CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
Anne Paxton
February 2019—A forgotten creditor. A poor relation. An envious rival. In the theater, one of these characters often emerges from the woodwork, ready to supply a plot twist just when the protagonist is riding highest. In the health care world, a new study shows, laboratories may be finding that the very popularity of hemoglobin A1c has magnified the underappreciated effect of fats known to interfere with this centrally important diabetes diagnostic. Enter, stage left: Lipemia.
‘Laboratories need to be aware that this interference may occur, especially if they’re using a photometric-based method.’ —Paul Yip, PhD, D(ABCC)
Interferences in general are an interest of Paul M. Yip, PhD, D(ABCC)—in particular interferences that affect front-line chemistry testing. “Many times users are unaware when there may be an interference present that can affect the final result, especially when serum indices are not checked,” says Dr. Yip, division head of biochemistry at Sunnybrook Health Sciences Centre and associate professor in the Department of Laboratory Medicine and Pathobiology, University of Toronto.
Reporting on results of a study he conducted with colleague Michelle L. Parker, PhD, Dr. Yip said in a poster presentation at the AACC annual meeting last July that lipemia interferes differently with HbA1c results depending on the assay method used and the extent of the lipemia (Parker ML, et al. Abstract 288).
To conduct the study, which received funding support from Bio-Rad Laboratories Canada, Drs. Yip and Parker used samples spiked with saline and/or Intralipid to generate triglyceride levels of 0, 5, and 20 g/L to investigate the concentration of Intralipid-sourced triglycerides that may cause significant interference on four commonly used HbA1c analytical methods, and they used clinically lipemic specimens to assess the performance of nine routine HbA1c platforms. “We included essentially all the major manufacturers’ platforms available in North America,” Dr. Yip says.
The platforms studied were: Bio-Rad D-100, Bio-Rad Variant II Turbo 2.0, and Bio-Rad VII HbA2/HbA1c Dual Program; Sebia Capillarys; Beckman Coulter AU; Siemens Dimension Vista; Roche Cobas c501 Tina-quant; Ortho Vitros; and Abbott Architect.
Most immunoassays and the enzymatic method for HbA1c, the study found, are susceptible to negative interference from elevated triglycerides, while chromatographic and electrophoretic methods are resistant. For the Intralipid-spiked specimens, similar results were obtained; the findings are consistent with an earlier study that also used triglyceride/cholesterol-spiked specimens but assessed fewer assays (Wu X, et al. Biochem Med [Zagreb]. 2016;26[3]:353–364).
The assays that involved more of the traditional separation of the specimens were ones that were less affected by lipemia, Dr. Yip says. “Meanwhile, the other methods that used large chemical auto-analyzers that do multiple tests and have non-separation–based assays and any photometric or optical method that uses light” were also thrown off by lipemia. “In the presence of cloudiness or turbidity, the lipids in the sample are obviously going to have an impact on the light,” he says.
To avoid reporting falsely low HbA1c measurements, the study concluded, laboratories should consider evaluating their assay performance for significant interference from clinical lipemia. In addition, the authors said, “Although further investigations are needed, our data suggest that a serum triglyceride threshold of approximately 10 mmol/L may warrant a cautionary note when reporting HbA1c or reflexive testing to a lipemia-resistant platform.”
Hemolysis, hyperbilirubinemia, and lipemia are serum indices, Dr. Yip notes, a general class of interferences that occur in serum or plasma samples and may arise because of the patient’s physiological state or the handling of the samples. While considerable attention has been paid to the ways that hemoglobin variants can interfere with HbA1c, matrix-related interferences like lipemia have been investigated less, he says.
Lipemia’s interference with HbA1c is an under-recognized problem, in his view. “HbA1c is available on many different automated platforms, but not much has been reported in terms of how lipemia can affect results,” Dr. Yip says. “No one has really encountered, at least to my knowledge, a result that was so discrepant that through their troubleshooting and investigation they determined it was due specifically to a lipid interference.”
Lipids in a patient specimen can vary considerably. “They can be produced within the body, they may arise from diet if you have had a fatty meal, and they are heterogeneous in composition, in terms of the size of the lipid particles and the different concentrations of individual lipids. All of those things together can have a very different impact on the analysis.”
When laboratories are reporting on the presence of lipemia, often they are dealing with a serum or a plasma sample, Dr. Yip notes. “Because the plasma and the cells in the blood have been separated, you can see whether or not the sample is clear or if it is cloudy or murky. HbA1c analysis, though, is using a whole blood sample in which everything appears red. It’s difficult if not impossible to tell whether or not lipemia is there.” Adding to that difficulty, “When users are putting a sample into an automated analyzer, they may not even think to look at the specimen. It’s just a matter of loading it on and letting the machine do the testing.”
Major diagnostics manufacturers generally document common interferences well, he says. “Pretty much all of them we found did make a claim in their package inserts as to the kind of interference and whether it was a robust interference.” It’s difficult to replicate lipemia in a manufacturer’s environment, he says. But it can be simulated through Intralipid, a nutritional supplement that is essentially an emulsion of oil and other lipid material. “You can do an artificial setup by adding specified amounts of Intralipid and checking to see if it has any interference on what you are analyzing.” He and Dr. Parker used that technique in their study.
Says Dr. Yip: “HbA1c has taken on such a primary role as a diagnostic test and we’ve been caught up in improving accuracy, reducing imprecision, standardizing the results, and doing a lot of important work to make the assay available and accessible.” Now that those improvements appear to be in place, “we can start setting our sights on some of the less appreciated issues that can have an impact on results.”
In the traditional HbA1c context of diagnosing diabetes, patients would probably see a primary care physician or address the disease on an ambulatory basis, he notes. “Now we see patients going into the ER and ER doctors ordering HbA1c because they want to diagnose diabetes or see what sort of diabetic state the patient is in. In an emergency, you are probably in an acute situation and there are other bodily changes happening at the same time, so you are testing in less than ideal conditions.”
HbA1c is highlighted as the go-to test for diabetes because it doesn’t require a fasting specimen. But in these emergency circumstances, a nonfasting specimen is even more likely and the potential for lipemia is going to increase along with that, Dr. Yip says. “For diabetics there is a greater incidence of dyslipidemia as well. If dyslipidemia is present at a sufficiently high level, that can also have an impact on the test result.”
Each laboratory needs to determine for itself what its risk of encountering an interference is, he says, and how it is going to mitigate that risk. At University Health Network in Toronto, where Dr. Yip worked previously and where the lipemia study was conducted, he estimated that about 0.2 percent of, or one in 500, samples were significantly lipemic. The method that the UHN laboratory used was not affected by lipemia. “But for any laboratory using a routine auto-analyzer, many if not all of the major chemistry auto-analyzers can do a serum indices check, which could give a crude estimate of how much lipemia might be present.”
Dr. Yip sees immediate clinical implications for this study. “That’s because we appreciate now that A1c has to perform so tightly, so well, the total allowable error is constantly getting smaller and smaller, and my feeling is that it will continue to tighten. We have various guidelines that specify diagnosis of diabetes at an A1c of 6.5 percent. Now we have to be very sure that that 6.5 percent is as accurate as possible. If you have significant lipemic interference with one of the methods and an error that can falsely lower results, you could be missing that diagnosis of diabetes.”
‘Laboratories should take note of this and discuss it with their clinicians and determine
what the impact is on their patient population.’ —James Nichols, PhD, D(ABCC)
“Any of the spectrophometric methods that we use in chemistry can be interfered with by lipids, by bilirubin, or by hemolysis. The HbA1c analyzed by HPLC or capillary electrophoresis did not show this problem because there you are separating hemoglobin from the constituents of plasma and you separate the different types of hemoglobin on those columns or the capillary. If you have high triglycerides or high lipids, those interferences are going to wash out in the solvent front at the beginning of the run, and then later you’ll see the separation of the hemoglobins.”