Diagnosis of Iron Overload

M. Domenica Cappellini, MDProfessor of Internal Medicine

University of MilanMaggiore Hospital

Milan, Italy

Iron Overload and Disease States

Feder JN, et al. Nat Genet. 1996;13:399.Porter JB. Br J Haematol. 2001;115:239.

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

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.

Conditions at Risk of Iron OverloadSources of Iron

Liver cirrhosis/ fibrosis/cancer

Diabetes mellitus

Growth failure

Capacity of serum transferrin to bind iron is exceeded

Iron overload

Cardiac failure

Infertility

Complications of 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

Courtesy of Dr. M. D. Cappellini.

Cohen AR & Porter JB. In: Steinberg MH, et al, eds. Cambridge University Press;2001:979–1027.

Consequences of Iron-Mediated Toxicity During Iron Overload

Cell death Fibrosis

Organelle damage TGF-β 1

Hydroxyl radical generation

Lipid peroxidation

Lysosomal fragility

Enzyme leakage

Collagen synthesis

LPI = labile plasma iron; TGF = transforming growth factor.

Increased LPI or “free” iron

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.

50

30

0

40

10

20

Increased risk of complications

Normal

0 20 5010 30 40

Age (years)

Thalassaemia major: transfusion without chelation

Homozygous haemochromatosis

Heterozygote

Optimal level in chelated patients

Hep

atic

iro

n,

mg

/g o

f li

ver,

dry

wei

gh

t

Threshold for cardiacdisease and early death

Olivieri N, et al. Blood. 1997;89:739.

Liver Iron and Risk from Iron OverloadH

epat

ic I

ron

(µ

mo

l/g

wet

wei

gh

t)

50

100

150

200

250

0

Assessing Iron Overload

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.

Transferrin Saturation

• Normal values: 16%–30%

• > 40%: iron overload

Monitoring—Plasma Ferritin

• Relatively noninvasive

• Inexpensive

• Routine laboratory assay

• Values confounded by

– Inflammation

– Liver function

– Ascorbate status

0 4000 8000 12000

24,000

12,000

8000

4000

0

Hepatic Iron(µg Fe/g liver)

Pla

sma

Fer

riti

n (

µg

/L)

Brittenham G, et al. Am J Hematol. 1993;42:81.

Sickle cell anaemia (n = 37)

Thalassaemia major (n = 74)

Serum Ferritin and Risk fromIron 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;95:26. 2. Olivieri NF, et al. N Engl J Med. 1994;331:574. 3. Telfer PT, et al. Br J Haematol. 2000;110:971. 4. Davis BA, et al. Blood. 2004;104:263. 5. Borgna-Pignatti C, et al. Haematologica. 2004;89:1187.

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

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;343:327. 2. Jensen P, et al. Blood. 2003;101:4632. 3. Angelucci E, et al. Blood. 2002;100:17. 4. Porter JB. Hematol/Oncol Clinics. 2005;S7. 5. Mariotti E, et al. Br J Haematol. 1998;103:916.

150

300

100

200

0

50

250

10 255 15 20

LIC = liver iron concentration.

r = 0.98

Angelucci E, et al. N Engl J Med. 2000;343:327.

To

tal

Bo

dy

Iro

n S

tore

s (m

g/k

g)

LIC (mg/g, dry weight)

r2 = 0.98

25 patientswith iron overload

and cirrhosis

≥1 mg dry weight liver sample

LIC Accurately Reflects Total Body Iron Stores

r = 0.98

Courtesy of Dr. J. Porter.

Approximate LIC, mg/g dry weight liver

>3.2~3.2>1.2~1.2<1.2

<1.2

<1.2

Heterozygous

>15<1.235>15<1.230

(Not surviving)>15<1.225>15~15<1.220>15>7<1.215

>15~7<1.210

>15>3.2<1.25

β-thalassaemia MajorHomozygousNormal

Age (years)

Haemochromatosis

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.

LIC and Prognosis

Estimation of LIC

Liver biopsy

• Distribution artifact

• Debate about safe levels

• Safety

• Patient acceptance

• Sample size

– ≥1 mg dry weight

– >4 mg wet weight

Photos courtesy of Dr. J. Porter.Porter JP. Br J Haematol. 2001;115:239.

2 cm

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

SQUID = superconducting quantum interface device; MRI = magnetic resonance imaging; SIR = signal intensity ratio.

1. Anderson LH, et al. Eur Heart J. 2001;22:2171. 2. St. Pierre TG, et al. Blood. 2005;105:855.3. Gandon Y, et al. Lancet. 2004;363:357. 4. Jensen, et al. Blood. 2003;101:4632.

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

Superconductive Quantum Interference Device

Josephson effect

V

I

Ic-Ic

IS S

0.8

0.060

0.1

0 42 6 8temperature (°K)

0.160.2

0.26

Conductive Superconductive

Superconductivity

Meissner effect

T>Tc T<Tc T<Tc

Normal(nonsuperconducting)

Flux expulsion(superconducting state)

Persistent current(superconducting state)

Res

ista

nce

(ohm

)

SQUID Biomagnetic Susceptometer

Courtesy of A. Piga, Turin Thalassaemia Centre.SQUID Thalassaemia Center. Turin, Italy

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.

T2 (heart, liver)Spin echo, gradient-echo sequences

Signal intensity ratio (SIR)

T2*(heart)Gradient-echo sequences

ms

R2 (liver)Gradient-echo sequences

s-1

Quantitative IronAssessment by MRI

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.

R2 MRI—A New Measure for LIC

St Pierre TG, et al. Blood. 2005;105:855.

30

20

40

50

0.5 1.0 1.5 2.0

Biopsy Iron Concentration (mg/g-1 dry tissue)

Mea

n T

ran

sver

se R

elax

ati

on

Rat

e <

R2

> (

s-1)

0

100

200

50

150

250

300

0 10 20 30 40 50

R2 MRI is a validated and standardized method for measuring LIC.

This technique is now approved by TGA and FDA and in the EU

Hereditary haemochromatosis

Hepatitis

β-thalassaemia

β-thalassaemia/ haemoglobin E

Wood JC, et al. Blood. 2005;106:1460.

R2* Measurement of LIC

HIC = hepatic iron concentration.

Estimated HIC (mg/g dry weight)

R2*

(H

z)

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

MRI = magnetic resonance imaging; LIC = liver iron concentration.

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

Assessing Cardiac Function and Iron Load

ECG = electrocardiogram; ECHO = echocardiogram; MUGA = multiple gated acquisition;MRI = magnetic resonance imaging; SIR = signal intensity ratio.

1. Davis BA, et al. Blood. 2004;104:263. 2. Anderson LH, et al. Eur Heart J. 2001;22:2171. 3. Jensen P, et al. Blood. 2003;101:4632.

Monitoring—Heart• Rhythm

– 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

T2* MRI: Emerging New Standard for Cardiac Iron

Photos courtesy of Dr. M. D. Cappellini.Anderson LJ, et al. Eur Heart J. 2001;22:2171.

LV

EF (

%)

0

50

70

40

30

20

10

60

80

90

0 20 40 60 9080 10010 30 50 70

Heart T2* (ms)

Cardiac T2* value of 37 in a normal heart

Cardiac T2* value of 4 in a significantly iron overloadedheart

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)

Westwood MA, et al. J Magn Reson Imaging. 2005;22:229.

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)

Anderson LJ, et al. Eur Heart J. 2001;22:2171.

No Correlation of Heart Iron Concentration with Liver Iron Concentration?

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.

Tools for MonitoringIron 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;84:3245. 2. Brittenham G, et al. N Engl J Med. 1994;331:567. 3. Davis BA, et al. Blood. 2004;104:263.

Tools for MonitoringIron 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.

Angelucci E, et al. Haematologica. 2008, in press.

Iron Overload EvaluationRecommendations

1. Do not use a single test alone for iron overload management

2. Exclude haemochromatosis

3. Serum ferritin is the basic parameter, buta) Do not use it alone

b) Be aware of its poor predictive value

c) Use the trend of repeated measures (iron load direction)

4. Measure liver iron concentration (iron load amount and “buffer reserve”)a) By biopsy, if indicated

b) By SQUID, where available

c) By MRI (method, calibration, error)

5. Assess the heart iron by MRI T2* (cardiac risk), at least oncea) If positive, use it as the main result to set treatment

b) If negative, do not exclude body iron overload

6. In transfused patientsa) Accurately record the iron input

b) Do iron balance, where feasible

7. Integrate available tests for effective management of iron chelation