Robin (00:01):

Hello and welcome to today's installment of Diagnostics in Practice, where we discuss new and innovative test offerings from Mayo Clinic Laboratories. I'm Robin Huiras, a senior marketing specialist with Mayo Clinic Labs, and today I'm joined by Dr. Tony Maus, who is the co-director of the Clinical Mass Spectrometry Laboratory at Mayo Cli nic. Our discussion today will focus on Mayo Clinic Laboratories' new insulin analogs assay. This test targets a longstanding diagnostic challenge, which is clarifying the cause of unexplained hypoglycemia. Thanks for joining us today, Dr. Maus.

Dr. Maus (00:36):

Thanks for having me. I'm very excited to be here.

Robin (00:38):

Now, before we get into our discussion, I'm wondering, can you share with our listeners a little bit about yourself and your experience at Mayo Clinic, particularly around this area of testing?

Dr. Maus (00:49):

Sure. I started at Mayo Clinic immediately after graduate school doing test development for the Clinical Mass Spectrometry Lab, or CMSL as I'll refer to it henceforth, and the Clinical Forensic Toxicology Lab, or CFTL. I served in various research and development roles supporting these laboratories for seven years. Two years ago, I had the opportunity to transition to a director role. I now have primary oversight of endocrine and vitamin testing in CMSL and provide technical support to the drug testing in CMSL and CFTL.

Robin (01:18):

Great. Thanks for sharing your background with us, Dr. Maus. Your experience in this testing space really does highlight the expertise that goes into test development at Mayo Clinic. Now, I'd like to start off our talk today at a really high level. Why is this test important, and what is the clinical problem it will solve?

Dr. Maus (01:37):

Primary use of this test is aiding in the evaluation of patients with unexplained hypoglycemia. Given the critical role insulin plays in regulating blood glucose level, measuring insulin is a critical part of the workup when a patient presents with hypoglycemia. These measurements have historically been performed nearly exclusively by immunoassays, but immunoassays have limitations that hinder the evaluation of hypoglycemia. This can create a situation where lab results don't align with the clinical presentation, and clinicians are left without a clear answer. This test is designed to close that gap by differentiating between and measuring human insulin and synthetic insulin analogs.

Robin (02:13):

Got it. Let's swing back to talk about those immunoassay limitations. Can you break those down for us?

Dr. Maus (02:19):

Absolutely. So to start, it's widely known that immunoassays can produce falsely high or falsely low results due to interferences from a myriad of things, such as heterophile antibodies, biotin, hemolysis, etc. This test can be used as an alternative means to measure insulin if such interferences are suspected. However, the primary insulin immunoassay limitation this test addresses is varying cross-reactivity with human insulin and insulin analogs. Across manufacturers, immunoassays vary widely in their cross-reactivity with insulin analogs. Some have a high degree of cross-reactivity, while some have no cross-reactivity at all. The situation is further complicated by the fact that even within a given manufacturer, the cross-reactivity can differ for the various insulin analogs that are available. The immunoassays are not able to differentiate between the analogs and/or endogenous human insulin, nor are they able to provide any indication that analogs are present. Thus, an immunoassay result may be elevated by the presence of an insulin analog or may be completely unable to detect it.

This inconsistency can make evaluation of hypoglycemic patients very challenging, especially across different labs or platforms.

Robin (03:24):

Yeah, that does sound complicated, Dr. Maus. So how does this new assay approach the challenge differently?

Dr. Maus (03:30):

The assay, which goes by the test ID INSAL, uses liquid chromatography, high-resolution, accurate mass- mass spectrometry to differentiate between and directly measure human insulin and the analogs aspart, lispro, glulisine, and glargine from a single sample in a multiplex manner. The combination of both liquid chromatography and high-resolution mass spectrometry is critical to accurately measuring these forms of insulin.

High-resolution mass spectrometry itself provides a high degree of specificity and is ideally suited for measurement of intact peptides like insulin. However, in the case of insulin analog analysis, not even high-resolution mass spectrometry detection is enough. Liquid chromatography separation enhances the specificity of the analysis overall, but in the case of insulin analogs, it is absolutely necessary for distinguishing between human insulin and the synthetic insulin lispro. The two differ only in the position of two amino acids, which are flipped in lispro compared to native human insulin.

Thus, the molecular weight is identical, and you cannot differentiate between them with a hypothetical mass spectrometer with unlimited mass resolution. In our method, we have chromatographically separated these two forms of insulin, ensuring accurate and specific measurement of all of the aforementioned insulin analogs.

Robin (04:44):

It's really great to hear that the new test provides more specific answers. Let's pivot a little bit and shift into the clinical utility of this test. When should physicians be thinking about ordering?

Dr. Maus (04:55):

As we discussed earlier, the primary application of this test is cases of unexplained hypoglycemia, and it is a powerful alternative assessment for any case when the clinical presentation does not align with insulin immunoassay results. This test is uniquely suited to assist in cases where factitious hypoglycemia is suspected due to insulin exposure that is not acknowledged or understood. As I mentioned before, it can also be used in cases where immunoassay interferences are a possible explanation for the unexpected results. An additional use case we have not talked about thus far that is more obvious, but worth mentioning nonetheless, is monitoring of treatment adherence to prescribed insulin therapy, a.k.a., treatment compliance.

Robin (05:35):

Thanks for sharing your thoughts on the use cases for this new test, Dr. Maus. If you don't mind, I'd like to dig into the topic of factitious hypoglycemia a little bit because to me, it sounds like these situations might be complex. How does this test help in these sensitive scenarios?

Dr. Maus (05:50):

Of course. Factitious hypoglycemia cases are relatively rare, sensitive, and diagnostically challenging situations where patients are exposed to insulin analogs outside of intended therapy. Unfortunately, there are instances where patients are harming themselves or relatives who want to harm them administer insulin covertly, making it difficult for clinicians to confirm drug present without this specialized assay. In cases where factitious hypoglycemia is suspected, this test provides objective evidence of exposure. It allows clinicians to confirm or exclude insulin analog involvement with a level of specificity that simply hasn't been available before.

Robin (06:27):

Thanks, Dr. Maus. That really does make a lot of sense, and it's great to know that this test offers a way to provide such critical insight in these sensitive cases. From a physician's perspective, I'm wondering, what's the biggest advantage here?

Dr. Maus (06:40):

Yeah, this test provides specific, accurate, and definitive measurements for human insulin and insulin analogs that up to this point have not been available. Managing hypoglycemic patients, especially those that have the potential to be exposed to insulin analogs, is inherently complex. This test greatly reduces that complexity by providing valuable insight into potential causes of hypoglycemia. It allows clinicians to confidently rule in or rule out potential explanations of hypoglycemia, which reduces the time needed to reach a final diagnosis.

Robin (07:10):

Yeah. And that's just so important when we're talking about these sensitive cases. And I appreciate you sharing your thoughts on that. I'm curious, how does that translate into patient impact?

Dr. Maus (07:21):

We've talked about a variety of potential scenarios where this test provides answers in a way no other test can. Regardless of whether the situation is really complex, like surreptitious use of insulin analogs or simply immunoassay interference, test results that provide diagnostic clarity faster translates to patients getting the most effective care more quickly. This enables appropriate treatment decisions, minimizes unnecessary or incorrect interventions, and allows the care team to quickly provide any additional support to the patient that is necessary. Ultimately, better diagnostic clarity leads to better care.

Robin (07:55):

Providing better care is just so critically important to any patient. Thanks for sharing your perspective on that, Dr. Maus. I'd like to shift gears a little bit again and talk about how the test result is reported for this assay. What information does a clinician actually receive in that report?

Dr. Maus (08:13):

The assay reports quantitative values for human insulin and the analogs aspart, lispro, glulisine, and the metabolites of glargine. Thus, instead of a single number, like you would get from an immunoassay, you are getting a panel of results that tells you exactly what forms of insulin are present and at what level, which can be really beneficial in the complex cases we talked about previously.

Robin (08:33):

Thanks for detailing that result report, Dr. Maus. I'm curious, are there nuances to this test report that clinicians should keep in mind when interpreting results?

Dr. Maus (08:43):

Absolutely. There are a few important ones. First, while the test provides quantitative data, there are no established therapeutic reference intervals for insulin analogs. Therefore, it is not intended for therapeutic drug monitoring in the traditional sense. Instead, interpretation is more focused on presence, absence, and the relative levels in the clinical context. Also, clinicians must be aware that some of the synthetic analogs have been designed for longer half-lives in circulation than native insulin. Thus, the detection windows can vary based on the specific analog as well as patient factors like metabolism. So the results should always be considered alongside the broader clinical picture.

Robin (09:17):

Thanks. And what about testing limitations? What should clinicians know?

Dr. Maus (09:21):

Yes, the panel does not currently include the very long-acting insulin analogs like degludec and detemir due to analytical challenges. It also cannot distinguish between endogenous and exogenous human insulin, and like all insulin assays, hemolysis can interfere with the results.

Robin (09:37):

Thanks for sharing those important details. This sounds like such a unique test. Are there other labs currently offering anything like this that you're aware of?

Dr. Maus (09:45):

No. At this point, this is the only lab offering a clinically validated assay like this commercially. This can, in part, be attributed to the technical complexity. The sample preparation protocol is sophisticated, and we are utilizing state-of-the-art, high-resolution mass spectrometry for this application. However, the unique and impactful answers this test is able to provide makes the complexity worthwhile. Not only does this create clinical value, but also positions the test as a leader in this space.

Robin (10:13):

Nice. It's great to hear that Mayo Clinic Labs has taken the lead in this space. I'd like to take a step back to touch on the amount of time this test has taken to develop. Can you talk about that journey, Dr. Maus?

Dr. Maus (10:24):

Absolutely. This assay has been in development for just over a decade. This is partly due to shifting priorities through time, which are an ever-present aspect of laboratory medicine that I'm sure many in the audience can relate to. There were also some critical reagents that were taken off the market when the test was nearing ready for implementation, which forced us to take several steps back. However, most of all, this reflects the level of analytical complexity involved. I've had a unique experience with this test. In my previous development roles, I was very involved hands-on in the early stages of development, and now I get to serve as the lab director and be part of the team that finally brings this to fruition. Therefore, I've had the pleasure of seeing this test evolve from concept to clinically available assay. However, it really was a team effort. Although countless people have made valuable contributions, I would especially like to thank Michelle Campbell, who finalized development of the assay, and the outstanding technical specialist team that finished the verification in CMSL.

Robin (11:17):

Wow, that's a really remarkable timeline. Beyond the challenges you just mentioned, were there others in bringing this test to market?

Dr. Maus (11:25):

Yeah, this test presents a plethora of challenges. The levels are relatively low, which necessitates purification of insulin with an antibody. Thus, we are performing so-called immunopurification, and it took us quite some time to identify the perfect antibody and optimize the sample preparation protocol. As I mentioned previously, the chromatographic separation of the insulin forms with similar amino acid sequences is challenging. So it took some time to ensure we could do that consistently over time. High-resolution mass spectrometry is also inherently a relatively complex technology. One time-consuming challenge we have yet to fully overcome is accurate measurement of the extremely hydrophobic longest-acting insulin analogs such as detemir and degludec. Overall, the timeline was necessary to ensure a robust, accurate, and clinically impactful test.

Robin (12:09):

Thanks for digging a little bit more into those challenges, Dr. Maus. Seems like in the clinical scenarios that we've talked about earlier, there's a great need for the sort of clarity the test provides. I'd love to talk a little bit more about the big picture and how this test might actually change clinical practice over time.

Dr. Maus (12:27):

I think this will become a standard part of evaluating unexplained hypoglycemia, particularly when exposure to insulin analogs is possible. Ultimately, this will reduce the time to diagnosis in the most challenging hypoglycemia cases, which will benefit patients and clinicians. Once awareness is spread, I expect it to become a go-to tool.

Robin (12:45):

That's just so great to hear, Dr. Maus, and I really appreciate you sharing your perspective on the potential of this test to change clinical practice. It does sound to me like Mayo Clinic and Mayo Clinic Labs is really closing a gap in this particular area of care. As we begin to think about wrapping up our conversation, I'm wondering, do you have any final thoughts on the benefit that this test offers to patients and physicians you'd like to share?

Dr. Maus (13:08):

To anyone out there listening, we love hearing about cases where our testing makes a positive impact. So if you have such a case, please reach out. We also welcome feedback on ways to improve our testing. I will also add that finding a solution for the long-acting insulin analogs is very much a future goal.

Robin (13:24):

Thanks. I look forward to hearing more about that test as time goes on, Dr. Maus. And I really appreciate, again, you walking us through the details of this test and for sharing the story behind the test development as well. It's clear to me, and I hope our listeners as well, that there's been a tremendous amount of work behind this assay and bringing it forward.

Dr. Maus (13:43):

Yeah, it has been a tremendous team effort and many years in the making to bring this forward. I very much appreciate the opportunity to speak to you about it today.

Robin (13:51):

And thanks to our listeners too for joining us today. I do hope you enjoyed our discussion and will tune in again for our next installment of Diagnostics in Practice.