Resultados MemN2N y KV-MemNN para WikiMovies

Figure 4.40: Haemoglobin electrophoresis at alkaline pH. Lane 1: Control; Lane 2: Normal or AA pattern; Lane 3: sickle-cell anaemia or SS pattern; Lane 4: sickle-cell trait or AS patern

Figure 4.41: High-performance liquid chromatography in (A) sickle-cell trait and (B) sickle-cell anaemia

A B

Neonatal Screening for Sickle-cell Anaemia

Screening can be carried out to identify those newborns who will later develop sickle-cell anaemia. The rationale behind this approach is that preventive measures can be taken to avert serious complications and reduce morbidity and mortality in later life.

Screening of newborn can be carried out in communities with increased frequency of sickle-cell gene. This approach is used in USA in African Americans.

In newborns, solubility test and sodium metabisulphite test cannot be used for screening since concentration of HbS is very small (< 10%). Widely used test for this purpose is citrate agar gel electrophoresis at acid pH. Haemolysate from cord blood sample is used. Newborns who will develop sickle-cell anaemia show predominance of HbF, some HbS and absent HbA; those with sickle-cell trait have HbF, HbS, and HbA.

Prenatal Diagnosis

Mothers from high-risk ethnic group should be screened in early pregnancy for HbS carrier state. If prospective mother as well as father are positive, they should be offered the option of prenatal diagnosis or of newborn screening. Two distinct approaches are available for prenatal diagnosis of sickle-cell anaemia: foetal blood analysis and foetal DNA analysis.

Foetal blood analysis: This involves globin chain synthesis studies in foetal blood using CM-cellulose chromatography. Abnormal globin chain is separated from normal globin chain and quantitated. Foetal blood sampling (by cordocentesis) can only be done after 18 weeks of gestation. Apart from prolonged waiting period, risk of procedure-related foetal loss is also comparatively greater.

Foetal DNA analysis: Foetal DNA may be obtained either from amniotic fluid cells or from chorionic villi (see prenatal diagnosis of thalassaemias). Chorionic villus biopsy is preferred because, if required, termination of pregnancy can be performed earlier.

Various methods are available for analysis of foetal DNA. Some of them are outlined below. (For details see “Prenatal diagnosis of thalassaemias”).

i. Southern blot analysis: A restriction enzyme called Mst II recognises three specific sites in normal β globin gene and cleaves DNA at these sites (Fig. 4.42). It produces two fragments of normal β globin gene: one measuring 1.15 kb and the other 0.2 kb. Mutation producing sickle haemoglobin causes a single base change A→T in the sixth codon of β globin gene. This mutation abolishes one cleavage site for Mst II in such a manner that only one large fragment 1 .35 kb long is produced after Mst II digestion. The technique consists of digestion of extracted DNA with Mst II followed by separation of fragments according to size by agarose gel electrophoresis. Fragments are denatured and then transferred onto nitrocellulose membrane. Radiolabelled 1.15 kb probe complementary to 5’ end of normal β globin gene is hybridised. On autoradiography, a single 1.15 kb band indicates normal β

globin genes on both homologous chromosomes (β/β), and a single 1.35 kb band indicates that both β globin genes have sickle mutation (i.e. β^S/β^S or sickle-cell anaemia). Presence of both 1.15 kb and 1.35 kb bands indicate heterozygous state for β^S gene (i.e. β^S/β or sickle-cell trait).

ii. Restriction fragment length polymorphism (RFLP) analysis: Principle of this technique is already outlined earlier (see “Prenatal diagnosis of thalassaemias”). Normal β globin gene is associated with 7.0 kb fragment while β^S gene is associated with 13.0 kb fragment in some populations, when restriction enzyme Hpa I is used. This polymorphism can be used to track the presence of β^S gene in a particular family.

iii. Methods employing DNA amplification:

a. Direct detection of mutation with restriction enzymes: The PCR-amplified DNA is digested with a restriction enzyme (such as Dde I). Fragments of different size are produced in normal β globin gene and in β^S globin gene as mutation abolishes a cleavage site in the latter.

b. Allele-specific oligonucleotide probe analysis: Two allele-specific probes are synthesised, one complementary to the normal sequence and the other to the abnormal (sickle mutation) sequence. Amplified DNA is dot blotted on to nylon membranes and probes are applied. Hybridisation occurs if sequences are complementary to each other.

Figure 4.42: Southern blot analysis of β globin gene using restriction enzyme Mst II. Normal β globin gene has three restriction sites for the enzyme Mst II (arrows on upper part of figure) with production of two fragments 1.15 kb and 0.2 kb. Sickle mutation results in abolition of one restriction site with formation of a large fragment 1.35 kb. Lower part of the figure shows Southern blot analysis. Both father (lane 1) and mother (lane 2) are heterozygous for sickle-cell mutation (sickle-cell trait); offspring in lane 3 is affected, while foetus in lane 4 also has sickle-cell anaemia

c. Colour DNA amplification: Normal β globin gene primer and mutant (β^S) globin gene primer are labeled with different fluorescent dyes. The resulting normal and abnormal amplified gene products are of different colours and can be easily identified.

Treatment

Treatment of cell anaemia is symptomatic and supportive. Patients with sickle-cell disease are best managed at a comprehensive care centre that has properly trained multidisciplinary staff. Main treatment modalities in sickle-cell anaemia are shown in Box 4.7.

1. Measures to prevent crises include early detection and treatment of infections and avoidance of exposure to extreme cold, stress, hypoxia, and dehydration. All infections should be treated intensively. Pneumococcal vaccine, influenza vaccine and penicillin prophylaxis are indicated during early childhood.

2. Treatment of vaso-occlusive episode involves relieving pain by analgesics, keeping patient warm, maintaining adequate fluid intake, oxygenation, and treatment of infections. Partial exchange transfusion reduces percentage of sickled cells and improves oxygenation; this may limit organ damage during acute vascular episode.

3. During pregnancy in sickle-cell anaemia, due to the increased risk of prematurity and stillbirth in foetus and of maternal vaso-occlusive crisis, close antenatal supervision is required. Folic acid and iron should be given routinely. Regular blood transfusion therapy has been advocated, but usefulness of this approach is not yet proved.

4. Oral contraceptive pill as a means of family planning should be avoided as it poses increased risk of thrombosis.

5. Exchange transfusion has been advised prior to surgery to reduce the risk of vaso-occlusive episodes by decreasing the percentage of HbS (to less than 30%).

During operation, hypoxia, dehydration and circulatory stasis and exposure to cold should be avoided.

6. Radiographic contrast media cause dehydration of red cells, increase MCHC and precipitate sickling. Exchange transfusion has been recommended prior to cerebral angiography.

7. Cerebrovascular accidents are managed with prompt exchange transfusion during acute episode to reduce HbS to less than 30%. This limits neurologic damage.

Box 4.7 Main treatment modalities in sickle-cell anaemia

exchange transfusion. Patients are given adequate analgesia, incentive spirometry to prevent further infiltrates, and broad-spectrum antibiotics.

9. Role of transfusion therapy in sickle-cell anaemia is summarised in Box 4.8. Regular blood transfusions merely to increase haemoglobin concentration are not indicated since they lead to increase in blood viscosity. Blood transfusions are indicated in certain situations as follows:

i. Packed red cell transfusion to improve oxygen-carrying capacity are required during symptomatic anaemia (i.e. causing breathlessness, impending CCF) aplastic crisis, acute splenic sequestration crisis, etc.

ii. Regular chronic transfusion therapy is employed to reduce the number of HbS containing cells (to less than 40%). This is indicated to prevent recurrence of strokes in cerebrovascular episodes. Role of this form of therapy prior to major surgery and during pregnancy is being investigated.

iii. Partial exchange transfusion is indicated during acute or impending attack of cerebrovascular episode or vaso-occlusive episode. Exchange transfusion reduces viscocity, avoids hypervolemia and improves oxygen-carrying capacity. The purpose behind this therapy is to limit or prevent the irreversible organ damage.

10. Hydroxyurea: A mainstay of treatment in sickle-cell anaemia is hydroxyurea (now called hydroxycarbamide). It has following benefits in sickle-cell anaemia:

(a) it increases production of HbF and reduces number and severity of crises (as HbF does not participate with HbF in sickling process, polymerisation of HbS is retarded); (b) it reduces white cell count thus causing anti-inflammatory effect;

Box 4.8 Blood transfusion in sickle-cell disease

• Main complications are iron overload in adults from chronic transfusion therapy, transmission of infections, and alloimmunisation

(c) it increases red cell volume and hydration thus reducing sickling and haemolysis;

(d) it decreases adhesiveness of red cells and leucocytes; and (e) it releases nitric oxide that causes vasodilatation.

Hydroxuurea reduces the number of painful crises, transfusion requirements, and incidence of acute chest syndrome. It is indicated in children, adolescents, and adults with sickle-cell anaemia who have frequent pain, severe vaso-occlusive events, and severe anaemia.

11. Haematopoietic stem cell transplantation is the only form of therapy that can cure the disease. Since it is associated with significant morbidity and mortality, it should be reserved for severely affected patients having HLA- matched sibling donor.

Prognosis

The course of sickle-cell anaemia is highly variable. Some patients have relatively mild disease with survival into adulthood while others die during infancy or early childhood from severe disease. Leading causes of death include severe sepsis, cerebrovascular episode, acute chest syndrome, and splenic sequestration crisis.