Sickle cell disease

Abstract

The haemoglobinopathies are the most common monogenetic diseases in the world. They include the thalassaemias and sickle cell syndromes. The sickle cell syndromes encompass several abnormal haemoglobin variants, of which homozygosity for the sickle cell gene – that is, sickle cell anaemia is the most common and most severe. Originally characteristic of the tropics and subtropics, recent mobility and migratory trends have meant that the prevalence of sickle cell disease (SCD) has significantly increased in the UK. It is important that GPs have an understanding of this disease, in order to help their patients deal with complications of every-day life. This article will address three main aspects of SCD: diagnosis, health maintenance, and some acute and chronic complications of SCD.

The GP curriculum and sickle cell disease

Clinical module 3.02: Genetics in primary care states that GPs should be able to:

Identify patients with, or at risk of, a genetic condition

Identify patients with genetic conditions, and families, who would benefit from being referred to appropriate specialist genetic services

Discuss results from the antenatal and new-born screening programmes, which are identifying carriers and people affected with genetic conditions

Demonstrate an awareness of the systemic manifestations and complications of genetic disease, and should be aware of management and treatment strategies (be it in the community or referral to specialist care)

Clinical module 3.04: Care of children and young people states that GPs should be able to:

Recognise and respond to the needs of children and young people in special circumstances through referral and joint working

Understand that a child’s and young person’s experiences in early life have a crucial impact on their adult health and life

Understand that health promotion in all contact with children, young people, and families is important

Pathophysiology of sickle cell disease

Structure and function of haemoglobin

Haemoglobin is the oxygen-carrying molecule of the body; it carries oxygen from the lungs to the tissues and carbon dioxide from the tissues to the lungs. The structure of haemoglobin consists of four globin chains; two alpha (α₂) and two beta (β₂) chains encasing a haem (iron-containing) molecule.

Sickle cell disease is the result of an amino acid substitution of valine for, the wild type, glutamic acid residue at position 6 of the β-globin chain on chromosome 11. The result is the dysfunctional HbS (α₂ β^s₂) which, upon de-oxygenation, forms hydrophobic communications with other HbS molecules that leads to the formation of polymers (Fig. 1). Polymerisation induces the sickle shape, which represents the molecular hallmark of sickle cell anaemia (SCA) (Schnog et al., 2004). Polymerisation is dependent on haemoglobin concentration, presence of haemoglobins other than HbS, blood oxygen saturation, pH, temperature, and 2-3-bisphosphoglycerate.

Figure 1.

Pathophysiology of sickle cell disease: Polymerisation of deoxygenated HbS.

From intrauterine life to adulthood, there are different haemoglobin molecules present. The predominant haemoglobin in adulthood is HbA (α₂ β₂). The production of HbA significantly increases from birth and reaches adult levels early in the first 1 to 2 years. The other haemoglobin molecules present in adult life are haemoglobin A₂ (HbA₂) and fetal haemoglobin (HbF). HbA₂ is a normal variant of HbA and exists in small amounts in adults (Schnog et al., 2004).

The predominant haemoglobin residue in utero is HbF. It has a high affinity for oxygen, thereby giving the developing fetus better access to oxygen from the maternal circulation. The concentration of HbF markedly decreases within the first 12 weeks after birth, reaching a final level of approximately 2% of the total haemoglobin concentration in non-sickle cell disease (SCD) adult patients (Fig. 2).

Figure 2.

Synthesis of globin chains during normal development.

HbF consists of two alpha and two gamma globin chains (α₂ γ₂). The synthesis of the γ-globin chain decreases rapidly after birth, whereas the synthesis of β ^S -globin rises to form tetramers with the α-globin chains (Fig. 2). Thus, it is rare for infants to manifest clinical signs and symptoms of SCD within the first 2 months of life until the HbF has been replaced by, the now predominant and defective, sickle (β ^S )- haemoglobin (National Institute of Health, 2002). It is, therefore, imperative that neonatal screening be performed prior to 2 months of age, so that appropriate treatment is started before signs and symptoms manifest. The percentage of HbF affects the outcome of disease severity in patients with SCD; it modulates the clinical and haematological features, with higher levels correlating with a milder disease phenotype (Akinsheye et al., 2011). It does this by limiting HbS polymerisation, which has led to the use of hydroxyurea (to induce HbF production) in these patients.

Sickle haemoglobin and other haemoglobin variants

There are over 450 β-haemoglobin variants, and over 200 α-haemoglobin variants. Most of these haemoglobin variants will not cause symptoms in individuals (i.e. they are not clinically significant diseases), are rare, and will not be detected by neonatal screening (Hoyer & Sheidt, 2005). Some of the other more common β-haemoglobin variants include HbC, HbD_Punjab, HbO_Arab and HbE. These variants combine with HbS to form ‘double heterozygous’ states that can cause clinically significant disease; HbS can combine with HbC to form HbSC disease, which causes sickling and is common in West Africa.

HbS can also combine with the thalassaemias (these are genetic mutations that result in reduced synthesis of α or β-globin genes). For example, HbS combines with β-thalassaemia to form varying degrees of sickling syndromes depending on the severity of the β-thalassaemia gene inherited. For example, a combination of HbS and β⁰-thalassaemia (no production of β-globin chains) will produce a sickling syndrome identical to homozygous HbSS, whereas a combination of HbS and β⁺-thalasaemia (some production of β-globin chains) may produce a less-severe sickling syndrome. Table 1 demonstrates different types of sickling syndromes, how they are represented in neonatal screening, and their haematological parameters.

Table 1.

Characteristics of some important sickling syndromes.

Haemoglobinopathy	Neonatal screening result	Hb separation by 2 months of age¹	Full blood count, reticulocytes	Haematological parameters by 2 years
				MCV² HbA₂³ HbF
HbSS	FS	FS	Haemolysis and anaemia by 6–12 months	Normal or increased less than 3.6%⁴ Less than 25%
HbSC	FSC	FSC	Mild or no anaemia by 2 years	Normal or not applicable⁵ Less than 15%
HbS/β⁰-thal	FS	FS	Haemolysis and anaemia by 6–12 months	Decreased more than 3.6% Less than 25%
HbS/β⁺-thal	FSA/FS⁶	FSA	Mild or no anaemia by 2 years	Normal or decreased more than 3.6% Less than 25%

MCV: mean cell volume; thal: thalassaemia; F: foetal haemoglobin; S: haemoglobin S; A: haemoglobin A; C: haemoglobin C

Table shows typical results; exceptions occur:

1. Haemoglobins reported in order of quantity (e.g. FSA = F > S > A)

2. Normal MCV: greater than 70 at 6–12 months, greater than 72 at 1–2 years

3. HbA₂ results vary somewhat depending on laboratory methodology

4. HbSS with co-existent α-thalassaemia may show ↓MCV and HbA₂ >3.6%

5. Quantity of HbA₂ cannot be measured by haemoglobin electrophoresis or column chromatography in the presence of HbC

6. Quantity of HbA at birth sometimes insufficient for detection

Adapted from National Institute of Health, 2002 , The management of sickle cell disease. www.nhlbi.nih.gov/files/docs/guidelines/sc_mngt.pdf .

Epidemiology of SCD

Heterozygosity for the sickle mutation (HbAS) confers a survival advantage against malaria; the so-called ‘malaria hypothesis’. Thus, the prevalence of the sickle gene (and hence SCD) reflects the distribution of falciparum malaria (Piel et al., 2010); with frequencies higher in areas where malaria is (or was) endemic as demonstrated in Fig. 3.

Figure 3.

Global distribution of the sickle cell gene and endemic malaria.

SCD is typically a disease of the tropics and subtropics; with high incidences in Africa, the Mediterranean, the Middle East, India, and the Caribbean. However, recent increases in migration levels have seen SCD become prevalent in the industrialised Western countries, and in particular, concentrations in urban areas. It is estimated that approximately 1 in 2000 infants are born with SCD in the UK, and more than 12 000 individuals are living with the disease. This makes SCD the fastest-growing genetic disorder in the UK (Streetly, Latinovic, & Henthorn, 2010).

Haematological parameters

People who are heterozygotes (HbAS), also known as sickle cell trait, have normal haematological values with a mostly benign phenotype. They may manifest clinical signs or symptoms in conditions of extreme physiological stress. Patients with SCA have abnormal haematological values with a haemoglobin level of between 60 and 100 g/L. It is characterised by a haemolytic anaemia with evidence of a raised lactate dehydrogenase, unconjugated hyper-bilirubinaemia, reticulocytosis (2–10%), and a decrease in serum haptoglobin levels. The anaemia is usually normochromic and normocytic. Sickle cells are visible on blood film analysis (Fig. 4). Patients with SCA usually have evidence of leucocytosis and thrombocytosis (even when asymptomatic), which is a result of their chronic inflammatory state.

Figure 4.

A light micrograph demonstrating characteristic sickle cells, polychromasia and target cells.

Diagnosis of SCD

The diagnosis of SCD should be suspected in individuals from high-risk communities. In families with a history of a haemoglobinopathy, further investigation is mandatory. Mostly patients present in infancy, usually with infection, jaundice, and failure to thrive. Full blood count (FBC) evaluation will indicate whether a patient is anaemic. Most patients will have a normocytic anaemia. Further evaluation will involve the assessment of the HbA₂ level and the HbF levels.

Definitive diagnosis of SCD is made in the laboratory using high-performance liquid chromatography and isoelectric focusing. Other methods of diagnosing SCD are less reliable and false positive results have limited their use.

Antenatal screening

Antenatal screening aims to detect the presence of an abnormal haemoglobin variant in expectant parents. Clinically significant haemoglobinopathies (such as HbSS, HbSC, and compound heterozygous states with β-thalassaemia) are identified early in antenatal screening, usually by 8–10 weeks of pregnancy, thereby providing parents information on which to base reproductive choice. For women who are known to be carriers of the sickle cell mutation, screening of the male partner should be offered as soon as possible.

There are two approaches to the offer of antenatal screening in SCD; this depends on the prevalence of SCD in an area and is divided into high- and low-prevalence areas. High-prevalence areas are defined as a rate of 1.5 SCD pregnancies per 10 000 births whereas a low-prevalence area is defined as that less than 1.5 SCD pregnancies per 10 000 births. In high-prevalence areas, all women are screened for abnormal haemoglobin variants. In low-prevalence areas, the decision to screen will depend on an assessment of risk based on laboratory tests and the family origin questionnaire. For further comprehensive details on antenatal screening in SCD and thalassaemia, refer to the NHS sickle cell and thalassaemia screening programme website.

Prenatal diagnosis

Women of reproductive age from high-risk groups should be offered genetic counselling and prenatal diagnosis (PND). PND identifies the type of haemoglobin variant that the neonate has inherited from its parents. There are three main forms of PND: chorionic villus sampling, amniocentesis, and fetal blood sampling. These tests are done at different stages of pregnancy and risk miscarriage to the fetus. The advantages and disadvantages of the tests must be discussed with the parents prior to the procedure. If the test is positive, mothers must be counselled and should be offered the option to consider terminating the pregnancy. The wishes of the mother and her partner (to terminate or proceed with the pregnancy) should be respected and supported. Pre-implantation genetic diagnosis is an option for mothers who wish to identify embryos that may be at risk.

Neonatal screening

Penicillin prophylaxis reduces the risk of pneumococcal sepsis and improves morbidity and mortality in SCD. This finding provided a powerful incentive for the implementation of neonatal screening for SCD (Gaston et al., 1986). The roll-out of universal screening in the United Kingdom has been supervised by The National Sickle Cell and Thalassaemia Screening Programme.

When neonatal screening is combined with a timely diagnosis, parental education, and comprehensive medical care, the risk of morbidity and mortality from SCD is markedly reduced in children. It is, therefore, imperative that GPs pay specific attention to infants born from high-risk groups and ensure that these infants have been screened for SCD. Young infants migrating to the UK from high-prevalence countries may not have been adequately investigated at birth; therefore, it is important that GPs ensure that these patients have a haemoglobinopathy screen, so that they are properly managed.

The screening programme is not foolproof; some infants may go undiscovered due to extreme prematurity, blood transfusion prior to screening, clerical errors, or loss to follow-up. Any high-risk infant that has not been screened by 2 months of age should be screened immediately. This is important, as this is the age at which patients with SCD begin to present or show clinical manifestations of disease. They are protected within the first 2 months by the high percentage of HbF, which rapidly declines in the first 2 months and is replaced by the defective HbS.

Clinical manifestations of SCD

Although SCD is due to a single gene mutation, there is considerable heterogeneity in disease course and progression; some patients have clinically unapparent disease whereas others are fraught with frequent complications (Montalembert, 2008). SCD is mainly characterised by features of vaso-occlusion and haemolysis (Sebastiani et al., 2007). It affects the musculoskeletal system (acute painful crises, dactylitis, and avascular necrosis), pulmonary system (acute chest syndrome, pulmonary emboli, and pulmonary hypertension), cardiovascular system (heart failure secondary to haemosiderosis), neurological system (acute cerebrovascular events), hepatic system (gallstones, hepatic sequestration, hepatic siderosis, and hepatitis), the spleen (sequestration crises and hyposplenism) and the kidneys. Furthermore, these patients are at risk of overwhelming sepsis (due to splenic dysfunction) and crises (vaso-occlusive, sequestration, and aplastic) that have significant effects on morbidity and mortality. An in-depth discussion of the multiple complications associated with SCD is beyond the scope of this article, and so discussion will be restricted to some important disease manifestations, namely: acute painful (or vaso-occlusive) crises, infections, stroke, and acute chest syndrome. For further reading please refer to the National Institute of Health (2002).

Vaso-occlusive painful crisis

Painful crises represent the hallmark of SCD and are the commonest clinical presentation (Preboth, 2000). On deoxygenation, the red blood cell adopts a rigid sickle shape that occludes the post-capillary venules. The result is tissue hypoxia, infarction and, ultimately, pain. Vaso-occlusion may affect any tissue in the body; however, it generally affects the chest, lower back and extremities. These painful crises may be exacerbated by infection, dehydration, cold weather, hypoxia and stress. However, in more than 50% of cases, a precipitant is not found (Yale, Nagib, & Guthrie, 2010).

The aim of treatment in acute painful crises is the immediate control of pain. This is achieved through rapid assessment and administration of adequate analgesia. Patients are usually aware of analgesics that help with their pain and will only present to medical centres when pain control at home is sub-optimal.

Analgesics are the foundation for management of sickle cell pain (Preboth, 2000). The pain ladder provides a good structure for pain management; however, by the time these patients have presented to hospital they have tried simpler analgesics, and are likely to require more potent medications. Paracetamol and non-steroidal anti-inflammatory drugs (NSAIDs) are effective in mild pain.

Opioid analgesia should be administered if NSAIDs and paracetamol are not controlling the pain. A major barrier to the chronic use of opioids (from patients and medical personnel alike) is the fear surrounding tolerance and dependence; these are recognised side effects of chronic opioid use and should not be confused with addiction. Patients with SCD are no more likely than any other patient to develop addiction (National Institute of Health, 2002). Thus, denial of opioids due to fear of addiction is gratuitous and leads to inadequate pain control. Education surrounding this issue is important, so that it does not disrupt the administration of effective analgesia. Patient-controlled analgesic devices are also effective in severe painful crises.

Analgesia should be supported with hydration, rest and regular re-assessment of pain. Best treatment is achieved at specialist centres with day-units and where the patients have written protocols. Analgesia should be given within 30 minutes of presentation, and pain should be adequately controlled within 60 minutes of admission (Benjamin, Dampier, Jacox, & Odesina, 1999). These measures are put in place to help manage the morbidity associated with vaso-occlusive painful crises.

Infections in SCD

Despite dramatic improvements in survival, infections remain a frequent cause of morbidity and mortality in SCD worldwide (Ricera, Di Girolamo, & Rund, 2009). Patients with SCA are prone to a wide spectrum of infections. Their susceptibility to these infections stems from therapies associated with management such as splenectomy and the main disease process itself. Since the introduction of blood product screening in 1992 the risk of blood-borne infection (BBI) has dramatically declined in the UK, however, it still remains a major issue in resource-poor countries (Di Marco et al., 2010).

The incidence of bacterial infections in SCD has declined since the introduction of penicillin prophylaxis (PP) and routine immunisations against Haemophilus influenzae Type B (HIB) and Streptococcus pneumonia (Quinn, Rogers, McCavit, & Buchanan, 2010). Bacterial infections, however, are still some of the commonest infections encountered. This is mostly secondary to splenic dysfunction (Quinn et al., 2010). The spleen is the first organ to be damaged in SCD (Brousse, Buffet, & Rees, 2014). Fifty percent of patients with SCA are functionally asplenic by 2 years of age and 94% by 5 years of age (Cober and Phelps, 2010). Mortality is significantly higher in children when compared with adults (with S.pneumonia causing the majority of infections). This is followed by HIB and Neisseria Meningitides (Lynch and Kapila, 1996). Salmonella osteomyelitis is another important bacterial infection encountered in SCA. Splenic dysfunction, due to functional asplenia or splenectomy, increases the susceptibility to encapsulated bacterial infections and overwhelming sepsis. Parental education regarding splenic palpation and recognition of septic signs, alert bracelets to warn clinicians that a patient is hyposplenic, vaccinations, and PP as demonstrated in the Prophylactic Penicillin Study Group I and II (PROPS I and PROPS II) trials, have been valuable in the prevention of bacterial infections (Quinn et al., 2004).

Splenectomised and hyposplenic patients must receive routine vaccinations. All children are vaccinated with the 23-valent pneumococcal polysaccharide vaccine (PPV-23; Pneumovax) at 2 and 5 years of age. The more immunogenic polysaccharide conjugated pneumococcal vaccine (PCV-7; Prevanar) is administered to infants. Although the contribution of vaccinations to the prevention of bacterial infections is appreciated, it is important to acknowledge that vaccinations do not completely prevent bacterial infections. Further prevention of infection is accomplished by chemoprophylaxis. Penicillin V 125 mg and 250 mg given twice daily in newborns and in patients 3–5 years of age, respectively, prevents up to 80% of life-threatening cases of S.pneumonia species (Gaston et al., 1986; Quinn et al., 2010). The question on duration of antibiotic prophylaxis in hyposplenic SCA patients was addressed by the PROPS II trial, which demonstrated the safety of chemoprophylaxis discontinuation after 5 years of age. However, some clinicians remain cautious and continue prophylaxis well beyond this age.

A high index of suspicion is important in patients who present with symptoms suggestive of infection. These patients should receive a full septic screen. Management will depend on the offending organism grown in culture, and on their symptoms. Patients presenting acutely unwell and septic should be stabilised in secondary care and treated immediately with empiric intravenous antibiotics. Further management of these patients must involve a holistic approach (involving nurse specialists, haematologists and psychologists) where help with PP compliance, education regarding splenic palpation and sepsis recognition, and compliance with immunisation are addressed.

Viral infections in SCA patients are common (Di Marco et al., 2010). The majority of these infections are acquired through blood transfusions, particularly in resource-poor countries (Costagliola, Girot, Rubella, & Lefrere, 1992; Di Marco et al., 2010). Patients with SCD require regular transfusions, either chronically for survival or intermittently to overcome an acute insult. The hepatitis B virus, hepatitis C virus, and human immunodeficiency virus are among the more common entries on an exhaustive list of viral infections (Ricera et al., 2009). The hepatitis viruses are a major cause of morbidity worldwide. They represent an additional insult to multi-transfused patients, who may already suffer with chronic liver disease secondary to hepatic haemosiderosis. GPs must enquire about previous transfusion history (particularly in patients who have emigrated from SCD-endemic areas) and be proactive to ensure that patients are screened for these viruses.

Parvovirus B19 (PVB19) is a common childhood infection. It causes transient red cell aplasia in patients with SCA. This can be fatal and so prompt identification and management, with blood transfusion, is essential. Diagnosis is made on blood count analysis, which shows anaemia with no reticulocytosis, and serological tests that detect human PVB19 immunoglobulins (IgG and IgM) in blood.

Stroke in SCD

A stroke is a relatively common complication of SCD; it affects up to 11% of patients by 20 years of age and 24% of patients by 45 years (Verduzco and Nathan, 2009). Both haemorrhagic and ischaemic stroke are seen in SCD (albeit with variable prevalence at different stages of life).

The risk of a stroke is most significant in childhood (due to their more fragile vessels) between the ages of 2 and 5 years (Ohene-Frempong et al., 1998). Due to the significant effects of morbidity and mortality associated with a stroke in SCD, screening of children using transcranial Doppler (TCD) measurements was implemented. Children are screened and risk stratified depending on TCD readings. Evidence from the stroke prevention trial in SCA (STOP) suggests that chronic blood transfusion is valuable in the primary and secondary prevention of a stroke; thus, high-risk individuals identified from TCD screening are considered for chronic blood transfusion therapy.

The decision to begin chronic blood transfusions is not straightforward. Although its efficacy has been proven in the primary and secondary prevention of strokes, chronic blood transfusion therapy is associated with significant complications of its own; iron overload (with effects on the heart, liver and endocrine glands) and the inconvenience associated with iron chelation therapy, alloimunisation, and the possible risk of BBIs. It can also be an inconvenience for patients who will need blood transfusions every 2 to 4 weeks. In addition, not all patients with abnormal TCD readings will develop a stroke; therefore, it can be a hard decision for parents to make when deciding whether their child should start chronic transfusions (and risk the complications associated with it) when, in fact, their child may never have otherwise developed a stroke. It is important that GPs are aware of this screening programme.

Recognising that stroke is a major complication of SCD and that it can cause considerable morbidity can enable patients to have primary prevention within primary care. Most initial counselling will be provided by sickle cell units, however, GPs can reinforce health advice. SCD is a chronic health condition and is therefore associated with depression and anxiety, which can be supported in primary care. An understanding of the treatments available for iron overload is also important. Iron chelation therapy saves lives in chronically transfused patients; however, issues surrounding compliance with medication hinders effective treatment. GPs should explore these issues and can support patients in making sure they are receiving the correct treatment.

Acute chest syndrome

Acute chest syndrome (ACS) is the second-most-common cause of hospitalisation (Miller, 2011) and is the leading cause of mortality in SCD (Platt et al., 1994). It is characterised by respiratory symptoms in the presence of a new pulmonary infiltrate on a chest radiograph. The diagnosis may not be immediately apparent, as it mimics the common chest infection and may be dismissed as such. Indeed, infection is one of its most common aetiologies; however, the treatment of ACS will require more intensive therapies compared with a standard lower respiratory tract infection. Therefore, a high index of suspicion is important in making the diagnosis. The pathology of ACS stems from intra-pulmonary sickling, intra-pulmonary inflammation, and consequently alveolar consolidation. The cause is often multifactorial; however, infection and pulmonary fat embolism (PFE) are the most common aetiologies (Miller, 2011). Infection is more common in paediatric patients with atypical bacteria (Mycoplasma pneumonia and Chlamydia pneumonia) and viruses being the predominant offending organisms. PFE is usually more common in adult patients.

The progression of ACS can be variable; some patients may rapidly develop respiratory failure requiring intubation and ventilation whereas others may have a milder disease course, where oxygen support and antibiotics is all that is needed. However, ACS must be regarded as a medical emergency, as it is difficult to predict into which category of ACS severity patients may fall. Treatment involves pulmonary support (in the form of oxygenation and incentive spirometry) and blood transfusion. The aim of transfusion in the management of ACS is to reduce the concentration of circulating sickled cells and thus increase the affinity of circulating blood for oxygen, which is achieved by simple top-up or exchange transfusion. Further supportive care involves adequate pain control (which helps to prevent chest splinting), the use of nebulised bronchodilators and antibiotics. The GP must be clinically astute in assessing these patients and must refer them immediately for specialist treatment if the diagnosis is suspected.

Key points

SCD is now a relatively common genetic disorder in the UK due to increases in migration from SCD-endemic countries

Mothers should be made aware of the services available to them (such as PND and genetic counselling) and they should be supported accordingly when making potentially distressing decisions such as terminating a pregnancy

Neonatal screening is important and GPs should ensure that no infants from high-risk backgrounds have missed screening for SCD to ensure that they get the best holistic treatment available

PP and Pneumovax and HIB vaccinations save lives in SCD; GPs are in a good position to help promote education and awareness of the importance of these and address issues surrounding compliance

The safety of discontinuation of penicillin chemoprophylaxis after 5 years of age has been demonstrated in trial data, although some clinicians remain cautious about discontinuing this approach

SCD patients who present with symptoms suggestive of infection should have a full septic screen and be treated with empirical intravenous antibiotics in secondary care, and ACS, if suspected, must be treated as a medical emergency

References

Akinsheye

Alsultan

Solovieff

(2011) Fetal haemoglobin in sickle cell anaemia. Blood 118(1): 19–27.

Benjamin, L. J., Dampier, C. D., Jacox, A. J., & Odesina, V. (1999).Guideline for the management of acute and chronic pain in sickle cell disease. American Pain Society Clinical Practice Guidelines, 12.

Brousse

Buffet

Rees

(2014) The spleen and sickle cell disease: The sickled spleen. British Journal of Haematology 166(2): 165–176. doi: 10.1111/bjh.12950.

Cober

M. P.

Phelps

S. J.

(2010) Penicillin prophylaxis in children with sickle cell disease. The Journal of Paediatric Pharmacology and Therapeutics 15(3): 152–159. Retrieved from www.ncbi.nlm.nih.gov/pmc/articles/.

Costagliola, D. G., Girot, R., Rebulla, P., & Lefrere, J. J. (1992). Incidence of AIDS in HIV1 infected thalassaemia patients. European and Mediterranean WHO Working Group on Haemoglobinopathies and Cooleycare. British Journal of Haemoatology, 81, 109–112.

Di Marco

Capra

Angelucci

Borgna-Pignatti

Telfer

Harmatz

Kattamis

(2010) Management of chronic viral hepatitis in patients with thalassaemia: Recommendations from an international panel. Blood 116: 2875–2883. Retrieved from www.bloodjournal.org/content/bloodjournal/116/16/2875.full.pdf.

Gaston

M. H.

Verter

J. I.

Woods

Pegelow

Kelleher

Presbury

(1986) Prophylaxis with oral penicillin in children with sickle cell anaemia. A randomized trial. New England Journal of Medicine 314: 1593–1599. Retrieved from www.ncbi.nlm.nih.gov/pubmed/3086721.

Hoyer

J. D.

Scheidt

R. M.

(2005) Identification of haemoglobin variants by HPLC. Clinical Chemistry 51(7): 1303–1304. doi: 10.1373/clinchem.2005.049577.

Lynch

A. M.

Kapila

(1996) Overwhelming post splenectomy infection. Infectious Disease Clinics of North America 10: 693–707. Retrieved from www.ncbi.gov/pubmed/8958164.

10.

Miller

S. T.

(2011) How I treat acute chest syndrome in children with sickle cell disease. Blood 117(20): 5297–5305, doi: 10.1182/blood-2010-11-261834.

11.

Montalembert

(2008) Management of sickle cell disease. British Medical Journal 337: 1397, doi: 10.1136/bmj.a1397.

12.

National Institute of Health. (2002). The management of sickle cell disease. Retrieved from www.nhlbi.nih.gov/files/docs/guidelines/sc_mngt.pdf.

13.

Odievre

Verger

Silva-Pinto

A. C.

Elion

(2011) Pathophysiological insights in sickle cell disease. Indian Journal of Medical Research 134(4): 532–537. Retrieved from www.ncbi.nlm.nih.gov/pmc/articles/PMC3237253.

14.

Ohene-Frempong

Weiner

S. J.

Sleeper

L. A.

Miller

S. T.

Embury

Moohr

J. W.

Wethers

D. L.

(1998) Cerebrovascular accident in sickle cell disease: Rates and risk factors. Blood 91(1): 288–294. Retrieved from www.bloodjournal.org/content/bloodjournal/91/1/288.full.pdf.

15.

Piel

P. B.

Patil

A. P.

Howes

R. E.

Nyangiri

O. A.

Gething

P. W.

Williams

T. N.

Weatherall

D. J.

(2010) Global distribution of the sickle cell gene and geographical confirmation of the malaria hypothesis. Nature Communications 1, 104). doi: 10.1038/ncomms1104.

16.

Platt

O. S.

Brambilla

D. J.

Rosse

W. F.

Milner

P. F.

Castro

Steinberg

M. H.

Klug

P. P.

(1994) Mortality in sickle cell disease. Life expectancy and risk factors for early death. New England Journal of Medicine 330: 1639, doi: 10.1056/NEJM199406093302303.

17.

Preboth

(2000) Management of pain in sickle cell disease. American Family Physician 61(5): 1544–1550. Retrieved from www.aafp.org/afp/2000/0301/p1544.html.

18.

Quinn

C. T.

Rogers

Z. R.

Buchanan

G. R.

Buchanan

G. R.

(2004) Survival of children with sickle cell disease. Blood 103(11): 4023–4027. doi: 10.1182/blood-2003-11-3758.

19.

Quinn

C. T.

Rogers

Z. R.

McCavit

T. L.

Buchanan

G. R.

(2010) Improved survival of children and adolescents with sickle cell disease. Blood 115: 3447–3452. doi: 10.1182/blood-2009-07-233700.

20.

RCGP. Clinical module 3.02: Genetics in primary care. Retrieved from www.rcgp.org.uk/gp-training-and-exams/~/media/Files/GP-training-and-exams/Curriculum-2012/RCGP-Curriculum-3-02-Genetics-In-Primary-Care.ashx.

21.

RCGP. Clinical module 3.04: Care of children and young people. Retrieved from www.rcgp.org.uk/~/media/Files/GP-training-and-exams/Curriculum-2012/RCGP-Curriculum-3-04-Children-and-Young-People.ashx.

22.

Rees

D. C.

Williams

T. N.

Gladwin

M. T.

(2010) Sickle cell disease. The Lancet 376: 2018–2031. doi: 10.1016/S0140-6736(10)61029-X.

23.

Ricera

B. M.

Di Girolamo

Rund

(2009) Infections in thalassaemia and haemoglobinopathies: Focus on therapy-related complications. Mediterranean Journal of Haematology and Infectious Diseases 1, 1). doi: 10.4084/MJHD.2009.028.

24.

Schnog

J. B.

Duits

A. J.

Muskiet

F. A. J.

Ten

C. H.

Rojer

R. A.

Brandjes

D. P.

(2004) Sickle cell disease: A general overview. The Journal of Medicine 62(10): 364–374. Retrieved from www.ncbi.nlm.nih.gov/pubmed/15683091.

25.

Sebastiani

Nolan

V. G.

Baldwin

C. T.

Abad-Grau

M. M.

Wang

Adewoye

A. H.

McMahon

L. C.

(2007) A network model to predict the risk of death in sickle cell disease. Blood 110: 2727–2735. Retrieved from www.bloodjornal.org/content/bloodjournal/110/7/2727.full.pdf.

26.

Smith-Whitley, K., Zhao, H., Hodinka, R. L., et al. (2004). Epidemiology of human parvovirus B19 in children with sickle cell disease. Blood, 103, 422–427.

27.

Streetly

Latinovic

Henthorn

(2010) Positive screening and carrier results for the England-wide universal newborn sickle cell screening programme by ethnicity and area. Journal of Clinical Pathology 63(7): 626–629. doi: 10.1136/jcp.2010.077560.

28.

Verduzco

L. A.

Nathan

D. G.

(2009) Sickle cell disease and stroke. Blood 114(25): 5117–5125. doi: https://dx-doi-org.web.bisu.edu.cn/10.1182/blood-2009-05-220921.

29.

Yale

S. H.

Nagib

Guthrie

(2000) Approach to the vaso-occlusive crisis in adults with sickle cell disease. American Family Physician 61: 1349, Retrieved from www.ncbi.nlm.nih.gov/pubmed/10735342.