Dr Ebru Tuğrul Sarıbeyoğlu

1. Hemolytic anemia ( immun/ nonimmun) and Hemorrhagic disease of the newborn Dr Ebru Tuğrul Sarıbeyoğlu

2. Goals: • Define hemolysis • Understand causes of hemolysis • Clinical presentation of hemolysis • Immune and non immune mechanisms of hemolysis • Diagnostic tests for immun hemolysis • pathogenesis, clinical course and treatment of the hemorragic disease of the newborn

4. Hemolysis • Premature!!! (normal RBC survival time:110-120 days) destruction of red blood cells (RBCs) • Anemia results when the rate of destruction exceeds the capacity of the marrow to produce RBCs

6. Evidence of RBCs break down • Serum bilirubin level↑ • Serum haptoglobulin level ↓ • Plasma hemoglobin level ↓ • Urinary uroblinogen ↑ • Hemoglobinuria • Blood smear: red cell fragments (schistocytes, spherocytes, target cells)

7. Evidence of increased erythropoeisis • Reticulocytosis • Increased MCV ( as a result of normoblasts and reticulocyts) • Erythroid hyperplasia of the bone marrow • Chronic: expansion of marrow space

8. Intra/Extravascular hemolysis • The site of hemolysis may be intravascular→ the erythrocyte is destroyed in the circulation, • or extravascular →the red cell destruction occurs within macrophages in the spleen, liver, or bone marrow

10. Intravascular hemolysis is typically severe and results from mechanical damage to the red cell due to prosthetic valves, the presence of fibrin within the vasculature (microangiopathic hemolytic anemia), or thermal injury to the erythrocytes from serious burns; infections or toxins, such as Clostridium perfringens bacteremia, severe falciparum malaria, or certain snake venoms; or complement-mediated damage to red cells, as with paroxysmal nocturnal hemoglobinuria, ABO-incompatible blood transfusions, and cold agglutinins.

11. Laboratory Observations • Laboratory abnormalities seen with hemolysis: • increased RBC destruction, • increased erythropoiesis by the bone marrow, • disease-specific findings. • The unconjugated bilirubin is elevated, accounting for more than 80% of the total bilirubin, and is not excreted in the urine. • Unlike in liver disease, in patients with hemolysis pruritus is usually absent.

12. Intravascular hemolysis liberates hemoglobin into the bloodstream, where it binds to haptoglobin. The haptoglobin/hemoglobin complex is then removed by the liver. A reduced serum haptoglobin level is one of many findings in intravascular hemolysis, but it also occurs in extravascular hemolysis. Haptoglobin is an acute-phase reactant and levels increase in response to inflammation, infection, and malignancy. One needs to be aware of nonhemolytic conditions that can result in low haptoglobin levels (liver disease, hereditary haptoglobin deficiency after red cell transfusions), and normalized haptoglobin levels despite hemolysis (acute phase surges) during work-up of such patients.

13. When the amount of free hemoglobin in the circulation exceeds the binding capacity of haptoglobin, it makes the plasma pink and is filtered through the kidneys. The urine may become red, and urine iron levels increase. The urine proves positive for blood upon dipstick testing in the absence of erythrocytes on urine microscopy (not hematuria!!!!). Other than hemolysis, only in hemochromatosis and nephrotic syndrome can one detect increased urinary iron levels.

16. RBC Membrane Disorders • Hereditary Spherocytosis • Hereditary Elliptocytosis • Acanthocytosis usually seen with chronic liver disease, and is the result of cholesterol accumulation in the red cell membrane. • RBC Metabolic Disorders: Pyruvate Kinase Deficiency, G6PD Deficiency • Wilson’s Disease copper overload. • Congenital Hemoglobinopathies: Sickle Cell Anemia, Hemoglobin C Disease, Hemoglobin SC Disease, Hemoglobin E Disease, Thalassemias

17. Another classification of hemolytic anemias distinguishes between disorders intrinsic to the red cell, generally hereditary, and those extrinsic to the red cell, generally acquired. The intrinsic disorders: abnormal hemoglobins( HbS or HbC), enzyme defects( deficiencies in G6PD), membrane abnormalities (hereditary spherocytosis or elliptocytosis). The extrinsic abnormalities: immunologic—alloantibodies (ABO incompatibility), autoantibodies (warm (IgG) or cold (IgM) antibody hemolytic anemias), drug-induced antibodies, mechanical factors ( trauma from prosthetic valves or fibrin deposition in small vessels, as in microangiopathic hemolytic anemias), infections and toxins( falciparum malaria or certain snake venoms),

18. Most hemolytic disorders are extravascular. The causes of extravascular hemolysis include • infections, • drugs, • immunologic processes • red cell membrane defects, such as hereditary spherocytosis; • erythrocyte metabolic defects, such as deficiencies in pyruvate kinase or G6PD; • hemoglobin structural defects, such as sickle cell anemia or hemoglobin C.

19. Most of the disorders that lead to hemolysis are not specific to any race. It can occur in persons of any age. Most cases are not sex specific. Autoimmune hemolytic anemia is slightly more likely to occur in females than in males. G6PD deficiency is an X-linked recessive disorder. Males are usually affected, and females are carriers. Hereditary disorders are usually evident early in life.

20. Chronic Congenital Hemolytic Anemias Even though there are numerous congenital hemolytic disorders, their clinical features are very similar. Chronic congenital hemolytic anemias are usually characterized by anemia, jaundice, periodic crises, splenomegaly, and black pigment gallstones ( family history!!) Other than during a crisis, symptoms are usually mild to moderate because of compensation by the bone marrow. Chronic symptoms may become severe at times of crisis. (Aplastic crisis with human parvovirus type B19 ,splenic crisis).

21. Acquired Hemolytic Anemias If hemolytic anemia develops acutely, as in hemolytic transfusion reaction or G6PD deficiency, the symptoms may suggest an acute febrile illness with skeletal pains, headache, malaise, fever, and chills. Symptoms of shock, renal failure, jaundice, and anemia may be evident in severe cases.

23. G6PD catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate while concomitantly reducing the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) to nicotinamide adenine dinucleotide phosphate (NADPH). NADPH, a required cofactor in many biosynthetic reactions, maintains glutathione in its reduced form. Reduced glutathione acts as a scavenger for dangerous oxidative metabolites in the cell. With the help of the enzyme glutathione peroxidase, reduced glutathione also converts harmful hydrogen peroxide to water. Red blood cells rely heavily upon G6PD activity because it is the only source of NADPH that protects the cells against oxidative stresses; therefore, people deficient in G6PD are not prescribed oxidative drugs, because their red blood cells undergo rapid hemolysis under this stress

24. Erythrocytes manufacture ATP through glycolysis. A deficiency in pyruvate kinase,which potentiates the last step of glycolysis (phosphoenolpyruvate converted to pyruvate), results in RBCs with decreased energy.The events leading to hemolysis are still not well understood, but it seems that the lack of ATP impairs the Na+/K+-ATPase and other ATP dependent processes leading to a cellular loss of K+ and water. This causes rigidity of the RBC and eventual splenic hemolysis

25. Clinical presentation depends on whether the onset of hemolysis is gradual or abrupt and on the severity of erythrocyte destruction. A patient with mild hemolysis may be asymptomatic. In more serious cases, the anemia can be life threatening, The clinical presentation also reflects the underlying cause for hemolysis.

26. A patient with mild hemolysis may have normal hemoglobin levels if increased production matches the rate of erythrocyte destruction. Skull and skeletal deformities can occur with a marked increase in hematopoiesis, expansion of bone in infancy, and early childhood disorders such as sickle cell anemia or thalassemia.

27. Blood smear is the single most valuable test in defining the underlying disorder causing hemolysis. Spherocytes are the hallmark of hereditary spherocytosis, sickle cells of sickle cell anemia, target cells of thalassemia, schistocytes of RBC fragmentation, erythrophagocytosis of red cell surface damage by complement-fixing antibodies and infections, auto agglutination of cold agglutinin disease, and elliptocytes of hereditary elliptocytosis. The bone marrow usually shows erythroid hyperplasia.

28. IMMUNE HEMOLYSİS • Autoimmune hemolytic anemia (AIHA) is caused by autoantibodies that react with RBCs at temperatures ≥ 37° C (warm antibody hemolytic anemia) or < 37° C (cold agglutinin disease). • Hemolysis is usually extravascular.

29. AIHA is diagnosed by detection of autoantibodies with the direct antiglobulin (direct Coombs') test. Antiglobulin serum is added to washed RBCs from the patient; agglutination indicates the presence of immunoglobulin or complement bound to the RBCs. Generally IgG is present in warm antibody hemolytic anemia, and C3 (C3b and C3d) in cold antibody disease. The test is ≤ 98% sensitive for AIHA; false-negative results can occur if antibody density is very low or if the autoantibodies are IgA or IgM. In general, the intensity of the direct antiglobulin test correlates with the number of molecules of IgG or C3 bound to the RBC and, roughly, with the rate of hemolysis.

30. Coombs’ test The Coombs' test looks for antibodies that may bind to your red blood cells and cause hemolysis. There are two forms of the Coombs' test: direct and indirect.

31. The direct Coombs' test is used to detect antibodies that are already bound to the surface of red blood cells. Many diseases and drugs (quinidine, methyldopa, and procainamide) can lead to production of these antibodies. These antibodies sometimes destroy red blood cells and cause anemia. The direct antiglobulin test detects the presence of antibodies and complement on erythrocytes by using a reagent that contains antibodies directed against human immunoglobulin and complement components (primarily C3). This test is nearly always positive in warm AHA, but when IgG is present in very small quantities, other diagnostic techniques may be necessary to detect them.

33. A positive direct Coombs' test means you have antibodies that act against your red blood cells. This may be due to: Autoimmune hemolytic anemia without another cause Chronic lymphocytic leukemia or other lymphoproliferative disorder Drug-induced hemolytic anemia (many drugs have been associated with this complication) Erythroblastosis fetalis (hemolytic disease of the newborn) Infections: infectious mononucleosis, Mycoplasmal infection, Syphilis Systemic lupus erythematosus or another rheumatologic condition Transfusion reaction, such as one due to improperly matched units of blood

34. Autoantibodies unattached to erythrocytes may be present in the serum and are detectable by incubating the patient’s serum or plasma with normal red cells, to which the antibodies then attach. These erythrocytes are then tested for the presence of autoantibodies with the Coombs reagent. This is the indirect antiglobulin or Coombs test. The indirect Coombs' test looks for unbound circulating antibodies against a series of standardized red blood cells. The indirect Coombs' test is used to determine whether a person might have a reaction to a blood transfusion.

36. A positive indirect Coombs' test means you have antibodies that will act against red blood cells your body views as foreign. This may suggest: Autoimmune or drug-induced hemolytic anemia Erythroblastosis fetalis hemolytic disease Incompatible blood match (when used in blood banks)

37. Warm antibody hemolytic anemia:Warm antibody hemolytic anemia is the most common form of AIHA; it is more common among women. Autoantibodies in warm antibody hemolytic anemia (AHA), generally react at temperatures ≥ 37° C. In AHA, RBC life span decreases because autoantibodies (usually polyclonal IgG) are optimally active against erythrocytes at body temperature. These warm antibodies account for about 80% to 90% of acquired AHA, and only about 10% are caused by antibodies maximally active at lower temperatures (cold-reactive autoantibodies).

38. Immune Hemolysis Warm-Antibody Acquired AHA In warm antibody AHA, hemolysis occurs primarily in the spleen. It is often severe and can be fatal. Most of the autoantibodies in warm antibody hemolytic anemia are IgG. In about one-half of cases, an underlying disorder is present—most commonly a lymphoproliferative disease such as chronic lymphocytic leukemia or lymphoma, but also systemic lupus erythematosus, other inflammatory conditions, some infections (cytomegalovirus), and nonlymphoid malignancies. Certain drugs can also play a role in the development of these warm antibodies and hemolysis: levodopa (Dopar, Larodopa) and penicillin can cause IgG, and quinidine can cause IgM-type antibodies and AHA.

39. In warm AHA, IgG coats many red cells with or without complement. Macrophages in the spleen and Kupffer cells in the liver trap these erythrocytes, sometimes ingesting them whole. The reticulocyte count is usually increased, as are the serum indirect bilirubin and LDH levels.

40. Cold Agglutinin Disease Cold agglutinins are IgM antibodies that bind red cells at cold temperatures. Nearly all healthy people have low titers of clinically insignificant cold agglutinins. Cold AHA can be idiopathic or secondary (infection, lymphoma). EBV and Mycoplasma pneumoniae are the two most common causes; Hemolysis is usually intravascular and in the liver. Agglutination is seen when blood is cooled. In certain infections, transient, high titers of cold agglutinins appear, causing the abrupt onset of an anemia that is short-lived, but occasionally severe. Cold agglutinin disease is mediated by a complement-fixing monoclonal IgM antibody either in an acute (mycoplasma or Epstein-Barr virus infection) or chronic setting (lymphoproliferative disorders).

41. Cold Agglutinin Disease In cold agglutinin disease, RBCs clump on the peripheral smear, and İn addition to anemia, the automated blood count often reveals an increased mean corpuscular hemoglobin concentration (MCV), reflecting the presence of the spherocytes. and spuriously low Hb due to such clumping; hand warming of the tube and recounting result in values significantly closer to normal. Warm antibody hemolytic anemia can often be differentiated from cold agglutinin disease by the temperature at which the direct antiglobulin test is positive; a test that is positive at temperatures ≥ 37° C indicates warm antibody hemolytic anemia, whereas a test that is positive at lower temperatures indicates cold agglutinin disease.

43. Hemorrhagic Disease of Newborn • Vitamin K is an essential cofactor for γ -glutamyl carboxylase enzymatic activity that catalyses the γ -carboxylation of specific glutamic acid residues in a subclass of proteins.3 These vitamin K–dependent proteins are known as Gla-proteins. Media file 1 outlines the vitamin K cycle.

44. Hemorrhagic Disease of Newborn • Coagulation factors II, VII, IX, and X and other Gla-proteins (eg, protein C, protein S, protein Z) also depend on the presence of vitamin K for their activity. • In vitamin K deficiency, des-carboxylated proteins are formed that are functionally defective because they can not bind calcium and phospholipid. These abnormal coagulation factors are called protein-induced by vitamin K absence (PIVKA). PIVKA-II is des-carboxylated prothrombin.

45. Currently, the following 3 forms of vitamin K are known: K1: Phylloquinone is predominantly found in green leafy vegetables, vegetable oils, and dairy products. Vitamin K given to neonates as a prophylactic agent is an aqueous, colloidal solution of vitamin K1. K2: Menaquinone is synthesized by gut flora. K3: Menadione is a synthetic, water soluble form that is no longer used medically because of its ability to produce hemolytic anemia.

46. Vitamin K supplementation given after the birth for early onset vitamin K deficiency bleeding may be too late to prevent this disease, especially if vitamin K supplementation was not provided during pregnancy. • Numerous maternal medications and/or exposure to toxins during pregnancy are associated with vitamin K deficiency bleeding in neonates (eg, anticonvulsants [eg, phenytoin, barbiturates, carbamazepine], antitubercular drugs [eg, rifampin, isoniazid], vitamin K antagonists [eg, warfarin, phenprocoumon]).

47. Hemorrhagic Disease of Newborn • Newborn infants are at risk of developing vitamin K deficiency, and this coagulation abnormality leads to serious bleeding. • Placental transfer of vitamin K is very limited, and phylloquinone (vitamin K1) levels in umbilical cord blood is very low. • the storage of vitamin K in neonatal liver is also limited. • This makes the newborn infant uniquely vulnerable to hemorrhagic disorders unless exogenous vitamin K is given for prevention of bleeding immediately after birth.Once the infantile gut is colonized with bacterial flora, the microbial production of vitamin K results in a lower risk of infantile vitamin K deficiency bleeding. • A gut-related microbial source of vitamin K is particularly important if dietary phylloquinone is restricted.

48. Hemorrhagic Disease of Newborn • The newborn infant’s intestinal tract is relatively sterile and takes some time to colonize with bacteria, which may have a role in synthesizing vitamin K2 (menaquinones). • Because Bacteroides species are among the most common bacteria that inhabit the human intestinal tract, and because strains such as Bacteroides fragilis synthesize vitamin K, Bacteroides species are more significant in producing human vitamin K in the intestine than Escherichia coli.

49. Hemorrhagic Disease of Newborn • Breast milk is a poor source of vitamin K (breast milk levels are 1-4 μ g/L). The recommended dietary intake of vitamin K is 1 μ g/kg/d. • Breastfed infants have intestinal colonization with lactobacilli that do not synthesize vitamin K; thus, reduced production of menaquinones increases the neonatal risk of developing a hemorrhagic disorder if not supplemented with vitamin K. • Formula-fed infants have higher fecal concentrations of vitamin K1 because of dietary intake and significant quantities of fecal menaquinones, reflecting the gut’s microflora. Preterm infants who are receiving total parenteral nutrition (TPN) are not at risk because they are receiving vitamin K via the multivitamin additive to the TPN.

50. Hemorrhagic Disease of Newborn vitamin K deficiency bleeding is usually classified by 3 distinct time periods after birth, • Early-onset vitamin K deficiency bleeding in the newborn • Classic vitamin K deficiency bleeding in the newborn • Late-onset vitamin K deficiency bleeding in the newborn