DOT/FAA/AM-02/14

Arvind K. Chaturvedi

Dudley R. Smith

John W. Soper

Dennis V. Canfield

James E. Whinnery

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

August 2002

Final Report

This document is available to the public

through the National Technical Information

Service, Springfield, VA 22161.

Characteristics and Toxicological

Processing of Postmortem Pilot

Specimens From Fatal Civil

Aviation Accidents

Office of Aerospace Medicine

Washington, DC 20591
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 thereof.
i

Technical Report Documentation Page

1. Report No. 2. Government Accession No. 3. Recipient's Catalog No.

DOT/FAA/AM-02/14

4. Title and Subtitle 5. Report Date

August 2002 Characteristics and Toxicological Processing of Postmortem Pilot

Specimens From Fatal Civil Aviation Accidents 6. Performing Organization Code

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

Chaturvedi, A.K., Smith, D.R., Soper, J.W., Canfield, D.V., and

Whinnery, J.E.

9. Performing Organization Name and Address 10. Work Unit No. (TRAIS)

FAA Civil Aerospace Medical Institute

P.O. Box 25082 11. Contract or Grant No.

Oklahoma City, OK 73125

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

Office of Aerospace Medicine

Federal Aviation Administration

800 Independence Ave., S.W.

Washington, DC 20591 14. Sponsoring Agency Code

15. Supplemental Notes

This work was accomplished under the approved tasks, AM-B-01-TOX-202 and AM-B-02-TOX-202.

16. Abstract

Autopsied biological samples from civil aviation accident pilot fatalities are submitted to the Civil Aerospace

Medical Institute (CAMI) for toxicological evaluation. However, such evaluation is dependent upon types

and amounts of submitted samples, and obtaining suitable samples is governed by the nature of an accident.

Characteristics of those samples and associated toxicological processing have not been well documented in

the literature. Therefore, the CAMI Toxicology Database was searched for these aspects. CAMI received

samples from the pilot fatalities (CAMI cases) of approximately 80% of the 1990-2000 aviation accidents

reported by the National Transportation Safety Board. Accidents and cases during June-September were

higher than the other months, and more than half of the received cases had multiple samples in sufficient

amounts. For example, out of 1,891 cases processed for the 1996-2000 accidents, 1,211 had at least

adequate amounts of blood, urine, and/or vitreous humor; 324 had inadequate amounts of blood and urine;

and 356 had no blood or urine. Muscle, liver, lung, and/or kidney samples were submitted in 90% of the

cases, while cerebrospinal fluids were submitted in only 8% of the cases. The toxicologically preferred

samples, blood and urine, were available in 78% and 56% of the 1,891 cases, respectively. Out of 51 cases

containing only one sample type, 46 had muscle and the remaining 5 had other sample types. Samples were

primarily analyzed for combustion gases, alcohol/volatiles, and drugs. Generally, the presence of analytes is

demonstrated in at least 2 different sample types by using 2 different analytical techniques for reporting a

particular case as "positive." An effective quality-assurance/quality-control is maintained throughout the

process. In the majority of the aviation accidents, sufficient amounts and types of biological samples were

submitted for toxicological evaluation.

17. Key Words 18. Distribution Statement

Forensic Sciences, Toxicology, Pilot Fatality Specimens,

Civil Aviation Accident Investigation

Document is available to the public through the

National Technical Information Service

Springfield, VA 22161

19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No. of Pages 22. Price

Unclassified Unclassified 14

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

1

CHARACTERISTICS AND TOXICOLOGICAL PROCESSING OF POSTMORTEM PILOT

SPECIMENS1 FROM FATAL CIVIL AVIATION ACCIDENTS

INTRODUCTION

For the investigation of aircraft accidents occurring within the jurisdiction of the United States,

autopsied biological samples collected from the victims of fatal civil aircraft (air carrier and general

aviation) accidents are submitted to the Federal Aviation Administration’s (FAA’s) Civil Aerospace Medical Institute (CAMI) for toxicological evaluation (2).

Such sample submission is coordinated through the

FAA’s Office of Accident Investigation by the National Transportation Safety Board (NTSB), which is

responsible for investigating all United States civilian

aircraft accidents. In the majority of cases, the submitted postmortem samples are from pilots and copilots.

However, depending upon the nature of an accident—for example, an accident involving fire—

samples from passengers and other crewmembers are

sometimes submitted.

The submitted biological samples are routinely

analyzed for the presence of combustion gases, alcohol/volatiles, and drugs (7,8,10,16). Most of the

drugs present in the samples from pilots/copilots are

in the subtherapeutic to therapeutic concentration

range (7,8,16), which is consistent with the nature of

the postmortem aviation toxicology and could basically be referred to as the human-performance associated postmortem forensic toxicology (13). Therefore,

multiple types of postmortem specimens in sufficient

amounts are needed for the toxicological analysis;

however, this requirement frequently cannot be

achieved in accidents wherein victims’ bodies are

scattered, disintegrated, commingled, contaminated,

and/or putrefied.

Limited information is available on characteristics

and toxicological processing of postmortem samples

from aviation pilot/copilot fatalities (cases) and associated accidents. Therefore, the CAMI Toxicology

and the FAA National Aviation Safety Data Analysis

Center (NASDAC) databases were searched to obtain

the applicable information.

MATERIALS AND METHODS

Databases

Since 1990, a toxicology database for civil aircraft

accident fatalities has been maintained at CAMI (Oklahoma City, OK). This CAMI Toxicology Database

was searched to ascertain the number of aviation

accidents and associated fatalities (cases), occurring

between 1990 and 2000, from which CAMI received

postmortem biological samples. In this paper, such

accidents are referred to as “CAMI accidents” and

related fatalities as “CAMI cases.” These fatalities

primarily consisted of pilots and copilots. Therefore,

the “pilot fatalities” phrase used in this paper means

pilot, as well as copilot, fatalities. Because of early

database limitations, the data pertaining to the types

and amounts of received-samples and other related

information could not be completely retrieved for the

accidents occurring prior to 1996. However, this

information was effectively obtained from the database for the period of 1996-2000 and is accordingly

incorporated in this research report. The number of

fatal aviation accidents reported by the NTSB for the

1990-2000 period was obtained from the FAA’s

NASDAC Database, Washington, D.C.

Postmortem Aviation Forensic Toxicology

Samples

Biological specimens collected during autopsy are

packed with refrigerant packs in the styrofoam box of

the FAA’s TOX-BOX evidence containers. These

containers with specimens are subsequently shipped

to CAMI by an air carrier service for next-day delivery. It is strongly recommended to the TOX-BOX

users that they use a single TOX-BOX evidence container for shipping samples from a particular fatality.

Shipping samples from another fatality requires a

separate TOX-BOX evidence container.

The supply of newly assembled TOX-BOX evidence containers with kits is maintained at various

FAA Flight Standard District Offices. These containers with kits are regularly supplied by CAMI in advance

1

 The synonym “sample” is interchangeably used for “specimen”

throughout the report.
2

to those offices, so that those boxes/kits are readily

available in an event of an accident. If additional

boxes/kits are needed at a particular site, CAMI ships

the requested number of boxes/kits by an air carrier

service for next-day delivery.

Details of the contents of the TOX-BOX kit are

given in Table I. Gray-top and green-top glass tubes

are for blood samples; red-top glass tubes are for

vitreous humor, spinal fluid, and/or bile samples.

Plastic bottles are for urine, gastric content, and/or

bile samples; plastic bags are for solid tissue samples.

Recommended quantities for different types of biological samples required by CAMI for toxicological

evaluation are provided in the kit instruction sheet

(Table II).

It is assumed that the external specimen chain-ofcustody (ESCOC) and accident information forms

are properly completed following the instructions

provided with the kit, and all sample containers are

properly sealed and labeled. Necessary information—

such as name of victim, date and time of sample

collection, type and amount of sample, and initials of

sample collector—must be legibly written on the

labels of the respective sample containers. The names

and addresses of the coroner or medical examiner and

of the accident investigator should also be included on

the forms.

Sample Accessioning

Fatality samples are received in the accessioning

room of CAMI’s Toxicology and Accident Research

Laboratory. Only authorized individuals are allowed

in this restricted area. Individuals involved in performing analyses do not have access to the area.

Unauthorized individuals are always escorted by an

authorized employee.

Each fatality is given a unique CAMI case number,

and an internal chain-of-custody (ICOC) form is

prepared for the case. This process entails obtaining

and incorporating information for the pilot/copilot

and for the associated accident from the completed

ESCOC and accident information forms, the FAA

Administrator’s Daily Alert Bulletin, the NTSB

Website, the airman and medical certification records,

and other available authentic and reliable sources.

Examples of the ICOC preparation items are: name,

birth date, airman and medical certificate numbers,

and social security number of the pilot/copilot; date,

place, and manner of the accident; aircraft tail number; and NTSB reference number. The ICOC form

also includes a complete cataloging of the samples

received, which encompasses sample types and any

characteristic appearances (bloody, burned, cherry

red color, coagulated, clotted, green, putrefied, etc.),

sample container types and their weights with samples;

markings and notes on containers; and any other

relevant information. Known medical conditions of

the pilots/copilots and/or any reported medications

are noted on the ICOC form.

Analytical Batch Preparation

Depending upon the types and amounts of samples

received, carboxyhemoglobin (COHb), blood cyanide (CN ̄), alcohol/volatile, glucose, and hemoglobin A1c (HbA1c) analysis and drug screening batches

are prepared and submitted for analysis. Batches are

prepared by transferring a portion (aliquot) of the

specimens of a case into test tubes. Generally, batches

are comprised of samples from more than one case.

Each batch also contains aliquots from at least 2

quality-assurance/quality-control (QA/QC) blind

samples—1 negative QA/QC blind sample and 1

positive QA/QC blind sample. Only on rare occasions, 2 negative QA/QC blind sample aliquots are

used. This generally happens when a positive QA/QC

blind sample for a particular analyte is not available,

primarily because of the newness, uniqueness, and/or

rareness of the analyte. Amounts of aliquots used for

a particular batch depend upon the type of the batch.

The location of the blind sample aliquots is randomly

changed from batch to batch. Similarly, where applicable and possible, concentrations and types of analytes

in the QA/QC blind samples are also changed. The

QA/QC blind sample aliquots are consistent with

batch types, case aliquot matrices, and analytes.

Analysts do not know the location of and the type

or amount of analyte(s) present in the QA/QC blind

aliquots of a particular batch. Batch aliquots are

analyzed by using standard operating procedures and

practices established for the laboratory. After the

completion of analyses, the documentation for the

batches with the analytical data is submitted by the

analysts for independent review.

If the initial analysis/screening is positive, additional samples are analyzed for confirmation or

quantitation of the analyte(s). For such analyses, new

confirmatory/quantitative batches are prepared with

necessary QA/QC blind sample aliquots and are submitted for analysis. Most of the time, an aliquot from

the sample type in which the analyte was detected

during the initial analysis/screening is included in the

confirmatory/quantitative batches. These batches also

contain an aliquot from another sample type of the

case, provided that is available. Specimens of a par-
3

Table II. Types, Amounts, and Analytical Suitabilities of Specimens∗ Required by CAMI For Toxicological

Evaluation

Specimen Type†

Optimal

Amount‡ Analysis Type Suitability

Blood 40 mL Green-top tube blood for carboxyhemoglobin (COHb), blood cyanide (CN ̄ ),

hemoglobin A1c (HbA1c), and drug analyses

Gray-top tube blood for ethanol and drug analyses

Urine 100 mL Ethanol, drug, and glucose analyses

Vitreous Humor 2 mL Ethanol and glucose analyses

Spinal Fluid Available

Amount

Ethanol and, perhaps, drug analyses

Bile 10 mL Abused drug§

 radioimmunoassay (RIA) screening, when urine is not available

Gastric Contents¶ 100 g Need-based drug analysis

Liver 500 g Ethanol and drug analyses

Muscle 300 g Ethanol and drug analyses

Spleen 150 g Ethanol and drug analyses

Lung 100 g Ethanol and drug analyses

Kidney 100 g Ethanol and drug analyses

Brain 100 g Ethanol and drug analyses

Heart 50 g Ethanol and drug analyses

∗

The given types and amounts of specimens are for the ideal situation wherein multiple specimen types are available

in enough amounts. It is understood that this requirement may not always be met since it could be possible that some

sample types are available in optimal or adequate amounts, while others may not be available at all. In such situations,

analyses are performed on selected sample types. The sample selection is based on the intrinsic nature of the

analytical method, analysis (qualitative, confirmatory, and/or quantitative), and analyte. Therefore, all available

samples should be submitted.

†

Drugs/substances (tablets, caplets, capsules, powder, and/or liquid) found at the accident scene are also occasionally

submitted. Analyses of drugs/substances may be helpful in the investigation of accidents, particularly when they are

also found in the submitted biological samples.

‡

An adequate amount considered is half of the optimal amount. Any specimen submitted in the amount less than the

adequate amount is considered to be inadequate.

§

Amphetamine, methamphetamine, phencyclidine, cocaine, opiates, cannabinoids, barbiturates, and benzodiazepines.

¶

All gastric contents should be submitted. In those fatalities (cases) wherein the submission of all gastric contents

could not be possible, any available amount of gastric contents should be submitted. By knowing the total amount of

the gastric contents, the amount of the drug/substance present in the contents can be determined.

Table I. Contents of CAMI's Tox-Box Kit

Description of Contents

One instruction sheet

One external specimen chain-of-custody (ESCOC) form with completed example

One accident information form with completed example

Three 10-mL gray-top glass tubes*

 (Vacutainers) in a styrofoam holder/protector with identification (ID)

labels affixed

Three 10-mL green-top glass tubes†

 (Vacutainers) in a styrofoam holder/protector with ID labels affixed

Three 10-mL red-top glass tubes‡

 (Vacutainers) in a styrofoam holder/protector with ID labels affixed

Two 120-mL plastic specimen bottles§

 with caps and 2 specimen bottle ID labels

Seven whirl-pack plastic specimen bags§

 and 7 specimen bag ID labels

Three 20-cc disposable plastic syringes¶

Three 18Gx11⁄2"-needles¶ and 1 16Gx4"-needle¶

Eight ziplock bags and 1 marking pen

One large black plastic bag with 3 sheets of liquid absorbent material

Two reusable refrigerant packs, 1 security seal, 1 yellow plastic tape roll, and necessary tags

One preprinted return address label for the CAMI laboratory

*

Each tube is sterile and contains 20 mg of potassium oxalate and 100 mg of sodium fluoride.

†

Each tube is sterile and contains sodium heparin.

‡

Sterile, but without additives.

§

Without additives.

¶

Sterile.
4

ticular case utilized during the analytical process are

automatically tracked and recorded on the batch-bybatch basis using the internal specimen-chain-of-custody form of the case.

Specimen Selection and Analytical Methodology

Green-top tube blood is preferred for the analysis

of COHb, CN ̄, and HbA1c. If this blood is not

available in sufficient amount, then gray-top tube

blood is used. HbA1c analysis in blood is conducted

when glucose values are > 100 mg/dL in urine and/or

> 125 mg/dL in vitreous fluid or upon the request of

the accident investigator. COHb, CN ̄, and HbA1c

determinations in blood are made by spectrophotometric, colorimetric, and latex immunoagglutination

inhibition methodologies, respectively. Glucose levels

in urine and vitreous fluid are measured enzymatically.

Alcohol/volatile analysis is performed by headspace

gas chromatography (GC). The order of preference of

specimens for the alcohol/volatile analysis is vitreous

fluid, urine, gray-top tube blood, brain, muscle, and

other tissues. If the ethanol concentration in liquid

samples—for example, vitreous fluid, urine, and

blood—is >20 mg/dL by the GC method, then those

liquid sample aliquots of the batch are also analyzed

by the radiative energy attenuation method to reconfirm the presence of ethanol in the liquid samples.

Attempts are always made to save some blood for

confirmatory/quantitative drug analyses. Urine is preferentially used for the screening of prescription and

over-the-counter drugs by high performance liquid

chromatography (HPLC) and by gas chromatography/mass spectrometry (GC/MS) and of abused

drugs—amphetamine, methamphetamine, phencyclidine, cocaine, opiates, cannabinoids, barbiturates,

and benzodiazepines—by fluorescence polarization

immunoassay (FPIA). Certain additional drugs—for

example, acetaminophen, salicylate, quinidine, theophylline, phenytoin, and propoxyphene—are also

screened in urine by FPIA.

In the absence of urine, other specimens are screened

for abused drugs by radioimmunoassay (RIA). The

order of specimen preference for the RIA-based abuseddrug screening is bile, blood, liver, kidney, and other

tissues. Bile is not used for the screening of prescription and over-the-counter drugs by HPLC and GC/

MS; instead, blood or other tissues are used. In those

cases wherein bile is not available, blood or any other

tissue is used for the RIA-based abused-drug screening, and the same sample type used for RIA is preferentially used for the HPLC and GC/MS-based drug

screening, as well. In other words, with the exception

of bile, it is preferred to use the same specimen type for

the RIA-based abused-drug screening and for the

HPLC and GC/MS-based drug screening. Blood

samples are also screened for acetaminophen, salicylate, quinidine, theophylline, and phenytoin by FPIA.

As mentioned earlier, confirmatory/quantitative

batches contain aliquots of the sample type, which

was determined as positive during the initial analysis/

screening, and of at least one additional sample type

of the case. Toxicologically preferred samples for

these types of batches are blood and urine. Depending

upon the type of analyte, these batches are analyzed by

GC/MS, liquid chromatography/mass spectrometry,

or any other specific technique. Confirmatory/quantitative batches for COHb, CN ̄, HbA1c, and glucose

are generally prepared with new aliquots of the applicable sample type of the case and appropriately reanalyzed by using the methods mentioned previously in

the text. Recently, a new GC method has been adopted

in the laboratory for the COHb confirmatory/quantitative batch to analyze carbon monoxide in blood

utilizing a thermal conductivity detector.

Reporting

After the successful analyses and reviews of all the

batches, an analytical report for the case is generated.

Subsequently, the entire case folder, along with the

report, is reviewed for the complete case. This final

review process entails accessioning, scientific, and

QA/QC level reviews. After the final review, the

report is signed, reviewed once more at the clerical

level, and then disseminated to the appropriate authorities and agencies. A general description of the

processing of aviation forensic toxicology specimens

is outlined in Figure 1.

RESULTS

Biosamples from aviation accident pilot fatalities

are submitted to CAMI by the NTSB for toxicological

analysis. Such submissions are coordinated through

the FAA’s Office of Accident Investigation and the

local coroner/medical examiner offices. Samples are

primarily shipped in TOX-BOX evidence containers.

As is outlined in Figure 1, the toxicological evaluation

process involves initial analyses/screenings and confirmatory/quantitative analyses. After carefully reviewing analytical findings, toxicology reports are

sent to the NTSB, FAA’s Office of Accident Investigation, and other authorized agencies, such as coroners, medical examiners, and law enforcement agencies.

Case-related records and analytical data are maintained
5

Figure 1. A flowchart for a typical toxicological processing of postmortem biological samples. Details are

given in the Materials and Methods section. Carboxyhemoglobin (COHb), blood cyanide (CN ̄), alcohol/

volatile (A/V), glucose, and hemoglobin A1c (HbA1c) analysis and drug screening batches are prepared

and submitted for analysis. Batches are generally comprised of samples from more than one case. Each

batch contains portions (aliquots) of case specimens and quality-assurance/quality-control (QA/QC)

blind samples. Combustion gases carbon monoxide, as COHb, and hydrogen cyanide, as blood CN ̄,

are measured since these gases are considered the 2 primary toxic gases that are produced during a

fire. Drugs include prescription, over-the-counter, and abused drugs. Amphetamine, methamphetamine,

phencyclidine, cocaine, opiates, cannabinoids, barbiturates, and benzodiazepines are considered as

abused drugs. Abbreviations: AAI, FAA’s Office of Accident Investigation; A/V, Alcohol/Volatile; CN ̄,

Cyanide; COHb, Carboxyhemoglobin; HbA1c, Hemoglobin A1c; ME, Medical Examiner; NTSB, National

Transportation Safety Board; QA/QC, Quality-Assurance/Quality-Control; RFS, Regional Flight Surgeon.

If negative, report is generated.

SPECIMENS

Blood, Urine, Vitreous Humor, Bile, and

Solid Tissues

(liver, lung, kidney, spleen, muscle, brain, and heart)

ACCESSIONING

Case Number Assignment

Internal Chain-of-Custody

Specimen Chain-of-Custody

BLOOD

COHb, CN ̄ ,

HbA1c, A/V, and

Drugs

URINE/VITREOUS HUMOR

A/V and Glucose

URINE

Drugs also

SOLID TISSUES

A/V and Drugs

BILE

Abused Drugs only

ANALYSIS RESULT REVIEW

If positive, CONFIRMATORY/

QUANTITATIVE ANALYSES in specimens

are requested.

BATCH PREPARATION AND ANALYSES

ANALYSIS RESULT REVIEW

AND REPORT GENERATION

WHOLE CASE REVIEW

Accessioning, Scientific, and QA/QC Levels

REPORT SIGNING, CLERICAL REVIEW, AND

REPORT DISSEMINATION (NTSB, AAI, RFS, ME, etc.)

BATCH PREPARATION FOR COHb, CN ̄ , HbA1c, A/V, AND GLUCOSE

ANALYSES AND DRUG SCREENING
6

at CAMI. Unless there is pending litigation, samples

of negative and positive cases are properly destroyed

after 2 and 5 years, respectively, from the date of their

receipt at CAMI.

As is evident from Figure 2, CAMI received samples

from the majority of the aviation accidents that occurred during 1990-2000. Over this period, a total of

5,051 aviation accidents were reported by the NTSB.

Out of these 5,051 accidents, 237 were foreign aviation accidents. The 11-year averages were 459 (408-

512) for NTSB-reported all fatal accidents, 438

(365-509) for NTSB-reported United States fatal

accidents, and 354 (324-400) for NTSB-reported

United States fatal accidents from which CAMI received samples (CAMI accidents). For the 1990-2000

period, samples were received from the pilot fatalities

(CAMI cases) of approximately 80% of the total

United States aviation accidents (4,814) reported by

the NTSB. The percentage of the 1990 NTSB-reported

United States fatal accidents from which biological

samples were submitted to CAMI was 73%. This

percentage slowly increased in the ensuing years and

reached 92% for the year 2000. The gap narrowing

between the number of accidents reported by the

NTSB (NTSB accidents) and the number of accidents

from which postmortem pilot samples were submitted to CAMI (CAMI accidents) is exhibited in Figure

2 (also, see the figure insert). The 11-year average of

CAMI cases was 473 (396-585), with the highest

number of 585 cases in 1991. Monthly averages for

the 1990-2000 aviation accidents from which CAMI

received samples and associated CAMI cases indicated the highest number of accidents and cases during June-September (Figure 3).

Optimal, adequate, and inadequate amounts of

various sample types are mentioned in Table II. Although it is not always possible to obtain all samples

in optimal amounts, more than half of the cases

Figure 2. Numbers of the NTSB-reported 1990-2000 United States fatal aviation accidents (NTSB

accidents) versus numbers of those aviation accidents (CAMI accidents) from which biological

samples of the fatalities (CAMI cases) were received at CAMI. Insert: Percentage of the total 1990-

2000 United States fatal aviation accidents reported by the NTSB (NTSB accidents) from which

samples were received at CAMI (CAMI accidents).

200

400

600

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

Year

Number

CAMI Accidents

NTSB Accidents

CAMI Cases

60

80

100

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

Year

% NTSB Accident

CAMI Accidents
7

received at CAMI generally had multiple samples in

sufficient amounts to allow toxicological analyses

(Figures 4 and 5). For example, out of 1,891 cases

processed for the 1996-2000 accidents, 1,211 cases

had adequate amounts of blood, urine, and/or vitreous humor; 324 cases had inadequate amounts of

blood and urine; 356 cases had no blood or urine.

Muscle, liver, lung, and/or kidney samples were submitted in 90% of the cases, while cerebrospinal fluid

was submitted in only 8% of the cases (Figure 5). The

toxicologically preferred samples, blood and urine,

were available in 78% and 56% of the 1,891 cases,

respectively. Out of 51 cases containing only 1 sample

type, 46 cases had muscle, 3 cases had vitreous humor,

brain, or spleen, and the remaining 2 cases had unidentified body tissues. Optimal amounts of heart,

brain, kidney, lung, blood, and vitreous fluid samples

were submitted in 64, 56, 53, 49, 39, and 37% of the

1,891 cases, respectively (Figure 5). Considering

CAMI’s sample requirements (Table II), urine samples

were submitted in optimal amounts in only 2% of the

1,891 cases and in adequate amounts in 16% of the

1,891 cases. After combining “adequate” amounts

with “optimal” amounts, it was determined that heart,

brain, kidney, lung, and blood samples were submitted in 60-80% of the 1,891 cases.

DISCUSSION

One of the important components of aviation

accident investigations is forensic toxicology of postmortem biological samples collected from pilot fatalities. In the United States between 1990 and 2000,

biosamples from approximately 80% of all civilian

aviation accidents were submitted to CAMI. Failure

to receive the samples from the remaining accidents

could be attributed to (i) unrecoverable pilot remains

from accident sites, (ii) religious belief of the deceased

pilot’s family in not performing autopsy and postmortem analysis, and (iii) accident investigator-incharge’s decision not to seek forensic toxicology or

not to send samples to CAMI. The increase in the

number of CAMI accidents seen for the 2000 accidents in relation to the 1990 accidents is probably the

result of concerted professional interactions of CAMI

accessioning staff with the NTSB contact person(s)

and FAA’s Regional Flight Surgeons. The enhanced

interaction has been achieved by providing these

groups weekly, by E-mail, a list of those aviation

accidents from which samples were not received at

CAMI. These messages led the respective E-mail receivers to determine the reasons for not sending the

samples and, if necessary, to expedite the sending of

20

40

60

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Monthly Averages of CAMI Accidents and Cases

Cases

Accidents

Figure 3. Monthly averages of the United States aviation accidents and associated cases from

which postmortem samples were received at CAMI. These accidents occurred during the 11-

year period of 1990-2000.
8

Figure 5. Optimal, adequate, and inadequate amounts of various postmortem biological

specimens received at CAMI from the 1,891 cases (fatalities) of the United States fatal aviation

accidents that occurred during 1996-2000.

0%

20%

40%

60%

80%

100%

Blood

Urine

Vitreous Fluid

Spinal Fluid

Bile

Gastric Contents

Liver

Lung

Kidney

Spleen

Muscle

Brain

Heart

CAMI Cases

Optimal Adequate Inadequate

Figure 4. Specimen type and amount-based categorizations of postmortem toxicology cases

received at CAMI from the United States fatal aviation accidents that occurred during 1996-

2000. A total of 1,891 cases (fatalities) were received.

216 CASES

Adequate Blood and 

Urine With Vitreous

Fluid and Any Other 

Sample Types

22 CASES

Optimal Blood and 

Urine With Vitreous Fluid and 

Any Other Sample Types

356 CASES

No Blood or Urine

(Only Solid Tissues)

973 CASES

Either Adequate Blood 

or Urine With or

Without Any Other 

Sample Types

324 CASES

Inadequate Blood 

and Urine With or

 Without Any Other 

Sample Types
9

samples. This increased contact of CAMI was initiated in late 1998, and its positive effect has become

clearly evident in 2000.

The higher number of “CAMI cases” in relation to

the respective number of “CAMI accidents” was obviously attributed to more than 1 occupant in some of

those accident aircraft. The highest number of CAMI

cases as seen for the 1991 accidents was, at least, partly

attributed to the samples from the fatalities of the 01

February 1991 Los Angeles accident involving 2 aircraft (15,17) in which there was a fire. Therefore, in

addition to the samples from pilots/copilots, samples

from other occupants were also submitted for COHb

and CN ̄ analyses. In that 2-airplane runway collision, samples from a total of 33 fatalities were submitted. The observed pattern of the increased accidents

and associated cases during the summer months (JuneSeptember) could be ascribed to the higher aviation

activities during these months.

Each aviation accident is different, and so is the

postmortem toxicology of the accident-associated fatalities (cases). Obtaining multiple sample types in

sufficient amounts is not always possible since it is

governed by the nature of the accident. Differences

from case to case are attributed to the availability of

sample types, amounts of samples submitted for analysis, numbers and concentrations of analytes present in

those samples, and chemical nature of the analytes.

Instead of obtaining all the desired sample types in

optimal amounts for an accident, only certain sample

types are sometimes available in optimal or adequate

amounts, while others in inadequate amounts or not

available altogether. In those scenarios, analyses are

performed on selected sample types, and their selection is based on the intrinsic nature of the analytical

method, analysis type (qualitative, confirmatory, and/

or quantitative), and analyte.

Pharmacological agents in pilot fatalities are generally present in the subtherapeutic to therapeutic concentration range (7,8,16). This observation is

consistent with the very fundamental difference between general forensic toxicology and aviation forensic toxicology (9). The former field—wherein

toxic-to-lethal levels of drugs are often involved—

primarily deals with the cause and manner of poisoning/death, whereas the latter field with the cause of

accident. In this way, “postmortem aviation forensic

toxicology” is basically a human-performance associated postmortem forensic toxicology endeavor (13).

Having multiple sample types in sufficient amounts is

essential for postmortem aviation forensic toxicology

because this facilitates the analyte and/or analytical

method dependent selection of sample types. For

example, blood from the green-top (heparin) tube is

more suitable for COHb, CN ̄, and HbA1c analyses

since it is more homogeneous than the blood from the

gray-top (potassium oxalate and sodium fluoride)

tube. Blood from the gay-top tube is appropriate for

ethanol analysis because of the antibacterial properties of sodium fluoride. The presence of sodium

fluoride prevents the bacteria-mediated in vitro production of ethanol after blood collection. Ethanol

analysis in vitreous fluid, urine, and brain is essential

for establishing the postmortem production of ethanol (6,14,18). Since vitreous fluid and brain are

anatomically preserved, the potential for their exposure to bacteria is usually less. Also, the bacterial

growth is relatively less in urine because of its physiological nature. Thus, the potential for putrefaction

and subsequent postmortem ethanol production is

less in vitreous fluid, urine, and brain than in other

biofluids or tissues, including blood. Glucose analysis

can be conducted more accurately in vitreous fluid

and urine than in postmortem blood. However, HbA1c

concentration determination in blood is crucial in

establishing the existence of hyperglycemia and overall glycemic control (5,20).

Depending upon distribution characteristics, certain drugs may not be present in appreciable amounts

in some body compartments, whereas they may be

present in considerable amounts in other compartments (3,4,9,12,19). Conjugates of drugs and their

metabolites are generally found in appreciable amounts

in bile. Therefore, this matrix is suitable for detecting

those drugs, which may not be present in appreciable

amounts in other compartments. Bile can effectively

be used for detecting drugs, including abused drugs,

by RIA. However, this sample type may not be suitable for the screening of prescription and over-thecounter drugs by HPLC and GC/MS because this

matrix produces extraneous peaks on the chromatograms, thereby interfering with the analysis. Urine is

an appropriate matrix for finding drug metabolites, as

well as basic drugs. In blood, drug metabolites usually

do not accumulate much and basic drugs are generally

present in low concentrations. Compared with basic

drugs, neutral and acidic drugs are commonly present

in higher concentrations in blood. Demonstrating the

presence of drugs and/or their metabolites in multiple

sample types provides convincing scientific evidence
10

for the exposure of fatal accident victims to those

drugs. Such findings are important when metabolites are

not available as drugs, suggesting that the metabolites

were biotransformed in vivo from the parent drug.

The present study documents that sufficient

amounts and types of biological samples are being

submitted from the majority of the aviation accidents

to ensure thorough toxicological evaluation. As a

standard practice at CAMI, the presence of analytes

must generally be established in at least 2 different

sample types by using 2 different analytical techniques for reporting a particular case as “positive,”

provided sufficient amounts of samples are available.

If there is only 1 sample type in a particular presumptive positive case, 2 different portions of the sample

are usually analyzed prior to reporting as a positive

case. Similarly, a different aliquot of samples is reanalyzed for specimen specific analysis, such as COHb

and CN ̄ analyses in blood. Initial analysis/screening

and subsequent confirmatory/quantitative analysis

could be considered 2 types of analyses.

During the whole toxicological process from the

case specimen receiving in the accessioning area to the

analytical result reporting and the data/record keeping, an effective QA/QC program is enforced by

multilevel reviews. The CAMI laboratory participates

in the forensic urine drug and whole blood alcohol/

volatile proficiency-testing surveys and in the accreditation/certification program of the College of American Pathologists (Chicago, IL) to ensure the highest

level of integrity. Additionally, the laboratory participates in the FAA’s postmortem forensic toxicology

proficiency-testing program (11), which is one of the

few proficiency-testing programs recognized by the

American Board of Forensic Toxicology Laboratory

Accreditation Program (1).

In conclusion, CAMI receives postmortem pilot

specimens from the majority of the United States fatal

civil aviation accidents. Such submitted samples are

generally of multiple types in sufficient amounts.

Toxicological evaluation is conducted by using selective and sensitive analytical procedures in multiple

sample types. During the entire process, the highest

level of integrity and quality is maintained.

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2

 This document can be obtained through the National Technical Information Service, Springfield, VA 22161. It is also available at the

Civil Aerospace Medical Institute’s Web site: www.cami.jccbi.gov.
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