High Performance Liquid Chromatography (HPLC) for Identification of Hemoglobin Subtypes

Introduction

Hemoglobinopathies, a group of genetic disorders that affect the structure or production of hemoglobin, the oxygen-carrying protein in red blood cells, are among the most common single-gene disorders worldwide. These disorders arise from mutations in the genes encoding hemoglobin subunits, leading to alterations in the protein’s structure, function, and stability. Hemoglobinopathies encompass a wide spectrum of conditions, ranging from mild anemia in asymptomatic cases to severe, life-threatening complications.

Hemoglobinopathies are broadly classified into two main categories: thalassemias and structural hemoglobin variants.

  • Thalassemias: Thalassemias result from mutations in the genes encoding globin chains, leading to either reduced or absent production of specific globin chains. This imbalance in globin chain synthesis disrupts hemoglobin formation, resulting in anemia, a condition characterized by low red blood cell count or hemoglobin levels.
  • Structural hemoglobin variants: Structural hemoglobin variants arise from mutations in the genes encoding globin chains, leading to alterations in the amino acid sequence of the protein. These alterations can affect hemoglobin’s structure, stability, and oxygen-binding affinity, leading to various clinical manifestations.

Among the most prevalent hemoglobinopathies are:

  • Sickle cell disease (SCD): SCD is caused by a single mutation in the beta-globin gene, resulting in the production of abnormal hemoglobin S. Hemoglobin S, under low oxygen conditions, can polymerize within red blood cells, causing them to sickle and become rigid, leading to pain, fatigue, and other complications.
  • Beta-thalassemia: Beta-thalassemia results from mutations in the beta-globin gene, leading to reduced or absent production of beta-globin chains. The severity of beta-thalassemia depends on the number of mutated genes inherited.
  • Alpha-thalassemia: Alpha-thalassemia arises from mutations in the alpha-globin gene, leading to reduced or absent production of alpha-globin chains. Alpha-thalassemia can range from mild, asymptomatic cases to severe, transfusion-dependent anemia.

Diagnosis of hemoglobinopathies typically involves a combination of family history, clinical evaluation, and laboratory tests. Accurate identification of hemoglobin subtypes is essential for diagnosing and managing these conditions. High-performance liquid chromatography (HPLC) has emerged as a valuable tool for hemoglobin subtype identification due to its high sensitivity, specificity, and ability to separate and quantify various hemoglobin variants.

Principle of HPLC

High-performance liquid chromatography (HPLC) is a versatile and powerful analytical technique widely used in various scientific disciplines, including chemistry, biochemistry, pharmaceutical science, and environmental science. Its ability to separate and quantify complex mixtures of compounds with high sensitivity and resolution has made it an indispensable tool for a wide range of applications.

The Essence of HPLC: Separation and Detection

At the heart of HPLC lies the principle of separation based on the differential distribution of solutes between a stationary phase and a mobile phase. The stationary phase is typically a solid material packed inside a chromatographic column, while the mobile phase is a liquid solvent that flows through the column. As the sample mixture is injected into the column, the solutes interact with the stationary phase and the mobile phase to varying degrees, causing them to travel through the column at different speeds. This separation is achieved based on the interplay of various factors, including the solute’s molecular size, polarity, and affinity for the stationary phase.

The Stationary Phase: The Scaffold for Separation

The stationary phase plays a crucial role in the separation process. It provides a surface for solutes to interact with and influences their distribution between the stationary and mobile phases. Common stationary phases include silica gel, alumina, and bonded phases, which have specific functional groups attached to their surface to enhance selectivity for certain types of solutes.

The Mobile Phase: The Driving Force for Separation

The mobile phase, in the form of a liquid solvent, continuously flows through the chromatographic column, carrying the solutes along its path. The choice of solvent and its composition significantly impact the separation process. The solvent should be compatible with the stationary phase and the solutes to ensure proper interaction and separation.

Detection and Quantification

As solutes elute from the column, they are detected and quantified using a variety of detectors. Common detectors include UV-visible absorbance detectors, which measure the absorbance of light at specific wavelengths, and mass spectrometers, which identify and quantify solutes based on their mass-to-charge ratio.

HPLC Application Hemoglobin Subtype Identification

The ion exchange chromatography for hemoglobin separation relies on the selective interaction between charged groups on the silica-based exchange material (stationary phase) and the charged groups on the hemoglobin molecule (mobile phase). The silica surface is modified with carboxyl groups, imparting a weak cationic charge, enabling the separation of hemoglobin (Hb) molecules based on their charge differences. When a hemolysate containing a mixture of hemoglobins is introduced onto the resin, the elution rate of different hemoglobins is governed by the pH and ionic strength (ion concentration) of the buffered elution solution. The pH and ionic strength of the eluent are carefully controlled to achieve selective elution of different Hb variants.

Separation Mechanism

The separation of hemoglobin variants in HPLC is primarily driven by two factors:

  1. Charge: Hemoglobin variants differ in their net charge due to variations in their amino acid sequences. The stationary phase can be designed to selectively interact with hemoglobin variants based on their charge, allowing for their separation.
  2. Hydrophobicity: Hemoglobin variants also exhibit differences in their hydrophobicity, the tendency of a molecule to repel or attract water. The stationary phase can be tailored to selectively interact with hemoglobin variants based on their hydrophobicity, further enhancing separation.

Detection and Quantification

As hemoglobin variants elute from the column, they are detected using a UV-visible absorbance detector. Hemoglobin absorbs light at specific wavelengths, and the intensity of the absorbed light is proportional to the concentration of the hemoglobin variant. This information is used to quantify the relative proportions of different hemoglobin subtypes in the sample.

Advantages of HPLC for Hemoglobin Subtype Identification

HPLC offers several advantages for hemoglobin subtype identification:

  1. High Sensitivity: HPLC can detect even small amounts of hemoglobin variants, making it suitable for analyzing samples with low hemoglobin concentrations.
  2. Specificity: HPLC can distinguish between closely related hemoglobin variants, providing accurate subtype identification.
  3. Quantification: HPLC allows for the quantification of different hemoglobin variants, providing insights into their relative proportions in the sample.
  4. Automation: HPLC methods can be automated, reducing manual labor and improving consistency.

VARIANTTM II Beta Thalassemia Short Program (Bio-Rad Laboratories Inc., Hercules, CA, USA)

The VARIANTTM II Beta Thalassemia Short Program employs the principles of ion-exchange high-performance liquid chromatography (HPLC) to accurately identify and quantify hemoglobin variants. This automated method utilizes the VARIANTTM II Sampling Station (VSS) for sample preparation and injection and the VARIANTTM Chromatographic Station (VCS) for separation and detection.

Sample Preparation and Injection

The VSS automates the sample mixing, dilution, and injection process, ensuring precision and consistency. Prepared samples are then injected onto the analytical cartridge for separation and analysis.

Separation and Detection

The VCS employs dual pumps to deliver a precisely controlled gradient of increasing ionic strength to the analytical cartridge. This gradient facilitates the separation of HbA2 and HbF based on their differential ionic interactions with the cartridge material. The separated hemoglobin variants pass through a flow cell equipped with a filter photometer, which measures the absorbance changes at 415 nm. An additional filter at 690 nm compensates for background absorbance.

Data Analysis and Interpretation

The VARIANTTM II CDM (CDM) Software processes the raw data collected from each analysis. To aid in result interpretation, predefined windows have been established for commonly encountered hemoglobins based on their characteristic retention times. For each sample, the CDM generates a sample report and a chromatogram, providing detailed information on the eluted hemoglobin fractions, their retention times, peak areas, and fractional values.

Materials

  • EDTA peripheral blood sample
  • VARIANTTM Automated HPLC system
  • Diluent buffer
  • 2 phosphate buffers of different pH and ionic strengths
  • HbA2/Hb F calibrator
  • Lyphochek® Hemoglobin A2 Control, Bilevel (2 each of 2 levels) 

Protocol

  1. To ensure the accuracy of the subsequent analyses, the HbA2/HbF Calibrator is analyzed at the start of each run to generate calibration factors for both hemoglobin A2 and F. These calibration factors will then be applied to calculate the area percentages for HbA2 and F in all subsequent analyses within the run. Additionally, the Lyphocheck Hemoglobin A2 Control (Levels 1 and 2 from Bio-Rad) is analyzed to verify that the concentration values of HbA2 and HbF remain within acceptable limits.
  2. A total of 5 uL EDTA blood sample is diluted with a 1 mL diluent buffer and followed by an analysis time of 6.5 minutes per sample. 
  3. For individuals with low hemoglobin readings, they must be pre-diluted in the ratio of 1:100 wash buffer in order to fit the total area within 1,000,000 to 3,000,000 µvolt·second to be considered as valid results.  

Interpretation

Peripheral whole blood can be effectively resolved into hemoglobin A, A2/E, F and other haemoglobins variants. This program has two calibrated areas (%), namely peak F and A2, whereby the HbF and HbA2 concentration can be measured.

Manufacturer assigned windows for Bio-Rad VARIANTTM HPCL System

Peak NameWindow (minutes)Retention Time (minutes)
F window1.00 – 1.301.15
P2 window1.30 – 1.601.45
P3 window1.60 – 1.901.75
A0 window1.90 – 3.302.60
A2 window3.30 – 3.903.60
D window3.90 – 4.304.10
S window4.30 – 4.704.50
C window4.90 – 5.305.10
HPLC chromatogram indicating elevated HbA2 levels, potentially suggesting homozygous Hb E.
This HPLC chromatogram depicts an individual with an elevated HbA2 level, a potential indication of homozygous Hb E. HbA2, a minor hemoglobin component, typically constitutes 1.5-3.5% of total hemoglobin in normal individuals. However, in individuals with homozygous Hb E, HbA2 levels can increase significantly, reaching up to 20% or more.

Disclaimer: This protocol is intended for informational purposes only and may need to be modified depending on the specific laboratory procedures and patient circumstances. Always consult with a qualified healthcare professional for guidance. See additional information.

References

  1. George E, Jamal AR, Khalid F, Osman KA. High performance liquid chromatography (HPLC) as a screening tool for classical Beta-thalassaemia trait in malaysia. Malays J Med Sci. 2001 Jul;8(2):40-6. PMID: 22893759; PMCID: PMC3413648.
  2. PK Gupta, H Kumar, S Kumar, M Jaiprakash. Cation Exchange High Performance Liquid Chromatography for Diagnosis of Haemoglobinopathies. Medical Journal Armed Forces India, 2009;65(1):33-37. https://doi.org/10.1016/S0377-1237(09)80051-8.
  3. M Dogaru, D Coriu, T Higgins. Comparison of two analytical methods (electrophoresis and HPLC) to detect thalassemias and hemoglobinopathies. Revista Română de Medicină de Laborator 2007; 9(4):39-48.

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