Normocytic Normochromic Anemia PDF

Normocytic normochromic anaemia Sef lucrari Dr Mihaela Andreescu UTM – Facultatea de Medicina  Anemia classifications Clinical elements Reticulocite count Acute/Chronic Hereditary/Aquired Hyper-r...

Normocytic normochromic anaemia Sef lucrari Dr Mihaela Andreescu UTM – Facultatea de Medicina  Anemia classifications Clinical elements Reticulocite count Acute/Chronic Hereditary/Aquired Hyper-regenerative Hypo- regenerative Excessive Decreased erythrocyte Bleeding erythrocyte production Erythrocyte volume destruction Macrocytic Microcytometry Normally  ANEMIA THROUGH DECREASED RED BLOOD CELL PRODUCTION HEREDITARY ACQUIRED Stem cell inhibition anemia Stem cell inhibition anemia An. Fanconi Aplastic anaemia Sdr Swachman Anemias from leukemias and myelodysplastic sdr Dyskeratosis congenita Anaemias associated with medullary infiltration Post chemotherapy Inhibition of erythroid progenitors: Inhibition of erythroid progenitors: Sdr. Diamond Blackfan Single red series aplasia (parvovirus, drugs, thymomas) Congenital dyserythropoietic syndromes Endocrine diseases Acquired sideroblastic anaemia (Md, copper deficiency) Functional alteration of erythroid progenitors due to nutritional/other causes: Functional alteration of erythroid progenitors due to nutritional/other causes: Megaloblastic anemias (B12 malabsorption - selective deficiency, congenital IF deficiency, transcobalamin II Megaloblastic anaemia (B12, folate deficiency, acute nitrous oxide deficiency, deficiencies in cobalamin or folate anaemia) metabolism) Fe deficiency Deficiencies in purine or pyrimidine metabolism Other nutritional causes Anaemia from chronic diseases and chronic inflammation Diseases of iron metabolism (hereditary atransferinaemia, DMT1 mutation) Anaemia from renal failure Anaemia from poisoning (Pb) Hereditary sideroblastic anaemia Acquired thalassaemia (clonal haematopoietic B.) Thalassaemia AC anti-erythropoietin  ANEMIA THROUGH INCREASED RED BLOOD CELL DESTRUCTION ACQUIRED HEREDITARY Mechanical causes: Haemoglobinopathies Macroangiopathies (March hemoglobinuria, artificial heart valves) Sicklemia Microangiopathies (DIC, PTT, vasculitis) Unstable Hb Parasites and microorganisms: malaria, Cl. Perfringens) Mediated by AC: Diseases of the H membrane Hemolytic anemia with warm antibodies Cytoskeletal membrane diseases Cryopathic sdr. (cold agglutinins, HPN) Membrane lipid diseases (abetalipoproteinemia, hereditary Post transfusion reactions stomatocytosis) Membrane diseases associated with Ag H abnormalities H membrane diseases: (McLeod sdr, Rh deficiency sdr) Spur cell haemolysis Membrane diseases associated with abnormal transport (hereditary xerocytosis) Acanthocytosis and acquired stomatocytosis Erythrocyte enzyme defects Chemical factors: As, Cu, Chlorate, Venoms (Pyruvate kinase, 5 nucleotidase, G6PDH) Porphyries Physical factors: heat, radiation, oxygen  Diagnostic algorithm normocytic anemia  THE CLASSIFICATION OF ANEMIAS ACCORDING MCV 1) Microcytic anemia 1) Iron deficiency anemia (Fe) (MCV < 80 fl) 2) Thalasemia (α and β) hypochromic 3) Anemia from chronic disease (MCHC < 27 pg) 4) Sideroblastic anemias 1) Haemolytic anaemias 2) Anemias secondary to recent bleeding 1) Normocytic anemia (MCV 80 – 2) Anaemia in chronic diseases 100 fl) normochromic 3) Aplastic anaemia and other conditions causing medullary infiltration (MCHC > 27 pg) 4) Most of non-haematin deficiencies 5) Combined deficiency (Fe causing microcytosis + B12/folate causing macrocytosis) 1) Folate or B12 deficiency 2) Hemolytic anemia 1) Machrocytic anemia 3) Liver disease (MCV > 100 fl) 4) Marros dysplasia and marrow failure including aplastic anemia 5) Anemia secondary to some antimetabolic drugs (e.g. : hidroxiuree) 6 Haemolytic anaemias  Globin, the protein component of haemoglobin, is divided into amino acids, which end up back in the bone marrow, to ERYTHROCYTE DESTRUCTION be used in the production of new red blood cells. Hb that is not phagocytosed is divided in the circulation, releasing alpha and beta chains that are removed from the circulation by the kidneys. Iron from the hem portion of Hb can be stored in the liver or spleen, primarily as ferritin or haemosiderin, or transported through the bloodstream via transferrin to the bone marrow for recycling into new erythrocytes. The iron-free portion of Hb is degraded to biliverdin, a green pigment, and then to bilirubin, a yellow pigment. Bilirubin binds to albumin and travels through the blood to the liver, which uses it to make bile, a compound released in the intestines to help emulsify dietary fats. In the large intestine, bacteria separate bilirubin from bile and convert it into urobilinogen and then stercobilin, which is eliminated from the body in the faeces. The kidneys also remove any circulating bilirubin and other related metabolic products, such as urobilins, and secrete them in the urine.  Hemolysis removal of erythrocytes from circulation Physiological haemolysis Pathological haemolysis removal of aged erythrocytes acts on all erythrocytes (over 120 days old) in circulation regardless of their age from circulation Intratissue extravascular haemolysis ⇒ premature destruction - in the spleen of an increased number of erythrocytes Pathological ⇒ shortening the lifespan of haemolysis erythrocytes compensatory increase in erythrocyte production by the bone marrow Compensated haemolysis = chronic destruction fully compensated by erythrocyte production (no anaemia, only increased reticulocytes) Hyperplasia of the erythrocyte series resulting => increased reticulocyte count. Decompensated hemolysis = destruction is greater than erythrocyte production→ anemia  Extravascular: involves erythrocytes with reduced deformability and altered surface properties that are ERYTHROCYTE DESTRUCTION ingested by macrophages. Pathologically, decreased deformability occurs in hereditary spherocytosis and hereditary elliptocytosis through decreased surface-to-volume ratio. Increased intracytoplasmic viscosity occurs in sickle cell disease and HbC. Membrane alteration occurs in many forms of anaemia, such as autoimmune haemolytic anaemia, hereditary xerocytosis (membrane alteration leads to loss of K). Intravascular: physiologically reduced in quantity, can become important in hemolytic anemias or PNH- paroxysmal nocturnal hemoglobinuria (in which the complement creates holes in the Er membrane), in hemolysis induced by contact with mechanical heart valves, and in microangiopathic hemolytic anemia. Hb binds to haptoglobin (1:2) and is transported to the liver, where the hem is converted to biliverdin, which is then catabolised to bilirubin. EXTRAVASCULAR HEMOLYSIS Extravascular haemolysis occurs when damaged or abnormal erythrocytes are removed from the circulation by spleen, liver and bone marrow cells, similar to the process by which senescent erythrocytes are removed. The spleen usually contributes to haemolysis by destroying slightly abnormal erythrocytes or cells coated with hot antibodies. An enlarged spleen can even sequester normal erythrocytes. Erythrocytes that are very much altered or those coated with cold antibodies or complement (C3) are destroyed in the circulation and in the liver, which (because of its high blood flow) can efficiently remove damaged cells.  EXTRAVASCULAR HEMOLYSIS Protoporphyrin inside the macrophage comes under the action of hemoxygenase which breaks the porphyrin ring and produces biliverdin. The lungs excrete a secondary product of that reaction, CO. Green biliverdin is reduced to bilirubin. Because it is hydrophobic, bilirubin binds to albumin for transport in the plasma to the liver. The bilirubin-albumin complex enters the liver parenchymal cells, where bilirubin dissociates from albumin and is conjugated with glucuronic acid by glucuronyl transferase forming bilirubin diglucuronide, which is also called conjugated bilirubin. Bilirubin initially released by macrophages lacking these sugars is called unconjugated. Conjugated bilirubin is excreted as bile acid in the intestines. Conjugated bilirubin is oxidised by intestinal bacteria into various water-soluble compounds, collectively called urobilinogen. The majority of urobilinogen is further oxidised to stercobilin, which is the final pathway for protoporphyrin excretion. Because they are water soluble, some bilirubin and urobilinogen conjugate molecules are absorbed from the intestines and can be detected in plasma. The portal circulation transports blood directly to the liver, so that most of the absorbed conjugated bilirubin and urobilinogen is recycled directly into the bile again. Some, however, remains in the plasma and is filtered by the renal glomerulus and excreted in the urine. The yellow colour of the urine is due to urobilin, a derivate of urobilinogen. INTRAVASCULAR HEMOLYSIS Intravascular haemolysis occurs when the cell membrane has been severely damaged by any of the following mechanisms: autoimmune phenomena, direct trauma (e.g. march haemoglobinuria), shear stress (e.g. defective mechanical heart valves) and toxins (e.g. clostridial toxins, venomous snake bite). It results in hemoglobinemia when the amount of Hb released into plasma exceeds the Hb-binding capacity of plasma-binding protein haptoglobin, a globulin normally present in concentrations of about 1.0 g/L in plasma (decrease in serum haptoglobin). In haemoglobinaemia, unbound Hb dimers are filtered out in the urine and reabsorbed by renal tubular cells; haemoglobinuria occurs when the reabsorption capacity is exceeded. Iron is incorporated into haemosiderin inside the tubular cells; some of the iron is taken up for reuse and some ends up in the urine when the tubular cells break down - haemosiderinuria.  INTRAVASCULAR HEMOLYSIS When free in plasma, Hb exists bound to haptoglobin. By binding to haptoglobin, Hb avoids filtration at the glomerulus and is saved from urinary loss. The complex is transported to the liver, where it is bound to macrophage receptors and internalised, iron is rescued, and the remaining protoporphyrin is converted to bilirubin. If lysis is accelerated, haptoglobin is depleted, as hepatic haptoglobin production does not increase in response to increased hemolysis. A secondary mechanism of iron rescue involves haemopexin. In non- haptoglobin-bound Hb, iron is oxidised, forming methaemoglobin. The heme molecule dissociates from globin and binds hemopexin. This binding saves Fe from urinary loss and prevents oxidation of cell membranes. Hemopexin-metheme binds to hepatocyte receptors, is internalised, iron is rescued, and protoporphyrin converted to bilirubin. Unlike haptoglobin, hemopexin is recycled into plasma from the hepatocyte. Although its production is constant as it is recycled into plasma, haemopexin levels do not fall dramatically during periods of increased intravascular haemolysis. A third mechanism of iron rescue is the metheme-albumin system. Metheme can bind to albumin when haemopexin is saturated, which further reduces urinary losses. If the above systems are overloaded, excess haemoglobin or haem iron will leak into the urine. However, iron can be retained in the kidneys. When inside the renal tubular cell, iron is separated from protoporphyrin. There it is stored as ferritin or haemosiderin. When the amount of haemoglobin presented to the kidneys exceeds what can be reabsorbed, the excess is then detectable in the urine as haemosiderinuria.  Hemolysis Extravascular haemolysis Intravascular haemolysis Haemoglobinaemia - plasma analysis Jaundice plasma Haemoglobinuria- „rubine” urine (indirect bilirubin↑) Hemosiderinuria Hyperchromic urine Low levels of haptoglobin (urinary urobilinogen ↑) Methaemalbuminemia  DIAGNOSIS OF HAEMOLYTIC ANAEMIA mild jaundice Non-specific signs and in uncompensated haemolysis - symptoms of anaemia: pallor, symptoms: asthenia, dizziness. 1) indirect bilirubin increase Confirmation of haemolysis: 2) increased urinary urobilinogen 3) reticulocytosis 4) erythroblastic hyperplasia of bone marrow Mechanism of haemolysis Extravascular, Intravascular plasma in VSH tube (pink in intravascular hemolysis) / icteric in extravascular hemolysis Inspection dark yellow urine (urobilinogen ↑) in extravascular haemolysis / ruby in intravascular haemolysis  ANAEMIA + HYPERRETICULOCYTOSIS DIFFERENTIAL DIAGNOSIS HEMOLYSIS ACTIVE REGENERATION Acute post-hemorrhage Recovery after appropriate treatment 2 main groups of markers BIOCHEMICALS - E hyperdistruction a) INDIRECT BILIRUBIN ↑ (marker of protoporfin degradation) b) Urobilinogen in urine present MORPHOLOGICALS - Compensatory hyperregeneration a) Medullary erythroid hyperplasia; b) Hyperreticulocytosis OTHER MARKERS: a) LDH; b) Consumption of free haptoglobin from plasma TWO HAEMOLYSIS MECHANISMS: EXTRAVASCULAR INTRAVASCULAR Haemoglobinaemia Haemoglobinuria Haemosiderinuria 19  HAEMOLYTIC ANAEMIAS (HA)) INTRACORPUSCULAR EXTRACORPUSCULAR Hereditary intracorpuscular hemolytic anemias  AH INTRACORPUSCULAR (EREDITED) MEMBRANE DEFECT METABOLIC DEFECTS HEREDITARY SPHEROCYTOSIS Deficiency of HEREDITARY STOMATOCYTOSIS PIRUVAT KINAZA HEREDITARY ELLIPTOCYTOSIS GLUCOZO-6- HEREDITARY PYROPOIKILOCYTOSIS PHOSPHATE DEHYDROGENASE HAEMOGLOBIN DEFECTS Microcytic anaemia DEFECTS OF SYNTHESIS -THALASSEMIA ABNORMAL HAEMOGLOBINS ( Hb S, Hb C, INSTABLE Hb) Hereditary intracorpuscular hemolytic anemias Membrane defect - Spherocytosis erditarum  Inherited intracorpuscular hemolytic anemias by membrane defect Erythrocyte membrane disorders result from changes in the quantity or quality (or both) of individual proteins and their dynamic interactions with each other. Disruption of the vertical protein-protein interactions of the membrane, i.e. the spectrin-ankyrin-3 band, the binding or interaction of the 3-protein 4.2 band, leads to uncoupling of the membrane skeleton from the lipid bilayer. This leads to membrane instability with loss of lipids and some integral membrane proteins, resulting in loss of membrane surface and spherocytosis phenotype. Disruption of horizontal interactions of membrane scaffold proteins, including disruption of spectrin self-association or protein-protein junctional complex interactions, leads to membrane instability, membrane deformability and altered mechanical properties and the elliptocytosis phenotype. The development of biochemical techniques has enabled the separation and quantification of membrane proteins and the detection of membrane protein abnormalities in many inherited red cell disorders. Progression in molecular biology have allowed accurate determination of genetic defects in many cases.  HEREDITARY SPHEROCYTOSIS (MINKOWSKI – CHAUFFARD DISEASE) The most common hereditary hemolytic anemia without hemoglobin abnormality. Onset at different ages from newborn to school child. Autosomal dominant inheritance (abnormality is present in one parent even if asymptomatic). Pathogenesis Erythrocyte membrane abnormality (spectrin synthesis defect) resulting in two pathological features of erythrocytes: Excessive sodium and water penetration into the erythrocyte leading to depletion of the Na- ATP pump (large amounts of glucose and ATP are used to remove excessive Na+ and water from the cell), premature ageing of erythrocytes, their sequestration and destruction in the spleen. increased fragility of erythrocytes and decreased membrane deformability.  HEREDITARY SPHEROCYTOSIS (MINKOWSKI – CHAUFFARD DISEASE) Clinic Paraclinic Onset: Moderate anaemia (9-10g/dl), reticulocytosis neonatal: intense jaundice, anemia, (5-20%), microspherocytes (20-50%), splenomegaly normal leukocyte and platelet counts. in childhood: insidious with jaundice, pallor, asthenia, splenomegaly Medulogram: typical erythroid hyperplasia; Evolving: Low erythrocyte osmotic resistance is the main acute attacks of deglobulization (triggered diagnosis test (starts at 0.8% NaCl and is total at by viral or bacterial infections, accompanied 0.4% NaCl). by fever, jaundice, abdominal pain) Bone marrow aplasia episodes(sometimes) Normal values of osmotic resistance 0.35-0.45% NaCl.  HEREDITARY SPHEROCYTOSIS (MINKOWSKI – CHAUFFARD DISEASE) The autohemolysis test is greatly increased reaching 10-50% (normal 0.5-3.5%), but normalizes with the addition of glucose and ATP; Coombs test negative (differential diagnosis with AHAI); Hemoglobin electrophoresis normal; Indirect bilirubin, LDH - markers of hemolysis - and sideremia elevated. Paraclinic Differential diagnosis Positive diagnosis: It is done particularly with the acquired spherocytosis: family survey autoimmune hemolytic anemias or microspherocytes on peripheral blood by feto-maternal isoimmunization in the Rh or ABO smear system. osmotic fragility test Differentiation is mainly based on the Coombs autohemolysis test test.  HEREDITARY SPHEROCYTOSIS (MINKOWSKI – CHAUFFARD DISEASE) Treatment Complications Exsanguinotransfusion and phototherapy are used in newborns; Severe jaundice in the neonatal period Folic acid supplementation is necessary; Aplastic episodes precipitated by In acute deglobulization and aplastic crises, isogroup infection isoRh erythrocyte mass transfusion may be Medullary megaloblastosis required. Biliary lithiasis Splenectomy is the treatment of choice: Post-plenectomy infectious - Age over 6 years (lower infectious risk); complications - penicillin or amoxicillin prophylaxis daily 1-2 years postoperatively; - pneumococcal, meningococcal and Haemophilus influenzae vaccination is required. Evolution and prognosis Favorable if splenectomy was done at the right time. After splenectomy, anaemia, reticulocytosis and jaundice disappear; microspherocytes and low osmotic resistance persist without clinical consequences. The risk of biliary lithiasis depends on the timing of surgery. Hereditary intracorpuscular hemolytic anemias Metabolic defects - G6PDH deficiency  Glucose phosphate dehydrogenase (G-6PD) deficiency Definition Hereditary hemolytic anemia G-6-PD glycolytic enzyme deficiency Acute hemolytic episodes secondary to oxidant exposure Metabolic overload of heme Etiopathogenesis R X-linked manifest in males ~ 3000 enzyme variants Hb precipitation modified blood = Heintz bodies  Analgesics, antipyretics Haemolytic substances in people with G-6-PD NSAIDs: acetylsalicylic acid deficiency phenacetin Antibiotics Antimalarial Chloramphenicol quinine primaquine, mepacrine HIN Sulfamide, sulphone Nalidixic acid sulfasoxazole sulphanilamide biseptol Naphthalene Nitrofurans Quinidine nitrofurantoin Probenecid Methylene blue Raw beans Synthetic vitamin K Vicia fava  G-6P deficiency Clinical symptoms Two distinct clinical forms moderate (A -) black race severe (M or B -) in patients of Mediterranean origin Clinical manifestations depending on the level of enzyme activity haemolytic crises 1-2 days after exposure to oxidising substances in the course of infections Favism severe form of acute haemolysis (M or B -) ingestion of raw Vicia fava beans Southern Europe  G-6PD deficiency - treatment - complications - evolution Treatment Complications Acute haemolysis: Severe jaundice, neo-natal period Erythrocyte mass transfusions oxidizing agents ~ neonatal period Between episodes: Severe episodes of deglobulation Avoiding oxidants severe anaemia + HF, AKI, shock acidosis + hyperthermia (infections) Evolution acute episodes of deglobulation, induced by oxidizing agents between clinical and biological episodes = normal severe in marked deficiency (M) self-limited in the form (A -) rarely chronic evolution Prognosis improved by avoiding exposure to oxidants Intracorpuscular hemolytic anemias acquired Paroxysmal nocturnal hemoglobinuria  Paroxysmal nocturnal haemoglobinuria Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haematological disorder caused by somatic mutations of the PIGA gene in haematopoietic stem cells. Such mutations result in impaired production of glycosyl phosphatidyl inositol (GPI), an anchor molecule for many different cell membrane proteins. The latter include CD55 (also called decay- accelerating factor, DAF) and CD59 (membrane reactive lysis inhibitor, MIRL), which are natural complement inhibitors and are lost on the membrane of HNP cells. Their absence leads to suboptimal complement inhibition and complement- mediated hemolysis of red blood cells.  Paroxysmal nocturnal haemoglobinuria ØThe classical pathway is initiated by immune complexes interacting with C1q, C1r and C1s, acting on C2 and C4, resulting in the formation of the C4bC2a complex (C3 convertase). ØThe lectin pathway is activated by mannose-binding lectins found on the surface of pathogens and generates the same C3 convertase. ØThe alternative pathway is spontaneously activated at a low rate by a mechanism called C3 twitching, through activation of factors B and D and stabilisation by the plasma protein properdin. This mechanism also generates a C3 convertase with a strong amplification loop. ØC3b initiates the terminal complement cascade by forming the C5 convertase, which cleaves C5 and then C6, C7, C8 and C9. This results in the formation of the terminal membrane attack complex (MAC), which forms pores in the erythrocyte membrane favouring cell lysis (intravascular haemolysis). During complement activation, potent anaphylatoxins capable of inducing chemotaxis, cell activation, inflammation and extravascular haemolysis by immune effectors are produced. Thrombotic events and a variable degree of bone marrow failure occur in an unpredictable proportion of patients and may also change over time. Extracorporeal haemolytic anaemias EXTRACORPUSCULAR HAEMOLYTIC ANAEMIAS Anemias characterized by the synthesis of autoantibodies that react specifically with certain erythrocyte antigens (with or without serum complement fixation) and cause hemolysis of erythrocytes. I. BY ANTIBODIES: ALLOANTICORPI: post-transfusion, haemolytic disease of the newborn. AUTOANTICORPI: autoimmune haemolytic anaemia (AHAI) with hot and cold antibodies, idiopathic and secondary II. BY INFECTIOUS AGENTS: malaria, toxoplasma, Clostridium Welchii III. BY DRUGS IV. BY CHEMICALAGENTS: Pb, Cu, Zn, As, V. BY PHYSICALAGENTS: burns, ionising radiation VI. THROUGH TRAUMATIC FACTORS: valve prostheses, DIC, HUS, PTT, haemoglobinuria of March VII. HYPESPLENISM  IMMUNE HEMOLYTIC ANEMIAS Allo-Ac formation = normal immune response to foreign antigens Auto-Ac formation = disease state, Ac is produced against own Er Ag. Hemolyzes both own erythrocytes and isocompatible erythrocytes (low transfusion yield) EXTRACORPUSCULAR AH PATHOGENY EXTRACORPUSCULAR HA by autoantibodies Two mechanisms: alteration of own antigenic structures (by action of viruses and bacteria) decreased immune tolerance with disruption of the immune system's ability to recognise 'self'. EXTRACORPUSCULAR HA by allo-antibodies Erythrocyte autoantibodies can be of the "warm" (Ig G), "cold" (Ig M) or "cold-warm" (Donath-Landsteiner) type. Autoimmune extracorporeal hemolytic anemias AUTOIMMUNE HAEMOLYTIC ANAEMIA (AHAI) Autoantibody (auto-Ac) binding to erythrocyte membrane antigens (Ag) Initiation of hemolysis WARM antibodies- IgG mediated - EXTRAVASCULAR HEMOLYSIS COLD antibodies - mediated by two types of auto-Ag: Cold agglutinin disease (BAR) auto-Ac Ig M anti Ii - EXTRAVASCULAR HAEMOLYSIS Paroxysmal cold haemoglobinuria (PCH) - biphasic auto-Ac Donath- Landsteiner type Ig G, anti P INTRAVASCULAR HEMOLYSIS  Autoimmune haemolytic anaemias AUTOIMMUNE HA WITH WARM ANTIBODIES AUTOIMMUNE HA WITH COLD ANTIBODIES IDIOPATHIC 10% IDIOPATHIC 50% SECONDARY 90%: SECONDARY 50% Diseases with autoimmune substrate: SLE Diseases with autoimmune substrate: collagen diseases, myasthenia, ulcerative colitis, thyroiditis, Biermer anaemia Lymphoid neoplasms: LLC, NHL , WM Lymphoid neoplasms: Other tumours: lung cancer, colon cancer, ovarian cancer, Kaposi's sarcoma CLL, NHL, MM, HL Infections: CMV, MI, hepatitis, chickenpox, influenza, Other tumours: ovarian dermoid cyst, ovarian, caecal, renal, breast Mycoplasma pneumonia, HIV, spirochete, trypanosoma cancer Infections: CMV, MI, hepatitis, chicken pox, influenza Medications: -metl dopa, levodopa,  AUTOIMMUNE HA WITH WARM ANTIBODIES Clinic: Laboratory examinations: signs of underlying disease CBC : anaemia + reticulocytosis insidious triadic onset: anaemia, subicterus, Peripheral blood smear: polychromatophilic erythrocytes hyperchromic urine (reticulocytosis), erythrocyte changes: microspherocytes Biochemical markers of haemolysis: BI, LDH, urobilinogenuria rare explosive onset: hyperthermia, adynamia, dyspnoea, pallor, jaundice, +/- haemoglobinuria Bone marrow examination is mandatory: (viral infection) - erythrocyte hyperplasia, haemolysis triggered by stress: surgery, pregnancy, - moderate (immune dysregulation) or pronounced (lymphoproliferative mental overstrain disease) lymphoid infiltration 1/3 of cases splenomegaly.  AIHA WITH WARM ANTIBODIES - diagnostic algorithm - The DAT test or Coombs test is the basis of diagnosis and allows the different forms of autoimmune haemolysis to be distinguished. Warm antibody autoimmune haemolytic anaemia (wAIHA) is the most common form, accounting for 60% to 70% of all cases; DAT is positive with anti-IgG antiserums (70% of all wAIHA) or anti-IgG plus C at low titre. Cold agglutinin disease (CAD; 20% to 25% of all AIHA) is characterized by DAT positivity with anti-C antiserum and high titre cold agglutinin. In mixed forms (5% to 10% of all AIHA), DAT is positive for IgG plus C and cold agglutinins are present in high titre. Atypical forms (~10% of all AIHA) include DAT-, IgA-, and hot-actin autoimmune anemias that are IgM. Finally, the very rare form called paroxysmal cold hemoglobinuria (1% to 3% of all AIHA) supported by biphasic Donath-Landsteiner hemolysin is required.  Coombs Test (DAT) Direct Coombs test: is used to determine if there are autoantibodies (IgG) or complement (C3) on the erythrocyte membrane. The patient's erythrocytes are incubated with antibodies to IgG and C3. If IgG or C3 binds to RBC membranes, agglutination occurs - a positive result. A positive result suggests the presence of autoantibodies in the patient's erythrocytes. If the patient has received a transfusion within the last 3 months, a positive result could also represent alloantibodies in transfused RBCs (which usually occur in acute or delayed hemolytic reaction) Indirect Coombs test: is used to detect IgG antibodies against red blood cells in a patient's serum. The patient's serum is incubated with reactive RBCs; then Coombs serum (antibodies to human IgG or anti-human IgG) is added. If agglutination occurs, IgG antibodies (autoantibodies or alloantibodies) against erythrocytes are present. This test is also used to determine the specificity of an alloantibody.  AIHA WITH HOT ANTIBODIES Treatment: blocking the haemolysis mechanism, the treatment targets being » macrophages - suppression of receptors on macrophages with corticosteroids, Danazol, Ig i.v. » Ac-producing lymphocytes with corticosteroids, cyclophosphamide, azathioprine Prednisone/corticosteroid therapy: attack dose: 1-2 mg/kg/day promising responses in 5-7 days in 80% of cases clinical improvement, Hb increase. doses are maintained 3-4 weeks slowly decreasing doses by 5-10 mg/week to 10-15 mg in 3-4 months from start of treatment. discontinue treatment when Combs test is negative. Remissions are durable in approx. 1/5 of cases. Immunosuppressants (Cyclophosphamide, Chlorambucil): Corticosteroid-resistant forms (rather in AHAI with cold antibodies). Splenectomy: Physically removes macrophages responsible for clearance of auto-Ac coated erythrocytes and a large sample of immunocompetent lymphocytes; neumococcal, meningococcal and haemophilus vaccine before splenectomy.  Clinical: Auto-Ab accumulation in plasma causes vascularity and blood flow disorders vaso-occlusion in territories exposed to cold COLD AGGLUTININ DISEASE paresthesia, acrocyanosis, severe tegumentary changes, Raynaud-like syndrome, trophic tegumentary changes (cadaveric appearance, necrosis) BAR –fenomen Raynaud COLD AGGLUTININ DISEASE  Treatment Primary forms: rest in a warm atmosphere immunosuppression :- Cyclophosphamide 100-200mg/day p.o. - Chlorambucil 2-4mg/day Anti-CD 20 monoclonal antibodies - Rituximab Plasmapheresis Prednisone is not effective (does not influence Kupffer cell activity) Transfusions should be avoided - risk of worsening haemolysis (transfused red blood cells are not "wrapped" in C3d will be rapidly haemolyzed). Perform compatibility tests at 37C, administering small amounts that will be warmed.  Auto-Ac IgG anti P biphasic "Donath-Landsteiner" Cold IgG + Complement + Erythrocyte PAROXYSMAL warm auto-antibodies are released from erythrocytes, but complement-activated molecules remain, activating the intravascular hemolysis attack HAEMOGLOBINURIA AT Direct positive Coombs test with anti complement serum. REFRIGERATION (HPF) Donath-Landsteiner biphasic Ac test: patient's serum incubated on ice 60' with normal erythrocytes in the presence of complement after heating the sample to 37C, hemolysis occurs. PAROXYSMAL HAEMOGLOBINURIA AT REFRIGERATION (HPF) 1-2% of AHAI clinical forms: primary - very rare secondary - occur in children after viral infections (measles, mumps, chickenpox, infectious mononucleosis) and in adults after secondary or tertiary syphilis - attacks of haemoglobinuria triggered by exposure to cold, Treatment: treatment of the underlying disease + warming of the patient transfusions with washed red blood cells Alloimmune extracorporeal haemolytic anaemias HEMOLYTIC ANEMIAS PRODUCED BY ALLOANTIBODIES post-transfusion hemolysis hemolytic disease of the newborn  transfusion incompatibility in the AB0 system occurs at the first contact of the organism with the respective Ag. Post-transfusion haemolysis transfusion incompatibility in the Rh system requires anti Rh immunization, occurs at the second exposure of the body to the respective Ag and is IgG mediated. Incompatibility in the AB0 system is most severe / mediated by alloAb type IgM and complements severe intravascular events: agglutination Agglutination haemolysis activated by C’ DIC sudden onset with extreme anxiety, low back pain, haemoglobinuria, BP, fever, kidney damage (anuria), shock. Rh system reactions are milder and slower: hypothermia/fever, chills. Treatment: stop transfusion, combat shock and DIC, provide osmotic diuresis, corticosteroids, dialysis..  Maternal-fetal incompatibility = haemolytic disease of the newborn (HNB) fetal erythrocytes express erythrocytic Ag inherited from the father, which are missing from the mother's antigenic range passage of fetal erythrocytes across the placenta into the mother's bloodstream => induction of isoimmune Ac synthesis for the mother the process begins in the third month of gestation, and is amplified during labour (especially during obstetric manoeuvres) the most frequent immunisations occur in the AB0 system (2/3 of cases) and Rh (especially D which is highly immunogenic), but also in other systems (Kell, Kidd, Duffy) anti A or anti B immunization occurs exclusively in group 0 mothers anti Rh immunization occurs in Rh negative mothers after pregnancies with Rh positive fetuses firstborns do not get this haemolytic disease after each pregnancy with Rh-positive babies the mother becomes more and more intensively immunised and the next babies get increasingly severe haemolytic disease severe forms cause intrauterine death of fetuses with the appearance of anasarca (hydrops fetalis)- prophylaxis of isoimmunization of the Rh-negative mother. Non-immune extracorporeal haemolytic anaemias Aplastic anaemia  Aplastic anaemia Aplastic anaemia (AA) is a rare condition of bone marrow failure which, if severe and not treated properly, is fatal. AA is characterised by morphological features of the marrow, namely medullary hypocellularity and peripheral cytopenias. The molecular pathogenesis of AA is not fully understood and there is no common cause for all cases of aplastic anaemia. Altered T-cell function and possibly the involvement of autoimmune mechanisms cause damage to hematopoietic stem cells which ultimately determines the pathological features of the disease. Defective telomerase function and repair may also play a role in some cases. Simple chronic anaemia  Moderate anemia (normocytic or microcytic, normochromic or hypochromic) Slightly low sideremia Low or normal TIBC Sequestered iron in medullary macrophages Sideroblasts↓; Chronic disease anemia Serum ferritin N or ↑ Diagnosing a true iron the report is deficiency in a chronically ill Ferritin saturation not useful in patient can be particularly and ferritin change in case of difficult because: opposite directions kidney during iron chronic disease can cause the most useful deficiency, this ratio disease or hyposideremia despite the diagnostic is extremely sensitive hemodialysis, presence of iron in stores parameter to iron metabolism when and seems to be the and can differentiate transferrin can cause an increase in transferrin/ferritin anemia from chronic saturation saturation ratio diseases from true