B. Mechanical trauma to red cells 1. Microangiopathic hemolytic anemias: - thrombotic thrombocytopenic purpura, DIC 2. Cardiac traumatic hemolytic anemia C. Infections: malaria D. Chemical injury: lead poisoning E. Sequestration in mononuclear phagocyte system Hypersplenism
Impaired Red Cell Production
I. Disturbance of proliferation and differentiation of stem cells: Aplastic anemia Pure red cell aplasia Anemia of renal failure Anemia of endocrine disorders
II. Disturbance of proliferation and maturation of erythroblasts A. Defective DNA synthesis: 1. Deficiency or impaired use of vitamin B12 and folic acid (megaloblasticanemias) B. Defective hemoglobin synthesis 1. Iron deficiency 2. Thalassemias C. Unknown or multiple mechanisms: Sideroblastic anemia, Anemia of chronic infections, Myelophthisicanemias due to marrow infiltrations
Hemolytic Anemias CHARACTERISTICS: Shortening of the normal red cell life span Accumulation of the products of hemoglobin catabolism A marked increase in erythropoiesis within the bone marrow
pathogenETICclassification of hemolytic anemia Extracorpuscular mechanism Intracorpuscular defect). Hereditary and Acquired disorders.
Hereditary disorders are due to intracorpuscular defects
Acquired disorders to extrinsic factors such as autoantibodies.
pathogenETICclassification of hemolytic anemia INTRAVASCULAR/ EXTRAVASCULAR HEMOLYTIC ANEMIA
features NO Hemoglobinemia, Hemoglobinuria, and other related intravascular changes POSITIVE Anemia and Jaundice Splenomegaly Plasma Haptoglobin levels are Invariably reduced (some Hgb escape phagocytes )
Standard morphologic changes in the hemolytic anemias ( BOTH types ): 1. Marked increase in the numbers of normoblasts in the marrow 2. May lead to extramedullaryhematopoiesis. 3. The accelerated compensatory erythropoiesis leads to a prominent reticulocytosis in the peripheral blood. 4. The elevated levels of bilirubin, when it is excreted through the liver = pigment gallstones (cholelithiasis). 5. With chronicityhemosiderosis
Intrinsic defect in the red cell membrane Renders erythrocytes spheroidal, Less deformable, Vulnerable to splenic sequestration and destruction
Molecular Pathology A deficiency of spectrin The spectrin content of these cells varies from 60% to 90% of normal and correlates closely with the severity of spherocytosis.
Molecular Pathology Mutations diminish "vertical" interactions that serve to connect the membrane cytoskeleton to the overlying lipid bilayer Resulting in Loss membrane fragment Spherocyte.
Loss of membrane fragments during exposure to shear stress in the circulation
MORPHOLOGY Spheroidal shape of the red cells, apparent on smears abnormally small cells lacking their central zone of pallor (Fig. 14-6) . Spherocytosis, although distinctive, is not pathognomonic, since it is also seen in autoimmune hemolytic anemias.
Clinical Manifestation 1. Anemia Chronic Hemolytic Anemia Mild to moderate severity. Aplastic / Hemolytic crisis 2. Splenomegaly Moderate splenic enlargement is characteristic of HS (500 to 1000 gm); 3. Jaundice
Most patient have a Chronic Hemolytic Anemia, usually of mild to moderate severity. May be punctuated by an aplastic crisis (triggered usually by a parvovirus infection of the marrow red cell precursors Sudden Worsening of Anemia due to temporary suppression of red cell production
Some show “ Hemolytic Crisis" resulting from accelerated red cell destruction, but clinically it is less significant than the aplastic crisis
Diagnosis of HS is based on family history, hematologic findings, and laboratory evidence of spherocytosis manifested by osmotic fragility since there is little margin for expansion of red cell volume without rupture.
Laboratory Similar to Chronic Extravascular Hemolysis Direct Antiglobulin test is Negative Increased Osmotic Fragility test Spherocytes in the Smear Decreased or Absent central Pallor MCV normal (Normocytic Anemia) MCHC Often Increased reflecting a Decrease in Cell Surface
Osmotic Fragility Test RBC are suspended in a series of tubes contaning hypotonic sol’n of NaClvarting from 0.9% - 0.0% Then incubated at room temp for 30 mins Then centrifuged % Hemolysis in the supernatant soln is measured
Osmotic Fragility Test Spherocytes have limited capacity to expand in Hypotonic Solutions Lyze at a higher concentration of NaCl than biconcave RBC Test is NOT specific for HS It may occur in Acquired Spherocyticanemias Autoimmune Hemolysis
Review of rbc biochemistry Normally G6PD is highest in young rbc and Decreases as it ages in person with G6PD deficiency G6PD – Defense against oxidant injury
The reduced glutathione so generated protects against oxidant injury by catalyzing the breakdown of oxidant compounds like H2 O2 .
G6PD DEFICIENCY- Pathology
The Hemolytic susceptibility Increase Greatly during : 1. Intercurrent Illness 2. Exposure to Various Drugs that have oxidant properties SULFAMETHOXAZOLE NITROFURANTOIN PRIMAQUINE The hemolysis in G6PD deficiency is both intravascular and extravascular.
Effects of Heinz to rbc Decrease erythrocyte deformability. pass through the splenic cords pluck out the Heinz bodies appear to have a bite of cytoplasm removed The resultant loss of membrane more membrane damage induces the formation of spherocytes. All these changes predispose the red cells to become trapped in splenic cords and destroyed by erythrophagocytosis.
clinical features Acute Intravascular Hemolysis present upon exposure to the oxidant injuries 2-3 days lag Only senescent red cells are lysed, Episode is self-limited and hemolysis stops when only the younger red cells remain in the circulation (despite continued administration of the oxidant drug). Mediterranean variant have much lower levels of G6PD, their anemia is more severe.
2 Forms : G6PD A- In Black the variant A is prevalent with normal activity BUT 11% 0f Blacks have type A with only 5-15% of normal enzyme activity More susceptible to HEMOLYSIS after ingestion Oxidant drugs/ Infection G6PD Mediterranean Middle East Protects against Malaria The level of G6PD activity in affected males is LOW (<1%) BUT MAY HAVE A MORE SEVERE AND NON-SELF LIMITED HEMOLYTIC ANEMIA WITH INFECTIONS & WIDER VARIETY OF DRUGS
LABORATORY FINDINGS ACTIVE HEMOLYSIS SIMILAR TO HEMOLYTIC ANEMIA ( Both Typres ) HEINZ BODIES- methyl violet stain Often attach to rbc membrane QUANTITATIVE ASSAY OF G6PD
RECOVERY G6PD Deficiency The recovery phase is heralded by reticulocytosis, as in the case of other hemolytic anemias. Since hemolytic episodes related to deficiencies of G6PD occur in most patients only when there is oxidant injury, the morphologic changes encountered in most chronic hemolytic anemias are rarely present.
PATHOLOGY Point mutation Substitution of valine for glutamic acid at 6th AA position in Beta Globin chain
PATHOGENESIS: On deoxygenation, the HbS molecules undergo aggregation and polymerization. This change converts hemoglobin from a freely flowing liquid to a viscous gel, Leading ultimately to formation of HbS fibers and resultant distortion of the red cells, which acquire a sickle or holly-leaf shape
Effect: HbS Polymerization of B globin chain during Deoxygenation Sickling
Pathology: Sickling of red cells is initially a reversible phenomenon; with oxygenation, HbS returns to the depolymerized state. However, with repeated episodes of sickling and unsickling, membrane damage ensues and the cells become irreversibly sickled. These deformed cells retain their abnormal shape even when they are fully oxygenated and despite deaggregation of HbS
Incidence: Highest – African Decent About 8% of black Americans are heterozygous for HbS. Heterozygote 40% is HbS & 60% Normal hemoglobins. Offer slight Protection against falciparum malaria
incidence: In Homozygous HbS disease Almost all the hemoglobin in the erythrocyte is HbS. A Serious Chronic Hemolytic Anemia Manifest Early childhood Often fatal before 30y/o
DESCRIPTION : Hemoglobin, as you recall, is a tetramer of four globin chains comprising two pairs of similar chains, each with its own heme group. The hemoglobin in the adult is composed of 96% HbA (a2b 2 ), 3% HbA2 (a2d2 ), and 1% fetal hemoglobin (a2g2 ). Structurally abnormal (defective) Hgb
The precipitation of HbS fibers also has deleterious effects on the red cell membrane. Damage both sickled & normal rbc With membrane injury, the red blood cells Rbc loaded w/ Ca++ Ca++ Activates K ion channel Efflux of potassium and water and at the same time gain calcium Rbc Dehydrated with increased MCHC More dense Renders cell more sticky Microvascular occlusion
Factors that Affect Sickling The rate of HbS polymerization is also significantly affected by the hemoglobin concentration per cell, that is, the mean corpuscular hemoglobin concentration The higher the HbS concentration within the cell, the greater are the chances of contact and interaction between HbS molecules. Thus, dehydration, which increases the MCHC, greatly facilitates sickling and vascular occlusion
Factors that Affect Sickling In Heterozygous 40% of the hemoglobin is HbS, the rest being HbA, which interacts only weakly with HbS during the processes of gelation. Therefore, the heterozygote has little tendency to sickle, except under conditions of severe hypoxia. In contrast, the homozygote, with virtually undiluted hemoglobin of the S type, has full-blown sickle cell anemia.
Factors that Affect Sickling Finally, a fall in pH, by reducing the oxygen affinity of hemoglobin, can increase sickling because it enhances the amount of deoxygenated HbS. Hemolysis Occurs Extravascularly & some Intravascularly due to increased mechanical fragility
CLINICAL MANIFESTATION 1st several months of life – Initially Asymptomatic ( HbF) Severe Anemia Bone changes due to BM hyperplasia Expansion of marrow space Thinning of the Cortex Radial Striations in Skull ( x-ray ) Leg Ulcers Chronic Hyperbilirubinemia
CLINICAL MANIFESTATION Vasoocclusive Complications Painful crises, Chest, Abdomen, Bone Episodes of hypoxic injury and infarction Associated with infection, dehydration, and acidosis (all of which favor sickling) has been noted
CLINICAL MANIFESTATION Increased Susceptibility to infection (1) splenic function is impaired because erythrophagocytosis interferes with the ability of the spleen to clear bacteria; (2) in later stages, total splenic fibrosis removes an important filter of blood-borne microorganisms; and (3) defects in the alternative complement pathway impair opsonization of encapsulated bacteria such as pneumococci and Haemophilusinfluenzae. Septicemia and meningitis caused by these two organisms are the most common causes of death in children with sickle cell anemia.
COMPLICATIONS: HAND-FOOT SYNDROME Bilateral painful swelling of the hands due to capillary stasis SEQUESTRATION CRISIS Sudden pooling of Blood and Rapid Splenomegaly Result to Hypovolemic Shock May occur in Early childhood
COMPLICATIONS: FUNCTIONAL ASPLENIA Inadequate Antibody Responses Impaired Ability of the RES to Clear Bacteria Increase risk to Infection acute chest syndrome VASO-OCCLUSIVE EPISODES Progressive Infarction Fibrosis Contraction of Spleen ( AutoSplenectomy ) Bone Necrosis APLASTIC CRISIS Temporary cessation of bone marrow activity, usually triggered by parvovirus infection of erythroid progenitor cells. Reticulocytes disappear from the peripheral blood, and there is sudden and rapid worsening of anemia.
BLOOD PICTURE Normochromic & Normocytic Anemia Numerous target cells Howell-Jolly bodies Sickledrbc Hematocrit is Unreliable estimate of anemia Due to Air Trapping Osmotic Fragility is Decreased Neutrophilia & Thrombocytosis BM Hyperplasia
Thalassemia syndromes Heterogenous group of Inherited D/O Genetic Lesions Decreased Synthesis of either a or b- globin chain of HbA (a2b2)
FEATURES: Beta Thalassemia Deficient synthesis of beta chain Fress alpha chains tend to aggregate INSOLUBLE INCLUSIONS Alpha Thalassemia Deificient alpha chain As a consequence, free chains tend to aggregate into insoluble inclusions within erythrocytes Premature destruction of maturing erythroblasts within the marrow (ineffective erythropoiesis) Insoluble Inclusions rigid rbc Lysis of mature red cells in the spleen –(hemolysis).
beta-Thalassemias Total lack or Reduction in the synthesis of structurally normal beta -globin chains with unimpaired synthesis of alpha chains Types : (1) beta0 -thalassemia, associated with total absence of beta-globin chains in the homozygous state; and (2) beta+ -thalassemia, characterized by reduced (but detectable) beta-globin synthesis in the homozygous state.
PATHOGENESIS: Most of these result from point mutations. Impaired beta-globin synthesis contributes to the pathogenesis of anemia by two mechanisms . 1. Lack of adequate HbA formation, (MCHC) is lower Cells are hypochromic. Much more important 2. Decreased survival of red cells and their precursors, resulting from an imbalance between alpha- and beta-chain synthesis.
PATHOGENESISdecreased survival of rbc Damage to the RBC Inclusion Damage a) Cell membrane damage Loss of K+ and Impaired DNA synthesis b) Reduces its plasticity Sequestered by spleen ( Hemolytic Component ) Apoptotic death of red cell precursors within the bone marrow, a phenomenon called ineffective erythropoiesis
TYPES: Beta Thalassemia Major Homozygous for beta-thalassemia genes Beta+/beta+ or beta 0/beta 0 Have Severe Transfusion Dependent Anemia
Beta- thalassemia minor or beta- thalassemia trait. The presence of one normal gene in the heterozygotes (beta+ /beta or beta0 /beta) usually leads to enough normal beta-globin chain synthesis so that the affected individuals are usually asymptomatic with only a mild anemia Clincal manifestation :
Clinical Manifestation: Beta- thalassemiaintermedia. Intermediate degree of severity Patients have severe anemia, but not enough to require regular blood transfusions.
Thalassemia major Manifest 6 to 9 months after birth When hemoglobin synthesis switches from HbF to HbA. Anisocytosis (variation in size) with many small and virtually colorless (microcytic, hypochromic) red cells.
Growth retardation and die at an early age from the profound effects of anemia unless transfused Blood transfusions not only improve the anemia but also suppress secondary features related to excessive erythropoiesis. Thalassemia major
Cardiac disease resulting from progressive iron overload and secondary hemochromatosis is an important cause of death even in patients who can otherwise be supported by blood transfusions Thalassemia major
Thalassemia major Enormous expansion of BM Thinning of Cortex a) Frontal & Maxillary Bossing - thinning of the cortical bone with new bone formation on the external aspect, giving rise to the "crew- cut" appearance on x-rays Marked hepatosplenomegaly Hemosiderosis or hemochromatosis a) compensartory increased dietary iron uptake ( Tx iron chelators )
Iron chelators. & Transfusion With transfusions and iron chelation, many patients survive into the third decade, but the overall outlook continues to be grim. Bone marrow transplantation from an HLA-identical sibling is currently the only therapy that offers a cure. Prenatal diagnosis is possible by molecular analysis of DNA. Prognosis poor – No known Cure Thalassemia majorManagement
Low Hemoglobin Increased RBC Low MCV & MCHC – MicrocyticHypochromic Anisocytosis with N-rbc Retic count is LOW <10% Electrophoresis High HgF & HgA2 Limited amount of HgA Thalassemia major
Target cells (so called because the small amount of hemoglobin collects in the center), Stippled red cells, and Fragmented red cells, are common. Reticulocyte count is lower than would be predicted from the severity of anemia. Due ineffective erythropoiesis, Thalassemia major
This is the most severe form of alpha-thalassemia, Resulting from the deletion of all four alpha-globin genes. In the fetus, excess gamma-globin chains form tetramers (hemoglobin Bart) have extremely high oxygen affinity but are unable to deliver the oxygen to tissues. With intrauterine transfusion, many such infants can be saved. The fetus shows severe pallor, generalized edema, and massive hepatosplenomegaly similar to that seen in erythroblastosisfetalis
PAROXYSMAL NOCTURNAL HEMOGLOBINURIA
GPI – Linked Membrane Proteins
GPI-linked proteins that regulate complement activity 1. Decay-accelerating factor, or CD55; 2. Membrane inhibitor of reactive lysis, or CD59; 3. C8 binding protein CD59 is the most important because it limits spontaneous in vivo activation of the alternative complement pathway by rapid inactivation of C3 convertase.
PATHOLOGY : REVIEW Because several GPI-linked proteins inactivate complement, their absence renders blood cells unusually sensitive to lysis by endogenous complement.
FEATURES: Acquired Abnormality of Multipotential Stem Cell Mutation of Phosphatidylinositolglycan-class A gene Chromosome Xp22.1 Leads to DISRUPTION OF PROTEIN ANCHORING on RBC Membrane Deficient in : 1. Decay Accelerator Factor CD55 2. Membrane Inhibitor of Reactive Lysis CD59 3. C8 Binding Protein Leads to EPISODIC COMPLEMENT –MEDIATED INTRAVASCULAR HEMOLYSIS
Episodic Normocytic or Macrocytic Anemia Reticulocytosis Often Leukopenia & Thrombocytopenia - Platelets and Granulocytes are also more sensitive to lysis by complement. Hemoglobinuria 20 to Intravenous hemolysis Flow Cytometry FEATURES: LABORATORY
CLINICAL: 25% ( Intermittent )Paroxysmal & Nocturnal IntravascuarHemolysis 75% chronic hemolysis without dramatic hemoglobinuria Hemosiderinuria with loss of iron eventually leads to iron deficiency. Increased risk for Thrombosis & Bleeding - Fatal in 50% May progress to Leukemia and Aplastic anemia
IMMUNOHEMOLYTIC ANEMIA Caused by Extracorpuscular Mechanisms
Classification: Warm Antibody type Cold Agglutinin type Cold Hemolysins (Paroxysmal Cold Hemoglobinuria)
Warm Antibody Type The antibody is of the IgG type, does not usually fix complement, and is active at 37°C. Primary or idiopathic Secondary to Lymphomas and leukemias Other neoplastic diseases Autoimmune disorder (SLE) Drugs
Warm Antibody Hemolytic Anemia. Most common 40-70% Immune hemolytic anemia About 50% are Idiopathic or primary Most of the autoantibodies are of the immunoglobulin (Ig) G class Mostly Extravascular Hemolysis- Recognized by Fc receptors on Splenic Macrophages Producing Spherocytes Moderate Splenomegaly
Common in Women Drug – Induced Theory: 1. Hapten model( penicillin & cephalosphorin ) Induce antibody directed against the cell-bound drug 2. Autoantibody model ( methyldopa ) Initiates the prodn. of Ab that are directed against intrinsic red cell antigens, in particular the Rh Warm Antibody Hemolytic Anemia.
Blood Picture & Tx: Normocytic or Macrocytic Anemia Spherocytes (+) Direct Coombs test Patient’s rbc + Anti-human globulin serum Incubated Agglutination Corticosteroids Refractory cases : - With Splenectomy or Transfusion
Cold Agglutinin Type- Igm Less common than warm Ab Acute – Mycoplasma, IM Chronic / Idiopathic Lymphoma The antibodies are IgM and are most active in vitro at 0 to 4°C.( Agglutinate rbc at low temp). Antibodies dissociate at 30°C or above. The antibody fixes complement at warmer temperatures, but agglutination of cells by IgM and complement occurs only in the peripheral cool parts of the body.
Cold Agglutinins: IgM type Bind to & agglutinate rbc in cooler peripheral blood and can fix Complement Extravascular Hemolysis Due to binding by Complement receptors in liver Seen in warmer blood of central circulation Intravascular Hemolysis Due to Complement Activation
Cold Hemolysin: IgG type Idiopathic Secondary to infection/ Lymphoma Generally Do Not demonstrate Prominent Hemolysis Most develop Peripheral vascular symptoms on cold exposure- thrombosis ( Raynaud’s phenomenon ) Laboratory: Rbc agglutinate at room temp Lead to: Falsely High MCV & MCHC Falsely Lowered Hematocrit Warming blood to 37C Reverses Agglutination
Cold Hemolysin Hemolytic Anemia. Paroxysmal cold hemoglobinuria, Acute intermittent massive hemolysis, frequently with hemoglobinuria, after exposure of the affected patient to cold Least common Lysis is clearly complement dependent. AutoAntibodyare IgG type that bind P blood group antigen on rbc at low temp. Also known as Donath Landsteiner Ab
Complement mediated intravascular hemolysis does not occur until the rbcrecirculaye to warm central regions as complements ‘ enzymes are more efficient at 37 degrees. Unknown Autoantibodiesprod’n. Hx of infections such as mycoplasmal pneumonia, measles, mumps, and some ill-defined viral and "flu" syndromes Cold Hemolysin Hemolytic Anemia.
HEMOLYTIC ANEMIA RESULTING FROM TRAUMA TO RED CELLS
Pathogenesis: 1. Cardiac valve prosthesis ( Traumatic) Rbc shear stresses 20 Turbulent blood flow and Abnormal pressure gradients caused by the valves.
2. Narrowing or Obstruction of vasculature Mechanical damage to the red cells a) Most often caused by widespread deposition of fibrin in the small vessels in association with disseminated intravascular coagulation Pathogenesis:
b) Other causes of microangiopathic hemolytic anemia -Malignant hypertension, - SLE -Thrombotic Thrombocytopenic purpura (TTP) - Hemolytic-Uremic Syndrome (HUS) - Disseminated Cancer.