Serum Electrophoresis of Proteins (SPEP) is commonly performed using capillary zone electrophoresis (CZE) to detect monoclonal proteins when monoclonal gammopathy (MG) is suspected. During CZE, proteins migrate under an electroosmosis flow with direct integration of the fractions by spectrophotometry (Mondol et al., 2018). It has been reported that CZE can fail to detect 5% of MG and 40% of light chain multiple myeloma (LCMM). When small free light chains (FLC) pass in the urine, the residual concentration in serum is too weak to be detected by CZE. Serum Immunofixation (sIFE), Bence Jones proteinuria and serum free light chains (sFLC) quantification assays perform better sensibility for the detection of free light chains. We report a case of serum free light kappa chains at high concentrations that failed to be detected by CZE but detected by sFLC assays and sIFE.

Case Report

In September 2025, we received a sample of a 69-year-old woman for tests that were part of the follow-up of a light chain multiple myeloma (LCMM). Laboratory investigations from the sender laboratory showed a hemoglobinemia of 8.4 g/dL (reference interval (RI): 11.8 – 15.8 g/dL) with associated thrombocytopenia 20 G/mm3 (RI: 150 G/mm3 – 450 G/mm3), total serum protein: 67 g/L (RI: 64 - 83 g/L). Creatinine blood levels 4.20 mg/dl (RI: 0.5 mg/dl-1 mg/dl), the estimated GFR by 2021 CKD-EPI Creatinine was 11 ml/min/1.73 m² (RI: greater than 90). Erythrocyte sedimentation rate accelerated at 92 mm.

SPEP by CZE and immunotyping were performed by using respectively Capillarys system and Capillarys immunotyping on Capillarys Octa (Sebia, Lisses, France). They showed no abnormalities, as no monoclonal spikes on the electropherogram (Fig. 1) and no susbtraction on all immunotyping frames (Fig. 2). Serum Immunofixation was performed using HYDRASYS 2 scan system (Sebia, Lisses, France). It showed free kappa lights chains (Fig. 3). sFLC was performed on the Architect c4100 instrument (Abbott) using the Diazyme turbidimetric immunoassay (Diazyme Laboratories). Results showed an elevated serum FLC-kappa: 6392.8 mg/L (RI: 2.37 – 20.73 mg/L), serum FLC-lambda: 107.1 mg/L (RI: 4.23 – 27.69 mg/L), and a high FLC-kappa/FLC-lambda ratio of 59.70 (RI: 0.37 – 3.10). 

Monoclonal gammopathies (MG) are defined by the increased production of a monoclonal protein (MP), following an abnormal production of a plasma cells clone.  The MP can be an intact immunoglobulin (with both a heavy chain and a light chain) or an isolated light chain referred to as free light chain (FLC). MG consist in a wide range of diseases such as: MGUS (Monoclonal Gammopathy of Undetermined Significance), multiple myeloma, Waldenstrom's macroglobulinemia, light chain amyloidosis, light chain deposition disease, solitary plasmacytoma and other plasma cell leukemias and lymphomas. 

Fig. 1: The CZE electropherogram showing no abnormalities.

Serum protein electrophoresis (SPEP) is a common test used once a monoclonal gammopathy is suspected. SPEP separates serum protein based on their size and charges. CZE has been widely adopted to perform SPEP as the technique is automated and sensitive (Bossuyt et al., 2003). Monoclonal protein appears as a peak, usually in the gamma region. While it can detect and quantify the size of a monoclonal protein, it has its limitations. The CZE by Sebia we used can detect a monoclonal protein as low as 19 mg/dL (Sebia, 2022.). The manufacturer also states that this sensitivity may vary according to the mobility of the monoclonal component and polyclonal background. A well-known pitfall of CZE is its limited ability to detect monoclonal FLCs which are the sole products of LCMM (Dispenzieri et al., 2010). 

It is indeed known that CZE produces false negatives for serum FLC. This has been attributed to weak serum concentrations below the sensitivity range (Bakshi et al., 2005a; Biaz et al., 2022; Ellidag et al., 2015; Jenner, 2014). Free chains monoclonal proteins have low serum concentrations due to FLC readily passing in urine. The false negative in CZE might also be a consequence of the FLC's low molecular weight affecting their electrophoretic mobility. They can produce a modest peak or can be invisible if they co-migrate in other normal fractions (Carrère et al., 2019; Ellidag et al., 2015). It has been suggested that due to their low molecular weight, they can migrate before the start of the reading signal or remain immobile in the capillary (Biaz et al., 2022).

Fig. 2: The patient's serum immunotyping is normal; there is neither substraction nor change of symmetry visible in the kappa frame.

In our case, the concentration of monoclonal kappa free chains of 6392.8 mg/L is above the level of detection of the manufacturer. Capillary techniques (CZE and immunotyping) were strictly normal. This observation suggests that the failure to detect serum FLC with CZE is not strictly related to their low serum levels. On the other hand, sIFE which has the 10-fold sensitivity of SPEP was able to detect the monoclonal FLC-kappa as a thick band in the kappa lane.  Our case illustrates that FLC at concentrations over CZE level of detection can remain invisible on the electropherogram, as well as in immunotyping. It also indicates the added value of sFLC quantifications assays in the panels of laboratory tests for MG patients. SFLC quantifications assays are replacing the Bence Jones proteinuria test. 

It highlights the need to comply with the recent guidelines for laboratory detection and initial diagnosis of MG as well as diagnosis and follow-up of multiple myeloma. In a screening situation, performing CZE alone would have resulted in a missed or delayed diagnosis. We also suggest clinicians comply by ordering tests accordingly. Algorithms involve at least SPEP and sFLC quantification assays (Keren et al., 2022; Rajkumar, 2022) and sometimes sIFE is added (Dimopoulos et al., 2021; Mohammad et al., 2020; Keren et al., 2022) to increase detection sensitivity. Additionally, for laboratories, combining SPEP and sFLC into a unique test order has shown to increase compliance with the International Myeloma Working Group (IMWG) guidelines (OBrien et al., 2023). Such changes improved the detection of MG, where early diagnosis is associated with better clinical outcomes  (Bakshi et al., 2005; Dimopoulos et al., 2021; Holding et al., 2007). 

Fig. 3: The Serum Immunofixation gel showing a monoclonal band in Kappa.

CZE is a widely adopted test in clinical laboratories for its speed, its automation and sensitivity. It is vastly prescribed when a clinician suspects a monoclonal gammopathy as a screening test as well as for monitoring therapy for already diagnosed patients because CZE allows the quantification of the monoclonal protein. However, CZE has inherently limits detecting serum free lights chains. This pitfall is frequently explained by the lower concentration of MP constituted of serum free lights chains readily passing in the urine. In our case report, CZE and Capillary Immunotyping failed to detect free light chains kappa MP although the concentrations are over the limit of detection stated by the manufacturer. Our observation illustrates that the false negatives for sFLC in capillary electrophoresis are not only a matter of concentrations but might also involve of a particular interaction of sFLC during the capillary electrophoresis procedure. Overall, such false negatives should be avoided for patients in order not to miss diagnosis or delay treatments. Clinicians should comply with international recommendations when ordering tests by combining serum electrophoresis with sIFE or quantification of sFLC anytime MG is evoked. sIFE and serum quantifications perform better detecting MP constituted of FLC. Clinical laboratories could help enhance such compliance by combining those tests into one. Meanwhile, in clinical setups where compliance with international recommendations is not mandatory, CZE clinical laboratory reports should include a note reminding clinicians that SPEP alone can fail to detect monoclonal protein. 

We declare that all text, data, figures/tables, or other illustrations presented in the manuscript are completely original and do not contain or include material taken from other copyrighted sources and affirm that the work does not contravene any proprietary or own rights of others. We state that the article submitted has neither been previously published nor submitted elsewhere in any form or for simultaneous consideration in any other journal, and we will not reuse this previously published work without making substantial alterations and without desire to address. We state that we have substantially participated in the research and in preparation of the manuscript and that it represents original work adequate to claim authorship, and we agree that no author can publish anywhere else unless it has been changed substantially.

K.K.: analysis, data collection, figure preparation, manuscript writing. M.A: Manuscript review and final approval. B.Y.: Manuscript review and final approval.

We are very grateful to the authority of the institutions and reviewers for their review recommendations to finalize our present work.

The authors declare that they have no conflicts of interest, financial or otherwise, that could have influenced the conduct or outcomes of this study.