RBC Morphology and Anemias

CLBT 2200: CLT - CERTIFICATION REVIEW HEMATOLOGY REVIEW - RBC MORPHOLOGY & ANEMIA

RBC Morphology

  • Anisocytosis

    • Definition: Variation in cell size.

    • Measurement: Evaluated by Mean Corpuscular Volume (MCV) and Red Cell Distribution Width (RDW).

  • Anisochromia

    • Definition: Variation in cell color.

    • Measurement: Evaluated by Mean Corpuscular Hemoglobin Concentration (MCHC).

  • Poikilocytosis

    • Definition: Variation in cell shape.

  • Polychromasia

    • Definition: Variation in cell color, typically presenting as pink with a bluish tint.

RBC Indices

  • Mean Cell Volume (MCV)

    • Description: Average size of red blood cells.

    • Normal Range: 80 – 100 fL.

  • Mean Cell Hemoglobin (MCH)

    • Description: Amount of hemoglobin in an individual red blood cell, influencing the color.

    • Normal Range: 27 – 33 pg.

  • Mean Cell Hemoglobin Concentration (MCHC)

    • Description: Ratio of hemoglobin to cell size.

    • Normal Range: 32 – 36%.

Red Cell Distribution Width (RDW)

  • Definition: Measure of anisocytosis, variations in red blood cell size.

  • Platelet Cell Distribution Width (PDW)

    • Definition: Measure of variation in size of platelets.

Hemoglobin

  • Function: Gas transport, particularly O2.

    • Influences: Blood pH, 2,3-diphosphoglycerate (2,3-DPG) levels, and iron valence.

  • Types of Hemoglobin:

    • Oxyhemoglobin: Iron in +2 oxidation state (Fe +2) bonded to O2.

    • Deoxyhemoglobin: Iron in +2 oxidation state (Fe +2).

    • Carboxyhemoglobin: Iron in +2 oxidation state (Fe +2) bonded to CO.

    • Carbaminohemoglobin: Iron in +2 oxidation state (Fe +2) bonded to CO2.

    • Methemoglobin: Iron oxidized to +3 state (Fe +3).

    • Sulhemoglobin: Presence of sulfur in the hemoglobin structure.

Hematocrit

  • Definition: Pack cell volume, generally three times the hemoglobin level (Rule of 3).

  • Methods:

    • Manual: Spun hematocrit using a capillary tube; measure the percentage of RBCs.

    • Automated: Calculated based on MCV and total RBC count.

Normal Hematocrit Values

  • Females: 42 +/- 5.

  • Males: 47 +/- 7.

  • Hgb-HCT Relationship: Hgb X 3 = HCT +/- 3.

Reticulocyte Count

  • Definition: Measures bone marrow activity and should not exceed 2% in adults.

    • Seen as polychromatophilia on Wright stain.

Method of Reticulocyte Counting

  • Manual Method:

    • Stain RNA with supravital stain (Methylene Blue or New Methylene Blue).

    • Prepare smear and air dry; count number of retics in 1000 RBCs.

  • Automated Method:

    • Use fluorescent stain to tag RNA and measure with flow cytometry.

Reticulocyte Counting Calculations

  • Uncorrected Count: % of reticulocytes to total RBCs.

  • Corrected Count: Uncorrected count x Patient hematocrit / Normal hematocrit.

  • Retic Production Index (RPI): Corrected count / Maturation time of shift retics.

RBC Morphology Overview

  • Anisocytosis: Variation in size.

  • Poikilocytosis: Variation in shape.

  • Anisochromia: Variation in color.

Hypochromia vs Hyperchromia

  • Normal cells and variations; differences in stain perceivable degrees.

Sickle Cells vs Hemoglobin C Crystals

  • Various shapes and structures for abnormal hemoglobin formations like sickle forms and Hemoglobin C crystals.

Acanthocytes vs Schistocytes

  • Distinctions between normal RBCs and these abnormal shapes that arise under pathological conditions.

Ovalocytes vs Tear Drop Cells

  • Normal structures and pathological variations such as ovalocytes and teardrop (dacryocytes) forms.

Stomatocytes vs Target Cells

  • Identification of normal cells compared to stomatocytes and codocytes (target cells).

Rouleaux vs Agglutination

  • Identifying the presence of stacked RBCs (rouleaux) vs true agglutination which indicates immunologic activity.

Helmet vs Bite & Blister Cells

  • Characteristics of helmet cells versus normal blister cells in pathology.

Echinocytes vs Bite Cells

  • Definitions and distinctions among these abnormal RBC shapes (echinocytes and bite cells).

RBC Inclusions

  • Types and Causes:

    • Wright Stain Inclusions:

    • Basophilic stippling: Precipitated ribosomes and RNA.

    • Pappenheimer bodies: Precipitated iron.

    • Howell-Jolly bodies: Nuclear remnants (DNA).

    • Hgb S or Hgb C crystals: Abnormal Hgb.

    • Cabot rings: Nuclear remnant.

  • Supravital Stains Inclusions:

    • Siderocytes: Precipitated iron known as Pappenheimer bodies.

    • Heinz bodies: Denatured hemoglobin.

    • Reticulocytes: Ribosomal RNA visible.

Basophilic Stippling vs Howell-Jolly Bodies

  • Visual distinctions and pathological implications of two types of inclusions seen on a smear.

Pappenheimer Bodies vs Siderocytes

  • Comparing these iron-containing inclusions found in RBCs and confirming with Prussian blue stain.

Reticulocytes vs Heinz Bodies

  • Caution against confusing reticulocyte patterns with basophilic stippling.

RBC Inclusions - Parasites

  • Most common parasitic infections seen in RBCs:

    1. Malaria

    2. Babesia microti

    3. Leishmania

Anemia Evaluation

  • Types of Anemia:

    • Iron deficiency, hypochromic anemias.

    • Megaloblastic anemias.

    • Aplastic anemias.

    • Hemolytic anemias.

    • Anemia associated with non-hematological disorders.

Causes of Iron Deficiency

  • Increased Loss of Iron:

    • Conditions: Menstrual bleeding, GI bleeding, malignancy, ulceration, diverticulosis, parasites, hemorrhoids.

  • Inadequate Dietary Intake:

    • Deficient iron in diet.

  • Inadequate Maternal Stores:

    • During pregnancy.

  • Malabsorption:

    • Celiac disease, defective gastric function, achlorhydria, copper deficiency.

Iron Deficiency Anemia

  • Also known as microcytic, hypochromic anemia.

  • Characteristic: Most RBCs are smaller than the nucleus of a small lymphocyte, indicating microcytic, hypochromic nature.

Anemia of Chronic Disease

  • Characteristics: Usually less severe than iron deficiency; RBCs appear normal; serum iron and Total Iron Binding Capacity (TIBC) decreased.

  • Examples:

    • Inflammatory rheumatoid arthritis, Crohn’s disease, immune hemolysis, infectious mononucleosis, Mycoplasma pneumonia infection.

Hemochromatosis

  • Cause: Autosomal recessive disease, typically manifesting in adults aged 50-60 years.

  • Pathology: Absorption of up to 4 g/day of iron, leading to deposits in tissues and organs (pancreas, liver, spleen).

  • Bronze Diabetes: Development of adult-onset diabetes due to pancreatic damage from iron deposits.

  • Treatment: Therapeutic phlebotomy or iron chelating agents.

Sideroblastic Anemia

  • Cause: Can be inherited (sex-linked) or acquired (e.g., hepatosplenomegaly).

  • Laboratory Findings: Mild anemia, variable RBC indices, increased RDW; presence of Pappenheimer bodies observed (siderotic granules), indicating iron overload.

  • Treatment: Vitamin B6 (pyridoxine).

Lead Poisoning

  • Pathology: Lead interferes with iron storage in mitochondria, damaging enzymes involved in heme synthesis, leading to microcytic, hypochromic anemia with basophilic stippling.

  • Exposure Sources: Children from lead-based paint, adults from occupational exposure related to batteries and pottery.

Macrocytic, Normochromic Anemia

  • Profile: MCV ranging from 100-160 fL; there may be an increase in MCH; MCHC remains normal.

    • RBC usually low with a decreased retic count.

    • WBC normal to low with hypersegmented neutrophils (>5 lobes).

    • Platelets: Normal to low.

Megaloblastic Anemia

  • Characteristics:

    • MCV > 100 fL with increased RBC precursors in the bone marrow resulting in decreased release into peripheral blood (PB).

    • Decreased reticulocytes; fragile RBCs leading to increased serum bilirubin and lactate dehydrogenase (LDH).

Pernicious Anemia

  • Cause: Gastric parietal cell atrophy results in lack of intrinsic factor (IF).

    • More common in Scandinavian, English, and Irish descendants, particularly after age 50.

  • Pathology: Congenital absence of IF, involvement of autoantibodies targeting parietal cells and IF.

Other Causes of Vitamin B12 Deficiency

  • Examples:

    • Gastrectomy (removal of parietal cells).

    • Blind loop syndrome (bacterial overgrowth in the small intestine).

    • Fish tapeworm (Diphyllobothrium latum).

    • Drugs (e.g., alcohol, anti-tuberculosis).

Aplastic Anemia

  • Definition: Group of disorders characterized by cellular depletion and fatty replacement of bone marrow.

  • Features:

    • Decrease in white blood cells (WBC), red blood cells (RBC), and platelets (pancytopenia).

  • Causes: Can be due to drugs, chemicals, irradiation, infections, or immune dysfunction.

Clinical Manifestations of Aplastic Anemia

  • Gradual onset of symptoms; includes fatigue, dyspnea, palpitations, bleeding tendency, infections, pallor, petechiae, purpura, ecchymoses, and mucosal bleeding.

Laboratory Examination for Aplastic Anemia

  • Findings:

    • Generally, Hgb < 7.0 g/dL, normocytic/normochromic morphology, moderate anisocytosis and poikilocytosis.

    • Reticulocyte count: normal to low; white cells and platelets low; no specific morphologic abnormalities observed.

Hereditary Spherocytosis

  • Inheritance: Usually autosomal dominant; 25% of families may exhibit autosomal recessive patterns.

  • Etiology: Results in a decreased surface-to-volume ratio due to loss of surface area, classifying it as a red blood cell membrane defect leading to spherocytes.

Osmotic Fragility Test

  • Purpose: Measures surface-to-volume ratio of red cells.

  • Test Results: Increased fragility with spherocytes which lyse more readily than normal cells.

  • Procedure: Standard blood samples added to varying concentrations of saline, examining lysis of cells.

    • Screening results: Normal (0.85% and 0.45%) show no lysis; spherocytosis shows lysis.

Sickle Cell Anemia

  • Most Common Type of Severe Hemoglobinopathy: Incidence of 1:375 in African American live births.

  • Cause: Autosomal recessive trait for hemoglobin S due to a single amino acid substitution of valine for glutamic acid at the 6th position of beta chains.

Hemoglobin Inheritance

  • Normal Pattern: Homozygous (A/A), Sickle Cell Disease (S/S), Sickle Cell Trait (A/S).

Sickle Cell Anemia Pathophysiology

  • Functionality: Oxygenated Hemoglobin S performs normally. Deoxygenated Hemoglobin S becomes insoluble, leading to polymerization forming sickle shapes.

  • Sickling: Depends on oxygenation, pH, and dehydration of patient; can be reversible initially, but repeated sickling causes membrane damage.

Clinical Features of Sickle Cell Anemia

  • Signs include chronic hemolytic anemia with hemoglobin levels ranging 6-8 g/dL, asthenic physique, mild jaundice, vaso-occlusive episodes causing ischemic tissue injury.

  • Affected Organs: Primarily spleen, kidney, and bone marrow.

  • Diagnosis Method: CBC, reticulocyte count, peripheral blood smear, hemoglobin electrophoresis findings indicating NC anemia, anisocytosis, poikilocytosis, target cells, fragments, polychromasia, nucleated red blood cells, sickle cells, and siderotic granules.

Hemoglobin Electrophoresis for Diagnosis

  • Confirmation Tests: Cellulose acetate at alkaline pH and citrate agar at acid pH.

    • Sickle Cell Disease will show Hgb S and increased Hgb F.

    • Sickle Cell Trait will show decreased Hgb A1, and increased Hgb S and Hgb F.

Hemoglobin C Disease & Trait

  • Etiology: Amino acid substitution of lysine for glutamic acid at the 6th position of beta chains.

  • Clinical Signs: Mild chronic hemolytic anemia, NC anemia with target cells, micro spherocytes, fragments, Hgb C crystals which yield a “gold brick” appearance of RBCs.

  • Electrophoresis Findings: Hgb C 95%, Hgb F < 7%, no Hgb A.

Hemoglobin D & E Disease

  • Hgb D: Peripheral blood typically appears unremarkable; may show some targets; migrates with Hgb S on electrophoresis.

  • Hgb E: Presents little to no anemia but displays microcytic hypochromic cells with target cells; electrophoresis shows roughly 70% Hgb A and 30% Hgb E.

Fetal Hemoglobin (Hgb F)

  • Composition: Predominantly consists of 2 alpha chains and 2 gamma chains.

  • Screening Method: Kleihauer-Betke test used to detect fetal hemoglobin wherein fetal cells staining orange-red do not dissolve in acid, in contrast to adult cells which dissolve entirely, creating ghost cells for counting.

Thalassemias

  • Historical Background: Described in 1925 by Cooley as "Cooley’s Anemia"; initially cases observed primarily in those of Mediterranean descent.

  • Types: Categorized based on affected chains; includes alpha and beta thalassemia.

Thalassemias Explained

  • Definition: Genetic defects lead to quantitative defects in synthesis of globin chains resulting in imbalances, decreased hemoglobin levels, with excessive production of one chain type.

Genetics of Hemoglobin Synthesis

  • Chromosome #16: Contains alpha cluster genes that produce alpha chains but do not do so at equal rates; abnormalities may result from crossing over or mispairing leading to gene deletions.

  • Chromosome #11: Contains beta, delta, and gamma genes; mutations typically lead to point mutations resulting in varied expressions of hemoglobin.

Beta Thalassemias

  • Known as: Also referred to historically as Cooley’s anemia.

  • Physiology: Characterized by unbalanced globin chain synthesis resulting in reduced or absent beta chains, presence of alpha chains, and compensatory increases in gamma and delta chains, inflating levels of Hgb F and Hgb A2.

Beta Thalassemia Variants

  • Beta Thalassemia Major: Defined as severe hypochromic microcytic anemia with hemoglobin < 7 g/dL; characterized mainly by Hgb A2 and Hgb F; features trajectory indicating skeletal changes in long bones and skull upon X-ray.

  • Heterozygous Beta Thalassemia: Described as a mild form of chronic hypochromic microcytic anemia also termed as thalassemia minor, with Hgb levels ranging from 10-14 g/dL.

Alpha Thalassemia

  • Genetic Aspects: Typically, 2 functional alpha genetic sequences available on each chromosome; mutations result in varying severities of disease based on numbers of deletions.

Alpha Thalassemia Conditions

  • Hgb H Disease: Results from deletions that produce insoluble inclusions in mature RBCs known as Hgb H, which subsequently stain with brilliant cresyl blue giving a distinct appearance reputed as “golf ball” appearance.

  • Hgb Bart: A condition whereby only gamma chains exist in high amounts, associated with severe outcomes incompatible with life.

Hemoglobin Lepore

  • Description: Resultant fusion of delta and beta chains due to a fused gene from unequal crossing over; patients homozygous for Hgb Lepore express severe anemia.

  • Lab Diagnosis: Microcytic hypochromic anemia with decreased Hgb, Hct, MCV, MCH readings and variations in RBC count.

Anemia of Inflammation

  • Etiology: Characterized by decreased RBC lifespan, impaired iron metabolism, including faulty iron release from reserves, and reduced erythropoietin levels from macrophage and lymphocyte cytokines.

  • Characteristics: Presents mild anemia, maintains normocytic and normochromic characteristics, with low retic count and normal to low levels of serum iron and TIBC, even while storing iron in red cells.

Anemia of Infection

  • Etiology: Similar to anemia of inflammation; involves suppression of erythropoiesis driven by cytokine activity with bone marrow histiocytes engaging in phagocytizing RBCs and WBCs and potential effector steps mediated by HIV in the destruction of RBC precursors in bone marrow.