturer


former fragments are more prone to degradation than
the latter fragments. The number of base pairs at the
six loci decrease in the following order: DQA1 (239/
242) > LDLR (214) > GYPA (190) > HBGG (172) >
D788 (151) > GC (138). The visibility of loci smaller
than 214 base pair—GYPA, HBGG, D7S8, and GC—
in the Bag 3 sample further confirmed the proneness
for the DNA degradation. Despite the limited typing,
one can infer that the sample in Bag 3 possibly
belonged to the same source as the sample in Bag 1,
since the four visible loci in the former matched with
that of the latter. The Bag 4 sample was a mixture
containing biological material from two genetically
different sources, since three alleles (1.2, 3, 4.1) on the
DQAI strip and imbalanced dot intensities on the
PM strip were well evident. In a particular locus, an
allele common in both Bag 1 and Bag 2 samples was
more intense than the one present in only one sample
(Table 1). Based on these DNA results, toxicological
findings were reported on only those specimens deter-
mined to be from a single individual. Although
toxicological evaluation failed to disclose the presence

of commonly used drugs in either of the analyzed
muscle samples (Bag 1 and Bag 5), 16 mg/dl ethanol
was detected in the fluid from only the Bag 1 sample.
Volatiles found in another tissue (Bag 4) were ex-
cluded from the reporting, as the sample was geneti-
cally determined to be a mixture of tissue originating
from two different individuals.

The DQA1 and PM genetic profiles of the samples
in the cyanide batch, together with the known positive
and negative blind controls, are presented in Table 2.
DNA profiles of Sample 1 and Sample 2 matched with
those of the known positive and negative controls,
respectively. This finding is consistent with the
accessioning records that Sample 1 was, indeed, the
positive blind control and Sample 2, the negative
blind control. These observations implied that the
error did not take place during the accessioning or
batch preparation but occurred during the analysis.
Accordingly, proper corrective measures were taken at
the analytical level to prevent the recurrence of such
faulty analysis.

Table 2: DQA1 and PM profiles of the samples from Situation 2 and of
the known positive and negative blind controls.

Genetic Markers
Sample DQA1 LDLR GYPA HBGG _  D7S8 Gc
1 1.2; 4.1 AB AB AB AA AC
2 1.1541 AB AA AB AB cc
3 4.1541 AB BB AA AB cc
4 2:3 AB AB BB AA BC
5 23441 AB BB AA BB AB
6 1.2;3 BB AB BB AB cc
7 1.2;2 AB AA AB AB cc
Positive 1.2; 4.1 AB AB AB AA AC
Negative 1.1; 4.1 AB AA AB AB cc



DOT/FAA/AM-98/18

Office of Aviation Medicine
Washington, D.C. 20591

DNA Profiling as an
Adjunct Quality Control/
Quality Assurance in
Forensic Toxicology

Arvind K. Chaturvedi
Nicole T. Vu

Roxane M. Ritter
Dennis V. Canfield

Civil Aeromedical Institute
Federal Aviation Administration
Oklahoma City, Oklahoma 73125

July 1998

Final Report

This document is available to the public
through the National Technical Information
Service, Springfield, Virginia 22161.

U.S. Department
of Transportation

Federal Aviation
Administration




The PCR-based DNA profiling can be an effective,
useful tool for a QC/QA program of a forensic toxi-
cology laboratory because of its specificity, simplicity,
and ability to analyze putrefied samples. However, the
applicability of the DNA analysis will be limited to
only those toxicology laboratories where in-house
DNA facilities are readily available.

REFERENCES

AmpliType® (1995), PM+DQA1 PCR amplification
and typing kits, package insert, Roche Molecular
Systems, Inc., Alameda, CA.

Baselt RC, Cravey RH (1995). Disposition of toxic drugs
and chemicals in man. 4th ed. Foster City, CA:
Chemical Toxicology Institute, pp 63-5.

Budowle B, Waye JS, Shutler GG, Baechtel FS (1990).
Hae III—A suitable restriction endonuclease for
restriction fragment length polymorphism analy-
sis of biological evidence samples. Journal of Fo-
rensic Sciences 35:530-6.

Budowle B, Baechtel FS, Adams DE (1991). Validation
with regard to environmental insults of the RFLP
procedure for forensic purposes. In: Farly MA,
Harrington JJ, editors. Forensic DNA technology.
Chelsea, MI: Lewis Publishers Inc., pp 83-91.

Hochmeister MN, Budowle B, Borer UV, Eggmann U,
Comey CT, Dirnhofer R (1991). Typing of deox-
ytibonucleic acid (DNA) extracted from compact
bone from human remains. Journal of Forensic

Sciences 36:1649-61.

Moriya F, Hashimoto Y (1997). Postmortem diffusion
of tracheal lidocaine into heart blood following
intubation for cardiopulmonary resuscitation. Jour-
nal of Forensic Sciences 42:296-9.

uantiblotTM (1993), Human DNA quantitation kit,
q
package insert, Roche Molecular Systems, Inc.,
Alameda, CA.

Reynolds R, Sensabaugh G (1991). Analysis of genetic
markers in forensic DNA samples using the poly-
merase chain reaction. Analytical Chemistry 63: 2-

15.

Sajantila A, Strom M, Budowle B, Karhunen PJ, Peltonen
L (1991). The polymerase chain reaction and
post-mortem forensic identity testing: Applica-
tion of amplified D1S80 and HLA-DQa. loci to
the identification of fire victims. Forensic Science
International 51:23-34.

Singer-Sam J, Tanguay RL, Riggs AD (1989). Use of
chelex to improve the PCR signal from a small
number of cells. Amplifications: A forum for PCR
Users 3:11.

Walsh PS, Metzger DA, Higuchi R (1991). Chelex®
100 as a medium for simple extraction of DNA for
PCR-based typing from forensic material.
Biotechniques 10:506-13.

Walsh PS, Erlich, HA, Higuchi, R (1992). Preferen-
tial PCR amplification of alleles: Mechanisms
and solutions. PCR Methods and Applications
1:241-50.

TUS. GOVERNMENT PRINTING OFFICE: 1998 - 660-088/80001


DNA to the impregnated probes on the nylon strips
was accomplished by a reverse dot plot procedure
(AmpliType®, 1995). The hybrids were tagged with
biotin, which facilitates colorimetric detection upon
its binding to the HRP-SA enzyme conjugate. After
the removal of unbound DNA and the addition of the
chromogen 3,3',5,5'-tetramethylbenzidine (TMB),
hybridized DNA was detected as blue precipitate due
to catalytic oxidation of the colorless substrate (TMB)
by peroxidase.

RESULTS AND DISCUSSION

In our present approach, six independently inher-
ited genetic loci are included. They are considered to
be sufficient for establishing the fidelity of the samples
in question within the scope of a particular situation.
According to Perkin-Elmer’s population genetic stud-
ies, the power of discrimination for these loci ranges
from 99.93 to 99.98%, depending upon the ethnic
background of an individual (AmpliType®, 1995).
Although such discriminating power may not be as
compelling as what is generally cited for a matching
within an ethnic population, it will be statistically
sufficient for the matching within a particular foren-
sic toxicology situation, involving a relatively well-
defined, limited number of samples.

From the tissue mismatching/commingling related
results (Table 1), it is clearly evident that the samples
in Bag 1 and Bag 2 originated from different individu-
als, as both samples had different genotypes. The
genotype of the specimen in Bag 2 was consistent with
that of the specimen in Bag 5, confirming that these
samples originated from the same individual. Results
from the sample in Bag 3 were inconclusive, as out of
six loci, only four loci could be successfully visualized:
DQA\] and LDLR could not be typed. The “C” dot on
DQAI strip and “S” dot on PM strip were also not
developed. The reamplification with an increased
amount of DNA or the organic extraction (Budowle
etal., 1990) in place of Chelex® did not produce any
conclusive typing results. The preferential amplifica-
tion of the alleles in the sample from Bag 3 was further
supported by the visibility of only the corresponding
four DNA bands in the PCR product gel electro-
phoresis. Such partial amplification could be attrib-
uted to the presence of some unknown contaminant(s)/
inhibitor(s) in the Bag 3 sample, and could further be
ascribed to the quality (degraded) of DNA present in
the sample (putrefied). It has been established that, in
samples wherein DNA is degraded, some but not all
alleles are amplified (Walsh et al., 1992). Loci associ-
ated with longer base pair sequences are more affected
than those with shorter base pair sequences, as the

Table 1: DQA1 and PM profiles of samples from Situation 1.

Genetic Markers

Bag Specimen DQA1 LDLR GYPA HBGG_ D7S8 GC
1 Muscle 333 AB AA AB BB AC
2 Liver 1.2;4.1 AB AB AA AB cc
3 Lung - - AA AB BB AC
4 Kidney 1.2; 3; 4.1 AB AB AB AB Ac
5 Muscle 1.2; 4.1 AB AB AA AB cc

*Dashes denote that the loci were not detectable.

®Bold lettering in the sample profile exhibits relatively high intensity dot observed within

a particular locus.




Technical Report Documentation Page

T. Report No, 2. Government Accession No. 3, Recipients Catalog No.
DOT/FAA/AM-98/18

4 Title and Subiitle 3, Report Date

DNA Profiling as an Adjunct Quality Control/Quality Assurance in July 1998

Forensic Toxicology 6. Performing Organization Code

7, Author(s) 8. Performing Organization Report No.

Chaturvedi, A.K., Vu, N.T., Ritter, R.M., and Canfield, D.V.

9. Performing Organization Name and Address TO. Work Unit No. (TRAIS)

FAA Civil Aeromedical Institute

P.O. Box 25082 11. Contract or Grant No.
Oklahoma City, Oklahoma_73125

12, Sponsoring Agency name and Address 13. Type of Report and Period Covered

Office of Aviation Medicine
Federal Aviation Administration
800 Independence Ave., S.W.

Washington, D.C. 20591 14. Sponsoring Agency Code

15, Supplemental Notes

This work was accomplished under the approved task AM-B-97-TOX-202.

16. Abstract

To investigate aircraft accidents, multiple postmortem biological samples of victims are submitted to the Civil Aeromedical Institute
for toxicological evaluation. However, depending upon the nature of a particular accident, body components are often scattered,
disintegrated, commingled, contaminated, and/or putrefied. These factors impose difficulties on victim identification, tissue
matching, and thereby authentic sample analysis and resule interpretation. Nevertheless, these quality control/quality assurance
(QCIQA) related limitations can be overpowered by DNA profiling. In this regard, three situations are hereby exemplified where
DNA analysis was instrumental in resolving a tissue mismatching/commingling issue, pinpointing an accessioning/analytical error,
and interpreting an unusual analytical result. Biological samples from these cases were examined for six independently inherited
genetic loci using polymerase chain reaction (PCR) suitable for analyzing degraded DNA generally encountered in
putrefied/contaminated samples. In the first situation, three of five specimen bags from one accident were labeled with two different
names. DNA analysis revealed that one of these bags actually had commingled specimens, originating from two different individuals,
Therefore, the sample was excluded from the final toxicological evaluation. In the second situation, an unacceptable blind control
result was reported in a cyanide batch analysis. By comparing DNA profiles of the batch samples with those of the known positive
and negative blind controls, it was concluded that the error had occurred during the analysis instead of accessioning. Accordingly,
preventive measures were taken at the analytical level. The third situation was related to the presence of atropine at toxic
concentrations in the blood (318 ng/ml) and lung (727 ng/g) with its absence in the liver, spleen, and brain—a pattern inconsistent
with the general poisoning of drugs. DNA analysis of the blood and liver samples exhibited their common identity, ensuring that the
submitted samples had indeed originated from one individual. The selective presence of atropine was attributed to its possible
administration into the thoracic cavity by the emergency medical personnel at the accident site for resuscitation, but circulatory
failure prevented its further distribution. These examples clearly demonstrate the applicability of the PCR-based DNA profiling in a
QCIQA program to enhance the effectiveness of forensic toxicology operation. However, such applicability will be feasible only in
those setups where in-house DNA facilities are accessible.

17. Key Words 18, Distribution Statement

Quality Control/Quality Assurance, DNA Profiling, Document is available to the public through the

Polymerase Chain Reaction, Cyanide Analysis, Atropine National Technical Information Service

Administration, Aircraft Accident Investigation Springfield, Virginia 22161

19. Security Classi. (oF this report) 20. Security Classif, (ofthis page) 2. No. of Pages 22. Price
Unclassified Unclassified 10

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized



NOTICE

This document is disseminated under the sponsorship of
the U.S. Department of Transportation in the interest of
information exchange. The United States Government
assumes no liability for the contents or use thereof.