Annals o f Clinical & Laboratory Science, vol. 30, no. 2, 2000 191

Chromatographic Measurements of A2 in Samples that Contain Sickle Hemoglobin

Masih Shokrani, 1 Falicia Terrell, Ernest A. Turner, 2 and Maria del Pilar Aguinaga3 Comprehensive Sickle Cell Center, Meharry Medical College, Nashville, Tennessee; 1 Department of Microbiology, Immunology & Genetics, Department of Pediatrics, 3 Department of Obstetrics & Gynecology

Abstract. In the sickle cell syndromes, Hb A2 measurements aid in the differential diagnosis of sickle cell anemia from sickle-beta-. The purpose of this study is to assess the Hb A2 levels in samples containing sickle hemoglobin (Hb S) by the use of an automated high performance liquid chromatography system (HPLC- Variant (3-thalassemia Short Program). The blood samples analyzed were from individuals of African descent living in the state ofTennessee who had either sickle cell trait (Hb AS), (Hb SS), or sickle cell- disease (Hb SC). Interestingly, the Hb A2 levels determined by HPLC were found elevated in samples containing Hb S. The Hb A^ mean in Hb AS samples (n=l46) is 4.09% (SD + 0.42, range 2.20 to 5.20%); in Hb SS samples (n=33) it is 3.90% (SD ± 1.08, range 0.60 to 5-90%); and in Hb SC samples (n=27) it is 4.46% (SD ± 0.70, range 2.30 to 5.91%). The Hb A2 mean by HPLC in normal individuals (Hb AA, n=70) is 2.57% (SD ± 0.25, range 2.1 to 3.0%), and the Hb A2 range in (3-thalassemia carriers is 4 to 9%. Our results show that the Hb A2 levels in Hb S-containing samples partially overlap with those expected from (3- thalassemia carriers. The laboratory should be aware of this apparent elevation in Hb A2 levels determined by HPLC in individuals carrying Hb S. Other factors, such as family history and clinical symptoms, should be taken into account before a diagnosis of sickle cell trait, sickle-beta-thalassemia, or sickle cell anemia is made.

Keywords: Hemoglobin A2, hemoglobin S, high-performance liquid chromatography, thalassemia diagnosis

Introduction (3-thalassemia when determined by capillary IEF. The purpose of this report is to assess Hb A2 levels by HPLC The quantitation of Hb A2 is important for the in liquid blood samples containing Hb S. We have accurate diagnosis of hemoglobin disorders such as found that Hb A2 levels quantitated by HPLC are the (3-, or hemoglobin variants in elevated in samples from non-thalassemic individuals combination with (3-thalassemia (eg, sickle-beta- that carry Hb S. We have not observed this increase thalassemias). Hb A2 is known to be elevated in (3- in Hb A2 levels in samples that contain only Hb AA. thalassemia trait [1], sickle-beta-thalassemia [2], and HPLC reference ranges for Hb A2 in sickle cell trait, sickle cell disease with a-thalassemia [3]. In sickle sickle cell anemia, and Hb SC disease should be cell trait, Hb A2 determined by microchromatography established in order to prevent an erroneous association has been found slightly elevated [4], and significantly of these disorders with (3-thalassemia. elevated when determined by capillary isoelectric focusing electrophoresis (IEF) [5]. However, no Methods overlap in Hb A2 levels was seen between Hb S- containing samples and samples from patients with Blood specimens were collected in heparinized capillaries or in ethylene diaminetetraacetare (EDTA) Address correspondence to Maria del Pilar Aguinaga, Ph.D., microvettes (Sarstedt, Inc., Newton, NC). The criteria Comprehensive Sickle Cell Center, Meharry Medical College, for sample inclusion were: (1) presence of Hb S, (2) 1005 Dr. D. B. Todd Jr. Bvld., Nashville, TN 37208; tel 615 327 sample age < 7 days after collection, (3) symmetrical 6528; fax 615 327 5511; e-mail [email protected].

0091-7370/00/0200-0191 $1.00; © 2000 by the Association of Clinical Scientists, Inc. 192 Annab o f Clinical & Laboratory Science

Hb A2 chromatogram peak (Fig. 1), and (4) subjects cation exchange HPLC. The resin of the column has from 1 to 60 years of age. Samples from subjects being a negatively charged surface group that will bind the treated with hydroxyurea, a medication prescribed for positively charged protein (Hb), while the more treatment of sickle cell disease that increases Hb F negatively charged molecules will elute readily from levels, were excluded from this study, as were samples the column. A higher cation concentration will be diagnosed as sickle-beta-thalassemia (Fig. 1) or samples required to elute the positively charged hemoglobin with asymmetrical Hb A2 peak (Fig. 2). Blood from the column. The ionic strength of the elution specimens were received in the laboratory by postal buffer mixture is increased by raising the percent service, courier, or walk-in. Red cell hemolysates were contribution of elution buffer #2. As the ionic strength run by alkaline electrophoresis (pH 8.6) (Helena of the mixture increases, more strongly retained Laboratories, Inc., Beaumont, TX) and by isoelectric elute from the column [8], focusing electrophoresis (IEF) (Wallace Laboratories, Inc., Akron, OH) before HPLC analysis. The Bio- Results Rad Variant (3-thalassemia Short Program was used for Hb quantitations. The Variant (Bio-Rad Labora­ For this study, 146 Hb AS samples, 33 Hb SS samples, tories, Inc., Hercules, CA) is a fully automated HPLC and 28 Hb SC samples were analyzed by HPLC. system that separates a hemoglobin mixture into its Results showed that in Hb AS samples, the mean for individual components by pumping liquids through a Hb A^ is 4.09% (SD ± 0.42, range 2.20 to 5.2%). In column at high pressure. It is a fast and reproducible Hb SS samples, the mean for Hb A 2 is 3.90% (SD + method that can be used to separate and determine 1.08, range 0.60 to 5.90%. In Hb SC samples, the area percentages for various hemoglobins, including mean for Hb A^ is 4.46% (SD ± 0.70, range 2.30 to Hb F and Hb Ar It can also provide qualitative 5.91%). The corresponding hemoglobin quantitations determinations of abnormal hemoglobins. The (3- for Hb F, Hb A, Hb S, and Hb C are shown in Tables thalassemia Short Program utilizes the principle of 1, 2, and 3 for the sample populations mentioned

Elution Time (min.) E lu tion T im e (m in.)

Fig. 1. Bio-Rad HPLC chromatogram showing a typi­ Fig. 2. Bio-Rad HPLC chromatogram showing an cal Hb A2 peak (symmetrical peak). This patient was atypical Hb A2 peak (asymmetrical peak, having a diagnosed as sickle-beta-thalassemia. shoulder). This patient was diagnosed as sickle cell trait. HPLC analysis ofHb A 2 in blood that contains Hb S 193

above. The reference range for Hb A2 in normal adults It has been postulated that, in the presence of is 2.1 to 3.0%, with a mean of 2.57% [8] using the P-chain hemoglobin variants, the association of the Bio-Rad Variant system. OC- chain with the (3-globin chain variant is impaired, which fosters the association of CX-chains with Discussion 8-chains, increasing the Hb A^ levels [10,11]. Other factors that play a role in this increase are the The sensitivity of HPLC has significantly contributed accumulation of Hb S adducts and sample aging. The to the accurate quantitation of hemoglobin variants. Hb S adducts include glycohemoglobin [11] and other In this report we have established reference ranges for post-translational modification products of Hb S. The Hb A2 quantified by HPLC (Bio-Rad Variant (3- effect of sample aging is observed in the accumulation thalassemia Short Program) in samples containing of glycohemoglobin in blood samples over time [7]. Hb S. We found that blood samples containing Hb S If Hb S—containing blood samples are stored for 2 usually have increased Hb A2 levels when compared to weeks, Hb A2 levels are increased, possibly due to previously published HPLC reference ranges [8,9]. The accumulation of glycohemoglobin [7,12]. We did not HPLC manufacturer states that Hb S byproducts could determine glycohemoglobin in our samples. Since the coelute with Hb A2 [8]. We have not observed increased blood samples used for this study were relatively fresh Hb A2 values by HPLC in normal Hb AA samples. (1 week) and we still observed a marked increase in Hb A2, we postulate that other factors may be involved in the increase of Hb A2 in Hb S-containing samples Table 1: Hemoglobin percentages in Hb AS samples when determined by HPLC. (sickle cell trait), based on HPLC analysis. Concurrently elevated levels of Hb A2 and Hb F offer a practical way to diagnose carriers of the Hb N Mean ± SD (%) Range (%) p-thalassemia gene or other Hb disorders that lead to A 146 53.80±2.93 (47.70 - 63.60) an increase in this hemoglobin. The HPLC manu­ S 146 34.66±3.71 (21.2 0 - 42.10) facturer indicates that a range for Hb A^ of 4 to 9% is 4.09±0.42 a 2 146 (2.20 - 5.20) typical of heterozygous P-thalassemia [8]. Our results F 98 0.98±0.67 (0.20 - 3.90) show that in samples containing Hb S, the Hb A^ values are elevated above the normal reference ranges given by the manufacturer [8] and also above those that we previously reported in a healthy African American Table 2: Hemoglobin percentages in Hb SS samples population (Hb A^ range = 0.05 to 3.4%, mean 1.2%) (sickle cell disease), based on HPLC analysis. [9]. With the system used in the present study, Hb A2 Hb N Mean ± SD (%) Rang«; (%) levels in samples containing Hb S partially overlap with those expected for P-thalassemia carriers. When Hb S 33 86.74±3.62 (80.40 -- 94.70) A2 quantitations in Hb S—containing samples are 33 3.90±1.08 (0.60 -- 5.90) A, reported, it should be recognized that the apparently F 33 6.80±2.95 (1.30- 11.40) elevated Hb A2 values may well be within the normal range for those samples. The elevated Hb A2 values should be correlated with the clinical picture and family Table 3: Hemoglobin percentages in Hb SC samples studies to help establish the diagnosis. (hemoglobin SC disease), based on HPLC analysis.

Hb N Mean ± SD (%) Range(%) Conclusions S 28 46.46±1.27 (43.70 - 49.20) C 28 44.25±1.74 (40.40 - 46.90) The HPLC Hb A2 levels in subjects older than 1 year and younger than 60 years of age having sickle cell A, 27 4.46+0.70 (2.30-5.91) F 27 2.17+1.51 (0.50-6.10) trait (Hb AS) or sickle cell disease (Hb SS and Hb SC) have been assessed. These results confirm the elevation 194 Annals o f Clinical & Laboratory Science

of Hb A2 levels in individuals with Hb AS, Hb SS, 3. Balias SK, Gay RN, Chehab FE Is Hb A2 elevated in and Hb SC, as determined by the BioRad Variant adults with sickle-alpha-thalassemia (beta(S)/beta(S); HPLC System. We conclude that in Hb AS, levels of -alpha/-alpha)? Hemoglobin 1997;21:405-450. Hb A2 lower than 5.2% are normal. In Hb SS and Hb 4. Huisman THJ, Shroeder WA, Brodie AN, Mayson SM, SC samples, levels of Hb A2 lower than 5.9% may also JakwayJ. Microchromatography of hemoglobins, III: a simplified procedure for the determination of Hb Aj- be normal if there is no other sign of P-thalassemia. J Lab Clin Med 1975;86:700-702. If further increased Hb A2 levels are found in a sickle 5. Craver RD, Abermanis JG, Warrier RP, Ode DL, cell disease patient, one should also consider family Hempe JM. Hemoglobin A2 levels in healthy persons, history, clinical symptoms, drug treatment, and sample sickle cell disease, sickle cell trait, and P-thalassemia by aging before the diagnosis of an associated Pi- capillary isoelectric focusing. Am J Clin Path 1997; thalassemia disorder is made. 107:88-91. 6. Ou CN, Buffone GJ, Reimer GL, Alpert AJ. High- Acknowledgments performance liquid chromatography of human hemoglobins on a new cation exchange. J Chromatog The authors thank Dr. Mark J. Koury for critical 1983;266:197-205. 7. Kutlar A, Kutlar F, Wilson JB, Headlee MG, Huisman reading of the manuscript, Ms. Mai Williamson for THJ. Quantitation of hemoglobin components by technical assistance, Ms. Deborah Majors for statistical high-performance cation-exchange liquid chrom­ analyses, and Ms. Tasha White and Ms. Carolyn Tansey atography: its use in the diagnosis and in the assessment for typing the manuscript. Supported by grants from of distribution of hemoglobin variants. Am J Hematol the Tennessee Department of Health and Environment 1984;17:39-53. (GR-6 10510-600), and the National Heart, Lung and 8. BioRad. Variant Beta-Thalassemia Short Program Blood Institute (P60 HL-38737 and K14 HL-03141). Procedure Manual. BioRad Laboratories, Inc., Hercules, CA, 1994. References 9. Roa PD, Turner EA, Aguinaga MdP. Reference ranges for hemoglobin variants by HPLC in African Americans. Ann Clin Lab Sci 1995;25:228-235. 1. Huisman THJ. Levels of Hb A2 in heterozygotes and 10. Whitten WJ, Rucknagel DL. The proportion of Hb homozygotes for beta-thalassemia mutations: influence A is higher in sickle than in normal homozygotes. of mutations in the CACCC and ATAAA motifs of the 2 Hemoglobin 1981;5:371-378. beta-globin gene promoter. Acta Haematologica 1997; 11. Huisman, THJ. Chromatographic separation of Hb 98:187-194. A and quantities of Hb in patients with AC trait, 2. Sweeting I, Serjeant BE, Thomas PW, Serjeant GR. 2 A j CC disease, and C-. Clin Chim Acta Microchromatographic quantitation of Hb A2 levels in phenotypes of sickle cell-beta(+) thalassemia. J 1972;40:159-163. 12. Suh DD, Krauss J S, Bures K. Influence of Hb S adducts ChromatogB 1997;700:269-274. on hemoglobin Hb A2 quantification by HPLC. Clin Chem 1996;42;1113-1114.