“SOLUBLE TRANSFERRIN RECEPTOR – FERRITIN INDEX IN THE DIAGNOSIS OF ANAEMIA OF CHRONIC DISEASE IN PATIENTS
WITH RHEUMATOID ARTHRITIS”
Dissertation submitted for
M.D. BIOCHEMISTRY BRANCH – XIII DEGREE EXAMINATION
THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY CHENNAI – 600 032
TAMILNADU
MAY 2020
BONAFIDE CERTIFICATE
This is to certify that this dissertation work entitled “SOLUBLE TRANSFERRIN RECEPTOR –FERRITIN INDEX IN THE DIAGNOSIS OF ANAEMIA OF CHRONIC DISEASE IN PATIENTS WITH RHEUMATOID ARTHRITIS” is the original bonafide work done by Dr.
M.V.PREETHI, Post Graduate Student, Institute of Biochemistry, Madras Medical College, Chennai under our direct supervision and guidance.
Dr .R. JAYANTHI, MD., FRCP(Glasg) DEAN
Madras Medical College and Rajiv Gandhi Government General Hospital,
Chennai - 600 003.
Prof. Dr. K. RAMADEVI. M.D., PhD (Guide)
Director & Professor, Institute of Biochemistry Madras Medical College Chennai-600 003.
DECLARATION
I, Dr.M.V.PREETHI , Post Graduate , Institute of Biochemistry, Madras Medical College, solemnly declare that the dissertation titled “SOLUBLE TRANSFERRIN RECEPTOR –FERRITIN INDEX IN THE DIAGNOSIS OF ANAEMIA OF CHRONIC DISEASE IN PATIENTS WITH RHEUMATOID ARTHRITIS” is the bonafide work done by me at Institute of Biochemistry, Madras Medical College under the expert guidance and supervision of our Director and Prof. Dr. K.RAMADEVI M.D,.Ph.D.,, Institute of Biochemistry, Madras Medical College. The dissertation is submitted to the Tamil Nadu Dr. M.G.R Medical University towards partial fulfillment of requirement for the award of M.D., Degree (Branch XIII) in Biochemistry.
Place: Chennai
Date: Dr.M.V.PREETHI
SPECIAL ACKNOWLEDGEMENT
The author gratefully acknowledges and sincerely thanks
Dr .R. Jayanthi, MD., FRCP(Glasg)Dean, Madras Medical College and
Rajiv Gandhi Government General Hospital, Chennai, for granting her
permission to utilize the facilities of this Institution for the study.
ACKNOWLEDGEMENT
The author expresses her warmest respects and profound gratitude to Dr. K. Ramadevi, M.D,.Ph.D.,, Director and Professor, Institute of Biochemistry, Madras Medical College, Chennai, for her able guidance, constant encouragement, support and valuable time but for which this dissertation could not have been made possible.
The author expresses her heartfelt gratitude to her co -guide Dr. B. Sudha Prassana, Assistant Professor, Institute of Biochemistry, Madras Medical College, Chennai, for her constant and valuable guidance, unfailing support, encouragement and inspiration throughout the period of her study.
The author in particular, is extremely thankful to Dr. Balameena M.D, Senior Assistant Professor, Institute of Rheumatology, Rajiv Gandhi Government General Hospital, Chennai, for granting permission to obtain blood samples from the patients.
The author expresses her sincere gratitude to the Professors Dr.R.Chitraa, Dr.K.Pramila, Dr. Amuthavalli.V, Dr.Sumathy.S & Dr. Chelladurai, Dr. R. Panimathi, Institute of Biochemistry, Madras Medical College, for their guidance and support.
The author expresses her warm respects and sincere thanks to Dr.B. Lavanya Devi, Dr. Nirmala Devi. P, Dr. Chithra Sivasankari. C, Dr. Yogeswari. V, Dr. Veena julliette, Dr. Siva. S, Dr. Uma. P and Dr. K.R.Minu Meenakshi Devi, Assistant Professor, Institute of Biochemistry,
Madras Medical College for their guidance regarding the practical issues of research which is beyond the scope of textbooks.
The author expresses her respects and sincere thanks to all other Assistant Professors Institute of Biochemistry, Madras Medical College, for their guidance and support.
The author expresses warm respects to the members of the Institutional Ethics committee for approving the study. The author is indebted to the patients and persons from whom blood samples were collected for conducting the study.
The author expresses her special thanks to Biochemistry Laboratory Staff, for their timely help and cooperation during sample collection..
The author gratefully acknowledges the help rendered by Dr.Balaji M.D., for the statistical analysis of the study.
The author expresses her special thanks to her Parents Mr. M. Vijaya Mohan and Mrs. Jayanthi, her husband Dr. A.J. Sai Karthik, her mother-in-law Mrs. Rajeshwari Jeganathan , her daughter Baby S. Joshitha, her collegues Dr.R.P.Vinod Kumar & Dr. Geethanjali. R and other family members for the moral support and encouragement extended by them.
The author expresses her thanks to all her colleagues in the institute, for their constant encouragement throughout the study period.
Above all, the author is grateful to the Almighty for providing this opportunity, without whose grace nothing could be accomplished.
CONTENTS
S.
NO TITLE PAGE
NO.
1 Introduction 1
2 Review of Literature 4
3 Aim and Objective of the study 45
4 Materials and Methods 46
5 Statistical Analysis 61
6 Results 62
7 Discussion 83
8 Conclusion 88
9 Limitations of the Study 90
10 Future Scope of study 91
11 Bibliography 92
12 Annexures
ABBREVIATIONS
1. RA - Rheumatoid Arthritis
2. ACD - Anaemia of Chronic Disease 3. IDA - Iron Deficiency Anaemia 4. RES - Reticuloendothelial System 5. sTfR - Soluble Transferrin Receptor.
6. IL-6 - Interleukin
7. IL-6R - Interleukin 6 Receptor 8. TNF-α - Tumour Necrosis Factor 9. Hb - Hemoglobin
10. RBC - Red Blood Cells 11. Hct - Hematocrit
12. DNA - Deoxy Ribonucleic Acid.
13. NADPH - Nicotinamide Adenine Dinucleotide Phosphate 14. DMT - Divalent Metal Transporter
15. Dcytb - Duodenal Cytochrome b 16. HCP - Heme Carrier Protein
17. PNH - Paroxysmal Nocturnal Hemoglobinuria 18. IFN - Interferon
19. EPO - Erythropoietin
20. ROS - Reactive Oxygen Species 21. LPS - Lipopolysaccharide
22. NSAIDs - Non Steroidal Anti Inflammatory Drugs
23. TIBC - Total Iron Binding Capacity 24. TfR - Transferrin Receptor
25. FeOOH - Ferric Oxyhydride
26. cAMP - cyclic Adenosine Mono Phosphate 27. mRNA - messenger Ribonucleic Acid 28. IRP - Iron Regulatory protein 29. IRE - Iron Responsive element 30. UTR - Untranslated Region.
31. OD - Optical Density
32. ELISA - Enzyme linked Immuno Sorbent assay 33. HRP - Horse Raddish Peroxide
34. MCV - Mean Corpuscular volume 35. MCH - Mean Corpuscular Hemoglobin
36. MCHC - Mean Corpuscular Hemoglobin Content 37. CRP - C-Reactive Protein
38. ESR - Erythrocyte Sedimentation Rate.
39. CV - Coefficient of Variation
40. ROC - Receiver Operating Characteristics 41. CBC - Complete Blood Count
42.
sTfR-F - Soluble Transferrin Ferritin IndexIntroduction
1
INTRODUCTION
Anaemia is the most common cause of morbidity in Rheumatoid arthritis (RA)patients(1). It has been found that about 60% of Rheumatoid arthritis patients are anaemic(2). The causes of anaemia in RA may be due to anaemia of chronic disease (ACD), iron deficiency anaemia (IDA), anaemia due to vitamin B12 or folate deficiency(3).
Anaemia of chronic disease can be due to a very broad spectrum of diseases like infections, malignancies and inflammatory disorders (3).
In ACD, the limiting factor of erythrocyte haemoglobinisation is functional iron deficiency.
“Functional iron deficiency is defined as an imbalance between the iron needs of the erythroid marrow and iron supply, which is not maintained at a rate sufficient to allow haemoglobinisation of the erythrocytes”(4).
In iron deficiency anaemia (IDA) the iron supply depends on the amount of iron stores, whereas in ACD, the supply depends on the rate of mobilisation of iron from the Reticuloendothelial (RE) system. In ACD, functional iron deficiency may occur even in the presence of large iron stores when release is impaired.
The prevalence of iron deficiency in RA is 50-70% (5).
2
The various parameters used to study iron status are 1. Serum iron which reflects body iron stores, 2. Transferrin, that transports iron in the blood and 3. Ferritin, a storage form of iron in tissues.
However each of these above parameters have some disadvantages.
Serum iron shows diurnal variation and may increase after ingestion of meat or intake of iron supplements.
The hepatic transferrin synthesis is decreased in chronic disease(6).
Ferritin is an acute phase reactant and is increased independently of iron status in inflammation, malignancy, or liver disorder(7).
For accurate measurement of body iron stores, the gold standard is stainable iron in bone marrow aspirate. However, bone marrow examinations cannot not be performed routinely for the sole purpose of diagnosing IDA because it is invasive, expensive and time consuming. Moreover, the quantitation of stainable iron depends on technique and interpretation.
Soluble transferrin receptor (sTfR) is the truncated form of transferrin receptor that is involved in cellular iron homeostasis.
It is used to detect
Iron deficiency in inflammatory disorders
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Anaemia of chronic disease
To monitor erythropoietin treatment efficacy.
It reflects both tissue iron status and erythropoietic activity. The sTfR level is not affected by inflammation and it is not an acute phase reactant. Hence stfR level can be used to differentiate between IDA and ACD(8).
Serum ferritin reflects body iron stores and sTfR reflects cellular iron.
Hence its ratio sTfR/log ferritin is used as an estimate of body iron stores(9).
This ratio is used to differentiate anaemia of chronic disease from combined iron deficiency anaemia and ACD in RA patients.
Review of Literature
4
REVIEW OF LITERATURE
2.1 RHEUMATOID ARTHRITIS
Rheumatoid arthritis (RA) is a chronic progressive autoimmune disorder causing inflammation in the lining of the joints(10) It is more common in women than men mostly in the age group 35-50 years. Rheumatoid Arthritis affects nearly 24.5 million people worldwide(11). It accounts for 0.5 to 1% of adults in developed world(12). Cause of RA is unknown, but it is believed to be due to environmental and genetic factors(13). Chronic symmetric erosive arthritis of peripheral joints is the characteristic feature(14). It affects not only the joints but also has many extra- articular manifestations.
Pathogenesis of Rheumatoid Arthritis involve inflammatory mediators like cytokines, chemokines, growth factors, matrix metalloproteinases and adhesion molecules. TNF-α, IL-6 and IL-1 are the mediators of cell migration and causing inflammation in RA(15). IL-6 acts on neutrophils through IL-6 receptor (IL-6R) and secrete proteolytic enzymes and reactive oxygen intermediates causing inflammation and joint destruction(16) . IL-6 promote neutrophil recruitment by activated fibroblast(17).
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2.1.1 DIAGNOSIS OF RHEUMATOID ARTHRITIS:
Diagnosis of rheumatoid arthritis is based on clinical examination of the patients with the supporting evidence on history and examination
CRITERIA FOR DIAGNOSIS AND CLASSIFICATION OF RHEUMATOID ARTHRITIS- REVISED AMERICAN RHEUMATISM ASSOCIATED CRITERIA(18).
1. Early morning stiffness in and around the joints soon after arising from bed that lasts for a minimum of 1 hour
2. Arthritis or soft tissue swelling in 3 or more joints.
3. Arthritis of small joints like metacarpophalangeal joint, interphalangeal joints or wrist joints
4. Rheumatoid nodules
5. Rheumatoid factor positivity 6. Symmetrical swelling of joints
7. Radiographic findings suggestive of erosions and periarticular osteopenia in hand or wrist joints.
Symptoms are to be present for atleast 6 weeks and rheumatoid arthritis is defined by positivity of 4 or more out of 7 criteria.
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Anaemia is one of the extra articular manifestations of rheumatoid arthritis.
Next to cardiovascular disease, the most common extra articular manifestation is anaemia(19). In early RA, IL-6 is increased in patients with anaemia than in patients without anaemia(20). In RA, chronic inflammation causes increased Hepcidin levels which causes anaemia of chronic disease where iron is sequestered in macrophages and also poorly absorbed.
Anaemia usually occurs during the early stages of disease, where hepcidin levels are significantly elevated. IL-6 induces hepcidin during inflammation and causes hypoferremia(21). Anaemia is usually considered as a symptom due to the underlying inflammatory disorder and are thus neglected. But it is a complex medical symptom that needs specific diagnosis and treatment. Though inflammation induced changes in erythropoiesis and iron metabolism has a major role in pathogenesis of anaemia in RA, there are other factors like chronic blood loss, vitamin deficiency, treatment related adverse effects etc., can also be involved in development of anaemia(22).
2.2 ANAEMIA
According to World Health Organization, criteria for diagnosis of anaemia is Haemoglobin (Hb) concentration <13g/dL or Haematocrit (Hct) <39% in adult males and Hb <12 g/dL or Hct <37% in adult females(23).
Anaemia is caused either if red blood cell (RBC) production is inadequate or RBC lifespan is reduced from blood loss or destruction.
7
There are various classification of anaemia. Anaemia may be due to various red cell defect (24) namely,
Production defect (aplastic anaemia),
Defect in haemoglobin synthesis (IDA),
Maturation defect (megaloblastic anaemia),
Abnormal haemoglobin synthesis (haemoglobinopathies),
Loss of red blood cells (haemolytic anaemias)
In anaemia, body lacks the required amount of red blood cells to meet the body‟s oxygen demand.
2.2.1 IRON DEFICIENCY ANAEMIA
Iron deficiency anaemia is the common cause of nutritional anaemia. Iron deficiency is seen in more than 2 billion people worldwide(25).
2.2.1.1 FUNCTIONS OF IRON
Main function of iron in our body is to carry oxygen with haemoglobin.
Iron is incorporated into a variety of structural proteins like heme proteins. Heme proteins are a group of proteins that have a heme moiety incorporated into them.
Examples are haemoglobin, myoglobin, cytochrome p450, mitochondrial cytochromes, catalase and tryptophan 2,3-dioxgenase. Iron is involved in many biological function namely respiration , DNA synthesis, energy production and cell proliferation(26).
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Iron in haemoglobin delivers oxygen from lungs to peripheral tissues and in myoglobin acts as an oxygen binder. Nearly two third of the iron is utilised for this purpose.
Iron in mitochondrial cytochromes and in cytochrome P450 helps in transfer of electrons in electron transport chain and between substrates for drug metabolism respectively.
Iron other than heme is present as iron-sulphur clusters whose function apart from electron transfer is that it acts as catalytic centre and sensors of iron and oxygen.
Proteins with iron -sulphur clusters are NADPH-ubiquinone oxidoreductase, cis-aconitase and succinate-ubiquinone.
2.2.1.2 BODY IRON CONTENT AND ITS DISTRIBUTION
Adult human body contains nearly 3-4 grams of iron(27). Almost 60-70 % of iron is present in the erythron which includes the red blood cells and their progenitors in the marrow. The rest 30-40% is present in the Reticuloendothelial system of spleen and the liver. Daily iron requirement in the diet depends on age, gender, with greater amounts for children and pre-menopausal women, to make up for menstrual loss(28). The main route of excretion is through insensible loss via the desquamation of epithelial cells of intestine, urogenital loss, respiratory tract and skin(29,30).
9
FIGURE 1: Body Iron Distribution
(Image coutersy: https://forum.facmedicine.com/threads/understanding -iron- deficiency-anemia.19249/)
TABLE 1: Composition of iron in various substances(31).
DISTRIBUTION IRON CONTENT IN mg
Haemoglobin and reticulo endothelial
cells 2500 mg
Myoglobin 300 mg
Transferrin 3-4 mg
Ferritin 1000 mg
Absorption 1 mg
Losses 1 mg
10 2.2.1.3 IRON METABOLISM
The three cell types involved in iron homeostasis are 1. Duodenal enterocytes-that absorbs iron
2. Macrophages-recycle iron from erythrocytes 3. Hepatocytes-stores iron
Regulation of iron is at the level of absorption so “it is a one-way element”. Absorption is enhanced when iron stores in the body are depleted.
Absorption is decreased when adequate quantity of iron is stored. This is called
“mucosal block theory”(32) (33).
The various forms of iron in human diet are heme, ferritin and ferric form of iron complexed with macromolecules(27). Iron is released partially from these compounds by the acids in the stomach and digestive enzymes. Heme and non-Heme iron are absorbed by separate mechanism.
Elemental iron is absorbed in the luminal brush border of proximal duodenum and upper jejunum in the ferrous form(Fe2+ )(34) . An apical membrane transportor of inorganic iron called divalent metal transportor -1(DMT-1) helps in the uptake of iron in the enterocytes. DMT-1 also transport divalent metal cations like cobalt, manganese, nickel, lead, copper, cadmium and zinc(35). DMT-1 transports iron in ferrous form but most dietary iron is in ferric form. Inorganic iron from the diet in the ferric form is reduced to ferrous form by membrane bound ferrireductase, duodenal cytochrome b (Dcytb) or by STEAP ferrireductase
11
proteins.. Vitamin C, gastric acidity and many reducing agents help in this reduction(36).
Transport of heme iron is by heme carrier protein -1(HCP-1) and heme responsive gene-1(37).
Inside the enterocytes, iron is either stored in ferritin, an iron storage protein or transferred across the basolateral membrane by ferroportin or iron regulatory protein 1(IREG1), an iron exporter protein(38). Within the enterocytes, ferrous form is converted to ferric form that binds with apoferritin that forms ferritin. After export across basolateral membrane ,ferrous state is converted into ferric state by a copper containing ferroxidase, Hephaestin(34,39).
FIGURE 2: IRON ABSORPTION AND METABOLISM
(Image courtesy: Robbins Basic Pathology 8 edition.)
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Apotransferrin, a transport protein transports iron in ferric form in circulation. Each molecule of apotransferrin can bind two atoms of ferric iron.
Each site can bind one ion of HCO3 along with one ion of Fe3+.
Apotransferrin-Fe3+ complex is called Transferrin. Uptake of transferrin bound iron by the cell is mediated by transferrin receptor. It involves formation of clathrin-coated pits and its internalization into the cytoplasm. Transferrin and transferrin receptor complex are internalized by receptor mediated endocytosis to form a vacuole that gets acidified and releases the iron from transferrin(40). The clathrin coated vesicle losses its clathrin due to acidification by H+ ATPase pump to a pH of 5.5. This causes dissociation of iron bound transferrin to release iron.
Iron is released from interior of endosome to cytosol through endosomal DMT1.
Iron can be used for incorporation into haemoglobin or for storage. The transferrin without the iron is now called apotransferrin, remains bound to the transferrin receptor since it has high affinity at acidic pH(41). The apotransferrin without iron is then transported back to the surface of the cell and at neutral pH it dissociates from its receptor and is released, where it picks up further iron for tissue delivery.
This is called transferrin cycle(32).
13
FIGURE 3: UPTAKE OF IRON BY THE CELLS.
(Image courtesy: Harper Illustrated Biochemistry 31st edition)
2.2.1.4 IRON STORAGE AND RECYCLING
The iron absorbed by the intestine represents only a part of the circulating iron. Major circulating iron is due to recycling of iron from heme breakdown. Old RBCs are taken up by macrophages of spleen. The Heme of haemoglobin is metabolised to release Fe2+ by the enzyme heme oxygenase. Fe2+ is exported out of macrophage by ferroportin in to circulation(42). The iron released is stored as ferritin within the macrophages. The major storage organ of iron is liver(43). The iron stored in hepatocytes and macrophages of reticuloendothelial cells are released into circulation when needed such as in case of increased
14
erythropoiesis(44). There is increased expression of ferroportin on the surface of macrophages(45). Ferroportin transports ferrous iron out of the cell with the help of ceruloplasmin(ferroxidase) and /or Hephaestin (mainly in intestinal cells) that oxidises ferrous to ferric state so that it can bind to ferritin in the extracellular medium or bind to transferrin(26). It has been shown that expression of ferroportin causes mobilization of iron from ferritin and this causes degradation of ferritin by proteasomal pathway(46).
FIGURE 4: RECYClING OF IRON
(Image courtesy: Panwar B et al., Semin Nephrol. 2016;36(4):252–61.)
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2.2.1.5 REGULATION OF IRON METABOLISM Regulation of systemic iron balance involves
1. Controlled storage of iron in hepatocytes and macrophages.
2. Regulation at intestinal iron absorption
3. Effective recycling of iron from senescent RBCs.
All these events are controlled by hepcidin. Hepcidin is a peptide hormone synthesized in the liver. It regulates plasma iron and tissue distribution by inhibiting iron absorption in the enterocytes, recycling of iron by macrophages and mobilisation of iron from hepatic stores. It acts by inhibiting cellular efflux of iron by binding and inducing the degradation of ferroportin, the only sole cellular exporter of iron. Hepcidin synthesis is increased in iron overload states and decreased in anaemia and hypoxia. It is also increased in infection and inflammation that causes decrease in serum iron level and hence anaemia of inflammation(47).
16
TABLE: 2 CAUSES OF IRON DEFICIENCY ANAEMIA(27).
Cause Example
Physiologic
Increased demand Infancy, rapid growth during adolescence blood donation, menstrual blood loss, pregnancy
Environmental Insufficient intake due to poverty, malnutrition, vegan diet
Pathologic
Decreased absorption Gastrectomy, bariatric surgery, Helicobacter pylori infection, atrophic gastritis, celiac sprue, inflammatory bowel diseases (e.g., ulcerative colitis, Crohn‟s disease)
Chronic blood loss Esophagitis, peptic ulcer, erosive gastritis, diverticulitis, intestinal cancer, angiodysplasia, hemorrhoids, hookworm infestation, menorrhagia, intravascular hemolysis
(e.g. autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria (PNH), march hemoglobinuria, microangiopathic hemolysis damaged heart valves).
Systemic bleeding due to hemorrhagic telangiectasia, chronic schistosomiasis, Munchausen‟s syndrome.
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Drug-related Salicylates, NSAIDs, Proton-Pump Inhibitors, Glucocorticoids.
Genetic Iron-refractory iron-deficiency anemia
Iron restricted erythropoiesis Anemia of chronic disease, chronic kidney disease, Treatment with erythropoiesis- stimulating agents.
TABLE 3: INVESTIGATIONS TO DETECT IRON DEFICIENCY
PARAMETERS OBSERVATION
CBC Reduced Hb, MCV< 90 fL
Peripheral smear Microcytic, hypochromic anaemia
Bone marrow examination with
Prussian blue Decreased staining of iron.
Serum iron Decreased
Serum total iron binding capacity Increased
Transferrin saturation Decreased
Erythrocyte protoporphyrin Increased. It is a precursor of heme and accumulates in depleted iron stores(48)
Serum ferritin Decreased
Soluble transferrin receptor Increased
Stainable bone marrow iron is gold standard and most reliable.
2.2.2 ANAEMIA OF CHRONIC DISEASE
Chronic Infection or inflammation leads to anaemia called as anaemia of inflammation or anaemia of chronic disease. It usually causes mild to moderate
18
anaemia (Hb 7-12 g/dL). It develops in the setting of inflammatory disorders, infections or malignancies.
Anaemia of Chronic Disease is considered to be the second most common anaemia next to iron deficiency anaemia(49,50). It is the most common anaemia in hospitalized patients. Many diseases like congestive cardiac failure, obesity, renal failure and diabetes that are not considered inflammatory are associated with ACD type of anaemia and cytokine abnormalities.
2.2.2.1 PATHOGENESIS
The pathogenesis of ACD is multifactorial, that is linked to the underling chronic disease, but it is mainly due to alterations in iron balance which is derived from the immune activation(51).
There are various mechanism that contribute to the pathogenesis of ACD.
They are diminished erythropoiesis, diversion of iron traffic, diminished response to erythropoietin, erythrophagocytosis and invasion of bone marrow by tumour cells (52). Anaemia of Chronic Disease is a heterogeneous disease characterized by low serum iron and disturbances in iron homeostasis. There is no true iron deficiency but iron is present in the macrophages. Hence total body iron may be normal or increased.
Pathophysiology involves three factors namely 1. Reduction in lifespan of RBC
2. Decreased proliferation of erythroid progenitor cells
19
3. Increased uptake and retention of iron in the reticuloendothelial cells(53).
The molecular basis includes cytokines and acute phase reactants that disrupt the iron homeostasis regulation and erythropoiesis. Other factors that are involved in development of anaemia are disease and treatment related adverse effects, hemolysis, and vitamin deficiency. Anaemia in ACD can also be influenced by other factors not related to underlying disease, like age and gender particularly in elderly people (54,55).
a) REDUCED LIFE SPAN OF RBC
It is suggested that increased cytokines like interleukin-1(IL-1),produced by macrophages that are activated in patients with rheumatoid arthritis could ingest and destroy red cells that causes hemolysis (56).
Cytokines like IFN-γ and TNF-α cause retention of iron within macrophages of the Reticuloendothelial system by increasing DMT -1 expression and ferroportin repression
b) REDUCED PROLIFERATION OF ERYTHROID PROGENITOR CELLS
Proliferation and differentiation of erythroid precursors in ACD are impaired by 1. Decreased erythropoietin production
2. Inhibitory effects of inflammatory mediated cytokines on bone marrow(57).
Erythropoietin (EPO) expression is decreased and its level is reduced mainly due to cytokine mediated Reactive Oxygen Species (ROS) formation(58).
20
This then affects the binding affinity of EPO-induced transcription factors and damages EPO producing cells. A reduction in EPO production leads to iron retention as it is one of the negative regulators of hepcidin production.
In RA patients the number of erythroid burst-forming units are reduced thus the haemoglobin levels correlate inversely with circulating TNF(2,59).
c) INCREASED UPTAKE AND RETENTION OF IRON IN RES
In ACD , there is hypoferremia with adequate iron stores due to impaired mobilization of iron and retention within the cells.
Thus this diversion of iron leads to availability of little iron for erythropoiesis.
The major factor responsible for this is hepcidin. It is a small peptide produced by liver that functions as a systemic iron-regulatory hormone.
Hepcidin that was previously termed as liver-expressed antimicrobial peptide-1(LEAP-1) acts as a key regulator of intestinal iron absorption and iron recycling in macrophages. It binds to ferroportin in the macrophages and duodenal enterocytes, causing its internalization and degradation(60). This results in accumulation of iron as it is not exported out. Hepcidin expression is increased by inflammatory cytokines like IL-1 and IL-6, infectious stimuli like LPS(21,61). Thus, hepcidin excess causes reduced absorption of iron, reduced serum iron and increased iron in the reticuloendothelial system.
21 2.2.2.2 DIAGNOSIS
Diagnosis of ACD is both clinical and laboratory based(62).
The challenge is mainly to differentiate ACD and combination of ACD with iron deficiency.
There is mild to moderate anaemia with haemoglobin values between 9 to 10 g/dL
Normocytic normochromic anaemia in the peripheral smear study
Low reticulocyte index.
Serum iron pattern is important in ACD as it rules out iron deficiency. But it shows diurnal variation.
A true iron deficiency can occur simultaneously with ACD and it is difficult to diagnose true iron deficiency in patients with acute or chronic inflammation as most of the markers for iron metabolism are deranged by acute phase reaction.
Causes of iron deficiency in ACD are chronic gastrointestinal loss from GI bleeding, treatment modalities like NSAIDs and glucocorticoids, impaired absorption of iron due to celiac disease, autoimmune gastritis etc and reduced intake of iron due to restrictions of diet(63,64).
Ferritin levels are normal or increased due to increased iron stores and immune activation.
22
Diagnosis of iron deficiency is made when serum ferritin is less than 15 ng/mL(65). Serum iron and transferrin saturation are decreased in both ACD and IDA indicating iron sequestration in RES and impaired iron release in ACD and true iron deficiency in IDA.
Soluble transferrin receptor (sTfR), helps in diagnosis of patients with pure ACD and with both ACD and iron deficiency. It is decreased in ACD and increased in ACD with IDA.
sTfR to log ferritin is used to diagnose ACD associated with iron deficiency.
Index is less than 1 in ACD and more than 2 in combination.
EPO levels are no longer used since it is inadequate for the degree of anaemia and it is difficult to interpret.
Hepcidin levels are increased in ACD and decreased or normal in combination with iron deficiency. But since it is expensive, it is not routinely done.
Folic acid and vitamin B12 should be evaluated considering the pathogenesis involving erythropoiesis in ACD.
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TABLE 4: LABORATORY PARAMETERS TO DIFFERENTIATE IRON DEFICIENCY ANAEMIA, ANAEMIA OF CHRONIC DISEASE AND COMBINED ACD WITH IDA(49).
PARAMETERS IDA ACD ACD AND IDA
Hb Low Low Low
Serum Iron Low Low Low
Transferrin saturation Low Low Low
TIBC Raised Low Low/normal
Serum ferritin Low Normal/increased Normal
Serum hepcidin Low Raised Normal
Inflammatory markers Negative Raised Raised Soluble transferrin receptor Raised Normal/low Raised
stfR/log ferritin Raised Low Raised
2.3.1 TRANSFERRIN AND ITS RECEPTOR
Transferrin, a bilobed glycoprotein is synthesized in the liver. It has two iron binding sites and thus exists in two forms. They are diferric with two iron atoms and monoferric with single iron atom. Transferrin is a monomeric glycoprotein with a molecular weight of 80kDa(66).It has two lobes N and C and the lobes are connected by a short peptide(67). The two lobes are made up of two tyrosine, one histidine, arginine and aspartic acid. Iron binds at the cleft between the two lobes. Transferrin has a very high affinity to ferric iron than ferrous iron.
It transports iron to various tissues like liver, bone marrow and spleen. It is a
24
biomarker of body iron status. Transferrin-iron complex turnover rate is nearly ten times a day, that is essential to meet the daily demands for erythropoiesis. It balances between reticuloendothelial iron release and uptake by bone marrow. A carbonate (CO) is needed to attract iron to transferrin for creating opposite repulsive charges. It also acts as a ligand to stabilize the iron in transferrin.
Release of iron from its binding site in transferrin is favoured by acidic environment of pH 5.6.
FIGURE 5: STRUCTURE OF TRANSFERRIN WITH N AND C LOBES
(Image courtesy: Jeremy Wally et al., Biometals, 2007, Jan 11;20(3):249.)
Transferrin concentration in plasma is nearly 300mg/dl, that is sufficient to carry approximately 300μg of iron per decilitre of plasma. This constitute Total Iron binding capacity(TIBC) of plasma. Totally 30% of iron binding sites are
25
occupied in transferrin. This is reduced to 16% in severe IDA and increased to 45% in iron overload(31).
Transferrin is used to asses iron level in our body, to determine the cause of anaemia , iron carrying capacity of blood and to know about iron metabolism.
High transferrin indicates low iron since liver increase the production of transferrin for homeostasis. Significance of transferrin is mainly to detect iron deficiency and monitor erythropoiesis. Transferrin is lowered in liver damage, kidney injury, infection and malignancy. It is decreased in anaemia of chronic disease.
2.3.1.1 STRUCTURE OF TRANSFERRIN RECEPTOR
Transferrin receptor is a transmembrane glycoprotein and there are two receptors namely transferrin receptor 1(TfR1) and transferrin receptor 2 (TfR2).
TfR1 is widely distributed and TfR2 is predominantly expressed in liver and crypt cells of small intestine(31). TfR2 has lower affinity for Tf-Fe than TfR1 ,that serves as a sensor rather than importer of iron.
TfR is a homodimeric type II transmembrane protein. It has a small cytoplasmic domain, a large extracellular domain and a single pass transmembrane region. The dimeric Tfr ectodomain is butterfly shaped with each monomer having distinct domains namely, a protease like domain which is proximal to the membrane, a distal apical domain and a helical domain that is responsible for all the dimer contacts. The receptor has two disulphide bond between the cysteines residues at 89th and 98th position. The receptor has 760
26
amino acids with an extracellular domain of 671 amino acids at its C-terminal, 2 amino acids in the transmembrane domain and 61 amino acid in the cytoplasmic domain at its N-terminal(68).
FIGURE 6: STRUCTURE OF TRANSFERRIN RECEPTOR WITH THREE DOMAINS
(Image cortesy: Andrea Denardo et al., researchgate.net)
The figure 6 shows the structure of TfR which is composed of a cytoplasmic domain, ectodomain containing a protease domain which is red in colour, an apical domain which is green in colour and a helix domain (yellow) and a transmembrane segment (black).
27
FIGURE 7: BIOCHEMICAL STRUCTURE OF TRANSFERRIN RECEPTOR
(Image courtesy: Paul William et al., Med Microbiol Immunol (Berl).
1992;181:301–22.)
2.4.1 SOLUBLE TRANSFERRIN RECEPTOR
Kohgo et al., in 1986 first reported transferrin receptors in plasma by immunoassay(69)(70).
Soluble transferrin receptor is a truncated form of transferrin receptor with a molecular weight of 85kDa. It is a single polypeptide chain formed by the proteolytic cleavage of the extracellular domain of the receptor at arginine -100 and leucine -101 by metalloproteinase. Hence a truncated monomer of soluble transferrin receptor circulates in the blood. This truncated form of receptor contains extracellular domain without the transmembrane and cytoplasmic
28
domain. The gene for soluble transferrin receptor 2 (TFR2) is located in the chromosome 7q22(71).
FIGURE 8: STRUCTURE OF sTfR
(Image courtesy: https://www.rndsystems.com/resources/articles/soluble-transferrin- receptor-stfr)
The figure 8 shows the TfR molecule changing to sTfR. (C - cysteine; F- phenylalanine; R- arginine; E- glutamate; L- leucine; M- methionine;). Arrows indicate the site of proteolytic cleavage.
The plasma concentration of sTfR is proportional to the amount of transferrin receptors expressed on cell‟s membrane. Thus it reflects the cellular requirement of iron. Erythroblast contains the highest number of transferrin receptors nearly 3,00,000 to 4,00,000 per cells. As the number of cellular receptors increases in iron deficiency, sTfR concentration rises as erythropoiesis become iron-limited. The total iron stores in the body and the expression of receptor is inversely related to each other.
29
The production of transferrin receptor is regulated at the level of transcription of mRNA and is based on the intracellular content of iron.The sTfR measurements are used as an indicator for the rate of erythropoiesis and iron deficiency.
FIGURE 9: FORMATION OF TRANSFERRIN RECEPTOR COMPLEX
(Image cortesy: https://doi.org/10.1371/journal.pbio.0000051)
The reference range of sTfr for Indian population in serum and plasma is 0.3-2.9 mg/l. Unlike ferritin it does not show any significant difference between males and females. The cut off value for diagnosing iron deficiency anaemia is
>2.2mg/L.
30
TABLE 5: sTfr VALUES IN VARIOUS CLINICAL CONDITION(72). sTfR concentration Clinical conditions
Increased Iron deficiency anaemia
Increased erythropoiesis Pregnancy
Decreased tissue iron
Autoimmune hemolytic anaemia HbH disease
Polycythaemia vera Sickle cell disease Beta Thalassaemia
Decreased Chronic renal failure
Post bone marrow transplantation Aplastic anaemia
Normal Haemochromatosis
Acute and chronic myeloid luekemia Anaemia of chronic disease
Solid tumours
Plasma sTfR reflects the density of receptors on the cells and the number of cells that express the receptor. Hence it is related to cellular demands for iron and erythroid proliferation rate. Unlike serum ferritin and transferrin, sTfR is unaffected by concurrent chronic inflammation and disease. Thus, it is used to differentiate IDA and ACD and especially mixed IDA & ACD from pure ACD.
Serum sTfR is increased in IDA but not in ACD.
31 2.5 FERRITIN
Serum ferritin was discovered early in 1937 by French scientist Laufberger in horse spleen which contained 23% of iron by dry weight and developed as a clinical test in 1970‟s by immunoassay techniques(73). Addison et al..
demonstrated in 1972, that ferritin could be detected in serum by immunoradiometric assay(74). Ferritin in present in most tissues as cytosolic protein. Recently mitochondrial form and nuclear localization has been described.
It plays an important role in intracellular iron storage.
2.5.1 STRUCTURE
Ferritin is a highly conserved three dimensional structural protein. Ferritin is a 24-subunit globular protein with a molecular weight of 444kDa forming a nanocage with multiple metal protein interactions. It keeps the iron in soluble and non toxic form as free iron is highly toxic to the cells .The storage form of iron is ferritin and hemosiderin. The subunits are of two types H and L.
H refers to isolation of ferritin isoform from human heart that are rich in H subunit or its slower electrophoretic mobility since it is heavier of the two subunits. L subunit refers to the isoform isolated from human liver that are rich in lighter subunit. . Ferritin gene FTH and FHL for heavy and light chains are present in chromosome 11q12-q13 and 19q13.3-q13.4 respectively(75).
32
FIGURE 10: STRUCTURE OF FERRITIN
(Image courtesy: Bou-Abdallah et al., Biochimica et Biophysica Acta (BBA) General Subjects 2010, 1800 (8), 719–731.)
There are also multiple pseudo genes for the two subunits. H chains has 182 aminoacids with molecular weight of 21000 d & L chains has 174 aminoacids with molecular weight of 18500 d and this varies from tissues to tissues.
Ferritin is present both intra and extracellularly. Apoferritin is the form of ferritin without iron. Apoferritin forms a shell and with the iron in its core forms holoferritin or simply ferritin. Hence ferritin contains an apoferritin protein shell and an interior crystalline core made up of ferric oxyhydroxide (FeOOH)x. The composition of FeOOH core differs across species and in humans it is (5Fe2O3•9H2O) (6). The subunits of ferritin is arranged in 432 symmetry with 80A◦ diameter cavity which is capable of storing 3000 to 4500 ferric ions as inorganic complex.
33
FIGURE 11: SHOWS MINERALISATION OF FERRITIN
(Image courtesy: http://www.sciencedirect.com/science/article/pii/S0277538719302281)
It resembles a sphere or cube consists of six facets and each facet consists of four apoferritin monomers with a pore of 10 A◦ diameter in the centre with two iron binding sites. Ferritin containing 90% H type have low iron content <1000 Fe atoms /shell are present in tissues having high ferroxidase activity like brain and heart and with 90 % L type have more iron content >1500 Fe atoms are present in tissues having storage function like spleen and liver. The H monomer has ferroxidase centre that catalyses Fe2+oxidation(76).
FIGURE 12: FERRITTIN SUBUNITS AND ITS MINERALISATION
(Image courtesy: Carmona et al., Chem Commun. 2014 Nov 11;50(97):15358–61.)
34 2.5.2 PLASMA AND TISSUE FERRITIN
Plasma ferritin have a low affinity to iron compared to tissue ferritin and hence tissue iron content is more than plasma ferritin.
Circulating ferritin are mostly in the L form and do not contain iron. Nearly 50-80% of the plasma ferritin are glycosylated and glycosylated ferritin has a much longer half-life of approximately 50 hours than non-glycosylated form which has half-life of 5 hours. Plasma ferritin is synthesized in the rough endoplasmic reticulum and is glycosylated in the golgi apparatus before being secreted from the cell(77). There is a correlation between internal iron storage and external secretory ferritin so that plasma ferritin reflects ferritin concentration within the cells(77).
2.5.3 FUNCTIONS OF FERRITIN
Ferritin is the principal iron storage protein in tissues. It is involved in iron uptake, accumulation and release in to the cells. It protects the body from the toxic effects of iron by sequestering it in its bio-available form. Ferritin is present in mostly all cells and most iron is stored in hepatocytes, bone marrow and spleen making readily available iron for haemoglobin and haem protein synthesis.
2.5.4 IRON UPTAKE BY FERRITIN Only ferrous iron can be taken up by ferritin.
Steps in iron uptake and storage within the ferritin molecule are 1. Fe(II) oxidation
2. Fe(III) migration
35
3. Nucleation and growth of iron core mineral.
H chains help in Fe(II) oxidation and L chains in core formation(78).
2.5.5 REGULATION OF FERRITIN
Ferritin is regulated not only by iron but also there are many factors that regulate ferritin synthesis such as cytokines, oxidants, growth factors etc.
TABLE:7 REGULATION OF FERRITIN BY VARIOUS MECHANISM(79)
MECHANISM EFFECTOR
Transcriptional regulation
TNF, cAMP, iron, oxidative stress, chemopreventive agents, , hematopoetic differentiation c-myc
Posttranscriptional regulation via modulation of IRP proteins
Iron, nitric oxide, superoxide and hydroxyl radicals, phorbol ester.
Posttranscriptional regulation
independent of IRP IL-1β, phorbol ester, hemin
Release of iron from
intracellular compartments Oxidant stress (GSH depletion, menadione)
36
2.5.6 IRON MEDIATED REGULATION OF FERRITIN
The amount of cytoplasmic ferritin is regulated by translation of ferritin mRNA in response to intracellular iron. When iron levels are low, ferritin synthesis is decreased and conversely when levels are raised, synthesis of ferritin is increased.
Ferritin is regulated post transcriptionally by a process that involves binding of iron regulatory protein (IRP) in the cytoplasm to iron -responsive element (IRE) in the 5′ untranslated region of mRNA of ferritin.
Iron responsive element is the region in the untranslated region of ferritin mRNA that has a “stem loop” secondary structure. Iron responsive element regulates the translation and stability of these mRNA. Iron regulatory protein are RNA binding proteins that bind to the stem loop structure and thus inhibit ferritin mRNA translation. There are two iron regulatory proteins, IRP-1 and IRP-2. Iron regulatory protein -1, an iron -sulphur protein exists in two forms. It exists as a cytosolic aconitase when iron is abundant. With the loss of iron atom in iron- sulphur cluster when iron is scarce, it assumes an open configuration and thus can bind the stem loop in IRE and inhibits translation.
Thus, it acts as a repressor. IRP-2 is regulated by degradation. It is abundant in iron scarcity and degraded in iron excess.
37
Both IRP1 and IRP2 proteins binds to IRE and inhibits translation of ferritin mRNA. These two proteins are tissue specific. Iron regulatory protein -1 abundant in liver, kidney, intestine and brain while IRP2 abundant in pituitary and pro B lymphocytic cell line. Activation of IRP is through activation of signal transduction pathway like activation of protein kinase C that phosphorylates IRP.
Iron Responsive Element sequences are also present in the 3` untranslated region (UTR) of transferrin receptor. Thus IREs are present in many mRNA coding different proteins. They are of 30 nucleotide length RNA that form a special stem loop structure either in the 3‟-UTR (untranslated region) or 5‟-UTR of an mRNA.
These IREs are present in the RNA that codes,
Ferrittin H and L subunits
Transferrin
Transferrin receptor expressed on plasma membrane
Ferroportin, and iron exporter.
Divalent metal transporter 1 (DMT1)
Erythroid aminolevulinic acid synthetase, which catalyses the first step in heme synthesis.
Alzheimer's amyloidprecursor protein.
Succinate dehydrogenase
Mitochondrial aconitase
38
Ferritin released directly causes potential toxicity, so it is prevented by degradation of ferritin in the membrane bound lysosomes. Iron can also be stored in hemosiderin in liver, spleen and bone marrow. Iron is slowly released from hemosiderin and has higher iron content than ferritin. It has slower turnover rate than ferritin.
2.5.7 STfR1 AND FERRITIN SYNTHESIS ARE RECIPROCALLY REGULATED(31)
Synthesis of sTfR1 and ferritin are linked reciprocally to intracellular iron levels. When intracellular iron is low, sTfR1 synthesis increases and ferritin synthesis is decreased. The reverse occurs when iron is abundant. IRPs bind IRE only when iron is low. When IRP bind to 3‟ Untranslated Region of the Transferrin receptor-1(TfR1) mRNA , it stabilizes and increases TfR1synthesis.
When IRP bind to the IRE at the 5′Untranslated Region of ferritin mRNA, translation is blocked.
When iron is high, IRPs dissociate. Hence translation of ferritin mRNA is increased and sTfR1 mRNA is degraded.
39
FIGURE 13: TRANSLATION OF FERRITIN AND STFR.
(Image courtesy: Harper‟s Illustrated Biochemistry 31e.,)
TABLE 8: REFERENCE INTERVAL FOR FERRITIN
ng/mL
NEWBORN 25 to 200
1 MONTH 200 to 600
2 TO 5 MONTHS 50 to 200
6 MONTHS – 15 YEAR 7 to 140
ADULT MAN 20 to 250
ADULT WOMAN 20 to 200
IRON OVERLOAD ADULT MAN ADULT WOMAN
>400
>200
Source : Textbook of clinical chemistry and mlecular diagnostics by Tietz…
40
2.5.8 USES OF FERRITIN IN CLINICAL CONDITIONS(80,81) a) IRON DEFICIENCY ANAEMIA
Serum ferritin indirectly measures total body iron stores and low serum ferritin is specific for iron deficiency anaemia. Serum ferritin less than 12ng/mL is highly diagnostic of depleted iron stores and is widely accepted. Hypothyroidism and ascorbate deficiency also cause reduced serum ferritin.
Ferritin is assayed after evaluating haemoglobin, haematocrit and peripheral smear and found to decline in IDA even before changes in haemoglobin, red cell size and serum iron are manifested. A normal ferritin level cannot exclude iron deficiency as ferritin is affected by infection and inflammation.
It is used to monitor the response to iron therapy. Serum ferritin is measured before starting iron therapy to know the body‟s iron reserve and measured periodically to check the improvement in therapy.
Apart from IDA, other hypochromic anaemia like thalassemia, sideroblastic anaemia, tumour or infection makes ferritin normal or elevated but not low(82).
41 b) IRON OVERLOAD
Ferritin >400ng/mL indicates iron overload. Iron overload is seen when there is increased dietary absorption of iron, after blood transfusion or after administration of iron(83). It can be primary due to hereditary haemochromatosis or secondary due to repeated blood transfusion
c)ANAEMIA OF CHRONIC DISEASE
The most common cause of elevated ferritin other than iron overload is inflammation. In these cases, iron is shifted from erythrocytes to tissues(77). Increase in ferritin may be due to increase in its rate of synthesis due to increase in cellular metabolism like hyperthyroidism. Elevated ferritin in malignancy is mainly due to inflammation and tissue necrosis(84). Ferritin is an acute phase reactant and it is increased in chronic inflammation. It is a measure of body iron stores when inflammation is ruled out. In ACD serum ferritin is increased due to retention of iron in reticulo-endothelial cells and increased production in response to inflammation(85).
Increased plasma ferritin in various conditions mask iron deficiency anaemia. They are acute and chronic inflammation, alcoholic liver disease treatment with iron supplements etc…
d) ACUTE INFLAMMATORY MARKER
It is used as an acute inflammatory marker in fever, acute infection etc.
42
TABLE 9: ABNORMAL FERRITIN VALUES
DECREASED INCREASED
Iron deficiency anemia Ascorbate deficiency Hypothyroidism
Acute infection
Chronic inflammatory conditions Rheumatoid arthritis
Fever
Hereditary hemochromatosis Cataracts
Hodgkin‟s lymphoma Leukemia
Acerrloplasminemia Atransferrinemia GRACILE syndrome Multiple blood transfusions
2.6 sTfR - FERRITIN INDEX
Ferritin is decreased and sTfR is increased in iron deficiency anaemia.
Since there is an inverse relationship between sTfR and ferritin in IDA, its ratio sTfR/log of ferritin is used to diagnose iron deficiency with more accuracy(86). It is mainly used to differentiate iron deficiency from anaemia of chronic disease. It is very difficult to differentiate pure ACD from combined ACD with IDA. sTfR ferritin index is used for the above.
Ferritin detects the reduced iron stores at an earlier stage, but it is an acute phase reactant. Hence it cannot be used as a marker for iron deficiency when inflammation is present.
43
The sTfR measurement is used as an indicator for erythropoiesis rate and thus iron deficiency(87). If ferritin is below 15 ng/mL, iron stores are depleted and hence more amount of transferrin receptors are expressed. Thus, sTfR level reflects the amount of TfR which in turn is the measure of body‟s iron stores.
Majority of transferrin receptors are located in the erythropoietic cells. The sTfR in contrast to ferritin is not an acute phase reactant and not affected in liver or malignant disorders.
Thus sTfR levels in our body reflects the functional iron status and ferritin reflects the iron storage status of the body.
In iron deficiency, sTfR level rises well before significant decrease in hemoglobin level occurs.
2.7 ANAEMIA IN RHEUMATOID ARTHRITIS
Anemia in rheumatoid arthritis is due to both iron deficiency and inflammation. Iron deficiency is mainly due to chronic blood loss from non- steroidal anti-inflammatory cells (NSAIDs) that cause damage to the intestinal mucosa that results in ulceration and chronic blood loss. In rheumatoid arthritis patients, synovial membrane is characterized by increased vascularity, hyperplasia and infiltration of inflammatory cells mainly CD4 T cells. There are many studies to show that inflammation plays a major role in contributing to anemia in rheumatoid arthritis.
44
Anemia in RA is not very severe, but it is associated with disease activity and has a negative impact on symptoms and quality of life. It is important to differentiate ACD from combined ACD and IDA in RA since IDA can be easily treated with iron therapy(1). Without iron deficiency, iron therapy in RA may aggravate arthritic symptoms and fail to treat anemia(88). Stainable iron in the bone marrow is the gold standard for the differentiation of IDA and ACD in rheumatoid arthritis patients. However, it is invasive and causes discomfort to the patients.
Red cell indices and iron parameters such as serum iron, transferrin, transferrin saturation and total iron binding capacity show considerable overlap(1). Serum ferritin is an acute phase reactant and increases nonspecifically in many inflammatory disorders. In inflammatory disorders, ferritin is increased despite the presence of iron deficient states. Serum transferrin receptor, a truncated form of transferrin receptor in the cell surface, is increased in IDA than ACD(89). There is upregulation of transferrin receptor synthesis to compete for iron deficiency in IDA(90). There is a increase in erythroblast TfR efficiency for uptake of iron to compensate for low plasma levels that results in normal sTfR levels in ACD.
There is increase in sTfR value in RA patients with concomitant iron deficiency as erythroblasts respond to additional worsening of iron supply(91). There are studies to indicate that logarithmic transformation of ferritin and calculation of sTfR/log ferritin ratio (sTfR-F index) indicates iron depleted state(9,92).
Aim and Objective
of the study
45
AIM AND OBJECTIVE OF THE STUDY
1. To assess the anaemia in patients with Rheumatoid Arthritis
2. To differentiate anaemia of chronic disease from iron deficiency anaemia using stfr ferritin index
Materials and Methods
46
MATERIALS AND METHODS
The study protocol was approved by the Institutional Ethical committee of Madras Medical College, Chennai and a copy of it has been enclosed.
STUDY CENTRE
Institute of Biochemistry
Institute of Rheumatology and Institute of Internal medicine
Madras Medical College & Rajiv Gandhi Government General Hospital, Chennai – 3
STUDY PERIOD
DECEMBER 2017-NOVEMBER 2018(one year from the date of ethical committee clearance)
STUDY DESIGN
Cross sectional Case Control study INCLUSION CRITERIA
GROUP A:
Rheumatoid arthritis patients with anaemia as per WHO criteria, Hb value - Males <13 g/dL and females < 12 g/dL (23)
Age :25-55 yrs.
47 GROUP B :
Patients with iron deficiency anaemia as per WHO criteria, Hb value - Males
<13 g/dL and females < 12 g/dL (without any chronic disease condition)
Age 25-55 yrs
GROUP C:
Age and Sex matched healthy controls
EXCLUSION CRITERIA
Abnormal renal function
Haematological disorders
Recent blood transfusion
Any malignancy
Liver disorders
Thyroid disorders
Any other inflammations and infections causing anaemia
SAMPLE SIZE
Group A Rheumatoid arthritis patients 30
Group B Iron Deficiency Anaemia patients 30
Group C Controls 30
48 INVESTIGATIONS
Complete blood counts using cell counter
Serum sTfR using ELISA
Serum ferritin using ELISA
Peripheral smear
C-Reactive protein and ESR in RA patients.
SAMPLE COLLECTION
5 mL of blood sample were collected from all the subjects. 3 mL was transferred to serum tube and 2 mL to K2 EDTA tubes. After adequate time for clotting, serum tube is centrifuged. The serum that gets separated in the red capped tube is aliquoted in to two tubes. The aliquoted sample was stored at - 20◦C
It is used for testing of CRP, ferritin and soluble transferrin receptor levels. EDTA tubes were used for analysis of complete blood count and erythrocyte sedimentation rate. For peripheral blood smear analysis, a drop of blood was collected from the patients by needle prick. CBC, ESR, CRP and peripheral smear were processed on the same day and serum sample that was stored was used for analysis of ferritin and soluble transferrin receptor.
1.ESTIMATION OF SERUM sTfR LEVELS METHOD
Enzyme Linked Immuno Sorbent Assay (ELISA): Sandwich method
49 PRINCIPLE
The ELISA wells in the kit are pre coated with sTfR monoclonal antibody.
After addition of the Sample to the microtitre wells, the sTfR in the sample binds to the antibody that is precoated on the well. Biotin conjugated with anti human sTfR antibody is added to the wells. It binds to the human sTfR present in the sample. After incubation the unbound antibody is washed using wash buffer.
Streptavidin –HRP is then added to the wells and it binds to the biotin conjugated with antihuman sTfR antibody. After incubation the unbound Streptavidin- HRP is washed. Substrate solution is then added to all the wells and colour develops.
Stop solution is added to terminate the reaction. Stop solution contains acid.
Absorbance is read at 450nm.The intensity of the colour developed is proportional to the amount of sTfR present in the sample
REAGENTS
1. Standard solution (4.8 mg/L) and Standard Diluent 2. ELISA plate with precoated wells
3. Anti sTfR antibody coated with biotin 4. Streptavidin HRP
5. Substrate solution A 6. Substrate solution B
7. Wash buffer concentrate (30x) 8. Stop solution
50 REAGENT PREPARATION
All reagents are brought to room temperature before use.
WASH BUFFER
20ml of wash buffer concentration is diluted with deionized water to make up to 500ml of wash buffer. If crystals are formed in the wash concentrate then mix it gently until the crystals are dissolved completely.
STANDARD PREPARATION Standard no. Standard
concentration Preparation
Standard 5 2.4mg/L 120μl Original Standard + 120μl Standard diluents
Standard 4 1.2mg/L 120μl Standard No.5 + 120μl
Standard diluents
Standard 3 0.6mg/L 120μl Standard No.4 + 120μl
Standard diluent
Standard 2 0.3mg/L 120μl Standard No.3 + 120μl
Standard diluent Standard 1 0.15mg/L 120μl Standard No.2 + 120μl
Standard diluent
4.8mg/L 2.4mg/L 1.2mg/L 0.6mg/L 0.3mg/L 0.15mg/L
51 ASSAY PROCEDURE
1. All reagents, standard solution and wash buffer are prepared as instructed by the kit.
2. 50μl of standard solution is added to standard wells 1 to 6. No biotin antibodies are added as the standard solution contain biotin labelled antibody.
3. To the sample well to be tested, 40μl sample is added and then 10μl sTfR antibodies
4. 50μl streptavidin-HRP is added to all the wells. Then covered the plate with the sealer given by the manufacturer and incubated the plate at 60 minutes at 37℃.
5. Washing: The sealer was removed carefully from the membrane, and washed with ERBA washer 5 times with wash buffer. Wells were soaked with 0.35ml wash buffer for 1 minute. Then the well is blotted on to the filter paper.
6. Colour development: 50μl chromogen solution A is added to each well and then 50μl chromogen solution B is added to each well. It is then shaked gently to mix them up. Incubate the plate for 10 minutes at 37℃ in the dark for colour development.
7. 50μl Stop Solution is added to each well. The blue colour changes into yellow colour immediately at that moment.
52
8. The absorbance (OD) of each well is measured one by one under 450nm wavelength, within the 10 minutes after adding the stop solution using reader.
DERIVING RESULTS USING STANDARD GRAPH:
The concentration of the standards are marked at the x axis and the OD at the y axis. The corresponding values are plotted and the standard graph obtained is used to estimate the unknown concentration of the samples by calculating it using the measured OD .
S.No Concentration of standard (mg/L)
Absorbance (OD)
1. 0 0.0238
2. 0.15 0.0823
3. 0.3 0.1155
4. 0.6 0.2436
5. 1.2 0.5503
6. 2.4 0.9119
53 STANDARD GRAPH
SENSITIVITY OF THE ASSAY
Minimum sTfR detectable limit is： 0.005mg/L.
Assay range：0.01mg/L→4mg/L.
ESTIMATION OF SERUM FERRITIN LEVELS METHOD:
Enzyme Linked ImmunoSorbent Assay (ELISA), sandwich method.
PRINCIPLE
The wells are pre-coated with streptavidin. The standard, sample and the biotinylated Anti-ferritin antibody are added to the designated wells. Ferritin in
