Redes neuronales y de memoria
3.1. Redes Neuronales Artificiales
TREATMENT
To plan appropriate treatment, it is necessary to grade HS into mild, moderate, and severe forms (based on haemoglobin, reticulocyte count, and serum bilirubin). Treatment of severe HS is splenectomy. Splenectomy corrects haemolytic anaemia (though underlying skeletal defect and spherocytosis persist) and prevents complications such as gallstones. Although splenectomy is associated with increased life-long risk of sepsis from pneumococci and other encapsulated bacteria, risk is more in children and for this reason splenectomy is deferred until the child is 6 years old and is carried out only if the disease is severe or moderate. The risk of post splenectomy infections can be reduced by immunising children with polyvalent pneumococcal, H. influenzae, and meningococcal vaccines and by penicillin prophylaxis. Administration of folate is necessary in moderate or severe disease to prevent megaloblastic anaemia due to increased erythrocyte turnover.
HEREDITARY DISORDERS OF HAEMOGLOBIN
GENERAL FEATURES AND APPROACH TO DIAGNOSIS
Inherited disorders of haemoglobin are the commonest genetic disorders in the world.
In many countries, they constitute a public health problem.
Classification
These disorders are divided into three broad groups:
• Haemoglobinopathies
• Thalassaemias
• Hereditary persistence of foetal haemoglobin (HPFH)
Inherited disorders of haemoglobin due to structural alteration of the globin poly-peptide chain are called as haemoglobinopathies. Majority of haemoglobinopathies result from substitution of a single amino acid in globin chain due to a point mutation in the β globin gene. The most frequent haemoglobinopathies are HbS, HbC, and HbE.
Inherited disorders of haemoglobin due to reduced synthesis of one or more globin chains are known as thalassaemias. The two common types of thalassaemias are α and β. HPFH is characterised by failure of normal neonatal switch from haemoglobin F to haemoglobin A.
Both haemoglobinopathies and thalassaemias are common in India (Box 4.3 and Table 4.1).
Geographic distribution of haemoglobinopathies and thalassaemias parallels the distribution of Plasmodium falciparum. Heterozygotes with these disorders are relatively resistant to P. falciparum malaria.
It is predicted that frequency of inherited haemoglobin disorders is likely to increase because of following factors: (1) natural selection of heterozygotes due to protection afforded against malaria, (2) practice of consanguineous marriages in some affected ethnic groups, (3) increased survival of affected patients due to improvements in social conditions and public health, and (4) rapidly increasing population size in general in India.
Correct diagnosis of haemoglobin disorders is essential for proper management, for genetic counselling of prospective parents, and for prenatal diagnosis and decision regarding termination of pregnancy.
Different inherited disorders of haemoglobin are as follows:
Haemoglobinopathies
Haemoglobins with reduced solubility: Some point mutations in the β globin gene cause alteration in the solubility of haemoglobin. For example, point mutation GAG
→ GTG at the 6th codon of β globin gene leads to the formation of sickle haemoglobin
HbS Heterozygous
Homozygous
Asymptomatic Severe
HbDPunjab Heterozygous
Homozygous
β thalassaemia minor Heterozygous Asymptomatic
β thalassaemia major Homozygous Severe
C. Double heterozygous states
HbS/β+ thalassaemia Mild
HbS/β0 thalassaemia Like sickle-cell anaemia
HbS/HbDPunjab Like sickle-cell anaemia
HbE/β thalassaemia Like β thalassaemia major
HbDPunjab/β thalassaemia Mild
Box 4.3 Hereditary disorders of haemoglobin in India
• β thalassaemias: North, West, and East India, with highest prevalence in Sindhis, Punjabis, Gujaratis, and Bengalis
• HbS: Central and Southern India
• HbD: North India (especially Punjab)
• HbE: North East India
(HbS). Upon deoxygenation HbS polymerises causing formation of sickle cells;
these cells are less deformable than normal, cause vascular occlusion and are also phagocytosed in spleen.
Another example is HbC, which is formed by substitution of lysine for glutamic acid at position 6 of globin chain (β6Glu→ Lys). Crystallisation of HbC increases rigidity of red cells, which are destroyed in spleen.
Unstable haemoglobins: Instability of haemoglobin molecule arises from mutations that interfere with structural relationship between globin chains and haem. Instability results in precipitation of haemoglobin with formation of Heinz bodies that attach to the red cell membrane. The red cells become rigid, and are sequestered and destroyed in the spleen.
Haemoglobins with low oxygen affinity: Some mutations in globin gene stabilise the haemoglobin in deoxygenated state. There is higher than normal oxygen delivery to the tissues so that oxygen demands are met at a lower haemoglobin concentration (“pseudoanaemia”). A large percentage of deoxygenated haemoglobin can cause cyanosis.
Haemoglobins with increased oxygen affinity: An example is Hb Chesapeake in which a mutation at α1β2 interface stabilises the haemoglobin in the oxygenated or relaxed state. Oxygen is not released readily to the tissues resulting in tissue hypoxia and compensatory erythrocytosis.
Haemoglobin M: M haemoglobins arise from mutations that stabilise iron of haem in the nonfunctional ferric state. Such haemoglobins are unable to bind oxygen and produce cyanosis.
Structural haemoglobin variants caus ing phenotype of thalassaemia: Some mutations produce an abnormal haemoglobin molecule as well as reduced synthesis of globin chains, e.g. haemoglobin E (β26 Glu→ Lys) which is associated with the phenotype of β thalassaemia.
Nomenclature of haemoglobinopathies: More than 700 haemoglobinopathies have been reported so far. Majority of them are clinically insignificant. Variant haemoglobins are denoted by various methods like letters, name of the place where first discovered, residence of the prepositus, or family name of the index case. More systematic nomenclature consists of denoting the type of polypeptide chain, position, and the amino acid substitution, e.g. HbS is denoted by β6 Glu-Val, which represents substitution of valine for glutamic acid at position 6 of β globin chain.
Thalassaemias
Molecular lesions in thalassaemias cause reduced or absent synthesis of one or more of the globin chains. Imbalance in globin chain synthesis causes precipitation of unpaired globin chains, ineffective erythropoiesis and haemolysis.
obtaining clinical details including patient’s ethnic origin and family history, performing few basic haematological investigations, followed by confirmatory studies, which are guided by clinical data and results of baseline studies. Haemoglobin disorders are widely prevalent among certain population groups and knowledge of this may allow one to make an easier diagnosis in a relevant case, e.g. β thalassaemias are frequent in Mediterranean areas, Middle East, and in parts of North India; sickle-cell disease is common in tropical Africa and in certain tribes in Central and Southern India.
Presence of a similar disease or a relevant positive laboratory test in a close relative is a strong evidence of hereditary nature of the disease and helps in making the diagnosis in doubtful cases.
Various laboratory tests used in the evaluation of haemoglobin disorders are as follows:
(1) Measurement of haemoglobin or haematocrit, red cell count, and red cell indices; (2) Examination of blood smear; (3) Electrophoretic identification of abnormal haemoglobins—cellulose acetate electrophoresis at alkaline pH, citrate agar electrophoresis at acid pH, (4)
High-performance liquid chromatography, (5) Immunoassay for haemoglobin variants, (6) Globin chain electrophoresis; (7) Tests for HbS—slide test using reducing agent, solubility test, (8) Quantitation of haemoglobin A2, (9) Quantitation of haemoglobin F, (10) Determination of distribution of HbF in red cells, (11) Tests for inclusion bodies, (12) Globin chain synthesis studies (Fig. 4.4 and Box 4.4).
Figure 4.4: Investigations in inherited disorders of haemoglobin
Box 4.4 Laboratory diagnosis of disorders of haemoglobin
Presumptive diagnosis
• Haemoglobin electrophoresis
• Isoelectric focusing
• High-performance liquid chromatography
• Capillary electrophoresis Definitive diagnosis
• DNA analysis
• Protein analysis by mass spectrometry
Initial Peripheral Blood Examination
Generally, anaemia is severe in homozygous states whereas in heterozygotes, anaemia may be mild or absent.
Red cell indices are of critical importance in diagnosis of thalassaemias, in which MCV and MCH are characteristically low. Red cell distribution width (RDW, a measure of variation in size of red cells) is increased in iron deficiency anaemia, while it is normal in thalassaemia. Peripheral blood smear will show characteristic morphologic abnormalities such as: permanently sickled cells (sickle-cell diseases), numerous target cells (Haemglobin C disease), etc.
Haemoglobin Electrophoresis at Alkaline pH
The initial screening test in the evaluation of haemoglobinopathies is electrophoresis at alkaline pH (8.5) using a Tris EDTA-borate buffer. Various supporting media are used to achieve separation of haemoglobins such as filter paper, starch gel, or cellulose acetate membranes. Most widely used medium is cellulose acetate because the method is simple, only a small quantity of blood is needed, separation of haemoglobins is rapid, quantitation of different haemoglobins is possible, and strips can be stored permanently.
Principle: The migration of molecules having a net charge in an electric field is known as electrophoresis. Different haemoglobins have different net charge because of variations in their structure. In an alkaline buffer solution, haemoglobins migrate from cathode (-) to anode (+) and various haemoglobins have different rates of migration due to differences in their charge. Haemoglobins having more positive charge than HbA are nearer the cathode while haemoglobins having more negative charge are nearer the anode in relation to HbA. Identification of different haemoglobins is based on their relative positions on cellulose acetate strip.
Procedure
1. Red cells are haemolysed and a solution is prepared (haemolysate).
2. Haemolysate is applied near one end of cellulose acetate strip (point of origin).
3. Cellulose acetate strips are placed in the electrophoresis chamber containing Tris -EDTA-borate buffer with point of origin towards the cathode.
4. Electric current is applied till adequate separation is achieved.
5. The cellulose acetate strips are removed from the chamber, stained with a protein stain such as Ponceau S, and dried.
The test sample should be compared with a control sample containing known normal and abnormal haemoglobins. Usually a control sample known to contain haemoglobins A, F, S, and C is always included in every electrophoresis and is applied to each strip next to the test sample.
Relative mobilities of some haemoglobins using cellulose acetate electrophoresis at alkaline pH are shown in Figure 4.5.
Normal electrophoresis pattern in adults is reported as AA. While reporting abnormal haemo globins, haemoglobin with the highest concentration is written first followed by haemoglobin that is in lesser amount. Thus AC or AS indicate that concentration of HbA is higher than HbC or HbS (i.e. HbC or HbS trait respectively) while CA or SA denote that amount of HbC or HbS exceeds that of HbA (i.e. HbC-β thalassaemia or sickle-cell-β thalassaemia).
Electrophoresis at alkaline pH allows provisional identification from cathode to anode of following haemoglobins—C/A2/E/O-Arab, S/D/G/Lepore, F, A, J, Bart’s, H. Thus, haemoglobin variants A2, C, E and O-Arab migrate to the same position on cellulose acetate electrophoresis at alkaline pH. Similarly, haemoglobins S, D, G, and Lepore have identical migration. These co-migrating haemoglobins cannot be differentiated from each other only on the basis of cellulose acetate electrophoresis at alkaline pH. For this purpose, a second procedure is needed. Presence of HbS can be confirmed by sickling test using 2% sodium metabisulphite or solubility test. If test for HbS is positive, then idea about genotype is gained by assessing relative proportions of HbA, HbF, and HbS. If proportion of HbA is more than HbS, it indicates AS genotype (sickle-cell trait). If HbS exceeds HbA, then it is indicative of HbS-β+ thalassaemia;
and complete absence of HbA in the presence of HbS and HbF occurs in sickle-cell anaemia and HbS β0 thalassaemia. Family studies are helpful in arriving at the correct diagnosis.
For differentiating other identically migrating haemoglobins, citrate agar electro-phoresis at acid pH needs to be performed.
Figure 4.5: Cellulose acetate electrophoresis at alkaline pH. Lane 1: Control (AFSC); control is prepared by mixing blood from normal infant and persons with sickle-cell trait and HbC trait. Lane 2: AA (Normal person); Lane 3: SS (sickle-cell anaemia); Lane 4: AS (sickle-cell trait). Positions of various haemoglobins are shown at the top of cellulose acetate membrane for comparison
Elevation of HbF or HbA2 in the absence of any abnormal haemoglobin suggests thalassaemia. In such cases, alkali denaturation test for quantitation of HbF, estimation of HbA2, and family studies are helpful in making the diagnosis.
Citrate Agar Electrophoresis at Acidic pH
This is a useful method for further characterisation of haemoglobin variants after electrophoresis at alkaline pH.
As seen from Figure 4.6, the haemoglobin variants C, S, A, and F migrate to different locations. Citrate agar electrophoresis at acid pH provides separation of haemoglobins that have similar mobilities on cellulose acetate at alkaline pH. Thus HbS can be distinguished from HbD and HbG, and HbC from HbE and HbO-Arab. However, haemoglobin variants D, E, G, Lepore, and H have migration identical to HbA.
In cellulose acetate electrophoresis at alkaline pH, large proportion of HbF can obscure HbS. However with citrate agar at acid pH, clear separation of HbA and HbS from HbF is obtained. Therefore, citrate agar electrophoresis at acid pH is well suited for neonatal screening of sickle-cell anaemia.
High-Performance Liquid Chromatography (HPLC)
This technique is used as a screening test for (1) detection, identification, and quantification of haemoglobin variants, and (2) quantitation of HbA2 and HbF. It is also well suited for neonatal screening since it can detect small amounts of haemoglobin and needs small amount of blood. Various automated HPLC systems are available
Figure 4.6: Citrate agar gel electrophoresis at acidic pH. Lane 1: Control (A, F, S, C); Lane 2:
AA (Normal); Lane 3: SS (Sickle-cell anaemia); Lane 4: AS (Sickle-cell trait). Positions of various haemoglobins are shown above the agar gel for comparison
commercially. Haemoglobins A, F, S, C, E/A2, DPunjab, O-Arab, and DPhiladelphia can be separated and identified with HPLC.
In this automated technique, blood sample (haemolysate) is introduced into a column packed with silica gel. Different haemoglobins get adsorbed onto the resin.
Elution of different haemoglobins is achieved by changing the pH and ionic strength of the buffer. Haemoglobin fractions are detected as they pass through a detector and recorded by a computer (Fig. 4.7).
Isoelectric focusing: This refers to separation of haemoglobins according to their isoelectric points or pI (i.e. the point at which they do not have a net charge) in an electric field through a pH gradient. Precast polyacrylamide or agarose gels containing ampholytes with varying pI values are available commercially. The ampholytes produce a stable pH gradient (pH 6.0 at anode to pH 8.0 at cathode).
When haemolysate is applied near cathode, haemoglobins migrate through the gel and become stationary when their isoelectric points are reached to produce a distinct sharp band. As compared to electrophoresis, the technique has higher resolution and certain haemoglobins which co-migrate on cellulose acetate electrophoresis can be separated. However, quantitation of haemoglobin A2 is not possible and the technique is expensive (Fig. 4.8).
Capillary electrophoresis: This is either zone electrophoresis or isoelectric focusing carried out in a capillary tube. The technique is rapid, needs only a small sample, and quantitation is possible.
Immunoassay for Haemoglobin Variants
Commercial kits are available for detection of haemoglobin variants. These assays use monoclonal antibodies against specific haemoglobin variants. Currently HbS, HbC, HbE, and HbA can be detected by this method.
Figure 4.7: High-performance liquid chromatography. (A) Retention time of various haemoglobins (B) HPLC in normal adult (C) HPLC in normal newborn
Globin Chain Electrophoresis
α and β globin chains are separated from each other by the addition of 6 M urea and 2- mercaptoethanol to the buffer. When subjected to electrophoresis these chains migrate differently. The procedure is performed at both acid and alkaline pH and reveals characteristic patterns of migration of abnormal α and β chains.
This method provides a means of identifying abnormal haemoglobin variants that cannot be identified by routine electrophoretic methods (i.e. cellulose acetate at alkaline pH and citrate agar at acid pH.). It is especially helpful when variants other than S and C are present and which have identical migration on both cellulose acetate and citrate agar systems.
Tests for Haemoglobin S
Two types of tests are available :
Sickling test: When red cells containing HbS are subjected to deoxygenation, they become sickle-shaped while cells that do not contain HbS remain normal. Certain reducing chemical agents such as 2% sodium metabisulphite or sodium dithionite can deprive red cells of oxygen.
Blood and a reducing agent are mixed on a glass slide and a cover slip is placed over it that is sealed with petroleum jelly-paraffin wax mixture. Amount of HbS in red cells and degree of deoxygenation influence the speed and extent of sickling.
Sickling is usually evident after 30 minutes; if it is not then the slide is re-examined after allowing it to stand overnight. The sickled cells have minimum of two pointed projections (Fig. 4.9).
Causes of false-negative test
• Inactive, outdated reagents (incomplete reduction of oxygen tension)
• Blood samples containing low proportion of HbS (e.g. young infants, some cases of sickle-cell trait).
• High concentration of sodium metabisulphite
• Carryover from positive sample due to inadequate washing of pipette
• Mistaking crenated red cells for sickled cells Limitations of sickling test
• This test simply detects presence of HbS and does not differentiate sickle-cell anaemia from sickle-cell trait or other sickling syndromes.
• This test cannot be used for mass screening, as an experienced microscopist is required for interpretation.
Solubility test: Small amount of blood is added to a solution that contains high-phosphate buffer, a reducing agent (sodium dithionite) and saponin. Red cells are haemolysed and HbS, if present, is reduced by dithionite. Reduced HbS forms insoluble polymers, which refract light, and solution becomes turbid. A reader scale is held at the back of the tube; in negative test lines will be clearly seen since HbA is soluble in phosphate buffer, while lines will not be seen in positive test due to formation of polymers of HbS (Fig. 4.10). Positive result is also obtained with HbS Travis, and HbC Harlem. The solution remains clear in the presence of HbA, HbF, HbC, HbD, HbG, and HbO-Arab.
Figure 4.9: Sickling test (Wet preparation)
Figure 4.10: Sickle-cell solubility test. In negative test, lines of the reader scale kept behind the tubes are clearly visible. In positive test, lines are not seen due to turbidity
Causes of false-negative test
• Use of old or outdated reagents
• Low concentration of HbS as in young infants or in severe anaemia. (Solubility test should not be performed in infants <6 months to avoid getting misleading results).
• Following blood transfusion
Normally HbA2 (α2δ2) comprises of only a small proportion (1.5–3.0%) of total haemo-globin in adults. A raised HbA2 (3.5–7%) is a characteristic feature of thalassaemia minor. Estimation of HbA2 is also useful for distinguishing sickle-cell anaemia (HbA2
< 4%) from sickle-cell β0 thalassaemia (HbA2 > 4%).
In some cases of β thalassaemia minor, HbA2 is normal (called as normal HbA2 β thalassaemia). When iron deficiency complicates β thalassaemia minor, HbA2 is usually normal and therefore, it is not possible to make diagnosis of β thalassaemia minor until iron deficiency is adequately treated. HbA2 percentage is normal or low in δβ thalassaemia and in α0 thalassaemia trait.
There are two methods for estimation of HbA2: elution from cellulose acetate and micro column chromatography. A newer method is high-performance liquid chromatography (HPLC).
Estimation of HbA2 by elution from cellulose acetate: Cellulose acetate electropho-resis at pH 8.9 is carried out to separate HbA2 from other haemoglobins. Zones of HbA2 and of other haemoglobins are cut and eluated separately into different amounts of buffer. Absorbance of HbA2 eluate from remaining haemoglobins is measured in a spectrophotometer and percentage of HbA2 is calculated. This technique, however, is labour-intensive if a large number of samples are to be tested.
Estimation of HbA2 by micro-column chromatography: In this method, a glass tube or a column is filled with a supporting medium such as anion exchange resin DEAE cellulose and blood sample is introduced into the column. Mixture of haemoglobins gets adsorbed on to the resin. HbA2 is selectively eluted by using a buffer with specific pH and ionic strength. Other haemoglobins are eluted by using a buffer with different pH and ionic strength. Eluted HbA2 and other haemoglobins are spectrophoto-metrically measured and percentage of HbA2 is calculated.
For mass scale screening of HbS, solubility test is the most widely used, rapid,
For mass scale screening of HbS, solubility test is the most widely used, rapid,