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Published: August 2008Print Record of Viewing
With the introduction of Mayo’s expanded serum monoclonal protein panel, which includes quantitation of the kappa and lambda free light chains, urine monoclonal protein studies are no longer required as part of the initial diagnostic screen for monoclonal gammopathies. Dr. Katsmann discusses Mayo’s approach to the diagnosis of monoclonal gammopathies and the use of serum free light chain assays.
Presenter: Jerry Katzmann, PhD,
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With the introduction of Mayo's expanded serum monoclonal protein panel, which includes quantitation of the kappa and lambda free light chains, urine monoclonal protein studies are no longer required as part of the initial diagnostic screen for monoclonal gammopathies.
Jerry Katzmann, PhD, from the Division of Clinical Biochemistry and Immunology at Mayo Clinic, discusses Mayo's approach to the diagnosis of monoclonal gammopathies and the use of serum free light chain assays.
I will be talking about three tests: protein electrophoresis, immunofixation electrophoresis, and serum free light chain quantitation. There are of course lots of other diagnostic tests used for monoclonal gammopathies; hemoglobin for anemia, creatinine for renal function, bone x-rays for bony lesions, for example. In all of the tests, in the bottom portion of the slide are to help identify and characterize the particular disease associated with the monoclonal gammopathy.
There are at least a dozen different plasma cell proliferative disorders. On this slide I’ve listed the incidents of these in our Mayo Clinic practice. By far the most common disorder is monoclonal gammopathy of undetermined significance or MGUS. This is a premalignant condition and has no clinical symptoms; no anemia, no renal disease, no bone lesions. This is followed in incidence by other diseases such as multiple myeloma or primary amyloidosis or macroglobulinemia. All of these diseases listed here will have different clinical presentations and may require different treatments.
The testing we’re talking about are tests to indentify virtually all of these plasma cell proliferative disorders by indentifying the presence of monoclonal gammopathy. That is, diagnosing a monoclonal immunoglobulin. In addition to diagnostic uses PEL, IFE and free light chain assays may be useful for prognosis of either progression or survival and also may be used for disease monitoring.
This slide is an example of a normal serum analyzed by protein electrophoresis or PEL and immunofixation electrophoresis or IFE. The upper portion of this slide is a picture of an agarose gel with the five fractions identified; albumin, Alpha 1, Alpha 2, Beta, and Gamma. Above the gel is the gel-scan or electropherogram, which allows us to quantitate each fraction.
The lower portion of the slide has a picture of an immunofixation gel. In this assay duplicate gel lanes are electrophoresed and then specific antisera is used to precipitate or immunofix the various immunoglobulin molecules in the agarose. We can see that all these molecules are distributed as smears rather then as discreet bands.
This next slide is an analysis of serum from a patient with multiple myeloma. On the PEL gel we see a dense discreet band representing the monoclonal immunoglobulin. And on the IFE gel we see this monoclonal protein has an IgG heavy chain and kappa light chain. So this patient has a monoclonal plasma self-proliferative disorder that is secreting an IgG kappa molecule.
The PEL also allows us to quantitate the amount of this protein. And from the large M-Spike we can predict that this patient most likely has multiple myeloma and we can monitor the affects of therapy by monitoring the size of the M-Spike. These are straight forward assays; the problem is that not all monoclonal gammopathies are this obvious on serum PEL & IFE.
Although Multiple Myeloma is a malignancy with a large number of colonel plasma cells and should be easy to detect by these assays. This slide points out the problem. Approximately, 75% of myeloma patients secrete intact immunoglobulins that will have large M-spikes. But approximately, 25% secrete only light chains or nothing at all.
In this next slide, illustrates the analysis of serum from a patient with light chain multiple myeloma. We see that PEL has only a minimally abnormal pattern and the IFE has a small amount of monoclonal lambda light chain. Although this is a myeloma, it is very different from the IgG kappa seen on the previous slide.
The urine however, is a different story. Here we see a large M-spike on PEL and an intense monoclonal lambda band on IFE. It is because of these sorts of patients, that is, light chain monoclonal gammopathies that we have always recommended PEL & IFE on both serum and urine.
In 2002 an assay for free light chain quantitation became available. These antisera do not bind the light chain contained in intact immunoglobulins. They recognize epitopes on light chains that are hidden within the binding to the heavy chains.
When the light chains are in solution and not bound to heavy chain these epitopes are available and can be quantitated. There are two separate assays, one for kappa free light chain and one for lambda free light chain and they each have their reference range. In addition the kappa lambda free light chain ratio has a reference range, and this is useful diagnostically. If this ratio was high, it implies a clonal expansion of cells producing kappa free light chain and conversely if it’s low, it suggests an expansion of lambda-producing cells.
This next slide illustrates free light chain results from normals and patients. In the bottom corner are results from a series of normal donors. As we age, (illustrated by the red dots) renal clearance goes down, and if we have a polyclonal elevation of immunoglobulins (illustrated by the green dots) there may be an increase in kappa and lambda free light chains, but the ratio remains in the reference range illustrated by the rectangular box.
In patients with lambda light chain multiple myeloma or with kappa light chain multiple myeloma, we see that all the sera are outside the reference range but the median ratios are about 100 times normal. I’ve also included on this slide results from patients with non-secretory myeloma. These myeloma patients, remember, have normal PEL & IFE assays. But you can see that approximately two-thirds of them have abnormal serum free light chain results. So this assay seems exquisitely sensitive for monoclonal free light chain diseases. Now if we can identify these monoclonal light chain diseases with the serum free light chain assay, it raises the question of whether we still need urine in our screening panel.
This slide illustrates how we tested this hypothesis. We identified 428 patients with the monoclonal protein in the urine, and who also had serum studies that included PEL, IFE and free light chain. We wanted to test whether this combination of serum assays would detect all these patients with abnormal urine studies. This slide lists the distribution of diagnosis of this cohort, approximately one-third have multiple myeloma, approximately one-third have primary amyloidosis and approximately one-third have premalignant conditions of MGUS or smoldering multiple myeloma.
Now we see on this slide that if we tested serum with PEL & IFE we missed almost 7% of the cases. The 28 cases that are missed are almost all serious disorders that require intervention and treatment. It is this kind of experience that has led to the recommendations, that both serum and urine should be tested in the diagnostic screen.
If we now include the serum free light change in our analysis, on this slide we see that the combination of serum, protein electrophoresis, immunofixation electrophoresis and free light chain quantitation detect almost all of the patients detected by urine studies, only 2 patients out of 428 were missed and neither required intervention. And this has led us to conclude that we should add serum free light chain to our diagnostic screen and eliminate urine studies from this initial testing.
Now in addition to being clinical appropriate and easier on the patient it can also make it economic sense. Although adding the free light chain assay adds costs (illustrated on this slide) removing the urine tests more then offsets these costs.
So if we include the serum free light chain assay as part of the initial diagnostic screen, how will we use it?
Well obviously it becomes part of our diagnostic panel and association with PEL and IFE. In addition, although I haven’t talked about this, baseline values of the free light chain assay have been shown to have prognostic value for predicting progression in patients with MGUS and smoldering myeloma, as well as for overall survival, in patients with multiple myeloma, plasmacytoma & primary amyloidosis. And lastly, in patients with no M-spike, it can be used to monitor therapy and disease course.
So in summary and in conclusion, we recommend using serum protein electrophoresis, immunofixation electrophoresis and free light chain quantitation as the initial diagnostic screening panel for patients suspected of having a plasma cell proliferative disorder. This panel provides sufficient diagnostic sensitivity without the need for urine studies.
In addition if the patient does indeed have a monoclonal gammopathy, this panel provides prognostic information in many plasma cell proliferative disorders. And lastly, if there is no M-spike the serum free light chain assay may provide a quantitative value for disease monitoring.