Diagnosis of sickle cell disorders

INTRODUCTION — Sickle cell disease (SCD) is an inherited group of disorders characterized by the presence of hemoglobin S (Hb S), either from homozygosity for the sickle mutation (Hb SS) or compound heterozygosity with another beta globin variant (eg, sickle-beta thalassemia, Hb SC disease). The hallmarks of SCD are vaso-occlusive phenomena and hemolytic anemia. Sickle cell trait is a benign carrier condition.

Screening and diagnosis of sickle cell disorders are discussed here.

Discussions of the clinical manifestations and management of sickle cell disorders are presented separately:

●Sickle cell trait – (See "Sickle cell trait".)

●Sickle-beta thalassemia, Hb SC disease, and other compound syndromes – (See "Overview of compound sickle cell syndromes".)

●Clinical manifestations – (See "Overview of the clinical manifestations of sickle cell disease".)

●Management – (See "Overview of the management and prognosis of sickle cell disease".)

●Children – (See "Sickle cell disease in infancy and childhood: Routine health care maintenance and anticipatory guidance".)

●Transition from pediatric to adult care – (See "Sickle cell disease (SCD) in adolescents and young adults (AYA): Transition from pediatric to adult care".)

TERMINOLOGY — We use "sickle cell disorders" to refer to all conditions in which an individual carries the sickle cell variant at the beta globin locus (HBB).

●If the other beta globin gene is normal, the individual has sickle cell trait, a benign carrier condition. (See "Sickle cell trait".)

●The combination of the sickle cell variant at one HBB allele and one alpha globin gene mutation results in two benign carrier conditions, sickle cell trait and alpha thalassemia trait (also called alpha thalassemia minima), and no clinical disease. (See "Pathophysiology of thalassemia", section on 'Terminology and disease classification'.)

●"Sickle cell disease (SCD)" is an umbrella term that includes all combinations of the sickle cell variant plus another HBB variant at the other allele, the combination of which causes clinical sickling. The other HBB variant could be the sickle cell variant (Hb SS) or a different variant (beta thalassemia, Hb C, or others). Individuals with SCD have a disorder associated with vaso-occlusion of varying severity. (See "Overview of compound sickle cell syndromes" and "Overview of the clinical manifestations of sickle cell disease".)

OVERVIEW OF DIAGNOSTIC TESTING — The methods for SCD diagnosis vary with the age of the patient.

DNA-based testing is used for prenatal diagnosis. (See 'Reproductive testing and counseling' below.)

After birth, protein-based methods such as electrophoresis are the primary initial test for diagnosing sickle disorders. Protein electrophoresis separates hemoglobin species according to amino acid composition and their electrical charge. Confirmatory DNA testing may be necessary in unclear cases.

Characterization of adult hemoglobins in the fetal and newborn periods can be difficult because of the predominance of hemoglobin F (Hb F), which confounds detection of hemoglobin S (Hb S) by solubility testing.

Clinical manifestations of SCD are not present at birth, and usually begin to become apparent after the first few months of life as the concentration of Hb S rises and Hb F declines. Sickled cells can be seen in the peripheral blood of children with SCD at three months of age, and moderately severe hemolytic anemia is apparent by four months of age.

REPRODUCTIVE TESTING AND COUNSELING — Individuals with sickle cell disorders should be offered preconception counseling to determine the risk of having a child with a sickle cell disorder. Individuals at risk should be offered hemoglobinopathy testing early in pregnancy and the opportunity for prenatal diagnosis where appropriate. (See "Hemoglobinopathy: Screening and counseling in the reproductive setting and fetal diagnosis".)

Prenatal testing may include obtaining fetal DNA samples by chorionic villus sampling at 8 to 10 weeks gestation [1]. Other diagnostic approaches such as in vitro fertilization (IVF) with preimplantation genetic testing or testing fetal cells isolated from maternal blood have been reported in sickle cell disorders but are not routinely used [2-4].

Reproductive centers in the United States and Europe are developing protocols for preimplantation testing for sickle cell disorders [5,6].

NEWBORN SCREENING — Infants with SCD generally are healthy at birth and develop symptoms only when fetal hemoglobin levels decline later in infancy or early childhood.

The goals of newborn screening (NBS) include [7]:

●Early recognition of affected infants

●Early medical intervention to reduce morbidity and mortality, particularly from bacterial infections

●Institution of regular and ongoing comprehensive care through a multidisciplinary sickle cell clinic, in collaboration with the primary care physician, whenever feasible

●Access for families/caregivers of children with SCD to accurate information about the diagnosis, clinical manifestations, treatment options, and age-appropriate anticipatory guidance toward the management of these emerging issues

As an example of the effectiveness of these programs, the use of prophylactic penicillin and the provision of comprehensive medical care have reduced the mortality of SCD during the first five years of life from approximately 25 percent to less than 3 percent [8-10].

The magnitude of the problem in the United States is demonstrated by the frequency of carrier conditions for hemoglobinopathies:

●Data obtained in California from 1998 to 2006 found that the genetic trait for sickle cell and/or thalassemia occurred in 1 in 75 California births [11,12].

●The gene frequency is much higher in the African-American population: 4 percent for Hb S, 1.5 percent for Hb C, and 4 percent for beta thalassemia [13]. The gene frequency is also high in individuals from other racial and ethnic groups. In a study from California, 12.5 percent of infants with Hb S/beta thalassemia births were born to parents who self-identified as having Hispanic ethnicity on the newborn screening form [12].

These frequencies predict the occurrence of 4000 to 5000 pregnancies per year at risk for SCD in the United States [13].

Although there have been advances in the diagnosis and management of SCD in high-income countries, much remains to be learned about the optimum implementation of NBS programs and medical management of patients in low-income countries [14]. For example, a strategy that utilized a team of specially trained midwives at two large maternity services in Cotonou, Republic of Benin, resulted in almost 80 percent of women informed of the risk for SCD agreeing to testing of their offspring. Eighty-five percent of offspring testing positive enrolled in a sickle cell program, and more than 80 percent of these infants were still followed by the program at five years. The mortality rate for this cohort of children under five years of age with SCD was 10 times lower than the general rate recorded in the Republic of Benin [15].

Types of screening programs — Historically, two programs have been used for newborn screening (NBS): selective screening of infants of high-risk parents, and universal testing of newborns.

Universal testing seems preferable due to its economy and superiority of detection [16-18]. This approach has been endorsed by a consensus panel convened by the National Institutes of Health [19]. A cost-effectiveness analysis found that targeted screening of African-American newborns for SCD is cost-effective [20]. However, universal screening identifies more infants with disease and prevents more deaths. The advantages of this approach can be illustrated by the following:

●Observational studies have demonstrated that targeted screening does not identify all individuals. As examples:

•Targeted screening missed 20 percent of African-American newborns with SCD born in the state of Georgia [21].

•The number of SCD diagnoses doubled when screening was changed from targeted to universal in the state of Connecticut [22].

●Self-identification of race is a fluid definition that may change based on local social dynamics.

As of 2008, screening for SCD in newborns is mandated in all 50 states of the United States and the District of Columbia, regardless of birth setting [23]. In fact, the importance of NBS programs is being increasingly recognized worldwide. Due to changes in immigrant migration patterns, several European and African countries are initiating NBS programs. These programs face logistical and economic challenges for comprehensive implementation and patient outreach [24].

In the United States, any high-risk infant should have documentation of newborn screen results by one to two months of age so that confirmatory testing, if necessary, can be performed, and parental education, penicillin prophylaxis, and referral for comprehensive care can be implemented [25]. (See 'Approach to a positive result from newborn/infant screening' below.)

The methods for implementation of NBS programs vary somewhat from state to state. In Texas, as an example, "heel stick" filter paper screen is performed within 72 hours of life, with a second screen required at one to two weeks. Testing is by isoelectric focusing, and abnormal specimens are confirmed by repeat testing or by DNA analysis. Tandem mass spectrometry (MS/MS) is an alternative method for newborn screening as it is able to detect hemoglobin (Hb) peptides following digestion of blood spots with trypsin [26]. (See 'Hemoglobin patterns' below.)

Abnormal results are reported to the NBS program, data are maintained on a computer registry, and case management services are initiated to ensure medical follow-up. In a review of newborn screen outcomes from 1992 to 1998, 2,292,698 live births were recorded in Texas and 94 percent had specimens collected. The overall prevalence of SCD by ethnic group per 10,000 live births was: 29.91 African American, 0.11 White American, 0.29 Hispanic American, and 2.47 other/unknown [27]. Despite these measures, other states have reported gaps in compliance with early medical intervention, parental education, and provision of comprehensive health services [28].

In California, a two-tier approach to universal NBS is used. First, NBS is performed on dried blood spots utilizing high performance liquid chromatography (HPLC). Abnormal hemoglobin findings are then referred to the Hemoglobin Reference Laboratory (HRL), where additional testing, including DNA sequencing, may then be performed for final confirmation. With this approach, of approximately 530,000 annual NBS, approximately 2118 samples were referred for HRL over an eight-year period. Hemoglobin (Hb) genotypes included: sickle hemoglobinopathies (32 percent), alpha thalassemia conditions (24 percent), beta thalassemia conditions (4 percent), and other Hb variants, including traits (41 percent) [12,29].

Despite high percentages of NBS completions by states and high rates of follow-up testing where abnormal findings were detected, there are ongoing problems with timely follow-up and implementation of comprehensive care. One study suggests that there is a wide variation in stakeholder notification (physician, hospital, families/caregivers, hematologists), which can lead to alteration in early intervention [30]. Other states have reported gaps in compliance with early medical intervention, parental education, and provision of comprehensive health services [28].

Additional information and resources related to newborn screening results are discussed separately. (See "Overview of newborn screening".)

Methodology and diagnostic errors — The recommended approach to neonatal testing is to obtain blood samples by heel stick or cord blood and spot the sample onto filter paper for stable transport and subsequent electrophoresis, thin-layer isoelectric focusing, or HPLC [31,32]. Additional information about these methods is discussed separately. (See "Methods for hemoglobin analysis and hemoglobinopathy testing".)

A requirement for tests used in NBS is the capability to distinguish among Hb F, S, A, and C.

Diagnostic errors that can occur during neonatal testing include the following:

●Solubility testing may not be valid, because of the large amount of Hb F present in fetal blood.

●Hb electrophoresis testing after transfusion of red cells can result in an incorrect diagnosis. In such cases, SCD diagnosis should utilize DNA testing or be postponed for at least four months after transfusion.

●In very premature babies, Hb A may not be detected, resulting in misdiagnosis. Very premature babies with sickle cell trait may be found to have Hb S levels greater than Hb A, resulting in the incorrect diagnosis of hemoglobin S/beta⁺ thalassemia [33].

Hemoglobin patterns — According to convention, the patterns of Hb present are described in descending order according to the quantities detected (table 1):

FS pattern — Newborns with homozygous sickle cell anemia (Hb SS) have predominantly Hb F with a small amount of Hb S and no Hb A (ie, the FS pattern). An FS pattern is also found in newborns who have sickle cell-beta⁰-thalassemia, sickle cell-hereditary persistence of fetal hemoglobin, and, because Hb D and Hb G have the same electrophoretic mobility as Hb S, sickle cell-hemoglobin D disease and sickle cell-hemoglobin G disease. Family studies are confirmatory; in the newborn with sickle cell-beta⁰-thalassemia, for example, one parent has sickle cell trait and the other beta thalassemia minor. When family members are not available, the diagnosis is established by DNA-based testing or repeat hemoglobin analysis at three to four months.

FAS and FSA patterns — The newborn with sickle cell trait will have Hb F, Hb A, and Hb S (ie, the FAS pattern). The quantity of Hb A is greater than that of Hb S. If the quantity of Hb S exceeds that of Hb A (the FSA pattern), the presumptive diagnosis is sickle cell-beta⁺-thalassemia. Alternatively, the FAS pattern may be seen in a newborn with Hb SS who has received a red blood cell transfusion prior to the hemoglobin analysis; this is often the case for premature infants admitted to the Neonatal Intensive Care Unit. Based on the ambiguity of the diagnosis, we treat all newborns with FAS with a prior red blood cell transfusion as having SCD until further evaluations can be made. Obtaining the hemoglobin analysis on the parents, DNA-based testing on the child, or repeat hemoglobin testing at age three to six months may be helpful.

In the future, PCR-based diagnosis from blood spotted onto a filter paper may be used to detect sickle cell genes directly [34].

Approach to a positive result from newborn/infant screening — Informing the parents of the results of newborn screening has value for the child and potentially other family members (table 2). It is important that the clinician fully understand the findings and their implications in preparation for discussing them with the parents.

●FS pattern (suggests sickle cell disease) – An FS pattern suggests homozygous sickle cell anemia or sickle-beta⁺ thalassemia, both of which are also referred to as sickle cell disease (SCD). Both of these are associated with risks of many acute and chronic complications and require comprehensive management. (See 'FS pattern' above.)

Upon diagnosis of SCD, there is an immediate obligation to implement a program of comprehensive care for the affected child and family [25,35,36]. This requires medical professionals with special expertise in SCD and access to multidisciplinary teams, including social workers, psychologists, nurses, genetic counselors, and nutritionists. The patient should be referred to a hematologist with expertise in managing SCD or a hemoglobinopathy center.

The following is appropriate while awaiting specialist referral:

•Inform the family of the diagnosis of an SCD

•Initiate prophylactic penicillin (125 mg orally twice daily)

•Inform the family that they should seek immediate medical attention if the infant has a fever of 101°F (38.3°C) and inform the treating clinician of the SCD diagnosis

•Teach the family how to examine the spleen and document whether the size is normal. Provide instructions on how to determine if splenic sequestration is occurring, and, if so, emphasize the need for prompt evaluation in an emergency department for management

•Provide information regarding the genetic inheritance of the disorder

Additional details of management are presented separately. (See "Sickle cell disease in infancy and childhood: Routine health care maintenance and anticipatory guidance" and "Overview of the management and prognosis of sickle cell disease".)

●FSA pattern (suggests a compound state) – An FSA pattern suggests a compound state involving a sickle cell mutation and another mutation such as beta⁺-thalassemia. (See 'FAS and FSA patterns' above.)

As with the FS pattern, the patient requires comprehensive care from a hematologist or hemoglobinopathy center. The following is appropriate while awaiting specialist referral:

•Inform the family of the diagnosis of a sickle disorder.

•Provide information regarding the genetic inheritance of the disorder.

•Repeat hemoglobin electrophoresis in three to six months to further characterize the hemoglobinopathy.

●FAS pattern (suggests sickle cell trait) – For infants with an FAS pattern, the family should be informed of the likely diagnosis of sickle cell trait. For those with an FAS pattern after receiving a blood transfusion, additional evaluations are needed to determine the diagnosis.

The following is also appropriate:

•Provide education regarding the inheritance of sickle cell diseases, and reassurance that the infant does not have a chronic blood disorder.

•Explain that under settings of significant hypoxia (eg, high altitude unpressurized aircraft, very strenuous exercise with dehydration), sickling can occur and there is a risk of hematuria, worsening of traumatic hyphema, and a very rare type of renal cancer. (See "Sickle cell trait", section on 'Clinical findings'.)

•Counsel patients on the reproductive consequences of sickle cell carrier status (eg, potential for SCD if the other parent is also a carrier). (See "Sickle cell trait", section on 'Reproductive issues'.)

•Offer hemoglobinopathy screening to other family members who may carry a sickle cell mutation and are unaware of their carrier status and referral for genetic counseling or hematologic consultation if further information is desired.

Additional information regarding the sickle cell trait (carrier status) is provided by the Centers for Disease Control and Prevention.

OLDER CHILDREN AND ADULTS — Despite newborn screening, many patients with sickle cell disease (SCD) or sickle cell trait may be undiagnosed, in part due to immigration of young, unscreened patients from other countries [37].

Laboratory methods — Diagnosis of the sickle cell disorders can be made with several methods, which are discussed in more detail separately. (See "Methods for hemoglobin analysis and hemoglobinopathy testing".)

●High performance liquid chromatography (HPLC) is the preferred method of diagnosis. HPLC is a highly precise technique for identification and quantification of hemoglobins and can be fully automated. In California and the majority of the United States, newborn samples are screened with HPLC. This technique detects most hemoglobin variants by their different retention times. It is highly sensitive and specific and provides both quantitative and qualitative interpretation [12]. Major disadvantages are the initial cost of the apparatus and reagents.

●Improvement in high voltage capillary electrophoresis makes it a comparable technique to HPLC.

●Thin-layer isoelectric focusing is a highly accurate and cost effective tool for the diagnosis of sickle or other hemoglobin variants [18]. The bands on isoelectric focusing are sharper than those on electrophoresis and can distinguish some hemoglobins not seen on standard electrophoresis. Isoelectric focusing is more complicated because it is also sensitive to the presence of methemoglobin and glycosylated hemoglobins.

●Cellulose acetate electrophoresis at pH 8.4 is a standard method of separating hemoglobin S (Hb S) from other Hb variants. However, Hb S, G, and D have the same electrophoretic mobility with this method (figure 1).

●Citrate agar electrophoresis at pH 6.2 separates Hb S from Hb D and G, which co-migrate with Hb A in this system.

●There is no clinical situation in which a sodium metabisulfite test (Sickledex) is clinically indicated in the screening or management of sickle cell disorders (trait or disease). The test characteristics of the Sickledex confer a high likelihood of diagnostic error [38].

Thin-layer isoelectric focusing will separate Hb S, D, and G. Alternatively, the combination of cellulose acetate electrophoresis with either citrate agar electrophoresis or a solubility test allows a definitive diagnosis of a sickle cell disorder. Even with thin-layer isoelectric focusing, it is still necessary to use a confirmatory solubility test for Hb S.

In certain regions of the world, high-throughput molecular protocols (DNA testing) are being developed for hemoglobin disorders that may eventually replace the HPLC. DNA testing may be especially useful in cases such as those in which high concentrations of Hb F raise the possibility of hereditary persistence of fetal hemoglobin [39]. (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Hereditary persistence of fetal hemoglobin (HPFH)'.)

Available DNA-based methods are discussed in detail separately. (See "Methods for hemoglobin analysis and hemoglobinopathy testing", section on 'Molecular genetic (DNA-based) methods'.)

Of note, the "sickle cell prep" using metabisulfite or dithionite is no longer in routine clinical use and is largely of historical interest. Rare exceptions are presented separately.

Point of care (POC) diagnostics — Point of care (POC) diagnostics are of potential use in low-resource countries with an at-risk population. Unlike the standard laboratory-based methods listed above, POC testing does not require expensive laboratory equipment and infrastructure.

Several promising techniques for simple, rapid, inexpensive diagnosis of SCD are being developed, including paper-based tests that quantify sickle hemoglobin, a density-based rapid test using aqueous multiphase systems, and lateral flow immunoassays [40,41]. Another approach to POC testing involves development of monoclonal antibodies directed against common hemoglobin variants [42]. These techniques appear to have excellent sensitivity and specificity to diagnose sickle cell anemia using dry blood and/or liquid samples. Pilot data indicate these tests can be performed in a clinical setting by minimally trained medical staff [42]. They can be used in remote areas of the world where hemoglobinopathies are common but trained clinicians, technical staff, and laboratory equipment are lacking [43,44].

These techniques are undergoing field testing and validation in newborn screening programs in resource-poor, high-risk areas [45,46]. Additional details about POC testing is presented separately. (See "Methods for hemoglobin analysis and hemoglobinopathy testing", section on 'Point-of-care assays'.)

Hemoglobin A2 measurement — Measurement and interpretation of levels of the minor hemoglobin, Hb A2, has traditionally been considered valuable for the diagnosis of concomitant beta thalassemia. However, interpretation requires some expertise.

Beta thalassemia heterozygotes generally have an increased Hb A2 level (table 1). However, a cross-sectional study demonstrated that most clinical diagnoses of sickle cell-beta⁰-thalassemia in African Americans with SCD did not match the associated genotype [47]. Most of the individuals considered to have a sickle cell-beta⁰-thalassemia phenotype based on hemoglobin analysis had elevated Hb A2, high MCV, and high red blood cell count; on genetic analysis, these individuals actually had Hb SS and concurrent alpha chain deletions. As an example, 13 of 18 (72 percent) of individuals with a genotype of Hb SS and with one alpha chain deletion were misclassified as having sickle cell-beta⁰-thalassemia. Another 11 of 12 (92 percent) of those with Hb SS and two alpha chain deletions were misclassified as having sickle cell-beta⁰-thalassemia.

These findings illustrate why Hb A2 measurement alone (above or below a certain threshold) is not a definitive approach to distinguish Hb SS from sickle cell-beta⁰-thalassemia phenotype. The high frequency of concomitant unrecognized alpha thalassemia accentuates this misclassification. In the rare event that distinguishing the two SCD genotypes may be important for genetic counseling, gene analysis is required to have an accurate diagnosis.

Heterozygotes with delta-beta (δβ) thalassemia have a normal or decreased Hb A2 level with a high Hb F level. With many laboratory techniques, Hb A2 levels are influenced by closely migrating hemoglobin variants such as Hb E. Of importance, using HPLC and electrophoresis, Hb A2 is overestimated in the presence of Hb S, while severe iron deficiency reduces the Hb A2 level; this is a particular problem in pregnant women who may be thalassemia carriers. When Hb S is not present, microcolumn chromatography or HPLC accurately quantifies Hb A2. Electrophoresis with scanning densitometry does not accurately quantitate Hb A2 levels [48].

Findings in sickle cell anemia — The chronic hemolysis in those with sickle cell anemia (ie, Hb SS) is usually associated with a mild to moderate anemia (hematocrit 20 to 30 percent), reticulocytosis of 3 to 15 percent (accounting for high or high-normal mean corpuscular volume [MCV]), unconjugated hyperbilirubinemia, and elevated serum lactate dehydrogenase (LDH) and low serum haptoglobin. The peripheral blood smear reveals sickled red cells (picture 1), polychromasia indicative of reticulocytosis, and Howell-Jolly bodies reflecting hyposplenia (picture 2). The red cells are normochromic unless there is coexistent thalassemia or iron deficiency. If the age-adjusted MCV is not elevated, the possibility of sickle cell-beta-thalassemia, coincident alpha thalassemia, or iron deficiency should be considered. (See "Diagnostic approach to anemia in adults".)

The Hb F level is usually slightly to moderately elevated and Hb A is absent (figure 1). The amount of Hb F is a function of the number of reticulocytes that contain Hb F, the extent of selective survival of Hb F-containing reticulocytes to become mature Hb F-containing erythrocytes, and the amount of Hb F per red cell. Each variable is separately regulated and the expression of each shows interpatient variability [49]. In some patients with sickle cell anemia alone, values are as high (1 to 4 percent) as the modest elevations seen in heterocellular hereditary persistence of fetal hemoglobin [49]. (See "Overview of compound sickle cell syndromes".)

In addition, certain beta globin haplotypes appear to be related to factors that regulate production of Hb F. As examples, the Arab-Indian and Senegal haplotypes are associated with higher levels of Hb F (over 20 percent in some cases), probably due to linkage with important gamma globin regulatory sequences in the locus control region [50-52]. In one study of Senegalese patients, the mean Hb F was 8.2 percent, and approximately one-half of patients had a benign form of sickle cell anemia [52]. (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Sickle cell disease'.)

Diagnostic patterns in other sickle cell disorders — Several of the sickle cell disorders may have similar results with electrophoresis or isoelectric focusing. Additional information from examination of the peripheral smear often helps to separate the sickle cell diseases. (See "Overview of compound sickle cell syndromes".)

●The diagnosis of Hb SC disease is made by HPLC, isoelectric focusing, or hemoglobin electrophoresis, which demonstrates Hb S and Hb C in approximately equal amounts (or slightly more Hb S than Hb C), with no Hb A present (figure 1). Two independent Hb analysis techniques are necessary to distinguish Hb SC from Hb SC Harlem and other compound heterozygotes. The peripheral blood smear shows a predominance of target cells, with rare sickled cells that may be canoe-shaped (picture 3).

●Results from electrophoresis or thin-layer isoelectric focusing (IEF) are similar in sickle cell anemia and sickle cell-beta⁰-thalassemia, as nearly all the hemoglobin consists of Hb S, with no Hb A evident. Differences in the levels of Hb F and Hb A2 and in the peripheral blood smear may be useful in distinguishing these disorders (table 1). In those with sickle cell anemia, both sickled and target cells are seen; red cell indices are generally normal. In sickle cell-beta⁰-thalassemia, sickled cells, target cells, and hypochromic microcytic discocytes are prominent. If one parent does not have sickle cell trait, this is a useful indicator of the presence of sickle cell-beta⁰-thalassemia in the child, rather than sickle cell anemia.

●Sickle cell-beta⁺-thalassemia and sickle cell trait both have substantial amounts of both Hb A and Hb S. Sickle cell trait is not associated with anemia or microcytosis and has a Hb A fraction that exceeds 50 percent, along with 35 to 45 percent Hb S (figure 1) [53]. Sickle cell-beta⁺-thalassemia is associated with anemia, microcytosis, and a Hb A fraction that ranges between 5 and 30 percent [54]. Sickle cell trait in combination with alpha thalassemia can be suspected when there is less than 35 percent Hb S (table 3). (See "Overview of compound sickle cell syndromes", section on 'Sickle-beta thalassemia' and "Overview of compound sickle cell syndromes", section on 'Sickle-alpha thalassemia'.)

●For over 20 years, solubility tests (Sickledex and others) have been recognized as inadequate tests to determine the presence of SCD or sickle cell trait [38,55]. Further, the American College of Obstetricians and Gynecologists (ACOG) recommends that only hemoglobin analysis should be done prenatally in pregnant women to determine sickle cell trait status or other hemoglobinopathy trait status. Despite the acknowledged limitations of the solubility test, obstetricians continue to use the solubility test to screen for sickle cell trait and SCD in prenatal clinics [56].

There are some SCD variants that are uncommon yet important [57]. (See "Overview of compound sickle cell syndromes".)

●Sickle cell-hemoglobin D-Punjab (D-Los Angeles) and sickle cell-hemoglobin O-Arab are moderate to severe diseases characterized by anemia, reticulocytosis, and often, macrocytosis. Many of these patients experience the same frequency and severity of vaso occlusive events as individuals with hemoglobin SS.

●Sickle cell-hemoglobin C-Harlem is slightly milder than sickle cell anemia with a similar blood film.

Sickle hemoglobin O-Arab disease is a severe disorder similar to sickle cell anemia, with laboratory findings of low hemoglobin, elevated reticulocyte count, and moderate macrocytosis. (See "Hemoglobin variants including Hb C, Hb D, and Hb E", section on 'Hb O-Arab'.)

●Sickle cell-hemoglobin Lepore, also a moderately severe disease, is more characterized by microcytosis and a blood smear comparable to sickle cell-beta-thalassemia.

●Sickle cell-hereditary persistence of fetal hemoglobin is usually asymptomatic or extremely mild. Mild anemia and reticulocytosis may be noted. The electrophoresis may be misread as sickle cell anemia with an elevated Hb F. Definitive diagnosis requires family studies or DNA analysis. (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Sickle cell disease'.)

●Sickle cell-hemoglobin E is a clinically mild disease. The blood film shows targeting and variable microcytosis.

Approach to a positive result in older children or adults

●Sickle cell trait – Older children or adults may be diagnosed with sickle cell trait as part of screening of asymptomatic family members or prenatal screening. Individuals with sickle cell trait should be reassured that they do not have a chronic blood disease. They should understand the risks of complications of significant hypoxia, the effect on the accuracy of Hb A1c in monitoring diabetes, and the reproductive implications of sickle cell trait status [58].

All individuals with a child with SCD should be offered the opportunity to receive individualized genetic counseling regarding the risk of having another child with SCD. Cultural sensitivity and clinical competency are required regarding individual genetic counseling after the SCD status of both the child and the parent is acknowledged. Both cultural sensitivity and clinical competence are required because the hemoglobin analysis of the father may raise the possibility of non-paternity. Understanding the complex genetics of maternal uniparental disomy and SCD is a critical component of assessment of genetic counseling of the paternity status [59,60]. Additional information regarding the sickle cell trait (carrier status) is provided by the Centers for Disease Control and Prevention. Additional details regarding management are discussed in detail separately. (See "Sickle cell trait".)

●Sickle cell disease – Older children or adults diagnosed with sickle cell disease (SCD) may be at risk for many acute and chronic complications. They require comprehensive evaluation and management by a hematologist with experience in SCD or a hemoglobinopathy center. (See "Sickle cell disease in infancy and childhood: Routine health care maintenance and anticipatory guidance", section on 'Age five years to adolescence' and "Sickle cell disease in infancy and childhood: Routine health care maintenance and anticipatory guidance", section on 'Older adolescents' and "Overview of the management and prognosis of sickle cell disease".)

Once a diagnosis of SCD has been made, there are a number of additional evaluations that are helpful in predicting disease course and identifying early markers of disease complications. This may include testing to identify common coinheritance or genetic polymorphisms that influence the severity of SCD:

One example is alpha thalassemia trait (one alpha globin gene deletion), which occurs in approximately 35 percent of individuals with Hb SS, or two alpha globin gene deletions, occurring in approximately 5 percent of the individuals with Hb SS [61].

Molecular (DNA-based) techniques are required to detect alpha thalassemia trait. The presence of alpha thalassemia trait can decrease the imbalance between alpha and beta globin chains, which in turn decreases abnormal hemoglobin polymerization and reduces anemia and clinical complications.

Fetal hemoglobin (Hb F) level is a strong predictor of severity. Genetic determinants of high Hb F levels are discussed separately. (See "Fetal hemoglobin (Hb F) in health and disease", section on 'Sickle cell disease'.)

In the newborn period, hemoglobin Barts can be quantitated as an indication of the presence of alpha thalassemia trait by protein electrophoresis or HPLC. Quantitative Barts is often performed as part of the newborn screening program and reported to providers. (See "Diagnosis of thalassemia (adults and children)", section on 'Overview of subtypes and disease severity'.)

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Sickle cell disease and thalassemias".)

INFORMATION FOR PATIENTS — UpToDate offers two types of patient education materials, "The Basics" and "Beyond the Basics." The Basics patient education pieces are written in plain language, at the 5^th to 6^th grade reading level, and they answer the four or five key questions a patient might have about a given condition. These articles are best for patients who want a general overview and who prefer short, easy-to-read materials. Beyond the Basics patient education pieces are longer, more sophisticated, and more detailed. These articles are written at the 10^th to 12^th grade reading level and are best for patients who want in-depth information and are comfortable with some medical jargon.

Here are the patient education articles that are relevant to this topic. We encourage you to print or e-mail these topics to your patients. (You can also locate patient education articles on a variety of subjects by searching on "patient info" and the keyword(s) of interest.)

●Basics topics (see "Patient education: Sickle cell trait (The Basics)" and "Patient education: Sickle cell disease (The Basics)" and "Patient education: When your child has sickle cell disease (The Basics)")

PATIENT PERSPECTIVE TOPIC — Patient perspectives are provided for selected disorders to help clinicians better understand the patient experience and patient concerns. These narratives may offer insights into patient values and preferences not included in other UpToDate topics. (See "Patient perspective: Sickle cell disease".)

SUMMARY AND RECOMMENDATIONS

●Settings for diagnosis – The diagnosis of sickle cell disorders can take place in several settings: prenatal testing, newborn screening, diagnosis of symptomatic individuals, and testing of relatives. (See 'Reproductive testing and counseling' above and 'Newborn screening' above and 'Older children and adults' above.)

●Methodology – Diagnosis of a sickle cell disorder is generally made via high performance liquid chromatography (HPLC), isoelectric focusing (IEF), or gel electrophoresis techniques. Polymerase chain reaction (PCR) or DNA sequencing may also be used. (See 'Methodology and diagnostic errors' above and "Methods for hemoglobin analysis and hemoglobinopathy testing".)

●Sources of error

•Newborns/infants – Only general patterns of hemoglobin production are available during the newborn period because beta globin production is not fully developed (table 1). (See 'Hemoglobin patterns' above.)

In very premature infants, adult hemoglobin (Hb A) may not be detected. Very premature infants with sickle cell trait may have sickle hemoglobin (Hb S) levels greater than Hb A, resulting in the incorrect diagnosis of sickle cell-beta⁺ thalassemia. (See 'Methodology and diagnostic errors' above.)

If questions arise as to interpretation, the tests should be repeated at age three to six months. DNA testing can be performed if clinically indicated. (See 'FAS and FSA patterns' above.)

•Transfusions – Protein-based testing (HPLC electrophoresis) after transfusion can result in an incorrect diagnosis due to excess hemoglobin A (Hb A) from transfused red blood cells. In such cases, DNA testing can be done, or testing can be postponed for ≥4 months after transfusion.

●Diagnostic confirmation – For children and adults, the combination of HPLC and IEF allows for a definitive diagnosis of a sickle cell disorder. Sickledex or solubility testing is not adequate for diagnosis. (See 'Older children and adults' above.)

●Common disorders – The most common sickle cell disorders are as follows (table 1) (see 'Findings in sickle cell anemia' above and 'Diagnostic patterns in other sickle cell disorders' above):

•Sickle cell trait – The usual pattern is >50 percent Hb A, 35 to 45 percent Hb S, and <2 percent Hb F. If there is <35 percent Hb S, alpha thalassemia may be present.

•Sickle cell anemia (Hb SS) – There is 0 percent Hb A, <2 percent Hb F, normal Hb A2, and the remainder Hb S.

•Hemoglobin SC disease – Hb S and Hb C are both present.

•Sickle cell-beta⁺-thalassemia – There is 5 to 30 percent Hb A, increased Hb A2, with the remainder Hb S. Target cells and hypochromic red cells are present.

•Sickle cell-beta⁰-thalassemia – There is 0 percent Hb A, variable Hb F, increased Hb A2, with the remainder Hb S. Target cells and hypochromic microcytic red cells are present.

●Counseling - Informing the parents of newborn screen results (table 2) has value for the child and potentially other relatives, as does informing adults with a new diagnosis of SCD or sickle cell trait in themselves or a child. (See 'Approach to a positive result from newborn/infant screening' above and 'Approach to a positive result in older children or adults' above.)

●Management – Management of SCD, including post-diagnostic evaluations, is discussed separately. (See "Overview of the management and prognosis of sickle cell disease", section on 'Routine evaluations and treatments' and "Sickle cell disease in infancy and childhood: Routine health care maintenance and anticipatory guidance".)

ACKNOWLEDGMENT — UpToDate gratefully acknowledges Stanley L Schrier, MD (deceased), who contributed as Section Editor on earlier versions of this topic and was a founding Editor-in-Chief for UpToDate in Hematology.

The UpToDate editorial staff also acknowledges extensive contributions of Donald H Mahoney, Jr, MD to earlier versions of this topic review.