Clin Chem Lab Med 2002; 40(10):1014–1018 © 2002 by Walter de Gruyter · Berlin · New York
and Joris Delanghe
Department of Chemical Pathology, University of Zimbabwe
Medical School, Harare, Zimbabwe
Department of Clinical Chemistry, Ghent University, Gent,
Transferrin concentration and total iron binding capac-
ity (TIBC) are currently used to assess iron status. Al-
though correlation between TIBC and transferrin is
generally considered as good, conversion factors be-
tween the two analytes found in literature show large
Although the price per test is lower for TIBC, there
are a number of analytical advantages of serum trans-
ferrin. Due to binding of iron to other plasma proteins
(mainly albumin), TIBC methods generally overesti-
mate the iron binding capacity of transferrin. More-
over, no generic reference values are available for
TIBC. In contrast to TIBC, internationally accepted in-
terim reference ranges are available for serum trans-
ferrin. The introduction of the international CRM 470
protein standard material has lead to a significant re-
duction in interlaboratory variation for transferring
measurements. In view of these observations, deter-
mination of transferrin concentration, rather than
TIBC, is recommended. However, in non-European
populations characterized by a marked genetic varia-
tion in transferrin (TF BC and TF CD variants), in certain
cases, immunochemical determination of transferrin
may lead to errors. In these populations, TIBC mea-
surements may be preferred. Clin Chem Lab Med 2002;
Key words: Hemochromatosis; Iron; Iron overload; To-
tal iron binding capacity; Transferrin.
Abbreviations: CRM, certified reference material; SNP,
single nucleotide polymorphism; TF, transferrin; TIBC,
total iron binding capacity; UIBC, unsaturated iron-
Both iron deficiency and iron overload are important
public health problems. Iron deficiency is the most
prevalent nutritional problem in the world today, and
as much as 500 million people may be affected (1). On
the other hand, the presence of increased iron stores
with associated organ damage is also common. In peo-
ple of European descent, hereditary hemochromatosis
is the most prevalent monoallelic genetic disease (2)
and the utility of screening the general population is
being examined (3). In Africans, iron overload affects
up to 10% of some rural populations (4, 5).
Several laboratory tests are presently available for
the assessment of iron status (6, 7). The description by
Ramsay in 1957 of a practical way of determining the
total iron binding capacity (TIBC) of serum or plasma
provided a diagnostic test for both iron deficiency and
iron overload (8). Since then, TIBC has been used as a
parameter to assess the maximal capacity of serum to
transport iron. Since under physiological conditions
iron is exclusively bound by the β-globulin transferrin
(TF), determination of TF concentration in serum offers
a newer alternative for assessing the TF binding capac-
ity. The combination of total serum iron and either TIBC
or TF measurements allows the calculation of the occu-
pied and available TF binding sites.
TF saturation (calculated as a ratio of iron concentra-
tion and TIBC multiplied by 100), together with serum
ferritin are reliable screening tests for iron overload (9,
10). TF concentration has been used to derive TIBC and,
indirectly, TF saturation (11). TF index calculated as
serum iron divided by TF concentration has been sug-
gested as a better alternative to saturation in screening
for iron overload (12).
Both TIBC and TF assays have improved consider-
ably in the last decades (Table 1). In this review article
we discuss the advantages and disadvantages of the
use of TIBC and serum TF in the assessment of iron sta-
tus. We also address the implications of the genetic
variation of TF on the usefulness of these tests (13).
Iron Binding Capacity
In the plasma, iron is predominantly bound to TF, and
TIBC depends on the concentration of this globulin.
The fraction of TF to which iron is not actually bound is
known as the “unsaturated iron-binding capacity”
(UIBC). The sum of serum iron concentration and the
UIBC gives the TIBC.
Estimation of Unsaturated Iron-Binding Capacity
UIBC may be deduced from TIBC and serum iron con-
centration or measured directly by an isotope method.
In the latter method, the amount of radioactive iron re-
*E-mail of the corresponding author:
Total Iron Binding Capacity and Transferrin Concentration
in the Assessment of Iron Status
Kasvosve and Delanghe: Iron binding capacity, transferrin and iron status 1015
quired to saturate the serum (i.e. the UIBC) is measured
directly. In the original method of Bothwell et al.,
ammonium citrate was used and the absorption car-
ried out on Amberlite resin (14).
Total Iron Binding Capacity Determination
The method for TIBC measurement was first described
by Ramsay (8) and later refined by the International
Committee for Standardisation in Haematology and
described as a recommended procedure (15). This was
revised in 1990 (16). The chemical measurement of
TIBC consists of three steps. The first step involves ad-
dition of supraphysiological amounts of FeCl
rate the free binding sites on TF; the second is the re-
moval of unbound excess iron by adsorption onto solid
magnesium carbonate (8), charcoal or an ion-exchange
resin (17); the third is the determination of iron that is
dissociated from TF at acidic pH. Recently, direct TIBC
assays have been described (18, 19). The addition of
excess amounts of FeCl
results in nonspecific binding
of iron to albumin and other plasma proteins, leading
to overestimation of TIBC especially at low TF concen-
trations, as seen, for instance, in liver disease and in
nephrotic syndrome (20).
One of the major drawbacks of the TIBC test is the
large variation: reference ranges differ by as much as
35% between commercial methods (11), therefore a
generic reference interval can no longer be stated. Ex-
isting UIBC-based methods generally exhibit a signifi-
cant negative bias compared with TIBC methods (21).
Furthermore, the chemical methods for TIBC require a
relatively large sample volume and are sensitive to
contamination of the laboratory ware with iron (22).
Serum Transferrin Determination
Immunological methods for the measurement of
serum TF concentration are now available. The earlier
immunodiffusion methods (23) have been largely re-
placed by either automated immunoturbidimetric (24)
or immunonephelometric procedures (25). Although
the cost per test is lower for TIBC, determination of
serum TF offers some technical advantages. In addi-
tion, the sample volume needed for analysis of TF is
Recently, CRM 470 standardization was adopted for
fourteen plasma proteins (26). For TF, the reference
range was reported to be 2.0–3.6 g/l. Recently, refer-
ence intervals were determined in a Japanese popula-
tion using the same standardization (27). The results
obtained were more or less similar (1.90–3.20 g/l),
suggesting that racial differences are not very pro-
nounced. The introduction of the international refer-
ence material for serum proteins has resulted in a sig-
nificant reduction of the between-laboratory variance
for TF (28).
Coefficients of variation are very low, and well below
the requirements based on the large biological varia-
tion of iron (29). International interim reference ranges
have been proposed for a number of plasma proteins,
including TF (30) which have been accepted by the In-
ternational Federation of Clinical Chemistry and Labo-
ratory Medicine (IFCC) and many national scientific so-
Molecular Variation of TF
Like many other plasma proteins, TF is characterized
by genetically determined polymorphism. In Cau-
casians, individuals are homozygous for TF CC (C=
common). In less than 1% of Caucasians, TF CD (catho-
dal variants) and TF BC (anodal variants) heterozygotes
are found. In African and indigenous populations of
Oceania, prevalence of TF variants is often higher than
Isoelectric focusing of TF allows for further possibili-
ties to distinguish between TF CC subtypes. Recently,
single nucleotide polymorphisms (SNP) in the TF 5’
flanking region have been associated with differences
in TIBC. In patients with Parkinson’s disease, a disorder
in which there is abnormal iron deposition in the brain,
the presence of TF haplotype 3 was in slight excess
over the normal population (32). In cases of problems
of interpretation of TF results, the possibility of a TF
variant might be considered. In such situations the de-
termination of TIBC by chemical methods better re-
flects the patient’s iron status. The heterogeneity of the
carbohydrate moiety of TF is probably of no impor-
tance for the determination of serum TF. No correlation
has been found between the relative amount of carbo-
hydrate-deficient TF and the total TF concentration
Table 1 Milestones in total iron binding capacity and transferrin assays.
Year Milestone Reference
1957 TIBC determination 8
1959 Determination of UIBC using radioactive iron 14
1965 Radial immunodiffusion technique for transferrin measurement 23
1976 Immunoturbidimetric assay for serum transferrin 24
1978 Immunonephelometric assay for serum transferrin 25
1994 CRM 470 standard for 14 plasma proteins (including transferrin) 26
1996 Interim reference values based on CRM 470 standard 30
1016 Kasvosve and Delanghe: Iron binding capacity, transferrin and iron status
Derivation of TIBC from Serum TF Concentration
Since 1 mol of TF (molecular mass 79 570 Da) has the
capacity to bind two atoms of iron (atomic mass 56),
formulas for calculating TF saturation from TIBC and
vice versa have been proposed. Serum TF concentra-
tion may be estimated from TIBC by the following rela-
Serum TF (g/l) = 0.007 × TIBC (µg/l) (11).
TIBC (µg/dl) = TF (mg/dl) × 1.41
TIBC (µmol/l) = TF (g/l) × 25.2
TF saturation (%):
Serum iron (µg/dl)/TF (mg/dl) × 70.9
Serum iron (µmol/l)/TF (mg/dl) × 398 (34).
The above mathematical derivations have weaknesses.
Because some of the iron is bound to other proteins
(especially albumin) (35, 36) the relation between
serum TF concentration and TIBC is not completely lin-
ear. Due to binding to other plasma proteins, the calcu-
lated TIBC values are a few micromoles per liter higher
than the TF-bound iron. In the elderly, iron binding ca-
pacity correlated with serum albumin (35). Moreover,
change in calibrator yields TF concentrations that are
13% lower than those obtained with the older calibra-
tor (21). While early estimates suggested a value of
90000 Da as the molecular mass of TF (20, 37), more re-
cent estimate based on the amino acid sequence gives
a calculated molecular mass of 79570 Da (34, 38). Be-
cause of uncertainty as to the molecular mass of TF,
various values for TF molecular mass are being used
resulting in different conversion factors applied by
different laboratories (12). Therefore, a number of ma-
nufacturers recommend that the experimentally deter-
mined factor of 1.27 be applied instead of the theore-
tically derived 1.41 (39). This can be regarded as a
mathematical compensation for the non-TF bound
Several articles suggest that, contrary to the theory,
the relation between TIBC and TF is not fixed, espe-
cially when results are outside the reference range (40,
41). The reported mean ratio between TIBC and TF has
ranged from a low of 11.3 to a high of 26.1 (21). As a
consequence, the development of a universal algorithm
for the conversion of TF values into TIBC is not possible
A body of evidence exists supporting the hypothesis
that screening for hereditary hemochromatosis may be
cost-effective, given the low-cost, low-risk therapeutic
options available for the affected individuals (42). It is
crucial to diagnose hemochromatosis before irre-
versible organ damage (hepatic cirrhosis, diabetes) de-
velops. A DNA-based test for the HFE gene is now
available but it is only useful for the detection of he-
mochromatosis in the family members of patients with
a proven case of the disease (3). The protocol for
screening for hemochromatois entails determination
of serum TF saturation and serum ferritin level (3). In
contrast to iron deficiency, in which the diagnostic sen-
sitivity of serum ferritin is superior to TF saturation, TF
saturation has the highest predictive value in screening
for hereditary hemochromatosis (39). Elevation in
serum TF saturation occurs before significant iron
Persistently elevated TF saturation is the earliest bio-
chemical evidence of hemochromatosis. Fasting TF
saturation ≥45% is used as the screening threshold be-
cause it identifies 98% of affected persons while pro-
ducing relatively few false-positive results (3, 43). Since
TF saturation is obtained by dividing serum iron con-
centration by TIBC and expressing it as a percentage,
the determination of TIBC is necessary to compute TF
saturation. It is clear that the cost-effectiveness of he-
mochromatosis screening programmes will partly de-
pend on the method and the standardization of deter-
mining TF saturation.
Use of TIBC may be more problematic in cases of
atransferrinemia. Congenital atransferrinemia is a rare
genetic condition characterized by the absence of TF. In
these cases, extremely low values or even complete
absence of serum TF are found. Acquired atransfer-
rinemia has been described in association with the
nephrotic syndrome probably due to loss of TF through
the kidney (20, 44, 45). One patient with functional dis-
order of TF due to the presence of TF-IgG-TF immune
complexes has been described (46). In such patients,
positive values for TIBC (up to 69 µg/dl) have been re-
ported despite TF concentration readings of 0 mg/dl
(47, 48). This paradoxical finding could be attributed to
the increased nonspecific binding of iron to other
plasma proteins. Indeed, when TF is more than 50%
saturated, some of the iron is bound to other plasma
There are no generic reference values available for
TIBC. Intralaboratory variation increases due to varia-
tion of the TIBC method. In contrast, the introduction
of automated immunoassays and the universal CRM
470 protein standard has led to acceptable interlabo-
ratory variation for serum TF concentration. Gener-
ally, TF determinations have several analytical advan-
tages compared to TIBC determinations. Because the
TF assay is more precise, more readily automated
either by immunoturbidimetry or immunonephelome-
try, and less labor-intensive than the TIBC method,
many laboratories are now measuring TF rather than
TIBC. However, in certain cases attention should be
paid to the consequences of genetically controlled
polymorphisms of TF, especially in non-European
populations. In case of discordant laboratory findings
in the assessment of iron status due to the TF variants,
non-immunological determination of TF (e.g. by capil-
lary electrophoresis) (13) or chemical measurement of
TIBC is preferred.
Kasvosve and Delanghe: Iron binding capacity, transferrin and iron status 1017
This work was supported in part by an IFCC Scientific Ex-
change Fellowship Programme 13.5.15, 1999. We thank Erna
Vanbekbergen and Freddy Lason for their kind support during
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Received 25 June 2002, revised 30 August 2002,
accepted 9 September 2002
Corresponding author: Prof. J.R. Delanghe,
Department of Clinical Chemistry, Ghent University,
185 De Pintelaan, 9000 Gent, Belgium
Phone: + 32 9 2402956, Fax: + 32 9 2404985