Haemoglobin Electrophoresis

Medical Analysis

Understanding Haemoglobin Electrophoresis: A Comprehensive Clinical Guide

Haemoglobin electrophoresis is widely recognized as the most useful diagnostic method for the demonstration of abnormal haemoglobin [1, 2]. The technique operates on the principle that haemoglobin molecules can be separated on a variety of supporting media based on differences in their electric charge [11]. This process is highly valuable in identifying specific haemoglobin variants and determining their potential clinical significance in various patients [7]. There are two primary types of this diagnostic test: cellulose acetate electrophoresis, which is performed at a pH of 8.6, and agar gel electrophoresis, which is conducted at a pH of 6.0 [1, 4].

What is Hb Electrophoresis?

Haemoglobin (Hb) electrophoresis utilizes the principle of electrophoretic mobility to successfully separate and identify different haemoglobin variants [11]. This diagnostic procedure is essential for identifying various haemoglobinopathies, such as Sickle cell disease and Thalassemia [3, 7].

The Structure and Function of Haemoglobin

Haemoglobin is a specialized protein found within red blood cells (RBCs) that is responsible for carrying oxygen throughout the body [2, 9]. It is composed of multiple polypeptide chains [6]. Specifically, adult haemoglobin (HbA) is a tetramer composed of two alpha (α) and two beta (β) subunits [9]. Furthermore, each subunit contains a heme group, which is an iron-containing porphyrin ring that binds to oxygen [2].

The primary function of haemoglobin is to carry oxygen from the lungs to the body’s tissues, and in return, it transports carbon dioxide from the tissues back to the lungs for exhalation [6]. Additionally, haemoglobin plays a critical role in maintaining blood pH by acting as an effective buffer within the circulatory system [6].

Clinical Indications for Performing Hb Electrophoresis

Healthcare providers order haemoglobin electrophoresis for several specific clinical indications, including [7]:

• The evaluation of unexplained haemolytic anaemia [1].

• The confirmation of both Thalassemia Major and Thalassemia Minor [3, 12].

• The investigation of abnormal red cell features identified in a peripheral smear [4].

• When a patient presents with microcytic anaemia that is not caused by iron deficiency, chronic disease, or lead toxicity [12].

• The presence of a positive family history of haemoglobinopathy [7].

• Confirmation following positive sickle cell tests, or when there is a raised foetal Hb or an abnormal Reticulocyte Count [7, 14].

• Routine or diagnostic neonatal screening [7].

Proper Blood Sample Collection Procedures

Accurate results depend on correct sample collection [1, 4]. The procedure is as follows:

• Collect 2.0 to 2.5 ml of blood via venipuncture [1].

• Transfer the sample immediately into an EDTA tube, typically identified by a lavender top [1].

• Ensure the tube is mixed well [1].

• As far as possible, the sample should be sent to the laboratory immediately for processing [1].

Technical Methods: Cellulose Acetate and Citrate Agar Electrophoresis

1. Cellulose Acetate Electrophoresis at Alkaline pH

The principle behind this method is that at an alkaline pH, haemoglobin is negatively charged [4]. When subjected to electrophoresis, it migrates toward the anode (+) [4]. Consequently, structural variants that have a change in the surface charge of the molecule at an alkaline pH will separate from normal HbA [4].

This method is advantageous because it is simple, reliable, reproducible, and rapid [1, 4]. However, a significant disadvantage is that Hb S, D, G, and Hb A2, C, E, and O-Arab migrate very closely together, making differentiation between these specific haemoglobins impossible using only this method [4]. Several factors can affect the results, including the amount of haemoglobin applied, the positioning of the origin, any delay between sample application and the commencement of electrophoresis, delays in staining after the procedure, and inadequate blotting of the acetate prior to application [1, 4].

2. Citrate Agar Electrophoresis at pH 6.0

This method provides ready separation of haemoglobins that migrate together on cellulose acetate, such as separating S from D and G, and C from E and O [4]. Agarose gel electrophoresis is also commercially available as a substitute for both alkaline or acid separation systems, particularly in laboratories that process small numbers of samples [4, 11]. The principles are similar to citrate agar electrophoresis, but there are significant differences in the mobility of some variants of haemoglobin [4].

Interpretation of Results

Satisfactory separation can be achieved for Hb C, S, F, and A [1, 4]. Interpretation guidelines include:

• If both Hb S and Hb A bands are seen, but the Hb A band is darker than the Hb S band, it indicates Sickle Cell Trait [14].

• If only the Hb S band is seen, it indicates Sickle Cell Disease [14].

• If both Hb S and Hb A bands are seen, but the Hb S band is darker than the Hb A band, it indicates Sickle Cell Disease with a recent transfusion [1].

Major Haemoglobin Fractions

Clinical Interpretation of Haemoglobin Types

Clinical Applications and Significance

Haemoglobin electrophoresis has broad clinical applications, including the diagnosis of haemoglobinopathies, diagnosis of Thalassemias, newborn screening for early detection of sickle cell anaemia, monitoring therapy by tracking HbF levels, carrier screening for genetic disorders, and the evaluation of unexplained anaemia [7, 8]. It helps in the early and accurate diagnosis of abnormal haemoglobins and assists in differentiating Thalassemia Minor from iron deficiency anaemia and lead poisoning [12]. Furthermore, it is essential for the early diagnosis of infants, serves as a screening test in geographical areas with specific haemoglobin prevalence, and is vital for prenatal testing and genetic counselling [7, 8].

For Non-Medicos: Understanding Blood Haemoglobin Tests

If your doctor has suggested a “Haemoglobin Electrophoresis” test, it is a specialized blood test used to check the types of haemoglobin in your red blood cells. Haemoglobin is the protein that carries oxygen throughout your body. While most people have a standard type of haemoglobin (HbA), some people inherit different, abnormal types that can cause blood disorders [9].

Why is this test done?

This test is the “gold standard” for finding out if you have inherited blood conditions, such as:

• Sickle Cell Disease: Where red blood cells become misshapen, causing health issues [14].

• Thalassemia: A condition where the body makes an abnormal form of haemoglobin, leading to anaemia [12].

  Doctors often order this test if you have unexplained anaemia, a family history of blood disorders, or if routine blood tests show abnormal red blood cells [7]. It is also used to screen newborns to catch these conditions early and to monitor patients who are already receiving treatment [7, 8].

What to expect

The test is simple and quick. A healthcare professional will draw a small amount of your blood (usually about 2.5 ml) into a tube [1]. It is important that this sample is handled correctly and sent to the lab quickly so that the results are accurate [1].

How it works

Inside the lab, scientists use electricity to separate the different types of haemoglobin in your blood sample [11]. Because different haemoglobin types have different electrical charges, they move at different speeds across a special gel or strip. This creates a pattern that acts like a fingerprint, allowing experts to identify exactly which types of haemoglobin you have [4].

What the results mean

• Normal: Most adults have mostly HbA (about 95-98%) and very small amounts of other types like HbA2 and HbF [2].

• Abnormal: If the test finds other types like HbS (linked to Sickle Cell) or higher-than-normal levels of others, it helps your doctor understand if you are a “carrier” or if you have the disease itself [7, 9].

References:

1. Bain, B. J., Bates, I., & Laffan, M. A. (2016). Dacie and Lewis Practical Haematology. Elsevier.

2. Hoffman, R., Benz, E. J., Silberstein, L. E., Heslop, H. E., Weitz, J. I., & Anastasi, J. (2017). Hematology: Basic Principles and Practice. Elsevier.

3. Clarke, G. M., & Higgins, T. N. (2000). Laboratory investigation of hemoglobinopathies and thalassemias: Review and update. Clinical Chemistry, 46(8), 1284–1290.

4. Wild, B. J., & Bain, B. J. (2004). Investigation of abnormal haemoglobins and thalassaemia. Journal of Clinical Pathology, 57(2), 124–136.

5. Weatherall, D. J., & Clegg, J. B. (2001). The Thalassaemia Syndromes. Blackwell Science.

6. Bunn, H. F., & Forget, B. G. (1986). Hemoglobin: Molecular, Genetic and Clinical Aspects. W.B. Saunders.

7. Ryan, K., Bain, B. J., Clark, D., Comfort, A., De La Salle, B., & Hyde, K. (2010). Significant haemoglobinopathies: Guidelines for screening and diagnosis. British Journal of Haematology, 149(1), 35–49.

8. Old, J. M., Harteveld, C. L., Traeger-Synodinos, J., Petrou, M., Angastiniotis, M., & Galanello, R. (2016). Prevention of thalassaemias and other haemoglobin disorders: Technical approaches. Thalassaemia International Federation.

9. Steinberg, M. H., Forget, B. G., Higgs, D. R., & Weatherall, D. J. (2009). Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. Cambridge University Press.

10. Huisman, T. H. J., & Carver, M. F. H. (1998). The Hemoglobinopathies. Marcel Dekker.

11. Greene, D. N., & Vaughn, C. P. (2017). Hemoglobin electrophoresis. Clinics in Laboratory Medicine, 37(1), 1–18.

12. Muncie, H. L., & Campbell, J. S. (2009). Alpha and beta thalassemia. American Family Physician, 80(4), 339–344.

13. Giardine, B., Borg, J., Higgs, D. R., Peterson, K. R., & Hardison, R. C. (2014). HbVar: Database of hemoglobin variants and thalassemia mutations. Human Mutation, 35(11), 1284–1291.

14. Pearson, H. A., & O’Brien, R. T. (1975). Sickle cell testing programs. Journal of Pediatrics, 87(3), 361–365.

FAQ’s:

• What is hemoglobin electrophoresis used for?
  It is the most useful method for demonstrating and identifying abnormal hemoglobin variants and related disorders.

• What is the hemoglobin structure?
  It is a protein in red blood cells made of four polypeptide chains with iron-containing heme groups.

• What does hemoglobin do?
  It transports oxygen from lungs to tissues, carries carbon dioxide back, and maintains blood pH levels.

• When is this test indicated?
  It is indicated for unexplained hemolytic anemia, thalassemia screening, neonatal screening, and investigating abnormal red cell features.

• How is blood sample collected?
  Collect 2.0 to 2.5 ml by venipuncture into an EDTA lavender-top tube and mix it well.

• How does cellulose acetate work?
  At alkaline pH, hemoglobin is negatively charged and migrates toward the anode to separate from Hb A.

• What are the method advantages?
  Cellulose acetate electrophoresis is simple, reliable, reproducible, and provides rapid results for identifying hemoglobin variants.

• Why use citrate agar?
  It provides necessary separation for hemoglobin variants that migrate together on cellulose acetate, such as S, D, and G.

• What is HbA2 significance?
  HbA2 levels are typically 2 to 3 percent and are elevated in beta-thalassemia trait cases.

• What clinical information is gained?
  It enables early diagnosis of hemoglobinopathies, carrier screening, prenatal testing, and monitoring of various hemoglobin disorder treatments.

Related Tests

• Complete Blood Count (CBC)

• Sickle Solubility Test

• Reticulocyte Count