Printer Friendly

HPLC: its continuing role in diabetes monitoring.

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 thalassemias (5)--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, [beta], [gamma], or [delta] 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 [beta]-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.

Continuing Education

To earn CEUs, see test on page 20 or online at under the CE Test tab.


Upon completion of these articles, the reader will be able to:

1. Identify the history of HbA1C testing.

2. Identify the current role of HbA1C testing for diagnosis and monitoring of diabetes patients.

3. Describe HbA1c testing limits with hemoglobin variants.

4. Identify organizations leading the process of standardizing HbA1C testing.


(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:// 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. 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 HbSIc in non-diabetic Nigerians. Trop GeogrMed. 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, 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. interf.asp. Updated Sept. 2014. Accessed March 20,2015.

By Priya Sivaraman, PhD, and Nilam Patel, MT(ASCP)SH

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.
COPYRIGHT 2015 NP Communications, LLC
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2015 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:DIABETES; high performance liquid chromatography
Author:Sivaraman, Priya; Patel, Nilam
Publication:Medical Laboratory Observer
Article Type:Report
Geographic Code:1USA
Date:Jun 1, 2015
Previous Article:Clarification.
Next Article:Glycated hemoglobin in the diabetic patient.

Terms of use | Privacy policy | Copyright © 2022 Farlex, Inc. | Feedback | For webmasters |