1. Diagnosis of Iron Overload
M. Domenica Cappellini, MD Professor of Internal Medicine University of Milan Maggiore Hospital Milan, Italy

2. Iron Overload and Disease States

3. Causes of Iron Overload
Primary (hereditary)
Resulting from a primary defect in the regulation of iron balance, eg, hereditary haemochromatosis
Secondary (acquired)
Caused by another condition or by its treatment
Anaemias requiring repeated blood transfusion (eg, thalassaemia, sickle cell disease, and myelodysplastic syndromes)
Ineffective erythropoiesis
Toxic ingestion
Feder JN, et al. Nat Genet. 1996;13:399.
Porter JB. Br J Haematol. 2001;115:239.

4. Conditions at Risk of Iron Overload Sources of Iron
Absorption Transfusion Redistribution
Haemochromatosis +++
Thalassaemia major + +++
Thalassaemia intermedia +++ +
Sideroblastic anaemia ++ ++
CDA ++ ++
Aplasias +++
Chronic haemolytic anaemias +
Myelodysplasias ++
Off-therapy leukaemias +
Bone marrow transplant + +
Liver disease +
Porphyria cutanea tarda +
Neonatal iron overload +++
Atransferrinaemia +++
Aceruloplasminaemia
Dietary iron overload ++
Iatrogenic iron overload ++
Dialysis patients ++
Courtesy of A. Piga.

5. Complications of Iron Overload
Capacity of serum transferrin to bind iron is exceeded
Iron overload
Non–transferrin-bound iron circulates in the plasma
Excess iron promotes the generation of free hydroxyl radicals, propagators of oxygen-related tissue damage
Insoluble iron complexes are deposited in body tissues and end-organ toxicity occurs
Cardiac failure
Liver cirrhosis/ fibrosis/cancer
Diabetes mellitus
Growth failure
Infertility
Courtesy of Dr. M. D. Cappellini.

6. Consequences of Iron-Mediated Toxicity During Iron Overload
Increased LPI or “free” iron
Hydroxyl radical generation
Lipid peroxidation
Organelle damage
TGF-β 1
Lysosomal fragility
Collagen synthesis
Enzyme leakage
Cell death
Fibrosis
LPI = labile plasma iron; TGF = transforming growth factor.
Cohen AR & Porter JB. In: Steinberg MH, et al, eds. Cambridge University Press;2001:979–1027.

7. Organ Systems Susceptible to Iron Overload
Clinical sequelae of iron overload
Pituitary → impaired growth
Heart → cardiomyopathy, cardiac failure
Liver → hepatic cirrhosis
Pancreas → diabetes mellitus
Gonads → hypogonadism, infertility
Courtesy of Dr. M. D. Cappellini.

8. Liver Iron and Risk from Iron Overload
Thalassaemia major: transfusion without chelation 50 50
100
150
200
250
Homozygous haemochromatosis 40
Heterozygote
Hepatic Iron (µmol/g wet weight) 30
Hepatic iron, mg/g of liver, dry weight
Threshold for cardiac disease and early death 20
Increased risk of complications 10
Optimal level in chelated patients
Normal 10 20 30 40 50
Age (years)
Olivieri N, et al. Blood. 1997;89:739.

9. Assessing Iron Overload

10. Diagnosis of Iron Overload
Established
% transferrin saturation
Ferritin
Liver iron concentration (biopsy)
Investigational
Biomagnetic liver susceptometry (SQUID)
Magnetic resonance imaging
SQUID = superconducting quantum interference device.

11. Transferrin Saturation
Normal values: 16%–30%
> 40%: iron overload

12. Monitoring—Plasma Ferritin
Relatively noninvasive
Inexpensive
Routine laboratory assay
Values confounded by
Inflammation
Liver function
Ascorbate status
24,000
Sickle cell anaemia (n = 37)
Thalassaemia major (n = 74)
12,000
Plasma Ferritin (µg/L)
8000
4000
4000
8000
12000
Hepatic Iron
(µg Fe/g liver)
Brittenham G, et al. Am J Hematol. 1993;42:81.

13. Serum Ferritin and Risk from Iron Loading
Change in serum ferritin over time reflects change in liver iron concentration
Sequential evaluation of ferritin provides good index of chelation history1
Maintenance of serum ferritin <2500 µg/L significantly correlates with cardiac disease-free survival2-5
1. Gabutti V, et al. Acta Haematol. 1996;95: Olivieri NF, et al. N Engl J Med. 1994;331: Telfer PT, et al. Br J Haematol. 2000;110: Davis BA, et al. Blood. 2004;104: Borgna-Pignatti C, et al. Haematologica. 2004;89:1187.

14. Measuring and Interpreting Serum Ferritin
Advantages
Disadvantages
Easy to assess
Inexpensive
Repeat measures are useful for monitoring chelation therapy
Positive correlation with morbidity and mortality
Indirect measurement of iron burden
Fluctuates in response to inflammation, abnormal liver function, metabolic deficiencies
Serial measurement required

15. Monitoring—Why LIC?
Liver iron concentration (LIC) predicts total body storage iron1
Absence of pathology
Heterozygotes of hereditary haemochromatosis where liver levels <7 mg/g dry weight
Liver pathology
Abnormal ALT if LIC >17 mg/g dry weight2
Liver fibrosis progression if LIC >16 mg/g dry weight3
Cardiac pathology at high levels
Liver iron >15 mg/g dry weight association with cardiac death
All of 15/53 thalassaemia major patients who died4
Improvement of left ventricular ejection fraction with venesection post bone marrow transplantation5
1. Angelucci E, et al. N Engl J Med. 2000;343: Jensen P, et al. Blood. 2003;101:4632.
3. Angelucci E, et al. Blood. 2002;100: Porter JB. Hematol/Oncol Clinics. 2005;S7.
5. Mariotti E, et al. Br J Haematol. 1998;103:916.

16. LIC Accurately Reflects Total Body Iron Stores
300
r = 0.98
r = 0.98
r2 = 0.98
250
200
Total Body Iron Stores (mg/kg)
150
25 patients with iron overload and cirrhosis
100
≥1 mg dry weight liver sample 50 5 10 15 20 25
LIC (mg/g, dry weight)
LIC = liver iron concentration.
Angelucci E, et al. N Engl J Med. 2000;343:327.

17. Approximate LIC, mg/g dry weight liver
LIC and Prognosis
Approximate LIC, mg/g dry weight liver
Haemochromatosis
Age (years)
β-thalassaemia Major
Normal
Heterozygous
Homozygous 5
<1.2
<1.2
>3.2
>15 10
<1.2
<1.2 ~7
>15 15
<1.2
<1.2
>7
>15 20
<1.2
~1.2
~15
>15 25
<1.2
>1.2
>15
(Not surviving) 30
<1.2
~3.2
>15 35
<1.2
>3.2
>15
3.2–7 (adequate iron chelation)
7–15 (increased risk of complications)
15 (cardiac disease and early death)
LIC changes are presented for patients without phlebotomy or iron chelation therapy. LIC = liver iron concentration.
Courtesy of Dr. J. Porter.

18. Estimation of LIC Liver biopsy Distribution artifact
Debate about safe levels
Safety
Patient acceptance
Sample size
≥1 mg dry weight
>4 mg wet weight
2 cm
Photos courtesy of Dr. J. Porter.
Porter JP. Br J Haematol. 2001;115:239.

19. Measuring LIC by Liver Biopsy
Advantages
Disadvantages
Direct measurement of LIC
Validated reference standard
Quantitative, specific, and sensitive
Allows for measurement of nonheme storage iron
Provides information on liver histology/pathology
Positive correlation with morbidity and mortality
Invasive, painful procedure associated with potentially serious complications
Risk of sampling error, especially in patients with cirrhosis
Requires skilled physicians and standardized laboratory techniques

20. Noninvasive Measurement of Liver Iron
SQUID
Measures paramagnetic properties of liver iron
4 operational machines worldwide
MRI techniques
Potentially widely available
Gradient echo (T2*)
Insensitive at levels >15 mg/g1
Spin echo (T2)(R2)
Linear over larger range, longer acquisition time2
Gradient with SIR3
Spin echo with SIR4
SQUID = superconducting quantum interface device; MRI = magnetic resonance imaging; SIR = signal intensity ratio.
1. Anderson LH, et al. Eur Heart J. 2001;22: St. Pierre TG, et al. Blood. 2005;105: Gandon Y, et al. Lancet. 2004;363: Jensen, et al. Blood. 2003;101:4632.

21. Superconductive Quantum Interference Device
SQUID Biomagnetic Susceptometer
Superconductive Quantum Interference Device
Josephson effect V I Ic
-Ic S
0.8
0.06
0.1 4 2 6 8
temperature (°K)
0.16
0.2
0.26
Conductive
Superconductive
Superconductivity
Meissner effect
T>Tc
T<Tc
Normal (nonsuperconducting)
Flux expulsion (superconducting state)
Persistent current (superconducting state)
Resistance (ohm)
SQUID Thalassaemia Center. Turin, Italy
Courtesy of A. Piga, Turin Thalassaemia Centre.

22. LIC Assessment by SQUID
Advantages
Disadvantages
Linear correlation with LIC assessed by biopsy
May be repeated frequently
Indirect measurement of LIC
Limited availability
High cost
Highly specialized equipment requires dedicated technician
Not validated for LIC assessment and may underestimate levels
LIC = liver iron concentration; SQUID = superconducting quantum interference device.

23. Quantitative Iron Assessment by MRI
T2 (heart, liver)
Spin echo, gradient-echo sequences
Signal intensity ratio (SIR)
R2 (liver)
Gradient-echo sequences
s-1
T2*(heart)
Gradient-echo sequences ms

24. Liver R2 images and distributions for a healthy volunteer and 3 iron-loaded subjects with sequentially increasing liver iron concentrations
St Pierre TG, et al. Blood. 2005;105:855.

25. R2 MRI—A New Measure for LIC
300
250
Hereditary haemochromatosis
200
β-thalassaemia
Mean Transverse Relaxation Rate <R2> (s-1)
150
β-thalassaemia/ haemoglobin E 50
100
Hepatitis 40 30 50 20
0.5
1.0
1.5
2.0 10 20 30 40 50
Biopsy Iron Concentration (mg/g-1 dry tissue)
R2 MRI is a validated and standardized method for measuring LIC.
This technique is now approved by TGA and FDA and in the EU
St Pierre TG, et al. Blood. 2005;105:855.

26. R2* Measurement of LIC R2* (Hz) Estimated HIC (mg/g dry weight)
HIC = hepatic iron concentration.
Wood JC, et al. Blood. 2005;106:1460.

27. MRI Assessment of LIC Advantages Disadvantages
Assesses iron content throughout the liver
Potentially widely available
Pathologic status of liver and heart can be assessed in parallel
Indirect measurement of LIC
Requires MRI imager with dedicated imaging method
Liver iron levels can be assessed using a technique known as R2 (spin echo) MRI, which is a validated and standardized method for measuring LIC
MRI = magnetic resonance imaging; LIC = liver iron concentration.

28. Assessing Cardiac Function and Iron Load

29. Monitoring—Heart Rhythm Left ventricular function Heart “iron”
Resting or exercise ECG
24-hr Holter monitoring
Left ventricular function
ECHO
Quantitative sequential (MUGA or MRI)1
Wall motion abnormalities
Heart “iron”
T2*2
SIR (T2 weighted)3
ECG = electrocardiogram; ECHO = echocardiogram; MUGA = multiple gated acquisition; MRI = magnetic resonance imaging; SIR = signal intensity ratio.
1. Davis BA, et al. Blood. 2004;104: Anderson LH, et al. Eur Heart J. 2001;22: Jensen P, et al. Blood. 2003;101:4632.

30. T2* MRI: Emerging New Standard for Cardiac Iron90 80 70 60 50
LVEF (%)
Cardiac T2* value of 37 in a normal heart 40 30 20 10 10 20 30 40 50 60 70 80 90
100
Heart T2* (ms)
Cardiac T2* value of 4 in a significantly iron overloaded
heart
Relationship between myocardial T2* values and left ventricular ejection fraction (LVEF). Below a myocardial T2* of 20 ms, there was a progressive and significant decline in LVEF (R = 0.61, P < .0001)
Photos courtesy of Dr. M. D. Cappellini. Anderson LJ, et al. Eur Heart J. 2001;22:2171.

31. Cardiac T2* and Risk for Cardiac Dysfunction
In a study of 67 patients with thalassaemia major, 5 had systolic dysfunction LVEF <56%
All 5 patients also had myocardial T2* significantly <20 msec (the lower limit of normality)
Westwood MA, et al. J Magn Reson Imaging. 2005;22:229.

32. No Correlation of Heart Iron Concentration with Liver Iron Concentration?
Anderson LJ, et al. Eur Heart J. 2001;22:2171.

33. MRI Assessment of Cardiac Iron
Advantages
Disadvantages
Rapidly assesses iron content in the septum of heart
Iron levels can be quantified reproducibly
Functional parameters can be examined concurrently
Pathologic status of liver and heart can be assessed in parallel
Indirect measurement of cardiac iron
Requires MRI imager with dedicated imaging method
Technically demanding
Methodology remains to be standardized and validated
Cardiac iron levels can be rapidly and effectively assessed using a technique known as T2* (gradient echo) MRI, which is becoming the new standard method
MRI = magnetic resonance imaging.

34. Tools for Monitoring Iron Overload
Prognostic significance demonstrated
Serum ferritin (= body iron)1
Liver iron (= body iron)2
Heart function (LVEF)3
1. Olivieri NF, et al. Blood. 1994;84: Brittenham G, et al. N Engl J Med. 1994;331:567.
3. Davis BA, et al. Blood. 2004;104:263.

35. Tools for Monitoring Iron Overload
Prognostic significance not yet demonstrated
Cardiac iron (T2*), linked to LVEF1
NTBI, LPI
LPI measures the redox-active component of plasma iron2
Can form reactive radicals responsible for many clinical consequences of iron overload2
1. Anderson LJ, et al. Eur Heart J. 2001;22:2171.
2. Esposito BP, et al. Blood. 2003;102:2670.

36. Iron Overload Evaluation Recommendations
Do not use a single test alone for iron overload management
Exclude haemochromatosis
Serum ferritin is the basic parameter, but
Do not use it alone
Be aware of its poor predictive value
Use the trend of repeated measures (iron load direction)
Measure liver iron concentration (iron load amount and “buffer reserve”)
By biopsy, if indicated
By SQUID, where available
By MRI (method, calibration, error)
Assess the heart iron by MRI T2* (cardiac risk), at least once
If positive, use it as the main result to set treatment
If negative, do not exclude body iron overload
In transfused patients
Accurately record the iron input
Do iron balance, where feasible
Integrate available tests for effective management of iron chelation
Angelucci E, et al. Haematologica. 2008, in press.