Abid 4

Best Practice & Research Clinical Haematology
Vol. 15, No. 2, pp. 329±368, 2002

doi:10.1053/beha.2002.0214, available online at http://www.idealibrary.com on
Monitoring chelation therapy to achieve

optimal outcome in the treatment of
thalassaemia
John B. Porter MA, MD, FRCP, FRCPath

Professor of Haematology
Bernard A. Davis MRCP, MRCPath

Clinical Research Fellow
Department of Haematology, University College London, 98 Chenies Mews, London, WC1E 6HX, UK
Eective management of iron overload in thalassaemia requires monitoring both for iron
toxicity and the eects of excessive chelation. Careful monitoring together with adherence to
established regimens using desferrioxamine (DFO) results in a 78% survival rate at 40 years of
age at UCLH, with steadily improving survival as progressive cohorts receive chelation
earlier in life. By contrast, survival is considerably below this in non-specialist centres. The
prognostic signi®cance of the measures being used in monitoring should be known so that
decisions about chelation management are evidence-based. Serum ferritin measurement,
although easy to perform frequently, is subject to variability and falsely high or falsely low
values in relation to body iron are frequently obtained. However, there is evidence that
persistently high ferritin values above 2500 mg/l have poor prognostic signi®cance in patients
treated with DFO. Liver iron predicts total body iron in a more predictable way than serum
ferritin in thalassaemia. Liver iron concentrations of 15 mg/g dry weight appear to predict
those patients who develop heart failure in subjects treated with DFO. The prognostic
signi®cance of this measurement or indeed other measurements of iron overload in patients
treated with other chelation regimens is not known. Recent advances with MRI imaging have
aroused interest in its use for monitoring patients with thalassaemia. A recent publication
suggests a relationship between left ventricular ejection fraction and cardiac T2*, the value of
which shortens with increasing iron concentrations in the liver and hence by inference in the
heart. The prognostic value of this technique has not yet been demonstrated in prospective
studies and hence changes in therapy based on this measurement alone should be considered
with caution at this time. The value of monitoring to decrease morbidity from iron overload
is also discussed, particularly with reference to the estimation of iron deposition in the
pituitary.
Key words: transfusional iron overload; chelation therapy; desferrioxamine; deferiprone;

monitoring tissue damage; cardiac function; MRI; SQUID.
All correspondence to Professor J. B. Porter. E-mail: j.porter@ucl.ac.uk

1521±6926/02/$ - see front matter *
c 2002 Published by Elsevier Science Ltd.
330 J. B. Porter and B. A. Davis
GOALS AND OPTIMAL OUTCOMES WITH CHELATION AND

MONITORING
The primary goal of chelation therapy is to achieve tissue iron concentrations that do
not cause tissue damage: this requires a chelation regimen that will eliminate body
iron at a rate equal to, or in excess of, iron input from red cell transfusion plus iron
absorption from the gut. The iron concentration and the duration of exposure to
excess iron that will cause pathology is unlikely to be the same for all tissues. In iron
overload, the majority of body iron is sequestered as ferritin and haemosiderin, which
are not chelated at useful rates at clinically achievable chelator concentrations. Clinical
chelation therapy relies on the binding of iron pools that are transient and ®nite.
Therefore, once iron has accumulated, normalization of tissue iron takes many months
or years to achieve. A second goal of chelation therapy in the face of established
overload is to detoxify iron until safe levels of tissue iron have been achieved. The rapid
reversal of heart failure with continuous desferrioxamine (DFO) infusion is likely to be
due to this second mechanism. More detailed discussions of the mechanisms of
interaction of chelators with iron pools to achieve iron balance and iron detoxi®cation,
while minimizing chelator toxicity, have been given recently elsewhere.1±3
Eective management of iron overload in thalassaemia requires monitoring both for
iron toxicity and for the eects of excessive chelation. Careful monitoring together
with adherence to established regimens using DFO can result in 78% survival at 40
years of age, with a steadily improving survival as progressive cohorts receive chelation
earlier in life (Figure 1).4 By contrast, survival is considerably below this in non-
specialist centres.5 Optimal monitoring will allow the adjustment of the chelation
regime so as to minimize iron toxicity whilst decreasing the toxicity from the
chelators themselves. This chapter will focus on the objectives of chelation therapy and
the tools available for monitoring in order to minimize toxicity from excess iron and
also from excessive chelation therapy.
MONITORING OF IRON OVERLOAD AND ITS TREATMENT
Monitoring is important in deciding when treatment should be started, how it should

be modi®ed as iron loading increases or falls, in identifying patients most at risk of iron
mediated organ damage and in adjusting chelation treatment accordingly. With the
emergence of new chelator regimens, the future challenge will be to apply monitoring
techniques so as to tailor chelator regimens to an individual's requirements. The
ultimate goal will be not just to minimize mortality from iron overload but to decrease
morbidity, particularly from iron deposition in the anterior pituitary, still further so
that maximal growth, sexual development and fertility are achieved in all patients.
Estimation of iron balance

The rate of iron loading
The rate of iron loading will determine when chelation should start as well as the
chelation regimen to be adopted. It can be estimated from the volume of blood
transfused and iron accumulated from gastrointestinal (GI) absorption. To achieve iron
balance, chelation must excrete enough iron to match iron input from blood
transfusion and absorption from the GI tract. Iron accumulates from transfusion and
Monitoring chelation therapy in thalassaemia 331
A 1
0.75
Survival probability
0.5
0.25
0
0 10 20 30 40
Years from birth
B
1975 — 1997
1
1965 — 1974
0.75
Survival probability
1955 — 1964
0.5
0.25
0
0 10 20 30 40
Years from birth
Figure 1a and b. Survival of thalassaemia major at University College London Hospital (UCLH). The
Kaplan±Meier estimate of overall survival of the entire cohort as at 30th November 2000 is 78% at 40 years of
age. Sub-group analysis by ten-year birth cohort reveals a probability of survival of 69% at 40 years among
subjects born 1955±1964 (n 21) and of 78% at age 35 years among those born 1965±1974 (n 39). There
have been no deaths among patients born after 1974 (n 43).
from increased GI absorption. The iron content of each transfusion can be calculated
from the formula:
Iron content of transfusion mg volume ml haematocrit 1:16
In splenectomized patients with thalassaemia major, maintaining a mean haemo-

globin (Hb) of 12 g/dl, requires 100±200 ml of pure red cells per kg body weight per
year6, which is equivalent to 0.32±0.64 mg of iron per kg per day.7 Splenectomy
decreases the annual iron loading by an average of 39% if the transfusion requirement
exceeds 250 ml/kg of packed red cells per year.8 Anaemia results in erythropoietin-
induced erythroid expansion, which also increases iron absorption.9 In thalassaemia
intermedia, iron absorption is increased by 5±10 times of the 1±1.15 mg/day of healthy
individuals, which is equivalent to 0.1 mg per kg per day.10±12 This may increase
further following splenectomy13, possibly secondary to hypoxia associated with micro-
pulmonary emboli.14 In thalassaemia major, however, such increased iron absorption is
inversely proportional to the mean transfusional Hb obtained. If the pre-transfusion
Hb is allowed to fall to 9 g/dl rather than 10.5 g/dl, transfusional iron loading is
decreased while the erythron is expanded only threefold9, a level where GI iron
absorption does not exceed iron loss and there is little or no impairment of bone
metabolism.9,15
It is helpful to keep a record of blood intake and to calculate this on a yearly basis in
order to reveal trends in net blood consumption. A mismatch between cumulative
DFO dose and blood consumption that is unacceptably high (e.g. as a result of
hypersplenism) may explain why some patients are more iron-overloaded than would
be expected despite good compliance with chelation therapy. Conversely, other
patients with a low net blood consumption and hence iron accumulation, may have a
milder thalassaemia phenotype than predicted from the genotype.
Rate of iron excretion by chelators

Only a fraction of total body iron is immediately available for chelation, as has been
demonstrated in experimental animals16,17 and in thalassaemia patients.18 Therefore,
escalation of chelation dose has a limited impact on iron excretion and risks increasing
toxicity from excess chelator. The major source of chelatable iron is derived from
catabolism of haemoglobin in macrophages in the liver, spleen and bone marrow
amounting to 20±30 mg/day in non-haemolytic conditions19 and up to seven times this
value in thalassaemia intermedia.12 In the absence of chelators, this iron will bind to
transferrin and is delivered to the erythron or it will bind to citrate and albumin to
form non-transferrin bound iron (NTBI).20,21
Most urine iron excretion with DFO appears to be derived from iron released from
macrophages.16±18,22 This interception probably occurs extracellularly with DFO23 and
also with diethylenetriaminepentacetic acid (DTPA), which has little intracellular
penetration.24 In advanced iron overload, the hepatocyte is the major cell of iron
storage and is therefore key to normalizing iron stores. Since chelators do not interact
with ferritin and haemosiderin at clinically relevant rates25 they only access the fraction
of hepatocellular iron that is present in the labile iron pool (LIP). This pool is mainly
derived from the catabolism of ferritin within lysosomes26 but iron uptake into the cell
from transferrin or non-transferrin iron will also contribute to this pool. Chelation of
hepatocellular iron by DFO results in faecal excretion of iron and accounts for about
half of all iron excreted by DFO.18,27 Urine iron excretion with DFO increases in
proportion to iron loading from blood transfusions28 and the dose of chelation
used.29,30 However, the proportion of iron excreted in faeces increases with the dose
of DFO given.27 The value of measuring urinary iron in the monitoring of chelation
therapy depends on the chelation regimen being used. Other chelators result in
dierent routes of iron excretion: for example, deferiprone excretes iron almost
exclusively in the urine31; by contrast the new oral chelator developed by Novartis,
ICL670A32 appears to excrete iron exclusively in the faeces in preclinical studies and in
clinical iron balance studies.33
The measurement of urinary iron in monitoring patients on chelation therapy is
generally not pivotal, but can be useful with individual patients or when assessing new
treatment regimens. A dose titration of urinary iron excretion with DFO has been
advocated so as to adjust the optimal dose to the point where dose increments do not
increase iron excretion signi®cantly.34 This scheme is not widely used today, since with
standard DFO regimens, a dose of 40 mg/kg ®ve times a week is generally sucient to
keep a patient in iron balance without signi®cant drug toxicity. One of the problems
with urinary iron measurement is that there is a large day-to-day variation in iron
excretion and several samples at any given dose will be required in order to obtain a
meaningful estimation of iron excretion.7 Apart from the inherent variability of this
measurement, other factors aect urine iron excretion with DFO: iron excretion with
DFO is increased in patients given ascorbate supplements27 but not with deferiprone.
The point in the transfusion cycle27 is also important, with urinary iron excretion
being proportionally higher in the urine as the Hb level falls, presumably re¯ecting
increased iron turnover from catabolism of short lived endogenous red cells.
Monitoring iron loading to adjust chelation therapy

Serum ferritin
Serum ferritin level increases proportionately with iron loading and the test has the
advantage that it can be performed at frequent intervals but it also has several
limitations. First, the correlation between serum ferritin and liver iron, although
undoubtedly present, is not suciently precise to allow ferritin to be used as a `stand
alone' monitoring modality.12,35±37 It is clear that while many studies show
correlations, the spread of values is such that, in clinical practice, reliance on serum
ferritin alone as an estimate of body iron is unwise.38,39 A second limitation of serum
ferritin is that it is disproportionately raised when in¯ammation or tissue damage is
present40 and falsely depressed when patients are scorbutic, due to the rapid oxidation
of vitamin C.41 At high levels of iron loading, serum ferritin is contributed to by
hepatocellular injury, whereas at lower levels of iron loading, ferritin re¯ects iron
stores.42 More recent work on the response of serum ferritin to iron chelation
supports this contention: in patients commencing continuous intravenous (i.v.) DFO
chelation, the rate of fall in serum ferritin was proportional to the starting serum
ferritin value (r 0.9) when the serum ferritin exceeded 3000 mg/ml. However, the
rate of fall of serum ferritin was linear (133 mg per l per month) and independent of
the serum ferritin value when this fell below 3000 mg/l.43
A further limitation of serum ferritin is that the relationship with liver iron (and
hence total body iron44, diers with the cause of iron loading. Hence, in genetic
haemochromatosis the serum ferritin appears to be lower relative to liver iron than in
iron overload secondary to haematological disease (see Chapter 5 and Prieto et al42). In
thalassaemia intermedia, serum ferritin tends be lower relative to the degree of iron
overloading than in thalassaemia major.45 In sickle cell disorders, the serum ferritin
used as an estimate of iron loading is disproportionately increased in relation to iron
loading for several weeks after a painful sickle crisis46,47 and correlates poorly with
hepatic iron concentration (HIC).48,49 Finally the relationship between liver iron con-
centrations and serum ferritin has not been compared systematically for dierent
chelator regimens; it would seem advisable to establish such a relationship for any new
chelator being introduced into clinical practice.
Despite these limitations, serum ferritin is a useful tool for monitoring patients
receiving chelation treatment with DFO (Table 1) partly because it can be measured
more frequently than other estimates of iron loading such as liver iron. Studies of
patients followed for 15 years showed signi®cantly better survival in DFO treated
patients who maintained serum ferritin at 52500 mg/l on at least two-thirds of all
testing occasions50,51, although analysis of cardiac function has stronger predictive value
(see below). Thus, patients who consistently fail to achieve serum ferritin values below
2500 mg/l are at increased risk and this can be used to modify the chelation regimen.
A target ferritin of approximately 1000 mg/l is generally recommended as standard
practice in thalassaemia major7, although this approach is based on current clinical
practice with DFO rather than on evidence obtained from clinical trials. Serum ferritin
can also be a useful tool for dose adjustment of DFO using the therapeutic index (see
below).52 The prognostic value of serum ferritin measurement with other chelation
regimens is unknown, as is the value of adjusting the dose in relation to ferritin values
in order to decrease chelator toxicity.
Liver iron concentration by biopsy

In iron overload, over 70% of body iron is found in the liver44,53 and liver iron has been
shown to correlate closely with total body iron, which is approximately 10.6 times the
hepatic iron concentration (mg/g liver, dry weight).44 The case for using HIC to monitor
Table 1. Possible prognostic indicators to monitor in iron overload.
Measurement Finding Outcome Reference

Serum ferritin 52500 mg/l with s.c.signi®cantly better outcome; Olivieri et al (1994)50
DFO on 2/3 occasionsquestionable value if used
over 15 y f.u. alone
Liver iron 415 mg/g dry weight 9/32 patients died from Brittenham et al (1994)83
cardiac failure
Liver iron 47 mg/g dry weight levels found in HH Olivieri & Brittenham
heterozygotes without (1997)39
pathology
LVEF below reference range signi®cantly worse prognosis Davis et al (2001)51
(45% with MUGA) if no intervention with DFO
DFO frequency of use infusions 5 (250/y) signi®cantly reduced prognosis Gabutti & Piga (1996)85
5 5x/week 12% @ 30 yrs old
4 5/week 95% @ 30 yrs old
Cardiac T2* 5 20 ms all patients with abnormal Anderson et al (2001)64
LVEF had T2* below this
value N.B. Prognostic value
NOT yet shown
LVEF, left ventricular ejection fraction; DFO, desferrioxamine; s.c., subcutaneous; MUGA, multi-gated
acquisition scan; HH, hereditary haemochromatosis; f.u., follow-up.
DFO treatment is also based on the strong link between HIC and prognosis in patients
treated with DFO (see above). The measurement of liver iron can, in principle, be
estimated invasively using liver biopsy or non-invasively using a superconducting
quantum interface device (SQUID), magnetic resonance imaging (MRI) or with
computed tomography (CT) scanning.
Needle biopsy of the liver in thalassaemia patients, if performed following an
established methodology54, on samples of adequate size (at least 1 mg dry weight) and in
the absence of cirrhosis, has an acceptable coecient of variation on duplicate specimens
of 6.6%.55 In cirrhotic livers, the variability in measurable iron is greater because of the
uneven ®brotic content; multiple biopsy samples from patients with cirrhosis have
shown that samples weighing more than 4 mg dry weight give a signi®cantly lower
coecient of variation (20%) than smaller samples (29%).56 The safety and reliability of
liver biopsy is well established, particularly if performed under ultrasound guidance.
Even without ultrasound guidance, the procedure is eective and safe: in 1184
consecutive biopsies performed in thalassaemia major patients without ultrasound
guidance before bone marrow transplantation (BMT), there were no fatalities and a
complication rate of only 0.5% (haemoperitoneum, peri-cholecystic haematoma, kidney
haematoma, or bile peritonitis). In this series an adequate sample was obtained in 81%
of patients with a mean age of 11 years.57 In advanced liver disease there is uneven
distribution of liver iron56,58, although in non-cirrhotic livers an autopsy study59 found
that samples taken from inferior and superior sites in the midaxillary, anterior axillary
and midclavicular line had similar concentrations of non-haem iron.
In recent years there has been a trend in clinical practice towards the increased use
of liver biopsy, both to quantify liver iron and to assess liver in¯ammation and damage.
Because of the increased use of HIC for monitoring treatment, there is a need to
harmonize both the techniques used to measure HIC and the units in which it is
expressed. The measurement of liver iron can be performed on wet or dried samples
and the conversion of one to the other varies according to the drying and measuring
techniques used in the laboratory. An agreed international standardization of
measurement and reporting would be helpful for practising clinicians.
Liver iron by superconducting quantum interface device

The most sensitive, accurate and reproducible measurement of liver iron is obtained
using the SQUID60, which relies on the principle that ferritin and haemosiderin have
paramagnetic properties. The paramagnetic property of a tissue is determined by the
strength and direction of the magnetic response produced by placing that tissue in a
magnetic ®eld. In most biological materials, the application of a magnetic ®eld induces
a diametrically opposed magnetic ®eld in the tissue (diamagnetism). However, this
diamagnetic response is less that 10 6 of the applied ®eld. Ferromagnetic materials
such as magnets are not present in human tissues (unless metal objects are present)
and they respond to an applied ®eld by producing a ®eld in the same direction and
which is as strong or stronger than the applied ®eld (ferromagnetism). Paramagnetic
materials, such as ferritin and haemosiderin, also produce a magnetic ®eld in the same
direction as the applied ®eld but which is only a fraction as strong (10 4) as the applied
®eld strength. The size of the paramagnetic response is proportional to the number
of iron atoms present in ferritin or haemosiderin. Other iron-containing proteins, such
as haemoglobin, do not contribute a signi®cant paramagnetic signal.
In practice, a measurement is made with patients in a supine position with a
water bag above the liver to simulate the diamagnetic properties of non-ferritin or
non-haemosiderin containing tissues. The patients are lowered on to a wooden bed at

about 6 mm/s in the magnetic ®eld (maximum ®eld strength 25 mT) of a super-
conducting coil. The magnetic ¯ux change during the movement is registered by two
detector coils over a period of about 13 seconds. Current detectors need liquid
helium, operating at a temperature of 48K. Liver iron is then calculated from the signal
obtained and the volume of liver and spleen measured independently by bedside
sonography. The relationship between liver iron content and magnetic susceptibility is
linear over a wide range.60±62 Unlike MRI, the SQUID has limited spatial resolution
and cannot dierentiate between parenchymal and reticulo-endothelial (RE) iron.
Iron measurements using SQUID show an excellent correlation with liver iron as
measured by biopsy in haemochromatosis patients (r 0.98)60 and thalassaemia
patients without cirrhosis (r 0.98) provided samples exceed 1 mg dry weight.44 A
less impressive correlation has been obtained by other investigators in thalassaemic
patients63, but in that report liver iron biopsies were measured using a variety of
methods in several dierent centres. Both the SQUID and chemically determined iron
varies with the degree of liver ®brosis present.
SQUIDs are currently expensive to build, run and operate since they require a
dedicated technician and a building that is relatively free from paramagnetic interference.
Also metal objects, such as jewellery or pacemakers, will interfere with measurements.
Only four instruments are in use worldwide (USA, Germany and Italy) and these have
been calibrated using dierent approaches. Devices that are smaller and use liquid
nitrogen rather than liquid helium would be cheaper to build and to maintain.
Liver iron by magnetic resonance imaging

The potential of using MRI to quantify body iron and image its distribution has been
investigated for many years but a universally recognized technique of adequate
sensitivity and reproducibility has yet to emerge. Such an MRI technique would have
clear advantages over SQUID since MRI facilities now exist in most hospitals. The
additional capital outlay would be relatively modest compared with that for a SQUID. A
further potential advantage of MRI is that, in principle, information on organs other
than the liver can be obtained during the same scanning procedure: in particular
accurate and reproducible information about right and left ventricular ejection fractions,
ventricular volumes and ventricular wall motion plus information about heart iron
content64 could be obtained. However, there remains much debate about which
acquisition mode is most appropriate for obtaining measurements in dierent organs.
Iron overload in tissues results in shortening in the longitudinal axis (T1) and
transverse (T2) tissue relaxation times on which MRI is based and leads to a reduction in
signal intensity. The decrease in the intensity of spin echo images with iron overload is
thought to derive from decrements in the T2 relaxation times65,66 and is due mainly to
the paramagnetic properties of ferritin iron, although low molecular weight cytosolic
iron66,67 and iron bound to desferrioxamine (ferrioxamine) may also contribute.68 Thus,
the iron species present in tissues will have dierential eects on T1 and T2 relaxation
times.69
Early evaluations using MRI lacked sensitivity, but as shorter echo sequences have
become available the sensitivity has increased. Using spin echo (T2) analysis, an inverse
relationship between liver iron concentration and T2 measurements in iron overloaded
patients has been known for over a decade.70±73 Several approaches have been
investigated to circumvent the limitations of a simple spin echo technique. It is clear that
the shorter the spin echo time, the greater the sensitivity74 and provided spin echo
times are 20 ms or less, liver iron concentrations of 2 mg/g of liver tissue should be
detectable. Another approach is to compare the tissue of interest with a tissue that does
not load excess iron. Jensen et al72 found that using a standard spin echo MRI system at
1.5 T with an echo time of 25 ms, the signal intensity ratio (SIR) of liver to skeletal
muscle (which accumulates almost no excess iron) was dependent on the applied
repetition time in iron overloaded subjects. Adjustment of the SIR to a constant
repetition time was achieved and a close correlation between chemically determined
liver iron and MRI signal was found. Other authors have continued to use spin echo
sequences (1/T2 or R2) but have developed methodologies to circumvent problems
induced by motion artefact.75 A simple smoothing technique is used to accommodate
the image intensity perturbations caused by abdominal motion. The relaxivity maps
obtained are consistent with the variation of iron concentration throughout the liver
and this may be a useful approach to dealing with the heterogeneity of liver iron
deposition in the presence of ®brosis.
Gradient echo techniques have been investigated by a number of centres to improve
sensitivity, with or without SIRs to skeletal muscle.76±78 The coecient of variation
using this technique was low (12%). Angelucci et al79 investigated the relationship of
gradient echo pulse sequences of T1-weighted images to the SIR of liver iron to muscle
and found a linear correlation (0.84) and a closer correlation (0.99) in patients without
®brosis. They concluded that in the presence of ®brosis, this MRI technique was too
variable to be of practical value.
A further approach to gradient echo imaging is to measure T2*. T2 is related to T2*
by the formula 1/T2* 1/T2 1/T0, where T2 is the tissue relaxation and T0 is the
magnetic heterogeneity of the tissue being analysed. There are several advantages to this
approach: T2* does not require the analysis of SIRs, relying instead on multiple echos
over a shorter acquisition time period than spin echo techniques. This gives greater
sensitivity compared with spin echo T2 sequences at low and normal levels of tissue
iron, and greater reproducibility than results derived from SIRs. A further advantage is
the shorter acquisition time compared with spin echo sequences, thereby reducing
motion artefact which is a particular problem when analysing the heart. T2* was ®rst
used for imaging iron deposits in the brain.80,81 It was then used by Siegelman82 on the
liver. Ernst et al77 compared the use of gradient echo T2*-weighted sequences with T1-
weighted sequences. Using T2*, the best correlation with liver iron concentrations by
biopsy was seen for liver iron concentrations 5 100 mmol/g, (r 0.71). Recently T2*
has been applied to liver and heart (see below) by Anderson et al.64 Echo times of
2.2±20.1 ms were used for liver analysis using a single slice through the centre of the
liver. A good reproducibility (coecient of variation 3.3%) and a correlation of 0.93
between liver T2* and hepatic iron concentration was observed in thalassaemic patients
provided samples with ®brosis were excluded.
Use of liver iron in monitoring of chelation treatment

Whatever the method used to estimate liver iron concentration, the ultimate aim is
to decide when chelation treatment should be started, intensi®ed or reduced. This
statement implies that we have a full understanding of the relationship between liver
iron and prognosis: however, this is currently incomplete. The only clear data linking
tissue iron levels to prognosis in thalassaemia major has been obtained using liver iron
measurements.83 These authors followed 32 thalassaemia major patients over 15 years
of age, and treated with DFO for variable periods of time. All patients who died with
cardiac complications had HIC values 4 15 mg/g dry weight (Table 1).83
However, since heart failure was the cause of death in all patients, it is likely that liver
iron was not the primary determinant of cardiac free survival but represented an
association of risk. As toxicity from chronic iron overload is an insidious process, the
duration of exposure to excess iron and the age at which toxic levels are reached must
also be taken into account as prognostic determinants. It has also been suggested that
realistic target levels of liver iron in treated patients should be less than 7 mg/g dry
weight since heterozygotes with hereditary haemochromatosis (HH) can reach such
levels without signi®cant pathology resulting (see Table 1).39 This analogy assumes that
the distribution of iron between heart and liver is similar in heterozygotes with HH and
for patients with transfusional iron overload treated with chelators. This necessarily
cannot take into account that such levels take a lifetime to accumulate in heterozygotes
with HH but are reached in early childhood in transfusion-dependent patients.
At University College London Hospital (UCLH), we have observed many patients
on little or no chelation from the mid-1960s through to the late 1970s who had liver
iron values considerably and consistently above 15 mg/g dry weight before the
chelation era. They are alive and well today and now have low levels of liver iron
(57 mg/g) because of a sustained chelation policy with sub-cutaneous (s.c.) or i.v. DFO
for over 20 years. This and other reports show that a single measurement of liver iron
has little relevance to clinical management or prognosis.84 Thus, risk strati®cation
based on tissue iron levels must incorporate duration of exposure to a high body
iron burden as well as the chelation history in order to be meaningful. Provided iron
levels can be subsequently reduced, a poor outcome does not inevitably result. It is
conceivable that high levels of liver iron may exist without signi®cant `spill' of iron into
the heart. Indeed preliminary ®ndings from our patients with sickle disorders,
commenced on transfusion after childhood, show high liver iron values (415 mg/g dry
weight) with normal heart T2* values. What is important to survival is sustained
compliance with DFO over many years83,85and the total exposure over time to high
levels of body iron. This requires regular monitoring of liver iron.
The question arises as to how target levels of liver iron should be used to start or
adjust chelation regimens. Since liver iron is a more reliable estimate of body iron than
serum ferritin, it should be possible to adjust the dose of DFO given, based on the liver
iron determination: such a scheme has been suggested.39 By analogy with heterozygotes
with HH, a conservative target should be to maintain liver iron at 57 mg/g dry weight
at all times.39 We hypothesize, however, that once values above this have been reached
(from poor compliance or late commencement of chelation), it may be necessary to
achieve lower levels of liver (and hence total body) iron in order to prevent pathology in
organs such as the heart and the pituitary. Monitoring liver iron should, in principle, be
the best way to minimize toxicity from excess chelation. In order to minimize DFO
toxicity, a dose could be calculated based on the liver iron, using an index similar to the
ferritin therapeutic index.52 Indeed, Fischer86 has shown that the ferritin therapeutic
index correlates with liver iron and with an index of DFO dose divided by liver iron.
However, as yet, there are no published studies showing that such an approach
minimizes DFO toxicity and exactly what index should be aimed for.
Monitoring myocardial iron

Cardiomyopathy is the major cause of death from iron overload in thalassaemia
major.87,88 Broadly speaking, the risk increases with total body iron burden as measured
by liver iron content and decreases if compliance to DFO chelation is adhered to83,85
Little is known about the relationship between cardiac iron content and risk of heart
failure or arrhythmia in the post-chelation era. In patients with thalassaemia major dying
with cardiac failure, the concentration in myocardium, although signi®cantly more than
in skeletal muscle, is generally less than a ®fth of that in the liver and substantially less
than in many endocrine organs such as the parathyroid.53 In the pre-chelation era, post
mortem examination of the heart showed a correlation (r 0.76) between heart and
liver iron.89 Stainable cardiac iron was present in 100% of patients who had received
200±300 units of blood, in 60% of patients who had received 75±200 units and in 28% of
those who had received 51±75 units.89 Iron deposits were greatest in the epicardial third
and least in the middle third of ventricular walls, initially in the perinuclear areas of
myo®bres and spreading to the entire ®bre in heavily loaded myocytes.89 Atrial iron
deposition generally occurs later than in the ventricles. Iron deposition in conducting
tissue is occasionally found but is less common than in myocytes.89,90 Marked interstitial
®brosis in the heart was present in most patients with thalassaemia at post mortem in
the pre-chelation era.90 Iron deposition in the myocytes consists of small (74( + 12)AÊ)
super-paramagnetic particles of ferritin and haemosiderin.91 The crystalline structure of
ferritin cores in thalassaemia appears to be organ-speci®c in unchelated patients, being
larger and more crystalline in the heart than in the spleen and liver92 but with
surprisingly similar isoferritin pro®les.93 Biopsies from thalassaemia patients with heart
failure show disrupted myocytes with loss of myo®bres, dense nuclei and a variable
number of cytoplasmic, pleomorphic, electron dense, iron containing granules.94
Although intuitively one might expect a worse prognosis in patients with a high
myocardial iron content, the precise prognostic relevance of this iron content is not
known. A simple relationship between iron concentration and prognosis is unlikely; this
is not consistent with the marked improvement in clinical heart failure, left ventricular
ejection fraction and cardiac arrhythmias observed soon after commencing i.v. DFO43,95
when changes in total body iron are small. Furthermore, the concentration of iron
causing serious tissue injury is not the same for all organs.53 It has also been argued that
myocarditis is as important a determinant of heart failure as the iron content per se96
and there may be genetically determined variables aecting susceptibility to heart
failure in the presence of iron overload.97,98 Unfortunately, it is generally impractical to
answer how cardiac iron content, myocarditis, heart function and overall prognosis
relate to each other using endomyocardial biopsy. This is partly because of the relative
inaccessibility of the heart to biopsy compared with the liver but, more importantly, the
marked heterogeneity of iron deposition in iron overloaded patients and a poor
correlation with heart failure.99,100 Studies of the iron distribution in haemochromatotic
myocardium demonstrate that the subendocardial myocardium contains only half the
iron content of the subepicardial layer and there is a large sampling variation.
There has, therefore, been considerable interest in developing an MRI technique to
monitor myocardial iron loading73 and there have been a number of clinical studies
examining a variety of dierent MRI techniques in iron overloaded patients. Mavrogeni
et al101 compared two dierent MRI techniques: the T2 relaxation time and the heart/
skeletal muscle SIR. Such spin echo techniques suered from poor sensitivity at low
concentrations of tissue iron, with motion artefacts and poor signal-to-noise ratios at
longer echo times making quanti®cation of myocardial signals particularly unsatisfac-
tory. Jensen et al102 used spin echo images to determine the myocardium/muscle SIR,
which was converted to tissue iron concentration, based on a modi®ed calibration curve
from the liver model. They demonstrated signi®cant positive linear relationships
between the MRI-derived myocardial iron concentration, the serum ferritin
concentration and the MRI-determined liver iron concentration. The myocardial MRI
iron concentrations showed a signi®cant correlation with the number of blood units
given and the aminotransferase serum concentration. In another study, regression

analysis between T2 and measurements of ferritin demonstrated a reversed linear
relationship.103 Wielopolski & Zaino104 used nuclear resonance scattering to examine
the relationship between heart and liver iron deposition in thalassaemia major. They
found a good correlation between liver iron measured by biopsy and liver iron by
nuclear resonance scattering, as well as a good correlation between liver and heart
nuclear resonance scattering signals. This agrees with early post mortem correlations
between liver and heart iron concentrations in the pre-chelation era.89 A correlation
between liver and heart iron and that of other tissue such as skin has also been
demonstrated in an animal model of iron overload using X-ray ¯uorescence.105
The above ®ndings suggest a relationship between liver and heart iron using three
dierent techniques. In contrast, Anderson et al64 using T2* rather than a spin echo
technique, found no correlation at all between liver T2* or serum ferritin and heart T2*
in thalassaemia major patients on DFO or deferiprone. T2* gave a high degree of
reproducibility in both the liver and the heart (coecient of variations of 3.3% and 5.0%,
respectively). Heart T2* was shortened in 58% of patients despite long-term chelation
treatment and there was a linear decline in left and right ventricular ejection fraction
associated with shortening of myocardial T2*. The reason for the dierence between
the ®ndings of Jensen and those of Anderson, using two dierent techniques is unclear.
One possibility is that the techniques described by Jensen and Anderson may be
sensitive to dierent iron pools, since there is a large increase in the haemosider-
in:ferritin ratio (5:1) in iron overloaded myocardium compared with the normal heart
(2:1).99 Another possibility is the eect of chelation, since the patients in Anderson's
study had generally been exposed to more chelation therapy than those studied by
Jensen et al. Since DFO removes hepatic iron eciently, this could result in the loss of an
association between liver and heart iron which existed before chelation. Ferrioxamine
may be measured using the T2* technique, since the patients were on DFO therapy.
Ferrioxamine has a strong MRI signal and indeed has been used as a contrast agent in
MRI imaging.68 Contrary to the misconceptions of some clinicians, DFO rapidly
penetrates heart tissue due to its positive charge. We have observed rapid ferrioxamine
formation within myocardium within 2±4 hours of a subcutaneous infusion of DFO at
40 mg/kg to rats ( J. B. Porter & T. Hair, unpublished results). However, once within
cells the positively charged iron-bound ferrioxamine will egress relatively slowly from
cells unless there is an active excretion pathway as is present in hepatocytes.3 Thus at the
end of a DFO infusion, ferrioxamine may remain in extra-hepatic tissues for hours or
even days after treatment has ceased, possibly contributing to the T2* signal. Although
this hypothesis requires systematic investigation, imaging of ferrioxamine solutions up
to 10 mM appears to give no signi®cant T2* signal (M. Westwood, pers. comm.). Another
factor that could aect the T2* signal is the presence of myocardial ®brosis. Whatever
the factors aecting the heart T2* signal, the ®ndings of Anderson et al also showed a
link between cardiac T2* and left ventricular ejection fraction (LVEF), suggesting that
whatever is being measured, it may be of prognostic relevance. All patients with
signi®cantly reduced ejection fractions had shortened T2* values (520 ms), although
the converse was not the case: i.e. the majority of patients with shortened cardiac T2*
had normal LVEFs (Table 1).64 Furthermore, preliminary ®ndings show that in
prospective studies with i.v. DFO106 and in retrospective studies with deferiprone107,
heart T2* improves. Prospective studies on the prognosis of patients with shortened
T2* in the heart would be wise before recommendations about changing chelation
therapy based on this measurement are made.
Monitoring iron in pituitary and brain

The anterior pituitary appears to be the most sensitive tissue to the early eects of
iron overload because hypogonadotrophic hypogonadism is the most important cause
of poor growth and sexual development in otherwise optimally transfused and
chelated patients.108,109 The eects of in®ltration of the anterior pituitary with iron
appear decades before complications with cardiac failure develop. A simple method of
quantifying early in®ltration of the anterior pituitary, possibly before biochemical
markers show evidence of dysfunction, would be of value in planning chelation
strategies.
Soon after the advent of MRI as a diagnostic modality in the 1980s, it was recognized
that some of the contrast found in brain imaging correlated with patterns of iron
deposition. In healthy individuals, iron concentration is highest in speci®c nuclei of the
basal ganglia and some associated structures, and increases with age until a relatively
constant level is reached at the age of 20±30 years.110 In genetic haemochromatosis,
MRI or CT imaging suggests increased iron deposition in the lentiform nucleus.111 In
transfusional iron overload, the brain is relatively spared of iron loading, the caudate
nucleus and anterior (not posterior) pituitary being exceptions.
Much of the interest in brain imaging for iron has focused not on transfusional
overload, but on conditions where abnormal iron distribution occurs in the brain with
neurodegenerative disorders such as Alzheimer's disease (AD) and Huntington's
disease (HD) and Parkinson's disease. Early studies used T2-weighted MRI to show
attenuated signals from the basal ganglia, such signal attenuation being more evident at
high magnetic ®eld strengths of 1.5 T, while the relaxation rate, 1/T2 was strongly
dependent on the applied static ®eld.112 T2 values in Parkinson's disease patients are
reduced in the substantia nigra, caudate nucleus and putamen, although there is
considerable overlap with control subjects.113 In both AD and HD, increases in basal
ganglia (caudate, putamen and globus pallidus), ®eld dependent relaxation rate increase
(FDRI) were seen compared to normal control groups, suggesting that basal ganglia
ferritin iron is increased.114 Thus interest in iron distribution in the brain in conditions
other than transfusional overload, has demonstrated that MRI is a potentially powerful
tool for monitoring the distribution and content of brain iron.
In thalassaemia patients, Metafratzi et al115 assessed the T(2) relaxation rate (1/T(2))
of the grey matter and found that 1/T(2) values for the cortex, putamen and caudate
nucleus were higher in patients compared with the controls. Chatterjee et al109
examined three dimensional T1-weighted volume scans to correlate the selective loss
of anterior pituitary volume by MRI with biochemical markers of anterior pituitary
dysfunction in seven male patients with thalassaemia major or intermedia. The loss of
T1-weighted signal appeared to be mainly secondary to the loss of pituitary volume.
There was a positive correlation between biochemical markers of hypothalamic±
pituitary insuciency and loss of pituitary volume. Berkovitch et al116 examined 33
patients above 15 years of age with beta-thalassaemia major and found that anterior
pituitary function (gonadotrophin releasing hormone (GnRH) stimulation test)
correlated well with MRI results. Argyropoulou et al117 examined the T2 relaxation
rate (1/T2) as a marker of pituitary siderosis in transfusional iron overload and found a
positive correlation between the 1/T2 value and the serum ferritin, with a higher 1/T2
value in patients with hypogonadotrophic hypogonadism compared with that of
patients without any pituitary dysfunction.
Thus, MRI appears to be a promising technique for assessing iron deposition in the
pituitary. A prospective study is needed, linking such measurements to the growth,
sexual development and fertility of patients treated with standard iron chelation
regimens as well as with newer chelation regimens. Such studies could allow a rational
basis for deciding when to commence or intensify chelation therapy, thereby
improving the outcome further.
Monitoring iron induced tissue damage to adjust chelation therapy

Iron induced toxicity will broadly parallel body iron content. However, the duration of
exposure to excess iron, its distribution within the body and previous chelation history
are also important, but incompletely understood, factors contributing to iron toxicity.
Markers of oxidative damage are elevated in iron overload conditions, but it has not
been established how to use these to modulate chelation therapy. Monitoring for
evidence of damage to target organs is, therefore, key to successful management,
irrespective of the perceived iron loading in the liver or elsewhere (Table 2).
Monitoring heart function

Monitoring of the heart in thalassaemia should ideally include a yearly full assessment
by a cardiologist with a special interest in the cardiomyopathy of iron overload. In
addition to clinical examination, echocardiography and electrocardiography118, 24 hour
Holter monitoring is of value in patients with suspected dysrhythmias, while exercise
ECG may unmask latent dysrhythmias.119 Atrial ®brillation often responds to
continuous DFO infusion: in a recent study this was reversed in 6 out of 6 patients
within 12 months and in one patient, cardioversion occurred within 5 days without
the need for conventional anti-arrhythmic drugs.43
Table 2. A scheme for monitoring iron induced tissue damage.
System Monitoring Frequency

Cardiac complications ECG + exercise Yearly
24 h tape More frequently if complications
Assessment of LVEF with Yearly
MUGA, ECHO or MRI More frequently if complications
Growth Height and weight Each clinic visit until growth ceases
Sitting and standing height 6 monthly
Bone Dexa scan for osteoporosis Yearly after 8 years
Bone age (X-ray, knees wrists) At start of chelation treatment & every 1±2
years until growth completed
Spine X-ray Every 2±3 years
Sexual development Tanner staging Yearly from 10 years
FSH, LH, oestradiol, testosterone Yearly
Diabetes Urine for glucose Each visit
GTT Yearly
Hypothyrodism T4, TSH 6 monthly
Hypoparathyoidism Ca, PO4 3 monthly
PTH and vitamin D low Ca
ECG, electrocardiogram/electrocardiography; LVEF, left ventricular ejection fraction; MUGA, multi-
gated acquisition scan; ECHO, echocardiography; MRI, magnetic resonance imaging; FSH, follicle
stimulating hormone; LH, luteinizing hormone; GTT, glucose tolerance test; TSH, thyroid stimulating
hormone; PTH, parathyroidf hormone.
Data gathered prior to the adoption of DFO regimens had shown that death
from heart failure was predicted by an abnormal LVEF on echocardiography in the
preceding 6±12 months.120,121 This suggested a rationale for regular cardiac
monitoring, provided eective improvements could be demonstrated with the
introduction of DFO. Longitudinal monitoring of ventricular function has often been
considered to be of limited clinical value because systolic dysfunction is a late event in
advanced disease.118,122,123 This view has been largely derived from early echocardio-
graphic studies performed on un-chelated or poorly chelated patients.120,121,124±127
Echocardiography does not lend itself very well to longitudinal analysis, because it is
dicult to obtain reproducible quantitative data that is free from observer variability.
Furthermore, the prognostic signi®cance of diastolic abnormalities detectable by
echocardiography, which appear early in the disease process, is unclear.128 However,
systolic abnormalities that are clearly demonstrable by echocardiography are seen
late120,121,126,129, often at a time when symptomatic congestive heart failure has already
developed.
Early publications disagree about the risk factors, signi®cance and most useful
measurements using echocardiography. Valdes-Cruz et al130 found that the left
ventricular posterior wall in children with thalassaemia major showed abnormalities,
even in patients without clinical symptoms. Kremastinos et al131 found that systolic and
diastolic left ventricular posterior wall behaviour were the same in patients with and
without congestive heart failure; changes in left ventricular dimensions, shortening of
the internal left ventricular diameter and the peak velocity of circumferential ®bre
shortening occurred only in patients with congestive heart failure. Senior et al132 found
no dierences in fractional shortening and ejection fraction but did ®nd an increase in
the early relaxation period in thalassaemics compared with controls. In a longitudinal
study, Lau et al127 found that the left ventricular fractional shortening and ejection
fraction were normal in those thalassaemics who survived but diminished in those who
died. Intragumtornchai et al133 found a signi®cant decrease in both ejection fraction
and ®bre fractional shortening in patients with transferrin saturation levels 4 80%, but
no dierence in the parameters between patients with serum ferritin levels above or
below 1500 mg/l. Hou et al134 found the presence of an abnormal diastolic total period
and the duration of the early diastolic ¯ow-velocity peak values correlated well with
prognosis.
Some authors have found that additional abnormalities can be detected earlier using
exercise testing or the infusion of the sympathomimetic b-1 agonist dobutamine.135±137
In our experience and that of other workers, poorer tolerance among thalassaemic
patients to dynamic exercise124,138, coupled with the wide overlap in LVEF responses to
dynamic exercise among patients with normal and diseased hearts139 limit the clinical
value of dynamic exercise tests. Stress testing in response to dobutamine has been
reported to produce better image quality and more reproducible LVEF results in
thalassaemia major patients undergoing radionuclide angiography.140
Radionuclide angiography using multi-gated acquisition (MUGA) has been used in
the management of thalassaemia major for over 20 years.43,135,141±144 The advantage of
MUGA scanning over M-mode echocardiography as a sensitive indicator of declining
ventricular function was ®rst shown by Engle et al126 who found ejection fraction
abnormalities before they were apparent on echocardiography and before cardiac
decompensation had occurred. While giving no useful information about diastolic
ventricular function or of right ventricular function, this approach gives highly
reproducible quantitative measurements of the LVEF both at rest and in response to
dynamic exercise.145,146 The excellent reproducibility of MUGA scanning occurs
because, unlike echocardiography, few assumptions about ventricular geometry are

necessary, thereby reducing operator-dependent variability and making the technique
particularly suitable for serial studies in the same patient.147 Studies have also shown
that abnormalities of systolic function (both on exercise and at rest) may precede
congestive heart failure. This allows timely intensi®cation of DFO, either intravenously
or with continuous subcutaneous infusion, often with sustained improvement in LVEF
values141,143,144 and good long-term survival.43
At UCLH we have found that sequential monitoring of cardiac function using MUGA
scans is a valuable tool for assessing cardiac risk and can be used for modulating chelation
therapy.51 Since 1981, yearly quantitative monitoring of LVEF using MUGA has been
used for monitoring and adjusting chelation therapy, having a better intra- and inter-
observer standard error (1.8 and 2%) than echocardiography. Our ®ndings show that a
fall in the LVEF of 4 10 percentage points or to an absolute value of 545% (the lower
reference limit at our centre) is associated with a signi®cantly increased risk of cardiac
death (P 5 0.01) unless treatment intensi®cation is successfully eected. These criteria
were used as a trigger for intensi®cation of DFO chelation either by improved dosing
and adherence to s.c. DFO or by instituting a 24-hour continuous i.v. regimen through a
Port-a-Cath. Survival was signi®cantly better in patients who complied with
intensi®cation (P 5 0.001) suggesting that longitudinal monitoring of LVEF is valuable.
All patients who complied with intensi®ed treatment lived, while all who failed to
comply died.
The recent development of cardiac MRI measurement has provided an opportunity
to quantify both right and left ventricular function at the same time.64,106 The ®ndings
show that decline in LVEF and RVEF broadly parallel each other in thalassaemia major
patients. In thalassaemia intermedia, pulmonary hypertension leads to a relatively
selective failure of right ventricular function.148 MRI has also allowed an investigation of
the relationships between ventricular function and changes in MRI signal, presumed to
be due to iron. This has recently been reported by Anderson et al64,10 whose ®ndings
suggested that patients with a right or left ventricular ejection fraction below control
values tended to have the lowest T2* values and, by implication, the highest
myocardial iron. All patients with resting LVEF values below the reference range had
T2* values less than 20 ms, although conversely most patients with T2* values
520 ms had normal LVEFs (see above). How this parameter (T2*) can best be used in
the monitoring and treatment of patients requires further prospective study but an
example is given in Figure 2 in a patient who developed biventricular failure and was
treated successfully with i.v. DFO. What is striking is that acute heart failure
developed despite an apparently normal liver iron and in the context of improving
heart T2*. It is also clear that the rate of improvement in liver T2* is more rapid than
that in the heart, with a predicted correction of heart T2* to 4 20 ms occurring 3
years after normalization of liver iron. This patient's T2* is improving linearly with s.c.
DFO therapy, and it is clear from the normal T2* values obtained in patients who
started on DFO late (in their second decade), that this parameter can be normalized
with conventional therapy if used in a sustained way over several years. However,
some patients at UCLH still have shortened T2* values despite sustained therapy over
several years. Two such patients with currently shortened T2* values despite normal
liver iron concentrations, have had episodes of an acute fall in LVEF approximately one
decade previously. It needs to be established how such patients dier from patients
with normal T2* values prognostically and with respect to other variables such as
human leukocyte antigen (HLA) type or the presence of myocardial ®brosis, possibly
following acute myocarditis. It is premature at this point, however, to conclude that a
Figure 2. continued over page.

B
After 2 Weeks I.V. DFO 50—80 mg/kg/day with monitoring
20
18
16
14
12
10
2
1 mg/g/dw
0
50 60 80
10
0
50 60 80
T2* = 6.5 ms LVEF = 48% Dose mg/kg
Figure 2. continued over page.
change of therapy is necessary in all such cases, until more is understood about what
such measurements mean. These issues will be clari®ed only with a carefully controlled
prospective evaluation of this technique.
Whether cardiac function is monitored by echocardiography, MUGA or by MRI, the
intention is to use the information gained to make decisions about the commencement,
continuation or intensi®cation of chelation therapy. The ®rst demonstration of a link
between chelation therapy and observations of cardiac function was the bene®cial eect
of s.c. DFO on cardiac function in small groups of thalassaemia patients with sub-clinical
and symptomatic heart disease.141±144 In a more recent study, 25 i.v. devices were
Rate of change of heart T2* with sc DFO 24h/day, 6 days/week

22
21
20
19 Possible threshold for increased risk of fall in LVEF
18 (Anderson et al, 2001 (106))
17
16
15
Heart T2* (ms)
14
13
12
11 y = 4.61 + 0.36x R^2 = 0.9
10
9
8
7
6
5
4
3
2
1
Predicted time to heart T2* >20ms
0
0 6 12 18 24 30 36 42 48
Time (months)
Figure 2. This illustrates the challenges of monitoring, using MRI and other novel approaches, for dose
adjustment in a patient who developed acute heart failure. (A) The patient presented in 1995 aged 26 from
another hospital with gross iron overload, a serum ferritin level of 4 8000 mg/l and diabetes. A regimen of
continuous s.c. desferrioxamine (DFO) was started using 3 g over 24 h 6/week with balloon pumps. By the
time of the ®rst MRI assessment in 9/99, the serum ferritin level had fallen to 4100 mg/l, the liver iron was
estimated at 12.5 mg/g dry weight using MRI and 10.1 mg/g dry weight using biopsy. Although the left
ventricular ejection fraction (LVEF) was satisfactory at 63%, the T2* in the heart showed marked shortening
(3.2 ms). Eighteen months later, the serum ferritin had fallen to below 1000 mg/l and the liver iron to
2.3 mg/g dry weight. The heart T2* had also improved but was still substantially below 20 ms. The patient
presented with acute biventricular failure 4 months (9/01) later despite the ®nding of further improvement
in the heart T2* (to 6.5 ms) and an estimated liver iron of only 1.7 mg/g dry weight. No de®nitive evidence
of myocarditis was found. (B) The patient was started on incremental doses of i.v. DFO continuously (50, 60
and 80 mg/kg), leading to an improvement in LVEF from 31% to 48% over a period of 2 weeks. No changes in
non-transferrin bound iron (NTBI) were seen with dose escalation but the ratio of free DFO to iron bound
ferrioxamine (FO) increased in the blood at doses above 60 mg/kg suggesting a possible maximum dose for
sustained treatment. The patient then returned to s.c. DFO at this dose and remained well with an LVEF of
50%, 5 months later, a liver iron of 0.6 mg/g dry weight (dw) and further improvements in heart T2* to
7.2 ms with a further improvement to 8.4 ms at 6 months. (C) The rate of change and estimated time for
return of heart T2* to 4 20 ms is shown. At the doses of DFO given of 50±60 mg/kg over 24 h 6 days/week,
the rate of improvement in heart T2* is linear at 4.32 ms/year, giving a time to achievement of a
T2* 4 20 ms of 42 months since regular compliance with the above regime began, assuming the relationship
remains linear. This is 2±3 times slower than the initial rate of change in liver T2*.
inserted over a 16 year period into 17 patients and DFO (50 mg per kg per day) was
infused continuously over 24 hours, 6±7 days a week.43 Resting LVEF improved
signi®cantly from 36% to 49% in 7 out of 9 patients with a previously documented
deterioration in LVEF, and it stabilized in the remaining 2 patients (P 0.002).
Improvements in LVEF shown using MUGA were documented within 6±12 months of
commencing treatment, and were often clinically apparent within days to weeks of
starting infusion. Improvement was sustained on long-term follow up. Survival
probability was 61% at 13 years in the group as a whole and 62% at 13 years in those
with demonstrable cardiac disease.
Recently there have been suggestions that genetic factors may contribute to cardiac
risk in thalassaemia, such as the apolipoprotein E epsilon4 allele.97 This allele of the
apolipoprotein E (APOE) is associated with decreased antioxidant activity and
therefore iron-loaded patients possessing this allele might be at more risk of oxidative
damage. Indeed it was found that thalassaemia patients with left ventricular failure had
a signi®cantly higher frequency of APOE 4 than controls. Thus the APOE 4 allele may
represent an important genetic risk factor for the development of organ damage in
homozygous beta-thalassaemia. Kremastinos et al98 have also suggested that certain
HLA types (e.g. HLA-DQA1*0501) are more common in those thalassaemia patients
who are most at increased risk of congestive heart failure (odds ratio 14). These
authors speculated that this, or other certain HLA types, might predispose to an
increased risk of myocarditis. Genetic polymorphisms could also aect iron uptake
into the heart and screening prospectively for these and other markers might identify
higher risk patients.
Importantly it is now clear that although iron chelation with DFO leads to rapid
improvement in LVEF and in arrythmias, in the long term cardiac performance can
also be improved by decreasing body iron levels, without the need for direct chelation
from the heart. Thus, in thalassaemia major patients post-transplant, phlebotomy alone
was sucient to improve cardiac function as monitored by sequential echocardio-
graphy.149 Phlebotomy led to a normalization of indices of contractility and diastolic
function without the need for chelation. This is a very important observation because
it suggests that it is not necessary to remove iron directly from the heart to ultimately
normalize cardiac function and that normalization of body iron (leading possibly to
iron re-distribution) is sucient. This means that iron chelators may not need to access
intra-myocyte pools directly to normalize cardiac function in the long term. In the
acute situation, however, the continual removal of toxic intra-cellular iron species
appears to be critical in the initial reversal of deranged cardiac function.43
Monitoring endocrine function

Monitoring of the hypothalamic±pituitary±gonadal axis. The anterior pituitary is
probably the most sensitive tissue to iron overload, which results in poor growth,
poor sexual maturation and contributes to osteoporosis. The value of regular monitoring
for disorders of glucose tolerance, calcium metabolism and thyroid function is well
recognized.
The eects of iron overload on the hypothalamic±pituitary±gonadal (HPG) axis are
well recognized, resulting in hypogonadotrophic hypogonadism108,150±152, defects of
growth hormone secretion153 and reduced responsiveness of growth hormone to its
release hormone.154 Defects of the growth hormone receptor155±157 and de®ciency
of insulin-like growth factor158,159 have also been described. Iron deposition in the
anterior pituitary is causative to hypogonadotrophic hypogonadism as shown
histologically by its selective iron deposition in pituitary gonadotropes160 and by the
®nding of loss of anterior pituitary volume in iron overloaded thalassaemia patients on
MRI scanning.109 The precise level of body iron associated with pituitary iron loading is
not known, but a signi®cant correlation does exist between moderate-to-severe iron
deposition in the anterior pituitary and the risk of anterior pituitary failure.161 Primary
gonadal failure appears to be less important than defective function of the
hypothalamic±pituitary axis, although iron deposition in the testes and ovaries may
play a role in some cases of hypogonadism.162 Clinically, these HPG axis abnormalities
present as a failure of growth and sexual development or as premature secondary

failure of fertility.
With DFO chelation, growth failure and hypogonadotrophic hypogonadism can be
prevented in the majority of patients, as ®rst described in a group of thalassaemia
patients who had been optimally chelated with DFO since mid-childhood.163 Studies
in Italy show a somewhat less good outcome: arrested puberty was found in 16% of
males and 13% of females treated with standard DFO regimens; secondary
amenorrhoea was found in 23% and oligomenorrhoea or irregular menstrual cycles
in a further 16%.164 In a UK study, secondary amenorrhoea occurred in 25% of
thalassaemic women with previously normal pituitary function108, despite apparently
adequate chelation therapy. Fertility in both men and women with thalassaemia has
improved88,164,165 as a result of improved chelation therapy, but there are, as yet, no
reports of reversal of established impotence, infertility, low testosterone levels or
oligospermia by DFO therapy.
Monitoring growth and sexual development requires great skill because both
insucient chelation and excessive DFO chelation can result in poor growth. Other
factors such as anaemia in inadequately transfused patients further complicate the
picture. In children and adolescents who are not fully-grown, growth charts including
measurement of both sitting and standing height are essential. Formal assessment
of bone age to determine whether this has fallen in excess of 2 years behind
chronological age is necessary as well as a regular review of pubertal status,
development and fertility by an interested and experienced endocrinologist. Markers
of the integrity of the HPG axis in common clinical use include plasma levels of
luteinizing hormone, follicle stimulating hormone, testosterone, oestradiol and sex
hormone binding globulin (SHBG). Loss of pulsatility of GnRH release is an early and
sensitive indicator of HPG dysfunction, but is not practical in routine clinical
practice.108
Monitoring for diabetes mellitus. Diabetes mellitus is a relatively common complication

in poorly chelated patients with thalassaemia with a frequency of 4±6% in patients on
current standard therapy with transfusion and DFO.164 Iron-induced damage to the
islet cells is the major factor, as shown by the early and progressive loss of pancreatic
b-cell mass166,167 and the association between severity and duration of iron overload
and the development of diabetes.168 Standard s.c. DFO therapy reduces the risk of
diabetes mellitus and glucose intolerance in compliant patients.83,85,169 Furthermore,
some improvements in glucose tolerance with a decreased need for oral
hypoglycaemic agents have been reported following intensive DFO.170 In addition to
random testing for urine sugar in the clinic, regular monitoring of the glucose
tolerance test (at least yearly) is recommended so that impaired glucose tolerance can
be detected early and dietary advice plus intensi®cation of chelation given where
appropriate.
Monitoring thyroid and parathyroid function. Mild to fully developed clinical hypothyr-
oidism is well recognized in thalassaemia major.171±176 Persistently high serum ferritin
levels correlate with the presence of thyroid dysfunction165, so that patients with high
levels of iron loading should be monitored for hypothyroidism more often.
Improvement in thyroid function after intensive DFO therapy has been reported150,172
but not after frank hypothyroidism has developed.85 Clinical and sub-clinical
hypoparathyroidism are also well documented in thalassaemia patients176±181 and
require monitoring with measurement of serum calcium and phosphate, and
parathormone where appropriate. There have been no reports of reversal of

parathyroid and adrenal dysfunction with DFO. These complications are all preventable
in patients managing at least 260 infusions/year of s.c. DFO.85
Plasma markers of oxidative damage

While it is clear that iron overload induces oxidative damage in a number of tissues,
and there is good evidence in both animal and clinical studies for such mechanisms, it is
currently unclear how to apply these various markers to treatment adjustment or
how they could be useful prognostically. Nevertheless a number of markers are of
interest and merit investigation as to how chelation therapy aects them and what
prognostic value they have.
Regarding plasma markers, there is evidence for increased conjugated dienes and
malondialdehyde (MDA) in patients with beta-thalassaemia, being approximately twice
the levels of healthy controls and correlating with serum ferritin values.182 Modi®ed
low-density lipoproteins (LDLs: conjugated dienes) have been found to be three times
those of controls in thalassaemia intermedia.183 Protein carbonyls, oxidatively modi®ed
serum proteins, are increased twofold in HH patients compared with controls184 and
levels normalize with phlebotomy. In the plasma, there is also evidence of antioxidant
depletion in iron overload conditions. Both water-soluble antioxidants (vitamin C) and
lipophilic antioxidants such as vitamin E185,186 are depleted. Levels of vitamin E appear
to be inversely related to serum ferritin182 and non-transferrin bound iron (NTBI).187
We are conducting a prospective study that aims to identify a link between markers of
oxidative damage and the presence of NTBI in plasma.
These plasma markers of oxidative stress are supported by links of liver damage with
oxidative stress. In thalassaemia major, transaminases correlate inversely with vitamin E
and lycopene.182 ApoE 4 polymorphism, which is thought to lead to decreased
antioxidant capacity, may be associated with an increased risk of ventricular failure in
thalassaemia major.97
While the above observations show clearly a link between oxidative stress and iron
overload, it is not yet clear how these can, or might, be used in the monitoring of iron
overloaded patients. Neither is it clear whether supplementing the diets of patients
who are depleted of antioxidants is bene®cial. A cautionary note, derived from
experiences with vitamin C acting as a pro-oxidant when given in excess, is
sounded.188
Monitoring compliance with chelation therapy

The single most important prognostic determinant in thalassaemia major is the
frequency of use of DFO.83,85 The younger the age at which cohorts of patients begin
eective chelation with DFO, the better the prognosis (Figure 1).5,85,88 This means an
infusion frequency of not less than 5 times/week (Table 1). This may not be achieved if
the patient cannot aord or gain access to DFO or pumps or because of diculties
with taking DFO, either from practical issues such as allergy or from poor motivation.
The latter is often referred to as poor compliance (adherence) to the recommended
therapy. Poor motivation may result from external issues such as inadequate support
from family, friends or from the lack a dedicated thalassaemia centre.189 Internal issues
such as poor self esteem and adverse life events such as a bereavement, divorce, illness,
unemployment may impact adversely on a patient who has been hitherto compliant
with treatment.
One of the principal functions of a thalassaemia unit is to identify when patients are
beginning to cope badly with treatment or other aspects of life. When setting up a
programme for the care of such patients, the provision of a nurse specialist, doctor and
psychologist who have the time to listen to patients, identify those who are complying
poorly with treatment and intervene, if necessary, is critical.
Poor compliance is hard to identify early in a patient who is in denial about the
consequences of poor compliance or who wishes to mislead clinicians about chelation
use. One approach is to give patients a calendar, in which each infusion is noted during
treatment. Another scheme that has been used is for patients to return used vials
and for these to be counted at the clinic. Some infusion pumps have a tamper-free
electronic recorder. In the long term a clear trend of increasing ferritin or liver iron
will identify patients who are not complying with the treatment protocol.
An experienced member of the thalassaemia healthcare team will also be able to
identify and intervene with practical issues that can adversely aect compliance. Local
reactions at the site of DFO infusion are quite common with mild to moderate pain,
skin itching, erythema and induration. Persistent local reactions may be reduced by
varying the site of injection, by lowering the infusate concentration to 410%, or by
adding 5±10 mg of hydrocortisone to the infusion mixture in severe cases. The choice
of needles is also important to compliance: many patients prefer ®ne gauge needles
that are inserted vertically and have self adhesive tape (such as `Thallaset' needles), to
the traditional 25 gauge butter¯y needles that are inserted at about 458 to the skin.
The siting of the needle insertion is also important; the optimal position is generally in
the abdomen, although the deltoid and later the thigh can be used if patients are
shown how to avoid important vessels, nerves or organs. A variety of infusion devices
are now available (e.g. Cronoject) that are smaller, lighter and quieter than the
traditional Graseby pump. Pre®lled disposable balloon pumps encourage compliance
because the DFO does not have to be prepared by the patient.190
DFO is a particularly onerous form of chelation and treatment with a tablet clearly
has advantages for compliance. In a study with deferiprone using medication event
monitoring system (MEMS) devices to monitor when the container was accessed,
compliance with the thrice daily regimen was 80%. It is anticipated that compliance
with an orally active chelator that is taken only once daily, such as ICL67033, should be
better than this.
Monitoring to minimize chelator induced toxicity

Toxicity from iron chelation may result from the eects of the iron free ligand or from
the iron complexes of that ligand. There is inevitably a balance that must be struck
between maximizing the eects of iron excretion with a chelator regimen and
inducing toxicity from chelation of iron or other metals from pools that are key to
physiological requirements. The structure and properties of a chelator will impact
critically on such eects (see Chapter 6).
As iron loading falls with chelation treatment, the risk of toxicity from excess free
ligand is likely to increase unless the dose is reduced. While it is clear that dose
reduction decreases many of the toxicities seen with DFO, such as eects on growth
and audiometric and retinographic changes52,191,192, such a relationship has not been
demonstrated between the risk of agranulocytosis and dose of deferiprone193, except
in laboratory animals.194 Deferiprone and other chelators interact with key metal-
loenzymes and can deplete critical intracellular iron and zinc pools, thereby inducing
apoptosis.195 Recently, hydroxypyridinones designed to interact at a reduced rate with
enzymes have been shown to have decreased anti-proliferative actions and eects on
apoptosis, while maintaining their ability to mobilize intracellular iron.196
The great stability of the hexadentate DFO means that iron redistribution is
prevented and toxicity resulting from incomplete complexation of iron (III) is very
unlikely. By contrast, bidentate chelators such as deferiprone can dissociate and form
incomplete complexes with iron more easily. This is likely to explain the higher joint
toxicity of this drug in more iron overloaded patients197 and may be the mechanism of
DNA damage found using in vitro models.198
DFO toxicity
For a drug taken in gram quantities daily throughout life, DFO has a remarkably low
toxicity and the key risk factors are now largely known: namely, high doses, low
degrees of iron loading and young age. By adjusting the dose for age and degree of iron
overload, toxicity can be minimized. However, this does not exclude the need for
regular and careful monitoring.
Recommended monitoring includes assessment of growth, bone development,
audiometric and retinal function (Table 3).7 Dosing can be reduced as ferritin falls by
reference to the therapeutic index (mean daily dose divided by the serum ferritin).52
However, in sickle disorders or when tissue in¯ammation falsely increases serum
ferritin, this index is not recommended. The maximum recommended dose should not
exceed 40 mg/kg before growth ceases and even lower doses are advisable below 3 years.
Eye toxicity. Retinal toxicity was ®rst described in patients receiving very high doses
(4120 mg per kg per day) with i.v. therapy.199 Features include one or more of the
following: night blindness, annular ®eld loss with defects of dark adaptation, loss of
central acuity and colour vision, central scotomata, electro-oculographic (EOG) and
electroretinographic (ERG) changes. EOG, ERG and visually evoked response
abnormalities are characteristic of degeneration aecting the cones. Retinal pigmenta-
tion may also be found, making the combined features indistinguishable from those of
retinitis pigmentosa.
Table 3. Monitoring for desferrioxamine tolerability.
Eect Monitoring Frequency

Local skin reactions History & examination Clinic visits
Ototoxicty Audiometry Yearly unless symptomatic
Therapeutic ratio 3 monthly with ferritin results
Retinal toxicity Clinical History Clinic visits
Fundoscopy Yearly
Electro-retinography Yearly if available especially important if:
high dose
diabetic
Growth Height (sitting/standing) see table 2
Dose/therapeutic index 3 monthly
Bone X-ray, knees, wrists, spine see table 2
Sitting standing height
Yersinia infection Stool/blood culture, serology Clinical suspicion
It is rare to detect ocular toxicity at standard doses given as 8±12 hour infusions. It
is not clear whether the risk increases with continuous 24 hour treatment. In a review
of 17 cases given moderate to high dose continuous i.v. treatment we found only one
patient developed eye toxicity, when the therapeutic index was brie¯y exceeded.43
Full recovery was observed on resumption at a lower dose. This patient also had
diabetes, which was identi®ed as another possible risk factor by Arden et al200, possibly
because this is associated with increased permeability of the blood±retinal barrier and
increased monitoring is recommended in such patients. Non-iron overloaded patients
are particularly vulnerable to ocular toxicity, which has been reported in renal dialysis
patients after the administration of single doses (50 or 100 mg/kg i.v.) for the
treatment of aluminium overload.201±203 Visual disturbances have also been reported in
patients with rheumatoid arthritis and related disorders after only a few doses of
50 mg per kg per day.204,205
If patients develop visual symptoms, treatment must stop and ERG and EOG
assessment should be performed. Provided treatment is stopped early, full recovery is
possible in as little as 4 weeks.192 If time is allowed for recovery of symptoms, and
normalization of electro-retinography, then treatment can usually be started cautiously
at a lower dose. Cataracts may occur206 and may be dose-related, being reported in
patients receiving very high doses (4200 mg per kg per day).53,199 Complete resolution
of the cataracts usually occurs on stopping DFO, which may then be safely resumed at a
lower dose. When monitoring is ®rst performed, it is worth remembering that sub-
capsular punctiform lens opacities may result from the iron overload per se.207
Ototoxicity. DFO-induced ototoxicity is typically a bilateral, symmetrical, high

frequency, sensorineural hearing loss that may or may not be symptomatic and may
or may not be accompanied by tinnitus.52,192,206±212 The onset is usually insidious,
though occasionally acute.192 Rarely, a vestibular component may be present.52
Complete or partial recovery of hearing may occur on discontinuation or reduction
of dosage of DFO52,192, but stabilization of the hearing loss on reduced doses of DFO is
the more usual ®nding. Some patients treated with high doses in the late 1970s and
early 1980s have a de®cit that is severe enough to require the use of hearing aids.
Cases as severe as this should now be avoidable with careful monitoring and dose
adjustment. The risk of ototoxicity can be reduced by the use of the therapeutic index:
Therapeutic index mean daily dose mg=kg=current serum ferritin mg=l
Although it has been applied to prevent other potentially toxic DFO eects, it was
®rst elaborated for ototoxicity52, which was not seen in patients in whom this ratio
remained below 0.025 at all times.
Audiometric studies are much more sensitive than auditory brain-stem responses in
diagnosing ototoxicity.192 The frequency of monitoring for audiometric toxicity will
depend on the doses being used, the age of the patient and the feasibility of the tests
being performed. In general it is advisable to check audiometry yearly in all children
on regular DFO, in all patients where iron loading and serum ferritin values are low or
in patients given high intensity treatment. In patients with previously identi®ed
audiometric disturbance, more frequent testing may be advisable. The sensorineural
de®cit typical of DFO toxicity must be distinguished from conductive hearing loss,
which has a higher incidence in this group of patients compared with the general
population.52
Growth and skeletal complications. Maximizing growth in thalassaemia major perhaps

requires more skill and experience than any other aspect of monitoring. This is
because growth retardation may result both from iron overload in the anterior
pituitary leading to hypogonadotrophic hypogonadism (see above) and from excess
dosing with DFO. The objective is to allow the maximum proportion of patients to
attain their full height potential, enter puberty and attain full sexual maturation. This
requires a chelation regimen that minimizes iron loading in the anterior pituitary. It is
not clear from present evidence whether the quality of chelation treatment is the key
factor in determining the degree of hypogonadotrophic hypogonadism or whether
other factors such as transfusion history or as yet unidenti®ed genetic polymorphisms
are more important. Risk factors for DFO induced growth retardation include: a
young age of starting treatment (5 3 yrs)213,214, doses 435 mg/kg in very young
children38 and doses in excess of 40 mg per kg per day before growth has ceased.191,215
Growth velocity resumes rapidly when the dose is reduced to 540 mg per kg per day
and does not respond to hormonal treatment. The retardant eect of DFO on growth
results in disproportionate truncal shortening and loss of sitting height191,216, which is
probably due to the eect of DFO on spinal cartilage.38,217,218 However, it is not
certain how much this disproportion is due to DFO: in a recent multicentre study in
thalassaemic patients aged 3±36 years where about 18% exhibited short stature with
disproportion between the upper and lower body segments in 14%, the authors
concluded that this was a function of the disease itself rather than its treatment.219
Clinically evident bony lesions, typical of DFO over-treatment, are seen in only about
3% of patients.220 Bony histological changes (such as abnormal chondrocytes, alteration
of cartilage staining pattern, irregular columnar cartilage and lacunae in the
cartilaginous tissue) are seen in all thalassaemic patients irrespective of the presence
of clinically evident changes.220
Skeletal changes include rickets-like bony lesions and genu valgum in association
with metaphyseal changes, particularly in the vertebrae, giving a disproportionately
short trunk.85,214,215 Vertebral growth retardation or milder changes involving
vertebral demineralization and ¯atness of vertebral bodies have also been noted in
well-chelated patients.85 Other radiographic features include rickets and/or scurvy-like
lesions of the long bones221, circumferential metaphyseal osseous defects, sharp zones
of provisional calci®cation and widened growth plates.213 Marked abnormalities of the
metaphyseal growth plate may occur in the distal ulnar, radial and tibial metaphyses214
with platyspondyly of the vertebral bodies.221
In order to balance the risks of DFO-related skeletal toxicity against those of iron
overload, it is usual to commence chelation treatment at about 3 years of age (not
before) and to restrict the dose of DFO to a maximum of 35 mg per kg per day in
children under 5 years and 40 mg/kg thereafter until growth is complete. It is
advisable to monitor height velocity as well as sitting and standing height twice yearly
and adjust dosing as necessary. Growing patients should have annual radiological
assessment of the thoracolumbar-sacral spine as well as the forearm and knees, and the
dose of DFO should be reduced if signi®cant changes are noted since they are
irreversible.222 Until the relative roles of the disease itself and DFO are better
understood, careful monitoring of height with downward dose adjustment in those
with changes in height velocity or truncal height is the most practical approach.
Osteoporosis is increasingly recognized in up to 70% of adult patients. The major
cause of osteoporosis in thalassaemia major is hypogonadism. A possible link between
DFO metabolism and osteoporosis was suggested by pilot studies by our group223 but
a subsequent study showed no intrinsic dierence in DFO metabolism between
patients with and without osteoporosis.224 It must also be appreciated that backache is
not always caused by osteoporosis in these patients: intervertebral disc degeneration
being a common cause (pers. obs.).
Monitoring for other toxicities. Systematic monitoring for other toxicities is not usually
necessary, since they are very rare at recommended doses. Knowledge of their
existence is necessary, however, so that they can be recognized if they occur. Local
reactions, with skin reddening and soreness at the site of subcutaneous infusions are
often caused by solutions of DFO above the recommended concentration of 10%. If
dilution of DFO fails to improve skin reactions, small doses of hydrocortisone
(5±10 mg) mixed in with the DFO solution may be eective. Absorption of DFO from
the infusion site may be impaired due to subdermal ®brosis, giving painful lumps,
which may last for several days. Rotating the site of infusion is usually eective in
preventing this type of reaction. Rarely, systemic reactions, with fever, muscle aches
and arthralgia occur and anaphylaxis may occasionally be seen. Desensitization
regimens have been described225,226, which are usually successful, but may need to be
repeated. Occasionally, severe local skin reactions at the site of subcutaneous DFO
infusion can respond to such a desenstitization regimen (our unpublished results).
Rare events include: renal impairment in occasional patients given high doses227 and
reversible changes in the glomerular ®ltration rate (GFR) at lower doses228; a fatal
respiratory syndrome229 and pulmonary in®ltrates230 at very high doses (10±20 mg per
kg per h). Recently, sensorimotor toxicity in two patients with b-thalassaemia on high
dose (120 mg per kg per day) i.v. DFO has been reported231, which resolved on
discontinuation of DFO. In non iron-overloaded patients, DFO may potentiate the
action of the phenothiazine derivative prochlorperazine, leading to reversible coma in
two patients.204 Bolus i.v. doses may lead to nausea, vomiting, hypotension with acute
collapse or even transient aphasia.232 Thrombocytopenia has been reported in two
patients on renal dialysis.233
There is an increased risk of Yersinia infection in iron overloaded patients and this
risk increases further with DFO treatment since Yersinia does not make a natural
siderophore and uses iron from ferrioxamine to facilitate its growth.234 Patients
presenting with diarrhoea, abdominal pain or fever should stop DFO until Yersinia
infection can be reasonably excluded by stool samples, blood cultures and serological
testing. If Yersinia is identi®ed as the cause of the fever, DFO can generally be restarted
once the symptoms have settled with antibiotic treatment: the current treatment of
choice is cipro¯oxacin. The growth of other organisms (e.g. Klebsiella)235,236 may also
be facilitated by ferrioxamine and it is wise to withhold DFO in a febrile patient until
the source of the fever has been identi®ed and treated if necessary.
Minimizing DFO toxicity: conclusions. Overall, DFO toxicity is unlikely at the doses
currently recommended (30±50 mg/kg, 5±7 days a week). The toxic eects of excess
DFO dosing can be minimized by limiting the maximum dose before growth has
ceased to 5 40 mg/kg7,39,85 and giving even smaller doses in very young children,
particularly if DFO is started below the age of 3 years.39 Downward dosing adjustment
is necessary as iron loading falls particularly when serum ferritin levels are below
1000 mg/l. By taking measurements of ferritin every 1±2 months, a reduction of the
mean daily dose to achieve a therapeutic index of 5 0.025 reduces the risk of DFO
toxicity in thalassaemia major.52 This index is not applicable in sickle cell disease or in
other conditions where the ferritin may be falsely elevated. As discussed above, dose
adjustment based on liver iron has theoretical advantages over the therapeutic index
using ferritin; however, there are no studies showing what levels or ratios of DFO to
liver iron are optimal.
A scheme for monitoring is shown in Table 3. Patients with additional risk factors,
such as diabetes200 should be monitored more closely.
In pregnancy, DFO is not recommended by the manufacturers but there have been
over 40 pregnancies reported where DFO was used at various stages without
teratogenic eects.237 It is our practice to give low dose DFO (approximately 30 mg/kg)
in the ®nal trimester to mothers who have a perceived high cardiac risk and to most
mothers in the 24 hours prior to delivery and continued in the peri-partum period.
Since DFO is not absorbed in the gut and concentrations in breast milk are small, breast
feeding should not be contra-indicated.
Deferiprone toxicity
Reviews of deferiprone pharmacology, ecacy and use have been published recently2,3
and are summarized in Chapter 6. The relationship between dose and the toxicity of
deferiprone has not been established in humans, making recommendations about dose
adjustment as a result of monitoring speculative. The drug is less eective at removing
liver iron and hence total body iron than desferrioxamine238,239 at doses of 75 mg per
kg per day, the only dose at which the safety pro®le has been formally tested.240 This
drug is licensed in Europe as second line therapy for those patients who cannot take
DFO but is not licensed in the USA. One problem in monitoring for toxicity is that,
unlike desferrioxamine, it is not known whether the most serious toxicity of this drug,
agranulocytosis which occurs in between 0.6 and 4% of patients, is related to the dose
given. The relationship of other toxicities to dose is also unclear: arthralgia and
arthritis risk appears highest in the most iron overloaded patients, but it is unclear
how dose increments might aect other toxicities.
Neutropenia and agranulocytosis. Agranulocytosis (absolute neutrophil count

50.5 109/L) is the most serious unwanted eect of deferiprone. Initially this was
found in 3±4% of patients lasting from 4±124 days240,241, with mild neutropenia being
found in an additional 4%.242,243 More recently, a prospective multicentre safety trial in
187 patients at a dose of 75 mg/kg in three divided doses sponsored by the manufacturers
Apotex, showed that agranulocytosis occurred in only 0.6%240 provided the blood count
was monitored weekly and treatment stopped at the ®rst signs of neutropenia.
If neutropenia is found in a patient receiving deferiprone, it is dicult to know
whether this is due to inherent ¯uctuation in the neutrophil count (which is more
likely in unsplenectomized patients) or is the ®rst sign of deferiprone-induced
agranulocytosis. Weekly monitoring of the blood count is necessary to minimize the
risk from serious neutropenia, so that the drug can be stopped rapidly at the ®rst signs
of neutropenia. It is not clear whether increasing the dose given will increase the risk
of agranulocytosis since the mechanism by which it occurs in humans is not known.
Agranulocytosis is associated with bone marrow hypoplasia possibly through a
cytotoxic mechanism.192,241,244,245 It is, therefore, unclear whether increasing the dose
above 75 mg/kg in non-responders, as has been recommended by some investi-
gators246, is a safe approach. Growth factors have been used in some cases of severe or
protracted neutropenia but their value is uncertain. Reintroduction of deferiprone
leads to a rapid fall in the neutrophil count in some patients and, therefore, is
contra-indicated. Avoidance of deferiprone in patients in whom stem cell or
progenitor function is compromised would also seem advisable, such as those with
Diamond±Blackfan anaemia.241
Monitoring for arthropathy. Painful swelling of the joints, particularly the knees has
been reported in 6±39% of patients.197,240,242 Arthropathy seems most common with
high degrees of iron overload197,240, suggesting that solubilized iron complexes in the
joints might be responsible for the problems. Arthritis usually, but not always, resolves
after stopping therapy but often recurs on reintroduction of treatment.
Monitoring for other unwanted eects. Zinc de®ciency occurs in about 14% of
patients243 and one study has suggested that diabetic patients are particularly at risk.247
Serum zinc can be unreliable as a monitor of zinc de®ciency since it can ¯uctuate
depending on recent food ingestion and white cell zinc measurement may be a more
reliable method of screening.
Isolated cases of disturbances of immune function such as fatal systemic lupus,
increased antinuclear antibodies and rheumatoid factors248 and a fatal varicella
infection249 have been reported, but have not been found in longitudinal studies.38,240
Progression of audiometric disturbances was reported in 5 out of 9 patients switched
from desferrioxamine to deferiprone, but in 7 patients without audiometric
disturbances prior to switching no new abnormalities occurred.250 A variety of other
unwanted eects include nausea (8%) and ¯uctuation in liver function tests (44%).243
Treatment was discontinued in 13±30% of patients in various studies.239,242,243
Whether ¯uctuations in liver function tests re¯ect any signi®cant underlying liver
disease has been the subject of some controversy. Liver ®brosis was reported to
progress in 5 out 12 patients on deferiprone with a median time to progression of 3.2
Practice points
. serum ferritin is of limited value if used as a stand-alone marker for monitoring
iron overload
. in patients treated with desferrioxamine (DFO), a serum ferritin level
consistently above 2500 mg/l identi®es patients at highest risk of cardiac disease in
thalassaemia
. measurement of liver iron predicts total body iron as well as patients at risk of
iron induced heart disease in thalassaemia and should be performed yearly
. liver iron values 415 mg/g dry weight identify patients at greatest risk of heart
disease
. liver iron values below 7 mg/g dry weight should be aimed for
. sequential quantitative monitoring of heart function (e.g. using MUGA ) identi®es
patients at risk of heart disease and who require intensi®cation of DFO therapy
. compliance with DFO treatment is perhaps the strongest indicator of prognosis
and this variable requires careful monitoring by doctors with expertise in this
area
. the prognostic value of serum ferritin and liver iron measurement has only been
demonstrated for patients receiving DFO treatment
. the impact of deferiprone on prognosis is not known and the use of markers is
also unknown
Research agenda
. prospective studies of the prognostic signi®cance of T2* or other indirect
measurements of heart iron are required before recommendations about how
best to use this measurement in modulating chelation therapy can be made
. studies of the signi®cance of markers of iron overload are required for chelators
other than DFO, such as deferiprone, before recommendations can be made
about safe levels of ferritin, liver and body iron
. prospective studies on the eect of increasing the dose of deferiprone (above
75 mg per kg per day) on ecacy (liver iron concentration) and toxicity
(frequency of agranulocytosis) are required
. a prospective study comparing liver ®brosis over a period of 5±10 years in patients
treated with DFO or deferiprone, in patients both with and without evidence of
persistent hepatitis C infection would clarify uncertainties about the long term
use of deferiprone
years and in none of 12 age-matched control subjects receiving regular desferriox-

amine.238 Four out of ®ve patients were seropositive for hepatitis C but the frequency of
antibody to hepatitis C was the same (6/14) in the deferiprone-treated group and the
desferrioxamine-treated group (5/12) where no progression was observed. There are, as
yet, no prospective studies comparing pre-treatment and post-treatment ®brosis scores
in hepatitis C positive and negative patients, which address this issue. Retrospective
analysis of liver biopsies taken after at least 2 years treatment but without baseline
biopsies has been interpreted by the investigators as suggesting a lack of deferiprone
eect on ®brosis: liver biopsies taken at 2±4 years of drug exposure showed no ®brosis in
11/12 patients and mild ®brosis in 1/12 patients who were hepatitis C RNA negative,
whereas cirrhosis was present in 5/5 patients who were hepatitis C RNA positive.239 No
baseline biopsies and no DFO control group were included. Another study found
hepatic ®brosis in 7 out of 11 patients on long-term deferiprone, which was greater in
the hepatitis C positive than the hepatitis C negative group.251 There have been a large
number of abstracts reporting a lack of progression of ®brosis, the most recent being a
3.5 year follow up in 56 patients.252 This issue will only be resolved if prospective studies
are carried out. In the meantime it would seem wise to undertake baseline and follow
up liver biopsies where possible on both hepatitis C positive and negative patients.
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Best Practice & Research Clinical Haematology

Vol. 15, No. 2, pp. 329±368, 2002

Monitoring chelation therapy to achieve

John B. Porter MA, MD, FRCP, FRCPath

Bernard A. Davis MRCP, MRCPath

Key words: transfusional iron overload; chelation therapy; desferrioxamine; deferiprone;

All correspondence to Professor J. B. Porter. E-mail: j.porter@ucl.ac.uk

GOALS AND OPTIMAL OUTCOMES WITH CHELATION AND

MONITORING OF IRON OVERLOAD AND ITS TREATMENT

Monitoring is important in deciding when treatment should be started, how it should

Estimation of iron balance

Iron content of transfusion mg  volume ml haematocrit 1:16

In splenectomized patients with thalassaemia major, maintaining a mean haemo-

Rate of iron excretion by chelators

Monitoring iron loading to adjust chelation therapy

Liver iron concentration by biopsy

Table 1. Possible prognostic indicators to monitor in iron overload.

Measurement Finding Outcome Reference

Liver iron by superconducting quantum interface device

non-haemosiderin containing tissues. The patients are lowered on to a wooden bed at

Liver iron by magnetic resonance imaging

Use of liver iron in monitoring of chelation treatment

Monitoring myocardial iron

given and the aminotransferase serum concentration. In another study, regression

Monitoring iron in pituitary and brain

Monitoring iron induced tissue damage to adjust chelation therapy

Monitoring heart function

Table 2. A scheme for monitoring iron induced tissue damage.

System Monitoring Frequency

because, unlike echocardiography, few assumptions about ventricular geometry are

Figure 2. continued over page.

Figure 2. continued over page.

Rate of change of heart T2* with sc DFO 24h/day, 6 days/week

Monitoring endocrine function

present as a failure of growth and sexual development or as premature secondary

Monitoring for diabetes mellitus. Diabetes mellitus is a relatively common complication

parathormone where appropriate. There have been no reports of reversal of

Plasma markers of oxidative damage

Monitoring compliance with chelation therapy

Monitoring to minimize chelator induced toxicity

Table 3. Monitoring for desferrioxamine tolerability.

Eect Monitoring Frequency

Ototoxicity. DFO-induced ototoxicity is typically a bilateral, symmetrical, high

Therapeutic index  mean daily dose mg=kg=current serum ferritin mg=l

Growth and skeletal complications. Maximizing growth in thalassaemia major perhaps

Neutropenia and agranulocytosis. Agranulocytosis (absolute neutrophil count

years and in none of 12 age-matched control subjects receiving regular desferriox-

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Iron content of transfusion mg volume ml haematocrit 1:16

Eect Monitoring Frequency

Therapeutic index mean daily dose mg=kg=current serum ferritin mg=l