Band 54, Heft 6 - Special issue: Protein electrophoresis and serum free light chains in the diagnosis and monitoring of plasma cell disorders: laboratory testing and current controversies / Guest Editors: Jillian R. Tate, Maria Stella Graziani, Peter Mollee and Giampaolo Merlini

Monoclonal immunoglobulins are markers of plasma cell proliferative diseases and have been described as the first (and perhaps best) serological tumor marker. The unique structure of each monoclonal protein makes them highly specific for each plasma cell clone. The difficulties of using monoclonal proteins for diagnosing and monitoring multiple myeloma, however, stem from the diverse disease presentations and broad range of serum protein concentrations and molecular weights. Because of these challenges, no single test can confidently diagnose or monitor all patients. Panels of tests have been recommended for sensitivity and efficiency. In this review we discuss the various disease presentations and the use of various tests such as protein electrophoresis and immunofixation electrophoresis as well as immunoglobulin quantitation, free light chain quantitation, and heavy-light chain quantitation by immuno-nephelometry. The choice of tests for inclusion in diagnostic and monitoring panels may need to be tailored to each patient, and examples are provided. The panel currently recommended for diagnostic screening is serum protein electrophoresis, immunofixation electrophoresis, and free light chain quantitation.

The disease causing agent in systemic AL amyloidosis is a monoclonal immunoglobulin free light chain, or fragments thereof, circulating in the blood. It is not surprising, therefore, that measurement of serum free light chains plays a central role in the management of this disorder. In this paper, we review the utility of the serum free light chain assay in the investigation, prognostication and monitoring of AL amyloidosis. Data on the two currently available commercial assays is compared and some practical applications of the assay’s use are presented. While there are limitations, it is clear that the availability of the free light chain assay in the laboratory is a major advance and plays an essential role in the management of patients with AL amyloidosis.

Recently, monoclonal gammopathy of renal significance (MGRS) reclassified all monoclonal (M) gammopathies that are associated with the development of a kidney disease but do not meet the definition of symptomatic multiple myeloma (MM) or malignant lymphoma. The purpose was to distinguish the M gammopathy as the nephrotoxic agent independent from the clonal mass. The diagnosis of MGRS obviously depends on the detection of the M-protein. More importantly, the success of treatment is correlated with the reduction of the M-protein. Therefore, familiarity with the M-protein tests is a must. Protein electrophoresis performed in serum or urine is inexpensive and rapid due to automation. However, poor sensitivity especially with the urine is an issue particularly with the low-level M gammopathy often encountered with MGRS. Immunofixation adds to the sensitivity and specificity but also the cost. Serum free light chain (sFLC) assays have significantly increased the sensitivity of M-protein detection and is relatively inexpensive. It is important to recognize that there is more than one assay on the market and their results are not interchangeable. In addition, in certain diseases, immunofixation is more sensitive than sFLC. Finally, novel techniques with promising results are adding to the ability to identify M-proteins. Using the time of flight method, the use of mass spectrometry of serum samples has been shown to dramatically increase the sensitivity of M-protein detection. In another technique, oligomeric LCs are identified on urinary exosomes amplifying the specificity for the nephrotoxic M-protein.

Background: The measurement of circulating free light chains (FLC) is of utmost importance in immunoglobulin light chain (AL) amyloidosis, being a fundamental part of the diagnostic workup, prognostic stratification and assessment of response to therapy. Renal failure is a common feature of AL amyloidosis and can considerably affect the concentration of FLC. Methods: We assessed the impact of renal failure on the clinical performance of the Freelite assay in 982 consecutive, newly diagnosed patients with AL amyloidosis, 822 with estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73 m 2 , and 160 with eGFR <30 mL/min/1.73 m 2 . Results: The diagnostic sensitivity of the κ/λ FLC ratio was lower for λ amyloidogenic FLC in patients with renal failure (81% vs. 60%, p<0.001) and the FLC concentration had no independent prognostic significance in patients with severe renal dysfunction. However, FLC response to chemotherapy could still discriminate patients with better outcome. Conclusions: Renal failure is a relevant interference factor when using the Freelite assay for the identification of the amyloidogenic light chain and for prognostic assessment in patients with AL amyloidosis and renal failure.

The measurement of monoclonal protein (M-protein) is vital for stratifying risk and following individuals with a variety of monoclonal gammopathies. Direct measurement of the M-protein spike by electrophoresis and immunochemical measurements of specific isotypes or free light chains pairs has provided useful information about the quantity of M-protein. Nonetheless, both traditional electrophoresis and immunochemical methods give poor quantification with M-proteins smaller than 10 g/L (1 g/dL) when in the presence of polyclonal immunoglobulins that co-migrate with the M-protein. In addition, measurements by electrophoresis of M-proteins migrating in the β- and α-regions are contaminated by normal serum proteins in those regions. The most precise electrophoretic method to date for quantification involves exclusion of the polyclonal immunoglobulins by using the tangent skimming method on electropherograms, which provides a 10-fold improvement in precision. So far, however, tangent measurements are limited to γ migrating M-proteins. Another way to improve M-protein measurements is the use of capillary electrophoresis (CE). With CE, one can employ immunosubtraction to select a region of interest in the β region thereby excluding much of the normal proteins from the M-protein measurement. Recent development of an immunochemical method distinguishing heavy/light chain pairs (separately measuring IgGK and IgGL, IgAK and IgAL, and IgMK and IgML) provides measurements that could exclude polyclonal contaminants of the same heavy chain with the uninvolved light chain type. Yet, even heavy/light results contain an immeasurable quantity of polyclonal heavy/light chains of the involved isotype. Finally, use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) looms on the horizon as a means to provide more consistent and sensitive measurements of M-proteins.

The reliable detection of paraprotein in serum and urine is the primary purpose of electrophoretic procedures in clinical laboratories. Screening immunofixation electrophoresis (sIFE) employs a single application of antisera directed against heavy and light chains that facilitates the detection of paraproteins that migrate in the non-γ region or that are below the detection limit of protein electrophoresis. These paraproteins that are missed by routine electrophoresis occur in up to 27.3% of newly investigated and 13.6% of monitored patients. Small paraproteins missed by conventional electrophoretic techniques are clinically important in the diagnosis and monitoring of malignant plasma and B-cell disorders. The superior diagnostic performance of sIFE makes it suitable as the initial laboratory procedure to investigate paraproteins in complex serum and urine matrices.

Monoclonal gammopathies are characterised by the production of a monoclonal immunoglobulin or free light chains by an abnormal plasma cell or B-cell clone and may indicate malignancy or a precursor (MGUS). There is currently no consensus on the initial test or combination of tests to be performed in suspected monoclonal gammopathies but serum protein electrophoresis and urine protein electrophoresis are commonly requested as initial investigations. If abnormal, immunofixation electrophoresis is then performed to confirm the presence of paraprotein and to determine its heavy and light chain type. Recently, some groups have developed simplified “screening” IFE methods for use in parallel to SPEP for the detection monoclonal gammopathies. We argue here that screening IFE may be of benefit in clinical laboratories using SPEP with poor resolution in the β-region, assisting in the detection of mainly IgA paraprotein, but may be of less benefit in laboratories utilising higher resolution gels. Further it may increase the detection of trace bands of questionable clinical significance, representing transient phenomena in infectious and auto-immune conditions or very low risk MGUS. The increased detection of these bands using screening IFE would require further patient follow up, possibly causing unnecessary patient anxiety and additional follow up healthcare costs.

During the last decade, surveys by questionnaire in Canada, Australia and New Zealand revealed wide variation in reporting practices by laboratories and individual practitioners in the interpretation of serum and urine protein electrophoresis (PE). Such variation has potential to adversely impact patient outcomes if report structure is inconsistent or if the messaging is incorrectly perceived by the receiving physician. Concerted efforts have been initiated to promote harmonization in the use of interpretative comments. The primary goal is to add value through clear communication with requesting physicians in the interest of quality patient care. Resistance to a harmonized approach largely reflects longstanding personal reporting habits and preferences but change can be more readily embraced if the new system is intuitive, easy to use and saves time in reporting.

The objective of this study was to demonstrate the necessity of using different methods for amyloidogenic light chain detection. Serum and urine agarose gel electrophoresis and immunofixation, as well as serum free light chain (FLC) immunoassay measurements, were evaluated in a patient with verified multiple myeloma and consequent AL amyloidosis confirmed by Congo red staining and immunofluorescence techniques. Conventional chemistry tests [serum and urine electrophoresis (SPE and UPE); serum and urine immunofixation (SIFE and UIFE)] were inconclusive. Only quantitative FLC immunoassay (serum free light chain immunoanalysis, SFLC) provided correct diagnostic information. A combination of gel-based SIFE and UIFE with more novel quantitative FLC immunoassays appears necessary when searching for monoclonal immunoglobulin light chain-related diseases.

A 62-year-old diabetic man with prostate cancer first presented to our clinical laboratory in 2003 with a normal serum protein electrophoresis and immunofixation. In March 2009 he was diagnosed with an IgG κ myeloma. He underwent treatment and went into remission with the original paraprotein band being undetectable. Over the following 5 years, he developed oligoclonal bands and then eventually relapsed. Serum protein electrophoresis and immunofixation were inconclusive, however, isoelectrofocusing identified the oligoclonal pattern then the return of the original band, indicating relapse. This case illustrates the usefulness of an isoelectric focusing method to correctly determine clonality of small abnormal protein bands. It also highlights the need for appropriate commenting on reported results so that they are not confusing for clinicians.

Multiple myeloma (MM) is characterized, in about 80% of cases, by the production of monoclonal intact immunoglobulin and more than 95% of them have elevated concentrations of involved (i.e. of the same class of intact immunoglobulin) free light chain (FLC). The introduction of novel therapeutic strategies has changed the natural history of the disease, leading to new manifestations of relapse. Light chain escape (LCE) is a pattern of relapse in which the FLC increase is not accompanied by a concomitant raise of the original monoclonal component (MC). Here we present a case of a 55-year-old man with an IgG kappa MM stage III diagnosed in September 2007. At presentation an IgG kappa MC and urine Bence Jones protein (BJP) kappa were present. Bone marrow biopsy (BMB) showed the presence of 80% monotypic kappa plasma cells (PCs). The patient received bortezomib, thalidomide, dexamethasone before undergoing a double autologous stem cell transplantation (ASCT) in October 2008 and April 2009. In May 2011 he relapsed showing the same pattern of presentation and treatment with lenalidomide and dexamethasone was started. ln May 2013 serum and urine immunofixation and FLC became negative. In September 2014, an increase of kappa FLC was observed, while serum and urine immunofixations remained negative until January 2015, when urine immunofixation became positive. Eventually, in February 2015, serum immunofixation revealed the presence of a free kappa MC. After a new BMB showing 80% of monotypic kappa PCs, a LCE relapse was diagnosed and the patient started the treatment with bendamustine, bortezomib and dexamethasone. In the present case, the increase of kappa FLC has indicated relapse 4 and 5 months earlier than urine and serum IFE, respectively. Our observation confirms that it is advisable to routinely perform FLC or BJP during follow up of MM patients undergoing ASCT and/or treatment with biological drugs to ensure that LCE is not missed.

Serum free light chain (FLC) assays have been incorporated into routine clinical practice and their use is recommended in international guidelines for the management of monoclonal gammopathies. Given that FLCs are not simple analytes, laboratories should be aware of potential analytical issues when using FLC assays, including antigen excess, lot-to-lot variation and non-linearity. Whilst manufacturers of monoclonal antibody-based assays claim that they overcome such issues, the evidence available to date does not support this. Here we review and compare the technical performance of both polyclonal and monoclonal antibody-based assays. The evidence suggests that the Freelite assay, based on polyclonal antisera, gives a broader recognition of monoclonal FLCs than the N Latex assay, based on monoclonal antisera, and despite being cited as a technical concern, we show that lot-to-lot variation of the Freelite assay is good. Both non-linearity and antigen excess are characteristic of FLC analysis and laboratories should be aware of these phenomena regardless of the assay system they use. Comparisons of the absolute values of sFLCs determined using monoclonal and polyclonal antibody-based assays show poor quantitative agreement and, because current guidelines have been established using the polyclonal antibody-based Freelite assay, it should not be assumed that assays utilizing monoclonal antibodies will give compliance with these guidelines.

Recently, serum free light chain (FLC) assays incorporating anti-kappa (κ) and anti-lambda (λ) FLC monoclonal antibodies have become available: N Latex FLC assay (Siemens) and Seralite ® (Abingdon Health). The purpose of this review is to provide an overview of these two new monoclonal antibody-based methods. In doing so, the review will outline the performance characteristics of each method, including a summary of: assay principles, antibody specificity, analytical performance and assay performance in disease. Additionally, the review will describe the potential user benefits of adopting these new generation FLC assays, which are designed to overcome the established limitations of existing polyclonal antibody based FLC assays.

The measurement of the serum free light chains (FLC) is of paramount importance in the management of patients with plasma cell dyscrasias (PSD). The immunoassays for FLC measurement require adequate precision, accuracy, specificity and reproducibility between batches to prevent under or over estimation of FLC concentration and for an adequate patient monitoring. Considering the peculiarity of the measurand (monoclonal proteins), the optimization of any analytical aspect is difficult to achieve. Three methods are currently available for the assay. The first one has been on the market for over 15 years, and it is based on polyclonal antibodies. The vast majority of the clinical studies demonstrating the utility of the serum FLC measurement have been performed using this assay. A second method based on monoclonal antibodies (mAbs) was marketed in 2011; a third one, also employing mAbs and allowing the simultaneous measurement of κ and λ FLC is in the process of publication. These methods show relevant differences in the type of antibodies used and in the assay design and it is not possible to identify an immunoassay that is superior to the others in any analytical aspect. The comparison studies show that the three methods differ significantly in terms of quantitative values, especially when samples containing monoclonal proteins are compared. Hence the methods cannot be used interchangeably, in particular when the assay is used to monitor the patient response to therapy. In the absence of an international standard for FLC measurement, it is impossible, at this stage to establish, which method shows the best accuracy.

The serum free light chain (FLC) assay has proven to be an important complementary test in the management of patients with monoclonal gammopathies. The serum FLC assay has value for patients with plasma cell disorders in the context of screening and diagnosis, prognostic stratification, and quantitative monitoring. Nonetheless, serum FLC measurements have analytical limitations which give rise to differences in FLC reporting depending on which FLC assay and analytical platform is used. As the FLC measurements are incorporated in the International Myeloma Working Group guidelines for the evaluation and management of plasma cell dyscrasias, this may directly affect clinical decisions. As new certified methods for serum FLC assays emerge, the need to harmonise patient FLC results becomes increasingly important. In this opinion paper we provide an overview of the current lack of accuracy and harmonisation in serum FLC measurements. The clinical consequence of non-harmonized FLC measurements is that an individual patient may or may not meet certain diagnostic, prognostic, or response criteria, depending on which FLC assay and platform is used. We further discuss whether standardisation of serum FLC measurements is feasible and provide an overview of the steps needed to be taken towards harmonisation of FLC measurements.

Measurements of immunoglobulins and serum free light chains (sFLC) are frequently used in patients with monoclonal plasma cell dyscrasia (PCD). For optimum patient care, well-defined performance standards or goals for the measured concentrations of immunoglobulins and sFLC are required. Generally, data based on biological variation is a good and reliable method for setting desirable performance standards; this also applies for the measurements of paraprotein and sFLC. The benefits of this approach are several. Among others, it is independent of the clinician, and it provides us with information about reference change value and index of individuality. Several studies on biological variation of both immunoglobulins and sFLC have been published, and mostly the studies are well performed. The studies normally show small within-subject biological variation resulting in strict analytical goals, which in most cases are difficult to meet. Nevertheless, we still need further information on biological variation of immunoglobulins and sFLC in patients with PCD and in the elderly, which are the main target populations for the two measurands. Furthermore, to improve data on biological variation of immunoglobulins and sFLC, studies accounting for number of individuals, samples, and replicates, as well as time length of the studies are needed.

The detection and quantification of amyloidogenic monoclonal light chains are necessary for the diagnosis and evaluation of response to treatment in AL amyloidosis. However, the amyloid clone is often small and difficult to detect. We report the case of a 68-year-old man who was referred to our Center in April 2013 after syncope and the identification of left ventricular hypertrophy at echocardiography, suspected for amyloidosis. A commercial agarose gel electrophoresis immunofixation (IFE) did not reveal monoclonal components in serum and urine. The κ serum free light chain (FLC) concentration was 21.5 mg/L, λ 33 mg/L (κ/λ ratio 0.65), NT-proBNP 9074 ng/L (u.r.l. <332 ng/L) and an echocardiogram confirmed characteristic features of amyloidosis. The abdominal fat aspiration was positive and the amyloid typing by immune-electron microscopy revealed λ light chains deposits. A high-resolution (hr) IFE of serum and urine showed a faint monoclonal λ component in the urine. A bone marrow biopsy showed 8% plasma cells (BMPC) and a kappa/lambda light-chain restriction with λ light chain on immunofluorescence. The diagnosis of AL (λ) amyloidosis with cardiac involvement was made. In May 2013, patient was started on cyclophosphamide, bortezomib and dexamethasone. After six cycles, serum and urine hr-IFE were negative, the bone marrow biopsy showed 3% BMPC without light chain restriction by immunofluorescence, and a decrease of NT-proBNP was observed (5802 ng/L).Thus, treatment was discontinued. In this patient the amyloid clone could be detected only by in house hr-IFE of urine and bone marrow examination. The detection of the small dangerous amyloidogenic clone should be pursued with a combination of high-sensitivity techniques, including assessment of BMPC clonality. Studies of novel tools, such as mass spectrometry on serum and next-generation flow cytometry analysis of the bone marrow, for detecting plasma cell clones in AL amyloidosis and other monoclonal light chain-related disorders are warranted.

Background: Serum free light chain (FLC) analysis with ratio and urine immunofixation electrophoresis (IFE) are both available for routine use in helping to detect plasma cell dyscrasia and related diseases. Cases: Case reports showing one serum positive for serum FLC but that showed a hook effect and overestimated the amount of monoclonal FLC while urine IFE was negative for Bence Jones protein, and a second serum that showed elevated FLC κ and λ but a normal κ/λ ratio, while urine IFE was positive for Bence Jones protein. Conclusions: These two techniques complement one another. Neither of the techniques is truly quantitative, and both exhibit methodological defects.

Background: Quantification of serum free light chains (FLC) is important in the diagnosis of plasma cell diseases where an abnormal kappa:lambda ratio infers a population of monoclonal plasma cells. The Freelite™ and N Latex assays have been validated in populations without kidney disease but there is a paucity of data relating to the use of these assays in end stage kidney disease (ESKD). The aim of the study was to compare FLC assay performance in ESKD patients on haemodialysis. Methods: Cross-sectional multi-centre study comparing the performance of the two assays on 112 haemodialysis patients without known paraproteinaemia. We quantified FLC pre- and post-dialysis using both the N Latex and the Freelite assays. Results: FLC levels were elevated by both assays. Lambda FLC levels were considerably higher by the N Latex assay. Using the proposed renal reference range for Freelite (0.37–3.1) all but one patient had normal kappa:lambda FLC ratios. In contrast, there were no abnormal FLC ratios pre-dialysis using the N Latex assay. This was due to lambda FLC reading significantly higher by the N Latex assay. Kappa and lambda FLC levels decreased with dialysis but remained elevated above the normal range. The excess of lambda FLC by N Latex persisted post-dialysis but was somewhat attenuated. Dialysis adequacy and dialysis modality predicted clearance of kappa and lambda FLC by both assays. Conclusions: The N Latex assay reported significantly higher pre-dialysis lambda FLC concentrations compared with the Freelite assays. Clinicians should be aware of the need for a separate renal reference range for interpreting FLC ratio using the Freelite assay but not for the N Latex assay in ESKD patients.

Accurate measurement of IgA monoclonal proteins presents a significant challenge to laboratory staff. IgA heavy/light chain (Hevylite, HLC) analysis is an alternative methodology for monoclonal protein assessment, giving an independent measure of IgAκ and IgAλ concentrations. Clonality is assessed by calculating the ratio of involved immunoglobulin to background uninvolved immunoglobulin concentrations (e.g. IgAκ/IgAλ in an IgAκ patient). Here we discuss the challenges faced by the laboratory in IgA monoclonal protein assessment, and compare the performance of Hevylite assays with electrophoresis and total IgA results. We present data which validates the use of Hevylite for response assessment: in most cases, Hevylite provides comparable response assignment to that provided by serum protein electrophoresis (SPE) and total IgA; in other cases Hevylite provides additional information, such as detection of residual disease or relapse.

Serum protein electrophoresis (SPE), serum immunofixation (s-IFE), free light chain measurement (FLC) and nephelometric measurements of total immunoglobulin in serum (IgTot) are some of the laboratory tests required for the management of plasma cell proliferative disorders. The monoclonal protein is usually visible on SPE as a spike (M-spike) in the γ region and the derived densitogram is used to quantify it relative to serum total protein concentration. IgA M-protein, however, often migrates in the β region on SPE and its quantification can be masked by other serum proteins that migrate in this region. The immunoassay Hevylite™ (heavy/light chain, HLC) seems to solve this problem: it quantifies the involved/uninvolved isotype, calculating the ratio IgAκ/IgAλ, considered indicative of clonal proliferation. However, this test seems redundant in the case of artifacts on SPE such as obvious hemolysis or lipemia, or if the IgA M-spike is clearly visible in the β region. In conclusion whereas the IgA HLC assay does not represent an alternative to SPE and s-IFE in the diagnostic patient workup, it may prove to be an alternative to SPE, s-IFE and total IgA quantification in risk stratification and evaluation of response to therapy in patients affected by MM and other monoclonal plasma proliferative disorders.

Background: POEMS syndrome is defined by Polyneuropathy, Organomegaly, Endocrinopathy, Monoclonal gammopathy and Skin changes. The vascular endothelial growth factor (VEGF) appears to play a key role in the pathogenesis of the syndrome, and its concentrations are deemed to correlate to disease activity. The aim of the present study was to verify whether other biochemical markers including serum free light chains (FLC) and heavy/light chains (HLC) would be of value in monitoring POEMS patients. Methods: Fifty-three serum samples were collected from seven POEMS patients at diagnosis and during a follow-up period (range 14–56 months). VEGF was measured using an ELISA method, while FLC and HLC concentrations were measured using Binding Site reagents on a BNII (Siemens) nephelometer. Results: At diagnosis all patients presented high VEGF concentrations, while the κ/λFLC ratio (FLCr) was within the reference range. Four patients had abnormal HLC, HLCκ/HLCλ (HLCr) and FLC values. The relationship between the trend of VEGF and both HLC and FLC during the follow-up was analysed by means of Cohen’s κ coefficient. VEGF and HLC values displayed a significant κ-Cohen (0.537, p=0.002) in all chemotherapy-responder patients while in non-responders it did not. Conversely, in both responders and non-responders, VEGF and FLC values did not attain a significance on κ-Cohen analysis. In three out of four responders HLCr values increased, thus reflecting an improved clinical condition. Conclusions: The findings made in the present study indicate that HLC, either as intact immunoglobulin or as HLCr, may provide useful information, particularly in identifying responders and confirm that the role of FLC is unreliable in monitoring patients with POEMS syndrome.

Background: Serum immunoglobulin free light chains (FLC) are secreted into circulation by plasma cells as a by-product of immunoglobulin production. In a healthy individual the population of FLC is polyclonal as no single cell is secreting more FLC than the total immunoglobulin secreting cell population. In a person with a plasma cell dyscrasia, such as multiple myeloma (MM) or light chain amyloidosis (AL), a clonal population of plasma cells secretes a monoclonal light chain at a concentration above the normal polyclonal background. Methods: We recently showed that monoclonal immunoglobulin rapid accurate mass measurement (miRAMM) can be used to identify and quantify a monoclonal light chain (LC) in serum and urine above the polyclonal background. This was accomplished by reducing immunoglobulin disulfide bonds releasing the LC to be analyzed by microLC-ESI-Q-TOF mass spectrometry. Here we demonstrate that the methodology can also be applied to the detection and quantification of FLC by analyzing a non-reduced sample. Results: Proof of concept experiments were performed using purified FLC spiked into normal serum to assess linearity and precision. In addition, a cohort of 27 patients with AL was analyzed and miRAMM was able to detect a monoclonal FLC in 23 of the 27 patients that had abnormal FLC values by immunonephelometry. Conclusions: The high resolution and high mass measurement accuracy provided by the mass spectrometry based methodology eliminates the need for κ/λ ratios as the method can quantitatively monitor the abundance of the κ and λ polyclonal background at the same time it measures the monoclonal FLC.

Background: The use of therapeutic recombinant monoclonal antibodies (mAbs) has triggered concerns of mis-diagnosis of a plasma cell dyscrasia in treated patients. The purpose of this study is to determine if infliximab (INF), adalimumab (ADA), eculizumab (ECU), vedolizumab (VEDO), and rituximab (RITU) are detected as monoclonal proteins by serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE). Methods: Pooled normal sera were spiked with various concentrations (ranging from trough to peak) of INF, ADA, ECU, VEDO and RITU. The peak concentration for VEDO and RITU was also added to samples with known monoclonal gammopathies. All samples were analyzed by SPEP (Helena Laboratories) and IFE (Sebia); sera containing peak concentrations of mAbs were reflexed to electrospray-time-of-flight mass spectrometry (AbSciex Triple TOF 5600) for the intact light chain monoclonal immunoglobulin rapid accurate mass measurement (miRAMM). Results: For all mAbs tested, no quantifiable M-spikes were observed by SPEP at any concentration analyzed. Small γ fraction abnormalities were noted on SPEP for VEDO at 300 μg/mL and RITU at 400 μg/mL, with identification of small IgG κ proteins on IFE. Using miRAMM for peak samples, therapeutic mAbs light chain accurate masses were identified above the polyclonal background and were distinct from endogenous monoclonal gammopathies. Conclusions: MAbs should not be easily confounded with plasma cell dyscrasias in patients undergoing therapy except when a SPEP and IFE are performed within a couple of days from infusion (peak). In ambiguous cases the use of the miRAMM technology could precisely identify the therapeutic mAb distinct from any endogenous monoclonal protein.

Background: Monoclonal antibodies are promising anti-myeloma treatments. As immunoglobulins, monoclonal antibodies have the potential to be identified by serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE). Therapeutic antibody interference with standard clinical SPE and IFE can confound the use of these tests for response assessment in clinical trials and disease monitoring. Methods: To discriminate between endogenous myeloma protein and daratumumab, a daratumumab-specific immunofixation electrophoresis reflex assay (DIRA) was developed using a mouse anti-daratumumab antibody. To evaluate whether anti-daratumumab bound to and shifted the migration pattern of daratumumab, it was spiked into daratumumab-containing serum and resolved by IFE/SPE. The presence (DIRA positive) or absence (DIRA negative) of residual M-protein in daratumumab-treated patient samples was evaluated using predetermined assessment criteria. DIRA was evaluated for specificity, limit of sensitivity, and reproducibility. Results: In all of the tested samples, DIRA distinguished between daratumumab and residual M-protein in commercial serum samples spiked with daratumumab and in daratumumab-treated patient samples. The DIRA limit of sensitivity was 0.2 g/L daratumumab, using spiking experiments. Results from DIRA were reproducible over multiple days, operators, and assays. The anti-daratumumab antibody was highly specific for daratumumab and did not shift endogenous M-protein. Conclusions: As the treatment of myeloma evolves to incorporate novel monoclonal antibodies, additional solutions will be needed for clinical monitoring of patient responses to therapeutic regimens. In the interim, assays such as DIRA can inform clinical outcomes by distinguishing daratumumab from endogenous M-protein by IFE.

Daratumumab is a fully human anti-CD38 IgG1-κ monoclonal antibody (mAb) currently being evaluated in several Phase 2 and 3 clinical studies for the treatment of multiple myeloma (MM). In this clinical case study we demonstrate that daratumumab can be detected as an individual monoclonal band in serum immunofixation electrophoresis (IFE). M-protein follow-up by IFE is part of the International Myeloma Working Group (IMWG) criteria to assess treatment response. Therefore, it is crucial that the daratumumab band is not confused with the endogenous M-protein of the patient during IFE interpretation. Moreover, a significant number of IgG-κ M-proteins co-migrate with daratumumab. Co-migration introduces a bias in the M-protein quantification since pharmacokinetic studies show that daratumumab peak plasma concentrations reach up to 1 g/L. More importantly, co-migration can mask clearance of the M-protein by IFE which is necessary for classification of complete response by IMWG criteria (negative serum IFE). For optimal M-protein monitoring the laboratory specialist needs to be informed when patients receive daratumumab, and it is essential that the laboratory specialist is aware that a slow migrating band in the γ-region in those patients may be derived from the daratumumab. A daratumumab specific IFE reflex assay (DIRA) has been developed and can be utilized to abrogate interference. The here described mAb interference is not limited to daratumumab, and as therapeutic antibodies gain approval and enter into common clinical practice, laboratory specialists will need additional processes to characterize IFE interference and distinguish endogenous M-protein from therapeutic antibodies.