Noninvasive methods for quantitative assessment of transfusional iron overload in sickle cell disease

Noninvasive Methods for Quantitative Assessment of Transfusional Iron Overload in Sickle Cell Disease Guvy 111.Brittenhum, Sujit She& Christopher J. Allen, and D&d

E. FaweZZ

Because optimal management of iron chelation therapy in patients with sickle cell disease and transfusional iron overload requires accurate determination of the magnitude of iron excess, a variety of techniques for evaluating iron overload are under development, including measurement of serum ferritin iron levels, x-ray fluorescence of iron, magnetic resonance imaging, computed tomography, and measurement of magnetic susceptibility. The most promising methods for noninvasive assessment of body iron stores in patients with sickle cell anemia and transfusional iron overload are based on measurementof hepatic magnetic susceptibility, either using superconducting quantum interference device (SQUID) susceptometry or, potentially, magnetic resonance susceptometry. Semin Hematol38(suppll):37-56. Copyright 0 2001 by W.B. Saunders Company.

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HIS REVIEW specifically examines methods for the assessment of transfusional iron overload in patients with sickle cell disease, but many of the conclusions may be applied to other disorders with an increased body iron burden. A quantitative means of measuring body storage iron that is accurate, safe, noninvasive, and readily available is needed for optimal treatment of patients with sickle cell disorders who have received repeated red cell transfusions for the prevention or treatment of acute and chronic complications. Patients with sickle cell disease have long received intermittent red cell transfusions in preparation for major surgical procedures and for management of bone marrow aplasia, skin ulcer, intractable painful crisis, priapism, hypoxia with the acute chest syndrome, and other problems. Longterm transfusion therapy has been used for prevention of recurrent stroke100’12” and in some other special circumstances. Recently, long-term transfusion has been found to be effective for prevention of first stroke in patients with sickle cell anemia with abnormal findings on transcranial Doppler ultrasonography.’ This result will greatly increase the number of patients who receive long-term transfusions. Because the body lacks an effective means of eliminating excess iron without chelating therapy, the iron within transfused red cells is progressively deposited in the liver,*’ pancreas,45,111 and other endocrine organs,ll* as well as the heart*O and other tissue+ of patients with sickle cell disease. Seminars

in Hematology,

Comprehensive studies of the consequences of iron overload in sickle cell disease are lacking, but the available data suggest that patients are at risk for all of the complications of transfusional iron overload found in thalassemia and other forms of refractory anemia, including liver disease culminating in cirrhosis, diabetes, other endocrinopathies, and heart disease.93 A recent comparative study of children and adolescents with thalassemia and sickle cell disease who regularly received transfusions documents the similarities between the two groups of patients with respect to impaired growth and pubertal delay or failure.l14 Exchange transfusion and erythrocytapheresis can retard iron accumulation in some patients7’J1*; in others, phlebotomy therapy’** can be used. Nonetheless, for most patients with sickle cell disorders who repeatedly receive transfusions, management of iron overload requires treatment with an iron-chelating agent. From the Departments of Pediatrics and Medicine, Cohnbia University, New York, NY; and the Department of Physics, Case Western Reserve University, Cleuebnd, OH. Supported in part by research grants from The Aplastic Anemia Foundation, the Cooley’s Anemia Foundation, and National Institutes of Health gfpants HL62882 and DK49108. Address reprint requests to Gary M. Brittenham, MD, Columbia University College of Physicians and Surgeons, Hardness Pavilion, Room HP550, 630 W 168th St, New York, NY 10032. Copyright 0 2001 by W.B. Saunders Company 0037-1963/01/3801-1007$35.00/0 doi:10.1053/shem.2001.20143

Vol 38, No 1, Suppl 1 (January),

2001:

pp 37-56

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Deferoxamine B mesylate, the only agent available for clinical use in patients with transfusional iron overload, can control body iron and alleviate hepatic, cardiac, and endocrine dysfunction, improve growth and sexual maturation, and extend survival

22,32,51,53,93,129

Optimal Body Iron Level in Patients With Sickle Cell Disease Until the effects of iron excess in patients with sickle cell disease have been systematically examined, a quantitative approach to the management of transfusional iron overload is best guided by our understanding of other forms of iron overload that have already been studied more thoroughly. In patients with sickling disorders and transfusiona iron overload, as in thalassemia, the magnitude of the body iron burden is likely to be the principal determinant of the severity of iron toxicity and clinical outcome.l’ Therefore, the most important therapeutic goal of iron chelation therapy in patients with sickle cell diseaseis to maintain the body iron level in an optimal range that prevents iron toxicity from inadequate chelation therapy while avoiding the side effects of excessive chelator administration. With stable transfusion requirements and in the absence of other confounding factors, the lower the level of body iron desired, the higher the dose of iron chelator needed. With most of the adverse reactions encountered with deferoxamine, including visual and auditory neurotoxicity,95ai05 growth failure, bony changes,4aJ0,96J04and pulmonary toxicity, 48 the higher the dose, the greater the risk. As a consequence,therapy to maintain normal body iron levels might minimize the likelihood of iron toxicity but would greatly increase the risk of dose-related chelator toxicity. At the opposite extreme, with high body iron burdens, deferoxamine toxicity is rare but the risk of iron-induced cardiac disease and early death is greatly increased. In the absence of prospective clinical trials in patients with sickle cell disease and transfusional iron overload, the best available evidence of the risk of complications associated with modest increases in body iron comes from clinical experience with heterozygotes for hereditary hemochroma-

tosis. This autosomal recessive disorder is most often the result of a mutation in the HFE gene that produces a lifelong inappropriate increase in iron uptake.41 Homozygotes develop a chronic progressive increase in body iron stores. Minor iron loading develops in about one fourth of those heterozygous for hereditary hemochromatbsis, but body iron stores in these individuals do not seem to increase beyond approximately 2 to 4 times the upper limit of norma1.23~30 Body iron loads of the magnitude found in heterozygotes for hereditary hemochromatosis have no apparent ill effects and are associated with a normal life expectancy.23 In contrast, homotygotes who develop greater iron burdens have an increased risk of cardiac disease, hepatic fibrosis, diabetes mellitus, endocrine abnormalities, and other complications of iron overload. In hereditary hemochromatosis, as in transfusional iron overload, the greater the body iron excess, the higher the risk of adverse consequences.23Jo~31~74~89 The analogy between the iron overload produced by transfusion and that derived from increased iron absorption in hereditary hemochromatosis is limited.p3 Nonetheless, the considerations above suggest that a conservative goal for iron chelation therapy in patients with sickle cel1 disease is to maintain an “optimal” body iron level corresponding to hepatic storage iron concentrations of approximately 18 to 38 pmol iron/g liver, wet weight (-3.2 to 7 mg iron/g liver, dry weight), approximately the range found in heterozygotes for hereditary hemochromatosis. The risks of deferoxamine toxicity associated with regimens to maintain body iron within this range are minor but almost certainly increase as the body iron burden decreasesbelow this range. Patients with higher body iron burdens, up to approximately 80 pmol iron/g liver, wet weight (a 15 mg iron/g liver, dry weight), are considered to have an increased risk of hepatic fibrosis, diabetes mellitus, and other complications and need more intensive iron chelation therapy.93 Patients with even higher body iron burdens are recognized as having a greatly increased risk of cardiac disease and early death and are candidates for continuous intravenous ambulatory deferoxamine administration or other special

39

Assessmentof Iron Overload

0 0

10

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40

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Age

Figure 1. The solid line is derived from studies of patients with thalassemia major treated with red cell transfusion alone5; the broken lines are derived from studies of patients heterozygous or homozygous for hereditary hemochromatosisz3 The ranges of hepatic iron concentrations shown are (1) those found in normal individuals,18 bottom-most band; (2) those reported in heterozygotes for hereditary hemochromatosis and associated with a normal life expectancy,23 labeled “optimal in transfusional iron overload”; (3) those associated with an increased risk of iron-induced complications in patients with transfusional iron overload,lg labeled “increased risk of complications”; and (4) the concentration corresponding to a greatly increased risk for cardiac disease and early death in patients with transfusional iron overload,i9,94 labeled “threshold for cardiac disease and early death.” Adapted from Olivieri and Brittenham with permission of the American Society of Hematology.93

programs of management.93 These thresholds are shown graphically in Fig 1. Although these treatments were originally developed for transfusional iron overload in thalassemia,aj their application to sickle cell diseaseseemsprudent. The widespread vasoocclusive manifestations and chronic organ damage of sickle cell disease appear unlikely to increase the body’s tolerance for iron toxicity.

Quantitative Assessment Body Iron

of

If the goal of iron chelation therapy in patients with sickle cell diseaseis to maintain the body iron in an optimal range, then means for accurate measurement of the magnitude of iron overload are needed to guide treatment. The most quantitative means of determining the body iron burden lo1 in patients with transfusional iron overload and the reference method for comparison with other techniques is direct biochemical measurement of the nonheme iron concentration in a specimen of liver obtained at biopsy (of adequate size, in the absence of

cirrhosis or focal lesions of the liver). The discomfort, lack of acceptability to patients, and risk of biopsy have prompted a search for noninvasive alternatives for the evaluation of body iron. Ideally, an alternative measure of body iron for patients with transfusional iron overload should (1) provide reliable discrimination between body iron burdens above and below the optimal range, (2) provide reliable discrimination between body iron burdens above and below the threshold for an increased risk of cardiac complications and early death, and (3) be applicable over the entire range of body iron burdens found in patients with sickle cell diseasein order to permit monitoring of the progress of iron chelation therapy in all patients. Both indirect and direct methods are potentially useful as measures of body iron stores in sickle cell disorders. After summarizing the merits of established techniques, we examine new approaches to the noninvasive measurement of iron overload that are now under investigation, including determination of plasma ferritin iron, plasma transferrin receptor, x-ray fluorescence, magnetic resonance imaging (MRI), nuclear resonant scattering of x-rays (NRS), computed tomography (CT), and measurement of hepatic magnetic susceptibility. Because the greatest clinical experience with noninvasive assessmentof body iron has been gained with measurement of hepatic magnetic susceptibility, this approach is considered in greater detail.

Indirect

Measures of Body Iron Stores

Indirect measures of body iron use some indicator that is influenced by the amount of body iron stores instead of measuring tissue or body iron itself. Indirect methods offer the advantages of ease of use and are generally readily acceptable to patients but are semiquantitative and lack specificity, sensitivity, or both. Each indirect indicator may be affected by conditions or factors other than body iron, such as infection, inflammation, liver disease, ascorbate deficiency, malnutrition, malignancy, some coexisting disorders, and other factors.

40

B&e&am

Plasma Ferritin Protein Concentration Ferritin, the principal intracellular iron storage protein, circulates in the plasma in small amounts. Plasma ferritin is synthesized by the rough endoplasmic reticulum and is then glycosylated by the Golgi apparatus before release from the ce11.64,134 In contrast, the much larger amount of ferritin that is retained intracellularly is produced by the smooth endoplasmic reticulum and is not glycosylated.64,102 The physiologic function of the ferritin secreted into plasma remains uncertain2* but in normal individuals the small quantities released into plasma seem to be proportional to the much larger amount of cellular ferritin produced in the internal iron-storage pathway. As a consequence, the plasma ferritin concentration is influenced by the magnitude of cellular iron stores. In the absence of complicating factors, plasma ferritin concentrations decrease with depletion of storage iron and increase with storage iron accumulation. The plasma ferritin concentration seemsto have a logarithmic relationship with body iron stores, but some evidence suggests that this relationship differs between individual+‘; that is, that identical changes in body iron stores are associated with quantitatively different changes in plasma ferritin concentrations in different individuals. A variety of conditions may alter the relationship of plasma ferritin to body iron stores. Plasma ferritin is an “acute phase reactant. “8o3122Intracellular ferritin synthesis is controlled, at least in part, by the iron-regulatory proteins, IRP-1 and IRP-2,io8 whose activity is in turn altered by ascorbate,12i oxidative stress, and nitric oxide,67z79and in erythropoietic cells by erythropoietin.l3O Accordingly, ascorbate deficiency,42a107 acute and chronic inflammatory or infectious condi24 tions,9J4 and increased erythropoietin, may all perturb the relationship between plasma ferritin and body iron stores. Plasma ferritin levels may also be increased by the release of tissue ferritins caused by damage to the liver and other ferritin-rich tissues as well as by hepatic dysfunction, which impedes the clearance of circulating ferritin. For patients with a plasma ferritin concentration greater than 4,000 pg/L, the correlation between serum

et al

ferritin and iron stores has been suggested to result from “fortuitous addition of the effects of iron levels on ferritin synthesis and the effect of cell damage on ferritin release from the T.134 liver. Patients with sickling disorders who regularly receive red cell transfusions are at increased risk for many of the conditions that affect the relationship of plasma ferritin to body iron stores, especially acute and chronic infection, inflammatory responses to microvascular infarcts, liver disease, and chronic hemolytic anemia with erythroid hyperplasia. In an earlier study specifically examining patients with sickling disorders, we found that body iron stores, as assessedby hepatic iron concentration, accounted for only 57% of the variation in plasma ferritin concentrations.17 These results are shown in Fig 2, plotted in a format identical to that in Fig 1 to facilitate comparisons between different methods and show graphically the reliability of discrimination between different levels of hepatic iron over the entire range of concentrations found in patients with sickle cell disease. In this study, the 95% prediction intervals for hepatic iron concentration, given the plasma ferritin concentration, were so broad as to make a single determination of plasma ferritin an unreliable predictor of body iron stores. To our knowledge, no study has shown that repeated determinations improve the utility of the plasma ferritin concentration as a predictor of body iron stores. Overall, variabil-

*50)50

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10,000 @g/L)

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Figure 2. Relationship between hepatic storage iron concentration as determined by magnetic susceptometry and plasma ferritin concentration in 37 patients with sickle cell disease.17 To facilitate comparisons between different methods for the assessment of body iron stores, the original data have been replotted in the format of Fig 1.

Amwnent

ity resulting from factors other than iron status limits the clinical usefulness of the plasma ferritin level as a predictor of body iron stores in patients with sickle cell disease who have received transfusions.i7

Plasma Ferritin Iron Concentration Measurement of the plasma ferritin iron, rather than protein, concentration has been proposed as an improved means of estimating body iron.68 The underlying hypothesis is that plasma ferritin protein produced in the acutephase response would have less iron than that produced in response to iron loading, thus avoiding the confounding effects of inflammation. The iron content of ferritin in patients with the acute-phase response seems to be lower than that in healthy volunteers,li7 but the extent to which measurement of the plasma ferritin iron will improve the sensitivity and specificity of the determination of body iron stores remains uncertain. Preliminary studies have found a significant correlation between body iron stores and plasma ferritin iron concentrations, but the correlation was similar to that found with plasma ferritin protein.i3” This result, if confirmed in further studies, suggests the importance of factors other than inflammation that alter the relationship of body iron stores to plasma ferritin, such as the release of tissue ferritins from damage to the liver and other organs, release of red cell ferritin by hemolysis, and the effects of impaired clearance of circulating ferritin because of hepatic dysfunction.

Plasma Transferrin Concentration

Receptor

The soluble transferrin receptor circulating in plasma is a truncated form (M, 85,000) of the cellular transferrin receptor that consists of the N-terminal cytoplasmic domain that has been proteolytically released from the cell membrane.28 Plasma transferrin receptors are derived from the total body mass of cellular transferrin, with 80% or more originating in the erythroid marrow in normal individuals. Immunoassays that can detect the soluble truncated form of the transferrin receptor in human plasma are

of IranOverLoad

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now available for clinical use. Because children seem to have higher concentrations of plasma transferrin receptors than adults, age-specific reference ranges should be used.i23 Although some reports indicate that plasma transferrin receptor concentrations are decreased in the presence of iron overload,78,85 no quantitative relationship between the magnitude of iron overload and the transferrin receptor concentration has been described. Furthermore, in patients with sickle cell disease, erythroid hyperplasia increases the plasma transferrin receptor concentration.29 Consequently, the plasma transferrin concentration is not useful as a means of measuring iron overload in sickling disorders.

Other Tests Using Plasma, Blood, or Urine Elevated plasma iron concentration or transferrin saturation suggest parenchymal iron overload but do not quantitatively reflect body iron stores. The plasma transferrin concentration (or plasma total iron-binding capacity) is usually decreased with iron overload but not quantitatively related to the body iron burden. Non-transferrin-bound plasma iron58S69370,103 probably is a critical factor in tissue iron loading and injury in transfusional iron overload but is not quantitatively related to the total body iron burden. The erythrocyte zinc protoporphyrin provides an indicator of iron supply to erythroid precursors but is of no value in evaluating iron overload. Iron overload produces no diagnostic abnormalities on inspection of erythrocyte morphology or in measurements in the peripheral blood, including the hemoglobin concentration, hematocrit, red cell indices, red cell volume distribution, and reticulocyte count. In populations of patients homozygous for hereditary hemochromatosis, mean hemoglobin, mean hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) values may be detected by comparison with mean values in control populations of normal individuals,6 probably as a result of increased iron uptake and hemoglobin synthesis by immature erythroid cells. Nonetheless, these red cell changes are of no help in the measurement of body iron stores,

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especially in patients with sickle cell disease who have received transfusions. The measurement of urinary iron excretion with chelating agents, usually either deferoxamine or diethylenetriamine pentaacetate (DTPA),~T*,~~ offers another means of assessing body iron stores. The chelatable iron pools accessible to either deferoxamine or DTPA are subject to many of the same influences as described for plasma ferritin above, including ascorbate deficiency, inflammation, infection, liver disease,hemolysis, and other factors. With deferoxamine, the relative proportion of iron excreted into the urine and stool varies not only with the body iron burden but also with the dose of deferoxamine administered and erythroid activity. lo1 As a result, urinary iron excretion after chelator administration is an unreliable means for the quantitative assessment of body iron stores. Radioisotope methods (radioiron dilution or absorption, cobalt absorption) have research applications but are not used clinically in the evaluation of iron overload.

Magnetic Resonance Imaging Equipment for MRI is widely available, and the striking changesin the proton resonancebehavior of tissue water produced by the presence of iron have led to a number of attempts to apply MRI to the measurement of iron in the liver and other tissues. A variety of instruments, magnetic field strengths, imaging sequences(spin-echo, gradient recalled-echo), and parameters (longitudinal {Tl] and transverse {T2) relaxation times and signal intensity ratios as measured in proton, Tl-, T2or T2?-weighted images) have been exam&d,2,12,13,33,34,52,55,61,75-77,97,106,118,119

but

no

standard or generally accepted method has been adopted for clinical application. Noise from motion artifacts has been reduced with shorter imaging sequences but remains a problem in quantitative magnetic resonance studies.6h Comparison of absolute signal intensities from one magnetic resonance instrument to another is unreliable because of substantial interinstrumental variability. l2 Consequently, most methods use a ratio of signal intensities in the liver to another structure on the same image (for example, muscle, fat, spleen, or air) to increase Dermal X-ray Fluorescence reliability, although the interinstrumental variability of these ratios has yet to be examined. A variety of methods have been described that use No means of standardizing magnetic resonance x-ray fluorescence to noninvasively measure iron measurements from one facility to another have and other trace elements in the skin.35,*9,5@7J35 been developed. The skin is irradiated with a focused monochroEstimates of hepatic iron derived from MRI matic x-ray beam that excites elements in the are indirect, resulting from the effect of ferritin skin area to emit a characteristic x-ray fluoresand hemosiderin iron on the proton resonance cence that can be analyzed. This procedure can behavior of tissue water. This interaction is estimate dermal iron content 35 and has been complex and still poorly understood,12 involvused in studies of patients with thalassemia ing factors such as the number and sizes of the major and intermedia. ~~35 Because skin is not iron cores in ferritin and hemosiderin,i2* the a tissue used for iron storage, dermal iron distribution of iron and water within the tissue content does not reflect body iron stores. The examined, tissue hydration, and the water difdeterminants of dermal iron deposition have fusion coefficient,125 as well as the applied field not been established, but amounts in the skin strength, repetition time used in the imaging seem to be influenced by the rate of iron loading sequence, and other technical aspects of the (or removal). Moreover, the epidermis, the measurement procedure.21 In almost all magcellular part of the skin, is a very thin structure netic resonance methods, increased iron concencontaining layers of cells at different stages of tration reduces the signal intensity of the liver differentiation, complicating the analysis of to such an extent that discrimination between bulk measurements of iron content.*’ Measuredifferent concentrations becomes impossible. In ment of skin iron seemsunlikely to be clinically most studies, quantitative determination of useful in the assessmentof body iron in patients hepatic iron content is not possible in patients with sickling disorders. with signal intensity ratios corresponding to

Assessmentof Iron Ovedoad

hepatic iron concentrations that are greater than approximately 50 to 60 pmol iron/g liver, wet weight (-9 to 11 mg iron/g liver, dry weight). In clinical terms, this finding means that MRI could not be used during treatment to follow up patients with the most severe iron loading. The number of patients subject to this limitation depends on the population examined, but in patients with sickling disorders who receive regular transfusions, this proportion is substantial. For example, in our earlier study of patients with sickle cell diseasewho receive transfusions, more than half of those examined had hepatic iron concentrations greater than those that could be measured by magnetic resonance techniques (>60 pmol iron/g liver, wet weight)” (Fig 2). In a recent study, another factor restricting the precision of estimates of hepatic iron concentration by MRI was identified: the presence of hepatic fibrosis .2 In this study, the presence of fibrosis did not seem to affect the pattern of the relationship between hepatic iron and the signal intensity ratio; no significant difference was found in either the slope or the intercept of the regression equation between these factors in patients with or without fibrosis. Instead, the effect of fibrosis seemed to be to increase the variability of the relationship. In patients with fibrosis of any degree, variability in hepatic iron concentration accounted for only approximately 70% of the variation in signal intensity ratio.2 The clinical consequences of the limitation imposed by the presence of fibrosis on the precision of MRI studies may be appreciated by examining the 95% prediction intervals found in this study. For example, for a patient with a signal intensity ratio of about 1.0 and no fibrosis, the prediction interval extends from approximately 35 to 48 pm01 iron/g liver, wet weight (-6.5 to 9.0 mg iron/g liver, dry weight), and could be interpreted clinically to indicate that the liver iron concentration is near the upper limit of the “optimal” range shown in Fig 1. By contrast, if the patient with a signal intensity ratio of approximately 1.0 has hepatic fibrosis, the prediction interval is then widened considerably to about 9 to 65 pmol iron/g liver, wet weight (ml.7 to 12.0 mg iron/g liver, dry

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weight). This prediction interval, from near normal to the upper portion of the range of hepatic iron concentrations associated with an increased risk of the clinical complications of iron overload, is so broad that it is of little clinical use. In an individual patient with sickle celi disease, in the absence of a biopsy to determine if fibrosis is present, the prediction interval for an estimate of the hepatic iron measurement derived from MRI must be assumed to be so large that it is of little practical value in the management of transfusional iron overload. The physical basis for the effect of fibrosis on MRI is unknown; hepatic fibrosis may increase the extent of microheterogeneity in iron and water distribution within the liver and thereby exaggerate measurement variability. These factors could intrinsically limit the accuracy of determinations of iron by magnetic resonance methods’ and make this technique inherently unable to discriminate reliably between levels of hepatic iron that are needed for patients with transfusional iron overload. To illustrate this point, Fig 3A and B compares the results of MRI and the biochemical measurement of hepatic iron in two recent studies. In both studies, the investigators concluded that the magnetic resonance studies would be useful in the evaluation of patients with hereditary hemochromatosis.12J2 In contrast, for patients with sickle cell anemia and transfusional iron overload, the results suggest that neither would be very helpful in the management of iron chelation therapy. In Fig 3A, for example, magnetic resonance studies5’ provide little discrimination between patients whose hepatic iron concentrations are above the range labeled “optimal level in transfusional iron overload.” With a signal intensity ratio (liver to fat) of approximately 0.1, the hepatic iron concentration can range from about 50 to 150 pmol iron/g liver, wet weight. Similarly, in Fig 3B, patients with an ln(SNR) of approximately 1.0 may have hepatic iron concentrations of approximately 60 to 110 pmol iron/g liver, wet weight. Conversely, patients with hepatic iron concentrations of

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Brittenham -

et al

Direct

-2.0

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Intensity

F&~

(L.iver%at)

B 250 2 200 o’.g

Measures of Body Iron Stores

The direct methods for assessing body iron stores measure some physical property of tissue or body iron itself. Direct measures of body iron stores avoid the vulnerability to extraneous influences shown by indirect methods and are potentially superior in sensitivity and specificity. Direct techniques can provide quantitative, specific, and sensitive measures of amounts of storage iron but may involve tissue biopsy, repeated phlebotomy, or instrumentation that is not generally available.

:; 150 '3 z-SF

Conventional Methods

5% =E

As emphasized above, the most quantitative means of determining the body iron burden in patients with transfusional iron overload and the reference method for comparison with other techniques is direct biochemical measurement of the hepatic nonheme iron concentration in a specimen obtained at biopsy. Liver biopsy is the definitive test for assessing iron deposition and tissue damage, permitting not only quantitative determination of the hepatic nonheme iron

100 s 50 0 4.0

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Figure 3. (A) Comparison of hepatic iron concentration as determined by magnetic resonance imaging and by chemical analysis of liver tissue obtained at biopsy.52 The hepatic iron concentration is shown on the vertical axis and the liver-to-fat signal intensity ratio is shown on the horizontal axis. (6) Comparison of hepatic iron concentration as determined by magnetic resonance imaging and by chemical analysis of liver tissue obtained at biopsy. I* The hepatic iron concentration is shown on the vertical axis and the natural logarithm of the ratio of the signal intensity (SI) of the liver to the SD of the background air, or noise (SNR) is shown on the horizontal axis. GRE, gradient-recalled echo; TR, repetition time; TE, echo time. To facilitate comparisons between different methods for the assessment of body iron stores, the original data have been replotted inthe format of Fig 1.

50 to 60 pmol iron/g liver, wet weight, may have ‘ln(SNR) values that range from 1.0 to 3.0. It remains to be determined whether an improved understanding of the effect of ferritin and hemosiderin iron on the proton resonance behavior of tissue water or further developments in instrumentation, or both, can sufficiently improve the accuracy of estimates of hepatic iron to make them clinically useful for the management of transfusional iron overload in patients with sickle cell disease.

concentration but also histochemical determination of the cellular distribution of iron and pathologic assessment of the extent of injury. Quantitative phlebotomy provides a direct measure of total mobilizable storage iron (calculated as the amount of hemoglobin iron removed, with corrections for the hemoglobin deficit and estimated gastrointestinal iron absorption during the course of phlebotomy) but usually cannot be used in patients with sickle cell disease who regularly receive transfusions. Bone marrow aspiration and biopsy can provide information about (1) macrophage storage iron (by semiquantitative grading of marrow hemosiderin stained with Prussian blue or, if needed, by chemical measurement of nonheme iron), (2) iron supply to erythroid precursors, and (3) the general morphologic features of hematopoiesis. For the evaluation of iron overload in sickle cell disease, bone marrow aspiration and biopsy are of limited applicability because no information about the extent of parenchymal iron deposition is provided.

Assessmentof Iron Overlad

Nuclear Resonant Scattering of X-rays In this method, a noninvasive estimate of hepatic iron is provided by using gamma rays to raise >“Fe to its first excited state. Subsequent decay back to the ground state by resonant emission in turn produces gamma rays that may be counted using a suitable detector. Body (or organ) iron content will then vary linearly with resonant counts.132 Wielopolski et al,i3* using the Brookhaven Medical Research Reactor, used a [56Mn)CI, gamma ray source in a concrete, steel, and lead brick-shielded irradiation-detection facility with Ge(Li) detectors. Phantom studies suggest a limit of detection for the system of approximately 1,000 pg iron/g tissue with a radiation dose to the liver of 1 rad. The position of the liver is determined by ultrasound. Although a significant correlation of the results of nuclear resonant scattering studies and iron determined in specimens obtained by liver biopsy was found in more recent studies,i33 this method is limited both by the short half-life of the source (such that studies can be carried out only in close proximity to a reactor), and the levels of radiation exposure required. As a result, NRS seems unlikely to have a clinical application for the evaluation of transfusional iron overload in sickle cell disease.

Computed Tomography Iron overload can also produce striking changes on images of the liver derived from CT, and a number of attempts have been made to exploit this phenomenon for measurement of hepatic iron concentration. CT can detect the increase in x-ray density (higher CT number) caused by the greater electron density of iron compared with other liver constituents. An initial study, in which the usual single energy technique (a tissue slice is scanned at 1 energy level) was used, was unsuccessful, apparently because the wide variation in normal liver x-ray attenuation values obscures differences attributable to iron unless massive overload is present.88 In studies of patients with thalassemia major who received transfusions, Mitnick et al” suggest that the CT density of the liver related more to the

45

degree of hepatic fibrosis and cirrhosis than to the amount of iron. Although some investigators were more optimistic,26~73,84 most investigators have concluded that single-energy CT is of little clinical value in the measurement of iron overload.13,60 To diminish the influence of hepatic tissue on the measurement, a dualenergy technique can also be used.25,s2 The same tissue slice is scanned at two different energy levels, and the difference in CT number (the dual-energy differential) is calculated. Iron, with a higher atomic number than most other elements in the liver, will absorb relatively more at lower energy levels while the x-ray absorption of other liver materials is changed little. The difference in absorption at the two energy levels, the dual-energy differential, is then principally influenced by the presence of iron. Despite promising data in animals,54z90 both theoretical92 and laboratory studies in vitro lo9 suggest that the dual-energy technique remains vulnerable to a variety of sources of error (photon noise, variation in tissue composition, especially artifacts caused by fat),” and that both better instrumentation and more information on variations in body tissue are needed before this technique can be clinically useful.

Hepatic Magnetic Susceptibility Measurement of magnetic susceptibility provides the only direct and noninvasive means of determining hepatic iron stores that has been calibrated, validated, and used extensively in studies of patients with iron overload. First proposed and developed by Bauman and Harris,8 the magnetic technique uses the only storage compartment, the liver, whose iron content is invariably increased in all forms of systemic iron overload and detects increases in both parenchymal and reticuloendothelial storage iron levels.i4 After reviewing the theoretical basis for the use of the magnetic properties of ferritin and hemosiderin for the measurement of iron stores, we will consider the practical development of the method, its current state, and the prospects for future improvements in the instrumentation used to measure magnetic susceptibility.

Brittenbam et al

46

Figure 4. Elements of a susceptometric measurement of hepatic storage iron. The body is positioned so that the liver lies directly beneath the susceptibility probe, then lowered and the field change measured by the field detector (see text).

Theoretical Basis for Use of Magnetic Susceptibility for Measurement of Hepatic Iron Magnetism is one of the fundamental physical properties of matter. 36 All matter has measurable magnetic characteristics resulting from the e!ectronic structure of the constituent atoms and molecules. The magnetic susceptibility of tissue is determined by the strength of the magnetic response evoked in the tissue by application of a steady magnetic field. This property is much simpler than the resonance behavior that results from application of the oscillating magnetic fields used in MRI. When placed in a steady applied magnetic field, all materials respond with a steady induced magnetic field of their own. This fundemental response may be exploited diagnostically because the magnitude of the induced magnetic field varies greatly in different materials. In most human biological materials, this induced field is in the opposite direction from the applied field. This diamagnetic response is weak (approximately lop6 of the applied field), and its detection requires sensitive instrumentation (see below). By contrast, ferromagnetic materials, such as the common bar magnet, respond with an induced field that is as strong as or even stronger than the applied field and in the same direction. No known human tissues are ferromagnetic. Intermediate between the diamagnetic and ferromagnetic extremes is the paramagnetic response of the iron in ferritin and hemosiderin. The paramagnetic responseis in the same direction as the applied field but is typically about 1O-* smaller. This paramagnetic response is directly proportional to the number of iron atoms present in tissue iron storage compounds. In a measurement of hepatic magnetic susceptibility in vivo, the op-

posing diamagnetic (tissue) and augmenting paramagnetic (storage iron) responsesare superimposed. Taking into account the small and nearly constant diamagnetic effect of the liver tissue, the observed resultant magnetic susceptibility may be used to determine the number of storage iron atoms present.18 The contributions of other paramagnetic materials (oxygen, deoxyhemoglobin, some trace metals) to the hepatic magnetic susceptibility are so small that magnetic measurements are highly specific for ferritin and hemosiderin iron. The elements of a magnetic system for the measurement of hepatic iron are illustrated schematically in Fig 4. The susceptibility probe contains a localized field source (permanent magnet or current-carrying coils) and a magnetic field detector. The patient is placed directly under the probe, with the liver positioned in the region to which the field detector is maximally sensitive, and then lowered away from the probe, as shown in Fig 4. Because of the localized character of both the magnetic field and the response of the field detector, the field change observed, B, contains 3 principal contributions: B = BL -I- Bs + BN, (Equation 1) where B, B,, and B, are, respectively, the contributions from the liver; subcutaneous tissues, skin, ribs, and intercostal muscle (and nearby tissues, including the lung); and instrumental noise. As discussedlater, instrumental noise, B, is generally negligible for a superconducting instrument. Assuming that the instrumental noise can be neglected, equation 1 then becomes B = B, + Bs. (Equation 2)

Assessmentof Iron Overload

47

lsceptibi probe Figure 5. Differential measurement of hepatic storage iron is made by lowering the subject from the initial position and replacing the body by water.

Consequently, the chief measurement uncertainty derives from the contribution made by the subcutaneous tissues, skin, ribs, intercostal muscle, and nearby organs, including the lung. In principle, knowing the geometry and magnetic susceptibility of these regions, B, may be calculated to any desired accuracy. In practice, obtaining the geometrical information would make the clinical procedure time-consuming and expensive. To circumvent this problem, our laboratories introduced the differential measurement technique for the measurement of hepatic magnetic susceptibility, a method that has been adopted by all other workers in the field. This technique relies on the fact that, with the important exceptions of iron-loaded liver and air-filled lungs, the susceptibility of most biological material is near that of water. In the differential measurement technique, the patient is lowered, as described above, but the body directly under the detector is replaced by water rather than air, using a water bellows arrangement like that shown schematically in Fig 5. When the body is lowered in air (Fig 4), the susceptibility of the various regions under the detector are measured. When the measurement is made differentially, the detector’s response is proportional to the difference in susceptibility between the organs and water. Because the tissues overlying the liver have a magnetic susceptibility very close to that of water, the water bellows method largely cancels out the contribution of the tissues overlying the liver. The lungs (and possibly gas-filled intestines) can have a susceptibility substantially different from that of water but are not directly under the detector, so their contribution is small. To date, clinical measurements of hepatic

- bellows

magnetic susceptibility have relied on superconducting magnetometers, so the role of superconductivity in magnetic measurements of iron stores are briefly reviewed here. The defining property of a superconductor is the loss of electrical resistance below a certain temperature, called the transition temperature (T,). All superconducting materials known before 1986 had T, not far above absolute zero; a bath of liquid helium (4.2’K) was required to keep their temperature below T,. The recently discovered high-T, materials have T, values well above that of liquid nitrogen (77’K), allowing this readily available and inexpensive cryogen to be used as a refrigerant. The superconducting quantum interference device (SQUID) is a superconducting loop incorporating a “weak link” known as a Josephson junction, an arrangement that provides the most sensitive and stable magnetic detector known. The observed field change, B, in equation 2 is very small-less than one millionth of the magnetic field of the earth. Furthermore, this minute change occurs in the presence of a steady field that is applied to the region of the liver that can itself be a thousand million times larger than B. These conditions create a dual requirement for the field detector-extreme sensitivity and the stability to resolve a tiny field change in the presence of a large background. Those requirements are satisfied only by superconducting detectors, embodied as the SQUIDS and flux transformers discussed below. The operation of these devices is not accounted for by the simple absence of resistance, but relies on an additional quantum mechanical property possessedby superconductors termed “ fl ux quantization.“12’ Flux quantization is unique to superconductivity, giving supercon-

48

Brittenham et al

,t,

IaA6

B+A Figure 6. Principle of the superconducting flux transformer. B is the applied field, and I is the current induced by a change in the field, AB.

ducting detectors a stability that is possessedby no other field-sensing device. As shown schematically in Fig 6, suppose a superconducting coil (called a flux transformer) is cooled down in a field, B, produced by a steady localized magnetic field-the situation realized in superconducting instrumentation. Under these circumstances, the magnetic flux through loop 1 will be equal to the applied magnetic field multiplied by the area of the loop. Because no current is induced in the transformer when it enters the superconducting state, the flux is trapped in place by the flux quantization constraint. Furthermore, as long as the material remains superconducting, this flux can never change from the initial value. If the total field in the vicinity of loop 1 changesby a small amount, AB (due, for example, to the introduction of the body of a patient), a current must appear in the transformer to keep the total flux trapped at its initial value. The flux generated by this current is equal to but opposite of the flux generated by the change in the magnetic field, the net flux change being zero. The flux produced by the current in loop 2 can be detected by a SQUID, located in a field-free (shielded) location. Therefore, the function of the flux transformer is to take a flux change and transport it to a field-free location, where it can be measuredby the SQUID. The critical point is that a flux transformer respondswith a current proportional to the change in flux in a detector coil, not the total flux. In device terms, the purely super-

conducting property of flux quantitation ensures that the field measurement is intrinsically differential. The field change attributable to a normal complement of iron is extremely small (-1 part in a billion of the applied field), so a powerful differential technique is required for any practical liver-iron instrument. From a physics point of view, then, SQUID susceptometry of liver iron representsthe practical application of a fundamental quantum phenomenon.3*

Practical Development of Magnetic Susceptibility Measurements of Hepatic Iron In this section, we briefly review the development of magnetic susceptometry as a means of measuring hepatic iron stores from original concept,s through initial demonstration of feasibility in small animal and pathologic studies with magnetometers using conventional electronics,s7 to the design, development, and use of low-Tc SQUID susceptometers (operating at 4”K)36,38-40 that have since been used in investigative studies with hundreds of patients with all forms of iron overload. The use of the magnetic properties of ferritin and hemosiderin for the measurement of iron stores was first proposed and developed by Bauman et al8 more than 3 decadesago. This original work outlined the theoretical basis for the technique and succeededin demonstrating its practicality with small animal experiments in vivo, results subsequently confirmed by others.l16 Studies of human hepatic tissue in vitro showed that magnetic susceptibility was not significantly affected by pathologic conditions in diseasedhuman livers with a normal iron content (acute and chronic passive congestion, fatty metamorphosis, alcoholic, cardiac and postnecrotic cirrhosis, metastatic carcinoma, Wilson’s disease)and was in the same range as that found in normal livers with a normal iron content.87 In contrast, in diseased livers with increased iron stores (hemosiderosis, postnecrotic cirrhosis, leukemia, hemochromatosis),magnetic susceptibility and iron content were linearly related with a highly significant correlation (r = 0.99) over a range from twice to more than 60 times the normal liver iron storess7Thus, the presenceand amount of hepatic storage iron

49

Assessmentof Iron Overload

could be assessedaccurately in iron overload without interference from other pathologic conditions. An effort was made to construct a susceptometer for use in human studies, but further progress was prevented by the limitations of the room-temperature instrumentation. By a decade later, the introduction of low-T, SQUID magnetometers operating at 4’K had instrument sensitivity and stability increased more than a thousandfold. Our laboratories then made the first observations of humans in vivo using a SQUID magnetic gradiometer to estimate hepatic iron stores.63With the support of the National Institutes of Health (NIH), a prototype single-channel SQUID susceptometer was designed, constructed, and initially used for studies in normal subjects and patients with liver disease, iron deficiency, hereditary hemochromatosis, or transfusional iron overload.i* Results of magnetic measurements of liver nonheme iron in vivo were closely correlated with those of chemical measurements in liver biopsy specimens in vitro (Y = 0.98, P < 10e5) up to 115 pmol/g of liver tissue, wet weight, or more. Subsequently, with continued NIH support, the prototype single-channel susceptometer was replaced by a computer-enhanced, dual-channel SQUID instrument that has since been in continuous use in our laboratories. The design of this instrument forms the basis of all commercially produced instruments.99 In part because of the cost (more than $1 ,OOO,OOOper instrument) and complexity (requirement for physics and bioengineering expertise; need for liquid-helium cooling) of the low-T, device, clinical adoption of the method has been limited. At present, the only susceptometers in clinical use are the instrument in our own laboratory at Columbia University and another d$z;lr,e at the University of Hamburg, An additional low-T, susceptomGermany. eter was recently installed at the University of Turin in Italy.

Current

State of Biomagnetic Susceptometry

In our laboratories, the low-T, dual-channel SQUID susceptometer has been used extensively in studies of patients with various disor-

50

250

E

40

200

0

3

0 0

50 100 150 200 250 Hepatic Iron (Magnetic), pm&g, wet weight

Figure 7. Comparison of hepatic iron concentration as determined by magnetic susceptometry and chemical analysis of liver tissue obtained by clinically indicated biopsy. To facilitate comparisons between different methods for the assessment of body iron stores, the original data have been replotted in the format of Fig 1. Magnetic and biochemical measurements were made within 1 month; patients with cirrhosis those whose biopsy specimens were less than 5 mg, wet weight, were excluded.

ders of iron metabolism. This susceptometric method in effect provides an automated magnetic “biopsy” of liver ferritin and hemosiderin iron. In patients with iron overload, the results of magnetic measurements of hepatic nonheme iron obtained in this manner are quantitatively equivalent to those obtained by chemical analysis of tissue obtained by biopsy (Fig 7). Magnetic measurements of hepatic storage iron have proven valuable in the early detection of homozygotes for hereditary hemochromatosis,i5 permitting prophylactic phlebotomy to prevent the development of disease manifestations. As described earlier, we examined the relationship between hepatic iron stores and plasma ferritin concentration in individuals treated with red cell transfusion and iron chelation therapy in a series of patients with thalassemia major16 and sickle cell disease.17 These results provided evidence that variability resulting from factors other than iron status limits the clinical usefulness of the plasma ferritin concentration as a predictor of body iron stores. The ability of the magnetic method to provide frequent serial measurements of hepatic storage iron permitted a decade-long prospective evaluation of the effectiveness of deferoxamine iron chelation therapy. We prospectively studied patients with thalassemia major treated with regular parenteral infusions of deferoxamine for transfusional iron over1oad.i” This experience pro-

50

Brittenham et al

vided a unique opportunity to examine quantitatively the relationships between transfusional iron load, cumulative deferoxamine use, body iron burden, and clinical outcome. The results identified an apparent threshold for deaths from cardiac causes equivalent to approximately 80 pm01 iron/g liver, wet weight. Overall, the results of magnetic measurements of hepatic iron stores and proportional hazards analysis indicated that greater use of deferoxamine in relation to the transfusional iron load effectively decreasedthe body iron burden and reduced the risk of impaired glucose tolerance, diabetes mellitus, cardiac disease,and death. This study was the first to show an independent effect of deferoxamine in decreasing the relative risk of death in patients with transfusional iron overload.iY The use of magnetic susceptometry for the measurement of human hepatic storage iron has been examined by a seriesof other investigators. Efforts have been made (1) to usean air interface65 rather than the differential measurement technique described above; (2) to employ an alternating current field from large Helmholtz coi1s7,Y8,i15 rather than a localized direct current field from field coils; or (3) to utilize MRI to measure susceptibility changes in an external phantom adjacent to the liver,72 but none of these approacheshave proven clinically useful. Our design has been adopted as the basis for a commercially available susceptometer99that has been used extensively by the researchgroup at the University of Hamburg. These investigators have confirmed the usefulness of magnetic susceptometry in screening for iron overload in patients with hereditary hemochromatosislO and studies in patients with transfusional iron overload treated with iron-chelating agents.**,9l An excellent review of their program has recently been published.*3 In summary, measurement of magnetic susceptibility to quantify paramagnetic ferritin and hemosiderin iron provides the only direct and noninvasive means of determining hepatic iron stores that has been calibrated, validated, and utilized extensively in studies of patients with iron overload. Only a limited number of the low-Tc SQUID susceptometersare currently in use, in part becausethese instruments are expensive and technically demanding to maintain and

operate. Nonetheless, the results obtained with the low-T, SQUID devices have validated magnetic susceptometry as a safe, noninvasive, and quantitative approach to the measurement of hepatic iron stores in patients with iron overload.

Future Prospects for Biomagnetic Susceptometry Recent technological advances may make possible improvements in some components of the biomagnetic susceptometer while greatly decreasing the cost of each unit. For example, inexpensive high-sensitivity magnetoresistive magnetometers that operate at room temperature are now available.ii3 These transducers respond to a change in magnetic field with a change in transducer resistance, causing a change in the output voltage of the transducer that is directly proportional to the change in magnetic field. The use of a room-temperature magnetoresistive sensor for the susceptibility probe instead of the SQUID detection coils has been proposed and is under investigations1 Another approach, also under investigation, is to continue to exploit the unique advantages of superconductivity (stability and sensitivity) but to develop an instrument in which costly and inconvenient liquid helium is replaced by inexpensive liquid nitrogen, whose use is also technically much less demanding. The current low-T, susceptometer (operating in liquid helium at 4’K) has 3 elements that use superconductivity: (1) the SQUID; (2) the field coils that produce a localized steady magnetic field near the liver; and (3) the detection coils and flux transformer. Technically, the replacement, redesign, and refinement of each of these low-Tc elements, cooled by liquid helium, is now possible with components able to function when cooled by liquid nitrogen. Recently, to provide “proof of principle,” we constructed and operated a prototype high-T, susceptometer (operating in liquid nitrogen at 77°K)37 with (1) a high-T, SQUID; (2) a NdFeB (neodynium-iron-boron) permanent magnet providing a strong localized magnetic field; and (3) detection coils and flux transformer fashioned from a high-T, Y,Ba,Cu307., film deposited on a flexible substrate.46 The use of

Anessment of Iron Overlodd

high-T, flux transformers would both reduce the distance separating the detector coils from the liver and allow addition of surface detector coils that could provide better discrimination between the liver and the tissues overlying the liver. The enhanced resolution of the high-T, susceptometer should further improve the accuracy, sensitivity, and specificity of magnetic measurements of the hepatic iron concentration while making the instrumentation inexpensive and technically simpler to operate. Still another approach is what might be called “magnetic resonance susceptometry,” the use of magnetic resonance instrumentation to measure magnetic susceptibility and determine the hepatic iron concentration. As discussed earlier, most attempts to use MRI have attempted to measure ferritin and hemosiderin iron indirectly by determining the effect of the paramagnetic iron on the proton resonance behavior of tissue water. The accuracy of this approach may be inherently limited becauseeach atom of ferritin or hemosiderin iron does not contribute equally to the proton resonanceeffects. By contrast, magnetic resonance techniques might be used to measuretissue magnetic susceptibility. As emphasized above, the change in magnetic susceptibility produced by the presence of (paramagnetic) storage iron is directly proportional to the number of iron atoms present in ferritin and hemosiderin, ie, each contributes equally to the change in susceptibility. Several techniques have been described with demonstrations of feasibility in vitro. One method uses the phase difference of magnetic resonance images to measuredifferences in susceptibility.131 Another method is to estimate the susceptibility of the liver by using the MRI field distortions in an external reference water bath placed next to a subject.T2 Another technique analyzes macroscopic susceptibility inhomogeneities to determine the susceptibility of a material relative to another reference material.ll Recently, still another magnetic resonance imaging method was described that probes the magnetic susceptibility difference between two adjacent homogeneous macroscopic compartments exploiting a resonant frequency discontinuity between the two regions.127One material with a known susceptibility (such as blood) may then be considered as a reference to obtain the susceptibility of the second

51

material (such as liver or heart). Although all of these approaches face substantial practical obstacles, their development will be facilitated by the widespread availability of magnetic resonanceinstruments. Further evolution of biomagnetic susceptometry would be possible with the development of magnetic susceptibility tomography43J10 to provide s-dimensional images and measurements of iron deposition not only in the liver but also in the heart, pancreas, thyroid, pituitary, and elsewhere. Although at present only a theoretical approach to this method has been advanced,llO with continued instrumental development, detailed examination of the distribution of iron stores in patients with iron overload could become feasible.

Conclusion At present the most promising approach to the noninvasive assessmentof body iron stores in patients with sickle cell anemia and transfusional iron overload is based on measurement of hepatic magnetic susceptibility. Biomagnetic susceptometry using low-Tc SQUID technology provides the only direct and noninvasive means of measuring hepatic iron stores that has been calibrated, validated, and used extensively in studies of patients with iron overload. General clinical use of this method has been retarded by the expense and technical demands of the instrumentation needed, but recent technological advances promise to decrease the cost and complexity of the equipment and make magnetic measurements of hepatic iron more readily available. Alternatively, means of using magnetic resonance instruments for susceptibility studies may be feasible and are under investigation. By whatever means, the development of affordable, readily usable instrumentation for the noninvasive measurement of hepatic iron would be a major advance in the diagnosis and management of patients with sickle cell anemia and others with forms of iron overload that would find immediate and widespread clinical use worldwide.

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