CME INDIA Presentation by Dr. N. K. Singh, MD, FICP, Director – Diabetes and Heart Research Centre, Dhanbad; Editor – CME INDIA.

Hb1Ac Laboratories use many different methods for measuring A1C. As clinicians, we must be aware that some of these methods can give inaccurate results especially when the patient has a hemoglobin variant such as sickle cell trait or if there is an elevated level of fetal hemoglobin (HbF).

Occasionally we see such reports of Hb1Ac

How to interpret this graph provided with Hb1Ac report? Should we accept a hb1ac report only with a graph? What is the utility of Point of Care machine you use in your clinic?

Dr. Swati Srivastava, Prof. of Medicine, SMS, Medical College, Jaipur says: This is a HPLC chromatogram. The HbA1c peak is shaded. Hemoglobinopathies have impact on HbA1c reading., high HbF leads to falsely low HbA1c. Normal HbF is less than 1 % in adults. The reading of chromatogram is complex, Involving peak, retention time, area. We usually see the value and proceed.

Points to Consider

1. Hemoglobin A1c (HbA1c) testing is the gold standard  for testing and monitoring diabetes, specifically the type 2 diabetes.
2. Analysis of glycated haemoglobin (HbA1c) tells the story of  individual’s average blood glucose levels during the previous two to three months, which is the predicted half-life of red blood cells (RBCs).
3. But monitoring the can be inaccurate in many conditions, especially in  persons with elevated amounts of Haemoglobin F, or with abnormal haemoglobins found in sickle cell trait, HbC trait and HbE trait.
4. Variant haemoglobins might cause a false increase or decrease in HbA1c.
5. The design of the method affects whether or not it is influenced by increased non-A haemoglobin, so that HPLC from manufacturer X may be affected, but HPLC from manufacturer Y may not be. Similarly, immunoassay from manufacturer W may be affected, but immunoassay from manufacturer Z may not.
6. The National Glycohemoglobin Standardization Group (NGSP) keeps an up-to-date table on their website.
7. Haemoglobin A1c inaccuracies in the presence of haemoglobin variants are method dependent. A patient who is tested by two different laboratories or two different methods may have clinically significantly different results
8. Haemoglobin A1c does not always estimate average blood glucose (eAG) for every patient.
9. There is a very predictable relationship between HbA1c and Average glucose (AG).
10. Fasting glucose should be used with caution as a surrogate measure of AG.
11. It is important to remember that HbA1c is a weighted average of glucose levels during the preceding 3 months. Unless the patient’s glucose levels are very stable month after month, quarterly measurement is needed to ensure that a patient’s glycemic control remains within the target range.

The major methods used to assay HbA1c

We have basically 3 methods:

1. Immunoassay
2. HPLC
3. Boronate affinity

1. Immunoassays (Point of care)

• Immunoassays use antibodies to detect an analyte.
• Immunoassays for HbA1c are often used in point-of-care testing.
•  DCA 2000 (Siemens) is a popular example.
• Testing can be done from a fingerstick blood sample.
• Results are available within 10 minutes. The percentage of the glucose-hemoglobin moiety as the numerator to the total hemoglobin denominator equals the HbA1c% result.
• This is what, most of physicians using in their clinic. Haemoglobinopathies issues cannot be tackled. No graph generated.
• Thus we get  only an A1c value and no additional information about the presence of haemoglobin variants or labile and carbamylated Hb.
• Glycated HbF is not detected by the assay because the immunoassay antibodies only recognize HbA1c and not glycated HbF as it does not contain the b-chain that characterizes HbA1c.

2. HPLC

• High Performance Liquid Chromatography (HPLC) separates hemoglobin moieties by charge. The whole blood sample is hemolyzed and injected into a column filled with charged resin which binds the hemoglobin.
• Retention Time: The column is washed with buffers of different ionic strength at certain times. The various hemoglobin release or remove from the column at a specific time related to their charge and to the ionic strength of the buffer. This is referred to as the retention time.

• As the hemoglobin elutes, its concentration is measured by a detector.
• The graph of this timed process is called a chromatogram, and the axes of the chromatogram are concentration versus retention time.
• Only this method generates graph and see AREA UNDER GRAPH, if more than 25 percent, abnormal Hb needs consideration.
• HPLC is method of choice.

• Few authorities say, initial hb1ac detection, must be by this method.
• So, point to highlight is that Hemoglobin A1c inaccuracies in the presence of hemoglobin variants are method dependent. A patient who is tested by two different laboratories or two different methods may have clinically significantly different results.
• HPLC-based methods produce accurate results (the methods used in this study do not exhibit interference from HbF b 25-30%).
• It should be appreciated that initial HbA1c testing should be performed by HPLC in order to allow identification of hemoglobinopathies that may cause interference.
• If a variant interfering with HPLC is identified, analysis should always be performed by immunoassay.

• Bio-Rad HPLC-HbA1c results are valid and reportable on the VARIANT II TURBO System in the presence of HbF up to 25%.
• The VARIANT II TURBO detects the presence of HbF (22.4%) and its chromatogram provides a complete patient profile to clinicians.

3. Boronate Affinity

• Boronate Affinity Chromatography is not affected by most alternate hemoglobins
• This technique is being used in many reference laboratories as well as in point-of-care testing.
• It does not directly measure HbA1c.
• The benefit of this method is that normal and variant glycated hemoglobins are measured, so HbS, HbC, and HbE do not chemically interfere with the result ( Interference with A1c results in the presence of HbF above 15 percent is seen in this method).
• Results are given as ‘glycated hemoglobin’, which can be confusing to the clinician, but the corresponding (calculated) HbA1c is also provided.
• As the glycated and non-glycated products elute through a column, the hemoglobin interacts with boronic acid giving 2 peaks: glycated (GHb) and non-glycated hemoglobin.
• This method is less prone to interferences with variants; however, it lacks the ability to presumptively identify variant hemoglobins.

CME INDIA Discussion

• Dr. J. K. Sharma, Sr. Diabetologist, Delhi: Very pertinent and Important information and academic input.
• Dr. Somnath, Hyderabad: Which method should we use in our diabetic set up both without compromising quality of test and financial feasibility with testing load of average 15-20.
• Dr. NKS: HPLC is not feasible, machine costs over 8 lacs and needs many samples Point of care machines. Ok, if only one reading you get by labs doing with graph.
• Dr. Vijay Balaji K., Bangalore: This is very new for us. Didn’t know this was so valuable. All that we did was to only see HbA1c.
• Dr. Indurkar Sanjeev, Diabetologist, Aurangabad: Very Informative and useful. I am using BIORAD D10 and TOSHO HPLC machines, both are good and accurate.
• Dr. N K Singh: If you see 10 reports, how many you see with graph?
• Dr. Brij Mohan, Kanpur: Hardly 1-2 out of 10.
• Dr. A.K. Virmani, Jamshedpur: Almost all good labs are now giving the graph.
• Dr. Sandeep S Desai, Physician and Diabetologist, Valsad, Gujarat: Alere afinion by Abbott uses Boronate affinity and used by many as POC for A1C.

CME INDIA Learning Points

• HbA1c provides a reliable measure of chronic glycemia.
• It correlates well with the risk of long-term diabetes complications.
• It is currently considered the test of choice for monitoring and chronic management of diabetes.
• Along with the type 2 diabetes, the HbA1c is also used to diagnose, manage, and monitor the type 1 diabetes as well.
• The International HbA1c Consensus Committee has recommended that the HbA1c levels must be reported in terms of System International (SI) units (millimoles per mole, with no decimal places), which relate better scientifically to a valid measure of HbA1c.
• The NGSP still recommends using the units in terms of the percentage with one decimal place. In India we use in terms of percentage.
• There are some pitfalls in interpreting the result. In anaemic patients or those with shorter RBC lifespan (glucose-6-phosphate dehydrogenase deficiency, sickle-cell disease, etc.), the HbA1c levels may be compromised indicating a false “good” result.
• The excessive use of vitamin C, B, and E supplements and increased levels of cholesterol, liver, and kidney diseases can also present abnormally high levels of HbA1c.
• The increased reliability of point-of-care (POC) devices for A1c testing has been shown to improve individual monitoring of blood glucose levels, because they can be used directly at physician chamber.
• There is a shortage of information comparing the different machines. When using POC testing, one should keep in mind that POC values are often below results reported by a laboratory test, with the mean difference being -0.5%.
• Compared to a centralized laboratory test, the use of the POC-A1c device in a healthcare unit increases the chance of the early control of type 2 diabetes and reduced costs in relation to DM-related outcomes.
• For an HbA1c test to classify as normal, or in the non-diabetic range, the value must be below 5.7%. Anyone with an HbA1c value of 5.7% to 6.4% is considered to be prediabetic, while diabetes can be diagnosed with a HbA1c of 6.5% or higher.
• Tests should be sent to a laboratory certified by the NGSP to ensure results are standardized.
• Laboratories can use several methods to determine HbA1c. High performance liquid chromatography (HPLC) method is one of the most popular methods because it can eliminate labile components that other methods such as immunoassay or affinity chromatography use.
• Patients with elevated HbF due to HPFH (Hereditary Persistence of Fetal Hemoglobin) will have falsely low HbA1c results by immunoassay, whereas many HPLC-based methods produce accurate results (the methods used in this study do not exhibit interference from HbF b 25-30%).
• It has been suggested that initial HbA1c testing should be performed by HPLC in order to allow identification of hemoglobinopathies that may cause interference. If a variant interfering with HPLC is identified, analysis should always be performed by immunoassay.

CME INDIA Tail Piece

• HbA1c was first isolated by Huisman et al. in 1958.
• It was characterized by Bookchin and Gallop in 1968, as a glycoprotein.
• The elevated levels of HbA1c in diabetic patients were reported by Rahbar et al in 1969.
• Bunn et al. identified the pathway leading to the formation of HbA1c in 1975.
• Using the HbA1c as a biomarker for monitoring the levels of glucose among diabetic patients was first proposed by Koenig et al. in 1976
• About 6% of total HbA is termed HbA1, which in turn is made up of HbA1a1, HbA1a2, HbA1b, and HbA1c fractions, defined by their electrophoretic and chromatographic properties.
• HbA1c is the most abundant of these fractions and in health comprises approximately 5% of the total HbA fraction.
• The formation of the glycated hemoglobin is a normal part of the physiologic function cycle.
• As the average plasma glucose increases, the amount of glycated hemoglobin in the plasma also increases.
• The specific characteristic of the hemoglobin biomarker is utilized for estimating the average blood glucose levels over the previous two to three months.

Table comparing different techniques/Machines

@ In the absence of specific method data, it can generally be assumed that immunoassay methods do not have clinically significant interference from HbE and HbD because the E and D substitution are distant from the N-terminus of the hemoglobin beta chain (1).

$ In the absence of specific method data, it can generally be assumed that both immunoassay and boronate affinity methods show interference from HbF levels above ~10-15% (2,3).

# When HbE trait is detected there is no significant interference. In cases where the presence of HbE trait is not detected, HbA1c results are artificially lowered.

↑ Interference causes a higher result
↓ Interference causes a lower result

Source: http://www.ngsp.org/interf.asp

References

1. Article  in  Clinical laboratory science: journal of the American Society for Medical Technology · March 2011 DOI: 10.29074/ascls.24.2.71 · 
2. Lorena de Sousa Rosa^,Sóstenes Mistro et al.Cost-Effectiveness of Point-of-Care A1C Tests in a Primary Care Setting. Front. Pharmacol., 19 January 2021 | https://doi.org/10.3389/fphar.2020.588309
3. Gilstrap LG, Chernew ME, Nguyen CA, Alam S, Bai B, McWilliams JM, Landon BE, Landrum MB. Association Between Clinical Practice Group Adherence to Quality Measures and Adverse Outcomes Among Adult Patients with Diabetes. JAMA Netw Open. 2019 Aug 02;2(8):e199139.
4. https://www.bio-rad.com/applications-technologies/why-bio-rad-hplc?ID=5e7728d6-0f31-0631-ee35-0830b31efd85

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