Vol. 25 • Issue 9 • Page 28
Large clinical trials (DCCT, UKPDS) established the link between the glycemic control of diabetes patients, the HbA1c levels and the occurrence of diabetes complications.1,2 These trials established the importance of HbA1c as a useful test for monitoring known diabetic patients. Today, individual goals for glycemic control are HbA1c ≤ 6.5% for patients without concurrent serious illness and at low hypoglycemic risk.3 Furthermore, HbA1c levels > 7% are considered as a call for action to initiate or change therapy with the goal of reaching <7% HbA1c level.4
Interestingly, the use of HbA1c was expanded to the screening of diabetes in 2010 by the American Diabetes Association, and since then many other National Organizations, as well as the World Health Organization, have endorsed HbA1c ≥6.5% as a diagnostic criterion for diabetes.5,6
So, today, there is no question in the value of this test. There are over 100 commercial assays available to measure HbA1c that can be divided into separation methods (in which the Hb profile of the sample is displayed along with the percentage or mmol/mol value of HbA1c) or chemistry methods where the user only gets a percentage or mmol/mol value without the Hb profile.
Separation methods include ion exchange chromatography methods (IEC-HPLC), capillary zone electrophoresis (CZE or CE) and, to some extent, affinity chromatography (AC), whereas chemistry methods are immunoassays (IA) and enzymatic assays (EA).
Click to view larger graphic.
Click to view larger graphic.
HbA1c tests have improved tremendously in the last decade due to efforts from:7,8,9
• Standardization committees, like NGSP and IFCC
• Regulators, requiring that labs enroll in EQA programs or Proficiency tests
• Manufacturers, who are developing better and more resolutive tests (second/third generation)
The NGSP website regularly updates the performance of the different analytical instruments and lists common interferences, including Hb variants.10
In regards to POCT analyzers, it is worth noting that, although convenient to use in certain clinical situations, those methods lack stringent control procedures, can be possibly used by untrained or unqualified personnel and have an overall higher imprecision than the laboratory-NGSP-cleared methods mentioned above.11,12
If analytical interferences are present, the HbA1c value reported is not the correct value due to incorrect measurement.
In a case of clinical interference, the HbA1c value reported is analytically correct, but doesn’t reflect the patient’s level of glycemic control because of an alteration in the RBC metabolism. The target value used for therapeutic decision would not be applicable, and this leads to a misinterpretation of the results.
As examples of analytical interferences, a boronate affinity method would report a HbA1c value in all cases-even with an Hb E homozygote. But the reported value represents the percent of glycated Hb E and not the HbA1c level (Figure 1).
Similarly, on HPLC methods, certain Hb variants are known to co-elute with the HbA1c fraction, and therefore the result that will be reported is Hb X + HbA1c (approximately 50%), also a false HbA1c result (Figure 2).
The newest commercial separation method for HbA1c measurement is capillary electrophoresis.14 This method had the advantage of giving precise and accurate HbA1c results, even in the presence of Hb variants (traits). Since this method discriminates between homozygous and heterozygous variants, no erroneous results are reported in the event of a homozygous variant. Below an example with Homozygous Hb S (Figure 3).15
With regards to the clinical interferences, HbA1c will be affected by any condition impacting the hemoglobin metabolism. Whenever the erythrocyte lifesup is substantially shorter than normal, the HbA1c results will be low and not representative of the level of diabetes control. The opposite is also true-conditions that increase red blood cell lifesup, such as iron deficiency anemia, vitamin B12 or folate deficiency, will lead to falsely elevated HbA1c results.
Hemoglobin variants may cause clinical and analytical interference as described below:
• Alteration of the glycation rate: in a variant Hb (HbX) with a quaternary structure that differs from HbA’s structure, the production of HbX1c or stable adduct of glucose onto the N-terminal valine of the beta globin may not be the same as HbA1c. In such samples, chemistry/ enzymatic methods or affinity chromatography that cannot differentiate the HbA from HbX and HbA1c from HbX1c could give an erroneous HbA1c result. An ongoing debate in the scientific community still needs to clearly address this issue.16
Click to view larger graphic.
• Alteration of the red blood cell (RBC) lifesup: the average erythrocyte lifesup is 120 days in heterozygous HbAS individuals-93 days, and in those with HbAC-87 days. Beta thalassemia can affect RBC lifesup as well; it has been described that the RBC lifesup in patients with beta-thal is 81-93 days.16 If HbA1c is 7% with a lifesup of 120 days, the HbA1c level would be 6.4% at 110 days and 7.6% at 130 days.17
How to overcome erroneous and/or misleading HbA1c results
As a first step, it is important for clinicians, as well as laboratory managers, to know the advantages and disadvantages of the instruments they are using from the analytical standpoint in order to limit those errors. As a second step, they need to devise a strategy to minimize these occurrences and provide correct and accurate HbA1c results or glycemic control indicators in all circumstances.
The incidence of hemoglobinopathies is geographically variable, and therefore, knowing the various endemic hemoglobin disorders affecting the local community is primordial to make sure the analyzer in the laboratory is able to perform correctly in this circumstance. In areas known to have a population with a high prevalence of Hb variants, analytical methods that are capable of accurately measuring HbA1c and correctly identifying the presence of presumptive Hb variants should be used and may be preferred over immunoassay or borate affinity chromatography.19,20 The goal is to avoid reporting to clinicians unintended ðmisleading results that they’d use to adapt treatment because overtreatment and under-treatment of patients would either expose them to hypoglycemia episodes or to the chronic microvascular and microvascular complications of hyperglycemia.21
Additionally, in areas of high hemoglobinopathy prevalence, newly diagnosed diabetic patients should be screened initially by a simple hemoglobin electrophoresis test in order to determine if the patient carries a hemoglobin disorder that could potentially affect the HbA1c result. Patients identified as having hemoglobinopathies should be referred to another HbA1c testing facility that is using a method known not to have any interference from the specific Hb variant that was found.20,22
For known diabetic patients who had been followed for a while, but have never undergone a hemoglobin electrophoresis test, and who display doubtful HbA1c results (very high or very low values of HbA1c, discrepancy between the glucose values and the HbA1c level, different values of HbA1c when the patient changes testing facilities or testing methods, etc.), a hemoglobin electrophoresis test should also be performed to rule out a hemoglobinopathy affecting the HbA1c result.23
Other important tests that can help complete the clinical picture are: complete blood count (CBC) and reticulocyte count, sometimes also fructosamine or glycated albumin, which are markers of glycation that are not affected by the RBC metabolism.
The proposed algorithm takes into account these concerns (Figure 4).24
HbA1c remains a central test in the diagnosis and monitoring of diabetes. In most cases, NGSP approved lab instruments report accurate and precise results. However, challenging samples from patients with Hemoglobin-altering conditions could lead to erroneous and misleading reports.
In order to provide the best care to their communities, it is important for labs to recognize those situations and flag those results as a first step and, as a second step, elaborate a strategy to monitor those patients appropriately.
Dr. Maakaroun is director of medical affairs, Sebia Inc., Norcross, GA.
References are available online at laboratory-manager.advanceweb.com
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2. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). UK Prospective Diabetes Study (UKPDS) Group. Lancet 1998; 352:837-53.
3. American Association of Clinical Endocrinology/American College of Endocrinology- Comprehensive Type 2 Diabetes Management Algorithm (2016). Alan Garber et al
4. Medical management of hyperglycemia in type 2 diabetes: a consensus algorithm for the initiation and adjustment of therapy: a consensus statement of the American Diabetes Association and the European Association for the Study of Diabetes. Diabetes Care, 2009 Jan; 32(1):193-203
5 American Diabetes Association. Standards of medical care in diabetes-2010. Diabetes Care. 2010; 33 (suppl 1):S11-S61
6. WHO, Use of glycated haemoglobin (HbA1c) in the diagnosis of diabetes mellitus. Available at: www.who.int/diabetes/publications/report-hba1c_2011.pdf
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9 Cas Weykamp. HbA1c: A Review of Analytical and Clinical Aspects. Ann Lab Med 2013; 33:393-400
11. Miller CD, Barnes CS, Phillips LS, Ziemer DC, Gallina DL, Cook CB, et al. Rapid A1c availability improves clinical decision-making in an urban primary care clinic. Diabetes Care 2003; 26:1158-1163.
12. Lenters-Westra E and Slingerland RJ. Six of eight haemoglobin A1c point-of-care instruments do not meet the general accepted analytical performance criteria. Clin Chem 2010; 56:44-52.
13. S. Fellahi, M. Henderson, J. Capeau, J.-P. Bastard. Haemoglobin Hope and glycated haemoglobin: One peak may or may not hide the other, depending on the assay. Diabetes & Metabolism 38 (2012) 276-279
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15. Winter W, Schatz D, Faix J. Challenging Case studies in Lab Medicine – AACC webinar 24 June 2016: “A patient with diabetes who lacked HbA1c”.
16. Vandewiele A, Genbrugge K, Delanghe J. Spuriously high HbA1c due to the presence of haemoglobin Raleigh: a case report and review of the literature. Acta Clin Belg. 2010 Sep-Oct; 65(5): 336-40
17. JM Rhea, D Koch, J Ritchie, HV Singh, AN Young, T Burgess, RJ Molinaro. Unintended reporting of misleading Hb A1c values when using assays incapable of detecting hemoglobin variants. Arch Pathol Lab Med. 2013;137:1788-1791
18. David B.Sacks; The Pros and the Cons of using HbA1c for Diabetes Diagnosis. AACC Webinar April 10th 2012.
19. Rhea J, Molinaro R et al. Rare presumptive Hb variant misidentification prevents appropriate Hb A1c result Clinica Chimica Acta 431 (2014) 111-112
20. Bry L, Chen PC, Sacks DB. Effects of hemoglobin variants and chemically modified derivatives on assays for glycohemoglobin. Clin Chem 2001;47:153-63
21. Rhea JM, Roberts-Wilson TK, Molinaro RJ. Impact of hemoglobin variants on Hb A1c interpretation: Do we assume too much? MLO – June 2012
22. Rhea JM, Molinaro R. Pathology consultation on HbA1c methods and interferences. Am J Clin Pathol 2014; 141:5-16
23. National Institute of Health. Sickle Cell Trait and Other Hemoglobinopathies and Diabetes: Important Information for Providers. NIH Publication No. 14-6287 June 2014
24. (Adapted from) MD Yang Feh Chu, Hb A1c : The Right Result At The Very First Time, Taipei IFBLS world congress-Taipei, September 2013