Analysis of blood for asphyxiant toxicants -- Carbon monoxide and hydrogen cyanide

ISO 27368:2008 details analytical methods suitable for analysing the two primary toxic combustion gases, carbon monoxide (CO) and hydrogen cyanide (HCN), in blood samples collected from fire casualties. In blood, CO is measured as carboxyhaemoglobin (COHb) and HCN as cyanide ion (CN-). Although numerous methods are reported in the literature for performing blood COHb and CN- analyses, the analytical methods included herein are based upon their suitability for performing the analysis on ante-mortem and post-mortem blood samples from fire casualties. The analytical principle, analysis time, repeatability, reproducibility, robustness, effectiveness and instruments used are considered for those methods. Some of the methods described in ISO 27368:2008 might not be suitable for analysing putrid or clotted blood. Burned (solid) blood can be analysed after homogenization.

Analyse du sang pour substances toxiques asphyxiantes -- Monoxyde de carbone et acide cyanhydrique

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Published
Publication Date
10-Aug-2008
Current Stage
6060 - International Standard published
Start Date
28-Jul-2008
Completion Date
11-Aug-2008
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INTERNATIONAL ISO
STANDARD 27368
First edition
2008-08-15
Analysis of blood for asphyxiant
toxicants — Carbon monoxide and
hydrogen cyanide
Analyse du sang pour substances toxiques asphyxiantes — Monoxyde
de carbone et acide cyanhydrique
Reference number
ISO 27368:2008(E)
ISO 2008
---------------------- Page: 1 ----------------------
ISO 27368:2008(E)
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ii © ISO 2008 – All rights reserved
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ISO 27368:2008(E)
Contents Page

Foreword............................................................................................................................................................ iv

Introduction ........................................................................................................................................................ v

1 Scope ..................................................................................................................................................... 1

2 Normative references ........................................................................................................................... 1

3 Terms and definitions........................................................................................................................... 2

4 Symbols and abbreviated terms ......................................................................................................... 5

5 Blood samples ...................................................................................................................................... 6

5.1 General................................................................................................................................................... 6

5.2 Sample condition.................................................................................................................................. 6

5.3 Sample collection ................................................................................................................................. 6

5.4 Sample storage ..................................................................................................................................... 7

5.5 Sample analysis.................................................................................................................................... 7

6 Materials ................................................................................................................................................ 7

7 Common quality analytical elements.................................................................................................. 7

7.1 General................................................................................................................................................... 7

7.2 Qualitative, quantitative and confirmatory analyses ........................................................................ 7

7.3 Replicate analyses................................................................................................................................ 7

7.4 Analytical batch .................................................................................................................................... 8

7.5 Open controls........................................................................................................................................ 8

7.6 Calibrators ............................................................................................................................................. 8

8 Measurement of CO in blood as COHb............................................................................................... 8

8.1 COHb by whole-blood oximeters ........................................................................................................ 8

8.2 COHb by palladium chloride reduction ............................................................................................10

8.3 COHb by visible spectrophotometry (using calibration curve) ..................................................... 12

8.4 COHb by visible spectrophotometry (with CO saturation)............................................................. 14

8.5 COHb by visible spectrophotometry (without CO saturation) ....................................................... 16

8.6 COHb by headspace gas chromatography — Nickel-hydrogen reduction and flame

ionization detection ............................................................................................................................ 19

8.7 COHb by headspace gas chromatography — Thermal conductivity detection........................... 22

9 Measurement of HCN in blood as CN .............................................................................................. 23

9.1 CN by colourimetric method (p-nitrobenzaldehyde and o-dinitrobenzene)................................ 23

9.2 CN by visible spectrophotometry.................................................................................................... 25

9.3 CN as HCN by headspace gas chromatography — Nitrogen phosphorous detection.............. 29

9.4 CN by headspace gas chromatography — Electron capture detection ...................................... 31

9.5 CN by spectrophotofluorimetry or high-performance liquid chromatography using a

fluorescence detector......................................................................................................................... 33

9.6 CN by high-performance liquid chromatography–mass spectrometry....................................... 37

Annex A (normative) Analytical report pro forma......................................................................................... 41

Annex B (informative) Additional aspects of analytical methods ............................................................... 43

Annex C (informative) Interpretation of results............................................................................................. 47

Bibliography ..................................................................................................................................................... 52

© ISO 2008 – All rights reserved iii
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ISO 27368:2008(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies

(ISO member bodies). The work of preparing International Standards is normally carried out through ISO

technical committees. Each member body interested in a subject for which a technical committee has been

established has the right to be represented on that committee. International organizations, governmental and

non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the

International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. ISO shall not be held responsible for identifying any or all such patent rights.

ISO 27368 was prepared by Technical Committee ISO/TC 92, Fire safety, Subcommittee SC 3, Fire threat to

people and environment.
iv © ISO 2008 – All rights reserved
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ISO 27368:2008(E)
Introduction

Carbon monoxide (CO) and hydrogen cyanide (HCN) are two of the primary toxic combustion gases present

in fire atmospheres. Upon burning, carbon-containing substances generate CO, whereas nitrogen-containing

substances also produce HCN. Since structures surrounding human beings are composed of polymeric

materials containing carbon and nitrogen elements as their constituents, these materials generate CO and

HCN upon burning and fire victims are exposed to these gases by inhaling smoke. Although ISO 19701

documents methods for the analysis of CO and HCN in fire effluents, the actual toxic insult to exposed

persons can be assessed only by the analysis of the fire casualties' blood for CO as carboxyhaemoglobin

(COHb) and HCN as cyanide ion (CN ). These analytical findings are useful for
⎯ estimating life-threatening characteristics of fire atmospheres,
⎯ evaluating the degree of toxicity caused by smoke inhalation in fire victims,
⎯ determining the cause and manner of death of fire victims,
⎯ improving understanding of the direct causes of fire injury and death,

⎯ enhancing understanding of acute and delayed adverse effects of smoke on fire casualties,

⎯ administering immediate treatment for smoke poisoning and monitoring delayed adverse effects of smoke,

⎯ choosing appropriate emergency, long-term and/or follow-up treatments for surviving fire casualties,

⎯ setting priorities for emergency treatment of multiple fire casualties,

⎯ establishing relationships between the concentrations of CO and HCN in a fire atmosphere, blood COHb

and CN levels, and the degree of toxicity and performance impairment,

⎯ achieving correlations between concentrations of the two gases in fire atmospheres and of COHb and

CN in blood in order to improve tenability models,

⎯ identifying deficiencies with materials, products, assemblies, structures and escape routes, and

⎯ improving forensic toxicology analytical processes and procedures.

Compliance with this International Standard can help ensure a consistent data set for use in a variety of fields

such as

a) fire statistics, which themselves are frequently used to develop regulatory policy,

b) international collaboration on improved design, materials and use of habitable structures, and,

c) ultimately, improvement of international relations and trades.

Such compliance can further assist in developing better and safer fire-safety instruments and structures

(residential and commercial buildings; locomotive passenger vans, automobiles, aerospace vehicles and other

vehicular structures).

Various different methods are currently used for obtaining blood analysis data for these two fire toxicants and

the lack of standardized procedures can result in a wide variation of interpretation. It is, therefore, proposed to

set out best-practice, standardized procedures for blood sample collection, sample storage, sample

processing/preparation, sample treatment and transfer to analytical instrumentation, analytical instrumentation

© ISO 2008 – All rights reserved v
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ISO 27368:2008(E)

and techniques, data presentation and reporting, and guidance for data interpretation. The analytical methods

included herein are based upon their suitability for performing an analysis on ante-mortem and post-mortem

blood samples from fire victims and are commonly used in forensic toxicological analytical operations.

This International Standard is structured as follows.
⎯ Clause 1 describes the scope of this International Standard.
⎯ Clause 2 cites the normative references.
⎯ Clause 3 provides terms and their definitions.
⎯ Clause 4 lists symbols and abbreviated terms.

⎯ Clause 5 provides a general description of collecting, storing and analysing blood samples.

⎯ Clause 6 covers the quality of materials used during an analysis.
⎯ Clause 7 summarizes common quality analytical elements.
⎯ Clause 8 describes analytical methods for measuring CO as COHb.
⎯ Clause 9 delineates analytical methods for measuring HCN as CN in blood.

⎯ Annex A (normative) lists the information crucial for reporting blood analysis results.

⎯ Annex B (informative) outlines additional aspects of analytical methods.

⎯ Annex C (informative) discusses the interpretation of results, including the interactive effects of CO and

HCN.
⎯ The bibliography includes references cited in this International Standard.
vi © ISO 2008 – All rights reserved
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INTERNATIONAL STANDARD ISO 27368:2008(E)
Analysis of blood for asphyxiant toxicants — Carbon monoxide
and hydrogen cyanide

SAFETY PRECAUTIONS — Due consideration shall be given to the fact that both the blood samples

for the analyses of asphyxiant toxicants, carbon monoxide (CO) and hydrogen cyanide (HCN), and

many of the reagents used for their analyses can be biohazardous and/or toxic and can thereby pose

serious health hazards. It is recommended that the collection of blood samples from fire victims be

performed by medical practitioners and in accordance with best practices established by the medical

authorities in the area. Additionally, it is assumed that the procedures described herein are carried out

by suitably qualified professional personnel, adequately trained in the hazards and risks associated

with the handling of biological samples and such analyses and aware of any safety regulations that

can be in effect. Consideration shall also be given to the safe and ecologically acceptable disposal of

all biological samples and chemicals used for analyses. This can require extensive and specific

treatment prior to release of the waste into the environment. Again, it is assumed in this International

Standard that the personnel responsible for the safe disposal of such bio-samples and reagents are

suitably qualified and trained in these procedures and techniques and are aware of the regulations

that can be in force.
1 Scope

This International Standard details analytical methods suitable for analysing the two primary toxic combustion

gases, carbon monoxide (CO) and hydrogen cyanide (HCN), in blood samples collected from fire casualties.

In blood, CO is measured as carboxyhaemoglobin (COHb) and HCN as cyanide ion (CN ). Although

numerous methods are reported in the literature for performing blood COHb and CN analyses, the analytical

methods included herein are based upon their suitability for performing the analysis on ante-mortem and post-

mortem blood samples from fire casualties. The analytical principle, analysis time, repeatability, reproducibility,

robustness, effectiveness and instruments used are considered for those methods. Some of the methods

described herein might not be suitable for analysing putrid or clotted blood. Burned (solid) blood can be

analysed after homogenization.
2 Normative references

The following referenced documents are indispensable for the application of this document. For dated

references, only the edition cited applies. For undated references, the latest edition of the referenced

document (including any amendments) applies.

ISO 3696:1987, Water for analytical laboratory use — Specification and test methods

ISO 13344, Estimation of the lethal toxic potency of fire effluents

ISO/TS 13571, Life-threatening components of fire — Guidelines for the estimation of time available for

escape using fire data
ISO 13943, Fire safety — Vocabulary
ISO 19701, Methods for sampling and analysis of fire effluents
© ISO 2008 – All rights reserved 1
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ISO 27368:2008(E)
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 19701, ISO 13344, ISO/TS 13571,

ISO 13943, ISO 3696, and the following apply.
3.1
analyte

substance that is being identified or determined in a specimen during an analysis

EXAMPLES COHb and CN .
3.2
analytical batch

set of aliquots taken out from the specimens associated with various cases (fire casualties) and from negative

and positive blind controls for performing a particular type of analysis
3.3
asphyxiant

toxicant causing loss of consciousness and ultimately death resulting from hypoxic (deficiency-of-oxygen)

effects, particularly on the central nervous and/or cardiovascular systems
3.4
blind controls
open controls but their identity is unknown to the analysts
See open controls (3.20).
3.5
calibrator

material that is based on, or traceable to, a reference preparation or material and whose values are

determined by acceptable reference methods
3.6
carboxyhaemoglobin
compound formed when CO combines with haemoglobin

NOTE Haemoglobin has an affinity for binding to CO that is approximately 245 times higher than that for binding to

oxygen; thereby the ability of haemoglobin to carry oxygen is seriously compromised during CO poisonings (see C.3.3 and

Reference [73]).
3.7
Cheyne-Stokes respiration

breathing pattern characterized by rhythmic waxing and waning of the depth of respiration, with regularly

recurring periods of breathing cessation
3.8
cutaneous blood vessels
blood vessels relating to, or affecting, the skin
3.9
cyanogenic glycosides
group of molecules containing a sugar moiety and a cyanide (CN) group

NOTE Cyanogenic glycoside can release the poisonous HCN gas if acted upon by some enzyme.

EXAMPLE Amygadlin from almond.
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ISO 27368:2008(E)
3.10
cyanomethaemoglobin
compound formed when CN combines with methaemoglobin

NOTE During the treatment of CN poisonings, haemoglobin is chemically converted to methaemoglobin, which

easily binds with CN , producing cyanomethaemoglobin. The formation of cyanomethaemoglobin is an essential and

critical step in the CN detoxification process (see Reference [71]).
3.11
cyanosis
bluish discoloration of the skin caused by the lack of oxygen in the blood
3.12
deoxyhaemoglobin

form of haemoglobin without oxygen, the predominant protein in the red blood cells

NOTE Haemoglobin forms an unstable, reversible bond with oxygen. The oxygen-bonded haemoglobin is known as

oxyhaemoglobin. In the oxygen-unloaded form, it is called deoxyhaemoglobin and is purple-blue.

3.13
fire effluent

totality of gases and/or aerosols, including suspended particles, in the atmosphere resulting from combustion

or pyrolysis
3.14
fractional toxic concentration
FTC

ratio of the percent of COHb in a blood sample to 70 % COHb (FTC ) or of the concentration of CN ,

COHb
expressed in micrograms per millilitre, in a blood sample to 3,0 µg/mL CN (FTC )

NOTE It is considered that CO at 70 % COHb or HCN at 3,0 µg/mL CN individually can cause lethality. For an

additive effect of a mixture of the two gases, FTC plus FTC should be equal to unity. However, the above concept

COHb CN¯
does not rule out other additive effects of these gases (see Clause C.5).
3.15
haemoglobin

biological substance in the red blood cells made up of iron and protein and involved in carrying oxygen to

various parts of the body
NOTE Deoxyhaemoglobin or reduced haemoglobin is also referred as to haemoglobin.
3.16
isobestic point

wavelength at which the spectra of various species of a substance have the same absorbance

EXAMPLE The substance haemoglobin and its species oxyhaemoglobin and COHb.
3.17
methaemoglobin
particular type of transformed haemoglobin that is unable to bond with oxygen

NOTE Haemoglobin is converted to methaemoglobin by the oxidation of haemoglobin iron(II) (ferrous iron) into

iron(III) (ferric iron). This oxidized form of haemoglobin is in firm union with water and is chemically unable to associate

with oxygen; thus, it is ineffective for respiration. Large-scale conversion of haemoglobin to methaemoglobin can cause

blueness of skin due to lack of oxygen.
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ISO 27368:2008(E)
3.18
methanation unit

unit capable of chemically converting CO into methane (CH ) by using hydrogen in the presence of nickel as a

catalyst
3.19
mydriasis
dilatation of the pupil
3.20
open controls

specimens prepared for the purpose of being used as a control and known to the analysts

3.21
oxyhaemoglobin

oxygen-bonded form of haemoglobin, the predominant protein in the red blood cells

NOTE Haemoglobin forms an unstable, reversible bond with oxygen. In its oxygen-loaded form, it is called

oxyhaemoglobin and is bright red.
3.22
polymeric materials
materials composed of polymers

NOTE A polymer is a large molecule made up of many smaller repeating chemical units bonded together. These

units are known as monomers. Some polymers are naturally occurring, while others are synthetically manufactured.

3.23
post-mortem interval
period after death
EXAMPLE Time between death and blood sample collection from a dead body.
3.24
putrefaction

decomposition of organic matter, especially protein, by microorganisms, resulting in the formation of

substances of less complex constitution with the evolution of ammonia, hydrogen sulfide and other

substances and, thus, in the production of foul-smelling matter
NOTE This process is usually characterized by the presence of malodorous smell.
3.25
pyocyaneous organisms
group of microorganisms capable of producing CN
3.26
reduced haemoglobin

haemoglobin in the red blood cells after the removal of oxygen from oxyhaemoglobin or after the reduction of

iron(III) (ferric iron) in methaemoglobin to iron(II) (ferrous iron)
3.27
sulfaemoglobin

product formed by the action of hydrogen sulfide (or sulfides) on iron(III) (ferric iron) in methaemoglobin

NOTE This haemoglobin product is also known as sulfmethaemoglobin.
3.28
tachycardia
excessive rapidity in the action of the heart
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ISO 27368:2008(E)
3.29
tachypnea
excessive rapidity of respiration
3.30
thermostatization

process of automatic temperature regulation, especially wherein the expansive force of metals or gas acts

directly upon the source of heat, ventilation or the like, or controls them indirectly by opening and closing an

electric circuit
NOTE Derived from the term “thermostat”.
3.31
toxicants

poisonous substances capable of causing adverse, unwanted or undesired effect(s) on a living system

NOTE For the purpose of this International Standard, these substances are CO and HCN.

3.32
toxic insult

adverse, unwanted or undesired effect(s) on a living system due to, pertaining to, or of the nature of a poison

4 Symbols and abbreviated terms
A Area
α Absorbance
C Concentration
CBI 1-Cyano-2-benzoisoindole or 1-cyano[f]benzoisoindole
ClCN Cyanogen chloride
CN Cyanide
CN Cyanide ion
CO Carbon monoxide
COHb Carboxyhaemoglobin
ECD Electron capture detector
EDTA Ethylenediaminetetraacetate
F Factor
FED(s) Fractional effective dose(s)
FID Flame ionization detector
FTC Fractional toxic concentration
HCN Hydrogen cyanide
HHb Deoxyhaemoglobin
HPLC High-performance liquid chromatograph
© ISO 2008 – All rights reserved 5
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ISO 27368:2008(E)
IEC International Electrotechnical Commission
ISO International Organization for Standardization
MetHb Methaemoglobin
MSD Mass spectrometric detector
NDA 2,3-Naphthalenedialdehyde
NPD Nitrogen phosphorus detector
OxyHb Oxyhaemoglobin
R Ratio
TCD Thermal conductivity detector
tHb Total haemoglobin
TIC Total-ion chromatogram
V Volume
w Mass fraction
5 Blood samples
5.1 General

For the analyses of COHb and CN , blood from fire victims should be properly collected as soon as possible,

preserved, stored and analysed as quickly as possible. See also C.3.1 and C.4.1.
5.2 Sample condition

Fresh blood samples can be easily obtained from live fire victims, but collecting quality blood samples from

fire fatalities can frequently be challenging. This challenge is linked to the condition of the body, which is

affected by the severity of burn, the time between the death and the discovery of the body (post-mortem

interval), and the environmental factors, such as temperature and humidity. There are reports of the condition

of blood, for example, fresh or putrid blood, having an impact on the outcome of the analyses. Therefore, the

documentation of the history, condition and characteristics of the blood samples is crucial, and this information,

along with the blood samples, should be submitted to the analytical laboratories performing analyses.

5.3 Sample collection

It is recommended that blood samples from fire casualties be preferably collected in 10 ml (or smaller size)

sterile glass tubes containing heparin, or 20 mg of potassium oxalate and 100 mg of sodium fluoride, to

[1]

prevent blood clotting and/or to preserve the specimens . Some analytical methods use heparinized blood,

while other methods can use blood treated with either heparin or potassium oxalate-sodium fluoride. The

headspace in the tubes should be kept to a minimum and the tubes containing the blood samples should be

airtight sealed to minimize dissociation of CO and HCN and to prevent any escape of these gases from the

collected blood. Post-mortem blood samples can be collected from the heart, though no statistically significant

difference has been observed between the COHb levels in post-mortem heart blood and peripheral blood

[2]

specimens . Regardless of the blood collection site, however, it is recommended that the sample collection

site be mentioned in the documents submitted with the blood samples for analysis.

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ISO 27368:2008(E)
5.4 Sample storage

The blood specimens should be stored at 4 °C in the airtight, sealed containers to prevent the loss of CO,

[3],[4],[5],[6],[7],[8]

denaturation of haemoglobin and release of HCN . If it is necessary to store samples for a long

[3],[4],[5],[6],[9],[10],[11],[12],[13],[14]
period prior to analysis, then the samples should be frozen .
5.5 Sample analysis
[9],[15],[16]

Analyses should be performed as quickly as possible after the collection of blood . It is essential for

the analysis of COHb to homogenize those blood samples that are not homogeneous. A similar

recommendation has also been made for CN that autopsy blood should be homogenized before the

[17]
analysis .
6 Materials

All reagents, solvents, gases, and chemicals used in analyses should be of analytical grade quality and of the

highest available purity. Water used should be as defined in ISO 3696:1987, quality 3.

7 Common quality analytical elements
7.1 General

Forensic blood samples are precious. Depending upon the nature of the fire accident and condition of the fire

victim, a blood sample might, or might not, have been submitted in a large amount for analyses. Once the

blood samples are consumed during analyses, it might not be possible to obtain additional samples from the

sample submitters. Therefore, it is customary in forensic toxicological operations to use samples submitted for

analyses conservatively and cautiously.

Unless stated otherwise, all blind and open controls and calibrators used for analysis shall be prepared in

human whole blood. It is important that blood be collected from healthy human subjects who are not smokers

and are not exposed to CO. In other words, the collected human blood shall be free from CO and CN .

7.2 Qualitative, quantitative and confirmatory analyses

It is recommended that a qualitative analysis (screening) be performed initially on a portion (aliquot) of the

blood sample collected from each victim. On the qualitatively positive (presumptive positive) samples, a

quantitative analysis should be conducted. Although qualitative and quantitative analyses in some methods

can be simultaneously conducted on the same aliquot, it is preferred that the quantitative analysis be

performed on a different aliquot of the submitted sample than that which was used during the initial qualitative

analysis.

Additionally, quantitative analytical results should be confirmed on a different aliquot of the blood sample by a

second method based upon a analytical principle different from the method used during the first quantitative

analysis. Such confirmatory analyses can be qualitative or quantitative.
...

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