`
`
`
`MAGMA
`tic Resonance Materials in
`Physics, Biology and Medicine
`
`
`T2 relaxation time study of iron overload in b-thalassemia
`
`S.I Mavrogeni**, E.D. Gotsis >, V. Markussis °, N. Tsekos °, C. Politis 4, E. Vretou ¢,
`D. Kremastinos *
`
`* Onassis Cardiac Surgery Center, 50 Esperou Street, 175-61 P.Faliro, Athens, Greece
`> EncephatosInstitute, Athens, Greece
`° University of Minnesota, Minneapolis. USA
`* Drakopoulion Hospital, Athens, Greece
`
`Received 25 September 1997; received in revised form 15 December 1997; accepted 21 January 1998
`
`Abstract
`
`Myocardial iron deposition occurs as a result of blood transfusion therapy in b-thalassemia major patients. Since this deposition
`causes various cardiac complications,it is of interest to assess the iron content of the myocardiuminrelation to the clinical picture
`of the patients. Two different MRI indices were used to achieve this purpose: the T2 relaxation time and the heart/skeletal muscle
`signal intensity ratio. ECG gated spin echo images were obtained from 54 adult thalassemic patients, with a mean age of 26
`(18-44) years, at TE = 22 ms and 60 ms, using a 1.5 T system. Patients were divided into 2 groups (A and B), according to their
`serum ferritin levels (> or < 2000 ng ml~'). Results were compared with nine controls, with a mean age of 25 (18-43) years.
`Heart T2 relaxation time in controls (44.3 + 3.5 ms) was higher than in group A (29.9+5.7 ms, P<0.001) and group B
`(33.4 + 6.8 ms, P <0.01). T2 was measurable in 66%of group A and 83%of group B patients. The heart/muscle signal intensity
`ratio in group A (0.45 + 0.27) was lower than in group B (0.82 + 0.33, P < 0.001) and the controls (1.15 + 0.20, P< 0.001). The
`heart/muscle signalintensity ratio was measurable in 94%of the patients and demonstrated an inverserelationship with the serum
`ferritin levels (ry = — 0.52, P<0.01). This study indicates that the heart/muscle ratio is a sensitive index of iron overload andit
`can be measured in the majority of patients, irrespective of tissue iron concentration, thereby offering an advantage over the use
`of T2 relaxation time. © 1998 Elsevier Science B.V. All rights reserved.
`
`Keywords: b-Thalassemia; Heart; T2 relaxation time
`
`to be
`where the circumstances are appropriate for it
`performed. Gene therapy offers a bright outlook to the
`future treatment and annulment ofthe disease and its
`b-Thalassemia major is a genetic hemoglobinopathy
`complications. Despite the benefits of iron-chelation
`which is especially prevalent
`in Mediterranean coun-
`therapy, iron deposition occurs in various organs, as a
`tries. It is characterised by various degrees of ineffective
`result of inadvertent iron overloading from the trans-
`hemopoiesis and intramedullary hemolysis. Current
`fused blood. Iron deposition in the heart and other
`therapy includes regular blood transfusions with simul-
`organs is the causative factor of the main complications
`taneous iron chelation therapy, mainly employing des-
`in b-thalassemia major [1-5]. Excessive iron is primar-
`
`ferrioxamine, avoid—secondaryin order to
`
`
`
`ily retained in the reticuloendothelial system and, when
`hemosiderosis. Bone marrow transplantation offers an
`the capacity of this system is exceeded, secondary depo-
`effective alternative to constant blood transfusions,
`
`sition in parenchymal organs follows [6].
`The estimation of iron stores in each individual or-
`gan and the total iron burden in b-thalassemic patients
`
`+30 1 9882797;
`
`fax: +30 1
`
`1. Introduction
`
`* Corresponding author. Tel.:
`9882797; e-mail: som@aias.net.gr
`
`1352-8661/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved.
`PIT S1352-8661(98)00003-9
`
`Apotex Tech.
`Ex. 2033
`
`Apotex Tech.
`Ex. 2033
`
`
`
`8
`
`Su. Mavrogeniet al. / Magnetic Resonance Materials in Biology, Physics and Medicine 6 (1998) 7-12
`
`is useful in evaluating the efficacy of chelation therapy.
`This is a difficult task since hemosiderin and ferritin,
`the iron storage proteins, are mainly intracellular. On
`the other hand, serum ferritin levels are highly corre-
`lated with the amount of iron deposited in the tissues,
`and are hence considered a very sensitive index [7].
`However, the levels may be affected by factors such as
`fever,
`inflammation, etc.
`[8,9]. At present,
`the most
`accurate way to estimate the total body iron deposition
`is the direct measurementofiron contentin liver biopsy
`specimens[10], an invasive procedure which cannot be
`applied during routine follow-up, and which does not
`offer any information concerning iron deposition in the
`heart.
`The ability of stored intracellular tissue iron to en-
`hance magnetic susceptibility provides the basis by
`which it can be detected by magnetic resonance imaging
`(MRI). Recent studies in experimental animals have
`shownthat the T2 relaxation timeis linearly correlated
`with the total iron content for all organs, including the
`heart [11]. However, T2 relaxation time calculation may
`prove to be impossible in severely iron-overloaded pa-
`tients, due to a very low signal intensity whichis similar
`to background noise [12].
`The aim of this study was to assess heart, liver and
`skeletal muscle iron content
`in b-thalassemia major
`patients using two different indices (T2 relaxation time
`of the heart, liver and skeletal muscles, and the heart/
`muscle and liver/muscle MRI signal intensity (SI) ra-
`tios), and to correlate these parameters with the
`corresponding serum ferritin levels.
`
`2. Patients and methods
`
`2.1. Patient population (Table 1).
`
`Fifty four thalassemic patients, 24 males and 30
`females, with a mean age of 26 (18—44) years, were
`studied. Thirty nine of them had no symptomsofheart
`failure (NYHA I) whereas 15 were diagnosed with
`
`Table 1!
`Study groupstratification (values as x + SD or range)
`
`
`
`Group BGroup A Group C
`
`
`
`heart failure (NYHA II-IID. All patients were iron-
`chelated regularly and 40 of them were splenectomized.
`A serum ferritin level below 2000 ng ml~' was consid-
`ered to be the target value for a patient successfully
`treated with deferoxamine [27]. Patients were stratified
`into two groups, according to their average serum
`ferritin values (> or < 2000 ng ml~') over the previous
`5 years: Group A (n = 30) with mean ferritin levels of
`4150 + 1653 ng ml~' and group B (» = 24) with mean
`ferritin levels of 1240 + 366 ng ml~ '. The total number
`of transfusions andyears ofiron-chelation therapy were
`similar in both groups. The results from thalassemic
`patients were compared with those of nine normal
`volunteers (Group C; controls) with a mean age of 25
`(18-43) years. An informed consent was obtained from
`all participants.
`
`2.2. MRI techniques
`
`studies were conducted using a 1.5 T
`All MRI
`Siemens system (Magnetom, SP), with an ECG gated
`spin echo sequence (acquisition matrix = 256 x 256,
`FOV = 40 x 40 cm’, numberofslices, eight, TR equal
`to RR interval, and TE = 22 and 60 ms). The oblique
`orientation of the imaging slices was determined from
`scout coronal anatomical images (parameters as above
`with TE = 22 ms), in order to depict a short axis view
`of the heart. These images included the liver and a
`section of skeletal muscle (latissimus dorsi). The signal
`intensities of the heart (SI), the liver (SI,) and the
`skeletal muscle (SIy) were determined using circular
`regions ofinterest (ROIs). Two indices were employed:
`(a) The T2 relaxation time of the heart (T2,,),
`liver
`(T2,,) and skeletal muscle (T2,,), calculated from the
`images collected at
`the two TE values [13];
`(b) The
`heart/muscle (SIj;/SIy,) and liver/muscle (SI,/SI,4) sig-
`nal intensity ratios at TE = 22 ms. Theskeletal muscle
`was used as an internal standard to form this ratio since
`iron deposition is minimal in skeletal muscle [12].
`
`2.3. Statistical analysis
`
`The results are expressed as x + SD and were com-
`pared by means of the unpaired two-tailed Student |
`test. The Chi square test was used to compare percent-
`ages between groups. Correlations between various
`parameters were sought by employing Pearson’s corre-
`lation coefficient. Statistical significance was considered
`for P< 0.05.
`
`3. Results
`
`The T2 relaxation time of the left ventricle of the
`heart (T2),) in the high ferritin group (group A) was
`29.9 + 5.7 ms. In the low ferritin group (group B), the
`Apotex Tech.
`Ex. 2033
`
`Subjects (1)
`Age (yrs)
`Ferritin (ng ml~')
`Blood units transfused
`(n)
`12.74513.3444 _—
`Chelation (years)
`
`30
`26 (18-36)
`4102 +1541
`1025+274
`
`24
`26 (18-44)
`1150+3818
`9654429
`
`9
`25 (18-43)
`——
`—
`
`
`
`
`
`Group A: high ferritin population.
`Group B: low ferritin population.
`Group C: controls.
`“P<0.01.
`
`Apotex Tech.
`Ex. 2033
`
`
`
`SL. Mavrogeniet al. / Magnetic Resonance Materials in Biology, Physics and Medicine 6 (1998) 7-12
`
`9
`
`Table 2
`T2 relaxation time in the study groups (values expressed as x + SD)
`
`Group A
`
`Group B
`
`Group C
`
`Subjects (1)
`T2 heart (ms)
`T2 liver (ms)
`
`30
`29.9+5.7 (20)
`24.341.8(3)
`
`T2 muscle
`(ms)
`
`28.44+2.7 (30)
`
`9
`24
`33.4468 (20) 44.343.5 (9)
`29.04 10.3
`30.0 + 7.7 (9)
`(12)
`29.24+2.4 (24)
`
`30.0+0.9 (9)
`
`The numbers in the brackets denote the number of subjects in whom
`the T2 relaxation time was measurable.
`Group A: high ferritin population.
`Group B: low ferritin population.
`Group C: controls.
`* P<0.001, compared to group A.
`6 P<0.01, compared to group B.
`
`T2 relaxation time was 33.45+6.8 ms and in the
`controls (group C),
`the time was 44.343.5 ms. The
`T2,, in group C was higher compared both to group A
`(P <0.001) and group B (P <0.01). There was no
`statistically significant difference between groups A and
`B (Table 2).
`intensity of the myocardium (SI,,) was
`The signal
`only measurable at both echo times in 20/30 (66.7%) of
`the group A patients and in 20/24 (83.3%) of the group
`B patients (P:NS) (Fig. l(a, b)Fig. 2(a, b)). As a result
`the T2,;, could only be calculated in these patients.
`Seven additional group A patients and the remaining
`four group B patients had measurable signal intensities
`at TE=22 ms but not at TE=60 ms. Thesignal to
`noise ratio was too low at both echo times to permit T2
`and signal
`intensity ratio calculation in the final re-
`maining three group A patients. Twelve group A pa-
`tients
`(40%) and three group B patients
`(12.5%)
`manifested heart failure (NYHA II-III). Out of these
`patients, T2H was measurable only in four out of 12 of
`the group A population and in one out of three patients
`belonging to group B.
`There was no difference in the T2 relaxation time of
`the liver (T2,) between groups A, B and C. Thesignal
`intensities of the liver (SI,) at both echo times and,
`consequently, the calculated T2,, were only measurable
`in three out of 30 (10%) group A patients and 12 out of
`24 (50%) group B patients (P < 0.01, chi square test).
`Twelve additional patients of group A and nine group
`B subjects had measurable signal intensities of the liver
`at TE = 22 ms, but not at TE= 60 ms.
`The T2 relaxation time of the skeletal muscle (T2,,)
`was measurable in all subjects and was similar in the
`three groups.
`In group A, no correlation was docu-
`mented between T2,,, T2, or T2,, and ferritin levels,
`nor between the number of transfusions or iron-chela-
`tion time. In group B, an inverse correlation was found
`between the average ferritin levels and T2,, (r= — 0.49,
`P <0.05).
`
`The heart/muscle signal intensity ratio (SI,,/SIy) in
`group A (0.45+0.27) was lower
`than in group B
`(0.82 + 0.33, P<0.001) and group C (1.15+0.2, P<
`0.001) (Table 3). Similarly, the liver/muscle signal inten-
`sity ratio (SI,/SIy) in group A (0.17 + 0.13) was lower
`than in group B (0.37+0.31, P<0.01) and group C
`(1.28 + 0.16, P<0.01). Patients with heart failure, as a
`subgroup, had a lower SI,,/SI,, ratio (0.33 + 0.19) com-
`pared to the mean group A (P <0.001) and group B
`(P < 0.001) values. Ferritin levels were inversely corre-
`lated with both SI,,/SI,, (= — 0.52, P< 0.01) (Fig. 3)
`and SI_/SIy ratios (* = ~ 0.41, P< 0.01). In group B, a
`positive correlation (r= 0.55, P<0.01) was found be-
`tween the SI,,/SIy ratio and T2,, values.
`
`
`
`
`Fig. 1. (a and b) Short axis view of the heart of a thalassemic patient
`with mild iron overload, using TE = 22 ms (a) and TE = 60 ms (b).
`Apotex Tech.
`Ex. 2033
`
`Apotex Tech.
`Ex. 2033
`
`
`
`10
`
`SL. Mavrogeni et al. / Magnetic Resonance Materials in Biology, Physics and Medicine 6 (1998) 7-12
`(a)
`
`significant difference was found in the heart T2 relax-
`ation time between the high and low serum ferritin
`groups. Serum ferritin levels were only inversely corre-
`lated to heart T2 relaxation times (T2,,) in the low
`ferritin group. These observations result from the fact
`that the T2 of several patients in the high ferritin group
`could not be measured dueto lowsignalto noiseratio.
`On the contrary, signal
`intensity measurements were
`feasible in all patients,
`irrespective of iron burden.
`Significant differences were recorded between the two
`patient groups, and an inverse correlation was found
`between heart/muscle and liver/muscle SI
`ratio and
`serum ferritin levels
`in the thalassemic population.
`Since there is a significant error involved in the calcula-
`tion of the T2 relaxation time when the tissue signal
`intensity (SI) is low, heart SI (normalized in proportion
`to skeletal muscle employed as the control standard) is
`a more appropriate index for correlation with iron
`deposits.
`serious complication of b-tha-
`a
`failure,
`Heart
`lassemia, was present in both ferritin groups, although
`it was more prevalent in the severely iron-overloaded
`group. When heart failure patients from groups A and
`B were analysed as a separate sub-group, the T2 relax-
`ation time of the heart was measurable only in one
`third of the population, and the heart/muscle signal
`intensity ratio was even lower than the values observed
`in the high ferritin group. Similarly, the T2 relaxation
`time of the liver was measurable in half of the patients
`in the Jow ferritin group, whereas in the majority of the
`patients
`in the high ferritin group, measurements
`proved to be impossible. When the liver/muscle signal
`intensity ratio was employed, however, measurements
`in all patients were achieved and differences were man-
`ifested between the high and low ferritin groups, and
`the control subjects.
`
`Fig. 2. (a and b) Short axis view of the heart of a thalassemic patient
`with severe iron overload and impaired LV function, using TE = 22
`ms(a) and TE = 60 ms(b). In the latter image, the myocardial signal
`intensity is equal to the background noise.
`
`4. Discussion
`
`The presence of iron affects the tissue Tl and T2
`relaxation times [14,15]. However,
`the effect on TI]
`relaxation time is not as significant
`[15], and most
`studies use the T2 time for characterization of iron
`deposition. This effect is proportional to the concentra-
`tion of iron in the tissue and depends on the applied
`magnetic field (Bo) [16]. This concept was employed to
`evaluate a group of adult thalassemic patients.
`In this study, the heart and liver T2 relaxation times
`were significantly reduced, with higher iron concentra-
`tions resulting in lower T2 values. The signal intensities
`(SI) of different tissues demonstrated similar trends. No
`
`Table 3
`
`Signal intensity ratios (SI) in the study groups (values expressed as
`x+SD)
`
`
`
` Group B Group C Group A
`
`Subjects (7)
`SIy/SIy
`
`SIL/Sly
`
`
`24
`30
`0.45 + 0.27 (27) 0.82 + 0.33
`24)"
`0.17+0.13 (15) 0.37 +0.31
`(21°
`
`9
`1.15 +.0,20 (9)
`
`1.28 +0.16 (9)°
`
`
`intensities measured at TE =22 ms (H: heart, M: skeletal
`Signal
`muscle, L: liver). The numbers in the brackets denote the number of
`subjects in whom thesignal intensity ratios were measurable.
`Group A: high ferritin population.
`Group B: low ferritin population.
`Group C: controls.
`*P<0.001.
`5 P<0.01, compared to group A.
`* P<0.001, compared to groups A andB.
`
`Apotex Tech.
`Ex. 2033
`
`Apotex Tech.
`Ex. 2033
`
`
`
`heart/muscleSIratio
`
`S.J. Mavrogeniet al. / Magnetic Resonance Materials in Biology, Physics and Medicine 6 (1998) 7-12
`
`11
`
`1.60 7
`
`.
`
`s
`
`%
`
`¢
`
`+
`
`°
`
`*
`
`oe
`
`.
`
`+
`
`.
`
`.
`
`.
`
`+
`1000
`
`+
`2000
`
`t
`3000
`
`4000
`
`+
`5000
`
`t-
`6000
`
`+
`7000
`
`t
`8000
`
`t$—-
`9000
`
`1
`10000
`
`Ferritin (ng/ml)
`
`
`
`1.40 +
`
`1.20 +
`
`1.00 +
`
`0.80 +
`
`0.60 +
`
`0.40 +
`
`0.20 +
`
`0.00
`
`0
`
`Inverse relationship between the heart/muscle signal
`Fig. 3.
`(Y= — O54, 2<38b:
`
`intensity (SI) ratios and the average ferritin levels of the thalassemic patients
`
`Serum ferritin is currrently considered the most accu-
`rate index of body iron content. Variations in serum
`fertrim mainty correspond io chanpes in the reticuioen-
`dothelial system storage iron levels and not to changes
`in the parenchymal
`iron content
`[17]. Liver disease,
`inffammattion. wifection and assay
`camntications
`[8,9,18] have been reported to influence measurements.
`In this study, in an attempt to overcome these limita-
`tious,
`the average ferritin levels af the preceding five
`years were employed.
`There is a controversy over the precise relationship
`between the quantity of iron present in the myocardium
`and the degree of heart dysfunction. Correlations be-
`tween heart T2 relaxation time and cardiac biopsy are
`absent since right ventricular biopsies are subject
`to
`serious sampling errors and iron deposition is patchy
`(noi nniiorm) $59) A good correlation was Found ve-
`tween iron content and T2 relaxation time only when
`whole rat hearts were sampled [11]. Under these cir-
`cumstances, MRI appears to be useful
`in addressing
`this issue.
`Recently, studies of thalassemic patients at 0.5 T
`in@icaied Ynal carhhac commycavons are rAaied Jo ow
`T2 times [20]. The heart/muscle signal
`intensity ratio
`was also found to be sensitive to iron content
`in a
`gronp of pabenis realed by mUUpe dood Iranslusions
`[12]; anda trend’ tor worse Heart tinction was evident
`in the more heavily-transfused patients, even though
`there was no cormabon with serum Jerfiion Jeveds. do
`addition, Zaino et al.
`[21], employing a new in vivo,
`non-invasive method for measuring iron utilizing nu-
`clear resonancescattering (NRS), detected symptomatic
`cardiac disease in the patients with the more elevated
`cardiac tran tevets.
`Liver iron load, as studied by MRI, has been consid-
`ered aS an alternative to fiver biopsy for the assessment
`
`of total iron burden [22]. In vitro measurements of T2
`relaxation time of liver samples, from iron-overloaded
`Tats [14] and spieen sampies from thaiassemic patients
`{15}, demonstrated a linear correlation between relax-
`ation rate (1/T2) and iron content. The reticuloendothe-
`hal system oreferentiatlv accumulates tran from the
`breakdown of transfused red blood cells before iron
`deposition occurs in other parenchymal organs such as
`the heart. The data presented here are consistent with
`the study by Buja et al.
`[23], which indicates that
`cardiac iron deposition is accompanied by excessive
`hepatic iron deposits.
`Although the T2 relaxation time is generally a reli-
`able index of iron deposition, being well-correlated with
`liver biopsy information,
`it may be unmeasurable in
`severely iron-overloaded patients, due to low signal
`intensity equal ta background naise. Far this reason,
`some authors consider the application of the T2 relax-
`ation time to assess heart
`iron deposition as being
`inaccurate [12]. The use of a 0.5 T machine, where the
`magnetic susceptibility phenomenon is less prominent
`[16],
`the use of shorter echo times (TE) [15] or the
`application of MR spectroscopy [22], cauld he mate
`promising in the study of severe iron-overloaded pa-
`tients. Other non-invasive techniques such as dual en-
`ergy computed tomography [24,25],
`superconducted
`quantum. interference device (SQUID), anplicatianM4h
`and nuclear resonance scattering (NRS) can overcome
`this limitation, but are not available on a widescale.
`is study suggests that MRI could prove to be an
`effective non-invasive method in performing tissue
`characterization. MRI,
`in particular, provides
`the
`means for the simultaneous examination of heart iron
`deposition and feart
`function. Moreover, measure-
`ments are quantifiable, enabling te repeated examina-
`tions of thalassemic patients and evaluation of the
`iron-chelation therapy efficacy.
`Apotex Tech.
`Ex. 2033
`
`Apotex Tech.
`Ex. 2033
`
`
`
`12
`
`S.1. Mavrogeni et al. / Magnetic Resonance Materials in Biology, Physics and Medicine 6 (1998) 7-12
`
`In conclusion, T2 relaxation time is a very useful
`index in the monitoring of tissue iron deposition. How-
`ever,
`it
`is not always measurable using commercially
`available systems. As a consequence, the use ofsignal
`intensity ratio appears to be a valuable alternative for
`patient screening and follow-up.
`
`Acknowledgements
`
`We are indebted to Sonja Phillips, BSc. MSc., for
`proofing the manuscript.
`
`References
`
`{1] Kattamis C, Touliatos N, Haidas S, Matsaniotis N. Growth of
`children with thalassemia: effect of different
`transfusion regi-
`mens. Arch Dis Childhood 1970;45:502-6.
`[2] Propper RD, Cooper B, Rufo RR, et al. Continuous subcuta-
`neous administration of deferoxaminein patients with iron over-
`load. N Engl J Med 1977;297:418-23.
`Iron state and
`[3] Aldouri MA, Wonke B, Hoffbrand AV, et al.
`hepatic disease in patients with thalassemia major treated with
`long-term subcutaneous
`desferrioxamine.
`J Clin
`Pathol
`1987;40: 1353-9.
`[4] Engle MA. Cardiac involvement in Cooley’s anemia. Ann NY
`Acad Sci 1969;119:694—702.
`[5] Kremastinos DT, Tsiapras D, Tsetsos G, Rentoukas E, Vretou
`H, Toutouzas P. Left ventricular diastolic Doppler characteris-
`tics in bthalassemia major. Circulation 1993:88:1127~35,.
`[6] Jacobs A. The pathology of iron overload. In: Jacabs A, Wor-
`wood M, editors. Iron in biochemistry and medicine, H. New
`York: Academic Press, 1980:439—52,
`[7] Jacobs A, Miller F, Worwood M, Beamisch R, Wardrop C.
`Ferritin in the serum of normal subjects with iron def ~ ciency
`and iron overload. Br Med J 1972;XX:206-211.
`[8] Crosby WH. Serum ferritin fails to indicate hemochromatosis:
`nothing gold can stay. N Engl J Med 1976;294:333-4.
`(9] Green R, Watson LR, Saab GA, Crosby WH. ‘Normal’ serum
`ferritin: a caution. Blood 1977;50:545-7.
`[10] Ridson RA, Barry M, Flynn DN. Transfusional iron overload:
`the relationship between tissue iron concentration and hepatic
`fibrosis in thalassemia. J Pathol 1975;116:83-95.
`{11} Liu P, Henkelman M, Joshi J, Waien S, Butany J, Olivieri N.
`Quantification of myocardial
`tissue iron contents using NMR
`relaxation. Validation in a novel murine thalassemia model.
`JACC 1992;19(3):187A (abstract 935-976).
`
`{12] Johnston DL, Rice L, Vick W, Hedrick TD, Rokey R. Assess-
`ment of tissue iron overload by nuclear magnetic resonance
`imaging. Am J Med 1989;87:40—-7.
`[13] Sechtem U, Higgins C. Relaxation measurements and contrast
`media.
`In: Underwood R, Firmin D, editors. Magnetic Reso-
`nance of
`the
`cardiovascular
`system. London: Blackwell,
`1991:89--10L.
`
`[14] Stark DD, Moseley ME, Bacon BR, et al. Magnetic resonance
`imaging and spectroscopy of hepatic iron overload. Radiology
`1985;154:137-42.
`{15] Gomori JM, Grossman RI. Drott HR. MR relaxation times and
`iron content of thalassemic spleens: an in vitro study. AJR
`1988;150:567-9.
`[16] Vymazal J, Brooks RA, Zak O, McRill C, Shen C, Di Chiro G.
`Ti and T2 offerritin at different field strengths: Effect on MRI.
`Magn Res Med 1992:27:368-74.
`[17] Gomori JM, Horev G, TamaryH, et al. Hepatic iron overload:
`Quantitative MR imaging. Radiology 1991;179:367-9,
`[18] Worwood M, Cragg SJ, Jacobs A, McLaren C, Ricketts C,
`Economidou J. Binding of serum ferritin to concanavallin A:
`Patients with homozygous beta-thalassemia and transfusional
`iron overload. Br J Haematol 1980:46:409- 16.
`{19] Olson LJ, Edwards WD, McCall JT, Ilstrup DM, Gersh BJ.
`Cardiac iron deposition in idiopathic hemochromatosis: histo-
`logic and analytic assessment of 14 hearts from autopsy. JACC
`1987;10:1239—43.
`[20] Liu P, Olivieri N, Sullivan H, Henkelman M. Magnetic reso-
`nance imaging of cardiac iron overload in thalassemia: Detection
`of iron content and association with cardiac complications.
`JACC 1993:21(2):409A (abstract 948-119).
`[21] Zaino EC, Wielopolski L. In vivo measurement of hepatic and
`cardiac iron in Cooleys anemia by nuclear resonance scattering.
`Ann NYAcad Sci 1990;612:573-6.
`[22] Dixon RM, Styles P, Al-Refaie F, et al. Assessment of hepatic
`iron overload in thalassemic patients by magnetic resonance
`spectroscopy. Hepatology 1994:19:904-10.
`(23] Buja LM, Roberts WC. Iron in the heart: Etiology and clinical
`significance. Am J Med 1971:51:209-21.
`[24] Houang MT, Skalicka A, Arozena X, Huehns ER, Shaw DG.
`Correlation between computed tomographic values and liver
`iron content in thalassemia major with iron overload. Lancet
`1979;1:1322-33.
`[25] Olivieri NF, Grisaru D, Daneman A, Martin DJ, Rose V,
`Freedman MH. Computed tomography scanning of the liver to
`determine eff~ cacy of iron chelation therapy in thalassemia
`major. J Pediatrics 1989;114:427—30.
`[26] Brittenham G, Griffith PM, Nienhuis AW, et al. Efficacy of
`deferoxamine in preventing complications of iron overload in
`patients with thalassemia major. N Engl J Med 1994:331:567—
`73.
`
`[27oa
`
`Olivieri NF, Nathan DG, MacMillan JH, Wayne AS, Liu PP,
`McGee A, et al. Survival
`in medically treated patients with
`homozygous b-thalassemia. N Engl J Med 1994;331:574-8.
`
`Apotex Tech.
`Ex. 2033
`
`Apotex Tech.
`Ex. 2033
`
`