notes hematology

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Ramadas NayakMBBS MDProfessor

Department of PathologyKasturba Medical College

Manipal University

Mangalore, Karnataka, India

[email protected]

Sharada RaiMBBS MDAssociate Professor

Department of Pathology

Kasturba Medical College

Manipal University

Mangalore, Karnataka, [email protected]

Foreword

AR Raghupathy

JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD

New Delhi London Philadelphia Panama


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Website: www.jaypeebrothers.com

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2014, Jaypee Brothers Medical Publishers

All rights reserved. No part of this book may be reproduced in any form or by any means without the prior permission of

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This book has been published in good faith that the contents provided by the authors contained herein are original, and is

intended for educational purposes only. While every effort is made to ensure accuracy of information, the publisher and the authors

specically disclaim any damage, liability, or loss incurred, directly or indirectly, from the use or application of any of the contents

of this work. If not specically stated, all gures and tables are courtesy of the authors. Where appropriate, the readers should

consult with a specialist or contact the manufacturer of the drug or device.

Rapid Review of Hematology

First Edition: 2014

ISBN 978-93-5090-961-4

Printed at

Headquarters

Jaypee Brothers Medical Publishers (P) Ltd

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Dedicated to

Students who inspired us,

patients who provided the knowledge,

our parents and family members who

encouraged and supported us.


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It gives me great pleasure to write a short foreword for this new book on Rapid Review of Hematology. Tis is a well-written concise but precise and student-friendly text that will be highly valuable to medical students. It

will help in revising and reinforcing the fundamental concepts in hematology. It is very well organized with optional and

correct usage of good pictures, schematic diagrams and flow charts. Every essential topic has been discussed giving opt

importance and stress on salient features. Each statement mentioned in the text is well written as it carries the required

essential points.

In short, this book provides within one volume a user-friendly review of the basic essential concepts in hematology.

It will be of great help to not only second year MBBS students, but also for students preparing for entrance examinations,

and students of allied sciences.

Tis book will certainly serve as a valuable gift and a valuable addition to the students library and the user will

definitely appreciate the content and presentation of the information in this book.

In conclusion, I am sure, this book brought out by Dr Ramadas Nayak and Dr Sharada Rai will be a very usefulcompendium for second year MBBS students, the students preparing for entrance examinations, and students of allied

health sciences.

I hope the reader of this new book will get as much pleasure and knowledge as I did.

DEPARTMENT OF PATHOLOGY BANGALORE MEDICAL COLLEGE

VICTORIA HOSPITAL COMPLEX

BENGALURU 560 002

Phone: 670 1150 Ext.: 314, 315, 316, 317

AR RaghupathyMBBS MD PGDHHM (IGNOU)Professor and Head

Department of PathologyBangalore Medical College and Research Institute

Bengaluru, Karnataka, India

Foreword


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Hematology is one of the rapidly expanding fields of medicine and emerging as a clinical specialty in its own right.

Hematology is difficult to teach at the undergraduate level, as it is a part of the curriculum in Pathology, during which

undergraduate students do not have enough exposure to diseases of blood. Tis results in less attention to hematological

diseases at undergraduate level. After many years of teaching undergraduates, we found that undergraduate students

either neglect hematology or find it difficult to understand the subject. It is a nightmare for many students especially

during examinations. Tere are many hematology textbooks, but undergraduates face difficulties to refresh their

knowledge of hematology during examinations. Tis encouraged us to write a book to fill the niche, to provide basicinformation to an undergraduate in a nutshell. With this view in mind, Rapid Review of Hematologyis intended for the

undergraduates from medical, dental and paramedical fields. Most students are fundamentally visually oriented. As

the saying one picture worth thousand words, it encouraged us to provide many illustrations (e.g. etiopathogenesis,

clinical presentation, complications, peripheral blood smear and other relevant laboratory tests).

Organization

Tis book is organized into four sections namely disorders of red cells, disorders of white cells, disorders of hemostasis

and clinical scenario.

Te final section deals with common clinical scenario encountered during theory examination.

How to use this book

We recommend that this book not to be used as a hematology textbook rather than a supplement to Essentials in

Hematology and Clinical Pathology (Authored by Dr Ramadas Nayak, Dr Sharada Rai and Dr Astha Gupta). Te

concepts of hematology have been oversimplified in this book, but all the information, the student will ever need to

know, have been provided. Te readers are requested to give more emphasis on word in bold letters that represents

the key words to be remembered. Te peripheral smear and bone marrow findings have been highlighted in colored

background. Boxes have been provided at the sides of main text. Tese include some of the key points as well as commonly

expected questions during examinations. Tis book can serve as a source of rapid review of hematology.

Ramadas Nayak

Sharada Rai

Preface


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Our sincere thanks to Ms Prathiba Bhat for her untiring efforts, patience and excellent support in creating many

illustrations for this book.

Acknowledgments are also due to Dr Astha Gupta (Consultant Pathologist, New Delhi, India), Dr Rakshatha

(KS Hegde Medical college, Mangalore, Karnataka, India), Ms Rekha Nayak, Ms Rashmitha Nayak, and Mr Ramnath Kini

for their contribution in the preparation of the manuscript.

Our sincere thanks to Dr AR Raghupathy, Professor and Head, Department of Pathology, Bangalore Medical College

and Research Institute, Bengaluru, Karnataka, India, for his support and guidance.

We are grateful to Dr K Ramnarayan, Vice Chancellor of Manipal University, Manipal, Karnataka, India, and

Dr M Venkatraya Prabhu, Dean, Kasturba Medical College, Mangalore, Manipal University, Karnataka, India, for their

encouragement.

We are grateful to all our friends, undergraduate and postgraduate students who have inspired and supported us.

We wholeheartedly thank Shri Jitendar P Vij (Group Chairman), Mr Ankit Vij (Managing Director), Mr arun Duneja

(Director-Publishing), Ms Chetna Malhotra Vohra (Sr Manager, Business Development) of M/s Jaypee Brothers

Medical Publishers (P) Ltd, New Delhi, India, for publishing the book in the same format as wanted well in time.

We acknowledge the wonderful work done by Ms Sunita Katla (Publishing Manager), Ms Samina Khan

(PA to Director), Mr KK Raman (Production Manager), Mr Rajesh Sharma (Production Coordinator), Ms Seema

Dogra (Cover Designer), Mr Sarvesh Kumar Singh (Proofreader), Mr Rajesh Ghurkundi (Graphic Designer), andMr Raj Kumar (DP Operator) of M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India.

We thank especially Mr Venugopal V and Mr Vasudev H of M/s Jaypee Brothers Medical Publishers (P) Ltd,

Bengaluru Branch, Karnataka, India, for taking this book to every corner of Karnataka.

Acknowledgments


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Section 1: Disorders of Red Cells

1. Anemias of Impaired Red Cell Production 3

Anemia 3

Red cell indices 4

Iron deficiency anemia 5

Megaloblastic anemia 8 Pernicious anemia 11

Aplastic anemia 13

2. Hemolytic Anemias Due to Red Cell Membrane and Enzyme Defects 16

Hemolytic anemia 16

Hereditary spherocytosis 17

Glucose-6-phosphate dehydrogenase deficiency 20

3. Thalassemia Syndrome 22

Classification of hereditary defects in hemoglobin 22

Thalassemia syndrome 22

b-thalassemia 22

b-thalassemia major 23

b-thalassemia minor/trait 27

a-thalassemia 28

4. Sickle Cell Disease 29

Sickle cell disease 29

Sickle cell anemia 29

Sickle cell trait 34

5. Other Anemias 36

Immunohemolytic anemias 36

Hemolytic disease of the newborn 36

Antiglobulin (Coombs) test 39

Autoimmune hemolytic anemia 40

Fragmentation syndrome 41

Paroxysmal nocturnal hemoglobinuria 41

Anemias of blood loss 41

Sideroblastic anemias 42

Contents


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Rapid Review of Hematologyv

Section 2: Disorders of White Cells

6. Quantitative and Qualitative Disorders of Leukocytes 45

Normal differential leukocyte count (DLC) 45

Quantitative disorders of leukocytes 45

Qualitative disorders of leukocytes 50 Infectious mononucleosis (Glandular fever) 51

7. Acute Leukemia 52

Acute leukemia 52

Acute lymphoblastic leukemia/lymphoma 55

Acute myelogenous leukemia 57

Myeloid sarcoma 59

8. Myelodysplastic Syndromes 60

Myelodysplastic syndromes 60

9. Myeloproliferative Neoplasms 62

Myeloproliferative neoplasms (MPN) 62 Polycythemia or erythrocytosis 63

Polycythemia vera 63

Essential thrombocythemia 65

Primary myelofibrosis 66

10. Chronic Myelogenous Leukemia 68

Chronic myelogenous leukemia 68

Natural history of chronic myeloid leukemia 70

11. Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma 73

Chronic lymphocytic leukemia 73

Hairy cell leukemia 75

12. Plasma Cell Neoplasms 76

Plasma cell myeloma (multiple myeloma) 76

Plasmacytoma 80

Immunoglobulin deposition disease 80

Monoclonal gammopathy of uncertain significance (MGUS) 80

13. Lymphoid Neoplasms 81

Classification of lymphoid neoplasms 81

Follicular lymphoma (FL) 82

Diffuse large B cell lymphoma (DLBCL) 83

Burkitt lymphoma (BL) 83 Mature T cell and NK cell neoplasms 85

14. Hodgkin Lymphomas 87

Definition 87

Classification 87

Morphology of neoplastic cells 88

Classical Hodgkin lymphoma 88

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) 92

Etiology and pathogenesis of Hodgkin lymphoma 93


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Contents

Laboratory findings 93

Staging of Hodgkin lymphoma 94

Differences between Hodgkin lymphoma and non-Hodgkin lymphoma 94

15. Langerhans Cell Histiocytosis/Histiocytosis X 95

Morphology 95

Laboratory findings 95

Section 3: Disorders of Hemostasis

16. Disorders of Primary Hemostasis 99

Normal hemostasis 99

Classification of hemostatic disorders 99

Bleeding disorders caused by vessel wall abnormalities 99

Bleeding disorders due to abnormalities of platelet 100

Thrombocytopenia 100

Immune thrombocytopenic purpura 102

Thrombocytosis 104 Qualitative platelet disorders 104

17. Bleeding Disorders: Due to Abnormalities of Coagulation/Clotting Factor 105

Classification of coagulation disorders 105

Hereditary coagulation disorders 106

Hemophilia 106

Hemophilia A (Factor VIII deficiency) 106

Hemophilia B (Christmas disease, factor IX deficiency) 108

von Willebrand disease (vWD) 108

Acquired coagulation disorders 109

Disseminated intravascular coagulation 110

18. Thrombotic Disorders: Hypercoagulable State 113

Hypercoagulable state (Thrombophilia) 113

Inherited hypercoagulable states 114

Acquired hypercoagulable states 114

Section 4: Clinical Scenario

19. Clinical Scenario 119

Symptoms and signs that suggest a blood disease 119

Patterns strongly suggestive of a blood disease 120

Appendix 127

Bibliography 133

Index 135


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Anemias of Impaired Red Cell Production CHAPTER 1

SECTION 1

Disorders ofRed Cells


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TABLE 1.1: Morphological classification of anemia

Type of anemia Microcytic hypochromic Normocytic normochromic Macrocytic

Size of RBCs Smaller than normal Normal Larger than normal

Central pallor in RBCs More than 1/3 Normal Normal

Mean corpuscular

volume (MCV)

Reduced (< 80 fL) Normal (8298 fL) Increased (>100 fL)

Mean corpuscular

hemoglobin

concentration (MCHC)

Reduced (< 30 g/dL) Normal (3136 g/dL) Normal (3136 g/dL)

Examples Iron deficiency anemia,

thalassemia

During blood loss, anemia of

chronic diseases

Deficiency of vitamin

B12and folic acid

Morphology of RBC

Q. Classify anemia.

Spurious anemia is theterm used when RBCconcentration decreasesdue to hemodilution asseen in third semester ofpregnancy.

CHAPTER

1Anemias of ImpairedRed Cell Production

ANEMIA

Definition Decreasebelow normal of the hemoglobin concentration (Hb)/RBC count/hematocrit

(packed cell volume).

Reduction of the total circulating red cell massbelow normal limits.

Decrease in the oxygen-carrying capacityof the blood, which leads to tissue hypoxia.

Anemia may be absolute (decreased RBC mass), or relative (associated with a higher plasmavolume). Anemia is conventionally used for absolute anemia.

Classification of Anemia1. Morphological classification (able 1.1): it is based on:

a. Red cell size(normocytic, microcytic, or macrocytic), and

b. Degree of hemoglobinization(normochromic or hypochromic).

Q. Define anemia.

WHO criteria for anemia:adult males Hb


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SECTION 1 Disorders of Red Cells

2. Etiological classification:Te etiological classification of anemia is listed in able 1.2.

TABLE 1.2: Etiological classification of anemia

1. IMPAIRED RED CELL PRODUCTION

Disturbed Proliferation and Maturation of Erythroblasts

Defective DNA synthesis Megaloblastic anemias due to deficiency or impaired utilization of vitamin B12and folic acid

Anemia of renal failure due to deficiency of erythropoietin

Anemia of chronic disease due to iron sequestration and relative erythropoietin deficiency

Anemias of endocrine disorders

Defective hemoglobin synthesis

Defective heme synthesis: iron deficiency, sideroblastic anemia

Defective globin synthesis: thalassemias

Marrow Replacement

Primary hematopoietic neoplasms: acute leukemia, myelodysplastic syndromes

Marrow Infiltration (myelophthisic anemia)

Metastatic neoplasms

Disturbed Proliferation and Differentiation of Stem Cells

Aplastic anemia, pure red cell aplasia

2. INCREASED RED CELL DESTRUCTION (HEMOLYTIC ANEMIAS)

Intrinsic (Intracorpuscular) Abnormalities

Hereditary

Membrane abnormalities: spherocytosis, elliptocytosis

Enzyme deficiencies: glucose-6-phosphate dehydrogenase, pyruvate kinase

Disorders of hemoglobin synthesis

Deficient globin synthesis: thalassemia syndromes

Structurally abnormal globin synthesis (hemoglobinopathies): sickle cell anemia

Acquired

Membrane defects: paroxysmal nocturnal hemoglobinuria

Extrinsic (Extracorpuscular) Abnormalities

Antibody-mediated

Isohemagglutinins: transfusion reactions, Rh disease of the newborn

Autoantibodies: idiopathic (primary), drug-associated, systemic lupus erythematosus Mechanical trauma to RBCs:

Microangiopathic hemolytic anemia: disseminated intravascular coagulation

Infections: malaria

3. BLOOD LOSS

Acute: trauma

Chronic: lesions of gastrointestinal tract (e.g. carcinoma colon), gynecological disturbances

Anemia is the expressionof underlying disease andfrom treatment point, thecause of anemia must beidentified.

Causes of anemia:1. Decreased RBC

production2. Increased RBC

destruction (hemolysis)or

3. Blood loss.

Iron deficiency anemia isthe most common anemia.

RED CELL INDICESRed cell indices are useful in morphological characterization and diagnosis of anemias. Tey

are either directly measured or automatically calculated by specialized instruments. Red cellindices include:

1. Mean Corpuscular Volume (MCV) MCV is indicative of average volume of the RBC and is expressed in femtoliters (fL).

It is used for classification and differential diagnosis of anemias.

Normal range: 8298 fL.

MCV = PCV 1000

= 0.45 1000/5 = 90 fLRBC count in millions

Q. Write short notes on red cell

indices.

Red cell indices: MCV, MCH,MCHC and RDW.

Microcytic anemiahave MCV < 80 fL andmacrocytic anemia haveMCV> 100 fL.


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2. Mean Corpuscular Hemoglobin (MCH) MCH indicates the amount of Hb (weight) per RBC and is expressed as picograms (1 pg

= 10-12g).

It is of limited value in differential diagnosis of anemias.

Normal range: 2732 pg

MCH = Hb (in g/L)/RBC (in millions/L) = 15

10/5 = 30 pg

3. Mean Corpuscular Hemoglobin Concentration (MCHC) MCHC denotes the average concentration of hemoglobin in the RBC taking volume into

account. It is expressed as g/dL (earlier it was expressed as %).

It is a better indicator of hypochromasia than MCH.

Normal range: 3135 g/dL.

MCHC = Hb (in g/dL)/PCV = 15/0.45 = 33 g/dL

4. Red Cell Distribution Width (RDW) RDW is a quantitative measure of anisocytosis.

Normal RDW is 11.5% to 14.5%.

A normal RDW indicates that RBCs are relatively uniform in size. A raised RDW indicatesthat red cells are heterogeneous in size and/or shape. In early iron deficiency anemia,RDW increases along with low MCV while in thalassemia trait, RDW is normal with lowMCV.

RDW = (Standard deviation mean cell volume) 100

IRON DEFICIENCY ANEMIAIron deficiency anemia (IDA) is the most common nutritional disorder.

Etiology (Table 1.3)IDA is due to deficiency of iron causing defective heme synthesis.

MCH < 26 pg is seen inmicrocytic anemia andMCH > 33 pg is seen inmacrocytic anemia.

MCHC36 g/dL is anindication of hyperchromicRBCs.

RDW is useful fordifferentiating anemiadue to iron deficiency and

thalassemia.

Q. Discuss the etiopathogenesis

of iron deficiency anemia.

TABLE 1.3: Causes of iron deficiency anemia

1. Dietary deficiency/lack

Milk-fed infants

Elderly with improper diet and poor dentition

Low socioeconomical sections

Vegetarians (contains poorly absorbable inorganic iron)

2. Impaired absorption

Total/partial gastrectomy

Intestinal absorption is impaired in sprue, other causes of intestinal steatorrhea and chronic diarrhea Specific items in the diet, like phytates of cereals, tannates, carbonates, oxalates, phosphates and drugs

can impair iron absorption

3. Increased demand/requirement

Growing infants, children and adolescents

Pregnancy and lactation

4. Chronic blood loss: due to bleeding from the

Gastrointestinal tract (e.g. peptic ulcers, gastric carcinoma, colonic carcinoma, hemorrhoids, hookworm

infestation or nonsteroidal anti-inflammatory drugs)

Urinary tract (e.g. renal or bladder tumors)

Genital tract (e.g. menorrhagia, uterine cancer)

Respiratory tract (e.g. hemoptysis)

Dietary deficiency is thecommonest cause of IDA.

Iron is absorbed in theduodenum.

In adult men andpostmenopausal women,deficiency may be due tochronic gastrointestinalblood loss.

Infants who consume largeamounts of cow's milk aresusceptible to develop IDA.


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Pathogenesis of Iron Deficiency Anemia

It is due to decreased synthesis of heme and can be divided into 3 stages. Stage 1 (Iron depletion): iron adequate to maintain normal hemoglobin level and only

serum ferritin decreased.

Stage 2 (Iron deficient erythropoiesis): lowering of serum iron and transferrin saturation

levelswithout anemia (Hb, MCV and MCH within normal range). Bone marrow shows irondeficient erythropoiesis.

Stage 3 (Iron deficiency anemia): low serum iron, serum ferritin and transferrin saturation.Impaired hemoglobin production. Morphologically,first reduction in the size (microcytic)and later increase in the central pallor (hypochromia) of RBCs.

Laboratory Findings

Peripheral Blood Hemoglobin and hematocrit (PCV):decreased

Red cell indices:

MCV:


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Bone Marrow Cellularity:moderately hypercellular.

M:E ratio: varies from 2:1 to 1:2 (normal 2:1 to 4:1).

Erythropoiesis: hyperplasia and micronormoblastic maturation.

Myelopoiesis:normal.

Megakaryopoiesis:normal. Absence of bone marrow iron:Gold standard test, demonstrated by negative Prussian blue reaction.

Bone marrow showsmicronormoblasticeythroid hyperplasia.Marrow iron is absent.Prussian blue reactionnegative.

Serum Iron Profile (Table 1.4)

TABLE 1.4: Serum iron profile in IDA

Normal range Value in IDA Observation

Serum ferritin 15300 g/L


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Causes of Microcytic Hypochromic Anemia Iron deficiency anemia

Talassemia major

Anemia of chronic disorders

Others: alcohol, lead poisoning and drugs

Sideroblastic anemia (rare cause).

MEGALOBLASTIC ANEMIAAnemias characterized by defective/impaired DNA synthesis and distinct megaloblasts inthe bone marrow. Megaloblastic anemias are common among anemias due to impaired redcell production.

Etiology of Megaloblastic Anemia (Table 1.5)

TABLE 1.5: Causes of megaloblastic anemia

VITAMIN B12DEFICIENCY

1. Decreased Intake:inadequate diet, pure vegetarians (vegans)

2. Impaired Absorption

Gastric: deficiency of gastric acid or pepsin or intrinsic factor

Pernicious anemia

Post-gastrectomy

Intestinal

Loss of absorptive surface

Malabsorption syndromes

Diffuse intestinal disease, e.g. lymphoma, systemic sclerosis

Ileal resection, Crohn disease

Bacterial or parasitic competition for vitamin B12

Bacterial overgrowth in blind loops and diverticula of bowel Fish tapeworm infestation

3. Increased Demand:pregnancy, hyperthyroidism, disseminated cancer

FOLIC ACID DEFICIENCY

1. Decreased Intake:inadequate dietalcoholism, malnutrition

2. Impaired Absorption

Malabsorption states: nontropical and tropical sprue

Diffuse infiltrative diseases of the small intestine (e.g. lymphoma)

Drugs: anticonvulsant phenytoin and oral contraceptives

3. Increased Loss:hemodialysis

4. Increased Demand:pregnancy, infancy, disseminated cancer, markedly increased hematopoiesis

5. Impaired Utilization:folic acid antagonists, such as methotrexate

Pathogenesis of Megaloblastic Change

1. Impaired DNA synthesis: megaloblastic anemia is commonly due to deficiency ofvitamin B12 (cyanocobalamin) or folic acid. Both are required for the synthesis of DNA.

a. Delayed maturation of nucleus. Te nuclear maturation lags behind the cytoplas-mic maturation and results in abnormally large nucleated erythroid precursorsnamed as megaloblasts.

b. Cytoplasm matures normally. RBCs are larger than normal macrocytes.

c. Affects all rapidly dividing cells of the body (including skin, GI tract, and bone marrow).

2. Ineffective erythropoiesis: megaloblast precursors undergo intramedullary destruction.

Q. Enumerate the causes of

microcytic hypochromic anemia.

Q. Discuss the causes and

pathogenesis of megaloblastic

anemia.

Vitamin B12is present inanimal products.

Deficiency of vitamin B12and folic acid are the maincauses of megaloblasticanemia.

Folic acid is absorbed inthe jejunum.

Deficiency of vitamin B12and folic acid delayednuclear maturationmegaloblast macrocyte.

Ineffective erythropoiesisand hemolysis areresponsible for anemia.


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Laboratory Findings of Megaloblastic Anemia

Blood findings in vitamin B12and/or folic acid deficiency are similar.

Peripheral Blood Hemoglobin and hematocrit (PCV): reduced

Red cell indices

MCV:above 100 fL (normal 8298 fL)

MCH (normal 2732 pg)

Normal MCHC(3136 g/dL)

Peripheral smear (Figs 1.3 and 1.4):pancytopenia(decreased RBC, WBCs and platelets).

RBCs:

Macrocytic and oval (egg-shapedmacro-ovalocytes)-diagnostic.

Most macrocytes lack the central pallor(Figs 1.3 and 1.4).

Markedvariation in the size and shape of red cells (anisopoikilocytosis).

Evidence of dyserythropoiesis: basophilic stippling, Cabot ring and Howell Jolly bodies.

WBCs:

DecreasedWBC count (leukopenia).

Hypersegmented neutrophils(more than five nuclear lobes): first and specific morphologicalsign of megaloblastic anemia. These neutrophils are also larger than normal (macropolys).

Platelets:decreased.

Megaloblastic anemia Pancytopenia Macro-ovalocytes Hypersegmented

neutrophils Macropolys.

Reticulocyte count:normal or low.

Dimorphic Anemia Combined vitamin B12/folic acid and iron deficiency.

Peripheral smear shows two populations of RBCs namely: macro-ovalocytes and microcytic

hypochromic (Fig. 1.5).

Q. Write short note on

the laboratory findings in

megaloblastic anemia.

In megaloblastic anemiadue to vitamin B12deficiency, reticulocytecount may be normal orlow and high reticulocytecount is seen on 7th dayfollowing vitamin B12therapy.

Fig. 1.3: Peripheral blood smear showing macro-ovalocytes (arrows) andhypersegmented neutrophil (inset )

Fig. 1.4: Diagrammatic peripheral blood smear showingmacro-ovalocytes (thick arrows) and hypersegmented neu-trophil (thin arrow )


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Bone Marrow Cellularity:moderately tomarkedly hypercellular.

M: E ratio:due to marked erythroid hyperplasia, M: E ratio is reversed ranging from 1:1 to 1:6 (normal

2:1 to 4:1).

Erythropoiesis:megaloblastic type (Figs 1.6 and 1.7)

Megaloblasts: large, abnormal counterparts of normal normoblasts. Megaloblast shows asyn-

chrony of nuclear and cytoplasmic maturation.The cytoplasm shows normal hemoglobinization.

Ineffective erythropoiesis: developing megaloblasts die in marrow (intramedullary hemolysis).

Myelopoiesis:

Myeloid cells adequate in number. Granulocytic precursors display nuclear-cytoplasmic asynchrony in the form of giant metamyelo-

cytes and band forms.

Megakaryopoiesis:normal or increased in number.

Bone marrow iron:moderately increased.

Megaloblastic anemia-bone marrow: Megaloblasts Giant metamyelocytes.

Megaloblast are large,abnormal precursors ofRBCs seen in the bonemarrow of patients withmegaloblastic anemia.

Te differences between normoblasts and megaloblasts are shown in able 1.6

TABLE 1.6: Differences between normoblast and megaloblast

Characteristics Normoblast Megaloblast

Cell size Normal Larger than corresponding normoblast

Nuclear chromatin Normal Open sieve-like

Nuclear maturation Normal Lags behind cytoplasmic maturation

Mitosis Normal Increased and abnormal

Maturation in bone

marrow

Normal (Late >

intermediate > early

normoblast)

Increased proportion of more primitive erythroid cells

(Late < intermediate < early megaloblast)

Evidence of

dyserythropoiesis

Absent Present (irregular nuclei, Howell Jolly bodies)

Myelopoiesis Normal Shows giant metamyelocytes

Found in Normal bone marrow Bone marrow of megaloblastic anemia

Q. List the differences between

normoblast and megaloblast.

Megaloblasts: Nuclear maturation lags

behind cytoplasmicmaturation.

Nuclei have open sieve-like chromatin.

Fig. 1.5: Diagrammatic peripheral blood smear of dimorphic

anemia showing macro-ovalocytes and microcytes

A mixture of microcytichypochromic andmacrocytic RBCs is termedas dimorphic picture andoccurs in mixed deficiencyof iron and folic acid orvitamin B12.


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Biochemical Tests for Megaloblastic AnemiaCommon for both vitamin B12and folic acid deficiencyDeoxyuridine suppression test:it is a sensitive measure of deficiency of 5, 10-methylene HF,

which occurs in both folic acid and vitamin B12deficiency.

Serum homocysteine

Serum bilirubin: mild increase causes mild jaundice

Serum iron and ferritin

Plasma lactate dehydrogenase (LDH)

Serum vitamin B12/folate decreased.

Diagnostic tests for vitamin B12deficiency

Serum vitamin B12levels: decreased

Serum methylmalonic acid

Urinary excretion of methylmalonic acid

Schilling testfor vitamin B12absorption (Refer page 12).

Specific tests for folic acid deficiency

Serum folic acid levels: decreased

FIGLU in urine: excessively excreted.

PERNICIOUS ANEMIAPernicious anemia (PA) is an autoimmune disease due to deficiency of intrinsic factorcausingimpaired absorption of vitamin B12and megaloblastic anemia.

Rare in India. A genetic predisposition is suspected.

Age: older agefifth to eighth decadesof life

Sex: females are more involved than males (F: M is 1.5: 1).

Deoxyuridine suppressiontest is abnormal evenbefore the morphologicalchanges.

Schilling test determinesthe cause of vitamin B12deficiency.


and morphology of pernicious

anemia.

Vitamin B12is absorbedin terminal ileum andrequires IF.

Fig. 1.6: Bone marrow aspirate showing megaloblastic precursors(arrows) in varying stages of maturation (inset shows early megalo-blast)

Fig. 1.7: Diagrammatic picture of bone marrow aspirate showingmegaloblastic precursors (thick arrows) in varying stages of maturation


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SECTION 1 Disorders of Red Cells2

Etiopathogenesis An autoimmunedisease due to destruction of gastric mucosa.

Stomach shows damage to parietal cells, dense infiltration by lymphocytes and plasmacells chronic atrophic gastritisfailure of production of intrinsic factor.

Presence of autoantibodies:two major typesof autoantibodies

Anti-intrinsic factor (IF) antibody

ype I (blocking) antibody:blocks the binding of vitamin B12to IF. Present in 5075%of the cases.

ype II (binding) antibody:attaches to the IFvitamin B12complex and prevent itsbinding to receptors in the ileum. Present in about 40% of patients.

Parietal cell (Type III) antibody:neither specific for PA nor other autoimmune disorders.It is found in 90% of patients.

Morphology

Alimentary System Atrophic glossitis: tongue shiny, glazed and beefy.

Stomach:

Diffuse chronic atrophic gastritis and impaired secretion of hydrochloric acid, pepsinand intrinsic factor.

Histologically atrophy of the glands, with loss of both chief cells and parietal cells.

Nuclei of mucosal cells look similar to that of megaloblasts.

Dense infiltration by lymphocytes and plasma cells.

Intestinal metaplasia.

Central Nervous SystemFound in 75% of cases.

Demyelination in the dorsal and lateral tracts: subacute combined degeneration

Peripheral neuropathy.

Laboratory Findings (Fig. 1.8)

Blood, bone marrow and biochemical test findings are similar to those described earlier formegaloblastic anemias (Refer page 9 to 11).

Specific Diagnostic Tests for Pernicious Anemia Schilling test for vitamin B12absorption: abnormal

Radioactive vitamin B12is used to assess the status of intrinsic factor (IF) and vitamin B12.

Helps in distinguishing megaloblastic anemia due to IF deficiency (pernicious anemia)from other causes of vitamin B12deficiency.

Serum antibodies to intrinsic factor are highly specific for pernicious anemia

Achlorhydriawith histamine/pentagastrin stimulation.

Severe deficiency of intrinsic factor.

PA: autoimmune disease Atrophic gastritis IF deficiency Autoantibodies.

Atrophic gastritis maypredispose to carcinomastomach.

Q. Write short note on laboratory

findings in pernicious anemia.

Schilling test: diagnosticof PA but now veryinfrequently performed.


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Clinical Features of Megaloblastic Anemia

Te clinical features of vitamin B12deficiency anemia and pernicious anemia are:

Onset: insidiousand progresses slowly. Classic triad of presentation:weakness, sore throat and paresthesias.

ongue: painful redbeefy tongue.

Neurological manifestations:

Bilateral peripheral neuropathy:glove and sock distribution of numbness or paresthesia

Demyelination of spinal cord: subacute combined demyelination/degenerationof dorsal and lateral tractsataxia, uncoordinated gait, impairment of vibration andposition sense.

Atherosclerosis: serum homocysteine level is raised and is a risk factor for atherosclerosisand thrombosis.

APLASTIC ANEMIAHematopoietic stem cell (HSC) disordercharacterized by: Pancytopenia(anemia, neutropenia and thrombocytopenia)

With markedly hypocellular bone marrow(less than 30% cellularity).

Etiology

Te most common causes associated with aplastic anemia are shown in able 1.7.

Q. Mention the various clinical

features of megaloblastic anemia.

Folate deficiency anemiapresents with features ofmegaloblastic anemia dueto vitamin B12. Unlike withvitamin B12deficiency,neurological symptomsdoes not occur.

Nonmegaloblastic causesof macrocytic anemia:1. Alcohol2. Liver disease

3. Myxedema4. Cytotoxic drugs5. Myeloma6. Aplastic anemia7. Reticulocytosis8. Red cell aplasia.

Q. Write short notes on aplasticanemia.

Fig. 1.8: Clinical features and laboratory findings in pernicious anemia

PA patients sometimeshave a lemon-yellow colorowing to a combinationof pallor and mild

jaundice caused by excessbreakdown of hemoglobin.

Pernicious anemiapresent with features ofmegaloblastic anemia dueto vitamin B12deficiency.In addition, it may showfeatures of atrophicgastritis and achlorhydria.


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Fig. 1.9: Pathogenesis of aplastic anemia

TABLE 1.7: Common causes of aplastic anemia

1. ACQUIRED

Idiopathic

Acquired defects in stem cell

Immune mediated

SecondaryChemical Agents

Cytotoxic drugs: alkylating agents, antimetabolites Benzene

Inorganic arsenicals Chloramphenicol

Idiosyncratic

Chloramphenicol Phenylbutazone

Penicillamine Carbamazepine

Gold salts Organic arsenicals

Methylphenylethyl hydantoin

Physical Agents: whole-body irradiation

Viral Infections: hepatitis virus, Epstein-Barr virus, cytomegalovirus , herpes zoster ( Varicella zoster) , HIV

2. INHERITED: fanconi anemia, telomerase defects

6 I s of the causes ofaplastic anemia:1. Idiopathic2. Ingestion of drugs and

chemicals3. Idiosyncratic4. Irradiation

5. Infections and6. Inherited.

Pathogenesis (Fig. 1.9)Pathogenesis: Direct damage to the

hematopoietic stemcells and progenitorcells.

Immune-mediateddestruction.

Primary stem cellabnormalityinheriteddefect in the stem cells.

Clinical Features Any age of both sexes

Insidious

Progressive weakness, pallor and dyspnea due to anemia

Frequent (mucocutaneous bacterial infections) or fatal infections due to neutropenia


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Bleeding manifestations in the form of petechiae, bruises and ecchymoses due tothrombocytopenia.

Laboratory Findings

Peripheral Blood Hemoglobin

PCV

Reticulocyte count: markedly decreased.

Peripheral smear: pancytopenia, i.e. decreased red cells, neutrophils and platelets.

RBCs:normocytic normochromic anemia

WBCs:total leukocyte count decreased. Neutrophils markedly diminishedand neutropenia is a

reflection of the severity of aplasia. Initial stages, lymphocytes normal in number as the disease

progresses their count decreases.

Platelets:count is decreased.

Bone Marrow Marrow aplasiabest appreciated in a bone marrow (trephine) biopsy

Cellularity:marked hypocellularity.

Hematopoiesis:paucity of all erythroid, myeloid and megakaryocytic precursors.

Other cells:lymphocytes and plasma cells are prominent.

Bone marrow elementsare replaced by fat andaspiration usually yieldsdry tap.

No Splenomegaly

Diagnosis: diagnosis is made withperipheral blood andbone marrow biopsy findings.

Differential Diagnosis Should be distinguished from other causes of pancytopenia (able 1.8)

TABLE 1.8: Causes of pancytopenia

Decreased bone marrow function

Aplastic anemia Idiopathic

Secondar y Inherited

Myelodysplastic syndromes

Bone marrow infiltration with

Leukemia Lymphoma

Myeloma Tumors (carcinoma) Granulomatous diseases (e.g. tuberculosis, sarcoidosis)

Nutritional deficiencies: Megaloblastic anemia (vitamin B12and folic acid deficiency)

Paroxysmal nocturnal hemoglobinuria Myelofibrosis (rare) Hemophagocytic syndrome

Increased peripheral destruction

Hypersplenism

Prognosis: unpredictable.

Reticulocyte countis markedly low inaplastic anemia and ischaracteristic feature.

Absence of splenomegalyand in its presence thediagnosis of aplasticanemia should not be

made.


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6

HEMOLYTIC ANEMIA

Definition

Hemolytic anemias are due to increase in the rate of red cell destruction(hemolysis).

Classification of Hemolytic Anemias (Table 2.1)

Depending on: Location of hemolysis:intravascular and extravascular

Source of defect causing hemolysis: intracorpuscular defect and extracorpusculardefect

Mode of onset: hereditary and acquired disorders.

TABLE 2.1: Classification and causes of hemolytic anemia

Intrinsic (intracorpuscular) abnormalities Extrinsic (extracorpuscular) abnormalities

Hereditary

RBC membrane abnormalities

Membrane skeletal abnormalities:

spherocytosis, elliptocytosis

Membrane lipids: abetalipoproteinemia

Enzyme deficiencies

Enzymes of hexose monophosphate shunt:glucose-6-phosphate dehydrogenase

Glycolytic enzymes: pyruvate kinase

Disorders of hemoglobin synthesis

Deficient globin synthesis: thalassemia

syndromes

Structurally abnormal globin synthesis

(hemoglobinopathies): sickle cell anemia

Acquired

Membrane defects: paroxysmal nocturnal

hemoglobinuria

Antibody-mediated

Isohemagglutinis: Rh disease of the new-born,

transfusion reactions

Autoantibodies: idiopathic (primary), drug-

associated, systemic lupus erythematosus

Mechanical trauma to RBCs

Microangiopathic hemolytic anemia: disseminatedintravascular coagulation

Defective cardiac valves

Infections:malaria

Drugs, chemicals and toxins

Drugs: oxidant drugs, primaquine, dapsone, etc.

Chemicals: naphthalene, nitrites, nitrates, etc.

Toxins: snake venom, lead poisoning, clostridial

sepsis

Q. Define and classify hemolytic

anemia.

Normal lifespan of redcell is about 120 days.In hemolytic anemiasRBC survival time isconsiderably shortened.

Breakdown of normal RBCsoccurs in the macrophagesof the bone marrow, liverand spleen.

Decreased red cell survivaldoes not always causeanemia as there is acompensatory increase inred cell production by thebone marrow.

Hemolytic Anemias Due to

Red Cell Membrane and

Enzyme DefectsCHAPTER

2


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Hemolytic Anemias Due to Red Cell Membrane and Enzyme Defects CHAPTER 2

Location of Hemolysis

It may be intravascular and/or extravascular. Te differences between these two types arelisted in able 2.2.

TABLE 2.2: Differences between extravascular and intravascular hemolysis

Characteristics Extravascular hemolysis Intravascular hemolysis

Site of hemolysis RE system (spleen, bone marrow) Within circulation

Splenomegaly Usual Uncommon

Laboratory findings

Serum bilirubin-unconjugated

Serum haptoglobin

Hemoglobinemia

Moderately raised

Normal

Not seen

Mildly raised

Decreased

Positive

Urine

Hemoglobinuria

Hemosiderinuria

Absent

Absent

Present

Present

Examples Thalassemia, sickle cell anemia G6PD deficiency, PNH

HEREDITARY SPHEROCYTOSISHereditary spherocytosis (HS) is a rare inherited hemolytic anemiaresulting from the defectin the red cell membrane.

Normal structure of RBC membrane is depicted in Figure 2.1.

Etiopathogenesis Autosomal dominantdisorder

RBC membrane protein defect caused by various mutations. Most common mutations

involve ankyrin, band 3, spectrin, or band protein 4.2.

Mechanism of Hemolysis in HS (Fig. 2.2) Young HS RBCs are normal in shape. But as they age, they undergo loss of membrane

fragments in the circulation. Tese small RBCs assume a spherical shape(spherocytes).

Spherocytes are rigid, inflexible and less deformable. Tey get trapped in the spleenleading to premature destructionof spherocytes.

In most hemolytic anemiasred cell destruction isextravascular.

Q. List the differences between

extravascular hemolysis and

intravascular hemolysis.

Q. Describe the etiopathogenesis

of hereditary spherocytosis.

HS, is due to defect in theRBC membrane protein.

The common mutationsinvolve ankyrin, band 3,

spectrin or band protein4.2.

HS: intrinsic defect of RBCmembrane-extravascularhemolysis.

Fig. 2.1: Structure of the red cell membrane


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Laboratory Findings

Peripheral Blood Hemoglobin:decreasedand level depends on degree of hemolysis.

Red cell indices:

MCV: reduced (normal 8298 fL)

MCHC: raised and > 35 g/dL (normal 3136 g/dL).

Peripheral smear: very importantfor diagnosis (Figs 2.3 and 2.4).

RBCs:

Spherocytes are most distinctive but not pathognomonic. Spherocytes are small, dark-staining(hyperchromic) RBCs without any central pallor.

Polychromatophilia due to reticulocytosis.

WBCs:total leukocyte count (TLC) increased.

Platelets:normal.

Spherocytes may alsobe seen in autoimmunehemolytic anemia andburns.

Reticulocyte count: increased (Fig. 2.5).

Bone Marrow Cellularity:markedly hypercellular

Erythropoiesis:erythroid hyperplasia

Myelopoiesis:normal

Megakaryopoiesis:normal.

Bone marrow showserythroid hyperplasia.

Q. Write short notes on laboratory

findings in HS.

In hereditary spherocytosisMCHC is > 35 g/dL.

Spherocytes andreticulocytosis areobserved in the peripheralblood.

Fig. 2.3: Peripheral blood smear with numerous spherocytes (arrows) Fig. 2.4: Diagrammatic peripheral blood smearwith numerous spherocytes (arrows)

Fig. 2.2: Mechanism of hemolysis in hereditary spherocytosis


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Biochemical Findings Serum bilirubin:mildlyraised.

Urine urobilinogen: increased.

Serum haptoglobin:decreased.

Osmotic Fragility Test

Osmotic fragility is increased and there is shift of the curve to the right (Fig. 2.6).

Clinical Features Age: anytime from the neonatalperiod to adulthood.

Family history: most (75%) are inherited as autosomal dominant trait.

Anemia: mild to moderate.

HS: osmotic fragility isincreased with a shift ofcurve to the right.

Clinical features ofintermittent jaundice,splenomegaly andspherocytes in theperipheral smear is highlysuggestive of HS.

Fig. 2.5: Smear shows reticulocyte with blue filamentous/granularmaterial (new methylene blue stain) (arrows)

Fig. 2.6: Osmotic fragility test. Normal curve (blue) and increasedosmotic fragility in hereditary spherocytosis


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Jaundice: intermittent attacks, precipitated by pregnancy, fatigue, or infection.

Splenomegaly: moderate(500 to 1000 g).

Gallstones: pigmentgallstones.

Aplastic crises: may be triggered by an acute parvovirusinfection.

GLUCOSE-6-PHOSPHATEDEHYDROGENASE DEFICIENCY

Hemolytic disease due to red cell enzyme defects.

In G6PD deficiency, RBCs are susceptible to oxidative injuryby free radicals.

It is an X-linked recessive disorderand its full expression is seen only in males.

Tere are different subtypes.

Role of G6PD (Fig. 2.7)

Reduced glutathione (GSH) in the normal RBCs protects them against oxidant injury by

breakdown of compounds such as H2O2 to H2O. Te housekeeping enzyme, G6PD isrequired for normal GSH.

Sequence of Events in G6PD Deficiency

In G6PD deficiency, oxidants can cause both intravascular and extravascular hemolysis. In G6PD deficiency, there is decreased synthesis of reduced glutathione.

RBCswhen exposed tooxidant stress (during infections, exposure to drugs or chemical,fava beans) accumulate H2O2. Itdamages RBC membrane causing hemolysis.

Hemolyzed red cellsliberate hemoglobin.

Te hemoglobin is oxidized by oxidants leading to formation of methemoglobin, which

forms Heinz bodies(Fig. 2.8) in the cytoplasm of RBCs.

G6PD deficiency is anintrinsic defect andhemolysis is primarily

intravascular.

In G6PD, RBCs exposedto oxidant stress, thehemoglobin is oxidizedto methemoglobin whichforms Heinz bodies in thecytoplasm of RBCs.

Fig. 2.7: Role of G6PD against injury by oxidants


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Heinz bodies removed from RBC membrane by macrophages in the spleen and producebite cells. Tese bite cells areremoved via erythrophagocytosis in the spleen.

Clinical Presentation

G6PD deficiency manifests in several distinct clinical patterns. Usually present as acute self-limited acute intravascular hemolytic anemiafollowing exposure to oxidative stress.

Laboratory FindingsPeripheral Blood

Hemoglobin: decreased.

Reticulocyte count: increased.

Peripheral smear:

RBCs: moderate anisopoikilocytosis with polychromatophilia, microspherocytes and bite cells

(Fig. 2.8). Heinz bodiesidentified with a supravital stainand are best seen during active hemolysis.

WBCs:mild leukocytosis.

Platelets:normal.

Self-limited hemolysis:primarily the old red cells are hemolyzed, hence hemolysis is self-limited.

Urine

Hemoglobinuriawill be found during hemolysisand may last for about 16 days.

RBC Enzyme Analysis

Tests for G6PD deficiency are positive and should be assessed a few weeks after the acutehemolytic episode.

G6PD deficiency has aprotective effect againstPlasmodium falciparummalaria.

G6PD deficiencyoxidantdamage to RBC Bite cells

Heinz bodies.

G6PD: enzyme analysisconfirmatory test.

Fig. 2.8: Peripheral blood smear in G6PD deficiency with bite cells

(arrows). Inset shows Heinz bodies (supravital stain)


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2

CLASSIFICATION OF HEREDITARYDEFECTS IN HEMOGLOBIN

Hemoglobin defects may be quantitative (reduced production of normal hemoglobin) orqualitative (production of abnormal hemoglobin).

Quantitative defect: genetic mutations in the globin loci (e.g. thalassemia) may quan-titatively reduce the synthesis of a-globin or b-globin chain. It leads to net reduction ofhemoglobin.

Qualitative defect: genetic mutations in the a-globin or b-globin locus may produceabnormal hemoglobin (e.g. sickle cell anemia). Te abnormal hemoglobin may be func-

tionally normal, but its physical or physiologic properties differ from normal hemoglobin.

THALASSEMIA SYNDROME Tese are group of inherited disorders due to abnormality of globin production.

It is characterized by decreased or absence of synthesisof either aor b-globin chain ofadult hemoglobin, HbA (a2b2).

Classification

Tey are mainly classified as:

b-Talassemia syndromes:impaired synthesis of b-chainsof globin. a-Talassemia syndromes:impaired synthesis of a-chainsof globin.

Miscellaneous thalassemia syndromes.

b-THALASSEMIA Autosomal recessivehereditary disorder

Diminished synthesisof b-globin chains and normal synthesis of a-chains.

Q. Classify hereditary disorders of

hemoglobin.

The termhemoglobinopathyis usually used for aqualitative hereditarydisorder of hemoglobin.

Q. Classify thalassemia

syndromes.

In b-Thalassemia, thereis decreased/absence ofsynthesis of b-chains.

In a-Thalassemia, thereis reduced/absence ofsynthesis of a-chains ofglobin.

Thalassemia Syndrome

CHAPTER

3


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Thalassemia Syndrome CHAPTER 3

Molecular Pathology b-globin chains are encoded by a single gene.

Te molecular errors over 200 genetic defects leading to b-thalassemia have been identified.

Different types of mutations in b-globin gene can occur but mainly point mutationsrather than gene deletions (unlike in a-thalassemia). Te mutations result in defects in

transcription, RNA splicing and modification, translation via frame shifts and nonsensecodons. Mutations leading to aberrant RNA splicing are the most common cause.

Clinical and Genetic Classification (Table 3.1)

TABLE 3.1: Clinical and genetic classification of b-thalassemias

Clinical syndromes Genotype Clinical features

b-thalassemia major Homozygous (b0/b0,b+/b+)

or double heterozygous ( b0/b+)

Severe form, severe anemia

and transfusion dependent

High level of HbF in the blood

b-thalassemia intermedia Variable (b0/b+, b+/b+, b0/b, b+/b) Moderately severe and nottransfusion dependent

b-thalassemia minor/b-thalassemia trait Heterozygous (b0/b, b+/b) Mild anemia and asymptomatic

b-THALASSEMIA MAJOR It is a hereditary hemolytic anemia due to absence of synthesis of b-globin chain of

hemoglobin. Te synthesis of a-globinchainis not affected.

Homozygous form of b0/b0or b+/b+or double heterozygous b0/b+ (able 3.1)

Most common in Mediterranean countries, parts of Africa and South East Asia.

Hemolytic anemiais of severe degree.

Pathophysiology of b-thalassemia Major (Fig. 3.1)

Consequence of Defective or Absent b-chains Severe hemolytic anemiadue to:

1. Absence of b-globin chain:results in absence of synthesis of HbA(a2b2). Tis producesRBCs that are poorly hemoglobinized (hypochromic) and small in size (microcytic).

2. Ineffective erythropoiesis: unpaired and excess a-chains aggregate into insolubleprecipitates, which bind to and damage the membrane of erythroid precursors. Teseerythroid precursors fail to mature and undergo apoptosisin the marrow.

3. Extravascular hemolysis:RBCs with a-chain inclusions are removed by macrophages of

spleen (extravascular hemolysis). Synthesis of fetal hemoglobin (HbF):the -globin chain synthesis continues even 6 months

after birth and combines with a-globin leading to increased levels of HbF(a22). Te levelof HbF varies from 30% to 90%.

Consequences of Ineffective Erythropoiesis Changes in bone marrow: marked erythroid hyperplasia.

Changes in bone:

Skull X-ray: hair on end (crew-cut) appearance (Fig. 3.2)

ypical facies: thalassemic facies (Fig. 3.3)prominent forehead, cheekbones andupper jaw.

Point mutations leadingto aberrant RNA splicing isthe most common cause ofb-thalassemia.

b0= Total absence ofb-globin synthesis;

b+= Markedly reducedor diminished b-globinsynthesis;

b= normal b-globinsynthesis.

b-thalassemia is thecommonest quantitativedisorder of hemoglobin.

b-thalassemia major alsocalled Mediterranean orCooleys anemia.

Q. Describe the pathophysiology/

pathogenesis of b-thalassemia

major.

b-thalassemia major Absence of synthesis of

HbA produces severemicrocytic hypochromicanemia

Increased synthesis ofHbF.

b-thalassemia major Thalassemic facies Crew cut appearance on

skull x-ray Splenomegaly.


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Extramedullary hematopoiesis: in liver and spleen consequent hepatosplenomegaly.

Cachexia: develops in untreated patients.

Iron Overload and its Consequences Causes of iron overload:

1. Increased absorption of dietary iron from duodenum

2. Hemolysis

3. Repeated transfusions (usual mode of treatment).

Consequences: iron overload produces hemosiderosis andsecondary hemochromatosisand damages to parenchyma of organs (e.g. heart, liverand pancreas).

Clinical Features Age:infants develop moderate to severe anemia69 months after birth.

Growth and development: untreated/untransfused children fail to thriveand die within45 years of age.

Bone changes:those who survive longer develop distortion of skull and facial bones. X-rayskullshows hair on end appearance(Fig. 3.2) and face shows a characteristic thalassemicfacies(Fig. 3.3).

Marked splenomegaly: up to 1500 grams due to hyperplasia and extramedullaryhematopoiesis.

Extramedullary hemopoiesis: liver and lymph nodes may show extramedullaryhematopoiesis.

b-thalassemia major Iron overload damgaes

parenchymal organsdue to hemosiderosisand secondaryhemochromatosis.

Failure to thrive, retardedgrowth, monogoloid face,and hepatosplenomegalyare clinical features ofb-thalassemia major.

Fig. 3.1: Pathogenesis of -thalassemia major and its consequence


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Iron overload: multiple blood transfusions may lead to iron overload and result inhemosiderosis and secondary hemochromatosis (heart, liver and pancreas).

Laboratory Findings

Peripheral Blood Hemoglobin (ranges from 3 to 8 g/dL)and hematocrit(ranges from 8 to 23%): markedly

reduced

RBC count increased/normal (in contrast to iron deficiency anemia).

Reticulocyte count increasedand in the range of 5 to 15%.

Red cell indices: MCV decreased andin the range of 4570 fL (normal range 8298 fL).

MCHC decreasedand in the range of 2230 g/dL (normal range 3135 g/dL).

MCH decreased and in the range of2028 pg (normal range 2732 pg).

RDW-within normal limits (in contrast to iron deficiency anemia where it is increased).

Peripheral smear:

RBCs:

Microcytic hypochromicanemia

Moderate to marked anisocytosis and poikilocytosis

Many target cells(Figs 3.4 and 3.5)

Basophilic stippling

Nucleated red cell precursors(normoblasts) in variable numbers (540%). WBCs:leukocytosis with mild left shift.

Platelets:normal.

Q. Write short note on peripheral

smear findings in b-thalassemia

major.

b- thalassemia major: RDWnormal. The peripheral

blood smear showsmicrocytic hypochromicanemia, target cells andanisopoikilocytosis.

Bone Marrow Cellularity:markedly hypercellular.

M: E ratio:reversed to 1:1 to 1:5 depending upon the degree of erythroid hyperplasia.

Erythropoiesis:normoblastic with marked erythroid hyperplasia.



Bone marrow iron:markedly increased due to increased dietary absorption and hemolysis.

Bone marrow in b-thalassemia major showsmarked normoblasticerythroid hyperplasia.Marrow iron is markedlyincreased.

Q. Mention the laboratory

findings in b-thalassemia major.

RDW normalMCV, MCH and MCHCdecreased.

Fig. 3.2: X-ray appearance of skull in b-thalassemia showing hair-on-end appearance (Courtesy:Dr Nuthan Kamath)

Fig. 3.3: Appearance of typical thalassemic facies(Courtesy:Dr Nuthan Kamath)


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Biochemical Findings Bilirubin: increasedmainly of unconjugated type.

Urine urobilinogen: increased

Serum haptoglobin:markedly reduced.

Serum iron status:

Serum iron, serum ferritin and transferrin saturation are markedly increased

otal iron binding capacity (IBC): reduced.

Special Tests Fetal hemoglobin (HbF): increased to 30% to 90% (normal range 0 1%).

Hemoglobin electrophoresis (able 3.2): b+thalassemia (b+/b+or b0/b+genotypes): demonstrates bands of both HbA and HbF.

bo thalassemia (b0/b0genotype): since no b-chains are formed, there is no HbA. Majorhemoglobin is HbF with normal or low HbA2.

High performance chromatography(HPLC):HbF is increased (3090%). HPLC measuresvarious fractions of hemoglobin (Hb) and is used for confirmation of diagnosis.

Prenatal diagnosis by molecular analysis of DNA.

Estimation of globin chains:normally a: bratio is 1:1. Lack of b chain alter this ratio to530:1

Reduced/absence ofsynthesis of b-chains; theexcess a-chains combine

with-chains leading toincreased HbF.

Fig. 3.4: Peripheral blood smear in -thalassemia showing targetcells (arrows)

Fig. 3.5: Diagrammatic appearance of peripheral blood smear in -thalassemiashowing target cells (short arrows) and nucleated red cells(long arrows)

TABLE 3.2: Hemoglobin F and A2percentage in thalassemia syndromes

Type HbF HbA2

b-Thalassemia major (homozygous) 3090% < 3.5%

b-Thalassemia intermedia (double heterozygous) 1030% < 3.5%

b-Thalassemia minor/trait (heterozygous) 05% 3.68%

Note:normal adult cell

contains 96% HbA (a2b2),3% HbA22(a2d2) and 1%HbF(a22).


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b-THALASSEMIA MINOR/TRAIT More common than b-thalassemia major.

Most patients are heterozygous for thalassemic gene.

Usually asymptomaticand anemia is mild.

Laboratory Findings in b-Thalassemia Minor Peripheral blood:microcytosis, hypochromia,basophilic stippling and target cells.

Bone marrow: mild erythroid hyperplasia.

Hemoglobin electrophoresis: increase in HbA2 (a2d2) to 4 to 8% of the total hemoglobin(normal 2.5 0.3%). HbF levels may be normal or slightly increased.

NESROF test(Naked eye single tube red cell osmotic fragility test): positive. In this test, 0.02 mL of patients blood is added to 5 mL of 0.35% saline in a test tube.

After half an hour white paper with a dark black line is held behind the tube.

Te microcytic hypochromic RBCs of thalassemia minor are resistant to lysis thannormocytic normochromic RBCs.

Hence, the black line on the paper is not clearly visible through the test tube comparedto normal cells.

Estimation of HbA2:HPLC is used for accurate estimation. HbA2estimation is diagnosticand level ranges from 4% to 8%.

NESTROF test positivebecause the microcytichypochromic RBCs ofb-thalassemia minor areresistant to lysis thannormocytic normochromicRBCs.

TABLE 3.3: Differences between iron deficiency anemia and b-thalassemia major

Character Iron deficiency anemia b-thalassemia major

Etiology Deficiency of iron Reduced synthesis of bchain

Laboratory findings

RBC count Decreased (< 5 million/cu mm) Increased (> 5 million/cu mm)

Peripheral smear

Type of RBCs

Anisopoikilocytosis

Target cells

Microcytic hypochromic

Mild to moderate

Absent

Microcytic hypochromic

Severe

Present

Bone marrow iron Absent Markedly increased

Serum iron profile

Serum ferritin

Serum iron

TIBC

Reduced < 15 g/L

Reduced

Increased

Increased (300 1000 g/L)

Increased

Normal

Fetal hemoglobin (HbF) Normal (01%) Markedly increased (3090%)

RDW Increased Normal

Clinical features

Age Any age Presented < 2 years of age

Growth and development Normal Retarded

Hepatosplenomegaly Absent Present

X-ray findings Nil Hair on end appearance

Abbreviations:RDW, red cell distribution width; TIBC, total iron-binding capacity.

b-thalassemia major

should be differentiatedfrom iron deficiencyanemia. Treatment withiron in b-thalassemia majorworsens the iron load andits consequences.

b-thalassemia intermedia:it is a clinical entityintermediate betweenthalassemia trait andthalassemia major.

Differences between Iron Deficiency Anemia andb-Thalassemia Major (Table 3.3)


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TABLE 3.4: Differences between iron deficiency anemia and b-thalassemia minor/trait

Character Iron deficiency anemia b-thalassemia minor

Etiology Deficiency of iron Reduced synthesis of bchain

Laboratory findings

Peripheral smear- RBCs Microcytic hypochromic Microcytic hypochromic

Serum iron profile

Serum ferritin

Serum iron

TIBC

Reduced < 15 g/L

Reduced

Increased

Normal /slightly incresaed

Normal

Normal

HbA2level Normal or decreased (2.5 + 0.3 %) Increased (48 %)

RBC count < 5 million/cu mm >5 million/cumm

RDW Increased Normal

a-THALASSEMIA Inherited disorders characterized by reduced or absent synthesis of a-globin chains.

Autosomal recessive disorder.

Molecular Pathology

In contrast to a single gene coding b-globin chain, each a-globin chain are encoded by twogenes. Deletion of a-gene is the most common causeof reduced a-chain synthesis.

Clinical Syndromes

Four genes control a-chain synthesis. Severity of a-thalassemia varies greatly depending onthe number of a-globin genes deleted (able 3.5). Each of the four a-globin genes normallycontributes 25% of the total a-globin chains.

TABLE 3.5: Clinical syndromes associated with a-thalassemia disorders

Clinical syndrome No. of

a-globin

deleted

Clinicopathological features

Silent carrier state 1 Asymptomatic

a-Thalassemia trait 2 Usually asymptomatic. Normal hemoglobin level or minimal anemia

Hemoglobin H disease 3 Moderate microcytic hypochromic anemia

Hydrops fetalis (Hb Barts) 4 Severe form, fatal and usually results in intrauterine death

b-thalassemia trait shouldbe differentiated from irondeficiency (Table 3.4).

a-Thalassemia:anemia due to Lack of adequate

hemoglobin Effect of excess

unpaired non-a-chains(b,, d).

a-thalassemia is one ofthe cause of non-immunehydrops fetalis.

Immune hydrops fetalisis a hemolytic diseasecaused by blood groupincompatibility betweenmother and fetus.


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SICKLE CELL DISEASE

Definition

Sickle cell disease (SCD) is a group of hereditary disordersof hemoglobin characterized byproduction of defective hemoglobincalled sickle hemoglobin (HbS).On low oxygen tensionor deoxygenation, HbS imparts sickle shape to RBCs. HbS is produced due to qualitativedefectin hemoglobin production caused by mutation in-globin gene.

Classification of Sickle Cell Disease (Table 4.1)

TABLE 4.1: Classification of sickle cell disease

Sickle cell anemia (SS) Sickle cell trait (AS)

Homozygous stateboth the -globin chains are

abnormal/defective

Heterozygous stateone gene is defective

(for HbS) and while the other gene is

normal (for HbA)

Other sickling syndromes (Compound heterozygous)

If both the -globin chains have different abnormalities, (e.g. Hb SC, Hb S--thalassemia)termed as

compound heterozygous

SICKLE CELL ANEMIACharacteristic Features

Autosomal recessive disorder manifests early in life.

Homozygous state(SS) caused by a mutation in the -globin gene.

HbSconstitutes more than 70% of hemoglobinin their RBCs with no HbA.

Etiopathogenesis Production ofabnormal hemoglobincalled sickle hemoglobin (HbS).

Sickle cell diseases arehemoglobinopathiescharacterized byqualitative defect inhemoglobin synthesis.

Sickle cell anemia is a

homozygous state inwhich both -globinchains are abnormal.

Sickle cell trait: one

-globin chain is abnormaland other -globin chain isnormal.

Sickle cell anemia:autosomal recessivedisorder with extravascularhemolysis.

HbS provides protectionagainst falciparum malaria.


of sickle cell anemia.

CHAPTER

4Sickle Cell Disease


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Missense point mutation: in HbS, there is substitution of glutamic acid by valine in the6th position the -globinchain of hemoglobin (Fig. 4.1). It alters the solubility or stability

of the hemoglobin and produces hemolytic anemia. HbS is responsible for the characteristics of the disease.

Molecular Basis of Sickling (Fig. 4.2) During low O2 tension or deoxygenation, HbS molecules undergo aggregation and

polymerization.

Replacement of theglutamic acid residue by

valine in 6th position of-globin chain.

Fig. 4.1: Replacement of glutamic acid with valine in the sixth position of -globin

Fig. 4.2: Pathogenesis of sickle cell anemia

RBCs in sickle cell anemiahave shorter lifespan andcauses hemolytic anemia.

During low oxygen tensionor deoxygenation RBCsassume sickle shape andpredisposes to vesselocclusion.


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Sickle Cell Disease CHAPTER 4

If deoxygenation continues, the aggregated HbS molecules form long needle-like fibers(or pseudocrystalline structures known as tactoids) within RBCs.

Te tactoids growin length beyond the diameter of RBCs and distort RBC shape.

RBCbecome elongated and assumes a shapelike sickle (or crescent moon or holly-leaf orboat) and predisposes to stasis and vascular occlusion.

When the oxygen tension returns to normal, the sickled red cell returns to normalshape.

Recurrent sicklingcauses red cell membrane damage and these RBCs become irreversiblysickled cells (ISC).

Factors Affecting Sickling (Table 4.2)

TABLE 4.2: Factors affecting sickling

Factors Favors sickling Hinders sickling

Type of other associated

hemoglobins - HbA

- HbF

HbC -

Transit time in microvasculature Slowing of bloodstream -

MCHC Increased MCHC Decreased MCHC

Intracellular pH Decreased pH -

Other factors Temperature above 37C -

Infections -

Abbreviation:MCHC, mean corpuscular hemoglobin concentration.

Mechanism of Red Cell Damage HbS polymerization:when HbS polymerizes, it grows beyond the RBC membraneandproject through it.

Dehydration: repeated episodes of sickling leads to increased dehydration of RBCs. TeseRBCs become more rigid and nondefromable(irreversible sickled cells).

Percentage of ISC: degree of the hemolysis correlateswiththe percentage of irreversiblysickled cells.

Impaired cation homeostasis: structural changes in the RBC membrane causes the influxof Ca+ions, which activate an ion channel resulting in the efflux of K+and H2O.

Pathogenesis of the Microvascular Occlusions

Most serious clinical features are due to occlusion of microvasculature. Deformability: sickle cells are rigid and tend to aggregate. Te aggregated sickle cells block

the small blood vessels.

Factors that slow the blood flow: RBC cytoskeletal damageslow the movement of RBCsthrough microvascular beds.

Higher expression of adhesion molecules:sickle cells express higher levels of adhesionmolecules and thus become abnormally sticky to the endothilium.

Inactivation of nitric oxide: lysed sickle cellsliberate free hemoglobin, which binds andinactivates nitric oxide (NO). Tis narrows the vesselsand produces microvascular stasisand sickling.

In sickle cell anemia, HbFhinders sickling.

With repeated sickling the

RBCs become irreversiblysickled cells (ISC) and leadsto RBC membrane damageand hemolysis.

Most serious clinicalfeatures of sickle cellanemia are due tomicrovascular occlusion.


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Clinical Features (Fig. 4.3) Presence of HbF in the first 6 monthsof life has a protectiverole.

Symptoms appear after 6 monthsof age as the HbF disappears.

Infants and children present with acute problems like severe infection, acute chestsyndrome, splenic sequestrationand stroke.

Chronic hypoxia in children is responsible for generalized impairment of growth anddevelopment. Adults manifest with chronic organ damage.

Chronic Hemolytic Anemia Lifelong hemolysis (mainly extravascular) and causes chronic hemolytic anemia, which

is of moderate degree. Tis produces raised unconjugated (indirect) bilirubin, andpredisposes to pigmentbilirubin gallstones(cholelithiasis) and cholecystitis.

Crises

Four typesof crises are encountered. Tese are:

1. Sickling crisis (vaso-occlusive/pain/painful/infarctive crisis)

Most common

Blockage of microcirculationby sickled red cells produces hypoxic injury and infarction.

Bone: manifest as the hand-foot syndrome, dactylitisof the bones of the hands or feet or both.

Lung: acute chest syndrome (dangerous).

Spleen: acute abdominal paindue to infarcts of abdominal viscera caused by occlusionof vessels. Recurrent splenic infarctionresults in autosplenectomy.

The cardinal clinicalfeatures are due tochronic hemolytic anemia,crises (recurrent painful

episodes), infections andchronic organ damage.

Four crises encounteredin sickle cell anemia:

sickling crisis, hemolyticcrisis, aplastic crisis andsequestration crisis.

Recurrent splenicinfarction due tosickling crisis lead toautosplenectomy.

Fig. 4.3: Various effects of vascular occlusion and hemolysis in sickle cell anemia

Most common cause ofdeath in adults is acutechest syndrome.

Infants most commonlypresent with dactylitis.


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2. Hemolytic crisis

Rare type and presents with marked increase in hemolysis.

3. Aplastic crisis Associated withparvovirus B19. Reticulocytopenia.

4. Sequestration crisis

Usually occurs in children. Sudden trapping of bloodin spleen or liver causes rapid enlargement of the organ and

drop in hematocritleading to hypovolemic shock.

Other crises encountered rarely are hypoplastic crisis and megaloblastic crisis (due toinadequate folate).

Increased Susceptibility to Infections Common infections are pneumonia due to Pneumococcus, meningitis due to S.pneumoniaeandosteomyelitisdue toSalmonella. Increased frequency of osteomyelitis isdue to bone infarcts, which act as a nidus for infection.

Septicemiaand meningitisare the most common causes of death in children.

Causes of susceptibility to infections: Hypofunction of spleen:

In children: due to congestion and poor blood flow.In adults: due to multiple infarctsand resultant autosplenectomy.

Defects in the alternative complement pathway.

Impairs opsonization of encapsulated bacteria such as pneumococci andHaemophilus influenzae.

Chronic Organ Damage

Particularly seen in the spleen, bones, kidneys, heart, lungs, brain and skin.

Spleen Children after 6 months of life present with splenomegaly (up to 500 g). After 56 years of age, the spleen gets fibrosed and gradually reduces in the sizedue to

multiple infarcts. Gradual loss of splenic functionsecondary to infarcts results in autosplenectomy.

Bone: osteomyelitis, particularly with Salmonella typhimurium Extremities: skin ulcersover the lower extremities

Laboratory Findings in Sickle Cell Anemia

Peripheral Blood Hemoglobin: decreased.

Hematocrit (PCV): decreased. ESR: reduced. Reticulocyte count: increased and range from 3% to 10%.

Peripheral smear

RBCs:

Normocytic normochromic to mildly hypochromic. Moderate to severe degree of anisopoikilocytosis. Characteristic cell isthe sickle cellappear as long, curved cells with pointed ends (Figs 4.4

and 4.5); may also show target cells (due to red cell dehydration) and ovalocytes.

Polychromatophilia due to reticulocytosis.

WBCs:mildly increased with shift to left.

Platelets:mildly increased.

Peripheral smear showscharacteristic sickle cellsnumber of which varies.

Reticulocytopenia isseen in aplastic crisisand reticulocytosis in

sequestration crisis.

Susceptible to acuteinfections withencapsulated organisms.

Common pathogens:S. pneumonia,SalmonellaandPneumococcus.

SCA: severe hemolyticanemiaSickling crisis

Autosplenectomy.

Q. Laboratory findings in sickle

cell anemia.

Sickle cell anemia: ESR isreduced because sicklecells do not form rouleaux.


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Bone Marrow Cellularity:hypercellular.

Erythropoiesis:compensatorynormoblastic erythroid hyperplasia, which expands the marrow and

causes resorption of bone and secondary new bone formation.



Iron stores:usually increased.

In severe cases, skullbone shows crew-cut appearance inroentgenograms.

Serum Findings

Serum bilirubin: raised andpredisposes to pigment gallstones. Iron status: raised serum iron, serum ferritin and transferrin saturation.

Serum haptoglobin: reduced.

Urine Urobilinogen: increased.

Diagnostic/Confirmatory Tests Sickling test:

Sickling is induced by adding a reducing (oxygen-consuming) agent like 2% sodiummetabisulfite or sodium dithionite to blood sample.

Red cells with HbS show sickled(Fig. 4.6)and holly leafappearance.

It is diagnosticof sickle cell anemia.

Hemoglobin electrophoresis: HbS is a slow moving compared to HbA and HbF. Estimation of HbF: in homozygous state constitutes about 1030% of hemoglobin.

HPLC: useful for confirmation of diagnosis.

Prenatal diagnosis:by analysis of fetal DNA obtained by amniocentesis or chorionic villousbiopsy, to detect the point mutations.

SICKLE CELL TRAITHeterozygous state for the hemoglobin S mutation and shows both HbA and HbS (HbAS). Onedefective gene (from one parent with HbS) and while the other gene is normal.

Extramedullaryhematopoiesis can also

develop as a compensatorymechanism.

Sickle cell anemia:HbS7090%, HbF 1030%,no HbA.

Fig. 4.5: Diagrammatic peripheral blood smear with sickle cells (arrows)Fig. 4.4: Peripheral blood smear with sickle cells (arrows)


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Pathogenesis

In sickle cell trait, the hemoglobin A in RBCs prevents hemoglobin S polymerization. However,RBCs may sickle under extreme conditions (e.g. flight at high altitude in unpressurized aircraft,deep sea diving).

Clinical Features

Usually asymptomatic. Normal growth and development, lifespan and life expectancy.

Laboratory Findings

Peripheral Blood Hemoglobin: normal or mildly decreased.

Peripheral smear:

RBCs:normocytic normochromic picture with very few target cells and mild degree of anisopoi-

kilocytosis.

WBCs:normal.

Platelets:normal.

Bone MarrowHypercellular because of a compensatory normoblastic erythroid hyperplasia.

Diagnostic Tests Hb electrophoresis:demonstrates two bands of HbS and HbA.

Sickling test:sickling test is positive.

High-performance liquid chromatography (HPLC):useful for confirmation of diagnosis.

Sickle cell trait: Usually no anemia No significant clinical

features Amount of HbS varies

from 25% to 40% Hb A in RBCs prevents

polymerization of Hb S.

In sickle cell trait: HbS4045% and HbA 5560%.

Fig. 4.6: Sickling test. Sickled red cells (arrows) induced by reducing agent(2% sodium metabisulfite)

Sickling test is a diagnostictest for sickle cell anemia.


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IMMUNOHEMOLYTIC ANEMIASAnemias due to premature RBC destruction(hemolysis) mediated by antibodiesthat bind toRBCs. Te antibodies may be either allo or auto type.

Classification of Immunohemolytic Anemias (Table 5.1)

TABLE 5.1: Classification of immunohemolytic anemias

Alloimmune hemolytic anemia

Hemolytic disease of the newborn

Hemolytic transfusion reactions: mismatched blood transfusion

Autoimmune hemolytic anemia

Warm antibody type (IgG antibodies active at 37C)

Primary (Idiopathic)

Secondary: autoimmune disorders (systemic lupus erythematosus), drugs, lymphomas

Cold agglutinin type (IgM antibodies active at 4C18C)

Acute: mycoplasmal infection, infectious mononucleosis

Chronic: idiopathic, lymphomas

Cold hemolysin type (Donath-Landsteiner antibodies)

Alloimmune Hemolytic Anemia Production of antibody against foreign antigen not present on individuals red blood cell. Allo-antibodies are present either in the serum or bound to red cells.

HEMOLYTIC DISEASE OF THE NEWBORN It is an allo-immune hemolytic anemia developing in the fetus and newborn baby.

Hemolysis is extravascular.

HDN develops when the IgG antibodies against blood group of fetus passes from mother tofetus through the placenta.

Immunohemolyticanemias are characterizedby the destruction ofRBCs by either allo or autoantibodies.

Immunohemolyticanemias are mainlyclassified as:1. Alloimmune and2. Autoimmune hemolytic

anemia.

Hemolytic transfusionreactions are due to ABOmismatch. The antibodiespresent in the recipientsserum coat donors RBCsand lead to intravascularhemolysis.

Q. Write short notes on hemolytic

disease of newborn.

Other Anemias

CHAPTER

5


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Other Anemias CHAPTER 5

Occurs in two forms:

Rh incompatibility in which mother is Rh negative and fetus is Rh positive. Te anti-Dantibodies are responsible for the hemolytic anemia.

ABO incompatibility in which mothers blood group is O and fetus is either of A or Bblood group. Either anti-A or anti-B antibodies cause hemolysis.

Rh Hemolytic Disease of the Newborn (Fig. 5.1)

Rh hemolytic disease of the newborn is more important than due to ABO incompatibility.

Pathogenesis Occurs when mother is Rh (D antigen) negative and fetus is Rh positive.

Sensitization occurs when fetal Rh positive RBCs enter into Rh negative mothers. Rhnegative mother develops anti-Rh antibodies.

Sensitization occursonly at the time of delivery or during miscarriage. So, it does notmanifest in the first pregnancy.

HDN may be either due toRh or ABO incompatibilitybetween mother and fetalRBCs.

HDN usually does notmanifest during firstpregnancy. Sensitizationdevelops during deliveryor miscarriage.

Fig. 5.1: Pathogenesis of Rh hemolytic disease of the newborn

Rh HDN develops whenmother is Rh-ve and fetusis Rh+ve.


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In subsequent pregnancy, anti-Rh antibodies from mother cross placenta and coat theRh positive fetal red cells. Tese antibodies cause immunedestruction of fetal red cellsresults in severe hemolytic anemia leading tojaundiceof the newborn.

Fetus may develop cardiac failurehydrops fetalis(immune type).

Clinicopathological Features Infants may have jaundice at birth.

When the disease is severe, the levels of unconjugated bilirubinin the blood are highandbilirubin can pass the blood brain barrier.

Bilirubin is deposited in the central nervous system(especially the basal ganglia) producingneurological damageand is known as kernicterus (yellow coloration of cerebellum andbasal ganglia due to bilirubin deposition). It can cause death of the infant.

Prevention of Rh HDN: by the prophylactic removal of fetal cells entering the maternalcirculation before sensitization develops, by injecting anti-Dinto the Rh D negative mother.

Laboratory Findings

Peripheral blood Hemoglobin: decreased.

Reticulocyte count: increased.

Peripheral smear:

RBCs:normocytic normochromic anemia with numerous nucleated RBCs, polychromatophils

and occasional spherocytes.

WBCs:normal.

Platelets:normal.

Peripheral smear:normocyticnormochromic anemiawith nucleated RBCs andpolychromatophils.

Antiglobulin test (Coombs test): antibodies in the mother and baby are detected by indirectand direct Coombs test respectively (Fig. 5.2).

Hydrops fetalis is fatalcondition, characterizedby left and right-sidedheart failure producinggeneralized edema andmay result in death.

In Rh HDN, high levels ofunconjugated bilirubin cancross blood brain barriercausing kernicterus anddeath of infant.

Fig. 5.2: Direct and indirect methods of antiglobulin test (Coombs test)

In direct antiglobulintest, patients RBCs areused where as in indirect

antiglobulin test patientsserum is used for the test.


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Other Anemias CHAPTER 5

Serum findings Serum bilirubin: increased.

Lactate hydrogenase (LDH): increased.

Haptoglobin: decreased.

ABO Hemolytic Disease of the Newborn It is less severe.

Te fetus may be affected in the first pregnancy of a mother with blood group O.

Te IgG antibodies to A or B from maternal blood cross placenta and enter the fetalcirculation. Tese anti-A or anti- B antibodiesreactwith A and B antigenic determinantspresent in fetal fluids and tissues.

Tis results in consumption of major portion of the maternal IgG and the small portion,which is left combines with fetal red cells causing only mild hemolysis.

ANTIGLOBULIN (COOMBS) TESTIt is useful to detect the presence of incomplete antibody (IgG) and/orcomplement on theRBC membrane.

Principle RBCs coated with incomplete antibody (IgG) or C3 complement does not cause aggluti-

nation of RBCs.

Coombs reagent contains antibodies (antiglobulins) against human IgG/IgM/complement.

If the RBCs coated by incomplete antibody or complement, are treated with Coombsreagent, the antiglobulins in the reagent will induce agglutinationof such RBCs.

Types of Antiglobulin Test (Fig. 5.2) Direct (Coombs)antiglobulin test (DA)

Indirect (Coombs)antiglobulin test (IA)

Direct Antiglobulin Test (Fig. 5.2)

Direct antiglobulin test (DA) (direct Coombs test) detects antibodies(IgG) and/or comple-ment coated on the surface of patients RBCmembrane.

Patients RBCs are taken in a test tube and washed three times in normal saline.

Coombs (anti-globulin) reagent is added and observed for agglutination.

Agglutination indicates the presence of antibody on the RBC membrane and interprets aspositive DA.

Uses of Direct Antiglobulin Test Hemolytic disease of the newborn (HDN), in which direct Coombs test is performed on the

newborn babys red cells from the cord blood.

Autoimmune hemolytic anemia: to demonstrate in vivoattachment of antibodies to redcells.

Drug induced red cell sensitization.

Investigation of hemolytic transfusion reaction.

Antiglobulin test is usefulfor diagnosis of HDN.

ABO HDN is more commonbut less severe. It may beseen in first pregnancy.

Q. Write short notes on Coombs

(antiglobulin) test.

There are 2 types ofantiglobulin test: directand indirect.

Patients red cells are usedin direct antiglobulin test.


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In HDN, newborn babys RBCs from cord blood is used for direct antiglobulin test, whichwill be positive.

Indirect Antiglobulin Test (Fig. 5.2)

Indirect antiglobulin test (IA) (indirect Coombs test) detects the presence of incomplete(IgG) antibodies and/or complement in the patients serum.

In this test, patients serum is taken and O Rh positive cell suspension of any normalindividual is added.

O Rh positive RBCs are coated with (lgG) anti-Rh antibodies (if present) in the patients serum.

Add Coombs (antiglobulin) reagent and examine for agglutination.

Agglutination of RBCs indicates the presence of antibodies in the patients serum and test isreported

notes hematology

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