Cell counts, ESR, Retic Counts, Hgb and Hemoglobinopathies.

CLBT 2200: CLT CERTIFICATION REVIEW

• Unit 2 Topics: Cell counts, Hct (Hematocrit), ESR (Erythrocyte Sedimentation Rate), Retic Counts, Hgb (Hemoglobin) and Hemoglobinopathies.

Anticoagulants

• Ethylendiaminetetraacetic Acid (EDTA)

  • Action: Calcium chelator

  • Use: For cell counts and morphology examination

  • Preparation: Prepare smear within 2 hours of collection

  • Note: Platelets may clump in vitro (platelet satellitism).

• Sodium Citrate

  • Action: Calcium chelator

  • Use: Coagulation studies

  • Blood to Anticoagulant Ratio: 9:1

• Heparin

  • Action: Antithrombin

  • Use: Reserved for special testing, limited use in hematology.

Manual Cell Counts

• Formula for Cell Counts:
  $ext{Number of cells counted} imes ext{VCF} imes ext{Dilution factor}$

• Cells Counted:

  • Red Blood Cells (RBC): Count squares of 0.2 mm x 0.2 mm

  • White Blood Cells (WBC): Count squares of 1 mm x 1 mm

  • Platelets: Count in the center of a W square which contains 25 R squares.

• Neubauer Ruled Counting Area:

  • Small square area: 1/400 sq. mm

  • Loading well chamber depth: 0.1 mm

Coulter Counter Principle

• Measurement of Cells:

  • Saline conducts electricity better than cells (RBCs, WBCs, platelets).

  • Each cell passing through a test opening causes a drop in electrical current; the number of cells is proportional to the drop in electrical current.

Measurement of MCV (Mean Cell Volume)

• Method:

  • Volume displacement by red cells passing through the test opening (aperture) displacing an equal volume of saline corresponding to the size of the red cell.

Histograms

• Histogram of RBCs:

  • Example values:

  • RBC: 4.63 M/uL

  • Hgb: 13.7 g/dL

  • Hct: 40.3%

  • MCV: 87.1 fL

  • MCH: 29.6 pg

  • MCHC: 34.0 g/dL

  • RDW: 15.1%

  • Population: Middle-aged individuals

  • Distribution: Plotted representing RBC population with platelets and younger cells.

• Histogram of WBCs:

  • Classification of WBC types is based on size, displaying a full range of WBC types.

• Scatterplot Visualization:

  • Resembles a hiker: Head indicates neutrophils, Legs indicate lymphocytes, Backpack indicates monocytes, Arms indicate eosinophils, Belt indicates basophils, Feet indicate nucleated RBCs.

Red Blood Cell (RBC) Indices & Formulas

• Mean Cell Volume (MCV):
  ext{MCV} = rac{ ext{Hct ext{%}}}{ ext{RBC count (uL)}} imes 10

• Mean Cell Hemoglobin (MCH):
  $ext{MCH} = rac{ ext{Hgb (g/dL)}}{ ext{RBC count (uL)}} imes 10$

• Mean Cell Hemoglobin Concentration (MCHC):
  ext{MCHC} = rac{ ext{Hgb (g/dL)}}{ ext{Hct ext{%}}} imes 100

• Red Cell Distribution Width (RDW):

  • Measures the coefficient of variation of RBC volume.

Definitions of RBC Indices

• MCV (Mean Cell Volume):

  • Average size of red blood cells; normal range: 80-100 fL.

• MCH (Mean Cell Hemoglobin):

  • Amount of hemoglobin per red blood cell (indicates cell color); normal range: 27-33 pg.

• MCHC (Mean Cell Hemoglobin Concentration):

  • Ratio of hemoglobin to cell size; normal range: 32-36%.

• RDW (Red Cell Distribution Width):

  • Measure of anisocytosis, or variation in red blood cell size.

RBC Morphology

• Anisocytosis: Measure of variation in red cell size, assessed with MCV & RDW.

• Anisochromia: Variation in red cell color, measured with MCHC.

• Poikilocytosis: Variation in cell shape.

• Polychromasia: Variation in cell color (usually pink with bluish tint).

Hemoglobin Structure

• Composition:

  • Heme: 4% (4 heme groups)

  • Globin: 96% (composed of 1 polypeptide chain consisting of 2 pairs of globin chains or monomers).

• Heme:

  • Tetracarbon compound with a 4-carbon, 4-nitrogen structure (tetrapyrrole ring).

  • Iron (Fe) in the +2 oxidation state at the center.

• Globin:

  • Tetramer composed of paired polypeptide chains (2 alpha chains & 2 beta chains).

Globin Chains Description

• Zeta Chain:

  • Precursor to alpha chains, present in embryonic hemoglobin before 12 weeks.

• Gamma Chain:

  • Beta chains with amino acid substitution at the 136th position (either alanine or glycine), seen in embryonic hemoglobin & Hgb F (fetal hemoglobin post-12 weeks).

• Delta Chain:

  • Beta chains with 8 amino acid substitutions, found in embryonic & adult Hgb A2 only.

• Beta Chain:

  • A 146 amino acid chain with histidine in the 92nd position, found in embryonic & adult hemoglobin A1 only.

Stages of Hemoglobin Formation

• Mesoblastic Phase: Formation of embryonic hemoglobin begins in yolk sac prior to week 12.

• Hepatic Phase: Fetal hemoglobin replaces embryonic hemoglobin after week 12.

• Medullary Phase: Adult hemoglobin becomes predominant; small amounts of Hb F are produced.

Abnormal Hemoglobins and Their Diseases

• Hgb S:

  • Abnormal beta chains (valine substitution at the 6th amino acid position of beta chains).

  • Results in Sickle Cell Anemia.

• Hgb C:

  • Abnormal beta chains (lysine substitution at the 6th amino acid position of beta chains).

  • Causes Hemoglobin C Disease & Hemoglobin S/C Disease.

• Hgb E:

  • Abnormal beta chains (lysine substitution at the 26th amino acid position of beta chains).

  • Results in Hemoglobin E Disease.

• Hgb H:

  • Composed of 4 beta chains (no alpha chains).

  • Associated with Alpha Thalassemia Intermedia.

• Hgb M:

  • Tyrosine is substituted for histidine on either alpha or beta chains, leading to Hemoglobin M Disease (autosomal dominant).

Function of Hemoglobin

• Role: Gas transport (Oxygen - O2).

• Influencing Factors: Blood pH, 2,3-DPG level, and valence of iron ions.

• Types of Hemoglobin Forms:

  • Oxyhemoglobin (Fe+2 - O2)

  • Deoxyhemoglobin (Fe+2)

  • Carboxyhemoglobin (Fe+2 - CO)

  • Carbaminohemoglobin (Fe+2 - CO2)

  • Methemoglobin (Fe+3)

  • Sulfhemoglobin (sulfur attached)

Abnormal Forms of Hemoglobin

• Sulfhemoglobinemia:

  • Condition characterized by the presence of sulfur in hemoglobin, resulting in green blood.

  • Example: A patient taking 200 mg/day of Alsometreptan for migraines developed green-colored blood due to sulfhemoglobinemia.

• Methemoglobin:

  • Iron in hemoglobin in Fe+3 state, oxidized, unable to bind O2.

  • Levels > 10% associated with cyanosis (blue color), especially in lips & fingers; seen in Hemoglobin M Disease.

Measurement of Hemoglobin

• Cyanmethemoglobin Method:

  • Measures methemoglobin, carboxyhemoglobin, oxyhemoglobin, but does not measure sulfhemoglobin.

• Reaction Principle:

  • Use of a lysing agent to free hemoglobin which is measured at an absorbance of 540 nm.

  • All forms of hemoglobin except sulfhemoglobin react to form cyanmethemoglobin (the red color).

Normal Hemoglobin Ranges

• Women: 14 +/- 2 g/dL (11.5 – 15.5 g/dL)

• Men: 16 +/- 2 g/dL (13.5 – 17.5 g/dL)

• Child (<1 year): 11.0 - 13.5 g/dL

• Newborn: 15.0 - 21.0 g/dL

Hematocrit (Hct)

• Definition: Packed cell volume, traditionally estimated as 3 times the hemoglobin.

  • Formula: $ext{Hgb} imes 3 = ext{Hct} +/- 3$

• Normal Hematocrit Levels:

  • Females: 42 +/- 5%

  • Males: 47 +/- 7%

• Methods for Measurement:

  • Manual: Spun Hct (capillary tube centrifuged, and % of RBCs measured).

  • Automated: Calculated from MCV and total RBC count.

Clinical Significance of Hematocrit Levels

• High Hematocrit Levels (Causes):

  • Polycythemia, heart or kidney issues, anabolic steroid intake, dehydration (including diarrhea), lung problems, burns, smoking, high altitudes.

Reticulocyte Count

• Measure of Bone Marrow Activity:

  • Should not exceed 2% in adults, representing activity within the past 24 hours.

  • Seen as polychromatophilia or polychromasia on Wright stain.

• Description of Reticulocytes:

  • Immature red blood cells that are non-nucleated but contain some RNA, turning bluish on Wright stain and are larger than mature RBCs.

  • Also referred to as a shift cell.

Clinical Significance of Reticulocyte Counts

• Normal Range: 0.5 - 1.5 %

  • Counts less than 0.5% indicate no bone marrow activity.

  • Counts greater than 2% indicate increased bone marrow activity due to conditions like blood loss, hemolytic anemias, autoimmune issues, sickle cell disease, hemolytic transfusion reactions, iron deficiency anemia, or thalassemias.

• Decreased Count (< 0.5%):

  • Associated with radiation, immunosuppressive drugs, aplastic anemia, pernicious anemia, leukemia (late stage).

• Increased Count (> 2.0%):

  • Associated with blood loss, hemolytic anemias, autoimmune responses, sickle cell disease, hemolytic transfusion reactions, iron deficiency anemia, and thalassemias.

Reticulocyte Counting Methods

• Manual Counting:

  • Stain RNA with supravital stain such as methylene blue or new methylene blue.

  • Prepare a smear and air dry; count number of reticulocytes per 1000 RBCs.

• Automated Counting:

  • Employs fluorescent stains to tag RNA, measured using flow cytometry.

• Correction Factors:

  • Uncorrected: % retics to total RBCs.

  • Corrected: Uncorrected count multiplied by patient Hct divided by normal Hct.

  • Retic Production Index (RPI) formula: $rac{ ext{corrected count}}{ ext{maturation time of shift retics}}$.

Miller Disc Method

• Method Description:

  • Utilizes an ocular micrometer with a small square (B) within a large square (A) in a 1:9 ratio.

• Counting Process:

  • Count reticulocytes in 20 large squares.

  • Count red blood cells in 20 small squares.

Osmotic Fragility Test

• Purpose:

  • Measures the ratio of surface area to volume of red blood cells.

  • Increased fragility with spherocytes, as they lyse more readily than normal cells.

• Procedure:

  • Standard amount of blood added to varying concentrations of saline to assess lysis.

• Screening Procedure Results:

  • 0.85% and 0.45%: Normal shows no lysis; spherocytosis may result in lysis.

Sickle Cell Anemia

• Pathophysiology:

  • Oxygenated Hgb S functions normally; deoxygenated Hgb S is insoluble leading to polymerization of beta chains, causing cells to take a sickle shape.

  • Sickle shape depends on factors such as oxygenation level, pH, and patient dehydration. Sickle formation can reverse up to an extent, but repeated sickling damages cell membrane.

• Clinical Features:

  • Characterized by chronic hemolytic anemia with Hgb levels between 6-8 g/dL, ashen physique, mild jaundice, and vaso-occlusion that can lead to ischemic tissue injury, particularly in spleen, kidney, and bone marrow.

• Diagnosis:

  • Involves CBC, reticulocyte count, peripheral blood smear, hemoglobin electrophoresis, and examining peripheral blood smear for normocytic normochromic anemia combined with anisocytosis, poikilocytosis, target cells, fragments, polychromasia, nucleated RBCs, sickle cells, siderotic granules, and Howell-Jolly bodies. Sickle cell trait presents a fairly normal smear.

Sodium Metabisulfite Test

• Test Description:

  • A tube solubility test that uses reagents to reduce oxygen, causing insoluble hemoglobin to precipitate, and turbidity is read.

  • Warning: Do not use on infants; false negatives can occur.

Normal Hemoglobin Type Ranges

• Hgb A1: 96% (2 alpha + 2 beta chains)

• Hgb A2: 2 - 3% (2 alpha + 2 delta chains)

• Hgb F: 1 - 2% (2 alpha + 2 gamma chains).

Hemoglobin Electrophoresis

• Analysis Method:

  • At alkaline pH (cellulose acetate): Hgb A1 near anode; Hgb F/G next; Hgb S/D next; Hgb A2 next; Hgb C/E closest to cathode.

• At acid pH (citrate agar): Hgb F nearest anode; Hgb A1/G/D & E next; Hgb S next; Hgb C closest to cathode.

Confirmation Tests for Sickle Cell

• Tests:

  • Hgb electrophoresis in both cellulose acetate at alkaline pH and citrate agar at acid pH.

  • Sickle cell disease shows Hgb S and increased Hgb F, while sickle cell trait shows decreased Hgb A1 and increased Hgb S and F.

Hemoglobinopathies Overview

• Sickle Cell Anemia: Presence of Hgb S and Hgb F (no Hgb A1 or A2).

• Types of Sickle-cell Conditions:

  • Sickle-cell Trait: Genotype Hb S/Hb A.

  • Sickle-cell Disease: Genotype Hb S/Hb S (anemia).

Fetal Hemoglobin (Hgb F)

• Characteristics:

  • Predominant hemoglobin in fetus composed of 2 alpha chains and 2 gamma chains.

  • Screening method: Kleihauer-Betke test detects fetal hemoglobin since Hgb F is insoluble in acid solutions (low pH), leading to distinct staining patterns.

  • Counts the number of fetal cells versus the number of ghost cells in a set volume.

HPFH (Hereditary Persistence of Fetal Hemoglobin)

• Overview:

  • Continued production of increased amounts of Hgb F into adulthood.

  • Can be homozygous (100% Hgb F) or heterozygous (30% Hgb F).

Hgb C Disease & Trait

• Etiology:

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

• Clinical Features:

  • Generally mild chronic hemolytic anemia characterized by normocytic normochromic blood with the presence of target cells and microspherocytes.

• Electrophoresis Findings:

  • Hgb C approximately 95%, Hgb F < 7%, Hgb A is absent.

Hgb D & E Disease

• Hgb D:

  • Peripheral blood shows unremarkable features with few target cells, migrates with Hgb S during electrophoresis.

• Hgb E:

  • Little or no anemia; characterized by microcytic hypochromic RBCs with target cells; shown predominantly in Southeast Asian populations.

Hemoglobin Lepore

• Overview:

  • Named after the first family noted for its occurrence.

  • Result of fusion between delta and beta chains due to unequal crossing over.

• Clinical Features:

  • Patients may be transfusion-dependent with disorders similar to homozygous beta thalassemia.

Thalassemias

• General Overview:

  • Described in 1925 by Cooley, referred to as “Cooley’s Anemia.” Commonly presented in Mediterranean descent individuals.

• Types:

  • Based on affected chains: beta thalassemia, alpha thalassemia.

• **Genetics of Hgb Synthesis:

  • Chromosome 16 known for alpha genes; both a1 and a2 genes produce alpha globin at differing rates. Deletions or mutations can lead to thalassemias.

  • Chromosome 11 contains beta, delta, gamma & epsilon genes; mutations mainly involve point mutations leading to familial thalassemias.

Beta Thalassemias

• Details:

  • Also known as Cooley's anemia, characterized by unbalanced globin chain synthesis, leading to reduced or absent beta chains and excess of gamma & delta leading to elevated Hgb F and Hgb A2.

• Thalassemia Major:

  • Severe hypochromic microcytic anemia (Hgb < 7 g/dL); characterized by anisocytosis, poikilocytosis, and target cells.

• Skeletal Changes:

  • Expansion in bone marrow space, skeletal abnormalities noted on X-rays; manifestation includes features like hair-on-end appearance.

Heterozygous Beta Thalassemias

• Chronic Condition:

  • Mild chronic hypochromic microcytic anemia known as thalassemia minor.

  • Blood levels: Hgb 10 – 14 g/dL with mildly elevated Hgb F and Hgb A2.

Alpha Thalassemias

• Gene Characteristics:

  • Normally have 2 functional alpha genes on each chromosome (a1, a2).

  • Mutations cause a spectrum leading to varying severity:

  • - - (2 deletions)

  • a2 / - (1 deletion)

  • - / - (three deletions) -> incompatible with life.

• Hgb H Disease:

  • Characterized by the presence of Hgb H (formed through the aggregation of non-alpha chains) presenting inclusions detectable with staining.

  • Hgb H shows a golf ball appearance in mature RBCs due to the insoluble inclusions.

  • Different Hgb types developed, including Hgb Bart (4 gamma chains).

Hematology Review: Morphological Features

• Poikilocytes: Variation in RBC shape distinct in various disorders.

• Thalassemia: Notable presence of target cells, nucleated RBCs, bizarre shapes observable on microscopy.

• Polychromasia: Variation indicating increased reticulocyte levels.