Approach To TheDiagnosis of Anemia Gerald A. Soff M.D.
Red Blood Cells (RBCs)• Nutrient Factors – Iron – Folic acid – B12• Life Cycle and Breakdown – Finite life span 120 ± 20 days – Removal of RBCs performed by the reticuloendothelial (RE) system
Generally, Two Different Systems Have Been Used To Classify Anemia.• The Morphologic System; – (1) Microcytic (MCV < 80), Hypochromic (MCH < 30) – (2) Macrocytic (MCV >100), Normochromic (MCH 30–34) – (3) Normocytic (MCV 80–100), Normochromic.• Red Cell Kinetics – Hypoproliferative (Decreased Production) vs. – Increased Destruction or Blood Loss
Normal Blood Smear Central Pallor normally approximately 1/3 of cell diameter.
Reticulocyte Stain• Precipitated ribosomal RNA in young red cells.• Normally Approximately 1-2% of total red cells.• ~50-100,000/ul absolute count• If reticulocytes are not elevated, (in setting of anemia), then it is low, i.e. hypoproductive.• Elevated reticulocyte index indicates response to hemolysis or blood loss.
Reticulocyte Index• Reticulocyte “Count” as a percent of red cells. Only obtained when ordered. (Similar, but distinguished from polychromatophilia.)• Reticulocyte Index = (Retic %)*(Pt. Hgb/Nl Hgb)• (In severe anemia, reticulocytes are released prematurely, so further divide retic. index by 2.)• Absolute reticulocyte number= – (red cell number) X (reticulocyte percent) – (Normal; approximately 50,000-100,000/ul)• If reticulocytes are not elevated, (in setting of anemia), then it is low, i.e. hypoproductive.• Elevated reticulocyte index indicates response to hemolysis or blood loss.
Microcytic/Hypochromic Red Cells • Processes which affect Hemoglobin Synthesis; – Reduced Globin chain synthesis (Thalassemias) – Iron deficiency – Some Myeloproliferative/ Myelodysplastic Syndromes (Deficiencies in porphyrin and heme synthesis) – Some anemia of chronic disease
Hemoglobin Synthesis • Key Components • Globin chain (protein) • Heme/porphyrin ring • Iron
Iron Deficiency• Nutritional deficiency rare after childhood. – May be seen in adolescent females, especially with onset on menses.• Essential to identify putative source of blood loss, i.e. Gyn, GI.• Rare for iron deficiency from hematuria or hemoptysis.• Decreased iron absorption seen with chronic acid neutralization in stomach from PPIs, due to reduced oxidation of ferrous to ferric form.
Iron DeficiencyPale conjunctiva,pale palm creases Koilonychia
Thalassemia; (Decreased Production of Globin Chains)• -Thalassemia; Decreased Production of - globin chains• -Thalassemia; Decreased Production of - globin chains.• Results in decreased hemoglobin synthesis and a microcytic/hypochromic anemia• Severity of disease is depending upon the number of mutant genes.
Production of Globin Chains • Hemoglobin is a Globin Tetramer • Hemoglobin can consist of two alpha chains and 2 beta class chains of globin. • 2 2; Hgb A • 2 2; Hgb A2 • 2 2; Hgb F
-Thalassemia Syndromes Normally four genes for -Globin, two on each of chromosome 16. – +; One normal/one mutant on chromosome. – 0; Both mutant genes on one chromosome. (Mostly in Asians) If two genes mutant, Thalassemia minor – Hemoglobin electrophoresis normal. If three genes mutant; Hemoglobin H Disease – Hgb H; 4 Tetramer If all four genes mutant; Hydrops Fetalis; – Hgb Barts; 4 Tetramer
-Thalassemia• Two types of mutant alleles;• 0; Absence of expression. Typically a mutation in coding region of gene (On chromosome 11)• +; reduced expression of globin gene. Typically promoter mutation.
Differentiation of Microcytic AnemiaIn iron deficiency, red cell number, Hemoglobin, and MCVare all proportionally reduced.In Thalassemia, the MCV is disproportionately low, and redcell number is “spared.” Iron Deficiency Thalassemia NormalRed Cell Number 3.8 5.8 4.5-6.5 (Male) (109/ml) 3.9-5.6 (Fem.) MCV fl 67 60 80-99 Hgb (g/dl) 7.5 10 13.5-17.5 (M) 11.5-15.5 (F)
- vs -Thalassemia• Since only the -chain is synthesized of the -class of globin chains, the ratio of Hgb A, A2, and F is not altered in -Thalassemia.• In -Thalassemia, Hgb A2 ( 2 2 ) and Hgb F ( 2 2) levels as % of total Hgb are increased due to selective loss of -globin.• Hemoglobin electrophoresis can distinguish between - and -Thalassemia. (best for quantitation by column chromatography).
Thalassemia Syndromes• Depending on genetic severity;• Chronic anemia, high out-put cardiac states• Hepatosplenomegaly due to extramedullary hematopoiesis.• Transfusion-Dependent (Thalassemia major) – Need for iron chelation.• Due to increased marrow turnover, need for folic acid replacement.
Anemia Of Chronic Disease- Chronic Inflammatory Block• Anemia due to decreased red cell production, related to relatively insufficient levels of erythropoietin, for the degree of anemia.• Seen in chronic inflammatory diseases (Connective Tissue Disease), chronic infection, etc.• Classic in chronic renal insufficiency (often before dialysis requirement).
Anemia of Chronic Disease Prussian Blue (Iron) Stain In bone marrow, absent red cell precursor iron, with adequate storage iron. Can’t make diagnosis in absence of iron.
Distinguishing Between Iron Deficiency And The Anemia Of Chronic Disease Iron deficiency Anemia of Chronic Disease Serum Iron Decreased Decreased Serum Transferrin Increased Decreased Serum Iron Binding Increased Decreased Capacity Serum Ferritin Decreased Normal or Increased Bone Marrow Iron Absent Present Stores Absent Cannot definitively diagnose Anemia of Chronic Disease inabsence of adequate iron stores.
Macrocytic Anemias Macrocytic anemias may arise from abnormal DNA synthesis, producing megaloblastic changes in the bone marrow. The major causes include deficiencies in vitamin B12 and folate. Alcoholism, (even in the absence of folate or B12 deficiency), apparently from a direct effect of ethanol on the bone marrow. Hypothyroidism, (unknown mechanisms), mild macrocytic anemia. Myelodysplasia, abnormal maturation of red cell precursors often produces macrocytosis.
Incidence of Various Macrocytic Anemias Alcohol abuse 36 % Vitamin B12 or folate deficiency 21 % Drug intake 11 % Accelerated erythropoiesis 7% Liver disease 6% Myelodysplasia 5% Hypothyroidism 2% Unexplained 12%Colon-Otero G, et al. Med Clin North Am. 1992; 76:581-597.
Megaloblastic Anemia All cell lines affected. Macrocytic Anemia (MCV > 100). Coexisting iron deficiency, thalassemia, inflammation, may prevent macrocytosis. Hypersegmented PMN (any 6 lobe, >5% 5 lobed). Role of folate and vitamin B12 in DNA synthesis. – Additional role for B12 responsible for neurological and other manifestations.
Subacute Combined Degeneration of Spinal Cord in Pernicious Anemia
Vitamin B12 Absorption Gastric acid separates B12 from food. Parietal cells produce Intrinsic Factor: Binds B12 B12:IF complex absorbed in terminal ileum
The Schilling Urinary Excretion Test of Vitamin B12 AbsorptionSubjects B12 Given Alone B12 Given with Intrinsic FactorNormal 18 (9-36) -Pernicious anemia 0.5 (0-1.2) 13 (6-31)Malabsorption 3.6 (0-19) 3.3 (0-10)*
Pernicious Anemia Due to AutoimmuneReaction to Intrinsic Factor/Parietal Cells Immunofluorescence for Pernicious Anemia anti-parietal cell antibodies Atrophy, loss ofNormal parietal cells,Stomach gastric glands
Megaloblastic Erythroid Precursors Normal Megaloblastic
Normocytic/Normochromic Anemias• Intrinsic bone marrow disease• (1) Hypoplasia, (aplastic anemia or pure red cell aplasia)• (2) Infiltration by abnormal tissue, as in multiple myeloma, leukemia, fibrosis, or metastatic malignancy• (3) Myelodysplastic disorders, in which abnormal maturation occurs.• Diminished Erythropoietin (Anemia of Chronic Inflammatory Block or Anemia of Chronic Disease)
Hemolytic Anemia• Premature destruction of red blood cells – Red cells normally circulate approximately 100 to 120 days.• Intravascular vs. extravascular hemolysis.• May be immune or non-immune mediated.
Hemolytic Anemia, Laboratory Findings• Reticulocyte index is >3% and the absolute reticulocyte count is >100,000/mm3.• Immature erythrocytes prematurely leave bone marrow. The indirect bilirubin is elevated and accounts for >80% of the total bilirubin.• The serum LDH level is increased.• Serum haptoglobin is diminished.
Causes of Hemolysis (by Site of Abnormality)• Intrinsic to the red cell Extrinsic to the red cell• Abnormal hemoglobins Immunologic – Warm antibody – Sickle cell anemia – Cold antibody – Hemoglobin C, E, etc. – Drugs• Enzyme defects Mechanical – Glucose-6-phosphate – March hemoglobinuria dehydrogenase deficiency – Traumatic cardiac hemolytic anemia – Pyruvate kinase deficiency, etc. – Microangiopathic hemolytic anemia• Membrane abnormalities Infectious – Hereditary spherocytosis, – Malaria elliptocytosis – Clostridium perfringens infection – Acanthocytosis Chemicals – Paroxysmal nocturnal Hypersplenism hemoglobinuria
Spherocytes• Decreased surface-to-volume ratio• Loss of cellular membrane – Inherited membrane defect – Acquired via warm (IgG) autoantibodies
Hereditary Spherocytosis• Due to Abnormality of red cell cytoskeleton – Most mutations in Ankryn, Band 3 (Also Spectrin, Band 4.2)• Common autosomal Dominant – Approx 1/5000 (Not just Northern Europeans)• Clinically; Anemia, Jaundice, splenomegaly• Osmotic Fragility Test• Management; Splenectomy
Glucose 6-PhosphateDehydrogenase Deficiency • Enzyme defect in hexose- monophosphate shunt pathway. • Normally responsible for generation of reducing agents in cells (Reduced glutathione). • RBC sensitive to oxidant stress, drugs, infections, malaria, etc. • Heinz bodies; denatured (oxidized) hemoglobin, usually seen on reticulocyte stain. • X-Linked inheritance; – Mediterranean (Severe) – African (Mild) – Asian (Varied)
Leukoerythroblastosis/ Myelophthisic • Marrow invasion with abnormal cells (carcinoma, advanced myeloproliferative disease, fibrosis, etc.) • Immature red and white cells (promyelocytes, myelocytes, metayelocytes, teardrop cells.Nucleated rbc Myelocyte
Teardrop Cells Most prominently in thalassemias, marrow infiltration by fibrosis or malignancy. – From distortion of the erythrocytes as they travel through the vasculature of an abnormal bone marrow or spleen.
Approach To Anemia• Have a system, based on rates of production/destruction and morphology – Morphology • (1) Microcytic/Hypochromic • (2) Macrocytic • (3) Normocytic/Normochromic. – Red Cell Kinetics • Hypoproliferative (Decreased Production) vs. • Increased Destruction or Blood Loss• LOOK AT YOUR PATIENTS’ SMEARS.• Do not treat anemia with empiric iron therapy.
Burr Cells (Echinocytes)• Blunt, fairly symmetrical projections.• Prominent in renal failure from any cause – (may also occur with liver diseases, especially when uremia coexists.)• (Can develop as a storage artifact)
Spur Cells (Acanthocytes)• Several irregularly sharp projections of unequal length.• Most of the cells are also small and lack central pallor.• Prominent in liver disease, usually alcoholic cirrhosis, with an increase in the cholesterol:phospholipid ratio in the red cell membrane, leading to hemolysis.
Bite Cells (Degmacyte)• A semicircular defect in their edge that resembles a bite mark.• Oxidative destruction/precipitation of hemoglobin, often in patients with a deficiency of the enzyme glucose-6-phosphate dehydrogenase (G6PD) or unstable hemoglobins.
Heinz-Body Preparation• Fragments of Denatured Hemoglobin, usually bound to red cell membrane.
Red Cell Agglutination• Red Cells coated with antibodies – Typically IgM, but can be IgG• Extensive clumping typically develops with high levels of IgM, which are present in Waldenstrom’s macroglobulinemia or in cold-agglutinin hemolytic anemia from such causes as infectious mononucleosis and Mycoplasma pneumoniae infection.