Curt L. Rohlfing

Status of Hemoglobin A1c Measurement and Goals for Improvement: From Chaos to Order for Improving Diabetes Care

BACKGROUND The Diabetes Control and Complications Trial (DCCT) and United Kingdom Prospective Diabetes Study (UKPDS) established the importance of hemoglobin A(1c) (Hb A(1c)) as a predictor of outcome in patients with diabetes mellitus. In 1994, the American Diabetes Association began recommending specific Hb A(1c) targets, but lack of comparability among assays limited the ability of clinicians to use these targets. The National Glycohemoglobin Standardization Program (NGSP) was implemented in 1996 to standardize Hb A(1c) results to those of the DCCT/UKPDS. CONTENT The NGSP certifies manufacturers of Hb A(1c) methods as traceable to the DCCT. The certification criteria have been tightened over time and the NGSP has worked with the College of American Pathologists in tightening proficiency-testing requirements. As a result, variability of Hb A(1c) results among clinical laboratories has been considerably reduced. The IFCC has developed a reference system for Hb A(1c) that facilitates metrological traceability to a higher order. The NGSP maintains traceability to the IFCC network via ongoing sample comparisons. There has been controversy over whether to report Hb A(1c) results in IFCC or NGSP units, or as estimated average glucose. Individual countries are making this decision. SUMMARY Variability among Hb A(1c) results has been greatly reduced. Not all countries will report Hb A(1c) in the same units, but there are established equations that enable conversion between different units. Hb A(1c) is now recommended for diagnosing diabetes, further accentuating the need for optimal assay performance. The NGSP will continue efforts to improve Hb A(1c) testing to ensure that clinical needs are met.

The effect of elevated fetal hemoglobin on hemoglobin A1c results: five common hemoglobin A1c methods compared with the IFCC reference method.

Hemoglobin A1c (HbA1c) is an important indicator of risk for complications in patients with diabetes mellitus. Elevated fetal hemoglobin (HbF) levels have been reported to interfere with results of some HbA1c methods, but it has generally been assumed that HbA1c results from boronate-affinity methods are not affected by elevated HbF levels. None of the previous studies used the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method as the comparative HbA1c method. We, therefore, measured HbA1c in samples with normal and elevated HbF levels by several common assay methods and compared the results with those of the IFCC reference method.HbF levels of more than 20% artificially lowered HbA1c results from the Primus CLC 330/385 (Primus Diagnostics, Kansas City, MO), Siemens DCA2000 (Siemens Healthcare Diagnostics, Tarrytown, NY), and Tosoh 2.2+ (Tosoh Bioscience, South San Francisco, CA), but not the Bio-Rad Variant II (Bio-Rad Laboratories, Hercules, CA) and Tosoh G7. Physicians and laboratory professionals need to be aware of potential interference from elevated HbF levels that could affect HbA1c results, including those from boronate-affinity methods.

Standardization of C-Peptide Measurements

BACKGROUND C-peptide is a marker of insulin secretion in diabetic patients. We assessed within- and between-laboratory imprecision of C-peptide assays and determined whether serum calibrators with values assigned by mass spectrometry could be used to harmonize C-peptide results. METHODS We sent 40 different serum samples to 15 laboratories, which used 9 different routine C-peptide assay methods. We also sent matched plasma samples to another laboratory for C-peptide analysis with a reference mass spectrometry method. Each laboratory analyzed 8 of these samples in duplicate on each of 4 days to evaluate within- and between-day imprecision. The same 8 samples were also used to normalize the results for the remaining samples to the mass spectrometry reference method. RESULTS Within- and between-run CVs ranged from <2% to >10% and from <2% to >18%, respectively. Normalizing the results with serum samples significantly improved the comparability among laboratories and methods. After normalization, the differences among laboratories in mean response were no longer statistically significant (P = 0.24), with least-squares means of 0.93-1.02. CONCLUSIONS C-peptide results generated by different methods and laboratories do not always agree, especially at higher C-peptide concentrations. Within-laboratory imprecision also varied, with some methods giving much more consistent results than others. These data show that calibrating C-peptide measurement to a reference method can increase comparability between laboratories.

Effects of Hemoglobin C and S Traits on the Results of 14 Commercial Glycated Hemoglobin Assays

Glycated hemoglobin is widely used in the management of diabetes mellitus. At least 300,000 Americans with diabetes mellitus have the hemoglobin (Hb) C or S trait. The accuracy of HbA1c methods can be adversely affected by the presence of these traits. We evaluated the effects of HbC and HbS traits on the results of 14 commercial HbA1c methods that use boronate affinity, enzymatic, immunoassay, and ion exchange methods. Whole blood samples from people homozygous for HbA or heterozygous for HbC or HbS were analyzed for HbA1c. Results for each sample type were compared with those from the CLC 330 comparative method (Primus Diagnostics, Kansas City, MO). After correcting for calibration bias by comparing results from the homozygous HbA group, method bias attributable to the presence of HbC or HbS trait was evaluated with a clinically significant difference being more than 10% (ie, 0.6% at 6% HbA1c). One immunoassay method exhibited clinically significant differences owing to the presence of HbC and HbS traits.

Effects of hemoglobin C, D, E, and S traits on measurements of HbA1c by six methods.

Glycated hemoglobin is hemoglobin that has been irreversibly modified by addition of glucose through a non-enzymatic process and provides a weighted average of plasma glucose concentration over the erythrocyte lifespan. HbA1c is a specific glycated hemoglobin that is modified at the N-terminal valine of the Hb beta chains. HbA1c therefore provides a useful estimate of mean glycemia in patients with diabetes that has been shown to be directly related to risks for diabetes complications. Treatment goals for HbA1c have been established, and more recently the test has been recommended for use in diagnosing diabetes [1,2]. Therefore, accurate and precise measurement of HbA1c is extremely important. The most common hemoglobin variants worldwide are HbS, HbE, HbC and HbD traits. Previous studies have shown method-specific analytic interference with HbA1c results from these heterozygous hemoglobin variants. This study was approved by the University of Utah Institutional Review Board. Whole blood samples from individuals homozygous for HbA (n=42) and heterozygous for HbC, HbD, HbE, or HbS trait (n=23, n=41, n=76, n=40, respectively) were collected in EDTA tubes. Samples were frozen at −70 °C in small aliquots and shipped on dry ice to 4 sites for HbA1c analysis. Hemoglobin variants were identified by inspection of chromatograms obtained with a Bio-Rad Variant analyzer (Bio-Rad Laboratories, Hercules, CA) using the β Thal Short Program; samples with HbF >5% were excluded. Samples with HbA1c concentrations of 4–12% were included in this study. The evaluation included four ion-exchangeHPLCmethods: Variant II Turbo 2.0 (Bio-Rad Laboratories, Hercules, CA), ADAMS A1c HA-8180V (ARKRAY, Japan), and G7 and G8 (Tosoh Biosciences, San Francisco, CA), one boronate affinity method: In2it (Bio-Rad) and one capillary electrophoresis method: Capillarys2 Flex Piercing HbA1c (Sebia, Lisses, France). A boronate affinity HPLC method: ultra (Trinity Biotech, Kansas City, MO) was used as the comparative method since it has previously been shown to be unaffected by the presence of hemoglobin variants [3]. An overall test of coincidence of 2 least-squares linear regression lines was performed using SAS Software (SAS Institute, Cary NC) to determine whether the presence of each hemoglobin variant caused a statistically significant difference (Pb0.05) in results relative to the comparative method. Deming regression analysis was performed

Measurement of Hba1C in patients with chronic renal failure

BACKGROUND Carbamylated hemoglobin (carbHb) is reported to interfere with measurement and interpretation of HbA(1c) in diabetic patients with chronic renal failure (CRF). There is also concern that HbA1c may give low results in these patients due to shortened erythrocyte survival. METHODS We evaluated the effect of carbHb on HbA(1c) measurements and compared HbA(1c) with glycated albumin (GA) in patients with and without renal disease to test if CRF causes clinically significant bias in HbA(1c) results by using 11 assay methods. Subjects included those with and without renal failure and diabetes. Each subjects estimated glomerular filtration rate (eGFR) was used to determine the presence and degree of the renal disease. A multiple regression model was used to determine if the relationship between HbA(1c) results obtained from each test method and the comparative method was significantly (p<0.05) affected by eGFR. These methods were further evaluated for clinical significance by using the difference between the eGRF quartiles of >7% at 6 or 9% HbA(1c). The relationship between HbA(1c) and glycated albumin (GA) in patients with and without renal failure was also compared. RESULTS Some methods showed small but statistically significant effects of eGFR; none of these differences were clinically significant. If GA is assumed to better reflect glycemic control, then HbA(1c) was approximately 1.5% HbA(1c) lower in patients with renal failure. CONCLUSIONS Although most methods can measure HbA(1c) accurately in patients with renal failure, healthcare providers must interpret these test results cautiously in these patients due to the propensity for shortened erythrocyte survival in renal failure.

Point-of-Care Assays for Hemoglobin A1c: Is Performance Adequate?

To the Editor: The routine measurement of hemoglobin A1c (Hb A1c)1 has become an essential component of the standard of care for patients with diabetes and has been recommended by major clinical diabetes organizations. A small amount of evidence shows that having the Hb A1c result at the time of the doctors visit is beneficial (1). Recently, there has been discussion about whether the quality of point-of-care (POC) testing for Hb A1c is sufficient to meet clinical needs (2). The National Glycohemoglobin Standardization Program (NGSP) certifies methods annually at the manufacturer level, with only 1 lot of reagents being used at any point in time. Although College of American Pathologists proficiency testing provides an excellent snapshot of the performance of each method in the clinical laboratory, POC methods are CLIA waived; thus, users are not required to participate in proficiency testing. Inadequate performance of some of these methods in the hands of experienced users (2) has raised concerns about the ability of these methods to perform well enough for diabetes monitoring. The manufacturer of the A1cNow device (Bayer HealthCare) noted (3) that the use of EDTA-containing blood in …

Effects of hemoglobin C, D, E and S traits on measurements of hemoglobin A1c by twelve methods

BACKGROUND Hemoglobin C, D Punjab, E or S trait can interfere with hemoglobin A1c (HbA1c) results. We assessed whether they affect results obtained with 12 current assay methods. METHODS Hemoglobin AA (HbAA), HbAC, HbAD Punjab, HbAE and HbAS samples were analyzed on one enzymatic, nine ion-exchange HPLC and two Capillary Electrophoresis methods. Trinity ultra(2) boronate affinity HPLC was the comparative method. An overall test of coincidence of least-squared linear regression lines was performed to determine if HbA1c results were statistically significantly different from those of HbAA samples. Clinically significant interference was defined as >7% difference from HbAA at 6 or 9% HbA1c compared to ultra(2) using Deming regression. RESULTS All methods showed statistically significant effects for one or more variants. Clinically significant effects were observed for the Tosoh G8 variant mode and GX (all variants), GX V1.22 (all but HbAE) and G11 variant mode (HbAC). All other methods (Abbott Architect c Enzymatic, Bio-Rad D-100, Variant II NU and Variant II Turbo 2.0, Menarini HA-8180T thalassemia mode and HA-8180V variant mode, Sebia Capillarys 2 and Capillarys 3) showed no clinically significant differences. CONCLUSIONS Several methods showed clinically significant interference with HbA1c results from one or more variants which could adversely affect patient care.

Effects of 49 Different Rare Hb Variants on HbA1c Measurement in Eight Methods

Background: Previous studies have shown interference with HbA1c measurement from the 4 most common heterozygous Hb variants (HbAS, HbAE, HbAC, and HbAD) with some assay methods. Here we examine analytical interference from 49 different less common variants with 7 different HbA1c methods using various method principles. Methods: Hb variants were screened using the Bio-Rad Variant or Variant II beta thal short program, confirmed by alkaline and acid electrophoresis, and identified by sequence analysis. The Trinity ultra2 boronate affinity high-performance liquid chromatography (HPLC) method and Roche Tinaquant immunoassay were used as primary and secondary comparative methods, respectively, since these methods are least likely to show interference from Hb variants. Other methods included were the Tosoh G7 and G8, Bio-Rad D-10 and Variant II Turbo, Diazyme Enzymatic, and Sebia Capillarys 2 Flex Piercing. To eliminate any inherent calibration bias, results for each method were adjusted using regression verses the ultra2 with nonvariant samples. Each method’s calibration-adjusted results were compared and judged to be acceptable if within the 99% prediction interval of the regression line for nonvariant samples. Results: Almost all variant samples were recognized as such by the ion-exchange HPLC methods by the presence of abnormal peaks or results outside the reportable range. For most variants, interference was seen with 1 or more of the ion-exchange methods. Following manufacturer instructions for interpretation of chromatograms usually, but not always, prevented reporting of inaccurate results. Results: Laboratories must be cautious about reporting results when the presence of a variant is suspected.

The Effect of Increased Fetal Hemoglobin on 7 Common Hb A1c Assay Methods

To the Editor: Hemoglobin variants and increases in circulating fetal hemoglobin (Hb F),1 (defined as an Hb F level >2%) have been reported to interfere with some assay methods for Hb A1c (1–3). In view of the relatively common occurrence of such Hb F increases (approximately 1.5% of the US population) and the fact that individuals with an increased Hb F level are usually asymptomatic, it is important to know whether Hb A1c methods show Hb F interference and, if so, at what level of Hb F. In this report, we evaluate the presence of Hb F interference with several HPLC methods. This study was approved by the Institutional Review Board of the University of Utah and Johns Hopkins Medical Institutional Review Board, where the samples originated. The following commercial HPLC methods were evaluated: the G7 and G8 Variant Mode (Tosoh Bioscience); the D-10™ Hemoglobin A1c Program (D-10), the VARIANT™ II Hemoglobin A1c Program (VII NU), the VARIANT™ II TURBO Hemoglobin A1c Program (VII Turbo), and the VARIANT™ II TURBO HbA1c Kit – 2.0 (VII Turbo 2.0) (all Bio-Rad Laboratories); and the ultra2™ A1c boronate affinity method (ultra2) (Trinity Biotech). The Hb F level was estimated from the G7 or G8 Hb F percentage. A small number of samples (n = 24) with and without an increased Hb F level were analyzed by both the IFCC capillary electrophoresis (CE) method (4) and the G7 method to verify …