1. Update on Imaging: Detection of Iron in Liver and Heart Tim St. Pierre, BSc, PhD Professor School of Physics The University of Western Australia Crawley, Australia

2. Iron Loading Is Different in Different Organs

3. Why Is Measurement of Liver Iron Concentration Important? A patient’s liver iron concentration (LIC) value is the best measure of total body iron stores A patient’s LIC value enables better informed decisions on when to –Initiate chelation therapy –Increase chelation dose –Decrease chelation dose –Change mode of chelator delivery (eg, IV mode)

4. LIC Is a Reliable Measure of Total Body Iron Stores in Patients with Thalassaemia Major There is a very strong correlation between LIC and total body iron stores in thalassaemia major patients Abbreviation: LIC, liver iron concentration. With permission from Angelucci E, et al. N Eng J Med. 2000;343:327-331.

5. LIC Thresholds and Associated Risks LIC Threshold (mg Fe/g dry weight) Clinical Relevance 1.8 Upper 95% of normal 3.2 Suggested lower limit of optimal range for LICs for chelation therapy in transfusional iron overload 1 7.0 Suggested upper limit of optimal range for LICs for transfusional iron overload and threshold for increased risk of iron-induced complications 1 15.0 Threshold for greatly increased risk for cardiac disease and early death in patients with transfusional iron overload 1 1. Olivieri NF, Brittenham GM. Blood. 1997;89:739-761.

6. LIC and Long-Term Prognosis Initial LIC (mg Fe/g dw) 13-Year Cardiac Disease–Free Survival Patients (n)Group <793.3% (SE 6.4)15(i) 7–1571.4% (SE 17.1)7(ii) >1550.0% (SE 15.8)10(iii) Telfer PT, et al. Br J Haematol. 2000;110:971-977. 32 thalassaemia major patients followed for median period of 13.6 years after single biopsy LIC measurement

7. Why Is Measurement of Heart Iron Important? Heart iron measurements (by cardiac MRI) have been shown to have a high sensitivity and specificity for predicting cardiac failure within 12 months for thalassaemia major patients In a study of 652 thalassaemia major patients –83% of patients who developed arrhythmia had a cardiac T2* of <20 ms –98% of patients who developed heart failure had a cardiac T2* of <10 ms Kirk P, et al. Circulation. 2009;120: in press.

8. Relationship Between Liver and Heart Iron

9. Heart Iron Changes Generally Lag Behind Liver Iron Changes With permission from Noetzli LJ, et al. Blood. 2008;112:2973-2978.

10. Strengths and Weaknesses of Various Imaging Methods

11. Methods of Monitoring Iron Loading Serum ferritin Liver biopsy Biomagnetic susceptometry MRI

12. Methods of Monitoring Body Iron Stores

13. Is Serum Ferritin a Reliable Indicator of LIC? Cross-sectional study of 37 patients with sickle cell anaemia and 74 patients with thalassaemia major Only 57% of the variability in plasma ferritin concentration could be explained by the variation in hepatic iron stores The 95% prediction intervals for hepatic iron concentration, given the plasma ferritin, were so broad as to make a single determination of plasma ferritin an unreliable predictor of body iron stores Eg, given a plasma ferritin of 1000 ng/mL, the 95% prediction interval for hepatic storage iron was 0–6.948 mg Fe/g liver, wet weight Brittenham GM, et al. Am J Hematol. 1993;42:81-85.

14. Serum Ferritin in Thalassaemia Major and Intermedia Serum ferritin has almost no sensitivity or specificity for iron stores in thalassaemia intermedia With permission from: Taher A, et al. Haematologica. 2008;93:1584-1585. With permission from: Origa R, et al. Haematologica. 2007;92:583-588. Abbreviations: TI, Thalassaemia intermedia; TM, Thalassaemia major.

15. Serum Ferritin Serum ferritin can be used for monitoring trends in patient transfusional iron loading Serum ferritin does not give reliable information on degree of patient iron loading

16. Measuring Liver Iron Concentration by Biopsy Siegel CA, et al. Cleve Clin J Med. 2005;72:199-224. Methods –Percutaneous –Laparoscopic –Transjugular Risk of Complications –Death1:10,000–1:12,000 –Bile leak 1:1,000 –Bleeding 1:100 –Any pain 1:4 –Significant pain1:10–1:20

17. Heterogeneity of Iron Concentration Throughout the Liver Sample Size and TypeCV of LICPathologySource Needle biopsy (<4 mg dw) 19% NormalEmond, et al. 1999 Kreeftenberg, et al. 1984 Needle biopsy (<4 mg dw) >40% End-stage liver disease Emond, et al. 1999 Kreeftenberg, et al. 1984 Needle biopsy (9 mg dw) 9% NormalBarry, Sherlock. 1971 “Cubes” (200–300 mg wet weight) 17% 24%  -thalassaemia Noncirrhotic Ambu, et al. 1995 “Cubes” (1000–3000 mg wet weight) 19%  -thalassaemia Part-cirrhotic Clark, et al. 2003 Abbreviations: CV, coefficient of variation; dw, dry weight; LIC, liver iron concentration. Ambu R, et al. J Hepatol.1995;23:544-549. Barry M, Sherlock S. Lancet.1971;1:100-103. Clark PR, et al. Magn Reson Med. 2003;49:572-575. Emond MJ, et al. Clin Chem.1999;45:340-346. Kreeftenberg HG, et al. Clin Chim Acta. 1984;144:255-262.

18. Noninvasive Methods of Tissue Iron Measurement Biomagnetic Liver Susceptometry (SQUID)

19. Biomagnetic Liver Susceptometry Liver Bellows Cryogenic package Liquid helium Fischer R. In: Magnetism In Medicine: A Handbook. Wiley-VCH;1998:286-301.

20. Needle Biopsy LIC vs Biomagnetic Liver Susceptometry There is a good correlation between LIC by biopsy and LIC by SQUID up to 3.5 mg Fe/g wet tissue Above 3.5 mg Fe/g wet tissue, correlation decreases, most likely because of increased sampling error on biopsy Fischer R. In: Magnetism In Medicine: A Handbook. Wiley-VCH;1998:286-301.

21. Noninvasive Methods of Tissue Iron Measurement Magnetic Resonance Imaging (MRI)

22. Principles of MRI Clark PR, St. Pierre TG. Mag Res Imaging. 2000;18:431-438. Magnetic field and radio signal pulses Initial pulse excites protons in tissue Signal received from tissue decays with time after initial pulse Rate of decay different for different tissues Rate of decay highly influenced by presence of iron Rate known as either R2 or R2* depending on data acquisition technique Characteristic time of decay known as T2 or T2* depending on data acquisition technique

23. Calculating Tissue Iron from MRI Measurements Typical non–iron-loaded tissue Effect of increasing iron loading Intensity ratio methods Relaxometry methods, eg R2 or R2* The rate at which signal decays is known as R2 or R2* The characteristic time of decay is known as T2 or T2* Echo Time (ms) Signal Strength 100 80 60 40 20 0 1520 05 10 St. Pierre TG. Ann N Y Acad Sci. 2005;1054:379-385. Graphic courtesy of Dr. Tim St. Pierre.

24. Methods of Measurement of Tissue Iron Concentrations with MRI Relaxometry measurement of R2 is the most widespread method for measurement of liver iron concentration Relaxometry measurement of T2* is the most widespread method for assessing iron in the heart

25. Proton Transverse Relaxation Rate (R2) Image and Distribution With permission from St. Pierre TG, et al. Blood. 2005;105:855-861. R2 (s -1 ) Transverse Relaxation Rate R2 (s -1 ) LIC = 7.7 mg.g -1

26. Liver R2 Images and Distributions Non–iron-loaded subject 3 iron-loaded subjects R2 distribution shifts to higher values as LIC increases With permission from St. Pierre TG, et al. Blood. 2005;105:855-861.

27. Mean R2 vs iron concentration for 32 cubes of liver dissected from a single iron–loaded liver postmortem Dissected Liver Samples With permission from Clark PR, et al. Mag Res Med. 2003;49:572–575. Iron Concentration (mg/g dw) Mean Transverse Relaxation Rate (s -1 )

28. Relationship Between R2 and Needle Biopsy LIC (dw) With permission from St. Pierre TG, et al. Blood. 2005;105:855-861. Biopsy Iron Concentration (mg/g dry tissue) Mean Transverse Relaxation Rate (s -1 )

29. R2-MRI Is a Reliable Measure of LIC High sensitivity and specificity over entire range of LIC encountered Unaffected by presence of fibrosis/cirrhosis With permission from St. Pierre TG, et al. Blood. 2005;105:855-861. Fibrosis stages: 0–1 =  2–4 =  5–6 = 

30. Example—R2-MRI Measurements to Monitor Iron Chelation Therapy High iron Low iron LIC map Before chelation therapy intervention Mean LIC = 16.0 After 12 months of chelation therapy intervention Mean LIC = 1.6 Graphic courtesy of Dr. Tim St. Pierre.

31. Methods of Monitoring Heart Iron

32. Methods for Heart Iron Assessment T2* methods are used to assess heart iron loading With permission from Westwood M, et al. J Magn Reson Imaging. 2003;18:33-39. Echo time increasing

33. Relationship Between T2* and Cardiac Function With permission from Anderson LJ, et al. Eur Heart J. 2001;22:2171-2179.

34. Relationship Between R2* and Cardiac Function With permission from Anderson, LJ, et al. Eur Heart J. 2001;22:2171-2179. Transform using R2* = 1/T2* Graphic courtesy of Dr. Tim St. PierreAbbreviation: LVEF, left ventricular ejection fraction.

35. Calibration of Cardiac T2*/R2* Against Tissue Iron Concentration Preliminary calibration over small iron concentration range obtained from a single human heart With permission from Ghugre, et al. Magn Reson Med. 2006;56:681-686.

36. Implementing These Methods at Your Institution

37. MRI data acquisition –Relatively simple for liver –More involved for heart  Requires extra hardware and software on scanner MRI data analysis –Problematic for liver  High risk of erroneous analysis due to low signal to noise ratios; need to account for background noise, etc. –Relatively simple for heart

38. Implementing These Methods at Your Institution MRI data acquisition –Liver  No face-to-face training required –Heart  May require expert training of technicians MRI data analysis –Liver  ISO9001 Quality Assurance should be implemented, or data analysis should be outsourced to quality assured core lab –Heart  Technicians should receive training from experts

39. When to Measure Iron in the Liver vs the Heart Patients on regular blood transfusion –Measure liver iron annually –Measure heart iron annually after 20 units have been transfused Patients with hereditary haemochromatosis –Measure liver iron at diagnosis if >40 years of age and serum ferritin >1000 ng/mL Patients with thalassaemia intermedia –Measure liver and heart iron annually after age 10 years –If the baseline cardiac T2* in normal range, subsequent cardiac T2* no more frequent than 3–5 years unless there is difficulty controlling the liver iron

40. Conclusions It is now possible in most major hospitals to monitor iron in the liver and the heart using magnetic resonance imaging The ability to measure iron in these 2 organs provides the basis for making better informed decisions concerning the need to adjust patients’ chelation regimens