A 17-year-old African American male presented to the hematology clinic for treatment of sickle cell disease (SCD).1 He had received the diagnosis of hemoglobin (Hb) S/C disease at an outside hospital at the age of 6 years; the diagnosis was confirmed in house at 11 years of age. His disease course had been severe, with frequent pain crises of increasing intensity and 2 episodes of acute chest syndrome requiring hospitalization and multiple blood transfusions.
The patient’s physical examination was unremarkable: blood pressure, 120/64 mmHg; pulse, 83 beats/min; temperature, 36.9 °C. Laboratory results were as follows: white blood cell count, 11.8 × 109/L [reference interval (RI), 3.9–10.3 × 109/L]; Hb, 6.39 mmol/L (RI, 8.68–10.8 mmol/L); packed cell volume, 0.28 (RI, 0.42–0.50); red blood cell count, 3.59 × 1012/L (RI, 4.5–6.0 × 1012/L); platelet count, 417 × 109/L (RI, 135–370 × 109/L); mean corpuscular volume, 78 fL (RI, 83–102 fL); mean corpuscular Hb, 28.7 pg (RI, 27–31 pg); mean corpuscular Hb count, 368 g/L (RI, 320–340 g/L); red cell distribution width, 17.3% (RI, 11.5%–14.5%); and absolute reticulocyte count, 0.115 (RI, 0.02–0.10). A peripheral blood smear showed scattered target and sickle cells, rare nucleated red cells, and mild anisopoikilocytosis. Results for the qualitative sickle cell solubility test were positive. Considering the severe disease course, Hb analysis by HPLC and isoelectric focusing (IEF) was ordered (Fig. 1⇓ ).
The family of SCDs, which is characterized by Hb S (Glu6Val substitution in the β-globin protein), is prevalent among African Americans. This substitution decreases the solubility of deoxygenated Hb and leads to the formation of rigid polymers that induce red cell sickling. Sickle cells undergo hemolysis and cause microvascular occlusions that lead to ischemic injury. Inheritance of one Hb S mutation, sickle cell trait, is clinically silent. Inheritance of 2 βS alleles, sickle cell anemia (S/S disease), is debilitating, with severe pain crises, increased susceptibility to infection, cerebrovascular events, and chronic organ damage. Patients with S/S disease have severe anemia (Hb, 3.7–6.2 mmol/L) with sickle and target cells on peripheral blood smears. The SCD family also includes hemoglobinopathies of varying severities in which Hb S is coinherited with Hb C, Hb D, Hb E, or Hb O (1). SCD is treated with hydroxyurea, which effectively reduces pain crises and other clinical manifestations. The US Food and Drug Administration has approved hydroxyurea for use in adults, and its efficacy has been demonstrated in adolescents as well (1).
SCD is diagnosed by the measurement of substantial amounts of Hb S by at least 2 separation methods, including HPLC and an electrophoretic method, such as IEF, cellulose acetate, or citrate agar. Because many Hbs coelute or comigrate on HPLC or IEF, respectively, it is crucial that multiple methods be used to confirm suspected hemoglobinopathies. The presence of hemolytic anemia with sickle and target cells on a blood smear and a positive result in a sickle cell solubility test in an African American is consistent with SCD. The present patient’s severe clinical course early in life and his Hb profiles suggested that his Hb S/C diagnosis was incorrect.
Analysis by IEF showed the presence of Hb S and another Hb migrating near the position for Hb C. An HPLC analysis revealed the following: Hb A, <1% (RI, >94%); Hb A2, 3.5% (RI, 2.0%–3.8%); Hb F, 5.9% (RI, <2.0%); Hb S, 42.1% (RI, none); and Hb Other, 47.5% (RI, none). The other Hb eluted in the C window at 4.93 min. The distinctive Hb profiles in the IEF and HPLC analyses suggested 3 potential compound heterozygous hemoglobinopathies; Hb S/C, Hb S/CHarlem, or Hb S/OArab. Further discussion with the patient revealed a family history that included a brother with a recent diagnosis of Hb S/OArab, which had originally been misdiagnosed as Hb S/C disease.
In both cases, the methods used for the initial diagnoses are unknown. Hb C and Hb OArab comigrate on IEF and cellulose acetate electrophoresis (Table 1⇓ ), and past HPLC methodologies were not capable of separating the 2 Hb variants. Citrate agar electrophoresis was the only method capable of differentiating Hb C and Hb OArab. The misdiagnoses of Hb S/C disease in both brothers could have been avoided had citrate agar electrophoresis been used for diagnosis and confirmation in each case.
The diagnosis was Hb S/OArab disease.
hb s/c disease
Like the Hb S trait, Hb C (βGlu6Lys) heterozygotes have no clinical symptoms. Homozygotes have mild anemia without sickling. Hb S/C disease (coinheritance of Hb S and Hb C) is clinically significant. The red cells of S/C disease are severely dehydrated, causing mild microcytosis and crystal formation (2). Hb S is concentrated and polymerized in dehydrated red cells, and this process leads to complications. Generally, the clinical course of S/C disease is less severe than S/S disease. Painful episodes begin later in life, occur at less than half the frequency of S/S disease, and appreciable pathology typically manifests after 20 years of age (3).
Patients with S/C disease have mild anemia and distinctive peripheral blood smears with target cells, “boat-shaped” cells, and S/C poikilocytes. Results for sickle cell solubility tests are positive. S/C disease is diagnosed by detection of Hb S and Hb C in a 1:1 ratio. Hb OArab and Hb CHarlem appear similar to Hb C by IEF and HPLC (Table 1⇑ ); therefore, citrate agar electrophoresis should be used to distinguish these variants (4)(5). There is no specific treatment for S/C patients.
Hb CHarlem (βGlu6Val, Asp73Asn) is a rare double mutation of the β-globin gene that produces sickling disorders in homozygous and compound heterozygous (Hb S/CHarlem) individuals. Both disorders are clinically severe (5).
Hb S/CHarlem patients have moderate hemolytic anemia, and blood smears show target and sickle cells (4). S/CHarlem disease is diagnosed by the detection of equal amounts of Hb S and Hb CHarlem via multiple separation techniques (Table 1⇑ ) (4). The severity of Hb S/CHarlem disease may prompt physicians to treat patients with hydroxyurea.
Hb OArab (βGlu121Lys) has a prevalence of 1 in 30 000 (4). Hb OArab heterozygotes are asymptomatic, and homozygous individuals have hemolytic anemia with febrile illnesses. Coinheritance of Hb S and Hb OArab produces clinically severe disease, with hemolytic anemia, jaundice, vaso-occlusive complications (pain crises and stroke), pneumonia, acute chest syndrome, and sepsis (4)(6). Sickle and target cells, polychromasia, and sometimes Howell–Jolly bodies are detected on peripheral blood smears. Results of sickle cell solubility tests are positive. HPLC, IEF, and citrate agar electrophoresis (Table 1⇑ ) all detect Hb S and Hb OArab in equal amounts (4). Because clinicians expect a more severe disease course in S/OArab disease, treatment may be more readily escalated to the use of hydroxyurea compared with S/C disease, which typically features fewer and more mild complications, particularly before 20 years of age.
pathophysiology of hb s/oarab disease
Hb S/OArab and Hb S/S diseases are clinically similar. Hb OArab copolymerizes with Hb S in red cells. Like Hb S/S, Hb S/OArab has reduced oxygen affinity and a lower gelling point for concentrated deoxygenated Hbs (7)(8). When deoxygenated, Hb S/OArab induces irreversible sickling of red cells (6)(7), which are hemolyzed or cleared through the reticuloendothelial system. Sickled cells block the narrow capillaries, causing membrane damage and vaso-occlusive events (4).
resolution of the case
Distinguishing Hb S/C, Hb S/OArab, and Hb S/CHarlem diseases in the laboratory is challenging. Dehydrated red cells of S/C disease are typically microcytic (2), whereas patients with S/OArab disease are often normocytic. As with our patient, microcytosis is seen in some patients with S/OArab disease (6).
IEF revealed Hb S in equal proportion with another Hb that comigrated near Hb C (Fig. 1A⇑ ). Hb A2, Hb E, Hb CHarlem, and Hb OArab all migrate in the same area. Hb A2 rarely constitutes >10% of the total Hbs. HPLC can differentiate Hb OArab from Hb C, Hb CHarlem, and Hb E. The patient’s HPLC profile (Fig. 1B⇑ ) shows 2 main Hbs eluting in the S and C windows. The Hb in the C window eluted at 4.93 min, compared with 5.19 min for the Hb C standard. The retention times of Hb variants on the Bio-Rad Laboratories Variant II system are 4.91 min for Hb OArab vs 5.18 min for Hb C (9). Hb E and Hb CHarlem elute at 3.69 min (9) and 4.89 min (personal communication), respectively (Table 1⇑ ). A minor peak of unknown significance appears after Hb A2 on chromatographs of Hb OArab patients (10) and in the profile of our patient, but not in Hb CHarlem patients. Distinguishing Hb OArab from Hb CHarlem requires citrate agar electrophoresis, in which Hb OArab migrates between the A and S calibrators, whereas Hb CHarlem migrates with Hb S (Table 1⇑ ).
In 2002, the patient was misdiagnosed with S/C disease. The patient’s Hb profile was determined by IEF and HPLC with the Bio-Rad Variant I instrument, which could not separate Hb C and Hb OArab. Citrate agar electrophoresis should have been used to confirm the diagnosis. Differentiation between S/C and S/OArab diseases is now possible with newer HPLC systems (10). Patients whose diseases were diagnosed before implementation of this technology may have received the wrong diagnosis if citrate agar electrophoresis was not used. Sequencing of the present patient’s β-globin gene confirmed a heterozygous mutation at nucleotide 414 (G→A) associated with Hb OArab.
After the rediagnosis, the patient was started on hydroxyurea (1000 mg/day) in accordance with recent NIH consensus documents recommending hydroxyurea treatment in several sickle cell syndromes, including S/C and S/OArab, to reduce such severe disease manifestations as pain crises and acute chest syndrome (1). At follow-up, the patient reported improved health. His anemia had improved slightly (Hb, 7.1 mmol/L; packed cell volume, 0.31; mean corpuscular volume, 84 fL).
We recommend that patients with abnormally severe S/C disease before the age of 20 years be evaluated for Hb S/OArab. These 2 diseases can be differentiated in the laboratory when both new-generation HPLC and either IEF or citrate electrophoresis are used. Sequencing of the β-globin gene confirmed the diagnosis. In this case, an accurate diagnosis, although not essential, prompted a change in treatment strategy. An earlier diagnosis of S/OArab disease may have encouraged clinicians to treat the disease more aggressively and might have reduced the patient’s morbidity substantially.
POINTS TO REMEMBER
Symptoms of Hb S/OArab disease such as pain crises, infection, and hemolytic anemia are severe and begin early in life. Complications of Hb S/C are significantly fewer in number and have a later onset.
Patients with a diagnosis of Hb S/C disease and an unusually severe disease course early in life should be evaluated for Hb S/OArab or Hb S/CHarlem disease.
Hb OArab can be differentiated from other hemoglobinopathies with new-generation HPLC profiling in combination with IEF or citrate agar electrophoresis.
β-Globin sequencing can confirm a suspected diagnosis of Hb S/OArab disease.
Whereas S/C disease is usually mild and normally does not require invasive treatments before 20 years of age, S/OArab disease is a severe sickling disorder, and patients often receive treatment similar to those with S/S disease.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors’ Disclosures of Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
↵1 Nonstandard abbreviations: SCD, sickle cell disease; Hb, hemoglobin; RI, reference interval; IEF, isoelectric focusing.
- © 2009 The American Association for Clinical Chemistry