of
Hematology
of
Rapid Review
of
Hematology
Rapid Review
of
Hematology
Ramadas NayakMBBS MD Professor Department of Pathology Kasturba Medical CollegeManipal University Mangalore, Karnataka, India
Sharada Rai MBBS MD Associate Professor Department of Pathology Kasturba Medical College
Manipal University Mangalore, Karnataka, India
Foreword AR Raghupathy
JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD
Website: www.jaypeebrothers.com Website: www.jaypeedigital.com
© 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 the publisher.
Inquiries for bulk sales may be solicited at: [email protected]
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 specifically 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 specifically stated, all figures 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
Jaypee Brothers Medical Publishers (P) Ltd 4838/24, Ansari Road, Daryaganj
New Delhi 110 002, India Phone: +91-11-43574357 Fax: +91-11-43574314
Email: [email protected]
Overseas Offices
J.P. Medical Ltd
83 Victoria Street, London SW1H 0HW (UK) Phone: +44-2031708910 Fax: +02-03-0086180 Email: [email protected]
Jaypee-Highlights Medical Publishers Inc City of Knowledge, Bld. 237, Clayton Panama City, Panama
Phone: +507-301-0496 Fax: +507-301-0499
Email: [email protected] Jaypee Brothers Medical Publishers (P) Ltd 17/1-B Babar Road, Block-B, Shaymali Mohammadpur, Dhaka-1207
Bangladesh
Mobile: +08801912003485 Email: [email protected] Jaypee Brothers Medical Publishers (P) Ltd
Shorakhute, Kathmandu Nepal
Phone: +00977-9841528578 Email: [email protected] Jaypee Medical Inc
The Bourse
111 South Independence Mall East Suite 835, Philadelphia, PA 19106, USA Phone: + 267-519-9789
Dedicated to
Students who inspired us,
patients who provided the knowledge,
our parents and family members who
encouraged and supported us.
It gives me great pleasure to write a short foreword for this new book on Rapid Review of Hematology.
This 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.
This 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 useful compendium 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 Pathology Bangalore Medical College and Research Institute Bengaluru, Karnataka, India
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. This 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. There are many hematology textbooks, but undergraduates face difficulties to refresh their knowledge of hematology during examinations. This encouraged us to write a book to fill the niche, to provide basic information to an undergraduate in a nutshell. With this view in mind, Rapid Review of Hematology is 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
This book is organized into four sections namely disorders of red cells, disorders of white cells, disorders of hemostasis and clinical scenario.
The 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). The
concepts of hematology have been oversimplified in this book, but all the information, the student will ever need to know, have been provided. The readers are requested to give more emphasis on word in bold letters that represents the key words to be remembered. The peripheral smear and bone marrow findings have been highlighted in colored background. Boxes have been provided at the sides of main text. These include some of the key points as well as commonly expected questions during examinations. This book can serve as a source of rapid review of hematology.
Ramadas Nayak Sharada Rai
Preface
• 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 Tarun 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), and Mr Raj Kumar (DTP 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.
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
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
Contents xv
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
SECTION
1
Disorders of
Red Cells
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 (82–98 fL) Increased (>100 fL) Mean corpuscular
hemoglobin concentration (MCHC)
Reduced (< 30 g/dL) Normal (31–36 g/dL) Normal (31–36 g/dL)
Examples Iron deficiency anemia,
thalassemia
During blood loss, anemia of chronic diseases
Deficiency of vitamin B12 and folic acid Morphology of RBC
Q. Classify anemia.
Spurious anemia is the term used when RBC concentration decreases due to hemodilution as seen in third semester of pregnancy.
CHAPTER
1
Anemias of Impaired
Red Cell Production
ANEMIA
Definition
• Decrease below normal of the hemoglobin concentration (Hb)/RBC count/hematocrit
(packed cell volume).
• Reduction of the total circulating red cell mass below normal limits.
• Decrease in the oxygen-carrying capacity of the blood, which leads to tissue hypoxia. Anemia may be absolute (decreased RBC mass), or relative (associated with a higher plasma
volume). Anemia is conventionally used for absolute anemia.
Classification of Anemia
1. Morphological classification (Table 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 <13 g/dL and adult female Hb <12 g/dL. Grading of anemia: mild (Hb 9.1–10.5 g/dL), moderate (Hb 6.0–9.0 g/dL) and severe (Hb < 6.0 g/dL). Anemia is characterized by decreased oxygen carrying capacity of blood. Shows decreased Hb and PCV.
Classification: anemias are mainly classified based on 1) morphology and 2) etiology.
2. Etiological classification: The etiological classification of anemia is listed in Table 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 B12 and 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 expression of underlying disease and from treatment point, the cause of anemia must be identified. Causes of anemia: 1. Decreased RBC production 2. Increased RBC destruction (hemolysis) or 3. Blood loss.
Iron deficiency anemia is the most common anemia.
RED CELL INDICES
Red cell indices are useful in morphological characterization and diagnosis of anemias. They are either directly measured or automatically calculated by specialized instruments. Red cell indices 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: 82–98 fL.
MCV = PCV × 1000 = 0.45 × 1000/5 = 90 fL RBC count in millions
Q. Write short notes on red cell indices.
Red cell indices: MCV, MCH, MCHC and RDW.
Microcytic anemia have MCV < 80 fL and macrocytic anemia have MCV> 100 fL.
Anemias of Impaired Red Cell Production CHAPTER 1 5
2. Mean Corpuscular Hemoglobin (MCH)
• MCH indicates the amount of Hb (weight) per RBC and is expressed as picograms (1 pg = 10-12 g).
• It is of limited value in differential diagnosis of anemias. • Normal range: 27–32 pg
MCH = Hb (in g/L)/RBC (in millions/μL) = 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: 31–35 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 indicates that 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 low MCV.
RDW = (Standard deviation ÷ mean cell volume) × 100
IRON DEFICIENCY ANEMIA
Iron 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 in microcytic anemia and MCH > 33 pg is seen in macrocytic anemia.
MCHC<31 g/dL is seen in hypochromic RBC such as IDA and thalassemia. MCHC >36 g/dL is an indication of hyperchromic RBCs.
RDW is useful for differentiating anemia due 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 the commonest cause of IDA.
Iron is absorbed in the duodenum.
In adult men and postmenopausal women, deficiency may be due to chronic gastrointestinal blood loss.
Infants who consume large amounts of cow's milk are susceptible to develop IDA.
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
levels without anemia (Hb, MCV and MCH within normal range). Bone marrow shows iron
deficient 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: <80 fL (normal 82–98 fL) – MCH: <25 pg (normal 27–32 pg) – MCHC: <27 g/dL(31–36 g/dL)
– RDW: increased and >15%. It is earliest sign of iron deficiency (normal 11.5–14.5%).
• Peripheral smear (Figs 1.1 and 1.2):
– RBCs: microcytic (small) and hypochromic (pale). Severe anemia shows ring/pessary cells.
Moderate anisocytosis and poikilocytosis pencil/cigar-shaped cells.
– WBCs: normal; eosinophilia in hookworm infestation. – Platelets: normal
Q. Write short notes on peripheral smear findings in iron deficiency anemia.
Peripheral smear shows microcytic hypochromic
RBCs. • Reticulocyte count: low for the degree of anemia. Stages of IDA in
sequence: absent of iron stores→decreased serum ferritin→decreased serum iron→increased TIBC → decreased iron saturation→ microcytic hypochromic anemia.
Q. Discuss the laboratory findings in iron deficiency anemia. MCV, MCH and MCHC are reduced. RDW is raised.
Fig. 1.1: Peripheral blood smear showing microcytic
Anemias of Impaired Red Cell Production CHAPTER 1 7
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 shows micronormoblastic eythroid hyperplasia. Marrow iron is absent. Prussian blue reaction negative.
Serum Iron Profile (Table 1.4)
TABLE 1.4: Serum iron profile in IDANormal range Value in IDA Observation
Serum ferritin 15–300 µg/L <15 µg/L
Serum iron 50–150 µg/dL 10–15 µg/dL
Serum transferrin saturation 30–40% <15%
Total plasma iron-binding capacity (TIBC) 310–340 µg/dL 350–450 µg/dL Serum transferrin receptor (TFR) 0.57–2.8 µg/L 3.5–7.1 µg/L
Red cell protoporphyrin 30–50 µg/dL >200 µg/dL
Reticulocyte Hemoglobin
It is decreased and is an early feature of IDA.
Clinical Features of IDA
Nonspecific and related to both severity and the cause of the anemia (e.g. gastrointestinal
disease)
• Onset: insidious.
• Nonspecific symptoms: fatigue, palpitations, breathlessness, weakness and irritability. • Pharyngeal/esophageal webs formed cause dysphagia.
• Patterson-Kelly or Plummer-Vinson syndrome: – Microcytic hypochromic anemia
– Atrophic glossitis – Esophageal webs
• Congestive heart failure in severe anemia.
• Central nervous system: pica-unusual craving for substances with no nutritional value like clay or chalk. Craving for ice (pagophagia) specific to iron deficiency. Pica may be the
cause rather than effect of IDA.
Physical Findings
Diminished tissue enzymes cause characteristic epithelial changes of iron deficiency anemia. • Angular stomatitis and glossitis
• Chronic atrophic gastritis • Koilonychia (spoon nails)
Reduced: serum iron, ferritin, % transferrin saturation.
Increased: TIBC, TFR and red cell protoporphyrin.
The earliest laboratory indicator of IDA is reduced reticulocyte hemoglobin.
Q. Mention the various clinical features of iron deficiency anemia.
Patterson-Kelly or Plummer-Vinson syndrome: microcytic hypochromic anemia, atrophic glossitis and esophageal webs.
Koilonychia (spoon nails) is a physical finding seen in iron deficiency. First fingernails become thin and flat-platonychia, then brittle and finally spoon shaped.
Causes of Microcytic Hypochromic Anemia
• Iron deficiency anemia • Thalassemia major
• Anemia of chronic disorders
• Others: alcohol, lead poisoning and drugs • Sideroblastic anemia (rare cause).
MEGALOBLASTIC ANEMIA
Anemias characterized by defective/impaired DNA synthesis and distinct megaloblasts in the bone marrow. Megaloblastic anemias are common among anemias due to impaired red
cell production.
Etiology of Megaloblastic Anemia (Table 1.5)
TABLE 1.5: Causes of megaloblastic anemiaVITAMIN B12 DEFICIENCY
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 diet—alcoholism, 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 of vitamin B12 (cyanocobalamin) or folic acid. Both are required for the synthesis of DNA.
a. Delayed maturation of nucleus. The nuclear maturation lags behind the cytoplas-mic maturation and results in abnormally large nucleated erythroid precursors
named 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 B12 is present in animal products.
Deficiency of vitamin B12 and folic acid are the main causes of megaloblastic anemia.
Folic acid is absorbed in the jejunum.
Deficiency of vitamin B12 and folic acid → delayed nuclear maturation → megaloblast → macrocyte.
Ineffective erythropoiesis and hemolysis are responsible for anemia.
Anemias of Impaired Red Cell Production CHAPTER 1 9
Laboratory Findings of Megaloblastic Anemia
Blood findings in vitamin B12 and/or folic acid deficiency are similar.
Peripheral Blood
• Hemoglobin and hematocrit (PCV): reduced • Red cell indices
– MCV: above 100 fL (normal 82–98 fL) – MCH (normal 27–32 pg)
– Normal MCHC (31–36 g/dL)
• Peripheral smear (Figs 1.3 and 1.4): pancytopenia (decreased RBC, WBCs and platelets). – RBCs:
◆ Macrocytic and oval (egg-shaped macro-ovalocytes)-diagnostic. ◆ Most macrocytes lack the central pallor (Figs 1.3 and 1.4).
◆ Marked variation in the size and shape of red cells (anisopoikilocytosis).
◆ Evidence of dyserythropoiesis: basophilic stippling, Cabot ring and Howell Jolly bodies. – WBCs:
◆ Decreased WBC count (leukopenia).
◆ Hypersegmented neutrophils (more than five nuclear lobes): first and specific morphological sign 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 anemia due to vitamin B12 deficiency, reticulocyte count may be normal or low and high reticulocyte count is seen on 7th day following vitamin B12 therapy.
Fig. 1.3: Peripheral blood smear showing macro-ovalocytes (arrows) and hypersegmented neutrophil (inset )
Fig. 1.4: Diagrammatic peripheral blood smear showing macro-ovalocytes (thick arrows) and hypersegmented neu-trophil (thin arrow )
Bone Marrow
• Cellularity: moderately to markedly 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 of RBCs seen in the bone marrow of patients with megaloblastic anemia.
The differences between normoblasts and megaloblasts are shown in Table 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 cytoplasmic maturation.
• Nuclei have open sieve-like chromatin.
Fig. 1.5: Diagrammatic peripheral blood smear of dimorphic anemia showing macro-ovalocytes and microcytes
A mixture of microcytic hypochromic and macrocytic RBCs is termed as dimorphic picture and occurs in mixed deficiency of iron and folic acid or vitamin B12.
Anemias of Impaired Red Cell Production CHAPTER 1 11
Biochemical Tests for Megaloblastic Anemia
Common for both vitamin B12 and folic acid deficiency
Deoxyuridine suppression test: it is a sensitive measure of deficiency of 5, 10-methylene THF,
which occurs in both folic acid and vitamin B12 deficiency.
• 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 B12 deficiency
• Serum vitamin B12 levels: decreased
– Serum methylmalonic acid
– Urinary excretion of methylmalonic acid
• Schilling test for vitamin B12 absorption (Refer page 12).
Specific tests for folic acid deficiency
• Serum folic acid levels: decreased • FIGLU in urine: excessively excreted.
PERNICIOUS ANEMIA
Pernicious anemia (PA) is an autoimmune disease due to deficiency of intrinsic factor causing impaired absorption of vitamin B12 and megaloblastic anemia.
Rare in India. A genetic predisposition is suspected.
Age: older age—fifth to eighth decades of life
Sex: females are more involved than males (F: M is 1.5: 1).
Deoxyuridine suppression test is abnormal even before the morphological changes.
Schilling test determines the cause of vitamin B12 deficiency.
Q. Discuss the etiopathogenesis and morphology of pernicious anemia.
Vitamin B12 is absorbed in terminal ileum and requires 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 showing megaloblastic precursors (thick arrows) in varying stages of maturation
Etiopathogenesis
• An autoimmune disease due to destruction of gastric mucosa.
• Stomach shows damage to parietal cells, dense infiltration by lymphocytes and plasma cells → chronic atrophic gastritis → failure of production of intrinsic factor.
• Presence of autoantibodies: two major types of autoantibodies— – Anti-intrinsic factor (IF) antibody
◆ Type I (blocking) antibody: blocks the binding of vitamin B12 to IF. Present in 50–75%
of the cases.
◆ Type II (binding) antibody: attaches to the IF–vitamin B12 complex and prevent its
binding 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, pepsin and 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 System
Found 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 for megaloblastic anemias (Refer page 9 to 11).
Specific Diagnostic Tests for Pernicious Anemia
• Schilling test for vitamin B12 absorption: abnormal
– Radioactive vitamin B12 is 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 B12 deficiency.
• Serum antibodies to intrinsic factor are highly specific for pernicious anemia • Achlorhydria with histamine/pentagastrin stimulation.
• Severe deficiency of intrinsic factor.
PA: autoimmune disease • Atrophic gastritis • IF deficiency • Autoantibodies.
Atrophic gastritis may predispose to carcinoma stomach.
Q. Write short note on laboratory findings in pernicious anemia.
Schilling test: diagnostic of PA but now very infrequently performed.
Anemias of Impaired Red Cell Production CHAPTER 1 13
Clinical Features of Megaloblastic Anemia
The clinical features of vitamin B12 deficiency anemia and pernicious anemia are:
• Onset: insidious and progresses slowly.
• Classic triad of presentation: weakness, sore throat and paresthesias. • Tongue: painful red “beefy” tongue.
• Neurological manifestations:
– Bilateral peripheral neuropathy: glove and sock distribution of numbness or paresthesia – Demyelination of spinal cord: subacute combined demyelination/degeneration
of dorsal and lateral tracts—ataxia, uncoordinated gait, impairment of vibration and
position sense.
• Atherosclerosis: serum homocysteine level is raised and is a risk factor for atherosclerosis and thrombosis.
APLASTIC ANEMIA
Hematopoietic stem cell (HSC) disorder characterized by:
• Pancytopenia (anemia, neutropenia and thrombocytopenia)
• With markedly hypocellular bone marrow (less than 30% cellularity).
Etiology
The most common causes associated with aplastic anemia are shown in Table 1.7.
Q. Mention the various clinical features of megaloblastic anemia.
Folate deficiency anemia presents with features of megaloblastic anemia due to vitamin B12. Unlike with vitamin B12 deficiency, neurological symptoms does not occur.
Nonmegaloblastic causes of macrocytic anemia: 1. Alcohol 2. Liver disease 3. Myxedema 4. Cytotoxic drugs 5. Myeloma 6. Aplastic anemia 7. Reticulocytosis 8. Red cell aplasia.
Q. Write short notes on aplastic anemia.
Fig. 1.8: Clinical features and laboratory findings in pernicious anemia
PA patients sometimes have a lemon-yellow color owing to a combination of pallor and mild jaundice caused by excess breakdown of hemoglobin. Pernicious anemia present with features of megaloblastic anemia due to vitamin B12 deficiency. In addition, it may show features of atrophic gastritis and achlorhydria.
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
Secondary
Chemical 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 of
aplastic anemia: 1. Idiopathic
2. Ingestion of drugs and chemicals 3. Idiosyncratic 4. Irradiation 5. Infections and 6. Inherited.
Pathogenesis (Fig. 1.9)
Pathogenesis:• Direct damage to the hematopoietic stem cells and progenitor cells.
• Immune-mediated destruction. • Primary stem cell
abnormality—inherited defect in the stem cells.
Clinical Features
• Any age of both sexes • Insidious
– Progressive weakness, pallor and dyspnea due to anemia
Anemias of Impaired Red Cell Production CHAPTER 1 15
– Bleeding manifestations in the form of petechiae, bruises and ecchymoses due to thrombocytopenia.
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 diminished and 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 aplasia—best 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 elements are replaced by fat and aspiration usually yields dry tap.
No Splenomegaly
Diagnosis: diagnosis is made with peripheral blood and bone marrow biopsy findings.
Differential Diagnosis
• Should be distinguished from other causes of pancytopenia (Table 1.8)
TABLE 1.8: Causes of pancytopenia
Decreased bone marrow function
• Aplastic anemia – Idiopathic – Secondary – Inherited
• Myelodysplastic syndromes • Bone marrow infiltration with
– Leukemia – Lymphoma – Myeloma
– Tumors (carcinoma)
– Granulomatous diseases (e.g. tuberculosis, sarcoidosis) • Nutritional deficiencies:
– Megaloblastic anemia (vitamin B12 and folic acid deficiency) • Paroxysmal nocturnal hemoglobinuria
• Myelofibrosis (rare) • Hemophagocytic syndrome
Increased peripheral destruction
• Hypersplenism
Prognosis: unpredictable.
Reticulocyte count is markedly low in aplastic anemia and is characteristic feature.
Absence of splenomegaly and in its presence the diagnosis of aplastic anemia should not be made.
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 extracorpuscular
defect
• 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: disseminated intravascular 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 red cell is about 120 days. In hemolytic anemias RBC survival time is considerably shortened.
Breakdown of normal RBCs occurs in the macrophages of the bone marrow, liver and spleen.
Decreased red cell survival does not always cause anemia as there is a compensatory increase in red cell production by the bone marrow.
Hemolytic Anemias Due to
Red Cell Membrane and
Enzyme Defects
CHAPTER
Hemolytic Anemias Due to Red Cell Membrane and Enzyme Defects CHAPTER 2 17
Location of Hemolysis
It may be intravascular and/or extravascular. The differences between these two types are listed in Table 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 PresentPresent
Examples Thalassemia, sickle cell anemia G6PD deficiency, PNH
HEREDITARY SPHEROCYTOSIS
Hereditary spherocytosis (HS) is a rare inherited hemolytic anemia resulting from the defect in the red cell membrane.
Normal structure of RBC membrane is depicted in Figure 2.1.
Etiopathogenesis
• Autosomal dominant disorder
• 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. These small RBCs assume a spherical shape (spherocytes).
• Spherocytes are rigid, inflexible and less deformable. They get trapped in the spleen leading to premature destruction of spherocytes.
In most hemolytic anemias red cell destruction is extravascular.
Q. List the differences between extravascular hemolysis and intravascular hemolysis.
Q. Describe the etiopathogenesis of hereditary spherocytosis.
HS, is due to defect in the RBC membrane protein. The common mutations involve ankyrin, band 3, spectrin or band protein 4.2.
HS: intrinsic defect of RBC membrane-extravascular hemolysis.
Laboratory Findings
Peripheral Blood
• Hemoglobin: decreased and level depends on degree of hemolysis. • Red cell indices:
– MCV: reduced (normal 82–98 fL)
– MCHC: raised and > 35 g/dL (normal 31–36 g/dL).
• Peripheral smear: very important for 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 also be seen in autoimmune hemolytic anemia and burns.
• Reticulocyte count: increased (Fig. 2.5).
Bone Marrow
• Cellularity: markedly hypercellular • Erythropoiesis: erythroid hyperplasia • Myelopoiesis: normal
• Megakaryopoiesis: normal.
Bone marrow shows erythroid hyperplasia. Q. Write short notes on laboratory findings in HS.
In hereditary spherocytosis MCHC is > 35 g/dL.
Spherocytes and reticulocytosis are observed in the peripheral blood.
Fig. 2.3: Peripheral blood smear with numerous spherocytes (arrows) Fig. 2.4: Diagrammatic peripheral blood smear
with numerous spherocytes (arrows)
Hemolytic Anemias Due to Red Cell Membrane and Enzyme Defects CHAPTER 2 19
Biochemical Findings
• Serum bilirubin: mildly raised. • 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 neonatal period to adulthood.
• Family history: most (75%) are inherited as autosomal dominant trait. • Anemia: mild to moderate.
HS: osmotic fragility is increased with a shift of curve to the right.
Clinical features of intermittent jaundice, splenomegaly and spherocytes in the peripheral smear is highly suggestive of HS.
Fig. 2.5: Smear shows reticulocyte with blue filamentous/granular material (new methylene blue stain) (arrows)
Fig. 2.6: Osmotic fragility test. Normal curve (blue) and increased osmotic fragility in hereditary spherocytosis
• Jaundice: intermittent attacks, precipitated by pregnancy, fatigue, or infection. • Splenomegaly: moderate (500 to 1000 g).
• Gallstones: pigment gallstones.
• Aplastic crises: may be triggered by an acute parvovirus infection.
GLUCOSE-6-PHOSPHATE
DEHYDROGENASE DEFICIENCY
• Hemolytic disease due to red cell enzyme defects.
• In G6PD deficiency, RBCs are susceptible to oxidative injury by free radicals. • It is an X-linked recessive disorder and its full expression is seen only in males. • There 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. The housekeeping enzyme, G6PD is
required 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.
• RBCs when exposed to oxidant stress (during infections, exposure to drugs or chemical, fava beans) accumulate H2O2. Itdamages RBC membrane causing hemolysis.
• Hemolyzed red cells liberate hemoglobin.
• The 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 an intrinsic defect and hemolysis is primarily intravascular.
In G6PD, RBCs exposed to oxidant stress, the hemoglobin is oxidized to methemoglobin which forms Heinz bodies in the cytoplasm of RBCs.
Hemolytic Anemias Due to Red Cell Membrane and Enzyme Defects CHAPTER 2 21
• Heinz bodies removed from RBC membrane by macrophages in the spleen and produce
bite cells. These bite cells are removed via erythrophagocytosis in the spleen.
Clinical Presentation
G6PD deficiency manifests in several distinct clinical patterns. Usually present as acute self-limited acute intravascular hemolytic anemia following exposure to oxidative stress.
Laboratory Findings
Peripheral Blood
• Hemoglobin: decreased. • Reticulocyte count: increased.
• Peripheral smear:
– RBCs: moderate anisopoikilocytosis with polychromatophilia, microspherocytes and bite cells (Fig. 2.8). Heinz bodies identified with a supravital stain and 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
Hemoglobinuria will be found during hemolysis and may last for about 1–6 days.
RBC Enzyme Analysis
Tests for G6PD deficiency are positive and should be assessed a few weeks after the acute
hemolytic episode.
G6PD deficiency has a protective effect against
Plasmodium falciparum malaria. G6PD deficiency–oxidant damage to RBC • Bite cells • Heinz bodies. G6PD: enzyme analysis– confirmatory test.
Fig. 2.8: Peripheral blood smear in G6PD deficiency with “bite cells”
CLASSIFICATION OF HEREDITARY
DEFECTS IN HEMOGLOBIN
Hemoglobin defects may be quantitative (reduced production of normal hemoglobin) or qualitative (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 of hemoglobin.
• Qualitative defect: genetic mutations in the a-globin or b-globin locus may produce abnormal hemoglobin (e.g. sickle cell anemia). The abnormal hemoglobin may be func-tionally normal, but its physical or physiologic properties differ from normal hemoglobin.
THALASSEMIA SYNDROME
• These are group of inherited disorders due to abnormality of globin production.
• It is characterized by decreased or absence of synthesis of either a or b-globin chain of adult hemoglobin, HbA (a2b2).
Classification
They are mainly classified as: •
• b-Thalassemia syndromes: impaired synthesis of b-chains of globin. •
• a-Thalassemia syndromes: impaired synthesis of a-chains of globin. • Miscellaneous thalassemia syndromes.
b-THALASSEMIA
• Autosomal recessive hereditary disorder
• Diminished synthesis of b-globin chains and normal synthesis of a-chains.
Q. Classify hereditary disorders of hemoglobin.
The term
hemoglobinopathy is usually used for a qualitative hereditary disorder of hemoglobin. Q. Classify thalassemia syndromes. In b-Thalassemia, there is decreased/absence of synthesis of b-chains. In a-Thalassemia, there is reduced/absence of synthesis of a-chains of globin.
Thalassemia Syndrome
CHAPTER
3
Thalassemia Syndrome CHAPTER 3 23
Molecular Pathology
• b-globin chains are encoded by a single gene.
• The 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 mutations
rather than gene deletions (unlike in a-thalassemia). The mutations result in defects in transcription, RNA splicing and modification, translation via frame shifts and nonsense codons. 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-thalassemiasClinical 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 not
transfusion 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. The synthesis of a-globin chain is not affected.
• Homozygous form of b0/b0 or b+/b+ or double heterozygous b0/b+ (Table 3.1)
• Most common in Mediterranean countries, parts of Africa and South East Asia. • Hemolytic anemia is of severe degree.
Pathophysiology of
b-thalassemia Major (Fig. 3.1)
Consequence of Defective or Absent
b-chains
• Severe hemolytic anemia due to:
1. Absence of b-globin chain: results in absence of synthesis of HbA (a2b2). This produces
RBCs that are poorly hemoglobinized (hypochromic) and small in size (microcytic). 2. Ineffective erythropoiesis: unpaired and excess a-chains aggregate into insoluble
precipitates, which bind to and damage the membrane of erythroid precursors. These
erythroid precursors fail to mature and undergo apoptosis in the marrow.
3. Extravascular hemolysis: RBCs with a-chain inclusions are removed by macrophages of spleen (extravascular hemolysis).
• Synthesis of fetal hemoglobin (HbF): the ϒ-globin chain synthesis continues even 6 months after birth and combines with a-globin leading to increased levels of HbF (a2ϒ2). The level
of 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)
– Typical facies: thalassemic facies (Fig. 3.3)—prominent forehead, cheekbones and upper jaw.
Point mutations leading to aberrant RNA splicing is the most common cause of b-thalassemia. •• b0 = Total absence of b-globin synthesis; •• b+ = Markedly reduced or diminished b-globin synthesis; •• b = normal b-globin synthesis. b-thalassemia is the commonest quantitative disorder of hemoglobin. b-thalassemia major also called Mediterranean or Cooley’s anemia.
Q. Describe the pathophysiology/ pathogenesis of b-thalassemia major.
b-thalassemia major • Absence of synthesis of
HbA produces severe microcytic hypochromic anemia • Increased synthesis of HbF. b-thalassemia major • Thalassemic facies • Crew cut appearance on
skull x-ray • Splenomegaly.
• 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 and secondary hemochromatosis and damages to parenchyma of organs (e.g. heart, liver and pancreas).
Clinical Features
• Age: infants develop moderate to severe anemia 6–9 months after birth.
• Growth and development: untreated/untransfused children fail to thrive and die within 4–5 years of age.
• Bone changes: those who survive longer develop distortion of skull and facial bones. X-ray
skull shows hair on end appearance (Fig. 3.2) and face shows a characteristic thalassemic facies (Fig. 3.3).
• Marked splenomegaly: up to 1500 grams due to hyperplasia and extramedullary hematopoiesis.
• Extramedullary hemopoiesis: liver and lymph nodes may show extramedullary
hematopoiesis.
b-thalassemia major • Iron overload damgaes
parenchymal organs due to hemosiderosis and secondary hemochromatosis.
Failure to thrive, retarded growth, monogoloid face, and hepatosplenomegaly are clinical features of b-thalassemia major.
Thalassemia Syndrome CHAPTER 3 25
• Iron overload: multiple blood transfusions may lead to iron overload and result in
hemosiderosis 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 increased and in the range of 5 to 15%.
• Red cell indices:
– MCV decreased and in the range of 45–70 fL (normal range 82–98 fL). – MCHC decreased and in the range of 22–30 g/dL (normal range 31–35 g/dL). – MCH decreased and in the range of 20–28 pg (normal range 27–32 pg).
– RDW-within normal limits (in contrast to iron deficiency anemia where it is increased).
• Peripheral smear: – RBCs:
◆ Microcytic hypochromic anemia
◆ 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 (5–40%). – WBCs: leukocytosis with mild left shift.
– Platelets: normal.
Q. Write short note on peripheral smear findings in b-thalassemia major.
b- thalassemia major: RDW normal. The peripheral blood smear shows microcytic hypochromic anemia, target cells and anisopoikilocytosis.
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.
• Myelopoiesis: normal. • Megakaryopoiesis: normal.
• Bone marrow iron: markedly increased due to increased dietary absorption and hemolysis.
Bone marrow in b- thalassemia major shows marked normoblastic erythroid hyperplasia. Marrow iron is markedly increased.
Q. Mention the laboratory findings in b-thalassemia major.
RDW normal MCV, MCH and MCHC decreased.
Fig. 3.2: X-ray appearance of skull in b-thalassemia showing
Biochemical Findings
• Bilirubin: increased—mainly of unconjugated type. • Urine urobilinogen: increased
• Serum haptoglobin: markedly reduced. • Serum iron status:
– Serum iron, serum ferritin and transferrin saturation are markedly increased – Total iron binding capacity (TIBC): reduced.
Special Tests
• Fetal hemoglobin (HbF): increased to 30% to 90% (normal range 0 – 1%). • Hemoglobin electrophoresis (Table 3.2):
•
– b+ thalassemia (b+/b+ or b0/b+ genotypes): demonstrates bands of both HbA and HbF.
•
– bo thalassemia (b0/b0 genotype): since no b-chains are formed, there is no HbA. Major
hemoglobin is HbF with normal or low HbA2.
• High performance chromatography (HPLC): HbF is increased (30–90%). HPLC measures various fractions of hemoglobin (Hb) and is used for confirmation of diagnosis.
• Prenatal diagnosis by molecular analysis of DNA.
• Estimation of globin chains: normally a: b ratio is 1:1. Lack of b chain alter this ratio to 5–30:1
Reduced/absence of synthesis of b-chains; the excess a-chains combine with γ-chains leading to increased HbF.
Fig. 3.4: Peripheral blood smear in β-thalassemia showing target
cells (arrows) Fig. 3.5:showing target cells (short arrows) and nucleated red cells Diagrammatic appearance of peripheral blood smear in β-thalassemia (long arrows)
TABLE 3.2: Hemoglobin F and A2 percentage in thalassemia syndromes
Type HbF HbA2
b -Thalassemia major (homozygous) 30–90% < 3.5%
b -Thalassemia intermedia (double heterozygous) 10–30% < 3.5%
b -Thalassemia minor/trait (heterozygous) 0–5% 3.6–8%
Note: normal adult cell
contains 96% HbA (a2b2), 3% HbA22(a2d2) and 1% HbF(a2γ2).
Thalassemia Syndrome CHAPTER 3 27
b-THALASSEMIA MINOR/TRAIT
• More common than b-thalassemia major.
• Most patients are heterozygous for thalassemic gene. • Usually asymptomatic and 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.
• NESTROF test (Naked eye single tube red cell osmotic fragility test): positive.
– In this test, 0.02 mL of patient’s 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.
– The microcytic hypochromic RBCs of thalassemia minor are resistant to lysis than normocytic normochromic RBCs.
– Hence, the black line on the paper is not clearly visible through the test tube compared to normal cells.
• Estimation of HbA2: HPLC is used for accurate estimation. HbA2 estimation is diagnostic
and level ranges from 4% to 8%.
NESTROF test positive because the microcytic hypochromic RBCs of b-thalassemia minor are resistant to lysis than normocytic normochromic RBCs.
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 b chain
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 µg/L Reduced Increased Increased (300 – 1000 µg/L) Increased Normal
• Fetal hemoglobin (HbF) Normal (0–1%) Markedly increased (30–90%)
• 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 differentiated from iron deficiency anemia. Treatment with iron in b-thalassemia major worsens the iron load and its consequences.
b-thalassemia intermedia: it is a clinical entity intermediate between thalassemia trait and thalassemia major.
