Hemoglobin Electrophoresis: Diagnosis & Methods

• Hemoglobin (Hb) electrophoresis measures the different types of Hb in the blood
• Electrophoresis involves the movement of charged particles in an electrical field
• This movement leads to the formation of bands as particles separate
• Hb protein electrophoresis is effective for diagnosing hemoglobinopathies
• Hemoglobinopathies arise from numerous mutations in globin genes
• Mutations are divided into:
• Qualitative: production of abnormal hemoglobin molecules (e.g., sickle cell disease)
• Quantitative: reduction in normal hemoglobin amount (e.g., thalassemia)
• Developmental anomalies, such as hereditary persistence of fetal hemoglobin (HPFH), are another group of hemoglobin disorders where fetal hemoglobin persists
• Hb electrophoresis confirms diagnoses of diseases involving abnormal hemoglobin forms, like sickle cell anemia and thalassemia
• It helps couples assess the likelihood of having a child with inherited forms of anemia

Hemoglobin Electrophoresis Protocol

• The protocol uses two systems
• Initial electrophoresis in alkaline buffers (pH 8.2-8.6), uses cellulose acetate for rapid separation of Hb-A, -F, -S, -C, and other mutants with minimal prep time
• Supplementary electrophoresis (citrate agar, pH 6.8) evaluates structurally similar hemoglobins, based on interactions with the electrophoretic buffer (acid pH) and agar

Citrate Agar Electrophoresis

• Citrate agar electrophoresis allows for better separation, as it separates:
• Hb-S from Hb-D, which appear on the same band in cellulose acetate electrophoresis
• Hb-C from Hb-E and Hb-O, which appear on the same band in cellulose acetate electrophoresis

Principle of Hb Electrophoresis

• Hemolysates from whole blood are applied to a Cellulose Acetate Plate
• Hemoglobin (Hb) separates via electrophoresis using alkaline buffer
• Hb variants move at rates based on their net negative charge
• Patterns are stained with Ponceau S Stain
• A scanning densitometer scans the patterns to determine the relative percent of each band

Specimen Requirements

• Whole blood should be collected in tubes with EDTA or heparin
• Whole blood samples can be stored for up to one week at 2–6°C

Reagents Used

Included in the reagents are:

• Supre-Heme Buffer (Tris-EDTA and boric acid)
• Hemolysate Reagent (0.005 M EDTA in deionized water with 0.07% potassium cyanide as a preservative)
• Ponceau S Stain
• Clear Aid (polyethylene glycol)
• 5% acetic acid
• Absolute methanol
• Cellulose acetate plates

Equipment

• Included in equipment are:
• Chamber Wicks
• Electrophoresis Chamber
• Power Supply
• Microdispenser
• Blotter Pads

Sample Preparation

• Prepare hemolysate of patient samples, as follows
• Using whole blood: Add 1 part whole blood to 3 parts Hemolysate Reagent (or distilled water), then mix well, and stand for 5 minutes
• Using packed cells: Mix 1 part packed red blood cells to 6 parts Hemolysate Reagent (or distilled water), then mix well, and stand for 5 minutes

Preparation of Cellulose Acetate Plates

• Soak the required number of plates in Supre-Heme Buffer for 5 minutes, then load them into buffer slowly and smoothly

Chamber Preparation

• Pour 100 mL of Supre-Heme Buffer into each of the outer sections of the Chamber
• Wet two chamber wicks in the buffer and fold one over each of the middle support bridge (with one edge immersed in the buffer) making sure that there are no air bubbles under the wicks
• Cover the chamber to prevent buffer evaporation and discard the buffer and wicks after use

Sample Application

• Place 5 μL of the patients’ hemolysates into the wells of the Sample Well Plates
• To prevent evaporation, cover the Sample Well Plate with a glass slide (if the samples are not used within 2 minutes)
• Check the performance of the sample applicator by depressing its tips into the sample wells 3 or 4 times and applying this loading to a piece of blotter paper, press the button down and hold it for 5 seconds (this makes the second loading in the cellulose plates much more uniform)
• Remove the wetted cellulose acetate plate from the buffer with the fingertips and blot once firmly between two blotters
• Place the plate in the aligning base (cellulose acetate side up) aligning the top edge of the plate with the black line marked “CATHODE APPLICATION”
• Before placing the plate in the aligning base, place a drop of water or buffer on the center of the aligning base to prevent the plate from shifting during the sample application
• Apply the sample to the plate by depressing the applicator tips into the sample well 3 or 4 times and promptly transferring the applicator to the aligning base (press the button down and hold it for 5 seconds)

Electrophoresis of Sample Plate

• Quickly place the plate in the electrophoresis chamber with the cellulose acetate side down, such that the sample end is toward the cathodic (-) side of the chamber, then place a glass slide on the plate to ensure contact with the wicks
• Place the cover on the chamber, and electrophorese the plate for 25 minutes at 350 volts

Staining the Hemoglobin Bands

• Remove the plates from the electrophoresis chamber and stain in Ponceau S for 5 minutes
• Destain in 3 successive washes of 5% acetic acid, then allow the plates to stay in each wash 2 minutes or until the background is white

Using Clear Aid Solution

• Dehydrate the plate by washing it twice in absolute methanol (for two minutes each wash) and allow the plate to drain for 5-10 seconds before placing it in the next solution
• Place the plate into the Clear Aid solution for 5-10 minutes
• Drain off excess solution, then place the plate (acetate side up) onto a blotter, and into drying oven at 50-60°C for 15 minutes

Hemoglobin Band Evaluation

• Qualitative evaluation: Hemoglobin plates may be inspected visually for the presence of abnormal hemoglobin bands (the Hemo Controls provide a marker for band identification)
• Quantitative evaluation: Determine the relative percent of each hemoglobin band by scanning the cleared and dried plates in the densitometer using a 525 nm filter

Stability of End Product

• Dried plates are stable for an indefinite period of time

Normal Adult Results

• Hb-A: 95-98% of total Hb
• Hb-A2: 1.5-3% of total Hb
• Hb-F: 0-2% of total Hb
• Hb-S: 0%
• Hb-C: 0%

Normal Child (Hb-F) Results

Included are:

• Newborn: 65-90%
• At 6 months of age: 8%
• More than 6 months of age: 1-2%
• Note: normal reference values can vary by each laboratory

Abnormal Results

• Note: abnormal reference values can vary by each laboratory

β-Thalassemia Major

• Hb-A: variable (may be 0%)
• Hb-A2: within normal limits
• Hb-F: up to 98%

β-Thalassemia Minor (Trait)

• Hb-A: < 95%
• Hb-A2: 3.5-8%
• Hb-F: 1-5%

Hb H Disease (β4, α-Thalassemia)

• Hb-A: ↓
• Hb-A2: < 2%
• Hb-H (in adults): 2-40%
• Hb-Barts (γ4) (in newborn): 25-40%

HPFH

• Hb-F: 5-35% (heterozygous)
• Hb-F: 100% (homozygous)

Sickle Cell Disease

• Hb-A: 0%
• Hb-A2: 2-5%
• Hb-F: 1-20%
• Hb-S: > 80%

Sickle Cell Trait

• Hb-A: 60%
• Hb-A2: slightly ↑
• Hb-F: within normal limits
• Hb-S: 40%

Hb S/β-Thalassemia

• Hb-S: > Hb A
• Hb-A2: ↑
• Hb-F: ↑
• Note: if Hb S/βº-thalassemia, Hb A is completely absent, which makes the pattern identical to that of sickle cell disease

Hb C Disease (Homozygous)

• Hb-A: 0%
• Hb-A2: 2%
• Hb-F: variable
• Hb-C: >90%

Hb SC Disease (Heterozygous)

• Hb-S = Hb C
• Hb-A2: 2-4%
• Hb-F: 1%

Hb C/β-Thalassemia

• Hb-C > HbA
• Hb-F: variable
• Note: if HbC/β°-thalassemia): Hb A is absent, which makes the pattern identical to that of HbC disease

Limitations

• Some abnormal hemoglobins have similar electrophoretic mobilities, requiring differentiation via other methodologies

Methodologies to note

Included within are:

• Citrate agar electrophoresis, which confirms abnormal hemoglobins detected on cellulose acetate
• Anion exchange column chromatography, which accurately quantifies HbA2, which is important for diagnosing β-thalassemia trait

Quality Control

• Use four controls for hemoglobin electrophoresis available from Helena Laboratories
• AA2 Hemo Control
• ASA2 Hemo Control
• AFSA2 Hemo Control
• AFSC Hemo Control
• Controls should be used as markers for identifying hemoglobin bands, and can be quantitated to verify procedure accuracy
• Use at least one of these controls on each plate run

Explore qualitative hemoglobinopathies and the role of hemoglobin electrophoresis in diagnosis. Understand supplementary electrophoresis purposes and suitable anticoagulants for sample collection. Learn about hemolysate preparation, staining techniques, and the use of cellulose acetate.