Expression of Hemoglobin Variant Migration by Capillary Electrophoresis Relative to Hemoglobin A 2. Improves Precision

Transcription

1 Hematopathology / Hb Variant Migration by CE Relative to Hb Expression of Hemoglobin Variant Migration by Capillary Electrophoresis Relative to Hemoglobin Improves Precision David F. Keren, MD,, Renee Shalhoub, MT(SCP), Ronald Gulbranson, MT(SCP), and Deborah Hedstrom, MT(SCP) Key Words: Hemoglobinopathy; Capillary electrophoresis; Hemoglobin S; Hemoglobin C; Hemoglobin ; Hemoglobin G; Hemoglobin D DOI:.39/JCPOF8VJJOPSVF bstract We report the precision of the mean migration position of hemoglobin (Hb)S, HbC, (Philadelphia), and (Los ngeles) in 93 samples of whole blood assayed by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). y expressing the migration of Hb variants by CE relative to that of Hb in the same sample, there was a significant improvement in the coefficient of variation for each variant studied. The potential usefulness of expressing Hb variants relative to that of Hb was evaluated by comparing the separation of closely migrating Hbs. When expressed by their initial migrations on CE, 5 of the 3 cases of and overlapped. However, when the migrations of these variants were expressed relative to the Hb in the same sample, the cases of separated completely from the 9 cases of. These findings suggest that expressing Hb variants relative to an internal standard, such as Hb, may be of value for establishing a library of variant Hbs evaluated by CE. Identification of structural hemoglobin (Hb) variants and thalassemias traditionally has relied on alkaline and acid gel electrophoresis and, more recently, high-performance liquid chromatography (HPLC). -8 These methods detect structural variants by electrophoretic migration or elution patterns. Precision of measurement of Hb is needed to detect β-thalassemia and can be helpful in cases of α-thalassemia and iron deficiencies. 7,8 Capillary electrophoresis (CE) has been shown to be a reliable alternative to HPLC and is one that provides a more user-friendly interpretive format. 9-7 ll 3 methods readily separate the most common variants such as HbS, HbC, and HbE. However, some variants, such as and, are more difficult to separate from each other because they have identical migration on gels, overlapping elution times by HPLC, and similar migration by CE. While and are readily distinguished owing to their being α and β variants, respectively, the ability to separate such closely eluting or migrating proteins can be used as a test of the precision of each method. To improve precision by HPLC, the Primus Ultra system (Trinity iotech, Kansas City, MO) elution times are expressed relative to the elution time of a standard (HbF, Hb, HbS, or HbC) that was run through the same column the same day. This approach presents data for the variant as an arithmetic ratio to the standard with the nearest elution time. Use of relative ratios by HPLC improves separation of closely migrating Hbs.,9 In this report, we describe our findings for the precision of the migration position for common Hb variants evaluated by CE. We have demonstrated that expressing the migration m J Clin Pathol ;37:- Downloaded from DOI:.39/JCPOF8VJJOPSVF

2 Hematopathology / Original rticle position relative to Hb improves the ability of CE to distinguish between closely migrating variant Hbs ( and ). Materials and Methods Specimens This was a retrospective study on 93 variant-containing clinical samples of whole blood collected in EDT that were submitted for routine evaluation of hemoglobinopathies. They were stored at C to 8 C and processed by HPLC and CE within 3 days following venipuncture. ll samples used in the study needed to contain Hb. We have included all cases that contained or more of the following Hbs: HbS, HbC,, or. Each sample was examined by HPLC and CE. Our study was conducted in accordance with a protocol (R-- 73) approved by the institutional review board of St Joseph Mercy Hospital, nn rbor, MI. High-Performance Liquid Chromatography HPLC was used to establish independently the initial identification of the Hb variant. It was performed using the Primus Ultra Resolution method, a cation exchange column. This method relates the retention time of unknown Hbs to that of a calibrating standard containing Hbs: HbF, Hb, HbS, and HbC. Whole blood specimens collected in EDT were lysed with the hemolyzing reagent (provided by the manufacturer) for injection into the HPLC column. Elution of adsorbed Hbs used a gradient formed by mobile phases of is-tris and mmol of potassium cyanide with different ph values and ionic strengths, as previously described. 3,5, Capillary Electrophoresis CE was performed using the Sebia Capillarys system (Sebia, Norcross, G). Manufacturer guidelines were followed in performing the analysis, as previously described. The Sebia Capillarys records Hb migration on the x-axis from to 3. On each sample, when present, Hb is standardized to migrate at position 5. When a sample lacks Hb, the instrument uses the archived information from the most recent samples passing through that column to estimate the position of Hb in order to generate the migration point. While this provides reliable quantitative data for the variant, in the absence of Hb, the migration position is imprecise. When Hb is not present in a sample, the manufacturer recommends that the sample be diluted : with a normal sample to obtain a better migration position of the Hbs present in that sample. In addition to determining the migration position, the presence of Hb generates a grid that divides the migration into zones. The manufacturer provides a table delineating the variants it has determined to be present within each zone. We recorded the positions of the peak of Hb and the variant Hb in each sample. This approach allowed us to express the actual migration of each variant and the migration of each variant relative to that of Hb. Gel Electrophoresis Selected variants were analyzed on alkaline and acid agarose gel electrophoresis using the Sebia Hydrasys method (Sebia) according to manufacturer s guidelines. Each sample was run with controls for Hb, HbS, HbF, and HbC. Statistics The Student paired t test was used to determine the significance of difference of coefficients of variation (CVs) between migrations measured directly compared with migrations expressed relative to that of Hb in the same sample. Results Use of Migration Position by CE and Elution Time by HPLC to Evaluate Variant Hbs y CE, the mean migration position (x-axis value) and SD for Hb, HbS, HbC,, and were quite reproducible with CVs that ranged from.79% to.% Table. The most closely migrating variants in this study were and. oth migrated in zone, with mean migrations of 5 and 8, respectively Figure. Despite the tight SDs, migration data alone were not able to consistently separate the cases of from the 9 cases of Figure. Indeed, 5 of the 3 samples of and overlapped. The absolute elution time recorded by HPLC was used to compare separation of and cases with that of CE. Similar to CE, by HPLC 3 of the 3 cases overlapped when the absolute elution time was measured Figure. Variant to Hb Ratio by CE and Relative Elution Time by HPLC to Evaluate Variant Hbs To evaluate whether an internal standard would improve precision, the migration positions by CE were calculated as an arithmetic ratio relative to that of Hb in the same Table Precision of Variant Location by Migration Position Hb HbS HbC (n = 93) (n = 9) (n = 5) (n = ) (n = 9) Mean * SD Coefficient of variation (%) Hb, hemoglobin. * Mean migration units (range, -3). Downloaded from m J Clin Pathol ;37:- DOI:.39/JCPOF8VJJOPSVF

3 Keren et al / Hb Variant Migration by CE Relative to Hb Hb Hb Hb Hb Figure Capillary electrophoresis pattern with zones above and migration numbers below the electrophoretogram., Hemoglobin (Hb)G migrates in zone and in zone. Fractions are as follows: Hb,.9%;, 33.%; Hb,.%; and,.7%., migrates in zone. Fractions are as follows: Hb, 55.%;,.5%; and Hb, 3.%. Table Precision of Variant Location Relative to Hb HbS/Hb HbC/Hb /Hb /Hb (n = 9) (n = 5) (n = ) (n = 9) Mean SD Coefficient of variation (%) Hb, hemoglobin. sample. The results shown in Table indicate that precision improved for all variants (P <.), as shown by a decrease in the CVs. When the ratio (variant/hb ) was used for and, there was complete separation of all 3 cases Figure 3. The counterpart of expressing the CE migration point relative to that of Hb is the expression of the HPLC elution time relative to that of HbS recommended by the manufacturer. This arithmetic ratio improved the separation Migration Position Elution Time (min) 5.8 Figure, Capillary electrophoresis migration position for cases of hemoglobin (Hb)G and 9 cases of., Highperformance liquid chromatography elution time for cases of and 9 cases of. m J Clin Pathol ;37:- Downloaded from DOI:.39/JCPOF8VJJOPSVF

4 Hematopathology / Original rticle Variant Migration Position/Hb Migration Position Variant Elution Time/HbS Elution Time.98 Figure 3, Capillary electrophoresis variant migration relative to hemoglobin (Hb) migration for cases of and 9 cases of., High-performance liquid chromatography elution time relative to HbS elution time for cases of and 9 cases of. of the and cases by HPLC Figure 3. Only cases still overlapped. Discussion HPLC is a reliable, rapid, precise procedure that has the advantage of a well-characterized library of Hb variants. -8,8 In people without the common variants HbS and HbE, HPLC provides an accurate measure of Hb. However, HPLC has the disadvantage of providing a complex elution pattern. This is due to several elution peaks from breakdown and posttranslational products of Hbs.,3 Some of these products, such as those from HbS, reside in the Hb peak, thereby decreasing the percentage of HbS measured in heterozygotes.,8 Other HbS products comigrate and increase measured Hb. 8,3,8,9 In addition, on the Primus Ultra HPLC, common variants, including HbE,, and, comigrate with Hb, eliminating the ability to accurately measure Hb in their presence. y HPLC, the closely migrating Hbs such as and can be distinguished from each other by virtue of the unique presence of the product of the variant α chain with δ ( ). However, the elution times of and themselves overlap in a few cases, even when expressed relative to that of HbS. 7 CE has been used to evaluate hemoglobinopathies for variant Hbs for years. - With early CE techniques, it was demonstrated that CE could provide an accurate measurement of Hb and was superior to microcolumn techniques for measuring Hb in patients who had HbS., Yet, inhouse methods and early commercial ventures were deemed unlikely to compete with the rapid, accurate HPLC methods available at that time. Developed in the past decade, the Sebia Capillarys has a superior throughput compared with HPLC systems, provides a more straightforward pattern for interpretation, and does not have the need to account for glycated and breakdown products when measuring the most common variant Hbs.,3,7,7 Previously, we reported that the percentage of HbS in heterozygotes is higher when measured by CE than by HPLC because of the inability of the latter to account for fractions of HbS that coelute with Hb and Hb. 3 Furthermore, Hb is not measurable by the Primus Ultra HPLC method in the presence of the common variants,, or HbE, while CE provides an accurate determination of Hb in these situations. 3,5,7 The present study sought to determine the precision of CE in the identification of relatively common and closely migrating Hb variants. We found that the CVs of HbS, HbC,, and hover around % from the direct migration data. However, despite these tight CVs, we were not able to separate and, finding that 5 of the 3 cases overlapped. These overlapping variants are used here to challenge the precision of CE and HPLC in separating closely migrating and relatively common variants. The Primus Ultra HPLC technique expresses the elution of Hbs relative to of standards (HbF, Hb, HbS, or HbC) to improve precision over expressing the result as its absolute elution time. 3,5 On the current 3 cases of and, the direct elution time was able to separate all but of the cases. To see if comparison with a standard would improve precision on CE, we expressed the migration of relatively Downloaded from m J Clin Pathol ;37:- 3 3 DOI:.39/JCPOF8VJJOPSVF 3

5 Keren et al / Hb Variant Migration by CE Relative to Hb common Hb variants relative to Hb as an internal standard. We found that this improved the precision of HbS, HbC,, and as measured by their CVs. Furthermore, when this arithmetic ratio was used for the closely adjacent Hbs, it resulted in complete separation of all 3 cases of and. These studies confirm the original observation by Louahabi et al demonstrating that precision of several common Hb variants was improved by expressing the migration relative to that of Hb in the same specimen. The results of the present study suggest that expression of other variants relative to the migration of Hb may be helpful in creating a more precise library of variants for CE. However, we note that Hb migrates relatively near the variants studied. It may be that variants with a more anodal migration may require a different internal standard to achieve a similar result. From Department of Pathology, The University of Michigan, nn rbor; and Warde Medical Laboratory, nn rbor. ddress reprint requests to Dr Keren: Dept of Pathology, The University of Michigan Hospital and Health Systems, 5 Medical Science, 3 Catherine, SPC 5, nn rbor, MI References. Wajcman H, Prehu C, ardakdjian-michau J, et al. bnormal hemoglobins: laboratory methods. Hemoglobin. ;5:9-8.. Riou J, Godart C, Hurtrel D, et al. Cation-exchange HPLC evaluated for presumptive identification of hemoglobin variants. Clin Chem. 997;3: Ou CN, Rognerud CL. Rapid analysis of hemoglobin variants by cation-exchange HPLC. Clin Chem. 993;39:8-8.. Fucharoen S, Winichagoon P, Wisedpanichkij R, et al. Prenatal and postnatal diagnosis of thalassemias and hemoglobinopathies by HPLC. Clin Chem. 998;: Ou CN, Rognerud CL. Diagnosis of hemoglobinopathies: electrophoresis vs HPLC. Clin Chim cta. ;33:8-9.. Gosselin RC, Carlin C, Dwyre DM. 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