Iron Studies

AACB Tutorial February 2012

 Iron Studies

• Iron Metabolism
– Stores
– Absorption
– RBC turnover
• Iron study parameters
• Methods
• Cases
• Pattern recognition
• Reference Ranges
 Iron Metabolism

Function
• One of the most abundant elements on earth – only
trace amounts present in living cell
• Most of iron located in porphyrin ring of haem which is
incorporated into proteins:
– haemoglobin, myoglobin, catalase, peroxidases, cytochromes
• There are also iron-sulphur proteins
– NADH dehydrogenase, succinate dehydrogenase
In which iron is present in clusters with inorganic sulphur

• It is the ability of iron to interact reversibly with oxygen

and to function in electron transfer that makes it
biologically indispensable
 IRON
Body Compartments - 75 kg man
Stores
1000mg

Tissue Absorption < 1 mg/day

3 mg
500 mg Excretion < 1 mg/day

Red Cells
2300 mg
 Absorption
• Average diet 10-30 mg/day – only 5-10% absorbed mainly in
duodenum and upper small intestine
• Most dietary iron in the Fe 3+ state
• Gastric acid converts Fe3+ to more absorbable Fe2+
• Ascorbic acid, sugars and amino acids increase absorption
• Phosphates in eggs, milk and cheese, oxalates and phytates and
tannates decrease absorption

• Haem iron mainly from meat and fish is processed differently

• After release from polypeptide chain haem is absorbed intact by the
mucosal cells
• Porphyrin ring split and iron liberated – process not affected by
dietary factors
• The major factors affecting iron absorption are body iron stores and
the rate of red blood cell production
 Red Blood Cell Turnover

• Absorbed iron represents only a fraction of the iron

required
• 20 – 25 mg/day from old blood cell destruction is
recycled
• Macrophages transfer iron to plasma transferrin
• Carried to the bone marrow for Hb synthesis
• Stored as ferritin or hemosiderin
• In the gut stored iron can be mobilized or lost with
mucosal shedding
 Interpretation of Iron Studies

• Disorders of iron homeostasis is common

• Can be difficult to interpret
• Many biological influences on each
component
• Different age and sex related reference
ranges
 Serum Iron
• Measure of the amount of iron in the blood
• Usually does not fall until iron stores (serum ferritin) have
been depleted

• Diagnostic value
– Substantial day-to-day fluctuations
– Increased levels due to iron ingestion or ineffective
erythropoiesis
– Decreased levels due to infection, inflammation,
malignancy, menstruation and Fe deficiency
– Needs to be taken into consideration with TIBC
and/or transferrin to calculate TSAT
 Serum Ferritin
• Ferritin stores iron in a non toxic form – large spherical molecule protein
shell with core of ferric hydroxide phosphate
• Apart from Fe storage serum ferritin is also an acute phase reactant protein

• Two types of ferritin

– Intracellular
– Plasma
• Rate of synthesis of intracellular and plasma ferritin is similar

• Diagnostic value
– Is considered one of the most useful estimates of body iron storage

– Low levels are usually due to iron deficiency or response to erythropoietin

therapy

– High levels can be due to malignancies, infection, and/or inflammation

 Transferrin
• Is the principle plasma protein for the transport
of iron
• Increases with iron deficiency, as the body tries
to capture more iron
• Diagnostic value
– High levels indicate iron deficiency, also high
in normal pregnancy
– Low levels may indicate inflammation,
infection, malignant tumours, or malnutrition
– Needs to be taken into consideration with
serum iron to calculate TSAT levels
 Transferrin Saturation
TSAT—measurement of the iron bound to transferrin
– Indicates how much iron is immediately available to support
erythropoiesis

– Diagnostic value

• Low TSAT can indicate iron deficiency, erythropoiesis, infection, or

inflammation
• Elevated TSAT can indicate recent ingestion of dietary iron,
ineffective erythropoiesis, haemochromatosis or liver disease

• TSAT values may fluctuate by 30% or more in a day

 TSAT Calculation

TSAT % = Serum iron x 100 ÷ transferrin x 26.1

26.1 converts transferrin to an equivalent TIBC

conc.
 Previous TIBC Method

• Sample treated with ferric chloride

• Unbound iron absorbed by MgCO3

• Insoluble complex – centrifuged

• Sample and supernatant measured for iron

 Soluble Transferrin Receptor
• Transferrin receptors transport iron into cells
• Truncated portion of transferrin receptors coming mostly
from the bone marrow
• sTfR levels increase if bone marrow lacks iron
• Diagnostic value
– Elevated sTfR levels indicate iron deficiency in the
bone marrow or enhanced erythropoiesis
– sTfR levels are not affected by inflammation
– Differentiates patients with iron deficiency from
patients with anaemia of chronic disease
 Soluble Transferrin Receptor

• Initial research suggested sTfR would be a good

discriminator of Fe deficiency in difficult cases ie
inflammatory disorders or CRF

• Very difficult to standardise

• Not routinely available - monthly turnaround in IMVS
• Not considered useful
 Iron Methods
• Reaction Principle

• Transferrin 2(Fe 3+) + Buffer → 2(Fe3+ ) + Apo-transferrin

• 2 Fe3+ + Ascorbic Acid + 2H2O → 2Fe2+ + Dehydroascorbic Acid + 2 H3O +

• Fe 2+ + TPTZ → Fe2+-complex (blue coloured complex)

(600/800 nm) Olympus
• Fe 2+ + Ferrozine → Fe2+-Ferrozine complex
(571/658 nm) Advia

TPTZ (2,4,6-Tri(2-pyridyl)-5-triazine)
 Transferrin Methods

• Immuno-turbidimetric test

• Sample +R1(buffer)+R2(antiserum) →Insoluble

aggregates
Olympus 380/800nm

PEG enhanced Advia 596/694nm

Absorbance is proportional to transferrin concentration

Olympus Ferritin Method
• Immuno-turbidometric assay
• Latex particles coated with rabbit anti-human ferritin
• Immune complexes formed causing light scatter
• Measure the reduction of incident light at 660nm

Advia Method
• Two-site sandwich immunoassay
• Polyclonal goat anti-ferritin labelled with acridinium ester
• Solid phase monoclonal mouse on magnetic particles
• Chemiluminescent measurement
 Tests
Serum iron Transferrin Transferrin Sat Ferritin
μmol/L g/l % μg/L
Notes Very variable Transports iron Assessement of Most useful test
Diurnal changes Deposits iron to “transport iron” Acute phase
Best at 8 am. cells with TF protein
Not useful in receptor
assessing iron stores
Low Diurnal Chronic disease Iron deficiency Iron deficiency
result Intercurrent illness Chronic disease
Chronic disease

High Diurnal Iron deficiency Iron therapy Acute phase

result Iron overload Oestrogen Iron overload Iron overload
Iron therapy therapy Liver disease
Pregnancy Malignancy
 Iron deficiency

• 8-30 Fe 7
• 20-200 Ferritin 10
• 2.00-4.00 Transferrin 6.04
• 10-55 Trans Sat 5
• 115-155 Hb 109
• 80-98 MCV 81.9
• 27-33 MCH 25.3

• Result is consistent with iron deficiency

 Iron deficiency

• 8-30 Fe 1
• 20-200 Ferritin 3
• 2.00-4.00 Transferrin 1.81
• 10-55 Trans Sat 2

• 34-48 Albumin 17
• 65-85 TP 43

• Results are consistent with iron deficiency. Severe protein loss or

liver disease may be contributing to the abnormal iron studies
 ? Iron deficiency

• 8-30 Fe 9
• 20-200 Ferritin 55
• 2.00-4.00 Transferrin 3.2
• 10-55 Trans Sat 9

• Low iron and low saturation in the presence of normal ferritin do not
exclude Fe deficiency if inflammation or chronic disease is present
 Iron Overload

• 8-30 Fe 32
• 20-200 Ferritin 495
• 2.00-4.00 Transferrin 2.11
• 10-55 Trans Sat 60

• Raised ferritin with borderline high saturation. This pattern may

indicate early iron overload. Suggest repeat Fe studies in 3 months.
• Suggest haemochromatosis gene studies and review for signs of
organ damage
 Inflamation

• 8-30 Fe 22
• 20-200 Ferritin 546
• 2.00-4.00 Transferrin 3.24
• 10-55 Trans Sat 27

• The raised ferritin is likely due to inflammation or other underlying

illness. The transferrin saturation does not support a diagnosis of
iron overload.
 Anaemia of chronic disease
• 8-30 Fe 3
• 20-200 Ferritin 485
• 2.00-4.00 Transferrin 1.81
• 10-55 Trans Sat 7
• 115-155 Hb 101
• 80-98 MCV 93.7
• 27-33 MCH 30.4
• 0-60 GTT 81
• 30-110 ALP 297

• Raised ferritin with low transferrin. This pattern is consistent with

anaemia of chronic disease
 Notes

• No significant iron excreted from the body

• Iron absorption increased by anaemia even if not Fe deficient
• Plasma Fe levels vary considerably under physiological
circumstances
• Plasma Fe levels fall in many cases of anaemia not due to Fe def
• Fe carried in plasma bound to transferrin
• TIBC rises in Fe deficiency and falls in overload
• TIBC falls in many cases of anaemia
• A low ferritin is indicative of Fe deficiency
• A cheap, quick and informative test is the CBE and blood film
– microcytic and hypochromic picture
 Pattern Recognition

26y ♂ 18y ♀ 41y ♂ 67y ♀ 15y ♀ 21y ♀

Iron 18► 7▼ 36▲ 5▼ 3▼ 17►

TF 3.0► 5.0▲ 2.9► 2.1▼ 3.0► 4.8▲
TF% 24► 9▼ 90▲ 7▼ 10▼ 23►
Ferritin 245► 8▼ 1050▲ 400▲ 149► 190►
Cause Normal Iron Iron Chronic Intercurrent Oestrogen
deficient overload disease illness therapy or
Fe stores pregnant
likely normal Fe stores
likely normal
Pattern All normal ▼Ferritin ▲Ferritin ▲Ferritin Ferritin & TF ▲TF
▲TF ▲%sat ▼all ► Others
others ▼Iron normal
 Reference Ranges

Iron TF TF Saturation Ferritin

μmol/L g/L % μg/L
Male 8 - 30 2.00 – 4.00 10 - 55 20 - 300

Female 8 - 30 2.00 – 4.00 10 - 35 15 - 250

Children <12 Yrs 8 - 27 1.75 – 3.18 5 - 45 20 - 200