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A patient with a previous diagnosis of hemoglobin S/C disease with an unusually severe disease course.

CASE

A 17-year-old African American male presented to the hematology clinic for treatment of sickle cell disease (SCD). (3) 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[degrees]C. Laboratory results were as follows: white blood cell count, 11.8 x [10.sup.9]/L [reference interval (RI), 3.9-10.3 x [10.sup.9]/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 x [10.sup.12]/L (RI, 4.5-6.0 x [10.sup.12]/L); platelet count, 417 x [10.sup.9]/L (RI, 135-370 x [10.sup.9]/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).

DISCUSSION

The family of SCDs, which is characterized by Hb S (Glu6Val substitution in the [beta]-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 [[beta].sup.s] 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.

PATIENT FOLLOW-UP

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, <I% (RI, >94%); Hb [A.sub.2],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/[C.sub.Harlem], or Hb S/[O.sub.Arab]. Further discussion with the patient revealed a family history that included a brother with a recent diagnosis of Hb S/[O.sub.Arab], which had originally been misdiagnosed as Hb S/C disease.

[FIGURE 1 OMITTED]

In both cases, the methods used for the initial diagnoses are unknown. Hb C and Hb [O.sub.Arab] 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 [O.sub.Arab]. 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.

DIAGNOSIS

The diagnosis was Hb S/[O.sub.Arab] disease.

Hb S/C DISEASE

Like the Hb S trait, Hb C ([beta]Glu6Lys) 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 [O.sub.Arab] and Hb [G.sub.Harlem] 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 S/[C.sub.Harlem]

Hb/[C.sub.Harlem] ([beta]Glu6Val, Asp73Asn) is a rare double mutation of the [beta]-globin gene that produces sickling disorders in homozygous and compound heterozygous (Hb S/[C.sub.Harlem]) individuals. Both disorders are clinically severe (5).

Hb S/[C.sub.Harlem] patients have moderate hemolytic anemia, and blood smears show target and sickle cells (4). S/[C.sub.Harlem] disease is diagnosed by the detection of equal amounts of Hb S and Hb [C.sub.Harlem] via multiple separation techniques (Table 1) (4). The severity of Hb S/[C.sub.Harlem] disease may prompt physicians to treat patients with hydroxyurea.

Hb S/[O.sub.Arab]

Hb [O.sub.Arab] ([beta]Glu121Lys) has a prevalence of 1 in 30 000 (4). Hb [O.sub.Arab] heterozygotes are asymptomatic, and homozygous individuals have hemolytic anemia with febrile illnesses. Coinheritance of Hb S and Hb [O.sub.Arab] 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 [O.sub.Arab] in equal amounts (4). Because clinicians expect a more severe disease course in S/[O.sub.Arab] 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/[O.sub.Arab], DISEASE

Hb S/[O.sub.Arab] and Hb S/S diseases are clinically similar. Hb [O.sub.Arab] copolymerizes with Hb S in red cells. Like Hb S/S, Hb S/[O.sub.Arab] has reduced oxygen affinity and a lower gelling point for concentrated deoxygenated Hbs (7, 8). When deoxygenated, Hb S/[O.sub.Arab] 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/[O.sub.Arab,] and Hb S/[C.sub.Harlem] diseases in the laboratory is challenging. Dehydrated red cells of S/C disease are typically microcytic (2), whereas patients with S/[O.sub.Arab] disease are often normocytic. As with our patient, microcytosis is seen in some patients with S/[O.sub.Arab] disease (6).

IEF revealed Hb S in equal proportion with another Hb that comigrated near Hb C (Fig. 1A). Hb [A.sub.2], Hb E, Hb [C.sub.Harlem,] and Hb [O.sub.Arab] all migrate in the same area. Hb [A.sub.2] rarely constitutes > 10% of the total Hbs. HPLC can differentiate Hb [O.sub.Arab] from Hb C, Hb [C.sub.Harlem], 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 [O.sub.Arab] vs 5.18 min for Hb C (9). Hb E and Hb [C.sub.Harlem] elute at 3.69 min (9) and 4.89 min (personal communication), respectively (Table 1). A minor peak of unknown significance appears after Hb [A.sub.2] on chromatographs of Hb [O.sub.Arab] patients (10) and in the profile of our patient, but not in Hb [C.sub.Harlem] patients. Distinguishing Hb [O.sub.Arab] from Hb [C.sub.Harlem] requires citrate agar electrophoresis, in which Hb [O.sub.Arab] migrates between the A and S calibrators, whereas Hb [C.sub.Harlem] 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 [O.sub.Arab]. Citrate agar electrophoresis should have been used to confirm the diagnosis. Differentiation between S/C and S/[O.sub.Arab] 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 [beta]-globin gene confirmed a heterozygous mutation at nucleotide 414 (G [right arrow] A) associated with Hb [O.sub.Arab].

After the rediagnosis, the patient was started on hydroxyurea (1000 mg/day) in accordance with recent NIH consensus documents recommending hydroxy urea treatment in several sickle cell syndromes, including S/C and S/[O.sub.Arab], 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/[O.sub.Arab]. 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 [beta]-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/[O.sub.Arab] 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/[O.sub.Arab] 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/[O.sub.Arab] or Hb S/[C.sub.Harlem] disease.

* Hb [O.sub.Arab] can be differentiated from other hemoglobinopathies with new-generation HPLC profiling in combination with IEF or citrate agar electrophoresis.

* [beta]-Globin sequencing can confirm a suspected diagnosis of Hb S/[O.sub.Arab] disease.

* Whereas S/C disease is usually mild and normally does not require invasive treatments before 20 years of age, S/[O.sub.Arab] 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.

References

(1.) Brawley OW, Cornelius LJ, Edwards LR, Gamble VN, Green BL, Inturrisi CE, et al. NIH consensus development statement on hydroxyurea treatment for sickle cell disease. NIH Consens State Sci Statements 2008;25:1-30.

(2.) Ballas SK, Larner J, Smith ED, Surrey S, Schwartz E, Rappaport EF. The xerocytosis of Hb SC disease. Blood 1987;69:124-30.

(3.) Nagel RL, Fabry ME, Steinberg MH. The paradox of hemoglobin SC disease. Blood Rev 2003;17:167-78.

(4.) Hoyer JD, Kroft SH, eds. Color atlas of hemoglobin disorders: a compendium based on proficiency testing. Northfield (IL): College of American Pathologists; 2003.

(5.) Moo-Penn W, Bechtel K, Jue D, Chan MS, Hopkins G, Schneider NJ, et al. The presence of hemoglobin S and C Harlem in an individual in the United States. Blood 1975;46:363-7.

(6.) Zimmerman SA, O'Branski EE, Ross@ WF, Ware RE. Hemoglobin S/[O.sub.Arab]: thirteen new cases and review of the literature. Am J Hematol 1999;60:279-84.

(7.) Milner PF, Miller C, Grey R, Seakins M, DeJong WW, Went LN. Hemoglobin O arab in four negro families and its interaction with hemoglobin S and hemoglobin C. N Engl J Med 1970;283:1417-25.

(8.) McCurdy PR, Mahmood L, Sherman AS. Red cell life span in sickle cell-hemoglobin C disease with a note about sickle cell-hemoglobin 0 ARAB. Blood 1975;45:273-9.

(9.) Joutovsky A, Hadzi-Nesic J, Nardi MA. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60 000 samples in a clinical diagnostic laboratory. Clin Chem 2004;50:1736-47.

(10.) Joutovsky A, Nardi M. Hemoglobin C and hemoglobin O-Arab variants can be diagnosed using the Bio-Rad Variant II high-performance liquid chromatography system without further confirmatory tests. Arch Path Lab Med 2004;128:435-9.

(3) Nonstandard abbreviations: SCD, sickle cell disease; Hb, hemoglobin; RI, reference interval; IEF, isoelectric focusing.

Elizabeth K. O'Keeffe, [1] Melissa M. Rhodes, [2] and Alison Woodworth [1] *

[1] Departments of Pathology and [2] Pediatric Hematology-Oncology, Vanderbilt University Medical Center, Nashville, TN.

* Address correspondence to this author at: Department of Pathology, Vanderbilt University Medical Center, 4918EA TVC, 1301 Medical Center Dr., Nashville, TN, 37232-5310. Fax 615-343-9563; e-mail Alison.Woodworth@Vanderbilt. Edu.

Received June 11, 2008; accepted December 4, 2008.

DOI: 10.1373/clinchem.2008.112326

Commentary

Carlo Brugnara

Much progress remains to be made in our understanding of the factors underlying the clinical manifestations of sickle cell disease. The severity of the disease has long been known to be highly variable, with some patients experiencing extremely severe disease and major organ-specific complications early in their lifetimes, and other patients with clinically silent disease who receive their diagnoses much later in life and have an almost normal life expectancy.

Epidemiologic and clinical data have suggested that increased concentrations of hemoglobin F (Hb F) are associated with a decreased severity of the disease. White blood cell counts are also an important predictor of mortality and morbidity--both in sickle cell disease and in the general population-with a shortened life expectancy being associated with higher counts.

The case presented in this issue of Clinical Chemistry demonstrates the remarkable effect of double heterozygosity for Hb S and Hb [O.sub.Arab] on the clinical severity of the disease, compared with the more frequently observed Hb S/C double heterozygote. The worse effect of Hb [O.sub.Arab] has been attributed to enhanced polymerization of Hb S and to the accompanying dehydration of erythrocytes imposed by the presence of the positively charged Hb [O.sub.Arab] variant. This case also highlights the important role that the laboratory plays in the diagnosis and treatment of this disease. It is only thanks to the astute clinical and laboratory insights of the team taking care of this patient that the initial diagnosis of Hb S/C disease was questioned and the appropriate studies were conducted to reach the final, proper diagnosis. Although substantial progress has recently been made in modeling the risk for stroke (1) or death (2) in individual patients with sickle cell disease, this case highlights the crucial role of clinical reasoning and proper laboratory investigation when dealing with unexpected complications in particular patients.

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.

References

(1.) Sebastiani P, Ramoni MF, Nolan V, Baldwin CT, Steinberg MH. Genetic dissection and prognostic modeling of overt stroke in sickle cell anemia. Nat Genet 2005;37:435-40.

(2.) Sebastiani P, Nolan VG, Baldwin CT, Abad-Grau MM, Wang L, Adewoye AH, et al. A network model to predict the risk of death in sickle cell disease. Blood 2007;110:2727-35.

Department of Laboratory Medicine, Children's Hospital Boston, Boston, MA. Address correspondence to the author at: Department of Laboratory Medicine, Children's Hospital Boston, 300 Longwood Ave. 760, Boston, MA 02115. Fax 617-730-0383; e-mail Cario.Brugnara@childrens.harvard.edu.

Received January 21, 2009; accepted February 6, 2009.

DOI: 10.1373/clinchem.2009.123943

Commentary

James Hoyer

This case illustrates 2 very important points about laboratory testing for hemoglobin (Hb) disorders. The first is that most methods used for lib identification are not sufficient as single, stand-alone methods. The only exceptions to this rule are DNA sequencing and mass spectrometry, but these methods are not used by many clinical laboratories because of the expense involved. It has long been a requirement in the College of American Pathologists checklist for hematology that Hb variants, particularly in the S and AZ positions, be confirmed by a second method. As illustrated by this case, performance of acid electrophoresis would clearly have shown that the variant in the Hb AZ position was not Hb C (Fig. 1). Acid electrophoresis is easily able to distinguish between the 3 major variants that migrate in the AZ position: Hb C, Hb E, and Hb [O.sub.Arab]. Other combinations of methods, however, can accomplish the same result.

Second, this case emphasizes that Hb analysis should not be interpreted in isolation but must be correlated with the patient's clinical situation and other laboratory findings. The severe clinical course seen in this patient is highly unusual for Hb S/C disease. Furthermore, as was outlined, Hb S/C disease has very characteristic morphologic findings in peripheral blood smears. True sickle cells are rare in Hb S/C disease; their presence in this case was another reason to question the diagnosis. In cases such as this one, reconfirmation of a reported diagnosis is warranted, particularly if previous records are not available or if there are clinical inconsistencies.

[FIGURE 1 OMITTED]

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.

Mayo Clinic, Rochester, MN.

Address correspondence to the author at: Mayo Clinic, 200 1st St. SW, Rochester, MN 55905. Fax 507-284-5115; e-mail ihoyer@mayo.edu.

Received January 28, 2009; accepted February 6, 2009.

DOI: 10.1373/clinchem.2009.123950
Table 1. Separation profiles of Hb variants C, [C.sub.Harlem],
and [O.sub.Arab].

 Relative migration
 positions

 HPLC retention Isoelectric
 time, min focusing

Hb C 5.18 Cathodal to [A.sub.2]
Hb [C.sub.Harlem] 4.89 Comigrates with [A.sub.2]
Hb [O.sub.Arab] 4.91 Comigrates with [A.sub.2]

 Relative migration positions

 Citrate agar Acetate
 electrophoresis electrophoresis

Hb C Anodal to S Comigrates with [A.sub.2]
Hb [C.sub.Harlem] Comigrates with S Comigrates with [A.sub.2]
Hb [O.sub.Arab] Between S and A Comigrates with [A.sub.2]
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Title Annotation:Clinical Case Study
Author:O'Keeffe, Elizabeth K.; Rhodes, Melissa M.; Woodworth, Alison
Publication:Clinical Chemistry
Article Type:Case study
Geographic Code:1USA
Date:Jun 1, 2009
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