Indirect evidence for the potential ability of magnetic resonance imaging to evaluate the myocardial iron content in patients with transfusional iron overload

E L S E V I E R Magnetic Resonance Materials in Physics, Biology and Medicine 12 (2001) 153-166

MAGMA Magnetic Rt~')nance Materials in Physics, Biolo,r and M~icirm

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Indirect evidence for the potential ability of magnetic resonance imaging to evaluate the myocardial iron content in patients with

transfusional iron overload

P . D . J e n s e n ~,* , F .T . Jensen ~ , T. C h r i s t e n s e n c, L. H e i c k e n d o r f f b, L . G . J e n s e n b J. E l l e g a a r d ~

" Department of Hematology, Amtssygeluzset, Aarhus EOziversity Hospital, Tage Hansensgaa~, 2, DK-8000 Aarhus C, Denmark b Department o J Clinical Biochemistry, Aarhus Amtss3,gehus, Aarhus University Hospital, Aarhus, Denmark

Center Jor Nuclear Magnetic Resonance, Skejby 5),gehus, Aarhus Unit'ersity Hos)oital, Aarhus, Denmark

Received 5 May 2000; received in revised form 21 August 2000; accepted 7 January 2001

Abstract

The purpose of this study was to evaluate the potential ability of magnetic resonance imaging (MRI) for evaluation of myocardial iron deposits. The applied MRI technique has earlier been validated for quantitative determination of the liver iron concentration. The method involves cardiac gating and may, therefore, also be used for simultaneous evaluation of myocardial iron. The tissue signal intensities were measured from spin echo images and the myocardium/muscle signal intensity ratio was determined. The SI ratio was converted to tissue iron concentration values based on a modified calibration curve from the liver model. The crucial steps of the method were optimized: i.e. recognition and selection of the myocardial slice for analysis and positioning of the regions of interest (ROIs) within the myocardium and the skeletal muscle. This made the myocardial MRI measurements sufficiently reproducible. We applied this method in 41 multiply transfused patients. Our data demonstrate significant positive linear relationships between different iron store parameters and the MRt-derived myocardial iron concentration, which was significantly related to the serum ferritin concentration (p = 0.62, P <0.0001) and to the MRI-determined liver iron concentration (p =0.36, P =0.02). The myocardial MRI iron concentrations demonstrated also a significant positive correlation with the number of blood units given (p = 0.45, P = 0.005) and the aminotransferase serum concentration (/)= 0.54, P = 0.0008). Our data represents indirect evidence for the ability of MRI techniques based on myocardium/muscle signal intensity ratio measurements to evaluate myocardial iron overload. (�9 2001 Elsevier Science B.V. All rights reserved.

Kevwor&': MR imaging: Human heart: Hemosiderosis: Iron overload

1. I n t r o d u c t i o n

Deposition of iron within the myocardium is the most serious complication caused by multiple blood transfusions. If the iron is not removed by iron chelation, it leads to myocardial disease, a complication that shortens the life expectancy of patients with thalassemia major. However, non-thalassemic multiply transfused patients may also develop myocardial iron deposits as first reported by Buja and Roberts [1]. In 16 autopsy hearts from hematological patients, they found that

* Corresponding author. Tel." +45-894-97551" fax: + 45-894- 97599.

patients who had received more than 100 units of blood had extensive cardiac iron deposits, while patients who had received fewer blood units only had deposits if they also had hepatic fibrosis. Unfortunately, cardiac im- pairment caused by iron overload becomes clinically manifest only when left ventricular dysfunction and arrhythmia 's develop [2,3]. Electrocardiograms or rest- ing echocardiograms may be normal late in the course of myocardial disease, and are, therefore, not sufficiently sensitive for ear ly detection of iron-induced myocardial disease [4-6]. The most obvious way of detecting and monitoring myocardial iron deposits seems to be by direct assessment of the myocardial tissue iron concentration in endomyocardial biopsies.

1352-8661 01/$ - see front matter ::C? 2001 Elsevier Science B.V. All rights reserved. PII" S1352-8661(01)00112-0

154 P.D. Jensen et al./Magnetic Resonance ?,LJteria,ls in Physics, Biology and Medicine 12 (2001) 153-166

But due to possible complications, endomyocardial biopsy is only seldom performed, and repeated biopsy is not feasible for figllow-up investigations. However, such assessment has been performed in small series of patients [7,8]. Unfortunately, these studies, as well as post mortem studies of patients with transfusional iron overload [1,7,9] have shown that iron deposits are distributed inhomogeneously within the myocardium: The iron concentration differs between various regions of the heart, and i t tends to be lower in the endorriy- ocardial than in the epimyocardial part of the myocardial wall. A direct determination of the cardiac iron content by examination of endomyocardial biopsies may, therefore, be flawed by sampling error. Currently, the only non-invasive method potentially suitable for measuring myocardial iron in various clinical settings seems to be magnetic resonance imaging (MRI). This technique allows quantification of the hepatic iron concentration in a way that is sufficiently precise and reproducible for clinical use [10-12], but until now, only very few data have been published concerning the use of MRI for quantification of cardiac iron. Apart from the study of Mavrogeni et al. [13], only a few clinical case reports seem to indicate that changes in myocardial T2 relaxation time and the spin echo image signal intensity (SI) expressed as a ratio of cardiac to skeletal muscle might parallel developments in cardiac iron content [14,15]. Mavrogeni et al. [13], found a significantly higher heart T2 relaxation time in controls than in a group of iron loaded ~-thalassemic patients. Unfortunately, the T2 relaxation time calculation was impossible in severely iron-overloaded patients (25% of all patients) due to very low signal intensity which was similar to background noise. In contrast, the heart/ muscle SI ratio was measurable in 94% of the patients and was also significantly different for patients and controls. In none of the mentioned studies, an approach of validating the applied MRI technique is given, but recently, the relationship between tissue T2 relaxation rate (I/T2) and the cardiac iron concentration was studied in thalassemic iron overloaded mice, demonstrating a close linear correlation between l/T2 heart and the cardiac tissue iron concentration [16].

We have recently presented a validated MRI technique for quantification of the hepatic iron concentration [11]. This technique involves ECG-gated spin echo sequences, thereby offering a possibility for a simultaneous evaluation of hepatic and cardiac iron content by measuring heart/muscle and liver/muscle SI ratios. With this method we have monitored the hepatic iron concentration during iron chelation therapy in patients with transfusional iron overload [17] and during a venesection program in patients with hereditary haemochromatosis [I 1].

The aim of the present study was to investigate, if our MRI method, validated for quantitative determina-

tion of hepatic iron, could also be useful for evaluation of the myocardial iron concentral.ion after optimizing the image analysis for the myocardial wall. As direct validation of this method fi~r quantification of cardiac iron is not feasible because reliable chemical determinations of the cardiac tissue iron concentration cannot be obtained by endomyocardial biopsies, we investigated the relationship between the myocardial MRI values and different iron store related parameters (ferritin-, iron-, transferrin serum concentration; transferrin iron saturation, number of blood units given) in multiply transfused patients in order to validate the potential ability o f M R I for evaluation of myocardial iron indirectly. Moreover, we wanted to convert the heart/muscle SI ratios to tissue iron concentrations in order to improve the interpretation of the MR! values and we also wanted to investigate the repeatability and the variability of the MRI-derived cardiac iron concentration measurements within the myocardial wall (endo- and epimyocardial layers) and between different slices. A further aim was to investigate, if clinical characteristics (biochemical liver status, iron store status, hepatitis C status, presence of mutations in the HFE gene caus- ing hereditary hemochromatosis) were of significance Ibr the myocardial iron concentration in order to study if clinical characteristics may delineate subgroups of patients with outstanding myocardial iron deposits compared with the number of blood units given.

2. Patients and methods

2. I. Patients

Our study was approved by the local ethical commit- tee. It included 42 consecutive blood transfusion-dependent hematological, non-thalassemic patients (15 women, 27 men) with various degrees of transfusional iron overload (5-240 blood units), who had never received iron chelation treatment and who had had no episodes of significant blood loss. One patient was excluded because of possible urinary iron loss (paroxys- mal nocturnal hemoglobulinuria), leaving 41 evaluable patients and 15 normal controls. Twenty-seven of the patients had a confirmed diagnosis of myelodysplastic syndrome at various stages. Three patients had aplastic anaemia, one had red cell aplasia, tour had acute myeloid leukemia in complete remission after au- tologous bone marrow transplantation, and one had chronic hemolysis for unknown reasons. Finally, two patients had chronic myeloid leukemia, one had chronic lymphatic leukaemia, one had chronic myeloprolifera- tive syndrome, and one had myelofibrosis. Of 41 patients, 38 were investigated for the C282Y mutation and the H63D variant within the HFE gene, round in up to 90'!,/,, of patients with hereditary hemochromatosis [18],

P.D. Jensen et al./Magnetic Resonance Materials" in Physics, Biology and Medicine I2 (2001) 153-166 155

because the deposition of transfusional iron within the myocardium might be increased in these patients. The analyses were performed by PCR based restriction frag- ment length analysis [18]. In three cases, no material was available for analysis. We neither found patients who were homozygous for the C282Y mutation nor compound heterozygous for the C282Y mutation and the H63D variant, indicating that no patients with primary hemochromatosis were included in this study. We did, however, observe seven patients heterozygous for H63D (18.4%) and two patients for C282Y (5.3%). Table 1 summarizes further characteristics of the patients.

2.2. MRI techniques ~

We used a sequence giving T 1-weighted images at normal and slightly elevated tissue concentrations but predominantly T2-weighted images at highly elevated iron concentrations [19], in order to improve the sensi- tivity for the quantification of low grade iron overload. All patients were studied with a Philips Gyroscan, S 15-HP, operating at 1.5 T. Images were obtained using the following ECG-gated spin echo sequence, Echo time (TE) 25 ms, repetition time (TR) equal to the heart rate (500-960 ms). FOV = 450 mm 2 matrix = 256 x 256 pixels. The daily calibration of the MRI system com- prised control of resonance frequency of the system and test of the signal-to-noise ratio using a performance phantom. The total acquisition time for one complete study was 12-15 min. Oblique images (15-25 slices, with a slice thickness of 8 mm) were recorded, in order to visualize the left ventricle, the right liver lobe and the posterior vertebral muscles in the same slice [20]. The oblique orientation of image slices was determined from coronal anatomical images. The mean signal intensity (SI) for liver tissue (Sic), myocardium (Sin) and skeletal muscle (SIM) was obtained with the use of operator

Table 1 Clinical characteristics of the patients (N = 41)~'

Blood units 65 Serum ferritin concentration 2380

(~tg/I) Serum iron concentration 38

(p.mol/1) Liver iron concentration (gmol 401

Feig dry tissue) Serum ALAT concentration 56

(t0-/i) Serum ASAT concentration 31

(U/l) Age Anti-HCV antibody positive

54 One of 38 investigated patients

(5-240) (193- I 0 400)

(11-57)

(39-626)

(9-270)

(14-101)

(18-79)

~' Values shown are median, and range (in brackets).

defined regions of interest (ROIs) which were always greater than 50 pixels. A SI-ratio was calculated between the SI of the tissue of interest and the skeletal muscle, used as an internal standard. The use0alness of the skeletal muscle has been documented earlier [11,13].

2.3. Image analysis for determination of the liver iron concentration

For the measurements of SI L, a slice was chosen located one slice above the slice visualizing the upper border of the right kidney. The SIM of the paraspinous muscle was measured in the same slice. Afterwards the SIc/SIM ratio was calculated and the SIL/SIM ratio was adjusted for variable values of TR as described by Jensen et al. [11]. Our validation experiments have shown a tight linear inverse and semi-logarithmic relationship (Re _ _ 0.98 P < 0.0001) between the SIc/SIM ratio and the liver iron concentration determined by chemical analysis of liver biopsies within the range 5-650 l.tmol Fe/g dry weight [11]. The reference range is 1-15 (mean +_ 2S.D.)p, mol Fe/g dry weight. The inter- recording variation between different days (normal controls) is 2.9 +_ 2.7 (mean + S.D.) I.tmol Fe/g.

2.4. hnage amtlysis Jbr determinatiotz oJ" the cardiac iron concentration

Sin values of the myocardium were measured using the same set of images as those used for the measurement of the Sit. values, but different slices. Using our oblique plane imaging protocol, we always had at least tour slices (slice 2-5) displaying the left ventricle wall that could be candidates for choice of ROIs. In order to perform the image analysis on the same slice in all patients, a 'key slice' had to be detected (see Appendix A), allowing the enumeration of the relevant slices. The myocardial ROI (HI, Fig. 1) was placed within the lateral wall of the left ventricle. The skeletal muscle ROI (M7, Fig. 1) included the left paraspinous muscle and a section of m. latissimus dorsi. The selection of the most appropriate ROIs to be used for the image analysis was based on the determination of the ROIs with the smallest coefficient of variation within 15 normal controls (see Appendix A). SIH/SIM ratios were calculated for slice 2-5. Correction for variable TR was not performed. The SI ratios were converted to iron concentration values based on a modified calibration curve from the liver model (see Appendix A).

2.5. Data analysis

All data sets were analyzed for compatibility with the normal distribution by the Kolmogorov,Smirnov good- ness-of-fit test (Lilliefors modification). Unless other- wise indicated, normal data are given as mean _+ S.D.

156 P.D. Jensen et al./Magnetic Resommce Jd%lteriaA" 1)7 Physics, Biology and Medicine 12 (2001) 153-166

dependent variable not explained by the independent variable was normally distributed with constant variance (studied in residual plots). Spearman's rank correlation coefficient (p) was calculated in case of non-normality. Relationships between subgroups of patients and paraclinical findings (ferritin-, iron-, transferrin serum concentration; transferrin iron saturation; number of blood units given and liver iron concentration) were studied by one-way analysis of variance (ANOVA). Fisher's Protected Least Significant Differ- ence (PLSD) posthoc test was performed to compare subgroups. Wilcoxon's signed rank test or Mann- Whitney U-test was used when comparing data between groups in case of non-normal distribution. All statistical tests were two-tailed. The level of significance was 0.05. Statistics were performed by the Stat View 5.0 software for Macintosh (1992-1998 SAS Institute Inc., USA).

Fig. 1. (a) Representative slice 3 in a normal control. The slice displays the left ventricle as a ring (white lines) with constant wall thickness all around. The enumeration of the slices is based on the recognition of this 'key slice'. (b) Slice 2 of the same normal control used tbr determination of the cardiac iron concentration, displaying the cardiac ROIs H 2 - H 4 and the skeletal ROIs M 1 - M 6 . (c) Slice 2, displaying the cardiac ROIs H1, 5 and 6, and the skeletal ROI MT. The ROI H1 includes the areas of ROI H5 and H6.

and were compared by the use of paired or unpaired Student t-test. Simple linear regression analysis (least square method) was pertbrmed if the proportion of the

3. Results

3. I. Methodological aspects

After conversion of the myocardial S I H / S [ M ratios to iron concentrations (data given for slice 2), we found a wide range of the cardiac iron concentrations fi'om 1.3 to 93.0/amol Fe/g (mean, 14.5 _+ 15.3 ~tmol Fe/g) within the 41 patients and a small range from 1.1 to 6.7/arnol Fe/g (mean, 3.7 +_ 1.5 btmol Fe/g) within the 15 normal controls. The myocardial iron concentration was significantly higher in the patients than in the normal controls (P <0.0001, Mann-Whitney U).

3.2. Variation in iron content between slices oj" the myocardium

The cardiac iron concentration for slices 2-5 in all 41 patients is displayed in Fig. 2. The data are shown log-transformed in order to obtain a normal distribution and were then divided into two concentration intervals ( _< 8 and > 8 taInol Fe/g), in order to facilitate discrim.ination between the individual curves at low and high iron concentrations. Fig. 2 reveals considerable individual variation in the concentration values between the slices in several patients (up to 40 btrnol Fe/g). At low cardiac iron concentrations (Fig. 2a), there is obviously no difference of the log-transformed cardiac mean iron concentration between slices, but the spread of the iron concentration values clearly increases with the slice number (0.61 _+ 0.23, 0.70 4- 0.31, 0.69 + 0.38, and 0 .7_ 0.47 lamol Fe/g). For high myocardial iron concentrations (Fig. 2b), the statistical comparison of the means of the log-transformed cardiac iron concentration of all tour slices with each other revealed a significantly lower iron concentration in slice 4 than in

P,D. Jensen et al . / Magnetic Resonance MateriaLs in Physics, Biology amt Medicine 12 (200I) 153--166 157

slice 2 (P = 0.015, paired t-test) but no significant dif- s between the other slices. The spread of the cardiac iron concentration values was approximately the same in the different slices (1.23 _+ 0.26, 1.16 +_0.30, 1.04_ 0.27, and 1.14 +_ 0.33 l.tmol Fe/g). In 15 normal controls the mean cardiac iron concentrations in slices

2-5 were not significantly different (3.7 +_ 1.5, 3.6 _+ 1.8, 4.0 +_ 2.5, and 4.5 +_ 2.4 btmol Fe/g, not log-transformed values), but the spread of the iron concentration values seemed also to increase with the slice number.

3.3. Repeatabilio; of the myocardial iron determination

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Fig. 2. Variation of the MRI-derived cardiac iron concentration between 4 adjacent slices (slice 2-5) within two concentration intervals: (a) < 8 I.tmol Fe/g; (b) >_ 8 btmol Fe/g.

We investigated the significance of placing the ROIs within the myocardial wall and within the skeletal muscle for the repeatability of the myocardial iron measurements. The repeatability was assessed by calcu- lating the repeatability coefficient (twice the S.D. of the differences between two repeated measurements). The same trained operator repositioned the ROIs within slices 2-5 in all 41 patients and 15 normal controls in random order. For slices 2-5, the coefficients were 4.6, 15.4, 8.2 and 12.8 l.tmol Fe/g in the patients and 1.7, 1.6, 3.4, 2.4 lamol Fe/g in the normal controls. These coefficients indicate that the repeatability was dependent on the slice number in the patients, but not in the normal controls. In the patients the repeatability was best in slice 2. For this slice, a plot of the difference between two repeated measurements and their mean is given (Fig. 3). We then studied the repeatability of the cardiac iron concentration determinations in nine normal controls, when repeating the whole MRI examination at different occasions (Fig. 3c). The repeatability coefficient was lowest for slice 2 (4.4 ~tmol Fe/g), but only slightly larger for slices 3-5 (5.2, 6.4 and 6.0 }.tnlol Fe/g).

3.4. Di/.]et'ences betweelz the endomyocardial and eT)hl~yocara'ial regions

The homogeneity of the iron distribution within the same slice of the myocardial wall was studied by ana- lyzing slices 2-5 in all patients by dividing the ROI H1 into an epimyocardial and an endomyocardial half (Fig. 4). The epimyocardial iron concentration was significantly higher than the endomyocardial iron concentration in slice 2, 3 and 4 (P=0.003, 0.0007 and 0.01, Wilcoxon Signed Rank Test). Slice 2 was the slice in which most patients had the highest epimyocardial iron concentration (31/41 =75%), being up to 75% higher than the endomyocardial concentration (mean 33 _ 18%). Also, in the normal controls, the epimyocardial iron concentration was significantly higher in slice 2-5 (P=0.005, 0.005, 0.001 and=0.004 Wilcoxon Signed Rank Test).

3.5. Clinical resuhs

3.5. I. Cardiac iron and number of blood units given In the clinical results, the cardiac iron concentration

is obtained from the mean of two repeated positionings of the ROI H1 performed in random order by the same

158 P.D. Jensen et al. / 'Magnetic Resonance Materials in Physics, Biology and Medicine I2 (2001) 153-166

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Fig. 3. Repeatability of the MRl-derived determination of the cardiac iron concentration illustrated by scatter plots of the difference between two measurements against their mean. (a) Repeatability of positioning the ROIs H1 and M7, investigated in 41 patients and in 15 normal control (b). (c) Repeatability when repeating the whole MRI examination at a later occasion in nine normal controls.

trained operator. The patient with the highest cardiac iron concentration (93 ~.mol Fe/g) had received most blood (240 units), but the cardiac iron concentration was normal in the patient who had received least blood (5 units). Fig. 5 illustrates the relationship between the MRI-derived cardiac iron concentration and the number of blood units given divided into four clinically relevant groups (1 - 50, 51 - 100, 101-150, > 150 blood units) and the normal controls for comparison. Simple linear regression analysis revealed a significantly positive overall relationship between the log-transformed cardiac iron concentration and the number of blood units given (as continuous independent variable) ( R 2 -

0.27, P = 0.005).

3.5.2. Relationship between cardiac iron and iron status parameters

The investigation was performed for slices 2-5 separately (Table 2). We found a significant positive

overall correlation between the serum ferritin concentration and the MRI-derived cardiac iron concentration for all slices, but the level of statistical significance was highest for slice 2 (p = 0.62, P < 0.0001, Spear- man's o). For the same slice, the liver iron concentration, the number of blood units given and the transferrin iron saturation, but not the serum iron concentration, were all positively and significantly correlated with the cardiac iron concentration, but at lower levels of significance than the serum ferritin concentration. These relationships are displayed in Fig. 6. An inspection of the scatter plot of the serum t~rritin concentration plotted against the log-transformed cardiac iron concentration (Fig. 6a) reveals that the linear relationship between both variables only ex- ists for cardiac iron concentrations above the normal range. A separate linear regression analysis showed a tight, positive and linear relationship ( R 2 = 0 .68 , P < 0 .0001) .

P.D. Jensen et al./ Mqgnetic Resonance Materials in Physics, Biology and Medichw 12 (2001) 153-166 159

3.5.3. Cktrdiac iron and liver-related parameters"

A significant, positive and linear relationship between the cardiac iron concentration and the aminotransferase concentration in serum (ASAT) was found for cardiac iron concentrations above the upper reference limit (R 2 =0.31, P<0 .0001) (Fig. 6b). Moreover, we found a significant inverse relationship between the transferrin serum concentration and the cardiac iron concentration (R2= -0 .18 , P = 0.026) for concentrations above the normal range (Fig. 6c).

3.5.4. Cardiac iron and H F E gene status

Patients being heterozygous for the mutations C282Y or H63D did not have significantly higher myocardial iron concentrations .,than patients without these mutations, neither when the cardiac iron concentration was corrected for the number of given blood units (cardiac iron/the number of blood units) (Wilcoxon's Signed Rank test).

3.5.5. Relationship between cardiac and liver iron

concentrations

We found a significant positive linear overall correlation between the log-transformed myocardial iron concentration and the liver iron concentration. This correlation was only of moderate strength (p = 0.36, P = 0.02). However, inspection of the scatter plot (Fig. 7) reveals two well-separated subgroups (A and B) f\~r

cardiac iron concentration values above the upper limit of the normal range (group C). Within these subgroups regression analysis revealed close, positive, linear and significant relationships between the cardiac and the liver iron concentrations ((A) R 2 = 0.93, P < 0.0001; (B) R 2= 0.40, P = 0.002) with different intercepts, but similar slopes. Calculations based on the regression lines show that the cardiac iron concentration in group A patients is from 2.5 to 6 times higher than in group B patients and up to 40 times higher than in group C patients at comparable liver iron levels. The different extent of myocardial iron deposits compared with the liver iron concentration between the subgroups could not be ascribed to significant differences between the subgroups concerning the underlying hematological disease, hepatitis C status or HFE gene status. Moreover, the ANOVA test disclosed no systematic differences between the groups concerning the iron status (serum iron, serum transferrin iron saturation, liver iron, number of blood units or serum ferritin) or liver status (ALAT, ASAT). However, the calculated serum ferritin-to-liver-iron concentration ratio (Fig. 7b) was significantly higher in group A than in group B and group C (P=0.0002, and 0.0005, Fisher's PLSD) and the serum ASAT-to-liver-iron concentration ratio was also significantly higher in group A than in group B and C (P =0.03 and 0.01, Fisher's PLSD).

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Fig. 4. Comparison of the MRI-derived epimyocardial and the endomyocardial iron concentration investigated by use of the ROI H-1 and M-7 for slices 2-5. The differences between the epimyocardial and endomyocardial half of ROI H-1 are expressed in percent of the epimyocardial iron concentration N = 41. Positive values indicate higher epimyocardial iron concentrations.

160 P.D. Jensen et a l . / Me<q-netic Resommce Materials in Physics, Biolog3, amt Medicine 12 (2001) 153-166

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Fig. 5. Relationship between the MRI-determined cardiac iron concentration (data t\~r slice 2) and the amount of blood units transt;ased in 4I patients with transfusional iron overload. Horizontal bar represents upper limit ot" normal range (6.7 btmol Fe/g).

4. Discussion

4.1. Selection oj" the spin-echo sequence

The presence of iron in the tissue affects the T1 and T2 relaxation times [12,20]. This effect is depen-

dent of the magnetic field strength and of the tissue iron concentration. In most studies the T2 relaxation time, respectively, T2-weighted images have been used for characterization of tissue iron, because the effect of iron on T2 relaxation time is regarded to be stronger than the effect on T1 relaxation time. How- ever, data from recent studies performed on large groups of iron-loaded patients [12,22] by use of gradi- ent echo sequences seem to demonstrate that T1 relaxation is more sensitive at lo;~v tissue iron overload than T2 relaxation, while T2 relaxation is more sensitive at high tissue iron concentration. These data are in agreement with the results of earlier in vitro MR studies on ferric (Fe 3+) solutions [19], showing a more rapid decline in T1 relaxation time than T2 relaxation time for low concentrations of ferric iron. Due to these observations, we used a spin echo sequence for MR-derived quantification of iron within the heart and the liver, generating T 1-weighted images at normal and slightly elevated tissue iron concentrations, but generating predominantly T2-weighted images at high tissue iron concentrations. We use this sequence in order to increase the sensitiv- ity of our method for the quantification of low-grade iron overload compared with methods using exclu- sively T2-weighted images. The disadvantage of this sequence is an iron concentration dependent influence of" TR on the SI ratio. For liver tissue we have shown that this influence of TR on the SIL/SI M is small or absent at hepatic iron concentrations around the normal range but increasing with rising liver tissue iron concentrations [11]. We have developed an algorithm for the correction of SIL/SIM for variable TR to be used for the determination of the liver iron concentration. The iron concentrations fi~und within the myocardium for patients with iron overload are low compared with iron concentrations in the liver (up to 15 times, Buja and Roberts [1]). Therefore, we did not expect and did not find a significant correlation between TR and the SIH/SIM ratio. Accordingly, a correction of the SIH/SIM ratio for variable TR was not developed.

Table 2 Relationships between iron status parameters and the MRI-determined myo-cardial iron concentration within different slices ~'

Slice 2 Slice 3 Slice 4 Slice 5

Serum fierritin (btg/1) 0.62 <0.0001 0.49 0.002 0.32 0.05 0.32 0.05 Serum iron (I.trnol/1) - 0 . 14 0.36 0.02 0.91 0.13 0.44 0.06 0.71 Liver iron (gmol Fe/g) 0.36 0.02 0.21 0.19 0.22 0.18 0.26 0.11 Serum Tf (pmol/1) - 0 . 5 4 0.0006 -0 .32 0.05 -0 .18 0.27 -0.11 0.51 Iron saturation (%) 0,39 0,01 0.39 0,01 0.40 0.01 0.29 0.06 Blood units 0.45 0,005 0.28 0.08 0.25 0.12 0.33 0,04 Serum ALAT (U/l) 0.46 0.004 0.52 0.001 0,28 0.08 0,30 0.02 Serum ASAT (U/l) 0.54 0,0008 0.53 0.0009 0.31 0.05 0,39 0.02

~' Correlation analysis by Spearman's p. Values of p, left columns: P-values, right columns. Image analysis by use of the ROIs HI and M7.

P.D. Jensen et al. ,,'Magnetic Resonance Materials in Physics, Biology and ktedichTe 12(2001) 153 ...... 166 161

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Fig. 6. Linear regression analysis of relationships between the MRI-derived cardiac iron concentration and iron status and liver-related parameters and inter-relationships between these parameters in 41 patients with transfusional iron overload. Data points marked as rectangles represent cardiac iron concentrations within the normal range (0.7-6.7 ~tmol Fe/g). They are excluded from regression analysis.

4.2. ;STceletal muscle as an internal s tandard

Like others (e.g. [12,13,21,23]), we use skeletal muscle as an internal standard. In order to examine the usefulness of the skeletal muscle as an internal standard, we

have earlier measured the T2 and T1 relaxation times [11] for the skeletal muscle in iron-loaded patients with liver iron concentrations up to 600 lxmol Fe/g. The relaxation times were within the reference range, and no significant correlation with the liver iron concentra-

162 P.D. Jensen et al./Magnetic Resonance Materials in Physics, Biology and Medicine 12 (200I) I53-166

tion or the serum ferritin concentration could be estab- lished. Recently, Mavrogeni et al. [13] have also demonstrated normal T2 relaxation times for skeletal muscle in 54 iron-loaded thalassemic patients. No correlation was documented between T2 of the skeletal muscle and the serum t:erritin levels nor between the number of transfusions or iron-chelation time. However, it cannot completely be excluded that iron is deposited within the skeletal muscle in patients with very severe iron overload.

4.3. Selection of the R OI within the no, ocardial wall

The known inhomogeneity of the myocardial tissue and of the myocardial iron concentration made it nec- essary to empirically optimize the location of the RO! within the myocardial wall. First, we found a way of

recognizing and enumerating the relevant MRI slices. This procedure was not unequivocal in all patients, possibly due to small differences in the positioning of the patient in the scanner, or due to anatomical varia- tions. We then investigated the significance of the position of the ROI within the left ventricle wall and within the skeletal muscle in the normal controls and found which combination yielded the least variation of the calculated SI ratio between myocardial tissue. It could of course be asked, why the ROI with the smallest coefficient of variation should best represent the inho- mogenous distribution of iron within the myocardium. Unfortunately, the information in the literature concerning the local distribution of the iron deposits within the myocardium is not detailed enough to tell us, if the position of the RO! within the myocardial wall we have selected may give a reflection of the myocardial iron

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Fig. 7. (a) Relationship between the cardiac and hepatic iron concentration determined simultaneously by MRI in 41 patients with transfusional iron overload. Inspection of the scatter plot reveals two well-separated patient groups (A and B) outside the normal range (group C). Linear regression analysis is pert\~rmed for subgroup A and B. (b and c) Comparison of the mean (_+ S.E.M.) of the serum ferritin-to-liver-iron concentration ratio (b) and the mean (_+ S.E.M.) of the serum ASAT-to-liver-iron concentration ratio (c) between the subgroups A, B, C performed by Fischer's PLSD post hoc test. Two patients (marked by arrows) were excluded from the statistical analysis as they had the highest serum ASAT-to-liver-iron concentration ratios of all patients, although they had received fewest blood unit.

P.D. Jensen et al. / Magnetic Resonance M'aterl, als in Physics. Biology and Medicine 12 (2001) 153-166 163

concentration representative for the whole left ventricle. However, the ROI with the smallest coefficient of variation, which was determined within normal control, has an obvious advantage: It represents the ROI with the smallest inter-individual variation of the SIH/SIM ratio within the controls and will, therefore, represent the ROI with the lowest level of background noise due to other factors than iron, affecting the relaxation process. It may, therefore, be the best ROI to discriminate between normal and slightly elevated iron concentrations. The reason for why one ROI had least variation is not clear but may be due to anatomical differences of the myocardial tissue within the compared ROIs.

juvenile hereditary hemochromatosis undergoing heart transplantation [24]. This patient had very severe cardiac iron depositions. MRI showed a distinct dark ring formation corresponding to the left ventricular epimyocardial layer representing a region of high iron content. Visible epimyocardial ring formation was also encountered in some of our most iron-loaded patients, but the phenomenon was much more discrete due to lower iron concentrations. The reason for the dominat- ing epimyocardial iron deposition remains unexplained.

4.6. Conversion of the Sl~/Slivt ratio to the myocardial iron concentration

4.4. Variation of the iron concentration between slices

This variation was recognized as an important prob- lem when assessing the cardiac iron by our MRI technique. Only a minor part of this variation can be explained by the intra-observer variation in positioning the ROIs, or by the inter-recording variation of the scanner. Our data show that slice-related differences of the inter-individual variability of the myocardial iron values are not only detectable in normal controls but also in the patients with myocardial iron concentrations around the normal range. A significant difference of the mean cardiac iron concentration between slices (slice 2 and 4) was only found t\~r patients and only at high myocardial iron levels. As mentioned before our data do not shed light on why the slice 2 displays the lowest variability. But as increasing variability of the iron concentration values by the slice number was observed both in normal controls and patients, the finding cannot be related to the distribution of iron. The significantly higher iron concentration in slice 2 than in slice 4 may be explained by local inhomogeneity of the myocardial iron deposition within the tissue, but tissue inhomogeneity secondary to iron overload (e.g. fibrosis) also may be of importance. Our data point at the recognition of the proper slice as a crucial step. Improv- ing this step, for example by improved image quality may improve the precision of the method even further. Another possible approach to overcome the differences in slice-dependent iron concentrations could be the application of a RO! covering the whole left ventricle wall.

4.5. h'on concentration d'ij/]?rence across the myocarctial wal l

Our finding of a higher epimyocardial than endomyocardial cardiac iron concentration is in accordance with an earlier histological study [1,7]. A similar distribution of iron within the myocardium has also been reported in iron overload due to hereditary hemochromatosis [9]. Recemly, we reported the first case of

As standard endomyocardial biopsy techniques do not seem sufficiently reliable for a direct calibration of MRI scanners for quantitative determination of cardiac iron, we decided to use our own MRI-based method, which has been validated for the quantification of the liver iron concentration. After optimizing the image analysis for the myocardial wall, the reproducibility of the cardiac iron concentration measurements seem to be acceptable for clinical use. In order to make the interpretation and the comparability of the myocardial SIH/SIM ratio values easier we used the equation describing the relationship between SIu/SIM ratio and the chemically determined liver tissue iron concentration [1 I] for the conversion of the SI~/SIM ratio values to cardiac tissue iron concentrations. When doing so, we assumed that the general pattern of the relationship between the tissue iron concentration and relaxation properties in liver tissue and myocardial tissue is the same. Although this has been shown in a murine model [16], we have no proof for this assumption in humans. Moreover, the need for a correction factor to adjust the MRI derived cardiac iron concentration values within the controls to the chemically iron concentrations measured in normal controls from. the autopsy does not support our assumption. The chemically determined normal range of the myocardial iron concentration was only based o n 5 autopsy patients. The number of controls is limited and may have to be expanded. Moreover, it cannot be excluded that there may be a difference in the tissue iron content in-vivo and post mortem. However, the myocardial iron concentrations we found in our controls are close to the concentrations t:ound in earlier autopsy studies [9,25]. Despite all those problems, the obtained myocardial iron concentration values in our patients spread within the expected range suggesting that the assumption still could be reason- able. Thus, the range of the MRI-based cardiac iron concentrations in our patients (1.3-93 J.tmo1 Fe/g), who had received from 5 to 240 blood units, is very similar to the results of Buja and Roberts [1], who found from 9 to 87 j.tmol Fe/g in autopsy hearts of patients who had received from 65 to 359 units.

164 P.D. Jensen et al./Magnetic Resonance Materials" in Physics, Biology aml MedichTe 12 (2001) 153-166

4. 7. Myocardial iron concentration and the serum f erritin concentration

We found a significant overall relationship between the serum ferritin concentration and the log-transformed cardiac iron concentration. The coefficient of determination (R 2) was 0.45, which indicates that approximately half of the variation in the cardiac iron concentration can be accounted for by measuring the serum ferritin concentration. The relationship was even tighter at cardiac iron concentrations outside the normal range. The relationship is clearly stronger than in the study of Mavrogeni et al. [13], who found a R 2 at 0.25 for the linear overall relationship between serum ferritin and the myocardial SIn/SIM ratio, although both studies are conducted at 1.5 T systems, using comparable MRI techniques. The improved image analysis in our study may be one reason for the stronger relationship. The strong relationship is an important finding t;or the indirect validation of the method, indicating a connection between ferritin syn- thesis in response to iron overload and the myocardial iron content. The relationship between the MRI- derived cardiac iron concentration and the other iron parameters were clearly weaker than that of" serum ferritin. However, these observations are also important, because they also represent further indirect evidence I:or the potential ability of MRI to evaluate the cardiac iron concentration.

4.8. Myocardial iron concentration and the total ntmTber o/" blood units given

When looking at the relationship between the nun> ber of blood units given and the cardiac iron concentration we found the same pattern of a significant linear relationship only for elevated cardiac iron concentration values, which is quite different to the relationship between the liver iron concentration and the number of blood units which is tight and semi-logarithmic over the whole range of blood units. These observations may reflect the Pact that the liver is a natural iron storage organ, while the heart is not.

4.9. Myocarah'al iron concentration and the liver iron concentration

The finding of two well-defined distinct groups of patients outside the normal range displaying a close positive linear relationship between the cardiac and the liver iron concentrations is of clinical interest because it delineates a subgroup of patients with increased risk of myocardial iron loading compared with the burden of blood transfusions given. Unfortunately, we were not able to identify the responsible risk factor(s), but it could be excluded that differences in the underlying

hematological disease or the treatment status, HFE genotypes or hepatitis C antibody status were of importance. The groups did not display any significant differences in iron storage parameters, but the serum ferritin-to-liver-iron and the serum ASAT-to'liver-iron concentration ratios were significantly higher in group A than in groups B and C. These findings may suggest that the hepatocytes of patients in group A are more sensitive to iron toxicity than the hepatocytes of patients in the other groups, and that the degree of liver cell damage may promote the myocardial iron-loading. Such a hypothesis is also compatible with the demonstrated significant relationship between the cardiac iron concentration and the ASAT serum concentration in our patients. Furthermore, the data support earlier observations suggesting that hepatic cirrhosis is a clinical feature that identifies myocardial siderosis [1,8]. Our finding of a significant inverse correlation between the serum transferrin concentration and the cardiac iron concentration may also support the hypothesis that the iron loading of the myocardium depends on the liver function. Almost all transferrin is synthesized by the liver, which plays a central role in protecting tissues from iron toxicity by rapidly clearing non-transferrin- bound iron from the plasma [26]. Unfortunately, the liver histology has not been studied in our multi-transfused patients.

Overall, our data suggest indirectly that myocardial MRI values obtained by use of the described method may reflect the myocardial iron concentration encountered in patients with transfusional iron overload. It should, however, be remembered that the method is not calibrated directly and the iron concentration values may not be identical to the true myocardial iron concentration. However, the method seems to be sufficiently repeatable to warrant the clinical use, e.g for monitoring the cardiac iron content during iron chelation. Additional evidence is needed to document the clinical value of the method, for example by document- ing a normalization of myocardial MRI values during iron chelation for patients with elevated myocardial iron levels. Although our method may need further improvement, the presented results may encourage others to expand on the usefulness of this technique.

Appendix A. Appendix for method optimization

Enumeration of the slices: using our oblique plane imaging protocol, we always had at least five slices displaying the left ventricle wall that could be candidates for choice of ROIs. By studying the shape of these slices obtained from 200 MRI examinations of the heart, we looked for a slice being easily recognizable in all patients. This slice was represented by the first slice encountered when looking at the relevant slices in

P.D. Jensen et al. /Magnetic Resonance Materials in Physics, Biology amt Medicine 12 (2001) 153-166 165

descending succession showing the left ventricle as a ring of almost equal wall thickness all around (Fig. la). After recognition of this 'key slice' (slice number 3), the other slices (number 2-5) could be enumerated. We studied the repeatability of recognizing this slice (by the same trained person in random order). We found agreement between the first and second recognition in 36 of the 41 patients.

Positioning of the ROIs: the position of the myocardial and skeletal ROIs within a slice was optimized by determining that combination of the myocardial and the skeletal ROI which yielded the SIw/SIM ratio with the smallest coefficient of variation in the normal controls. For this purpose we designed six different ROIs placed within the w.all of the left ventricle and seven different ROIs within the skeletal muscle (Fig. l b and c). The SI~/SIM ratio based on the ROIs H1 and M7 had the smallest coefficient of variation (9%) out of 42 possible ratios.

The significance of the repetition time (TR): we exam- ined the relationship between the SIH/SIM ratio and TR ['or slices 2--5 in our patients (N=41) and in the normal controls ( N = 15)by use of linear regression analysis. We found no significant relationship between the SIH/SIM ratio and TR, neither in the patients nor in the normal controls in any of the slices (data not shown). Accordingly, a correction of the SI~/SIM ratios for variable TR was not performed.

Conversion of cardiac SIH/SI~ ratio to cardiac tissue iron concentrations: a study on a murine thalassemia iron overload model suggests similar linear relationships between the tissue iron concentration and relaxation parameters in liver tissue and myocardial tissue [16]. We assumed that the general pattern of the relationship is also the same for human liver and cardiac tissue and used the same semi-logarithmic regression curve that we use routinely for the conversion of SIL/ SIM ratios into liver iron concentrations, describing the relationship between the SIL/SIM ratio and the chemically determined liver iron concentration [11]. Accord- ingly, the cardiac iron concentration (CIC) could be calculated as:

CIC [/lmol Fe/g dry tissue]

= ( l0 A (SIH/(SIM/k) x TR) - 493"8) --_ ~ . ~ x 100

TR = To = 684 ms. The value of the correction i~tctor (k = 1.55) was adjusted empirically until a match was obtained between the distribution of the MRI-determined cardiac iron concentration values of 15 healthy controls (mean, 3.7 +_+ 1.5 p.mol Fe/g, ranging from 1.1 to 5.7 larnol Fe/g) and the distribution of the chemically determined cardiac iron concentration found in five normal autopsy controls (mean, 3.8_+ 1.0 gmol Fe/g ranging fi'om 2.8 to 5.3 lamol Fe/g). The controls

(39-83 years at age) had died fi'om non-hematological diseases and had never had any blood transfusions. None of these patients had recently been on oral iron supplementation or had a history of alcohol abuse. Fresh tissue samples were taken from the lateral wall of the left ventricle. The specimens were dried to constant weight, re-weighted, and wet ashed by use of a method described by Torrance and Bothwell [27]. Quality control studies of this method using standard reference material of the National Bureau of Standards (bovine liver, 1577a) showed agreement within a 5% range.

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Indirect evidence for the potential ability of magnetic resonance imaging to evaluate the myocardial iron content in patients with transfusional iron overload