Undergraduate Clinical Pathology Book 2024 PDF

MEDICAL STUDENT’S CLINICAL PATHOLOGY BOOK MEDICINE III, BLOCK: MCI-525 2nd Edition By Staff Members Of Clinical Pathology Department Sohag Faculty of Medicine 2024 “Improper patient preparation (such as fasting, exercise...

MEDICAL STUDENT’S CLINICAL PATHOLOGY BOOK MEDICINE III, BLOCK: MCI-525 2nd Edition By Staff Members Of Clinical Pathology Department Sohag Faculty of Medicine 2024 “Improper patient preparation (such as fasting, exercise, stress, smoking, caffeine intake, or certain medications), incorrect sample collection (like wrong timing, wrong tube, or improper posture), and poor sample handling (such as improper storage or delayed transport) represent the majority of lab result errors. These simple mistakes can greatly affect result accuracy, leading to incorrect diagnosis, false results, or delayed treatment, highlighting the importance of proper preparation and handling before testing begins”. "Analyte reference ranges may vary between laboratories due to differences in the population being tested, as well as the methods and equipment used. Factors such as age, sex, altitude, and ethnicity can also influence these ranges." Page | ii  Table of Contents Page Content Page Content 1 Section I : Hematology and Transfusion Medicine 66 Section III : Clinical Chemistry 2 Red Blood Cells 67 Hepatobiliary Disorders 4 Microcytic Hypochromic Anemias 70 Diabetes Mellitus 6 Macrocytic Anemias 76 Disorders of Plasma Lipids and Lipoproteins 7 Normocytic Normochromic Anemias 79 Renal Diseases 18 Polycythemia 88 Acid-Base Disturbances 19 Erythrocyte Sedimentation Rate 89 Electrolyte Imbalance 20 White Blood cells 90 Acute Pancreatitis 20 Leucocytic Count 91 Acute Myocardial Infarction 20 Terminology 93 Tumor Markers 21 Abnormal Differential Leucocytic Count 94 Disorders of Calcium and Phosphate 23 Acute Leukemia 98 Disorders of Thyroid Gland 24 Chronic Leukemias 29 Plasma Cell Myeloma 101 Section IV : Clinical Microbiology 31 Pancytopenia 102 Diagnosis of Infectious Diseases 32 Hemostasis 102 Meningitis 32 Laboratory Evaluation of Hemostatic Function 104 Blood Stream Invasion 34 Bleeding Disorders 106 Urinary Tract Infections (UTIs) 39 Thrombophilia 107 Upper Respiratory Tract Infections 40 Transfusion Medicine 108 Lower Respiratory Tract Infections 40 Blood Cell Transfusion-Related Antigens 110 Gastrointestinal Tract Infections 41 Criteria of Blood Donor Selection 111 Pyrexia of Unknown Origin 42 Testing of Donor's Blood 114 Wound Infections 43 Blood Components 115 Anaerobic Infections 44 Complications of Blood Transfusion 116 Lower Genital Tract Infections 48 Modification of Blood Products 117 Congenital Infections 48 Massive Transfusion 118 Antibiotics and Antibiotic Resistance 120 Antimicrobial Stewardship 49 Section II : Clinical Immunology 121 Infection Prevention and Control 50 Immunological diagnosis of Infectious Diseases 121 The Chain of Infection 50 Viral Hepatitis 122 Healthcare-Associated Infections (HAIs) 54 Infectious Mononucleosis 124 Hand Hygiene 55 Acquired Immunodeficiency Syndrome 125 Respiratory Hygiene and Cough Etiquette 57 Hypersensitivity Reactions 126 Prevention of Infectious Agents Transmission 57 Type I: Immediate (Anaphylactic) Hypersensitivity 127 Waste Disposal 58 Type II: Cytotoxic (Cytolytic) Reaction 129 Blood and Infectious Fluid Exposures 59 Type III: Immune Complex-Mediated Reaction 60 Type IV: Cell-Mediated (Delayed) Hypersensitivity Reaction 61 Autoimmune Diseases 61 Type 1 Diabetes Mellitus 62 Rheumatoid Arthritis 63 Systemic Lupus Erythematosus 64 Antiphospholipid syndrome 64 Celiac Disease 65 Autoimmune Hepatitis Page | iii  SECTION I HEMATOLOGY AND TRANSFUSION MEDICINE  Red Blood Cells (Erythrocytes) Erythropoiesis Erythropoiesis is the process involving the formation, proliferation, maturation, and release of red blood cells (RBCs) into the bloodstream. In adults, this process primarily occurs in the bone marrow (BM). The typical lifespan of an RBC is about 120 days. Erythropoietin (EPO), a hormone produced mainly in the kidneys in response to low O2 levels (hypoxia), regulates RBC production. At the end of their lifespan, RBCs are removed from circulation through intravascular or extravascular hemolysis, with their components either recycled or excreted. Normal Mature Red Blood Cells Shape: Biconcave disc with central pallor, lacking a nucleus. Diameter: 7–8 µm. Function: Contains hemoglobin, which gives the cells their red coloration and is essential for O2 transport. A normal peripheral blood smear (film) shows minimal variation in RBC shape and size. Reticulocytes Reticulocytes are immature RBCs. They can be visualized under a light microscope using special stains, such as brilliant cresyl blue or new methylene blue. The normal reticulocyte counts in adults ranges from 0.5% to 2.5%. Reticulocytosis: An increased reticulocyte count occurs in conditions such as hemorrhage, hemolysis, and in response to hematinic treatment for anemia. Reticulocytopenia: A decreased reticulocyte count is observed in conditions like aplastic anemia. Table 1-1. Normal RBC Parameters for Adults Abbreviation Parameter Reference Range Unit RBCs RBCs Count Male: 4.5 - 5.5 ×106/mm3 Females: 3.8 – 4.8 ×106/mm3 Hb Hemoglobin Level Male: 13 – 17 g/dL Female: 12 – 16 g/dL HCT (PCV) Hematocrit (Packed Cell Volume) Male: 40 – 50 % Females: 35 – 45 % MCV Mean Corpuscular Volume 80 – 100 fL MCH Mean Corpuscular Hb 28 – 34 pg MCHC Mean Corpuscular Hb Concentration 32 – 36 g/dL RDW Red Cell Distribution Width 11.5 – 14.5 % Page | 2 Common Abnormalities of RBCs 1. RBCs Shape Abnormalities (Poikilocytosis) Spherocytes Round, small cells Hereditary spherocytosis Lacking the biconcave shape Immune hemolytic anemia Elliptocytes Elongated, oval-shaped cells Hereditary elliptocytosis Iron deficiency anemia Target Cells Cells with a central bull’s eye appearance Liver disease Thalassemia Hemoglobinopathies Sickle Cells Crescent or sickle-shaped RBCs Sickle cell anemia Schistocytes Fragmented RBCs Hemolytic anemias DIC Acanthocytes RBCs with irregular, spiny projections Liver disease Abetalipoproteinemia Echinocytes RBCs with evenly spaced projections Uremia Liver disease Sometimes as an artifact Teardrop Cells Teardrop-shaped RBCs Myelofibrosis Thalassemia Iron deficiency anemias 2. RBCs Size Abnormalities (Anisocytosis) Microcytes Smaller-than-normal RBCs iron deficiency anemia (MCV < 80 fL) Thalassemia Chronic diseases Macrocytes Larger-than-normal RBCs Megaloblastic anemia (MCV > 100 fL) Liver diseases Alcoholism Megalocytes Large, oval-shaped RBCs Megaloblastic anemia 3. Abnormalities in RBC Color (Hemoglobin Content) Hypochromia Pale RBCs with increased central pallor Iron deficiency anemia Thalassemia Hyperchromia RBCs with increased hemoglobin content Hereditary spherocytosis Polychromasia RBCs with a bluish tint (indicating immaturity) Hemorrhage Hemolysis 4. RBC Inclusions Howell-Jolly Small, round DNA remnants within RBCs Megaloblastic anemia Bodies Post-splenectomy Basophilic Fine or coarse granules (RNA remnants) in RBCs Lead poisoning Stippling Thalassemia Megaloblastic anemia Pappenheimer Iron-containing granules in RBCs Sideroblastic anemia Bodies Hemolytic anemia Post-splenectomy Heinz Bodies Denatured hemoglobin precipitates G6PD deficiency Unstable hemoglobinopathies Cabot Rings Ring-like structures in RBCs, Megaloblastic anemia (remnants of the mitotic spindle) Severe anemias Malaria Parasites Parasites within RBCs Malaria Anisocytosis is estimated by red cell distribution width (RDW) Page | 3  Anemias Anemia is diagnosed when the hemoglobin concentration, red blood cell (RBC) count, and/or the hematocrit (HCT) (also known as packed cell volume, PCV) fall below the established normal reference ranges for a person’s age and sex. Morphological Classification of Anemias Based on red cell indices and red cell morphology in the stained film. 1. Microcytic hypochromic anemias. 2. Macrocytic anemias. 3. Normocytic normochromic anemias. I. Microcytic Hypochromic Anemias Microcytic hypochromic anemias are characterized by RBCs that are smaller than normal (microcytic) and have reduced hemoglobin content (hypochromic). Causes 1. Iron deficiency anemia (IDA) The most common cause of microcytic anemia. IDA results from insufficient iron, which is needed for hemoglobin synthesis. Causes include inadequate dietary intake, chronic blood loss (e.g. gastrointestinal bleeding, heavy menstruation), or poor absorption. 2. Thalassemias (Alpha and Beta, Major and Minor) Thalassemias are genetic disorders that result in reduced or absent production of one or more of the globin chains that make up hemoglobin (alpha or beta). This imbalance leads to ineffective erythropoiesis and increased destruction of abnormal RBCs. 3. Anemia of Chronic Disease (ACD) A type of anemia that occurs with long-term illnesses like infections, inflammation, or cancer. In these conditions, the body’s inflammatory response prevents proper use of iron and reduces the production of RBCs, although there may be enough iron stores. 4. Sideroblastic Anemia A rare disorder where the bone marrow produces ringed sideroblasts (abnormal RBC precursors with iron-loaded mitochondria). It is caused by defects in heme synthesis, leading to ineffective erythropoiesis. 5. Lead Poisoning Lead interferes with several enzymes in the heme synthesis pathway, leading to defective hemoglobin production and microcytic anemia. It is more common in children exposed to lead-based paints or contaminated environments. Table 1-2. Differential Diagnosis of Microcytic Hypochromic Anemias Thalassemia Thalassemia Sideroblastic Parameter IDA ACD Minor Major Anemia Serum Iron ↓ ↓ Normal ↑ ↑ Serum Ferritin ↓ ↑ Normal ↑ ↑ TIBC ↑ ↓ Normal N N BM iron stores Depleted ↑ Normal ↑ ↑+ RS Hb electrophoresis Normal N ↑ HbA2 ↑ HbF Normal RS; Ring Sideroblasts Page | 4 Laboratory Diagnosis of Iron deficiency anemia IDA occurs when iron intake and stores do not meet the needs for RBC production (e.g. inadequate intake, increased iron requirement, defective absorption) or in chronic blood loss. 1. Complete Blood Count Varies with the severity of depletion. RBCs, Hb and HCT are ↓ due to insufficient iron for normal RBCs production. ↓ MCV (microcytic) and ↓ MCH (hypochromic). Blood film: RBCs appear smaller with increased central pallor (RBCs lacking Hb). 2. Iron Studies Serum iron: ↓ (although it may be normal in the early stages). Serum Ferritin: ↓ (reflecting depleted iron stores). Total iron-binding capacity (TIBC): ↑ (as the body tries to absorb more iron). Transferrin Saturation: ↓ (i.e. a lower proportion of transferrin is bound to iron). BM iron stores: ↓ (indicating lack of iron available for RBCs production). 3. Investigation for the cause Ask about history of chronic blood loss. Request further laboratory test such as stool analysis for parasites, occult blood in stools especially in elderly, urine analysis for evidence of hematuria …etc. Laboratory Diagnosis of Sideroblastic Anemia Sideroblastic anemia may be either hereditary or acquired and results from defect in heme synthesis (ineffective erythropoiesis) 1. Complete Blood Count Results vary depending on the severity and type (hereditary or acquired). RBCs, Hb, and HCT: May be ↓ due to ineffective erythropoiesis. ↓ MCV (microcytic) and ↓ MCH (hypochromic). Blood film: Shows the presence of dimorphic RBCs—one hypochromic and the other normochromic, reflecting a mix of defective and normal erythropoiesis. 2. Iron Studies ↑ Serum Iron (due to ineffective use of iron in heme synthesis). ↑ Serum Ferritin (as iron accumulates in the body). TIBC: Normal or ↓ (as iron is not being used effectively despite high stores). 3. Bone marrow examination A bone marrow biopsy is sometimes performed to confirm the diagnosis and reveal: ↑ BM iron stores: Reflecting the inability to use available iron effectively. The presence of ring sideroblasts which are RBCs precursors that have iron-loaded mitochondria surrounding the nucleus, a hallmark of sideroblastic anemia. Laboratory Diagnosis of Anemia of Chronic Inflammation (Disease) This anemia characterized by the presence of adequate or even abundant iron stores, but the body is unable to properly use this iron for RBCs production due to inflammatory processes. 1. Complete Blood Count Anemia is usually mild in severity. RBCs, Hb, HCT may be slightly ↓ with normal or ↓ MCV and MCH. 2. Iron Studies ↓ Serum Iron (due to impaired iron mobilization from stores). ↑ Serum Ferritin (or may be normal), ↓ TIBC (transferrin production is reduced in response to inflammation). ↓ Transferrin Saturation (reflecting lower iron availability for erythropoiesis). Page | 5  II. Macrocytic Anemias Macrocytic anemias are characterized by the presence of abnormally large RBCs caused by impaired RBCs maturation, leading to ineffective erythropoiesis. Macrocytic anemias are broadly classified into megaloblastic and non-megaloblastic types based on the underlying cause. A. Megaloblastic Macrocytic Anemia Caused by defective DNA synthesis in RBC precursors, leading to large, immature RBCs (called megaloblasts) in the bone marrow. Causes: 1. Vitamin B12 deficiency: Due to malabsorption (e.g. pernicious anemia, GIT surgery). 2. Folate deficiency: Caused by poor diet or increased requirements (e.g. pregnancy). 3. Medications: Drugs that interfere with DNA synthesis (methotrexate, anticonvulsants). Note: Pernicious anemia is a type of megaloblastic anemia caused by vitamin B12 deficiency. It occurs when the body cannot absorb enough vitamin B12 due to the lack of intrinsic factor, a protein produced by the stomach that is essential for vitamin B12 absorption in the small intestine. B. Non-Megaloblastic Macrocytic Anemia Macrocytic anemia resulting from other factors affecting RBC production. Common Causes: 1. Liver disease: Impaired lipid metabolism affects RBC membrane development. 2. Hypothyroidism: Slowed metabolism affects erythropoiesis. 3. Aplastic anemia: BM failure to produce all blood cells, including RBCs. 4. Hemolysis: Increased RBCs turnover leading to compensatory macrocytosis. 5. Alcoholism: Direct toxic effects on bone marrow and RBCs production. Laboratory Diagnosis of Megaloblastic Anemia 1. Complete Blood Count: RBCs count and HCT are ↓, MCV is ↑, and MCH and MCHC are normal. Leucopenia and thrombocytopenia may be present in severe cases. Blood film shows: − Macrocytic RBCs (oval macrocytes), along with anisocytosis and poikilocytosis. − Hypersegmented neutrophils (neutrophils with more than 5 nuclear lobes) are typically seen, which is a characteristic feature of megaloblastic anemia. − RBC inclusions e.g. basophilic stippling and Howell-Jolly bodies may be present. 2. Bone marrow examination (if needed) Shows a picture of ineffective erythropoiesis in the form of: Hypercellularity, trying to compensate for ineffective erythropoiesis. The presence of megaloblasts (large immature RBC precursors with abnormal nuclear maturation due to defective DNA synthesis). 3. Serum Vitamin B12 Levels Low in cases of vitamin B12 deficiency (pernicious anemia, malabsorption, etc.). 4. Serum Folate Levels Low in folate deficiency (poor dietary intake, alcoholism, pregnancy … etc.). 5. Autoantibodies of pernicious anemia Such as anti-parietal cells and intrinsic factor antibodies (in cases of B12 deficiency). 6. Therapeutic trial: A specific dose of vitamin B12 and folate is administered, and the response is monitored through the reticulocyte count. In successful cases, the reticulocyte count typically begins to increase by the 3rd day and reaches a peak around the 7th day. Page | 6  III. Normocytic Normochromic Anemias A type of anemia characterized by a decreased number of RBCs while the RBCs remain normal in size and hemoglobin content. This condition occurs when RBCs are either lost, destroyed, or inadequately produced, resulting in anemia, but the RBCs themselves appear structurally normal. Common causes include acute blood loss, anemia of chronic disease, chronic kidney disease, and hemolytic anemia. Causes of Normocytic Normochromic Anemia A. Blood Loss Acute Blood Loss: Trauma, surgery, gastrointestinal bleeding, or hemorrhage. B. Decreased RBC Production 1. Anemia of Chronic Disease: Chronic infections, inflammation, or malignancies. 2. Chronic Kidney Disease (due to ↓ erythropoietin production). 3. Aplastic Anemia: BM failure due to autoimmune destruction, toxins, or radiation. 4. BM Disorders: Myelodysplastic syndromes, leukemia, or infiltration by cancer. 5. Endocrine Disorders: Hypothyroidism, hypopituitarism, adrenal insufficiency. 6. Early Stages of Nutritional Deficiency: Early iron, vitamin B12, or folate deficiency (before progressing to microcytic or macrocytic anemia). C. Increased RBC Destruction (Hemolysis) Hemolytic Anemia caused by autoimmune hemolysis, hereditary conditions (e.g. sickle cell anemia, G6PD deficiency), or external factors like toxins, infections, or medications. Laboratory Diagnosis of Aplastic Anemia Aplastic anemia is a rare disorder characterized by the failure of the BM to produce sufficient blood cells, leading to pancytopenia (a deficiency of blood cells: RBCs, WBCs, and platelets). The laboratory diagnosis of aplastic anemia involves a combination of blood tests and BM marrow examination to confirm the condition 1. Complete Blood Count Pancytopenia: ↓ RBCs, WBCs, and platelets counts. ↓ Hb and HCT (due to ↓ RBCs production). MCV: Usually normal, although it can sometimes be slightly elevated due to the release of immature RBCs (which are larger in size). Reticulocytopenia (reflecting BM failure to produce new RBCs). 2. Bone Marrow Biopsy and Aspiration Hypocellular bone marrow: The hallmark of aplastic anemia. The BM is replaced by fat and stromal cells, with less than 25% of normal cellularity. No evidence of fibrosis or malignancy. 3. Serum Iron and Ferritin Since the BM is not using iron for erythropoiesis, serum iron and ferritin levels may be normal or elevated. 4. Investigations for the cause: Viral Serologies for Hepatitis, EBV, CMV, HIV as these infections can be associated with secondary aplastic anemia. Autoimmune and Genetic Testing may be considered, especially in younger patients or in cases where congenital aplastic anemia is suspected. Page | 7  Hemolytic Anemias Hemolytic anemia is a type of anemia that results from an increased rate of RBCs destruction, which can occur either intravascularly (within the blood vessels) or extravascularly (within the reticuloendothelial system, primarily in the spleen and liver). Clinical Features of Hemolysis 1. Anemia (pallor, fatigue, dizziness, tachycardia, shortness of breath) 2. Jaundice 3. Dark urine (hemoglobinuria) Complications of chronic hemolysis 1. Splenomegaly. 2. Pigment stones. 3. Leg ulcers. 4. Iron overload. Laboratory Evidence of Hemolysis 1. ↓ Hemoglobin level. 2. ↑ Indirect bilirubin (because of the breakdown of hemoglobin). 3. Reticulocytosis: Increased production of immature RBCs (a compensation for RBC loss). 4. ↓ Haptoglobin (Haptoglobin binds free Hb, and its levels drop in hemolysis). 5. ↑ LDH (LDH Released from lysed RBCs, indicating their destruction). 6. Presence of schistocytes (RBC fragments) and microspherocytes in peripheral blood. Laboratory Evidence for Intravascular Hemolysis 1. Laboratory Evidence of Hemolysis: As previously mentioned. 2. Plasma free hemoglobin: ↑ (due to Hb release from lysed RBCs in blood). 3. Hemoglobinuria and/or Hemosiderinuria: Caused by the presence of free hemoglobin in the urine due to RBCs destruction, with hemoglobinuria occurring shortly after hemolysis and hemosiderinuria developing later as iron is deposited in the kidneys. Laboratory Evidence for Extravascular Hemolysis 1. Laboratory Evidence of Hemolysis: As previously mentioned. 2. Direct Antiglobulin Test (Direct Coombs Test): Positive − Detects IgG or complement on the surface of RBCs. − Method: Coombs reagent (anti-human Ig antibody) is added to the patient’s RBCs; agglutination indicates a positive result. 3. Indirect Antiglobulin Test (Indirect Coombs Test): Positive − Detects antibodies in the serum that can recognize antigens on RBCs. − Method: Mix the patient’s serum with donor RBCs (group O) and Coombs reagent (anti-human Ig antibody); agglutination indicates a positive result. Classifications of hemolytic anemias Page | 8  Hereditary Hemolytic Anemia I. Hereditary Hemolytic Anemia caused by Enzyme Defects These hemolytic anemias are caused by inherited mutations that affect RBC enzymes, leading to premature destruction of RBCs. The most common deficiencies include: A. Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency (Favism) G6PD is an enzyme that protects RBCs from damage caused by oxidative stress, which can be triggered by conditions such as infections, medications (like sulfonamides, antimalarials, and nitrofurantoin), or foods such as fava beans. When G6PD levels are insufficient, RBCs tolerate this stress, leading to membrane damage and hemolysis. Laboratory Diagnosis 1. Complete Blood Count: ↓ Hb, ↓ RBCs count, ↓ HCT with normal MCV. Reticulocytosis (compensatory RBC production by the BM). Peripheral Blood Smear shows characteristic findings: a. Heinz bodies: These are RBCs inclusions composed of oxidized HB caused by oxidative damage. Supravital stains (e.g. brilliant cresyl blue) are used to identify Heinz bodies on a blood smear, as they are not visible on routine Wright’s or Leishman stain. b. Bite cells: These are RBCs that look like a small "piece" has been removed from their edge, giving them a bitten appearance. This occurs when splenic macrophages remove Heinz bodies from inside the RBCs (Splenic Pitting). c. Spherocytes and microspherocytes: These are formed when RBCs lose part of their membrane, making them smaller and round. As a result, they become less flexible and are more easily destroyed in the spleen. d. Polychromasia: A condition where RBCs appear bluish or grayish on a blood smear due to residual RNA, indicating increased production of immature RBCs by the BM. 2. Other Laboratory Evidences of hemolysis: ↑ Indirect Bilirubin (due to RBC breakdown). ↑ Lactate Dehydrogenase (LDH), reflecting RBC destruction. ↓ Haptoglobin (as Haptoglobin binds free Hb released from lysed RBCs). 3. G6PD Enzyme Assay The enzyme assay shows ↓ G6PD activity caused by G6PD deficiency. 4. Genetic Testing: Confirms mutations in the genes responsible for the enzyme defects. 5. Laboratory Tests to exclude other Causes of hemolysis: Direct Coombs Test: Negative (used to rule out immune- hemolytic anemia). Osmotic Fragility Test: Normal (used to exclude hereditary spherocytosis). B. Pyruvate Kinase (PK) Deficiency PK deficiency disrupts glycolysis, reducing ATP production in RBCs, leading to cell membrane damage and premature destruction in the spleen. Laboratory Diagnosis 1. Complete Blood Count: Normocytic anemia with normal RBCs morphology. 2. PK Enzyme Assay: Shows ↓ PK activity. Page | 9 II. Hereditary Hemolytic Anemia caused by RBCs Membrane Defects Refers to a group of inherited disorders characterized by defects in proteins that maintain the RBC membrane's shape and flexibility, leading to increased fragility and destruction of RBCs, resulting in chronic hemolytic anemia. Examples of these disorders include hereditary spherocytosis, hereditary elliptocytosis, and hereditary stomatocytosis. A. Hereditary Spherocytosis (HS) An autosomal dominant disorder caused by mutations in membrane proteins such as ankyrin, spectrin, band 3, or protein 4.2. The defective membrane proteins cause a loss of membrane surface area, resulting in spherical-shaped RBCs (spherocytes). These spherocytes are less flexible and are easily destroyed in the spleen. Laboratory Diagnosis 1. Complete Blood Count: Mild to moderate anemia. MCHC ↑. Reticulocytosis. Blood smear shows spherocytes (i.e. round RBCs without central pallor). 2. Osmotic Fragility Test: Increased osmotic fragility due to reduced membrane surface area, causing RBCs to lyse more easily in hypotonic solutions. 3. Eosin-5'-Maleimide (EMA) Dye Binding Test: The test uses flow cytometry to measure reduced dye binding to membrane proteins, particularly band 3, resulting in lower fluorescence and aiding diagnosis. 4. Autohemolysis Test: Measures increased RBCs breakdown when a whole blood sample is incubated at 37°C for 24-48 hours, indicating abnormal RBC fragility due to membrane defects. 5. Gel Electrophoresis (SDS-PAGE): Identify defects in membrane proteins e.g. spectrin, ankyrin, band 3, or protein 4.2. 6. Genetic Testing: Detects genes mutations associated with deficient proteins (ANK1, SPTB, SLC4A1). B. Hereditary Elliptocytosis An autosomal dominant disorder caused by mutations affect proteins like spectrin or protein 4.1, which are crucial for maintaining the cytoskeletal structure of RBCs. The structural abnormalities cause the RBCs to become elongated or elliptical (elliptocytes). These cells are fragile and prone to destruction, especially in the spleen. Laboratory Diagnosis 1. Complete Blood Count: Mild to moderate anemia, typically normocytic. Reticulocytosis. Blood smear shows increased elliptocytes (oval or elongated RBCs). 2. Osmotic Fragility Test: Osmotic fragility may be normal or mildly increased. 3. Gel Electrophoresis (SDS-PAGE): Identify defects in membrane proteins: spectrin, protein 4.1, or others. 4. Genetic Testing: Detects genes mutations associated with deficient proteins (SPTA1, SPTB, or EPB41). Page | 10 III. Hereditary Hemolytic Anemia caused by Hemoglobin Defects (Hemoglobinopathies) Hemoglobin genes 4 α-globin genes (2 on each copy of chromosome 16) 2 β-globin genes (1 on each copy of chromosome 11) 2 γ-globin genes (1 on each copy of chromosome 11) 2 δ-globin genes (1 on each copy of chromosome 11) Normal Hemoglobin Fractions 1. Hemoglobin A (HbA): − The most common type of hemoglobin in adults, constituting about 96–98%. − Composed of 2 α chains and 2 β chains (α2β2). 2. Hemoglobin A2 (HbA2): − Makes up about 1–4% of adult hemoglobin. − Composed of 2 α chains and 2 δ chains (α2δ2). 3. Hemoglobin F (HbF): − Fetal Hb, dominant in fetuses and newborns, replaced by HbA after birth. − Composed of 2 α chains and 2 γ chains (α2γ2). − Higher affinity for O2 than HbA, facilitating O2 transfer from mother to fetus. Between the ages of 3 to 6 months, HbA gradually replaces HbF, and by 1 year of age, HbA predominates, constituting 96–98% of total Hb. Additionally, HbA2 accounts for approximately 1–4% of adult hemoglobin. Normal adult hemoglobin fractions HbA : 96 – 98% HbA2 : 1 – 4 % HbF : Less than 1% A. Thalassemias Thalassemias are a group of inherited blood disorders characterized by the reduced or absent production of one or more globin chains in hemoglobin, leading to imbalanced hemoglobin synthesis and ineffective RBCs production (erythropoiesis). This results in chronic anemia, which varies in severity. Types of Thalassemia 1. Alpha Thalassemia: Caused by mutations or deletions in the genes responsible for producing the α- globin chains (HBA1 and HBA2 genes). Severity depends on the number of α-globin genes affected. − Silent carrier : 1 gene deleted, no symptoms. − α thalassemia trait : 2 genes deleted, mild anemia. − Hb H disease : 3 genes deleted, moderate to severe anemia. − Hydrops fetalis : 4 genes deleted, often fatal before or shortly after birth. 2. Beta Thalassemia: Caused by mutations in the HBB gene, leading to reduced or absent β-globin chain production. Severity depends on whether one or both beta-globin genes are affected. − β thalassemia minor: One gene is mutated, causing no or mild symptoms. − β thalassemia intermedia: Both genes are mutated but with some residual β- globin production, causing moderate anemia. − β thalassemia major (Cooley's anemia): Both genes are severely mutated or absent, resulting in severe anemia requiring regular blood transfusions. Page | 11  3. Other Types of Thalassemia: Delta (δ) Thalassemia, Delta-Beta (δβ) Thalassemia, and other rare Thalassemias that can affect combinations of globin chains or are associated with unusual mutations in globin genes. β Thalassemia β Thalassemia Major Caused by a defect in both β-globin gene alleles (homozygous, autosomal recessive) resulting in ineffective synthesis of HbA, which leads to ↓ erythropoiesis, hemolysis, and an increase in HbF due to the absence of the normal Hb switch. In beta thalassemia major, hemolysis occurs because the body cannot produce enough β-globin chains, leading to an imbalance between α and β chains. The excess unpaired α chains form toxic aggregates inside RBCs, damaging their structure and causing them to be destroyed prematurely in the spleen, resulting in hemolysis. Clinically − Jaundice and severe anemia: Usually becomes apparent at 3rd to 6th month of age. − Stunted growth of children and delayed puberty. − Bone deformities: Especially in the face and skull due to overactive BM marrow. − Splenomegaly: Enlarged spleen due to excessive RBC destruction. − Iron overload: From frequent transfusions, causing liver and heart problems. Radiologic Findings − Include "hair-on-end" skull appearance, expanded medullary cavities, cortical thinning, and bone deformities due to BM hyperplasia. Osteoporosis, pathologic fractures, and hepatosplenomegaly caused by extramedullary hematopoiesis are also common. Laboratory Investigations 1. Complete Blood Count: Low hemoglobin level (usually 100×109/L; often > 300 ×109/L. (Sometimes decreased) Mostly mature cells Mostly immature cells Blast predominance (neutrophils, bands, and myelocytes) (promyelocytes, myeloblasts) Blasts 2.0 mg/dL A Anemia Hemoglobin