H
HAEMOGLOBINS (HEMOGLOBINS)
R A Sherwood, King’s College Hospital, London, UK
& 2005, Elsevier Ltd. All Rights Reserved.
Iron
Iron has diverse functions in biology and medicine,
with the result that both deficiency and excess have
severe consequences in humans. Indeed, iron defi-
ciency is the most prevalent nutritional problem
in the world today. Most of the iron in the body
(on average 50 mg kg
1 in the adult male and
40 mg kg
1 in the female) is contained in hemoglo-
bin, which is essential for the transport of oxygen to
the tissues. About 15% of the iron is found in
myoglobin, which acts as a reservoir of oxygen
in muscles. Iron not required immediately is stored in
the form of ferritin or hemosiderin, both of which
can bind large numbers of iron atoms. A very small
proportion of body iron (0.2%) is bound to trans-
ferrin, which transports iron between sites of need.
The measurement of iron, hemoglobin, and a com-
bination of the proteins described above is necessary
for the diagnosis and monitoring of iron deficiency or
excess as in hemochromatosis.
Inherited variations in hemoglobin structure exist,
some of which have a significantly reduced oxygen-
carrying capacity (e.g., sickle-cell anemia and
thalassemia). Identification of these hemoglobins is
essential for correct treatment. Additionally, the hemo-
globin molecule is glycated by circulating glucose and
the measurement of the proportion of glycated
hemoglobin in blood has proved invaluable in the
monitoring of diabetes mellitus.
Measurement of Plasma, Urine, and Tissue Iron
Plasma iron is derived mainly from the breakdown
of hemoglobin in the reticuloendothelial system.
Transferrin reversibly binds iron very firmly – there
are no free iron ions present in plasma as they would
be very toxic. Molecules of the transferrin–iron com-
plex combine with receptor sites on erythroblasts
and are then internalized, releasing iron into the
cytosol. Consequently, the first stage in the measure-
ment of iron in plasma (or serum) is dissociation of
the iron from transferrin, either with or without
protein precipitation. A number of chemical agents
have been successfully used for this process:
trichloroacetic acid (TCA), hydrochloric acid, thor-
ium(IV), acetate buffer and heat, and chloroform.
The iron released may be measured using a variety
of methods based on two main techniques: atomic
absorption spectrometry (AAS) (flame or furnace) and
ultraviolet (UV)–visible spectrophotometry. Flame
AAS provides the reference method for determina-
tion of plasma iron. Protein precipitation with TCA
is followed by centrifugation and measurement of
iron in the supernatant by the absorption at
248.3 nm in an air–acetylene flame. While atomic
absorption methods are routinely used for urine iron
measurements, the need to remove protein and any
hemoglobin contamination restricts the use of this
technique in routine clinical chemistry for plasma
iron. Electrothermal atomization AAS methods are
typically used for determination of iron in tissues
although inductively coupled plasma (ICP) is beco-
ming more widely available.
A number of spectrophotometric/colorimetric
methods have been developed for use on the automat-
ed analyzers commonly available in clinical chemistry
laboratories. These methods typically involve several
steps: dissociation of iron from transferrin; reduction of
iron(III) to iron(II); formation of colored complexes
with iron-chelating chromogens.
Early methods utilized protein precipitation prior
to complex formation, while today direct methods
predominate. The method recommended by the In-
ternational Committee for Standardization in Hema-
tology requires protein precipitation with TCA,
reduction with thioglycolic acid, followed by com-
plex formation with ferrozine or ferene. Direct methods
have significant benefits in analytical rapidity and
automation. Dissociation of the transferrin–iron
complex may be achieved by means of strong inorganic
acids – typically hydrochloric acid – with solubiliza-
tion of plasma proteins by the use of detergents. The
dissociation is facilitated by the presence of a reducing
agent, with the added benefit of reducing iron(III) to
iron(II) at the same time. A variety of reducing agents
have been used including hydroquinone, hydrazine,
224 HAEMOGLOBINS (HEMOGLOBINS)
Table 1 Colorimetric reagents for the determination of serum
iron, with their wavelength of maximum absorption and their
molar absorptivity (e) at that wavelength
Reagent lmax (nm) e (mol l
1 cm
1)
2,20 -Bipyridyl 522 8650
1,10-Phenanthroline 510 11 100
2,20 ,200 -Tripyridyl 552 11 000
Nitroso R salt 720 20 000
Bathophenanthroline 535 22 140
Tripyridyl-s-triazine (TPTZ) 595 22 600
Ferrozine 562 27 900
Ferene-S 593 35 500
sulfite, ascorbic acid, dithionite, hydroxylamine,
thioglycolic acid, and mercaptoacetate.
Ascorbic acid is the most commonly used reducing
agent. The free iron can then be reacted with a
number of iron-chelating agents that form colored
complexes. The more common chelating chromogens
available are listed in Table 1 together with the
wavelength of maximum absorption and the molar
absorptivity. It can be seen that since the discovery of
2,20 -bipyridyl in 1936 there has been a fourfold
increase in the sensitivity of the available chromo-
gens. TPTZ and Ferene-S produce blue complexes
that have the advantage of being free from interfer-
ence from bilirubins or the carotenoids, thus obviat-
ing the need for a sample blank measurement. These
chromogens, however, are not entirely specific for
iron as there is some binding and complex formation
with copper, which can be removed by incorporating
agents such as neocuprine, thiosemicarbazide, or
thiourea that preferentially bind copper.
As all the methods mentioned above rely on the
measurement of free iron after its release from pro-
teins, iron released from hemoglobin would also be
measured. For this reason any hemolysis will lead to
falsely elevated plasma iron concentrations.
The reference range for plasma iron in the adult
male is 9–29 mmol l
1 with a slightly lower range for
females (7–27 mmol l
1). A circadian rhythm has
been described for plasma iron, with peak concen-
trations in the morning and a trough in late after-
noon. Plasma iron is typically higher in the neonate,
falling rapidly to adult levels, and typically results for
the elderly are lower than the adult reference range.
Various drugs, e.g., aspirin, allopurinol, and choles-
tyramine, can cause a decrease in plasma iron
concentrations.
Urine Iron Assays
Urinary iron excretion in normal individuals is rela-
tively low (0.08 mg day
1), producing concentrations
too low to be measured by the methods available for
plasma iron. AAS is the technique of choice for urine
iron measurements. Isolated measurements of urine
iron concentration are of little clinical value. Clini-
cally, the most valuable measurement is assay of the
urine iron excretion after administration of des-
ferrioxamine, an iron chelator. In normal subjects
urine iron is elevated up to 10 times the baseline
level, while in patients with genetic hemochromatosis
elevations of up to 50 times baseline can be seen.
Intermediate increases of 10–20-fold may be found
in patients with hemosiderosis or following treat-
ment of secondary iron overload from blood trans-
fusions, e.g., in thalassemia patients.
Tissue Iron
Tissue iron includes iron from heme-containing
enzymes of cellular metabolism, accounting for
B300 mg, and storage iron of 800–1000 mg in men,
with lower amounts in women. The storage iron is
predominantly in hepatocytes and in reticuloendo-
thelial cells as ferritin and hemosiderin. Ferritin is
located intracellularly and can sequester up to 4500
atoms of iron within its central core. Hemosiderin
appears to be an iron-dense material consisting of
multiple aggregates of ferritin molecules in which
part of the protein shell has been degraded so that the
iron cores coalesce. Ferritin is water soluble, whereas
hemosiderin is not. Ferritin is widely distributed in
liver, spleen, bone marrow, placenta, heart, pancreas,
skeletal muscle, and intestinal mucosa. A variety of
isoferritins exist specific to particular locations,
ranging from acidic to basic in nature.
Tissue iron can be visualized and localized on
histological sections by the use of Perl’s Prussian
blue stain, which is based on the reaction of acidic
hexacyanoferrate(II) principally with hemosiderin,
but also with ferritin. A semiquantitative scoring
system has been devised by estimating the iron load
in parenchyma cells or Kupffer cells. Quantitative
methods utilize both chemical and physical tech-
niques. The tissue is first digested with TCA–HCl at
651C for 20 h and the iron in the supernatant is
reacted with bathophenanthroline sulfate and
thioglycolic acid as in the plasma method. Although
suitable for crude assessment of iron status, the pre-
cision of the method is less than satisfactory. The
most frequently used alternative is AAS after nitric
acid digestion. ICP atomic emission spectrometry has
been used for assay of liver iron following nitric acid
– perchloric acid digestion. ICP-mass spectrometry
(ICP-MS) has been used as an online system for the
detection of 57Fe originally incorporated into trans-
ferrin. The liver iron was fractionated by liquid
chromatography (LC) and the eluate was passed to
the ICP-MS system operating with an argon plasma.

Even more exotic techniques such as Mossbauer
spectroscopy or electron diffraction measurements
have been used in the research environment for de-
tection, identification, and quantification of different
forms of hemosiderin.
Transferrin and Iron-Binding Capacity
Transferrin has two high-affinity binding sites for
iron and exists in plasma as a mixture of apotrans-
ferrin and the mono- and diferric forms. Transferrin
may be measured either directly by immunological
techniques or indirectly by its functional capacity to
bind iron. The most widely used technique for the
measurement of total iron-binding capacity (TIBC) is
based on the saturation of transferrin with a solution
of iron(III) ions in excess. The excess of iron is
removed with an iron adsorbent, usually magnesium
carbonate or an anion-exchange resin. The iron con-
centration in the supernatant is then measured using
standard methods for plasma iron. An alternative
approach is to measure the unsaturated iron-binding
capacity (UIBC). An excess, but known amount, of
iron is added and the unbound fraction is measured
using a reagent with specificity to unbound iron. The
difference between the known and measured
amounts of iron is that taken up by the protein.
Measurement of UIBC is becoming the more com-
monly used technique as it does not require manual
steps and is more suitable for automation on the
large clinical chemistry analyzers.
The reference range for TIBC is typically
50–70 mmol l
1. The combination of plasma iron
and TIBC measurements can be used to derive the
‘transferrin saturation’ ((plasma iron/TIBC)  100).
In a normal individual transferrin is 30–40% satu-
rated with iron, whereas in anemic subjects the
degree of saturation can fall as low as 5%. In patients
with hemochromatosis transferrin can be 100% satu-
rated and ‘free iron’ can be detected in plasma.
Pregnancy and oral contraception can increase the
TIBC to 90 mmol l
1.
Most of the common immunological techniques
(nephelometry, turbidimetry, etc.) can be used to de-
termine the concentration of transferrin directly.
Generally, enhanced turbidimetric assays are now
used for transferrin measurements on automated
analyzers. Transferrin is decreased in disorders
involving reduced or altered protein synthesis
(e.g., liver disease) and increased in iron deficiency
anemia. A rare condition of near complete absence
of plasma transferrin (atransferrinemia) has been
reported.
Transferrin has two bi-antennary carbohydrate
side chains that can contain up to six sialic acid
HAEMOGLOBINS (HEMOGLOBINS) 225
residues and the sialylation process appears to be
directly affected by excess alcohol intake. Examina-
tion of serum from chronic alcohol abusers has
revealed that in such subjects there is a greater pro-
portion of asialo-, mono-, and disialo transferrin
compared to normal subjects where the tri- and tetra-
sialo forms predominate. The desialylated forms
have been termed ‘carbohydrate-deficient transfer-
rin’ (CDT) and form the basis of a test for chronic
alcohol misuse. The effect of alcohol on transferrin
appears to be a direct consequence of alcohol inter-
fering with the sialylation process rather than as a
result of any liver damage affecting synthesis as
abstinence in alcoholics with liver disease results in
the percentage of CDT in plasma returning to normal
(o2.6%) within 10–14 days. CDT can be measured
using separation techniques such as isoelectric focus-
ing (IEF) or LC where the exact composition of
the isoforms can be determined. For routine use
in detecting or monitoring subjects misusing
alcohol, separation of CDT is usually achieved
using microcolumn ion-exchange chromatography
with measurement of total transferrin and the car-
bohydrate-deficient forms allowing calculation of the
percentage CDT. This has benefits over absolute
measurements as it avoids false negative or positive
results in cases of decreased or increased trans-
ferrin synthesis (e.g., protein malnutrition or advan-
ced liver disease causing decreased synthesis and
iron deficiency or pregnancy causing increased syn-
thesis). A family of inherited diseases is now known
in which proteins are deficient in carbohydrate
side chains – the carbohydrate-deficient glycoprotein
syndromes.
Serum Transferrin Receptor
The mechanism for the transfer of iron from trans-
ferrin to the intracellular compartment of cells is now
better understood. A specific transferrin receptor on
cell membranes has been identified to which circu-
lating diferric transferrin binds, after which endocy-
tosis of the ligand–receptor complex occurs releasing
iron into the cell. In iron deficiency there appears to
be upregulation of the transferrin receptors to enable
more efficient transfer of iron. The extracellular
domain of the transferrin receptor (sTfr) appears
to be released into the circulation by proteolytic ac-
tion during cell turnover and may be detected in
plasma (serum) by immunological techniques. Its
concentration is increased in iron deficiency anemia
(18–35 mg l
1 compared with a normal range of
3–9 mg l
1). Measurement of sTfr is beneficial for
distinguishing iron-deficiency anemia from the ane-
mia of chronic disease.
226 HAEMOGLOBINS (HEMOGLOBINS)
Ferritin
Ferritin consists of a large spherical shell of 24 single
protein subunits surrounding an inner core of
insoluble iron(III) oxide hydroxide with a portion
of iron(III) phosphate. The iron-free protein, apo-
ferritin, has a relative molecular mass of B450 000
that can double when fully saturated with iron.
Ferritin is measured immunologically with isotopic
or nonisotopic (enzyme or chemiluminescence)
immunoassays. In normal subjects there is a wide
range of serum ferritin concentrations (15–300 mg l
1),
predominantly due to a highly negative skew to the
ferritin results owing to the lower iron stores in
women. Ferritin is decreased in iron-deficiency an-
emia but is an acute-phase protein and may be nor-
mal or increased if there is coexisting infection or
tissue injury. Ferritin may also be produced by a
number of malignant tumors. A ferritin greater than
750 mg l
1 in the absence of an acute phase is indi-
cative of iron overload and if secondary causes can
be excluded, genetic hemochromatosis should be
considered. The disease causing mutation for hemo-
chromatosis (C282Y mutation in the HFE gene) is
now known and can be detected using PCR and re-
striction enzyme digest methods.
RBC Zinc Protoporphyrin
In iron deficiency where iron is unavailable for in-
corporation into heme in the developing reticulocytes
or where incorporation is inhibited as in lead toxic-
ity, protoporphyrin IX accumulates in the red cells.
This can be measured by extraction into ethyl ace-
tate–acetic acid and then back-extracted into HCl.
After neutralization, an ether extraction followed by
differential extraction into HCl allows separation of
protoporphyrin and coproporphyrin.
Hemoglobin
Iron is incorporated into the heme produced by the
porphyrin synthetic pathway by linkage to the four
nitrogen atoms of protoporphyrin. In hemoglobin,
four heme units are linked to four polypeptide
chains, consisting of two pairs of like chains. There
are four major types of hemoglobin (A, A2, F, and G)
with chain structures:
* HbA 97% of total Hb in an adult ¼ a2b2,
* HbA2 3% of total Hb in an adult ¼ a2d2,
* HbF fetal Hb ¼ a2g2, and
* HbG intrauterine Hb ¼ G1 x2e2 and G2 a2e2.
Disorders of hemoglobin resulting from structural
abnormalities of a globin chain are termed
hemoglobinopathies, with over 400 variants known,
the majority of which are clinically benign. The sepa-
ration of these variants will be discussed later.
The measurement of total hemoglobin concentra-
tion in blood is one of the most frequently requested
tests in medicine. Automated methods predominate
but the principle of the method is little changed
from the original manual techniques. The Fe(II) of
hemoglobin is oxidized to Fe(III) by the addition of
hexacyanoferrate(III) and then converted into stable
cyanmethemoglobin by the addition of potassium
cyanide. The absorbance of cyanmethemoglobin is
measured at 540 nm. The normal range for adult
males is 14–18 g dl
1 and for females 12–16 g dl
1.
Hemoglobin Derivatives
The function of hemoglobin is the transport of
oxygen to the tissues from the lungs. When oxygen is
associated with the molecule it is termed oxy-
hemoglobin (OHb), whilst in the absence of oxygen
it is termed deoxyhemoglobin or reduced hemoglo-
bin (RHb). In these forms iron is present as iron(II).
When the iron is present as iron(III), a brown pigment
is formed – methemoglobin. If carbon monoxide is
present, i.e., because of smoke inhalation, a hemoglo-
bin–carbon monoxide complex is formed that is re-
ferred to as carboxyhemoglobin (COHb). A final
derivative of clinical interest is the degradation prod-
uct sulfhemoglobin (SHb) that contains one more sul-
fur atom than normal hemoglobin. The iron is in the
iron(II) state but the binding of oxygen is inhibited.
For the assessment of the oxygenation state of a
patient, measurement of the proportion of oxy-
hemoglobin can be of value and a variety of com-
mercial instruments exist for the measurement of the
hemoglobin derivatives (oximeters, co-oximeters, and
hemoglobinometers). The hemoglobin derivatives
have differing absorption maxima (Figure 1) and
these instruments are based on the differential spec-
troscopic measurement of the main derivatives: OHb,
RHb, and COHb, plus in some cases SHb. The ab-
sorption maxima are known to vary with temperature
and most commercial instruments are fitted with a
temperature-controlled cuvette. COHb normally
constitutes less than 5% of total hemoglobin,
although this may be slightly higher in heavy smok-
ers. COHb concentrations greater than 20% from
smoke or carbon monoxide inhalation are usually
fatal unless treated by the use of a hyperbaric oxygen
chamber.
The normal concentration of methemoglobin is
1.5% of total hemoglobin, although in congenital me-
themoglobinemia due to methemoglobin reductase de-
ficiency this may increase to 10–20%. Methemoglobin

10

(mol −1 cm −1)
1 HbS / HbC
535.0
582.2
594.5
626.6
0.1
500 600
Wavelength (nm)
Figure 1 Absorption spectra in the region 500–650 nm of the
main hemoglobin derivatives: reduced hemoglobin (-  -), oxy-
hemoglobin (—), carboxyhemoglobin (- - -), and methemoglobin
(-?-). (Reproduced with permission from Instrumentation Labo-
ratories 482 co-oximeter manual.)
can be measured by its absorbance in the region of
630–635 nm, which is eliminated by the addition of
cyanide. Sulfhemoglobin is not a normal constituent in
blood but may be present owing to reaction to certain
drugs, e.g., phenacetin. Sulfhemoglobin has a broad
absorbance band in the region of 600–620 nm that is
not destroyed by cyanide.
Separation of Variant Hemoglobins
The best-known hemoglobinopathies are sickle-cell
anemia and thalassemia, which manifest clinically as
a chronic anemia, hemolytic in the former and micro-
cytic in the latter. Sickle cell disease is found pre-
dominantly in people of African descent and is
characterized by sickle-shaped erythrocytes due to
the homozygous inheritance of HbS in which valine
is substituted for glutamic acid at position 6 in the
b-chain. When deoxygenated HbS is much less soluble
than deoxygenated HbA and forms rod-like poly-
mers (tactoids), giving the characteristic sickle shape
to the red blood cells. Sickling also occurs with
HbC, in which lysine is substituted for the same
glutamic acid. Thalassemia occurs in subjects of
Mediterranean origin or Asian origin and is charac-
terized by defective Hb synthesis. Both a- and b-
thalassemia exist where a-chain or b-chain synthesis
is decreased.
The identification of the variant hemoglobins is
based on their separation by electrophoresis or chro-
matography utilizing the different overall charge
on the molecule caused by the amino acid substitu-
tions. Cellulose acetate electrophoresis is normally
used for screening and is carried out at pH 8.6, where
HbS has two more and HbC four more charges per
HAEMOGLOBINS (HEMOGLOBINS) 227
Tracks 1 2 3 4 5 6 7 8 9 10 11 12 13 14
HbC HbS
disease trait trait
Figure 2 Typical separation of hemoglobin variants using cel-
lulose acetate electrophoresis. Track 1 is a standard mixture of
HbA and HbS, tracks 2, 4, 7, 8, 10, 11, 13 are normals; tracks 5
and 6 are HbC traits; tracks 9, 12, 14 are HbS traits; and track
3 is HbS/HbC disease. (Reproduced with permission from the
Department of Haematology, King’s College Hospital, London.)
molecule than native HbA (Figure 2). Citrate gel
electrophoresis at pH 6.2 relies on the interactions of
the substituted amino acids with agaropectin. Those
variant hemoglobins with substitutions near the sur-
face, e.g., HbS and HbC, are retained by these in-
teractions while those with deeper substitutions, such
as HbD or HbG, are not. Chromatographic tech-
niques use ion-exchange columns (diethylamino-
ethyl-DEAE cellulose) to separate the variants on
the basis of the aforementioned charge differences.
Preliminary screening with cellulose acetate electro-
phoresis will permit the rapid identification of many
variant types of hemoglobin, but for some variants,
such as HbD, coelution occurs with HbS. Globin
chain electrophoresis can be used to separate these
variants. The heme group is dissociated from the
globin chain with dithiothreitol and urea followed by
cellulose acetate electrophoresis using Tris–EDTA–
borate buffer at pH 8.9 and pH 6.2. Excellent reso-
lution of hemoglobin variants can be achieved by
IEF. More recently, capillary electrophoresis has been
shown to be an effective technique for the separation
of hemoglobin variants. Similarly, there is consider-
able interest in the application of tandem MS to
hemoglobin separations.
Glycohemoglobin and Its Measurement
All the hemoglobin types previously discussed will
react with hexose sugars, the product being referred
to as glycohemoglobin (GHb). The predominant
adult hemoglobin HbA consists of two a- and two
b-chains in a tetramer. Condensation with hexoses
gives rise to five GHb derivatives, designated col-
lectively HbA1, HbA1a1, HbA1a2, HbA1b, HbA1c,
and HbA1d. In vivo condensation between glucose
and hemoglobin occurs throughout the lifespan of
erythrocytes in proportion to the circulating blood
glucose concentration. The GHb concentration in a
228 HAEMOGLOBINS (HEMOGLOBINS)
particular blood sample therefore reflects the mean
blood glucose concentration of the preceding weeks
to months. Measurement of GHb has been shown
to be a valuable means of monitoring long-term
glycemic control in diabetics and is widely used in
clinical practice. Since the report of the Diabetes
Control and Complications Trial (DCCT) clearly
demonstrated a reduction in long-term complications
(i.e., nephropathy, retinopathy, and neuropathy) if
HbA1c is kept below 7.5%, measurements of GHb
have been steadily standardized to HbA1c DCCT
aligned methods.
Methods for Measurement of GHb
A wide variety of techniques, particularly separation
techniques, have been used to measure GHb (Table 2).
Ion-exchange chromatography Cation-exchange
chromatography, generally involving the use of
Biorex 70, has enjoyed widespread popularity for
GHb measurements. ‘Minicolumns’ separate glycated
HbA (all fractions) from nonglycated HbA with
the hemoglobin concentration in the eluate being
measured at either 414 or 552 nm. Although com-
monly used, these methods suffer the drawback of
being sensitive to pH, ionic strength of the buffers
used, and the temperature of the column. The results
are expressed as HbA1 with a target range of 6–8%
for good glycemic control. Many of these methods
are now being standardized to DCCT aligned HbA1c
values.
Liquid chromatography The LC methods are also
based on the use of cation-exchange columns. Sepa-
ration of HbA1c from HbA1a and HbA1b can be
achieved using LC and dedicated automated systems
are commercially available. A significant disadvan-
tage occurs in populations with a high percentage of
abnormal hemoglobins, as to achieve an acceptable
throughput a separation time of 5–8 min is used,
which is too short to resolve the abnormal hemoglo-
bins, leading to potentially misleading results. Typi-
cally, results are expressed as HbA1c with a range of
4–6% for nondiabetic subjects.
Table 2 Interferences with GHb measurements in the most commonly used techniques
Ion exchange
Negatively charged hemoglobins, e.g., HbF Positive
Positively charged hemoglobins, e.g., HbC, HbS Negative or none
Lipemia Positive
Renal failure (carbamylated Hb) Positive
Aspirin (hemoglobin–acetylsalicylic acid adduct) Positive
Affinity chromatography Unlike the techniques
based on ion exchange, which rely on the small
charge differences between the glycated hemoglobins,
affinity chromatography utilizes the complex forma-
tion that occurs between boronate and the cis-diol
groups of sugars, particularly fructose. The boronic
acid is attached to a support matrix such as agarose
and will bind all the species of GHbs. These are then
eluted with sorbitol and the hemoglobin quantified
colorimetrically. The major advantage of this method
is its insensitivity to abnormal hemoglobins and
automated versions using LC are available commer-
cially with short cycle times (2–3 min). This method
can be used in the clinic setting using capil-
lary blood samples prior to the patient being seen
by the diabetologist. Although the affinity chro-
matography based methods measure all GHb frac-
tions, most are now calibrated to DCCT aligned
HbA1c values.
Agar gel electrophoresis Electrophoresis on agar
slabs separates glycated HbA (all forms) from non-
glycated HbA as in ion-exchange chromatography
but does not suffer from sensitivity to pH, ionic
strength, or temperature. The method will not, how-
ever, resolve HbF from the glycated fraction, leading
to gross overestimation in individuals with persistent
HbF. A disadvantage is the analysis time, which even
in automated versions, is usually too long for use in a
clinic setting.
Isoelectric focusing IEF has been used to achieve
separation of HbA0, HbA1c, HbS, and HbC. HbF is
also separated but acetylated HbF comigrates with
HbA1c. Although capable of excellent separation,
IEF is too cumbersome for routine use in clinical
chemistry.
Immunoassays Developments in the production of
monoclonal antibodies have led to the production of
monoclonal antibodies specific to HbA1c. These anti-
bodies have been used with a variety of immunoassay
platforms to produce automated methods. Care must
be taken to determine if the epitope of the antibody is
Boronate affinity Agar gel Immunoassay
None Positive Negative
None Negative or none Negative
None None None
None None None
None Positive None

within a region of hemoglobin that can be altered in
the hemoglobinopathies.
Reference method A reference method has been es-
tablished that involves cleavage of hemoglobin into
peptides by the endoproteinase Glu-C, followed by
separation and quantitation of the hexapeptides by
LC–electrospray ionization mass spectrometry or
LC–capillary electrophoresis.
See also: Blood and Plasma. Clinical Analysis: Over-
view. Immunoassays, Applications: Clinical.
Further Reading
Bry L, Chen PC, and Sacks DB (2001) Effects of hemo-
globin variants and chemically modified derivatives on
assays for glycohemoglobin. Clinical Chemistry 47:
153–163.
Brugnara C (2003) Iron deficiency and erythropoiesis: New
diagnostic approaches. Clinical Chemistry 49: 1573–1578.
Cook JD, Flowers CH, and Skikne BS (2003) The
quantitative assessment of body iron. Blood 101:
3359–3363.
HAIR
See FORENSIC SCIENCES: Hair
HEADSPACE ANALYSIS
Contents
Static
Purge and Trap
Static
J D Green, Beverley, UK
& 2005, Elsevier Ltd. All Rights Reserved.
Introduction
Headspace analysis is a technique for sampling and
examining the volatiles associated with a solid or
liquid sample. The actual headspace itself is the
HEADSPACE ANALYSIS / Static 229
International Committee for Standardization in Haema-
tology (1990) Recommended method for measurement of
iron in serum. British Journal of Haematology 75: 615–616.
Jeppsson JO, Kobold U, Barr J, et al. (2002) Approved
IFCC reference method for the measurement of HbA1c
in human blood. Clinical Chemistry and Laboratory
Medicine 40: 78–89.
Park CH, Valore EV, Waring AJ, and Ganz T (2001)
Hepcidin, a urinary antimicrobial peptide synthesized
in the liver. Journal of Biological Chemistry 276:
7806–7810.
Scouller K, Conigrave KM, Macaskill P, Irwig L, and
Whitfield JB (2000) Should we use carbohydrate-
deficient transferrin instead of g-glutamyltransferase for
detecting problem drinkers? A systematic review and
metaanalysis. Clinical Chemistry 46: 1894–1902.
Sherwood RA, Pippard MJ, and Peters TJ (1998) Iron
homeostasis and the assessment of iron status. Annals of
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volume of vapor or gas above the sample. For most
headspace analysis purposes the sample and its as-
sociated headspace are held within an enclosed con-
tainer (Figure 1). Often, it is the volatiles that are of
primary importance when, for example, the chemical
composition of the mixture giving rise to the aroma
of the sample is to be determined. In other circum-
stances, examination of the volatiles rather than the
whole sample greatly simplifies the analysis and still
gives information relating to the whole sample. In
effect, the analytical complications that are often
apparent as a result of a complex matrix are consid-
erably decreased.