hb electrophoresis

Basic Principles of Electrophoresis

  • Electrophoresis is the process of moving charged biomolecules in an electrolyte solution by applying an electrical field across the mixture.
  • Mobility depends on charge, size, and shape of the molecules; separation occurs on the basis of molecular size.
  • Applications include:
    • Analysis and purification of very large molecules (proteins, nucleic acids).
    • Analysis of simpler charged molecules (sugars, amino acids, peptides, nucleotides, and simple ions).
  • Typical setup components include electrolyte solution, power source, sample wells in a gel, a gel slab, and cloth wicks for buffering.

Basic Equipment Layout

  • Electrodes generate the electric field; power supply controls current/voltage.
  • Sample wells in gel allow loading of the analyte.
  • Gel slab provides a porous matrix for separation.
  • Cloth wicks help maintain buffer contact and stable current.
  • Common basic arrangement is shown in figures (Power source → Electrodes → Buffers → Gel).

Supporting Media and Mobility

  • Supporting media are porous solid matrices that separate molecules:
    • Paper
    • Starch
    • Cellulose acetate
    • Polyacrylamide
    • Agar/agarose
  • Molecules move through the porous matrix at different velocities depending on their properties.
  • Electrophoresis direction: Cathode (-) to Anode (+); charged species migrate toward their opposite charge.

Buffers

  • Functions of buffer:
    • Carries the applied current.
    • Establishes the pH.
    • Determines the electric charge on the solute.
  • High ionic strength buffers sharpen bands but generate more heat.
  • Common buffers include:
    • Barbitone buffer & Tris-EDTA for protein
    • Tris-acetate-EDTA & Tris-borate-EDTA (50 mmol/L50\ \mathrm{mmol/L}; pH 7.5−7.8\mathrm{pH} \ 7.5-7.8)

Hemoglobin Electrophoresis: Overview

  • Hemoglobin electrophoresis is a blood test to detect different types of hemoglobin.
  • It uses cellulose acetate and gel electrophoresis to separate the various hemoglobins.
  • Principle: different Hb types have different electrical charges; under electrophoresis they separate at different rates forming bands.
  • By comparing the pattern to a normal blood sample, clinicians assess the types and quantities of Hb present.

Hemoglobin: Normal and Abnormal Forms

  • Hemoglobin (Hb) variants exist in normal and abnormal forms.
  • Normal Hb forms:
    • HbA (most common in healthy adults)
    • HbA2
    • HbF (fetal Hb; predominant in fetuses/newborns but decreases after birth)
  • Abnormal Hb forms discussed include HbS, HbC, HbD, HbE, HbM, HbF variants, etc.
  • HbF is found in growing fetuses/newborns and is replaced by HbA after birth.
  • Abnormal Hb forms can lead to diseases if produced in excess or not replaced appropriately.

Normal Values (Globin Fractions)

  • Adults:
    • HbA1: 95%−98%95\%-98\%
    • HbA2: 2%−3%2\%-3\%
    • HbF: 0.8%−2%0.8\%-2\%
    • HbS: 0%0\%
    • HbC: 0%0\%
  • Infants/Children:
    • HbF (newborn): 50% to 80%50\%\ to\ 80\%
    • HbF (6 months): 8%8\%
    • HbF (over 6 months): 1%−2%1\%-2\%

Inherited Hb Abnormalities: Main Categories

  • Three main categories of inherited Hb abnormalities:
    1) Structural or qualitative (Hemoglobinopathy) – amino acid sequence is altered due to incorrect DNA code.
    2) Quantitative – production of one or more globin chains is reduced or absent (Thalassemia).
    3) Hereditary persistence of fetal haemoglobin (HPFH) – complete or partial failure of γ-globin to switch to β-globin.
  • Pathological variants include:
    • Hemoglobin H ( HbH, 34) – a tetramer of β chains; may be present in thalassemia variants.
    • Hemoglobin Bart’s (Hb Bart’s, 74) – a tetramer of γ chains; may be present in thalassemia variants.

Specific Hemoglobin Variants

  • HbS (a2βS2): single amino acid substitution where Glu (Glu) at position 6 is replaced by Valine; under low oxygen, polymerizes, causing sickling of RBCs and reduced elasticity. Sickle cells block small vessels, leading to local hypoxia and tissue damage.
  • HbC (a2βC2): Glu at position 6 replaced by Lys; causes mild chronic hemolytic anemia; moves more slowly than HbA on alkaline electrophoresis.
  • HbD (HbD Punjab): β121 Glu replaced by Gln; migrates similarly to HbS on alkaline pH electrophoresis.
  • HbE (a2βE2): variant with mild chronic hemolytic anemia.
  • HbAS: heterozygous for sickle cell trait (one HbA gene and one HbS gene).
  • HbSC disease: compound heterozygous for HbS and HbC.
  • Normal cord blood and other reference patterns used for comparison.

Laboratory Investigations to Detect Abnormal Hb

I) Red cell morphology:

  • Sickle cells (HbS), Target cells, Basophilic stippling, Teardrop cells, Microcytosis, etc.
    II) Hemoglobin Electrophoresis:
  • Methods include cellulose acetate at alkaline pH and citrate agar at acid pH.
    III) Isoelectric focusing (IEF):
  • pH gradient established by carrier ampholytes; Hb molecules migrate to the position where their net charge is zero (isoelectric point); concentration into sharp bands.
    IV) High-Performance Liquid Chromatography (HPLC):
  • Weak cation exchange column; eluting solution's ionic strength is gradually increased to separate Hb variants by retention time.

Principle of Cellulose Acetate Electrophoresis (Alkaline, pH ~8.2–8.6)

  • Hb is negatively charged at alkaline pH; migrates toward the anode (+).
  • Mobility depends on net charge, which is determined by amino acid composition of the Hb molecule.
  • Procedure uses a red cell hemolysate applied to a cellulose acetate membrane placed in an electrophoresis tray with buffer at pH 8.2–8.6.
  • One end of the strip is near the cathode; the other near the anode; current causes Hb to migrate toward the anode.

Requirements for Cellulose Acetate Electrophoresis

  • Haemolysate prepared from red cells.
  • TE B buffer (pH 8.4).
  • Whatman No. 3 chromatography paper.
  • Cellulose acetate membranes.
  • HbA control (NIBSC standard).
  • Protein stain: Ponceau S (0.5% in 5% TCA).
  • Destain solution: 5% acetic acid.
  • Note: The NIBSC control is a freeze-dried Hb solution stabilized with sucrose (200 mM), potassium cyanide (6 mM) and chloramphenicol (1 mg/dL); WHO standard since 1994.

Procedure: Cellulose Acetate Electrophoresis (Step-by-step)

1) Fill all tank compartments with TE buffer (about 500 mL total) and ensure equal levels across compartments.
2) Wet two pieces of Whatman No. 3 filter paper (20 × 7.5 cm) with buffer;