Hemoglobin (Hb) Electrophoresis Practical Guide PDF

**HAEMATOLOGY II** **HML2143** **[PRACTICAL 2]** **Hemoglobin (Hb) Electrophoresis** **Introduction** Hemoglobin (Hb) electrophoresis is a test that measures the different types of Hb in the blood. Electrophoresis is the movement of charged particles in an electrical field, resulting in formati...

**HAEMATOLOGY II** **HML2143** **[PRACTICAL 2]** **Hemoglobin (Hb) Electrophoresis** **Introduction** Hemoglobin (Hb) electrophoresis is a test that measures the different types of Hb in the blood. Electrophoresis is the movement of charged particles in an electrical field, resulting in formation of bands that separate toward one end or another in the field. Electrophoresis of Hb proteins is an effective diagnostic tool for hemoglobinopathies because Hb variants have different charges. Hemoglobinopathies are caused by a large number of mutations in globin genes. These mutations can be divided into two distinct types: 1. Qualitative: production of abnormal hemoglobin molecules (*e.g*., sickle cell disease). 2. Quantitative: reduction in amount of normal hemoglobin (*e.g*., thalassemia). Another group of hemoglobin disorders include developmental anomalies (*e.g*., hereditary persistence of fetal hemoglobin (HPFH), where there is a persistent expression of fetal hemoglobin). In summary the main purposes of Hb electrophoresis are to: 1. Confirm the diagnosis of diseases involving abnormal forms of hemoglobin (Hemoglobinopathies**)**, such as sickle cell anemia and thalassemia. 2. Help couples determine how likely they are to have a child with certain forms of anemia that can be passed from a parent to a child (inherited). The protocol for hemoglobin electrophoresis involves the use of two systems (Figure 1): 1. **Initial electrophoresis** performed in **alkaline buffers** (pH = 8.2-8.6): Cellulose acetate is the major support medium used here because it yields **rapid separation** of Hb-A, -F, -S and -C and many other mutants with **minimal preparation time.** 2. **Supplementary electrophoresis** (citrate agar electrophoresis, pH = 6.8): because of the electrophoretic similarity of many structurally different hemoglobins, the evaluation must be done by this procedure which measures some other property. (This method is based on the complex interactions of the hemoglobin with the electrophoretic buffer (acid pH) and the agar support). **Citrate agar electrophoresis allows better separation** for (figure 1): 1. Hb-S from Hb-D (in cellulose acetate electrophoresis both of them appear on the same band). 2. Hb-C from Hb-E and Hb-O (in cellulose acetate electrophoresis all of them appear on the same band).  **Figure 1** **Principle of Hb Electrophoresis** Very small samples of hemolysates prepared from **whole blood** are applied to the Cellulose Acetate Plate. The hemoglobins in the sample are separated by electrophoresis using an alkaline buffer in which they are negatively charged (Hb variants move at different rates depending on their *net negative charge),* and are stained with *Ponceau S* Stain. The patterns are scanned on a scanning densitometer, and the relative percent of each band is determined. **Specimen** - Whole blood collected in tubes containing **EDTA or heparin.** - Whole blood samples may be stored up to one week at 2 to 6°C. **Reagents** - Supre-Heme Buffer: contains Tris-EDTA and boric acid. - Hemolysate Reagent: contains 0.005 M EDTA in deionized water with 0.07% potassium cyanide added as a preservative. - *Ponceau S* Stain. - Clear Aid: contains polyethylene glycol. - 5% acetic acid. - Absolute methanol. - Cellulose acetate plates. **Equipments** a. Chamber Wicks. b. Electrophoresis Chamber. c. Power Supply. d. Microdispenser. e. Blotter Pads. f. Applicator Kit. g. Staining Set. h. Any high quality scanning densitometer capable of scanning a cleared cellulose acetate plate at 525 nm may be used. **Procedure** a. **Sample Preparation:** Prepare a hemolysate of the patient samples as follows: - Using whole blood: Add 1 part whole blood to 3 parts Hemolysate Reagent (or distilled water). Mix well and allow to stand for 5 minutes. - Using packed cells: Mix 1 part packed red blood cells to 6 parts Hemolysate Reagent (or distilled water). Mix well and allow to stand for 5 minutes. b. **Preparation of the cellulose acetate Plates:** - Soak the required number of plates in Supre-Heme Buffer for 5 minutes (load them into buffer slowly and smoothly) (figure 3, A). c. **Preparation of the Chamber:** - 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) and make sure that there are no air bubbles under the wicks. - Cover the chamber to prevent buffer evaporation. Discard the buffer and wicks after use (figure 3, B). d. **Sample Application:** - Place 5 μL of the patients' hemolysates into the wells of the Sample Well Plates (figure 3, C). - 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) (figure 3, D & E). - 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" (figure 3, F). - 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) (figure 3, G). e. **Electrophoresis of Sample Plate:** - Quickly place the plate in the electrophoresis chamber (cellulose acetate side down) such that the sample end is toward the cathodic (-) side of the chamber (place a glass slide on the plate to insure contact with the wicks). - Place the cover on the chamber, and electrophorese the plate for 25 minutes at 350 volts (figure 3, H). f. **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. Allow the plates to stay in each wash 2 minutes or until the background is white. g. **Using Clear Aid Solution:** - Dehydrate, by washing the plate twice in absolute methanol (for two minutes each wash). Allow the plate to drain for 5-10 seconds before placing 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. h. **Evaluation of the Hemoglobin Bands:** - Qualitative evaluation: The 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:** The dried plates are stable for an indefinite period of time. **Results Interpretation:** A. **Normal results:** normal reference values can vary by each laboratory, but are usually within the following ranges (figure 2). **Adults:** - 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% **Child (Hb-F):** - Newborn: 65-90% - At 6 months of age: 8% - More than 6 months of age: 1-2% A. **Abnormal results:** abnormal reference values can vary by each laboratory, but are usually within the following ranges. **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 (the pattern is 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% **HbC/β-thalassemia:** - Hb-C \> HbA - Hb-F: variable ***Note:** if HbC/β°-thalassemia): Hb A is absent (the pattern is identical to that of HbC disease).* **Limitations:** Some abnormal hemoglobins have similar electrophoretic mobilities and must be differentiated by other methodologies such as: 1. Citrate agar electrophoresis: may be a necessary follow-up test for confirmation of abnormal hemoglobins detected on cellulose acetate. 2. **Anion exchange column chromatography**: is the *most accurate method for HbA2 quantitation* which is considered one of the most important diagnostic tests in the diagnosis of β-thalassemia trait. **Quality Control:** Four controls for hemoglobin electrophoresis are available from Helena Laboratories: 1. AA2 Hemo Control. 2. ASA2 Hemo Control. 3. AFSA2 Hemo Control. 4. AFSC Hemo Control. - The controls should be used as markers for the identification of the hemoglobin bands, and they may be quantitated for verification of the accuracy of the procedure. - Use at least one of these controls on each plate run. -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Figure 2:** Pattern of hemoglobin electrophoresis from several different individuals. Lanes 1 and 5 are hemoglobin standards. Lane 2 is a normal adult. Lane 3 is a normal neonate. Lane 4 is a homozygous HbS individual. Lanes 6 and 8 are heterozygous sickle individuals. Lane 7 is a SC disease individual. -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- +-----------------------+-----------------------+-----------------------+ | **A** | **B** | **C** | +=======================+=======================+=======================+ | **D** | **E** | **F** | +-----------------------+-----------------------+-----------------------+ | **G** | **H** | | +-----------------------+-----------------------+-----------------------+ **Figure 3**