Normal Red Cell Production Approach to (Erythropoiesis)

Kidney Bone marrow Multi-potent Epo stem cell mRNA Erythroid Epo stem cell Oxygen sensor 3-4 Tzu-Fei Wang, MD days Assistant Professor Blood vessel Department of Internal Medicine Division of Peripheral The Ohio State University Wexner Medical Center blood

RBC survival 100-120 days

Laboratory Evaluation of Anemia Anemia • (including MCV) • Peripheral blood smear • Reticulocyte count Low/normal reticulocyte High reticulocyte • Relative reticulocyte count count count • Percent of all RBC (normal 0.8%–1.5%) • Decreased production • Increased • Absolute reticulocyte count • Nutritional deficits loss/destruction • Relative reticulocyte count x RBC count • Bone marrow • Blood loss • Normal 50,000–75,000/µl pathology • • Other workup depending on the suspected causes (i.e. nutritional deficiency, hemolysis, etc) I will discuss Dr. Desai will discuss

1 Anemia with low/normal reticulocyte count Iron Intake • Low MCV () • • Mean iron intake 10-15 mg/d Contributionto the iron of intakefood groups1992-3 • Anemia of chronic disease to the iron intake 1992-3 • Main source of iron intake is • Lead intoxication meat (especially red meat) Other Bread • Other16% Bread11% • Serum iron is NOT a marker 16% 11% • Normal MCV Vegetables of iron status (will change Vegetables • Anemia of chronic kidney disease 16% with even just one meal) 16% Cereals Cereals • Medications 39% Meat • Infections Meat Eggs 39% 15% Eggs 3% • High MCV (macrocytosis) 15% 3% • Vitamin B12 and/or folate deficiency • Medications • disease, ETOH • Thyroid disease Fairweather-Tait S.; Proc Nutrition Society, 200;63:519-528

Body Iron Distribution and Storage Causes of Iron Deficiency in Adults Iron deficiency is a symptom, not a disease • Duodenum absorption: ~1-2 mg a day • A world-wide problem • Iron loss (sloughed mucosal cells, menstruation, • 3% of toddlers age 1-2 years other blood loss): ~1-2 mg a day • Up to 10% of women of child bearing age • Total body iron storage: 3000-4000 mg • Plasma transferrin: 3 mg • Increased iron • Inadequate iron supply requirements • Muscle (myoglobin): 300-500 mg • Insufficient dietary iron • Blood loss • Impaired iron • Bone marrow: 300 mg • Gastrointestinal tract absorption • Reticuloendothelial macrophages: 600 mg • Menstrual periods • Gastric (bariatric) surgery • Blood donation • Circulating erythrocytes (Hgb): 1800-2500 mg • Atrophic gastritis • Pregnancy and • Intestinal lactation • Liver (storage iron as ferritin): 1000 mg • Celiac disease • H. pylori infection

Andrews, NC. N Engl J Med 1999;341:1986-1995

2 Neurologic syndromes Diagnosis of Iron Deficiency associated with iron deficiency • Laboratory Tests • Serum ferritin is the best value for iron deficiency (< 20 ug/L diagnostic of Fe deficiency anemia) • MCV- the second most reliable indicator of iron stores • Pica • Restless leg syndrome • TIBC- high in iron deficiency and low in ACD • Latin word for magpie, a • Common neurologic • Serum iron/iron saturation bird which is reputed to disorder • Doesn’t differentiate between iron deficiency and eat almost anything • Criteria for diagnosis: anemia of inflammation 1. An urge to move the legs • Peripheral blood smear • Definition: Compulsive usually accompanied by ingestion of a non-food uncomfortable substance such as starch, sensations clay, earth, ice 2. Sensation begins or • Ceases within days of worsens during periods therapy of rest 3. Sensations relieved by • Occurs in ~25% of patients movement with iron deficiency 4. Worse in the anemia from any cause evening/night • Occurs in ~10% of cases of Ed Uthman from Houston, TX, • More commonly in women iron deficiency anemia USA (CC BY 2.0)

Treatment With Iron: Principles Available Oral Iron Supplements

• Iron is absorbed best on an empty stomach Approx. cost Oral iron Typical Elemental • Ascorbic acid increases absorption and to give 5000 preparations dose (mg) iron (mg) toxicity mg Ferrous sulfate (also 325 mg tid 65 $10.00 • Reticulocytosis occurs <7days; Increased has elixir version) Hgb in 2-3 weeks Ferrous gluconate 300 mg tid 36 $7-8.00 Ferrous fumarate 100 mg tid 33 $8.00-9.50 • Maximum iron dose ~200 mg/day Iron polysaccharide 150 mg bid 150 $11.00 complex • Side effects: GI upset, constipation, black Carbonyl iron 50 mg tid 50 $18.00 stool • Encourage iron rich food

3 Inadequate Response to Oral Iron IV Iron Agents

. Intolerance/Noncompliance (~30% • Iron Dextran discontinue) • INFeD® . Persistent blood loss • Dexferrum® . Decreased iron absorption • Sodium ferric gluconate complex (SFGC) • Ferrlecit® . Chronic inflammation or bone marrow • Iron Sucrose damage • Venofer® . Chronic kidney disease • Ferumoxytol • FerahemeTM

Anemia of Chronic Disease Anemia of Chronic Disease • Impaired iron transfer from reticuloendothelial . Characteristics macrophages and duodenum to plasma  Immune-driven by inflammatory cytokines transferrin, then to liver  Diversion of iron into RE system (iron not in the “right place” Blunted erythropoietin response . Diagnosis- iron status

Parameter Anemia of Chronic Iron deficiency Disease anemia Serum Iron Reduced Reduced TIBC Reduced-Normal Increased % saturation Reduced Reduced (TSAT) Ferritin Normal-increased Reduced sTfR Normal Increased

Cytokine levels Increased Normal http://www.uptodate.com/contents/causes-and-diagnosis-of-iron-deficiency-anemia-in-the- adult?topicKey=HEME%2F7150&elapsedTimeMs=0&view=print&displayedView=full

4 Treatment Options for Anemia of Chronic Disease • Treat the underlying diseases • Defect in DNA synthesis • RBC Transfusions • RNA synthesis is relatively unimpaired • For anemia of chronic kidney disease: • RBC’s hemoglobin production is far ahead of nuclear maturation ̶ Erythroid-stimulating agents (ESA) and potentially iron supplementation (ferritin • Nuclear cytoplasmic dissociation <100 and/or iron sat <20%) • Ineffective erythropoesis • For selected cases of anemia related to • Intramedullary hemolysis or myelodysplatic syndrome • Decreased red cell survival/misshapen cells ̶ Consider ESA

Vitamin B12 and folate Signs/Symptoms of B12 Deficiency • Anemia, hypersegmented neutrophils • “Beefy Red” tongue, smooth surface of the tongue Vitamin B12 Folate • Neurologic • Sources: green leafy • demyelination of the posterior and lateral • Sources: dietary vegetables meat products columns of the spinal cord • Daily requirement • paresthesia, loss of position/vibratory • Daily requirement about 50-100 ug/day sense 2-5 ug/day • Total body reserves (5-10 mg) last only 3- • in advanced disease, neuropathy, muscle • Total body stores , and even CNS symptoms 2-4 mg 4 months (irritability, somnolence, psychosis) • If intake stops, • Heat labile and water takes 2-3 years for soluble storage to be • Absorbed in jejenum depleted and ileum

Wikipedia, https://en.wikipedia.org/wiki/Hypersegmented_neutrophil

5 Bone marrow process • Broad DDx, including Approach to Anemia • Acute or chronic leukemia • Myelodysplatic syndrome (MDS) • Myeloproliferative diseases (MPD) • Involvement of malignancies in the bone marrow • Disseminated infections in the bone marrow • Patients usually have more symptoms such as unexplained weight loss, petechiae, fever, Payal Desai, MD hepatosplenomegaly, etc Assistant Professor • More than one cell line is abnormal and could be Director of Sickle Cell Research severe Division of Hematology • Referral to hematology and bone marrow biopsy is The Ohio State University Wexner Medical Center needed for definitive diagnosis http://www.uptodate.com/contents/evaluation-of-bone-marrow-aspirate- smears?topicKey=HEME%2F4434&elapsedTimeMs=0&source=search_result&searchTerm=l eukemia+bone+marrow&selectedTitle=1%7E150&view=print&displayedView=full

Classification of Inherited Diseases of Hemoglobin Alpha-

• Production abnormalities Main mechanism is whole gene deletion: () / Normal -/ Silent carrier SE Asia, 28% African Americans. • Structural abnormalities Normal or slightly MCV;  HbH inclusions () -/- -thalassemia minor SE Asia, 3% of Black Americans, --/ Mediterranean No clinical disease No or mild anemia, MCV, target cells HbH inclusions may be seen Genetic counseling

6 Beta-thalassemia Anemia with high reticulocyte count

• Low MCV (microcytosis) • Hypochromic, • Hemoglobinopathies (such as some • Variants Major, Intermedia, Minor sickle cell) •  WBC, normal platelet count • Normal/High MCV • Iron studies -  serum Fe, transferrin saturation • Acute hemorrhage and ferritin • Bone marrow – erythroid hyperplasia • • Hemoglobin electrophoresis • Hemoglobinopathies • Minor – elevated HbA2 • Membranopathies/enzymopathies • Only HbF and HbA2 are present • Autoimmune • Variable amounts of HbA if transfused • Microangiopathic Hemolytic Anemia

Unstable Hemoglobins Unstable Hemoglobins • Denaturation and precipitation of globin chains in RBC’s  Heinz bodies which cling to membrane  removed in  • Rare disorders. Many variants described hemolysis • Autosomal dominant only heterozygotes exist (homozygous do not survive) • Disrupt contact between heme and globin • Alter amino acids at interface between  and  chains • Alter the shape or structure of the globin molecule

7 The Common Variants of Sickle Cell Anemia Pathophysiology

Name Genotype Percent • Manifestations of SCD are driven by: Homozygous SS S-S 65 (Sickle Cell Anemia) • Vaso-occlusion with ischemia- Heterozygous SC S- C 24 reperfusion injury • Hemolytic anemia Heterozygous S-+ thal S- + thal 7 • Endothelial Activation

Heterozygous S-° thal S- ° thal 3

Owusu-Ansah 2015

Mortality in Sickle Cell Disease Complications of SCD

Childhood mortality rates in SCD Sepsis • In 1973 – median survival of 14.3 years Priapism • CSSCD ~ 85% SS children and adolescents with Myocardial Infarction survived to age 20 Multiorgan Failure Transfusion Reaction • In 2004 survival analysis of SS and Sβ° subjects SCD-related survival 93.6% by age 18

Diggs LM. Anatomic lesions in sickle cell disease. Sickle cell disease: diagnosis, management, education, and research. St. Louis: C.V. Mosby, 1973:189-229 Leikin SL, Gallagher D, Kinney TR, Sloane D, Klug P, Rida W. Mortality in children and adolescents with sickle cell disease. Pediatrics 1989;84:500-508 Platt et al. Mortality In Sickle Cell Disease – Life Expectancy and Risk Factors for Early Death. N Engl J Med 1994; 330:1639-1644 Konotey-Ahulu FID. The Sickle Cell Disease. Clinical Manifestations Including the “Sickle Quinn C T et al. Survival of Children with Sickle Cell Disease. Blood 2004;103:4023-4027 Crisis”. Arch Intern Med. 1974;133(4):611-619.

8 Immune Hemolytic Auto-Immune Hemolytic Anemias • causing hemolysis can be • Autoimmune broken down into 2 general categories: • Warm -mediated warm and cold • Cold antibody-mediated • Warm antibodies react with RBCs best at • Paroxysmal Cold Hemoglobinuria 37° and typically do not agglutinate red • Drug-related hemolysis cells • Hemolytic transfusion reactions • Hemolytic disease of the newborn • Cold antibodies typically react best at <32° and do cause RBC agglutination • Paroxysmal Nocturnal Hemoglobinuria

Warm-Antibody Hemolytic Coomb’s Test Anemias Etiology

• The Direct Coomb’s = DAT (Direct • Primary or Secondary Antiglobulin Test) - tests for IgG or C3 DIRECTLY ON THE RED CELLS. • Drugs • Solid or hematologic malignancy • The Indirect Coomb’s - tests for IgG or C3 in the serum which react with generic • Infection normal red cells. This is also known as the • Collagen Disease antibody screen in blood-banking. • Pregnancy

• Can be associated with immune platelet destruction = Evan’s syndrome

9 Warm-Antibody Hemolytic Anemias Warm-Antibody Hemolytic Anemias Treatment Clinical Features • Patients may require red cell transfusions, if they are symptomatic with their anemia • , is usually present • However, immunosuppression is the mainstay of • Depending on degree of anemia and rate of fall in therapy hemoglobin, patients can have VERY symptomatic • First Line – Steroids anemia • 1mg/kg/d prednisone oral or • Lab Dx - methlyprednisolone IV • reticulocytes,  bili,  LDH, ↓haptoglobin • Continue until Hb > 10g/dL then taper • Continue with Vitamin D, Ca, + • Positive Coomb’s test - both direct and indirect bisphosphonates • Spherocytes are seen on the peripheral smear • Consider PCP prophylaxis • Supplement with Folic Acid

Lechner et al. How I Treat Autoimmne Hemolytic Anemia. Blood 2010. Crowther et al. Evidence-based focused review of the treatment of idiopathic warm immune hemolytic anemia in adults. Blood 2011

WAIHA: Treatment WAIHA: Treatment • Second Line Therapy • 2nd Line Therapy • Ineffective • Splenectomy therapies • 80% patient with CR or PR with • Rituxan prednisone • Other Therapies • Azathioprine • Danazol • 15-20% need higher than maintenance • Cyclophosphamide • BMT dose to stay in remission • Cyclophosphamide • IVIG • Mycophenolate Mofetil • <20% of patients stay in remission after • Cyclosporine • Plasma withdrawal of steroids • Vincristine Exchange • Alemtuzumab • Ofatumumab

Lechner et al. How I Treat Autoimmne Hemolytic Anemia. Blood 2010. Lechner et al. How I Treat Autoimmne Hemolytic Anemia. Blood 2010. Crowther et al. Evidence-based focused review of the treatment of idiopathic Crowther et al. Evidence-based focused review of the treatment of idiopathic warm immune warm immune hemolytic anemia in adults. Blood 2011 hemolytic anemia in adults. Blood 2011

10 Drug-Induced Immune Hemolysis Drug-Induced Positive Antiglobulin Tests Three general mechanisms Serum and Mechanism DAT Eluate • Innocent bystander Neoantigen C3 (sometimes IgG also) Serum reacts with rbcs -Drug +RBC complex only in the presence of • Quinine, Quinidine, Isoniazide drug; eluate non-reactive • Hapten • Penicillins, Cephalosporins Drug Adsorption (DA) IgG (sometimes C3 also) React with drug-coated -Drug binds to RBC RBCs but not untreated • True autoimmune RBCs- Ab to drug IgG (rarely C3 also) React with normal RBCs • Alpha-methyldopa, L-DOPA, -WAIHA 11-36% of pts in absence of drug Procainamide

Cold Agglutinin Disease

• Pathogenic antibodies are usually IgM • In the cold, IgM can • Bind to red cells in the cooler extremities, lead to red cell then fix complement • When red cells return to the warmer torso, • Red cells clumps IgM falls off cannot pass through • Complement-coated red cells can be lysed microvasculature, directly within the vessel (intravascular leading to cyanosis hemolysis) and ischemia in • Alternatively, complement-coated red cells extremities can be engulfed by complement receptors on macrophages within the liver (extravascular hemolysis) John Lazarchick, ASH Image Bank 2011; 2011-1053

11 Cold Agglutinin Disease Cold Agglutinin Clinical features DiseaseTreatment • Can be associated with infection with either • Treatment is to keep patient (especially the or Mononucleosis extremities) warm. Blood and IV fluids should be warmed. • Can also be idiopathic or associated with a • Immunosuppression with oral chemotherapy Lymphoproliferative disease may be required • Most commonly IgM monoclonal • Steroids and splenectomy are usually ineffective. gammopathy • Rituxan (PR in 20/27 patients) • (may only be BM • If Rituxan Refractory, can consider Eculizumab or Bortezomib involvement)

Treatment AIHA Microangiopathic WAIHA CAD Drug-IHA Hemolytic Anemia 1st Line Folate Folate Treat if hemolysis • Non-immune hemolytic anemia Avoid cold present Treat secondary cause • reticulocytes,  bili,  LDH, ↓haptoglobin

2nd Line Splenectomy Chlorambucil Folate • NEGATIVE Coomb’s Rituxan Stop drugs • Prosthetic Valves, Heart valve induced,

3rd Line Other Cytotoxic ? Corticosteroids- Pregnancy Associated Syndrome, HTN, Drugs severe cases Infections, Immune D/os, DIC Transfusion Transfuse – least Transfuse- I+, Transfuse Recommendation incompatible blood warmer • Thrombotic Microangiopathy s • TTP, aHUS, HUS, Drug-Induced TMA

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