what does it mean to have abnormal hemoglobin?

• Journal Listing
• Indian J Med Res
• v.134(4); 2011 October
• PMC3237254

Indian J Med Res. 2011 Oct; 134(4): 538–546.

Aberrant haemoglobins: detection & characterization

Henri Wajcman

INSERM, Université Paris-Est, Faculté de Médecine, Créteil, France

Kamran Moradkhani

^* AP-HP, Groupe Henri-Mondor Albert-Chenevier, Service de Biochimie-Génétique, Créteil, France

Abstract

Haemoglobin (Hb) abnormalities though quite frequent, are generally detected in populations during surveys and programmes run for prevention of Hb disorders. Several methods are now bachelor for detection of Hb abnormalities. In this review, the post-obit are discussed: (i) the methods used for characterization of haemoglobin disorders; (ii) the problems linked to diagnosis of thalassaemic trait; (iii) the strategy for detection of common Hb variants; and (iv) the difficulties in identification of rare variants. The differences between developing and industrialized countries for the strategies employed in the diagnosis of aberrant haemoglobins are considered. We mention the limits and pitfalls for each approach and the necessity to characterize the abnormalities using at least two different methods. The recommended strategy is to use a combination of cation-exchange high operation chromatography (CE-HPLC), capillary electrophoresis (CE) and when possible isoelectric focusing (IEF). Hard cases may demand further investigations requiring specialized poly peptide and/or molecular biology techniques.

Keywords: Haemoglobin variant, neonatal screening, RBC density, sickle cell, thalassaemia

Introduction

Haemoglobin (Hb) abnormalities are the most frequent genetic disease, affecting approximately seven per cent of the world population¹. Today, abnormal Hbs are generally discovered during a systematic study performed within programmes for prevention of thalassaemias or sickle cell disease. In several regions (India, Turkey, Irak, Iran, Gaza strip, Saudi Arabia, Cyprus, etc.), these are plant during a premarital screening. In other regions, like westward Europeans countries, the research for the main Hb disorders is often express to populations at gamble. It is washed either every bit preconceptional or neonatal screening programmes.

In all cases, also the study of the Hb, a commencement essential test that demand to be performed is a complete blood cell count (CBC) looking more often than not for anaemia, microcytosis and, hypochromia. If necessary, information technology should be completed past a control of the claret iron status. Several high throughput methods, now available, such as cation-exchange loftier performance chromatography (CE-HPLC)^{2 ,3} or more recently capillary electrophoresis (CE)⁴ are generally employed. Nevertheless, electrophoretic studies [cellulose-acetate electrophoresis or isoelectric focusing (IEF)] are still performed in many laboratories⁵. In some developing countries, the Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT), a cost effective, rapid and reliable screening test for detection of β-thalassaemia trait, is largely used equally a first approach⁶.

The investigation of a patient presenting with a haematological disorder such as a chronic haemolytic anaemia, an unexplained polycythaemia or a permanent cyanosis is another, completely unlike condition, leading to the discovery of an abnormal Hb.

In this review, nosotros will talk over (i) the methods used for characterization of haemoglobin disorders; (ii) the issues linked to diagnosis of thalassaemic trait; (three) the strategy for detection of common Hb variants; and (iv) the difficulties in identification of rare variants.

Methodological aspects: The main tests used for phenotype characterization

Electrophoresis, a test based on the migration of electrically charged molecules under an applied electrical field, occupies 1 of the virtually important places in the history of abnormal Hb detection. Hb S [β6 Glu>Val], the first abnormal Hb described, was discovered in 1949 by Pauling et al using moving boundary electrophoresis⁷. Zone electrophoresis performed on cellulose acetate strips (CAE) is notwithstanding used in many clinical laboratories. This technique has a resolution lower than that of IEF, simply considering of its simplicity remains among the more popular methods used in Hb screening. In this technique, the Hb molecules are separated at alkaline metal pH. Under these conditions all Hbs have a negative charge and migrate towards the cathode. Hb South, which has an boosted positive accuse compared to Hb A migrates more slowly^v.

In IEF, a pH gradient is established by carrier ampholytes submitted to an electric electric current and the Hb molecules migrate across this gradient until they reach the position where their cyberspace charge is zip (isoelectric indicate)⁸. The molecules will then concentrate in a abrupt ring as illustrated in Fig. ane. This technique allows separating molecules with isoelectric points differing only by 0.02 pH unit. Hb D-Punjab [β121 Glu>Gln], not detectable from Hb Southward by CAE, could easily be recognized by this technique. The disadvantage of this method is its relatively high cost and the requirement for a well-experienced laboratory staff.

Isoelectric focusing analysis of red blood cells lysates containing unlike Hb variants, technical details are available in Ref. five. Line 1: β thalassaemia trait; Line 2: Normal adult; Line 3: Homozygous β⁺ thal; Line 4: HbA/Hb Korle Bu; Line 5: HbA/HbD-Punjab; Line six: HbA/HbO-Arab; Line seven: Normal newborn; Line 8: HbA/HbE; Line 9: HbA/HbC; Line 10: HbA/HbS; Line eleven: HbS/HbC.

Electrophoresis of Hbs on agar at acidic pH has been introduced some forty years ago^ix. It is not primarily sensitive to the charge of the mutated residue only to structural modifications of positively charged regions of the Hb molecule interacting with the agaropectin independent in the gel^x. This property is of special involvement to distinguish Hb South from other variants displaying mobility close to that of Hb South on CAE or IEF.

Capillary electrophoresis has been recently adapted to Hb written report^xi. This technique shares several advantages with CE-HPLC: information technology is a high resolution method associated with semi-automation, on-line detection and straight quantification of normal and abnormal Hb fractions. The Capillarys® organisation (Sebia, Evry, France) is equipped with eight capillaries in parallel, assuasive multiple and simultaneous sample assay. Each capillary can be used at least 3000 times¹².

Cation-exchange HPLC is considered every bit the method of choice to quantify the diverse normal and abnormal Hb fractions¹³. This is the method of reference for measuring Hb A_1C for monitoring of diabetes mellitus. It is also of full general use for measurement of the levels of Hb A₂, Hb F and of several abnormal Hbs^{ii ,3}. Automatic appliance accept been adult since several years ago for large series measurement. Cation-substitution HPLC and CE are the two methods recommended for detection of thalassaemic traits necessary for genetic counselling since these are the ones assuasive a precise quantification of Hb A_two, Hb F and abnormal Hb fractions. To make the diagnosis of a thalassaemic trait or quantify the abnormal Hb fraction, the use of densitometry scanning of electrophoretic pattern is a likewise imprecise method to be used today.

Three methods are proposed for neonatal screening of sickle cell affliction (Fig. 2). The starting time one is an adaptation of IEF which allows the simultaneous study of most 90 samples on a single plate, using a central cathode and two anodes^xiv. At a glance, whatsoever ring with abnormal mobility is detected too as the absence of the normal Hb A ring. This method is considered as a first line test and requires a confirmation by a second one, normally CE-HPLC. Cation-exchange-HPLC equipment devoted to neonatal screening have been developed which, in a 2.5 min programme per sample, allow quantitative detection of Hb S and of the most common Hb variants¹⁵. This approach by CE-HPLC is also suitable for neonatal diagnosis of thalassaemic patients^xvi. Capillary electrophoresis is the third method that may exist successfully used for neonatal screening¹⁷. Characterization of rare Hb variants responsible for a haematological disorder requires a completely different approach. The presence of a variant is usually obtained by IEF, which shows either a band with a clearly different isoelectric bespeak or an abnormal aspect of the Hb A band suggesting that information technology contains two fractions (Fig. 1). Cation-exchange HPLC or CE may ostend the presence of this abnormal component or reveal an abnormal Hb, not detected by IEF. The use of additional methods is often required. For example, reversed-phase loftier-performance liquid chromatography (RP-HPLC) reveals differences in hydrophobicity allowing discrimination betwixt variants displaying identical charges. This approach also indicates which globin chain is affected¹⁸.

Neonatal diagnosis for Hb S and Hb D-Punjab. Analysis of RBC lysates obtained from a dried claret spot obtained during the neonatal screening programme done in our laboratory. Analysis by two methods is required for the diagnosis. (A) Isoelectric focusing: Hb Southward and Hb D-Punjab have close isoelectric points and the resolution betwixt the 2 bands is poor. The absenteeism of HbA and thickening of HbS band help to diagnose. (B) Cation-exchange HPLC shows ii abnormal peaks: one is eluted in S window and the other in the D window. (C) Capillary electrophoresis: (to a higher place): newborn heterozygous for Hb D-Punjab and (beneath): newborn heterozygous for Hb South. The two Hbs have different migrations.

In well-equipped laboratories of industrialized countries mass spectrometry methods may be used to determine the structural abnormalities. Electrospray mass spectrometry may reveal a globin chain with an aberrant mass and, from the mass difference observed, the type of amino acrid exchanged could often exist deduced. Methods such equally MALDI-TOF or mass-mass spectrometry in which the globin bondage are cleaved in modest fragments will show a unmarried fragment, or a family of fragments from which the exact substitution could exist determined^{nineteen ,20}. Today, the facility for doing a Deoxyribonucleic acid sequence analysis and its relatively depression cost changed completely the approach for characterization of some rare Hb variants associated with a severe pathological phenotype not detected past conventional methods.

In that location are many different PCR-based techniques that can exist used to diagnose the globin cistron mutations, including dot-absorb assay, reverse dot-blot analysis, the amplification refractory mutation system (Arms), high resolution melting (HRM), gap-PCR and restriction endonuclease assay^{21 ,22}. Microarray-based human globin gene mutation detection methods have been recently reviewed²³.

Problems linked to diagnosis of β-thalassaemia trait

The key element in the diagnosis of a β-thalassaemia trait is the presence of non-iron scarce microcytic hypochromia anaemia. The screening of thalassaemia trait in the areas with limited laboratory facilities is often washed by NESTROFT test^half-dozen. Despite its sensitivity and rapidity, in around one out of four cases of fe deficiency anaemia, this test leads to a faux positive consequence²⁴. The all-time arroyo to screen the thalassaemia traits is, therefore, determining the Hb A₂ level. The large bulk of β-thalassaemia carriers nowadays with a high Hb A_two level, and this often accompanied with a moderate increase in Hb F level²⁵.

Hb A_two level is correctly measured by CE-HPLC simply information technology is necessary to proceed in mind that the normal value depends upon the experimental procedures used²⁶. From one chromatographic system to some other slight differences may be observed and values inside the normal range in a given organisation may well fall out of the normal range in another arrangement. Thus the normal values of the laboratory where the test is performed should ever be specified.

The Hb A₂ level may be modified by many factors. The about frequent problem is the co-beingness of an iron deficiency which may even normalize the Hb A₂ level requiring a novel Hb analysis after iron deficiency treatment. In β-thalassaemia carriers presenting with a normal Hb A_two level, the nigh frequent cause is a co-inherited δ-globin abnormality. The thalassaemic δ alleles are not exceptional affecting at to the lowest degree more one per cent of general population²⁷. The well-nigh frequent δ thalassaemic variant is Hb Yialoussa [δ27 Ala>Ser] which is quite frequent in Mediterranean region²⁸. Other δ- and α-globin concatenation abnormalities lead to unlike type of Hb A_ii. In these cases one should add together the two Hb A_ii fractions to obtain the total Hb A₂ level. In some cases the abnormal Hb A₂ fraction may only be visualized by using some other analytical test. Conversely, falsely increased levels of Hb A₂ may outcome from the co-existence of a variant with electrophoretic or chromatographic properties close to that of Hb A₂. As a rule, this situation has to be verified when a level of Hb A_two higher than 8 per cent is observed. The presence of a thalassaemia will generally exist ruled out by because haematological data showing absenteeism of microcytosis and hypochromia. Withal, two mutual variants, which elute on CE-HPLC in the Hb A₂ window, are really thalassaemic Hbs. The first one is Hb Due east, frequent in populations from Due south Eastern asia, and the second is Hb Lepore. Both variants are easy to distinguish by their electrophoretic behaviour. Since many other thalassaemic defects are present in the same populations every bit Hb E, a big variety of clinical phenotype associated with Hb E is observed²⁹. These range from very mild ones, the simple heterozygous land for Hb E, to severe ones, which associate Hb E to β° thalassaemia.

Some other important element for the diagnosis of a β-thalassaemia trait is the presence of Hb F. In the past, the classical way to determine its percentage was to perform a resistance to alkali denaturation test. Equally other kinetic techniques, information technology needs to be performed under rigorous experimental weather condition with a precise respect of the timing of the reaction³⁰. This method measures together Hb F and its acetylated fraction, which amounts to well-nigh x per cent of information technology. Now CE-HPLC and CE allow a direct measurement of the Hb F fractions. Depending on the system used, another Hb adducts may elute together with Hb F and increment slightly the area of the elution meridian. In these systems acetylated Hb F elutes generally in another position than Hb F and is therefore, not included in the event. Thus, in the same sample the percentage of Hb F will differ co-ordinate to the method used. This is not really important since in an adult any value of Hb F higher than 1 per cent should be considered as unusual. Increased Hb F level may indicate a β-thalassaemia trait but it could also be the upshot of a hereditary persistence of foetal Hb (HPFH) or of another condition, such every bit diabetes mellitus, or pregnancy.

Diagnosis of an α thalassaemia will be suspected by haematological indices showing some microcytosis and hypochromia, with a degree depending upon the type of thalassaemia. The Hb A_two per centum is theoretically decreased in proportion with the number of defective α genes. Abnormal Hb tetramers are observed in the more severe forms: Hb Bart's (γ4) in newborns, and Hb H (β4) in adults. The presence of an α thal-2 in which but 1 of the four α genes is inefficient is one of the most common genetic modifications observed worldwide. An interaction of this state of affairs with other globin abnormalities should always exist considered every bit a possible modulator cistron³¹. In a carrier of a β- thalassaemic trait the co-existence of an α-thalassaemia will partially compensate the disequilibrium in globin synthesis; due to a lesser corporeality of toxic free α globin concatenation bound to the red jail cell membrane, the hypochromic anaemia volition be much better tolerated. Conversely the association of a β-thalassaemic trait with additional α genes may lead to a β-thalassaemia intermedia phenotype³².

Problems linked to diagnosis of mutual Hb variants

Hb S is, worldwide, the nearly frequent, clinically astringent Hb variant. Information technology is specially encountered in populations from African ancestry, essentially carried on chromosomes with Senegal, Benin or Bantou haplotypes³³. In India and Standard arabic countries, Hb S has oft another origin, since in many cases it is institute in a haplotype named Arabo-Indian and its feature is to exist linked to a relatively high Hb F level. In these regions, the Hb S allele being much less frequent than in Africa, the patients who appear as homozygous are frequently compound heterozygous for Hb Due south and a β° thalassaemia. The β^South allele in homozygous status is by and large establish in some tribal populations of India³⁴.

Historically the diagnosis of Hb S was performed by electrophoretic methods. If IEF is nevertheless used in many laboratories, it becomes the rule to exist accomplished past CE-HPLC or CE. Since Hb S shares its electrophoretic and chromatographic backdrop with several variants it is necessary, in the absence of familial history of Hb Southward, to practise a confirmatory test. In the by, the solubility exam was considered as the golden standard since Hb S is the only aberrant Hb, which is insoluble at high concentrations when deoxygenated. This test is at present commercially difficult to go, and the large bulk of the clinical laboratories do not want to set up their own reagents. Thus combination of several methods needs to be used to confirm the diagnosis. A good compromise is to perform CE-HPLC and CE, or acid agar electrophoresis.

Only in some exceptional cases in whom another mutation is associated in cis with that of Hb Due south the heterozygous carriers tin also present a severe pathologic phenotype. Examples are provided past the heterozygous carriers of Hb S-Antilles [β6 Glu>Val and β23Val>Ile]³⁵ or Hb S-Oman [β6Glu>Val and β121Glu>Lys]³⁶ in whom the second mutation enhances sickling. Recently, an unstable β allele (β^{Southward-San Martin}) associating in cis the β^S allele to a mutation causing instability of the haemoglobin was described³⁷.

A few common variants, which pb to sickle cell anaemia when associated to Hb S have to be imperatively characterized, e.g. Hb D-Punjab and O-Arab. Both bear upon position β121 (GH4) where the Glu is replaced past a Gln in the first case, and by a Lys in the 2d ane. This residual is located at the outside of the Hb tetramer in a region, which participates in the building of the Hb S cobweb during sickling. These two mutations facilitate polymerization and, equally a result, chemical compound heterozygotes may be, clinically, equally severe as homozygous Hb Southward. Hb D-Punjab cannot be easily recognized from Hb South past using conventional CAE. For this method the aforementioned is true for Hb O-Arab that may be confused with Hb E or Hb C. In dissimilarity, CE-HPLC or CE allows an like shooting fish in a barrel identification of these two variants. This consideration is especially important for neonatal screening (Fig. 2). Since Hb D-Punjab is the merely variant with Hb D mobility, which leads to a severe disease when associated with HbS, its identity must be recognized. Other variants such equally Hb D-Islamic republic of iran, Hb Korle-Bu are completely harmless. For these variants, typical CE-HPLC profiles are observed. Fig. three shows CE-HPLC elution profiles of carriers of Hb O-Arab in various different compound heterozygous states. In the heterozygous or homozygous forms, Hb D-Punjab or O-Arab have no clinical consequences but these need to exist identified in programmes for prevention of sickle cell anaemia. The individuals compound heterozygous for one of these abnormalities and a β-thalassaemia allele are haematologically identical to a simple β-thalassaemia carrier.

CE-HPLC: Analysis of haemolysates from patients presenting with different associations involving Hb O-Arab. (A) Compound heterozygous Hb O-Arab/β°thalassaemia. (B) Compound heterozygous Hb South/HbO-Arab. (C) Compound heterozygous Hb C/Hb O-Arab. (D) Heterozygote Hb O-Arab associated with G-Philadelphia. The hybrid Hb (α_two ^G-Philβ_ii ^O-Arab) elutes in C window.

Presence of Hb Due south in homozygous or compound heterozygous with Hb variants, involved in sickling, modifies the RBC density distribution which can exist studied hands using phthalate density gradient^{38 ,39}. Fig. 4 illustrates the dissimilar profiles that could explicate the clinical heterogeneity of patients presenting with sickle cell anaemia associated with different genotypes. Information technology has to be pointed out that this uncomplicated and low cost technique may exist helpful to diagnose different α or β-thalassaemia associated to other Hb variants.

Ruby-red blood cell density profiles of patients with Hb S associated to various genotypes (technical details are available in Ref. 38). The per centum of cells denser than the phthalate index (y axis) is plotted confronting the density of the phthalate mixtures (x axis) used expressed in g/ml. Limits of normal distribution are shown in each graph. (A) Homozygous Hb S. Dense cells and young cells (reticulocytes) are observed at the correct and left side of the distribution, respectively. (B) Compound heterozygous Hb Due south/Hb O-Arab. The high percentage of dense cells is observed explaining the severity of this clan. (C) Compound heterozygous Hb S/Hb C. (D) Homozygous Hb Southward associated to an α thal-2. (E) Homozygous Hb S associated to an α thal-i. (F) Compound heterozygote Hb Southward/β°-thalassaemia. The Curve is shifted to the left when Hb Due south is associated to a thalassaemic allele.

Hb C and Hb E are also two frequent variants. The outset 1, which originates from the region of Upper Volta is frequent in Africa, while the second i is common in S East asia⁴⁰. Since, both upshot from the replacement of a glutamic acrid past a lysine these variants share some electrophoretical properties and information technology is difficult to distinguish these by CAE. The diagnosis betwixt Hb C and Hb Due east can exist established by CE-HPLC and CE, which evidence a different profile for these two variants. In Thailand several cases, initially considered as Hb Eastward, were characterized finally every bit Hb C using higher up mentioned techniques⁴¹. Hb C and Hb Due east both lead to some degree of microcytosis. Every bit a consequence of low expression of β^{Due east} allele, Hb Due east is associated with hypochromia, whereas HbC with hyperchromia considering of some dehydration of the reddish blood cell. Regarding the high frequency of Hb South and Hb C in the population originated from Africa, compound heterozygotes are not rare. And due to increasing intracellular concentration of total Hb (high MCHC linked to Hb C) the compound heterozygotes Hb Due south/Hb C sickle. Because their low MCHC, the rare cases of Hb S/Hb Eastward have a mild degree of clinical symptoms. The distribution of RBCs of heterozygous individuals of Hb E and Hb C tin be likewise hands distinguished using phthalate density gradient.

Problems linked to diagnosis of rare Hb variants

Amid the rare Hb variants some may have a high local incidence as Hb C, Hb Southward, Hb Due east or the various thalassaemic defects because these bring some protective effect against malaria. This is probable the instance of these unstable α chain variants which display in the heterozygous land some mild α thalassaemic features. Amidst those the about frequent is maybe Hb Constant Spring resulting from a mutation of the termination codon, leading to a low expressed elongated unstable α chain⁴². Many variants belonging to this group are now known and some have been reported in Asian populations⁴³.

Among the other rare variants several groups may be individualized. The commencement group is fabricated by those variants which are without whatever clinical furnishings and are fortuitously found during screening programmes. The only reason that leads to characterize these is to eliminate a trouble of possible interaction with Hb S or with a thalassaemic syndrome. These volition be found considering of a different electrophoretic or chromatographic behaviour. By comparison their properties in a bombardment of tests (IEF, CE-HPLC, RP-HPLC, globin concatenation electrophoresis, electrospray mass measurement) it is ofttimes possible to place these without need for a complete protein sequence assay or DNA sequencing.

The second group is that of rare Hb variants causing haematological disorders. Fifty-fifty when present in the heterozygous land, these lead to ascendant manifestations. A dominant phenotype of haemolytic anaemia is always observed in the case of unstable Hbs. Historically, the concept of this group of Hbs arose some 50 years ago from the study of patients suffering from chronic non-spherocytic haemolytic anaemia. Their haemolysate displayed a normal electrophoretic picture, just when incubated at 50°C revealed the presence of a Hb component, which precipitated faster than Hb A⁴⁴. Blood smears, either spontaneously or after incubation with an oxidant dye, showed inclusion bodies, made from Hb precipitates. After information technology was demonstrated that these Hb variants displayed structural modifications in some specific regions of the Hb molecule such equally the haem pocket or the α1β1 interface. Other variants having together electrophoretic abnormalities and instability were found after on⁴⁵. From a biological and clinical point of view, unstable Hbs form a very heterogeneous group. Each of the mutations responsible for an unstable Hb affects only a express number of individuals in a few families, usually unrelated. De novo mutations are frequently observed since these mutations are always a cause of illness. These practise not bring any selective advantage and are, thus, eliminated in the forthcoming generations. In industrialized countries the founder furnishings are usually identified since any patient suffering from haemolytic anaemia volition be investigated until the exact aetiology of the disease will be explained.

Conclusions

Today, detection of Hb mutants is usually easily performed by automated methods of poly peptide written report (CE-HPLC and CE). This led to the discovery of an impressive listing of variants which are reported on the Hbvar database (http://globin.cse.psu.edu/hbvar/menu.html)⁴⁵. The majority of Hb variants fortuitously discovered are of minimal clinical interest. Conversely, those found during the class of a haematological disorder bring frequently the aetiological respond for the affliction. Often unusual clinical presentations may exist explained by the interaction of several Hb abnormalities and their identification may require farther investigations.

Acknowledgments

Authors thank Claude Prehu, Mireille Mathis, Jean Riou, Christian Godart and Didier Hurtrel of Hemoglobinopathies Laboratory of Henri Mondor Academy hospital for their contribution to this piece of work by providing some helpful data, and acknowledge Josiane Bardakdjian and Michel Bahuau for providing the data in neonatal screening.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3237254/

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