RBC Disorders Erythropoiesis:

Tues. 10/19/10

Goal: formation of RBCs oxygen transport Site: bone marrow Main growth factor: erythropoietin Duration: 5 days (from proerythroblast to reticulocyte) Lifespan of RBC: 120 days
Test q: The normal lifespan of RBCs in peripheral blood is: 120 days.

Anemia vs. Polycythemia: Anemia: Decrease in Hgb and hematocrit levels in relation to age- and sex-adjusted normal ranges. - Signs: Pallor, Fatigue, Dizziness, Tachycardia, Shortness of breath Polycythemia: Increase in hemoglobin and hematocrit as compared to normal ranges - Signs: Headache, Pruritus, Dizziness

Red cell indices: - Hemoglobin (Hb) : concentration, in g/dL - Hematocrit (Hct) : proportion of blood volume that is occupied by RBCs - Mean cell volume (MCV) : average volume of RBC expressed in femtoliters - Mean cell hemoglobin (MCH) : average mass of hemoglobin per RBC, in picograms - Mean cell hemoglobin concentration (MCHC) : average concentration of hemoglobin in dL of packed RBCs, g/dL - RBC distribution width (RDW) : CV of RBC volume, size variability Can separate RBCs from the rest of the blood.

Determining the Concentration of Hemoglobin: Figure: The potassium ferricyanide oxidizes hemoglobin to hemiglobin (Hi; methemoglobin), and potassium cyanide provides cyanide ions (CN-) to form HiCN, which has a broad absorption maximum at a wavelength of 540 nm. Figure: RDW Abnormal in anisopoikilocytosis (abnormally shaped RBCs).

RBC distribution width (RDW):

Adult Reference Ranges for RBCs: The reference ranges for the laboratory providing the result should always be used in interpreting the test result. Advantages of the electronic cell counter - Speed of performance - elimination of visual fatigue of the technician - Improved precision are decisive Morphologically discernable stages of erythroid development: Proerythroblast Basophilic erythroblast Polychromatophilic erythroblast Orthochromatic erythroblast Reticulocyte Mature RBC Maturation: Bone marrow

Blood

When passing through bone marrow sinus, nucleus is extruded.

Reticulocyte count: Enumeration of reticulocytes indicates production of new red blood cells by the marrow Manual using supravital dye to precipitate residual RNA Automated in a cell counter Corrected reticulocyte count: actual patient value corected with an expected release of reticulocytes in health Polychromasia: Reticulocyte: Wright-Giemsa

Supravital
Live-nonfixed cells Methylene blue/brilliant cresyl blue

Polychromasia (purple tinge)

Polychomatophilic more purplish/bluish Reticulocytes Supravital stain:

Substantia reticulofilamentosa (Amount of precipitated RNA decreases w/age of the reticulocyte.)

Automated Reticulocyte count: RNA binding dye

Reticulocyte count: Decreased Iron deficiency B12/folate deficiency Anemia of chronic disease Primary marrow neoplasms Aplastic anemia

Increased Hemolytic disorder Treatment of iron or B12/folate deficiency with response Bleeding

Test q: Reticulocytes are DECREASED in all of the following EXCEPT: Hemolytic anemia. (Other choices: Aplastic anemia, Iron deficiency anemia, Leukemia, and Folate deficiency.) Test q: A healthy 19F suffered blunt abdominal trauma in a motor vehicle accident. On admission to the hospital, her initial hematocrit was 33%, but over the next hour, it dropped to 28%. A paracentesis yielded serosanguineous fluid. She was taken to surgery, where a liver laceration was prepared and 1 L of bloody fluid was removed from the peritoneal cavity. She remained stable. A CBC performed 3 days later is most likely to show which of the following morphologic findings in RBCs in the peripheral blood? Reticulocytosis. (= increase in reticulocytes)

Anemia: Reduction of the total circulating red cell mass below normal limits reduces the oxygen-carrying capacity of the blood, leading to tissue hypoxia In practice, the measurement of red cell mass is not easy, and anemia is usually diagnosed based on a reduction in the hematocrit and the hemoglobin concentration of the blood to levels that are below the normal range

Anemia: Clinical features Pale, weakness, malaise, and easy fatigability O2 dyspnea, fatty change in the liver, myocardium, and kidney Hypoxia Myocardial angina pectoris, particularly when complicated by pre-existing coronary artery disease CNS headache, dimness of vision, and faintness

Anemia according to underlying mechanism: BLOOD LOSS INCREASED RED CELL DESTRUCTION (HEMOLYSIS) DECREASED RED CELL PRODUCTION

Anemia paleness of mucosa and skin.

Classification of anemia based on MCV and RDW:

MCV Microcytic <70 fL Normal RDW Thalassemia minor Anemia of chronic disease Hemoglobinopathy traits High RDW Iron deficiency Anemia of chronic disease Thalassemia minor Fragmentation Hemolysis Early iron def. Early B12/folate deficiency Sickle cell disease

Above: Different problems correlate with different steps in maturation.

Normocytic

Anemia of chronic disease Hereditary spherocytosis Hemoglobinopathy traits Acute bleeding Aplastic anemia Myelodysplastic syndromes

ANEMIAS OF BLOOD LOSS: Acute Blood Loss loss of intravascular volume massive -cardiovascular shock, and death. rate of hemorrhage, external or internal bleeding O2 erythropoietin from the kidney proliferation of committed erythroid progenitors (CFU-E) in the marrow 5 days reticulocytes

Macrocytic >100 fL

B12/folate deficiency Liver disease Alcohol Thyroid insufficiency Myelodysplastic syndromes

ANEMIAS OF BLOOD LOSS: Chronic Blood Loss Chronic blood loss induces anemia only when the rate of loss exceeds the regenerative capacity of the marrow or when iron reserves are depleted and iron deficiency anemia appears Microcytosis pattern: Normal RBC size ~ nucleus of small lymphocyte Most common causes: Iron deficiency Hemolysis Thalassemia (globin synthesis defect)

Iron deficiency: Most common cause of anemia across the age groups Most common nutritional deficiency world-wide US: toddlers, adolescent girls, and women of childbearing age Iron present as: Functional (hemoglobin, myoglobin. enzymes) Storage iron (ferritin, hemosiderin) 15-20% total Intracellular ferritin (aggregates form hemosiderin) and plasma ferritin Hemosiderin prominent in iron overload Transferrin as transporter of iron in plasma
Test q: Among the following, the most common cause of anemia is: iron deficiency. (Other choices: Bone marrow replacement by cancer, Intravascular hemolysis, Genetic defect in hemoglobin synthesis, and Vitamin deficiency.)

Microcyte: <6 mm MCV <70 fL

Most of Fe control occurs through intestinal absorption. Normal = 33% saturation = iron level of 100-120ug/dL = total iron binding capacity 300-350ug/dL Iron deficiency Ferritin (reflects iron storage pool) - low % saturation - <10% iron level - low total iron binding capacity (TIBC) - high

Hepcidin controls iron turnover.

Figure: Iron stain of bone marrow (Prussian blue stain) Macrophages laden with iron. Etiology: 1. dietary lack 2. impaired absorption 3. increased requirement 4. chronic blood loss(most importantly). normal iron balance, 1 mg of /day Many women consume marginally adequate amounts of iron Heme iron is much more absorbable than inorganic iron Infants: high risk due to the very small amounts of iron in milk The impoverished: suboptimal diets for socioeconomic reasons The elderly: restricted diets with little meat because of limited income or poor dentition. Teenagers: junk food.

Chronic blood loss: The most common cause of iron deficiency in the Western world. Iron deficiency in adult men and postmenopausal women in the Western world must be attributed to gastrointestinal blood loss until proven otherwise. Chronic Blood loss: GI source Hereditary telangiectasia
Test q: A 65M has experienced worsening fatigue for the past 9 months. On phys exam, he is afebrile and has a pulse of 91/min, respirations 18/min, and blood pressure 105/60 mm Hg. There is no organomegaly. A stool sample is positive for occult blood. Lab findings include Hgb 5.9 g/dL, hematocrit 3 17.3%, MCV 66m , platelet count 3 3 250,000/mm , and WBC count 7800/mm . The reticulocyte concentration is 3.9%. No fibrin split products are detected, and the direct and indirect Coombs test results are negative. A bone marrow biopsy specimen shows marked erythroid hyperplasia. Which of the following conditions best explains these findings? Chronic blood loss. (Other choices: Vitamin B12 deficiency, Aplastic anemia, Metastatic prostatic adenocarcinoma, Autoimmune hemolytic anemia.)

Polyp

Adenocarcinoma

Iron deficiency: Microcytic hypochromic anemia (hypochromia low MCH before microcytosis) Anisopoikilocytosis (increased RDW) Confirmation with iron studies: Low ferritin Low % saturation Low iron level High TIBC Decreased reticulocyte count
Test q: A 29F and mother of 7 children is short of breath and complains of fatigue. The youngest child was born two years ago. Her serum iron is low and the TIBC is increased. She has not been treated for her anemia. You expect the reticulocyte count to be: decreased. The patient has blood drawn for a CBC. You would expect the RDW to be: increased.

Stages of Iron Deficiency: Marrow RE Fe Plasma Fe TIBC Hgb Hypchromia Microcytosis Ferritin FEP Normal 2-3+ 75-150ug/dl 300-400 ug/dl 13-15 gm/dl 12-300 ng/ml 30-70 ug//dl Iron def no anemia 0 N or N or N <4 ng/ml N or Iron def mild anemia 0 N or N or <4 ng/ml >100 ug/dl Iron def severe anemia 0 <4 ng/ml >100 ug/dl

Hemolytic anemias: Premature destruction of RBCs (<120 days lifespan) Elevated EPO levels to compensate for RBC loss Lab results: Anemia AND Hemoglobinemia Hemoglobinuria Jaundice intravascular hemolysis Hemosiderinuria Decresed haptoglobin OR Jaundice extravascular hemolysis Decre ased haptoglobin Splenomegaly Hemoglobinemia: Jaundice

Above: Bone marrow aspirate smear with compensatory erythroid hyperplasia

Hemoglobinuria/Hemosiderinuria:

Very reddish-pink serum.

Hemolytic anemias: Hereditary Membrane defects: hereditary spherocytosis Abnormalities in red cell enzymes: G6PD deficiency Abnormalities of hemoglobin synthesis: thalassemias, hemoglobin S, hemoglobin C Acquired Immune: infections (malaria), alloantibodies (Rh incompatibilities, incompatible transfusion), autoantibodies (autoimmune disease, lymphomas) Non-immune: mechanical damage (artificial heart valves, DIC), membrane abnormalities (paroxysmal nocturnal hemoglobinuria)

Hereditary spherocytosis: RBC membrane disorder (mutations of genes encoding for membrane cytoskeleton membrane cytoskeletal protein) Cell membrane inflexibility-RBC becomes spheroid and less deformable (increased splenic sequestation)
Test q: A 30F has had a constant feeling of lethargy since childhood. On phys exam, she is afebrile and has a pulse of 80/min, respirations of 15/min, and blood pressure of 110/70 mmHg. The spleen tip is palpable, but there is no abdominal pain or tenderness. Lab studies 3 show Hgb 11.7 g/dL, platelet count 159,000/mm , and WBC count of 3 5390/mm . The peripheral blood smear shows spherocytosis. The circulating RBCs show an increased osmotic fragility. An inherited abnormality in which of the following RBC components best accounts for these findings? Membrane cytoskeletal protein. (Other choices: G6P dehydrogenase, -globin chain, -globin chain, heme) REPEATED x5

At every step, lose a piece of membrane to compensate. Proteins in RBC membrane:

Normal erythrocyte

Spherocyte (ankyrin defect)

Elliptocyte (spectrin defect)

Stomatocyte (tropomyosin defect)

Acanthocyte

Spherocyte:

In contrast to microcytic hypochromia, these are very round and dark due to higher Hgb concentration. Types of Membrane Loss Leading to Spherocyte Formation:

Anisocytosis, Spherocytes, Howell-Jolly bodies, MCHC

Pathophysiology of hereditary spherocytosis:

Young HS red cells are normal in shape, but the deficiency of membrane skeleton reduces the stability of the lipid bilayer, leading to the loss of membrane fragments as red cells age in the circulation. The loss of membrane relative to cytoplasm forces the cells to assume the smallest possible diameter for a given volume, namely, a sphere

Hereditary spherocytosis: Anemia Lifespan 10-20 days erythropoiesis (to compensate) insufficient to compensate Splenomegaly (reflection of normal spleen function) Splenectomy beneficial Jaundice Gallstones (pigment bilirubin) Diagnosis: Family history Osmotic lysis test Clinical Features: Compensated- in 20% to 30% of patients asymptomatic Aplastic crises Parvovirus kills red cell progenitors Because of the reduced life span of RBCs, cessation of erythropoiesis for even short time periods leads to sudden worsening of the anemia Hemolytic crises increased splenic destruction of red cells (e.g., infectious mononucleosis);

G6PD deficiency: G6PD deficiency is a recessive X-linked trait, Several hundred genetic variants G6PD- and G6PD Mediterranean, cause most of the clinically significant hemolytic anemias. G6PD- is present in about 10% of American blacks; G6PD Mediterranean, is prevalent in the Middle East.

Role of (G6PD) in defense against oxidant injury:

Test q: Three days after taking an anti-inflammatory medication that includes phenacetin, a 23y/o African American man passes dark reddish brown urine. He is surprised by this because he has been healthy all his life and has had no major illnesses. On phys exam, he is afebrile, and there are no remarkable findings. CBC shows a mild normocytic anemia, but the peripheral blood smear shows precipitates of denatured globin (Heinz bodies) w/supravital staining and scattered bite cells in the population RBCs. Which of the following is the most likely diagnosis? Glucose-6-phosphate dehydrogenase deficiency. REPEATED x2

G6PD deficiency: Episodic hemolysis exposures that generate oxidant stress: - infections - drugs - foods: fava bean, which generates oxidants when metabolized Favism Cross-linking of reactive sulfhydryl groups on globin chains, which become denatured and form membrane-bound precipitates known as Heinz bodies can damage the membrane

Sickle Cell Disease: Hereditary hemoglobinopathy Autosomal recessive th Point mutation in the 6 position of globin chain (ValineGlutamic acid) Aggregation and polymerization of HbS in low oxygen conditions 8% African Americans are heterozygous for this mutation (40% of the Hb is HbS) Figure: Anisocytosis, poikilocytosis, sickle cells (drepanocytes), target cells, Howell-Jolly bodies Sickle Cell Disease: Pathogenesis Clinical Course: - Anemia - Painful crises: microocclusions/infarcts of bone, lung, liver - Chronic hemolysis with hyperbilirubinemia - Splenic autoinfarction with infections with encapsulated bacteria (H. influenzae, S. pneumoniae) - Aplastic crisis associated with Parvovirus infection

Can result in infarct of various organs or microvascular occlusion.

Test q: A 40M who has had chronic anemia since childhood is admitted to the hospital w/fever and chest pain. On phys exam, his temp is 38.7C. A chest radiograph shows extensive patchy infiltrates. Lab studies include a blood culture positive for Streptococcus pneumoniae. Despite supportive therapy, he dies a few days later. At autopsy, there is a small, fibrotic, 5g spleen filled with deposits of iron and calcium and prominent expansion of the marrow space in the skull. Which of the following conditions is most likely to have resulted in these findings? Sickle cell anemia. Test q: A 4F of Mediterranean ancestry is diagnosed w/Sickle Cell Anemia. It is important that she be vaccinated ASAP for: Streptococcus pneumoniae. Test q: A 20M black sickle cell patient should have been vaccinated to prevent infection by: Streptococcus pneumoniae. Test q: A 17y/o black man w/a known history of sickle cell disease presents to the ER complaining of left upper quadrant pain suggestive of a splenic infarct. Microscopic exam of the spleen would most likely reveal: coagulative necrosis.

Spleen in sickle cell disease:

Sickle cells clogging up a sinusoid

Sickle Cell Disease laboratory diagnosis: SCD, homozygous

Hemoglobin electrophoresis: may only have 1 type of Hgb.

SC Disease: 90% HbS SC trait: 40% HbS and 60% HbA

Sickle cell Solubility Test:

Test q: A 35yo apparently healthy man undergoes a medical exam while applying for life insurance. He is not anemic. His hemoglobin electrophoresis is reported as: HbA 62%, HbS 35%, HbF 1%, HbA2 1%; no variant C, D, G, or H bands detected. The most likely diagnosis is which of the following? Sickle trait. Test q: In Sickle Cell disease (homozygous), the percentage of hemoglobin that is SS is: 90%. Test q: A 12M has a history of episodes of severe abdominal and back pain since early childhood. On phys exam, he is afebrile, and there is no organomegaly. Lab studies show Hgb 3 3 11.2 g/dL, platelet count of 194,000/mm , and WBC count of 9020/mm . The peripheral blood smear shows occasional sickled cells, nucleated RBCs, and Howell-Jolly bodies. Hemoglobin electrophoresis shows 1% Hgb A2, 6% HbF, and 93% HbS. Hydroxyurea therapy is found to be beneficial in this patient. Which of the following is the most likely basis for its therapeutic efficacy? Increase in production of HbF. (Other choices: Increase in production of HbA, decrease in overall globin synthesis, stimulation of erythrocyte production, increase in oxygen affinity of hemoglobin) Test q: A patient presents w/a microcytic hypochromic anemia. The PBS shows microcytic RBCs w/many target forms. Splenomegaly is present. The next diagnostic tests one should order is/are: TIBC and Hgb electrophoresis.

Above: Sickle cell solubility test. In this test, whole blood is added to a high phosphate buffer w/saponin and sodium dithionite, which causes the hemoglobin to become deoxyhemoglobin. Deoxyhemoglobin S is insoluble. The turbidity of the sample on the left indicates the presence of HbS. The clear sample on the right contains no HbS.

Thalassemia syndromes: Heterogeneous group of disorders leading to decreased synthesis of or chains of adult hemoglobin, HbA Thalassemias:

Thalassemias:

Globin Synthesis:

Thalassemia syndromes: Heterogeneous group of disorders leading to decreased synthesis of or chains of adult hemoglobin, HbA 0 + when homozygous associated with total absence of chain; some residual production of chain

Above: eta globin gene (arrows indicate mutations)