Context.—Current standards for laboratory accreditation from the College of American Pathologists state that when high-performance liquid chromatography (HPLC) is used as a screening test, all non-A, non-S abnormal hemoglobin (Hb) variants must be confirmed by an alternative method, including alkaline and acid electrophoresis.

Objective.—To determine whether confirmation of Hb C and Hb O-Arab variants by an alternative method is required when using the Bio-Rad Variant II HPLC system.

Design.—We reviewed 48 478 consecutive hemoglobin identification test results performed on the Bio-Rad Variant II HPLC system during the period November 15, 2000 to January 15, 2003.

Setting.—Special Hematology Laboratory, Department of Pathology, Bellevue Hospital Center, New York, NY.

Main Outcome Measures.—The chromatogram patterns and retention times (RTs) for specimens containing Hb C and Hb O-Arab were analyzed. We compared the results by the HPLC method with those by the confirmatory tests (alkaline and acid electrophoresis) for both variants.

Results.—We identified 3668 cases of abnormal hemoglobin variants, including 660 cases of Hb C trait (17%), 5 cases of Hb O-Arab trait (0.1%), and 1 case of Hb SO-Arab (0.03%). A unique pattern of separation on the chromatogram for Hb O-Arab was revealed, presenting as 2 distinct peaks in 2 different manufacturer-defined RT windows, namely, D and C. The chromatogram for Hb C did not show the D window in any of the reviewed cases. The RT in the C window (C-RT) revealed a statistically significant difference for Hb C and Hb O-Arab (5.18 ± 0.01 minutes and 4.91 ± 0.01 minutes, respectively; P < .001).

Conclusion.—According to our review, the identification of Hb C and Hb O-Arab is accurate using HPLC methodology, as performed by the Bio-Rad Variant II HPLC system. This method can be both confirmatory and diagnostic at the same time.

The most widely used method for hemoglobin (Hb) analysis is alkaline cellulose acetate electrophoresis at pH 8.6. It is rapid, reproducible, and capable of separating common hemoglobin variants, such as Hb S, Hb F, Hb A, and Hb C, but Hb C, Hb A2, Hb O-Arab, and Hb E are unresolved from each other, as are Hb S, Hb D, Hb G, Hb Lepore, and Hb Hasharon. In addition, there are many other hemoglobin variants with electrophoretic mobilities identical or similar to Hb S and Hb C. Consequently, acidic citrate agar electrophoresis at pH 6.2 is needed for identification of the aforementioned hemoglobin variants. Nevertheless, these electrophoretic methods will still not be able in most cases to separate Hb D from Hb G, Hb Lepore, and Hb Hasharon, and in some cases Hb E from Hb O-Arab. Often the hemoglobin variant is inferred from the electrophoretic mobility, the quantity of the hemoglobin variant, and the patient's ethnic background.1,2 

A review of the College of American Pathologists (CAP) Hemoglobinopathy Survey Reports3 indicates the number of laboratories using high-performance liquid chromatography (HPLC) technology for identification of hemoglobin variants has increased approximately 12.5-fold in the past 10 years. There are presently more than 100 laboratories using this technology to quantitate all hemoglobin fractions and to screen for hemoglobin variants. During this same time, the number of laboratories performing some form of hemoglobin electrophoresis has increased only 2-fold.

High-performance liquid chromatography is an excellent screening method for hemoglobinopathies and thalassemias.1,4,5 The simplicity of the automated system with internal sample preparation, superior resolution, rapid assay time, and accurate quantitation of hemoglobin concentration makes this an ideal methodology for the diagnosis of hemoglobin disorders in the routine clinical laboratory.6 A number of automated HPLC systems are now commercially available, and evaluations have been published.7–11 

Current standards for laboratory accreditation from the CAP require a confirmatory test for all samples with hemoglobin variants migrating in non-A, non-S positions on alkaline electrophoresis, isoelectric focusing, or HPLC to be further defined with electrophoresis at acid pH or with other acceptable methods where clinically and technically appropriate.12 

Both Hb C and Hb O-Arab are found in the African American population, along with Hb S and other β and α variants. It is of clinical significance to differentiate accurately between Hb C and Hb O-Arab, because their respective interactions with Hb S lead to clinically different diseases. Heterozygotes for either Hb C or Hb O-Arab are clinically asymptomatic and typically have no laboratory abnormalities other than the detection of the hemoglobin variant. Patients who are homozygotic for either Hb C or Hb O-Arab are clinically asymptomatic but may have a mild compensated hemolytic anemia.13,14 Hemoglobin SO-Arab disease presents with clinical and laboratory manifestations characteristic of a sickling disorder with painful episodes, hemolytic anemia, and jaundice similar to that found in Hb SS disease.15–17 Hemoglobin SC disease, however, is a clinically milder disorder with modest anemia and infrequent crises. Vitreous hemorrhages and aseptic necrosis of the femoral head or hematuria may be the only disability found in adults, and most patients have a normal life span.13,17 

The purpose of this study was to determine if the laboratory, using only HPLC technology, could accurately and reliably differentiate between Hb C and Hb O-Arab without the need for further confirmatory tests. We have extensively reviewed the laboratory data for patients with either of these variants. We conclude that Hb C and Hb O-Arab can be correctly identified by the Bio-Rad Variant II HPLC system (Bio-Rad Laboratories, Hercules, Calif) and do not require the confirmatory tests as stated in the current CAP standards.

Specimens were drawn into tubes containing potassium EDTA (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ). All specimens were analyzed by HPLC on the Bio-Rad Variant II HPLC system using the Variant II β-Thalassemia Short Program Reorder Pack, as described in the instruction manual for the kit. Briefly, in this system the samples are mixed by the Variant II sampling station, diluted with kit-specific hemolyzing/wash buffer, and injected into a kit-specific analytic cartridge. The Variant II dual pumps deliver a programmed buffer gradient of increasing ionic strength to the cartridge, where the Hb fractions are separated based on their ionic interaction with the cartridge material. The separated Hb fractions pass through a flow cell, where absorbance is measured at 415 nm; background noise is reduced with the use of a secondary wavelength at 690 nm. The raw data are integrated by the Clinical Data Management computer system (Bio-Rad) and a chromatograph/sample report is generated. The integrated peaks are assigned to manufacturer-defined windows (Table 1) derived from the retention time (RT) of hemoglobin fractions and common variants. If a peak elutes at an RT that is not predefined, it is labeled as an unknown.

A total of 48 478 tests for hemoglobin identification performed in the Bellevue Hospital Special Hematology Laboratory in a 26-month period were reviewed. For specimens that showed chromatographic patterns consistent with sickle trait, the presence of Hb S was confirmed using the sodium metabisulfite reduction test.18 All non-A, non-S hemoglobin variants were confirmed by alkaline cellulose acetate electrophoresis at pH 8.6 and acidic citrate agar electrophoresis at pH 6.2, using the Helena hemoglobin electrophoresis system according to the manufacturer's recommendations. Four of the 5 Hb O-Arab trait and the Hb SO-Arab specimens were forwarded to the Mayo Medical Laboratories (Rochester, Minn) for confirmation. Confirmation was achieved by performance of HPLC, alkaline and acid hemoglobin electrophoresis, isoelectric focusing, globin-chain electrophoresis, and unstable hemoglobin screen.

Fifty chromatograms of Hb AC, 4 of Hb AO-Arab, and 1 of Hb SO-Arab were closely analyzed. To perform precise statistical analysis of the RT in the C window, we chose the 5 results of Hb AC preceding and the 5 results following each case of Hb O-Arab observed. The pattern of peak distribution, RT, and percentage of the area in each manufacturer-defined RT window in which common variants have been observed to elute were analyzed.

All data analysis was performed using Minitab Statistical Software (Minitab Inc, State College, Pa).

There were 3668 specimens identified with either β- or α-globin variants. Of these, 660 cases of Hb AC, 4 cases of Hb AO-Arab, and 1 case of Hb SO-Arab were identified. Patients on hypertransfusion regimens for treatment of a sickling disorder or other transfusion-dependent diseases were not included.

Distinct peaks were identified in all chromatograms for the Hb AO-Arab specimens corresponding to the D and C windows (Figure 1). In the chromatograms for Hb AC, there were peaks in only the C window (Figure 2). The D window was not identified in any of the closely reviewed 50 chromatograms for Hb AC. The mean RT for the C window (C-RT) was 5.18 ± 0.01 minutes and 4.91 ± 0.01 minutes for Hb C and Hb O-Arab, respectively (Table 2). The difference in the RT was statistically significant (P < .001). For the Hb O variant, the mean RT for the D window (D-RT) was 3.99 ± 0.02 minutes (data not shown). Confirmatory electrophoresis was performed in all cases and was concordant with the original results predicted by HPLC for both Hb C and Hb O-Arab trait. The difference in the RT for these 2 variants was further demonstrated by mixing equal amounts of hemolysates from patients with Hb C trait and Hb O-Arab trait. The HPLC chromatograph clearly showed 2 peaks in the C window at the expected RT (Figure 3).

We also reviewed the percentage of the hemoglobin fraction in the C-window area. There was no statistically significant difference in the amount of the Hb C and Hb O-Arab variants (37.57 ± 3.22% and 34.15 ± 2.84%, respectively; P = .11; data not shown). The amount of the variant, therefore, does not contribute to the differential diagnosis between these 2 hemoglobin variants.

In our case of Hb SO-Arab, the pattern of peaks and RT by HPLC for the Hb O-Arab variant was exactly as seen in the specimens of Hb AO-Arab. A small peak preceding the major Hb S peak was present (Figure 4), but it was not integrated by the Clinical Data Management software. Its perceived RT, however, appeared to be similar to those in the D window. Based on the C-RT, the preliminary diagnosis of Hb SO-Arab was made. Hemoglobin electrophoresis was performed according to the manufacturer's recommendations. The confirmatory electrophoresis result, however, was misinterpreted by the laboratory staff as Hb SC-Harlem due to the poor separation of Hb S and Hb O-Arab seen on acid electrophoresis. The electrophoretic mobility of Hb C-Harlem is identical to Hb C on alkaline electrophoresis and identical to Hb S on acid electrophoresis. The electrophoretic mobility of Hb O-Arab, on the other hand, is identical to Hb C on alkaline electrophoresis and is similar to, but slightly behind, Hb S on acid electrophoresis. After further dilution of the specimen, the electrophoretic pattern of the acid electrophoresis was distinctive for the presence of Hb S and Hb O-Arab.

The CAP standards are designed to improve the quality of clinical laboratory services by examining preanalytic, analytic, and postanalytic aspects of quality improvement in the laboratory. With regard to the determination of hemoglobin variants, the standards are written to assure that clinically significant variants are detected and properly confirmed.

Standard HEM.38100 Phase II asks the following question:

Are all samples with hemoglobin variants migrating in “non-A, non-S” positions on alkaline electrophoresis, isoelectric focusing, or HPLC further defined with electrophoresis at acid pH or other acceptable methods where clinically and technically appropriate?12 

The importance of confirmation is crucial when alkaline and acid electrophoresis is used for the diagnosis of hemoglobin variants. Some of the clinically significant sickling disorders (Hb SS, Hb SD-Los Angeles, Hb SG-Philadelphia, Hb S-Lepore, Hb SC, Hb SE, and Hb SO-Arab) cannot be differentiated by a single hemoglobin electrophoretic technique. Although these are all described as sickling disorders, clinically these combinations of hemoglobin variants express varying manifestations and degrees of severity.13–17 Alkaline electrophoresis permits the provisional identification of Hb A, Hb F, Hb S/D-Los Angeles/G-Philadelphia/Lepore, Hb C/A2/E/O-Arab, and a number of less common hemoglobin variants. When a hemoglobin variant is detected by alkaline electrophoresis, it is necessary to confirm by an alternative technique, most commonly acid electrophoresis. Both techniques distinguish Hb S from Hb D-Los Angeles, Hb G-Philadelphia, and Hb Lepore, but do not distinguish between the latter 3 variants. Acid electrophoresis will distinguish Hb C from Hb A2, Hb E, and Hb O-Arab, and will help distinguish the latter 3 hemoglobin variants from each other.2 

In this study, we examined HPLC patterns for the 2 β-globin variants Hb C and Hb O-Arab to determine if these hemoglobin variants can be reliably and accurately detected and confirmed without the need to perform time-consuming, labor-intensive, and costly hemoglobin electrophoreses.

We demonstrated the statistically significant difference in RT using the Bio-Rad Variant II HPLC System for the peak in the C window for Hb C (5.18 ± 0.01 minutes) and Hb O-Arab (4.91 ± 0.01 minutes). We also observed the presence of a minor peak in the D window in all Hb O-Arab samples with a constant RT (3.99 ± 0.02 minutes) on chromatographs. Review of example chromatographs for Hb O-Arab in the published literature shows the presence of this minor peak.2,5 It is assumed that this minor peak is glycosylated Hb O-Arab or possibly a degraded product of this hemoglobin. Whichever explanation is correct, it is consistently present in the samples seen in this laboratory. The presence of such minor peaks can be reliable indicators of the variant or the type of variant present. For example, α-globin variants can be reliably predicted by the presence of an α2variantδ2 minor peak following the corresponding α-variant, just as Hb A22Aδ2) follows Hb A. This α2variantδ2 minor peak can sometimes be so minute that it is easily missed on examination of electrophoresis gels.5 

In addition, we had an important and challenging experience in the diagnosis of Hb SO-Arab. The HPLC chromatograph clearly indicated the presence of Hb S and Hb O-Arab in this specimen, based on the respective RTs. Although the D window for this specimen was not integrated by the computer software, examination of the chromatograph indicated a small peak at a perceived RT similar to that seen in Hb AO-Arab specimens. The presence of the D window was not identified by the system, probably due to the Hb S being eluted as a large and wide peak in the S window, causing consolidation of these 2 windows.

Although hemoglobin electrophoresis was performed according to the manufacturer's recommendations, the mobility of the hemoglobin bands was consistent with Hb SC-Harlem due to poor separation of Hb S and Hb O-Arab on alkaline electrophoresis. Zimmerman et al16 reported a similar incident. This experience further supports our conclusion that examination of the C-RT and the presence of the minor peak in the D window are highly reliable for the identification and differentiation of Hb SC and Hb SO-Arab, which is important for clinical management.

Ou et al4,5 demonstrated the complete separation of Hb S, Hb D-Los Angeles, and Hb G-Philadelphia from each other using a cation-exchange HPLC method, while traditional electrophoretic methods, even in combination, were not capable of completely separating these commonly encountered hemoglobin variants. In this report, similarly, we demonstrated complete separation of Hb C, Hb O-Arab, and Hb E (RT = 3.69 ± 0.07 minutes; data not shown) from each other using the same methodology, while traditional electrophoretic methods, even in combination, are not capable of completely separating these variants. With such excellent resolution, it raises questions about the validity of the CAP checklist question HEM.38100 Phase II.

The authors thank Margaret Karpatkin, MD, and James Donnelly, PhD, for their helpful advice in the preparation of this manuscript. We also thank the staff, especially Joan Hadzi-Nesic, of the Special Hematology Laboratory at Bellevue Hospital Center, New York, NY, for their technical expertise.

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The authors have no relevant financial interest in the products or companies described in this article.

Author notes

Reprints: Michael Nardi, MS, Department of Pediatrics, NYU School of Medicine, 550 First Ave, New York, NY 10016 ([email protected])