Review Article

Analysis of carbon monoxide

Brian Widdop

Abstract
Address The degree of exposure to carbon monoxide is most often assessed by measuring
Medical Toxicology Unit the blood carboxyhaemoglobin saturation. This measurement is relevant to
Guy’s and St Thomas’ Hospital
investigations of acute accidental or deliberate poisoning and of chronic exposure in
NHS Trust, Avonley Road
London SE14 5ER, UK a domestic or work place environment. Simple spectrophotometric methods based on
differential protein precipitation or dithionite reduction are prone to interference from
This article was prepared at the invitation of other haemoglobin pigments and are imprecise for low-level estimations. Automated
the Analytical Investigations Standing spectrophotometric devices (CO-oximeters) that estimate simultaneously total
Committee of the Association of haemoglobin, percentage oxyhaemoglobin and percentage carboxyhaemoglobin
Clinical Biochemists. have acceptable accuracy for carboxyhaemoglobin saturation levels of 45% and are
recommended for most clinical purposes. For the investigation of low-level exposure
Correspondence
Dr Brian Widdop and the detection of increased haemolysis in neonates, more sensitive methods
E-mail: brian.widdop@gstt.sthames.nhs.uk involving the release of carbon monoxide and its measurement by gas
chromatography are required. Gas chromatographic methods are also appropriate
when examining post-mortem blood samples where putrefaction or heat stress has
resulted in a signiŽcant change in haemoglobin composition.
Ann Clin Biochem 2002; 39: 378- 391

Introduction saturation of 5^6% and heavy smokers can reach

Sources of carbon monoxide
Endogenous carbon monoxide is produced by the
catabolism of haem and results in a background
carboxyhaemoglobin (COHb) saturation of 0¢4^0¢7%
in resting healthy subjects.1 The gas is an ubiquitous
product of incomplete combustion of materials
containing carbon, and many accidental poisonings
are due to faulty domestic heating appliances.2 Car
exhaust fumes can contain up to 10% of carbon
monoxide and deliberate self-exposure to these in an
enclosed space remains a common and very e¡ective
form of suicide.3,4 With the advent of new legislation to
cut down carbon monoxide emissions via catalytic
converters, there have been reports that this has
reduced the incidence of this manner of suicide,5 but
given a su¤cient length of time of exposure, death
from hypoxia may occur.6 Unintentional carbon
monoxide poisoning in vehicles also occurs due to
leaking exhausts and inadequate ventilation.7
Exhaust emissions are mainly responsible for non-
smokers living in urban areas having COHb satura-
tions of 1^2%. Tobacco smoke has about 4% carbon
monoxide content. Regular smokers develop a COHb
10%.8 Passive inhalation in smoke-¢lled rooms for
1¢5 h has been shown to increase the COHb saturation
in non-smokers by almost 40%.9 Burning charcoal
briquettes indoors for either cooking or heating has
also caused problems.10
A more surprising cause of carboxyhaemo-
globinaemia is exposure to dichloromethane (methy-
lene chloride) vapours. This solvent is widely used as a
paint stripper, degreasing agent and aerosol propel-
lant, and following inhalation is metabolized by
mixed-function oxidases to carbon dioxide and carbon
monoxide. COHb saturation levels of 5^15% are
usually found in subjects exposed to the vapours for
2^3 h, but levels of 40% and 50% have been reported
in individual cases.11,12
Mechanisms of toxicity
Carbon monoxide has an a¤nity for haemoglobin that
is approximately 220 times that of oxygen. It displaces
oxygen from oxyhaemoglobin and at the same time
changes the allosteric structure of haemoglobin such
that the a¤nity of the remaining sites for bound
oxygen increases. As a consequence, the oxygen-
carrying capacity of the blood is reduced and the
release of oxygen (and therefore its delivery) to the

378 © 2002 The Association of Clinical Biochemists


tissues is inhibited, leading to progressive asphyxia.
Areas of high metabolic activity (heart, brain) are
particularly susceptible to the resulting cellular
hypoxia. Carbon monoxide is also a cellular poison in
its own right as it competes with oxygen for other
haemoproteins such as myoglobin, peroxidase,
catalases and the cytochromes, although as the
a¤nity of oxygen for cytochrome oxidase is high the
e¡ect on this system may be quite small.13 Binding of
carbon monoxide to cardiac muscle myoglobin14 can
result in myocardial depression and hypotension
leading to ischaemia, which exacerbates the hypoxia
induced by the impaired oxygen delivery.
Clinical effects and correlation with
carboxyhaemoglobin saturation levels
The main features of acute carbon monoxide
poisoning are headache, nausea, confusion, stupor
and coma.15,16 Headache, nausea and fatigue are also
signs of chronic poisoning, but there is also an
insidious deterioration in intellectual capacity, with
di¤culties in concentration and decreased cognitive
function.17,18 More than 40% of patients still have
neurological problems 3 years after chronic exposure.
The condition is often misdiagnose d, and COHb
measurements are therefore particularly useful in this
situation.
Considerable controversy surrounds the diagnostic
and prognostic value of COHb measurements in acute
poisoning cases. The evidence for a correlation with
patient outcome in hospital is weak, although sending
blood samples to the laboratory after the patient has
been given oxygen clouds the issue as carbon
monoxide is dislodged from haemoglobin very quickly
such that the blood COHb concentration no longer
re£ects tissue exposure. Carbon monoxide does not
react very quickly with haemoglobin. Red cells shaken
within an atmosphere of 100% carbon monoxide take
20 min to saturate and only about 25% of haemo-
globin is converted to COHb after 5 min. As a
consequence, much of the inhaled carbon monoxide
(which dissolves quickly in plasma) has little time to
combine with haemoglobin before the blood reaches
other susceptible organs where it combines and
disrupts cellular enzymes. It is therefore the non-
haemoglobin-bound carbon monoxide that kills.
Carbon monoxide persists in tissues long after COHb
levels in blood have returned to normal19 and this is
one of the arguments used by protagonists of hyper-
baric oxygen therapy for acute carbon monoxide
poisoning. Hyperbaric oxygen increases the elimina-
tion rate of COHb, causes more oxygen to be dissolved
in the plasma and may help to disperse residual carbon
monoxide from the tissues. The primary objective of
the therapy is to prevent the onset of delayed neuro-
logical deterioration, which occurs 2^3 weeks after
Analysis of carbon monoxide 379
severe exposure, but it has not been established that all
patients will bene¢t and there are complications, such
as decompression sickness, associated with the treat-
ment.20,21
In forensic investigations, there are occasional
incidents in which the circumstances seem to point
irrefutably to carbon monoxide poisoning, but the
blood analysis shows low or normal COHb levels.22
These cases have included car-exhaust suicides where
the only reasonable explanation is that the car engine
stopped after a short time but had introduced
su¤cient carbon monoxide into the cabin to cause
irreversible coma. Over time, the atmosphere in the
car changes to normal, the victim continues to respire
so that COHb also reduces to normal values, but death
occurs due to cerebral anoxia 1^2 days later. It is also
important to bear in mind that elderly people,
particularly those who have frequent periods of
hypoventilation, may have a much lower proportion of
red cells carrying oxygen than younger people. As a
result, older people tend to be more susceptible to
carbon monoxide exposure and may die with relatively
low COHb levels.
Although there is no doubt that a normal COHb
saturation level does not rule out the possibility of
carbon monoxide poisoning, an elevated measure-
ment is a clear indication of exposure and, despite
signi¢cant individual variation, it is possible to
produce guidelines that link this parameter to signs
and symptoms (see Table 1).
Analysis
Applications
COHb measurements are used most often to diagnose
acute carbon monoxide poisoning. There are,
Table 1. Clinical signiŽcance of COHb saturation levels
COHb (%) Interpretation, signs and symptoms
0- 2 Normal levels in non-smokers
5- 6 Normal levels in tobacco smokers; impaired
driving skills and decreased exercise tolerance
in non-smokers
10- 20 Headache, fatigue
20- 30 Severe headache, nausea, vomiting, dizziness,
blurred vision, fainting
30- 40 Nausea, vomiting, fainting, increased heart and
respiratory rate, impaired neurological function
40- 50 Coma, convulsions, impaired cardiovascular and
neurological function
50- 60 Coma, convulsions, depressed respiration and
depressed cardiovascular status
60- 70 Coma, convulsions, cardiorespiratory depression,
bradycardia, severe hypotension
470 Respiratory failure and death
COHb ˆ carboxyhaemoglobin.
Ann Clin Biochem 2002; 39: 378- 391
380 Widdop
however, other applications such as in the detection of
increased haemolysis in newborns 23,24 and the inves-
tigation of the e¡ects of chronic exposure to low levels
of carbon monoxide on human health and perfor-
mance, particularly in the work-place.25 This has
stimulated the development of very sensitive methods,
some of which will be described later. The other major
application is in the investigation of fatalities due to
deliberate or accidental carbon monoxide poisoning.
In addition, it is a routine procedure for forensic
chemists to examine blood samples from ¢re victims
for COHb content.Whereas a saturation level of 450%
indicates carbon monoxide poisoning as the primary
cause of death, levels of 10^50% show that smoke was
inhaled, carbon monoxide could have been a contri-
buting factor and proves beyond doubt that the
deceased was alive when the ¢re started. If the value is
below 10%, either the individual was dead before the
¢re began or died shortly afterwards. This information
can be quite crucial in cases of civil litigation and in
criminal investigations. Similar situations can arise
when a fatal road accident could be linked to exposure
to carbon monoxide from a faulty exhaust. Toxico-
logical analyses usually form part of the investigation
protocol for fatal airplane accidents and it is routine to
carry out tests for carbon monoxide exposure in
samples from pilots.
In uence of post-mortem changes and choice
of method
Clinical chemists usually have the advantage of a fresh
blood sample and have concentrated on spectro-
photometric methods designed to estimate the
percentage of haemoglobin that has combined with
carbon monoxide to produce COHb. These measure-
ments can be unreliable in the presence of other
haemoglobin pigments, and estimations in old or post-
mortem blood samples are especially di¤cult in this
respect due to the spontaneous production of
methaemoglobin and sulphaemoglobin. Blood
samples that have been subjected to great heat can
show gross changes, notably thermocoagulation,
resulting in a signi¢cant decrease in total soluble
haemoglobin and the appearance of methaemoglobin.
For these reasons, forensic toxicologists have preferred
techniques that release carbon monoxide from the
blood sample and allow the concentration of the gas
itself to be measured. The amount of carbon monoxide
found has then to be related back to the original blood
by measuring either haemoglobin or total iron
content. It is di¤cult to extrapolate a reliable estimate
of the percentage haemoglobin in the ante-mortem
blood from one carried out on post-mortem blood that
has clotted. This has led to many instances in which
hospital biochemistry laboratory results and forensic
science laboratory results do not agree.
Ann Clin Biochem 2002; 39: 378- 391
Sample stability and storage
Carbon monoxide can be produced in decomposing
blood, probably due to the breakdown of haemoglobin.
Conversely, signi¢cant losses can also occur during
storage, although freezing the samples and thawing
them only at the time of analysis can halt this.26
Samples can also be stabilized to some extent by
adding sodium dithionite.27 In a recent study by
Kunsman et al.,28 COHb was found to be stable in post-
mortem blood samples stored in vacutainer tubes for
up to 2 years at 38C with or without preservative, and
they emphasiz ed that temperature was the most
important factor. This contrasts with the report by
Chace et al.29 that headspace volume, surface area and
initial COHb concentration had a signi¢cant in£u-
ence, as well as with the work of Vreman et al.,30 who
cited EDTA vacutainers as a cause of falsely elevated
saturation values, although this occurred only at very
low levels. The conclusion is that anticoagulated blood
samples should ideally be sealed into vials with a
minimum of air space and stored deep-frozen or at
least at 38C prior to assay.
Preparation of calibrants
Preparation of COHb calibrants requires careful
attention to detail, and fresh blood, preferably from
non-smokers, should be used. Blood more than a week
old takes much longer to become fully carboxylated.31
The usual procedure is to dilute the blood with saline,
centrifuge and then haemolyse the red cells with a
borate bu¡er. A 0% COHb calibrant is prepared by
bubbling air through one portion of the diluted and
haemolysed blood for about 10 min to dissociate any
COHb. To make a 100% COHb calibrant, pure carbon
monoxide is bubbled through another portion for
30 min. Any dissolved carbon monoxide is then
removed by passing nitrogen through the solution for
15 min. These two calibrants can then be mixed
together to give a range of intermediate concen-
trations. Commercial quality-control material (e.g.
from IL,Warrington, UK) in sealed glass ampoules is a
much more convenient means of calibration for
routine clinical assays. The material is prepared from
puri¢ed human haemoglobin and contains various
proportions of the clinically important haemoglobin
derivatives. It is quite suitable for most purposes as
long as the expiry dates are strictly observed.
External quality assessment
The United Kingdom External Quality Assessment
Scheme (UKNEQAS) for toxicology has been operating
since 1994 and has over 150 participating labora-
tories. The scheme includes quality assessment
material for percentage COHb measurement, which is
prepared from expired human red cells. One sample is
circulated every month and results are reported in the

format of consensus mean, standard deviation, co-
e¤cient of variation, standard deviation from the
mean, bias, Bias Index Score (BIS) and target value. A
12-month BIS summary is also included. A full
description of the scheme can be obtained from http://
www.ukneqas.org.uk /Directory/ CC/toxicol.htm.
Historical methods
Scientists have been interested in COHb for many
years and a variety of approaches were used prior to
the advent of modern spectrophotometric and
chromatographic equipment. Although most of the
methods described below are no longer in use in
Western countries, some still ¢nd a role in developing
countries where laboratories have meagre funds and
therefore rely on more rudimentary techniques.
Simple comparative colour test
Blood from a carbon monoxide victim sometimes has a
cherry red appearance due to the COHb content, but a
normal coloration by no means excludes poisoning.
More sensitive than visual examination is to compare
0¢5 mL of the blood sample diluted with 10 mL of
10 mmol/ L ammonia with the colour of a sample of
normal blood treated in the same way. A pink tinge as
opposed to the straw colour of normal blood suggests
COHb is present, although blood from a case of cyanide
poisoning may also be pink.
Hartridge reversion spectroscope
This was a simple device, now obsolete, that allowed
the absorption bands of COHb and oxyhaemoglobin to
be observed directly in a diluted blood sample. After
adding a few grains of sodium dithionite, the two
spectral bands of oxyhaemoglobin were reduced to
one (reduced haemoglobin), whereas the two bands
for the una¡ected COHb persisted. This method could
be used quantitatively and experienced operators
could achieve an accuracy of +10%.
Microdiffusion
This method uses a Conway microdi¡usion cell, which
is basically a petri dish with an outer and an inner
well, which can be made airtight by a glass cover-plate
covered with a viscous sealant material. The principle
is to liberate carbon monoxide from the blood placed in
the outer well using sulphuric acid and then allow it to
react with a solution of palladium chloride in the inner
well.32 This method requires skill and a great deal of
practice to derive a reliable quantitative result. It still
¢nds favour with some older forensic toxicologists and
those working in countries where more expensive
equipment is not available. However, in a modern
setting, it is most likely to ¢nd use as a fairly rapid
semi-quantitative test, which can detect COHb
saturation levels of 10% or above.
Analysis of carbon monoxide 381
Colorimetric methods
In addition to microdi¡usion, there are numerous
other methods, which rely on reacting liberated
carbon monoxide with a colour reagent, and some are
claimed to be capable of measuring endogenous
saturation levels as low as 1%. For example, in one
method33 the liberated gas mixed with air is led into a
£ask containing an alkaline solution of the silver salt
of p-sulphaminobenzoic acid which generates a
colloidal solution of silver. The absorbance of this
solution is related to the carbon monoxide concentra-
tion in the gas sample. This is a technically demanding
assay and not appropriate in a present-day laboratory.
Volumetric methods
This was a classic technique carried out with a Van
Slyke volumetric blood apparatus, which in its day was
regarded as the reference method for COHb measure-
ment.34 Even then, it was time -consuming and is
unlikely to be found in operation today.
Current methods
Differential protein precipitation
Whitehead and Worthington published their colori-
metric method in 1961 and it quickly became one of the
most widely used procedures for COHb measurements,
at least in UK clinical chemistry laboratories.35 The
method is based on a principle ¢rst applied by Wol¡.36
Oxyhaemoglobin is precipitated by heat at around pH 5,
whereas COHb is only partially precipitated. After
treatment and ¢ltration, the absorbance of the sample
at 555 nm can be read in a simple colorimeter and
compared with a sample of 100% carboxylated blood
treated in the same way. Whitehead and Worthington
were concerned that apparent levels of 2¢0^6¢5% were
present in normal blood samples. This was found to be
due to unprecipitated oxyhaemoglobin after heating, so
the pH, heating temperature and time of heating were
adjusted such that complete precipitation occurred
with approximately 100% of COHb remaining in
solution. The normal blood values were reduced to
1¢6^2¢2%; this background absorption was shown to
result from chromogenic material in the plasma and
could be removed by washing the red cells with normal
saline. Whitehead and Worthington evaluated their
method by distributing blood with10% COHb around12
UK clinical chemistry laboratories. The results were
quite reassuring, with a range of 7¢9^11¢6% and a mean
value of 9¢9% (standard deviation ˆ 1¢4). This is
surprising given the critical conditions needed to
ensure reproducible results.31 Although the method
still survives in some laboratories, a recent comparative
survey 37 carried out among136 UKNEQAS participants
concluded that the method was no longer acceptable
due to its poor precision.
Ann Clin Biochem 2002; 39: 378- 391
382 Widdop
Spectrophotometric methods
Measurement of the proportion of COHb in blood
samples by spectrophotometric methods is based on
comparing the absorbance spectra of COHb with that
of oxyhaemoglobin and/or reduced haemoglobin. In
1962, a review by Maehly 38 outlined the main
problems of this approach; these were the presence of
other pigments and the variation in accuracy of both
wavelength and photometry in the spectro-
photometers available at the time. Figure 1, which
shows the absorption spectra of four common haemo-
globin pigments together with those of sulphaemo -
globin and lipid, illustrates the complexities. In spite of
the problems having been pointed out, a number of
methods based on this principle followed, but failed to
be reliable or practicable due to interference of related
pigments 39 and the di¤culty of calibrating individual
spectrophotometers.
Later workers 40,41 exploited the fact that sodium
dithionite converts oxyhaemoglobin and methaemo-
globin in crude haemolysates to reduced haemoglobin.
Carbon monoxide has a far greater a¤nity for haemo-
globin than has oxygen and therefore COHb remains
unchanged. Newly available recording spectrophoto-
meters made such an approach quite attractive. The
method in its simplest form is as follows.
The blood sample is mixed with dilute ammonia and
the solution divided into three parts. One part (A) is
saturated with carbon monoxide (100% COHb
standard), oxygen is bubbled through another part (B)
to displace any carbon monoxide (0% COHb standard)
and the other part (C) is untreated. A small amount of
sodium dithionite is added to these solutions and each
is scanned between 500 and 640 nm in such a fashion
that the absorption curves are superimposed. Figure 2
illustrates the set of spectra that should appear.
Absorbances for each solution are measured at
Ann Clin Biochem 2002; 39: 378- 391
Figure 1. Absorption spectra of reduced haemo-
globin (HHb), carboxyhaemoglobin (COHb), oxy-
haemoglobin (O2Hb), methaemoglobin (MetHb),
sulphaemoglobin (SulphHb) and lipid. Reduced
haemoglobin is a problem in post-mortem
specimens which may have undergone some
putrefaction.
540 nm (the point of maximum di¡erence in absor-
bance) and at 579 nm (the isobestic point) and the
absorbance ratios (540/579 nm) derived.
The percentage COHb saturation value is calculated
from the following equation:
…ratio for C† ¡ …ratio for B†
Saturation …%† ˆ £ 100
…ratio for A† ¡ …ratio for B†
A typical value for the ratio of absorbance for the
saturated COHb sample is approximately 1¢5 and for
the reduced haemoglobin about 1¢1. This is good
enough to give a reasonable saturation measurement
in a case of overt carbon monoxide poisoning, but
the measurement is prone to interferences from
other haemoglobin pigments, such that results from
blood samples with a high lipid content are not reli-
able. The main weakness in the method lies in the
danger of the spectrophotometer not measuring
absorbance values at the exact isobestic point of
579 nm because here the rate of spectral decline is
very steep and a discrepancy of a few nanometers can
introduce gross inaccuracy.
Method sensitivity can be substantially enhanced
by taking measurements in the Soret band of the
spectrum (400^420 nm) where absorbance values for
haemoglobin pigments are about 10-fold higher than
in the visible region (Small et al.42). The method of
Rodkey et al.40 used the sodium dithionite reduction
principle to produce a two-component system (COHb
and reduced haemoglobin) and took absorbance
readings at 420 and 432 nm. This procedure was
subsequently re¢ned and made more robust by Beutler
and West,43 and was sensitive enough to give reliable
values on as little as 3 mL of blood.
The calculation of COHb saturation by the Beutler
and West method is derived from well-known methods

Figure 2. Spectra of carboxyhaemoglobin (curve A), reduced haemoglobin (curve B) and of a sample from a patient poisoned with
carbon monoxide (curve C).
for computing the amounts of two pigments in a
mixture given their molar absorbance values at two
wavelengths. The formula is as follows:
1 ¡ …AR £ F1 †
SCO ˆ £ 100
AR …F2 ¡ F1 † ¡ F3 ‡ 1
where SCO ˆ percentage COHb saturation, A R ˆ ratio
of absorbance values measured at 420 and 432 nm
(A 420/A 432), F1 ˆ eHb 432/eHb420, F 2 ˆ eCO 432/eHb420,
F3 ˆ eCO 420/eHb420, e ˆ molar absorptivity of the
pigment at the listed wavelength.
The values of the constants F1, F2 and F3 are
published values, where F1 ˆ 1¢3330, F 2 ˆ 0 ¢4787 and
F3 ˆ 1¢9939. For more precise analyses it is best to
determine the F values experimentally on the parti-
cular spectrophotometer in use. The formula is readily
assimilated into a software package and modern
instruments can be programmed to calculate A R
directly, insert the value and yield a print-out of the
percentage COHb saturation.
Note that the principle of this method removes the
need for standard solutions, although it is wise to keep
a careful check on the validity of the procedure by
including externally produced blood COHb quality-
control samples in each analytical run.
Analysis of carbon monoxide 383
There is an intrinsic weakness in this and similar
spectrophotometric methods in that a small change in
the A 420/A 432 ratio produces a more signi¢cant change
in the percentage COHb measured. As a result, they
tend to be inaccurate at lower levels of saturation. This
can be overcome by choosing an up-to-date very stable
spectrophotometer and careful calibration of the
instrument prior to analysis.
Derivative spectrophotometric methods
Interest in applying derivative spectral techniques for
measurements grew in the 1980s.44^47 This was
stimulated by a search for a more sensitive and reliable
means of determining COHb at low levels of saturation
and the fact that by 1980 most modern spectro-
photometers were able to generate derivative spectra.
In derivative spectroscopy, the rate of change of
absorbance with wavelength is measured. The ¢rst
derivative spectrum plots the spectral slope in terms of
units of absorbance per nanometer of wavelength
against wavelength. The second derivative spectrum
(the derivative of the ¢rst derivative) measures the
curvature of the spectrum and has units of absor-
bance per nanometer squared. Descriptions of the
theory behind derivative spectroscopy and of its
applications in clinical chemistry have been
published. 48,49 In short, derivative spectra-based
Ann Clin Biochem 2002; 39: 378- 391
384 Widdop

techniques can eliminate sources of non-speci¢c Automated differential spectrophotometry: CO-oximeters

interference and thereby bring about dramatic
changes in the sensitivity and accuracy of measure-
ments. Fukui et al.44 studied a derivative method based
on that of Rodkey et al.40 The characteristics of the
¢rst, second, third and fourth derivative spectra from
300 nm to 500 nm were examined for COHb, reduced
haemoglobin and a mixture of the two. This work
demonstrated that the higher the derivative order
the sharper the spectra became and the better the
separation from the haemoglobin band. By measuring
COHb absorbance at the maximum point for the
fourth derivative (419 nm) extreme sensitivity was
possible and the method compared very favourably
with a conventional spectrophotometric procedure
and with a gas-chromatographic method. The
method is particularly applicable to blood samples
taken from the bodies of ¢re victims, in which heat and
exposure to other toxic gases bring about de-
generation of the haemoglobins. Conventional two-
wavelength spectrophotometry is very susceptible to
alterations in haemoglobin spectra and also to
turbidity, which is usually quite pronounced in heat-
degenerated blood samples. The fourth derivative
technique overcomes most of these di¤culties in
measuring both COHb and total haemoglobin in this
type of sample.44
Perrigo and Joynt 50 carried out a detailed evaluation
of derivative methods applied to autopsy samples,
examining various factors. Samples with high satura-
tion levels were prone to losses due to air exchange,
whereas at lower saturations there were apparent
increases probably resulting from methaemoglobin
production as putrefaction set in. They also pointed
out that calibration procedures for autopsy samples
should use negative autopsy blood to prepare cali-
brants rather than clinical blood samples, in order to
compensate for the haemolysis commonly seen in
post-mortem blood. Moreover, the time at which
readings were taken after diluting the blood samples
needed to be standardized, a factor investigated by
Panzali et al.47
Malefant et al.51 described the principles of determining
total haemoglobin and percentage oxyhaemoglobin
and COHb saturation by simultaneous measurement
of the absorption of a blood haemolysate at di¡erent
wavelengths using an instrument that consisted of a
spectrophotometer linked to an analogue computer.
Blood was introduced directly into the device, auto-
matically haemolysed and drawn into a cuvette for
absorbance measurements. High-precision narrow-
wavelength interference ¢lters were used to measure
the absorbance values of total haemoglobin, oxyhae-
moglobin and COHb simultaneously at exact wave-
lengths. The computer then worked on the data,
calculating haemoglobin concentration and percen-
tage saturation of oxyhaemoglobin and COHb. The
instrument was subsequently marketed as the IL-182
CO-Oximeter, which monitored absorption at 548,
568 and 578 nm. Brunelle et al.52 discussed the
analytical principles behind CO-oximeter design at
length. The main factors that governed accuracy of
performance were the availability of reference mate-
rials of known purity, such that the extinction coe¤-
cients for each haemoglobin derivative could be
established with con¢dence, and the selection of
appropriate wavelengths. The wavelengths chosen
should not only maximize the accuracy of measure-
ment of the haemoglobin derivatives, but also reduce
interference from other blood constituents such as
bilirubin. Signi¢cant improvements have been
achieved by including wavelengths of measurement in
addition to the three needed to determine oxy-
haemoglobin, reduced haemoglobin, COHb and
methaemoglobin. These are usually described as over-
determined systems and treat interferences that
absorb in the wavelength range of the haemoglobins
being measured as either components or as back-
ground absorbance. By this means, interference from
bilirubin, sulphaemoglobin and fetal haemoglobin 53
can be taken into account by the calculation software
of the instrument. Turbidity is often a cause of in-
accuracy and can stem from incomplete haemolysis,

Table 2. CO-oximeters and monitored wavelengths

Instrument
IL 482 CO-Oximeter
OSM3 Hemoximeter
CCD 2500 CO-Oximeter
CCD 270 CO-Oximeter
AVL 912 CO-Oxylite
Manufacturer
Instrumentation Laboratory (UK) Ltd,
Warrington, Cheshire, UK
Radiometer Ltd, Crawley, UK
Chiron Diagnostics, MedŽeld, MA USA
Chiron Diagnostics, MedŽeld, MA USA
AVL Medical Instruments UK Ltd, Stone, UK
Monitored wavelengths (nm)
535¢2, 585¢2, 594¢5, 626¢6
535, 560, 577, 622, 636, 650
521, 535, 546, 585, 594, 627, 660
557, 577, 597, 605, 624, 635, 650
530, 536, 542, 548, 554, 560, 566, 572, 578, 590,
604, 612, 622, 630, 640, 648

Ann Clin Biochem 2002; 39: 378- 391

 Analysis of carbon monoxide 385

high lipid concentrations or thrombocytosis; the in comparison with a gas-chromatographic method,

in£uence of turbidity can also be reduced by the more CO-oximetry invariably gave higher results. Oritani
re¢ned instrumentation. A list of CO-oximeters, et al.60 examined the changes in COHb and other
manufacturers and their wavelengths of measure - haemoglobin measurements that occurred when fresh
ment is given in Table 2. blood containing 25^50% COHb was gradually heated
Wavelength selection between the di¡erent models to temperatures of 70^808C. They concluded that
is quite varied and, as pointed out by Yukawa et al.,54 when the temperature reached 758C there was a
the manufacturers rarely explain the mathematical pronounced increase in measured COHb content,
basis behind their operation. These authors were also accompanied by a rapid fall in total soluble haemo-
concerned about the validity of some of the algorithms globin. There was no in vitro production of COHb and
that may be in use and, in particular, how some of the the increase in the ratio of COHb to total soluble
parameters such as weighting factors that take haemoglobin was responsible for the apparent
account of redundant wavelengths are calculated. increase in COHb content.
However, CO-oximeters caught on quickly in the Neither of these papers dismisses CO-oximetry
clinical laboratory and performed well in comparison outright for forensic work.59,60 Both agree that it has
to other spectrophotometric methods and to gas- merits as a rapid and e¡ective screening test to distin-
chromatography.55,56 They are simple to use, little or guish samples with COHb levels of, say, 410%. Oritani
no sample preparation is needed and the results for the et al.60 go further than this in claiming that, by
relevant haemoglobin derivatives are obtained with measuring methaemoglobin, sulphaemoglobin and
great speed. Precise temperature control of the total haemoglobin as well as COHb, some idea of the
measuring cell is crucial to avoid serious errors,57 but likely time and temperature at which the sample was
this is a built-in feature of the apparatus. The later heated might be forthcoming.
models are reputedly so stable that long intervals
between calibrations (approximately 3 months) are Accuracy and precision of CO-oximeters and other
quite acceptable. There is always a need to be aware, optical methods
however, of possible sources of extraneous inter- Optical methods are used far more widely than any
ference. For example, methaemoglobinaemia can be other in the clinical laboratory setting and it is
treated by parenteral administration of methylene appropriate to review here some of the comparative
blue, which stimulates the reducing activity of investigations of their performance at some length.
NADPH-dependent methaemoglobin reductase in the Mahoney et al.61 compared the results from ¢ve CO-
red cells. This antidote exhibits strong absorbance in oximeters, including some with the over-determined
the 550^700-nm range (i.e. the same region as the design, with a gas-chromatographic reference method
haemoglobin derivatives). Gourlaine et al.58 evaluated and a cyanomethaemoglobin method. The instru-
the e¡ect of methylene blue on the measurement of ments used are listed in Table 2; these were calibrated
COHb and methaemoglobin in blood by six types of and maintained according to the manufacturers’
CO-oximeters and concluded that it was not safe to use instructions. Over the course of the study, 100 blood
CO-oximetry to evaluate the e¤cacy of treating samples drawn at random from patients needing
methaemoglobinaemia with methylene blue. routine blood-gas analysis were analysed. Forty of
However, marked di¡erences were also found for these, chosen at random, were tonometered with
COHb measurements. For example, addition of carbon monoxide to give a range of samples
100 mg/ L of methylene blue decreased the readings containing low, medium and high COHb saturation
for COHb on most of the instruments by up to 23¢4%,
whereas on one instrument there was an increase
Table 3. Results obtained by the analysis of commercial
of 14 ¢4%. Clearly, this is an extreme situation as it
COHb quality-control material using Žve CO-oximeters
would be rare to encounter a patient with both carbon
monoxide poisoning and methaemoglobinaemia. COHb (%)
Nevertheless, this work does emphasize the need for
Model Level 1 Level 2 Level 3
caution when using this and other forms of spectro-
photometry. IL 482 62¢3 (0¢4) 3¢1 (0¢4) 23¢3 (0¢2)
As with the other techniques, there has been R OSM3 58¢6 (0¢3) 3¢4 (0¢5) 21¢ 6 (0¢2)
concern about using CO-oximetry directly on post- CCD 2500 61¢ 9 (0¢3) 2¢9 (0¢2) 23¢2 (0¢2)
mortem blood from ¢re victims. These samples may CCD 270 60¢0 (0¢2) 2¢4 (0¢2) 21¢ 0 (0¢2)
contain high quantities of methaemoglobin and AVL 912 58¢2 (1¢ 0) 3¢5 (0¢5) 22¢5 (0¢1)
sulphaemoglobin. Levine et al.59 showed that pre- Results are expressed as mean (standard deviation) (n ˆ 15).
treating samples with sodium dithionite improved the COHb ˆ carboxyhaemoglobin. Reproduced from reference 61 with
accuracy of measurement, especially at low levels, but permission.

Ann Clin Biochem 2002; 39: 378- 391

386 Widdop

Table 4. Ranges of results for COHb in 100 blood samples using Žve CO-oximeters and a gas-chromatographic
method
COHb (%)
Model 52¢5 (n ˆ 51) 42¢545¢0 (n ˆ 9) 45¢0410¢0 (n ˆ 19) 410¢0 (n ˆ 21)
IL 482 0¢1- 2¢5 2¢5- 4¢9 4¢5- 9¢0 9¢1- 17¢6
CCD 2500 0¢8- 2¢9 2¢9- 5¢4 5¢3- 10¢1 9¢9- 18¢5
R OSM3 0¢5- 3¢0 2¢2- 3¢8 4¢5- 8¢9 8¢2- 16¢5
CCD 270 72¢8- 1¢ 8 73¢6- 3¢5 3¢4- 7¢8 8¢1- 15¢8
AVL 912 0¢0- 3¢1 0¢0- 5¢1 4¢9- 9¢1 8¢7- 16¢2
GC method 0¢3- 2¢1 2¢8- 4¢6 5¢1- 9¢8 10¢4- 17¢7
COHb ˆ carboxyhaemoglobin; GC ˆ gas chromatography. Reproduced from reference 61 with permission.

Table 5. Bias and imprecision data for the differences in percentage COHb as determined by Žve
CO-oximeters
COHb (%)
Model 52¢5 (n ˆ 51) 42¢545¢0 (n ˆ 9) 45¢0410¢0 (n ˆ 19) 410¢0 (n ˆ 21)
IL 482 ‡ 0¢3(0¢4) 70¢1 (0¢4) 70¢9 (0¢5) 71¢1 (1¢ 0)
CCD 2500 ‡ 0¢9(0¢4) 0¢4 (0¢4) 70¢8 (0¢5) 70¢3 (0¢9)
R OSM3 ‡ 0¢3(0¢3) 70¢5 (0¢4) 71¢ 2 (0¢5) 72¢0 (0¢9)
CCD 270 70¢4 (0¢7) 71¢ 6 (1¢ 8) 71¢ 8 (0¢6) 72¢2 (1¢ 0)
AVL 912 ‡ 0¢9 (0¢4) 70¢3 (1¢ 0) 70¢8 (0¢9) 71¢ 7 (0¢8)
Data are expressed as bias (standard deviation of the difference mean). COHb ˆ carboxyhaemoglobin. Reproduced from
reference 61 with permission.

levels. Table 3 shows comparative data derived by
analysing commercial quality-control material.
Table 4 lists the comparative data derived from the 100
patients’ blood samples. Mahoney et al. compared the
CO-oximeters and the gas-chromatographic reference
method by calculating the di¡erences in measured
percentage COHb for each instrument, and calculated
the bias (mean of the di¡erences) and the imprecision
of the biases (standard deviation of the di¡erence
mean) (Table 5). All the CO-oximeters gave quite
acceptable accuracy with samples representing
exogenous exposure to carbon monoxide (45%
saturation), but there was su¤cient magnitude and
variation of the bias below this level to rule out the
technique as a tool to investigate, for example,
neonatal and adult haemolysis or very low environ-
mental exposure to carbon monoxide.
In 1999, Barnett and Wilson 37 analysed data from
the UKNEQAS COHb monthly circulations during
1995^97; the results are shown in Table 6. The data
showed considerable di¡erences in the frequency of
outliers, with the Radiometer instruments performing
best in this respect. There was little di¡erence in
precision between the other types of dedicated instru-
ments or the second derivative spectrophotometric
method, but the spectrophotometric methods (with or
without sodium dithionite) and the Whitehead and
Worthington technique produced a much higher rate
of rejected results (410%) and the standard devia-
tions were signi¢cantly greater than those of the other
techniques. Accuracy varied signi¢cantly and it was
interesting that the over-determined AVL instrument,
which uses more wavelengths than the other models,
gave the highest readings, whereas those from all
other methods (apart from second derivative spectro-
photometry) were comparable. The authors concluded
that the method of Whitehead and Worthington 35 and
the spectrophotometric methods lacked precision and
could not be recommended. However, CO-oximeters,
whether of the low-throughput stand-alone variety or
those that form part of high-throughput blood gas
analysers, were equally appropriate for routine
clinical or toxicological work. This was supported in a
recent report by Lim and Tan,62 who were interested
primarily in ¢nding out if di¡erent anticoagulants had
any e¡ect on measurements of methaemoglobin and
COHb by CO-oximetry. The assays were carried out on
a dedicated CO-oximeter (AVL 912) and on a
combined-function analyser (ABL 520 pH/blood gas
analyser). Both instruments gave good accuracy and
precision for COHb over the ranges usually linked to
exposure. None of the anticoagulants tested (potas-
sium oxalate, EDTA, lithium heparin, £uoride
heparin) had any e¡ect on the stability of COHb.
Fourier transform infrared spectrophotometry
COHb has characteristic bands at wave numbers
1953 (cm71) and 1969 (cm71), thus Fourier transform

Ann Clin Biochem 2002; 39: 378- 391


Table 6. Results from an evaluation of COHb quantiŽcation by external quality assessment
Outliers*
Technique (%)
IL 282/482/682 CO-Oximeter (31)** 2¢1
Radiometer OSM2/3/ABL 520 (26) 0¢2
Chiron 270/800 CO-Oximeter (28) 2¢8
AVL 912 CO-Oxylite (7) 1¢4
Spectrophotometry, direct (12) 17¢3
Spectrophotometry, dithionite (20) 14¢5
Spectrophotometry, second derivative (2) 5¢3
Whitehead and Worthington (1) 37¢0
*DeŽned as 43 standard deviations from the sample mean. **Figures in parentheses are the mean number of measurements
made per sample. COHb ˆ carboxyhaemoglobin; SD ˆ standard deviation; SEM ˆ standard error of the mean. Reproduced
from reference 37 with permission.
infrared spectrophotometry (FTIR) can be used for
quantitative measurements in blood samples.63 The
technique is described as fast and devoid of the inter-
ference problems encountered with UV spectro-
photometry. However, the main interest of this
approach lies in its capability of determining the COHb
content of dried blood samples (e.g. on articles of
clothing) and it is therefore used predominantly in
specializ ed forensic laboratories.
Gas-chromatography
Numerous methods have been described in which a
reagent is added to whole blood to liberate carbon
monoxide which is then analysed either by chemical or
physicochemical means. The quantity of carbon
monoxide found is then related back to the haemo-
globin content of the sample as measured by, for
example, the routine cyanomethaemoglobin tech-
nique or by extrapolation from the total iron content.
The most sensitive and accurate methods are based on
gas-chromatography.
Liberating agents have included sulphuric and
hydrochloric acids, but potassium ferricyanide is by
far the most popular. These are mixed with haemolytic
agents such as detergents and saponin to bring about
complete lysis and fragmentation of the red cell
membrane. Blackmore 31 de¢ned the conditions for
complete decarboxylation.
Some of the earlier techniques for collecting the
liberated carbon monoxide and transferring it into a
gas-chromatograph were quite crude, but have been
improved by various modi¢cations to reaction vessel
design made over the years.64^67 Some workers have
preferred the simpler approach of headspace analysis.
Typical of these was the method of Guillot et al.,68 in
which whole blood (1 mL) was placed in a vial sealed
with a £uorosilicon septum. Using hypodermic
needles the vial was £ushed with helium, a solution of
Analysis of carbon monoxide 387
Mean variation
in results Mean difference
[SD (SEM)] from target value
(COHb%) COHb (SEM) (%)
1¢ 06 (0¢07) 70¢16 (0¢23)
1¢ 03 (0¢06) 70¢13 (0¢25)
1¢ 35 (0¢09) 70¢10 (0¢20)
0¢96 (0¢09) ‡ 0¢84 (0¢25)
2¢28 (0¢19) 70¢31 (0¢27)
2¢19 (0¢18) ‡ 0¢14 (0¢21)
1¢ 27 (0¢26) ‡ 0¢74 (0¢35)
2¢84 (0¢49) 70¢14 (0¢49)
potassium ferricyanide was added and the mixture
was heated for 1h at 908C. An aliquot of the head-
space gas was then injected directly on to a gas-
chromatograph.
A stainless steel packed column containing 60^80
mesh molecular sieve (5 — ) with helium as carrier gas
has been the standard system for separating carbon
monoxide from oxygen and nitrogen for many years.
Capillary molecular sieve columns are now available
and have been put to good use in improving the sensi-
tivity of carbon monoxide measurement. The method
of Van Dam and Daenens 69 has a detection limit of
50¢02% using a 1-mL blood sample and it is therefore
possible to obtain accurate results on volumes as small
as 50 mL. Methods that involved on-line extraction
chambers 67 had been found to degrade the resolution
of carbon monoxide from other gases because of the
gradual accumulation of water and carbon dioxide on
the column from previous injections. In the capillary
column method a split injection system with a split
ratio of 1/10 was used. This meant that only 20 mL of
headspace vapour was injected on to the column and
therefore accumulation was avoided.
The gas-chromatograph can be coupled either to a
thermal conductivity detector or to a £ame ionization
detector. In its basic form, a thermal conductivity
detector consists of two heated ¢laments, each of
which forms an arm of a Wheatstone bridge. The
column e¥uent and a stream of reference gas £ow over
the ¢laments and when a gas or vapour component
emerges the thermal conductivity changes. This
changes the temperature of the ¢lament and this in
turn changes the electrical resistance, unbalances the
bridge and produces a signal. Some of the early types
were rather insensitive for carbon monoxide detection
unless operated at high temperatures. The ¢laments
themselves deteriorated rapidly unless oxygen
was completely removed from the gas samples.70
Ann Clin Biochem 2002; 39: 378- 391
388 Widdop

Developments in design over the years have brought
the thermal conductivity detector back into fashion
and the micro thermal conductivity detector used by
Van Dam and Daenens 69 in conjunction with capillary
column separation allowed them to measure a level of
0¢4% saturation with only 50 mL of blood. The
alternative approach is to reduce carbon monoxide
catalytically to methane and then quantify this by
£ame ionization detection.71,72 Carbon monoxide and
the helium carrier gas emerge from the column, are
mixed with hydrogen and passed through the heated
nickel catalyst (approximately 3008C). The carbon
monoxide is reduced to methane and this passes on to a
£ame ionization detector. This type of detector conveys
great sensitivity; for example,Vreman et al.66 were able
to measure with considerable accuracy and precision
COHb saturation levels as low as 0 ¢2% in samples
collected either by venepuncture, ¢nger or heel stick.
In these methods, calculation of the percentage
COHb saturation can be made by saturating a portion
of the sample to 100% COHb by bubbling pure carbon
monoxide through it for at least 30 min followed by a
stream of pure oxygen or nitrogen to remove any
dissolve d gas. This is analysed alongside the test
sample and the percentage COHb is calculated by
comparison of peak areas as follows:
Percentage COHb saturation ˆ
peak area of carbon monoxide in sample6100
peak area of carbon monoxide in 100% carboxylated sample
An alternative method, and one that is favoured by
forensic toxicologists dealing with post-mortem blood
or tissue samples in which some decomposition has
taken place, is to measure the actual volume of carbon
monoxide released from the sample by reference to
known volumes of the pure gas. The calculation is as
follows:
Carbon monoxide volume ˆ
peak height of carbon monoxide in sample
peak area of carbon monoxide in calibrant
6 volume of carbon monoxide in calibrant
The haemoglobin content of the sample is also
measured either by a modi¢ed cyanomethaemoglobin
method73 or by determining the total iron content.31
This allows a theoretical calculation of the
carbon monoxide volume in a 100% carboxylated
sample.
Instead of manipulating pure carbon monoxide
gas to calibrate the gas-chromatograph, it is possible
to generate the gas on the basis of the stoichiometric

Table 7. Summary of current methods for estimation of COHb in blood

Method Reference(s) Principle Main application Problems
Differential protein 35 Heat precipitation of Clinical diagnosis of Poor precision in
precipitation oxyhaemoglobin at pH 5 carbon monoxide poisoning routine use
Spectrophotometry 39- 43 Dithionite conversion of oxy- Clinical diagnosis of carbon Spectral interference
and methaemoglobin to monoxide poisoning from other
reduced haemoglobin haemoglobin
pigments and
lipids; imprecise at
low levels
Derivative 44- 47 Use of derivative spectrometry Forensic investigations Strict control over
spectroscopy to eliminate non-speciŽc timing of readings
interference essential
CO-oximetry 53- 55 Automated simultaneous Clinical diagnosis of acute and Good precision for
measurements of absorbance chronic exposure; applicable clinical samples,
at different wavelengths to low levels but loses accuracy
when applied to
putreŽed post-
mortem samples
Fourier transform 63 Absorbance measurement at Forensic examination of dried Not generally
infrared characteristic bands blood samples available in clinical
spectrophotometry laboratories
Gas-chromatography 66- 69, 75 Chemical liberation of carbon Forensic investigations and Very precise, but
monoxide from blood and assessment of low levels complex and too
direct or indirect measurement of environmental exposure slow for
of the gas emergency clinical
work
COHb ˆ carboxyhaemoglobin.

Ann Clin Biochem 2002; 39: 378- 391


formation of carbon monoxide by the reaction
of formic acid with hot concentrated sulphuric
acid.74,75
Measurement of carbon monoxide in breath
Devices that measure carbon monoxide directly in air
or breath samples have been around for many years.
These are essentially electrochemical sensors that
depend on the oxidation of carbon monoxide at the
anode with subsequent generation of an electrical
signal:
CO ‡ H2 O ! CO2 ‡ 2H‡ ‡ 2e¡
At the same time air oxygen is reduced at the cathode:
2O2 ‡ 8H‡ ‡ 8e¡ ! 4H2 O
The current produced is ampli¢ed and displayed
digitally in parts per million (ppm) or, because the
alveolar breath carbon monoxide concentration can
be related to the blood carboxyhaemoglobin satura-
tion,76 as percentage COHb saturation. These devices
are quite speci¢c for carbon monoxide, unlike the
warning detectors used for domestic purposes which
rely on solid-state semi-conductor technology and
react to several other gases.
Instruments for measuring carbon monoxide in
breath were originally intended for smoking education
and cessation programmes,77 but they have also found
a role in the diagnosis of chronic and acute carbon
monoxide poisoning.78 The advantages are obvious.
Sampling is non-invasive and can be carried out on
small children, adults and unconscious patients.
Results are available within minutes and the equip-
ment is inexpensive, portable, easily calibrated and
can be used by non-technical personnel at the scene of
an incident. Surprisingly, this type of instrumentation
is, in fact, little used (at least in the UK) for reasons that
are not clear.
Conclusion
A summary of the current methods that are available
for estimating COHb in blood is presented in Table 7.
Carbon monoxide is an unusual poison in that its
measurement in blood has to be related in some way to
the extent to which it has reduced the oxygen supply to
the tissues. For fresh blood samples, spectrophoto-
metric techniques that can estimate total haemo-
globin, percentage oxyhaemoglobin and percentage
COHb simultaneously are ideal. There can be no doubt
that CO-oximeters, with their ease of operation,
freedom from spectral interferences and built-in soft-
ware that calculates the parameters listed above, will
remain the preferred instrument in hospital laborat-
ories for many years to come. They are sensitive and
precise enough to detect chronic exposure to carbon
Analysis of carbon monoxide 389
monoxide from, for example, faulty heaters. However,
for investigating low levels of environmental exposure
and COHb levels in habitual smokers, gas-chromato-
graphic methods are preferred. CO-oximeters are
expensive to buy and to maintain and, in the absence
of adequate funding, manual spectrophotometric
methods that include a preliminary sodium dithionite
reduction step are quite adequate for diagnosing
moderate to serious carbon monoxide exposure. In the
vast majority of fatal cases due to acute carbon
monoxide poisoning, CO-oximetry is an appropriate
means of con¢rming the cause of death. The exceptions
to this arise when post-mortem blood samples have
decomposed or their haemoglobin composition has
been changed by thermal stress. Spectral interference
mitigates accurate analysis of the COHb content, and
comparison of this with either total haemoglobin or
oxyhaemoglobin is not reliable. The best alternative is
to measure the carbon monoxide content of the blood
sample by gas-chromatography, measure the total
haemoglobin content by, for example, the conventional
cyanomethaemoglobin method, and calculate from
these values the percentage COHb saturation.
Finally, the belief that carbon monoxide binding
to haemoglobin is so strong that blood samples will
be stable whatever the storage conditions is a
complete fallacy. It is vital that samples that are not
to be dealt with immediately are stored correctly.
As little air as possible should be left in the tube
and the samples should be placed in a deep freeze
without delay, irrespective of the anticipated degree of
carboxylation.
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Accepted for publication 19 February 2002
Ann Clin Biochem 2002; 39: 378- 391