A 53-year-old woman presented to the orthopedic department with severe diffuse muscular and bone pain. An x-ray of her right upper extremity revealed a lytic destructive lesion in the right humerus. Computed tomography scans showed multiple lytic lesions in the spine and pelvis, and a biopsy confirmed the presence of 70% plasma cells, which were κ light chain restricted. The patient was referred to a hematologist. Although no monoclonal protein was detected in the serum by protein electrophoresis or by immunofixation electrophoresis (IFE),1 the κ free light chain (FLC) was increased at 47.2 mg/L (reference interval, 3.3–19.4 mg/L), with a κ/λ FLC ratio of 23 (reference interval, 0.26–1.65). Serum concentrations of β2-microglobulin and albumin were 248 nmol/L (reference interval, 59.5–153 nmol/L) and 37 g/L (reference interval, 34–47 g/L), respectively. The urine protein concentration was not increased, but protein electrophoresis revealed a small M (monoclonal) spike in the γ region (32 mg/24 h). In addition, IFE identified a monoclonal κ light chain (Bence Jones protein). On the basis of these findings, the patient was informed that she had stage I (International Staging System) oligosecretory/nonsecretory multiple myeloma (MM).
The patient underwent surgical repair of her right humerus and was evaluated 1 month after her surgery. At that point, a second serum FLC measurement showed a κ FLC concentration of 68.2 mg/L. The patient’s hematologist recommended close observation in lieu of initiating therapy because of her lack of symptoms. The patient’s serum κ FLC concentration was monitored monthly and remained <50 mg/L for the next 3 months. Five months after diagnosis, the patient began to complain of mild fatigue, shortness of breath, and palpitations. A 10-fold increase in the urinary M protein to 333 mg/24 h was noted, along with a decrease in the blood hemoglobin concentration to 79 g/L (reference interval, 120–155 g/L) and an increase in the calcium concentration to 2.80 mmol/L (reference interval, 2.22–2.52 mmol/L). Her hematologist recommended initiation of treatment, and the patient was enrolled in a clinical trial protocol consisting of lenalidomide (25 mg/day) and dexamethasone (40 mg/day) administered for 21 days of a 28-day cycle. At 1 month after initiation of treatment, the patient was noted to be tolerating the regimen well, with an increase in her hemoglobin to 91 g/L, a minimal decrease in serum IgG from a pretreatment value of 2.95 g/L to 2.71 g/L (reference interval, 6.00–15.00 g/L), and a reduction in her serum creatinine concentration from 115 μmol/L to 88 μmol/L (reference interval, 62–106 μmol/L). The patient also stated that she felt less fatigued. Her serum κ FLC concentration, however, was inexplicably increased to 2180 mg/L (Fig. 1⇓ ). By her next follow-up appointment a month later, the patient had completed 2 full cycles of the lenalidomide and dexamethasone regimen and showed evidence of continued response, as evidenced by a rise in the hemoglobin concentration to 105 g/L and a decrease in urinary protein excretion to 132 mg/24 h. The patient’s serum κ FLC concentration remained increased at 1500 mg/L, however.
QUESTIONS TO CONSIDER
Why was the patient’s high serum FLC initially not detected by IFE?
What are the potential causes of increased FLC after treatment?
How can light-chain escape be differentiated from FLC antigen excess?
MM, a hematologic malignancy belonging to a class of diseases known as plasma cell proliferative disorders, is characterized by the neoplastic proliferation of a single clone of plasma cells producing a monoclonal immunoglobulin. The clone proliferates in the bone marrow and often produces osteolytic bone lesions, osteopenia, osteoporosis, and/or pathologic fractures. The monoclonal proteins may cause further organ damage, often leading to renal insufficiency and/or renal failure. The median age of diagnosis for MM is 66 years; only 2% of patients are younger than 40 years (1).
MM patients often present with bone pain. Weakness and fatigue are also a common manifestation of MM and are often associated with anemia. Radiographic imaging of the skeleton reveals punched-out lytic lesions, diffuse osteopenia, osteoporosis, or fractures in nearly 80% of patients. Computed tomography and magnetic resonance imaging may be helpful for patients presenting with no abnormalities apparent on conventional x-rays. Examination of the bone marrow reveals bone marrow involvement by plasma cells, which constitute >10% of all nucleated cells in 96% of MM patients; this finding is confirmatory for MM. Laboratory findings at the time of diagnosis include anemia (73% of patients), increased serum creatinine (50%), hypercalcemia (28%), and leukopenia (20%). Serum β2-microglobulin may also be increased (75%) and serves as a prognostic indicator, with higher values predicting poorer survival prospects. M protein is detected by IFE in the serum and/or urine in 97% of patients (1).
quantification of m proteins in the diagnosis and management of mm
The serum FLC assay was developed in the early 2000s to detect light-chain epitopes that are exposed only when not bound to a heavy chain. The assay quantifies κ and λ FLCs and has come into routine use in the diagnosis and management of several plasma cell proliferative disorders, including monoclonal gammopathy of undetermined significance, light-chain amyloidosis, and MM. The clinical utility of the FLC assay has been reviewed elsewhere (2).
Despite its utility, the FLC assay is not without its limitations. Lot-to-lot variation in assay reagents can be an issue. In addition, both polyclonal and monoclonal serum FLCs have often been found to dilute in a nonlinear fashion, leading to underestimation in the absence of off-line dilution (3). False high results may occur due to polymerization of light chains (4)(5). Finally, falsely low serum FLC results may be obtained in cases of antigen excess (6).
what caused the increase in serum flc after treatment in this patient?
For our patient, the hematologist sought the consultation of the laboratory director after consecutive monthly measurements of the serum free κ chain produced values much higher than before the initiation of treatment. The sudden increase in serum FLC after treatment was worrisome to the hematologist because it may have been indicative of a phenomenon referred to as “light-chain escape from plateau phase” (LEPP) (7). LEPP was documented in a series of 3 MM patients who were noted to undergo a shift in secretion from intact immunoglobulin to FLCs after treatment with either thalidomide or lenalidomide. LEPP is thought to be a novel manifestation of relapsed disease due to selective pressure brought on by thalidomide/lenalidomide treatment. It typically follows an aggressive clinical course, with subsequent therapies offering only marginal benefits. Fortunately, the sample from the patient’s second pretreatment visit had been stored frozen. Retesting of this sample at a starting dilution of 1 part in 400 (i.e., 400-fold dilution) instead of 1 part in 100 (100-fold dilution) indicated the need for further sample dilutions and yielded a final κ FLC concentration of 12 700 mg/L instead of the initially reported 68.2 mg/L.
Nephelometric assays measure light scatter caused by the formation of immune complexes in solution and are subject to limitations inherent in antigen–antibody reactions. This method requires that antigen concentrations fall within a certain range known as the “antibody excess” of the Heidelberger–Kendall curve. Higher antigen concentrations produce falsely low readings. In this particular case, the patient had extremely high κ FLC concentrations that caused antigen excess and an artifactually low reading before treatment. Treatment with dexamethasone and lenalidomide caused a drop in this patient’s κ FLC value to within the region of equivalence for the assay, causing what was perceived to be a spurious increase in concentration.
what is the prevalence of antigen excess in the flc assay?
After this case, our laboratory identified an additional 4 MM patients (2 cases of κ light-chain MM, 1 of nonsecretory MM, and 1 of IgA κ MM) who had artifactually low serum κ FLC concentrations during the course of disease monitoring. This experience prompted us to further investigate the incidence of serum FLC antigen excess. During a 4-month period (August 1, 2006, to November 30, 2006), all clinical FLC assays ordered in our laboratory were performed at the recommended 100-fold dilution as well as at a second (400-fold) dilution (8). FLC assays were performed on a Dade Behring BN II nephelometer with FLC reagent sets from The Binding Site (Birmingham, UK). Of the 7538 serum FLC studies that were clinically ordered and assayed in duplicate during this period, no samples exhibited λ FLC antigen excess, but 9 patients (0.12%) were identified to have κ FLC excess. These 9 patients all had increased κ FLC concentrations and abnormal κ/λ FLC ratios when tested at the initial 100-fold dilution, but the instrument did not indicate a need for further dilutions. When retested at a 400-fold dilution, however, all 9 samples gave substantially higher results or indicated that additional dilutions were needed. Four of the 9 patients had 2 samples submitted for testing, and each pair of samples yielded similar results. The sample with the largest change in the κ FLC result was from 77 mg/L to 141 000 mg/L. On average, the concentrations of these samples increased approximately 200-fold when they were diluted. Diagnostically, the assay identified all 9 samples as abnormal. From a monitoring standpoint, however, the initial κ FLC results were misleadingly lower than the results obtained at a higher starting dilution.
Some nephelometers, including the BN II, have automated detection algorithms designed to detect antigen excess. Such algorithms may include monitoring the initial rate of the precipitin reaction as well as a separate prereaction step. The manufacturer’s protocol for the Freelite™ assay does not include a prereaction step and relies on whether the result lies beyond the linear portion of the calibration curve, as well as on the initial rate of precipitin formation, to determine if further dilutions are necessary. This protocol may have led to the analyzer missing certain cases of antigen excess.
Because of these findings, we have modified the laboratory operating procedure. All sera with a FLC κ/λ ratio >2 and a κ FLC concentration that has not been obtained at test dilutions >100-fold are retested at a serum dilution of 400-fold. In samples that are not in antigen excess, the 400-fold dilutions give concentrations that are 50% higher on average than those obtained with the 100-fold dilutions. For consistency, the 100-fold dilutions are reported. Our choice of the 100-fold dilution for reporting purposes is based on the fact that most samples have the 100-fold dilution within the linear portion of the calibration curve.
POINTS TO REMEMBER
Serum FLC is a sensitive test for the diagnosis of monoclonal FLC disease.
Increases in the FLC concentration after treatment for MM may be due to a light-chain escape phenomenon, referred to as “light-chain escape from plateau phase” (LEPP).
Our practice has a high percentage (55%) of abnormal FLC ratios in submitted samples, and approximately 0.1% of the sera in our practice are in κ FLC excess. No cases of λ FLC excess have been observed.
Because of the potential for κ FLC antigen excess, newly identified cases with abnormally high ratios should be retested with a further dilution.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors’ Disclosures of Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
Acknowledgments: FLC reagent sets for the dilution studies were provided at no charge by The Binding Site Ltd.
↵1 Nonstandard abbreviations: IFE, immunofixation electrophoresis; FLC, free light chain; MM, multiple myeloma; LEPP, light-chain escape from plateau phase.
- © 2010 The American Association for Clinical Chemistry