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THE USE OF RBC INDICES TO DIFFERENTIATE BETWEEN IRON DEFICIENCY ANAEMIA AND BETA THALASSEMIA

TRAIT AND TO FIND OUT THE PROPORTION OF BETA THALASSEMIA TRAIT AMONG THE PATIENTS WITH

MICROCYTIC HYPOCHROMIC ANAEMIA

Submitted in partial

DR.

THE TAMILNADU

INSTITUTE OF INTERNAL MEDICINE MADRAS MEDICAL COLLEGE

A dissertation on

THE USE OF RBC INDICES TO DIFFERENTIATE BETWEEN IRON DEFICIENCY ANAEMIA AND BETA THALASSEMIA

TRAIT AND TO FIND OUT THE PROPORTION OF BETA THALASSEMIA TRAIT AMONG THE PATIENTS WITH

MICROCYTIC HYPOCHROMIC ANAEMIA

Submitted in partial fulfilment of requirements for

DR. M.D. DEGREE BRANCH-1 GENERALMEDICINE

OF

THE TAMILNADU DR.M.G.R.MEDICAL UNIVERSITY CHENNAI.

INSTITUTE OF INTERNAL MEDICINE MADRAS MEDICAL COLLEGE

CHENNAI – 600 003

MAY 2020

THE USE OF RBC INDICES TO DIFFERENTIATE BETWEEN IRON DEFICIENCY ANAEMIA AND BETA THALASSEMIA

TRAIT AND TO FIND OUT THE PROPORTION OF BETA THALASSEMIA TRAIT AMONG THE PATIENTS WITH

MICROCYTIC HYPOCHROMIC ANAEMIA

fulfilment of requirements for

M.G.R.MEDICAL UNIVERSITY

INSTITUTE OF INTERNAL MEDICINE

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CERTIFICATE

This is to certify that this dissertation entitled “THE USE OF RBC INDICES TO DIFFERENTIATE BETWEEN IRON DEFICIENCY ANAEMIA AND BETA THALASSEMIA TRAIT AND TO FIND OUT THE PROPORTION OF BETA THALASSEMIA TRAIT AMONG THE PATIENTS WITH MICROCYTIC HYPOCHROMIC ANAEMIA” submitted by Dr. S. SUDHA appearing for M.D.Branch I - General Medicine Degree examination in MAY- 2020 is a bonafide record of work done by her under my direct guidance and supervision in partial fulfilment of regulations of The TamilNadu Dr. M.G.R.

Medical University, Chennai. I forward this to The Tamil Nadu Dr.M.G.R.

Medical University, Chennai, Tamil Nadu, India.

Prof. Dr. S. RAGUNANTHANAN, M.D., Guide & Research Supervisor,

Institute of Internal Medicine, Madras Medical College &

Rajiv Gandhi Govt. General Hospital, Chennai - 3.

Prof. Dr. S. RAGUNANTHANAN, M.D., Director (I/c) and Professor,

Institute of Internal Medicine, Madras Medical College &

Rajiv Gandhi Govt. General Hospital, Chennai - 3.

Prof. Dr. R. JAYANTHI, M.D, FRCP (Glasg) The Dean

MMC & RGGGH, Chennai - 03.

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DECLARATION

I Dr. S SUDHA, solemnly declare that the dissertation titled “THE USE OF RBC INDICES TO DIFFERENTIATE BETWEEN IRON DEFICIENCY ANAEMIA AND BETA THALASSEMIA TRAIT AND TO FIND OUT THE PROPORTION OF BETA THALASSEMIA TRAIT AMONG THE PATIENTS WITH MICROCYTIC HYPOCHROMIC ANAEMIA” is done by me at Institute of Internal medicine , Madras Medical College & Rajiv Gandhi Govt.

General Hospital, Chennai between April 2018 to March 2019 under the guidance and supervision of Prof. Dr. S. RAGUNANTHANAN, M.D. This dissertation is submitted to the Tamil Nadu Dr.M.G.R. Medical University towards the partial fulfilment of requirements for the award of M.D. Degree in General Medicine (Branch-I).

Place: Chennai-3 Signature of Candidate Date:

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ACKNOWLEDGEMENT

I sincerely thank our dean Prof. Dr. JAYANTHI M.D., FRCP (Glasg.) for allowing me to conduct this study in our hospital.

I hereby express my gratitude and sincere thanks to our unit Chief and the Director, Institute of Internal Medicine, Madras Medical College, Prof. Dr. S RAGUNANTHANAN M.D., for his guidance andadvice throughout the course of the study.

I would like to express my gratitude to Prof Dr. MARGARET.C M.D, D.M., Head of the Department, Department of Hematology, Madras Medical College & RGGGH , for her advice and guidance in conducting this study.

I thank my former professor Dr.S.TITO M.D., Assistant Professors Dr. SUBBURAGHAVALU M.D., Dr. RAMYA LAKSHMI M.D., and Dr. PRIYATHARCINI M.D., without whom this study would have been impossible.

I whole heartedly thank my family members, all my friends and fellow post graduates for their support and encouragement during the hardships of the study.

I am indebted to all my patients without whom this study would not have been possible.

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LIST OF ABBREVIATIONS

Hb - Hemoglobin RBC - Red Blood Cell HbA - Adult hemoglobin HbA2 - Hemoglobin A2 HbF - Fetal Hemoglobin ALA - AminoLevulinicAcid HMB - Hydroxy Methyl Bilane MCV - Mean Corpuscular Volume MCH - Mean Corpuscular Hemoglobin

MCHC - Mean Corpuscular Hemoglobin Concentration RDW - Red Cell Distribution Width

DMT - Divalent Metal Transporter IDA - Iron deficiency Anaemia BTT - Beta thalassemia trait

UIBC - Unsaturated Iron Binding Capacity TIBC - Total Iron Binding Capacity

RDWI - Red cell Distribution Width Index LBW - Lean Body Weight

NTBI - Non Transferrin Bound Iron EF - England & Fraser Formula MI - Mentzer’s Index

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CONTENTS

S.

NO. TITLE PAGE

NO.

1 INTRODUCTION 1

2 AIMS AND OBJECTIVES 3

3 REVIEW OF LITERATURE 4

4 MATERIALS AND METHODS 63

5 OBSERVATION AND RESULTS 66

6 DISCUSSION 92

7 CONCLUSION 97

8 LIMITATION OF STUDY 99

9 BIBLIOGRAPHY 10 ANNEXURES

- PROFORMA

- ETHICAL COMMITTEE APPROVAL - PLAGIARISM SCREENSHOT

- PLAGIARISM CERTIFICATE - INFORMATION SHEET

- CONSENT FORM - MASTER CHART

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Introduction

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1

INTRODUCTION

Among the total global population, approximately 5 to 7 % carry a pathological hemoglobin gene. This mainly includes different forms of thalassemia and the structural hemoglobin variants. Beta thalassemia is very common among the thalassemia and it is more prevalent in Italy, the Middle East, Greece and the Indian subcontinent 47.Around 1.5 % of the total population are beta thalassemia carriers of which 50 % are found in South east Asia 48. In India it is found that the consanguineous marriage remains the choice of estimated 10.4%

of the total population. Pre marital check up is not followed in our country despite the high prevalence of consanguineous marriage. Hence many genetic diseases are transmitted from the parents to the off springs, Beta thalassemia is one of them.

There is a 25 % chance of developing a beta thalassemia major child for every beta thalassemia trait couple. It is estimated that the 10 percent of global thalassemic patients are born in India every year 49. The average treatment cost per thalassemic child is Rs 1.25 lakhs per annum. Hence it is high time to develop screening programs to detect beta thalassemia carrier state in our general population which would help us to reduce the thalassemia major cases. Both beta thalassemia trait and iron deficiency anaemia will present as microcytic hypochromic anaemia in the clinical practice.

Therefore it is very much important to differentiate these two entities in order to detect and provide proper premarital counseling to the patients with beta

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thalassemia trait and to prevent excessive iron supplementation in these patients.

A battery of tests such as serum iron serum ferritin, serum total iron binding capacity, serum transferrin saturation percentage and hemoglobin electrophoresis are required to confirm these two diseases. It is both impractical and burdensome to both patients and practitioners to carry out these tests in the outpatient department in our country especially in the rural setup. It also increases the economic burden of the patients. Hence many indices and formulas using RBC indices derived from the automated analyzer such as hemoglobin, RBC count, MCV and Red cell distribution width were used to differentiate between these two clinical entities. The commonly used indices are Mentzer index, shine and Lal index, Srivastava index, England and Fraser index, Red cell distribution width index and Green & king index. There is a controversy regarding both the choice of RBC indices and their cut off value to distinguish between beta thalassemia trait and iron deficiency anaemia.

The screening reliability of these RBC indices varies from country to country. Hence this study has been carried out to find out the sensitivity , specificity , positive predictive value, negative predictive value and the diagnostic accuracy of the RBC indices such as Mentzer’s index, England and Fraser formula, red cell distribution width, red cell distribution width index and RBC count to differentiate between these two clinical entities in our population.

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Aims and Objectives

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AIMS AND OBJECTIVES

1) To study the use of RBC indices to differentiate between iron deficiency anaemia and beta thalassemia trait

2) To study the proportion of beta thalassemia trait among the patients with microcytic hypochromic anaemia.

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Review of Literature

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4

REVIEW OF LITERATURE

Anaemia is defined as a medical condition characterized by reduction in the red blood cell count (and consequently oxygen carrying capacity of the blood) that is not sufficient to match the physiological needs of the body 1.

Classification of anaemia based on Hb values:

Population Non anaemia

Mild anaemia

Moderate anaemia

Severe anaemia Non pregnant

women

12gm /dL or higher

11-

11.9gm/dL 8-10.9gm/dL less than 8gm/dL Pregnant

women

11gm/dL or higher

10-

10.9gm/dL 7-9.9gm/dL less than 7 gm /dL

Men 13gm/dL or

higher

11-

12.9gm/dL 8-10.9gm/dL less than 8gm/dL

The Most common cause of anaemia worldwide is iron deficiency anaemia.1 Other nutritional causes include vitamin B12, folic acid and vitamin A deficiency. Other causes of anaemia includes blood loss due to acute or chronic conditions , acute and chronic inflammation, parasitic infestations , congenital or acquired disorders causing defective hemoglobin synthesis , decreased red blood cell production or decreased red blood cell survival.

Red blood cells:

They are biconcave disks with a mean diameter of about 7.8 micrometers and a thickness of about 2.5 micrometers at the thickest point and 1 micrometer or less at the centre 2 The average RBC per cubic millimeter in the healthy male is

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5.2 million and in females its about 4.7 million per cubic millimeter. If the Hb is 100% saturated each gm of Hb can combine with about 1.34 ml of oxygen. Hence a normal man can carry about 20 ml of oxygen combined with Hb in each 100ml of blood and a normal woman can carry 19 ml of oxygen per 100 ml of blood 2.

Erythropoiesis:

RBC’s are produced in the yolk sac during the early week of embryonic life and liver being the major organ during the second trimester. During the last month of gestation and after birth bone marrow is the major site of production2. A progenitor is a marrow cell derived from pluripotent hematopoietic stem cell through the process of differentiation. Proerythroblast is the earliest precursor in the process of erythropoiesis 3. Basophilic erythroblasts are smaller in size when compared to proerythroblast. The cytoplasm is basophilic due to the continued presence of polyribosomes. During the mitotic division of basophilic erythroblasts

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into polychromatic erythroblast cytoplasm changes from intense blue to grey due to dilution of polyribosome content by hemoglobin. The erythroblast loses their mitotic potential at this point 3. The concentration of hemoglobin increases during the stage of orthochromic erythroblast. The nucleus gets extruded during the reticulocyte stage. Small amounts of ribosomes, mitochondria, centrioles are retained in the reticulocyte which forms aggregates and stains deep blue with supravital staining such as cresyl blue and new methylene blue. Maturation of reticulocyte takes about 2 to 3 days 3. The concentration of reticulocyte is less than 1 percent due to its shorter life span.

The factors that decreases the tissue oxygenation such as anaemia, low blood volume, poor blood flow and pulmonary disease stimulates the release of erythropoietin from the kidney. Erythropoietin increases the production of erythroblasts from the hematopoietic stem cell and hastens the other stages of erythropoiesis 2.

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Vitamin B12 and folic acid plays an important role in the synthesis of thymidine triphosphate, which is one of the essential components of DNA synthesis. Hence their deficiency causes failure of nuclear maturation and cell division 2.

Hemoglobin ;

Hemoglobin is a tetramer with heme and two pairs of globin chains.

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(The above figure shows the three dimensional structure of hemoglobin with alpha chains represented in darker shades of green and blue and beta chains in lighter shades of green and blue. Heme component is represented in red colour.)

Adult hemoglobin (HbA) contains one pair of alpha chain and one pair of beta chain. The chromosome 16 has two genes for alpha globulin and chromosome 11 has one gene for beta globulin. The heme group consists of a single molecule of protoporphyrin IX bound to a single ferrous ion and it is linked covalently to a specific site in the globin chain.

Hemoglobin A2 normally constitutes about 2 to 3 percent of the total hemoglobin in adults. It consists of two alpha chains and two delta chains. In patients with beta thalassemia trait percentage of HbA2 is raised and it can be as high as 7 to 9 percent. It is also slightly increased in patients megaloblastic anaemia. When beta thalassemia trait co exists with iron deficiency anaemia the HbA2 levels are less elevated but the values are still high for the condition 4. Decreased HbA2 levels are found in alpha thalassemia, iron deficiency anaemia and sideroblastic anaemia.

 Hb Gower I consists of two zeta chains and two epsilon chains.

 Hb Gower II has two alpha chains and two epsilon chains.

 Hb Portland has two zeta chains and two gamma chains.

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Hb Gower I, Hb Gower II and Hb Portland are the products of yolk sac erythroblasts found during 4 th week and 14th week of gestation. After 14 th week these are completely replaced by the fetal hemoglobin (HbF).

HbF consists of two alpha chains and two gamma chains. It starts appearing during the 8th week of gestation. HbF becomes minor hemoglobin several months after birth constituting less than 1 percent of total hemoglobin in the adult.

Heme synthesis;

The first step in heme synthesis takes place in the mitochondria. Succinyl CoA derived from citric acid cycle combines with the amino acid glycine to form delta amino levulinic acid in the presence of the enzyme ALA synthase. This the rate limiting step in the synthesis of heme. Pyridoxal phosphate is necessary for the activation of glycine. In the cytoplasm two molecules of ALA condenses in the presence of the enzyme ALA dehydratase to form one molecule of porphobilinogen and water. ALA dehydratase is inhibited by lead and the enzyme is a zinc containing enzyme 5. The next step in the synthesis of heme is the condensation of four molecules of porphobilinogen in a head to tail manner to form linear tetrapyrrole HydroxyMethylBilane. The enzyme catalyzing this step is uroporphyrinogen I synthase which is also named as PBG deaminase or HMB synthase.

HMB undergoes spontaneous cyclisation to form uroporphyrinogen I or it may be converted by the action of uroporphyrinogen III synthase into

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uroporphyrinogen III. Uroporphyrinogen III undergoes decarboxylation in the cytoplasm in the presence of uroporphyrinogen decarboxylase into coproporphyrinogen III. Coproporphyrinogen III enter into mitochondria where it is converted into protoporphyrinogen III in the presence of coproporphyrinogen oxidase. Protoporphyrinogen III is oxidized by protoporphyrinogen oxidase into protoporphyrin III. The final step in heme synthesis involves incorporation of iron in ferrous state into protoporphyrinogen catalyzed by ferro chelatase which is also called as heme synthase and it is a mitochondrial enzyme. Heme then combines with globin chains to form hemoglobin 5.

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The below picture explains the steps involved in heme synthesis

Classification of anaemia Pathological classification;

Anaemia can be classified based on the red cell mass into

● absolute anaemia ( decreased red cell volume )

● Relative Anaemia (normal total red cell volume in an increased plasma volume) 3.

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Absolute anaemia:

It can be further classified into 1. decreased red cell production 2. Increased red cell destruction.

3. Blood loss and blood redistribution.

Relative anaemia:

The causes include macroglobulinemia, pregnancy, athletes and post flight astronauts.

Morphological classification:

Based on the RBC indices such as MCV, MCH, MCHC the anaemia can be classified.

MCV - It is defined as the average volume of the individual RBCs in a given sample of blood. It is expressed in terms of femtolitres(fL).

MCV = Hematocrit /red blood cell count.

Normal range is 80 to 100 fL. If MCV is less than 80fL it is called as microcytic and if >100 fL it is macrocytic

MCH - It is defined as the average amount of hemoglobin present per red blood cell. it is expressed in terms of picograms(pg)

MCH = hemoglobin/ red blood cell count.

Normal range is 27-31pg/ cell.

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MCHC - it is defined as the average concentration of hemoglobin present per unit volume of red blood cells.

MCHC = hemoglobin / Hematocrit Normal range is 32 -36 gm/dL

RDW - It is defined as a measure of the variation of red cell volume.

Normal valve is 11.5 to 14.5 %

Microcytic anaemia:

The causes include

1. iron deficiency anaemia 2. Thalassemia

3. Anaemia of inflammation / anaemia of chronic disease 4. Sideroblastic anaemia ( congenital, lead , alcohol and drugs ) 5. Copper deficiency and zinc poisoning ( rare)

Normocytic anaemia:

1. Acute blood loss

2. Early iron deficiency anaemia

3. Anaemia of inflammation / anaemia of chronic disease 4. Bone marrow suppression

5. Chronic renal insufficiency

6. Endocrine dysfunction such as hypothyroidism and hypopituitarism

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14 Macrocytic anaemia:

1. Excessive ethanol use 2. Folate deficiency

3. Vitamin B12 deficiency 4. Myelodysplastic syndromes 5. Acute myeloid leukemia 6. Reticulocytosis

7. Drug induced anaemia ( hydroxyurea, AZT, chemotherapeutic agents ) 8. Liver disease

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15 IRON DEFICIENCY ANAEMIA:

Globally iron deficiency affects more than 2billio people 6. The calculated iron deficiency is twice as that of iron deficiency anaemia worldwide 6. Total quantity of iron in the body is about 4 to 5 grams of which 65% is present in the form of hemoglobin 2

Iron metabolism;

The daily absorption of iron is limited to 1 to 2 mg as the excess iron is toxic. Most of the daily required iron (25mg) is provided by macrophages through the phagocytosis of erythrocytes 7. Iron is absorbed from the small intestine. It combines with apo transferrin which is a beta globulin synthesized by the liver to form transferrin and transported in the plasma. Normally about one third of

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transferrin is saturated with iron. The major function of transferrin is to provide iron to cells especially erythroid precursors in the bone marrow to synthesize hemoglobin. Erythroid precursors have high affinity receptors for transferrin which play an important role in the iron transport into the cells through receptor mediated endocytosis 8.

As the free iron is highly toxic it is sequestered inside the cell as ferritin or hemosiderin. Highest levels of ferritin is found in the liver, bone marrow, skeletal muscles and spleen 8. Most of ferritin is stored within the parenchyma cells in liver. It is found inside macrophages in organs such as the spleen and bone marrow. Partially degraded ferritin inside the cell aggregates to form hemosiderin granules. Hemosiderin is chemically active and it stains blue black with potassium ferrocyanide present in the Prussian blue stain 8.

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Most of the Non heme iron in the gastrointestinal tract is present as ferric state. It is converted into ferrous state by the ferric reductase such as STEAP3, cytochrome b. Ferrous iron is transported by the divalent metal transporter 1 (DMT1) across the apical membrane of the duodenal mucosa. Heme iron is transported by transporters that are incompletely characterised through the apical membrane. Inside the cytoplasm of the mucosal cells heme is metabolized to release ferrous iron. Iron that is present in the mucosal cells can be either transported to the blood or stored as mucosal iron. Ferrous iron is oxidized by the iron oxidases such as hephaestin and ceruloplasmin into ferric iron which is then transported by the ferroportin across the basolateral membrane of the enterocytes.

Both the transporters DMT1 and ferroportin are distributed widely in the body and play an important role in iron transport in other tissues as well 8. Iron uptake from the intestine by DMT 1 Is increased by the hypoxia inducible factor 2 alpha 9.

If the hepatic iron stores increases the liver synthesis a small peptide hormone called hepcidin which decreases iron absorption of iron by binding to

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ferroportin transporter and inhibiting it. When the hepcidin levels increases iron gets trapped in the enterocytes as mucosal ferritin and it is lost as the enterocytes are sloughed. Hepcidin also suppresses the release of iron from macrophages which is an important source of iron for the erythroid precursors 8. Hepcidin also functions as an acute phase reactant. Inflammatory cytokines such as interleukin 6 increases the levels of hepcidin and this explains the reduced erythropoietic iron in patients with iron of chronic disease.

TMPRSS6 is a hepatic transmembrane serine protease which reduces the production of hepcidin when the hepatic iron stores are low. Mutation in this transmembrane serine protease results in very high levels of hepcidin which results in decreased iron absorption from gastro gastrointestinal tract. As a result of which there will be severe microcytic hypochromic anaemia that is resistant to oral iron therapy 8.

Causes of iron deficiency anaemia:

Iron deficiency can result from 6

1. Physiological ( increased demand ) 2. Environmental

3. Decreased absorption 4. Chronic blood loss 5. Drug related 6. Genetic

7. Iron restricted erythropoiesis

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19 1) Physiological

Increased demand of iron occurs during infancy, rapid growth (adolescence) , second and third trimester of pregnancy, blood donation.

2) Environmental

Insufficient intake resulting from poverty, malnutrition, strictly vegetarian diet

3) Decreased absorption

Post gastrectomy, duodenal bypass, bariatric surgery, helicobacter pylori infection, celiac sprue, atrophic gastritis, inflammatory bowel disease

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20 4) Chronic blood loss

GIT: oesophagitis, erosive gastritis, peptic ulcer, diverticulitis, benign tumours, intestinal cancer, inflammatory bowel disease, angiodysplasia, hemorrhoids, hookworm infestation, obscure source.

Genitourinary system: menorrhagia, intravascular hemolysis

Systemic bleeding: hemorrhagic telangiectasia, chronic schistosomiasis, Munchausen’s syndrome (eg self-inflicted hemorrhages)

5) Drug related

Glucocorticoids, salicylates, nonsteroidal anti-inflammatory drugs, proton pump inhibitors.

6) Genetic

Iron refractory iron deficiency anaemia

7) Iron restricted erythropoiesis

Treatment with erythropoiesis stimulating agents, anaemia of chronic disease, chronic kidney disease.

Clinical features:

Due to depletion of iron containing enzymes through out the body in severe and long standing deficiency the patient will have alopecia, koilonychia, atrophic changes in the stomach and gastric mucosa , malabsorption etc.. Pica

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(consumption of nonfood substances such Clay) occurs due to depletion of iron in CNS8.

Irrespective of the presence or absence of anaemia, iron deficiency itself has a negative impact on the quality of life in patients with heart failure 10. Severe iron deficiency anaemia during pregnancy has been associated with low neonatal weight, increased risk of preterm delivery and increased maternal and newborn mortality. Severe anaemia can also cause restless leg syndrome.

Laboratory investigations

(the above peripheral smear shows the microcytic hypochromic picture in a patient with iron deficiency anaemia)

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22 Serum iron:

Serum iron exhibits a diurnal variation physiologically. Its level is maximum between 7 am to 10am in the morning and decreases in the evening reaching the nadir at 9 pm. This diurnal variation rarely influences the diagnosis.

Serum iron levels are decreased during menstruation, during an acute or chronic inflammatory state, acute myocardial infarction. Conversely serum iron remains elevated during the chemotherapy for malignancy due to the inhibition of erythropoiesis and its related uptake of iron by the erythropoiesis by chemotherapeutic drugs. This effect is most commonly seen during the 3rd to 7th day after the initiation of chemotherapy 23. Before obtaining blood samples for iron studies, oral iron preparations should be withheld at least for about 24hours.

Parenteral iron dextran injection causes elevation of serum iron for several weeks.

Serum iron elevation following the infusion of iron sucrose or sodium ferric gluconate is only for shorter duration 24.

Reference range : Males - 65-175 microgram /dL Females -50-170 microgram/dL

Iron binding capacity and transferrin saturation:

Iron binding capacity is the measure of the amount of transferrin in the blood. Normally one third of transferrin is saturated with iron. The latent iron binding capacity or unsaturated iron binding capacity (UIBC) can be easily measured by the spectrophotometric techniques or with radioactive iron. The sum of the plasma iron and unsaturated iron binding capacity is the Total Iron Binding

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Capacity (TIBC). It can also be measured directly. UIBC and TIBC are increased and serum iron levels are decreased in iron deficiency anaemia resulting in a transferrin saturation of 15% or less. In case of anaemia of chronic Inflammation serum transferrin levels and TIBC are decreased resulting in a normal transferrin saturation and low serum iron levels 3.

TIBC reference range: Male -225 - 535 microgram /dL.

Female -215 -535 microgram/dL.

Transferrin saturation: 13 -45 %

Serum ferritin:

Serum ferritin is mainly secreted by macrophages, hepatocytes relatively contains only little iron yet for unknown reasons serum ferritin levels correlates well with the total body iron stores. Serum ferritin levels of less than 10 microgram/L is characteristic of iron deficiency anaemia. Increased serum ferritin levels are seen in certain malignancies, chronic kidney disease and in Inflammation states such as rheumatoid arthritis. In juvenile rheumatoid arthritis, Gaucher's disease and in various macrophage activation syndromes there will be massive iron loading of macrophages resulting in serum ferritin levels usually in range of thousands. This will mask iron deficiency in such conditions 3.

Reference range : Men - 22-322ngm/ml Women -10-291ngm/ml

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24 Erythrocytes zinc protoporphyrin;

It is increased in case of iron deficiency anaemia, sideroblastic anaemia and lead poisoning. It can be used for the large scale screening of the children to detect lead poisoning and iron deficiency anaemia. However it could not differentiate between iron deficiency Anemia and anaemia associated with malignant or inflammatory process 3.

Serum transferrin receptor;

Transferrin receptor circulates in blood bound to transferrin. It's level correlates well with the amount of cellular receptor and therefore it is proportional to the number of erythroblasts in bone marrow expressing the receptor. Its level is increased in iron deficiency anaemia. In chronic inflammation cytokines suppresses the synthesis of transferrin receptors leading to decreased levels in patients with anaemia of chronic inflammation 3.

Reticulocyte hemoglobin content:

This indicates the restriction of iron in hemoglobin synthesis 3 to 4 days before test.

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26 Treatment of iron deficiency anaemia:

All the patients with iron deficiency anaemia should be treated with iron supplementation. In patients from malaria endemic areas caution should be exercised while treating as treatment would reverse the protective effects of iron deficiency 11. Iron supplementation further increases the susceptibility co infections in those patients 12. It has been established in intro studies that Plasmodium falciparum is less effective in infecting iron deficient RBC’s than infecting iron rich RBC’s , a protection that can be reversed by iron supplementation 13.

A number of studies have found that iron supplementation in the form of intravenous iron have improved fatigue in women in whom serum ferritin were in the iron deficit range in the absence of anaemia 14.

Patients with cardiovascular symptoms such as angina or heart failure in the presence of severe iron deficiency anaemia should be treated with red cell transfusion. This not only correct the hypoxia rapidly but also the iron deficiency.

One unit of packed RBCs approximately provides about 200 mg of iron.

Though hepcidin measurements is not routinely in clinical practice, many studies have shown that the hepcidin levels would guide in selecting patients for oral iron therapy. Patients with low hepcidin levels will benefit from oral iron therapy and patients with normal or elevated levels will be resistant to oral therapy 15.

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The response to iron therapy may also be predicted by the change in hemoglobin content of reticulocytes after one week of oral iron therapy 16.

In patients refractory to oral iron supplementation measures should be taken to eradicate Helicobacter pylori infection. If celiac disease is suspected a gluten free diet has to be instituted. These steps will improve iron absorption and in some patients it may eliminate the need for iron supplementation.

Oral iron supplementation:

In patients with iron deficiency anaemia recommended daily dose is 100 to 200 mg of elemental iron and for children it is 3 to 6 mg per kg body weight. In Order to replete iron stores treatment should be continued for a period of about 3 to 6 months. Absorption of iron is enhanced by ascorbic acid, amino acids, sugars and citric acid and inhibited by carbonates, oxalates, tannates (found in tea), phosphates and drugs such as antacids, histamine receptor blockers, proton pump inhibitors.

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28 The commonly used iron preparations are

1. Ferrous fumarate 2. Ferrous gluconate 3. Ferrous sulfate

Other oral preparations available include heme iron polypeptide, carbonyl iron, ferric citrate, ferrous ascorbate, and ferrous succinate18.

Ferrous fumarate: It contains about 33% of elemental iron. For prevention of iron deficiency dosage is 30 to 60mg per day for three consecutive months in a year. In patients with iron deficiency Anemia it is 65 to 200mg per day administered in two to three divided doses.

Ferrous gluconate: It contains about 12 % of elemental iron.

Ferrous sulfate: It contains about 20% of elemental iron. Exsiccated ferrous sulfate (dried) contains about 30% of elemental iron.

Enteric coated preparations or sustained release preparations are not preferred as they deliver the iron more distally and it is poorly absorbed. In some cases scenarios intact capsules were excreted in the stool 19.

Increasing evidence suggest that alternate day dosing may result in better absorption of iron than daily dosing 20 21.

Elderly patients experience greater toxicity with oral preparations and they should be treated with lower doses 22.

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29 Side effects:

Gastrointestinal symptoms are more common with oral iron preparations. It includes nausea, flatulence, constipation, epigastric distress, diarrhea and vomiting. Patients may also complain of itching and blackmail/ green tarry stools.

The black coloured stools will not produce false positive results with the tests for occult blood. All these GI symptoms will be resulting lower compliance with oral preparations. Compliance can be improved by the following ways

● Increasing the interval between the dosing

● Taking along with food or milk although this may decrease the absorption

● Switching to a preparation with low levels of elemental iron

● Switching from a tablet form to liquid.

Iron salts will decrease the serum concentration of the following drugs when administered along with it

● Bictegravir

● Bisphosphonate derivatives

● Cefdinir. ( Red colour non bloody stools are formed due to formation of insoluble complex of iron with cefdinir)

● Dimercaprol

● Dolutegravir

● Eltrombopag

● Entacapone

● Levodopa

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● Levothyroxine

● Penicillamine.

● Phosphate supplements

● Quinolones

● Trientine

Intravenous iron:

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31 Currently available Intravenous formulations are

 Ferric carboxymaltose

 Ferric gluconate

 Ferumoxytol

 Iron sucrose

 Iron isomaltoside

 Low molecular weight iron dextran

Ferric carboxymaltose:

In iron deficiency anaemia, if patient is less than 50 kg dose is 15mg/ kg on day 1. Same dose may be repeated after 7 days. Repeat course of anaemia reoccurs. If patient is >50 kg dose is 750 mg on day 1 and the dose can be repeated after 7 days.

If iron deficiency anaemia is associated with inflammatory bowel disease the dose is 500 to 1000mg as iv infusion

In case of restless leg syndrome the dose is 500 mg on day 1. It may be repeated after 5 days.

No dosage adjustment is required in the presence of hepatic or renal disease.

Ferric gluconate:

In chronic kidney disease patients undergoing dialysis ferric gluconate is given at the dose of 125mg per dialysis session. For repletion of iron stores most

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of CKD patients will require a total dose of 1000mg over a period of 8 HD sessions. Clinically significant hypotension may occur but it usually resolves within one to two hours and it may aggravate the hypotension associated with hemodialysis. Hypersensitivity reactions are also common with this preparation.

Ferumoxytol:

In iron deficiency anaemia the dosage is 510mg as iv infusion on day 1 and the same dose can be repeated after 3 to 8 days.

The response may be assessed after 30 days of the second dose.

Ferumoxytol has been associated with very serious hypersensitivity reactions.

Hypersensitivity reactions can occur even in patients in whom it was previously well tolerated. It can alter the magnetic resonance imaging. Peak alteration occurs during the first 2 days following the infusion and it may persist till 3 months after the administration. However, it does not affect the positron emission tomography, single positron emission computed tomography or nuclear medicine imaging.

Iron sucrose:

In chronic kidney disease patients on hemodialysis, the dosage is 100 mg per day for a period of 10 days to reach a cumulative dose of 1000mg. In CKD patients dependent on peritoneal dialysis the dosage is two infusions of 300 mg of iron sucrose at about 2 weeks apart. It is followed by another dose of 400 mg after 2 weeks. In non-dialysis dependent patient dosage is 200 mg on 5 occasions over a period of two weeks.

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33 Iron isomaltoside:

Cumulative dosage is calculated based on the Ganzoni formula or the simplified table

Ganzoni formula: iron need (mg)= weight in kg *(target Hb - actual Hb) * 2.4 + depot iron in mg

Simplified table:

Hb (gm/dL) weight <70 kg weight > 70 kg

more than 10 1000mg 1500mg

less than 10 1500 mg 2000mg

Ganzoni formula is used in patients with chronic kidney disease whereas simplified table is used in patients with iron. Deficiency anaemia. It is contraindicated in patients with decompensated liver disease or in the presence of active hepatitis. It can also cause hypophosphatemia. Phosphate levels usually normalizes within four weeks.

Iron dextran:

In iron deficiency anaemia total dosage required is calculated by the following formula

Total dose (mL) = 0.0442 (desired Hb - observed Hb) * LBW + (0.26* LBW) LBW - Lean Body Weight in kg

Desired Hb is usually 14.8 gm /dL

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Replacement iron following blood loss (mg) = blood loss (mL) * hematocrit. 25 mg should be given as a test dose. Patients should be observed for one hour. Fatal reactions have occurred even in patients in whom test dose was tolerated. Daily dosage is limited to 100 mg per day. Iron dextran exacerbates cardiovascular disease related complications. Acute exacerbation of joint swelling and pain is seen in patients with rheumatoid arthritis.

ACE inhibitors will enhance the adverse effects of iron dextran.

Dimercaprol will enhance the nephrotoxic effect of iron dextran.

Calculation of total iron dose:

In clinical practice there is no evidence that the total doses more than 1000mg are useful clinically. A fixed dose of approximately 1000mg of elemental iron is generally sufficient to treat iron deficiency and to replenish iron iron stores. Whether it could be given as a single infusion or multiple infusion depends on the specific product. An example of a simple calculation is in the following table.

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Use of premedication and treatment of anaphylactic reactions:

A number of adverse reactions attributed due to intravenous iron infusion are in fact due to the premedications especially diphenhydramine.

Diphenhydramine causes somnolence, dizziness, flushing, hypotension, nasal congestion, irritability, supraventricular tachycardia and wheezing.

No premedication is recommended for a patient without a history of asthma or drug allergy. For patients with a history of more than one drug allergy

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or asthma, premedication with 125mg of methylprednisolone I.v prior to the administration of any intravenous iron preparations is recommended 25.

In patients with a history of inflammatory arthritis, premedication with 125 mg of methylprednisolone I.v is recommended along with a short course of oral prednisolone at the dose of 1mg/kg for 4 days.

I.V preparations are administered slowly at first and the patients are observed for the infusion reactions.

Arthralgia, transient fever, myalgia and flushing are seen generally in about 0.5 to 1 percent of infusions. If they are not associated with tachypnea, tachycardia, stridor, hypotension, wheezing or periorbital edema infusion is temporarily stopped. Antihistamines is generally not recommended as this would worsen the symptoms. If the symptoms resolved infusion can be resumed and completed. If patient deteriorates it is treated as a serious reaction.

Many infectious agents require iron as a growth factor. Therapeutic use of iron may be associated with an increased risk of infection 26.

Response to iron supplementation and follow up:

 If pica is present, it disappears well before the reticulocyte response.

 There will be a sense of well being within a few days of initiation of treatment.

 Reticulocytosis will be seen in patients with moderate or severe anaemia

 The hemoglobin level rises slowly and normalizes within 6 to 8 weeks.

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 In patients with iron deficiency anaemia, tongue papillation is decreased first over the top and lateral borders and then it moves centrally and posteriorly. Following iron supplementation, rapid correction is observed over a period of weeks to months.

For the patients on oral supplementation, hemoglobin is measured after two weeks of starting the therapy. For patients on intravenous iron therapy repeat hemoglobin is recommended after 4 to 8 weeks.

Cause for failure to respond to iron therapy

 Coexisting disease interfering with marrow response such as infection , inflammatory disorder , concomitant malignancy , coexisting folate / vit B12 deficiency

 Patient is not iron deficient , possible correct diagnosis include thalassemia , lead poisoning , anaemia of chronic inflammation, copper deficiency, myelodysplastic syndrome and refractory sideroblastic anaemia.

 Continued blood loss

THALASSEMIA:

Cooley and Lee in 1925 describes a severe form of anaemia in early stages of life, associated with bone changes and splenomegaly.

William L Bradford and George H whipped coined the name thalassemia.

Thalassemia is a group of inherited disorders associated with a defect in the

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production of globin chains. There will be disordered globin chain production, ineffective erythropoiesis, hemolysis and variable degree of anaemia.

A pair of alpha globin gene on chromosome no 16 encodes for the alpha globin chain. A single beta globin chain on chromosome no 11 encodes for beta globin chain. It is endemic in Mediterranean countries, tropical Africa, Indian subcontinent, in the Middle East and Asia. Heterozygous carriers are protected against malaria 27. Alpha thalassemia and beta thalassemia are the two major types.

BETA THALASSEMIA:

Molecular basis of beta thalassemia:

Over 200 mutations have been identified in association with beta thalassemia 28. They may be classified broadly into deletions of beta globulin

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gene and non deletion mutations resulting in defect in the processing, transcription or translation of beta globin messenger. About 20 alleles accents for majority of all beta thalassemia determinants.

β0 mutation is associated with absence of globin chain synthesis.

β+ mutation is associated with reduced globin chain synthesis

Indian 619bp deletion removes 3’ end of beta gene but leaves behind the 5’

end. Homozygotes of this deletion have β0-thalassemia. Heterozygotes for Indian deletion have increased levels of hemoglobin A2 and HbF.

The boundaries of introns and Exons are invariable marked by the dinucleotides, GT at the 5’ and AG at 3’. Single base substitutions that involve any of these splice junctions will totally affect the normal splicing of the RNA and result in β0-thalassemia phenotype.

Base substitution results in a nonsense mutation in which there will be change of the amino acid codon into chain termination codon. It prevents translation of mRNA and results in β0-thalassemia.

The insertion or deletion of nucleotides in the coding region of the beta globin gene results in a shift mutation. Insertion of one nucleotide between 8 and 9 codons & deletion of 4 nucleotides in 41 and 42 are the two common mutations in the Asian Indians. 29.

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Mutation in exon 3 of beta globin gene is associated with dominantly inherited beta thalassemia.

Pathophysiology of beta thalassemia:

Two major phenomena in beta thalassemia is 1. Ineffective erythropoiesis

2. Hemolysis

Ineffective erythropoiesis:

Due to the absence of beta chain the free alpha chains cannot form tetramers. They are highly unstable. Alpha globin chains aggregate and precipitate adjacent to the Red cell membrane 30. Precipitated alpha chains form large single inclusions in the circulating RBCs and RBC precursors.

There will be increased production of gamma chain and delta chains. But it is usually insufficient to restore the balance between beta like chains and alpha chains and to prevent alpha chain aggregation 31. The normal RBC cytoskeletal and membrane requires the assembly of band 3, band 4.1 and spectrin. In patients with beta thalassemia there will be disordered and discontinuous insertion of these proteins, especially in the areas of alpha chain aggregation and during the early

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stages of erythropoiesis. Because of theses abnormalities in the cytoskeleton there will be oxidative stress leading to the production of reactive oxygen species. This leads to apoptosis inside the bone marrow

In beta thalassemia characteristic stages of ineffective erythropoiesis is the expansion of early erythroid progenitor cells and their accelerated differentiation.

It is followed by the maturation arrest and apoptosis at the sarge of polychromatophilic erythroblast 32.

In patients with severely impaired erythropoiesis, RBC production takes place at the extra medullary sites such as liver, spine and spleen. Extramedullary erythropoiesis in spine can cause neurological deficits and significant pain.

Hemolysis:

Hemolysis in beta thalassemia patients is because of 1. Reduced deformability

2. Reduced survival

Reduced deformability:

In beta thalassemia RBC’s are dehydrated and dense leading to reduced deformability. This is especially seen in patients who have undergone splenectomy. There is also an increased membrane rigidity in these patients.

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42 Reduced survival of the RBC’S:

Reduced survival of the RBC’s is due to increased phagocytosis by the reticuloendothelial system in the spleen and liver because of the following reasons.

 Reduced sialic acid in the RBC'S membrane.33

 Macrophages recognizes phosphatidylserine as the signal for phagocytosis.

Normally it is expressed in the inner leaflet of the RBC membrane. In patients with beta thalassemia it is expressed on the outer cell membrane. 34

 Autoantibodies are formed against the abnormal proteins expressed on the RBC membrane in patients with beta thalassemia.

 Increased cytokines production will activate macrophages and monocytes.

Hypercoagulable state:

There is an increased risk of thromboembolism in patients with beta thalassemia. However the mechanisms have been poorly understood. Increased phosphatidylserine in the outer leaflet may promote thrombosis similar to its role in stimulating coagulation cascade on the surface of activated platelets.35

Iron overload:

Iron overload in beta thalassemia is because of the following reasons 1. Transfusion related iron overload

2. Increased erythroferrone causes increased iron absorption in these patients.36

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3. Because of anaemia and increased erythropoiesis, hepcidin levels will be suppressed which leads to increased iron absorption from the gastrointestinal tract. Because of increased absorption of iron from the GIT, there will be increased levels of stored iron as well as free iron which is also called as non-transferrin bound iron (NTBI). NTBI is associated with the production of reactive oxygen species which contributes to the end organ damage. In beta thalassemic patients with heart disease NTBI was found to be increased. Increase in the reactive oxygen species will interfere with erythropoiesis in the bone marrow.

Organ damage:

It is because of the combined effects of chronic hypoxia, iron overload, anaemia and chronic inflammatory state.

1. Renal diseases: It is because of the extra medullary erythropoiesis that involves the kidneys. Increased uric acid levels and other metabolic effects due to increased hematopoietic turnover also affects the kidney. Iron overload is directly toxic to kidney. Deferasirox which is used as the iron chelating agent is nephrotoxic.

2. Cardiac disease: It includes pulmonary hypertension and cardiomyopathy.

Causes are multifactorial. Iron toxicity plays an important role in cardiac dysfunction. Other factors include chronic anaemia, hemolysis, vascular changes, endocrinopathy and pulmonary disease.

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3. Diabetes: Because of iron deposition and subsequent oxidative damage to the pancreatic cells there will be diabetes and other endocrine and metabolic abnormalities.

Thalassemic patients are protected against Plasmodium falciparum. This effect is seen more in alpha thalassemia than beta thalassemia patients. The mechanism behind is poorly understood.

Yersinia enterocolitica is an iron loving organism. It adds to the morbidity of this patient. It usually presents as appendicitis like syndrome

Clinical and genetic classification of beta thalassemia:

Clinically it can be classified into three major types 1. Beta thalassemia major

2. Beta thalassemia intermedia 3. Beta thalassemia minor

Beta thalassemia major:

It is also called Cooley's anaemia or transfusion dependent thalassemia or Mediterranean anaemia. It is the most common form in Southeast Asia, parts of Africa and Mediterranean countries. As the hemoglobin switches from HbF to HbA around 6 to 9 months of age patients present with severe anaemia. Hb levels are usually between 3 to 6 gm / dL in untransfused patients. The major Hb in these patients is HbF. HbA2 levels are more often normal or low but sometimes may be high. Clinical features include hepatosplenomegaly, growth retardation,

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prominent cheekbones. With iron chelation and transfusion patient will survive till the third decade. In patients heavily transfused, there will be iron load leading to secondary hemochromatosis. Cardiac diseases resulting from secondary hemochromatosis is an important cause of death in these patients.

Beta thalassemia intermedia:

It usually presents during two to four years of age 37. It is characterized by the more heterogeneous clinical presentations. Spectrum includes patients with chronic hemolytic anaemia who do not depend on blood transfusion during early stage of life but later become transfusion dependent to those patients with mild to moderate anaemia who does not require transfusion. Clinical findings of extra medullary hematopoiesis may be present. Iron overload occurs in these patients also but the age in which it manifests is highly variable.

Beta thalassemia minor or beta thalassemia trait:

It is a carrier condition and the individuals are heterozygous for beta+ or beta0 thalassemia mutation. They have mild anaemia and they are usually asymptomatic. They exhibit marked microcytosis which may be mistaken for the iron deficiency Anemia. There will be marked. elevation of HbA2 to 4 to 8%.

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Early identification of this clinical entity is highly important in order to provide genetic counseling to the individuals

Clinical manifestations:

Anaemia:

There will be microcytic hypochromic anaemia and increased RBC count.

These patients are more susceptible to drugs, infections and nutritional deficits that interfere with the RBC production as there is a constant erythropoietic stress.

Examples include parvovirusB19 causing aplastic crisis and other infection causing hypoplastic crisis.

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47 Jaundice and pigment gallstones:

In patients with beta thalassemia major there will be chronic hemolysis, leading to pigment gallstones and inflammation of the biliary tract.

Skeletal changes:

Because of the ineffective erythropoiesis and extra medullary hematopoiesis skeletal changes are more prominent in thalassemia major and many patients with thalassemia intermedia.

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Facial deformity: It includes delayed pneumatization of sinuses, frontal bossing, overgrowth of maxilla, increased malar prominence, jumbling of upper incisors leading on to the characteristic “chipmunk facies “. There will be dental malocclusion.

Changes in the body habitus: The ribs and extremity bones become box shaped and eventually convex. There will be premature closure of the epiphysis leading on to the shortening of limbs, especially the arms.

Osteopenia and osteoporosis: It is very common due to the widening of the bone marrow spaces 38. Because of the widening of the diploid spaces in the skull there will be characteristic “hair on end “appearance in the x- ray of the skull. Osteoporosis if left untreated it leads to the impaired growth and fractures of the bones including vertebral fractures, back pain and spine deformities.

Bony masses: Erythroid bone marrow break through the bone, invade the bony cortex and set up masses of ectopic erythroid colonies in the pelvic or thoracic cavities and sinuses. The expanding masses behave like a tumour and cause spinal cord compression and other abnormalities.

Chronic hyper transfusion regimen especially early in childhood can partially prevent these skeletal changes. Once it has occurred hyper transfusion may sometimes reverse the skeletal changes but it will retain some sequelae that have already occurred.

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49 Growth impairment:

There is impaired growth and development in these patients because of the following reasons

 Chronic Anemia

 Ineffective erythropoiesis results in a hypermetabolic state

 Hypermetabolic state resulting in nutritional deficiency

 Toxicity associated with the iron chelation therapy

 Excess iron deposition results in endocrinopathies.

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50 Hepatosplenomegaly:

Chronic hemolysis along with the extra medullary hematopoiesis results in the hepatosplenomegaly. Viral hepatitis also contributes to the liver injury during the period when the routine screening of the blood products are not done. There is increased risk of hepatocellular carcinoma in these patients because of the iron overload and viral infection.

In patients with thalassemia minor spleen size is large when compared to the normal but it is not palpable.

Endocrine and metabolic abnormalities:

It is common in thalassemia major and thalassemia minor because of the iron overload.

Hypogonadism: It is because of the deposition of the excess iron in the pituitary. Development of the primary and secondary sexual characters is delayed in both girls and boys 39. Menarche is delayed and oligomenorrhea or amenorrhea is very common in these patients. Boys have no or sparse facial and body hair and there will be decreased libido

Hypothyroidism: Iron deposition both in the pituitary and thyroid gland contributes to hypothyroidism in these patients.

Insulin resistance and diabetes: Because of the iron deposition in the pancreatic islets there will be abnormal carbohydrate metabolism leading to glucose intolerance and diabetes.

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The presence of diabetes itself is an independent risk factor for cardiac complications in these patients. 40

Heart failure and arrhythmias:

Heart failure and / or fatal arrhythmias are the major cause of death in these patients. Heart disease in these patients is multifactorial and includes anaemia, concomitant diabetes, cardiac iron deposition, pulmonary arterial hypertension, vascular dysfunction due to oxidative stress, high cardiac output state because of increased pulmonary vascular resistance and tissue hypoxia and vitamin D deficiency.

Cardiac iron deposition results in the sterile pericarditis, restrictive cardiomyopathy, arrhythmias (supraventricular and ventricular) and heart failure.

Bradycardia and repolarization abnormalities (left shift of T wave axis and QT prolongation) are the strongest indicators of excess iron deposition in the heart41.

In 2013 American Heart Association emphasized the importance of using MRI to assess the cardiac iron load in patients with thalassemia and to initiate iron chelation therapy in patients with increased cardiac iron 42. MRI will show varying degree of myocardial fibrosis, myocardial iron deposition and necrosis.

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Pulmonary abnormalities and pulmonary hypertension:

The abnormalities in the pulmonary function tests include small airway obstructive disease, restrictive pattern, decreased maximal oxygen uptake, hyperinflation and abnormal anaerobic thresholds. The mechanism behind this is very poorly understood.

Thalassemia major patients will also develop pulmonary hypertension the cause of which is entirely not clear.

Management of beta thalassemia:

Dietary restrictions and supplementation:

Folic acid supplementation is recommended for patients with beta thalassemia in order to compensate for increased requirements due to increased RBC turnover. The dose is 1 to 2 mg. Beta thalassemia patients should be advised about the judicious intake of iron rich foods. Zinc supplementation is not given unless the patient has symptoms suggestive of the zinc deficiency such as impaired smell or taste or documented zinc deficiency.

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53 Regular transfusion:

Regular transfusion is indicated in patients with thalassemia major.

Chronic transfusion in these patients is referred as hyper transfusion. Suggested pretransfusion hemoglobin level in these patients is 9.5 to 10 gm /dL. This approach is directed towards attaining the optimal balance between minimizing the iron overload and suppressing the hematopoiesis. It is not practical to transfuse more than 2 units of PRBC at the same time. So, the interval between two transfusion is adjusted in order to maintain hemoglobin in the appropriate level. Post transfusion hemoglobin should be between 12 to 13gm /dL and not more than 15gm/dL.

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Patients with thalassemia intermedia requires transfusion during the time of erythropoietic stress such as periods of rapid growth, acute illnesses, pregnancy and surgery. Many patients with thalassemia intermedia will become transfusion dependent during the 3rd and 4th decade of life.

Assessment of iron stores and initiation of iron chelation:

Serum ferritin level is used for the serial testing to assess iron load and to monitor treatment with the iron chelating agents.

Baseline MRI is obtained in these patients. MRI based estimates of the liver and cardiac iron is used if serum ferritin levels are discordant.

MRI measurement of cardiac iron is usually done after 8 to 10 years of age.

Iron concentration in mg /gm dry weight = 45*(T2 *MRI in milliseconds raised to the power -1.22)

In patients with transfusion independent thalassemia or thalassemia intermedia the iron overload is mainly because of the increased gastrointestinal

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absorption. Iron gets deposited more in the liver than in the macrophages. Hence serum ferritin will underestimate the total iron overload in these patients 43. The patients with thalassemia major will present with features of iron overload during 10 to 15 years of age, whereas patients with thalassemia major usually presents at 2 to 3 years of age.

Iron chelation is started in one or more of the following situations

 When serum ferritin exceeds 1000ngm/dL

 When liver iron concentration exceeds 3mg iron per gram of dry weight.

 After transfusion of 20 to 25 units of packed cells.

 During the initiation of chronic transfusion program.

For the patients with beta thalassemia it is usually started before the age of 6 years.

The aim of iron chelation in these patients is to reduce the production of reactive oxygen species and preventing the organ damage so that the patient have reduced morbidity and increased survival. Each unit of blood contains about 200 mg to 250 mg of iron.

The three chelating agents used are

 Deferasirox

 Desferoxamine

 Deferiprone

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56 Desferoxamine:

In patients with chronic iron overload the dosage is 40 mg to 50 mg /kg/day over 8 to 12 hours for a period of 5 to 7 days per week. It is contraindicated in severe renal disease or anuria. No dosage adjustment is required in case of hepatic impairment. Urticaria, hypotension, flushing of skin and shock may occur following rapid I.v administration of the drug. Infusion related reactions can be avoided by limiting the rate of infusion to 15mg /kg / hour. In patients with poor compliance it may be given on the same day either prior to transfusion or after it. It should not be administered concurrently along with the transfusion. In case of induration or erythema topical glucocorticoid cream or anaesthetic agent can be used. Urine may change pink, reddish or orange with the use of this drug. It also causes dysplasia, thrombocytopenia and leukopenia. The patients have increased susceptibility to infections with yersinia and mucormycosis.

Desferoxamine especially when treated with higher doses have been associated ARDS.

Combining desferoxamine with ascorbic acid will impair cardiac function.

If ascorbic has to be given along with it, it should be initiated after one month of regular treatment with desferoxamine and the dose of ascorbic acid should not exceed 200 mg per day in adults.

It is also used in the diagnosis as well as the treatment of the aluminium induced toxicity in patients with chronic kidney disease.

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There is no significant difference in the amount of iron being reduced by deferiprone and desferoxamine.

Deferiprone:

It can cause agranulocytosis that leads to serious life threatening infections and death. Absolute neutrophil count is measured before starting the therapy and then weekly.

The dosage is oral formulation 75mg / kg / day in three divided doses maximum of 100 mg / kg / day.

No dosage adjustment is required in the presence of hepatic and renal failure.

Gastrointestinal adverse effects include nausea, vomiting, diarrhea, weight gain.

Elevation in the ALT values have also been observed. If there is persistent elevation of the enzymes treatment has to be discontinued.

Hypersensitivity reactions in the form of Henoch - Schoenlein purpura, periorbital edema with skin rashes and urticaria have also been reported.

It is teratogenic hence it should be avoided in patients planning for the pregnancy.

References

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