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In this paper, we investigate the feasibility of two typical techniques of Pattern Recognition in the classification for Thalassemia screening. They are the Support Vector Machine (SVM) and the K-Nearest Neighbour (KNN). We compare SVM and KNN with a Multi-Layer Perceptron (MLP) classifier. We propose a two-classifier system based on SVM. The first layer is used to differentiate between pathological and non-pathological cases while the second layer is used to discriminate between two different pathologies (α-thalassemia carrier against β-thalassemia carrier) from the first output layer (pathological cases). Using the parameters sensitivity (percentage of pathologic cases correctly classified) and specificity (percentage of non-pathologic cases correctly classified), the results obtained with this analysis show that the MLP classifier gives slightly better results than SVM although the amount of data available is limited. Both techniques enable thalassemia carriers to be discriminated from healthy subjects with 95% specificity, although the sensitivity of MLP is 92% while that of SVM is 83%.

A new silent hemoglobin variant, Hb Ozieri (alpha 71(E20)Ala-->Val), was observed in five apparently unrelated newborn babies during a screening for hemoglobinopathies on the island of Sardinia. This asymptomatic variant was detected by means of isoelectric focusing (IEF), isolated using IEF in an immobilized ultranarrow pH-gradient and characterized at the structural level using FAB- and electrospray-mass spectrometric techniques. A Val for Ala substitution was unambiguously detected at position 71 of the alpha-globin chain. This substitution indicates that a C to T transition occurred in the GCG codon for Ala which contains one of the 35 unmethylated CpG dinucleotides of the alpha-globin gene. This observation brings the number of variants due to a mutation in the alpha-globin gene CpGs (the third instance of a silent mutation) to 13 and raises the possibility that unmethylated CpGs might be hotspots for mutations as the methylated ones.

Thalassemias are pathologies that derive from genetic defects of the globin genes. The most common defects among the population affect the genes that are involved in the synthesis of α and β chains. The main aspects of these pathologies are well explained from a biochemical and genetic point of view. The diagnosis is fundamentally based on hematologic and genetic tests. A genetic analysis is particularly important to determine the carriers of α -thalassemia, whose identification by means of the hematologic parameters is more difficult in comparison with heterozygotes for β -thalassemia. This work investigates the use of artificial neural networks (ANNs) for the classification of thalassemic pathologies using the hematologic parameters resulting from hemochromocytometric analysis only. Different combinations of ANNs are reported, which allow thalassemia carriers to be discriminated from normals with 94% classification accuracy, 92% sensitivity, and 95% specificity. On the basis of these results, an automated system that allows real-time support for diagnoses is proposed. The automated system interfaces a hemochromo analyzer to a simple PC.

Modern proposals for pre-natal genetic analysis of Down's syndrome consist in isolating DNA from amniotic cells and amplifying a highly polymorphic small tandem repeat region of the chromosome 21-specific D21S11 marker. The polymerase-chain-reaction-amplified fragments are typically 5′-end labelled with a green or blue fluorescent reporter and data acquisition occurs on-lane in DNA sequencing gel-slabs and equipment. The following patterns are expected: for normal individuals, 1 peak or two peaks in a 1:1 ratio. In the case of trisomy 21, the following patterns are found: either three peaks in a 1:1:1 ratio or a two-peak profile with a 2:1 gene ratio. We have developed a capillary electrophoretic system, offering precise diagnostic value by exploiting the intrinsic DNA absorbance at 254 nm. The separation occurs in capillaries coated with an extremely stable and hydrophilic layer of poly(N-acroyloyl amino ethoxy ethanol) and filled with a background electrolyte consisting of 89 mM Tris-borate, 2 mM EDTA, 2.5 μM ethidium bromide and 8% short-chain, low-viscosity, replaceable, liquid, linear, sieving polyacrylamide. The technique offers high reproducibility and precise on-liine, automated peak acquisition and quantitation.

Amplification of the beta-globin gene by the PCR technique, followed by the enzymatic digestion of the DNA fragment obtained, was used to easily identify the human beta-globin variant Hb Hamilton which is characterized by the valine to isoleucine substitution at position 11. The result revealed the predicted G to A transition at codon 11 which abolishes a MaeIII restriction site. This mutation, which is rather common among Sardinians, is at the level of one of the five CpG dinucleotides of the beta-globin gene.

A new isoelectric focusing technique for the separation and quantitation of glycosylated haemoglobin (HbA1c) is described. By using an equimolar mixture of two separators (0.2 M beta-alanine + 0.2 M 6-aminocaproic acid) a 2-pH unit Ampholine range (pH 6-8) is transformed in a shallow, 0.6-pH unit span (pH 6.7-7.3). This brings about an increment of resolution between HbA and HbA1c by a factor of about three, thus allowing proper densitometric evaluation of the trichloroacetic acid-fixed MetHb bands by conventional gel scanners. Excellent agreement is found among microchromatography, isoelectric focusing followed by densitometry in situ, and isoelectric focusing followed by band excision, elution and spectrophotometric determination. The present method also allows full resolution between HbA1c and fetal haemoglobins (F and Fac bands).

Intact tetramers of foetal haemoglobin (G gamma, A gamma and the mutant A gamma T) can be separated by isoelectric focusing in immobilized pH gradients over a very shallow pH interval (pH 7.35-7.55). The G gamma tetramer exhibits a lower pI (7.450) than the A gamma tetramer (pI 7.453); the delta pI between the two species is barely 0.003 of a pH unit, close to the theoretical resolution limit of the technique, delta pI = 0.001. Haem-free, denatured gamma chains exhibit a reversal in pI order, the A gamma chain being more acidic than the G gamma chains: this is attributed to preferential binding of detergent micelles to the more hydrophobic A gamma polypeptide. The advantage of the present technique is the simultaneous analysis of several samples (30-40 per gel slab) and the recovery of intact, haemoglobin tetramers for subsequent studies.

It has been found that all commercially available carrier ampholytes (the amphoteric buffers used to create and stabilize the pH gradient in isoelectric focusing, IEF) (e.g., Ampholine, Pharmalyte) are extensively oxidized by persulphate during gel polymerization. These oxidized species are formed for all pH ranges, from acidic to basic, and exhibit chromophoric peaks in the 300–360 nm range. It is believed that these oxidized products are N-oxides (possibly even nitrosamines). Owing to their high redox potentials during IEF, especially in alkaline pH ranges, they are able to capture two H+ and two electrons and be reduced, while oxidizing in proteins SH group of Cys to —S—S— (possibly even to cysteic acid). This generates artefactual band heterogeneity, not present in the starting sample, which could never be eliminated as, on rerunning the zone, additional oxidation will take place. In such cases, the presentation of proof of sample homogeneity to agencies such as the FDA, for recombinant DNA products for human consumption, might be impossible. Such a cause of artefactual sample heterogeneity can be virtually eliminated by simply not exposing the carrier ampholytes to persulphate. In the proposed IEF protocol, “empty” gels are polymerized, washed, dried and reswollen in the desired pH interval.

A method for separating fetal hemoglobin Sardinia (AγT, a variant containing threonine in position 75 instead of isoleucine) from normal fetal hemoglobin is described, based on the use of shallow immobilized pH gradients (pH 7.30–7.55). The novelty of the fractionation technique is the direct analysis of intact, native hemoglobin tetramers, while present methods utilize only reverse-phase, high performance liquid chromatography of denatured, heme-free globin chains.

The applications of isoelectric focusing in immobilized pH gradients to the analysis of (i) human hemoglobin mutants, (ii) animal hemoglobin mutants (from cattle, sheep, dog and mouse), and (iii) tryptic digests of alpha and beta chains, are discussed and evaluated. Immobilized pH gradients appear to be an excellent tool for screening of genetic polymorphism and for detecting "silent mutants", i.e. those substitutions involving amino acids with nonionizable side chains. At present, not even capillary zone electrophoresis, claimed to have a resolving power equivalent to 1 million theoretical plates, has shown a resolution capability comparable to that of immobilized pH gradients, at least in the field of protein separation.

The detection of neutral amino acid mutants, by isoelectric focusing in immobilized pH gradients (IPGs), is exemplified by the separation of hemoglobin (Hb) Beirut (126 beta Val----Ala) from Hb A in a shallow pH 7.2-7.6 IPG gradient with 2% Ampholine pH 6-8. The mechanism of these separations appears to involve minute alterations in the pK values of ionizable groups bordering the mutation site, which are in turn reflected in tiny alterations in the net surface charge, delta pI. The delta pI values are of the order of 0.01 to 0.001 pH units, outside the resolving limits of conventional isoelectric focusing, and correspond to changes of the order of 0.1-0.01 unit charge (a proton or an electron).

As an alternative to the recently proposed screening for beta-thalassemias by isoelectric focusing (IEF) of denatured globin chains in urea-detergent gels [6], IEF of intact hemoglobins, obtained from umbilical cord blood in neonatal units, is reported here. For maximum separation, IEF should be performed in nonlinear pH gradients, in gels containing 0.2 M beta-alanine and 0.2 M 6-amino caproic acid, which flatten the pH gradient around pH 7, thus increasing the resolution between HbA and HbFac. The method can unambiguously detect homozygous and heterozygous beta-thalassemic conditions. A bimodal distribution of HbA at birth has been found: In heterozygous patients, HbA values of 9.02% (range 6.8-9.98%) have been found while in normal newborns HbA levels of 20.34% (range 11.02-30.6%) have been demonstrated.

A new method for antenatal diagnosis of thalassemias is reported based on the analysis of the major Hb components of fetal cord blood, sampled at week 18 of pregnancy under ultrasonic guidance, by isoelectric focusing in immobilized pH gradients (IPG). In an IPG gel encompassing a pH 6.7-7.6 span, HbA and HbFac are separated by a distance nine times greater than in a conventional carrier ampholyte pH 6-8 gel and three times greater than in an ampholine gel with separators (an equimolar mixture of beta-alanine and 6-amino caproic acid). Band evenness (in terms of uniform protein concentration within a zone) and straightness (in terms of parallel alignment of the bands to the electrodes), because of insensitivity of IPG gels to salt distortions, allows for accurate and reproducible quantitation of HbF, -A, and -Fac levels. The possibility of greatly overloading IPG matrices in total Hbs increases the sensitivity of the technique to the detection of only 0.5% HbA in the total Hb mixture, the lower limit of conventional IEF being only 2.5% HbA. Of 15 fetuses from couples at risk analyzed in the region of Ozieri, three were found to be homozygous beta-thalassemic, eight heterozygous, and four normal with no false-positives or -negatives.