Anemia and Its Laboratory Diagnosis PDF

ANEMIA And Its Laboratory Diagnosis Prof. Dr / Shaaban Redwan Helal Clinical Pathology – Hematology Unit. Assiut University 1  Objectives At the end of this Lecture you will be able to: Define anemia : Discuss the causes and clinical sig...

ANEMIA And Its Laboratory Diagnosis Prof. Dr / Shaaban Redwan Helal Clinical Pathology – Hematology Unit. Assiut University 1  Objectives At the end of this Lecture you will be able to: Define anemia : Discuss the causes and clinical significance of different types of anemia Describe the classification of anemia : Explain - Microcytic anemia - Macrocytic anemia - Normochromic normocytic anemia Discuss the laboratory findings for each category of anemia Perform basic laboratory tests for the diagnosis of 2 anemia  Introduction Definition of Anemia : Anemia is a decrease in the RBC count, Hb and/or HCT values as compared to normal reference range for age and sex (Also determined by alteration in plasma volume) – ‘True’ anemia: – decreased RBC mass and normal plasma volume – Pseudo or dilutional anemia: – normal RBC mass and increased plasma volume – An increase in plasma volume can occur in Pregnancy, volume overload (IVs) congestive heart failure – Low Hb and HCT values 3 Definition of Anemia cont’d 4  Definition of Anemia cont’d….. Anemia must also relate to the level of hemoglobin the individual normally possesses. – If an adult male usually maintains a hemoglobin level of 16g/dl, and over a period of days is noted to have decreased to 14 g/dl, this must be considered significant even though both values are within the normal range for an adult male. 5  Definition of Anemia cont’d….. Various diseases and disorders are associated with decreased hemoglobin levels. These include: – Nutritional deficiencies – External or internal blood loss – Increased destruction of RBCs – Ineffective or decreased production of RBCs – Abnormal hemoglobin synthesis – Bone marrow suppression by toxins, chemicals, or radiation & replacement by malignant cells – Infection 6. Definition of Anemia cont’d….. Functionally anemia is defined as tissue hypoxia (inability of the body to supply tissue with adequate oxygen for proper metabolic function) – There is an abnormal hemoglobin with an increased O2 affinity resulting in an anemia with normal or raised hemoglobin levels, hematocrit, or RBC count. Generally anemia is not a disease, but rather the expression of an underlying disorder or disease. 7  Definition of Anemia cont’d….. Anemia may develop: – When RBC loss or destruction exceeds the maximal capacity of bone marrow RBC production or – When bone marrow production is impaired 8  Hematologic Response to Anemia Tissue hypoxia causes increased renal release of erythropoietin (EPO) to accelerate bone marrow erythropoiesis The normal bone marrow can increase its activity 7-8 times normal – Marrow becomes hypercellular 9  Signs of Accelerated Bone Marrow Erythropoiesis The marrow becomes hypercellular due to a marked increase in RBC precursors (called erythroid hyperplasia) and the M:E ratio falls. Nucleated RBCs may be released into the blood circulation along with the outpouring of reticulocytes – NRBC number tends to correlate with the severity of anemia – Increased polychromasia on the Wright's- stained blood smear is seen due to increased number of circulating Retics. 10  If demand exceeds maximal bone marrow activity, RBC production may occur in extramedullary sites, liver, spleen (hepatosplenomegaly). 11  Physiologic Response to Anemia Ability to adapt to anemia depends on: – Age and underlying disease – Cardio/pulmonary function – Rate at which anemia develops (BM can compensate easier if the onset of anemia is slow), – Underlying disease 12  Clinical features: Symptoms of hypoxia: decreased oxygen delivery to the tissues/organs causes: – fatigue , faintness, weakness, dizziness, headaches, dyspnea, poor exercise tolerance, leg cramps. 13.Clinical features cont’d….: Signs of anemia general signs include pallor of mucous membrane, which occur if the Hgb concentration is less than 9 g/dl, specific signs are associated with particular types of anemia, for example, jaundice in hemolytic anemia, leg ulcer in sickle cell anemia 14  Diagnosis of anemia Before making a diagnosis of anemia, one must consider: – Age – Sex – Geographic location – Presence or absence of lung disease 15 Diagnosis of anemia cont’d…… How does one make a clinical diagnosis of anemia? A. Patient history –Dietary habits –Medication –Possible exposure to chemicals and/or toxins –Description and duration of symptoms 16 Diagnosis of anemia cont’d…… Patient history :……….. –Tiredness –Muscle fatigue and weakness –Headache and vertigo (dizziness) –Dyspnia (difficult or labored breathing) from exertion –G I problems –Overt signs of blood loss such as hematuria (blood in urine) or black stools 17 Diagnosis of anemia …. B. Physical exam – General findings Hepato or splenomegaly Heart abnormalities Skin pallor – Specific findings In vitamin B12 deficiency there may be signs of malnutrition and neurological changes In iron deficiency there may be severe pallor, a smooth tongue, and esophageal webs In hemolytic anemias there may be jaundice due to the increased levels of bilirubin from increased RBC destruction 18. Diagnosis of anemia …… C. Lab investigations : 1. A complete blood count (FBC ), CBC – RBC count : – Hematocrit (Hct) or packed cell volume – Hemoglobin determination – RBC indices calculation – Reticulocyte count 2. Blood smear examination to evaluate: – Poikilocytosis – Leukocytes or Platelets abnormalities 19  Diagnosis of anemia.. Lab investigation cont’d…… 3. A bone marrow smear and biopsy to observe: – Maturation of RBC and WBC series – Ratio of myeloid to erythroid series – Abundance of iron stores (ringed sideroblasts) – Presence or absence of granulomas or tumor cells – Red to yellow ratio – Presence of megakaryocytes 20  3.1.5. Diagnosis of anemia cont’d….. 4. Hemoglobin electrophoresis 21  Diagnosis of anemia….. Lab investigation cont’d…… 5. Antiglobulin testing 6. Osmotic fragility test 22  Methods of Anemia Classification Several schemes of classifying anemias exist 1. Morphologic – Based on RBC morphology – Anemia is divided into three groups mainly on the basis of the MCV (RBC indices) 2. Pathophysiologic – Anemia is divided using three main causes/mechanisms I. Impaired erythrocyte formation (Aplastic anemia, IDA, sideroblastic anemia, anemia of chronic diseases, megaloblastic anemia) – Retic count is low – The bone marrow fails to respond appropriately 23 due to disease or lack of essential supplies  Methods of Classification III. Increased blood loss (Acute, Chronic) – Retic count is typically high – Anemia results when red cell loss exceeds the bone marrow’s capacity to increase its activity IV. Increased destruction of RBCs (hemolytic anemias) – Retic count is typically high – Anemia results when red cell destruction exceeds the bone marrow’s capacity to increase its activity 24  Methods of Classification Morphologic Categories of Anemia 1. Normocytic Normochromic anemia (normal red cell indices) Blood loss anemia Hemolytic anemia Aplastic anemia Chronic diseaes Renal insufficciency 25  Methods of Classification cont’d Morphologic Categories of Anemia Microcytic hypochromic ( low red cell indices) Iron deficiency anemia Sideroblastic anemia Lead poisoning Thallassemia Chronic diseases 26 Methods of Classification cont’d Morphologic Categories of Anemia Macrocytic Normochromic ( high MCV and MCH, normal MCHC) Megaloblastic anemia Liver disease Post splenectomy Hypothyroidism Stress erythropoiesis 27  Morphologic Categories of Anemia 1 2 1 Microcytic/hypochromic 2 Macrocytic/Normochromic N.B. The nucleus of a small lymphocyte (shown by the arrow) is used as a reference to 3 a normal red cell size 28 3 Normocytic/Normochromic  Pathophysiological classification 1. A. B. 2. 29 1. Microcytic- Hypochromic Anemia 30  Microcytic- Hypochromic Anemia Many RBCs smaller than nucleus of normal lymphocytes Increased central pallor. Includes – Iron deficiency anemia – Thalassemia – Anemia of chronic disease – Sideroblastic anemia – Lead poisoning 31 The Cause of Microcytic-Hypochromic Anemia Iron Protoporphyrin Iron deficiency Chronic inflammation Sideroblastic anemia or malignant (ACD) Heme + Globin Thalassemia ( or ) Hemoglobin 32 Microcytic/Hypochromic Anemias Normoblastic RBC maturation  normocytic red cells Normal RBC maturation is shown for comparison RBC maturation in microcytic anemiasAbbott Manual 33 A. Iron Deficiency Anemia (IDA) Is a condition in which the total body iron content is decreased below a normal level This results in a reduced red blood cell and hemoglobin production More than half of all anemias are due to iron deficiency. 34  Iron Deficiency Anemia (IDA) Causes: – Nutritional deficiency – Malabsorption (insufficient or defective absorption) – Inefficient transport, storage or utilization of iron – Increased need – Chronic blood loss (GI bleeding, ulcer, heavy menstruation, etc) 35  DIETARY SOURCES OF IRON Inorganic Iron eg lentils Organic iron eg beef DAILY IRON REQUIREMENT 10-15mg/day (5-10% absorbed) 36 Daily Iron cycle (Fig) 37  Estimated daily iron requirements Units are mg/day Adult men 0.5-1 Post menopausal female 0.5-1 Menstruating female 1-2 Pregnant female 1.5-3 Children 1.1 Female (age 12-15) 1.6-2.6 38 Iron absorption, Transport and storage Iron absorbed from duodenum and jejunum in the GIT Moves via circulation to the bone marrow Incorporated with protoporpyirin in mitochondria of the erythroid precursor to make Heme 39  There are three proteins important for transporting and storage of iron: –Transferrin, –Transferrin receptors and –Ferritin Transport: Transferrin: transports iron from the plasma to the erythroblasts in the marrow for erythropoiesis The transferrin will bind to transferrin receptor on the erythrocyte membrane 40 Storage Hgb contains about two third of the body iron At the end of their life, RBCs are broken down in the macrophage of reticuloendothelial system and then iron is released from Hgb enters plasma and provided to transferrin. Some stored in reticuloendothelial cell as ferritin soluble protein – iron complex) and hemosiderin (37%) (degraded form of ferritin insoluble) iron is also found in muscles as myoglobin and in other cells as iron containing enzymes 41  The distribution of body iron Amount of iron Male Female % in average adult (g) (g) of total Hb 2.4 1.7 65 ferritin & hemosiderin 1.0 0.3 30 Myoglobin 0.15 0.12 3.5 Heme enzyme 0.02 0.15 0.5 Transferrin-bound 0.004 0.003 0.1 iron 42  Iron Deficiency Anemia (IDA) Sequence of iron depletion When iron loss or use exceeds absorption, there is a sequence of iron depletion in the body: 1. Storage iron decreases/ low serum ferritin; serum iron & TIBC are normal, no anemia, normal red cells. 2. Serum iron decreases/TIBC increases (increased transferrin); no anemia, normal red cells. 3. Anemia with microcytic/hypochromic red cells = IDA. 43  CLINICAL FEATURES IRON DEFICIENCY Symptoms eg. fatigue, dizziness, headache Signs eg. pallor, Tongue atrophy/ glossitis - raw and sore, angular cheilosis (Stomatitis) Angular Cheilosis or Stomatitis Koilonychia Glossitis 44  Clinical signs and symptoms – Spoon-shaped nails (koilonychia), brittle nails and hair. 45  Lab Investigation of IDA Iron tests ►Used to differentiate microcytic hypochromic anemia's or detect iron overload (hemochromatosis) – Iron circulates bound to the transport protein transferrin Transferrin is normally ~33% saturated with iron Iron tests include serum iron, Total Iron Binding Capacity (TIBC), serum ferritin 46  Lab Investigation cont’d Serum iron level – measures the amount of iron bound to transferrin – Does not include the free form of iron Total Iron Binding Capacity (TIBC) – Is an indirect measure of the amount of transferrin protein in the serum – Inversely proportional to the serum iron level If serum iron is decreased, total iron binding capacity of transferrin increased (transferrin has more empty space to carry iron) 47  Lab Investigation cont’d Serum ferritin – indirectly reflects storage iron in tissues – found in trace amount in plasma – It is in equilibrium with the body stores – Variation in the quantity of iron in the storing compartment is reflected by plasma ferritin concentration e.g. Plasma ferritin is decreases in IDA Plasma ferritin increases in ACD Limitation: During infection or inflammation Serum Ferritin increases like other acute phase proteins, and then it is not an accurate indicator in such situations. 48 Bone marrow iron (Tissue iron) Tissue biopsy of bone marrow Prussian blue stain Type of iron is hemosiderin 49 ABSENTiron Absent IRON STORES stores IN BONE in bone marrow MARROW IN IRON DEFICIENCY Normal control Iron deficiency 50  Iron Deficiency Anemia Blood smear Lab findings – Low RBC, Hgb, Hct – Low MCV, MCH, MCHC – Normal WBC and PLT 51  Iron Deficiency Anemia RBC morphology – Hypochromia – Microcytosis – Anisocytosis – Poikilocytosis Pencil cells (cigar cells) Blood smear Target cells – no RBC inclusions Iron parameters – Low serum iron, – High TIBC, 52 – Low serum ferritin  Iron Deficiency Ovalocytes - Pencil forms No RBC inclusions Wright’s stained blood smear 53  IDA cont’d Treatment – Identify the underlying cause – Oral iron is given; see increased Retic count post-therapy. – May see dimorphism following treatment a dual red cell population with older microcytic red cells along with the newly produced normocytic red cells. 54  B. Sideroblastic Anemia (SA) This group of anemias are characterized by defective protoporphyrin synthesis (blocks) resulting in iron loading and a hypochromic anemia due to deficient hemoglobin synthesis. Block(s) in protoporphyrin synthesis leads to iron overload and microcytic/hypochromic anemia 55 Sideroblastic Anemia (SA) 56  Terms: Siderocytes are mature RBCs in the blood containing iron granules called Pappenheimer bodies....abnormal. Sideroblasts are immature nucleated RBCs in the bone marrow containing small amounts of iron in the cytoplasm....normal. 57  SA Sideroblastic anemia is characterized by the – Accumulation of iron in the mitochondria of immature nucleated RBCs in the bone marrow; – Iron forms a ring around the nucleus  these are called ringed sideroblasts....abnormal. The iron accumulation in the mitochondria is the result of blocks in the protoporphyrin pathway. 58  SA cont’d Lab findings: Microcytic/hypochromic red cells, low MCV and MCHC; variable anemia, low retic. RBC inclusions: Basophilic stippling and Pappenheimer bodies (siderocytes). (May see target cells). High serum iron and high serum ferritin (stores); low TIBC and high % saturation. *Decreased transferrin synthesis occurs in iron overload states. Bone marrow: ringed syderoblasts (Hall mark of Sideroblastic Anemia) 59  Sideroblastic Anemia (SA) Blood Bone marrow Bone marrow Ringed Sideroblast Pappenheimer bodies Sideroblast RBC with iron NRBC with iron NRBC with ring of iron Wright’s stain Prussian blue stain Prussian 60 blue stain60  Sideroblastic Anemia (SA) Blood Blood Basophilic stippling/stippled RBCs Pappenheimer bodies Wright’s stain Blood Pappenheimer bodies Prussian blue iron stain 61  Sideroblastic Anemia (SA) Bone marrow findings (if done): 1. *Ringed sideroblasts demonstrated with Prussian blue stain. 2. Increased stainable iron in macrophages. Bone marrow 100x Bone marrow 10x Ringed Sideroblasts Increased stainable iron Prussian blue iron stain Prussian blue iron stain 62  Types of Sideroblastic anemia cont’d Primary - cause unknown (can't identify blocks) and are not reversible....called Idiopathic or primary Sideroblastic anemia. 1.Elderly, responds to no treatment. Requires transfusion support if severe anemia. 2.Characterized by a dimorphic red cell population - micro/hypo red cells with 3.normocytic and/or macrocytic red cells....MCV is variable and RDW is high. 4.Primary type of sideroblastic anemia is one of myelodysplastic syndromes called Refractory Anemia with Ringed Sideroblasts; may terminate in leukemia 63  Secondary Types of SA Alcohol inhibits vitamin B6/pyridoxine Anti-tuberculosis drugs inhibit vitamin B6 Lead causes multiple blocks – Inhaled or ingested – Abnormal lead level – Neurologic problems Stippled RBCs – Lead poisoning – Lead line (gums) – Chelation therapy (EDTA). Wright’s stained blood smear 64 65  C. Anemia of chronic disease Anemia of chronic disease (ACD) – inability to use iron and decreased response to EPO – Very common anemia, #2 Associated with systemic disease, including chronic inflammatory conditions: –Rheumatoid arthritis –Chronic renal disease –Thyroid disease –Malignancies –Tuberculosis –Chronic fungal infections etc 66  ACD pathogenesis Lactoferrin is an iron biding protein in the granules of neutrophils Its avidity for iron is grater than transferrin During infection or Inflammation, neutrophil- lactoferrin released into plasma and Scavenges available iron Bind to macrophage and liver cells (because they have receptor for lactoferrin Cytokines: Produced by macrophages during inflammation and contribute to ACD by inhibiting erythropoiesis 67  Lab Diagnosis Blood findings – Early stage: normocytic normochromic – Late stage: hypochromic microcytic, – Low serum iron, low TIBC, normal or high serum ferritin Leukocytosis Abundant storage of iron in macrophage (Prusian blue) 68  D. Thalassemias Inherited decrease in alpha or beta globin chain synthesis needed for Hgb A; quantitative defect – All have microcytic/hypochromic RBCs and target cells Genetic mutations classified by: – ↓ beta chains = beta thalassemia…Greek/Italian – ↓ alpha chains = alpha thalassemia…Asian Beta Alpha Target cells/Codocytes 69 69 Haemoglobin Molecule Consists of 4 globin chains + 4 heme groups Normally, each individual inherits 2α, 1β, 1γ, and 1δ gene from each parent.....so 4α, 2β, 2γ, and 2δ genes are inherited. Hgb A = 2α & 2β Hgb A2 = 2α & 2δ Hgb F = 2α & 2γ 97% 2% 1% 70  Thalassemia Impaired alpha or beta globin synthesis results in an unbalanced number of chains produced that leads to: – RBC destruction in beta Thalassemia major – Production of compensatory Hgb types in beta thals – Formation of unstable or non-functional Hgb types in alpha thals 71  Thalassemia Severity ranges from lethal, to severe transfusion-dependency, to no clinical abnormalities; severity depends on the number and type of abnormal globin genes inherited. 1.Major  severe anemia; no α (or β) chains are produced, so cannot make normal hemoglobin (s). 2.Minor/trait  mild anemia; slight decrease in normal hemoglobin types made. 72  Beta Thal Major (Homozygous) Both beta genes abnormal – Marked decrease/absence of beta chains leads to alpha chain excess…no Hgb A is produced – Rigid RBCs with Heinz bodies destroyed in bone marrow and blood (ineffective erythropoiesis) Heinz bodies Excess alpha chains Supravital stain 73  Beta Thal Major (Homozygous) Clinical findings Lab findings – Severe anemia, target cells, nucleated red cells – RBC inclusions – No hemoglobin A; compensatory Hgb F Target cell HJB NRBC Stippled NRBC Wright’s stained blood smear 74 Beta Thal Major (Homozygous) Blood smear Howell-Jolly body Target cells NRBC Pap bodies Target Transfused cell RBC Blood smear Transfused RBC Treatment – Transfusion – Splenectomy – Iron chelation Hypercellular Bone Marrow (10x) 75  Beta Thal Minor (Heterozygous) One abnormal beta gene – Slight decreased rate of beta chain production – Blood picture can look Stippled RBC similar to iron deficiency Lab findings – Mild anemia, target cells, no nRBCs, stippled RBCs Target cell – No Heinz bodies Ovalocytes – Normal iron tests Wright’s stained blood smear – Compensates with Hgb A2 76  Alpha Thal Major/Homozygous Deletion of all 4 alpha genes results in complete absence of alpha chain production – No normal hemoglobin types made Known as Barts Hydrops Fetalis – Die of hypoxia….Bart’s Hgb 77  Alpha Thal Intermedia = Hgb H Disease Three alpha genes deleted – Moderate decrease in alpha chains leads to beta chain excess…unstable Hgb H – Moderate anemia Heinz bodies Excess beta chains Supravital stain Target cells Wright’s stain blood smear 78  Alpha Thal Minor (Heterozygous) One or two alpha genes deleted (group) – Slight decrease in alpha chain production – Mild or no anemia, few target cells – Essentially normal electrophoresis; many undiagnosed 79 Beta Thalassemias 80 Alpha Thalassemias 81 Differential Diagnosis of Microcytic Anemia + HGB Synthesis Defects 82 2. Macrocytic Normocytic Anemias 83 Macrocytic Normocytic Anemias Characteristics ?????? Wright’s stained blood smear 84 A. MEGALOBLASTIC ANEMIA Vitamin B12 deficiency Folate deficiency Abnormal metabolism of folate and vit B12 B. Non megaloblastic anemia Liver disease Alcoholism Post splenoctomy Neonatal macrocytosis Stress erythropoiesis 85  A. Megaloblastic Anemia Macrocytosis due to a deficiency of vitamin B12 or folic acid that causes impaired nuclear maturation – Vitamin B12 & folate are DNA coenzymes necessary for DNA synthesis and normal nuclear maturation – Results in megaloblastic maturation…nucleus lags behind the cytoplasm and leads unbalanced growth called maturation asynchrony Both deficiencies cause enlarged fragile cells – Many cells die in the marrow (ineffective) – Show a similar blood picture and clinical findings Only vitamin B12 deficiency causes neurological symptoms…required for myelin synthesis 86 Megaloblastic Anemia Megaloblastic RBC maturation  macrocytic red cells Normoblastic RBC maturation  normocytic red cells RBC maturation in microcytic anemias…IDA 87  Lab Findings of Megaloblastic Anemia Mild to severe anemia, – Increased MCV & MCH, normal MCHC – Low RBC, HGB, WBC and PLT counts (fragile cells) due to ineffective hematopoiesis. – Low reticulocyte count – Macrocytic ovalocytes and teardrops; – Marked anisocytosis and poikilocytosis – Schistocytes/microcytes - due to RBC breakage upon leaving the BM – Erythroid hyperplasia - low M:E ratio (1:1) – Iron stores increased. 88  Howell-Jolly body Teardrop Schistocyte Blood NRBC Blood Macrocytic Ovalocytes Stippled RBC & Cabot Ring Giant Platelet Pap bodies Hypersegmented Neutrophil >5 lobes 89 Vitamin B12 (Cobalamin) Deficiency 90  Vitamin B12 deficiency Occur as a result of one of the following conditions 1. Nutritional Coballamin deficiency Strict vegetarianism 2. Abnormal intragasteric events ( i.e. inadequate proteolysis of food Coballamin) atrophic gastritis. 3. Loss or atrophy of gastric mucosa ( deficient IF) total or partial gasterectomy – May develop B12 and iron deficiency with macro and micro red cells…a dual (dimorphic) RBC population pernicious anemia 91  Cont….. 4. Abnormal events in the small bowel lumen – Inadequate pancreatic protease – Competing agents like fish tape worms (D.latum) – Disorders of ileac mucosa – Diminished or absent of IF – Coballamin receptor – Drug effects – Metabolic disorders ( coballamin is not used by cells) 92  Folate (Folic acid) Deficiency: Deficient intake. Increased needs: pregnancy, infant, rapid cellular proliferation, and cirrhosis Malabsorption (congenital and drug-induced) Two RBC populations Dimorphism Inherited DNA Synthesis Disorders: Deficient thiamine Macrocytic and factors (e.g. enzymes) RBCs responsible for folate metabolism. Microcytic Toxins and Drugs: RBCs 93  1. Pernicious Anemia it is defined as anemia resulting from defective secretion of IF associated with autoimmune attack on the gastric mucosa leading to atrophy of the stomach or Abs that block IF action. Abs block the site of IF where vit B12 binds. The diagnosis is confirmed by low serum B12 level and typically abnormal results of schilling test 94  Schilling test Used to diagnose pernicious anemia and determine if IF is available. If absorbed a portion of oral dose of vit B12 (not used by the body) ---- excreted in urine If not absorbed (malabsorption)…….. Not detected in urine but pass out in feces 95 Stage I. To diagnose malabsorption give the patient a radio active cobalt labeled vit B12 orally and Non labeled vit B12 intramuscularly to saturate liver If IF is present in the stomach (no other disease) vit B12 is absorbed and labeled vit B12 detected in urine If absorption is impaired labeled vit B12 not detected in urine instead in stool Diagnose malabsorption if it is not appear in urine by proceeding stage II. 96 Stage II. Differentiate cause of malabsorption Oral dose labeled vit B12 + IF Appear in urine…… pernicious anemia (give IF) If not appear in urine it is other cause of malabsorption 97 Stage III Performed to determine if the patient suffer from mal absorption of the IF – B12 complex secondary to small intestinal bacterial overgrowth. If this is the condition then tetracycline should normalize vit B12 absorption. 98  B. Non-Megaloblastic Anemia Macrocytosis that is NOT due to vitamin B12 or folate deficiency Accelerated erythropoiesis – Regenerating marrow or marked reticulocyte response following recent blood loss NRBC Polychromatophilic RBCs Wright’s stain 99  Non-Megaloblastic Anemia Liver disease and alcoholism – Complex & multiple problems – Degree of anemia varies, round macrocytes – Target cells/acanthocytes - due to abnormal lipid metabolism. – Echinocytes are also commonly found on the smear in liver disease. Target cells Echinocytes Stomatocytes, Alcoholic Acanthocytes 100  Differential Diagnosis of Macrocytic Anemia Megaloblastic and non-Megaloblastic – Perform B12 and folate levels – Specific morphology Blood smear 101 3. Normocytic Normochromic Anemia 102  3. Normocytic Normochromic Anemia Is a condition in which the size & Hgb content of RBCs is normal but the number of RBCs is decreased. It includes Aplastic anemia due to BM failure Blood loss anemia Hemolytic anemia 103  A. Aplastic Anemia – Condition of blood pancytopenia caused by bone marrow failure…decreased production of all cell lines and replacement of marrow with fat. – Due to damaged stem cells, damaged bone marrow environment or suppression – No extramedullary hematopoiesis 104 Types of aplastic anemia –Primary/idiopathic = 50% –Secondary/acquired….chemicals, drugs, infections, radiation = 50% –Congenital….Fanconi’s Aplasia plus dwarfism, skeletal abnormalities, mental retardation, abnormal skin pigmentation. 105  Lab diagnosis of Aplastic Anemia Normochromic –Normocytic Normal RBCs RBC (normal MCV & MCH) No Platelets Low reticulocyte count & Hgb Pancytopenia No abnormal cells Hypoplasia Bone marrow Blood Normal Serum iron, vitamin 10X B12 and folate levels Bone marrow, decreased # precursor cells 106  B. Hemolytic anemia Result from an increase in the rate of pre mature red cell destruction. Compensated hemolytic disease Uncompensated hemolytic disease It leads to – Erythropoietic hyperplasia – BM produces red cells 6 to 8X the normal rate – Marked reticulocytosis 107  Hemolytic anemia Two main mechanisms for RBC destruction in HA –Intravascular hemolysis: in the circulation –Extravascular hemolysis: in RE system (reticuloendothelial system) 108  Extravascular hemolysis Aged RBC 120 day Exstravascularly removed Abnormal RBC by Macrophage (RES) in BM, liver and spleen During destruction RBC releases Hgb Hgb Protoporphyrin Unconjugated bilirubin liver (glucuronic acid) Iron Globin reabsorbed conjugated bilirubin Amino acid gut reabsorbed & Excreted as urobilin & 109 Protein synthesis urobilinogen  Extravascular hemolysis Lab features –Increased RBC break down Serum bilirubin increase Stool urobilinogen increase Blood urobilinogen increase Urine urobilinogen increase 110  Intravascular hemolysis Red cells are destroyed in blood vessels and Hgb is released into the circulation: Free Hgb Saturates plasma haptoglobin Excess free Hgb is filtered by the glomerules (kidney) (if rate of hemolysis saturates renal reabsorption capacity) Free Hgb enters urine Fe is released in bladder tubule Renal tubule loaded with hemosiderin 111  Intravascular hemolysis Lab features – Hemoglobinemia and hemoglobinuria – Hemosiderin uria – Reduced/absent serum haptoglobin 112  1. Hereditary hemolytic anemia This is a congenital hemolytic anemia. some of which present at birth and other later in life, while still others may remain silent unless a physiological stress is super imposed Result of intrinsic red cell defects – Membrane defect (Hereditary Shperocytosis, Elliptocytosis and sickle cell anemia) – Metabolic defect : G6PDH and PK defic – Hgb chain defect (hemoglobinopatheis) : sickle cell anemia 113  A. Hemolytic Anemias due to Membrane Defects Most common is Hereditary Spherocytosis (HS) – Membrane defect is decreased spectrin and increased permeability of membrane to sodium ions Lab findings – Anemia varies – Few to many spherocytes Spherocytes on smear, high MCHC – Increased OF test 114  H Ovalocytosis/Elliptocytosis Membrane defect is H Ovalocytosis polarization of cholesterol or hemoglobin at ends and increased sodium permeability Over 25% ovalocytes Normocytic ovalocytes – Most asymptomatic – Mild anemia in 10- 15% 115  Hereditary Stomatocytosis Membrane defect is abnormal permeability to sodium and potassium Caused by edema 20-30% stomatocytes on H Stomatocytosis blood smear – Mild to severe hemolytic anemia 116  H Acanthocytosis Defect is increased membrane cholesterol due to abnormal plasma lipids Numerous acanthocytes on smear – Mild anemia – Also known as abetalipoproteinemia H Acanthocytosis = Abetalipoproteinemia 117  Osmotic Fragility Test (OF) Most commonly used to diagnose Hereditary Spherocytosis – Red cells are placed in hypotonic solutions Hypotonic 118 Osmotic Fragility Test (OF) Decreased Surface: Volume Ratio “Easy to Lyse” Increased Surface: Volume Ratio “Hard to Lyse” 119  B. Defect red cell metabolism (Enzyme defect) G6PD deficiency – G6PD is the only source of NADPH in red cell – NADPH is reduced for the production of reduced Glutathione. – Hgb and RBC membrane are usually protected from oxidant stress by reduced glutathione (GSH) – In G6PD deficiency NADPH and GSH synthesis is impaired, rendering the red cells vulnerable to oxidant stress. – Most individuals with G6PD are asymptomatic except during oxidant stress resulting from drugs or other causes. 120  B. Hemolytic Anemias due to Enzyme Defects Inherited enzyme deficiencies that lead to premature RBC death 121  Hemolytic Anemias due to Enzyme Defects PK Deficiency PK deficiency – ↓ATP impairs cation pump – Severe hemolytic anemia – Echinocytes Echinocytes G-6-PD Deficiency – Unable to protect Hgb due to G-6-PD Deficiency decreased NADPH – No clinical problems unless exposed to oxidants – Exposure to oxidants induce Heinz body formation and RBC destruction Normal RBCs if no exposure to oxidant 122  G-6-PD Deficiency Blood findings after oxidant exposure: – Mod to severe anemia – Schistocytes, spherocytes due to pitting out of Heinz bodies by spleen Enzyme assay G-6-PD deficiency after G-6-PD deficiency exposure to oxidant Hemolytic episode Heinz bodies - denatured Hgb Damaged RBCs 123 Supravital stain Wright’s stain  C. Normocytic anemias due to hemoglobinopathies Inherited hemoglobin defect with production of structurally abnormal globin chains; – All have target cells Beta chain amino acid substitution = variant Hgb – Hgb S = valine substituted for glutamic acid @ 6th of ß – Hgb C = lysine substituted for glutamic acid @ 6th of ß Target cells/Codocytes 124  Hemoglobin S Disorders A. Hemoglobin S disease/Sickle cell anemia/Hgb SS – Two sickle cell genes inherited (both beta Target cell chains are abnormal) – Symptomatic after 6 months of age Sickle cell Lab findings – Severe anemia HGB S Disease (Hgb SS) – Targets, sickle cells – NRBCs, inclusions – No Hgb A, >80% Hgb S, ↑F 125  Hemoglobin S Disorders B. Hemoglobin S trait/Sickle cell trait/Hgb SA – One sickle cell gene inherited Lab findings Target cells only NO – Asymptomatic, targets only Sickle cells – No anemia or sickle cells – ~60% Hgb A, ~40% Hgb S Potential problems if hypoxic HGB S Trait (Hgb SA) 126  Hemoglobin C Disorders A. Hemoglobin C disease/Hgb CC Two C genes inherited (both β chains are abnormal) C crystals polymerize differently and look like blocky Hgb packed rods in the red cells....intracellular. C crystals Lab findings – Mild anemia – Many target cells Target cell – Intracellular C crystals – No Hgb A, >90% Hgb C HGB C Disease (Hgb CC) – Decreased OF 127  Hemoglobin C Disorders B. Hemoglobin C trait/Hgb CA – One C gene inherited HGB C Trait (Hgb CA) Lab findings – Asymptomatic, no anemia – Targets, no C crystals – ~60% Hgb A, ~40% Hgb C – Normal Hgb A2 and F Target cells only NO C crystals 128  Hemoglobin SC Disease Hemoglobin SC disease/Hgb SC One sickle gene and one C gene inherited Double heterozygote- inherit sickle gene (S) from one parent and C gene from other parent; Both β chains are abnormal Lab findings – Intermediate in HGB SC Disease (Hgb S & Hgb C) severity between Hgb SS & SA – Several target cells – Many SC crystals SC Crystals – No Hgb A, ~50% Hgb S, ~50% Hgb C, ↑ F Target cells 129  2. Acquired hemolytic anemia A variety of acquired conditions result in shortened survival of previously normal red cells. These include immune mediated destruction, red cell fragmentation disorders, acquired membrane defects, spleen effects Result of extrinsic causes – Immune HA; warm HIHA, cold AIHA – Drug associated – Infection associated 130  Warm Autoimmune HA (WAIHA) Altered immune response causes production of an IgG warm autoantibody against ‘self’ RBC antigens – Antibody/complement attaches to RBC antigen…partially phagocytosed (loss of membrane)  spherocytes Cause: Primary (idiopathic) or secondary to disease Spherocytes & polychromasia 131 Blood  Warm Autoimmune HA (WAIHA) Lab findings – Mod to severe anemia, spherocytes, high MCHC – Erythrophagocytosis – Looks similar to H spherocytosis but positive DAT – Increased OF, bilirubin – Erythroid hyperplasia Ingestion of coated RBC Blood Electron Microscopy RBC Monocyte with ingested RBC RBC 132  Cold Autoimmune HA (CAIHA) Altered immune response causes production of an IgM cold autoantibody against ‘self’ RBC antigens – Antibody/C3 attaches to RBC antigen  agglutination (lysis by complement or macrophage) Primary (idiopathic) or secondary to disease 50x 100x 133 RBC Agglutination  Cold Autoimmune HA (CAIHA) Lab findings – Agglutination of red cells in extremities....ears, toes, nose  tissue damage  gangre – Severity varies with seasons….avoid the cold – IgM antibodies cause RBC agglutination – Reticulocytosis – Positive Direct Antiglobulin Test (detects complement) 134  Hemolytic Transfusion Reaction Incompatible blood transfusion – Recipient has antibodies to antigens on the donor red cells received – Donor cells are destroyed ABO worst – Intravascular hemolysis that is complement-induced lysis…immediate – Can be life-threatening 135  Hemolytic Disease of the Newborn Caused by maternal IgG antibodies directed against baby RBC antigens – Antibodies cross placenta and destroy fetal red cells HDN due to Rh incompatibility – Rh negative mother forms Rh antibody after exposure – HDN due to Rh Sever anemia Many nucleated red cells HDN due to ABO incompatibility – Mother’s ABO blood type is O; baby is type A or B – HDN due to ABO Mild, no anemia Spherocytosis 136  Hemolytic Anemias due to Trauma Fragmentation syndromes…most common finding on smear are schistocytes; anemia varies Types of trauma – Mechanical…prosthetic heart valves/cardiac abnormalities – Microangiopathic (MAHA)…small vessels (DIC.........bleeding) – March hemoglobinuria…forceful contact…. Schistocytes Schistocytes Fibrin Strands RBC 137 RBC fragmentation on fibrin strands  Normocytic/normochromic Hemolytic Anemias due to Trauma – Fragmentation Syndromes A.When RBCs are exposed to excessive trauma within the cardiovascular system, they may undergo fragmentation and lysis. Schistocytes are the most common finding on the smear; RBC destruction tests are abnormal. Severe trauma causes intravascular hemolysis. B.Three types of trauma: 1. Mechanical trauma A. Prosthetic heart valves or cardiac abnormalities fragment red cells. B. Mod to severe anemia with schistocytes and polychromasia. 138  Normocytic/normochromic Hemolytic Anemias due to Trauma – 2. Microangiopathic hemolytic anemia (MAHA) – trauma occurs in small vessels A. Disseminated Intravascular Coagulation (DIC) is a widespread clotting disorder initiated by conditions such as OB obstetrical) complications or sepsis. In DIC, clotting factors and platelets form fibrin  fibrin deposited in the microvessels fragment red cells. See anemia with schistocytes on smear, decreased platelets and depletion coagulation factors  leads to severe bleeding; can be fatal.. 139 2. Microangiopathic hemolytic anemia (MAHA) cont’d…….. B. Hemolytic uremic syndrome (HUS) - most often occurs in children following GI infection (E. coli); noted for renal failure  fibrin damages kidney; hemolytic anemia with echinocytes and schistocytes, decreased platelets; often requires dialysis; can be fatal 140  Normocytic/normochromic Hemolytic Anemias due to Trauma – 3. March Hemoglobinuria A. Transient, occurs after forceful contact of body with hard surfaces....joggers, soldiers after long march, bongo drum players. B. Hemoglobinuria; schistocytes may be present on smear. 141  Hemolytic Anemias due to Infectious Agents, and Thermal Burns Anemia varies, with severe hemolysis – Schistocytes and spherocytes on blood smear – Parasitize RBC, elaborate lytic toxins or cause direct damage to red cell membrane Malaria fever Closteridal infections..release toxins Schistocytes & Spherocytes 142  Normocytic/normochromic Hemolytic Anemia due to Infectious Agents A. Malarial infection 1. Damage to membrane occurs when parasite is pitted out of red cell by splenic macrophages or the entire RBC is removed; chills and fever as red cells rupture. 2. P. falciparum causes severe hemolysis called Blackwater fever. 143  Normocytic/normochromic Hemolytic Anemia due to Infectious Agents B. Clostridial infections - Clostridia elaborate toxins which damage RBC membrane causing severe intravascular hemolysis. Both malaria and clostridial infections are characterized by schistocytes and spherocytes on the blood smear; other infectious organisms can cause hemolysis, e.g. Toxoplasma, Bartonella, Babesia, Ehrlichia. 144  ‫زكاة العلم تعليمه‬ ‫إلى اللقاء فى محاضرات أخرى‬ ‫‪145‬‬