IHO: Anemias (Dr. McCary - 4 hrs)

Hemolytic Anemia - Sickle Cell Disease, Clinical Outlook/Treatment

90% survive to age 20, 50% survive beyond the 5th decade Treatment - hydroxyurea - inhibitor of DNA synthesis -Increases HbF by unknown mechanism -Anti-inflammatory effect - reduced leukocyte production Decreases crises in children and adults ?Cure - hematopoietic stem cell transplantation

Anemia - Definition and Categories

Anemia is defined as a reduction of the circulating red blood cell mass below normal limits -Measured by hemoglobin and hematocrit -Reduced oxygen carrying capacity results -Tissue hypoxia 3 broad categories of anemia -Blood loss anemia - acute or chronic -Hypoproliferative anemia -Hemolytic anemia

Hemolytic Anemia - Immunohemolytic Anemias - Warm Antibody Type, Peripheral Smear

Anisiocytosis Polychromasia Many spherocytes from partial loss of membrane by attempted phagocytosis of IG coated RBC

Anemia of Diminished Erythropoiesis -Megaloblastic Anemia - B12 Deficiency, Mechanism of Intake of B12

B12 bound to protein in food Pepsin frees B12 in the stomach B12 binds haptocorin from saliva Pancreatic proteases release B12 B12 binds intrinsic factor Ileal epithelium bind the B12/IF complex through IF receptor B12 binds transcobalmin II and secreted into plasma B12 transport to bone marrow, and GI epithelium (rapidly dividing cells) and liver

E) Increased RDW

A 52 year old female patient is on α-methyl dopa for hypertension and presents with fatigue. Her HgB is 8 g/dl and her Hct is 22% with the peripheral smear below. Her DAT is positive consistent with immune hemolytic anemia. Which is the most likely alteration of her RBC indices to be found? A) Decreased reticulocyte count B) Decreased RDW C) Decreased MCHC D) Increased MCV E) Increased RDW

D) Serum B12 and Folate (Look at the 7-segment portion)

A 58 year old woman presents with fatigue and you notice pale conjunctiva. Her HgB is 7.6g/dl, and her MCV is 125. The smear is shown below. What additional next test would be most helpful? A) Bone marrow biopsy B) Serum protein electrophoresis C) Serum ferritin D) Serum B12 and Folate E) DAT

RBC morphologic changes in most common anemia

A: Normal B: Rouleaux increased protein C: Agglutination - antibody mediated D: Polychromasia - RNA, reticulocyte E: Basophilic stippling - Immature vs lead F: Hypochromic microcytic RBC (iron) G: Macroovalocyte (folate/B12) H: Microspherocytes (HS) I: Eliptocytes (HE) J: RBC fragments - thermal injury/burn pt K: Nucleated RBC L: Howell-Jolly bodies (spleen absence) M: Pappenheimer bodies (sideroblastic anemia) N: Cabot ring (megaloblastic/MDS) O: Malaria rings P: Schistocyte (MAHA) Q: Tear drop (Marrow fibrosis) R: Echinocyte (Burr cell) liver disease S: Acanthocyte (spur cell) T: Bite cell (G6PDD) U: Sickle cell V: Hemoglobin C crystal W: target cell (Thalasemaia) X: Hemoglobin C Y: Heinz body (G6PDD)

Hemolytic Anemia - Glucose-6-Phosphate Dehydrogenase Deficiency

Abnormality in hexose monophosphate shunt or glutathione metabolism due to impaired enzyme function lead to reduced protection from oxidative injury and lead to hemolysis Hereditary deficiency of G6PD activity - the most common human enzyme defect, more than 400-500 million people worldwide, most asymptomatic -X-linked recessive trait -Males at higher risk -Hundreds of G6PD variants exist, most harmless -2 cause most of the clinically significant cases, G6PD-, and G6PD Mediterranean Part of the pathway for reduction of H2O2 Protective effect in malaria patients (Plasmodium falciparum) -However hemolysis has been observed with malaria drugs since the 1920's -Only older red cells were hemolyzing Most common forms -G6PD A- present in 10% of African Americans (90% of African cases) -G6PD Mediterranean prevalent in the Middle east Mutation results in misfolding of the protein -Half life of the protein is markedly reduced -No protein synthesis in RBC -Enzyme levels fall quickly below what is protective from oxidative stress -Older RBC more susceptible than younger RBC Episodic hemolysis characteristic due to exposures increasing oxidant stress A) Infection - oxygen-derived free radicals produced by leukocytes -Viral hepatitis, pneumonia, typhoid fever most common B) Drugs -Antimalarial, sulfonamides, nitrofurantoin C) Foods -Fava bean, generates oxidants when metabolized -Favism more common with G6PD Mediterranean than G6PD A-

*Blood Loss: Differentiate between the pathophysiology of acute and chronic blood loss*

Acute - effects due to loss of intravascular volume, if massive cardiovascular collapse, shock, death A) If patient survives, intravascular volume is replaced by shift of interstitial fluid, diluting blood and lowering hematocrit -Normochromic normocytic anemia -Decreased oxygenation triggers erythropoietin release stimulating RBC production -5 days until new RBC (reticulocytes) are released from the marrow B) Internal bleeding - iron recaptured C) External bleeding, vaginal bleeding, or lost through GI tract - iron is lost, iron deficiency can hamper recovery Chronic A) Anemia results when rate of loss exceeds rate of synthesis B) Rate of synthesis can be compromised by iron deficiency and Iron deficiency anemia may result -Microcytic hypochromic anemia -GI tract most common source of loss

Hemolytic Anemia - Immunohemolytic Anemias - Cold Agglutinin Type, Peripheral Smear

Aggregation and clumping of RBC is characteristic Cold agglutinin antibodies always autoantibodies

Pernicious Anemia

All racial groups, more prevelent in Scandinavian/Caucasian populations Older adults - age 60 is median Genetic predisposition suspected, family history common Antibodies against multiple self antigens A) Type I antibody - blocks binding of B12 to IF, in plasma and gastric juice B) Type II antibody - prevent binding of B12-IF to IF receptor - cubulin C) Type III antibody - recognize α and β subunits of the gastric parietal cell proton pump, not specific, also seen in 50% of adults with idiopathic gastritis not associated with pernicious anemia Autoantibodies are diagnostically useful but not causative of gastric injury Autoreactive T-cell response initiates gastric injury triggering formation of antibodies When intrinsic factor secreting cells fall below a threshold, anemia develops as B12 reserves are depleted Commonly associated with other autoimmune processes - thyroiditis and adrenalitis Associated findings A) Stomach - atrophic gastritis, nearly absent parietal cells, Megaloblastic epithelial cells B) Atrophic glossitis - shiny, glazed, red tongue C) CNS lesions in ¾ of florid cases -Demyelination of dorsal and lateral spinal cord tracts leading to spastic paraperesis, sensory ataxia, paresthesia

Hemolytic Anemia - Hereditary Spherocytosis

An inherited disorder due to intrinsic defect or RBC membrane cytoskeletal protein rendering the RBC spherical, less deformable, and vulnerable to splenic sequestration and destruction 75% autosomal dominant Various mutations in alpha spectrin, beta spectrin, band 4.2, band 3 weakening the RBC cytoskeleton RBC lifespan decreases from 120 days to 10-20 days Young cells normal in shape but destabilized membrane results in membrane shedding and formation of spheres Spheres are poorly deformable Clinical Features - Anemia, splenomegaly, jaundice Consider HS with unexplained splenomegaly, unexplained and unconjugated hyperbilirubinemia, and gallstones at a young age; transient anemia with acute with infection; or severe anemia with pregnancy Diagnosis based on family history, hematologic findings, laboratory tests HS red cells have increased MCHC due to loss of membrane, K+, and H2O Severity highly variable -20-30% nearly asymptomatic -Most have a mild to moderate chronic anemia -Generally stable +Aplastic crisis may be triggered by acute parvovirus infection which kills erythrocyte progenitors +No red cell production for 1-2 weeks until immune response, may need transfusion bridge -Hemolytic crisis - e.g. infectious mononucleosis and increased RBC destruction Splenectomy treats anemia but results in increased infection risk -Pneumococcus, heamophillus, and meningococcal immunization pre-splenectomy

Anemia of Diminished Erythropoiesis - Iron Deficiency Anemia, Clinical Manifestations (Summary)

Anemia - usual nonspecific findings Iron deficiency specific due to long standing deficiency and depletion of other iron-containing enzymes -Koilonychia -Alopecia -Atrophic change of tongue and gastric mucosa -Loss of CNS iron may lead to pica (consume non food substances -soil, clay) Plummer-Vinson Syndrome - triad of microcytic hypochromic anemia, esophageal web, and atrophic glossitis

Anemia of Diminished Erythropoiesis - Megaloblastic Anemias, Diagnosis

Biochemical testing, measure B12, folate and metabolites in stepwise process - metabolites may increase with renal failure B12 -Deficiency perturbs methionine synthase activity leading to substrate buildup (homocysteine) -Deficiency perturbs methylmalonyl-CoA mutase leading to elevated MMA levels -Elevated serum homocysteine and MMA are sensitive indicators of B12 deficiency Folate -Deficiency elevates homocysteine, normal MMA

Hemolytic Anemia - Sickle Cell Disease, Morphologic Manifestations

Blood -Variable numbers of irreversibly sickled cells -Reticulocytosis -Target cells from red cell dehydration -Nuclear remnants in some RBC (Howell-Jolly bodies) Spleen -Early childhood - enlarged to 500 gm - congestion -Chronic erythrostasis leads to splenic infarcts -Fibrosis -Atrophy -Autosplenectomy Bone marrow - compensatory erythroid hyperplasia -Secondary bone resorption and new bone formation Extramedullary hematopoiesis Bilirubinemia and gallstones

Hemolytic Anemia - Glucose-6-Phosphate Dehydrogenase Deficiency, Intravascular or Extravascular Hemolysis?

Both intravascular and extravascular hemolysis occur! Globin chain crosslinking of reactive sulfhydryl groups leads to denaturation and precipitation forming membrane bound precipitates called *Heinz bodies*. Direct membrane damage leads to intravascular hemolysis Spherocytes form due to loss of membrane area Spleen removes Heinz bodies as RBC pass through - bite cells Acute intravascular hemolysis begins 2-3 days following exposure to oxidants G6PD Mediterranean affected more severely than G6PD A- G6PD A- cells have only 5-15% of normal G6P activity which declines with age of RBC, and older RBC severely deficient Self limited as only older cells at risk, and lysed cells replaced by younger more resistant cells Recovery phase marked by reticulocytosis appears ~5 days Due to intermittent nature, splenomegaly and cholelithiasis absent

A patient presents with anemia, no hemoglobinuria, no hemosiderinuria and the presence of spherocytes on the peripheral smear. A sample of his blood is tagged with Cr51 and reinjected. Where would the greatest degree of radioactivity be found? A) Bone marrow B) Lymph node C) Spleen D) Liver E) Kidney F) Bladder

C) Spleen

Hemolytic Anemia - Immunohemolytic Anemias

Caused by antibodies that bind to RBCs leading to premature destruction Primary - idiopathic Secondary - autoimmune, infection, drug or neoplasm Classified based on antibody type -Warm Antibody type (70-80% of AIHA)- IgG active at 37C - Warm Antibody Hemolytic Anemia - usually polyclonal -Cold Agglutinin type - IgM active below 37C - Cold Antibody Hemolytic Anemia, bind at low temperature and cause lysis above 22C - usually clonal -Cold Hemolysin type - IgG active below 37C (AKA Donath-Landsteiner antibody) Paroxysmal Cold Hemoglobinuria - rare - polyclonal

Hemolytic Anemia - α-Thalassemia, Genetics

Clinical Syndromes determined and classified by number of genes deleted Each gene contributes 25% of α-globin chains A) Silent carrier - 1 gene deleted, asymptomatic, slight microcytosis B) Α-thalassemia trait - 2 gene deletions, same (α α /--) or different chromosomes (α-/α-), microcytosis, minimal anemia, no physical manifestations C) Hemoglobin H disease - deletion of 3 globin genes, β tetramers form -HbH high oxygen affinity, poor oxygen delivery -HbH precipitates facilitating splenic phagocytosis - extravascular hemolysis D) Hydrops fetalis - deletion all 4 α-globin genes - fatal in utero or shortly after birth. -Almost exclusively in Asians - Chinese, Cambodians, Thai, and Filipinos -Anasarca, anemia, hepatosplenomegaly -Hemoglobin Bart high oxygen affinity - poor delivery -Intrauterine transfusion and lifelong transfusion dependence - iron overload +Limb and urogenital defects, developmental delay, neurologic defects -Hematopoietic stem cell transplant curative

Hemolytic Anemia - Sickle Cell Disease, Diagnosis

Clinical findings Irreversibly sickled cells Testing A) Mixing blood with oxygen consuming agent to induce sickling if HbS is present B) Hemoglobin Electrophoresis C) Prenatal diagnosis by amniocentesis or chorionic biopsy

Hemolytic Anemia - Sickle Cell Disease

Common hereditary hemoglobinopathy caused by point mutation in β-globin, leading to polymerization of deoxyhemoglobin with several clinical consequences -red cell distortion -hemolytic anemia -microvascular obstruction -ischemic tissue damage Heterozygote (Trait) in 8-10% of African Americans ~2 million individuals Trait as high as 30% in some areas of Africa Homozygote (Disease) ~70,000 in USA High frequency due to HgS protective effect against malaria (falciparum) The sickle mutation has arisen independently at least 6 times in areas endemic for falciparum malaria - Darwinian selection Parasite density lower and malarial disease less severe in AS children than AA -Intracellular parasite consumes oxygen and decreases intracellular pH promoting sickling and more rapid clearance by splenic phagocytes -Sickling impairs formation of RBC membrane knobs containing a parasite derived protein involved in infected RBC adhesion to endothelium, a pathogenetic step in cerebral malaria

Anemia of Diminished Erythropoiesis - Iron Deficiency Anemia, Diagnosis

Depressed hemoglobin and hematocrit Hypochromic and microcytic -Low MCV, MCH, MCHC Low serum iron and ferritin High plasma iron binding capacity (reflects elevated transferrin) Transferrin saturation less than 15% Reduced iron inhibits hepcidin synthesis and serum level is low Oral iron produces reticulocytosis in 5-7 days followed by recovery in uncomplicated deficiency

Hemolytic Anemia - Immunohemolytic Anemias, Diagnosis

Diagnosis - detection of antibody +/or compliment on the patients RBC surface *Direct Coombs antiglobulin test (DAT)* - Patient RBC incubated with antibodies specific for human immunoglobulin or compliment. If immunoglobulin or compliment is present on the patients RBC, clumping of cells occurs Indirect Coombs antiglobulin test - patients serum is mixed with test RBC bearing defined antigens. This defines the antigen target and temperature dependence of the antibody

Your patient presents with fatigue and a 10 year history of Crohn's disease. You find a normocytic normochromic anemia. His serum iron and iron binding capacity are low, but his ferritin is elevated. Which of the following may best treat his anemia? A) Blood transfusion B) Oral iron C) Parenteral iron D) Prednisone E) Erythropoietin

E) Erythropoietin (Blood transfusion also plausable) (Prednisone also, treat underlying disease)

Hemolytic Anemia-Reticulocyte

Evidence of marrow RBC production Recently released RBC still contain RNA - polychromasia/basophilic stippling Reticulocyte stain precipitates the RNA All polychromatic cells are reticulocytes Not all reticulocytes are polychromatic, only younger ones Reticulocytes 24 hours in circulation or less Anemia with increased reticulocyte count suggests hemolysis with marrow response Bone marrow usually with erythroid hyperplasia and adequate iron stores

Hereditary Spherocytosis - Osmotic Fragility Test

Fragility usually increased in hereditary HS, but can be normal in mildly affected individuals Osmotic fragility almost always increased even in mildly affected individuals following a 24 hour incubation at 37C 1 - 1A - normal blood 2 - 2A - HS blood Increased fragility characteristic, but not specific, can also be seen in immune mediated hemolytic anemia

Hemolytic Anemia - Glucose-6-Phosphate Dehydrogenase Deficiency, Fluorescent Spot Test and Quantitative Assay

Fluorescent spot test - recommended test for screening -Patient blood and normal (control) blood are separately mixed with substrates which include NADP. -If G6PD is present the NADP is converted to NADPH which is fluorescent while NADP is not -A spot of each mixture is placed on filter paper and examined with UV light Quantitative assay of G6PD -Measures rate of reduction of NADP to NADPH

Anemia of Diminished Erythropoiesis - Megaloblastic Anemia - Folate Deficiency

Folate deficiency results in megaloblastic anemia with same blood and bone marrow morphology as B12 deficiency Folate functions in 1 carbon transfers -Purine synthesis -Conversion of homocysteine to methionine (also requires B12) -Deoxythymidilate monophosphate (dTMP) synthesis required for DNA synthesis Impaired DNA synthesis is the common denominator of Folate and B12 deficiency and is the immediate cause of megaloblastosis Etiology - 3 major causes A) Decreased folate intake -Inadequate intake - grossly deficient diet - chronic alcoholics, indigent, very old -Impaired absorption - sprue, intestinal lymphoma, drugs B) Increased folate requirement -Pregnancy, infancy, increased hematopoiesis, disseminated cancer C) Impaired folate utilization -Methotrexate and cancer chemotherapeutic agents

Hemolytic Anemia - Sickle Cell Disease - Pathogenesis

HbS molecules stack into polymers when deoxygenated -Cytosol changes from free flowing to viscous gel Long needle like fibers form with continued deoxygenation Sickle shape results HbA, and HbF interfere with HbS polymerization A) Sickle trait less severe than Sickle Cell Disease B) Symptoms in infants masked until 5-6 months when production of HbF normally falls -Less severe disease if hereditary persistence of HbF Variant hemoglobin HbC - 2-3% of African Americans heterozygous 1 in 1250 are compound heterozygotes HbS/HbC -These cells( HbSC) are 50% HbS rather than 40% in HbAS cells and lose salt and water increasing HbS concentration -Increases polymerization rate -Symptomatic sickling disorder, but less severe than sickle cell disease termed HbSC disease MCHC -Increased concentration increases probability of polymerization -Cell dehydration increases sickling -Co-existent alpha thalassemia reduces Hb concentration in patients homozygous for HbS leading to milder disease Intracellular pH -Decreased pH, leads to decreased oxygen affinity, increasing deoxygenated HbS, increasing sickling Microvascular transit time -Transit time in most microvascular beds too short to promote sickling -Oxygenated HbS RBC with increased endothelial adhesion

Hemolytic Anemia - Glucose-6-Phosphate Dehydrogenase Deficiency, Smear Morphology

Heinz bodies - inset - seen only with vital staining such as methyl violet or crystal violet. Deep blue to purple. Are precipitates of denatured hemoglobin attached to the cell membrane Bite cell center (top arrow) Sphereocyte/irregularly contracted cell - below bite cell=Heinz body removed by spleen

Hemolytic Anemia - Sickle Cell Disease, Proteins

Hemoglobin tetrameric protein, 2 pairs of globin chains each with its own heme group. A) HbBA - α2β2 B) HbBA2 - α2δ2 C) HbF - α2γ2 D) HbS - α2βs2 -HbS about 40%, HbA 60% in patient with Sickle cell trait -HbS nearly 100% in Sickle cell disease

***Hemolytic Anemias - Key Concepts***

Hereditary Spherocytosis -Autosomal dominant -Red cell membrane protein skeleton defect results in loss of membrane -RBC form spheres -Removed by spleen -Splenomegaly and anemia Thalassemia -Autosomal co-dominant mutations in α- or β-globin, decreasing adult Hb, microcytic hypochromic anemia -Unpaired α chains in β thalassemia aggregate and damage RBC precursors worsening poor erythropoiesis Sickle Cell Anemia -Autosomal recessive mutation in β-globin causing deoxyhemoglobin to form long polymers that sickle the RBC -Vascular blockage causes pain crises, tissue infarction - marrow and spleen -Membrane damage leads to moderate to severe hemolytic anemia G-6-PD deficiency - X-linked, mutations destabilize G-6-PD, leading RBC susceptible to oxidant damage Immune Hemolytic Anemia -Antibody to RBC antigen or antigen modified by drug (hapten) -RBC opsonization - extravascular hemolysis or compliment fixation and intravascular hemolysis

Hemolytic Anemia - Thalassemia syndromes

Heterogeneous group, inherited mutations, decreasing production of α-globin or β-globin chains that compose adult hemoglobin α2β2 - hemoglobin A Hemolysis results from imbalance of globin chain synthesis -Anemia -Tissue hypoxia -α-thalassemia - defect in α-globin -Β-thalassemia - defect in β-globin Hemoglobin deficiency and relative excess of the non affected globin chain -Reduced functioning Hb tetramers results in microcytic hypochromic RBC -Precipitation of homotetramers or unpaired chains -Unpaired subunits significant source of morbidity and mortality Endemic - Mediterranean, Africa, Middle East, India, Asia - *protective effect in heterozygote carriers* (1.5% of world population)-most common in endemic malaria areas

Anemia of Diminished Erythropoiesis - Megaloblastic Anemias

Impairment of DNA synthesis, ineffective erythropoiesis, abnormally large RBC and precursors 2 main types -Pernicious anemia (major form of vitamin B12 deficiency) -Folate deficiency anemia Common morphologic features -Macrocytic ovalocytes - lack central pallor, appear hyperchromic but MCHC not elevated -Anisiocytosis - varied size -Poikilocytosis - varied shapes -*Low reticulocyte count* -*Enlarged neutrophils with nuclear hypersegmentation*

Common morphology associated with anemia (hemolytic) and low tissue oxygen

Increased bone marrow erythroid precursors - normoblasts Usually normocytic anemia Increased reticulocytes in the blood (Polychromasia, can be macrocytic as reticulocytes are larger than normal RBC) Hemosiderin deposition predominantly spleen, liver, and bone marrow - hemosiderosis Severe anemia can lead to extramedullary hematopoiesis usually liver, spleen and lymph nodes When chronic, increased biliary excretion of bilirubin can lead to gallstones

Anemia - Clinical Findings

Independent of cause -Pallor -Weakness -Malaise -Easy fatigability -Dyspnea on mild exertion Hypoxia can lead to fatty change of liver, myocardium, kidney -Myocardial - cardiac failure, angina (concurrent CAD) -Renal - oliguria and anuria in the setting of acute blood loss and shock secondary to renal hypoperfusion

Hemolytic Anemia, Inherited and Acquired

Inherited -Membrane defects -Enzyme defect -Globin defects Acquired -Immune hemolytic anemia -Microangiopathic hemolytic anemia -Infection related -Drug related

Hemolytic Anemia - α-Thalassemia

Inherited deletions that cause reduced or absent synthesis of α-globin chains Normally 4 α-globin genes -Disease severity depends on number of genes affected Disease due to both lack of normal α2β2 hemoglobin and excess unpaired chains (βγδ) -Newborn - excess γ forms tetramers, hemoglobin Barts -Older children and adults excess β forms tetramers HbH Free β and γ are more soluble than free α thus hemolysis and ineffective erythropoiesis are less severe than in β-thalassemia

Hemolytic Anemia - Sickle Cell Disease, Factors affecting rate and degree of sickling

Interaction of HbS with other types of hemoglobin MCHC - increased concentration of HbS increases sickling Intracellular pH - decreased pH increases sickling Transit time through microvasculature - slow transit time increases sickling -Spleen, bone marrow, or inflamed vascular beds.

Nutrients Required for Erythropoiesis: Laboratory testing for what specific nutrients is is usually indicitive

Iron Folate Vitamin B-12

Anemia of Diminished Erythropoiesis - Iron Deficiency Anemia

Iron deficiency is the most common nutritional deficiency worldwide with clinical signs and symptoms related to inadequate hemoglobin synthesis Iron absorption is regulated by hepcidin, a circulating peptide synthesized and released by the liver in response to increases in liver iron levels Alterations in hepcidin have a central role in diseases of altered iron metabolism Etiology of Iron Deficiency A) Inadequate dietary intake - rare in developed countries B) Impaired absorption - sprue, steatorrhea, chronic diarrhea C) Increased requirement - growing infants, children and adolescents, pregnancy, D) Chronic blood loss -Most common cause in the Western world - assume GI loss until proven otherwise in adult men and postmenopausal women -External bleed, bleed into GI or Genital tract depletes iron reserves All causes produce a microcytic hypochromic anemia Iron reserves maintain normal hemoglobin and hematocrit initially during chronic blood loss Serum transferrin saturation level and iron level lowers prior to formation of anemia Anemia appears when iron stores are depleted and also shows low serum iron, ferritin, and transferrin saturation levels

Hemolytic Anemia - Hereditary Spherocytosis Smear Findings

Lab findings of chronic extravascular hemolysis: erythroid hyperplasia and reticulocytosis Dark (hyperchromic) spherocytes with no central pallor, smaller than normal RBC -MCV - low normal -MCHC often increased due to decreased cell surface area -Increased RDW Increased osmotic fragility Spherocytes are not pathognomic, common in hemolytic anemia Nuclear remnants - Howell-Jolly bodies Moderate splenomegaly 500-1000 gm Gallstones in 40-50% of adults from excess bilirubin

Intravascular Hemolysis

Less common Mechanical injury -Cardiac valve -Thrombotic narrowing of microcirculation Complement fixation -Secondary to antibody binding to red cell antigen Intracellular parasites -Falciparum malaria Exogenous toxins -Clostridia sepsis - enzyme release that digests RBC membrane Clinical manifestation -Anemia -Hemoglobinemia -Hemoglobinuria -Hemosiderinuria -Jaundice - hemoglobin - haptoglobin complexes converted to bilirubin Free hemoglobin bound by haptoglobin and removed by phagocytes Haptoglobin is depleted and free hemoglobin oxidizes to methemoglobin (brown) some of which passes into urine which is red/brown in color NO splenomegaly

Hemolytic Anemia - Immunohemolytic Anemias - Cold Agglutinin Type

Less common than warm antibody type IgM binds to red cells avidly at 0-4C Transient following infection (mycoplasma pneumonia, EBV, CMV, Influenza, HIV) self limited, rarely clinically significant hemolysis Chronic cold agglutinin hemolytic anemia -Idiopathic -B-cell neoplasm -Symptoms in exposed extremities when temperature is below 30C, pallor, cyanosis, Raynaud +IgM transiently binds when cold, releases when rewarms, but C3b opsonizes the RBC then removed by spleen

***Anemia Differential Diagnosis***

Mean cell volume (MCV): average RBC volume in femtolitres (fL) Mean cell hemoglobin (MCH): average mass of Hg per cell in picograms (pg) Mean cell hemoglobin concentration (MCHC): average concentration of Hg per volume of packed RBC in grams/deciliter (g/dL) Red cell distribution width (RDW): the coefficient of variation of red cell volume Reticulocyte count: measure of the marrow ability to make and release new RBC A) <2% or absolute count <100,000/ul = hypoproliferative anemia B) >2% or absolute count >100,000/ul = response to blood loss or hemolytic anemia

Anemia - Red Cell Indices/Measurements/Normal Ranges

Mean cell volume (MCV): average RBC volume in femtolitres (fL) Mean cell hemoglobin (MCH): average mass of Hg per cell in picograms (pg) Mean cell hemoglobin concentration (MCHC): average concentration of Hg per volume of packed RBC in grams/deciliter (g/dL) Red cell distribution width (RDW): the coefficient of variation of red cell volume Reticulocyte count: measure of the marrow ability to make and release new RBC A) <2% or absolute count <100,000/ul = hypoproliferative anemia B) >2% or absolute count >100,000/ul = response to blood loss or hemolytic anemia

MCV and RDW in the Diagnosis of Anemia

Mean cell volume (MCV): average RBC volume in femtolitres (fL) Mean cell hemoglobin (MCH): average mass of Hg per cell in picograms (pg) Mean cell hemoglobin concentration (MCHC): average concentration of Hg per volume of packed RBC in grams/deciliter (g/dL) Red cell distribution width (RDW): the coefficient of variation of red cell volume Reticulocyte count: measure of the marrow ability to make and release new RBC A) <2% or absolute count <100,000/ul = hypoproliferative anemia B) >2% or absolute count >100,000/ul = response to blood loss or hemolytic anemia

Anemia of Diminished Erythropoiesis- Key Concepts

Megaloblastic anemia -Caused by deficiency of folate or B12, leading to inadequate synthesis of thymidine and defective DNA synthesis/replication -Enlarged abnormal hematopoietic precursors (megaloblasts), ineffective hematopoiesis, macrocytic anemia, and usually pancytopenia -B12 deficiency also associated with neurologic damage, particularly posterior and lateral spinal cord tracts Iron deficiency anemia -Caused by chronic bleeding or inadequate iron intake -Insufficient hemoglobin synthesis -Hypochromic microcytic anemia Anemia of chronic disease -Caused by inflammatory cytokines which increase hepcidin level -Macrophage sequestration of iron -Suppressed erythropoietin levels Aplastic anemia A) Caused by bone marrow failure due to many causes -Toxins, drugs, radiation, viruses, inherited defects of telomerase and DNA repair Pure red cell aplasia -Acute - Parvovirus B19 -Chronic - Thymoma, large granular lymphocytic leukemia, neutralizing antibodies to erythropoietin

Anemia of Diminished Erythropoiesis and Hemolysis -Lead

Microcytic Hypochromic anemia similar to iron deficiency Peripheral smear with coarse basophilic stippling Marrow with few ringed sideroblasts Look for lead line along the gingiva Neurologic symptoms and behavior change

Hemolytic Anemia - β-Thalassemia minor Peripheral Smear

Mild asymptomatic hypochromic microcytic anemia - variable Mild anisiocytosis Some poikilocytosis Basophilic stippling - aggregates of polyribosomes Not so different from normal...

Hemolytic Anemia - Sickle Cell Disease, Clinical Presentation

Moderately severe anemia (HCT 18-30%) with reticulocytosis, hyperbilirubinemia, irreversibly sickled cells Vaso-occlusive crises also called pain crises A) Hypoxic injury and infarction B) Triggers include infection, dehydration, acidosis C) Most cases with no identifiable trigger D) Sites - bone - common in children - hand-foot syndrome E) Lung - Acute chest syndrome - fever, cough, chest pain, lung infiltrates - potentially fatal -Inflammation - stasis - sickling - hypoxia F) Penis - priapism in 45% of males post puberty G) CNS - stroke H) Eye - retinopathy Sequestration Crises - occurs in children with intact spleen -Massive entrapment of sickle cells -Rapid splenic enlargement -Hypovolemia and sometimes shock Aplastic crises -Parvovirus B19 infection of RBC precursor cells -Transient cessation of erythropoiesis and worsening of the anemia Chronic hypoxia -Growth impairment -Organ damage =Spleen, heart, kidney, lungs Infection risk from encapsulated organisms -altered splenic function and defect of alternate complement pathway -Pneumococcus and Haemophillus septicemia and meningitis -Vaccinate Severity varies widely

Extravascular Hemolysis

More common - phagocytic destruction Alteration making RBC less deformable RBC sequester in splenic cords and are phagocytosed Clinical features -Anemia -Splenomegaly -Jaundice -Decreased haptoglobin - binds free hemoglobin preventing urinary excretion Major site of RBC destruction is spleen Splenectomy beneficial

Hemolytic Anemia - β-Thalassemia minor

More common than β-thalassemia major, same ethnic groups Heterozygous Asymptomatic Mild hypochromic microcytic anemia - resembles iron deficiency Marrow with mild erythroid hyperplasia *Increase in HbA2 and serum iron* studies to exclude iron deficiency anemia

Hemolytic Anemia - Immunohemolytic Anemias - Warm Antibody Type

Most common immunohemolytic anemia -Primary 50%, idiopathic -Secondary - Autoimmune (SLE), drugs, lymphoid neoplasm -Usually IgG -Extravascular hemolysis as RBC bound IgG captured by phagocyte Fc receptor, partial phagocytosis reduces RBC membrane area, spherocytes form, removed in spleen -Moderate splenomegaly -Antibodies usually directed to Rh blood group antigens Secondary - Drug induced A) Antigenic drugs -Hemolysis follows large IV dose of drug 1-2 weeks after therapy begins -Penicillin and cephalosporin bind to RBC membrane -Usually act as opsin-phagocytosis-extravascular hemolysis -Occasionally bind compliment-intravascular hemolysis B) Tolerance breaking drugs -Antihypertensive α-methyldopa is prototype -Induces antibodies predominantly to Rh blood group antigens in 10% taking drug -1% develop clinically significant anemia

Hemolytic Anemia - β-Thalassemia major

Most common in Mediterranean countries, Africa and Southeast Asia In US typically in immigrants Manifests 6-9 months after birth -HbF converts to HbA at this time Early death if untreated Transfusion dependent - iron overload important cause of death must also use iron chelators - survival to third decade possible, current therapies allow some patients to reach 7th decade Janus 2 kinase (JAK2)-STAT5 pathway involved in disproportionate erythropoiesis -JAK2 inhibitors, hepcidin agonist, and transferrin may help restore normal erythropoiesis, iron metabolism, and reduce splenomegaly Hematopoietic stem cell transplantation is only hope for cure, better results in young patients with limited iron overload Splenectomy only if increased transfusion requirement due to hypersplenism Side note: -Blood transfusion treat anemia but contribute to iron overload -Maxillary marrow expansion and frontal bossing

Hemolytic Anemia - β-Thalassemia

Mutation that diminish synthesis of β-globin chains Clinical severity varies widely depending on specific mutation and gene dose - homozygous vs heterozygous 2 types of causative mutations -Β0 - No synthesis of β chains -Β+ - reduced synthesis of β chains Over 100 different mutations, mostly point mutations A) Splicing mutation - most common, no or reduced synthesis B) Promotor region mutations - reduce transcription 75-80% C) Chain terminator mutations - no synthesis -Frame shift or new stop codon Deficit in Hemoglobin A results in "underhemoglobinized" hypochromic microcytic RBC with subnormal oxygen carrying capacity RBC survival is diminished due to imbalance in α- and β -globin synthesis A) Unpaired α chains precipitate causing membrane damage -Precursors undergo apoptosis (75-80% of precursors) - ineffective erythropoiesis -Cells released from marrow have inclusions and membrane defects allowing splenic sequestration and destruction - extravascular hemolysis B) Excess absorption of dietary iron C) Ineffective erythropoiesis suppresses hepcidin, a negative regulator of iron absorption D) Repeated transfusions lead to iron overload - secondary hemochromatosis Clinical Syndromes based on severity - gene defect and dose -*β-Thalassemia major* -*β-Thalassemia minor or trait* -β-Thalassemia intermedia

Bone Growth in Hemolytic Anemia - β-Thalassemia Major

New bone formation Outer skull table Perpendicular radiations Secondary to marrow expansion Crew cut appearance Bone marrow expansion and cortical thinning result in fracture risk

Anemia of Diminished Erythropoiesis - Megaloblastic Anemia - B12 Deficiency, Summary

Non-pernicious etiology Achlorhydria and loss of pepsin - B12 not released from food proteins Gastrectomy - loss of IF Exocrine pancreatic loss - B12 not released from haptocorrin-B12 complexes Ileal disease - lack of IF receptors

Anemia of Diminished Erythropoiesis - Anemia of Chronic Disease, Summary

Normochromic normocytic to mildly hypochromic microcytic RDW usually WNL Increased bone marrow iron stores High serum ferritin Reduced TIBC Treat underlying condition to correct anemia Erythropoietin therapy benefits some patients - esp. those with cancer

Anemia of Diminished Erythropoiesis - Anemia of Chronic Disease

Normocytic normochromic anemia of underproduction Impaired RBC production associated with chronic diseases that produce systemic inflammation Second most common anemia after iron deficiency Most common anemia in hospitalized US patients A) Reduced RBC progenitors B) Impaired iron utilization C) 3 disease categories -Chronic microbial infections - osteomyelitis, bacterial endocarditis, lung abscess -Chronic immune disorders - RA, Regional enteritis (Crohn's) -Neoplasms - lung CA, breast CA, Hodgkin's lymphoma Persistent systemic inflammation A) Reduced RBC survival - not intrinsic to red cell but the milieu B) Altered iron metabolism -Low serum iron -Reduced total iron binding capacity -Abundant macrophage stored iron C) Cytokine direct inhibition of erythropoiesis and erythropoietin secretion Il-6 stimulates liver production of hepcidin -Reduces transfer of stored iron to marrow RBC precursors Erythropoietin levels low for degree of anemia -Hepcidin may be involved in lowering erythropoietin

Hemolytic Anemia - Pyruvate Kinase Deficiency

PK deficiency is less common than G6PD deficiency but more commonly causes hemolysis as most with G6PD deficiency never hemolyze -PKD high penetrance of hemolytic phenotype Hemolytic mechanism unclear -Defect in RBC ATP generation but other disorders with more significant defect do not hemolyze -Possible ineffective erythropoiesis and increased apoptosis -More rigid RBC, mainly extravascular hemolysis- splenomegaly often present No characteristic RBC morphology Highly variable clinical from fatal hydrops fetalis to compensated hemolysis without anemia

E) Mutations of ankyrin, spectrin, or band proteins

You have a 20 year old female who presents with jaundice. Your workup reveals a normochromic normocytic anemia. The DAT is negative. The peripheral smear is shown below. What is the most likely pathophysiology to explain the findings? A) Absence of β globin chains B) Point mutation of β globin C) Absence of α globin chains D) Autoimmune hemolysis E) Mutations of ankyrin, spectrin, or band proteins

Hemolytic Anemia - Paroxysmal Nocturnal Hematuria

PNH caused by an acquired mutation in phosphatidylinositol glycan complementation group A gene (PIGA) 2 to 5 per million Only hemolytic anemia caused by an acquired mutation Many normal individuals contain small numbers of bone marrow cells with this mutation Becomes clinically apparent when selective growth advantage exists such as autoimmune reaction to glycophosphatidylinositol (GPI) linked antigens Peripheral blood - present with anemia, mild macrocytosis, thrombocytopenia and/or neutropenia often present as well, reticulocytosis common PIGA codes an enzyme essential in synthesis of 3 compliment regulatory proteins -CD55 - decay accelerating factor -CD59 - membrane inhibitor of reactive lysis -C8 binding protein RBC deficient in these factors susceptible to lysis or injury by complement -RBC lysis with acidified serum, ?slight serum pH drop during sleep not validated -Manifests as intravascular hemolysis caused by the C5b-C9 membrane attack complex -Hemolysis is nocturnal and paroxysmal in only 25% -Hemolysis exacerbated by infection, surgery, exercise, excess alcohol Thrombosis can occur in 40% - usually venous, hepatic vein Budd-Chiari may be fatal Treated with monoclonal antibody that prevents conversion of C5 to C5a, Eculizumab

Anemia of Diminished Erythropoiesis - Megaloblastic Anemia - B12 Deficiency

Pernicious anemia is a specific form of megaloblastic anemia caused by an autoimmune gastritis that impairs the production of intrinsic factor - required for vitamin B12 uptake from the gut B12 required for 2 reactions, deficiency in humans in both reactions resulting in impaired DNA synthesis A) B12 is essential cofactor in conversion of homocysteine to methionine -Lack of B12 traps folate as N5-methylfolate and dUMP cannot be converted to dTMP a necessary DNA building block -Lack of methionine inhibits reactions creating folate polyglutamates B) Isomerization of methylmalonyl coenzyme A to succinyl coenzyme A requires B12 -Increased plasma and urine levels of methylmalonic acid can lead to incorporation of abnormal fatty acids into neuronal lipids, myelin breakdown, neurologic complications (?) -Hereditary deficiency of methylmalonyl coenzyme A do not result in B12 like neuronal complications Thus folate is the cause of anemia in B12 deficiency, and anemia improves with administration of folate - neurologic symptoms do not improve and may worsen

Anemia of Diminished Erythropoiesis - pure red cell aplasia

Primary marrow disorder with suppression only of red cell precursors Chronic - autoimmune with underlying neoplasm such as thymoma, drug exposure, or autoimmune disorder Acute - secondary to parvovirus B19 infection, self limited and recover in 1-2 weeks from infection and anemia resolves -Aplastic crises in people with preexisting hemolytic anemia -May be chronic if infection is not cleared, immunosuppressed patients

Anemia of Diminished Erythropoiesis - Alcohol

Ring Sideroblasts in marrow of anemic malnourished alcoholics Chronic consumption or binging in well nourished individuals do not have ringed sideroblasts Sideroblastic change is not the sole cause of anemia -Alcohol has a direct toxic effect on hematopoiesis Usually high MCV vacuolated precursors, and ring sideroblasts, 50% with folate deficiency as well Sideroblasts disappear over 4-12 days after alcohol withdrawal

Anemia - Lab testing - IDA (Iron Deficiency anemia)/ACD (anemia of chronic disease)

Serum Ferritin=extracellular ferritin A) Intracellular and extracellular ferritin are in equilibrium B) Low stored iron=low intracellular ferritin=low serum ferritin C) Normal intracellular iron =normal intracellular ferritin=NL serum ferritin D) Anemia of Chronic Disease -NL to increased iron = NL to increased serum ferritin -Iron deficiency anemia, low iron =low serum ferritin However -Ferritin is an acute phase reactant and is elevated with inflammation thus IDA with infection may have a low normal ferritin Serum iron -Reduced in IDA -NL or sometimes low in ACD Iron Binding Capacity - a direct measure of transferrin -Elevated in IDA -Normal or sometimes increased in ACD -Transferrin is also an acute phase reactant thus IBC overlaps sometimes in IDA/ACD Transferrin saturation - ratio of serum iron to IBC -NL is 1:3 -IDA usually decreased to 1:5 or lower -ACD usually NL sometimes decreased

Hemolytic Anemia

Shared features A) Shortened red cell life span - less than 120 days B) Elevated erythropoietin levels -Compensatory increase in erythropoiesis -Measured by elevated reticulocyte count C) Accumulation of hemoglobin degradation products from RBC hemolysis Mechanism A) Senescent RBC removed by splenic, bone marrow, and liver macrophages -Triggered by age dependent surface proteins B) Most hemolytic anemias from premature destruction of RBC - extravascular

Anemia of Diminished Erythropoiesis - Iron Deficiency Anemia, Peripheral Smear

Small pale RBC Thin peripheral rim of hemoglobin Few recently transfused normal RBC Low MCV Low MCH Low MCHC RDW increased

Anemia of Diminished Erythropoesis - Aplastic Anemia

Syndrome of chronic primary hematopoietic failure and *pancytopenia* (anemia, neutropenia, thrombocytopenia) Autoimmune most common Few with inherited or acquired abnormalities of hematopoietic stem cells Most cases idiopathic ~65% Chemical and drug exposure - dose related or idiosyncratic Viral infections Irradiation Inherited defect in telomerase 5-10% adult onset Short telomeres in 50% Extrinsic immune mediated suppression of marrow progenitors -Stem cell antigenically altered - drug, toxin, virus -Activated cellular immune response TH1 cells produce interferon-γ and TNF that suppress and kill hematopoietic precursors -60-70% of patients respond to immune suppressive drugs such as cyclosporine or antithymocyte globulin Intrinsic abnormality of stem cells -Occasional transformation to myelodysplasia or leukemia -Short telomeres Both pathways (extrinsic and intrinsic) involved Image: Normal (L), Aplastic (R)

Hemolytic Anemia - β-Thalassemia major Peripheral Smear

Target cells Nucleated RBC Hypochromic microcytic Anisiocytosis Poikilocytosis Reticulocyte count 2-8%, lower than expected due to intramedullary destruction

Hemolytic Anemias - Trauma to RBC

Trauma -RBC trauma from artificial heart valves (mechanical>>bioprosthetic porcine/bovine) -Shear forces from turbulent flow Microangiopathic hemolytic anemia -Luminal narrowing in microvasculature from fibrin and platelet deposition produce shear stress -Usually DIC (deceminated intervascular coagulation), also TTP, HUS, malignant hypertension, SLE, disseminated carcinoma Either process - same morphology - RBC fragments - Schistocytes -Burr cells, helmet cells, triangle cells

Anemia - Classifications

Underlying mechanism -Blood loss -Increased RBC destruction -Decreased RBC production RBC Morphology A) Red cell size -Normocytic -Macrocytic +Impaired marrow erythroid maturation -Microcytic +Disorders of hemoglobin synthesis B) Red cell color -Normochromic +Normal hemoglobin -Hypochromic +Low hemoglobin

C) Increased hemoglobin F and normal A2

Your 8 month old male Caucasian patient develops a severe anemia with the peripheral smear shown below. What is the most likely change related to the pathophysiology of his disease? A) Elevated hemoglobin level containing abnormal beta chains B) Beta chains containing fused parts of gamma and delta chains C) Increased hemoglobin F and normal A2 D) Decreased serum iron E) Increased hemoglobin H

E) Biliary obstruction, choledocholithiasis

Your patient is a 20 year old African American presenting with severe right upper quadrant pain and tenderness on exam. Hepatomegaly and splenomegaly are not found. His bilirubin is elevated, AST and ALT are normal, and his peripheral smear is shown. What is the most likely diagnosis for the pain? A) Abnormal bilirubin clearance due to hemolysis B) End stage liver disease C) Hepatic necrosis D) Splenic infarct E) Biliary obstruction, choledocholithiasis