Hemoglobinopathies: Analytic Diagnostic Techniques
Hemoglobinopathies and Thalassemia’s are a kind of Genetic disorders caused mainly by a mutation or deletion of Globin genes in Hemoglobin. Hemoglobin consist of α or β Amino acid chains. An alteration in α chain production leads to α Thalassemia and alteration in β chain causes β Thalassemia. Hemoglobinopathies on the other hand may be caused due to the structural abnormalities or alteration of amino acid sequences in the Globin genes. These are mainly hereditary and constitute to around 4 to 5% of the world population. (WHO Statistics, 2012) Each year around 250,000 to 300,000 new born children have either hemoglobinopathies or thalassemia. Both of these syndromes may lead to anemia. Different variants of hemoglobin have been studied over the years, the main types include:
Hb S, Hb C, Hb E, Hb D-Punjab, Hb O-Arab, Hb G-Philadelphia, Hb Constant Spring, Hb Hasharon, Hb Korle-Bu, Hb Lepore, Hb M and Hb Kansas. (Trent RJ, 2006, pp:27–38)
However this paper would involve Hemoglobinopathies and its analytical analysis. The methods involved should be precise and quantification of results is highly essential. The main methods used in this paper are high performance liquid chromatography (HPLC), Electrophoresis, Mass Spectrophotometry (MS) and Sickle solubility test. The principles and methodology of these techniques are widely studied globally. (Daniel, Y. A. 2005pp 635–643)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
Fully automated systems have replaced traditional methods like electrophoresis in screening of hemoglobinopathies. One of the widely used methods are cation-exchange HPLC. However the methodology to be implied will depend on the amount of workload, type of sample (dry blood or liquid blood spots), handling, Reproducibility. The other factors that may count include expertise and cost. The materials required for electrophoresis may although be cheaper, but labor cost definitely sets the cost margin high. Opting for HPLC may thus be cost neutral, faster and more precise.
Varied types of haemoglobins can be detected using high performance liquid chromatograph. Detection, identification and quantification are the basic principles which HPLC follows. HPLC usually quantifies Hb A2 in an accurate and precise manner. It is mainly used to detect β thalassemia. Haemoglobins A, A2, F, S, C, D-Punjab and G-Philadelphia are mainly separated using HPLC. Along with these, glycosylated and derivative forms of haemoglobin are separated which may hinder results and efficiency of the techniques. The chromatograms on every occasion should be double checked and surety of using controls on every batch is highly recommended. Maintaining and checking of widows in first stage analysis is essential. Adjusting the column temperature and flow rate may improvise the position on the windows. The levels of Hb A2 and Hb F is to be checked on a daily basis and controls should be used in every step. A known fact with use of HPLC is that variants of haemoglobin may co-elute with Hb A2 which may make separation and quantification of variants difficult.
(Shirley L. October 2009.)
The use of HPLC in detection and analysis of hemoglobinopathies has several advantages, usually in comparison to electrophoresis:
- Small samples (5 μl) are sufficient for analysis.
- Automated system enables to carry out large batches in lesser time.
- Separation and Quantification of varied haemoglobins in every sample.
- Identification of larger variants of haemoglobin can be done.
- δ chain variants detection is easier, which is an important detection in β thalassemia.
- Automated and lesser labor cost.
Use of HPLC for identification of large number of variants of Hemoglobin. Alternate back up methods should be conducted for confirmation.
(Kate Ryan, et al. 2010. pp35-49)
Mass Spectrophotometry
Mass spectrophotometry is an emerging techniques involved for reliable and accurate diagnosis of haemoglobinopathies. Most diagnostic methods involve double checking for positives or counter checking with other alternative methods. Mass spectrometry–mass spectrometry (MSMS) is potentially accurate, reliable and faster, it is widely used for detection of any inherited sickle trait or heamoglobin mutations.
(Daniel, Y. A. et al. 2005. pp635–643)
Characterization of haemoglobinopathies is possible through Electrospray mass spectrometry (MS) or Mass spectrophotometry- Mass spectrophotometry (MSMS). MS-MS technique is latest for detection of intact globin chains. This is done by initial MS-derived blood scans. The technique is also used for peptide analysis which is essential for haemoglobinopathies. Globin mutations can be easily detected through mass spectrophotometry. Clinically important variants of Globin can be detected through the patented MSMS technique.
In the last 30 years, Mass spectrometry (MS), a technique was widely used in clinical chemistry, research and medicinal laboratories. Lately it has been used in the field of Hb analysis. Tryptic analysis of Hb was deviced in the year 1981 by a premier scientist.
(Wada Y, et al. 1981. pp233-241.)
The study of intact globin chains can be done with the help of soft ionization techniques electrospray ionization (ESI) and MALDI. In early1990, the application of ESI MS involving intact Hb chains was reported by ESI or MALDI-TOF MS and later became a known method for intact globin chain analysis of Hb. (Falick AM et al. 1990.396-400.)
Around 60 mutations in intact globin chains of Hb can be detected through various means of MS. Also lately muatations or alterations can be analyzed using a nanoESI MS technique. The most advanced technique is the use of MALDI-TOF MS, it is said to have recognized any particular mutation in globin chains from a single red blood cell. Complete and final analysis of mutations or detection of Hb variants can be done through protein sequence analysis. MS techniques has also claimed to detect mutations in globin chains near to termini of the β-chain of Hb. ESI-MS is most frequently used for studying Hb variants and is also associated with tandem MS using peptide sequencing techniques. The drawback for this technique is its formation of multiple charged fragments. (Wild BJ, et al. 2001. pp691-704.)
Alternatively, MALDI-TOF MS produces single-charge peptide ions and is used to detect single mutation of Hb variants. The detection of multiple variants is not often seen with MALDI-TOF MS. (McCombs ME. et al.1998. 5142-5149.)
Studies have also shown that MALDI-TOF MS can be used to identify Hb variants along with abnormal globin chains analysis.
Most samples are introduced into a mass spectrophotometer. The most widely used mass spectrophotometers are SCIEX API 4000 (Applied Bio-systems, Warrington, UK) and QTRAP® 6500 (Applied Bio-systems, Warrington, UK)
Hb S, Hb C and Hb D variants of haemoglobin can be identified using mass spectrophotometry. The system has advantages like high precision, accuracy and lesser time. However large amount of samples cannot be carried out at the same time and this being a disadvantage in laboratories where workload is high. (Edwards RL et al. 2011pp.265–2270)
Electrophoresis (CAE) Cellulose Acetate Electrophoresis
Fractions of Hb variants can be quantified and qualitatively analyzed by traditional methods like electrophoresis. There are numerous electrophoresis methods which can be employed for eluting out varied fractions. Cellulose acetate electrophoresis (CAE) at an alkaline pH (8.2 to 8.4) or even basic agarose gel electrophoresis (AGE) at acidic pH (6.0 to 6.2) are carried out for separation of Hb A, Hb F, Hb S/D, Hb C/E/O-arab and other variants of Hemoglobin. After running the gel as mentioned in the kits, Hb bands can be visualized by staining with acid violet or amido black or similar stains. At alkaline pH variants of Hb like Hb C, Hb E, Hb A2, and Hb O are successfully separated. Co-migration of Hb S, Hb D, and Hb G can also be seen. Alternatively at acidic pH, separation of Hb C from Hb E, and Hb O and Hb S from Hb D and Hb G is seen. The major advantages of electrophoresis methods is because of their simplicity. However, CAE often requiring more time and labor is not preferred. Another drawback is the inability to quantify Hb A2. The use of HPLC can solve major of the problems in electrophoretic methods and hence it is much preferred. (Ryan K, et al. 2010. pp35-49.)
Another unique type of electrophoresis devised by Sebia, Capillary electrophoresis. The principle of liquid-flow electrophoresis is used for Hb analysis. Hemolysates are automatically prepared by the instrument. This is then followed by sample loading into the capillary. Migration of samples occurs simultaneously followed by quantification and separation of Hb fractions. A survey according to College of American Pathology (CAP) 2009 showed that clinical laboratories have reported hemoglobinopathies in 7% of cases using capillary electrophoresis. (Ryan K, et al. 2010 pp.35-49.)
SICKLE SOLUBILITY TEST
It is widely used analytical technique to determine Hb S. The technique is said to detect Hb S at very low concentrations. This method can this detect all kinds of sickle cell trait. A drawback of this techniques is the presence of false positives, in case of individuals with high plasma protein levels or anemic patients. This can be resolved by washing out red blood cells or using concentrated form of the blood sample.
All possible testing done through sickle solubility test should be confirmed by HPLC. This is often done to confirm the presence of Hemoglobin S variant and also to detect the sickle cell trait. Sickle solubility test is not performed in infants below 6 months in age and is widely used in emergency cases, mostly before anaesthesia. The emergence of HPLC has led to less use of Sickle solubility test, since it has limited use and interpretation of data is misleading because of the occurrence of false positives. (Ryan K, Bain BJ, et al. 2010 pp35-49.)
Sickle Solubility Test. Control (AA) AND Hb S Variant (AS)
Image Source: Wellcome images. [Electronic Print] Available at: < http://wellcomeimages.org/indexplus/result.html?*sform=wellcome-images&_IXACTION_=query&%24%3Dtoday=&_IXFIRST_=1&%3Did_ref=N0015517&_IXSPFX_=templates/t&_IXFPFX_=templates/t&_IXMAXHITS_=1 > [Accessed 16 December 2013]
Comparative Analysis of Equipment and Techniques
The techniques mentioned in the paper have been widely used in Clinical laboratories, pharmaceutical companies, industries and chemistry pathological laboratories. The techniques have been considered dependent on the type of work load, labor, amount of blood samples, and the emergence of a situation. Based on these and many factors, appropriate use of a particular technique depends on the work environment and facilities.
HPLC is the most widely used technique with many advantages following it. It can be used for large samples within lesser time compared to electrophoresis and isoelectric focusing techniques. However the use of HPLC may encounter certain misleading results and need to be counter checked with a second round or alternatively with another technique.
Electrophoresis is the simplest method for haematoglobiopathies screening.Setting up the equipment or kit is simple and handling is more efficient. However labor work increases the cost of this technique. Time is another factor where the technique falls back. Analysis of the data is quite simple and accurate separation of Hb variants is possible. Detecting abnormal Hb variants is crucial for screening in haemoglobinopathies.
Mass spectrophotometry is an emerging techniques involved for reliable and accurate diagnosis of haemoglobinopathies. Most diagnostic methods involve double checking for positives or counter checking with other alternative methods. Mass spectrometry–mass spectrometry (MSMS) is potentially accurate, reliable and faster, it is widely used for detection of any inherited sickle trait or heamoglobin mutations.
Sickle solubility test may not have the accuracy and precision compared to the above techniques but is used in emergency situations. The method is simple and quick.
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