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Unexpected hemoglobin [A.sub.1c] results.

CASE

A 52-year-old woman with a medical history of hepatitis B, hyperlipidemia, hypertension, anemia, and depression presented to the internal medicine clinic for a routine visit. Laboratory tests 3 months previously had revealed an impaired fasting glucose concentration of 5.9 mmol/L (106 mg/dL) [reference interval, 3.9-5.6 mmol/L (70-100 mg/dL)]. Therefore, a hemoglobin (Hb) [2] [A.sub.1c] analysis was performed. The initial Hb [A.sub.1c] evaluation by cation-exchange HPLC (CE-HPLC) (Hb [A.sub.1c] Program on the VARIANT II TURBO Link System; Bio-Rad Laboratories) showed an Hb [A.sub.1c] value of 115.8% (reference interval, 4.0%-6.0%) (Fig. 1).

In an effort to determine if the unusual Hb [A.sub.1c] result was due to potential hemoglobinopathies, we performed an Hb variant analysis with the Bio-Rad VARIANT CE-HPLC [beta]-Thalassemia Short Program. The analysis revealed the absence of Hb A and the presence of sickle cell Hb (Hb S) (37.4%), along with normal Hb [A.sub.2] (3.2%) and Hb F (<1.0%) (Fig. 2). Also evident was another large peak (53.0%) that eluted earlier than Hb A, which we called P2. This study suggested the presence of an Hb variant with a chromatographic retention time virtually identical to that of Hb [A.sub.1c], in addition to Hb S (Figs. 1 and 2). A subsequent Hb electrophoretic analysis at pH 6.0 (QuickGel Acid; Helena Laboratories) identified Hb S and another abnormal band with a mobility similar to Hb F (not shown).

PATIENT FOLLOW-UP

To identify the Hb variants, we investigated DNA sequences corresponding to the patient's [beta]-globin genes. This analysis identified a substitution at codon 6 [GAG to GTG (Glu to Val)] on one allele, corresponding to Hb S, and a substitution at codon 1 [GTG to GCG (Val to Ala)] on the other allele, corresponding to Hb Raleigh.

The presence of these hemoglobinopathies suggested that the spurious Hb [A.sub.1c] result obtained with the CE-HPLC method was due to the elution of Hb Raleigh, which has a retention time similar to that of Hb [A.sub.1c]. We evaluated the Hb [A.sub.1c] result with a turbidimetric inhibition immunoassay (Dimension[R] Clinical Chemistry System; Siemens) and obtained an Hb [A.sub.1c] value of 4.1%, which was not consistent with the impaired fasting glucose concentration of 5.9 mmol/L (106 mg/dL).

DISCUSSION

Hb [A.sub.1c] is produced by nonenzymatic addition of a glucose molecule to the N-terminal valine residue on the chain of Hb A. Glycation of the N-terminal residue changes its structure and decreases the positive charge of Hb A. The American Diabetes Association has recommended Hb [A.sub.1c] as an indicator of long-term glycemic control in patients with diabetes mellitus and for the screening and diagnosis of diabetes mellitus; an Hb [A.sub.1c] cutoff value of 6.5% has been suggested (1).

Methods of Hb [A.sub.1c] analysis can be divided into 2 categories: methods based on molecular charge and those based on structure. The former category includes CE-HPLC and electrophoresis, and the latter includes immunoassays, boronate affinity chromatography, and mass spectrometry (2). In CE-HPLC and electrophoresis assays, Hb [A.sub.1c] can be separated from Hb A because glycation of the N-terminal valine decreases the positive charge. Therefore, charge-based methods may be affected by posttranslational modifications (e.g., carbamylation and acetylation) (3) or by Hb mutations (2) that alter the charge. Common Hb traits such as Hb AS and Hb AC, however, do not interfere with the CE-HPLC Hb [A.sub.1c] assay used in this study (Bio-Rad VARIANT II TURBO) (4). Immunoassays use antibodies that target N-terminal glycated amino acids on the [beta] chain to quantify Hb [A.sub.1c], and the Hb [A.sub.1c] percentage is calculated from the Hb [A.sub.1c] and Hb concentrations (2). Thus, any factor that prevents glycation or any mutation in the epitope of the N-terminal amino acids that affects antibody recognition will produce erroneous results. Additionally, patients with increased Hb F (>10%) will have a falsely low Hb [A.sub.1c] value by immunoassay because the [gamma] chain shares only 4 of the first 10 amino acids with the [beta] chain of Hb A and has little to no immunoreactivity with most antibodies used in Hb [A.sub.1c] assays (2). In the boronate affinity chromatographic assay, boronic acid reacts with the cis diol groups created by glycation, thereby allowing glycohemoglobins such as Hb [A.sub.1c] to be separated from Hb A (2). On the other hand, Hb variants with excessive glycation, such as Hb Himeji, can interfere with boronate affinity chromatography (5). Mass spectrometric assay, an IFCC reference method, specifically measures the glycated N-terminal valine of the Hb A [beta] chain (6), but the prohibitive cost of a mass spectrometer and the complicated nature of its installation and operation are likely to preclude its use in most clinical laboratories in the near future (2).

The method initially used to analyze the patient's Hb [A.sub.1c] was a CE-HPLC assay. A CE-HPLC Hb variants analysis (Fig. 2), acid gel Hb electrophoresis, and DNA sequencing of i-globin genes demonstrated that the falsely increased Hb [A.sub.1c] value was due to Hb Raleigh, which eluted in the Hb [A.sub.1c] window (Fig. 1). Hb Raleigh is unique in that a mutation (T to C) at the second base of the codon encoding the first amino acid of the [beta] chain changes the N-terminal valine to an alanine residue. This substitution would not necessarily induce any change in Hb A charge except that N-terminal alanines are immediately acetylated to acetylalanines soon after translation (7, 8). This acetylation decreases the positive charge to one similar to Hb [A.sub.1c]. Therefore, the retention times of Hb [A.sub.1c] and Hb Raleigh are virtually identical; the elution peaks of these 2 Hbs fall in the same window in the chromatogram (Fig. 1). Thus, the presence of Hb Raleigh produces a falsely increased Hb [A.sub.1c] value when the latter is assessed by CE-HPLC. Chen et al. reported a case of falsely increased Hb [A.sub.1c] due to Hb Raleigh in the Bio-Rad VARIANT CE-HPLC Hb [A.sub.1c] assay (9). In their case, the patient was heterozygous for Hb A and Hb Raleigh, and the falsely increased Hb [A.sub.1c] value of 46% included a small fraction of real Hb [A.sub.1c]. In our case, the patient was heterozygous for Hb S, in which the N-terminal valine of the [beta] chain was glycated as Hb [S.sub.1c], and Hb Raleigh, in which the N-terminal acetylalanine of the [beta] chain could not be glycated. Therefore, Hb [A.sub.1c] did not exist in this patient. The spurious Hb [A.sub.1c] value (115.8%) primarily represented Hb Raleigh. Other Hb variants that produce similar interferences include Hb Graz, Hb Sherwood Forest, Hb South Florida, Hb Niigata, and carbamylated Hb A (2).

The turbidimetric inhibition immunoassay produced an Hb [A.sub.1c] value of 4.1%, which represents the Hb [S.sub.1c] percentage, an equivalent of the Hb [A.sub.1c] percentage. Hb [S.sub.1c] was underestimated because of the heterozygosity with Hb Raleigh. Hb S is characterized by a substitution at codon 6 (GAG to GTG) that leads to the replacement ofa glutamic acid residue in the [beta] chain by a valine residue. Although this substitution is at the sixth amino acid residue, the antibody used in the immunoassay still recognizes Hb [S.sub.1c]. The acetylated alanine at the N terminus of the Hb Raleigh [beta] chain, however, cannot be glycated and therefore prevents reaction with the antibody in the immunoassay. When the Hb [A.sub.1c] percentage is calculated, the numerator includes Hb [S.sub.1c] only, but the denominator consists of both Hb S and Hb Raleigh, as well as small amounts of Hb [A.sub.2] and Hb F. Therefore, the Hb [A.sub.1c] percentage for this patient (as measured by immunoassay) was underestimated by approximately 50%. This issue with the Hb [A.sub.1c] immunoassay has been discussed by Chen et al. (9) and Jain et al. (10). Similarly, boronate affinity chromatography assays also underestimate Hb [A.sub.1c] for patients with Hb Raleigh, because the N-terminal acetylalanine in the Hb Raleigh [beta] chain cannot be glycated. Hb Raleigh has a decreased affinity for boronate in the column (9), although the column can still interact with other glycated residues. For these patients, Chen et al. (9) and Jain et al. (10) recommended the use of fructosamine (9), multiple measurements of capillary glucose throughout the day, or continuous glucose monitoring of the glycemia (10). We did not perform these tests for our patient because of insufficient sample. For patients with the Hb Raleigh trait, the IFCC tandem mass spectrometric assay maybe the best method, because it measures Hb A and Hb [A.sub.1c] specifically. The IFCC reference method, however, would be useless for our patient unless a mass spectrometric assay were available to measure Hb S and Hb [S.sub.1c], because she had no Hb A or Hb [A.sub.1c].

The present case is an example of how Hb variants can interfere with Hb [A.sub.1c] assays to produce spurious results. When an aberrant Hb [A.sub.1c] value is generated and/or the value does not match the clinical impression, the possibility of interference by Hb variants should be considered, and the interpretation of Hb [A.sub.1c] values should be based on the patient's medical history and other laboratory results. Efforts should be made to identify the Hb variant, and alternative Hb [A.sub.1c] methods free of the interference should be selected to monitor the patient's glycemic control.

QUESTIONS TO CONSIDER

1. What are the various types of methods used for measuring Hb [A.sub.1c]?

2. How do Hb variants interfere with each of these Hb [A.sub.1c] methods?

3. What actions should be taken when a spurious Hb [A.sub.1c] result is present?

POINTS TO REMEMBER

* Hb [A.sub.1c] assays can be divided into methods that use molecular charge (CE-HPLC and electrophoresis) and methods that use molecular structure (immunoassays, boronate affinity chromatography, and mass spectrometry).

* Hb variants (or their glycated forms) may interfere with Hb [A.sub.1c] assays based on CE-HPLC and electrophoresis by coeluting/comigrating with Hb A and/or Hb [A.sub.1c]. If an Hb variant's amino acid substitution occurs within the N-terminal [beta]-chain epitope recognized by an anti--Hb [A.sub.1c] antibody or if a patient has a greatly increased Hb F percentage, Hb [A.sub.1c] immunoassays will be affected. Hb variants with decreased or increased glycation will interfere with boronate affinity chromatography. Mass spectrometry is the IFCC reference method and generally appears to be unaffected by the presence of genetic or chemical modifications to the Hb A molecule.

* When a spurious Hb [A.sub.1c] result is obtained, the possibility of interference by Hb variants should be considered, and the interpretation of Hb [A.sub.1c] results should be based on the patient's medical history and other laboratory results. In addition, efforts should be made to identify the Hb variant, and alternative Hb [A.sub.1c] methods that are free of the interference should be used. If there is no appropriate method for a particular Hb variant, fructosamine, daily multiple testing of capillary glucose, or continuous glucose monitoring may be used to monitor glycemic control. These alternative tests may also be used for patients who have an altered erythrocyte life span and changes in the degree of glycation. Hb [A.sub.1c] testing cannot be used for these individuals.

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 or 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.) American Diabetes Association. Standards of medical care in diabetes--2010. Diabetes Care 2010;33(Suppl 1):S11-61.

(2.) Bry L, Chen PC, Sacks DB. Effects of hemoglobin variants and chemically modified derivatives on assays for glycohemoglobin. Clin Chem 2001;47: 153-63.

(3.) Weykamp CW, Penders TJ, Siebelder CW, Muskiet FA, van der Slik W. Interference of carbamylated and acetylated hemoglobins in assays of glycohemoglobin by HPLC, electrophoresis, affinity chromatography, and enzyme immunoassay. Clin Chem 1993;39:138-42.

(4.) Mongia SK, Little RR, Rohlfing CL, Hanson S, Roberts RF, Owen WE, et al. Effects of hemoglobin C and S traits on fourteen commercial glycated hemoglobin assays. Am J Clin Pathol 2008;130:136-40.

(5.) Ohba Y, Miyaji T, Murakami M, Kadowaki S, Fujita T, Oimomi M, et al. Hb Himeji or fi 140 (H18) Ala--Asp. A slightly unstable hemoglobin with increased fi N-terminal glycation. Hemoglobin 1986;10:109-25.

(6.) Jeppsson JO, Kobold U, Barr J, Finke A, Hoelzel W, HoshinoT, et al. Approved IFCC reference method for the measurement of HbA1c in human blood. Clin Chem Lab Med 2002;40:78-89.

(7.) Marchis-Mouren G, Lipmann F. On the mechanism of acetylation of fetal and chicken hemoglobins. Proc Natl Acad Sci U S A 1965;53:1147-54.

(8.) Moo-Penn WF, Bechtel KC, Schmidt RM, Johnson MH, Jue DL, Schmidt DE Jr, et al. Hemoglobin Raleigh (beta 1 valine replaced by acetylalanine). Structural and functional characterization. Biochemistry 1977;16:4872-9.

(9.) Chen D, Crimmins DL, Hsu FF, Lindberg FP, Scott MG. Hemoglobin Raleigh as the cause of a falsely increased hemoglobin A1C in an automated ion-exchange HPLC method. Clin Chem 1998;44(Pt 1):1296-301.

(10.) Jain N, Kesimer M, Hoyer JD, Calikoglu AS. Hemoglobin Raleigh results in factitiously low hemoglobin A1c when evaluated via immunoassay analyzer. J Diabetes Complications 2011;25:14-8.

Commentary

Randie R. Little (1,2) *

The 4 most common hemoglobin (Hb) variants worldwide and in the US are Hb S, Hb E, Hb C, and Hb D. In addition, there are many other less common variants. These variants frequently go unrecognized in the heterozygous form (e.g., the Hb S trait) because they are usually clinically silent, but such a variant might still be clinically important if its presence can lead to erroneous test results. A large number of patients with diabetes have clinically silent Hb variants that may interfere with Hb [A.sub.1c] measurement by some methods. Most of the commonly used Hb [A.sub.1c] methods have been evaluated with the 4 most common Hb variant traits (see http://www.NGSP.org), and although one or more of these variants interfere with some methods, others do not. When an Hb variant causes a change in the erythrocyte life span or actually produces a change in Hb glycation, Hb [A.sub.1c] measurement may not give clinically useful results, regardless of the assay methodology. In the case of the patient described by Sofronescu et al., there were actually 2 Hb variants present, Hb S and Hb Raleigh. Because there is no Hb A, one cannot directly measure Hb [A.sub.1c]. One could consider using boronate affinity to measure the total glycated Hb, which would include glycated Hb S and Hb Raleigh, except that Hb Raleigh is glycated to much less of an extent (compared with Hb A) because of the specific amino acid substitution. A small number of cases require other measures of glycemic control, such as fructosamine or continuous glucose monitoring, instead of Hb [A.sub.1c] measurement, but such cases are rare. For the vast majority of patients with diabetes (and for those being tested for the presence of diabetes), Hb [A.sub.1c] measurement is the best way to assess long-term glycemic control, as long as an appropriate methodology is used. It is the responsibility of all manufacturers to clearly state their methods' limitations, and it is up to each laboratory to know--and to convey to their clinicians when appropriate--these limitations for the methods they use to measure Hb [A.sub.1c].

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 or 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) Departments of Pathology & Anatomical Sciences and (2) Child Health, University of Missouri School of Medicine, Columbia, MO.

* Address correspondence to the author at: Diabetes Diagnostic Laboratory M767, Department of Pathology & Anatomical Sciences, One Hospital Dr., Columbia, MO 65212. Fax 573-884-8823; e-mail littler@health.missouri.edu.

Received October 12, 2010; accepted October 20, 2010.

DOI: 10.1373/clinchem.2010.157610

Commentary

Simon J. Fisher *

Hemoglobin [A.sub.1c] (Hb [A.sub.1c]) is the standard test for assessing glycemic control over the immediately previous 3-month period (life span of a red blood cell). An increased Hb [A.sub.1c] value (i.e., [greater than or equal to] 6.5%) has also recently been approved by the American Diabetes Association as diagnostic for diabetes. In the case presented, a diagnostic Hb [A.sub.1c] test was ordered to distinguish the prediabetic state of impaired fasting glucose from overt diabetes. The commonly used cation-exchange HPLC Hb [A.sub.1c] assay reported a spuriously high Hb [A.sub.1c] value of 115.8% (reference interval, 4%-6%), which alerted the healthcare team and prompted an investigation. The authors' data demonstrated that the patient's falsely increased Hb [A.sub.1c] value was consistent with the presence of a hemoglobin variant, Hb Raleigh, which falsely increased the chromatogram band usually attributable to Hb [A.sub.1c]. For people with hemoglobinopathies, some Hb [A.sub.1c] assays give falsely high (or low) readings that can lead to the overtreatment or undertreatment of diabetes, respectively (see the National Glycohemoglobin Standardization Program Web site http://www.ngsp.org/interf.asp). In addition to hemoglobinopathies, disorders of erythrocyte life span are also common situations in which Hb [A.sub.1c], although accurately measured with conventional assays, falselyreflects glycemic control. Rapid red blood cell turnover (i.e., hemolysis or blood loss) and a prolonged red blood cell life span (i.e., kidney diseases) alter the time available for glycation, and these processes can produce Hb [A.sub.1c] values that underestimate or overestimate, respectively, glycemic control. Healthcare providers should be vigilant in suspecting hemoglobinopathies or erythrocyte life span disorders when (a) the Hb [A.sub.1c] value is discordant with the patient's recorded serum glucose concentrations or clinical presentation, (b)an Hb [A.sub.1c] result is >15%,or(c) Hb [A.sub.1c] test results change radically between assays. For situations in which Hb [A.sub.1c] values are unreliable, measurements of fructosamine, frequent daily glucose testing, or continuous glucose-monitoring systems should be used to monitor glycemic control more accurately in people with diabetes.

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 or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: S.J. Fisher, Merck.

Research Funding: None declared.

Expert Testimony: None declared.

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.

Department of Internal Medicine, Division of Endocrinology, Metabolism and Lipid Research, Washington University, St. Louis, MO.

* Address correspondence to this author at: Washington University, 660 S. Euclid Ave., Campus Box 8127, St. Louis, MO 63110. E-mail sfisher22@wustl.edu.

Received November 5, 2010; accepted November 16, 2010.

DOI: 10.1373/clinchem.2010.157636

Alina-Gabriela Sofronescu, [1] Laurie M. Williams, [1] Dorinda M. Andrews, (1) and Yusheng Zhu [1] *

[1] Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC.

[2] Nonstandard abbreviations: Hb, hemoglobin; CE-HPLC, cation-exchange high-performance liquid chromatography; Hb S, sickle cell Hb.

* Address correspondence to this author at: Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Ave., MSC 908, Suite 309, Charleston, SC 29425. Fax 843-792-0424; e-mail zhuyu@musc.edu.

Received August 26, 2010; accepted October 28, 2010.

DOI: 10.1373/clinchem.2010.155804
Fig. 1. CE-HPLC chromatogram for Hb [A.sub.1c] analysis.

Hb S and an aberrant Hb [A.sub.1c] value of 115.8% represented
the predominant Hb peaks in the chromatogram.

Peak name Calibrated Retention Peak area
 area,% Area, % time, min

[A.sub.1a] -- 0.4 0.106 6826
[A.sub.1b] -- 4.6 0.213 76457
[A.sub.1b] 115.8 * -- 0.405 816032
P3 -- 1.3 0.770 21144
Variant -- 4.2 0.860 70124
 window
S window -- 40.2 0.920 665 722

* Value outside of expected range Total area: 1656306

Fig. 2. Chromatogram of Hb variants analysis with the CE-HPLC
[beta]-Thalassemia Short Program.

Hb S (37.4%), wild-type Hb [A.sub.2] (3.2%), and Hb F (<1.0%) were
identified, but Hb A was not detected. A large peak, which we
designated P2, was detected at 53.0%.

Analyte ID Percent Time, min Area

PI 1.2 0.75 33627
F 0.5 0.94 16262
P2 53.0 1.30 1542147
[A.sub.0] 2.7 2.28 79887
Unknown 1 1.6 2.50 47682
[A.sub.Z] 3.2 3.68 108007
S window 37.4 4.45 1088414
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Title Annotation:Clinical Case Study
Author:Sofronescu, Alina-Gabriela; Williams, Laurie M.; Andrews, Dorinda M.; Zhu, Yusheng
Publication:Clinical Chemistry
Date:Feb 1, 2011
Words:3745
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