Comparison between free light chains and immunofixation electrophoresis: one-year data from a tertiary hospital

Introduction

A class of diseases known as monoclonal gammopathies is defined by the unchecked growth and build-up of abnormal plasma cells together with the overproduction of M-proteins [1]. These proteins, which can be found in the patients’ serum, urine, or both, are also known as monoclonal proteins, M-proteins, M-spikes, paraproteins, or immunoglobulinopathies. They comprise two light chains of either lambda (λ) or kappa (κ), and two heavy chains of the same class. Sometimes, plasma cells solely generate free light chains (FLCs), referred to as Bence-Jones proteins, and are mostly detected in urine [2], 3]. The World Health Organization has categorized the plasma cell dyscrasias, including multiple myeloma (MM), extraosseous plasmacytoma, solitary plasmacytoma of the bone, monoclonal immunoglobulin deposition diseases, and monoclonal gammopathy of unknown significance (MGUS) [4], 5]. The second most prevalent hematologic malignancy is MM [6]. MM accounts for approximately 10–15 % of all hematological malignancies and 1 % of all cancers worldwide [7]. In contrast to other age groups, MM is substantially less prevalent in people under 45; however, it is more common in those over 45 [8]. At diagnosis, the typical age is 65, and the current 5-year survival rate is around 46.6 % [9], 10]. The nonspecific signs and symptoms of MM might lead to its diagnosis based only on abnormal laboratory testing [11]. Diagnosing MM may begin with the detection of a monoclonal immunoglobulin in the bloodstream [12]. M-proteins must be found using a dependable, sensitive technique. Currently, agarose gel electrophoresis is commonly used in laboratories to identify M-proteins in serum and/or urine samples. This identification may be further verified using immunofixation electrophoresis (IFE) [1]. IFE, a technique that combines protein electrophoresis and immunoprecipitation, is used to detect and identify M-proteins. It plays a key role in diagnosing, monitoring, and treating monoclonal gammopathies by determining the specific type of M-protein produced [13]. In the presence of an M-protein detected on serum protein electrophoresis (SPE), an indistinct sharp band, hypogammaglobulinemia, and increased beta or gamma fraction of immunoglobulin heavy chain concentrations, the result is considered abnormal. Thus, serum and urine IFE evaluation, serum free κ and λ analysis, and bone marrow biopsy for confirmation are required to identify the M-protein [14]. A quantitative nephelometric method is used for the detection of FLCs. The test measures κ and λ light chains that circulate as monomers or dimers and are not bound to immunoglobulin heavy chains. Quantification of κ and λ FLCs, along with calculation of the FLC κ/λ ratio, provides a sensitive and specific approach for the detection of excessive monoclonal FLCs. Studies have demonstrated that this method can be used as a complementary tool to IFE. In addition to its diagnostic utility in FLC-related disorders, the test is also used for monitoring disease progression in conditions such as primary systemic amyloidosis (AL), light chain deposition disease (LCDD), light chain multiple myeloma (LCMM), and non-secretory multiple myeloma (NSMM), where a band may be detected that cannot be measured by protein electrophoresis in IFE [15].

In our study, we aimed to retrospectively analyse the IFE data studied in the Medical Biochemistry Laboratory of a tertiary training and research hospital in Türkiye in 2022 and to compare the detected paraprotein types with the current literature.

Materials and methods

In this study, the results of 1,169 serum IFE analyses performed in the Medical Biochemistry Laboratory of a tertiary training and research hospital in Türkiye in 2022 were retrospectively evaluated in terms of types of monoclonal gammopathies and their frequencies. Serum immunofixation samples are shown in Figures 1 and 2. Patients’ information was accessed from the laboratory information system. Multiple immunofixation results of the same patients were included in the study only once. For this study, we used a SAS-1 compact gel electrophoresis analyzer, a SAS-2 compact gel processor system, and SAS-1 IFE-4 Gels from Helena Biosciences Europe in our laboratory. All patient data was collected and analyzed using the Microsoft Office Excel^® 2007 software program (Microsoft Corp., Redmond, WA, USA). The obtained data were analyzed based on the relevant clinical departments and diagnoses. Additionally, simultaneous serum free κ and λ light chain levels, as well as serum free κ/λ ratios, were compared with IFE results. Then, concordance analysis was performed. Furthermore, serum free κ/λ values were compared between IFE-positive and IFE-negative groups to evaluate potential differences. Serum free κ and λ light chain levels were analyzed using the Siemens BN II nephelometer (Siemens Healthcare Diagnostics, Germany). For FLC analyses, the reference ranges provided by the manufacturer were used: κ FLC, 6.7–22.4 mg/L; λ FLC, 8.3–27.0 mg/L. The results were reported as FLC concentrations and FLC κ/λ ratio (reference range: 0.26–1.65). In patients with κ/λ ratios greater than 1.65, elevated κ FLC levels are observed, suggesting the production of clonal κ FLCs. In contrast, patients with ratios less than 0.26 exhibit elevated λ FLC levels, indicating the presence of clonal λ FLC production. Statistical analysis was performed using SPSS version 18.0 (IBM Corporation, NY, USA). The Shapiro-Wilk test was used to assess the distribution of the data. Mean and standard deviation values were reported for normally distributed variables; median and interquartile ranges (1st and 3rd quartiles) were used for non-normally distributed variables. The Mann–Whitney U test was employed to compare two independent groups with non-normally distributed data. The Pearson chi-square test was used to compare categorical variables between two groups. A p-value of <0.05 was considered statistically significant.

Figure 1:

An example of an immunofixation gel of a healthy person.

Figure 2:

This gel shows a monoclonal heavy chain band of immunoglobulin A that matched with a monoclonal kappa light chain band.

Results

In our study, 1,169 serum IFE results were retrospectively analyzed, and paraprotein bands were detected in 116 different cases. The frequency of detected paraprotein was determined as 10 %. IgG-κ in 23 cases (20 %), free λ light chain in 21 cases (18 %), IgG-λ in 19 cases (16 %), IgA-λ in 11 cases (9 %), IgA-κ in 10 cases (9 %), free κ light chain in 9 cases (8 %), IgG-κ/λ in 7 cases (6 %), IgM-λ in 4 cases (3 %), IgM-κ in 3 cases (3 %), IgG-κ/free λ light chain in 2 cases (2 %), IgG-κ/IgA-λ in 2 cases (2 %), biclonal IgA-λ in 1 case (0.8 %), biclonal IgG-λ in 1 case (0.8 %), We detected IgA-κ/free λ light chain in 1 case (0.8 %), IgA-κ/free λ light chain in one case (0.8 %), IgG-λ/IgA-λ in 1 case (0.8 %). Table 1 shows the distribution rates of paraproteins detected by serum IFE.

Among the patients with paraprotein bands, 63 (54 %) were male and 53 (46 %) were female. The mean age of males was 66.4 years (31–90 years), and the mean age of females was 64.6 years (26–87 years). The age and gender distribution of the patients with paraprotein bands are shown in Table 2. In our study, the median age was 67 years. The number of positive cases under 30 years of age was 1, and the positivity rate was 0.86 %. The number of positive cases under 40 years of age was 6, and the positivity rate was 5.2 %. The number of positive cases over 70 years of age was 45, and the positivity rate was 38.9 %. The age distribution of different paraprotein band types in males and females is also shown in Supplementary Tables 1 and 2.

IFE requests were analysed according to the pre-diagnoses of the patients (Supplementary Table 3). It was found that 338 requests were made with a prediagnosis of vitamin D deficiency, 131 with anaemia, 115 with leukocyte disorders, and 1,231 with various other diagnoses.

IFE requests were also evaluated based on the requesting clinical departments (Supplementary Table 4). The highest number of IFE requests originated from the hematology department, followed by internal medicine, nephrology, and neurology.

The normal and abnormal distributions of the κ/λ ratios in the IFE-positive and IFE-negative groups were determined, and concordance was calculated (Supplementary Table 5). Concordance was found to be 87 %. Serum κ results were compared with IFE κ monoclonal positivity and negativity; concordance was 93 %. A comparison of serum λ results with IFE λ monoclonal positivity showed that concordance was 94.5 %. Among 87 patients with positive IFE results, the κ/λ ratio was within the normal range in 58 cases and abnormal in 29 cases. In contrast, among 596 patients with negative IFE results, 566 had a normal κ/λ ratio, while 30 showed an abnormal ratio. Of the 32 patients with serum κ M-protein positivity, 16 had a normal and 16 had an abnormal κ/λ ratio. Among the 45 patients with serum λ M-protein positivity, an abnormal κ/λ ratio was detected in 13 patients, whereas 32 had a normal ratio (Supplementary Tables 6 and 7).

When κ/λ ratios were compared between IFE-positive and IFE-negative groups, a statistically significant difference was found (p=0.007), with higher κ/λ ratios observed in the IFE-positive group. No significant difference was found between the two groups in terms of gender distribution (p=0.117). However, a significant difference was observed in age between the groups; the mean age of the IFE-positive group was significantly higher (p<0.001).

Discussion

MM is defined by the invasion of cancerous plasma cells into the bone marrow and is linked to elevated levels of M-protein in the bloodstream and/or serum. The frequency of occurrence rises in correlation with age, with around 40 % of patients being diagnosed before the age of 60 and only 2 % of instances happening prior to the age of 40 [16]. The geriatric population has a prevalence rate of monoclonal gammopathy that can reach up to 8 % [17]. In our study among the patients with paraprotein bands, 63 (54 %) were male and 53 (46 %) were female. The mean age of males was 66.4 (31–90 years), and the mean age of females was 64.6 (26–87 years) in our study; the median age was 67 years. The number of positive cases under 30 years of age was 1 and the positivity rate was 0.86 %. The number of positive cases under 40 years of age was 6 and the positivity rate was 5.2 %. The number of positive cases over 70 years of age was 45 and the positivity rate was 38.9 %. Kyle et al. also obtained results similar to those of the data obtained in our study. Kyle R. A. et al. conducted a retrospective study of the medical records of 1,027 patients who were diagnosed with MM at the Mayo Clinic in Rochester, Minnesota, USA between January 1, 1985, and December 31, 1998. Their findings have demonstrated that 2 % of the patients were under the age of 40, while 38 % were 70 years or beyond. The age at which half of the population is older and half is younger was 66 years. Additionally, they documented that out of the total of 1,027 patients, 59 % were of the male gender. [11]. Tamimi et al. found that 35 % of the patients with MM were over 70 years of age. Our results are compatible with their data. However, while they obtained a rate of 15 % for patients under 40 years of age, we found a rate of 5 %, which is lower than their rate [1]. Cohen et al. demonstrated that the presence of monoclonal gammopathies is independently linked to male gender and advancing age [18]. There is a suggestion that the impact of age is connected to immunological dysregulation that occurs as a result of aging [18].

In our study, 1,169 serum IFE results were retrospectively analyzed, and paraprotein bands were detected in 116 different cases. The frequency of detected paraprotein was determined as 10 %. Our negative electrophoresis result was determined to be 90 %. Tamimi et al. detected monoclonal gammopathy with a rate of 6.3 %, and a negative electrophoresis result was 93 % in their study with 6,624 patients [1]. The data we obtained is similar. The rate of paraprotein band detection obtained by Dikker et al. as a result of retrospective examination of serum IFE results of 403 cases was 23 %. They found a higher rate than the positivity rate we obtained [13]. In our study IgG-κ in 23 cases (20 %), free λ light chain in 21 cases (18 %), IgG-λ in 19 cases (16 %), IgA-λ in 11 cases (9 %), IgA-κ in 10 cases (9 %), free κ light chain in 9 cases (8 %), IgG-κ/λ in 7 cases (6 %), IgM-λ in 4 cases (3 %), IgM-κ in 3 cases (3 %), IgG-κ/free λ light chain in 2 cases (2 %), IgG-κ/IgA-λ in 2 cases (2 %), biclonal IgA-λ in 1 case (0.8 %), biclonal IgG-λ in 1 case (0.8 %), We detected IgA-κ/free λ light chain in 1 case (0.8 %), IgA-κ/free λ light chain in one case (0.8 %), IgG λ/IgA λ in 1 case (0.8 %). The Ig types obtained by Tamimi et al. were defined as 41 % (n=171) IgG-κ, 19 % (n=79) IgG-λ, 7 % (n=29) IgM-κ, 3 % (n=13) IgM-λ, 2.6 % (n=11) IgA-κ, 2.9 % (n=12) IgA-λ, 14.7 % (n=61) FLC κ, and 9.6 % (n=40) FLC λ [1]. Dikker et al. detected IgG-κ (33 %) and IgG-λ (20 %) monoclonal paraprotein types with the highest frequency [13]. Ercan et al. reported monoclonal paraprotein types as IgG-κ with a frequency of 41.3 % and IgG-λ with a frequency of 35.6 % in a study evaluating the results of IFE in Ankara [12]. In our study, we detected IgG-κ (20 %) with the highest frequency, in accordance with the literature data. While Tamimi, Dikker, and Ercan detected IgG-λ in the second frequency in their studies, unlike them, we detected free λ light chains in the second frequency (18 %) and IgG-λ in the third frequency (16 %). Monoclonal gammopathy has been found in approximately 10 % of patients with polyneuropathy of unknown cause [19]. Determination of the paraprotein type is not only important for the treatment and prognosis of paraproteinemias, but also for their complications.

Upon reviewing the clinics where IFE was requested, the highest frequency of requests was found to originate from the hematology clinic. Patients referred to the hematology/oncology service constituted the largest group. Among patients without neoplastic disorders of the lymphoid or plasma cell systems, IFE testing was commonly requested by the internal medicine clinic for conditions such as obesity, diabetes, hypertension, gastroesophageal reflux disease, hyperlipidemia, hypothyroidism, and renal failure. Patients with neurological disorders represented one of the largest tested subgroups. SPE and serum immunofixation electrophoresis (SIFE) were performed in all patients with unexplained neuropathy, as well as in many with other disorders, including motor neuron disease and multiple sclerosis. Similar findings were also reported by Singh et al. [20].

The serum FLC (sFLC) assay provides a valuable contribution to the laboratory diagnosis of monoclonal immunoglobulin disorders. An altered κ to λ light chain ratio reflects an overproduction of one light chain type and may suggest a monoclonal proliferation of that specific light chain or an immunoglobulin containing it. It is generally accepted that appropriate screening for monoclonal immunoglobulins should include SPE/SIFE, and urine protein electrophoresis and urine immunofixation electrophoresis (UPEP/UIFE). However, urine-based evaluations are often not performed due to logistical challenges. In contrast, sFLC testing can be performed on the same serum sample used for SPE/SIFE, eliminating the need for a separate urine specimen [20].

In our study, we found that 95 % of patients without M-protein detected by IFE had a normal κ/λ FLC ratio. A substantial proportion of patients with monoclonal gammopathy (MG), as confirmed by IFE, also exhibited normal sFLC ratios. Among 87 IFE-positive patients, only 29 had an abnormal FLC ratio, while 58 displayed normal ratios.

These results suggest that κ and λ FLC assays are significantly less sensitive than IFE but are specific for the detection of serum M-proteins. Furthermore, the κ/λ ratio has limited value in routine screening, as the majority of sera with abnormal IFE patterns demonstrated normal FLC ratios.

Our findings are consistent with those reported by Jaskowski et al. and Wood et al. [21], 22], further supporting the limited clinical utility of this test in certain contexts.

IFE is a biochemical method used in the diagnosis, treatment, and follow-up of monoclonal gammopathies. In patients with suspected MM, amyloidosis, or other monoclonal gammopathies, SPE together with serum and urine IFE is recommended. These methods are highly sensitive and reliable [8]. One of the most important limitations of our study is the lack of urine immunofixation data. Another important limitation is that no statistical research can be performed with values such as albumin, total protein, calcium, sedimentation, urea, and creatinine.