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CXA332 HAEMATOLOGY 2 NOTES SEMESTER ONE, 2020

TABLE OF CONTENTS WEEK 1 2 3 4 5 6 7 8 9 10 11 12 13

TOPIC Introduction to Anaemias HA; Membrane Defects and Enzyme Deficiencies HA; Immune and Non-Immune Non/Megaloblastic Macrocytic Anaemias Haemoglobinopathies and Thalassaemias Conditions of Iron Deficiency and Iron Overload No Lecture Introduction to Haematopoietic Neoplasms; Myeloproliferative Neoplasms and MDSs Acute Myeloid Leukaemias Precursor Lymphoid Neoplasms Mature Lymphoid Neoplasms No Lecture

PAGE 2 8 12 19 24 32 37 42 52 59 62 -

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PART ONE ANAEMIAS

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WEEK 1 – INTRODUCTION TO ANAEMIAS General Aspects of Anaemia - Not a specific disease - Reflects an underlying disorder or condition - Important to diagnose exact cause - Prevalence is higher in; - Younger adult females (menstruation) - Older adults, especially males (iron deficiencies) - Low socioeconomic groups (nutrient deficiencies) - Anaemia results from red cell destruction in excess of red cell production Definition of Anaemia - Physiological; A decrease in the oxygen carrying capacity of the blood - Laboratory; a significant decrease in the haemoglobin below the normal reference interval for the age and sex of the patient Clinical Signs and Symptoms - Pale skin, sclera, gums, nail beds - Lethargy, shortness of breath - Myalgia - CNS symptoms (dizziness and headaches) Systems of Classification of Anaemia - Morphological - Size; Micro/Normo/Macro - Colour; Hypo/Normo/Hyper - Shape; Ovalo/Sphero/Spur/Sickle - Functional/Pathophysiological - RBC survival defects (increased loss) - Intravascular or extravascular haemolysis - RBC maturation defects (decreased production) - Nuclear or cytoplasmic - RBC proliferation defects (decreased production) - May be multifactorial, primary or secondary, and mild, moderate or severe Laboratory Investigation of Anaemia - Step 1; Clinical History - Age, sex, ethnicity, physiological condition - Clinical signs/symptoms - Can influence choice of laboratory tests - Assists in the interpretation of results (incidence of anaemia is increased in some groups) - Clinical signs - Classic Anaemia - Pale - Tachycardia - Megaloblastic Anaemia - Peripheral neuropathy (difficulty walking) - Smooth (beefy) tongue - Haemolytic Anaemia - Jaundice - Hepatomegaly - Splenomegaly - Haematuria 3

Laboratory Investigation of Anaemia (continued) - Step 1; Clinical History (continued) - Clinical Symptoms - Classic Anaemia - Tiredness/lethargy/weakness - Malaise/muscle cramps - Shortness of breath - Megaloblastic Anaemia - Forgetfulness - Numbness/tingling fingers/toes - Haemolytic Anaemia - Abdominal tenderness - Step 2; Laboratory Tests - FBC; Hb, MCV, MCH, RDW, RBC - Films; peripheral blood and bone marrow - Specific/further tests (haematological or biochemical) - Reticulocyte count (bone marrow activity) - Specific differential tests - Bone marrow examination - Other laboratory tests - Confirmation of FBC results - Number and morphology of - RBC, WBC, PLAT - Assessment of anaemia based on RBC morphology - Morphological and functional classification - Provisional diagnosis - Is anaemia present? - How sever is the anaemia? - Mild (95-110g/L), moderate (80-95g/L), severe(60 years - 15-20% inherited; Fanconi’s anaemia - 80-85% acquired - 70% idiopathic - 10-15% due to; - Prescription drugs - Chemicals/radiation - Post viral infection - Autoimmune disease - Paroxysmal nocturnal haemoglobinuria (PNH) - Pathogenic mechanisms - Cellular (intrinsic) - Quantitative/qualitative deficiency in CD34+ stem cells - Intrinsic stem cell defect -> damage to cellular DNA - Autoimmune component - Immunosuppressive therapy - Clinical features - Mucosal bleeding - Purpura, ecchymoses - URT/LRT infections - Mild-moderate chronic anaemia - Expected laboratory results - Thrombocytopaenia ( loss of RBC biconcave shape - Severity - 20% mild, 70% moderate, 10% severe - Clinical presentation - Mild HS - Compensated erythropoiesis - Diagnosed later in life (anaemia/infections/bleeding) - Moderate/Typical HS - Moderate-severe anaemia - Splenomegaly, jaundice, gall stones - Diagnosis in early childhood/neonatal period - Severe HS - Marked anaemia - Transfusion depended survival (iron overload) - Splenomegaly, jaundice, skeletal abnormalities - Laboratory tests - FBC - Decreased; MCV - Variable; Hb, RBC, HCT - Increased; MCHC, RETIC - Other differentiating tests - Increased conjugated bilirubin - Decreased haptoglobin - Direct antiglobulin test (DAT) negative - Diagnostic tests - Family history, clinical presentation - Spherocytosis - Hyperionic cryohaemolysis (HCH) - Flow cytometry (EMA binding to band 3) - Hereditary Elliptocytosis - Aetiology and Pathophysiology - Heterogenous disorder (autosomal dominant) - 1:4000 people affected - Usually milder than hereditary spherocytosis - Defects - Horizontal membrane defect (mechanical instability) - Spectrin dimer-dimer association - More common in Africa/Mediterranean - RETIC 5-20% - Clinical and Laboratory findings - Common HE; - Nil to mild anaemia, 30-90% elliptocytes, Hb usually normal - Stomatocytic HE; - Nil anaemia - Oval stomatocytes - Hereditary-Pyropoikilocytosis (HPP; rare form of HE) - Severe anaemia, elliptocytes, schistocytes, microspherocytes 10

Membrane Disorders (continued) - Paroxysmal Nocturnal Haemoglobinuria (PNH) - Aetiology and pathophysiology - Rare, acquired disorder - 2.5:1,000,000 - Clonal HSC mutation - Idiopathic or secondary to aplastic/hyperplastic/myelodysplastic syndromes - Most common in young-middle aged adults (30-40 years old) - Lack of CD55 and CD59 - Glycosyl phosphatidylipsilol (GPA) link proteins that anchor surface proteins to cell membrane - Complement inhibiting proteins - Abnormal sensitivity to complement, especially in microvasculature -> IVH - Tissue hypoxia -> decreased pH - Clinical presentation - Chronic IVH anaemia, mild to severe - 25% of cases have haemoglobinuria - Free Hb -> decreased NO - Oesophageal spasms, erectile dysfunction, abdominal pain - Thrombosis in hepatic vein - Recurrent infection - Spontaneous bleeding - Can develop into aplastic anaemia - Laboratory findings - Significant screening tests - Normocytic, normochromic anaemia - Variable MCV; - Reticulocytes, secondary IDA/folate DA, pancytopaenia - Occasional spherocytes - RETIC +/++ - Further differentiating tests - Decreased haptoglobins - Increased unconjugated bilirubin and LDH - Increased haemoglobinuria/haemosideriuria - DAT negative - Diagnostic test - Flow cytometry(CD55 and CD59 deficiency/defect Enzyme Disorders - G6PD - Glutathione (MMP shunt) - Powerful antioxidant maintained in reduced state (GSH) by NADPH - G6PD converts NADP to NADPH - No G6PD -> no GSH - Increase Hb oxidative stress, MetHb, Heinz bodies, EVH (bite cells), IVH in acute crises - Most common erythrocytic enzymopathy - RBC are sensitive to oxidative stress (especially older cells) - Prevalence; 5% global average - Varies between 3-30% geographically - Highest prevalence in areas endemic for malaria - Range of clinical presentation - Asymptomatic to incompatible with life - Chronic, compensated haemolysis (EVH) - Sudden, acute haemolytic episodes (IVH) 11

Enzyme Disorders (continued) - G6PD - Inheritance - >400 variants affecting 400 million people - Enzyme deficiency or functional abnormality (complete lack is fatal) - X-linked inheritance - Heterogenous females can have dual RBC populations - Lyonization, random inactivation of X-chromosome on HSC - Clinical presentation - WHO Classification - Class I; severe symptoms, IVH - Idiopathic or secondary to infections/lymphoproliferative disorders - ‘non-sense’ RBC results - RBC too low for Hb - Falsely increased MCV, MCH and MCHC, falsely low HCT - Resolve FBC results by warming to 37oC - Primary (60%); idiopathic - Secondary (40%) - Lymphoproliferative disorders - Infections - Autoimmune conditions - Drugs - Acute; severe, self-limiting anaemia (WAIHA) - Chronic; less severe anaemia (CAD) - Clinical signs and symptoms - WAIHA - Weakness, tiredness - Splenomegaly, jaundice - CAD - Numbness, tingling, pain and cyanosis in extremities - Haematuria, haemoglobinuria

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Immune Haemolytic Anaemias (continued) - Alloimmune haemolytic anaemias - Blood group mismatch - ABO (IgM) -> immediate IVH transfusion reaction - May result in recipient’s death - Other (IgG) -> EVH - Delayed HTR - Haemolytic disease of the foetus or newborn - Transplacental bleed -> maternal sensitisation -> antibody production -> cross placenta and attach to foetal RBCs -> sensitised cells removed by the foetal RES -> foetal haemolytic anaemia -> congestive cardiac failure (CCF) - Hydrops foetalis’ - Neonatal jaundice (kernicterus) - Drug related - Drug induced immune complex formation - Innocent bystander - Dug adsorption - Hapten - Drug alone is not antigenic - Drug-RBC complex - Autoantibody mediated - Drugs stimulate autoantibodies against RBC - Membrane modification - Non-immunologic protein adsorption and membrane modification - DAT positive, but rarely causes haemolysis Non-Immune Haemolytic Anaemia - Physical or mechanical trauma - Thrombotic microangiopathic anaemias (TMA) - AKA microangiopathic haemolytic anaemia (MAHA) - Common features - Haemolysis due to microcirculatory thrombosis - Organ damage (especially kidneys) - Endothelial lining of small blood vessels - Thrombocytopaenia - Platelet activation and fibrin deposition - Schistocytes - RBCs ‘sliced’ open -> IVH - RBCs ‘reseal’ -> schistocytes and EVH - Differentiating characteristics - Underlying aetiology - Primary (inherited) of secondary - Clinical presentation - WBC, PLAT coagulation screen and FDPs - Biochemical markers of haemolysis and others - Population most at risk - Age/sex/pregnancy - Organs affected - Kidney, heart, CNS, GIT, pancreas

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Non-Immune Haemolytic Anaemias (continued) - Physical or mechanical trauma (continued) - Thrombotic microangiopathic anaemias (TMA) (continued) - Thrombotic thrombocytopaenic purpura (TTP) - Inherited (race) or acquired (infections, pregnancy, drugs, neoplasms, connective tissue disease) - Adults 20-50 years old (F>M, 3:2) - Autoantibodies to ADAMTS-13 - Ultra-large vWF multimers - Thrombi in microcirculation - Brin, kidney, heart, pancreas - Severe CNS symptoms - Mild kidney dysfunction - IVH - Decreased Hb, RBC, HCT, PLAT, haptoglobins, - DAT negative - Reticulocytes, schistocytes, spherocytes - Coagulation profile unremarkable - Haemolytic uraemic syndrome (HUS) - Acquired condition of primary haemolysis - Similar blood picture to TTP - Young children ( intravascular platelet activation -> fibrin deposits -> TMA - Blood picture similar to other TMAs - Haemolysis and thrombocytopaenia are less severe, and liver disease - Increased AST, bilirubin (total and unconjugated), LDH - Decreased haptoglobins - Normal PT/APTT, until very late in disease - Mechanical trauma to RBC in vascular circulation - Exercise-induced haemoglobinuria - Transient - No cell fragments; decreased haptoglobins, increased haemoglobinuria - No anaemia; may be slight reticulocytotic - Thermal injury (burns) - Direct effect of heat on RBC membrane proteins - Altered metabolism of fatty acids/lipoproteins - RBC budding, schistocytes, spherocytes 15

Non-Immune Haemolytic Anaemia (continued) - Physical or mechanical trauma (continued) - Mechanical trauma (continued) - Traumatic cardiac haemolytic anaemia - Uncommon cardiac complication of prosthetic heart valve surgery - Significant RBC fragmentation due to ‘shear’ stress - IVH/EVH - PLAT not significantly decreased - Chemicals and drugs - IVH due to membrane damage - Hb denaturation -> Heinz bodies - Lead poisoning - Sideroblastic anaemia -> basophilic stippling - RBC membrane damage -> increased fragility - Pathophysiology - Inhibits haeme synthesis - Accumulation of iron in mitochondria - Cause of sideroblastic anaemia - Damages RBC membrane - Changes physiochemical composition - Increased oxidative stress - Cause of non-immune haemolytic anaemia - Populations most at risk - Children 1-3 years old; environmental exposure - Adults; industry exposure - Laboratory features - Moderate normocytic anaemia - Dimorphic - Normocytic, normochromic - Microcytic, hypochromic - Macrocytic, if increased RETIC - Increased RDW - Dual RBC histogram profiles - Basophilic stippling - Animal venoms - Snake and spider - Thrombosis -> DIC - IVH due to membrane damage - Infectious agents - Direct parasitisation of RBC (e.g. malaria) - Bacterial haemolytic toxins (e.g. Clostridium spp) Malaria - What is malaria - Febrile protozoan infection of vertebrates - 5 species of Plasmodium that infect humans - P. falciparum (most severe) - P. malariae - P. ovale - P. vivax (most common) - P. knowlesi (simian/monkey malaria)

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Malaria - DNA sequencing - P. falciparum - Central lakes of Africa - 165 million years ago - P. vivax and P. malariae - South east Asia - 200,000-300,000 years ago - Genetic mutations - Haemoglobinopathies - Thalassaemia - SCA - RBC enzyme deficiencies - G6PD resistant to P. falciparum - Duffy negative phenotype - Resistant to P. vivax but not P. ovale - Transmission - Bite of female Anopheles mosquito - Africa; A. gambezi - Australia; A. farauti - Aedes; ? another vector - Blood transfusion - Sharing of needles/needle stick injury - Disease process - Differs with species - Incubation period - Length of asexual life cycle - Severity of illness - Parasite load (cell age) - P. falciparum - P. knowlesi - Site of maturation - P. falciparum - Immune reaction of host - P. falciparum - Recurrent infection - P. vivax - P. ovale P. falciparum P. malariae P. ovale P. vivax P. knowlesi Incubation (days) 7-27 23-69 16-18 13-17 5-7 Life Cycle (hours) 36-48 72 48 48 24 Parasite Load All ages Older Young/reticulocytes All ages Severity Malignant tertian Benign quaternary Benign tertian Malignant - Clinical features - early symptoms resemble influenza - chills, fever, headache, dizziness, myalgia - most common symptoms - recurring fever and sweating - abdominal pain - altered mental state - Less common symptoms - jaundice, splenomegaly, hepatomegaly - haemolytic anaemia in 25% of cases - haemolysis is proportional to parasitation (P. falciparum/P. knowlesi) 17

Malaria (continued) - clinical symptoms (continued) - Less common symptoms (continued) - bleeding gums, epistaxis, needle sites (especially P. falciparum) - DIC - Thrombocytopaenia - Acute kidney injury (especially P. falciparum) - Black water fever - Laboratory diagnosis - Overseas travel/prior infection and fever - Thrombocytopaenia (1/3 RBC >1/3 RBC Shape Band form Schizont Frequency Vary rare Common Common Configuration Random Random Daisy head Number Gamete Shape Chromatin Size

P. knowlesi Single Delicate

P. ovale Single Single Dot

>1/3 RBC Band form

>1/3 RBC compact Common Daisy head

Small, round

8-24

12-24

8-24

Common Grape cluster 100fL - Large oval or round RBC - Classification - Megaloblastic anaemia - Impaired DNA synthesis -> megaloblastic BM morphology - E.g. folic acid or B12 deficiency - MCV >115fL (often 130-150) - Non-megaloblastic anaemia - Normal BM morphology - Reticulocytosis - Alteration to membrane cholesterol:phospholipid ratio - E.g. alcoholism, medications, liver disease - MCV ineffective erythropoiesis - Decreased M:E ratio - Megaloblastic changes - Large erythroblasts - Open chromatin pattern - Normal haemoglobinisation - Giant metamyelocytes - Hypersegmented megakaryocytes - Non-megaloblastic anaemia - Variable (normal – increased); shortened RBC lifespan - Normal or decreased M:E ratio - Normal cell maturation - Unless coexisting folic acid decrease - Other laboratory tests - B12 deficiency - Total serum B12 immunoassay - If low; - Holotranscobalamin level - Methyl malonic acid (MMA)(serums/urine) - Folic acid deficiency - Serum folate immunoassay - Red cell folate assay (if serum folate is low) - Pernicious anaemia - IF antibodies - Immunoassay (high specificity, low sensitivity) - Blocking antibodies (50-60% of patients) - Precipitation antibodies (30% of patients that are positive for blocking antibodies) Megaloblastic Anaemia - Role of folic acid in the body - Essential nutrient for methylation - Assists in metabolism of amino acids and nucleotides - Deficiency -> impaired cellular replication/metabolism - Role of B12 in the body - Methylation or homocysteine -> methionine - Isomerisation of methyl malonyl CoA -> succinyl CoA - Krebs cycle and fatty acid/phospholipid synthesis 21

Megaloblastic Anaemia (continued) - Role of B12 in the body (continued) - Deficiency leads to; - Nerve demyelination -> peripheral neuropathy - CNS dysfunction - Impaired DNA synthesis - Ineffective cellular division - B12/folate are required for normal DNA synthesis - Aberrant DNA synthesis -> asynchronous N:C maturation - Abnormal cellular division - Ineffective haemopoiesis - Fewer, larger cells -> pancytopaenia - Shortened RBC lifespan -> premature haemolysis - Epithelial disruption; lining of GIT - Hair loss and greying - Causes of B12 deficiency - Inadequate intake (vegans) - Increased requirements - Pregnancy, infants, children - Malabsorption - Failure to separate from food proteins - Low HCl (e.g. atrophic gastritis, chronic antacid use) - Failure to separate from haptocorrin - Decreased proteases (e.g. chronic pancreatic disease) - Lack of intrinsic factor (IF) - Pernicious anaemia (anti-IF) - Helicobacter pylori infection - Gastrectomy/bypass - Intestinal disease (e.g. coeliac) - Competition for available B12 - Diphyllobothrium latum (fish tapeworm) infection - Overgrowth of intestinal flora - Pernicious anaemia - Autoimmune disease - Immune destruction of gastric parietal cells - Anti-IF antibodies (specific to P.A) - Anti-parietal cell antibodies - Most commonly older women; insidious, takes decades until clinically evident - Treatment - Intramuscular B12 injection - High dose oral B12 - Life-long - Causes of folate deficiency - Inadequate intake - Increased requirement - Pregnancy, infants and children - Malabsorption - Intestinal disease - Deficiency of PCFT (rare) - Alcoholism - Impaired metabolism - Drugs; cytotoxic and anti-epileptic drugs - B12 deficiency; folate trap - Excessive loss via kidney dialysis 22

Megaloblastic Anaemia (continued) - Neural tube defects - Part of the embryo that becomes brain/spinal cord - Closure occurs around 3-4 weeks gestation - Neural tube defects - Anencephaly (brain, lethal) - Spina bifida (spine) - Incidence decreases with folic acid supplementation prior to, and during, pregnancy - Other causes of megaloblastic anaemia - Myelodysplastic syndrome (MDS) - Delayed cytoplasmic and nuclear maturation - Congenital dyserythropoietic anaemias (CDAs) - Childhood condition - Acute erythroid leukaemia - Myeloblasts in BM - Use of reverse transcriptase inhibitors - HIV treatment - Lack of response to B12/folate supplements - Treatment - Straight deficiency - B12 or folic acid (or both -> severe anaemia) - Pernicious anaemia - Ongoing B12 injections/oral megadose - 24-48 hour response - RBC abnormalities disappear quickly - Reticulocyte response after 2-3 days - Hypersegmented neutrophils linger - Avoid transfusion Non-Megaloblastic Anaemia - Macrocytic circulating RBCs - Round rather than oval - Alteration in cholesterol:phospholipid ratio - MCV usually blood loss - Nutritional deficiencies -> ineffective erythropoiesis - Suppression of haemopoiesis by alcohol - Thrombocytopaenia - Altered plasma lipids - Altered cholesterol:phospholidid in cell membranes - E.g. target cells, stomatocytes, spur cells - Shortened RBC lifespan (haemolysis) - Alterations to WBC/PLAT count/morphology

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Non-Megaloblastic Anaemia (continued) - Chronic alcohol abuse - Mild macrocytosis due to; - Alcohol related BM suppression (toxicity) - Especially thrombocytopaenia - Folate deficiency with or without iron deficiency - Nutritional deficiency or ethanol interference - Associated liver disease - Associated GIT bleeding or haemolysis - Blood picture - Same as liver disease - Mixed deficiency pictu...