Red Blood Cells & Anemias RBC: ~5 x 106/ml; Generate ~ 8,000,000,000/hour Andrew D. Leavitt, MD Departments of Laboratory Medicine & Medicine March 26, 2012

Outline – topics we will cover Erythropoiesis – names, faces, & numbers (review) RBC biology - Hemoglobin O2 dissociation curves Hemoglobin – fetal, adult RBC membrane Hemoglobin switching Anemias: Production vs. destruction vs. loss Membrane or contents – Hgb, enzymes Immune mediated Physical destruction Anemia of inflammation Ribosomopathies

CELL SIZE & CYTOPLASM COLOR Erythropoiesis Abbott Labs, Morphology of Human Blood Cells Pro- Erythroblast Basophilic Polychromatic Orthochromatic Reticulocyte RBC CHROMATIN STRUCTURE NUCLEAR SIZE & COLOR CELL SIZE & CYTOPLASM COLOR 5 stages in the marrow

Red Blood Cell (RBC) Delivers O2 from your lungs to all your tissues Takes CO2 from your tissues to your lungs Also – binds NO (a vasodilator) Their size: 8  [capillaries have ~3 M diameter] Their life-span: 120 days Mature RBCs have no nucleus Too few RBCs = anemia

What tells your body to make RBCs? EPO: Is not stored, but expressed in response to the kidney sensing oxygen in the blood It - 1. Increases # of E-committed progenitors 2. Increase GATA1 and FOG expression 3. Enhances anti-apoptotic gene expression 4. Increases transferrin receptor expression

Iron in a Porphyrin ring Hemoglobin Hemoglobin (Hgb) in the RBC carries O2, which is poorly soluble in water, to tissues & CO2 from tissues to lungs. ~640,000,000 Hemoglobin molecules/RBC Must coordinate: 1.heme and globin synthesis 2. a (Chromosome 11; 141 aa) & b (Chromnosome 16; 146) chains Colors of a bruise are globin breakdown products b6Glu to Val Tetramer of two heterodimers (a &b) Heme Iron in a Porphyrin ring Linus Pauling 1949 – molecular diagnosis 2012 – no therapy

Hemoglobin oxygen dissociation curve Arterial O2 tension Mean Venous HbF ; CO2

Erythropoiesis: Hemoglobin Switching (ALWAYS: 2 alpha & 2 “beta-like” [3 ‘b’ options]) Embryo Fetus Birth Adult 6 mo. Globin chain synthesis (%) 50 a b d g z e Alpha: Chromosome 16 Beta-like: Chromosome 11 HgF a2g2 (75) HgA a2b2 (25) HgA a2b2 (96-98) HgA2 a2d2 (2-3) HgF a2g2 (< 1) Hoffman et al. Hematology 2000

Schematic of red cell membrane depicting proteins crucial for normal membrane mechanical strength * * *mutations lead to hereditary spherocytosis

Normal Values for Hematology Tests - UCSF

Thinking about anemia…a clinical perspective Framework: RBC size (MCV) & is the marrow responding (reticulocyte count) Etiology: Cell intrinsic: 1. Hgb – too little; abnormal type; unequal a and b synthesis 2. Enzyme 3. Membrane Cell extrinsic: Immune mediated Physical destruction Blood loss Marrow does not produce Primary – leukemia, myelodysplasia Secondary – vitamin deficiency

Reticulocyte Stain – detects the ribonucleoprotein in the young RBC

#1.1 Too little Hgb: Iron Deficiency Anemia Small cells – microcytic (MCV < 80 fL) Young women – OK Older people – Worry about gastrointestinal blood loss normal peripheral blood (40x) Iron deficiency anemia, peripheral blood (40x) Lead poisoning can look like Fe deficiency – Lead blocks heme synthesis

Iron Deficiency Anemia peripheral blood, 100X Hypochromic RBCs Elliptocytes Iron stian of bone marrow – none found ~ 500 million people worldwide

The Iron Circuit – mostly a game of recycling Daily absorption: Duodenum ~ 1 mg Daily loss: urine, feces, skin, hair ~ 1 mg Transferrin Plasma (4 mg) Ineffective erythropoiesis Macrophage (0.5 – 1.5 gm) Bone Marrow Erythroblasts ( ~ 150 mg) Circulating RBCs (1.7 – 2.4 gm) Menstrual loss/hemorrhage

Iron Circuit in Disease Weiss & Goodnough NEJM 2005 Iron Circuit in Disease Infection/inflammation/malignancy IL-6/LPS hepcidin (25aa protein) absorption macrophage Fe storage Impairs Erythropoiesis: Decrease EPO Directly suppresses marrow Interferon – g TNF – a IL-1, IL-10

Iron stain of bone marrow aspirates Iron deficienyc anemia - iron stain Anemia of inflammation – iron stain Storage iron (blue)

#1.2 Abnormal Hgb: Sickle cell disease Multi-system disease Cardiovascular/strokes Kidneys Skin Lungs Immune system Treatment: Supportive care Transfusions Demethylating agents Stem cell transplants

#1.3 Abnormal a/b Globin ratio: Thalassemia Small cells – microcytic (MCV < 80 fL) Imbalance between a and b chains Name based on which chain is deficient [a or b thal] Make lots of RBCs, just unstable and lyse, so increased retics Alpha Thal: silent carrier -a / aa trait -- / aa or -a / -a Hgb H -a / -- Hydrops fetalis -- /-- Beta Thal: Minor Intermedia Major

Erythropoiesis: Hemoglobin Switching (ALWAYS: 2 alpha & 2 “beta-like” [3 ‘b’ options]) Embryo Fetus Birth Adult 6 mo. Globin chain synthesis (%) 50 a b d g z e Alpha: Chromosome 16 Beta-like: Chromosome 11 HgF a2g2 (75) HgA a2b2 (25) HgA a2b2 (96-98) HgA2 a2d2 (2-3) HgF a2g2 (< 1) Hoffman et al. Hematology 2000

Normal peripheral smear Alpha thal trait Normal peripheral smear Hemoglobin H, -- / -A Target cells Retics Beta-thalassemia major Nucleated RBC Target cells

Severe thalassemia Beta-thalassemia major (transfused) Nucleated RBC Target cells Beta-thalassemia major (transfused) Nucleated RBC Normal RBCs (transfused)

#2 Enzyme: G-6-PD deficiency (Heinz body hemolytic anemia) Reduced glutathione (GSH) is critical for RBCs to counter oxidative stress. Bite cells Arrows show Heinz bodies Sulfa drugs, fava beans, antimalarials..…

#3: Membrane: red cell membrane depicting proteins crucial for normal membrane mechanical strength * * *mutations lead to hereditary spherocytosis

Hereditary spherocytosis (HS) Reticulocytes spherocytes

Thinking about anemia…a clinical perspective Framework: RBC size (MCV) & is the marrow responding (reticulocyte count) Etiology: Cell intrinsic: 1. Hgb – too little; abnormal type; unequal a and b synthesis 2. Enzyme 3. Membrane Cell extrinsic: Immune mediated Physical destruction Blood loss Marrow does not produce Primary – leukemia, myelodysplasia Secondary – vitamin deficiency

Autoimmune hemolytic anemia Reticulocytes spherocytes

Testing for anti-RBC antibodies: attached or in circulation Hemolytic disease of the newborn

RBC destruction - extrinsic Erythroid Hyperplasia, TTP, bone marrow aspirate (100x) Thrombotic thrombocytopenic purpura (TTP) schistocytes (RBC fragments) Hemolytic uremic syndrome (HUS) schistocytes (RBC fragments) Retics

Megaloblastic anemia (MCV > 100) B12 deficiency Folate deficiency Affects DNA synthesis

Megaloblastic anemia (MCV > 100) Anemia yet hypercellular marrow – ineffective erythropoiesis

Ribosomopathies and hematopoietic disorders Our findings indicate that the erythroid lineage has a low threshold for the induction of p53, providing a basis for the failure of erythro- poiesis in the 5q- syndrome, DBA, and perhaps other bone marrow failure syn- dromes. Narla & Ebert 2010

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