Applying MASS-FIX to Urine Samples



David Murray, M.D., Ph.D.

David L. Murray, M.D., Ph.D.

Assistant Professor of Laboratory Medicine and Pathology
Division of Clinical Biochemistry
Mayo Clinic
Rochester, Minnesota



I am David Murray. I am a consultant in the Protein Immunology Lab at Mayo Clinic, and I'll be your presenter today as we talk about this "Hot Topic."


These are disclosures. I need to remind everyone that Mayo has filed patents on the use of mass spectrometry to detect monoclonal immunoglobulins, and these have been licensed to Thermo Binding Site.

Learning objective

Here's our learning objectives for today’s "Hot Topic." The first thing I want to do is bring awareness to our clients who order urine immunofixation testing of this new method that we will be implementing and why we did it. The second learning objective: I'd like to review the role of urine testing in this disease that we call monoclonal gammopathies, because a lot has changed over the years. And lastly, I want to talk about some test utilization issues that we've discussed here among our own physicians at Mayo Clinic.

Urine testing in monoclonal gammopathies

Let’s start by talking about what is the role of urine in testing for monoclonal gammopathies. Well, in terms of establishing a diagnosis, early on back in the 1800s, urine was one of the first methods used to detect multiple myeloma. There was a scientist physician, Bence Jones, who discovered that patients with multiple myeloma tend to have urine that if you heat it up it becomes cloudy, and if you cool it down it becomes clear. And this became known as the so-called Bence Jones proteinuria. Historically, this test was used for a long time, but truthfully now in modern clinical testing, it has a very limited role. So, urine doesn't really have a big role in terms of establishing the diagnosis of a monoclonal gammopathy. And that's evident by the most recent guidelines. When they talk about screening for a monoclonal protein, the College of American Pathologists recommends that both serum protein electrophoresis and serum-free light chain measurements should be used to screen for a monoclonal protein. The International Myeloma Working Group, because of other factors, adds that serum immunofixation electrophoresis should also be added to that screening algorithm. But as you see, urine is not really mentioned a lot in these screening algorithms except for when you suspect a patient has AL amyloidosis. In this case, if you screen all the serum and you don't find a monoclonal protein, but your clinical suspicion is still high that the patient may have AL amyloidosis then urine is suggested as a possible means to detect that monoclonal protein, and we'll discuss why in the next slide.

Monoclonal gammopathies: Primary systemic amyloidosis

This is a case I like to share with people who come through my lab to learn about screening for monoclonal gammopathies. And the disease, primary systemic amyloidosis, is a disease that really results from the precipitation or aggregation of light chains associated with monoclonal proteins within the organs of the tissue. This disease has a terrible outcome if left untreated as there is multiple organ failure that happens, but primary amyloidosis can present at very low M-protein levels. So, what I'm showing you here is a traditional serum protein electrophoresis on the top and traditional immunofixation electrophoresis on the bottom of a patient who has a diagnosis of primary amyloidosis. And as you can see it's not hard to appreciate any monoclonal protein within this testing. There's not the traditional M-spike that you would see in multiple myeloma. Sometimes when you go to the urine, as in this case, you can see here, now this is the traditional urine immunofixation and urine protein electrophoresis. I show here an arrow, there's a low indication of the monoclonal lambda light chain that is causing the primary AL amyloidosis in these patients. So sometimes urine testing can provide that extra needed sensitivity, especially in the case of primary systemic amyloidosis.

Kidney failure is very common among patients who have monoclonal proteins, but the disease is really mostly in the bone marrow. So why is it that kidney failure is so common among patients that have monoclonal gammopathies? 

And it really comes down to the fact that M-proteins can be toxic to the kidney, both the glomerulus or the kidney tubules, which we'll go over in just a second. And so, it's really the presence of these monoclonal proteins circulating in the serum when they pass through the kidney can cause the kidney damage. So even though the disease is primarily in the bone marrow, follow-up testing of kidney function in these patients with monoclonal proteins is highly warranted.

Urine testing in monoclonal gammopathies

When we look at the role of urine testing in these diseases, it really comes down to looking at patients who have established disease. So, if a patient is diagnosed with a monoclonal protein in their serum, they really should have some follow-up urine testing to evaluate their kidney function. And you can't just rely on urine total protein measurements. For one thing, dipstick urine testing for total protein is not very sensitive to the presence of immunoglobulin light chains, so you could underestimate the total protein that is in the urine. Sometimes high levels of urine protein may not actually be an indicator of kidney disease and we'll talk about those situations. So, the type of kidney failure really needs to be established using urine protein electrophoreses. And something like urine MASS-FIX can establish the presence of a monoclonal protein in urine which is important because as we discussed, especially light chains, are very toxic to the kidney. So, establishing those two factors will help in the care of the patients.

Serum and urine differences in Ig light chain concentration

So, as we look at the kidney, the kidney performs a function of filtration of the serum, removing toxins and other things that the body doesn't need, and this is really done through the nephron (which is shown here in the little round circle), which consists of a glomerulus, which is the filtration apparatus which filters the serum. And then the tubules are also an essential part because then the tubules will reabsorb certain species such as sodium or potassium or certain proteins that are necessary for the body. So, the tubules will reabsorb those proteins and the wastes will go out through the urinary bladder. This filtration barrier within the kidney is set so that albumin can't be filtered out of the serum. Albumin has a molecular weight of somewhere around 60,000 kilodaltons. So, the filtration barrier of the glomerulus is estimated somewhere between 40 and 50 kilodaltons. When you have a patient, like this patient, that has light chain multiple myeloma, again, you don't really see a traditional M-spike in this protein electrophoresis, but on immunofixation, you can see the presence of a monoclonal light chain in this patient. This monoclonal light chain has a mass somewhere in the vicinity of 23,000 kilodaltons. You can see that it can pass freely through the glomerulus, into the tubules and into the urine of the patient. It's not uncommon for some of these patients that have high levels of monoclonal light chains being produced in their serum upon urine testing. As you see here, a large spike is seen, which is the lambda light chains, and these are the result of light chains passing freely from the serum into the urine and giving you a high level of protein in the urine. But in this case, this isn't really indication of renal failure. This is more an indication of a high level of light chains in the patient's serum.

Types of renal failure in monoclonal gammopathies

So, this brings me to the types of renal failure that you can detect by using electrophoretic methods. 

The first one we just discussed; this is the so-called overflow proteinuria. This is the case in which the M-protein, which is usually the light chains, is so high in the patient's serum that once they get into the urinary space after filtration from the glomerulus, the tubules just simply can't keep up reabsorbing all of it, and this results in a lot of light chains in the patient's urine. But if you look at the typical findings of the electrophoresis shown here, you'll notice that there's not much albumin. There's not much of the other proteins present in serum, just a lot of light chains. This is the so-called overflow proteinuria. It's just an overflow of light chains. And in this case, there's not renal damage here, this is just a result of having too many light chains in your urine. So, this is a case where the protein may be high in the urine, but there really isn't renal failure.

The second type of pattern that we'll talk about is the glomerular pattern. As I show here, the glomerulus is the filtration barrier for the serum and sometimes, M-proteins can bind to this glomerulus resulting in destruction of the filtration barrier. When that happens, that is true renal failure for the patient, and what they end up with is a different looking pattern. In this patient, you can see there's a high level of albumin because that filtration barrier has been destroyed. But there really isn't much in terms of the monoclonal proteins here. And this is true renal failure that needs to be treated in some fashion. But I also remind you this can happen to other types of renal failure such as hypertension, drug use, or diabetes.

The next pattern is really what we call the tubular pattern. So here, the light chains which freely pass into the urinary space can be absorbed by the renal tubule cells resulting in their cell death and diminished reabsorption of proteins within the tubules themselves. Now this is a curious conundrum in that not all light chains will do this, but only certain light chains will do it and it's really hard to predict which light chains will produce this type of renal failure within patients with monoclonal gammopathies. And this is another important pattern to screen for. In this case, what you see is usually slightly lower levels of proteins. In terms of the concentration, you see albumin and a host of the other proteins that can be minutely filtered out of the serum and not reabsorbed by the tubules.

So, these are the three types of patterns that we talked about, and there are the so-called mixed pattern, where you can both have glomerular damage and tubular damage. But this is why urine protein electrophoresis is still important in the follow-up testing of patients with monoclonal gammopathies and can't totally be replaced by just urine total protein measurements.

MASS-FIX testing

As I said, not only is the type of pattern that’s there and is present and is important, but also establishing the fact that there may be light chains which could be toxic to the kidney are there. And this is traditionally the role of immunofixation testing was used, but MASS-FIX testing at Mayo Clinic is going to be used to replace traditional immunofixation testing for numerous reasons, which we'll talk about later. But I will just briefly describe MASS-FIX testing so you can be familiar with how it works and how it simulates immunofixation in our lab.

This is traditional immunofixation done on a gel where the patient's serum or urine is put on six different times. Each one of these lanes is a different application of the patient's urine in this case, then the electrophoresis is done. And MASS-FIX is laid over top and we fix on all the proteins that are present in the first lane, and then the GAM kappa lambda represents IgG, IgA, IgM, kappa, and lambda. And we're looking here for the establishment of a monoclonal protein.

In the MASS-FIX testing, this has just been replaced by immunopurification. So we take the patient's urine, we put it in with these beads that pull down the patient's IgG, A, M, kappa, and lambda in their urine. We spot this on a MALDI plate and then we do the data acquisition, and we overlie the spectrum looking for the presence of a monoclonal protein. In a patient without monoclonal protein, this is the typical result you expect to see where the blue represents all the lambda light chains in the patient. As you can see this has a normal polyclonal background with a broad distribution of immunoglobulin light chains. The kappa is in orange and represents all the kappa light chains that were pulled down out of the urine in this case. And then the IgG represents light chains that were once attached to an IgG, the A and the second figure down represent the light chains that were attached to an IgA, and on the bottom, the IgM. So, in this case, we see no peaks. We see just the broad polyclonal background, similar as we see on SPEP and immunofixation. But in a patient with a monoclonal protein as shown here, you can see now that the lambda in blue shows a peak, a restricted band which represents monoclonal Ig lambda because we see that also in the IgG pull-down. So, this is the basis of the tests. But what are the reasons? Why did we decide to switch from immunofixation to the MASS-FIX for urine? And it really comes down to a question of specificity. And I think the best way to show this is a case that came through our lab at one point.

Increased specificity for urine

This is a case of a 52-year-old man. He had a biopsy done of an organ and his fat, which showed a lambda-based AL amyloidosis. So, there was an underlying suspicion that he should have a monoclonal lambda light chain somewhere within his serum. Traditional serum immunofixation on this patient really just showed us an IgG kappa. We really didn't see any monoclonal IgG lambda in this patient, which we would expect to see. But when we did the serum MASS-FIX, with higher resolution, we could see more clones than what we could see by traditional immunofixation. So, what I'm showing you here is you can see in the pink, which represents the kappa, the teal which represents the light chains that were on IgG, you can see that IgG kappa is a small spike here on the MASS-FIX testing. But you can also see, which we can appreciate on the immunofixation and the IgG lambda, some more IgG lambdas, and then this one, which kind of looks like a free lambda because we don't see it within the IgG.

Well, eventually because of this, we did order urine. And this shows a case where urine was really helpful in detecting the monoclonal light chain associated with AL amyloidosis as you can see here. Here's our monoclonal lambda in the urine. When we perform the same MASS-FIX testing on that, we can see that peak right here, which is the monoclonal lambda. But unlike what we can do with immunofixation, we can line up the masses and then go back to the serum and actually track which clone in the serum is important. This is what we mean by increased specificity for urine. This is something we couldn't do with traditional immunofixation.

Blinded comparison of M-protein LC mass urine – Serum 

So, we looked at that. We looked at the difference of the mass of the light chain detection within the serum and the urine. And we looked at this blinded between multiple cases where we had both serum and urine testing. What I'm showing here on the top is the difference in the urine light chain mass from the serum. You can see that 95% of the time, the mass matches within plus or minus 20 daltons. And so that helps us in being assured that we're on the right clones. But also, it's helping us with quality control. So, if you look at this case here, this caused us to go back and look at what we were doing with the patient's serum in urine. What had happened was we had switched to tracking the monoclonal therapeutic within the serum, and actually the clone was showing up in the urine. And because of this mismatch in mass, we were able to use this as a quality control parameter to give better results to both our serum and urine test.

This just shows a case where you get the match. So, on the top is the patient's urine. You can see in this case we only pulled down light chains, kappa light chains, in the patient's urine. But the mass as shown here are the +2 charge state. If you look down at the serum, you can see that this matches very well.

Test code changes for urine

So, this brings me to the last topic for this "Hot Topic," and that is what is really changing for our clients. I want to start by what's not changing first. What's not changing: We're still going to accept 24-hour and random urine samples. That doesn't change. Urine protein electrophoresis that we've traditionally done and will still be available for ordering, and you can still order that without the immunofixation or the mass spec, and you can still order a panel where you both get the urine protein electrophoresis and urine MASS-FIX testing. So, that's what's not changing.

But what is changing? So, we had discussions with our hematologists and our nephrologists to talk about test utilization, and we came up with the idea that maybe we are going to do a reflex test. Talking about those types of proteinuria, we decided that we might start with urine MASS-FIX testing first, and if we don't detect a monoclonal protein, they felt like they actually could just use the urine total protein as a marker for the type of renal failure that was going on. But if we did detect a monoclonal protein by our urine MASS-FIX test, then we will perform UPEP. Looking at our cases that come through our labs, 70% of our urine testing is negative for a monoclonal protein. So, we felt like we could eliminate 70% of our urine protein electrophoresis using this reflex algorithm. So here at Mayo Clinic, this is going to be the preferred way of testing for urine among our hematologists and nephrologists in patients that have a monoclonal protein detected. And so, this will also be available for any of our clients who would also like to use this algorithm.

Thank you

And with that, I'd just like to thank you for tuning in to this "Hot Topic." Hopefully, you learned a little bit about urine testing in the setting of monoclonal gammopathies, and we're always here to answer your questions or to talk and discuss further our testing.


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This post was developed by our Education and Technical Publications Team.