Performance of immunotyping versus immunofixation for the detection of monoclonal gammopathies

Serum immunofixation (IF) is routinely used in clinical laboratories to confirm the presence of M-proteins and determine isotypes. For laboratories looking for a more automated method, immunotyping (IT) by capillary electrophoresis is also FDA-approved to confirm M-protein presence and isotype determination.

In this SelectScience webinar, now available on demand, Dr. Katie Thoren, Ph.D., DABCC, assistant attending clinical chemist at Memorial Sloan Kettering Cancer Center, discusses a recent study comparing the performance of IT and IF for the detection of M proteins, reviews the limitations of each test, and discusses potential strategies for mitigating these limitations.

Read on for highlights from the Q&A discussion and find out about challenges associated with switching from IF to IT.

Would you use IT instead of IF or would you use a combination of these techniques? If you suggest using a combination, when would you use each one?

KT: After doing this study, I'm very comfortable with IT so I think we would primarily use that for our samples. However, I do think there are situations where reflexing to IF would be helpful. IT would be the more automated, more efficient way of testing, and then in certain cases it would be beneficial to reflex to IF. I think it's also going to depend on whether the lab has patient history and access to clinical information as well.

Some of the limitations of IT are that it's not as good for the detection of really small bands in the beta region. For example, if we run IT on a patient that has a history of an M protein in the beta region, and we're not seeing it by IT, that would be a case where we would then reflex to IF.

Another example of where IT is not as good in detecting M proteins is when we have hypogammaglobulinemic samples, where there is a really low signal in the gamma region. If we're not seeing anything by IT, we can reflex to IF.

Are there any challenges associated with switching from IF to IT? And if so, what are they?

KT: We did this study in the winter of 2019 and were getting ready to switch to IT in February of 2020, but then the COVID-19 pandemic interrupted this process. So, unfortunately, we have not switched to IT yet, but I anticipate that the largest challenge is probably going to be just getting familiar with doing the interpretations of IT.

I think we get used to interpreting the IF and we forget what it was like when we learned IF, but it does take some time with both IF and IT to become familiar with how to interpret those results. Again, the hardest part is deciding when to call a small band positive and being able to distinguish between what's real and what's noise. That takes time and experience of practicing with the samples. It is helpful if you have patient history. In our case, we would be seeing patients longitudinally, so that's going to help us familiarize ourselves with how to read the IT results.

The training that we got from Sebia was really helpful in that it's a very methodical, stepwise approach to reading the traces. They walk you through the different steps of how to read the results, which makes it more approachable when you're first starting out to learn the IT interpretations. That was certainly really helpful. So, that's the biggest challenge I anticipate is the pathologist/technologist getting used to reading the results.

What was your gold standard for identifying M proteins with either IT or IF?

KT: We had huge amounts of data and you could compare this many different ways, and we did compare it to a lot of different things. When we were looking at whether IF or IT could detect the original clone, our gold standard was the combined results from all of the tests from IF, IT, and the free light chain, or the urine.

As we have patient history, we'd know with a sample that, for example, the patient had a history of a G Kappa. So, we looked at those results, and if any of them detected the G Kappa, we called that a true positive. If none of those tests detected the original clone, that was a true negative, because patients can be in remission and that protein could not be there. That's how we did the gold standard for the original clone detection and categorized whether the IF result was a true negative, false positive, etc.

When comparing techniques just by band, we didn't have a gold standard, we were doing concordance between the two. This is a little bit harder because if we look at it by band, for some of our samples, there's multiple bands. Due to our patient population, we have a lot of complex samples, including patients on monoclonal antibody drugs, so it’s hard to know which is right. We had thought about using a more sensitive analytical technique to serve as the gold standard but ended up using concordance between those when we analyzed the data just by band.

So, it was a tough thing to do, but our gold standard, at least for the original clones, ended up being a panel of test results.

Which of the two methods were most cost-effective for your study purposes?

KT: As I mentioned, we have not yet switched to IT, so we haven't looked at all the full details of the cost analysis here. It does get complex because we're talking about having different numbers of instruments, and there's a factor of technologists’ time. So, there's certainly a lot to consider that goes into this cost comparison.

For us, we’ve had a high turnover of technologists, and it takes a long time to train them. Therefore, I think having a more automated, less labor-intensive method for running these samples is really important, and that's something to factor in. In short, I think a big part of cost-effectiveness is going to be technologist time savings for IT.

If a doctor requests a serum protein electrophoresis (SPEP) and the profile looks like a pattern compatible with inflammation, but there are distortions in the beta or gamma regions, would you suggest IT even in the case where patient history may be unknown?

KT: Yes. Our approach is that if there are abnormalities in the SPEP, it's always better to reflex to IF or IT to confirm the presence or absence of a monoclonal protein, especially when the history is not known. We're at a cancer center where there's a high prevalence of monoclonal gammopathies so may take a slightly different approach as we're on high alert for the presence of M proteins. We actually don't really use SPEP for other indications. We don't tend to comment on inflammatory responses. We are really using these tests for the identification and confirmation of the presence of monoclonal proteins.

As you did not know any other clinical results of samples in the study, how did you select the dilutions for IT without knowing concentrations? If you had a sample with unknown concentrations, which dilution would you prefer for IT?

KT: For the study samples, we first ran the SPEP to then know the appropriate dilution for the IT. So, we did go off some data, we weren't just guessing up the concentration.

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