Since 1958, when hemoglobin A1c was first separated from other forms of hemoglobin using a chromatographic column,¹ high performance liquid chromatography (HPLC) has been considered the gold standard for the monitoring of glucose control in patients with diabetes. Over the course of 57 years, alternative methodologies have been developed, and they have merit, but HPLC still provides significant and unique benefits to laboratorians, patients, physicians, and hospitals. Historically, there has been one key impediment to its utilization, however: the labor- and time-intensive nature of HPLC. But this impediment no longer exists; in today’s modern laboratories, HPLC testing for hemoglobin A1c (also known as HbA1c or A1C) can be fully integrated on hematology automation platforms. This enables labs, if you will, to provide the gold standard without having to spend all the gold! This article will explore the state of the art in integrated HPLC testing in hematology from the perspective of benefits to the lab, the patient and physician, and the overall healthcare system.

New/old gold: the state of the art in A1C testing

Multi-discipline EDTA automation islands are gaining popularity in hematology. They can provide for 90 percent of the tests drawn on an EDTA tube through the integration of routine hematology testing (CBC, differential, reticulocyte, body fluid analysis, and smear preparation and review) with more specialized testing such as HPLC for HbA1c. Information management and workflow management are key components of these automation islands, enabling the combined systems to work in synchronicity. While there are other methods available for determining HbA1c, a key issue remains the wide array of variants, both genetic and chemical, that can cause false positives and false negatives, which HPLC avoids.

There are many different types of HPLC; ion-exchange and boronate affinity methods have both been used to measure HbA1c. Ion-exchange HPLC has the added advantage of being able to identify the presence of variants such as S, C, D, and E in their heterozygous state, as well as fetal Hb, CHb, and LA1c.

HbA1c measures the amount of glucose attached to hemoglobin (Hb) in red blood cells, and it is used to monitor the glucose levels of people with diabetes over the course of the red blood cell’s 120-day life span. Hemoglobin variants, elevated fetal hemoglobin, and chemically modified derivatives, however, can cause HbA1c results to be inaccurate. The effects vary depending on the specific Hb variant or derivative and the specific HbA1c method. NGSP (the National Glycohemoglobin Standardization Program now uses its acronym as its official name) provides updated tables containing information for most of the commonly used current HbA1c methods for the four most common Hb variants, elevated HbF and carbamylated Hb.² 

Any condition that shortens erythrocyte survival or decreases mean erythrocyte age (e.g., recovery from acute blood loss, hemolytic anemia) will falsely lower HbA1c test results regardless of the assay method used.³  Iron deficiency anemia, a major public health problem in developing countries, is associated with higher HbA1c and higher fructosamine.⁴ There are many hemoglobin variants that have been identified—more than 1,200, according to HbVar, a database of human hemoglobin variants and thalassemias⁵—but there are several variants that are commonly encountered, including HbS, HbC, HbD, and HbE, along with non-variant HbF (fetal hemoglobin) and derivatives such as LA1c (labile A1C) and CHb (carbamyl-Hb).⁶

Genetic variants and HbA1c results

Genetic variants arise from point mutations in the, ß, γ, or δ Hb chains.⁷ HbS and HbC are the most common, impacting upward of 383,000 people in the United States.⁸ Due to the prevalence of these genetic variants in regions with malaria, the estimates of incidence of HbS and HbC are up to one-third of all diabetes patients worldwide.⁹ Additionally, there are chemical modifications of Hb, such as carbamylated Hb, found in uremic (often diabetic) patients; high circulating fetal hemoglobin (HbF), seen in ß-thalassemia, pregnancy, leukemia, and hereditary conditions;¹⁰ and labile HbA1c,¹¹ found with pathologic conditions that affect red cell half-life such as hemolysis, blood loss, iron deficient anemia, and blood transfusions.¹²

Among the common hemoglobinopathies, HbS affects one in 12 African Americans and one in 100 Hispanic Americans. It also affects those from Mediterranean countries, India, and Saudi Arabia. In the heterozygous state (one abnormal gene and one normal gene) it is known as sickle cell trait (HbAS); in the homozygous state (two abnormal genes) it is known as sickle cell anemia (HbSS disease).9

Similarly, HbC affects 2.3% of African Americans and those of West African descent. It also has hetero- and homozygous states (HbAC and HbCC disease). There is also a combined variant, HbSC (known as sickle-hemoglobin C disease), or double heterozygote. Mongia et al. found that while HbS and HbC can statistically affect HbA1c results across the range of testing options, including HPLC ion-exchange chromatography and boronate affinity methods, enzymatic assays, and immunoassays, only ion-exchange HPLC allows the operator to identify the presence of an HbS or C variant.⁸ Other methodologies, including electrophoresis, isoelectric focusing and electrospray mass spectrometry, are infrequently used.⁹

HbD trait is clinically significant for HbD-Punjab, affecting those from that region of India. HbE, on the other hand, affects Asian Americans, particularly those from Southeast Asia, China, India, the Philippines, and Turkey. It is estimated to affect up to 30% of the population of Southeast Asia and has both hetero/homozygous states (HbAE/HbEE).

High HbF is a circulating fetal hemoglobin that affects 1.5% of the U.S. population and interferes significantly with HbA1c methods, resulting in an artificially low HbA1c value.¹³ Since these patients are usually asymptomatic, it is important for the testing system to indicate the presence of high HbF. Some HPLC methods are able to provide an accurate HbA1c result in the presence of levels of HbF as high as 25%, as well as detecting it, showing a peak in the HbF window.¹⁴

Hemoglobin derivatives such as carbamylated Hb (CHb) are present in hemodialysis patients who are uremic. In certain conditions CHb levels can be so high that they can interfere with HbA1c. HPLC is one of the methods that has been shown to have no interference of CHb on HbA1c results, with an added advantage of being able to detect CHb.¹⁴

Labile A1c (LA1c) is an intermediate in the synthesis of HbA1c. Methods need to ensure that HbA1c is accurate in the presence of LA1c, especially in exceptional cases where Labile A1c is high. HPLC enables users to view the LA1c as a distinct peak in the chromatogram.

Overall, the presence of these variants and derivatives can affect the accuracy of HbA1c results. Numerous studies show interferences across a number of platforms.¹² Ion-exchange HPLC remains the gold standard due to minimizing interferences and enabling the operator to identify the variants and derivatives.¹⁵ According to NGSP, “Only HPLC utilizing ion-exchange chromatography measures HbA1c. Affinity columns measure any hemoglobin that has glucose attached regardless of its attachment point or its structure because the column binds the glucose portion of the molecule. Any variant hemoglobin that is present will be detected as glycated products.”¹⁶

Enumerating the benefits

The benefits of accurate HbA1c measurement affect three key stakeholders: laboratorians, patients and physicians, and hospitals/hospital systems. They impact the healthcare public policy realm as well. The benefits fall across four key metrics: quality, efficiency, productivity, and cost.

For laboratorians: From the perspective of the laboratorian, quality is demonstrated through accuracy of results. The ability of ion-exchange HPLC to provide accurate HbA1c values in the presence of variants, along with its ability to see the variants in the chromatograms, is significant. While other methodologies will have interference from at least one variant, HPLC minimizes the interference from most common variants—from HbS/C/D/E, to fetal Hb—to derivatives such as CHb and labile A1c. More important, when there is a question about an HbA1c result that seems inconsistent either with glucose testing or previous HbA1c results, the ion-exchange HPLC chromatogram provides the lens to visualize where the interference is.

Efficiency, which is defined as reducing an input relative to a fixed output, is seen in the reduction of time required to produce and verify the result. Aside from a fast time to first result on modern HPLC systems, the average turnaround time for a sample with Hb variants is four hours (from draw to reporting of results), compared to over 24 hours for non-HPLC systems which require manual follow-up to identify the source of discrepant HbA1c results. 

Productivity, defined as producing more output (results) relative to fixed input (such as labor), is improved through the elimination of pre- and post-analytical sorting with the EDTA island of automation. With the integration of hematology and HPLC on one workstation, productivity increases as the lab is able to increase testing and sample management capacity (handle higher volumes) without increasing staff. With the dramatic increase in diabetes that is unfortunately anticipated in coming years, this will be crucial to keeping hematology workflow under control. 

Finally, cost is affected through the superior efficiency, productivity (labor), and quality (fewer repeats, etc.) of the HPLC system.

For patients and physicians. From the perspectives of the patient and physician, quality (accuracy) is critical. Physicians need to have confidence that they are initiating the right course of treatment. An important aspect of that is knowledge of the presence or absence of variants and their presumptive identification. The physician will not have that knowledge unless the method specifically calls out the presence of the variant-ion-exchange, and HPLC is one of the few methods that does.

The NDEP Criteria for Diagnosis demonstrates the consequences if a patient is misdiagnosed. For example, pre-diabetic ranges are 5.7 to 6.4%. If a person is actually a 6.4% but the method is wrongly measuring the A1c at 6.5% then the patient is automatically categorized as “diabetic.” According to the guidelines and recommendations for lab analysis in the diagnosis and management of diabetes, intra-laboratory CV should be < 2%. Since the presence of variants can play a very important role in affecting HbA1c values, it is important to identify them and provide the physician with the complete picture. Since the CVs of HbA1c are a range, small variations can cause over- or undertreatment. For the patient, this translates to avoiding the consequences of either—for example, suffering from hypoglycemia or overmedication or the long-term health consequences of under-controlled diabetes.

In terms of efficiency (time), having the correct HbA1c avoids having to hold the patient (in an inpatient setting) or hoping the patient returns for another consult in the outpatient setting. The ability to identify variants enables the patient to be released in a timely fashion, freeing the doctor’s and patient’s time. 

This also impacts productivity of the doctor and better patient care. Having HbA1c in one EDTA tube saves multiple tube draws (and reduces blood volume requirements, repeats, and call backs.) Finally, it is cost-effective for both patient and physician. Patients avoid unnecessary, costly medication, while  doctors minimize repeat consultations. Both avoid the long-term health consequences of failure to intervene correctly in a timely fashion.

For hospitals and healthcare public policy: Ultimately, using the best method from the start has benefits to the hospital and our health system overall. Accurate, quality HbA1c results mean improved patient outcomes, which reduces the long-term burden of diabetes. Such results also avoid the unintended consequences of failure to treat or over-medication. Efficiency in the system improves with the expedited communication of results, reducing the time necessary to treat a patient relative to the DRG (Diagnosis-Related Group). By correctly identifying variants and derivatives from the start, bed turnover/census can be improved and the hospital/hospital system can manage the anticipated higher volume of patients with existing infrastructure, improving productivity. Finally, managing diabetes correctly will reduce the overall cost and economic burden on the broader healthcare policy realm, avoiding potential consequences for inappropriate treatment.

Go for the gold

When it comes to testing for HbA1c, a convincing case can be made for using ion-exchange HPLC, integrated with automated hematology lines. EDTA tube management is a new reality that enables labs to utilize this methodology without incurring prohibitive ongoing costs. It is beneficial for labs to consider this unique combination of capabilities based on the benefits to all the stakeholders.

References

1. Huisman TH, Martis EA, Dozy A. Chromatography of hemoglobin types on carboxymethylcellulose. J Lab Clin Med. 1958;52(2):312–327.
2. NGSP, Factors that interfere with HbA1c test results, http://www.ngsp.org/factors.asp. Updated 9/2014. Accessed May 6, 2015.
3. Goldstein DE, LIttle RR, Lorenz RA, American Diabetes Association Technical Review of Glycemia. Diabetes Care. 1995;18:896-909.
4. Sundaram RC, Selvaraj N, Vijayan G, et al. Increased plasma malondialdehyde and fructosamine in iron deficiency anemia. Biomed Pharmacother. 2007;61:682-685.
5. HbVar. A database of human hemoglobin variants and thalassemias. Globin.bx.psu.edu/hbvar/menu.html. Accessed 4/14/15.
6. International Hemoglobin Information Center variant list. Hemoglobin. 1994;18:77-183.
7. Bry L, Chen PC, Sacks DB. Effects of hemoglobin variants and chemically modified derivatives on assays for glycohemoglobin. Clin. Chem. 2001;47(2):153-163.
8. Mongia SK, Little RR, Rohlfing CL, et al. Effects of hemoglobin C and S traits on the results of 14 commercial glycated hemoglobin assays. Am J Clin Path. 2008;130:136-140.
9. Reid HL, Famodu AA, Photiades DP, Osamo ON. Glycosylated haemoglobin HbA1c and HbS1c in non-diabetic Nigerians. Trop Geogr Med. 1992;44:126-130.
10. Shu  I, Devaraj S, Hanson SE, Little RR, Wang P. Comparison of hemoglobin A1c measurements of samples with elevated fetal hemoglobin by three commercial assays. Letter to the editor. Clinica Chimica Acta 2012;413:1712-1713.
11. Corbe-Guillard, Jaisson S, Pileire C, Gillery P. Labile hemoglobin A1c: unexpected indicator of preanalytical contraindications. Letter to the editor. Clin Chem. 2011;57(1):340.
12. National Diabetes Information Clearinghouse (NDIC). Sickle cell trait and other hemoglobinopathies and diabetes: Important information for providers. http://diabetes.niddk.nih.gov/dm/pubs/hemovari-A1C/index.aspx. Accessed March 14, 2015.
13. Little RR. The effect of increased fetal hemoglobin on 7 common HbA1c assay methods. Letter to the editor. Clin Chem. 2012;58(5):945.
14. Little RR, Rohlfing CL, Tennill AL. Measurement of HbA1c in patients with chronic renal failure. Clinica Chimica Acta. 2013;418:73-76.
15. Higgins TN, Ridley B, Tentative identification of hemoglobin variants in the Bio-Rad VARIANT II HbA1c method. Clin Chem. 2005;38(3):272-277.
16. NGSP, HbA1c assay interferences. http://www.ngsp.org/interf.asp. Updated Sept. 2014. Accessed March 20, 2015.Priya Sivaraman, PhD, serves as Senior Product Manager, US Sales and Marketing, for Bio-Rad Laboratories.

Priya Sivaraman, PhD, serves as Senior Product Manager, US Sales and Marketing, for Bio-Rad Laboratories.

Nilam Patel, MT(ASCP)SH, serves as Senior Product Manager, Automation Solutions, for Sysmex America, Inc.

Glycated hemoglobin in the diabetic patient

By Craig Foreback, PhD

The utility of glycosylated hemoglobin measurements in the management of patients with diabetes has been established for almost 40 years.^1-6 Hemoglobin A1c (HbA1c) is the most abundant minor hemoglobin in normal red cells and is elevated as much as threefold in people with diabetes. The term, “A1c” should be reserved for HbA, which has glucose attached to the N-terminus of the b-chain by a ketoamine linkage. Glucose is also linked in the same way to other sites of the hemoglobin molecule such as the N-terminus of the a-chain and certain lysine residues. In diabetic patients these glucose adducts are increased in parallel with HbA1c. HbA1c is formed slowly and continuously throughout the life of the red cell. Thus HbA1c levels should provide an integrated assessment of blood glucose levels over the preceding 90 to 120 days.

Glycated hemoglobin: strengths and limitations

Initially high-performance liquid chromatographie (HPLC) ion exchange and affinity chromatography mini-columns were primarily employed in most clinical laboratories as methodologies for measuring A1c. A comparison of the determination of glycosolated hemoglobin by affinity chromatography to ion-exchange methods has been published.⁷ The authors reported that, although the methods correlated well, they observed that the affinity technique showed higher percentages of glycated hemoglobins than would be obtained by using ion-change chromatography. The affinity support binds a greater range of glycated species, leading to the conclusion that affinity methods may more accurately represent the levels of glycosylation. (Methods of obtaining glycated hemoglobin values are expressed in detail in Table 1.)

Table 1. Glycated hemoglobin obtained by four different methods in the presence of homozygous and compound hetererozygous hemoglobin variants¹⁶

An automated POC immunoassay (Bayer DCA 2000, now Siemens) for glycated hemoglobin was developed and evaluated in the early 90s.^8-10  One evaluation of the Siemens DCA 2000 concluded that when compared to an HPLC method it had significant constant and proportional values.¹¹ At low concentrations of hemoglobin F the DCA 2000 underestimated HbA1c and at higher hemoglobin F concentrations tended to overestimate HbA1c. Recently the DCA vantage offered an improvement over the previous version and passed the National Glycohemoglobin Standardization Program (NGSP) criteria for acceptable performance. Later, immunoassays were also developed for automated multichannel chemistry analyzers, including the Abbott Architect/Aeroset, Siemens Dimension and Advia, Beckman Unicel DxC/Synchron and AU, Roche Cobas Integra and Ortho Clinical Diagnostics Vitros. Immunoassays quantify HbA1c by using antibodies that recognize the structure of the N-terminal glycated amino acids on the b chain of Hb A.

While many laboratories favored the affinity approach because it measured all glycated fractions, it became obvious that the two methods were producing different results, leading to two distinct normal ranges. This complicated the interpretation of results among different medical centers, which became more complex with the implementation of the Diabetes Control and Complications Trial (DCCT). The 29 centers involved sent samples from patients in the study to a central location at the Joslin Diabetes Center in Massachusetts. Ion-exchange HPLC was chosen as the method for the analysis of all samples. The study demonstrated the utility of measuring HbA1c to monitor and treat people with diabetes, negating any perceived advantage of determining total glycated hemoglobin. Since ion-exchange HPLC was used in the DCCT study, it became widely accepted and is commonly used for HbA1c measurement today.

First-generation assay epitopes typically span amino acids 4 to 10 of the Hb b chain, encompassing the altered amino acid at position 6 present in Hb S and Hb C variants. HbA1c measurement interferences in the presence of these Hb variants prompted the development of second-generation assays that used antibodies developed to epitopes spanning amino acids 1 to 4. Second-generation assay antibodies largely eliminated analytic interferences from Hb S and Hb C and improved the overall accuracy when compared to first-generation assays. The second-generation assay antibodies are thought to bind Hb S and Hb C variants similarly to Hb A, and thus provide clinically useful HbA1c values for patients with heterozygous trait conditions, where red blood cell life span is thought to be essentially normal.

Table 2. Average lifespan of RBCs for normal and variant hemoglobins

However, as shown in Table 2 this assumption is not always valid. In addition, these assays create an increased risk for reporting misleading HbA1c results for patients harboring homozygous Hb S, Hb SC, or Hb S–b-thalassemia, where red blood cell life span is decreased. These assays provide an ostensibly reportable HbA1c value without warning that the patients appeared to not have any Hb A. Although the values labeled as HbA1c, obtained by using these methods, may be fairly accurate, their clinical significance may be misleading and do not reflect glycemic status accurately. The ADA recommends against the use of HbA1c for patients with certain Hb variants, such as Hb SS, Hb SC, CC and Hb S–b-thalassemia. The HPLC affinity method can also be misleading because it also provides an HbA1c in the absence of Hb A. Table 1 illustrates how ion exchange, immunoassay, and affinity chromatography are affected by the presence of homozygous and compound heterozygous variants. It also indicates that ion exchange methods can provide obviously erroneous results in the presence of the variants.

Methods that detect other glycated fractions report an HbA1c value even though there is no hemoglobin A. Table 2 illustrates the variation of red cell half-life when homozygous, double or compound heterozygous, and heterozygous variants are present. Even heterozygous variants such as AS or AC have reduced half-lives. It is assumed that the shortened red cell life in such hemoglobins does not affect glycation rates, but questions have been raised about whether that is always true.

NGSP and standardization

The purpose of the NGSP is to standardize glycohemoglobin test results (HbA1c) so that clinical laboratory results are comparable to those reported in the DCCT where relationships to mean blood glucose and risk for vascular complications have been established. The methods and reagents are certified by the NGSP as having documented traceability to the DCCT reference method. Currently, 16 pages of methods and instruments are certified, encompassing more than 200 different combinations of instruments and reagents. The certificate of traceability expires after one year, and the NGSP recommends that manufacturers certify their methods yearly. The most recent certifications occurred in April 2015.

Testing for diabetes with HbA1c

To meet tightening College of American Pathologists (CAP) requirements and to provide accurate HbA1c medical decision information to physicians and patients, laboratories will need to use an HbA1c assay that is accurate and precise. The information on the performance of individual methods can be easily obtained by reviewing CAP GH2 Summary reports or from the NGSP website. In addition to greater accuracy and precision, it is important to use an HbA1c method that will identify abnormal hemoglobins that could impact HbA1c results. Improvements in methodologies used in regard to precision, accuracy, and the ability to detect Hb variants are preparing the market for a shift toward diabetes screening with HbA1c.

In January 2010, the American Diabetes Association (ADA) announced its long-awaited recommendations for diagnosis of diabetes. According to recommendations, testing for diabetes should be considered for all adults who are overweight (BMI 25 kg/m2) and have additional risk factors. The ADA established the threshold of diabetes to be ≥6.5% HbA1c. The HbA1c test should be performed using a method certified by NGSP and standardized to DCCT reference study. An HbA1c range of 5.7-6.4% identified individuals with a high risk for future or pre-diabetes. POC HbA1c assays are not sufficiently accurate at this time to use for diagnostic purposes.

For patients with Hb variants but normal red blood count (RBC) turnover, HbA1c assays without inferences from abnormal hemoglobins can be used. The problem is that the laboratory may not have the patient history indicating what Hb variant is present. As mentioned earlier, affinity and immunoassay methods cannot detect any of the variants. The NGSP website summarizes the effects of hemoglobin variants and elevated fetal hemoglobin on methods and instruments for HbA1c.¹⁴

Individualized Quality Control Plan

New quality control recommendations by Centers for Medicare and Medicaid Services (CMS) allow clinical laboratories to develop an Individualized Quality Control Plan (IQCP) based on risk management. In a recent report,¹⁵ sigma-metric values (total allowable error- %bias)/CV) were calculated for some commonly used HbA1c techniques. Sigma-metric directly relates to the predicted probability of producing an unreliable patient result; higher sigma-metric indicates better method performance. In CAP surveys, the total allowable error is currently set at 6% (previously, at 7%; it is projected to be brought down to 5% in the near future).

With the initiative to use glycated hemoglobin to test for diabetes, it is even more imperative that the assay for this analyte is accurate and precise. Manufacturers of testing that involves ion exchange and capillary zone electrophoresis claim that only these techniques actually measure glycation at the N-terminus of the beta chain; other methods, they assert, also include other glycated hemoglobins in the reported results, and in the presence of hemoglobin variants immunoassays and affinity methods may produce misleading results. Precision and accuracy are also extremely important, since the ADA has recommended any value above 6.5 as diagnostic for diabetes. Laboratories must carefully choose instruments and methods to perform HbA1c testing and have a clear understanding of what is really being measured. Finally, it is imperative that the laboratory effectively communicate the limitations of their method to care givers.

References

1. Bunn HF, Evaluation of glycosylated hemoglobin in diabetic patients.Diabetes. 1981; 30:613-617.
2. Koenig RJ., Peterson CM, Jones, RL, et al.  Correlation of glucose regulation and hemoglobin A,o in diabetes mellitus. NEJM. 1976;295:417-420.
3. Gabbay KH, Hasty K, Breslow JL, et al. Glycosylated hemoglobins and long-term blood glucose control in diabetes mellitus. J Clin Endocrinol Metab. 1977;44:859-864.
4. Gonen G, Rubenstein AH, Rochman H, Tanega SP, Horwitz D L. Haemoglobin A. an indication of the metabolic control of diabetic patients. Lancet 1977;2:734-737.
5. Dolhofer R, Stadele K, Wieland, OK. Clinical and biochemical studies on the significance and formation of hemoglobins A1e and A1a+b in diabetes mellitus. Klin Wochenschr. 1977;55:945-954.
6. Graf RJ, Halter JB, Porte D. Glycosylated hemoglobin in normal subjects and subjects with maturity-onset diabetes. Diabetes. 1977;27:834-839.
7. Klenk DC, Hermanson GT, Krohn RI, et al. Determination of glycosolated hemoglobin by affinity chromatography: Comparison with colorimetric and ion-exchange methods, and effects of common interferences. Clin Chem. 1982;28:2088-2094.
8. Ng RH, Sparks KM, Hiar CE. Rapid automated immunoassay system measuring hemoglobin A1c by using precalibrated, unitized reagent cartridges.Clin Chem. 1992;38;1647.
9. Guthrie R, Hellman R, Fineberg NS, et al. A multi physician’s office laboratory evaluation of an immunological method for the measurement of HbA1c, Diabetes Care.1992;15:1494-1498.
10. Marrero DG, Vandagriff JL, Gibson R, et al. Immediate HbA1c results , Performance of new HbA1c system in pediatric outpatient population, Diabetes Care,.1992;15:1045-1049.
11. Diem P, Walchli M, Primus ME, Marti U. Agreement between HbA1c measured by DCA 2000 and by HPLC: effects of fetal hemoglobin concentrations. Archives of Medical Research. 2004;35:145-149.
12. McCurdy PR. 32-DFP and 51-Cr for measurement of red cell life span in abnormal hemoglobin syndromes, Blood.1969;33(2):214-224.
13. Prindle KH, McCurdy PR. Red cell lifespan in hemoglobin C disorders (with special reference to hemoglobin C trait), Blood. 1970:36:14-19.
14. NGSPwebsite. www.ngsp.org. Accessed May 7, 2015.
15. Woodworth A, Korpi-Steiner N, Miller, JJ, et al. Utilization of assay performance Characteristics to estimate hemoglobin A1c result reliability. Clin Chem. 2014;60:1073-1079.
16. Rhea JM, Koch D, Ritchie J, et al. Unintended reporting of misleading HbA1c values when using assays incapable of detecting hemoglobin variants. Arch Pathol Lab Med. 2013; 137:P1788-1791.