Haemoglobinopathy Diagnosis Essay

Question 1

It is a group of hereditary disorders in the blood that results in the synthesis of hemoglobin molecules with abnormal structure (CDC, 2014). It arises due to the substitution (Hb S and Hb C), deletion (Hb Gun Hill), hybridization (Hb Lepore) or elongation (Hb Constant Spring) of amino acids in the DNA sequence (Uthman, n.d). Haemoglobinopathy Diagnosis Essay. This group of single-gene disorders is commonly found in the Mediterranean, Southeast Asian and African populations. Approximately, 7% of the world’s population are carriers, meaning, they carry the abnormal gene but do not experience pathologic effects.

Question 2

The most common and most clinically relevant among this group of disorders are the following – thalassemia and sickle cell anemia (NCPEG, 2013). Those who have thalassemia produce an abnormally low quantity of hemoglobin, a molecule that transports oxygen in to different organs. In some thalassemia cases, no haemoglobin molecule is produced at all. The two major groups of thalassemia include the following – alpha and beta. The former is the most common form and usually exhibits mild symptoms while the latter is the classic form. The most severe form of thalassemia which is called Cooley’s anemia is under beta thalassemia. Sickle cell anemia patients, on the other hand, have red blood cells that are elongated in shape.Haemoglobinopathy Diagnosis Essay.  This may cause poor oxygen transport as well as blockage of blood vessels (WHO, 2011). In addition, the following clinical findings may be observed in sickle cell anemia patients – jaundice, cholelithiasis, aplastic crisis, hemolytic crisis, dactylitis, autosplenectomy, priapism, renal pallilary necrosis, infarctive crisis, sequestration crisis and leg ulcers (Uthman, n.d).

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Question 3

The aim of this practical is to give students the experience of working through the laboratory process of finding and identifying abnormal hemoglobins. Furthermore, the students are expected to – 1) outline a scheme for investigating a suspected hemoglobinopathy, 2) suggest some suitable techniques for hemoglobin analysis, 3) explain the principles of high performance liquid chromatography, and how it can be used to separate different forms of hemoglobin, 4) explain the principles and methodology of the Hb S solubility test, 5) describe the role of electrophoresis in the detection of abnormal hemoglobins, and 6) outline some other techniques that may be employed to definitively identify abnormal hemoglobins.Haemoglobinopathy Diagnosis Essay.

Question 4

The hemoglobin variants are generally identified based on their relative mobilities (CHORI, 2010). These variants migrate from the positive to the negative terminal at different speeds (Wintrobe, 1968) in certain pH levels (Sadoun, n.d). In citrate agar electrophoresis, hemoglobin variants are separated based on their interactions between the agar and citrate buffer ions (Turgeon, 2011).

Question 5

The following are the common haemoglobin variants that can be identified by citrate agar electrophoresis – Hb F, Hb A1, Hb A2, Hb S and, Hb C – arranged from the most to least anodal (StudyBlue.com). Proteins, in general are anodal, meaning, they tend to migrate in to the negative terminal during electrophoresis. Hb F, the fastest, is normally found in foetuses. Its production is stopped in early infancy (about 6 months of age). When present in adults, it is an indication of abnormality. Hb A, particularly the A2 derivative, is significant in the diagnosis of beta thalassemia because it is slightly elevated in such cases. This is also useful in ruling out iron deficiency anemia because Hb A2 is normal or slightly decreased in such cases. In determining cases of sickle cell anemia, the presence of Hb S is needed to be established.Haemoglobinopathy Diagnosis Essay.  The existence of Hb C, on the other hand, results in the decrease of red blood cell life span. Target cells are usually found in persons with Hb C disease, and usually suffer from less severe pathologic manifestations than those with Hb S.

Question 6

These answers were inferred based on the blood count and blood film results only.
Sample A –No abnormal hemoglobin variant detected
Sample B – A possible thalassemia case, Hb H or Hb A2 was/were detected in HPLC Test.
Sample C – A possible case of sickle cell anemia or hemoglobin SC disease, Hb S and/or Hb C was/were detected. Abnormal Hb S/Hb C band/s was/were found in HPLC test result. Hb S and was confirmed using Sickle Solubility Test. Haemoglobinopathy Diagnosis Essay.

Question 7

Other techniques in the identification of variant hemoglobins include protein chemistry methods such as isoelectric focusing (IEF) and high-performance liquid chromatography (HPLC) as well as Sickle Solubility Tests (Lubin et al, 2015). To quantitatively determine differences in globin chain synthesis, isoelectric focusing technique is used. On the other hand, hemoglobins are separated based on their various affinities for the column in high performance liquid chromatography technique, making it an ideal technique for screening different variants of abnormal hemoglobins. If initially found in electrophoresis or high performance liquid chromatography tests, which serve as screening tests, the presence of Hb S in suspected sickle cell anemia cases is confirmed using sickle solubility test.

Question 8

Isoelectric focusing is used for analysis of whole blood samples, dried blood spots or haemolysates. It is also semi-automated that is why large number of samples can be screened. A good separation of Hb F from Hb A as well as other clinically significant hemoglobin variants can be observed. However, interpretation is more difficult because it separates hemoglobin into its post-translational derivatives, eg. Hb A can separate into Ao and A1 and other products (Ryan et al, 2010).
High-performance liquid chromatography (HPLC) is fully automated and requires a very small amount of blood sample, usually, 5uL. It is suitable for the diagnosis of beta thalassemia because it can detect gamma hemoglobins. HPLC separates Hb A, Hb A2, Hb F, Hb S, Hb C, Hb D-Punjab and Hb G-Philadelphia from each other, however it cannot separate hemoglobins that co-elute with each other. Haemoglobinopathy Diagnosis Essay. Another disadvantage is that it separates glycosylated forms of hemoglobin as well as hemoglobin derivatives, which can make interpretations difficult (Ryan et al, 2010).
Sickle Solubility Testsare commonly used to detect Hb S even beyond infancy period where concentrations are as low as 20%. However, false positive results may be obseved in patients with anemia and high levels of protein (Ryan et al, 2010).

Question 9

Hemoglobinopathies are usually diagnosed using electrophoresis and HPLC techniques but some are missed. In addition, mutant hemoglobins are not readily identified by these methods. These are the reasons why confirmatory tests are performed (ARUP.com, 2006). Suspected hemoglobin variants can be identified using polymerase chain reaction (PCR) or other DNA analysis methods (Kutlar, 2007). Hemoglobin instability tests, particularly isopropanol and heat tests, can detect unstable hemoglobins in cases of hemolytic anemia (Ithanet, 2012). Another confirmatory test used in polycythemia cases is the P50 test (Mayo Clinic, n.d). This test is also used in diagnosing patients with decreased oxygen affinity haemoglobin. Haemoglobinopathy Diagnosis Essay.

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References:

ARUP 2006, Hemoglobinopathies, ARUP Laboratories, viewed 23 February 2015, http://www.arupconsult.com/Topics/Hemoglobinopathies.html
CDC 2014, Hemoglobinopathies Monitoring, Centers for Disease Control and Prevention, Georgia, viewed 23 February 2015, http://www.cdc.gov/ncbddd/hemoglobinopathies/
CHORI 2010, Hemoglobin Separation by Citrate AgarElectrophoresis (CAE), viewed 23 February 2015, http://rbclab.com/Pages/200/210/113%20CAE6/210%20113.html
Ithanet 2012, Protocol: Hb Stability – Isopropanol and Heat Tests, Semantic MediaWiki, viewed 23 February 2015, http://www.ithanet.eu/ithapedia/index.php/Protocol:Hb_stability_%E2%80%93_isopropanol_and_heat_tests
Kutlar, F. 2007, ‘Diagnostic Approach to Hemoglobinopathies’, Hemoglobin, vol. 31, no. 2, pp. 243-250, viewed 23 February 2015, http://www.ncbi.nlm.nih.gov/pubmed/17486507
Lubin et al. 2015, ‘Laboratory Diagnosis of Hemoglobinopathies’, Clinical Biochemistry, vol. 24, no. 4, pp. 363-374, viewed 23 February 2015,  Haemoglobinopathy Diagnosis Essay.http://www.sciencedirect.com/science/article/pii/000991209180011Q
Mayo Clinic n.d, Oxygen Dissociation, P50, Erythrocytes, viewed 23 February 2015, http://www.mayomedicallaboratories.com/test-catalog/Clinical+and+Interpretive/9110
NCHPEG 2013, Hemoglobinopathies: Sickle Cell Disease, alpha-Thalassemia, and beta-Thalassemia, viewed 23 February 2015, http://www.nchpeg.org/index.php?option=com_content&view=article&id=427
Ryan, K. et al. 2010, ‘Significant haemoglobinopathies: guidelines for screening and diagnosis’, British Journal of Haematology, vol. 149, issue 1, pp. 35–49
Sadoun, H. n.d., Hemoglobin electophoresis, lecture Powerpoint Slides, viewed 23 February 2015, http://www.kau.edu.sa/Files/0007058/Files/63705_Hemoglobin%20Electrophoresis.pdf
Study Blue 2014, Porphyrins and Hemoglobin, lecture Flashcards, viewed 23 February 2015, https://www.studyblue.com/notes/note/n/porphyrins-and-hemoglobin/deck/4121929
Turgeon, M. 2011, Clinical Hematology: Theory and Procedures, 5th ed. Lippincott Williams & Wilkins, Philadelphia, Pennsylvania.
Uthman, E. n.d., Hemoglobinopathies and Thalassemias, viewed 23 February 2015, http://web2.airmail.net/uthman/hemoglobinopathy/hemoglobinopathy.html
Wintrobe, M. 1967, Clinical Hematology, 6th Edition. Lea and Febiger, Philadelphia.
WHO 2011, Sickle-cell disease and other haemoglobin disorders, viewed 23 February 2015, http://www.who.int/mediacentre/factsheets/fs308/en/. Haemoglobinopathy Diagnosis Essay.