Detecting monoclonal light chains in urine: microLC‐ESI‐Q‐TOF mass spectrometry compared to immunofixation electrophoresis (2014)

When urine protein electrophoresis (PEL) and immunofixation electrophoresis (IFE) are ordered to identify a monoclonal immunoglobulin, it is recommended that urine be adequately concentrated in order to increase diagnostic sensitivity (Keren, 1998; International Myeloma Working Group, 2003). Our laboratory concentrates urine samples up to 200-fold, depending on the urine protein concentration, and will dilute high protein samples in order to not overload the gels and overwhelm the antisera. The centrifugal concentrators take from 10 min (10-fold) to 45 min (200-fold), and large concentration factors may require the transfer of the specimen during the process. In addition, a sample volume of 20 ml is required for the 200-fold concentration and if 20 ml is not available, the specimen is rejected (Roden et al, 2008). We recently demonstrated that electrospray-ionization time-of-flight mass spectrometry (microLC-ESI-Q-TOF MS) is a viable alternative to PEL and IFE for identifying monoclonal light chains in serum and urine (Barnidge et al, 2014). We termed this methodology “monoclonal immunoglobulin Rapid Accurate Molecular Mass” or miRAMM. Our initial work demonstrated the principle that a monoclonal light chain found in serum has the same molecular mass as in urine and the exact mass of the light chain can be used to identify the clonal product for monitoring disease. The sensitivity of miRAMM in serum assays appears better than serum IFE, and in this study we wanted to understand the relationship of miRAMM with concentrated urine specimens. We obtained 157 waste urine specimens: 100 urine samples of known light chain positivity (50 Kappa and 50 Lambda) and an additional randomly selected 57 urine samples. We performed urine PEL and IFE according to the Clinical Immunology Laboratory protocols and urine and serum miRAMM according to our published miRAMM procedures (Barnidge et al, 2014). The miRAMM protocol used in this study reduces disulfide bonds, separates immunoglobulin heavy and light chains, and analyses total light chain (bound and free). Of the 157 urine samples tested by IFE, 42 were negative and 115 were positive (Table 1). Among the 42 samples that were negative by IFE, 41 were also negative by miRAMM. Mass spectrometry detected a monoclonal immunoglobulin light chain in one of the urine samples that had a normal IFE. This patient had a history of Waldenström macroglobulinemia, had a 9·1 g/l serum M-spike (IgM kappa), and had a 23·243 kDa monoclonal kappa light chain in both the serum and urine (Table 2). The 115 IFE-positive samples were sub-divided into three groups based on the urine PEL: (i) PEL with a quantifiable M-spike, (ii) PEL with a non-quantifiable ‘fuzzy band’, and (iii) normal PEL (Table 1). Of the 115 IFE-positive samples, 112 (97·4%) had monoclonal light chains as identified by miRAMM. All 49 of the samples with a quantifiable M-spike had a monoclonal light chain detected by miRAMM. The three negative samples were from patients with low protein concentrations that required 200-fold concentration prior to PEL and IFE (Table 2). Even though these samples were concentrated 200-fold, two of these three samples had no PEL abnormality and the third had a non-quantifiable, fuzzy band. The goal of this study is to expand upon our initial observation, which demonstrated that accurate molecular mass can be used to detect a monoclonal immunoglobulin in patient urine (Barnidge et al, 2014). We have coined miRAMM as the acronym for the rapid accurate molecular mass of monoclonal immunoglobulin light chains. Neat urine samples analysed by miRAMM were compared to concentrated urine analysed using standard PEL and IFE methods. Our results indicate that miRAMM of neat urine has a sensitivity that is close to IFE of urine that is concentrated up to 200-fold. Three samples with low protein concentrations and low concentrations of monoclonal immunoglobulin were not detected by miRAMM. Further work will need to focus on ways to prepare the miRAMM samples with smaller reagent volumes and thus smaller pre-analytic sample dilution. The ability to analyse neat urine will simplify handling and reduce time and costs. Although three of 115 IFE-positive samples were not detected by miRAMM, one of 42 IFE-negative samples contained a monoclonal light chain detected by miRAMM. This patient had an IgM kappa M-spike in the serum, and the monoclonal light chain in the serum had the same exact mass as the monoclonal light chain detected in the urine. The normal urine IFE result is therefore false-negative and, in conjunction with the three miRAMM false-negative samples, suggests that the sensitivity of miRAMM with neat urine is close to the sensitivity of IFE with 200-fold concentrated urine. In addition to the use of miRAMM as a urine screening test, the assay can be used for subsequent monitoring of the light chain exact mass. The resolution of mass spectrometry allows the specific malignant clonal product to be monitored and should remove any confusion with post-therapeutic small transient clones. We have described a new method to identify and monitor monoclonal urinary light chains. Further research is necessary to refine the method and precisely define the relationship to current assays, but the benefits are significant. By minimizing the need to concentrate and transfer specimen prior to testing, miRAMM improves efficiency and decreases possible errors. The minimal reagent needs of protein analysis by mass spectrometry should also lead to significant cost savings for the laboratory. All 4 authors contributed to the study design, data acquisition and analysis, and participated in the writing of the manuscript. The authors declare no conflict of interest.