High-Tc Susceptometer Project
The goal of this inter-disciplinary project is to design, build, and clinically validate, a
high-Tc
magnetic susceptometer to measure iron in the human liver. The program, which
started in October 2001, is supported by a five-year Bioengineering Consortium
grant from the NIH, with our consortium collaborators located at
Los Alamos,
Tristan Inc., and Columbia
University. This document provides a brief description of the medical and
instrumental aspects of the project.
Medical
Physicians
have a pressing clinical need for a quantitative means of measuring body storage
iron that is accurate, safe, non-invasive and readily available to improve the
diagnosis and management of patients with iron overload, including hereditary
hemochromatosis, thalassemia major, sickle cell disease, aplastic anemia,
myelodysplasia and other disorders. Three
recent developments have added urgency to the medical requirement for a
quantitative means of assessing body iron stores: (i) the discovery of HFE, the
gene responsible for most cases of hereditary hemochromatosis, (ii) the recent
demonstration that transfusion therapy is an effective means of preventing
stroke in patients with sickle cell disease, and (iii) the availability of new
iron-chelating agents for the treatment of transfusional iron overload in
patients with thalassemia major, myelodysplasia and other forms of refractory
anemia. In this section, the
pathophysiology of iron overload and the public health importance of the
development of a diagnostic device for accurate measurement of body storage iron
are summarized. The limitations of
clinically available means for estimating body iron will then be reviewed and
the theoretical basis for the use of the magnetic properties of ferritin and
hemosiderin for the measurement of iron
stores outlined.
(1) Pathophysiology and
health importance of iron overload:
Iron is an essential nutrient required by every human cell.
A transition metal (atomic number 26, atomic weight 55.85), iron can
serve as a carrier for oxygen and electrons and as a catalyst for oxygenation,
hydroxylation, and other critical metabolic processes, in part because of its
ability to reversibly and readily cycle between the ferrous (Fe+2)
and ferric (Fe+3)
oxidation states. The very
reactivity that is metabolically useful also makes iron potentially hazardous.
Ionic iron can participate in a number of reactions to produce free
radical species, which can in turn damage cellular constituents.
As a consequence, if too much iron accumulates (iron overload) and
exceeds the body's capacity for safe transport and storage, iron toxicity may
produce widespread organ damage and death.
The concentration of iron in the human body is normally about 40 to 50 mg
Fe/kg body weight; women typically have lower amounts, whereas men have higher
amounts. Most of this iron is
contained in essential iron compounds required for normal metabolic activity but
about 5 to 6 mg Fe/kg in women, 10 to 12 mg Fe/kg in men is storage iron in the
form of ferritin and hemosiderin, principally in hepatocytes and in macrophages
in the liver, bone marrow, spleen, and muscle, serving as a readily available
reserve in the event of blood loss. The
major pathways of iron absorption and loss and of internal iron exchange and
storage are shown schematically in Figure b-1.
Humans are unique in their lack of any effective means to excrete excess
iron. As a consequence, iron balance is physiologically regulated
by controlling iron absorption: iron stores and absorption are reciprocally
related, so that as stores decline, absorption increases.
Normally, iron exchange with the environment is extremely limited, with
less than 0.05% of the total body iron acquired or lost each day, making humans
unequaled among animals in the effectiveness with which iron is conserved.
Figure b-1:
Body iron supply and storage in the normal individual.
A schematic representation of the major iron pools (functional, storage
and transport) and the major routes of iron movement. The mean amount of iron in the storage compartment is about 1
gram in men and about 250 mg in women.
Iron overload is caused by
conditions that alter (hereditary hemochromatosis, refractory anemia with
ineffective erythropoiesis) or bypass (transfusional iron overload) the normal
control of body iron content by regulation of intestinal iron absorption.
Whether derived from increased absorption of dietary iron or from
transfused red blood cells, progressive iron accumulation eventually overwhelms
the body's capacity for safe storage. In
all varieties of iron overload, the development and severity of organ damage is
closely correlated with the magnitude of the body iron excess.
Symptomatic patients may present with any of the characteristic
manifestations of iron overload: liver disease with the eventual development of
cirrhosis and, often, hepatocellular carcinoma, diabetes mellitus, gonadal
insufficiency and other endocrine disorders, arthropathy and increased skin
pigmentation; iron-induced cardiomyopathy may be lethal.
Hereditary hemochromatosis:
The most common genetic disease known among Caucasian populations and the
most common form of iron overload in the United States is a genetically
determined disorder, the homozygous state for hereditary hemochromatosis,
occurring in as much as 0.5 percent of the population or more than 1 million
individuals. In hereditary
hemochromatosis, the underlying genetic abnormality results in an
inappropriately elevated iron absorption, with a chronic progressive increase in
body iron stores leading to parenchymal iron accumulation, initially in the
liver but later in the pancreas, heart, and other organs.
Despite marked parenchymal
iron deposition, macrophage iron in the bone marrow may be scant or even absent
(Figure b-2). By the time that
symptoms of parenchymal damage develop, usually in middle or late life, body
iron stores have typically increased from the normal range of 1 g or less to 15
to 20 g or more; further increments in the body iron may be fatal.
The traditional clinical means of establishing the diagnosis of
homozygous hereditary hemochromatosis has been through pathological examination
and measurement of the storage iron concentration in liver tissue obtained by
percutaneous or open biopsy. If
hereditary hemochromatosis is diagnosed before irreversible organ damage has
developed (cirrhosis, diabetes mellitus), then phlebotomy therapy can remove the
excess iron, prevent the development of disease manifestations and give the
affected individual a normal life expectancy.
Figure b-2: Changes
in body iron supply and storage with hereditary hemochromatosis. Despite massive
iron deposition in hepatocytes and other parenchymal cells, macrophage iron
stores in the bone marrow, liver and spleen may be normal or only modestly
increased. Measurement of the hepatic storage iron concentration provides the
best means of evaluating the extent of body iron excess.
The most
important recent advance in hereditary hemochromatosis has been the
identification in 1996 of the gene, now termed HFE, responsible for the majority
of cases. Mutations in this gene
are present in 85% or more of U.S. patients with hereditary hemochromatosis.
As a result, hereditary hemochromatosis seems a
near ideal candidate for population screening: an autosomal recessive disorder
that (i) has a high prevalence in the U.S. population, (ii) has a high frequency
of serious clinical manifestations affecting the homozygous genotype, (iii) can
be identified by safe and reliable screening and diagnostic tests, and, (iv)
after early diagnosis, can be treated effectively and inexpensively to prevent
later complications. Despite these
favorable factors, not all patients homozygous for HFE mutations have iron
overload and not all patients with iron overload have HFE mutations.
Thus, while phenotypic and genotypic screening and diagnostic tests can
identify those at risk for iron overload, a major factor limiting the institution of public health
programs screening for iron overload is the lack of a reliable, safe,
non-invasive and quantitative means of measuring body iron
(see below).
Transfusional
iron overload progressively develops in patients with chronic
refractory anemia who require red cell support.
Iron deposition resulting from transfusion initially has a predominant
pattern of iron deposition in macrophage sites followed by later redistribution
to parenchymal tissues (Figure b-3). In patients with severe congenital anemias,
such as thalassemia major (Cooley's anemia), transfusional iron loading begins
in infancy. Severe iron loading may
also develop in transfusion-dependent anemias that appear later in life, namely,
aplastic anemia, pure red cell aplasia, hypoplastic or myelodysplastic
disorders. If ineffective
erythropoiesis and erythroid hyperplasia complicate the underlying anemia,
increased absorption may contribute to the iron burden, but an adequate
transfusion regimen will help suppress erythropoiesis and may reduce iron
absorption to near normal levels. Adequately
transfused patients with thalassemia major or other congenital refractory
anemias grow and develop normally during the first decade of life. Thereafter,
without treatment for iron excess, growth fails, sexual maturation is delayed or
absent, and liver disease, diabetes mellitus, and other endocrine abnormalities
develop; patients usually die of heart disease in adolescence.
Transfusion-dependent forms of refractory anemia that are acquired later
in life, such as aplastic, myelodysplastic, or sideroblastic anemias, ultimately
follow a similar course. Heart
disease is the most frequent cause of death in patients with transfusional iron
overload. Patients with sickle cell
anemia or sickle cell-b thalassemia are
also at risk for iron overload if chronically given transfusions for the
prevention of recurrent complications. The
most important recent development in the management of patients with sickle cell
disorders has been the demonstration that transfusion greatly reduces the risk
of a first stroke in children with sickle cell anemia who have abnormal results
on transcranial Doppler. The
preventive use of red cell transfusions in patients with sickle cell disease
will greatly increase the number of patients in the U.S. with transfusional iron
overload.
Figure b-3:
Changes in body iron supply and storage with transfusional
iron overload. Marked macrophage iron deposition is present along with iron
overload in hepatocytes and other parenchymal cells. Measurement of the hepatic storage iron concentration also
provides the best means of evaluating the extent of body iron excess in patients
with transfusional iron overload.
In
patients with transfusional iron overload, the severity of the underlying anemia
usually precludes phlebotomy therapy as a means of removing toxic accumulations
of iron. Treatment with a chelating
agent capable of sequestering iron and permitting its excretion from the body is
the only other therapeutic approach now available.
Over the past three decades, deferoxamine B, a naturally occurring
trihydroxamic acid produced by Streptomyces
pilosus, has been found to be a generally safe and effective means of
managing iron overload that can prolong survival and avert or ameliorate
iron-induced organ damage. To avoid
the side effects of excessive deferoxamine administration (notably, blindness
and deafness) and to prevent iron toxicity from inadequate therapy, careful
monitoring of the body iron is required. The reference method for evaluation of body iron
stores is measurement of the hepatic iron concentration in a biopsy sample but
the discomfort and risk of biopsy preclude the use of biopsy for frequent serial
monitoring of the progress of iron-chelating therapy. Consequently,
the single greatest problem in the monitoring of iron-chelating therapy in
patients with iron overload is the lack of a safe, non-invasive, reliable and
quantitative means of measuring the body iron (see below).
(2) Limitations of available
methods for the estimation of human iron stores:
The
limitations of the currently available techniques for the estimation of body
iron stores may be briefly reviewed: both indirect and direct methods can be
used. The indirect
measures of body iron status have the advantages of ease and convenience,
but clinical experience has shown that these indirect methods may often be
misleading. All are subject to
extraneous influences and lack specificity, sensitivity, or both.
The measurement of plasma ferritin (protein) provides the most useful indirect estimate
of body iron stores, but there are many common clinical conditions
in which the plasma ferritin is not a reliable indicator of body iron
stores. Inflammation, infection,
liver disease, hemolysis, ineffective erythropoiesis and ascorbate deficiency
– common complications of hereditary hemochromatosis, transfusional iron
overload, or both -- all perturb serum ferritin levels independently of changes
in total body iron. In particular,
normal serum ferritin levels in precirrhotic hemochromatosis restrict its use as
a dependable means of detecting increased body iron in this disorder.
In patients with severe transfusional iron overload and thalassemia
major, the correlation between serum ferritin and iron stores appears to
result from a fortuitous addition of the effects of iron levels on
ferritin synthesis and the effect of cell damage on ferritin release from the
liver. Measurement of the plasma
ferritin iron has been proposed as an improved means of estimating
body iron but recent comparisons between the plasma ferritin iron and the
hepatic storage iron concentration have found no improvement over the
correlation found with plasma ferritin (protein) alone.
The plasma iron, transferrin
and transferrin saturation do not
quantitatively reflect body iron stores. The
lability of plasma iron and transferrin saturation, especially in early
hemochromatosis, limit their usefulness as a screening device.
Measurement of urinary iron excretion after injection of an iron chelator does not
quantitatively reflect the level of body iron stores. Erythrocyte
protoporphyrin levels or examination of the erythrocyte indices and
morphology are of no use in the detection of iron overload.
Equipment for magnetic resonance imaging (MRI) is widely accessible and
the striking changes in the proton resonance behavior of tissue water produced
by the presence of iron have led to a number of attempts to apply magnetic
resonance imaging to the measurement of iron in the liver and other tissues.
Various instruments, magnetic field strengths, imaging protocols, and
parameters (longitudinal [T1] and transverse [T2] relaxation times, signal
intensity ratios of liver to muscle or other tissues in proton, T1-, T2- or
T2*-weighted images), have been used but no satisfactory quantitative procedure
has been developed or clinically applied. Radioisotope
methods (radioiron dilution or absorption, cobalt absorption) remain
research procedures.
Diagnostic x ray spectrometry (DXS),
a technique that can estimate dermal iron content, does not provide a measure of
total body iron load..
The direct
measures of body iron status yield quantitative, specific, and sensitive
determinations of body or tissue iron stores16
. Quantitative phlebotomy
provides a direct measure of total mobilizable storage iron but is generally
acceptable only if the procedure is of therapeutic benefit and cannot be used in
transfusion-dependent patients with iron overload.
Tissue biopsy of the major iron
storage sites, the liver and bone marrow, may provide either qualitative (histologic)
or quantitative (chemical analysis) means of ascertaining iron status.
Quantitatively, measurement of the hepatic storage iron concentration
provides the best means of evaluating the extent of body iron excess in all
forms of iron overload, recognizing that the exact relationship between hepatic
iron and the total body iron burden depends on the underlying disorder.
At present, the detection and assessment of parenchymal iron overload (as
in hereditary hemochromatosis) is possible only with a liver biopsy.
The liver is the only storage compartment whose iron content is
consistently increased in all forms of systemic iron overload.
As shown in Figures b-2 and b-3, both parenchymal and reticuloendothelial
storage iron excess are detectable in the liver.
While biopsy
techniques with
chemical analysis of
tissue iron content provide the most quantitative direct measures of iron
status generally available, their discomfort, and for liver biopsy, risk, limit
their acceptability to patients and preclude their frequent use in serial
observations. Computed tomography (CT) theoretically should detect excessive
tissue iron deposition by an increase in tissue x ray absorption coefficients.
The usual single-energy CT estimates of hepatic iron have little clinical
utility
. While theoretically,
dual-energy CT would be expected to a better technique, a recent study has concluded that this
method is still subject to considerable error because of variations in tissue
composition. Nuclear
Resonant Scattering of X rays (NRS) studies must be carried out near a
nuclear reactor and seem unlikely to be clinically applicable
.
Instrumental
The mathematical and physical basis
of iron measurement was discussed in detail at a recent (April 2001) NIH
workshop. A panel overview from that workshop, "Session IV: Detection of Iron Overload by Susceptometry"
is given in pdf
file. A (low-Tc)
instrument based on those principles is available commercially (Tristan Inc.).
This is of proven value for clinical research and patient management in selected
locations. However, its cost, and the complications associated with the
provision of liquid helium, have prevented any widespread
clinical adoption. The objective of our NIH project is to exploit the technical
advantages conferred by high-Tc superconductivity so as to
reduce the cost and simplify the system to the point that it offers the
clinician a cost-effective and widely available means of iron measurement
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