1. Pakistan Society Of Chemical Pathologists Distance Learning Programme In Chemical Pathology (DLP-2) Lesson No 7 Disorders of Iron Metabolism By Brig Aamir Ijaz MCPS, FCPS, FRCP (Edin) Professor of Pathology / Consultant Chemical Pathologist AFIP Rawalpindi

2. Part I MCQs (One Best Type)

3. e. Parenchymal iron overload
Q.1: All of the following features are common to Hereditary Hemochromatosis and Iron Deficiency Anaemia EXCEPT: a. Depleted ferritin in enterocytes b. Depleted ferritin in macrophages c. Increased intestinal iron absorption d. Increases mucosal iron transfer e. Parenchymal iron overload
e. Parenchymal iron overload
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4. b. Increased transferrin saturation
Q 2. The hepcidin-ferroportin interaction controls systemic iron homeostasis. Which of the following is NOT an indicator of high hepcidin concentrations a. High iron in macrophages b. Increased transferrin saturation. c. Internalized ferroportin d. Iron accumulated in enterocytes, e. Trapped iron in hepatocytes
b. Increased transferrin saturation
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5. Slides Courtesy of Dr Sobia Irum(AFIP Rwp)
Hepcidin
Slides Courtesy of Dr Sobia Irum(AFIP Rwp)

6. Hepcidin
Hepcidin was first discovered as a small bactericidal peptide.
The name ‘hepcidin’ originates from the place of synthesis in hepatocytes (hep‐) and its antimicrobial activity (‐cidin)
The gene encoding hepcidin (HAMP, 19q13) is expressed in the liver, heart, lungs, brain, spinal cord, intestine, stomach, pancreas, adipocytes, skeletal muscles, testis and macrophages.

7. Hepcidin gene and peptide
The human hepcidin gene (HAMP) is located on chromosome 19q13.1
HAMP encodes a precursor of hepcidin – preprohepcidin
Preprohepcidin is cleaved and gives rise to hepcidin. There are three forms of hepcidin: 25 aa, 22 aa and 20 aa peptide. All three forms are detectable in urine, but only hepcidin‐25 and hepcidin‐20 are present in human serum.
Hepcidin‐25, is a major form of hepcidin,

8. Mechanisms of hepcidin action
It depends on hepcidin interactions with ferroportin.
Ferroportin is the only iron exporter, which is expressed on the surface of reticulo‐endothelial macrophages, hepatocytes, duodenal enterocytes and placenta cells.
Hepcidin binds to ferroportin and causes its internalization and degradation in endolysosomes, which in turn blocks the iron transport.
When iron stores are adequate or high, increased hepcidin expression inhibits intestinal iron absorption, release of recycled iron from macrophages and its transport across the placenta.
On the other hand, when iron stores are low, hepcidin production is suppressed, thus maintaining iron metabolism homeostasis

9. Mechanisms of hepcidin action
Interactions between duodenal enterocytes, hepatocytes, and macrophages in iron homeostasis regulated by hepcidin
Mechanisms of hepcidin action

10. Regulation of hepcidin synthesis
Positive regulators of hepcidin production
a. Inflammation
Hepcidin is not only iron‐regulatory hormone but also type II acute‐phase reactant. It means that its synthesis can be induced by inflammatory cytokine IL‐6.
Data shows that hepcidin could be the pathogenic mediator of anemia of chronic diseases (ACD).
b. Increase in iron stores

11. Regulation of hepcidin synthesis (Cont)
Negative regulators of hepcidin production a. Hypoxia, anemia, increased erythropoiesis Hypoxia and anemia regulate the erythrocytes production through erythropoietin (Epo) synthesis. Some authors claim that Epo is a hormone down ‐modulating hepcidin mRNA expression b. Decrease in iron stores

12. Hepcidin in the pathogenesis of iron disorders and other diseases
Hemochromatosis (HH), the most common form of genetic iron overload, is divided into two groups.
Associated with defective or suppressed hepcidin gene caused by mutation in four genes: HFE‐1, HJV, TfR‐2 and HAMP.
Ferroportin disorders.
The increased level of serum hepcidin is observed in many chronic inflammatory diseases such as chronic kidney diseases, thalassemia, glucose‐6‐phosphate dehydrogenase deficiency, sickle cell disease (SCD), coronary artery disease (CAD) and myelodysplasia.
The individuals with the anemia of inflammation, characterized by disturbance of iron absorption, hypoferremia
and hyperferritinemia

13. Hepcidin as a potential diagnostic and therapeutic tool
Distinguishing anemia of chronic diseases (ACD) from iron deficiency anemia (IDA), as it is known that hepcidin production is induced by inflammation in ACD and reduced in IDA.
Greatest practical application of hepcidin assay is the utilization of hepcidin in diagnosis and monitoring of hemochromatosis.

14. e. Increased hepcidin concentration
Q 3: An oral load of 65 mg of iron in a healthy volunteer will cause: a. Decreased ferroportin b. Decreased sensing of hepatocytes to transferrin c. Decreased signals from other cells to produce hepcidin d. Decreased transferrin saturation e. Increased hepcidin concentration.
e. Increased hepcidin concentration
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15. Q 4: Which of the following genetic mutations results into Hemochromatosis with mildest hepcidin deficiency: a. Autosomal recessive Hemojuvelin (HJV) b. Autosomal recessive Hepcidin (HAMP) c. Autosomal recessive Transferrin Receptor 2 (TfR2) d. Bone morphogenetic protein pathway (BMP6) e. Heterozygous HFE
e. Heterozygous HFE
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16. Q 5 : In Hemochromatosis which of the following cells are iron-overloaded: a. Bone marrow cells b. Enterocytes c. Erythrocytes d. Hepatocytes e. Macrophages
d. Hepatocytes
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17. e. Transferrin saturation
Q 6 : A 49 year male patient is a known case of hepatitis C (HCV-RNA PCR Positive) being evaluated for treatment options. His biochemical tests showed: • Serum bilirubin: 32 μmol/L (<17) • Serum ALT: 267 U/L (<42) • Serum Alkaline Phosphatase: 168 U/L (20-178) • Serum ferritin: ng/ml (Iron Overload > 400) At this point of time which of the following investigations will be most appropriate first line test in this patient: a. HFE mutation analysis b. Liver biopsy c. Serum iron d. TIBC e. Transferrin saturation
e. Transferrin saturation
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18. d. Soluble transferrin receptors
Q 7 : A 55 years female is a known patient of rheumatoid arthritis and acid peptic disease. She has to undergo endoscopy to evaluate the ulcerating lesion in the stomach. Gastroenterologist has referred the patient to you for a reliable assessment of her iron status. Which of the following laboratory investigations will be most helpful in this patient?: a. Serum Ferritin b. Serum Iron c. Serum TIBC d. Soluble transferrin receptors e. Transferrin saturation
d. Soluble transferrin receptors
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19. Soluble transferrin receptor (sTfR)
Slides Courtesy of Dr Uzma Ansari (AFIP Rwp)

20. Soluble transferrin receptor (sTfR)
The soluble transferrin receptor (sTfR) is a truncated form of membrane-associated transferrin receptor.
sTfR is present on the surface of all iron requiring cells in the body, with highest number on the surface of precursor erythrocytes.
The circulating sTfR concentration is proportional to cellular expression of the membrane-associated TfR and inversely related to iron status
elevates in response to iron deficiency
decreases in response to iron repletion.

21. Advantage of sTfR over ferritin
It is not an acute-phase reactant thus it can identify iron deficiency in hospitalized and chronically ill patient with
Inflammation
Infection
Chronic disease
Other conditions in which ferritin concentration does not correlate with iron status, e.g. Cystic fibrosis
It thus reduces the need for a bone marrow biopsy or trial of iron therapy

22. Limitation of sTfR  
Can not differentiate iron deficiency from ineffective erythropoisis as it also increased with increased erythropoisis e.g. thalessemia , sickle cell anemia and hereditary spherocytosis

23. e. Transferrin saturation
Q 6 : A 49 year male patient is a known case of hepatitis C (HCV-RNA PCR Positive) being evaluated for treatment options. His biochemical tests showed: • Serum bilirubin: 32 μmol/L (<17) • Serum ALT: 267 U/L (<42) • Serum Alkaline Phosphatase: 168 U/L (20-178) • Serum ferritin: ng/ml (Iron Overload > 400) At this point of time which of the following investigations will be most appropriate first line test in this patient: a. HFE mutation analysis b. Liver biopsy c. Serum iron d. TIBC e. Transferrin saturation
e. Transferrin saturation
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24. e. Relative iron deficiency
Q 8 : A 57 year male is a known patient of Chronic Kidney Disease. He is on periodic haemodialysis. His haemoglobin is low in spite of repeated erythropoietin injections. His recent biochemical profile shows: • Serum ferritin: ng/ml • Transferrin saturation: 17% What is the most probable cause of his low haemoglobin?: a. Absolute iron deficiency b. Acute phase reaction c. Anaemia of chronic disorder d. HFE hemochromatosis e. Relative iron deficiency
e. Relative iron deficiency
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25. Part II Match the answers (Extended Matching Questions)

26. Clinical, biochemical and genetic features of various disorders of iron metabolism Options
Anemia of chronic kidney diseases
Anemia of chronic inflammation
Bantu siderosis
Chronic liver disease
Haemochromatosis (haemojuvelin-HJV)
Haemochromatosis (HAMP)
Haemochromatosis (TFR2)
Hemochromatosis (Ferroportin disease)
Hemochromatosis (HFE)
Hypotransferrinemia
Iron deficiency anaemia
Iron-refractory iron deficiency anaemia
Sidroblastic anaemia
Thalassemia intermedia
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27. Slides Courtesy of Dr Qurat Ul Ain (AFIP Rwp)
Biochemical and genetic features in various disorders of Iron metabolism
Slides Courtesy of Dr Qurat Ul Ain (AFIP Rwp)

28. Biochemical and genetic features in various disorders of Iron metabolism
%Trasferrin saturation
Serum Ferritin
Serum Hepcidin
Gene Mutation
Iron Deficiency Anemia -
Iron Refractory Iron Deficiency Anemia
Normal or
TMPRSS6
Iron Loading Anemias*
Anemia of Chronic Disease
Hypotransferrinemia
or N
or N˦
or N˦
*Thalasemia Major, Sidroblastic, Aplastic or Chronic Hemolytic Anemias
˦ Iron overload is seen predominantly in macrophages

29. Biochemical and genetic features in various disorders of Iron metabolism (cont)
%Trasferrin saturation
Serum Ferritin
Serum Hepcidin
Gene Mutation
HFE Hemochromatosis
HFE
(C282Y)
Juvenile Hemochromatosis
HJV
TFR2 Hemochromatosis
TFR 2
Ferroportin Hemochromatosis
1. Ferroportin Disease
SLC40A1
2. Hepcidin Resistance
HAMP Hemochromatosis
(compound heterozygous)
C282Y + C78T
or N
or N˦
˦ Iron overload is seen predominantly in macrophages

30. h. Hemochromatosis (Ferroportin disease)
Q 9 : A 39 year male suffering from hepatomegaly with following biochemical and genetic features: • Serum ferritin: ng/ml (Iron Overload > 400) • Transferrin saturation: 54% • Serum Hepcidin: Increased • Mutation analysis: SLC40A1 What is the most probable Disorder of Iron Metabolism?
h. Hemochromatosis (Ferroportin disease)
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31. k. Iron deficiency anaemia
Q 10 :A 36 year old female with general weakness without any hepato-splenomegaly with following biochemical and genetic features:   Serum ferritin: ng/ml (Iron Def < 10) Transferrin saturation: 37% Serum Hepcidin: decreased Mutation analysis: not done   What is the most probable Disorder of Iron Metabolism?
k. Iron deficiency anaemia
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32. i. Hemochromatosis (HFE)
Q 11 : A 53 year male diabetic is having darkening of skin and following biochemical and genetic features:   Serum ferritin: ng/ml (Iron Overload > 400) Transferrin saturation: 62% Serum Hepcidin: Decreased Mutation analysis: C282Y (homozygous) What is the most probable Disorder of Iron Metabolism?
i. Hemochromatosis (HFE)
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33. Slides Courtesy of Dr Majid Latif (AFIP Rwp)
HFE Hemochromatosis
Slides Courtesy of Dr Majid Latif (AFIP Rwp)

34. HFE Hemochromatosis Most prevalent type of hemochomatosis
Occurs predominantly in Caucasians of European descent
It is the classical type of hemochomatosis, previously called hereditary or idiopathic hemochromatosis
Transmitted as autosomal recessive trait
Caused by common mutations in the HFE gene
The most common deleterious mutation is C282Y

35. HFE Hemochromatosis (Cont)
HFE protein associates with the transferrin receptor and modulates hepatic expression of hepcidin
Thus abnormal HFE protein caused by common HFE mutations can lead to augmentation of iron absorption by the small intestine
Clinically significant iron overload occurs almost exclusively in adults but is rarely severe before the fourth decade

36. HFE Hemochromatosis (Cont)
The biochemical penetrance of HFE C282Y homozygosity is fairly high
More than 50% of homozygotes have elevated transferrin saturation values and/or serum ferritin concentrations and about 10% have elevated ALT
A high proportion of homozygotes diagnosed in families of probands are seriously affected

37. L. Iron-refractory iron deficiency anaemia
Q 12 : A 31 year female is having low haemoglobin for the age and gender without any obvious source of chronic bleeding. Her biochemical and genetic features are:   Serum ferritin: ng/ml (Iron Def < 10) Transferrin saturation: 33% Serum Hepcidin: Increased Mutation analysis: Mutation in TMPRSS6 What is the most probable Disorder of Iron Metabolism?
L. Iron-refractory iron deficiency anaemia
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38. Iron-refractory iron deficiency anaemia
Slides Courtesy of Dr Amina Tariq (AFIP Rwp)

39. Iron refractory iron deficiency anemia (IRIDA)
Hereditary recessive anemia due to a defect in the TMPRSS6 gene encoding Matriptase-2 (MT-2)
MT-2 is a transmembrane serine protease; predominantly expressed on hepatocytes that cleaves Hemojuvelin (HJV); thus down-regulating hepcidin expression.
The anemia usually appears in the post-natal period, although in some cases it is only diagnosed in adulthood.
Hallmarks are:
Microcytic hypochromic anemia
Low transferrin saturation
Low reticulocyte count
Inappropriately normal / high serum hepcidin

40. How to differentiate IRIDA from other causes of IDA and other genetic microcytic anemias?:
Positive family history in IRIDA in contrast to acquired IDA.
Acquired IDA may result from chronic blood loss or decreased iron absorption, as in celiac disease; which can be picked by clinical data.
The age of onset may contribute to diagnosis since microcytic anemia is not present at birth in IRIDA; in contrast to other genetic conditions such as DMT1 mutation or atransferrinemia.
Iron overload is present in sideroblastic anemia, beta thalassemia, DMT 1 mutation and atransferrinemia.
Serum hepcidin level is high in IRIDA while it is low in nutritional IDA and other genetic microcytic anemias.

41. e. Haemochromatosis (haemojuvelin-HJV)
Q 13 : A 22 years male has low testosterone and low FSH and LH with following other biochemical and genetic features:   Serum ferritin: ng/ml (Iron Overload > 400) Transferrin saturation: 57% Serum Hepcidin: Decreased Mutation analysis: G320V What is the most probable Disorder of Iron Metabolism?
e. Haemochromatosis (haemojuvelin-HJV)
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42. f. Haemochromatosis (HAMP)
Q 14 : A 48 year male has hepatomegaly with following biochemical and genetic features:   Serum ferritin: ng/ml (Iron Overload > 400) Transferrin saturation: 66% Serum Hepcidin: Decreased Mutation analysis: C282Y + C78T   What is the most probable Disorder of Iron Metabolism?
f. Haemochromatosis (HAMP)
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43. j. Hypotransferrinemia
Q 15 : A 40 year male has severe microcytic hypochromic anemia with parenchymal iron overload on liver biopsy shows. His biochemical and genetic features are   Serum ferritin: ng/ml (Iron Def < 10) Transferrin saturation: 16% Serum Hepcidin: Decreased Mutation analysis: Not done What is the most probable Disorder of Iron Metabolism?
j. Hypotransferrinemia
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44. Part III Short Answer Questions:

45. Q. 16: Serum Iron, Serum TIBC and Transferrin Saturation are widely used for the investigation of iron disorders in our country. Please answer following questions related to these tests (this question carries THREE marks):   In reagent kits used on auto-analysers two separate reagent solutions are used for colorimetric analysis of Iron and TIBC with common standard. What is basic chemical difference in these two reagent solutions used for Iron and TIBC?   How TIBC is determined by this method. Give essential steps. You may use hypothetical data for calculations.   After determining Iron and TIBC, calculate Transferrin Saturation using the same hypothetical data.
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46. Serum Iron is measured in acidic pH
Q. 16 a. In reagent kits used on auto-analysers two separate reagent solutions are used for colorimetric analysis of Iron and TIBC with common standard. What is basic chemical difference in these two reagent solutions used for Iron and TIBC?
pH is the basic chemical difference in two reagent solution used for the analysis of Iron and TIBC.
Serum Iron is measured in acidic pH
Serum TIBC is measured in alkaline pH
R-1 is Liquid (iron saturating solution)
R-2 is Powder (Magnesium carbonate)

47. Serum Transferrin (approx) =0.007×TIBC
Q. 16 b. How TIBC is determined by this method. Give essential steps. You may use hypothetical data for calculations.
TIBC is indirect measurement of transferrin, and it is determined by following steps:
Measure serum Iron in acidic pH which release bound Iron
A known ferrous iron standard 105 µmol/L (586 µg/dL) incubated with serum at alkaline pH (pH7.9) saturates the available binding sites on serum transferrin. The unbound excess iron is then complexed with R2 and form a blue complex, which is measured.
The UIBC = total iron added- the excess iron measured.
TIBC=UBIC+ serum Iron
TIBC is the measurement of maximum concentration of Iron that transferrin can bound
Serum Transferrin (approx) =0.007×TIBC

48. Q. 16 c. After determining Iron and TIBC, calculate Transferrin Saturation using the same hypothetical data.
Using hypothetical data mentioned in above answer
Transferrin saturation = Iron/TIBC×100
Transferrin saturation=4/101×100
Transferrin saturation=3.9%
Opinion: Iron deficiency anaemia because low serum Iron, High TIBC and low transferring saturation)

49. b. Competitive ELISA. a. TOF
Q. 17. Dr Naushaba is a PhD scholar, working on various iron disorders. She wants to determine Hepcidin in urine and serum of her experimental animals. Please suggest her at least TWO methods of hepcidin after thorough literature search.
a. TOF
Cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS) using hepcidin analogue as internal standard for quantitation.
This method allows discrimination between the four naturally occurring isoforms, hepcidin-20, -22, -24 and -25 (BIOACTIVE), which are the result from differential processing at the hepcidin amino-terminus. 
b. Competitive ELISA.
This method measures the total hepcidin (sum of concentration of all) isoforms and is especially appropriate for large sample numbers.

50. Serum Hepcidin Profile on TOF
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51. Urine Hepcidin Profile on TOF
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52. Q. 18. Dr Ahmed is a Consultant Chemical Pathologists in a tertiary care hospital. He wants to improve Laboratory Service for diagnosis and monitoring of iron disorders by providing Serum Ferritin and Soluble Transferrin Receptors. Please help him selecting reagent kits which can be used on the same immunoassay autoanalyser. You can use commercial names in this question.
Serum Ferritin and Soluble Transferrin Receptor (sTfR) on same immunoassay autoanalyzer can be analysed by kits from Roche®Diagnostics:
Ferritin Roche – Tina –quant
sTfR Roche –Tina-quant
Roche Tina-quant method, principle is immunoturbidimetry using Roche kit on Hitachi modular P clinical analyzer.
Other manufacturers also offer kits for these two test on the same auto-analyser e.g. Beckman-Coulter

53. Thank You and Best Of Luck