HbA1c platforms studied for lipemia interference

“With the chemistry analyzers, you are mixing the patient’s whole blood with the reagent from the manufacturer, and the reaction is occurring in that same cuvette where the detection occurs. Everything is in the same cuvette, and you’re shining light through it, so if you have an interfering substance like lipids, it is going to interfere with that detection if it is the right wavelength.” The one method may be using a different wavelength than the other analyzers, he says, and thus there may be less interference for that reason.

Can results from different platforms be comparable then? That is the issue, in Dr. Nichols’ view. “If you are seeing a negative effect or impact, as Dr. Yip was showing with the triglycerides, it is significant for diabetics because most diabetics have problems with glucose metabolism. Because they are not metabolizing glucose, they need to metabolize protein and triglycerides or lipids for energy sources.”

Many people with diabetes have high cholesterol or high triglycerides. “Because of that, they are going to show, with these particular methods, a decrease in HbA1c. The HbA1c will make them look more compliant or better than they actually are,” Dr. Nichols says. “Their estimated average glucose over the past couple of months is going to look a little better than their actual average glucose. And they would be undertreated, in essence.”

If a laboratory is monitoring a patient over time with a chemistry platform that is falsely showing a decrease in HbA1c with triglycerides, he says, that is one thing. “But if you are monitoring a patient over time you are probably going to see a trend of increase or decrease overlaid with this interference. If you don’t switch methods, you can probably trend a patient over time because their lipids are not going to significantly change, unless there’s treatment with lipid-lowering drugs. And we’ve been managing patients currently pretty well based on these different methods.”

A visual inspection for lipemia in the laboratory is common with the automated platforms and chemistry analyzers, Dr. Nichols says. “We used to look at every sample visually and give an assessment of lipemia. Now, a visual inspection is either automated or it is ordered as a part of the chemistry panels. And we append comments on interferences at different levels.”
He suspects laboratories that are using the chemistry platforms studied by Drs. Yip and Parker can do a similar type of commenting. “If it was above a particular level that was significant, they would run a lipemia index and append a comment or note that the result may be falsely decreased because of the increase in lipids.”

Whether clinicians will notice is a different matter. “It depends on the electronic medical record system. A lot of times we append comments. Up front on the first EMR screen, you’ll see a number with an asterisk or a little arrow, but unless the clinicians click on the results on this EMR screen, they don’t see the comment. So, there can be issues with missing an important interference comment—and with a magnitude that is clinically significant.”

In addition, since the benefit of HbA1c is that the patient does not have to fast before the test, patients may have eaten just before the test and may have higher triglycerides, creating an even larger decrease in their HbA1c results.

High patient triglycerides are common enough to be a concern, Dr. Nichols says. “The study showed a pretty significant difference. The results were negatively biased by 10 percent and 25 percent at 5 g/L and 20 g/L of triglycerides.

“If you are looking at a 10 percent to 25 percent decrease in a result and you are sitting just above abnormal at an HbA1c of seven or eight, a 25 percent decrease will take you down to six, which would be considered intermediary, or even lower into the normal range. That could lead to incorrect treatment or a missed diagnosis.” This is more important for less serious cases of diabetes and maybe type 2 diabetes, for which clinicians screen patients in their physical exams, he adds, because if a person is widely out of range with their glucose, they will have HbA1cs of 10, 11, or in the teens. “A 10 percent decrease there is not going to be missed.”

Separately, there is already concern about using the chemistry methods for HbA1c because of the risk of missing hemoglobinopathies. “In other words, if you have a patient with sickle cells or high prevalence of hemoglobin variants in your patient population, you should be cautiously using a chemistry analyzer in the first place because the number you get won’t tell you that you have a hemoglobin variant, unless the patient knows they have a variant. So that chemistry analyzer number is going to be misleading for HbA1c to start with, depending on the type of hemoglobin variant and the specific method.” Independent of a direct interference, the presence of some hemoglobin variants can increase red cell turnover, affecting HbA1c levels.

Most of the chemistry methods’ package inserts list the common interferences, including hemolysis and triglycerides. “That factors in when they are developing the reagents and validation studies as part of the FDA submission data,” Dr. Nichols says, although, he adds, apparently the study showed that some of the manufacturers don’t have this data in their inserts.
The study results do not mean that laboratories should think right away about switching methods if they are showing a bias, Dr. Nichols says. Instead, “Laboratories should take note of this and discuss it with their clinicians and determine what the impact is on their patient population.”

In response to the study, Jeannine Holden, MD, Beckman Coulter’s chief medical officer, says lipemia is potentially an issue for any assay with a turbidimetric readout, not just HbA1c, and the study results Dr. Yip and colleague obtained are consistent with Beckman Coulter’s instructions for use for its HbA1c assay.

HbA1c assays are challenging in many respects, she says, and each of the common assay types has its benefits and drawbacks. “Immunoassays are generally more resistant to variant hemoglobins, so they may be preferable in areas with higher prevalence, and they don’t require a separate, dedicated instrument.”

Lipemia is underappreciated as a source of interference, Dr. Holden says. “In general, clinician awareness of lipemia as a potential interfering substance for assays is low, so laboratorians need to consider it when reporting any turbidimetric assay, not just HbA1c. Clinicians may also be unaware of the impact of red cell turnover on HbA1c results regardless of the assay type,” she adds, “and unlike lipemia, accelerated red blood cell turnover may be difficult to detect.”

Does Roche recommend the cautionary note suggested in the study? Günter Trefz, PhD, head of homogeneous immunoassay development, R&D Germany, Roche Diagnostics, points to the cautionary note on the Cobas c501Tina-quant package insert and notes that the limitations section clearly states: “Lipemia (Intralipid): No significant interference up to an Intralipid concentration of 600 mg/dL. There is poor correlation between triglycerides concentration and turbidity.”

“According to internal statistical data, the number of samples with an L-index greater than 200 mg/dL is less than 0.1 percent,” Dr. Trefz tells CAP TODAY. The number of samples with L-index greater than 500 mg/dL has been shown to be less than 0.02 percent in another study (Mainali S, et al. Pract Lab Med. 2017;8:1–9). “Therefore, it is rare to have samples with an L-index at or above 600 mg/dL.”

The company refers users to the NGSP website (ngsp.org/factors.asp) for full details, but says, “High-performance liquid chromatography methods for determination of HbA1c may be resistant to lipemia, but HPLC users must be aware of potential hemoglobin variant interference which exists at a higher prevalence and leads to inaccurate % HbA1c values.”

Roche also raises a question about the difference between the study’s methodology and CAP Surveys. “In this study, there appears to be a problem with how zero lipemia samples were measured. The baseline for zero lipemia samples appears to have been measured with an HPLC or CE method, resulting in bias with the Roche Tina-quant method. This finding is not seen in CAP Surveys, where the Roche Tina-quant method shows no significant bias. The correct procedure to detect lipemia interference would be to baseline each assay individually with the zero lipemia sample.”

Dr. Yip is continuing his research on lipemia interference by studying larger numbers of HbA1c samples on the various platforms. In the meantime, he says, “Laboratories need to be aware that this interference may occur, especially if they’re using a photometric-based method. And they should talk to the manufacturers about how they can approach the potential interference.”

Anne Paxton is a writer and attorney in Seattle.