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Hemoglobinopathies and Thalassemias

John Old, in Emery and Rimoin's Principles and Practice of Medical Genetics, 2013

71.9.9.8 δβ-Thalassemia/Hb S

Hb S/δβ-thalassemia is a milder form of sickle-cell disease than sickle-cell anemia, because the high percentage of Hb F (15–25%) produced by the δβ-thalassemia allele protects against red cell sickling by reducing the Hb S concentration and inhibiting its polymerization. Hb S/δβ-thalassemia has been described in Sicilian, Italian, Greek, Arab, and African-American individuals. Patients have a mild anemia with a Hb concentration in the range of 10–12 g/dl, a significantly reduced MCH and MCV, Hb S, Hb F, and a normal or low Hb A2 level.

Issues with Immunology and Serology Testing

Amer Wahed, Semyon Risin, in Accurate Results in the Clinical Laboratory, 2013

Hemoglobinopathy S

Hemoglobin S hemoglobinopathy is the most common hemoglobinopathy detected in the United States. Possible diagnoses of patients with Hb S hemoglobinopathy include sickle cell trait (Hb AS), sickle cell disease (Hb SS), and sickle cell disease status post RBC transfusion/exchange. Patients with sickle cell trait may also have concomitant α-thalassemia, and the diagnosis of Hb S/β-thalassemia (0/+/++) is also occasionally made. Double heterozygous states of Hb SC, Hb SD, and Hb SO are important sickling states that should not be missed.

Patients with Hb SS disease may have increased Hb F. The distribution of Hb F among the haplotypes of Hb SS is as follows: Hb F, 5–7% in Bantu, Benin, or Cameroon; 7–10% in Senegal; and 10–25% in Arab/Indian types [19]. Hydroxyurea also causes an increase in Hb F. This is usually accompanied by macrocytosis. Hb F can also be increased in Hb S/HPFH.

Hb A2 values are typically increased in sickle cell disease and more so by HPLC. This is because the post-translational modification form of Hb S, Hb S1d, produces a peak in the A2 window. This elevated value of Hb A2 may produce diagnostic confusion with Hb SS disease and Hb S/β-thalassemia. It is important to remember that microcytosis is not a feature of Hb SS disease, and patients with Hb S/β-thalassemia typically exhibit microcytosis.

Hb SS patients and Hb S/β-0-thalassemia patients do not have any Hb A, unless the patient has been transfused or has undergone red cell exchange. Glycated Hb S has the same retention time (approximately 2.5 min) as Hb A in HPLC [19]. This will produce a small peak in the A window and raise the possibility of Hb S/β+-thalassemia.

Hb S/α-thalassemia is considered when the percentage of Hb S is lower than expected. Classical cases of sickle cell trait are 60% of Hb A and approximately 35–40% of Hb S. Cases of Hb S/α-thalassemia will have lower values of Hb S, typically below 30% with microcytosis. A similar picture will also be present in patients with sickle cell trait and iron deficiency. Common challenges in hemoglobinopathy detection are summarized in Table 18.3.

Table 18.3. Common Challenges in Hemoglobinopathy Detection

Scenario Cause of Challenge
HPLC Carryover of sample from one to the next
Transfusion Transfusion from donors who are, for example, Hb AS (S trait) or Hb AC (C trait)
Medication Hydroxyurea raises Hb F levels
Iron deficiency Lowers Hb A2 levels, thus masking diagnosis of β-thalassemia trait
HIV Falsely increases Hb A2 levels
Agarose gel Small percentages of abnormal hemoglobins (e.g., Hb A2′) may be undetected
HPLC Hb A2′ elutes in the S window. Thus, in sickle cell disease or trait, Hb A2′ will be undetected
HPLC Fast hemoglobin variants (e.g., Hb H and Hb Bart’s) may not be quantified effectively. Hb F quantification will also be affected

Hematologic Arthritis

Philipp N. Streubel, Joaquin Sanchez-Sotelo, in Morrey's the Elbow and its Disorders (Fifth Edition), 2018

Pathogenesis

Hemoglobin S results from the inherited substitution of valine for glutamic acid as the sixth amino acid of the beta globin chain. This change produces profound alterations in the stability and solubility of the hemoglobin molecule.4 Hemoglobin S molecules polymerize in hypoxic and acidic environments, imparting a sickle shape to erythrocytes.

Sickle erythrocytes show increased adhesion. Interaction of sickle cells with adhesion proteins of the vascular endothelium initiates an inflammatory response, which further increases cellular adhesiveness. Increased adhesion and inflammation decrease blood flow, leading to further sickling. Repeated episodes of decreased blood flow may lead to impaired nourishment of critical structures. Vascular occlusion is responsible for the bone and joint manifestations of sickle cell disease, including avascular necrosis and increased risk for infection. Synovial biopsy specimens generally reveal microvascular thrombosis, with occasional intraluminal sickled cells, perivascular fibrosis, and mononuclear inflammatory cell infiltration.46

Haematological Diseases in the Tropics

Jecko Thachil, ... Imelda Bates, in Manson's Tropical Infectious Diseases (Twenty-third Edition), 2014

Introduction

Haemoglobin S (HbS) has a prevalence of 25–30% in many parts of Africa and also some areas in the Middle East (Figure 65.6). HbS tends to be common among ethnic groups that have traditionally had high exposure to Plasmodium falciparum malaria. In sub-Saharan Africa approximately 230 000 infants are born with sickle cell disease each year, mostly with HbSS. Sickle cell disease (SCD) is an autosomal recessive disorder characterized by production of an abnormal haemoglobin, sickle haemoglobin. Sickle haemoglobin (HbS) arises from a mutation in codon 6 of the β-globin gene resulting in replacement of the normal glutamic acid residue by a valine.68 SCD is most commonly caused by the co-inheritance of two sickle cell genes (homozygous Hb SS disease) but patients who are heterozygous for HbS and for another haemoglobin mutation such as HbC (haemoglobin SC disease) or β-thalassaemia (Sβ0 and Sβ+) can also present with features of SCD.69 SS disease and Sβ0 disease are more severe than SC disease and Sβ+ disease (Box 65.6).70 SCD can affect multiple organs and its clinical course is punctuated by episodes of acute illness on a background of progressive organ damage, especially of the central nervous system and the lungs.70

Figure 65.6. Distribution of the HbS allele.

(Rees DC, Williams TN, Gladwin MT. Sickle cell disease. Lancet 2010; 376: 2018–31. Copyright Elsevier 2010.)Copyright © 2010
Box 65.6

Clinical Varieties of Sickle Cell Disease Based on the Co-Existing Mutations in Haemoglobin

Severe

HbSS

HbS/β0-thalassaemia

Severe HbS/β+-thalassaemia

HbS/OArab

HbS/D Punjab

HbS/C Harlem

Moderate

HbS/C

Moderate HbS/β+-thalassaemia

HbA/S Oman

Mild Sickle-Cell Disease

Mild HbS/β++-thalassaemia

HbS/E.

Investigation of Variant Haemoglobins and Thalassaemias

Barbara J. Wild, Barbara J. Bain, in Dacie and Lewis Practical Haematology (Twelfth Edition), 2017

Haemoglobin S solubility test

Principle

Sickle cell haemoglobin is insoluble in the deoxygenated state in a high molarity phosphate buffer. The crystals that form refract light and cause the solution to be turbid.35

Reagents

Phosphate buffer. Anhydrous dipotassium hydrogen phosphate, 215 g; anhydrous potassium dihydrogen phosphate, 169 g; sodium dithionite, 5 g; saponin, 1 g; water to 1 litre.

Note: Dissolve the K2HPO4 in water before adding the KH2PO4, then add the dithionite and finally the saponin. This solution is stable for 7 days. Store refrigerated.

Method

1.

Pipette 2 ml of reagent into three 12 × 75 mm test tubes.

2.

Allow the reagent to warm to room temperature.

3.

Add 10 μl of packed cells (from EDTA-anticoagulated blood) to one tube, 10 μl of packed cells from a known sickle cell trait subject as a positive control to the second tube and 10 μl packed cells from a normal subject as a negative control to the final tube.

4.

Mix well and leave to stand for 5 min.

5.

Note: The blood reagent mixture should be light pink or red. A light orange colour indicates that the reagent has deteriorated.

6.

Hold tube 2.5 cm in front of a white card with narrow black lines and read for turbidity, in comparison with the positive and negative control samples.

7.

If the test appears to be positive, centrifuge at 1200 g for 5 min. A positive test will show a dark red band at the top, whereas the solution below will be pink or colourless.

Interpretation and comments

A positive solubility or sickling test indicates the presence of haemoglobin S and as such is useful in differentiating it from haemoglobins D and G, which migrate with haemoglobin S on CAE at alkaline pH, and similarly for the confirmation of the nature of a variant haemoglobin provisionally identified as S by HPLC or IEF. Positive results are also obtained on samples containing the rare haemoglobins that have both the haemoglobin S mutation and an additional mutation in the β chain. A positive solubility test merely indicates the presence of a sickling haemoglobin and does not differentiate between homozygotes, compound heterozygotes and heterozygotes. In an emergency, it may be necessary to decide if an individual suffers from sickle cell disease before the haemoglobin electrophoresis or HPLC results are available. In these circumstances, if the solubility test is positive, a provisional diagnosis of sickle cell trait can be made if the red cell morphology is normal on the blood film. If the blood film shows any sickle cells or numerous target cells, irrespective of the Hb, a provisional diagnosis of sickle cell disease should be made; many patients with sickle cell/haemoglobin C compound heterozygosity will have a normal Hb. Remember that the sickle test is likely to be negative in infants with sickle cell disease.

False-positive results have been reported in severe leucocytosis, in hyperproteinaemia (such as multiple myeloma) and in the presence of an unstable haemoglobin, especially after splenectomy. The use of packed cells, as described in this method, minimises the problem of false-positive results caused by hyperproteinaemia and hyperlipidaemia.

False-negative results can occur in patients with a low Hb and the use of packed cells will overcome this problem. False-negative results may also occur if old or outdated reagents are used and if the dithionite/buffer mixture is not freshly made. False-negative results are likely to be found not only in infants younger than age 6 months but also in other situations (e.g. post-transfusion) in which the haemoglobin S level is < 20%.

All sickle tests, whether positive or negative, must be confirmed by electrophoresis or HPLC at the earliest opportunity.

Anemia and Red Blood Cell Disorders

Randy Hurley, in Immigrant Medicine, 2007

Sickle cell disease:

Hb S results from the substitution of valine for glutamic acid at position 6 of the β globin chain. The resultant hemoglobin has reduced solubility at low oxygen tensions. Inheritance of one sickle globin gene leads to sickle trait whereas inheritance of two sickle globin genes leads to sickle cell anemia. Sickle trait is characterized by minimal anemia and hyposthenuria but no vaso-occlusive crisis unless severe hypoxia occurs (such as at extreme altitude, etc.). Sickle cell anemia is characterized by a moderate to severe chronic hemolytic anemia with recurrent painful vaso-occlusive crisis. The peripheral smear shows characteristic sickle-shaped cells and increased polychromasia (Fig. 46.4). The sickle cell gene can be coinherited with βthalassemia (sickle-β-thal).

Figure 46.4. Sickle cell anemia. There are occasional clumped sickle-shaped cells and target cells.

Hematologic Diseases

Stephen McKew, ... Imelda Bates, in Hunter's Tropical Medicine and Emerging Infectious Disease (Ninth Edition), 2013

Hemoglobin Sickle Cell (SC) Disease

Hemoglobin sickle cell (SC) results from the inheritance of HbS from one parent and HbC from the other. The highest prevalence is in West Africa. The clinical features are similar to those in HbSS disease but slightly less severe. Splenic perfusion remains intact into adulthood and so splenomegaly, splenic infarcts and splenic sequestration can present in adulthood. Regular ophthalmic review should be undertaken as proliferative retinopathy may start in the second decade of life.

Anemia is less marked in HbSC than in HbSS (8–14 g/dl). The blood film in HbSC differs from that in HbSS as there are fewer sickle cells and more target cells, and rhomboid HbC crystals may be seen within ghost cells. The sickle solubility test is positive owing to the presence of HbS and diagnosis can be confirmed by Hb electrophoresis or HPLC.

Hemoglobinopathies

Philip Lanzkowsky M.B., Ch.B., M.D., Sc.D. (honoris causa), F.R.C.P., D.C.H., F.A.A.P., in Manual of Pediatric Hematology and Oncology (Fifth Edition), 2011

Pathophysiology

Hemoglobin S arises as a result of a point mutation (A–T) in the sixth codon of the β-globin gene on chromosome 11, which causes a single amino acid substitution (glutamic acid to valine at position 6 of the β-globin chain). Hemoglobin S is more positively charged than Hb A and hence has a different electrophoretic mobility. Deoxygenated hemoglobin S polymerizes, leading to cellular alterations that distort the red cell into a rigid, sickled form. Vaso-occlusion with ischemia–reperfusion injury is the central event, but the underlying pathophysiology is complex, involving a number of factors including hemolysis-associated reduction in nitric oxide bioavailability, chronic inflammation, oxidative stress, altered red cell adhesive properties, activated white blood cells and platelets and increased viscosity. The following mechanisms are thought to be involved:

Sickle cells are prematurely destroyed, causing hemolytic anemia

Intravascular hemolysis reduces nitric oxide (NO) bioavailability by the following mechanisms (Fig. 8-1):

Release of arginase from the red cells consumes plasma L-arginine, a substrate for NO production

Free plasma hemoglobin reacts with NO, producing methemoglobin and nitrate, thereby depleting NO

Increased xanthine oxidase and NADPH oxidase activity in sickle cell disease leads to production of free oxygen radicals that consume NO.

Figure 8-1. Intravascular Hemolysis Reduces Nitric Oxide Bioactivity.

Nitric oxide is produced by isoforms of nitric oxide (NO) synthase, using the substrate L-arginine. Intravascular hemolysis simultaneously releases hemoglobin, arginase and lactate dehydrogenase (LDH) from red cells into blood plasma. Cell-free plasma hemoglobin stochiometrically inactivates NO, generating methemoglobin and inert nitrate (A). Plasma arginase consumes plasma L-arginine to ornithine, depleting its availability for NO production (B). LDH also released from the red cell into blood serum serves as a surrogate marker for the magnitude of hemoglobin and arginase release. NO is also consumed by reactions with reactive oxygen species (O2) produced by the high levels of xanthine oxidase activity and NADPH oxidase activity seen in sickle cell disease, producing oxygen radicals like peroxynitrite (ONOO−) (C). The resulting decreased NO bioactivity in sickle cell disease is associated with pulmonary hypertension, priapism, leg ulceration and possibly with nonhemorrhagic stroke. A similar pathobiology is seen in other chronic intravascular hemolytic anemias.

From: Kato GJ, Gladwin MT, Steinberg MH. Deconstructing sickle cell disease: Reappraisal of the role of hemolysis in the development of clinical subphenotypes. Blood Reviews. 2007;21, with permission.

NO normally regulates vasodilation, causing increased blood flow and inhibits platelet aggregation. Thus, reduced NO bioavailability is thought to contribute to vaso-constriction and platelet activation

Adhesion molecules are overexpressed on sickle reticulocytes and mature red cells. Increased red cell adhesion reduces flow rate in the microvasculature, trapping red cells contributing to vaso-occlusion

Sickle cells increase blood viscosity, which also contributes to vaso-occlusion

Sickle red cells may damage the endothelium leading to production of inflammatory mediators. Ischemia–reperfusion also causes inflammation

White blood cell counts are often elevated in sickle cell disease and these white cells have increased adhesive properties. White blood cells adhere to endothelial cells and may further trap sickled red cells, contributing to stasis

Activated platelets may interact with abnormal red cells, causing aggregation and vaso-occlusion

Hemoglobin F affects HbS by decreasing polymer content in cells. The effect of HbF on HbS may have direct and indirect effects on other RBC characteristics (i.e. percentage of HbF affects the RBC adhesive properties in patients with SCD). Elevated HbF concentration is associated with a reduction in certain complications of sickle cell disease.

The relative role of hemolysis or viscosity/vaso-occlusion is postulated to differ among different subphenotypes of sickle cell disease (Figure 8-2). In particular, hemolysis and NO depletion are thought to play an important role in priapism, leg ulcers and pulmonary hypertension, while viscosity/vaso-occlusion is thought to be more central in the pathophysiology of vaso-occlusive pain and acute chest syndrome; however, considerable overlap exists.

Figure 8-2. Model of Overlapping Subphenotypes of Sickle Cell Disease.

Published data suggest that patients with sickle cell disease with higher hemoglobin levels have a higher frequency of viscosity-vaso-occlusive complications closely related to polymerization of sickle hemoglobin, resulting in erythrocyte sickling and adhesion. Such complications include vaso-occlusive pain crisis, acute chest syndrome and osteonecrosis. In contrast, a distinct set of hemolysis-endothelial dysfunction complications involving a proliferative vasculopathy and dysregulated vasomotor function, including leg ulcers, priapism, pulmonary hypertension and possibly nonhemorrhagic stroke, is associated with low hemoglobin levels and high levels of hemolytic markers such as reticulocyte counts, serum lactate dehydrogenase, plasma hemoglobin and arginase, producing a state of impaired nitric oxide bioavailability. The spectrum of prevalence and severity of each of these subphenotypes overlap with each other. Patients with alpha-thalassemia trait tend to have less hemolysis and higher hemoglobin levels, tending to decrease the prevalence of hemolysis-endothelial dysfunction and tending to increase the prevalence of viscosity-vaso-occlusion. The effect of fetal hemoglobin expression or chronic red cell transfusion is more complex, simultaneously increasing hemoglobin level, but reducing sickling and hemolysis.

From: Kato GJ, Gladwin MT, Steinberg MH. Deconstructing sickle cell disease: Reappraisal of the role of hemolysis in the development of clinical subphenotypes. Blood Reviews. 2007;21, with permission.

Protein Structure and Function

John W. Pelley PhD, in Elsevier's Integrated Biochemistry, 2007

Structural Alterations in Hemoglobin

Sickle cell hemoglobin (HbS) is caused by a mutation that replaces glutamic acid at residue 6 in β-globin with valine (β6 Glu → Val). This amino acid substitution leads to the formation of linear polymers of deoxygenated HbS. Removal of O2 from HbS in the tissues exposes a complementary site that is also on the surface. The valine residue on the surface of HbS binds to the complementary site, linking the two tetramers together (Fig. 3-12). As more tetramers become linked, linear polymers are formed that convert the normally flexible red cells into stiff, sickle-shaped cells. The inelastic, sickle-shaped cells plug the capillary beds and precipitate the sickling crisis. Note that the complementary site is not exposed in oxygenated blood, so the sickling is initiated in the peripheral tissues and joints.

Figure 3-12. Formation of linear aggregates between molecules of sickle cell hemoglobin.

HbS is the most common hemoglobin variant worldwide, since the heterozygous form confers a resistance to malaria. It occurs primarily in the black population of the United States, affecting 1 in 500 newborns. When the mutation occurs on both chromosomes (chromosome 11), it produces sickle-cell disease; this has the most severe symptoms, since the RBC has no source of normal β-globin. With a mutation only on one chromosome (in heterozygotes), it produces sickle-cell trait (1 in 10 newborns); the production of nearly equal amounts of normal β-globin and βs-globin reduces the severity of the symptoms by lowering the degree of sickling that occurs.

Hemoglobin C is caused by a mutation at the same site (position 6) as sickle cell hemoglobin except the alteration is glutamate to lysine (β6 Glu → Lys). Since lysine has little or no tendency to bind the complementary site, no sickling occurs.

Hb Boston is caused by a tyrosine substitution (β58 His → Tyr) close to the heme iron; this stabilizes the heme iron in the oxidized form, preventing the binding of O2. Hb Boston is one of several hereditary methemoglobinemias that are characterized by cyanosis.

Hb Chesapeake is caused by a leucine substitution (α92 Arg → Leu) that weakens the salt bridges, causing them to break more easily. The resulting increase in O2 affinity, resulting from decreased sensitivity to negative allosteric effectors, makes it more difficult for RBCs to unload O2 in the tissues, creating hypoxia. This signals an increase in RBC production and leads to polycythemia.

Hb Köln is caused by a methionine substitution (β98 Val → Met) that produces an unstable β-globin. The denaturation of the hemoglobin eventually leads to RBC fragility and hemolytic anemia.

Hemostatic Aspects of Sickle Cell Disease

Kenneth I. Ataga MBBS, Richard Lottenberg MD, in Consultative Hemostasis and Thrombosis (Fourth Edition), 2019

The Red Blood Cell and Hemoglobin S Polymerization

Sickle hemoglobin (HbS) occurs when the normal β6 glutamic acid residue is replaced by valine (GAG to GTG mutation at codon β6). The polymerization that occurs when HbS (α2β2S) is deoxygenated is the primary event in the pathophysiology of SCD and results in damage to erythrocytes, tissues, and organs.12 Notwithstanding this straightforward molecular basis, the pathophysiology of clinical disease is exceedingly complicated. The rate and extent of HbS polymer formation is dependent on the intraerythrocytic HbS concentration, the degree of hemoglobin deoxygenation, and the intracellular concentration of fetal hemoglobin (HbF).12 The HbS polymer is a twisted, rope-like structure composed of 14 strands that distorts the red blood cell into the classic sickle shape. The Hb tetramer is oriented such that in one of the two β subunits, β6 valine forms a hydrophobic contact with a complementary site on a β subunit of the partner strand. There is evidence that the polymerization of HbS is extremely cooperative and can be regarded as a simple crystal-solution equilibrium.13 The lag period required for the formation of polymer is referred to as the delay time. As the range of transit times in the microcirculation is short relative to the range of delay times of sickle red blood cells (RBCs), polymers do not form in most of the cells.14 If, however, sickle RBCs are subjected to prolonged transit times, then HbS polymer would form in almost all the cells as a result of equilibration at the lower oxygen tension. Hb F inhibits the polymerization of Hb S, primarily owing to the glutamine residue at codon γ87,15 which prevents a critical lateral contact in the double strand of the sickle fiber.

The density distribution of sickle RBCs is very broad, due mainly to the high number of reticulocytes with a relatively low intracellular hemoglobin concentration and the presence of a high number of very dense cells. These cells appear dense on microscopy because of enhanced cellular dehydration following polymerization-induced damage to the cell membrane.12 As the rate of HbS polymerization is strongly dependent on the intracellular hemoglobin concentration,13 dense sickle RBCs are more likely than less dehydrated cells to polymerize and contribute to the hemolytic and vaso-occlusive aspects of SCD.

The major clinical manifestations of SCD appear to be driven by two major pathophysiologic processes: vaso-occlusion with ischemia–reperfusion injury and hemolytic anemia.16 Acute vaso-occlusive episodes are thought to be caused by the entrapment of RBCs and leukocytes in the microcirculation, with resultant vascular obstruction and tissue ischemia. These vaso-occlusive events are usually triggered by inflammatory stimuli, which increase adhesive interactions between both RBCs and leukocytes and the endothelium in postcapillary venules, resulting in vascular occlusion.17-20 The obstruction of precapillary venules by rigid and deformed RBCs also contributes to microvascular occlusion. Microvascular occlusion and tissue ischemia are usually followed by the restoration of blood flow, which promotes tissue injury mediated by reperfusion with increased oxidant stress, inflammatory stress, and increased expression of endothelial cell-adhesion molecules.16

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Summary | 1 Annotations
β6 valine forms a hydrophobic contact with a complementary site on a β subunit of the partner strand.
2020/01/29 04:07