Iron Metabolism and Folate Metabolism
Standard Answer:
1. Distribution of iron Compartment Amount Percentage Haemoglobin (RBCs) 2-2.5 g 65-70% Ferritin (stores) 0.5-1.0 g 15-25% Hemosiderin (stores) 0.3-0.5 g 5-10% Myoglobin 0.1-0.2 g 5-8% Transferrin (plasma) 3-4 mg 0.1% Tissue enzymes 8-12 mg <1% Normal total body iron: • Adult male: 3.5-4.5 g • Adult female: 2.5-3.0 g Daily requirements: • Adult male: 1-2 mg/day • Adult female (premenopausal): 2-3 mg/day • Pregnancy: 5-6 mg/day 2. Proteins involved in Iron metabolism and functions Protein Function Ferritin Intracellular iron storage protein; sequesters iron in soluble, non-toxic form Transferrin Plasma iron transport protein; binds Fe3+ and delivers to tissues Transferrin receptor (TfR1) Cell surface receptor; mediates iron uptake via receptor-mediated endocytosis Hepcidin Key regulatory hormone; inhibits iron absorption and release from stores Ferroportin Only known cellular iron exporter; releases iron from enterocytes, macrophages, hepatocytes HFE protein Regulates hepcidin expression; mutated in hereditary haemochromatosis Ceruloplasmin Ferroxidase; oxidizes Fe2+ to Fe3+ for transferrin binding Hephaestin Ferroxidase; facilitates iron export from enterocytes DMT1 (Divalent Metal Transporter 1) Imports Fe2+ across enterocyte apical membrane Dcytb (Duodenal Cytochrome b) Ferrireductase; reduces Fe3+ to Fe2+ at enterocyte brush border Functions of two proteins: • Hepcidin: Inhibits iron absorption by binding to ferroportin, causing its internalization and degradation; reduces dietary iron absorption and iron release from macrophages and hepatocytes • Transferrin: Binds Fe3+ with high affinity (Kd = 10^-23 M), transports iron to bone marrow for erythropoiesis and other tissues; delivers iron via transferrin receptor-mediated endocytosis 3. Events in Iron metabolism (All Sites) Dietary iron absorption (Duodenum): 1. Ferric iron (Fe3+) reduced to ferrous (Fe2+) by Dcytb 2. Fe2+ imported into enterocyte via DMT1 3. Either stored as ferritin or exported via ferroportin 4. Exported Fe2+ oxidized to Fe3+ by hephaestin 5. Fe3+ binds to transferrin in circulation Transport (Plasma): • Transferrin delivers iron to cells via TfR1 • Iron enters cells via receptor-mediated endocytosis • Iron released from endosome by DMT1 (Steap3 reduces Fe3+ to Fe2+) Storage (Liver, Spleen, Bone marrow): • Iron stored as ferritin (soluble) and hemosiderin (insoluble) • Ferritin = apoferritin shell + iron core Erythropoiesis (Bone marrow): • Iron incorporated into haemoglobin in erythroblasts • Heme synthesis occurs in mitochondria Macrophage iron recycling (Spleen, liver): • Phagocytosis of senescent RBCs • Haemoglobin degradation • Iron released via ferroportin Regulation: • Hepcidin controls ferroportin expression • Hypoxia and erythropoiesis suppress hepcidin • Iron overload and inflammation increase hepcidin 4. Iron metabolism Iron metabolism encompasses the processes of dietary iron absorption, transport in plasma, cellular uptake, utilization, storage, and recycling, tightly regulated by hepcidin to maintain iron homeostasis. Key features: • No active excretion mechanism • Regulation primarily at absorption (duodenum) and macrophage recycling (spleen) • Tight control by hepcidin (negative regulator) • Iron circulates bound to transferrin • Storage in ferritin and hemosiderin • Iron is utilized for haemoglobin synthesis in erythroid precursors • 90% of daily iron requirement comes from recycling of senescent RBCs 5. Causes of Iron Deficiency Anemia Dietary causes: • Inadequate dietary intake • Vegetarian/vegan diet • Poor socioeconomic status • Malabsorption Increased requirements: • Pregnancy • Lactation • Childhood/adolescent growth spurts Blood loss: • Menstrual blood loss • Gastrointestinal bleeding (peptic ulcer, colon cancer, diverticulosis) • Hookworm infestation • Hematuria • Frequent blood donation Malabsorption: • Coeliac disease • Gastric surgery (gastrectomy, bypass) • Achlorhydria (proton pump inhibitors) • Inflammatory bowel disease • Helicobacter pylori infection 6. Laboratory findings in iron deficiency anemia Peripheral blood film: • Microcytic (MCV < 80 fL) • Hypochromic (MCHC < 32%) • Anisocytosis (RDW increased) • Poikilocytosis • Target cells, pencil cells (cigar cells) • Elliptocytes Haematology: • Low Hb and PCV • Low MCV, MCH, MCHC • Increased RDW • Normal or low WBC • Normal or increased platelets Iron studies: • Low serum iron • Low serum ferritin (< 15-30 ng/mL) • High TIBC (> 360 ug/dL) • High transferrin (> 300 mg/dL) • Low transferrin saturation (< 15%) • Increased soluble transferrin receptor (sTfR) • Decreased serum hepcidin Bone marrow: • Decreased or absent stainable iron (Prussian blue stain) Others: • Elevated FEP (free erythrocyte protoporphyrin) • Elevated sTfR/log ferritin ratio • Low reticulocyte count 7. Classification of causes of iron deficiency anaemia I. Decreased intake/availability: • Dietary deficiency • Malabsorption (coeliac, gastrectomy, achlorhydria) • Increased requirements (pregnancy, growth) II. Increased loss: • Gastrointestinal bleeding • Menstrual bleeding • Urinary bleeding • Pulmonary bleeding • Chronic blood donation III. Increased utilization: • Pregnancy • Lactation • Growth in children IV. Defective utilization: • Iron-refractory iron deficiency anaemia (IRIDA) • Atransferrinaemia 8. Ten lab investigations for iron deficiency anemia 1. Full Blood Count (FBC) with differential 2. Peripheral blood film examination 3. Serum iron 4. Serum ferritin 5. Total Iron Binding Capacity (TIBC) 6. Transferrin saturation percentage 7. Serum transferrin 8. Soluble transferrin receptor (sTfR) 9. Reticulocyte count 10. Bone marrow aspirate with iron stain (Prussian blue) - gold standard 11. Free Erythrocyte Protoporphyrin (FEP) 12. Hepcidin levels 13. Stool occult blood testing 14. Haemoglobin electrophoresis (to exclude thalassemia trait) 9. Laboratory features of Iron deficiency anaemia • Haemoglobin: Low (mild to severe) • MCV: Low (< 80 fL) • MCH: Low (< 27 pg) • MCHC: Low (< 32%) • RDW: High (> 15%) • Platelets: Often elevated (reactive thrombocytosis) • WBC: Usually normal • Reticulocyte count: Low for degree of anaemia • Serum iron: Low (< 50 ug/dL) • TIBC: High (> 360 ug/dL) • Transferrin saturation: Low (< 15%) • Ferritin: Low (< 15-30 ng/mL) • sTfR: Elevated • FEP: Elevated (> 70 umol/mol haem) • Bone marrow iron: Absent or markedly reduced Blood film findings: • Microcytes, hypochromic cells • Anisocytosis, poikilocytosis • Pencil cells • Target cells (occasional) • Elliptocytes 10. Differentiation between Iron deficiency anaemia and Anaemia of chronic disease Parameter Iron Deficiency Anaemia Anaemia of Chronic Disease Serum iron Low Low Ferritin Low (< 15-30 ng/mL) Normal or high (> 100 ng/mL) TIBC High (> 360 ug/dL) Low or normal Transferrin saturation Low (< 15%) Low (15-20%) sTfR High Normal or low FEP High Normal or high Bone marrow iron Absent Present Hepcidin Low High Inflammatory markers Normal Elevated (CRP, ESR) Response to iron therapy Good Poor Underlying condition Blood loss, diet Inflammation, infection, malignancy 11. Iron Overload Iron overload is the pathological accumulation of iron in body tissues leading to organ damage. Primary (Genetic): • Hereditary Haemochromatosis (HFE mutations: C282Y, H63D) • Juvenile haemochromatosis (HJV, HAMP mutations) • African iron overload • Aceruloplasminaemia Secondary (Acquired): • Transfusional iron overload (thalassemia, MDS, aplastic anaemia) • Chronic liver disease • Ineffective erythropoiesis (thalassemia, sideroblastic anaemia) • Excessive dietary iron (African iron overload) • Porphyria cutanea tarda Pathophysiology: • Iron deposition in organs (liver, heart, pancreas, joints, skin, pituitary) • Oxidative damage via Fenton reaction • Lipid peroxidation • Fibrosis and organ failure Clinical features: • Fatigue, weakness • Arthralgia • Hepatomegaly, cirrhosis, hepatocellular carcinoma • Diabetes mellitus (bronze diabetes) • Cardiomyopathy, heart failure • Skin pigmentation (bronze skin) • Hypogonadism, impotence • Pituitary dysfunction Investigations: • Serum ferritin: elevated (> 300 ug/L males, > 200 ug/L females) • Transferrin saturation: elevated (> 45-50%) • Serum iron: elevated • Liver function tests • Liver biopsy with iron quantification (hepatic iron index) • MRI iron quantification • Genetic testing (HFE mutations) • Cardiac evaluation (ECG, echocardiogram) Treatment: • Phlebotomy (venesection) • Iron chelation (deferoxamine, deferasirox, deferiprone) • Dietary iron restriction • Avoid vitamin C supplements 12. Iron deficiency anemia in 58-year-old patient A. Five causes of iron deficiency in this patient: 1. Chronic gastrointestinal bleeding from peptic ulcer disease 2. Omeprazole-induced achlorhydria impairing iron absorption 3. Bleeding from gastric or duodenal ulceration 4. Gastric cancer (increased risk with long-term PPI use) 5. Coeliac disease (may be unmasked by PPI) 13. Short notes Hepcidin: • 25-amino acid peptide hormone produced by hepatocytes • Key regulator of systemic iron homeostasis • Inhibits iron absorption (duodenum) and iron release from macrophages and hepatocytes • Binds to ferroportin, causing internalization and degradation • Regulated by: iron status (increased by iron loading), erythropoiesis (suppressed by erythropoietic demand), hypoxia (suppressed), inflammation (increased via IL-6) • Mutations cause: iron overload (low hepcidin) or anaemia of inflammation (high hepcidin) Movement of dietary iron to erythron: 1. Dietary Fe3+ reduced to Fe2+ by Dcytb in duodenal brush border 2. Fe2+ imported into enterocyte via DMT1 3. Transported across enterocyte to basolateral membrane 4. Exported via ferroportin (Fe2+ -> Fe3+ by hephaestin) 5. Fe3+ binds to apotransferrin forming transferrin-iron complex 6. Transferrin delivers iron to erythroblasts via TfR1 7. Receptor-mediated endocytosis 8. Iron released from endosome (Steap3, DMT1) 9. Transported to mitochondria for haem synthesis 10. Haemoglobin synthesized in erythroid precursors 11. Regulated by hepcidin (blocks ferroportin when iron sufficient) 14. Hepcidin and control of expression Hepcidin expression control: Stimulators (increase hepcidin): • Iron loading (via BMP-SMAD pathway) • Inflammation (IL-6 via JAK-STAT pathway) • Lipopolysaccharide • High iron stores • Hepcidin itself Suppressors (decrease hepcidin): • Anaemia (HIF-2alpha) • Hypoxia • Erythropoietic activity (erythroferrone released by erythroblasts) • Iron deficiency • Androgens • Vitamin D Role of hepcidin in iron metabolism: • Negative regulator of iron absorption • Blocks ferroportin-mediated iron efflux • Reduces dietary iron absorption • Inhibits iron release from macrophages • Decreases iron mobilization from hepatocytes • Maintains iron homeostasis • Dysregulation causes: iron overload (deficiency) or anaemia (excess) 15. Hereditary haemochromatosis Definition: Autosomal recessive disorder characterized by excessive iron absorption and progressive iron deposition in parenchymal organs. Genetics: • Most common: HFE gene mutations (chromosome 6) • C282Y homozygosity (most severe) • C282Y/H63D compound heterozygosity • H63D homozygosity (milder) • Other mutations: HJV, HAMP, TFR2 (rare) Pathophysiology: • Mutant HFE protein fails to regulate hepcidin • Reduced hepcidin -> increased ferroportin -> increased iron absorption • Iron overload in liver, heart, pancreas, joints, skin Clinical features: • Early: fatigue, arthralgia, impotence • Late: cirrhosis, hepatocellular carcinoma, diabetes, cardiomyopathy, hypogonadism • Bronze skin pigmentation Diagnosis: • Elevated ferritin (> 300 ug/L men, > 200 ug/L women) • Elevated transferrin saturation (> 45-50%) • HFE genetic testing • Liver biopsy (if indicated) • MRI iron quantification Treatment: • Therapeutic phlebotomy (goal: ferritin < 50 ug/L) • Iron chelation (if phlebotomy not feasible) • Liver transplant (cirrhosis, HCC) • Annual monitoring for liver cancer 16. Causes of folate deficiency Dietary causes: • Inadequate intake (poverty, elderly, fad diets) • Poor diet (lack of green vegetables) • Overcooking vegetables (destroys folate) • Goat's milk diet in infants Increased requirements: • Pregnancy and lactation • Childhood growth spurts • Haemolytic anaemia (increased erythropoiesis) • Malignancy • Chronic inflammatory diseases Malabsorption: • Coeliac disease • Tropical sprue • Crohn's disease • Gastrectomy • Jejunal resection Drugs: • Methotrexate (dihydrofolate reductase inhibitor) • Phenytoin • Sulfasalazine • Trimethoprim • Oral contraceptives Increased losses: • Dialysis • Chronic haemorrhage Alcoholism: • Poor diet • Malabsorption • Impaired hepatic storage 17. Haematological findings of folate deficiency Peripheral blood: • Macrocytic anaemia (MCV > 100 fL) • Normochromic or slightly hyperchromic • Oval macrocytes • Hypersegmented neutrophils (> 5 lobes) • Neutropenia, thrombocytopenia (may occur) • Anisocytosis, poikilocytosis • Basophilic stippling Bone marrow: • Hypercellular • Erythroid hyperplasia • Megaloblastic maturation (nuclear-cytoplasmic asynchrony) • Giant metamyelocytes • Megakaryocytes with abnormal morphology • Increased iron stores Biochemistry: • Reduced serum folate (< 4 ng/mL) • Reduced RBC folate (< 150 ng/mL) - more reliable • Homocysteine elevated • Methylmalonic acid (MMA) normal (differentiates from B12 deficiency) • Increased LDH • Increased indirect bilirubin • Decreased haptoglobin Haematology: • Low Hb, PCV • High MCV, MCH • Low or normal MCHC • Reticulocyte count low (inadequate response) 18. Five major causes of folate deficiency with examples 1. Inadequate dietary intake • Poverty and malnutrition • Elderly with poor diet 2. Malabsorption • Coeliac disease • Tropical sprue 3. Increased requirements • Pregnancy • Haemolytic anaemia 4. Drug-induced • Methotrexate (DHFR inhibitor) • Phenytoin 5. Excessive loss • Chronic haemodialysis • Chronic blood loss
Anaemia
Standard Answer:
1. Anaemia Anaemia is a reduction in the haemoglobin concentration, red blood cell count, or packed cell volume below the normal reference range for age, sex, and physiological state, resulting in decreased oxygen-carrying capacity of blood. Normal values: • Adult males: Hb < 13 g/dL • Adult females: Hb < 12 g/dL • Pregnant females: Hb < 11 g/dL • Children: Age-dependent 2. Classification of Anemias Morphological classification (based on MCV): • Microcytic (MCV < 80 fL): Iron deficiency, thalassemia, sideroblastic anaemia, anaemia of chronic disease • Normocytic (MCV 80-100 fL): Anaemia of chronic disease, acute blood loss, haemolytic anaemia, aplastic anaemia, renal failure • Macrocytic (MCV > 100 fL): Megaloblastic (B12, folate deficiency), alcohol liver disease, MDS, hypothyroidism, reticulocytosis Pathophysiological classification: • Reduced production: • Iron deficiency • B12/folate deficiency • Bone marrow failure (aplastic anaemia, MDS) • Anaemia of chronic disease • Renal anaemia • Endocrine anaemia • Increased destruction (Haemolytic): • Inherited (membrane, enzyme, haemoglobin disorders) • Acquired (immune, microangiopathic, infection) • Blood loss: • Acute • Chronic Five tests for investigating anaemia: 1. Full Blood Count (FBC) with differential 2. Peripheral blood film 3. Reticulocyte count 4. Iron studies (serum iron, ferritin, TIBC, transferrin saturation) 5. Vitamin B12 and folate levels 6. Haemoglobin electrophoresis 7. Coombs test (direct and indirect) 8. Bone marrow aspiration and biopsy 9. Renal function tests 10. Liver function tests 3. Haemolytic anaemia Haemolytic anaemia is a type of anaemia caused by increased destruction (lysis) of red blood cells exceeding the bone marrow's compensatory erythropoietic capacity, leading to reduced RBC survival and accumulation of haemoglobin degradation products (bilirubin, LDH, decreased haptoglobin). 4. Inherited haemolytic anemias - classification and discussion Classification: 1. Membrane defects: • Hereditary spherocytosis (spectrin, ankyrin, band 3 defects) • Hereditary elliptocytosis (spectrin defects) • Hereditary stomatocytosis 2. Enzyme defects: • Glucose-6-phosphate dehydrogenase (G6PD) deficiency • Pyruvate kinase (PK) deficiency • Hexokinase deficiency 3. Haemoglobin defects: • Sickle cell anaemia (HbS) • Thalassemias (alpha, beta) • Unstable haemoglobins (HbE, HbF) • Haemoglobinopathies (HbC, HbD, HbE) Discussion of Sickle cell anaemia: • Genetic: Autosomal recessive; point mutation in beta-globin gene (GAG -> GTG; Glu -> Val at position 6) • Pathogenesis: Mutant HbS polymerizes under hypoxic/dehydrated conditions -> sickled RBCs -> vaso-occlusion, haemolysis • Clinical: Painful crises, acute chest syndrome, stroke, avascular necrosis, splenic sequestration • Diagnosis: Hb electrophoresis showing HbS > 80%, HbF elevated • Treatment: Hydroxyurea, blood transfusions, penicillin prophylaxis, stem cell transplantation 5. Acquired haemolytic anaemia - classification and examples Classification: 1. Immune-mediated: • Autoimmune (warm, cold, drug-induced) • Alloimmune (transfusion reactions, HDN) • Drug-induced immune haemolytic anaemia 2. Non-immune: • Microangiopathic (TTP, HUS, DIC, HELLP) • Mechanical (heart valves, march haemoglobinuria) • Chemical (lead poisoning, snake venom) • Infectious (malaria, Clostridium, Bartonella) • Paroxysmal nocturnal haemoglobinuria (PNH) • Thermal injury Examples: • Warm autoimmune haemolytic anaemia: Idiopathic, SLE, CLL, lymphoma • Cold haemagglutinin disease: Mycoplasma infection, EBV, lymphoma • Drug-induced: Penicillin, methyldopa, quinine • TTP: ADAMTS13 deficiency • Malaria: Plasmodium falciparum • PNH: Somatic mutation in PIGA gene 6. Four examples of haemolytic anaemia • Sickle cell anaemia • Hereditary spherocytosis • Autoimmune haemolytic anaemia (warm type) • G6PD deficiency 7. Laboratory features of autoimmune haemolytic anaemia Haematology: • Anaemia (variable severity) • Reticulocytosis (high) • Normal MCV (or elevated due to reticulocytes) • Platelets normal or low (Evans syndrome) Peripheral blood film: • Spherocytes • Polychromasia • Nucleated RBCs • Autoagglutination (cold type) • Schistocytes (if associated with microangiopathy) Biochemistry: • Elevated LDH • Elevated indirect bilirubin • Decreased haptoglobin • Elevated reticulocyte count • Increased urine urobilinogen Immunology: • Direct Coombs test (DAT): Positive • Cold agglutinin titres (cold type) • IgG and C3d coating on RBCs Bone marrow: • Erythroid hyperplasia (compensatory) 8. Inherited haemolytic anaemias - classification and discussion (Same as question 4 above) 9. Treatment of warm antibody autoimmune haemolytic anaemia First-line: • Corticosteroids (prednisolone 1-2 mg/kg/day) • Response in 70-80% patients within 2-3 weeks Second-line: • Splenectomy (after 3-6 months if no response) • Rituximab (monoclonal anti-CD20 antibody) • Immunosuppressants (azathioprine, cyclophosphamide, mycophenolate mofetil) Supportive: • Blood transfusion (if severe anaemia, for life-threatening) • Folate supplementation (due to increased erythropoiesis) • IVIG (immunoglobulin) in refractory cases • Avoid transfusion if possible (may worsen haemolysis) • Transfuse with least incompatible blood if necessary Newer therapies: • Complement inhibitors (eculizumab for complement-mediated cases) 10. Sickle cell disease and clinical syndromes Sickle cell disease: Group of inherited haemoglobin disorders characterized by the presence of haemoglobin S (HbS) due to a point mutation in the beta-globin gene (Glu6Val), causing sickling of RBCs under deoxygenated conditions. Clinical syndromes of sickle cell anaemia: 1. Vaso-occlusive crises: • Most common acute complication • Pain (bones, chest, abdomen, joints) • Triggered by infection, dehydration, hypoxia, cold, stress • Dactylitis (hand-foot syndrome) in infants 2. Acute chest syndrome: • Chest pain, fever, cough, hypoxia • Infiltrates on CXR • Leading cause of death in adults 3. Splenic sequestration: • Sudden enlargement of spleen • Severe anaemia, hypovolaemic shock • Most common in children (1-5 years) 4. Aplastic crisis: • Transient suppression of erythropoiesis • Usually triggered by Parvovirus B19 infection • Severe anaemia with absent reticulocytes 5. Stroke: • Overt stroke (2-4% by age 20) • Silent cerebral infarcts (more common) • Most common cause is MCA occlusion 6. Chronic complications: • Avascular necrosis of femoral humeral heads • Leg ulcers • Priapism • Renal dysfunction • Pulmonary hypertension • Retinopathy • Gallstones (pigment stones) • Osteomyelitis (Salmonella) 11. Clinical features, haematological findings, treatment of sickle cell anaemia Clinical features: • Chronic haemolytic anaemia (fatigue, pallor, jaundice) • Painful crises (bone, chest, abdomen) • Dactylitis (hand-foot syndrome in children) • Splenomegaly (children) -> autosplenectomy (adults) • Growth retardation • Delayed puberty • Leg ulcers • Priapism • Stroke (motor deficits) • Acute chest syndrome • Avascular necrosis Haematological findings: • Hb: 5-9 g/dL (steady state) • Peripheral blood film: sickled cells, target cells, Howell-Jolly bodies, polychromasia • Reticulocytosis: 10-20% • WBC: Elevated (due to inflammation) • Platelets: Elevated (reactive) • Bilirubin: Elevated (unconjugated) • LDH: Elevated • Haptoglobin: Decreased • Hb electrophoresis: HbS > 80%, HbF 5-15%, HbA2 normal, HbA absent • Sickling test: Positive • Solubility test: Positive Management options: 1. Symptomatic therapy: • Adequate hydration • Analgesia (NSAIDs, opioids) • Oxygen therapy • Warmth 2. Disease-modifying: • Hydroxyurea (increases HbF, reduces crises) • Chronic transfusions (for stroke prevention, severe disease) • Iron chelation (for transfusional iron overload) 3. Infections: • Pneumococcal vaccination (PCV13, PPSV23) • Haemophilus influenzae vaccination • Penicillin prophylaxis (children < 5 years) • Prompt treatment of infections 4. Complications: • Acute chest syndrome: exchange transfusion, antibiotics • Stroke: chronic transfusion, chelation • Splenic sequestration: emergency transfusion, splenectomy (recurrent) 5. Curative: • Haematopoietic stem cell transplantation (HSCT) • Gene therapy (CRISPR, lentiviral vectors) 6. Supportive: • Folic acid supplementation • Nutritional support • Psychological support • Genetic counselling 12. Causes of Sickle cell anaemia with examples Causes: 1. Homozygous HbSS: Inheritance of HbS gene from both parents 2. Compound heterozygous: • HbS + HbC (HbSC disease) • HbS + beta-thalassemia (HbS/beta-thalassemia) • HbS + HbD • HbS + HbE • HbS + HbO-Arab Examples for each: • HbSS: West African origin, autosomal recessive inheritance • HbSC: West African, milder than HbSS • HbS/beta0-thalassemia: Mediterranean, African, Asian, severe as HbSS • HbS/beta+-thalassemia: Milder, variable severity 13. Stages of development of anaemia and findings Stage Findings Stage 1: Iron depletion Low ferritin, normal iron, normal TIBC, normal Hb, normal MCV Stage 2: Iron-deficient erythropoiesis Low ferritin, low iron, high TIBC, low transferrin saturation, normal Hb, normal MCV Stage 3: Early iron deficiency anaemia Low ferritin, low iron, high TIBC, low transferrin saturation, low Hb, low MCV Stage 4: Severe iron deficiency anaemia Very low ferritin, very low iron, very high TIBC, very low transferrin saturation, very low Hb, very low MCV, hypochromic cells, target cells, pencil cells 14. Major clinical crises seen in sickle cell anaemia • Vaso-occlusive crisis (painful crisis) • Acute chest syndrome • Splenic sequestration crisis • Aplastic crisis (Parvovirus B19) • Haemolytic crisis (accelerated haemolysis) • Priapism crisis • Stroke (cerebrovascular crisis) • Hepatobiliary crisis (gallstones, hepatic sequestration) • Renal crisis (papillary necrosis) 15. 3-year-old boy with severe anaemia and splenomegaly A. Most likely diagnosis: Sickle cell anaemia (HbSS) B. Diagnosis approach: • History: Recurrent painful swelling of hands and feet (dactylitis) at 6 months (first manifestation of SCA) • Examination: Severe anaemia (pallor, tachycardia), splenomegaly, jaundice • Investigations: • Full Blood Count: Low Hb, elevated WBC, elevated platelets • Peripheral blood film: Sickled cells, target cells, Howell-Jolly bodies • Reticulocyte count: Elevated • Haemoglobin electrophoresis: HbS > 80%, HbF elevated, HbA absent • Sickling test: Positive • Solubility test: Positive • HPLC (High Performance Liquid Chromatography): Confirms diagnosis • DNA analysis (prenatal/newborn) C. Management options: 1. Acute crisis management: • Hospitalization • IV hydration • Analgesia (NSAIDs, morphine) • Oxygen therapy • Treat infection if present 2. Chronic management: • Penicillin prophylaxis (until age 5) • Pneumococcal vaccines • Hydroxyurea (reduce crises) • Folic acid supplementation • Regular monitoring (FBC, iron studies, organ function) 3. Complication-specific: • Blood transfusion (splenic sequestration) • Exchange transfusion (stroke, acute chest syndrome) • Splenectomy (recurrent sequestration) 4. Curative: • HSCT (haematopoietic stem cell transplantation) • Gene therapy (investigational) 5. Preventive: • Avoid triggers (dehydration, cold, infection, hypoxia) • Genetic counselling • Newborn screening 16. 9-month-old male with swollen hands, feet, jaundice A. Diagnosis: Sickle cell anaemia (HbSS) with dactylitis B. Pathophysiology: • Deoxygenation of HbS -> polymerization -> sickling of RBCs • Sickled RBCs are rigid, less deformable • Vaso-occlusion in small vessels of hands and feet • Microvascular occlusion -> Ischaemia -> Inflammation -> Painful swollen digits • Haemolysis -> Anaemia, jaundice (unconjugated hyperbilirubinaemia) • Spleen involvement -> Splenomegaly (sequestration) • HbS 85% (homozygous HbSS) • HbF 12% (fetal haemoglobin protective) C. Four treatment modalities: 1. Hydration and analgesia (IV fluids, analgesics) 2. Oxygen therapy (if hypoxemic) 3. Blood transfusion (if severe anaemia) 4. Hydroxyurea (long-term disease modification) 5. Antibiotics (if infection suspected) 6. Warmth to affected areas D. Ten possible complications: 1. Vaso-occlusive crises (recurrent pain) 2. Acute chest syndrome 3. Splenic sequestration 4. Aplastic crisis (Parvovirus B19) 5. Stroke 6. Avascular necrosis (hip, shoulder) 7. Leg ulcers 8. Priapism 9. Pulmonary hypertension 10. Chronic renal failure 11. Retinopathy (proliferative) 12. Gallstones (pigment stones) 13. Osteomyelitis (Salmonella) 14. Delayed growth and puberty 15. Cognitive impairment (silent stroke) 17. Ten clinical features and five complications of Sickle Cell Anaemia Ten clinical features: 1. Chronic haemolytic anaemia (pallor, fatigue, jaundice) 2. Painful vaso-occlusive crises (bone, chest, abdomen) 3. Dactylitis (hand-foot syndrome) in infants 4. Acute chest syndrome (chest pain, cough, hypoxia, infiltrates) 5. Splenomegaly (children) -> autosplenectomy (adults) 6. Growth retardation and delayed puberty 7. Leg ulcers (especially in older children/adults) 8. Priapism (males) 9. Stroke (motor deficits, seizures, cognitive impairment) 10. Avascular necrosis (femoral head, humeral head) Five complications: 1. Stroke (cerebrovascular accident) 2. Acute chest syndrome 3. Splenic sequestration crisis 4. Pulmonary hypertension 5. Chronic kidney disease (renal papillary necrosis) 6. Aplastic crisis (Parvovirus B19) 7. Osteomyelitis (Salmonella) 8. Gallstones (pigment stones) 9. Retinopathy 10. Delayed growth and puberty 18. Five important investigations to make a diagnosis 1. Full Blood Count (FBC): Low Hb, elevated WBC, elevated platelets 2. Peripheral blood film: Sickled cells, target cells, polychromasia 3. Reticulocyte count: Elevated (compensatory haemolysis) 4. Haemoglobin electrophoresis: HbS > 80%, HbF elevated, HbA absent 5. Sickling test/Solubility test: Positive (confirms HbS presence) 6. High Performance Liquid Chromatography (HPLC): Quantifies HbS, HbF, HbA2 7. DNA analysis: Confirms beta-globin gene mutation (prenatal/newborn) 8. Bilirubin, LDH, haptoglobin: Confirm haemolysis 19. Management options for sickle cell anaemia 1. Symptomatic/Supportive: • Hydration • Analgesia (NSAIDs, opioids) • Oxygen therapy • Warmth • Rest 2. Disease-modifying: • Hydroxyurea (HbF induction) • Chronic transfusion (stroke prevention, severe disease) • Iron chelation (deferoxamine, deferasirox) 3. Infection prevention: • Pneumococcal vaccination • Penicillin prophylaxis (children < 5 years) • Prompt infection treatment • Malaria prophylaxis (endemic areas) 4. Complication-specific: • Exchange transfusion (acute chest syndrome, stroke) • Blood transfusion (splenic sequestration, aplastic crisis) • Splenectomy (recurrent sequestration) • Cholecystectomy (symptomatic gallstones) 5. Curative: • Haematopoietic stem cell transplantation (HSCT) - matched sibling donor • Gene therapy (CRISPR-Cas9, lentiviral vectors) 6. Psychosocial: • Genetic counselling • Patient education (avoid triggers) • Support groups • School/employment support 20. Sickle cell crisis and habitus Sickle cell crisis: An acute episode of pain or organ dysfunction caused by vaso-occlusion of sickled red blood cells in patients with sickle cell disease, triggered by infection, dehydration, hypoxia, cold, or stress. Types: • Vaso-occlusive crisis (painful crisis) • Acute chest syndrome • Splenic sequestration • Aplastic crisis • Priapism • Stroke Sickle cell habitus: The characteristic physical appearance of patients with sickle cell anaemia, including: • Asthenic (slender) body build • Short stature (growth retardation) • Delayed puberty • Long extremities relative to trunk • Frontal bossing (skull expansion due to marrow hyperplasia) • Maxillary hyperplasia • Pigmented fundal scars (retinopathy) • Leg ulcers • Jaundice • Spleen not palpable (autosplenectomy in adults) 21. Difference between Sickle cell crisis and sickle cell disorder Feature Sickle Cell Crisis Sickle Cell Disorder Definition Acute episode of pain/organ dysfunction Chronic inherited haemoglobinopathy Duration Acute (hours to days) Lifetime Pathophysiology Vaso-occlusion/infarction HbS polymerization, haemolysis Types Vaso-occlusive, sequestration, aplastic, acute chest HbSS, HbSC, HbS/beta-thalassemia Clinical features Pain, fever, organ dysfunction Chronic anaemia, crises, complications Triggers Infection, dehydration, hypoxia, cold Genetic inheritance Treatment Analgesia, hydration, transfusion Disease-modifying, supportive, curative Prognosis Usually resolves with treatment Variable, lifelong management 22. Treatment of Vaso-occlusive Crisis 1. Analgesia: • Mild: Paracetamol/acetaminophen, NSAIDs (ibuprofen, diclofenac) • Moderate-severe: Opioids (morphine, pethidine, tramadol) • Patient-controlled analgesia (PCA) for severe pain • Regular pain assessment 2. Hydration: • IV fluids (0.9% normal saline or 5% dextrose) • 1.5-2 times maintenance • Monitor fluid balance (avoid overload) 3. Oxygen: • If SpO2 < 92% • 2-4 L/min via nasal cannula 4. Address triggers: • Treat underlying infection (antibiotics) • Maintain warmth • Rest 5. Additional: • Blood transfusion (if severe anaemia) • Exchange transfusion (if severe complications) • Inhaled nitric oxide (experimental) • Stem cell transplant (curative, not for crisis) 23. Pathophysiology of Vaso-occlusive crisis in sicklers 1. HbS polymerization: • Deoxygenation (low O2) • Dehydration (increased HbS concentration) • Acidosis (decreased pH) • Increased temperature 2. Sickling of RBCs: • HbS polymerizes into rigid, elongated fibres • RBCs lose deformability • Increased viscosity 3. Microvascular occlusion: • Sickled RBCs obstruct small vessels • Reduced blood flow • Ischaemia and infarction 4. Inflammatory response: • Endothelial activation • Leukocyte adhesion • Cytokine release (IL-1beta, TNF-alpha) • Pain and tissue damage 5. Vicious cycle: • Ischaemia -> Hypoxia -> More sickling -> More occlusion • Inflammation -> Endothelial activation -> More adhesion 24. Steady state Steady state in sickle cell anaemia refers to the baseline clinical and haematological status of a patient without acute complications or crises, characterized by: • Stable haemoglobin level (5-9 g/dL) • No acute pain • No fever • No organ dysfunction • Compensated chronic haemolysis • Reticulocytosis (10-20%) • No acute illness or infection • Baseline laboratory values 25. Differentiation between Macrocytic and Microcytic Anaemia Feature Macrocytic Anaemia Microcytic Anaemia MCV 100 fL < 80 fL MCHC Normal or slightly high Low Causes B12 deficiency, folate deficiency, alcohol, MDS, hypothyroidism, liver disease, reticulocytosis Iron deficiency, thalassemia, sideroblastic anaemia, anaemia of chronic disease RBC morphology Oval macrocytes, hypersegmented neutrophils Microcytes, hypochromic cells, target cells, pencil cells RDW Elevated Elevated (iron deficiency), normal (thalassemia) Reticulocyte count Low (unless haemolysis) Low or normal Bone marrow Megaloblastic changes, giant metamyelocytes Erythroid hyperplasia, absent iron stores Ferritin Normal or high Low (iron deficiency) B12/folate Low (in megaloblastic) Normal Pathophysiology Impaired DNA synthesis (megaloblastic) Reduced haemoglobin synthesis 26. Vitamin B12 Metabolism Dietary sources: • Meat, fish, eggs, dairy products • Bacteria synthesis • Not found in plants Absorption: 1. Dietary B12 released from protein by gastric acid and pepsin (stomach) 2. Binds to salivary R-binders (haptocorrin) 3. Intrinsic factor (IF) produced by parietal cells (stomach) 4. Pancreatic proteases digest R-binders, releasing B12 5. B12 binds to IF in small intestine 6. IF-B12 complex binds to cubilin (receptor) on ileal enterocytes 7. Absorbed via receptor-mediated endocytosis 8. Transports via transcobalamin II (TCII) in plasma Transport: • Transcobalamin I (haptocorrin): 20-25% of serum B12 • Transcobalamin II: 10-20% of serum B12, active transport to tissues • Transcobalamin III Metabolism: • Coenzyme in two reactions: 1. Methylmalonyl-CoA mutase (converts methylmalonyl-CoA to succinyl-CoA) 2. Methionine synthase (converts homocysteine to methionine) Storage: • Liver (1-5 mg, sufficient for 3-5 years) • Muscle Daily requirement: • 2-3 ug/day • Pregnancy: 3-4 ug/day Causes of deficiency: • Inadequate intake (vegans) • Malabsorption (pernicious anaemia, gastrectomy, ileal resection) • Drugs (metformin, proton pump inhibitors) • Increased requirements (pregnancy, haemolysis) • Inherited (transcobalamin II deficiency) 27. Megaloblastic Anaemia Megaloblastic anaemia is a type of macrocytic anaemia characterized by impaired DNA synthesis due to vitamin B12 or folate deficiency, resulting in: • Nuclear-cytoplasmic asynchrony in bone marrow • Enlarged erythroid precursors (megaloblasts) • Ineffective erythropoiesis • Giant metamyelocytes and hypersegmented neutrophils • Macrocytic anaemia 28. Biochemical basis of megaloblastic anaemia 1. B12 deficiency: • Inactive methionine synthase • Reduced methionine synthesis • Reduced tetrahydrofolate (THF) regeneration • Methyl-THF accumulates (trap hypothesis) • Reduced folate polyglutamate synthesis • Impaired DNA synthesis (decreased thymidine) • Nuclear maturation delay • Cytoplasmic maturation continues -> nuclear-cytoplasmic asynchrony 2. Folate deficiency: • Reduced THF • Reduced DNA synthesis • Nuclear maturation delay • Same final pathway as B12 deficiency 3. Consequences: • Megaloblastic changes in bone marrow • Ineffective erythropoiesis (intramedullary death) • Panels of cells: premature cell death • Macrocytosis (due to nuclear lag) 29. Six haematological findings in megaloblastic anaemia 1. Peripheral blood: • Macrocytic anaemia (MCV > 100 fL) • Oval macrocytes • Hypersegmented neutrophils (> 5 lobes) • Neutropenia, thrombocytopenia (severe cases) • Poikilocytosis • Howell-Jolly bodies (if splenic dysfunction) 2. Reticulocyte count: Low (inappropriate) 3. Bone marrow: • Hypercellular • Erythroid hyperplasia • Megaloblasts (nuclear-cytoplasmic asynchrony) • Giant metamyelocytes • Megakaryocytes with abnormal morphology • Increased iron stores 4. Biochemistry: • Elevated LDH • Elevated indirect bilirubin • Reduced serum B12 (B12 deficiency) • Reduced serum folate (folate deficiency) • Elevated homocysteine (both) • Elevated methylmalonic acid (B12 deficiency only) • Decreased haptoglobin (haemolysis) 5. Serum: • Intrinsic factor antibodies (pernicious anaemia) 6. Other: • Increased urinary methylmalonic acid excretion (B12 deficiency only) 30. Megaloblastic anaemias (B12 deficiency) - pathogenesis, clinical features, investigations, management Pathogenesis: • Reduced B12 -> impaired methionine synthase -> THF trap -> reduced DNA synthesis -> nuclear maturation delay -> ineffective erythropoiesis -> pancytopenia Clinical features: • Haematological: Fatigue, pallor, dyspnoea, palpitations, glossitis (beefy red tongue), jaundice • Gastrointestinal: Diarrhoea, anorexia, weight loss, malabsorption • Neurological: • Peripheral neuropathy (sensory loss, paraesthesia) • Subacute combined degeneration of spinal cord (demyelination of posterior and lateral columns) • Ataxia, positive Romberg's sign • Dementia, memory loss, depression • Optic neuropathy • Incontinence • Psychological: Irritability, depression, paranoia Investigations: • FBC: Macrocytic anaemia, neutropenia, thrombocytopenia • Blood film: Oval macrocytes, hypersegmented neutrophils • Reticulocyte count: Low • Serum B12: Low (< 200 pg/mL) • Serum folate: Normal (distinguishes from folate deficiency) • MMA: Elevated (B12-specific) • Homocysteine: Elevated (both B12 and folate) • Schilling test (partial): Defective absorption (pernicious anaemia) • Intrinsic factor antibodies: Type 1 (blocking), Type 2 (binding) • Parietal cell antibodies • MRI spine: T2 hyperintensity in posterior columns (subacute combined degeneration) Management: 1. Treatment: • B12 deficiency: • Cyanocobalamin 1000 ug IM daily for 1 week • Then weekly for 4 weeks • Then monthly maintenance (1000 ug IM) for life • For pernicious anaemia: Lifelong B12 replacement 2. Supportive: • Blood transfusion (if severe anaemia) • Potassium monitoring (hypokalaemia risk with treatment) • Iron supplementation (if concurrent iron deficiency) 3. Underlying cause: • Address cause (diet, gluten-free, treat infection, stop offending drug) • Pernicious anaemia: Monitor for gastric carcinoma 4. Prognosis: • Anaemia responds rapidly (reticulocytosis in 3-5 days) • Neurological improvement slower, may be incomplete 31. G6PD deficiency Definition: X-linked recessive enzyme deficiency of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the hexose monophosphate (pentose phosphate) shunt, which produces NADPH to maintain glutathione in reduced state (protective against oxidative stress). Genetics: • X-linked inheritance • Affects males (hemizygous) • Females: Heterozygotes (variable expression due to Lyonization), homozygotes (severe) • Most common: G6PD A- variant (African), G6PD Mediterranean (Caucasian, Asian) Pathogenesis: • Oxidative stress (drugs, infection, fava beans) -> Reduced NADPH -> Reduced glutathione -> Hb denaturation -> Heinz bodies -> RBC membrane damage -> Haemolysis Clinical features: • Usually asymptomatic • Acute haemolytic crisis (within 24-48 hours of exposure) • Anaemia, jaundice, dark urine (haemoglobinuria) • Neonatal jaundice (especially in G6PD Mediterranean) Triggers: • Drugs: Primaquine, dapsone, sulfonamides, nitrofurantoin, aspirin (high dose), quinine, isoniazid, chloramphenicol • Foods: Fava beans (broad beans) • Infections: Viral hepatitis, pneumonia, typhoid • Others: Diabetic ketoacidosis, neonatal jaundice Investigations: • FBC: Anaemia (normocytic, normochromic), reticulocytosis • Blood film: Heinz bodies (denatured Hb), bite cells (pockets where Heinz bodies removed), blister cells • Biochemistry: Elevated LDH, elevated indirect bilirubin, decreased haptoglobin • G6PD enzyme assay: Decreased activity (during acute episode and after recovery) • G6PD electrophoresis (genetic variants) Treatment: • Remove trigger (stop offending drug) • Supportive care (hydration) • Blood transfusion (severe anaemia) • Neonatal jaundice: phototherapy, exchange transfusion Prevention: • Avoid triggers (list of drugs to avoid) • Genetic counselling • Newborn screening (in high-prevalence areas) 32. Why aplastic anaemia is a misnomer Aplastic anaemia is a misnomer because: • "Aplastic" implies complete absence or failure of development of bone marrow • However, the bone marrow is not truly "aplastic" but rather: • Markedly hypocellular • With fatty replacement • But not completely devoid of haemopoietic cells • May show islands of haematopoiesis • More accurately described as "bone marrow failure" or "hypoplastic anaemia" • Peripheral blood shows pancytopenia (not just anaemia) • May have residual haematopoiesis 33. 68-year-old man with massive spleen, anaemia, thrombocytopenia, leucoerythroblastic picture A. Differential diagnoses: 1. Myelofibrosis (primary or secondary) 2. Chronic myeloid leukaemia (CML) 3. Myelodysplastic syndrome (MDS) with fibrosis 4. Acute myeloid leukaemia (AML) with fibrosis 5. Myeloproliferative neoplasm (MPN) - e.g., polycythaemia vera, essential thrombocythaemia 6. Metastatic carcinoma (bone marrow infiltration) 7. Lymphoma with bone marrow involvement 8. Hairy cell leukaemia 9. Granulomatous disease (TB, sarcoidosis) B. Further tests: 1. Bone marrow aspiration and biopsy (trephine) with reticulin stain 2. Cytogenetics (karyotype, FISH) 3. JAK2 V617F mutation analysis 4. BCR-ABL mutation analysis 5. Calreticulin (CALR) mutation 6. Flow cytometry (immunophenotyping) 7. Peripheral blood film (with careful examination) 8. Serum LDH 9. Imaging (CT chest, abdomen, pelvis) 10. Cytokine assays (if suspected) C. Prognosis based on IPSS (International Prognostic Scoring System) IPSS for MDS: Parameter Score Cytogenetics: Good (normal, -Y, del(5q), del(20q)) 0 Intermediate 0.5 Poor (complex, chromosome 7 abnormalities) 1.0 Bone marrow blasts: < 5% 0 5-10% 0.5 11-20% 1.5 21-30% 2.0 Cytopenias: 0-1 cytopenia 0 2-3 cytopenias 0.5 Risk groups: • Low risk: Score 0 • Intermediate-1: Score 0.5-1.0 • Intermediate-2: Score 1.5-2.0 • High risk: Score >= 2.5 Median survival: • Low: 5.7 years • Int-1: 3.5 years • Int-2: 1.2 years • High: 0.4 years Given the patient's age, massive spleen, leucoerythroblastic picture, and thrombocytopenia, likely prognosis would depend on specific diagnosis and IPSS score. 34. Aplastic Anaemia Aplastic anaemia is a bone marrow failure syndrome characterized by pancytopenia in the peripheral blood and hypocellular bone marrow with fatty replacement, in the absence of significant fibrosis or infiltration. 35. Aplastic Anaemia - Definition Aplastic anaemia is a rare, life-threatening disorder of bone marrow failure characterized by: • Pancytopenia (anaemia, neutropenia, thrombocytopenia) • Hypocellular bone marrow (< 25% cellularity) • Absence of abnormal cells or fibrosis • Impaired haematopoietic stem cell function • Usually acquired (immune-mediated) 36. Diagnosis and Treatment of Aplastic Anaemia Diagnosis: 1. History: Presentation with fatigue, infections, bleeding 2. Examination: Pallor, petechiae, ecchymoses, fever 3. Investigations: • FBC: Anaemia, neutropenia, thrombocytopenia • Reticulocyte count: Low (< 1%) • Bone marrow aspirate and trephine biopsy: Hypocellular (< 25%), fatty replacement, no abnormal cells • Cytogenetics: Normal karyotype (distinguishes from MDS) • Flow cytometry (PNH clone screening) • Viral studies (EBV, CMV, HBV, HCV, HIV, Parvovirus B19) • Vitamins B12, folate (to exclude deficiency) • Iron studies (to exclude deficiency) • Ham's test (PNH) • HLA typing (for HSCT evaluation) Severity classification (Camitta criteria): • Severe AA: Bone marrow cellularity < 25% + 2 of: Neutrophils < 0.5x109/L, Platelets < 20x109/L, Reticulocytes < 1% (or < 20x109/L) • Very severe AA: Same as severe + Neutrophils < 0.2x109/L • Moderate AA: Not fulfilling severe criteria but with pancytopenia Treatment: 1. Supportive care: • Transfusions (RBCs, platelets) • Infection prophylaxis (antibiotics, antifungals) • Prompt treatment of infections • Avoid contact sports (splenomegaly) 2. Definitive therapy: • HSCT: Curative, preferred for young patients (< 40 years), with matched sibling donor • Immunosuppressive therapy (IST): For elderly patients or without donor • ATG (anti-thymocyte globulin) + Cyclosporine A • Eltrombopag (TPO-RA) added to IST 3. Supportive: • G-CSF (for neutropenia) • Erythropoietin (for anaemia, limited use) 37. Ten aetiological factors of Aplastic anaemia 1. Idiopathic (most common) 2. Drugs: • Chemotherapeutic agents (alkylating agents) • Chloramphenicol • NSAIDs • Anticonvulsants 3. Chemicals/toxins: • Benzene • Pesticides • Heavy metals (arsenic) 4. Infections: • Viral hepatitis (non-A, non-B, non-C) • EBV, CMV, HIV • Parvovirus B19 5. Radiation: • Therapeutic (radiotherapy) • Accidental 6. Congenital: • Fanconi anaemia • Diamond-Blackfan anaemia • Dyskeratosis congenita • Shwachman-Diamond syndrome 7. Immune-mediated: • Eosinophilic fasciitis • Thymoma 8. Pregnancy: • Idiopathic (rare) 9. Bone marrow infiltration: • Metastatic carcinoma • Myelofibrosis 10. Autoimmune: • SLE, rheumatoid arthritis 38. Classification of severity of Aplastic anaemia Parameter Severe AA Very Severe AA Moderate AA Bone marrow cellularity < 25% < 25% Not fulfilling severe criteria Neutrophils < 0.5x109/L < 0.2x109/L 0.5x109/L Platelets < 20x109/L < 20x109/L 20x109/L Reticulocytes < 1% or < 20x109/L < 1% or < 20x109/L 1% Other criteria 2 of 3 criteria 2 of 3 criteria Pancytopenia but less severe 39. Complications of stem cell transplantation • Graft failure (primary/secondary) • Graft-versus-host disease (GVHD) - acute and chronic • Infections (bacterial, viral, fungal, protozoal) • Veno-occlusive disease (VOD/SOS) • Mucositis (oral, gastrointestinal) • Haemorrhagic cystitis • Idiopathic pneumonia syndrome • Interstitial pneumonia • Secondary malignancies • Endocrine dysfunction (growth, thyroid, gonadal) • Cataracts • Cardiotoxicity (e.g., anthracyclines) • Pulmonary fibrosis (radiation) • Osteoporosis • Avascular necrosis 40. Anatomic structure and functions of the spleen Anatomic structure: • Location: Left hypochondrium, behind the 9th-11th ribs • Weight: 150 g (adult) • Dimensions: 12 x 7 x 3 cm • Capsule: Connective tissue + smooth muscle • Two surfaces: Diaphragmatic (convex) and visceral (concave) • Hilum: Vascular pedicle (splenic artery, vein) • Two main regions: • White pulp: Periarteriolar lymphoid sheaths (PALS) with T cells, B cell follicles, marginal zone • Red pulp: Splenic cords (Billroth's cords), splenic sinuses, venous sinuses Functions: Red pulp functions: 1. RBC destruction: Senescent RBCs removed (RBC graveyard) 2. Iron recycling: Haemoglobin breakdown -> Iron recycled 3. Extramedullary haematopoiesis: In fetal life and in disease (myelofibrosis, thalassemia) 4. Removal of RBC inclusions: Howell-Jolly bodies, Heinz bodies, siderotic granules 5. Filtering: Removes particulate antigens, microorganisms White pulp functions: 1. Immune surveillance: T and B cell zones 2. Antibody production: Plasma cell differentiation 3. Response to encapsulated organisms: Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis Marginal zone functions: 1. Antigen trapping: Captures blood-borne antigens 2. Immune cell activation: Initiates immune responses Other functions: • Platelet reservoir: Up to 30% of platelet mass • Reticuloendothelial system: Phagocytosis of microorganisms • Lymphocyte production: T and B cell maturation 41. Ten causes of splenomegaly and five cases of massive splenomegaly Ten causes of splenomegaly: 1. Infections: • Malaria • Visceral leishmaniasis (Kala-azar) • Infectious mononucleosis (EBV) • Typhoid fever • Schistosomiasis • HIV/AIDS 2. Haematological: • Haemolytic anaemias (sickle cell, spherocytosis) • Thalassemia (major) • Myeloproliferative neoplasms (CML, polycythaemia vera) • Lymphomas (Hodgkin, Non-Hodgkin) • Leukaemias (ALL, AML, CLL, CML) 3. Infiltrative: • Amyloidosis • Gaucher disease • Sarcoidosis 4. Portal hypertension: • Cirrhosis • Hepatic vein thrombosis 5. Storage diseases: • Gaucher disease • Niemann-Pick disease 6. Rheumatological: • SLE • Rheumatoid arthritis (Felty's syndrome) 7. Endocrine: • Hyperthyroidism 8. Cystic: • Splenic cysts 9. Trauma: • Splenic rupture 10. Congenital: • Congenital splenomegaly (rare) Five causes of massive splenomegaly (> 1000 g): 1. Chronic myeloid leukaemia (CML) 2. Myelofibrosis 3. Visceral leishmaniasis (Kala-azar) 4. Gaucher disease 5. Thalassemia major 6. Polycythaemia vera 7. Hairy cell leukaemia 8. Lymphoma (splenic marginal zone, mantle cell) 42. 72-year-old vegetarian with pancytopenia, MCV 125 fL, oval macrocytes A. Likely diagnosis: Megaloblastic anaemia due to Vitamin B12 deficiency (pernicious anaemia or dietary deficiency) B. Five further tests to confirm diagnosis: 1. Serum Vitamin B12 level 2. Serum folate level 3. Serum methylmalonic acid (MMA) - elevated in B12 deficiency 4. Serum homocysteine - elevated in B12 and folate deficiency 5. Intrinsic factor antibody (IFAB) - for pernicious anaemia 6. Parietal cell antibody 7. Schilling test (if available) C. Five other findings in peripheral blood film: 1. Oval macrocytes (large, oval-shaped RBCs) 2. Hypersegmented neutrophils (> 5 lobes) 3. Anisocytosis (marked variation in size) 4. Poikilocytosis (variation in shape) 5. Howell-Jolly bodies (if asplenic) 6. Basophilic stippling 7. Giant platelets D. Five supportive treatment measures and reasons: 1. Vitamin B12 replacement: • Cyanocobalamin 1000 ug IM daily for 1 week, then weekly for 1 month, then monthly maintenance • Reason: To correct deficiency and prevent neurological progression 2. Blood transfusion: • For severe anaemia (Hb < 7 g/dL, symptomatic) • Reason: To improve oxygen delivery and alleviate symptoms 3. Potassium supplementation/monitoring: • Monitor K+ levels during treatment • Reason: B12 replacement causes rapid erythropoiesis, increased K+ uptake, hypokalaemia risk 4. Iron supplementation (if indicated): • Oral iron if iron deficiency confirmed • Reason: Rapid erythropoiesis may deplete iron stores 5. Supportive care: • Adequate nutrition • Reason: To improve overall nutritional status and support recovery 6. Neurological support: • Physiotherapy (if ataxia) • Reason: To manage neurological complications
Thalassaemias
Standard Answer:
1. Thalassemia and differentiation between Alpha and Beta Thalassemia Thalassemia: Inherited group of haemoglobin disorders characterized by reduced or absent synthesis of one or more globin chains (alpha or beta), leading to imbalanced globin chain production, ineffective erythropoiesis, and haemolysis. Alpha vs Beta Thalassemia: Feature Alpha Thalassemia Beta Thalassemia Affected chain alpha-globin (chromosome 16) beta-globin (chromosome 11) Gene defect Deletion common Point mutation common Types alpha0 (no alpha-chain), alpha+ (reduced alpha-chain) beta0 (no beta-chain), beta+ (reduced beta-chain) Excess chains beta chains (beta4 = HbH) alpha chains (alpha4 = alpha aggregates) Clinical severity Silent carrier, HbH disease, Hydrops fetalis Thalassemia minor, intermedia, major (Cooley's) Prenatal diagnosis DNA analysis (deletion analysis) DNA analysis (mutation analysis) Hb electrophoresis HbH (beta4) in HbH disease HbF increased (beta-thal), HbA2 increased RBC morphology Microcytic, hypochromic, target cells Microcytic, hypochromic, target cells, nucleated RBCs Iron status Normal to increased Normal to increased (transfused) 2. Classification of thalassemias Alpha Thalassemia: • Silent carrier (alpha-thal-2): Single alpha-globin gene deletion (-alpha/alphaalpha) • alpha-thal trait (alpha-thal-1): Two alpha-globin gene deletions (--/alphaalpha or -alpha/-alpha) • HbH disease: Three alpha-globin gene deletions (--/-alpha) • Hydrops fetalis (Hb Bart's): Four alpha-globin gene deletions (--/--) Beta Thalassemia: • beta-thalassemia minor (trait): One beta-globin gene defect (beta/beta0 or beta/beta+) • beta-thalassemia intermedia: Variable severity, often beta+/beta+ or compound heterozygotes • beta-thalassemia major (Cooley's anaemia): Two beta-globin gene defects (beta0/beta0 or beta+/beta+) 3. Differences between alpha and beta thalassemias Feature Alpha Thalassemia Beta Thalassemia Genes 2 genes on chromosome 16 1 gene on chromosome 11 Inheritance Autosomal recessive Autosomal recessive Prevalence Mediterranean, Africa, Asia, Middle East Mediterranean, Middle East, Asia Excess chains beta chains (HbH) alpha chains (aggregates) Prenatal detection Deletion analysis Mutation analysis Diagnostic markers HbH, Hb Bart's Elevated HbF, HbA2 Hydrops fetalis Yes (severe) No Skeletal changes Usually milder Prominent (crew cut, facial changes) Hepcidin Variable Low (due to ineffective erythropoiesis) Iron overload Variable (HbH may have iron overload) Common (transfusion + ineffective erythropoiesis) 4. Alpha thalassemia Alpha thalassemia is caused by reduced or absent alpha-globin chain synthesis due to deletions or point mutations in one or both alpha-globin genes on chromosome 16. Types: 1. Silent carrier (alpha-thal-2): -alpha/alphaalpha • Asymptomatic • Normal Hb, MCV, MCH • No clinical significance 2. alpha-thal trait (alpha-thal-1): --/alphaalpha or -alpha/-alpha • Mild microcytic, hypochromic anaemia • Hb normal or slightly reduced • MCV: 70-80 fL • Hb electrophoresis: Normal (or slight HbA2 < 2.5%) 3. HbH disease: --/-alpha • Moderate haemolytic anaemia • Hb: 7-10 g/dL • Microcytic, hypochromic RBCs • HbH (beta4) on electrophoresis (5-30%) • Splenomegaly • Iron overload possible 4. Hydrops fetalis (Hb Bart's): --/-- • Lethal in utero • Severe anaemia, oedema, hepatosplenomegaly • Hb Bart's (gamma4) > 80% • Usually fatal at birth Treatment: • Mild forms: No treatment • HbH disease: Transfusions (if needed), splenectomy (if severe), iron chelation • Hydrops fetalis: Prenatal diagnosis, intrauterine transfusion (experimental) 5. Thalassemias - classification, genetics, pathogenesis, clinical features, laboratory diagnosis, treatment Classification: (See question 2 above) Genetics/Inheritance: • Autosomal recessive • alpha-thalassemia: 2 genes (chromosome 16); deletions common • beta-thalassemia: 1 gene (chromosome 11); point mutations common • Compound heterozygous states (e.g., HbE/beta-thal) Pathogenesis: • Imbalanced globin chain synthesis • Excess alpha chains (beta-thal) precipitate -> membrane damage -> ineffective erythropoiesis -> haemolysis • Excess beta chains (alpha-thal) form HbH -> unstable -> haemolysis • Ineffective erythropoiesis -> marrow expansion -> skeletal changes Clinical features: Beta thalassemia minor: • Mild anaemia (Hb 10-12 g/dL) • Microcytic, hypochromic RBCs • Asymptomatic Beta thalassemia intermedia: • Moderate anaemia (Hb 7-10 g/dL) • Splenomegaly • Bone changes (mild) • Gallstones • Iron overload (less severe than major) Beta thalassemia major (Cooley's anaemia): • Severe anaemia (Hb < 7 g/dL) within 1-2 years of age • Failure to thrive • Hepatosplenomegaly • Skeletal changes: frontal bossing, maxillary hyperplasia, "crew cut" skull X-ray • Jaundice (haemolysis) • Transfusion dependence • Growth retardation • Gallstones (pigment stones) • Iron overload (due to transfusions) • Endocrinopathies (hypogonadism, delayed puberty, diabetes) Laboratory diagnosis: Blood findings: • Anaemia: microcytic, hypochromic • MCV: Low • MCHC: Low or normal • RDW: Normal (alpha-thal), elevated (beta-thal with transfusions) • Reticulocytosis (ineffective erythropoiesis) • Nucleated RBCs (beta-thal major) • Target cells, teardrop cells, basophilic stippling Hb electrophoresis: • beta-thal minor: • HbA2: 3.5-8% (elevated) • HbF: 1-5% (slightly elevated) • HbA: Reduced • beta-thal major: • HbF: 30-90% (elevated) • HbA2: Normal or elevated • HbA: Absent or minimal • alpha-thal: • HbA2: < 2.5% (low) • HbH (beta4): 5-30% (HbH disease) • Hb Bart's (gamma4): > 80% (hydrops fetalis) Other: • Serum ferritin: Elevated (transfused patients) • Serum iron: Elevated • TIBC: Normal or low Treatment: Beta thalassemia major: 1. Transfusion: • Regular blood transfusions to maintain Hb > 9-10 g/dL • Leukoreduced, irradiated blood 2. Iron chelation: • Deferoxamine (subcutaneous, IV) • Deferasirox (oral) • Deferiprone (oral) • To prevent iron overload (ferritin target < 1000 ug/L) 3. Splenectomy: • For hypersplenism (increased transfusion requirements) • After age 5-6 years (to prevent sepsis) 4. HSCT: • Curative option • Matched sibling donor preferred • Better outcomes in children 5. Supportive: • Folic acid supplementation • Infection prevention (vaccinations, antibiotics) • Endocrine monitoring • Bone density monitoring Beta thalassemia intermedia: • Transfusion as needed (not regularly) • Iron chelation (if iron overload) • Splenectomy (if hypersplenism) • Hydroxyurea (may increase HbF) 6. Sample for prenatal diagnosis of haemoglobinopathies • Chorionic villus sampling (CVS): At 10-13 weeks gestation (earlier) • Amniocentesis: At 14-16 weeks gestation • Fetal blood sampling (cordocentesis): At 18-20 weeks (rarely used now) • Pre-implantation genetic diagnosis (PGD): In IVF cycles • Cell-free fetal DNA (cffDNA): Non-invasive prenatal diagnosis (NIPD) - in development
Leukaemia
Standard Answer:
1. Distinguishing between Acute Lymphoblastic Leukaemia (ALL) and Acute Myeloblastic Leukaemia (AML) using investigations Investigation ALL AML Peripheral blood film Lymphoblasts (small, scant cytoplasm, nucleoli) Myeloblasts (larger, Auer rods, granules) Bone marrow 20% blasts (lymphoblasts) 20% blasts (myeloblasts) Cytochemistry PAS positive (block positivity) MPO positive, Sudan black B positive Immunophenotyping CD19, CD10, TdT positive (B-ALL); CD3, CD7 positive (T-ALL) CD13, CD33, CD34, MPO positive Cytogenetics Hyperdiploid, t(12;21), t(9;22), MLL rearrangements t(8;21), inv(16), t(15;17), complex karyotype Blast morphology Small to medium, scant cytoplasm Medium to large, granules, Auer rods Translocation Ph chromosome in ALL t(8;21), inv(16), t(15;17) TdT Positive (90%) Negative CD34 Positive (often) Variable MPO Negative Positive Auer rods Absent Present (in some) Cytoplasm Scant, agranular Abundant, granular Nuclei Round, convoluted Varied, with nucleoli 2. Ten differences between Acute Myeloblastic and Acute Lymphoblastic Leukaemia Feature AML ALL Age at presentation Peak adults (60+ years) Peak children (2-5 years) Auer rods Present (in 20-30%) Absent Cytochemistry MPO, Sudan black B positive PAS positive; MPO negative Immunophenotype Myeloid markers (CD13, CD33) Lymphoid markers (CD19, CD10, CD3) TdT Negative Positive (90%) Organomegaly Less prominent Hepatosplenomegaly, lymphadenopathy CNS involvement Less common More common Philadelphia chromosome Less common (except in M0, M7) Common (25% adults) Blast morphology Medium-large with granules Small-medium with scant cytoplasm Treatment Anthracyclines + Ara-C Vincristine, prednisolone, L-asparaginase Prognosis Better in younger patients Better in children Subtypes FAB M0-M7 FAB L1-L3 (WHO B-ALL, T-ALL) 3. Supportive treatment of Acute leukemias 1. Transfusion support: • RBC transfusion (to maintain Hb > 7-8 g/dL) • Platelet transfusion (to maintain > 10-20x109/L) • Leukoreduced, irradiated components 2. Infection control: • Antibiotics (empiric for fever) • Antifungals (prophylactic or therapeutic) • Antivirals (acyclovir for herpes) • Pneumocystis jirovecii prophylaxis (cotrimoxazole) • Neutropenic diet 3. Metabolic support: • Hydration (IV fluids, 2-3 L/day) • Allopurinol or rasburicase (for tumour lysis syndrome) • Electrolyte monitoring • Nutritional support 4. Growth factors: • G-CSF (to shorten neutropenia) • Erythropoietin (for anaemia) • Thrombopoietin (for thrombocytopenia) 5. Emotional/Psychological: • Psychological support • Counselling • Support groups 6. General: • Strict aseptic precautions • Central line care • Mouth care (to prevent mucositis) • Anti-emetics (for nausea/vomiting) • Pain management 4. Classification of Acute Leukaemia FAB Classification: AML (M0-M7): • M0: Undifferentiated AML • M1: AML without maturation • M2: AML with maturation • M3: Acute promyelocytic leukaemia (APL) • M4: Acute myelomonocytic leukaemia • M5: Acute monocytic leukaemia • M6: Erythroleukaemia • M7: Megakaryoblastic leukaemia ALL (L1-L3): • L1: Small blasts, scant cytoplasm, small nucleoli • L2: Larger blasts, more cytoplasm, prominent nucleoli • L3: Large blasts, vacuoles, prominent nucleoli (Burkitt-like) WHO Classification (current): • Based on cytogenetics, immunophenotype, genetics • AML with recurrent genetic abnormalities • AML with MDS-related changes • Therapy-related AML • AML not otherwise specified 5. Six Prognostic determinants (unfavourable) of ALL 1. Age >= 35 years (adults) 2. White blood cell count > 30,000/uL (B-ALL) or > 100,000/uL (T-ALL) 3. Cytogenetics: t(9;22) (Ph chromosome), MLL rearrangements (11q23) 4. Immunophenotype: T-ALL (worse than B-ALL in adults) 5. Slow response to induction therapy (day 14-28 blast clearance) 6. CNS involvement at presentation 7. Minimal residual disease (MRD) positivity after induction 8. Hypodiploidy (less than 44 chromosomes) 9. Near haploidy (23-29 chromosomes) 6. Four haematological investigations and findings in ALL 1. Full Blood Count: • Anaemia (normocytic, normochromic) • Leucocytosis (WBC > 50,000/uL) or leucopenia • Blasts present (varying percentage) • Thrombocytopenia (platelets < 100x109/L) 2. Peripheral blood film: • Lymphoblasts (small to medium, scant cytoplasm, fine chromatin, prominent nucleoli) • Smudge cells • May have pancytopenia 3. Bone marrow aspirate: • Markedly hypercellular (90-100%) • Blasts > 20% (lymphoblasts) • Normal elements suppressed (erythroid, myeloid, megakaryocytic) • Auer rods absent 4. Cytochemistry/Immunophenotyping: • PAS positive (block positivity) • TdT positive (90%) • CD19, CD10 positive (B-ALL) • CD3, CD7 positive (T-ALL) 7. FAB classification of Acute Leukemia and three features of each type AML: Subtype Morphological Features M0 (Undifferentiated) Myeloid appearance, MPO negative, CD13/CD33 positive M1 (Without maturation) 90% blasts, MPO positive (few), moderate granules M2 (With maturation) 20% blasts, MPO positive, maturation to promyelocytes M3 (APL) Hypergranular promyelocytes, Auer rods (faggot cells), t(15;17) M4 (Myelomonocytic) 20% blasts, monocytic and granulocytic differentiation M5 (Monocytic) Monoblasts, promonocytes, non-specific esterase positive M6 (Erythroleukaemia) 50% erythroblasts, > 30% myeloblasts M7 (Megakaryoblastic) Megakaryoblasts, CD41/CD61 positive, fibrosis ALL: Subtype Morphological Features L1 (Small blasts) Small cells, scant cytoplasm, fine chromatin, round nuclei, small nucleoli L2 (Large blasts) Larger cells, more cytoplasm, irregular nuclei, prominent nucleoli L3 (Burkitt-like) Large cells, basophilic cytoplasm, vacuoles, prominent nucleoli 8. Basis and significance in clinical practice Basis: Classification is based on morphology, cytochemistry, immunophenotyping, and cytogenetics. Significance: • Prognostication (different risk groups) • Treatment selection (different protocols) • Monitoring response (MRD) • Predicting relapse • Drug selection (targeted therapy: ATRA for APL, tyrosine kinase inhibitors for Ph+ ALL) 9. Supportive treatment of acute leukaemias (See question 3 above) 10. Treatment phases of Acute Lymphoblastic Leukaemia 1. Induction phase: (4-6 weeks) • Achieve remission (blasts < 5% in marrow) • Drugs: Vincristine, prednisolone, L-asparaginase, daunorubicin (adults) • Goal: Rapid reduction of leukaemic burden 2. Consolidation (Intensification) phase: (2-6 months) • Eliminate residual disease • Drugs: High-dose methotrexate, cytarabine, 6-mercaptopurine, cyclophosphamide • Goal: Eradicate MRD 3. CNS prophylaxis: • Intrathecal methotrexate, cytarabine, or hydrocortisone • Cranial irradiation (in high-risk patients) • Goal: Prevent CNS relapse 4. Maintenance phase: (2-3 years) • Low-dose daily/weekly therapy • Drugs: 6-mercaptopurine, methotrexate • Goal: Sustain remission and prevent relapse 5. HSCT: • For high-risk patients (Ph+, MLL rearrangements, MRD+) • Allogeneic HSCT 11. 21-year-old male with Acute Myeloblastic Anaemia A. Possible clinical features and why: 1. Anaemia features: • Fatigue, pallor, dyspnoea, palpitations • Why: Bone marrow failure -> reduced erythropoiesis 2. Bleeding: • Petechiae, ecchymoses, gum bleeding, easy bruising • Why: Thrombocytopenia (bone marrow failure) 3. Infection: • Fever, sore throat, pneumonia, sepsis • Why: Neutropenia (bone marrow failure) 4. Bone pain: • Sternum, ribs, long bones • Why: Marrow expansion, periosteal involvement 5. Extramedullary involvement: • Hepatosplenomegaly, lymphadenopathy • Why: Infiltration B. Five investigations and possible outcomes: 1. Full Blood Count: • Outcome: Anaemia, leucocytosis (or leucopenia), thrombocytopenia, circulating myeloblasts 2. Peripheral blood film: • Outcome: Myeloblasts (medium-large, Auer rods, granules), nucleated RBCs 3. Bone marrow aspiration and biopsy: • Outcome: Hypercellular (90-100%), > 20% myeloblasts, suppression of normal elements 4. Cytochemistry: • Outcome: MPO positive, Sudan black B positive (myeloid origin) 5. Immunophenotyping (Flow cytometry): • Outcome: CD13+, CD33+, CD34+, MPO+, TdT- 6. Cytogenetics/FISH: • Outcome: t(8;21), inv(16), t(15;17), complex karyotype 7. Molecular analysis: • Outcome: FLT3-ITD, NPM1 mutations (prognostic factors) 12. Six laboratory findings in CLL 1. Full Blood Count: • Lymphocytosis (WBC > 5x109/L, often > 50x109/L) • Anaemia (normocytic, normochromic) • Thrombocytopenia (in advanced stages) 2. Peripheral blood film: • Mature lymphocytes (small, round nucleus, condensed chromatin) • Smudge cells (debris of fragile lymphocytes) • Rouleaux (increased plasma proteins) 3. Bone marrow: • Hypercellular (> 30% lymphocytes, often > 40%) • Nodular, interstitial, or diffuse infiltration 4. Immunophenotyping (Flow cytometry): • CD19+, CD5+, CD23+, CD20+ (weak) • sIg (surface immunoglobulin) weak • CD10-, CD22- (weak) • FMC7- (weak or negative) 5. Cytogenetics: • del(13q) (good prognosis) • del(11q) (intermediate prognosis) • del(17p) (poor prognosis) • Trisomy 12 (intermediate) • IGHV mutational status: Unmutated (poor prognosis), mutated (good prognosis) 6. Biochemistry: • LDH: Normal or elevated • beta2-microglobulin: Elevated (prognostic indicator) • Direct Coombs test: Positive (autoimmune haemolytic anaemia in 10-15%) 13. Eight good prognostic features in CLL 1. Early stage (Rai 0, Binet A) 2. Female gender 3. Age < 65 years 4. IGHV mutated (> 2% mutation) 5. No del(17p) or TP53 mutation 6. No del(11q) 7. Isolated del(13q) as sole abnormality 8. Normal beta2-microglobulin 9. Low LDH 10. Good performance status 11. Lymphocyte doubling time > 12 months 12. No B symptoms 14. Forms of staging of CLL 1. Rai Staging System: • Rai 0: Lymphocytosis only • Rai I: Lymphocytosis + Lymphadenopathy • Rai II: Lymphocytosis + Hepatosplenomegaly • Rai III: Lymphocytosis + Anaemia (Hb < 11 g/dL) • Rai IV: Lymphocytosis + Thrombocytopenia (Platelets < 100x109/L) 2. Binet Staging System: • Binet A: < 3 lymphatic areas involved, Hb > 10 g/dL, Platelets > 100x109/L • Binet B: >= 3 lymphatic areas involved, Hb > 10 g/dL, Platelets > 100x109/L • Binet C: Anaemia (Hb < 10 g/dL) or Thrombocytopenia (< 100x109/L) 15. Staging CLL using RAI and BINET RAI Stage Clinical Features Binet Stage 0 Lymphocytosis only A (if < 3 areas) I + Lymphadenopathy A (if < 3 areas) II + Hepatosplenomegaly B (if >= 3 areas) III + Anaemia (Hb < 11) C (if Hb < 10 or Platelets < 100) IV + Thrombocytopenia C (if Hb < 10 or Platelets < 100) Lymphatic areas (Binet): 1. Cervical 2. Axillary 3. Inguinal 4. Spleen 5. Liver 16. Drugs for CLL and Bone Marrow findings Drugs for CLL: 1. Chemotherapy: • Fludarabine • Cyclophosphamide • Chlorambucil • Bendamustine 2. Chemoimmunotherapy: • FCR (Fludarabine + Cyclophosphamide + Rituximab) • BR (Bendamustine + Rituximab) 3. Targeted therapies: • Ibrutinib (BTK inhibitor) • Idelalisib (PI3K inhibitor) • Venetoclax (BCL-2 inhibitor) • Acalabrutinib (BTK inhibitor) 4. Anti-CD20 antibodies: • Rituximab • Obinutuzumab • Ofatumumab 5. Other: • Lenalidomide (immunomodulator) • Alemtuzumab (anti-CD52) Bone Marrow Findings in CLL: • Hypercellular (increased marrow cellularity) • Nodular, interstitial, or diffuse infiltration by small mature lymphocytes • <= 30% prolymphocytes • Normal or decreased haematopoiesis (in advanced stages) • Fibrosis (rare) 17. Leukaemoid Reaction Definition: Leukaemoid reaction is an exaggerated leukocytosis (WBC > 50,000/uL) with the presence of immature myeloid cells in the peripheral blood, resembling leukaemia, but occurring in response to an underlying non-haematological condition. Causes: • Infections (severe bacterial, tuberculosis) • Malignancies (metastatic carcinoma, lymphoma) • Tissue necrosis (myocardial infarction, burns) • Severe haemorrhage • Drugs (G-CSF, corticosteroids) • Inflammatory conditions (rheumatoid arthritis) • Haematological conditions (haemolytic anaemia, severe bleeding) Features differentiating from CML: • Normal LAP (leukocyte alkaline phosphatase) score (high in reactive, low in CML) • No Philadelphia chromosome • No BCR-ABL mutation • WBC count usually < 100,000/uL • Toxic granulation in neutrophils (reactive) • Döhle bodies (reactive) • Underlying cause present 18. Clinical features of Chronic Myeloid Leukaemia Early/Chronic phase: • Fatigue, weakness • Weight loss • Night sweats • Fever • Abdominal fullness (splenomegaly, hepatomegaly) • Bone pain • Anorexia Accelerated phase: • Progressive symptoms • Fever, night sweats, weight loss (B symptoms) • Anaemia • Thrombocytopenia • Splenomegaly worsens • Bleeding Blast crisis: • Acute leukaemic picture • Fever, infections • Bleeding • Bone pain • Anaemia • Lymphadenopathy • Extramedullary disease (CNS, skin) Physical findings: • Splenomegaly (massive in 70-90%) • Hepatomegaly • Pallor • Petechiae/ecchymoses • Lymphadenopathy (rare) 19. Chronic Myeloid Leukaemia - Aetiopathogenesis, Clinical features, Investigations, Treatment Aetiopathogenesis: • Acquired cytogenetic abnormality (Philadelphia chromosome: t(9;22)(q34;q11)) • BCR-ABL fusion gene on chromosome 22 • Constitutive tyrosine kinase activity (activated ABL) • Uncontrolled proliferation of myeloid cells • Stem cell origin • Progresses through chronic phase -> accelerated phase -> blast crisis • Risk factors: Radiation exposure, benzene, age (peak 50-60 years) Clinical features: (See question 18 above) Laboratory investigations: • FBC: • Leucocytosis (WBC > 50-100x109/L) • Anaemia (normocytic, normochromic) • Thrombocytosis (or normal, or low) • Peripheral blood film: • Granulocytosis (neutrophils, myelocytes, metamyelocytes, promyelocytes) • Basophilia (increased) • Eosinophilia • Maturation to mature neutrophils • Low LAP score • Bone marrow: • Hypercellular • Granulocytic hyperplasia • Megakaryocytic hyperplasia • Reduced erythroid series • No fibrosis (early) • Cytogenetics: • Philadelphia chromosome (t(9;22)) in > 90% • Molecular: • BCR-ABL transcript (quantitative PCR) • Biochemistry: • Uric acid: Elevated • LDH: Elevated • Vitamin B12: Elevated (transcobalamin I) • LAP: Low Treatment: Chronic phase: 1. Targeted therapy: • Tyrosine kinase inhibitors (TKIs) • Imatinib (first-line) • Nilotinib, Dasatinib (second-generation TKI, for resistant/intolerant) • Bosutinib, Ponatinib (for resistant cases) • Goal: Complete cytogenetic response, major molecular response 2. Allogeneic HSCT: • Curative option • For young patients (especially < 40 years) • For patients with resistant disease Accelerated phase/Blast crisis: • Higher-dose TKIs • Chemotherapy (like AML for blast crisis) • HSCT (if possible) Supportive: • Allopurinol (prevent tumour lysis) • Hydration • Transfusions (RBCs, platelets) • Infection prophylaxis 20. Bone marrow findings of CML • Cellularity: Hypercellular (increased) • Marrow infiltration: Granulocytic hyperplasia (myeloid series increased) • Blasts: < 5% in chronic phase • Megakaryocytes: Increased, often small/hypolobulated • Erythropoiesis: Reduced (relative) • Maturation: Present (granulocytes mature to segmented forms) • Fibrosis: Minimal or absent (early stages) • Reticulin: Normal or mild increase • Karyotype: t(9;22) present 21. Clinical features of Chronic Myeloid Leukaemia (See question 18 above) 22. Bone marrow findings of chronic myeloid leukaemia (See question 20 above) 23. Philadelphia Chromosome negative, BCR-ABL positive CML This is a rare variant of CML (approximately 5-10% of cases) where: • Conventional cytogenetics shows no Philadelphia chromosome (normal karyotype) • But molecular testing (FISH or PCR) detects BCR-ABL fusion gene • This is due to cryptic translocation or variant translocation • Has same clinical features, course, and treatment response as typical Ph+ CML • TKI treatment is equally effective • More common in younger patients 24. Induction Therapy Induction therapy is the initial, intensive phase of chemotherapy used to achieve remission in acute leukaemias, characterized by: • High-dose multi-agent chemotherapy • Goal: Eliminate > 99% of leukaemic cells (achieve morphological remission) • Takes 4-6 weeks • Followed by consolidation/maintenance • Associated with significant toxicity (myelosuppression, organ toxicity) • For AML: "7+3" (cytarabine 7 days + daunorubicin 3 days) • For ALL: VDP (vincristine + daunorubicin + prednisolone) + L-asparaginase 25. Possible causes of myeloma kidney 1. Light chain cast nephropathy (most common): Monoclonal light chains precipitate in renal tubules, forming casts, causing tubular obstruction and interstitial inflammation 2. Hypercalcaemia: Due to bone resorption; leads to nephrogenic diabetes insipidus, renal calcification 3. Amyloidosis (AL amyloid): Light chain deposition in glomeruli, interstitium 4. Light chain deposition disease: Non-amyloid deposition in glomeruli and tubules 5. Hyperuricaemia: Tumour lysis, renal impairment 6. Hyperviscosity: (IgA, IgG3) -> Renal hypoperfusion 7. Infection: Pyelonephritis, UTI 8. Dehydration: From hypercalcaemia, vomiting, polyuria 9. NSAID use: Renal toxicity 10. Contrast nephropathy: From investigations 11. Plasma cell infiltration: Direct renal infiltration (rare) 26. Classify Hodgkin's leukaemia (Note: Correct term is Hodgkin's lymphoma, not leukaemia) WHO Classification of Classical Hodgkin Lymphoma: 1. Nodular sclerosis Hodgkin lymphoma (NSHL): • Most common (70% of cases) • Lacunar cells, fibrosis bands • Older adolescents and young adults • Good prognosis 2. Mixed cellularity Hodgkin lymphoma (MCHL): • 20-25% of cases • Pleomorphic cellular infiltrate • Older patients • Associated with EBV • Intermediate prognosis 3. Lymphocyte-rich Hodgkin lymphoma (LRHL): • 5% of cases • Small lymphocytes, occasional HRS cells • Good prognosis 4. Lymphocyte-depleted Hodgkin lymphoma (LDHL): • Rare (< 5%) • Few lymphocytes, many HRS cells • Elderly, HIV-associated • Poor prognosis Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL): • Distinct entity • Popcorn cells (lymphocyte-predominant cells) • B-cell phenotype (CD20+, CD30-, CD15-) • Better prognosis
Lymphoma
Standard Answer:
1. Clinical features, classification, staging of Hodgkin's lymphoma Clinical features: • Painless lymphadenopathy: Cervical, supraclavicular, mediastinal (most common) • B symptoms: Fever (> 38 degreesC), night sweats, weight loss (> 10% in 6 months) • Pruritus: Generalized itching • Alcohol-induced pain: In lymph nodes (rare but specific) • Mediastinal mass: Cough, dyspnoea, superior vena cava syndrome • Hepatosplenomegaly (in advanced disease) • Pel-Ebstein fever: Cyclic fever pattern (rare) Classification: WHO Classification: 1. Classical Hodgkin Lymphoma: • Nodular sclerosis (NS) • Mixed cellularity (MC) • Lymphocyte-rich (LR) • Lymphocyte-depleted (LD) 2. Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) Staging (Ann Arbor): Stage Definition I Single lymph node region or single extranodal site II Two or more lymph node regions on same side of diaphragm III Lymph node regions on both sides of diaphragm IV Diffuse involvement of one or more extranodal organs (bone marrow, liver, lung) Modifiers: • A: No B symptoms • B: B symptoms present • E: Extranodal extension (limited) • S: Splenic involvement 2. Burkitt's lymphoma - clinical features, investigation, management Clinical features: • Endemic (African): Jaw and facial bone tumours (children), abdominal masses • Sporadic (Western): Abdominal masses, ileocaecal involvement, intussusception • Immunodeficiency-associated: HIV, post-transplant • Rapid growth: Large, rapidly enlarging mass • B symptoms: Fever, weight loss • CNS involvement (in advanced disease) Investigations: 1. Full Blood Count: Anaemia, thrombocytopenia 2. Peripheral blood film: Blasts (if leukaemic phase) 3. Biopsy: Staging 4. Histopathology: "Starry sky" appearance (tingible body macrophages) 5. Immunophenotyping: CD19+, CD20+, CD10+, CD22+, IgM+, BCL6+, Ki67 high (> 95%) 6. Cytogenetics: t(8;14) (most common), t(2;8), t(8;22) 7. Bone marrow: Involvement in advanced disease 8. Lumbar puncture: CNS involvement 9. Imaging: CT, MRI, PET-CT for staging 10. Serum LDH: Elevated (tumour burden) Management: 1. Chemotherapy: • CODOX-M/IVAC (cyclophosphamide, vincristine, doxorubicin, methotrexate, cytarabine) • DA-EPOCH-R (dose-adjusted etoposide, prednisolone, vincristine, cyclophosphamide, doxorubicin, rituximab) • R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone) - less aggressive 2. Rituximab: Anti-CD20 antibody 3. CNS prophylaxis: Intrathecal methotrexate, cytarabine 4. Tumour lysis prevention: Hydration, allopurinol, rasburicase 5. HSCT: For relapsed/refractory disease 6. Supportive: Blood products, antibiotics, G-CSF 3. Haematological findings and histological classifications of Hodgkin's lymphoma Haematological findings: • Peripheral blood: • Normocytic, normochromic anaemia • Neutrophilic leucocytosis • Lymphocytopenia (in advanced disease) • Eosinophilia (rare) • Platelets: Normal or elevated (reactive) • Reed-Sternberg cells (rarely seen) • Bone marrow: • Infiltration in advanced disease (stage IV) • Fibrosis • Biochemistry: • ESR: Elevated • LDH: Elevated (tumour burden) • Albumin: Low (acute phase) • Alkaline phosphatase: Elevated (bone involvement) • Acute phase proteins: CRP elevated • Immunology: • Slight immunodeficiency (defective cell-mediated immunity) Histological classifications: (See WHO classification in question 1 above) Reed-Sternberg cells: • Binucleate/multinucleate giant cells • Large eosinophilic nucleoli (owl-eye appearance) • CD30+, CD15+, CD45-, PAX5+ (weak) • EBV+ in some subtypes • Origin: Germinal centre B cells
Multiple Myeloma
Standard Answer:
1. Multiple Myeloma Multiple myeloma is a malignant plasma cell disorder characterized by: • Clonal proliferation of plasma cells in bone marrow • Production of monoclonal immunoglobulin (M-protein) in serum and/or urine • End-organ damage (CRAB criteria) • Bone lesions (lytic lesions, osteoporosis) • Anaemia, renal impairment, hypercalcaemia 2. Clinical features that characterize this condition • Bone pain (vertebrae, ribs, pelvis, skull) • Pathological fractures • Anaemia (fatigue, pallor, dyspnoea) • Renal impairment (proteinuria, renal failure) • Recurrent infections (pneumonia, UTI) • Hypercalcaemia (confusion, constipation, polyuria) • Weight loss • Hyperviscosity syndrome (headache, visual disturbance, bleeding) • Neurological symptoms (spinal cord compression, radiculopathy) • Bleeding (due to platelet dysfunction) • Amyloidosis (AL amyloid) 3. CRAB stands for • C: HyperCalcaemia (Ca > 11.5 mg/dL or > 2.75 mmol/L) • R: Renal impairment (Creatinine > 2 mg/dL or > 177 umol/L) • A: Anaemia (Hb < 10 g/dL) • B: Bone lesions (lytic lesions, osteoporosis, fractures) 4. Haematological condition associated with the disorder • Monoclonal gammopathy of undetermined significance (MGUS) • Smouldering (asymptomatic) multiple myeloma • Plasma cell leukaemia • AL amyloidosis • Waldenström's macroglobulinaemia • Heavy chain disease 5. Ten investigations and expected findings Investigation Expected Findings Full Blood Count Anaemia (normocytic, normochromic), leucopenia, thrombocytopenia Peripheral blood film Rouleaux, plasma cells (rare) Serum protein electrophoresis M-protein spike (IgG, IgA, light chains) Serum immunofixation Monoclonal immunoglobulin (IgG, IgA, or light chains) Urine protein electrophoresis Bence-Jones protein (light chains) Serum beta2-microglobulin Elevated (prognostic) Serum LDH Elevated (tumour burden) Serum calcium Elevated (hypercalcaemia) Serum creatinine Elevated (renal impairment) Bone marrow aspirate/biopsy Plasma cells > 10-20% (clonal), abnormal morphology Skeletal survey (X-ray) Lytic lesions, osteoporosis, fractures MRI/PET-CT Bone lesions, extramedullary disease 6. Multiple Myeloma - pathogenesis, clinical presentation, diagnosis, haematological findings, treatment Pathogenesis: • Clonal plasma cell proliferation in bone marrow • Cytogenetic abnormalities: t(4;14), t(11;14), del(13q), del(17p), trisomies • Myeloma cells produce M-protein, cytokines (IL-6, IL-1beta, TNF-alpha) • Bone disease: Osteoclast activation (RANKL, MIP-1alpha), osteoblast inhibition (DKK1, sFRP) • Renal disease: Light chain cast nephropathy, hypercalcaemia, amyloidosis • Immunodeficiency: Suppression of normal immunoglobulins Clinical presentation: • Bone pain (back pain, rib pain) • Anaemia, fatigue • Infections (recurrent) • Renal impairment • Hypercalcaemia symptoms Diagnosis (SLiM CRAB criteria): • C: Hypercalcaemia • R: Renal impairment • A: Anaemia • B: Bone lesions • S: Plasma cells > 60% • L: Light chain ratio > 100 • M: > 1 focal lesion on MRI Haematological findings: • Anaemia: Normocytic, normochromic • Rouleaux formation • Lymphocytopenia • Plasma cells (rare) • Bone marrow: Plasma cells > 10% (clonal) • M-protein: Serum (IgG, IgA) or urinary light chains Treatment: 1. For transplant-eligible (< 65 years, good performance status): • Induction: Bortezomib + Thalidomide/Lenalidomide + Dexamethasone (VTD, VRD) • Autologous HSCT (high-dose melphalan) • Maintenance: Lenalidomide or Bortezomib 2. For transplant-ineligible (> 65 years, comorbidities): • Bortezomib + Melphalan + Prednisone (VMP) • Lenalidomide + Dexamethasone (RD) • Lenalidomide + Bortezomib + Dexamethasone (RVD) 3. Relapsed/Refractory: • Carfilzomib, Pomalidomide, Daratumumab, Isatuximab • Anti-CD38 antibodies (Daratumumab, Isatuximab) • Elotuzumab (anti-SLAMF7) 4. Supportive: • Bisphosphonates (Zoledronic acid, Pamidronate) for bone disease • Erythropoietin for anaemia • IVIG for infections • Analgesia for pain • Renal protection (hydration, avoid nephrotoxins) 7. Lab diagnosis of myeloma 1. Serum protein electrophoresis (SPEP): Monoclonal spike 2. Serum immunofixation (IFX): Characterizes M-protein (IgG, IgA, IgM, light chains) 3. Urine protein electrophoresis (UPEP): Bence-Jones protein (light chains) 4. Serum free light chain (FLC) assay: Elevated kappa/lambda ratio (in light chain myeloma) 5. Bone marrow aspirate and biopsy: Plasma cells > 10% (clonal), morphology (plasmablastic, etc.) 6. Cytogenetics/FISH: Abnormal karyotype (t(4;14), t(11;14), del(17p), trisomies) 7. Serum beta2-microglobulin: Elevated (prognostic) 8. Serum LDH: Elevated (prognostic) 9. Serum albumin: Low (prognostic) 10. CRP: Elevated (inflammation) 11. Renal function: Elevated creatinine, urea 12. Serum calcium: Elevated (hypercalcaemia) 13. Imaging: Skeletal survey, MRI, PET-CT (bone lesions) 8. 68-year-old man with anaemia and severe low back pain A. Most likely diagnosis: Multiple myeloma B. Diagnosis approach: Clinical features: • Anaemia (fatigue, pallor) • Bone pain (low back pain, vertebral involvement) • Age > 65 years (typical) • Consider: Pathological fracture, renal impairment Investigations: 1. FBC: Anaemia, leucopenia, thrombocytopenia 2. ESR: Elevated 3. Serum protein electrophoresis: M-protein spike 4. Serum immunofixation: Monoclonal immunoglobulin (IgG, IgA) 5. Urine protein electrophoresis: Bence-Jones protein 6. Serum calcium: Hypercalcaemia 7. Serum creatinine: Renal impairment (light chain nephropathy) 8. beta2-microglobulin: Elevated 9. Bone marrow aspiration: Plasma cells > 10% 10. Skeletal survey: Lytic lesions, osteoporosis, vertebral collapse 11. Serum free light chain assay: Abnormal ratio Diagnostic criteria (SLiM CRAB): • Bone marrow plasma cells > 10% or biopsy-proven myeloma • Plus any of: • C: Hypercalcaemia • R: Renal impairment • A: Anaemia • B: Lytic bone lesions • S: Plasma cells > 60% • L: Free light chain ratio > 100 • M: MRI with > 1 focal lesion C. Four drugs used in treatment: 1. Bortezomib (proteasome inhibitor) 2. Lenalidomide (immunomodulatory drug) 3. Dexamethasone (steroid) 4. Melphalan (alkylating agent) 5. Daratumumab (anti-CD38 monoclonal antibody) 6. Carfilzomib (proteasome inhibitor) 7. Pomalidomide (immunomodulatory drug)
Blood Groups
Standard Answer:
1. Significant characteristics of the Bombay blood group Definition: Bombay blood group (Oh) is a rare blood group characterized by absence of A, B, and H antigens on red blood cells. Significant characteristics: • RBCs lack A, B, and H antigens • Serum contains anti-A, anti-B, and anti-H antibodies • Genotype: hh (non-functional FUT1 gene) • First described in Mumbai (Bombay), India • Extremely rare (< 1 in 10,000 in India, extremely rare elsewhere) • Cannot receive blood from any ABO group except Bombay group blood • Anti-H antibodies are clinically significant (can cause haemolytic transfusion reactions) • Detected by absence of H antigen (failure to agglutinate with anti-H lectin from Ulex europeus) 2. Bombay blood group (See question 1 above) 3. Six indications for ABO typing 1. Pre-transfusion compatibility testing (crossmatching) 2. Blood donor selection and compatibility 3. Prenatal testing (Rh incompatibility risk assessment) 4. Newborn blood grouping 5. Investigation of haemolytic disease of newborn (HDN) 6. Paternity testing (disputed paternity) 7. Organ transplantation compatibility 8. Pre-operative assessment 9. Investigation of transfusion reactions 4. Six characteristics of ABO antibodies 1. Naturally occurring: Present in serum without prior sensitization (except in Bombay) 2. Usually IgM (although IgG in some cases): Pentameric structure 3. Room temperature reactive: React best at 4-22 degreesC 4. Complement fixing: Can cause intravascular haemolysis 5. Blood group specific: Anti-A, Anti-B, Anti-A,B 6. Clinically significant: Cause haemolytic transfusion reactions 7. Titre varies: With age, immune stimulation 8. Not present in newborns: Develop by 3-6 months 9. Some are IgG: Can cross placenta (HDN) 10. React with soluble antigens: Found in secretions 5. ABO blood groups - Antigens and antibodies Blood Group RBC Antigens Serum Antibodies A A antigen Anti-B B B antigen Anti-A AB A and B antigens None O None (H antigen present) Anti-A, Anti-B 6. ABO compatibility for red cell transfusion Recipient Blood Group Can Receive from Donors A A, O B B, O AB AB, A, B, O (universal recipient) O O only (universal donor) 7. Clinical importance of ABO blood group 1. Blood transfusion: Compatibility essential (avoid haemolytic transfusion reactions) 2. Haemolytic disease of newborn (HDN): ABO incompatibility (less severe than Rh) 3. Organ transplantation: Compatibility required (especially kidneys, liver) 4. Forensic medicine: Paternity testing 5. Prenatal testing: Risk assessment 6. Epidemiology: Association with diseases (O with peptic ulcer, A with gastric cancer) 7. Cryptic antigens: In secretions (saliva, semen, milk) 8. Why is the Rhesus blood group clinically important 1. Haemolytic disease of newborn (HDN): RhD-negative mother with RhD-positive fetus -> Anti-D antibodies -> Haemolysis in fetus/newborn 2. Blood transfusion: RhD-negative patients should receive RhD-negative blood (to avoid alloimmunization) 3. Anti-D prophylaxis: Anti-D immunoglobulin given to RhD-negative mothers to prevent sensitization 4. Haemolytic transfusion reactions: Rh antibodies (IgG) cause extravascular haemolysis (delayed reaction) 5. Frequency: RhD-negative is common (15% in Caucasians) 6. Antigenicity: Highly immunogenic (anti-D is common) 7. Prenatal testing: Essential for Rh typing in pregnancy 8. Multiple Rh antigens: D, C, c, E, e (more than 50 antigens) 9. Rhesus haemolytic disease of the newborn Pathogenesis: 1. RhD-negative mother exposed to RhD-positive fetal RBCs (during delivery, abortion, trauma) 2. Sensitization -> Anti-D antibodies (IgG) produced 3. Subsequent pregnancy with RhD-positive fetus 4. Maternal IgG anti-D crosses placenta (via FcRn receptor) 5. Anti-D binds to fetal RhD-positive RBCs 6. Extravascular haemolysis (spleen) 7. Fetal anaemia, erythroblastosis fetalis Clinical features: • Fetus: Hydrops fetalis, ascites, pleural effusion, anaemia • Newborn: Jaundice (unconjugated bilirubin), hepatosplenomegaly, anaemia, oedema, pallor • Severe: Kernicterus (bilirubin encephalopathy) -> Brain damage Diagnosis: • Antenatal: Indirect Coombs test (maternal), fetal blood group (amniocentesis), ultrasound • Postnatal: Direct Coombs test (fetal), FBC, bilirubin, reticulocyte count Treatment: • Antenatal: Anti-D immunoglobulin (prophylaxis), intrauterine transfusion • Postnatal: Phototherapy, exchange transfusion, IVIG, blood transfusion Prevention: • Anti-D immunoglobulin at 28 weeks and 34 weeks • Post-partum within 72 hours of delivery (if RhD-positive infant) 10. Clinical importance of blood groups in medicine 1. Transfusion medicine: Compatibility (ABO, Rh, other systems) 2. Prenatal medicine: HDN prevention (Rh incompatibility) 3. Organ transplantation: ABO compatibility required 4. Haematology: Autoimmune haemolytic anaemia (DAT) 5. Forensic medicine: Paternity testing, crime investigation 6. Epidemiology: Disease associations 7. Immunology: Blood group antibodies in autoimmune diseases 8. Genetic studies: Population genetics, inheritance patterns 9. Drug development: Some blood groups affect drug response 10. Stem cell research: Blood group antigens on stem cells 11. Blood group antibodies in the foetus and newborn Foetus: • No antibodies (unless maternal IgG crosses placenta) • ABO antibodies not present (passive only) • Rh antibodies may be present (maternal IgG) Newborn: • ABO antibodies are absent (not developed yet) • Maternal IgG antibodies present (transferred across placenta) • Rh antibodies (if RhD-positive father, RhD-negative mother) • Direct Coombs test: Positive if maternal antibodies bound to fetal RBCs (HDN) 12. Five components of blood and their uses 1. Packed Red Blood Cells: • Symptomatic anaemia, acute blood loss, surgery 2. Platelets: • Thrombocytopenia, bleeding, prophylaxis 3. Fresh Frozen Plasma: • Coagulopathy, DIC, liver disease 4. Cryoprecipitate: • Hypofibrinogenaemia, haemophilia A, von Willebrand disease 5. Albumin: • Hypoalbuminaemia, burns, fluid resuscitation 6. IVIG (Immunoglobulins): • Immunodeficiency, autoimmune diseases 7. Factor concentrates: • Haemophilia A/B, specific factor deficiencies 8. Leukocyte-reduced components: • Prevention of transfusion reactions, CMV prevention 13. Disputed Paternity Definition: Forensic application of blood grouping to determine paternity. Principles: • A child inherits one allele for each blood group from each parent • If alleged father lacks an allele present in child, paternity is excluded • Exclusion of paternity: Father cannot donate an allele present in child • Inclusion: Father may be the father (not proof of paternity) • Using multiple blood group systems increases exclusion probability Systems used: 1. ABO blood group 2. Rh blood group 3. MN system 4. HLA typing 5. DNA profiling (most accurate, > 99.99% probability) Limitations: • Cannot prove paternity definitively • Only exclusions are definitive • Requires DNA for definitive proof
Bone Marrow Failure
Standard Answer:
1. Bone Marrow Failure Bone marrow failure is a heterogeneous group of disorders characterized by impaired production of one or more haematopoietic cell lineages, resulting in cytopenia(s) in the peripheral blood due to inadequate haematopoiesis. 2. Ten causes of Bone Marrow failure 1. Aplastic anaemia (idiopathic, immune-mediated) 2. Myelodysplastic syndromes (MDS) 3. Acute myeloid leukaemia (AML) 4. Myelofibrosis (primary/secondary) 5. Chemotherapy/Radiotherapy (myelotoxicity) 6. Drugs (chloramphenicol, NSAIDs, anticonvulsants) 7. Infections (viral hepatitis, EBV, CMV, Parvovirus B19, HIV) 8. Congenital (Fanconi anaemia, Diamond-Blackfan anaemia, dyskeratosis congenita) 9. Paroxysmal nocturnal haemoglobinuria (PNH) 10. Bone marrow infiltration (metastatic carcinoma, lymphoma, multiple myeloma) 11. Nutritional deficiencies (B12, folate) 12. Autoimmune (SLE, rheumatoid arthritis) 3. Six drugs of choice in Bone Marrow Failure For Aplastic Anaemia: 1. Anti-thymocyte globulin (ATG): Rabbit/horse ATG + Cyclosporine A 2. Cyclosporine A: Immunosuppressive 3. Eltrombopag: Thrombopoietin receptor agonist (TPO-RA) 4. Hematopoietic stem cell transplantation (HSCT): Curative For MDS: 1. Azacitidine: Hypomethylating agent 2. Decitabine: Hypomethylating agent 3. Lenalidomide: For 5q- syndrome 4. HSCT: Curative Supportive: 1. G-CSF: For neutropenia 2. Erythropoietin: For anaemia (limited) 3. Blood transfusions: RBCs, platelets 4. Clinical Presentations of Bone Marrow Failure Anaemia features: • Fatigue, weakness • Pallor • Dyspnoea on exertion • Palpitations • Headache, dizziness Bleeding/Thrombocytopenia features: • Easy bruising • Petechiae, ecchymoses • Gingival bleeding • Epistaxis • Menorrhagia • Gastrointestinal bleeding Neutropenia/Immunodeficiency features: • Recurrent infections (bacterial, fungal, viral) • Fever • Mouth ulcers • Pneumonia, sepsis General features: • Weight loss (if associated with malignancy) • Lymphadenopathy (if infiltration) • Splenomegaly (if associated with infiltration, myelofibrosis) 5. Differentiate between constitutional and inherited bone marrow failure; Pancytopenia and bone marrow failure Constitutional vs Inherited Bone Marrow Failure: Feature Constitutional Inherited Definition Present from birth (genetic) Genetically transmitted Age of onset Childhood/adolescence Variable (childhood to adulthood) Inheritance Autosomal recessive, autosomal dominant Autosomal recessive, X-linked Examples Fanconi anaemia, Diamond-Blackfan anaemia, Dyskeratosis congenita All are inherited Somatic features Present (short stature, skeletal anomalies, skin pigmentation) Present Bone marrow Hypocellular Hypocellular Cancer risk High (MDS, AML) High (MDS, AML) Treatment HSCT, supportive care HSCT, supportive care Pancytopenia vs Bone Marrow Failure: Feature Pancytopenia Bone Marrow Failure Definition Decreased all three cell lines Impaired haematopoiesis Cause Any cause (peripheral destruction, sequestration, failure) Specific to marrow dysfunction Examples B12 deficiency, hypersplenism, viral infection Aplastic anaemia, MDS, myelofibrosis Bone marrow Variable (may be normal, hypercellular, hypocellular) Hypocellular or fibrotic Treatment Treat underlying cause Immunosuppression, HSCT, supportive 6. Laboratory Findings in Myelofibrosis Peripheral blood: • Anaemia (normocytic, normochromic) - most common • Leucocytosis (WBC elevated, may be very high) • Thrombocytosis (or thrombocytopenia in advanced) • Leucoerythroblastic picture (nucleated RBCs, immature WBCs) • Teardrop cells (dacrocytes) • Poikilocytosis (varied shapes) • Anisocytosis • Basophilia Bone marrow: • Fibrosis (reticulin and/or collagen) • Megakaryocytic hyperplasia (large, atypical megakaryocytes) • Osteosclerosis (bone formation) • Hypercellular (early), hypocellular (late) • Aspiration often "dry tap" (no marrow cells obtained) Others: • LDH: Elevated • Uric acid: Elevated • ESR: Elevated • Alkaline phosphatase: Elevated (bone turnover) • Serum LAP: Low • Cytogenetics: JAK2 V617F (50-60%), CALR (20-25%), MPL (5-10%) 7. Immune antibodies Definition: Immunoglobulins (IgG, IgM, IgA, IgD, IgE) produced by B cells/plasma cells that recognize specific antigens. Types: • IgG: Most abundant, crosses placenta, opsonizes, activates complement • IgM: Pentamer, activates complement efficiently, ABO antibodies • IgA: Mucosal immunity, found in secretions • IgD: B cell receptor • IgE: Allergic reactions, parasitic infections Functions: • Neutralization (binds toxins/viruses) • Opsonization (enhances phagocytosis) • Complement activation • Antibody-dependent cell-mediated cytotoxicity (ADCC) Clinical significance: • Autoimmune diseases (autoantibodies) • Immunodeficiencies • Monoclonal gammopathies (myeloma) • Transfusion reactions (alloantibodies) 8. Immunopathology of Goodpasture syndrome and Pernicious anaemia Goodpasture syndrome: • Pathogenesis: Autoantibodies (IgG) against type IV collagen (alpha3 chain) in glomerular basement membrane and alveolar basement membrane • Target: Renal and pulmonary basement membranes • Pathology: Linear immunofluorescence on renal biopsy • Clinical: Rapidly progressive glomerulonephritis + pulmonary haemorrhage • Mechanism: Type II hypersensitivity reaction Pernicious anaemia: • Pathogenesis: Autoantibodies against: • Intrinsic factor (blocking and binding antibodies) • Parietal cells (antibodies to H+/K+ ATPase) • Target: Gastric parietal cells and intrinsic factor • Pathology: Type II hypersensitivity (antibody-mediated) • Clinical: B12 deficiency -> Megaloblastic anaemia, neurological symptoms • Mechanism: Autoimmune destruction of parietal cells -> Lack of intrinsic factor -> B12 malabsorption 9. 48-year-old female with PNH-like presentation A. Likely bone marrow failure syndrome: Paroxysmal Nocturnal Haemoglobinuria (PNH) B. Pathophysiologic mechanism: • Somatic mutation in PIGA gene (X-linked) in haematopoietic stem cells • PIGA gene encodes GPI anchor synthesis • Loss of GPI-linked proteins on RBCs, WBCs, platelets • Deficient proteins: • CD55 (decay-accelerating factor, DAF) • CD59 (membrane inhibitor of reactive lysis, MIRL) • CD16, CD24, CD14 • Pathophysiology: • Absence of CD55 and CD59 -> Loss of complement regulation • Uncontrolled complement activation -> Complement-mediated lysis of RBCs (intravascular haemolysis) • Haemoglobinuria (coke-coloured urine) • Bone marrow failure (in PNH) • Thrombosis (due to platelet activation) • Clinical: • Paroxysmal nocturnal haemoglobinuria (morning urine) • Iron deficiency (chronic haemoglobinuria) • Thrombosis (especially in hepatic veins) • Bone marrow failure (in PNH) C. Treatment options: 1. Eculizumab: Monoclonal antibody to C5 complement component (prevents MAC formation) 2. Supportive: • Blood transfusions (RBCs) for anaemia • Iron supplementation (for iron loss from haemoglobinuria) • Anticoagulation (for thrombosis) 3. HSCT: Curative (allogeneic) 4. Ravulizumab: Long-acting C5 inhibitor 5. Pegcetacoplan: C3 inhibitor 6. Danicopan: Oral complement inhibitor
Blood Transfusion and Blood Donor Selection
Standard Answer:
1. Criteria for a blood donor Age: 18-65 years (varies by country) Weight: > 50 kg (minimum) Hb/PCV: • Hb > 12.5 g/dL (males) or > 12.0 g/dL (females) • PCV > 38% (males) or > 36% (females) Health status: • No acute illness (fever, infection) • No chronic diseases (hypertension, diabetes if controlled) • No history of hepatitis, HIV, syphilis, malaria • No history of blood transfusion in last 12 months Medications: • No antibiotics (if acute infection) • No blood thinners (unless temporarily stopped) Lifestyle: • No high-risk behaviour (IV drug use, multiple partners) • No tattoos/piercings in last 3-6 months Pregnancy: • Not currently pregnant • Not in the postpartum period (< 6 months) Travel: • No travel to malaria-endemic areas (varies by country) Frequency: • Minimum 3 months between donations • Maximum 4-5 donations/year (males), 3-4 donations/year (females) Screening: • Negative for HIV, HBV, HCV, syphilis, malaria • No evidence of vCJD risk 2. Blood transfusion protocol and examples Definition: A set of standardised procedures and guidelines for the safe and appropriate administration of blood products, including patient identification, pre-transfusion testing, transfusion administration, and monitoring. Examples when protocols are needed: 1. Pre-operative blood transfusion (anaemia correction) 2. Emergency transfusion (massive bleeding) 3. Chronic transfusion (thalassemia, MDS) 4. Neonatal transfusion (neonates) 5. Transfusion in haematological malignancies (chemotherapy-induced anaemia) 6. Autologous transfusion (pre-operative) 7. Plasma exchange (TTP, myasthenia gravis) 8. Platelet transfusion (thrombocytopenia) 3. Blood transfusion reaction and prevention Blood transfusion reaction: Adverse reaction following transfusion of blood products, ranging from mild to life-threatening. Types: • Acute reactions (within 24 hours) • Delayed reactions (> 24 hours) Prevention steps: 1. Patient identification: Confirm identity, blood group, crossmatch (ABO, Rh) 2. Pre-transfusion testing: ABO/Rh grouping, antibody screening, crossmatching 3. Patient history: Previous transfusion reactions, pregnancy (Rh status) 4. Component selection: Use appropriate component for indication 5. Administration: • Use standard transfusion sets (with filter) • Pre-medicate (if indicated): Antipyretics, antihistamines • Start slowly (first 15 minutes) 6. Monitoring: • Vital signs before, during, after transfusion • Observe for signs of reaction • Document appropriately 7. Blood product storage: Correct temperature, proper handling 8. Leukoreduction: Remove WBCs (prevent febrile reactions) 9. Irradiation: Prevent GVHD (in immunosuppressed patients) 10. Antibody detection: Regular antibody screening 4. Management of acute blood transfusion reaction Immediate action: 1. STOP transfusion (immediately) 2. Keep IV line open: Infuse normal saline 3. Check patient identity: Confirm correct patient/product 4. Notify clinician (medical staff) 5. Assess ABCs (Airway, Breathing, Circulation) 6. Vital signs monitoring: Temperature, BP, pulse, respiratory rate 7. Treat symptoms: • Fever: Antipyretics (paracetamol), NOT aspirin (bleeding risk) • Hypotension: IV fluids, vasopressors • Anaphylaxis: Epinephrine, antihistamines, corticosteroids • Acute haemolytic reaction: Stop transfusion, IV fluids, diuretics, renal protection • Bacterial sepsis: Broad-spectrum antibiotics Investigations: • Blood sample from patient (post-transfusion) • Blood culture (if sepsis) • Coombs test (direct/indirect) • Haemoglobin, bilirubin, LDH • Renal function tests • Urine analysis (haemoglobinuria) Reporting: • Notify blood bank • Complete reaction report • Donor blood sample for culture/analysis 5. Complications of blood transfusion and Blood component therapy Complications: 1. Acute haemolytic reaction: ABO incompatibility 2. Febrile non-haemolytic reaction: Cytokines/antibodies 3. Allergic reaction: Urticaria, anaphylaxis 4. Transfusion-related acute lung injury (TRALI): Pulmonary oedema 5. Transfusion-associated circulatory overload (TACO): Volume overload 6. Bacterial sepsis: Contaminated blood products 7. Viral infections: HIV, HBV, HCV, CMV, EBV, HTLV 8. Parasitic infections: Malaria, Babesia, Chagas 9. GVHD: Immunosuppressed patients (irradiated components) 10. Iron overload: Multiple transfusions (thalassemia, MDS) 11. Immunosuppression: Post-transfusion 12. Anaphylaxis: IgA deficiency Blood component therapy advantages/disadvantages: (See question 6 below) 6. Autologous blood transfusion Definition: Blood donation and transfusion where the donor and recipient are the same person. Types: 1. Pre-operative autologous donation (PAD): Patient donates before surgery 2. Intra-operative blood salvage (cell salvage): Blood lost during surgery recovered, washed, and reinfused 3. Post-operative blood salvage: Drainage blood collected and reinfused Advantages: • No risk of transfusion-transmitted infections • No risk of alloimmunization • No risk of ABO incompatibility • Reduces demand on blood bank • Avoids immunosuppression Disadvantages: • Requires pre-operative planning • May not be feasible in emergencies • Cost of collection and storage • May not be possible in all patients (anaemia, sepsis) • Risk of bacterial contamination (if salvage) • May not provide enough volume for massive blood loss Indications: • Elective surgery (cardiac, orthopaedic, vascular) • Jehovah's Witnesses (religious reasons) • Patients with rare blood groups • Patients with history of transfusion reactions 7. Classification of blood transfusion reactions with examples Acute reactions (within 24 hours): 1. Immunological: • Acute haemolytic (ABO incompatibility) • Febrile non-haemolytic (WBC antibodies) • Allergic (Urticaria, anaphylaxis) • TRALI (anti-HLA, anti-granulocyte antibodies) 2. Non-immunological: • Bacterial contamination/sepsis • TACO (volume overload) • Hypothermia • Air embolism • Citrate toxicity Delayed reactions (> 24 hours): 1. Immunological: • Delayed haemolytic (extravascular) • Post-transfusion purpura • GVHD • Alloimmunization 2. Non-immunological: • Iron overload (haemosiderosis) • Transfusion-transmitted infections (HIV, HBV, HCV, malaria) Examples of each: • Acute haemolytic: ABO mismatch (O recipient receiving A blood) • Febrile non-haemolytic: Previous transfusion, multiparous women • Allergic: Previous allergies, IgA deficiency • TRALI: Multiparous donors (anti-HLA antibodies) • Bacterial sepsis: Contaminated platelet concentrates (room temperature storage) • TACO: Elderly patients with cardiac failure • Delayed haemolytic: Antibody formation (e.g., anti-E) • GVHD: Immunosuppressed patients • Iron overload: Thalassemia major (multiple transfusions) 8. Management of transfusion reaction (See question 4 above) 9. Transfusion Transmitted Infections Definition: Infections transmitted to a patient through the transfusion of infected blood products. Types: 1. Bacterial infections: • Contamination (platelets, RBCs) • Yersinia enterocolitica, Pseudomonas, Serratia 2. Viral infections: • HIV (1, 2) • HBV, HCV, HDV • CMV, EBV, HTLV (1, 2) • Parvovirus B19, West Nile virus, Dengue, Zika 3. Parasitic infections: • Malaria (Plasmodium spp.) • Babesia (B. microti) • Trypanosoma cruzi (Chagas disease) • Leishmania (visceral leishmaniasis) 4. Prion diseases: • vCJD (variant Creutzfeldt-Jakob disease) • Rare Examples: • Bacterial: Yersinia enterocolitica (RBCs), Pseudomonas (platelets) • Viral: HIV, HBV, HCV • Parasitic: Malaria, Babesia 10. Blood component therapy - advantages and disadvantages (See question 6 in Erythropoiesis section) 11. Blood component therapy and three types with indications Definition: Separation of whole blood into individual components to provide targeted therapy. Three types and indications: 1. Packed Red Blood Cells (PRBC): • Indications: • Symptomatic anaemia • Acute blood loss • Surgery with anticipated blood loss • Pre-operative optimization 2. Platelets: • Indications: • Thrombocytopenia (< 20x109/L) • Bleeding due to thrombocytopenia • Prophylaxis in chemotherapy patients • Surgical bleeding in thrombocytopenia 3. Fresh Frozen Plasma (FFP): • Indications: • Coagulopathy (DIC, liver disease) • Massive transfusion (dilutional coagulopathy) • Specific factor deficiencies (when factor concentrates unavailable) • Thrombotic thrombocytopenic purpura (TTP) - plasma exchange 4. Cryoprecipitate: • Indications: • Hypofibrinogenaemia (< 100 mg/dL) • Haemophilia A (factor VIII deficiency) • von Willebrand disease (if DDAVP fails) • Massive transfusion 12. Blood component therapy and various blood components with indications (See question 11 above) 13. Six indications for Fresh Frozen Plasma 1. Disseminated Intravascular Coagulation (DIC): With bleeding and coagulopathy 2. Liver disease: Coagulopathy (prolonged PT/APTT) 3. Massive transfusion: > 1 blood volume replaced (dilutional coagulopathy) 4. Specific factor deficiencies: When factor concentrates unavailable (factors II, V, VII, X, XI, XII) 5. Plasma exchange: TTP, haemolytic uraemic syndrome 6. Reversal of warfarin: When vitamin K is not available or urgent (prolonged INR) 7. Bleeding in vitamin K deficiency: If correction needed 8. Plasmapheresis: For patients on therapeutic apheresis 14. Blood components prepared in hospital and storage methods Components: 1. Packed Red Blood Cells (PRBC): • Storage: 1-6 degreesC (refrigerator) • Shelf life: 21-42 days (depending on additive solution) 2. Platelets: • Storage: 20-24 degreesC (room temperature, with agitation) • Shelf life: 5 days 3. Fresh Frozen Plasma (FFP): • Storage: -20 degreesC to -30 degreesC (frozen) • Shelf life: 1 year 4. Cryoprecipitate: • Storage: -20 degreesC to -30 degreesC (frozen) • Shelf life: 1 year 5. Cryo-poor plasma: • Storage: -20 degreesC to -30 degreesC • Shelf life: 1 year 6. Platelet-rich plasma (PRP): • Rarely used 7. Washed RBCs: • For IgA deficiency, anaphylactic reactions Storage methods: • PRBC: In additive solutions (AS-1, AS-3, AS-5, SAGM) • Platelets: Blood bags with constant agitation (rotating shaker) • FFP: Frozen within 8 hours of collection 15. Short notes Imatinib mesylate: • Tyrosine kinase inhibitor (TKI) • Used in CML (Philadelphia chromosome positive), Ph+ ALL, GIST (gastrointestinal stromal tumour) • Mechanism: Inhibits BCR-ABL tyrosine kinase • Dose: 400-600 mg/day (oral) • Side effects: Fluid retention, myelosuppression, nausea, oedema, muscle cramps Recombinant Factor 8 concentrate: • Used for haemophilia A (factor VIII deficiency) • Recombinant (rFVIII) - no plasma-derived (safer) • Forms: Octocog alfa, B-domain deleted (rFVIII-Fc) • Treatment: Prophylactic and on-demand • Side effects: Inhibitor development (neutralizing antibodies) Erythropoietin: • Glycoprotein hormone produced by kidney • Stimulates erythropoiesis • Indications: Anaemia of chronic disease, CKD, chemotherapy-induced anaemia • Dose: 50-150 U/kg/week (subcutaneous/IV) • Side effects: Hypertension, thrombosis, pure red cell aplasia 16. Irradiated packed red cells and cryoprecipitate Irradiated packed red cells: • RBCs exposed to gamma radiation (25-30 Gy) • Prevents lymphocyte proliferation (prevents TA-GVHD) • Indications: Immunosuppressed patients, HSCT, neonates, congenital immunodeficiency • Shelf life: 28 days (post-irradiation) • No change in RBC function Cryoprecipitate: • Precipitate formed from thawing FFP at 1-6 degreesC • Contains: Factor VIII, von Willebrand factor, fibrinogen, factor XIII, fibronectin • Volume: 10-15 mL per bag • Indications: Hypofibrinogenaemia, haemophilia A, von Willebrand disease, massive transfusion • Storage: -20 degreesC to -30 degreesC (frozen) • Shelf life: 1 year
Coagulation
Standard Answer:
1. Blood coagulation using cell-based model A. Role of extrinsic tenase enzyme complex: • Components: Tissue factor (TF) + Factor VIIa • Formation: TF exposed on damaged endothelium -> binds FVII -> FVIIa • Function: Activates Factor X (to Xa) and Factor IX (to IXa) • Location: On TF-bearing cells (smooth muscle, subendothelial cells) B. Role of intrinsic tenase enzyme complex: • Components: Factor IXa + Factor VIIIa + Calcium + Phospholipid • Formation: On platelet surface (activated platelets) • Function: Activates Factor X (to Xa) (amplification) • Location: On platelet surface (activated platelets) C. Role of prothrombinase complex: • Components: Factor Xa + Factor Va + Calcium + Phospholipid • Formation: On platelet surface • Function: Converts Prothrombin (Factor II) -> Thrombin (Factor IIa) • Location: On platelet surface • Significance: Thrombin generation -> Fibrin formation 2. Process of blood coagulation (See question 5 in Coagulation section below and Physiology of hemostasis above) 3. Process of blood coagulation (See Physiology of hemostasis above) 4. Coagulation factors Factor Name Vitamin K Half-life I Fibrinogen No 3-5 days II Prothrombin Yes 2-3 days III Tissue factor No - IV Calcium No - V Labile factor No 12-36 hours VII Stable factor Yes 3-6 hours VIII Anti-haemophilic factor No 8-12 hours IX Christmas factor Yes 24 hours X Stuart-Prower factor Yes 30-40 hours XI Plasma thromboplastin antecedent No 40-80 hours XII Hageman factor No 40-80 hours XIII Fibrin-stabilizing factor No 9-10 days Prekallikrein Fletcher factor No - HMWK Fitzgerald factor No - 5. Cell-based theory of coagulation Definition: The cell-based model of coagulation proposes that coagulation occurs in three overlapping phases on cell surfaces rather than in fluid phase (as in cascade model): Phase 1: Initiation (on TF-bearing cells): • TF exposed on damaged endothelium • TF binds FVII -> FVIIa (TF-FVIIa complex) • Activates FX -> FXa (small amount) • Activates FIX -> FIXa (small amount) • FXa activates FV -> FVa Phase 2: Amplification (on activated platelets): • Thrombin generated (from initiation phase) • Activates platelets (exposes phospholipid) • Activates FV -> FVa, FVIII -> FVIIIa, FIX -> FIXa • FXI -> FXIa • Prevents coagulation inhibitors (TFPI, antithrombin) Phase 3: Propagation (on platelet surface): • Intrinsic tenase complex (FIXa + FVIIIa) -> Activates FX -> FXa • Prothrombinase complex (FXa + FVa) -> Converts prothrombin -> Thrombin (large burst) • Thrombin -> Fibrinogen -> Fibrin • Thrombin activates FXIII -> FXIIIa -> Cross-links fibrin 6. Congenital and acquired coagulation disorders Congenital: • Haemophilia A (factor VIII deficiency) • Haemophilia B (factor IX deficiency) • von Willebrand disease (vWF deficiency) • Factor XI deficiency • Factor VII deficiency • Factor X deficiency • Factor V deficiency • Factor II deficiency • Fibrinogen deficiency • Factor XIII deficiency Acquired: • Liver disease (factor synthesis) • Vitamin K deficiency • DIC (consumption) • Anticoagulant therapy (warfarin, heparin) • Massive transfusion (dilution) • Autoimmune inhibitors (factor VIII inhibitors) • Uraemia • Amyloidosis • Thrombocytopenia 7. Coagulation disorders of Liver Diseases Pathogenesis: • Decreased synthesis of clotting factors (I, II, V, VII, IX, X, XI, XII, XIII) • Vitamin K deficiency (malabsorption, decreased stores) • Thrombocytopenia (hypersplenism, bone marrow suppression) • DIC (end-stage liver disease) • Impaired fibrinogen synthesis (dysfibrinogenaemia) Clinical: • Bleeding (GI bleeding, petechiae, ecchymoses) • Prolonged PT and APTT (especially PT due to FVII deficiency) • Low fibrinogen (in severe disease) • Thrombocytopenia Treatment: • Vitamin K (if deficiency suspected) • FFP (for bleeding with coagulopathy) • Factor concentrates (if available) • Platelet transfusion (for thrombocytopenia) • Tranexamic acid (for fibrinolysis) 8. Platelets and coagulation factors Platelets: • Function: Primary haemostasis, platelet plug formation, support secondary haemostasis • Normal count: 150-400x109/L • Life span: 7-10 days • Size: 2-4 um • Granules: alpha-granules (vWF, fibrinogen, PDGF), dense granules (ADP, Ca2+, serotonin) • Activation: Shape change, degranulation, aggregation Coagulation factors: • Function: Secondary haemostasis, fibrin clot formation • Vitamin K-dependent: II, VII, IX, X, Protein C, Protein S • Contact factors: XII, XI, Prekallikrein, HMWK • Co-factors: V, VIII, III (TF), IV (Ca2+) 9. Ten causes of DIC with examples 1. Sepsis (Gram-negative) • Example: Meningococcaemia 2. Obstetric complications • Example: Amniotic fluid embolism, placental abruption 3. Malignancy • Example: Acute promyelocytic leukaemia (APL), adenocarcinoma 4. Trauma • Example: Severe head injury, multiple fractures 5. Massive transfusion • Example: > 1 blood volume replaced 6. Snake envenomation • Example: Viper bites 7. Vascular malformations • Example: Giant haemangiomas (Kasabach-Merritt syndrome) 8. Infections • Example: Meningococcaemia, malaria 9. Liver failure • Example: Acute liver failure 10. Immunological • Example: Anaphylactic reactions, transfusion reactions 10. Type 2 von Willebrand's disease Definition: Qualitative defect of von Willebrand factor (vWF) with impaired platelet binding. Types: • Type 2A: Defect in high molecular weight multimers (decreased platelet-dependent function) • Type 2B: Increased affinity for platelets (increased platelet binding) • Type 2M: Defective platelet binding (normal multimer pattern) • Type 2N: Defective factor VIII binding (normal multimer pattern) Genetics: Autosomal dominant (most), autosomal recessive (rare) Clinical: • Mucocutaneous bleeding (similar to vWD type 1) • Easy bruising, epistaxis, menorrhagia • More severe than type 1 Investigations: • Bleeding time: Prolonged • vWF:Ag: Normal or low (type 2A) or normal (type 2B, 2M, 2N) • vWF activity (ristocetin cofactor): Low (type 2A, 2M) or low/normal (type 2B) • Multimer analysis: Abnormal pattern • Ristocetin-induced platelet aggregation: Low (type 2A, 2M), increased (type 2B) • Factor VIII: Reduced (type 2N) Treatment: • DDAVP (desmopressin): For type 2A, 2M, 2N (not for type 2B) • vWF concentrate (plasma-derived) • Antifibrinolytics (tranexamic acid) 11. Physiology of hemostasis (See question 15 in Erythropoiesis section)
Haematopoiesis and Growth Factors
Standard Answer:
1. Define hematopoiesis Hematopoiesis is the process of blood cell formation and development from hematopoietic stem cells (HSCs) in the bone marrow, leading to the production of all mature blood cells including erythrocytes, leukocytes, and thrombocytes. 2. Features/characteristics of haematopoietic stem cells (HSCs) • Self-renewal capacity • Multipotency (ability to differentiate into all blood cell lineages) • Plasticity • Quiescence (remain in G0 phase) • Ability to migrate and home to bone marrow niches • Expression of surface markers: CD34+, CD133+, CD90+, CD117 (c-Kit), Sca-1 • Lack of lineage-specific markers (Lin-) • Asymmetric division capability 3. Diagram illustrating the fate of HSC ``` [Diagram omitted] vv [Diagram omitted] vvvvvv [Diagram omitted] vvv [Diagram omitted] vvv [Diagram omitted] vvv [Diagram omitted] vv [Diagram omitted] v [Diagram omitted] ``` 4. Three regulators of haematopoiesis • Growth factors/Cytokines (e.g., EPO, G-CSF, GM-CSF) • Transcription factors (e.g., GATA-1, PU.1, C/EBPalpha) • Bone marrow microenvironment (stromal cells, extracellular matrix) 5. Three organs in which haemopoiesis can occur • Bone marrow (primary site in adults) • Liver (fetal haemopoiesis) • Spleen (fetal and extramedullary in adults) 6. Three haematological growth factors • Erythropoietin (EPO) • Granulocyte-Colony Stimulating Factor (G-CSF) • Thrombopoietin (TPO) 7. Bone marrow microenvironment The bone marrow microenvironment is the specialized three-dimensional structural and functional network within the bone marrow that supports and regulates haematopoiesis. It consists of: • Stromal cells (fibroblasts, endothelial cells, osteoblasts, adipocytes) • Extracellular matrix components (collagen, fibronectin, laminin) • Growth factors and cytokines • Cell-cell interactions • Nerve endings • Vascular sinuses • Hematopoietic stem cell niches (osteoblastic and vascular niches) 8. Indications of erythropoietin therapy • Anaemia of chronic kidney disease • Anaemia in cancer patients receiving chemotherapy • Anaemia in HIV patients on zidovudine therapy • Preoperative autologous blood donation • Anaemia of prematurity • Myelodysplastic syndromes with low-risk • Anaemia in chronic disease (rheumatoid arthritis, inflammatory bowel disease) 9. Differentiation Differentiation is the process by which a less specialized cell (stem cell or progenitor cell) undergoes progressive maturation to become a more specialized cell type with specific morphological, biochemical, and functional characteristics, accompanied by the expression of lineage-specific genes and loss of pluripotency. 10. Blood and its subcomponents Blood is a specialized connective tissue circulating in the cardiovascular system. Subcomponents: • Plasma (55% of blood volume): water, proteins, electrolytes, nutrients, hormones, waste products • Formed elements (45% of blood volume): • Erythrocytes (red blood cells) • Leukocytes (white blood cells): neutrophils, lymphocytes, monocytes, eosinophils, basophils • Thrombocytes (platelets) 11. Serum Serum is the liquid portion of blood remaining after clotting has occurred, which removes fibrinogen and other clotting factors. It contains water, electrolytes, proteins (albumin, globulins), hormones, and metabolic waste products, but lacks clotting factors (especially fibrinogen and prothrombin). 12. Haematocrit and its value in humans Haematocrit (Packed Cell Volume - PCV) is the percentage of blood volume occupied by red blood cells after centrifugation. Normal values: • Adult males: 40-52% • Adult females: 36-48% • Newborns: 45-61% • Children: 35-40% 13. Five functions of blood • Oxygen transport from lungs to tissues • Carbon dioxide transport from tissues to lungs • Nutrient transport (glucose, amino acids, lipids, vitamins) • Waste product transport to excretory organs • Immune defense and protection • Temperature regulation • Coagulation and haemostasis • Hormone transport • Acid-base balance maintenance 14. Five plasma proteins • Albumin • Globulins (alpha, beta, gamma) • Fibrinogen • Prothrombin • Transferrin • Ceruloplasmin • Haptoglobin • Complement proteins 15. Distribution of respiratory O2 and CO2 in plasma and haemoglobin Oxygen: • Dissolved in plasma: ~1.5% (2-3 ml O2/L blood) • Bound to haemoglobin: ~98.5% (200 ml O2/L blood) • Each Hb molecule binds 4 O2 molecules • Oxyhaemoglobin (HbO2) Carbon Dioxide: • Dissolved in plasma: ~7% • As bicarbonate (HCO3-): ~70% (converted by carbonic anhydrase) • Bound to haemoglobin as carbamino compounds: ~23% • Transported as carbamino-haemoglobin 16. Haemoglobin Haemoglobin is a tetrameric metalloprotein found in red blood cells composed of four globin polypeptide chains (two alpha and two beta chains in adult HbA) each containing a heme group with a ferrous iron (Fe2+) atom at its center. Functions: • Oxygen transport from lungs to tissues • Carbon dioxide transport from tissues to lungs • Hydrogen ion buffering • Nitric oxide scavenging and transport Types: HbA (alpha2beta2), HbA2 (alpha2delta2), HbF (alpha2gamma2), and various abnormal haemoglobins. 17. Rouleaux and Diapedesis Rouleaux: The stacking of red blood cells in a "coin-roll" formation due to increased plasma proteins (fibrinogen, globulins) causing reduced zeta potential and increased aggregation. Seen in multiple myeloma, inflammatory conditions, and infections. Diapedesis: The process by which leukocytes (particularly neutrophils and monocytes) migrate through the endothelial cell junctions of blood vessel walls to enter tissues in response to inflammatory signals. Involves margination, rolling, adhesion, and transmigration mediated by selectins, integrins, and chemokines. 18. Two main features of Haematopoietic stem cells. Mention two types and their characteristics. Two main features: • Self-renewal capacity • Multipotency Two types: • Long-term HSCs (LT-HSCs): Self-renew indefinitely, maintain haematopoiesis throughout life, predominantly quiescent • Short-term HSCs (ST-HSCs): Limited self-renewal capacity, more actively cycling, differentiate into multipotent progenitors 19. Hayflick limit The Hayflick limit is the finite number of cell divisions (approximately 40-60) that a normal somatic cell can undergo before entering senescence, due to progressive shortening of telomeres with each cell division. This does not apply to germ cells, cancer cells, and stem cells that express telomerase. 20. Road map of haematopoiesis The hierarchical organization of haematopoiesis from stem cells to mature cells: • HSC -> Multipotent Progenitor (MPP) • MPP -> Common Myeloid Progenitor (CMP) and Common Lymphoid Progenitor (CLP) • CMP -> Megakaryocyte-Erythroid Progenitor (MEP) and Granulocyte-Monocyte Progenitor (GMP) • MEP -> Erythrocytes, Platelets • GMP -> Granulocytes (neutrophils, eosinophils, basophils) and Monocytes/Macrophages • CLP -> T cells, B cells, NK cells 21. Renewal, proliferation, and differentiation Renewal: The ability of HSCs to self-replicate to maintain the stem cell pool through asymmetric or symmetric division. Proliferation: The process of increased cell division and expansion of progenitor cell populations in response to growth factors, leading to increased cell numbers. Differentiation: The progressive process by which cells become more specialized and committed to specific lineages, losing pluripotency while gaining lineage-specific characteristics, markers, and functions.
Growth Factor
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22. What is growth factor control Growth factor control refers to the regulatory mechanisms that govern the production, secretion, and activity of growth factors/cytokines in haematopoiesis. It involves: • Feedback loops (negative and positive) • Hormonal regulation • Inflammatory mediators • Hypoxic response (e.g., HIF-1alpha regulating EPO) • Cell-cell interactions • Autocrine and paracrine signaling • Receptor availability and signaling pathways 23. Eight characteristics of haematopoietic Growth factors • Glycoproteins (except some) • Act at very low concentrations (picomolar) • Bind to specific high-affinity cell surface receptors • Exhibit pleiotropy (multiple effects on different cell types) • Exhibit redundancy (multiple factors have overlapping functions) • Act synergistically with other growth factors • Short half-life in circulation • Produced by multiple cell types (stromal cells, immune cells) • Regulated by feedback mechanisms • Stimulate proliferation, differentiation, and survival 24. Mechanisms of Cytokine regulation • Transcriptional regulation (mRNA synthesis) • Post-transcriptional regulation (mRNA stability) • Translational regulation • Post-translational modification (glycosylation) • Receptor expression and affinity • Signal transduction pathways (JAK-STAT, PI3K/Akt, MAPK) • Negative feedback regulators (SOCS, CIS) • Soluble receptors (decoy receptors) • Receptor antagonists • Extracellular matrix binding and presentation 25. Two types of haematopoietic GFs • Myeloid growth factors: EPO, G-CSF, GM-CSF, M-CSF, TPO, IL-3, IL-5, IL-6 • Lymphoid growth factors: IL-2, IL-4, IL-7, IL-9, IL-15, IL-21 26. Clinical applications of Haematopoietic Gfs • Anaemia (EPO): CKD, chemotherapy-induced anaemia • Neutropenia (G-CSF): chemotherapy-induced, bone marrow transplant, congenital neutropenia • Stem cell mobilization (G-CSF, Plerixafor) • Thrombocytopenia (TPO-mimetics): ITP, chemotherapy • Myelodysplastic syndromes (EPO, G-CSF) • Aplastic anaemia (immunosuppression + G-CSF/EPO) • Radiation/chemical exposure (various) 27. Short note on haematopoietic Microenvironment with diagram The haematopoietic microenvironment (HME) is the specialized bone marrow niche that supports haematopoiesis through structural, cellular, and molecular components. ``` [Diagram omitted] vvv [Diagram omitted] vvvvvv [Diagram omitted] vvvvvv [Diagram omitted] vv [Diagram omitted] ``` The niche provides: • Cell-cell contact • Growth factor and cytokine production • Adhesion and homing • Regulation of HSC quiescence and self-renewal • Oxygen gradient regulation 28. Cytokines and their functions, production Cytokines: Small secreted proteins (~5-20 kDa) that mediate cell-to-cell communication and regulate immune responses, inflammation, and haematopoiesis. Functions: • Regulation of haematopoiesis • Immune response modulation • Inflammation • Cell proliferation and differentiation Production: Produced by various cells including: • Stromal cells • Macrophages • Lymphocytes (T cells, B cells) • Fibroblasts • Endothelial cells • Epithelial cells 29. Four effects of growth factors • Proliferation of progenitor cells • Differentiation into mature cells • Survival (anti-apoptotic) effects • Activation of mature cell functions • Chemotaxis and migration • Self-renewal regulation 30. Thrombopoiesis Thrombopoiesis is the process of platelet production from megakaryocytes in the bone marrow, regulated by thrombopoietin (TPO). Steps: • Megakaryocyte differentiation from HSC • Endomitosis (polyploidization) • Cytoplasmic maturation • Proplatelet formation • Platelet release into circulation 31. Haemopoietic stem cell transplantation (HSCT) HSCT is the intravenous infusion of haematopoietic stem cells to re-establish haematopoiesis in patients with bone marrow failure or following myeloablative therapy. Types: • Autologous HSCT: Stem cells from the patient themselves • Allogeneic HSCT: Stem cells from a donor (matched sibling, matched unrelated donor, haploidentical, cord blood) • Syngeneic HSCT: Stem cells from an identical twin 32. Six indications for HSCT • Acute leukemias (ALL, AML) • Chronic myeloid leukemia • Myelodysplastic syndromes • Aplastic anaemia • Lymphomas (Hodgkin, Non-Hodgkin) • Multiple myeloma • Thalassemia major • Sickle cell anaemia • Severe combined immunodeficiency (SCID) • Metabolic storage diseases
Erythropoiesis
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1. Orthopoeisis Orthopoeisis is the normal, orderly process of red blood cell (erythrocyte) production in the bone marrow, proceeding through sequential stages from erythroid progenitor to mature erythrocyte. Site Factors Erythropoiesis occurs in: • Embryo: Yolk sac • Fetus: Liver, spleen • Children: All bones (bone marrow) • Adults: Axial skeleton (vertebrae, sternum, ribs, pelvis, skull), proximal ends of long bones 2. Erythropoietin Source: Primarily produced by peritubular interstitial fibroblasts in the kidney (90%), with minor production by the liver (10%). Stimulus: Hypoxia (detected by HIF-1alpha), anaemia, decreased oxygen delivery, cobalt, androgen hormones. Mechanism of action: Binds to EPO receptor on erythroid progenitor cells -> JAK2 phosphorylation -> STAT5 activation -> increases transcription of anti-apoptotic genes (Bcl-xL) -> promotes survival, proliferation, and differentiation of erythroid progenitors. Use of erythropoietin: Treatment of anaemia in chronic kidney disease, chemotherapy-induced anaemia, anaemia of prematurity, pre-operative autologous blood donation, myelodysplastic syndromes. 3. Red cell concentrate preparation, storage, and indications Preparation: • Whole blood collected in anticoagulant (CPD, CPDA-1) • Centrifugation to separate RBCs from plasma and platelets • Removal of plasma and buffy coat • Addition of additive solution (AS-1, AS-3, AS-5, SAGM) to extend storage • Leukoreduction (filtration) optional Storage: • Temperature: 1-6 degreesC • Shelf life: 21-42 days depending on anticoagulant/additive solution • Contains: RBCs, additive solution, minimal plasma Types: • Packed red blood cells (PRBC): Standard RBC concentrate • Leukoreduced RBCs: Filtered to remove WBCs • Washed RBCs: Removes plasma proteins and antibodies • Irradiated RBCs: Prevents GVHD • Frozen RBCs: Glycerolized, stored at -80 degreesC, extended shelf life Indications: • Symptomatic anaemia (Hb < 7-8 g/dL) • Acute blood loss (>20-30% blood volume) • Surgery with anticipated major blood loss • Transfusion-dependent thalassemia • Aplastic anaemia • Bone marrow failure • Chemotherapy-induced anaemia 4. Five causes of acquired coagulation disorders • Liver disease (hepatic failure) • Vitamin K deficiency • Disseminated intravascular coagulation (DIC) • Anticoagulant therapy (warfarin, heparin) • Massive transfusion (dilutional coagulopathy) • Autoimmune inhibitors (factor VIII inhibitors) • Uraemia • Amyloidosis Investigations for suspected coagulation disorders: • Prothrombin time (PT) • Activated partial thromboplastin time (APTT) • Thrombin time (TT) • Fibrinogen level • D-dimer • Platelet count • Bleeding time • Mixing studies (50:50 with normal plasma) • Specific factor assays • Liver function tests • Vitamin K status (PIVKA-II) 5. Haemopoietic Stem cell Transplantation and cure for haematological disorders HSCT cures haematological disorders through: • Myeloablative conditioning: Eradicates malignant cells with high-dose chemotherapy/radiation • Graft-versus-leukaemia effect: Donor immune cells eliminate residual malignant cells • Replacement of defective haematopoiesis: Healthy stem cells establish normal haematopoiesis • Immuno-reconstitution: Restores functional immune system Complications: • Graft failure (primary/secondary) • Graft-versus-host disease (GVHD) - acute and chronic • Infections (bacterial, viral, fungal, protozoal) • Veno-occlusive disease (VOD/SOS) • Mucositis • Haemorrhagic cystitis • Idiopathic pneumonia syndrome • Secondary malignancies • Endocrine dysfunction (growth, thyroid, gonadal) • Cataracts • Cardiotoxicity • Pulmonary fibrosis 6. Blood component therapy Blood component therapy is the separation of whole blood into specific components (RBCs, platelets, plasma, cryoprecipitate) to provide targeted therapy, allowing optimal use of each donation and minimizing transfusion of unnecessary components. Advantages: • Optimal utilization of each donation (multiple patients from one donation) • Targeted therapy (patient receives only what is needed) • Reduced transfusion reactions • Reduced volume overload • Better preservation of components • Cost-effective • Reduced transmission of infection (per unit exposure) Disadvantages: • Requires specialized equipment and trained personnel • Increased risk of multiple donor exposures • Shorter shelf life of some components • Higher cost of processing • Risk of bacterial contamination (especially platelets) • Alloimmunization risk with multiple transfusions 7. Red cell membrane diagram ``` [Diagram omitted] ``` 8. Red cell membrane The red cell membrane is a complex, flexible structure consisting of: • Lipid bilayer: Phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine) and cholesterol • Membrane proteins: • Integral proteins: Band 3 (anion exchanger), Glycophorins A, B, C, Rh proteins • Peripheral proteins: Spectrin, Ankyrin, Band 4.1, Band 4.2, Actin • Glycocalyx: Carbohydrate-rich outer layer containing blood group antigens Functions: • Maintains biconcave discoid shape • Provides deformability for passage through microcirculation • Acts as osmotic barrier • Contains blood group antigens • Mediates gas exchange • Prevents cell lysis 9. Three proteins each involved in vertical and horizontal interactions Vertical interactions (between membrane and cytoskeleton): • Ankyrin (connects Band 3 to spectrin) • Band 4.1 (connects glycophorin C to spectrin) • Band 4.2 (stabilizes ankyrin-band 3 interaction) Horizontal interactions (within cytoskeleton): • Spectrin (alpha and beta chains) • Actin • Protein 4.1 (cross-links spectrin to actin) • Adducin 10. Two genetic disorders affected by vertical and horizontal interactions 1. Hereditary Spherocytosis: Defective proteins: Ankyrin, Spectrin, Band 3 Clinical features: • Anaemia (mild to severe) • Jaundice • Splenomegaly • Gallstones Lab investigations: • Blood film: spherocytes, polychromasia • Increased MCHC • Osmotic fragility: increased • Coombs test: negative • Reticulocytosis • Serum bilirubin: elevated (unconjugated) • Bone marrow: erythroid hyperplasia 2. Hereditary Elliptocytosis: Defective proteins: Spectrin (alpha or beta), Protein 4.1 Clinical features: • Usually asymptomatic • Mild anaemia • Occasionally haemolysis • Splenomegaly (mild) Lab investigations: • Blood film: elliptocytes (>25%) • Increased reticulocytes (if haemolysis) • Osmotic fragility: variable (normal or decreased) • Coombs test: negative • Mild anaemia 11. Short notes on platelet production, erythropoiesis, and spleen functions Platelet production (Thrombopoiesis): • HSC -> Megakaryocyte progenitor -> Megakaryocyte • Megakaryocytes undergo endomitosis (polyploidization) • Cytoplasmic maturation with granules • Proplatelet formation (extension of pseudopodia) • Platelet release into marrow sinusoids • Regulated by Thrombopoietin (TPO) from liver and kidneys • Maturation time: 4-5 days • Platelet life span: 7-10 days Erythropoiesis: • HSC -> BFU-E -> CFU-E -> Proerythroblast -> Basophilic erythroblast -> Polychromatic erythroblast -> Orthochromatic erythroblast -> Reticulocyte -> Erythrocyte • Regulated by Erythropoietin (EPO) • Requires: iron, vitamin B12, folate, vitamin B6, amino acids • Maturation time: 7-8 days • RBC life span: 120 days Functions of the spleen: • Red pulp: • Destruction of senescent RBCs (RBC graveyard) • Iron recycling • Extramedullary haematopoiesis (fetal/adult in disease) • Removal of RBC inclusions (Howell-Jolly bodies) • White pulp: • Immune surveillance (T and B cell zones) • Antibody production • Response to encapsulated organisms • Marginal zone: • Antigen trapping • Immune cell activation • Reservoir for platelets and WBCs 12. Role of RBC membrane in performing its functions • Deformability: Allows RBCs to pass through capillaries smaller than cell diameter • Osmotic stability: Prevents lysis in hypotonic environments • Gas exchange: Facilitates CO2 and O2 transport through membrane • Antigen presentation: Displays blood group antigens for immune recognition • Ion homeostasis: Maintains Na+/K+ balance via Na-K ATPase • Cell integrity: Prevents fragmentation and breakdown • Surface charge: Prevents aggregation (repulsion) 13. Substances of the RBC membrane and functions Lipids: • Phospholipids: Bilayer formation, membrane fluidity • Cholesterol: Membrane stability, fluidity modulation • Glycolipids: Blood group antigens, cell recognition Proteins: • Band 3: Anion exchange (Cl-/HCO3-), structural support • Glycophorin A: Sialic acid content, negative charge • Spectrin: Cytoskeletal framework, membrane strength • Ankyrin: Vertical linkage to band 3 • Protein 4.1: Stabilizes cytoskeleton, actin-spectrin binding • Protein 4.2: Stabilizes ankyrin-band 3 interaction • Actin: Cytoskeletal component • Na-K ATPase: Maintains Na+/K+ gradient • Ca-ATPase: Maintains low intracellular Ca2+ 14. Haematopoietic growth factors and their functions Definition: Haematopoietic growth factors are glycoprotein hormones that regulate the survival, proliferation, differentiation, and maturation of haematopoietic progenitor cells. Factors and functions: Growth Factor Produced by Function EPO (Erythropoietin) Kidney (peritubular cells), liver Stimulates erythroid differentiation, RBC production G-CSF (Granulocyte-CSF) Macrophages, endothelial cells, fibroblasts Stimulates neutrophil production and function GM-CSF (Granulocyte-Macrophage-CSF) T cells, macrophages, fibroblasts Stimulates granulocyte and monocyte production M-CSF (Macrophage-CSF) Macrophages, fibroblasts, endothelial cells Stimulates monocyte/macrophage production and function TPO (Thrombopoietin) Liver, kidney, bone marrow Stimulates megakaryocyte maturation and platelet production SCF (Stem Cell Factor) Stromal cells, fibroblasts HSC self-renewal, progenitor proliferation IL-3 (Interleukin-3) T cells, NK cells Multi-lineage progenitor stimulation IL-5 T cells Eosinophil production and activation IL-7 Stromal cells, thymic epithelial cells T and B cell development IL-2 T cells T cell proliferation and activation 15. Physiology of hemostasis Haemostasis is the physiological process that stops bleeding at the site of vascular injury through a coordinated sequence of events: Primary haemostasis (Platelet plug formation): 1. Vasoconstriction: Reflex contraction of smooth muscle in injured blood vessel 2. Platelet adhesion: von Willebrand factor (vWF) binds to exposed subendothelial collagen -> Platelets adhere via GPIb-IX-V receptor binding to vWF 3. Platelet activation: Shape change, granule release (ADP, thromboxane A2, serotonin) 4. Platelet aggregation: Activation of GPIIb-IIIa receptors -> Fibrinogen cross-linking -> Platelet plug formation Secondary haemostasis (Coagulation cascade): 1. Intrinsic pathway: Factor XII -> XI -> IX -> (with VIII) -> X 2. Extrinsic pathway: Tissue factor + VIIa -> X 3. Common pathway: Xa + Va (prothrombinase complex) -> Prothrombin -> Thrombin 4. Fibrin formation: Thrombin converts fibrinogen to fibrin -> Fibrin mesh stabilizes platelet plug Fibrinolysis: • Tissue plasminogen activator (tPA) converts plasminogen to plasmin • Plasmin digests fibrin clot -> D-dimer production Regulation: • Antithrombin III (inhibits thrombin and factors Xa, IXa, XIa, XIIa) • Protein C + Protein S (inactivates factors Va and VIIIa) • Tissue factor pathway inhibitor (TFPI) • Thrombomodulin (activates protein C) • Fibrinolysis (plasmin)
HIV/AIDS
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1. Modes of transmission of HIV infection • Sexual transmission: Heterosexual, homosexual (most common) • Parenteral: • IV drug abuse (sharing needles) • Blood transfusion (screened in developed countries) • Needle-stick injuries (healthcare workers) • Organ transplantation • Vertical transmission: • Mother-to-child: Intrauterine, intrapartum, breastfeeding • Other: • Less common: Mucosal exposure (oral sex, if open wounds) 2. Relevant preventive measures 1. Safe sex: • Condom use • Reduce number of partners • HIV testing and counselling 2. Needle safety: • Use sterile needles (for IV drugs) • Proper disposal of needles 3. Blood safety: • Blood donor screening (HIV testing) • Blood product screening (nucleic acid testing) 4. Prenatal care: • Universal screening of pregnant women • Antiretroviral therapy (ART) for HIV-positive pregnant women • Elective caesarean section (if indicated) • Avoid breastfeeding (in developed countries) 5. Post-exposure prophylaxis (PEP): • Within 72 hours of exposure (needle-stick, sexual assault) • ART for 28 days 6. Antiretroviral therapy (ART): • Treatment as prevention (TasP) • Pre-exposure prophylaxis (PrEP) for high-risk populations 7. Education and awareness: • Condom use, safe needle practices, regular testing 8. Vaccines: (under development) 3. Clinical features of HIV Acute HIV (2-6 weeks post-exposure): • Fever: Most common • Pharyngitis: Sore throat, painful swallowing • Lymphadenopathy: Generalized • Rash: Maculopapular, truncal • Arthralgia/Myalgia • Headache • Malaise • Vomiting, diarrhoea • Thrush • HIV seroconversion illness: 50-90% symptomatic Chronic/Asymptomatic HIV (years): • Asymptomatic • Persistent generalized lymphadenopathy • Recurrent infections Advanced HIV/AIDS (CD4 < 200 cells/uL): • Opportunistic infections: • Pneumocystis jirovecii pneumonia (PCP) • Toxoplasmosis (CNS) • Cryptococcal meningitis • Cytomegalovirus (CMV) retinitis • Mycobacterium tuberculosis • MAC (Mycobacterium avium complex) • Oral candidiasis (thrush) • Herpes zoster • Malignancies: • Kaposi sarcoma (HHV-8) • Non-Hodgkin lymphoma • Cervical cancer • Neurological: • AIDS dementia complex (HIV-associated dementia) • Peripheral neuropathy • Wasting syndrome: Weight loss (> 10%), fever, diarrhoea • Haematological: • Anaemia, neutropenia, thrombocytopenia • Dermatological: • Seborrhoeic dermatitis, Kaposi sarcoma
Haemolytic Disease of Fetus & Newborn
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1. Haemorrhagic Disease of the Newborn Definition: Bleeding disorder in newborns due to vitamin K deficiency, characterized by bleeding (GI, skin, umbilical, intracranial). Pathophysiology: • Newborns have low vitamin K stores • Vitamin K-dependent factors (II, VII, IX, X, Protein C, Protein S) are reduced • Breastfed infants are at higher risk (low vitamin K in breast milk) • Gut colonization inadequate (vitamin K synthesis) • Maternal medications (anticonvulsants, warfarin) worsen deficiency Clinical types: • Early onset (< 24 hours): Severe (due to maternal medications) • Classical (2-7 days): Most common, GI bleeding, umbilical bleeding, skin bleeding, intracranial bleeding • Late onset (2-12 weeks): Intracranial bleeding, commonly in exclusively breastfed infants with malabsorption Treatment: • Vitamin K (IV/IM/PO) - 1 mg (IV/IM) or 2 mg (PO) • FFP for severe bleeding • Vitamin K prophylaxis (IM at birth) 2. Differentiate between Haemolytic Disease Of the Newborn and Haemorrhagic disease of the newborn Feature HDN Haemorrhagic Disease Cause Rh/ABO incompatibility (maternal-fetal) Vitamin K deficiency Pathophysiology Antibody-mediated haemolysis Factor deficiency (II, VII, IX, X) Clinical features Jaundice, anaemia, oedema, hepatosplenomegaly Bleeding (GI, skin, umbilical, intracranial) Onset 1-5 days (usually) 2-7 days (classical) Jaundice Yes (unconjugated bilirubin) No Bleeding Minimal (may have petechiae) Prominent Haemolysis Yes No Investigations Coombs test (positive), bilirubin elevated, anaemia PT/APTT prolonged, normal platelet count, low factors Treatment Phototherapy, exchange transfusion, IVIG Vitamin K, FFP Prevention Anti-D immunoglobulin Vitamin K prophylaxis 3. Haemolytic disease of the newborn and immunological mechanisms Definition: HDN is a condition where maternal antibodies cross the placenta and cause destruction of fetal RBCs due to antigen-antibody incompatibility. Immunological mechanisms: 1. Sensitization: • RhD-negative mother exposed to RhD-positive fetal RBCs (during delivery, abortion, invasive procedures) • Fetal RBCs enter maternal circulation • Immune response -> Anti-D antibodies (IgG) produced 2. Antibody production: • B cells -> Plasma cells -> Anti-D antibodies (IgG) • IgG crosses placenta via FcRn receptor 3. Fetal RBC destruction: • IgG binds to RhD-positive fetal RBCs • Opsonization -> Extravascular haemolysis (spleen, liver) • Macrophages phagocytose antibody-coated RBCs • Haemoglobin breakdown -> Bilirubin -> Jaundice ABO HDN: • Mother (blood group O) has naturally occurring Anti-A, Anti-B (IgG) • Fetus (blood group A or B) • Less severe than Rh HDN Other antibodies: • Anti-C, Anti-c, Anti-E, Anti-e, Anti-Kell, Anti-Duffy 4. Hydrops Fetalis Definition: Severe form of HDN causing generalized oedema (anasarca), ascites, pleural effusion, pericardial effusion, and severe anaemia in the fetus, often fatal. Causes: • Immune: Rh HDN (most common), ABO HDN, other blood group incompatibilities • Non-immune: • Haemoglobinopathies (alpha-thalassemia major) • Congenital infections (Parvovirus B19, CMV, toxoplasmosis) • Cardiac abnormalities • Chromosomal abnormalities (Turner's, Down's) • Twin-twin transfusion Pathophysiology: • Severe anaemia -> High-output cardiac failure -> Oedema -> Hydrops • Hypoxia -> Anaerobic metabolism -> Acidosis -> Further heart failure Treatment: • Prenatal: Intrauterine transfusion (fetal blood transfusion) • Postnatal: Exchange transfusion, phototherapy, supportive care • Prevention: Anti-D immunoglobulin for Rh-negative mothers 5. Eight agents that may cause haemolytic anaemia in G6PD deficiency 1. Primaquine (antimalarial) 2. Dapsone (leprosy) 3. Sulfonamides (antibiotics) 4. Nitrofurantoin (urinary tract infections) 5. Aspirin (high dose, > 3 g/day) 6. Quinine (antimalarial) 7. Isoniazid (anti-TB) 8. Chloramphenicol (antibiotic) 9. Methylene blue (for methemoglobinaemia) 10. Nalidixic acid (antibiotic) 11. Fava beans (broad beans) 12. Infection: • Viral hepatitis • Pneumonia • Typhoid fever • Severe bacterial infections 13. Other: • Diabetic ketoacidosis • Neonatal jaundice • Phenylhydrazine 6. Clinical types/presentations of haemorrhagic disease of the newborn (Vit K deficiency bleeding) 1. Early onset (<= 24 hours): • Severe (often fatal) • Caused by maternal medications (anticonvulsants, warfarin) • Bleeding: Cephalohaematoma, intracranial, GI 2. Classical (2-7 days): • Most common • Breastfed infants (low vitamin K) • Bleeding: GI (melena, haematemesis), umbilical (oozing), skin (ecchymoses), intracranial (severe) • Increased PT, APTT, decreased factors II, VII, IX, X 3. Late onset (2-12 weeks): • Exclusively breastfed infants with malabsorption (biliary atresia) • Intracranial bleeding (common) • Neurological symptoms (lethargy, seizures) 7. Classify vit K deficiency bleeding and write short note Classification: • Early onset (<= 24 hours): Maternal medications • Classical (2-7 days): Breastfed infants • Late onset (2-12 weeks): Exclusively breastfed, malabsorption Short note on Classical HDN: • Onset: 2-7 days • Cause: Vitamin K deficiency (low stores, low in breast milk) • Clinical: GI bleeding (melena, haematemesis), umbilical bleeding, skin bleeding (purpura, ecchymoses), intracranial bleeding • Investigations: Prolonged PT (most sensitive), APTT; factors II, VII, IX, X reduced; platelet count normal • Treatment: Vitamin K (1 mg IM/IV); FFP for severe bleeding • Prevention: Vitamin K IM at birth 8. Five maternal prenatal medications that interfere with vit K metabolism 1. Warfarin (anticoagulant, vitamin K antagonist) 2. Phenytoin (anticonvulsant, induces microsomal enzymes) 3. Rifampicin (antibiotic, enzyme inducer) 4. Carbamazepine (anticonvulsant) 5. Chloramphenicol (antibiotic) - not commonly used in pregnancy 6. Cephalosporins (antibiotics, with methylthiotetrazole side chain) 7. Phenobarbitone (anticonvulsant) Mechanism: These medications interfere with vitamin K metabolism in the fetus by: • Inhibiting vitamin K epoxide reductase (warfarin) • Inducing cytochrome P450 enzymes (phenytoin, rifampicin) • Direct inhibition of vitamin K-dependent carboxylation 9. Coombs test and Kleihauer test Coombs test: • Direct Coombs test (DAT): Detects antibodies bound to RBCs (in vivo) • Indications: HDN, autoimmune haemolytic anaemia, transfusion reaction • Positive: Antibody-coated RBCs agglutinate with anti-human globulin • Indirect Coombs test (IAT): Detects antibodies in serum (in vitro) • Indications: Pre-transfusion screening, antenatal screening • Positive: Patient serum antibodies react with test RBCs Kleihauer test (Kleihauer-Betke test): • Purpose: Detects fetal RBCs in maternal circulation (feto-maternal haemorrhage) • Principle: Fetal haemoglobin (HbF) is resistant to acid elution; maternal HbA is eluted • Indications: • Rh-negative mother with suspected feto-maternal haemorrhage • Determine dose of Anti-D immunoglobulin required • Method: Acid elution -> Only fetal RBCs (with HbF) stain pink • Calculation: Number of fetal RBCs / 2000 maternal RBCs = % fetal haemoglobin • Quantification: Used to calculate Anti-D dose
DVT, DIC and Haemophilia
Standard Answer:
1. Risk factors for venous thrombosis Inherited: 1. Factor V Leiden (most common) 2. Prothrombin G20210A 3. Antithrombin III deficiency 4. Protein C deficiency 5. Protein S deficiency 6. Hyperhomocysteinaemia 7. Dysfibrinogenaemia Acquired: 1. Surgery (orthopaedic, abdominal, pelvic) 2. Trauma (fractures, head injury) 3. Immobilization (bed rest, long-haul flights) 4. Malignancy (solid tumours, haematological) 5. Pregnancy (and postpartum period) 6. Oral contraceptives (oestrogen) 7. Hormone replacement therapy 8. Obesity 9. Smoking 10. Heart failure 11. Respiratory failure 12. Polycythaemia vera 13. Myeloproliferative neoplasms 14. Antiphospholipid syndrome (APS) 15. Age > 60 years 2. Lab investigations for diagnosis of thromboembolism 1. D-dimer: Elevated (sensitive, not specific), negative D-dimer rules out VTE in low-risk patients 2. Venous duplex ultrasound (Doppler): DVT detection (compressibility, flow) 3. CT pulmonary angiography: PE diagnosis (gold standard) 4. VQ scan: PE diagnosis (if CT not available) 5. Compression ultrasound: Proximal DVT 6. MR venography: DVT (if ultrasound inconclusive) 7. Contrast venography: DVT (gold standard, rarely used) 8. ECG: Sinus tachycardia, right heart strain (PE) 9. Chest X-ray: PE (normal or non-specific) 10. Arterial blood gas: Hypoxia, hypocapnia (PE) 11. Echocardiogram: Right heart dysfunction (PE) 3. Five Causes of DIC with examples (See question 9 in Coagulation section) 4. DIC/Define Disseminated Intravascular Coagulation Definition: DIC is an acquired syndrome characterized by widespread activation of coagulation pathways leading to: • Intravascular deposition of fibrin thrombi • Consumption of clotting factors and platelets • Bleeding (due to depletion) • Organ failure (from microvascular thrombosis) Pathophysiology: • Excessive thrombin generation (systemic activation) • Fibrin deposition in microvasculature • Consumption of clotting factors (I, II, V, VIII, XIII) • Platelet consumption -> Thrombocytopenia • Activation of fibrinolysis (plasminogen) -> Fibrin degradation products (FDPs), D-dimer • Bleeding (due to factor depletion) and thrombosis (simultaneously) Clinical: • Bleeding: Petechiae, ecchymoses, GI bleeding, mucosal bleeding, surgical site bleeding • Thrombosis: Organ dysfunction (renal, hepatic, pulmonary) • Features of underlying condition: Sepsis, trauma, malignancy 5. Ten causes of DIC (See question 9 in Coagulation section) 6. Pathogenesis of DIC (See question 4 above) 7. Coagulation cascade (See Physiology of hemostasis above) 8. Blood transmissible infections and four examples Definition: Infections transmitted through transfusion of blood or blood products. Four examples: 1. HIV (Human Immunodeficiency Virus) 2. Hepatitis B Virus (HBV) 3. Hepatitis C Virus (HCV) 4. Malaria (Plasmodium spp.) 5. Syphilis (Treponema pallidum) 6. HTLV (Human T-cell Lymphotropic Virus) 7. CMV (Cytomegalovirus) 8. Parvovirus B19 9. Babesiosis (Babesia spp.) 10. Chagas disease (Trypanosoma cruzi) 9. Haemophilias Definition: Inherited bleeding disorders characterized by deficiency of specific clotting factors. Types: 1. Haemophilia A: Factor VIII deficiency (most common, 80-85%) 2. Haemophilia B: Factor IX deficiency (Christmas disease, 15-20%) 3. Haemophilia C: Factor XI deficiency (autosomal, less severe) Genetics: X-linked recessive (haemophilia A and B), autosomal recessive (haemophilia C) Clinical features: • Bleeding into joints (haemarthrosis) • Bleeding into muscles • Haematomas • Bleeding after trauma/surgery • Prolonged bleeding (nosebleeds, oral bleeding) • Intracranial haemorrhage (rare, severe) Investigations: • APTT: Prolonged • PT: Normal • Thrombin time: Normal • Platelet count: Normal • Factor VIII assay (Haemophilia A): Reduced • Factor IX assay (Haemophilia B): Reduced • Factor XI assay (Haemophilia C): Reduced • von Willebrand factor: Normal (to differentiate from vWD) • Inhibitor screening (antibodies) Treatment: • Factor VIII concentrate (Haemophilia A) • Factor IX concentrate (Haemophilia B) • Desmopressin (mild to moderate, Haemophilia A) • Antifibrinolytics (tranexamic acid) • Gene therapy (emerging) 10. Clinical presentation of classical haemophilia Haemophilia A (Factor VIII deficiency): Clinical features: • Haemarthrosis: Bleeding into joints (knees, elbows, ankles) - most common • Muscle haematomas: Deep muscle bleeding • Prolonged bleeding: After trauma, surgery, dental extraction • Spontaneous bleeding: Without obvious cause (in severe disease) • Oral bleeding: Bleeding gums, mouth ulcers • Haematuria: Blood in urine • Intracranial haemorrhage: Severe, life-threatening (head injury) • Retroperitoneal haematoma: Abdominal pain • Haemophilic arthropathy: Joint damage from recurrent bleeding Severity: • Severe: Factor VIII < 1% -> Spontaneous bleeding • Moderate: Factor VIII 1-5% -> Bleeding with minor trauma • Mild: Factor VIII 5-40% -> Bleeding with major trauma/surgery 11. Immunopathology of Goodpasture syndrome and Pernicious anaemia (See question 8 in Bone Marrow Failure section) 12. Haemophilia A and diagnosis Definition: Haemophilia A is an X-linked recessive bleeding disorder caused by deficiency of factor VIII (anti-haemophilic factor). Diagnosis: • History: Family history, bleeding (haemarthrosis, muscle haematomas) • Examination: Joint swelling, haematomas, bruising • Investigations: • APTT: Prolonged • PT: Normal • Thrombin time: Normal • Platelet count: Normal • Factor VIII assay: Reduced (< 50%) • Inhibitor screening: To detect antibodies • vWF assay: Normal (to exclude vWD) • Genetic testing: F8 gene mutation (for carriers, prenatal) • Cytogenetics: (Not required) 13. Ten clinical manifestations of Haemophilia A 1. Haemarthrosis (knees, elbows, ankles) 2. Muscle haematomas (calf, thigh) 3. Spontaneous bruising (ecchymoses) 4. Prolonged bleeding after trauma/surgery 5. Oral bleeding (gums, mouth) 6. Haematuria (blood in urine) 7. Intracranial haemorrhage (head injury) 8. Retroperitoneal haematoma 9. Haemophilic arthropathy (joint damage) 10. Bleeding after dental extraction 11. Epistaxis (nosebleeds) 12. Haematemesis (GI bleeding) 13. Dissecting haematoma (rare) 14. Compartment syndrome (muscle haematoma) 14. Two investigations for Haemophilia A 1. Factor VIII assay: Reduced (< 50% of normal) 2. APTT (activated partial thromboplastin time): Prolonged 3. Inhibitor screening: To detect anti-factor VIII antibodies 4. Genetic testing: F8 gene mutation (for diagnosis, carriers) 15. Five treatment modalities for haemophilia 1. Factor VIII/IX concentrate: Recombinant or plasma-derived 2. Prophylactic factor replacement: Regular dosing (to prevent bleeding) 3. On-demand factor replacement: For acute bleeding episodes 4. Desmopressin (DDAVP): For mild-moderate haemophilia A (releases vWF/FVIII) 5. Antifibrinolytics (tranexamic acid, EACA): For mucosal bleeding 6. Gene therapy: Emerging (factor IX gene therapy) 7. Bypassing agents: For patients with inhibitors (FEIBA, rFVIIa) 8. Supportive: Immobilization, ice, pressure, pain management 16. Treatment of a patient with haemophilia General principles: • Factor replacement: Give factor VIII/IX (recombinant or plasma-derived) • Prevention: Prophylactic factor replacement (in severe disease) • Acute bleeding: On-demand factor replacement • Dose: Based on factor level and severity • Mild bleeding: 10-20 IU/kg (target 30-40%) • Moderate bleeding: 20-30 IU/kg (target 40-60%) • Severe bleeding: 40-50 IU/kg (target 80-100%) Specific: • Haemarthrosis: Factor replacement + analgesia + immobilization • Muscle haematoma: Factor replacement + analgesia + rest • Surgery: Factor replacement before/during/after surgery • Mild-moderate: Desmopressin (DDAVP) can be used • Inhibitors: Bypassing agents (rFVIIa, FEIBA) 17. Treatment of haemophilia A and complications Treatment: 1. Factor VIII concentrate: Recombinant or plasma-derived 2. Prophylactic therapy: Regular factor VIII (50 IU/kg twice weekly) 3. On-demand therapy: For acute bleeding 4. Desmopressin (DDAVP): For mild-moderate haemophilia A 5. Antifibrinolytics: Tranexamic acid (for mucosal bleeding) 6. Gene therapy: (Under development) 7. Supportive: Physiotherapy, pain management Complications: 1. Inhibitor development: Neutralizing antibodies (most serious) 2. Joint damage: Haemophilic arthropathy (from recurrent bleeds) 3. Muscle atrophy: From disuse due to joint bleeding 4. Haemorrhage: Intracranial, retroperitoneal (life-threatening) 5. Transfusion-transmitted infections: (Plasma-derived products - now rare) 6. Allergic reactions: To factor concentrates 7. Anaemia: From chronic bleeding 8. Compartment syndrome: Muscle haematoma 18. Haemophilia - Mode of inheritance, Pathophysiology, Clinical features, Treatment plan Mode of inheritance: • X-linked recessive (haemophilia A and B) • Males affected (hemizygous) • Females: Carriers (heterozygous), affected (homozygous, rare) • No male-to-male transmission • All daughters of affected males are carriers • Sons of carrier females have 50% risk of haemophilia • Factor XI deficiency: Autosomal recessive Pathophysiology: • Deficiency of factor VIII (A) or factor IX (B) • Impaired tenase complex formation (FIXa + FVIIIa) • Reduced activation of FX -> Reduced thrombin generation • Delayed fibrin formation -> Bleeding • Joint bleeding -> Synovitis -> Cartilage damage -> Arthropathy Clinical features: (See question 13 above) Treatment plan: 1. Acute bleeding: • Factor replacement (dose based on severity) • IMMOBILIZATION of affected joint • Analgesia • Ice packs • Avoid NSAIDs 2. Prophylaxis: • For severe haemophilia • Regular factor replacement (2-3 times/week) • Prevent joint bleeds 3. Surgery: • Factor replacement (pre-operative, intra-operative, post-operative) • Close monitoring of factor levels 4. Inhibitor management: • Bypassing agents (rFVIIa, FEIBA) • Immune tolerance induction (ITI) - high-dose factor VIII 5. Gene therapy: • Emerging treatment (for factor IX deficiency) 6. Supportive: • Physiotherapy • Occupational therapy • Psychological support
Haematological Malignancies
Standard Answer:
1. The Log-kill hypothesis and its application in chemotherapy of Haematological malignancies Log-kill hypothesis: • Proposed by Skipper and Schabel (1964) • Chemotherapy kills a constant fraction (logarithmic) of tumour cells, not a constant number • Each course of chemotherapy kills a constant proportion (e.g., 99.9% = 3 logs) of remaining tumour cells • Tumour burden: 1 log = 1010 cells (approx. 1 cm diameter) Application: 1. Induction therapy: Reduces tumour burden from ~1012 to ~109-1010 cells (3-4 log kill) 2. Consolidation therapy: Further reduces to ~106-107 cells 3. Maintenance therapy: Eradicates residual disease (minimal residual disease, MRD) 4. Stem cell rescue: Allows higher doses (more log kill) with rescue Significance: • Repeated cycles needed to eradicate all cells • Dose intensity is important (higher dose = more log kill) • Combination therapy (synergy) • Drug resistance emerges with suboptimal treatment • MRD detection used to guide therapy 2. Factors implicated in the aetiology of haematological malignancies 1. Genetic: • Inherited syndromes (Down's, Fanconi, Bloom, Ataxia telangiectasia) • Familial (familial CLL, CML) 2. Infections: • Viruses: EBV (Burkitt's), HTLV-1 (ATLL), HIV (lymphoma), HHV-8 (Kaposi sarcoma), HCV (lymphoma) • Bacteria: H. pylori (gastric MALT) 3. Environmental: • Chemicals: Benzene, pesticides, herbicides • Radiation: Ionizing radiation (atomic bombs, radiotherapy) 4. Immunosuppression: • HIV/AIDS • Organ transplantation (post-transplant lymphoproliferative disorders) 5. Haematological disorders: • MDS -> AML • MGUS -> Myeloma • Myeloproliferative neoplasms -> AML 6. Age: • Peak incidence in elderly 7. Race/Ethnicity: • Geographical variation 3. 60-year-old patient with haematological malignancy - Supportive care, Cytotoxic drugs, and side effects General supportive care: 1. Infection prevention: • Antibiotic prophylaxis • Antifungal prophylaxis • Antiviral prophylaxis (acyclovir) • Pneumocystis jirovecii prophylaxis (cotrimoxazole) • Strict hand hygiene • Neutropenic diet 2. Haematological support: • Blood transfusion (RBCs, platelets) • Growth factors (G-CSF, EPO) 3. Nutritional support: • Nutritional assessment • Dietary support (enteral/parenteral if needed) 4. Symptoms management: • Anti-emetics • Analgesia • Antipyretics • Mouth care (mucositis) 5. Psychosocial support: • Counselling • Support groups • Psychological support Three classes of cytotoxic drugs, two drugs, and two side effects each: 1. Alkylating agents: • Drugs: • Cyclophosphamide • Melphalan • Side effects: • Myelosuppression • Nausea/vomiting • Secondary malignancies (leukaemia) • Bladder toxicity (cyclophosphamide) 2. Antimetabolites: • Drugs: • Methotrexate • Cytarabine (Ara-C) • Side effects: • Myelosuppression • Mucositis • Hepatitis • Renal toxicity (methotrexate) 3. Anthracyclines: • Drugs: • Daunorubicin • Doxorubicin • Side effects: • Cardiotoxicity (dose-dependent) • Myelosuppression • Alopecia • Vesicant (extravasation) 4. Vinca alkaloids: • Drugs: • Vincristine • Vinblastine • Side effects: • Neurotoxicity (peripheral neuropathy, vincristine) • Myelosuppression (vinblastine) • Alopecia • Constipation (autonomic neuropathy) 4. General supportive care in management of haematological malignancies (See question 3 above)
Myelodysplastic Syndrome
Standard Answer:
1. Myelodysplastic syndrome Definition: MDS is a heterogeneous group of haematopoietic stem cell disorders characterized by: • Dysplastic haematopoiesis (ineffective) • Cytopenias in peripheral blood (single or multiple) • Hypercellular bone marrow • Risk of progression to acute myeloid leukaemia (AML) • Clonal haematopoiesis Pathogenesis: • Clonal proliferation of abnormal HSCs • Apoptosis (programmed cell death) in bone marrow -> Cytopenias • Genetic mutations (SF3B1, TET2, ASXL1, DNMT3A, RUNX1, TP53) • Hypomethylation (epigenetic) • Chromosomal abnormalities (del(5q), del(7q), monosomy 7, complex karyotype) 2. Types of dysplastic cells seen in MDS 1. Dyserythropoiesis: • Megaloblastic changes (nuclear-cytoplasmic asynchrony) • Multinucleation (binucleate, multinucleate) • Nuclear fragments (Howell-Jolly bodies) • Ring sideroblasts (iron-loaded mitochondria) • Basophilic stippling • PAS-positive vacuoles 2. Dysgranulopoiesis: • Hypogranulation (pseudo-Pelger-Huet cells) • Hypersegmented neutrophils • Giant metamyelocytes • Ringed neutrophils • Lack of cytoplasmic granules 3. Dysmegakaryopoiesis: • Micromegakaryocytes (small, mononuclear) • Hypolobated nuclei • Multinucleation • Small hypolobated megakaryocytes • Megakaryocytes with bizarre nuclei 3. Classification of MDS based on FAB and WHO FAB Classification (French-American-British): Subtype Blasts Ring Sideroblasts Monocytes RA (Refractory Anaemia) < 5% < 15% < 1x109/L RARS (RA with Ring Sideroblasts) < 5% >= 15% < 1x109/L RAEB (RA with Excess Blasts) 5-20% Variable < 1x109/L RAEB-t (RAEB in Transformation) 21-30% Variable < 1x109/L CMML (Chronic Myelomonocytic Leukaemia) < 20% Variable 1x109/L WHO Classification (2016): Subtype Blasts Ring Sideroblasts Cytogenetics MDS with single lineage dysplasia (SLD) < 5% < 15% Normal MDS with multilineage dysplasia (MLD) < 5% < 15% Normal MDS with ring sideroblasts (MDS-RS) < 5% >= 15% Normal MDS with excess blasts-1 (MDS-EB1) 5-9% Variable Any MDS with excess blasts-2 (MDS-EB2) 10-19% Variable Any MDS with del(5q) < 5% Any del(5q) only MDS, unclassifiable (MDS-U) < 5% Variable Any
Thrombophilia
Standard Answer:
1. Thrombophilia A. Definition: Thrombophilia is an inherited or acquired predisposition to develop venous thromboembolism (VTE) due to abnormalities in coagulation proteins, fibrinolysis, or platelet function. B. Five causes of acquired thrombophilia: 1. Antiphospholipid syndrome (APS) - lupus anticoagulant, anti-cardiolipin, anti-beta2-glycoprotein I 2. Malignancy (solid tumours, haematological) 3. Pregnancy (hormonal changes, venous stasis) 4. Oral contraceptives/Hormone replacement therapy (oestrogen) 5. Myeloproliferative neoplasms (polycythaemia vera, essential thrombocythaemia) 6. Surgery/Trauma (immobilization, tissue factor release) 7. Obesity 8. Smoking 9. Nephrotic syndrome (loss of anticoagulant proteins) 10. Heparin-induced thrombocytopenia (HIT) C. Five causes of inherited thrombophilia: 1. Factor V Leiden mutation (G1691A) 2. Prothrombin G20210A mutation 3. Antithrombin III deficiency 4. Protein C deficiency 5. Protein S deficiency 6. Hyperhomocysteinaemia (MTHFR mutation - controversial) 7. Dysfibrinogenaemia (rare) 8. Factor XIII deficiency (rare) D. Three investigations useful in diagnosing thrombophilia: 1. Antithrombin III assay (functional) 2. Protein C assay (functional, antigenic) 3. Protein S assay (functional, antigenic) 4. Factor V Leiden mutation (DNA analysis) 5. Prothrombin G20210A mutation (DNA analysis) 6. Lupus anticoagulant (LA testing, dRVVT) 7. Anti-cardiolipin antibodies (IgG, IgM) 8. Anti-beta2-glycoprotein I antibodies (IgG, IgM) 9. Homocysteine levels 10. Factor VIII levels
Others
Standard Answer:
1. Short notes on: Tumour Lysis Syndrome: • Metabolic emergency caused by massive tumour cell destruction (spontaneous or chemotherapy) • Characterized by: • Hyperuricaemia (purine breakdown) • Hyperkalaemia • Hyperphosphataemia (phosphate release) • Hypocalcaemia (secondary to hyperphosphataemia) • Risk factors: Large tumour burden, high grade lymphomas (Burkitt's), ALL, AML, chemotherapy sensitivity • Prevention: Hydration, allopurinol, rasburicase (recombinant urate oxidase) • Treatment: Aggressive hydration, allopurinol/rasburicase, correction of electrolytes, renal replacement therapy ADCC (Antibody-Dependent Cell-Mediated Cytotoxicity): • Immune mechanism where target cells (e.g., tumour cells) are killed by immune cells (NK cells) through antibody-mediated recognition • Process: 1. Antibody (IgG) binds to target cell antigen 2. Fc region of antibody binds to FcgammaRIII (CD16) on NK cells 3. NK cells release cytotoxic granules (perforin, granzyme) -> target cell lysis • Clinical application: Monoclonal antibodies (Rituximab, Trastuzumab) mediate ADCC Principle of ELISA technique: • Enzyme-Linked Immunosorbent Assay (ELISA): Quantitative/qualitative immunoassay using enzyme-linked antibodies • Types: • Direct ELISA • Indirect ELISA • Sandwich ELISA • Competitive ELISA • Principle: 1. Antigen (or antibody) immobilized on solid phase (microplate) 2. Antibody (or antigen) added -> binds specifically 3. Enzyme-linked secondary antibody added 4. Substrate added -> Colour change (chromogenic substrate) 5. Colour intensity is measured (spectrophotometer) 6. Concentration proportional to colour intensity • Uses: HIV antibody detection, autoimmune antibodies, cytokines, hormones, tumour markers 2. Two types of cellular communication or messaging: • Autocrine: Cell responds to its own signal • Paracrine: Signal affects nearby cells • Endocrine: Signal travels through bloodstream to distant cells • Juxtacrine: Cell-cell contact (direct interaction) 3. Endocrine signalling - five stages of cellular messaging and response with examples 1. Signal synthesis: • Hormone production in endocrine gland • Example: Insulin produced by pancreatic beta-cells 2. Release of signal molecule: • Hormone secreted into bloodstream • Example: Insulin released into circulation 3. Transport of signal: • Travel via blood to target tissues • Example: Insulin travels to liver, muscle, adipose tissue 4. Signal binding to receptor: • Hormone binds to specific receptor on target cell • Example: Insulin binds to insulin receptor (tyrosine kinase) 5. Cellular response: • Intracellular signalling cascade -> Cellular response • Example: Glucose uptake, glycogen synthesis, protein synthesis 6. Signal termination: • Degradation of hormone/receptor • Example: Insulin degraded by liver/kidney 4. Platelet storage granules alpha-granules: • Proteins: P-selectin, vWF, fibrinogen, fibronectin, thrombospondin, PDGF, TGF-beta, PF4 • Functions: Adhesion, aggregation, angiogenesis, wound healing Dense granules (delta-granules): • Contents: ADP, ATP, Ca2+, serotonin, histamine, pyrophosphate • Functions: Platelet aggregation (ADP), vasoconstriction (serotonin) Lysosomal granules: • Contents: Acid hydrolases, cathepsins • Functions: Degradation Other: • alpha-granule deficiency -> Gray platelet syndrome • Dense granule deficiency -> Hermansky-Pudlak syndrome, Chediak-Higashi syndrome 5. Serine proteases Definition: Enzymes that cleave peptide bonds using a serine residue in their active site. Examples in coagulation: • Thrombin (Factor IIa) • Factor Xa • Factor IXa • Factor XIa • Factor XIIa • Factor VIIa • Plasmin Others: • Trypsin • Chymotrypsin • Elastase Function: • Coagulation cascade (serine proteases activate zymogens) • Fibrinolysis (plasmin) • Inflammation (complement system) 6. Polycythaemia vera Definition: Myeloproliferative neoplasm characterized by: • Increased red cell mass (erythrocytosis) • Increased haemoglobin and haematocrit • Usually with increased WBC and platelet counts • JAK2 V617F mutation in > 95% of cases Clinical features: • Ruddy complexion (plethora) • Headache, dizziness, visual disturbances (hyperviscosity) • Pruritus (after warm bath) - aquagenic pruritus • Splenomegaly • Erythromelalgia (burning pain in hands/feet) • Thrombosis (stroke, DVT, PE, hepatic vein thrombosis) Diagnosis: • Major criteria: • Hb > 16.5 g/dL (males) or > 16.0 g/dL (females) or PCV > 49% (males) > 48% (females) • JAK2 V617F mutation • Minor criteria: • BM biopsy: Hypercellular with trilineage hyperplasia (panmyelosis) • Serum erythropoietin: Low • Endogenous erythroid colony formation (EEC) Treatment: • Phlebotomy: To maintain PCV < 45% • Aspirin: 75-100 mg/day (reduce thrombosis risk) • Cytoreduction: • Hydroxyurea • Interferon alpha (in young patients) • Ruxolitinib (JAK2 inhibitor) • Anagrelide: For thrombocytosis (if needed) 7. T-cell ontogeny Definition: T-cell development from haematopoietic stem cells to mature T cells. Stages: 1. Bone marrow (origin): • HSC -> Common lymphoid progenitor (CLP) • CLP migrates to thymus (via circulation) 2. Thymus (maturation): • Pro-T cell (DN1, DN2): No TCR rearrangement (double negative, CD4-CD8-) • Pre-T cell (DN3, DN4): Beta chain rearrangement -> Pre-TCR • Double positive (DP): CD4+CD8+ -> TCR rearrangement (alphabeta) • Positive selection: Cortical epithelial cells present self-MHC -> Survival (beta-selection) • MHC-I restricted -> CD4-CD8+ (single positive) • MHC-II restricted -> CD4+CD8- (single positive) • Negative selection: Medullary epithelial cells present self-antigens -> Deletion of autoreactive T cells 3. Peripheral circulation: • Mature CD4+ (helper) and CD8+ (cytotoxic) T cells • Leave thymus to secondary lymphoid organs (lymph nodes, spleen, mucosal tissues) Regulation: • Notch signalling (thymic microenvironment) • Interleukins (IL-7) T cell subsets: • CD4+ T cells: Helper T cells (Th1, Th2, Th17, Treg) • CD8+ T cells: Cytotoxic T cells • gammadelta T cells: Non-MHC restricted • NKT cells: Innate-like
Practical Related
Standard Answer:
1. PCV (Packed Cell Volume) PCV (Packed Cell Volume) is the volume percentage of red blood cells in whole blood after centrifugation, expressed as a percentage, also known as haematocrit. 2. Five uses of haematocrit 1. Diagnosis of anaemia: To confirm and classify anaemia 2. Monitoring treatment: Response to treatment in anaemia (iron, B12, EPO) 3. Assessment of dehydration: Increased haematocrit in dehydration 4. Pre-transfusion assessment: Pre-operative optimization 5. Evaluation of polycythaemia: Elevated haematocrit in PV 6. Monitoring in chronic diseases: CKD, heart failure 3. Five things needed to measure haematocrit 1. Blood sample: Whole blood (anticoagulated with EDTA) 2. Haematocrit tube: Glass or plastic capillary tube (Wintrobe tube, microhaematocrit tube) 3. Centrifuge: Microhaematocrit centrifuge (high speed) 4. Sealant: For capillary tubes (for microhaematocrit) 5. Reader: Haematocrit scale/reader or ruler 6. Microscope: (for microhaematocrit) 4. 25-year-old female with low haematocrit (PCV-19%) Five causes of low PCV: 1. Iron deficiency anaemia (menorrhagia, poor diet) 2. Vitamin B12/folate deficiency (megaloblastic anaemia) 3. Chronic blood loss (GI bleeding) 4. Haemolytic anaemia (autoimmune, G6PD deficiency) 5. Bone marrow failure (aplastic anaemia, leukaemia) 6. Anaemia of chronic disease (renal disease) 7. Thalassemia (minor/major) How to go about management: 1. History: • Menstrual history (menorrhagia) • Dietary history (iron, B12, folate) • GI symptoms (bleeding, malabsorption) • Family history (haemoglobinopathies) • Drug history 2. Examination: • Pallor, jaundice, petechiae • Abdominal exam (hepatosplenomegaly) • Lymphadenopathy • Vital signs 3. Investigations: • FBC with differential: Anaemia type (microcytic, normocytic, macrocytic) • Peripheral blood film: RBC morphology, WBCs, platelets • Iron studies: Serum iron, ferritin, TIBC, transferrin saturation • B12, folate levels • Reticulocyte count: Bone marrow response • Haemoglobin electrophoresis: To exclude thalassemia/haemoglobinopathies • Coombs test: If haemolytic anaemia suspected • Renal function tests • Bone marrow examination: If aplastic anaemia or malignancy suspected 4. Treatment based on diagnosis: • Iron deficiency: Oral iron (ferrous sulphate) + treat underlying cause • B12/folate deficiency: Replace deficient vitamin • Haemolytic anaemia: Treat underlying cause (stop drug, immunosuppression) • Bone marrow failure: Supportive care, immunosuppression, HSCT • Anaemia of chronic disease: Treat underlying disease, EPO 5. Haematocrit (Packed cell volume) (See question 1 above) 6. Five causes of low PCV 1. Iron deficiency anaemia 2. Megaloblastic anaemia (B12/folate deficiency) 3. Haemolytic anaemia 4. Acute blood loss 5. Bone marrow failure 6. Anaemia of chronic disease 7. Renal failure (EPO deficiency) 8. Thalassemia 9. Aplastic anaemia 10. Leukaemia 7. Four blood products and two indications for each 1. Packed Red Blood Cells (PRBC): • Symptomatic anaemia (Hb < 7-8 g/dL) • Acute blood loss (> 20-30% blood volume) 2. Platelets: • Thrombocytopenia (< 20x109/L) with bleeding • Prophylaxis in chemotherapy-induced thrombocytopenia 3. Fresh Frozen Plasma (FFP): • Disseminated Intravascular Coagulation (DIC) • Coagulopathy in liver disease 4. Cryoprecipitate: • Hypofibrinogenaemia (< 100 mg/dL) • Haemophilia A (factor VIII deficiency) 5. Albumin: • Hypoalbuminaemia (severe) • Burns (fluid resuscitation)