Serum and Urine Protein Electrophoresis and Serum-Free Light Chain Assays in the Diagnosis and Monitoring of Monoclonal Gammopathies

Abstract

Background

Laboratory methods for diagnosis and monitoring of monoclonal gammopathies have evolved to include serum and urine protein electrophoresis, immunofixation electrophoresis, capillary zone electrophoresis, and immunosubtraction, serum-free light chain assay, mass spectrometry, and newly described QUIET.

Content

This review presents a critical appraisal of the test methods and reporting practices for the findings generated by the tests for monoclonal gammopathies. Recommendations for desirable practices to optimize test selection and provide value-added reports are presented. The shortcomings of the serum-free light chain assay are highlighted, and new assays for measuring monoclonal serum free light chains are addressed.

Summary

The various assays for screening, diagnosis, and monitoring of monoclonal gammopathies should be used in an algorithmic approach to avoid unnecessary testing. Reporting of the test results should be tailored to the clinical context of each individual patient to add value. Caution is urged in the interpretation of results of serum-free light chain assay, kappa/lambda ratio, and myeloma defining conditions. The distortions in serum-free light chain assay and development of oligoclonal bands in patients‘ status post hematopoietic stem cell transplants is emphasized and the need to note the location of original monoclonal Ig is stressed. The need for developing criteria that consider the differences in the biology of kappa and lambda light chain associated lesions is stressed. A new method of measuring monoclonal serum-free light chains is introduced. Reference is also made to a newly defined entity of light chain predominant intact immunoglobulin monoclonal gammopathy. The utility of urine testing in the diagnosis and monitoring of light chain only lesions is emphasized.

Introduction and Background

Arne Wilhelm Kaurin Tiselius invented protein electrophoresis for which he was awarded a Nobel Prize in 1948. Modifications to the method include introduction of paper and agarose gel as the matrix for electrophoresis, immuno-electrophoresis, and zonal electrophoresis with immunofixation. The same methods for serum are applicable to urine protein electrophoresis, except that urine should be concentrated prior to electrophoresis (1, 2).

Diagnostic methods for monoclonal gammopathy are serum protein electrophoresis (SPEP) and serum protein immunofixation electrophoresis (SIFE). The corresponding methods for urine are urine protein electrophoresis (UPEP) and urine protein immunofixation electrophoresis (UIFE). Additional testing includes total serum and urine proteins, estimation of concentration of major protein bands, and monoclonal immunoglobulin in SPEP, by densitometry (1–9,). Capillary zone electrophoresis (CZE) and immunosubstraction provide similar information (10, 11).

There is lack of standardization about terminology for monoclonal gammopathy interpretation. The terms, paraprotein, monoclonal immunoglobulin, monoclonal band, monoclonal peak, M-protein, M-band, and M-spike are often used interchangeably. It would be preferable to use the term monoclonal immunoglobulin with immunoglobulin type (e.g., monoclonal IgG kappa). The term monoclonal free light chains is recommended for denoting the findings in light chain monoclonal gammopathy; the historical term Bence-Jones protein should be avoided (1, 8, 9).

Quantification of serum IgG, IgM, IgA, and serum-free light chains (SFLC) are in common use and recommended by the international myeloma working group (IMWG). IMWG also recommends examination of 24-hour urine. Additional tests include complete blood cell count (CBC), basic or comprehensive metabolic profile, serum levels of β2 microglobulin, and lactate dehydrogenase (LDH), for staging purposes. Bone marrow examination, cytogenetic analysis, flow cytometry and imaging studies are also useful (2, 12, 13).

Serum and urine protein electrophoreses serve as screening studies in evaluation of monoclonal gammopathy and other disorders, such as nutrition, state of hydration, acute inflammation, chronic inflammation, alpha 1 anti-trypsin deficiency, intravascular hemolysis, nephrotic syndrome, cirrhosis, iron deficiency, C3 complement, proteinuria, renal tubular damage, and cryoglobulins. The primary use of SPEP, SIFE, UPEP, and UIFE is in the diagnosis and monitoring of monoclonal gammopathies. It is recommended that the pathologist review the medical record for a comprehensive report and to promote optimal utilization of SIFE, SFLCA, and other tests. Billing codes 84165, 86334, 84166, and 86335 are used for SPEP, SIFE, UPEP, and UIFE, respectively. Including the findings from a review of the medical record is an essential first step in the effort toward reporting the results as a comprehensive clinical pathology consultation and billing under code 80502 (1–11).

There is wide variation in practice about reporting of serum and urine protein studies. At one extreme, the view is that only presence or absence of monoclonal immunoglobulins needs to be addressed, whereas pathologists opt for more comprehensive reporting with review of medical records. One argument/rationale for abbreviated reporting could be the mismatch between workload and staffing. An additional issue is the qualifications of the person signing out the protein electrophoresis reports. A pathologist has an incentive to present the SPEP and SIFE findings in the context of the clinical status of the patient, taking into account other disorders that may affect serum proteins (e.g., cirrhosis, nephrotic syndrome, acute inflammation, iron deficiency, chronic inflammatory disorders such as rheumatoid arthritis, SLE and HIV infection), and to document chart review required for a clinical consultation (14,). A PhD scientist may limit the report to presence or absence of monoclonal immunoglobulin, in part due to inability to bill for the service. Chart review may also be used by a pathologist to ensure appropriateness of testing. At one institution, the pathologist, by presenting the local utilization data, was able to convince the medical executive board that clinical staff should be limited to ordering SPEP with reflex to SIFE only, and the pathologist would decide if SIFE was warranted (14). Chart review is also relevant in distinguishing monoclonal IgG kappa bands that may result from the use of therapeutic monoclonal antibodies.

Classical monoclonal gammopathies, also referred to as neoplastic monoclonal gammopathies (NMG) consist of monoclonal gammopathy of undetermined significance (MGUS), smoldering or asymptomatic myeloma (SMM), and plasma cell myeloma/multiple myeloma (MM) (15–17).

Analysis of SPEP involves visual examination and densitometric scans, which provide the concentrations of different normal protein bands and of monoclonal immunoglobulin (MIg). Accurate quantification of MIg peaks is compromised by the lack of standard preparations of purified monoclonal Ig that could be used to calibrate the instruments. Quantitative accuracy is further complicated by the variations in the resolution of the gels used for analysis. It is recommended that a gel capable of resolving the two major bands in beta region be used as the minimal level of resolution for SPEP (1, 2, 5, 10, 11).

SIFE, UPEP, and UIFE results are evaluated by visual examination to ascertain the heavy and light chain isotype of the MIg and the presence of monoclonal free light chains. Although capillary electrophoresis (CZE) is not addressed in this review, the issues pertinent to SPEP, SIFE, UPEP, and UIFE apply to CZE as well (10).

In addition to NMGs, there are other instances in which a monoclonal immunoglobulin may be detected in serum and/or urine. MIg may be present as part of the evolution of normal immune response, with process evolving through oligoclonal pattern, one apparent MIg, polyclonal increase in immunoglobulins to normal pattern (18,). A partial list of other disorders associated with MIg includes plasmacytoma; autoimmune disorders such as chronic inflammatory demyelinating neuropathy, myasthenia gravis, cold agglutinin disease, multiple sclerosis; Waldenström macroglobulinemia, and other lymphomas; CLL and other leukemias, including plasma cell leukemia; any neoplastic disorder; AL amyloidosis; light and/or heavy chain deposition disorders; and Polyneuropathy Organomegaly Endocrinopathy Monoclonal Plasma cell disorder and Skin changes (POEMS). Therapeutic monoclonal antibodies may yield an IgG Kappa band on SPEP/SIFE (1–4, 19, 20).

The heavy chains, gamma, mu, alpha, delta, and epsilon with either kappa or lambda chains, heavy chains alone, or light chains alone could result in a monoclonal band on SPEP/SIFE. IgG kappa, IgG lambda, IgA kappa, IgA lambda intact immunoglobulins (IIg), and kappa and lambda light chain monoclonal gammopathies account for 98%–99% of the monoclonal gammopathies. The remaining monoclonal gammopathic disorders may be biclonal, rarely triclonal, nonsecretory, or with IgD or IgE specificity. For the purposes of this monograph, the term monoclonal gammopathies will be limited to Ig lesions consisting of IgG kappa, IgG lambda, IgA kappa, IgA lambda, and light chain lesions of kappa and lambda light chain specificity. Nonsecretory lesions will be addressed as appropriate. About 85% of the MM are IIg lesions, with 13%–15% being light chain lesions only, and 2%–3% represented by biclonal and nonsecretory lesions (20).

Screening and Diagnosis of NMGs

Diagnostic work-up starts with SPEP that may be a screening test or be triggered by suspicion of multiple myeloma due to lytic bone lesions, osteoporosis, pathological fracture, elevated total globulins, renal failure, unexplained neuropathy, amyloidosis, anemia, or hypercalcemia. Additional testing should be employed in an algorithmic fashion (21).

For screening, SPEP and UPEP should both be ordered (the controversy about SFLCA replacing urine testing will be addressed later). The pathologist, based on the results of SPEP and UPEP, along with review of electronic medical record (EMR), should decide if SIFE and/or UIFE and SFLCA assays are warranted. If a monoclonal band is noted on SPEP, SIFE should be done to characterize the band, to ascertain that the band is in fact MIg, and to establish its heavy and light chain specificity. Pathologic states mimicking a MIg band include fibrinogen, elevated C-reactive protein and C3c band, all in anodal gamma region. An apparent peak in the beta region due to elevated transferrin levels is usually associated with iron deficiency and sometimes triggered by estrogen treatment; an apparent peak in the alpha region due to elevated alpha 2 macroglobulin are other examples that may require SIFE to rule out MIg. An artefact at the point of application, often due to cryoglobulins, is another cause of a spurious MIg band (1–4, 14, 21). Serum immunoglobulin quantification should be done to provide information about the state of uninvolved immunoglobulins, as well as a support/corroboration for the densitometric estimate of MIg concentration.

In the case of an IIg monoclonal band in serum, UIFE should be done to ascertain the presence of monoclonal light chains in urine as the latter are nephrotoxic. UIFE should also be done if the band noted on SPEP is a light chain only band. If an IIg monoclonal band or a light chain monoclonal band are detectable by serum and urine examination, there is limited need for SFLCA, though there is wide variations in opinions (20).

In the circumstance of a monoclonal band on SPEP that does not stain for gamma, mu, or alpha heavy chains, but a light chain is detectable on SIFE, the specimen should be examined for IgD or IgE MIg. Quantification for IgD and IgE may be sufficient (1–4,). In renal failure, a monoclonal band may be detectable on SPEP when a patient has only a light chain myeloma. The likelihood of a detectable monoclonal band on SPEP is enhanced with lambda light chain myelomas due to higher tendency of lambda monoclonal light chains to dimerize (22).

Scenarios producing spurious monoclonal bands include restricted IgG subclasses (e.g., IgG4 and the previously mentioned therapeutic monoclonal antibodies) (19, 23, 24,). The low concentration of the MIg band, <0.1 g/dL, in a specific location and history of treatment with MIg is generally sufficient for identification of therapeutic MIg bands. Specific antibodies to therapeutic MIg exist in case a positive identification of a band is needed (23).

In reporting results of SPEP/SIFE, changes in non-immunoglobulin proteins should be correlated with clinical findings from review of EMR. It is not infrequent that SPEP provides the first indication of a serious disorder such as cirrhosis, nephrotic syndrome, alpha 1 anti-trypsin deficiency, chronic inflammation, and nutritional deficiency (1–7).

Reporting Findings of Electrophoretic Analyses

There is lack of standardization for reporting SPEP/SIFE, despite attempts by multiple agencies and groups. The following is a suggested format for the report when a MIg is first detected

1. Identify the immunoglobulin type of MIg

2. Record concentration of MIg.

3. Record location of MIg band. Location may be recorded in general terms, such as cathodal end of gamma region, cathodal region, mid-gamma region, anodal gamma region, interface of beta-gamma region, beta region overlapping C3 band, between transferrin and C3 band, overlapping transferrin band, or alpha 2 region.

4. The rationale for recording the location is:

   • It provides a check on the integrity of the specimen on subsequent examinations (25).

   • If an oligoclonal pattern develops following treatment, location of the neoplastic MIg band is invaluable in distinguishing between the treatment-induced bands versus recurrence or persistence of the malignant clone (26).

5. Comment on the state of uninvolved/background immunoglobulins.

6. Issue one consolidated report with results of SPEP, SIFE and if applicable, UPEP and UIFE. Even if a preliminary report based on SPEP alone is issued, a consolidated report with SIFE, UPEP, and UIFE results is strongly recommended.

7. If MIg is detected for the first time and review of EMR suggests that NMG was not suspected, the pathologist should communicate directly with the attending physician so that results do not go unnoticed. This is not meant to imply that finding a monoclonal Ig constitutes a critical value, but is suggested as a value-added measure.

8. Other clinical parameters (e.g., age, and gender of the patient, anemia, hypercalcemia, hypoalbuminemia, and major diagnoses) should be included to document review of EMR.

If MIg detected is light chain only, there is even less standardization in reporting. If the monoclonal light chain is detectable on UPEP/UIFE only, concentration of the involved SFLC may provide an estimate of the tumor mass and aid in assessing response to treatment. SFLCA does not measure the concentration of monoclonal light chains, just the mass of total light chains. Thus, using the SFLCA results for monoclonal light chain analysis is akin to using the serum levels of the involved IIg rather than the concentration of the MIg peak.

Urine specimens, whether random, first morning, or 24-h collection, should be concentrated 10 to 100-fold to enhance the sensitivity of UPEP/UIFE.

Suggested Format for Reporting if No MIg Is Detected

Even though total serum protein and concentrations of the major components (i.e., albumin, alpha 1, alpha 2, beta 1 and beta 2 and gamma globulins) are included in the routine reports, comments on abnormalities in non-Ig proteins that may be increased or decreased, genetic variants, and any prominent lipid band should be recorded. Refer to the clinical findings and make a statement supporting or contradicting a clinical differential diagnosis. Comment on SFLC results, as these may be ordered routinely, despite a high false positive and false negative rate of findings. It is imperative to caution against over-reading the abnormal kappa/lambda ratios due to their poor sensitivity and specificity for NMG disorders (20, 21, 27). Patients categorized as having MGUS solely based on an abnormal kappa/lambda ratio have been observed. It has been noted, anecdotally, that patients have been subjected to bone marrow examination to resolve an abnormal kappa/lambda ratio, without testing urine, and without any other clinical indication for additional testing.

Indications for SIFE

If no peak/band is detected on SPEP, there may still be a need to perform SIFE to rule out MIg below the detection limit of SPEP. The following are indications for SIFE:

1. Hypogammaglobinemia: This may be a manifestation of a light chain MM.

2. Undiagnosed polyneuropathy: Neuropathy due to an autoantibody is susceptible to treatment by plasma exchange. These monoclonal autoantibodies are usually present in too low a concentration to be discernible on SPEP.

3. An abnormal or prominent band of normal proteins (e.g., a transferrin or C3 bands of higher than usual intensity).

4. A broader than usual normal protein band as this may be due to an adjacent MIg band.

5. History of lytic bone lesion, osteoporosis, or pathological fracture.

6. History of autoimmune disorder (e.g., myasthenia gravis, cold-agglutinin disease, hemolytic anemia, or SLE).

7. Patients status post-transplantation of hematopoietic stem cells or solid organs.

8. History of amyloidosis.

9. History of lymphoma or leukemia.

Appropriate rationale to avoid SIFE include lack of any of the indications listed above with normal SPEP. Polyclonal increase in gamma globulins without any suggestion of a specific peak is not an indication for SIFE, an abnormal kappa/lambda ratio notwithstanding. An abnormal kappa/lambda ratio on SFLCA per se is not an indication for SIFE (27).

If an SIFE is ordered but the attending pathologist considers it to be unwarranted, the latter may cancel the request (the negative implication for part B billing notwithstanding) and include a statement in the SPEP report that SIFE was not performed (8, 10, 14).

Indication for SIFE on Repeat Testing

Patients monitored for MGUS or SMM do not require repeat SIFE if the location of the peak has not changed. Reporting concentration of the peak without repeating SIFE is sufficient. In patients treated for MM, if MIg band is in the same location as before, reporting the concentration of MIg is sufficient. If no MIg is detectable on SPEP, SIFE is warranted as the latter has higher sensitivity. If there are one or more new bands following treatment, SIFE should be done to characterize the new bands. An oligoclonal SPEP/SIFE pattern develops in about 70% of patients treated with autologous stem cell transplantation (ASCT) (28). It may not always be feasible to distinguish between a post-treatment oligoclonal band and a recurrence/persistence of the malignant clone if both are of the same Ig type and in the same location.

In light chain MM, it is appropriate to repeat SFLCA to monitor the level of the involved light chain, keeping in mind that SFLCA does not measure monoclonal light chains only, but all light chains, benign or malignant. UIFE provides positive evidence for monoclonal light chains, if monoclonal light chain is detected (20, 27, 29).

Suggested Format for Reporting Repeat Examination by Protein Electrophoresis with Known NMG

For NMG, not being treated for multiple myeloma, a statement recording the following should suffice:

Previously reported monoclonal Ig (list type) is noted at the same location at a concentration of “x” g/dL. Concentration of the MIg was “y” g/dL on “z” date. A change in the background immunoglobulins should be documented. There is no need to perform an SIFE. However, if a urine sample was submitted a UIFE should be carried out and the results recorded in a consolidated report. It is appropriate to comment on other laboratory findings and changes in serum protein results in the context of the clinical status, as revealed by review of EMR.

For patients treated for multiple myeloma, the suggested format for repeat protein electrophoresis should include the following:

• The previously described monoclonal Ig (mention the Ig type) is noted at a concentration of “x” g/dL. The concertation of MIg was “y” g/dL on “z” date.

• Comment on background immunoglobulins.

• Presence of oligoclonal bands, if any, with a description of the Ig type and location.

• If MIg peak is noted in the same location as the original malignant spike, SIFE is not warranted. SFLCA in such patients does not add value.

• If no MIg is detectable on SPEP, an SIFE should be performed and presence or absence of MIg documented. SFLCA may be considered to detect rare instances of light chain escape. No cases of light chain escape were observed in a 10-year, retrospective review of about 500 cases at this institution. It is postulated that the recurrence of disease in light chain predominant multiple myelomas may present with monoclonal light chains prior to the detection of monoclonal IIg thus creating the appearance of light chain escape (30).

• In case of light chain myelomas, where no MIg was detectable on SPEP or SIFE at original examination, and no MIg is detected at repeat examination, a comment about the concentration of involved SFLC should be entered.

  • UIFE ought to be performed.

  • MALDI-TOF Mass-spectrometry or preferably quantification of monoclonal light chains by ultrafiltration immunofixation electrophoresis test (QUIET) may be performed on serum to detect and quantify monoclonal light chain (31, 32).

MIg Overlapping Normal Protein Band

If MIg clone overlaps a normal protein, usually in the beta region, it is appropriate to include a statement that MIg band overlaps C3 or transferrin and that the concentration of the 2 proteins is included in the quantitation per densitometry. Such values would be useful in monitoring the course of treatment in combination with the concentration of the involved Ig. It has been proposed that measuring heavy/light may be more accurate than trying to monitor the MIg concentration in the beta region; however, this does not seem to add meaningfully to monitoring the patient by measuring the MIg and relevant Ig concentration (33, 34).

Purported Uses of SFLCA

Screening for Monoclonal Gammopathy

The acknowledged gold standard for diagnosing monoclonal gammopathy is SPEP, SIFE, UPEP, and UIFE (35,). IMWG recommends testing for SFLCs. It has been proposed that SFLCA can obviate the need for urine testing, when employed with SPEP and SIFE. SPEP/SIFE, and using the proposed SFLC reference range of 0.26 to 1.65 does discern almost all cases of myeloma, including light chain MM. However, urine examination is also positive in 100% of the cases of light chain MM. When urine examination is positive for a monoclonal light chain, it is diagnostic of monoclonal gammopathy. An abnormal SFLCA result is not contributory in this instance and is falsely abnormal in about 36% of tertiary care patients (27,). More than 30% of patients with a detectable MIg in serum have a normal SFLCA result (20,). While using SPEP and SIFE with SFLCA will detect all significant NMGs, SFLCA will generate a large number of false positive results, entailing unneeded additional investigation, patient consternation and possible harm from unwarranted additional testing (13, 34, 36–42).

The rate of false positive kappa/lambda ratio is greater than 50% in patients with polyclonal hypergammaglobulinemia (27,). Providers sometimes diagnose patients with an abnormal κ/λ ratio, without any evidence of MIg as having MGUS and subject them to consultation by oncology (20, 27, 42, 43,). Polyclonal increase in gamma globulins may predispose to development of monoclonal gammopathy due to persistent inflammatory response (44,). Higher prevalence of kappa dominance in abnormal κ/λ ratio, without any evidence of MIg, reflects a biological preference for kappa chains. In normal serum, IgG kappa accounts for more than 60% of the total IgG. In NMGs the ratio of lesions associated with kappa vs. lambda light chains is about 2:1 (20, 27, 29, 45). This dominance of kappa preference is seen to an extreme in patients with polyclonal increase in gamma globulin. Kappa dominant, abnormal κ/λ ratio is noted in more than 90% of such cases. A similar preference for kappa chains has also been documented in the oligoclonal response noted following hematopoietic stem cell transplantation (45, 46).

The preference for kappa light chains appears to extend to NMGs. Though a normal κ/λ ratio is noted in more than 30% of the samples with a detectable MIg, this false negative rate is much higher for lambda chain associated lesions than that for kappa chain associated lesions. About 60% of kappa light chain associated MGUS samples have a normal κ/λ ratio whereas the corresponding frequency for lambda light chain associated lesions is almost 90% (20, 45,). The usefulness of SFLCA applies to monitoring of light chain myeloma and perhaps the newly defined entity of light chain predominant multiple myeloma (LCPMM). The assay could have a role in early detection of relapse in LCPMM (29, 30).

Though IMWG recommends electrophoretic evaluation of 24-h urine specimens, a concentrated random urine sample is usually adequate. First morning specimen is preferable due to it being naturally more concentrated (47).

While SFLCA is less expensive than UPEP/UIFE, SFLCA has limited utility in a screening algorithm, whereas a positive finding of MIg in UIFE is diagnostic (27, 36).

Prognostic Value of SFLC

The literature asserts that an abnormal κ/λ ratio in MGUS indicates a worse prognosis (48–53,). An abnormal κ/λ ratio is a nonspecific finding and indicates a state of chronic inflammation (53,). In patients with no MIg, an abnormal κ/λ ratio indicates a worse prognosis and the same is the case in Hodgkin disease and other lymphomas and individuals without any neoplastic disorders (51, 54,). An abnormal κ/λ ratio suggests an adverse outcome in a nonspecific way as do elevated ferritin, CRP, and RDW, and lower levels of serum albumin (52, 54–58).

Monitoring the Results of Treatment

It has been claimed that because light chains have a shorter half-life than IIg, changes in SFLCs could be used to ascertain response or lack of response to treatment faster than using level of neoplastic MIg. A retrospective review of patient records did not disclose any change in treatment based on SFLCA. A systematic examination did not reveal any benefit in using SFLCA over monitoring the level of neoplastic MIg (59, 60).

Monitoring the response of stem cell transplants with SFLCA is complicated by the emergence of oligoclonal bands in nearly 70% of cases (28, 45,). Oligoclonal bands tend to produce a kappa dominant abnormal κ/λ ratio, irrespective of the primary disorder. More than 15% of the abnormal κ/λ ratios in patients with lambda chain associated lesions (i.e., IgG lambda or IgA lambda MM), status post ASCT were kappa dominant. This is a conservative estimate of the false positive rate in that the plasma cells needed to produce sufficient excess kappa light chains to overcome the monoclonal lambda chain due to lambda chain associated malignancy, to result in kappa dominant abnormal κ/λ ratio. If this conservative 15% false positive kappa dominant abnormal κ/λ ratio is applied to patients with kappa chain associated MM, about 40% of the kappa dominant abnormal κ/λ ratios in these patients would be false positives, thus obliterating the utility of monitoring κ/λ ratio in status post ASCT patients (45, 61, 62,). There is lack of consensus about prognostic value of oligoclonal pattern (63, 64).

Following chemotherapy and ASCT, the original malignant clone may not be detectable. In such case a dominant oligoclonal band of the same immunoglobulin class may be mistaken for residual/recurrent disease. While it is helpful to note the location of the malignant Ig band to evaluate recurrence, in the absence of residual serum or electrophoretic profiles for comparison, this technique is not very accurate. The historic progression of MIg migration is made more manageable by the equipment manufacturers providing software for comparison with earlier gels (26).

It would behoove the equipment manufacturers to improve the resolution of gels to further facilitate separation of post-treatment clones from the original malignant Ig clone.

SFLCA in AL Amyloid and Non-Secretory MM

There may be a limited role for SFLCA in monitoring patients with AL amyloid and/or nonsecretory MM. Assays assessing propensity of light chains to form dimers may be a better indicator of risk for amyloidosis and in general a need for more sensitive methods has been emphasized (65–69).

Stringent Complete Response to Treatment for MM

IMWG has defined stringent complete response as meeting criteria for complete response plus a normal κ/λ ratio, however, a large number of false-positive kappa dominant abnormal κ/λ ratios with ASCT renders this criterion moot. It has been shown that lack of normal SFLCA result is not consequential to outcome of treatment (45, 70–75).

Myeloma Defining Condition

The IMWG has developed criteria for myeloma defining condition as having one of the following findings in a patient with SMM:

• More than 60% plasma cells in bone marrow.

• More than one bone lesion on MRI examination

• Involved serum-free light chain concentration of greater than 100 mg/L and ratio of concentration of involved to un-involved light chain >100.

It was observed that about 16% of the SMM patients meeting criterion “c” above progressed to MM. A 16% sensitivity is poor performance for a clinical assay, and suitability of early treatment has been disputed (76–78,). The referenced study did not segregate the patients by the type of light chain. This is imperative as it has been shown that on average, kappa chain associated NMGs produce 4 times more free light chains than do lambda chain associated NMGs. In addition, the uninvolved kappa light chains in lambda chain associated lesions are, on average, present at twice the concentration of lambda light chains in kappa chain associated NMGs. Thus, if one were to use criteria specific for light chain type of lesions, the criterion “c” above should be amended to require that level of involved kappa light chain is >100 mg/L and lambda light chain >25 mg/L. The ratio of involved to uninvolved light chains should be 100 for kappa and 12.5 of lambda chain associated lesions (79).

Objective evidence for treating SMM based on myeloma defining condition is lacking and standard of care remains observation of SMM till symptomatic MM develops (78, 80, 81,). Evidence to support treating patients with symptomatic MM early rather than late has not been forthcoming. Prolonged overall survival has been reported with ASCT and also disputed by others (81–86,). There is general agreement that ASCT prolongs progression free survival but not overall survival (86).

Monitoring the Course of Light Chain MMs

SFLCA offers no advantage over urine examination in diagnosis of light chain MM. A case could be made for using SFLCA for monitoring the course of light chain MMs. The SFLCA provides a number that could make comparison of serial measurements meaningful. Using 24-h urine to calculate the change in production of SFLCs would be inconvenient, cumbersome, and not too accurate. The erroneously abnormal κ/λ ratios seen in IIg MM would be applicable to light chain MM as well (20, 87–89,). UIFE may be sufficient in monitoring light chain MM (47).

Light Chain Escape

In rare instances, subsequent to treatment, an IIg myeloma may recur as a light chain only disorder. The detection and monitoring of such patients may be facilitated by SFLCA (90,). However, the newly defined entity of LCPMM may provide a better explanation for the rare phenomenon described as light chain escape (30).

Minimal Residual Disease

As and when curative treatment for MM becomes available, determining minimal residual disease (MRD), may be warranted. For now, tests for ascertaining MRD are only relevant in randomized controlled trials (90, 91).

Measuring SFLCs

There is lack of standardization, and correlation amongst the various methods for measuring SFLC is inadequate. Reagents from a single vendor, when used on different platforms, provide disparate results. There are variations in normal populations by ethnicity in addition to variations induced by non-NMG disorders (92–99).

Measuring Monoclonal Serum Free Light Chains

An affinity purification and MALDI-TOF mass spectrometry assay has been described to detect monoclonal serum-free light chains; however, the assay is not quantitative and would offer no advantage over UIFE (31).

The QUIET method for measuring monoclonal SFLCs consists of ultrafiltration of serum using a nominal 50 kDa cut-off membrane, concentrating the filtrate and testing the concentrated specimen by immunofixation electrophoresis, using antisera to kappa and lambda light chains. Densitometry of the IFE gel provides quantity of monoclonal light chain (32,). It is speculated that the QUIET method could be used to detect minimal residual disease in patients with any type of MM by monitoring the monoclonal SFLCs, by using antisera specific to SFLCs (32).

In summary, serum and urine protein electrophoretic tests remain the gold standards for diagnosis and monitoring of monoclonal gammopathic disorders. The pathologist should review the EMR and issue a comprehensive report of various laboratory studies and a clinical correlation of the laboratory results. SFLCA adds minimal value and may be useful only in monitoring patients with light chain MM and perhaps LCPMM patients. Newer quantitative assays for monoclonal SFLC may add value in monitoring monoclonal light chain disorders and possibly in determining minimal residual disease in MM.

Nonstandard Abbreviations: SPEP, serum protein electrophoresis; SIFE, serum proteinimmunofixation electrophoresis; UPEP, urine protein electrophoresis; UIFE, urine protein immunofixation electrophoresis; CZE, capillary zone electrophoresis; SFLC, serum-free light chains; SFLCA, serum-free light chain assay; IMWG, international myeloma working group; NMG, neoplastic monoclonal gammopathies; MGUS, monoclonal gammopathy of undetermined significance; SMM, smoldering or asymptomatic myeloma; MM, plasma cell myeloma/multiple myeloma; MIg, monoclonal immunoglobulin; EMR, electronic medical record; LCPMM, light chain predominant multiple myeloma; QUIET, Quantification by Ultrafiltration and Immunofixation Electrophoresis Testing.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 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; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.

Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: G. Singh, Diazyme Inc., HealthTap. Stock Ownership: None declared. Honoraria: None declared. Research Funding: None declared. Expert Testimony: None declared. Patents: G. Singh, patent application pending for QUIET.

REFERENCES

Nonstandard Abbreviations

 
• SPEP

  serum protein electrophoresis

 
• SIFE

  serum protein immunofixation electrophoresis

 
• UPEP

  urine protein electrophoresis

 
• UIFE

  urine protein immunofixation electrophoresis

 
• CZE

  capillary zone electrophoresis

 
• SFLC

  serum-free light chains

 
• SFLCA

  serum-free light chain assay

 
• IMWG

  international myeloma working group

 
• NMG

  neoplastic monoclonal gammopathies

 
• MGUS

  monoclonal gammopathy of undetermined significance

 
• SMM

  smoldering or asymptomatic myeloma

 
• MM

  plasma cell myeloma/multiple myeloma

 
• MIg

  monoclonal immunoglobulin

 
• EMR

  electronic medical record

 
• LCPMM

  light chain predominant multiple myeloma

 
• QUIET

  Quantification by Ultrafiltration and Immunofixation Electrophoresis Testing