Hemolytic Anemia

General stuff Hemolytic anemia occurs when there is increased destruction of RBCs, and leads to: elevated EPO (kidney tries to compensate increase RBC production), hyperactive marrow (also tries to throw out more RBCs), extramedullary hematopoiesis (bone marrow production in places besides bone marrow again trying to make more blood cells, most often in reticular endothelial system, liver, spleen). Can find reticulocytosis (immature blood cells in peripheral blood), and as RBCs are destroyed billirubin and lactate dehydrogenase (LDH) can be found in the blood (products of hemoglobin destruction). Histology

Anisopoikilocytosis Large cells (arrows) And abnormal shapes

Reticulocytosis w/ reticulin stain can see RNA inside See reticulocytes

Hypercellular marrow Bone marrow aspirate Dark, round nuclei are RBC precursors, see lots of them

Morphology changes: Pigmented gallstones, jaundice, splenomegaly Hemolytic anemias can be categorized in three ways: Intravascular vs. Extravascular Intracorpuscular (intrinsic, inside the RBC) vs. Extracorpuscular (extrinsic, outside) Hereditary vs. Acquired (main way used in the lecture)

Extravascular hemolysis Mechanism RBC can usually slip through endothelium, but with membrane abnormalities they cant do it (they get stuck and/or break on the way through). Blue thing is a macrophage dealing with RBC fragments. Hereditary anemias are all intrinsic and are caused by hemoglobin defects (sickle cell, thalassemia), membrane defects (HS), or enzyme defects (glucose6-phosphate dehydrogenase, G6PD). Acquired anemias are intrinsic (paroxysmal nocturnal hemouria = PNH) or extrinsic (immune, trauma, infection like malaria, toxins like lead)

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Hemolytic Anemia
Hemoglobin Defects Biochem review of structure: 2 alpha, 2 beta chains. Some variation in chains used based on age (always have alpha chain, but fetus uses gamma instead of beta until a few months after birth = HbF). Hb problems because of: structural abnormalities (sickle cell) or deficiency in a chain (thalassemia)

Sickle Cell Anemia Organize and be able to discuss sickle cell anemia in terms of incidence, clinical features, etiology, pathogenesis, morphology of bone marrow, peripheral smear and spleen, clinical presentation, laboratory diagnosis and complications/clinical course. Trait in 8% of African Americans, is usually silent (no symptoms), provides malaria resistance, is in 30% of native Africans. Homozygous have Hb at 6-8 g/dL (normal is 12 g/dL or higher) Pathogenesis: point mutation, have valine instead of glutamic acid @ 6th amino acid on beta chain forms globin polymers when RBC is deoxygenated. Usually forms sickle-shapes in microvascular beds, where blood flow is slow (spleen/marrow/inflamed areas) ischemic tissue damage. Spleen HbS cant get through splenic sinusoids trapped congestion. In kids: congestion splenomegaly; can lead to sequestration crisis if there are too many cells stuck there hypovolemia/shock (emergency). In adults: congestion vascular stasis, infarction/fibrosis of spleen and eventually autosplenectomy (spleen is really small, and doesnt really function, treat these patients like they dont have one). Clinical course and complications: Hemolytic anemia (the topic of the lecture), infection (decreased spleen function, cant fight encapsulated bacteria need vaccines). Sequestration crisis shock, vaso-occlusive crises depending on which tissue it occurs in (bone pain or dactylitis in kids, lungs acute chest syndrome, penis priapism, CNS seizures/strokes, skin ulcers). Also aplastic crises (acute worsening of anemia triggered by parvovirus B19), and impaired growth & development from chronic hypoxiaalmost all organs get involved (in addition to stuff already mentioned: liver cholestasis, gallstones; cardiac high output, pulmonary hypertension; eyes retinal vessel occlusion, neovascularization; pregnancy maternal mortality @ 1.5%) Diagnosis: Look @ smear see sickle cells, or sickle solubility test (screening), treat with reducing solution, HbS causes turbid solution so you cant see the black lines in the back as well (normal on left, positive on right), can ID trait and homozygotes

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Hemolytic Anemia
Metabisulfite slide test (screening): Blood w/ oxygen consuming reagent induce sickling (remember, the globin polymers form when deoxygenated), also ID trait/homozygous. Electrophoresis (for confirmation) Hb negatively charged, different chains have different charges, can spread em out on a gel. Other confirmation tests: Stability testing, DNA sequencing (these werent emphasized in lecture) Management: folate, vaccinations (cause of spleen dysfunction), eye exams (cause of poor circulation), hydroxyurea, bone marrow transplant HbC has target cells. C the target, picture right here

Thalassemia: Organize and be able to discuss the thalassemia disorders in terms of genetics - molecular changes, pathogenesis, clinical features, morphology of peripheral smear, spleen, liver, bone, and bone marrow, laboratory diagnosis, and clinical course/complications. Decreased production of a chain insufficient HbA and too much unpaired globin chains. Alpha thalassemia deficient synthesis of alpha chains; beta thalassemia deficient beta chain synthesis. Prevelant in Mediterranean, Africa, SE asia. Protecs vs. malaria. On the CBC: Inc. RBC, low MCV, low Hct, normal RDW. Mentzer index = MCV/RBC ratio, if index < 13 probably thalassemia, if >13 probably iron deficiency Beta Thalassemia. 2 beta globin genes, on chromosome 11. More severe than alpha excess unpaired alpha chains precipitate membrane abnormalities hemolysis, ineffective erythropoiesis, EMH and excess absorption of iron; splenic sequestration B0 = no beta-globin synthesis B+ = reduced, but still there, beta-globin synthesis Molecular cause for each: usually point mutations Most severe is B-thal major, least sever is minor

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Hemolytic Anemia
Clinical Manifestations B-thal major patients are transfusion dependent

Alpha Thalassemia. Decreased alpha chains, relative excess of gamma (fetus/infant) and beta globins, which can form tetramers. Hb-Barts = gamma tetramer, HbH = beta. Beta and gamma chains are more soluble than alpha chains alpha thal less severe than beta. Symptoms vary based on number of genes affected. Molecular basis: usually gene deletion. nRBCs are RBC w/ nuclei Membrane Defects: Organize and be able to discuss hereditary spherocytosis in terms of genetics, pathogenesis, clinical presentation, morphology of peripheral smear and spleen, laboratory diagnosis and clinical course-complications.

Need spectrin, actin, protein 4.1 for good membrane structure. In hereditary spherocytosis (absent central pallor and more spherical than usual). Cells less deformable vulnerable to splenic sequestration/destruction Hereditary Elliptocytosis cells in an ellipse shape

Enzyme deficiencies: Compare and be able to discuss G-6PD, paroxysmal nocturnal hemoglobinuria, mechanical hemolytic anemia and malaria in terms of clinical features, pathogenesis and laboratory diagnosis.

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Hemolytic Anemia
G6PD Enzyme needed for hexose-monophosphate shunt, which makes glutathione (an antioxidant, protective vs. cell injury). Is X-linked. Presentation: acute hemolytic anemia from oxidant stress (via drugs, infections, or fava beans). Prevelance highest in Africa & Mediterranean (gives a bit of malaria protection). Pathogenesis: Oxidative stress in RBC Hb denatures/precipitates Heinz bodies form direct RBC membrane damage (intravascular hemolysis) AND spends longer time in spleen bite cells. Clinical features: Self-limiting (from food/drugs, stop them and it goes awaydont eat fava beans), RBCs without enough G6PD die, younger ones make sufficient enzyme survive hemolysis stops. Since it isnt chronic no plenomegaly/gallstones. Paroxysmal nocturnal hemoglobinuria (PNH) is acquired, intrinsic hemolytic anemia. Its a stem-cell disorder where erythrocytes cant prevent complement-mediated hemolysis, affects RBCs, WBCs and platelets. Genetics: mutations in PIGA (enzyme needed for GPI-linked proteins). Causes deficiency in CD55 (DAF), CD59 (MIRL), or C8 binding protein increased complement sensitivity. In RBCs: intravascular hemolysis, chronic hemolysis w/o dramatic hemoglobinuria really only paroxysmal and nocturnal in 25% of patients In platelets: venous thrombosis leading disease-related death In WBCs: MDS/AML (5-10% of pts) Together

Autoimmune hemolytic anemia. Compare and be able to discuss warm and cold antibody immunohemolytic anemias in terms of etiology, pathogenesis, associated risks-diseases, laboratory diagnosis and complications.

Warm. IgG binds RBC @ body temp. IgG RBC recognized by splenic macrophages engulfed/destroyed. Spherocytes formed (picture to the left here). Can recognize treat w/ steroids decrease anemia. Lab tests: Coombs test. Direct = antisera for human IgG/complement added to pt RBC or Indirect = patient serum added to defined test RBCs. Positive if theres agglutination. Treatment: prednisone, danazol, immunosuppressive therapy, anti-CD20 antibody (rituximab), splenectomy

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Hemolytic Anemia
Cold. IgM binds RBCs, fixes complement intravascular hemolysis, or IgM recognized @ liver via Kupffer cells destroy the RBCs. Associated with: mycoplasma, lymphoid neoplasms, idiopathic. Can see RBC agglutination @ right here. Occurs most often @ cold body temp (affects extremities like fingers/toes/nose/ears most). Treatment: keep patient warm, anti-CD20 antibody. Extrinsic anemia: Microangiopathic hemolytic anemia. Red cells pass through fibrin strands and rupture. Associated w/ conditions like TTP/HUS, DIC, hypertension. Prosthetic heart valves can cause shearing blood cells. Leads to schistocytes (triangle-shaped cell, doesnt have pointy ends like sickle-cell though) Other Objectives In both of these remaining objectives, theyre asking you to know about the diagnosis/differentiation of each of the hemolytic anemias discussed based on lab tests/symptoms/disease progression. Theyre okay review questions. Categorize and be able to discuss laboratory test procedures used in the diagnosis of hemolytic anemias. List normal values and expected abnormal values of these laboratory test for the different types of hemolytic anemias (sickle cell anemia, beta thalassemia, alpha thalassemia, G-6PD, PNH, hereditary spherocytosis, immunohemolytic anemias, malaria and hemolytic anemia secondary to mechanical trauma. Compare and be able to discuss iron deficiency anemia, megaloblastic anemia, sickle cell anemia, thalassemia, aplastic anemia, hereditary spherocytosis, and anemia of acute blood loss in terms of etiology-pathogenesis, clinical manifestations and laboratory features. Be able to differentiate these diseases based on clinicopathologic data. Anemias not really mentioned in the lecture include anemia secondary to mechanical trauma, which is caused by shearing stress on RBCs from repetitive motion. Examples include marathon running, long marches, and (according to Wikipedia) bongo drumming. Microangiopathic hemolytic anemia is also an example of mechanical trauma since the RBCs physically break themselves, usually against prosthetic heart valves. Dr. Kahns 3 minute summary (about 55 minutes into the lecture on tegrity): With RBC destruction, determine congenital vs. acquired. If congenital, can be HbS (sickle cell) or HbC, membrane structure abnormality (spherocytosis), enzyme problem (G6PD, which is x-linked so almost always in men), or thallasemia. If acquired, then its immune or non-immune. Immune: antibodies vs. RBCsis warm (IgG, has spherocytes) or cold (IgM, occurs more in colder locations/seasons, see agglutination on smear). Nonimmune: PNH, Microangiopathic hemolytic anemia, RBCs break apart on fibrin strands, clostridia/malaria also can cause hemolysis.

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