Chromatography-for-Hemoglobinopathies table 5.pptx.pptx

Biochemistry practical

1. Chromatography for Hemoglobinopathies

2. Introduction to Hemoglobinopathies Hemoglobinopathies are a group of inherited blood disorders affecting the structure or production of hemoglobin. These disorders can have a wide range of effects, depending on the specific type and severity of the mutation. 1 What are Hemoglobinopathies ? Inherited disorders resulting from genetic mutations affecting the globin chains (alpha or beta) that constitute hemoglobin. 2 Diagnostic Importance Early and accurate diagnosis is crucial for personalized treatment plans, managing complications (e.g., vaso-occlusive crises in sickle cell disease), and providing genetic counseling to families to assess the risk of inheritance. 3 Forms of Hemoglobinopathies This broad category encompasses numerous disorders, including sickle cell anemia (characterized by abnormal hemoglobin S), beta-thalassemia (reduced or absent beta-globin chains), alpha-thalassemia (reduced or absent alpha-globin chains), and hemoglobin E disease (another common variant in Southeast Asia).

3. Basics of Chromatography 1 Definition A laboratory technique that separates a mixture's components based on their differential affinities for a stationary and a mobile phase. 2 Principles Separation relies on the differential partitioning of sample components between a stationary phase (e.g., a solid adsorbent or a liquid bound to a solid support) and a mobile phase (a liquid or gas). Components with higher affinity for the stationary phase move slower, while those with higher affinity for the mobile phase move faster, leading to separation.

4. Chromatography techniques for Hemoglobinopathy Diagnosis High-Performance Liquid Chromatography (HPLC): HPLC is the gold standard for hemoglobinopathy diagnosis, offering precise separation of different hemoglobin variants based on their charge and hydrophobicity. Ion-Exchange Chromatography (IEC) : Ion exchange chromatography (IEC) is a technique used to separate and identify different hemoglobin variants. IEC is generally less expensive and time-consuming compared to HPLC, but may offer slightly less resolutio n. Thin-Layer Chromatography (TLC) : Thin-layer chromatography (TLC) is a simpler, less expensive technique that can be used for preliminary screening or in resource-limited settings. It may not offer the same resolution as HPLC or IEC, it can still be useful.

5. Chromatography's Role in Diagnosis Identifying Hemoglobin va riants Chromatography, particularly High-Performance Liquid Chromatography (HPLC) and ion-exchange chromatography, separates hemoglobin molecules based on their net charge and other properties. This allows for the precise identification of abnormal hemoglobins such as HbS (sickle cell hemoglobin) and HbC, crucial for diagnosing sickle cell anemia and other hemoglobinopathies. Comparison with Other Diagnostic Methods Compared to methods like electrophoresis, which may have limitations in resolving closely related hemoglobin variants, chromatography offers higher resolution and sensitivity. This enhanced accuracy in identifying specific hemoglobin variants is vital for providing accurate diagnoses and tailoring effective treatment plans for patients with hemoglobinopathies.

6. High-Performance Liquid Chromatography (HPLC) 1 Blood Sample Collection and Preparation A blood sample is collected, and hemoglobin is carefully extracted and purified from red blood cells. This purification step removes any substances that could interfere with the analysis. 2 HPLC Column Selection The choice of HPLC column is crucial and depends on the specific hemoglobin variants being analyzed. 3 Sample Injection and Separation The purified hemoglobin sample is carefully injected into the HPLC system. A precisely controlled mixture of solvents (the mobile phase) flows through the column, carrying the hemoglobin molecules. Separation happens because different hemoglobin variants interact differently with the stationary phase inside the column, causing them to travel at different speeds and thus separate over time. 4 Detection and Quantification A UV-Vis spectrophotometer is commonly used as a detector, measuring how much light the eluting hemoglobins absorb at specific wavelengths. The detector's signal is directly proportional to the amount of each hemoglobin variant present. 5 Data Analysis and Interpretation The resulting chromatogram—a graph showing absorbance versus time—shows distinct peaks for each hemoglobin variant. The time it takes for each peak to appear (retention time) and the size of each peak (peak area) are used to identify and measure the amounts of the various hemoglobin variants, including abnormal ones like HbS, HbC, and HbE.

7. Case Studies: HPLC Analysis Hemoglobin Variant Chromatographic Findings & Interpretation HbS (Sickle Cell Hemoglobin) Hemoglobin HPLC showed raised fetal hemoglobin (HbF) and a variant peak in S window (71.9%) at retention time of 4.36 mins. Adult Hb (HbA) of 8.5% was noted. Diagnosis mainly relies upon identification of HbS Beta Thalassemia HPLC of index case with elevated HbA2 and abundant HbF at 95% without HbA. Elevated HbA2 and elevated HbF are seen in beta thalassemia . Nearly all hemoglobin is HbF in a thalassemia major while in thalassemia trait the levels of HbF are much lower with largely preserved HbA levels.

8. Ion Exchange Chromatography Ion exchange chromatography (IEC) is a powerful technique used to separate ions and polar molecules based on their charge. In hemoglobinopathy diagnosis, IEC uses a resin to separate different hemoglobin variants (HbA, HbS, HbC, etc.) due to their varying net charges at a specific pH. Stationary Phase & Mobile Phase The process uses a stationary phase (charged resin) and a mobile phase (buffer solution) carrying the hemoglobin mixture. Binding Process Hemoglobin variants with opposite charges to the resin bind, while others pass through. Selective Elution By adjusting the mobile phase's ionic strength or pH, bound hemoglobins are selectively eluted, separating the different hemoglobin types. This separation enables the identification and quantification of various hemoglobins, crucial for diagnosing hemoglobinopathies.

9. Advantages of Chromatography 1 High Sensitivity Detects even minuscule amounts of abnormal hemoglobin, crucial for early diagnosis and intervention, especially in cases where the presence of variant hemoglobin might be low. 2 High Specificity Accurately identifies specific hemoglobin variants, differentiating between various types of abnormal hemoglobins (like HbS, HbC, HbE, etc.) without confusion, thus enabling precise diagnosis and tailored treatment. 3 Rapid Results Offers a significantly faster turnaround time compared to older methods, expediting diagnosis and allowing for quicker initiation of appropriate management strategies for patients. 4 Quantitative Analysis Not only identifies the presence of different hemoglobins but also precisely quantifies their proportions. This is important for assessing disease severity and monitoring treatment response.

10. Challenges and Limitations of Chromatography in Hemoglobinopathy Diagnosis Sample Contamination Blood sample contamination from other substances can hinder accurate hemoglobin variant separation and detection. Careful sample preparation, including proper handling and filtration, is essential. Instrument Calibration and Maintenance Regular HPLC system calibration (detector and pump) ensures precise hemoglobin variant quantification. Poor maintenance, affecting column performance (pressure fluctuations, peak broadening), compromises accuracy and reliability. Complex Data Interpretation Chromatogram interpretation demands expertise to distinguish normal from abnormal hemoglobin peaks. Subtle variations in peak shapes or retention times can be challenging, potentially requiring additional analytical techniques.