Laboratory methods for analyzing monoclonal proteins

INTRODUCTION — The monoclonal gammopathies (paraproteinemias or dysproteinemias) are a group of disorders characterized by the proliferation of a single clone of plasma cells, which produces an immunologically homogeneous protein commonly referred to as a paraprotein or monoclonal protein (M protein, where the "M" stands for monoclonal). (See 'Definition of an M protein' below.)

A complete immunoglobulin (Ig) consists of two heavy polypeptide chains of the same class designated by a capital letter and a corresponding Greek letter:

●Gamma in IgG

●Alpha in IgA

●Mu in IgM

●Delta in IgD

●Epsilon in IgE

The heavy polypeptide chains are further subdivided: IgG has four subclasses (IgG1, IgG2, IgG3, and IgG4) and IgA has two (IgA1 and IgA2). To form the intact immunoglobulin, the paired heavy chains are associated with two light chains of the same type, either kappa or lambda, but not both (figure 1). (See "Structure of immunoglobulins".)

This topic will review the methods used to detect an M protein in the serum or urine and identify it according to its heavy chain class and light chain type. Further details about monoclonal gammopathies, and the diagnosis of multiple myeloma, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, immunoglobulin light chain (AL) amyloidosis, light and heavy chain deposition diseases, and heavy chain diseases are presented separately.

●(See "Diagnosis of monoclonal gammopathy of undetermined significance".)

●(See "Multiple myeloma: Clinical features, laboratory manifestations, and diagnosis".)

●(See "Epidemiology, pathogenesis, clinical manifestations, and diagnosis of Waldenström macroglobulinemia".)

●(See "Clinical manifestations, pathologic features, and diagnosis of lymphoplasmacytic lymphoma".)

●(See "Clinical presentation, laboratory manifestations, and diagnosis of immunoglobulin light chain (AL) amyloidosis".)

●(See "Monoclonal immunoglobulin deposition disease".)

●(See "The heavy chain diseases".)

DEFINITION OF AN M PROTEIN — An M protein (paraprotein, monoclonal protein, M component) is a monoclonal immunoglobulin secreted by an abnormally expanded clone of plasma cells in an amount that is above the polyclonal immunoglobulin background. This M protein is detected by immunofixation of serum and/or urine, or (rarely) other body fluids (eg, jejunal fluid in alpha heavy chain disease), or by the serum free light chain assay [1].

The M protein can contain:

●Heavy and light chains (an intact immunoglobulin)

●Light chains only (ie, light chain myeloma, light chain deposition disease, AL amyloidosis)

●Heavy chains only (ie, heavy chain disease, heavy chain deposition disease)

IMPORTANCE OF CLINICAL HISTORY IN TESTING

Communication between clinician and laboratory — Clear communication between the clinician managing the patient and the laboratory is an essential part of evaluating monoclonal proteins as clinical history influences the choice of tests performed and their interpretation. It is especially important to communicate if the clinician suspects AL amyloidosis or a plasma cell/lymphocytic disorder so that a greater effort can be made to detect an M protein, which may be present in such a low concentration that it is not detected by routine serum or urine protein electrophoresis, but may be detected by more sensitive techniques (eg, immunofixation, mass spectrometry, or free light chain assay).

A search for monoclonal proteins should be included in the evaluation of all patients suspected of having multiple myeloma, Waldenström macroglobulinemia, AL amyloidosis, or a related disorder. This evaluation should also be considered for any patient with an elevated total serum protein or otherwise unexplained signs and symptoms suggestive of the presence of a plasma cell disorder. (See 'Indications' below.)

An initial search for monoclonal proteins usually includes a serum protein electrophoresis (SPEP), serum immunofixation, 24-hour urine protein electrophoresis (UPEP), and urine immunofixation. Alternatively, a serum free light chain (FLC) assay may be used instead of the 24-hour UPEP as an initial test. The evaluation of monoclonal gammopathies is discussed separately. (See "Diagnosis of monoclonal gammopathy of undetermined significance".)

Causes — The presence of an M protein in the serum or urine indicates an underlying clonal plasma cell or lymphoproliferative disorder (table 1). In some cases, the clonal process producing the M protein is malignant and is associated with evidence of neoplastic disease infiltrating bone, lymph nodes, liver, spleen, or other organs (eg, multiple myeloma, solitary plasmacytoma, Waldenström macroglobulinemia). In other cases, the M protein is produced by a small limited premalignant clonal expansion, and causes no symptoms (eg, monoclonal gammopathy of undetermined significance [MGUS]) or limited symptoms (eg, monoclonal gammopathy of clinical significance (table 2)). (See "Diagnosis of monoclonal gammopathy of undetermined significance".)

Most premalignant disorders in medicine have limited clinical consequence until malignant transformation occurs. In contrast, premalignant clonal expansion of plasma cells can result in disabling or fatal disease related to the secreted immunoglobulin, termed monoclonal gammopathy of clinical significance [2].

Complications may reflect one or more adverse properties of the secreted M protein, such as [2]:

●Ability to agglutinate red cells (eg, cold agglutinin disease) (see "Cold agglutinin disease")

●Insolubility at low temperatures (eg, cryoglobulinemia) (see "Overview of cryoglobulins and cryoglobulinemia")

●Increased viscosity (eg, Waldenström macroglobulinemia) (see "Epidemiology, pathogenesis, clinical manifestations, and diagnosis of Waldenström macroglobulinemia")

●Deposition in tissues with resulting organ dysfunction (eg, AL amyloidosis or the immunoglobulin deposition diseases) (see "Clinical presentation, laboratory manifestations, and diagnosis of immunoglobulin light chain (AL) amyloidosis" and "Monoclonal immunoglobulin deposition disease")

●Neuropathy (paraneoplastic neuropathy; eg, MGUS, Waldenström macroglobulinemia, AL amyloidosis, POEMS syndrome) (see "POEMS syndrome" and "Clinical presentation, laboratory manifestations, and diagnosis of immunoglobulin light chain (AL) amyloidosis")

●Kidney impairment (eg, monoclonal gammopathy of renal significance, proliferative glomerulonephritis with monoclonal immunoglobulin deposits [PGNMID], acquired Fanconi syndrome)

The clinician seeing a patient with an M protein must therefore make the appropriate diagnosis and initiate effective therapy in a timely manner if appropriate, in order to prevent irreversible organ damage and/or shortening of life [3].

Interference with laboratory tests — The presence of a circulating M protein may interfere with one or more laboratory tests performed on liquid-based automated analyzers, by precipitating during the analysis, by volume displacement and change of sample viscosity, or by virtue of its specific binding properties.

The most common artifacts are a low value for HDL cholesterol, a high value for bilirubin, as well as altered measurement of inorganic phosphate [4-8]. Other examples include interference with measurement of LDL cholesterol, C-reactive protein, antistreptolysin-O, creatinine, glucose, sodium, chloride, bicarbonate [9], urea nitrogen, albumin, iron [10], and inorganic calcium (table 3).

Re-analysis using a different method or sample dilution can be employed for obtaining accurate measurements [4]. These events may occur in patients whose clinicians are unaware of the presence of an underlying M protein and might result in the mismanagement of patients with monoclonal gammopathy, especially as regards measurement of HDL and LDL cholesterol and estimation of cardiovascular risk [6,8].

ANALYSIS OF SERUM — Analysis of serum for the presence of M proteins, or for the evaluation of a patient with increased total serum proteins, is classically performed using electrophoretic techniques, supplemented with additional tests for protein quantification and methodologies to determine whether the protein arises from a single clone (ie, monoclonal) (table 4).

Serum protein electrophoresis (SPEP) — SPEP is an inexpensive and easy to perform screening procedure to detect an M protein. SPEP is usually done by the agarose gel method (agarose gel electrophoresis). The resulting M protein, if found, can be quantitated using a densitometer tracing of the gel (figure 2A-B). Thus, the SPEP serves two purposes: detect presence or absence of an M protein in the serum, and, in conjunction with the total protein concentration, enable measurement of the concentration ("size") of the M protein.

While variation across laboratories has been reported [11,12], the quantification is generally uniform across laboratories such that the size of a monoclonal spike measured at one laboratory can be compared with the size of the monoclonal spike from a different laboratory. However, this measurement can be impacted by the patient's hydration status and there is a certain degree of subjectivity in the quantification process. As such, we obtain sequential measurements from the same laboratory, whenever possible.

Capillary zone electrophoresis is an alternative and slightly more sensitive method of performing SPEP. It measures protein on-line via ultraviolet light absorbance techniques; protein stains are not necessary and no point of application is seen [13,14]. The electrophoretograms are similar to those produced with high resolution agarose gel SPEP. Mass spectrometry is another method available at some centers. (See 'Mass spectrometry' below.)

In the electrophoretic methodologies (agarose or capillary zone), proteins are classified by their final position after electrophoresis is complete into five general regions: albumin, alpha-1, alpha-2, beta, and gamma (figure 2A). These regions, which also use a Greek lettering system, do not refer to the immunoglobulin class to which an M protein may belong, and refer only to mobility through the support medium. The various immunoglobulin classes (IgG, IgA, IgM, IgD, and IgE) are usually located in the gamma region of the SPEP, but they may also be found in the beta-gamma and beta regions and may occasionally extend into the alpha-2 globulin area.

Indications — SPEP is indicated in all patients in whom multiple myeloma, Waldenström macroglobulinemia, primary amyloidosis, or a related disorder is suspected. The initial SPEP should always be performed in combination with serum immunofixation in order to determine and confirm monoclonality, and to determine the immunoglobulin heavy and light chain class if an M protein is identified. (See 'Serum immunofixation' below and "Multiple myeloma: Clinical features, laboratory manifestations, and diagnosis" and "Epidemiology, pathogenesis, clinical manifestations, and diagnosis of Waldenström macroglobulinemia".)

SPEP should also be considered in any patient with an elevated total serum protein or otherwise unexplained signs and symptoms suggestive of the presence of a plasma cell disorder. These include any one or more of the following [15]:

●Unexplained anemia, back pain, weakness, or fatigue

●Osteopenia, osteolytic lesions, or spontaneous fractures

●Kidney impairment with a bland urine sediment

●Heavy proteinuria in a patient over age 40 years

●Hypercalcemia

●Hypergammaglobulinemia

●Immunoglobulin deficiency

●Bence Jones proteinuria

●Unexplained peripheral neuropathy

●Recurrent infections

●Elevated erythrocyte sedimentation rate or serum viscosity

As an example, the presence of a localized band or spike on SPEP in adults with nephrotic syndrome, refractory heart failure, orthostatic hypotension, peripheral neuropathy, carpal tunnel syndrome, or malabsorption strongly suggests the possibility of primary amyloidosis (AL) and requires confirmation with immunofixation studies. (See 'Serum immunofixation' below and "Clinical presentation, laboratory manifestations, and diagnosis of immunoglobulin light chain (AL) amyloidosis".)

Monoclonal gammopathy — A monoclonal protein (M protein) usually presents as a single narrow peak, like a church spire, in the gamma, beta, or alpha-2 region of the densitometer tracing or as a dense, discrete band on the agarose gel (figure 2A-B). In approximately 5 percent of sera with an M protein, two M proteins are present (biclonal gammopathy) (figure 2B-C) [16]. Serum immunofixation must be performed in order to determine clonality (eg, monoclonal, biclonal, polyclonal). (See 'Serum immunofixation' below.)

A monoclonal protein can be present in a number of different disorders (monoclonal gammopathies), including B cell and plasma cell proliferations (algorithm 1). The most common of these are listed in the table (table 1).

Biclonal gammopathy — Occasionally a patient may have two different monoclonal proteins, and this is referred to as biclonal gammopathy (figure 3).

Biclonal gammopathies are typically detected when two different heavy or light chain classes of M proteins are detected on immunofixation (eg, an IgA kappa M protein and an IgM lambda M protein in the same patient). Sometimes, the presence of monomers and polymers of an M protein may produce two spikes on the SPEP and can resemble a biclonal gammopathy.

Patients with biclonal gammopathy can have a malignancy such as multiple myeloma or may just have a premalignancy. Patients with biclonal gammopathy of undetermined significance have the same clinical spectrum as those with monoclonal gammopathy of undetermined significance (MGUS) and should be followed in the same manner [16]. (See "Clinical course and management of monoclonal gammopathy of undetermined significance".)

Polyclonal gammopathy — A polyclonal increase in immunoglobulins is usually identified as a broad-based peak or band, typically of gamma mobility (figure 2B, 2D). Polyclonal gammopathies are most often due to infectious, inflammatory, or reactive processes. In chronic hepatitis, for example, the gamma component may reach 6 or 7 g/dL (60 or 70 g/L).

The cause is usually apparent based on history and review of initial laboratory tests (eg, liver disease, connective tissue disease, chronic infection, hematologic disorders and non-hematologic malignancy). Infrequently, polyclonal gammopathy presents without evidence of an underlying process.

Our initial laboratory evaluation of unexplained polyclonal gammopathy includes:

●Complete blood count with differential and platelet count

●Liver biochemical tests, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase

●Hepatitis C serology

●Human immunodeficiency virus (HIV) serology

Further testing for secondary causes is based on a comprehensive history and physical examination. Blood and urine cultures, and fluid cultures, are included if the patient is febrile or acutely ill. Autoantibody testing is included if autoimmune hepatitis is suspected. (See "Overview of autoimmune hepatitis", section on 'Autoantibodies'.)

In a retrospective cohort study of 148 patients seen at the Mayo Clinic, in whom a polyclonal gamma globulin level ≥3 g/L was found, a single associated medical disorder was present in 130 [17]. No patient developed multiple myeloma or a clonal plasma cell proliferative disorder. The most common disorders were:

●Liver disease – 61 percent

●Connective tissue disease – 22 percent

●Chronic infection – 6 percent

●Hematologic disorders – 5 percent

●Non-hematologic malignancy – 3 percent

●Other – 3 percent

Polyclonal gammopathy, along with bone marrow plasmacytosis, is a common finding in HIV-infected patients [18]. However, these patients also have an increased incidence of clonal plasma cell disorders (eg, MGUS, multiple myeloma, plasmablastic lymphoma) [19-21]. (See "HIV-related lymphomas: Epidemiology, risk factors, and pathobiology" and "HIV infection and malignancy: Management considerations", section on 'Plasma cell disorders'.)

Hypogammaglobulinemia — Hypogammaglobulinemia (<0.6 g/dL [6 g/L]) is characterized by a decrease in size of the gamma mobility component on SPEP, and should be documented by quantitation of serum IgG, IgA, and IgM levels. (See 'Quantitation of immunoglobulins' below.)

Hypogammaglobulinemia may be congenital, sex-linked, and/or part of a combined immunodeficiency state. It may also be acquired, as in multiple myeloma, AL amyloidosis, chronic lymphocytic leukemia, lymphoma, or the nephrotic syndrome (see "Primary humoral immunodeficiencies: An overview" and "Severe combined immunodeficiency (SCID): An overview"):

●Panhypogammaglobulinemia occurs in about 10 percent of patients with multiple myeloma. Most of these patients have a Bence Jones protein (monoclonal free kappa or lambda light chains) in the urine, but lack intact immunoglobulins in the serum [22].

●Panhypogammaglobulinemia is seen in approximately 20 percent of patients with AL amyloidosis, often associated with a nephrotic pattern (mainly albumin with nonselective globulin loss) in the urine.

The evaluation of unexplained hypogammaglobulinemia should include a 24-hour urine protein electrophoresis and immunofixation. Further testing for secondary causes is based on a comprehensive history and physical examination. (See 'Analysis of urine' below.)

Other SPEP patterns — A number of other patterns may be observed on the SPEP:

●A decrease in serum albumin and an increase in alpha-1 and alpha-2 globulins may be present in patients with infection or metastatic malignancy.

●Marked reduction of the alpha-1 globulin component is usually due to a deficiency of alpha-1 antitrypsin.

●Two albumin bands (bisalbuminemia) are rarely found. This familial abnormality produces no symptoms [23].

False negative results — A small M protein may be present even when quantitative immunoglobulin values, beta and gamma mobility components on SPEP, and total serum protein concentrations are all within normal limits. If a clonal plasma cell disorder is suspected, the most sensitive tests to screen for presence of a monoclonal protein are serum and urine immunofixation, mass spectrometry, and the serum free light chain assay. (See 'Serum immunofixation' below and 'Serum free light chains' below.)

The following caveats need to be kept in mind:

●In alpha heavy chain disease (HCD), which occurs in patients with a form of small intestinal lymphoma called immunoproliferative small intestinal disease (IPSID), one never sees a localized band or sharp peak, presumably because of the tendency of these chains to polymerize, or due to their high carbohydrate content [24-27]. In some patients these proteins can be found in jejunal fluid but not in the serum. (See "Clinical presentation and diagnosis of primary gastrointestinal lymphomas", section on 'Lymphoma of the small intestine' and "The heavy chain diseases", section on 'Alpha HCD'.)

●In mu HCD, panhypogammaglobulinemia is a prominent feature and a localized band is found in only 40 percent [28]. (See "The heavy chain diseases", section on 'Mu HCD'.)

●In an occasional patient with gamma HCD, the electrophoretic tracing may appear broad and heterogeneous rather than showing a localized band [26,29,30]. (See "The heavy chain diseases", section on 'Gamma HCD'.)

●An M protein may produce a broad band in the agarose gel, suggesting a polyclonal pattern. This can occur when an M protein complexes with other plasma components, or when there are IgM dimers and pentamers, IgA polymers, or IgG aggregates.

●Some patients make only monoclonal light chains (Bence Jones proteinemia), which are usually present in concentrations too low to be visible as a spike in the agarose gel, because of rapid excretion in the urine [31]. The serum concentrations will rise if such patients develop kidney failure.

●In some patients with IgD or IgE myeloma, the M protein spike may be small and easily overlooked.

False positive results — Serum protein elements other than immunoglobulins may falsely suggest the presence of an M protein. As examples:

●Fibrinogen (in plasma) is seen as a discrete band between the beta and gamma mobility regions. This is indistinguishable from an M protein; addition of thrombin to the specimen will produce a clot if fibrinogen is present. The presence of fibrinogen is established if the discrete band is no longer detected when electrophoresis is repeated after the addition of thrombin.

●Hemoglobin-haptoglobin complexes secondary to hemolysis may appear as a large band in the alpha-2-globulin region.

●High concentrations of transferrin in patients with iron-deficiency anemia may produce a localized band in the beta region.

●Nephrotic syndrome is often associated with increased alpha-2 and beta bands which can be mistaken for an M protein. Serum albumin and gamma globulin concentrations are usually reduced in this setting.

●Nonspecific increases in acute phase reactants or certain hyperlipoproteinemias may result in increases in alpha-1 bands.

●Patients with high levels of IgG4 can produce a relatively restricted band cathodic to the beta bands.

●A common artifact is the presence of a protein band at the point of application of the sample. A clue to the presence of this artifact is the presence of this band on simultaneously performed samples from multiple patients.

Autoantibodies do not cause a false positive M protein. Even though autoantibodies may target a specific antigen, they are polyclonal. Rarely, a very high rheumatoid factor titer can produce a relatively restricted band which can be over interpreted as an M protein [32].

Several therapeutic monoclonal antibodies used for the treatment of plasma cell dyscrasias (eg, daratumumab, isatuximab, elotuzumab) are human IgG kappa monoclonal antibodies that can be detected on SPEP. This is particularly important in patients with IgG kappa M protein as standard SPEP cannot differentiate between the therapeutic monoclonal antibody and the endogenous M protein. Laboratories should be notified if a patient has received one of these antibodies. The laboratory may be able to modify the assay or use another assay to better estimate the M protein level. As an example, a daratumumab-specific immunofixation electrophoresis reflex assay has been developed, which uses a murine anti-daratumumab antibody to shift the migration of daratumumab on electrophoresis to allow for differentiation between it and native IgG kappa [33,34]. Mass spectrometry may also be able to distinguish between therapeutic and endogenous M proteins. (See 'Mass spectrometry' below.)

In contrast, infliximab, rituximab, adalimumab, eculizumab, and vedolizumab do not routinely produce false positives on SPEP [35]. Small monoclonal IgG proteins may be identified in such patients directly following infusion. Thus, a small IgG kappa finding on immunofixation could be due to an exogenous drug.

Serum immunofixation — SPEP is a useful initial procedure to screen for an M protein but has two drawbacks. First, it is not as sensitive when M proteins are small. An M protein may be easily overlooked or an apparent M protein may actually represent a polyclonal increase in immunoglobulins or another protein. Second, if an M protein is present, the immunoglobulin heavy and light chain class cannot be determined from the SPEP. Consequently, the laboratory must perform serum immunofixation in order to ascertain the presence of an M protein and to determine its type. (See "Overview of therapeutic monoclonal antibodies".)

Serum immunofixation is critical for the differentiation of a monoclonal from a polyclonal increase in immunoglobulins. In traditional immunofixation, the patient's serum is electrophoresed into at least five separate lanes. Following electrophoretic separation of the serum proteins, each sample is overlaid with a different monospecific antibody, usually three for the heavy chain component and two for the light chain component (eg, anti-gamma, anti-alpha, anti-mu, anti-kappa, and anti-lambda, respectively). Precipitation of proteins (ie, the antigen-antibody complex) is allowed to occur, followed by washing (nonprecipitated proteins wash out) and staining of the remaining immunoprecipitates.

Immunosubtraction is an alternative procedure to serum immunofixation. In this procedure the serum sample is incubated with Sepharose beads coupled with anti-gamma, -alpha, -mu, -kappa, and -lambda antisera. After incubation with each of the heavy and light chain antisera, the supernatants are reanalyzed to determine which reagent(s) removed the electrophoretic abnormality. The immunosubtraction procedure is technically less demanding, is automated, and is therefore a useful alternative to serum immunofixation [13,14]. It has the same indications, and accomplishes the same goals, as serum immunofixation. (See 'Indications' below.)

An M protein is characterized on immunofixation by the combined presence of a sharp, well-defined band associated with a single heavy chain class and a sharp, well-defined band with similar mobility characteristics that reacts with either kappa or lambda light chain antisera, but not both (figure 4). In comparison, an M protein is characterized on immunosubtraction by the elimination of the electrophoretic abnormality by incubating with an antiserum to a single heavy chain class and with either anti-kappa or anti-lambda, but not both.

Several therapeutic monoclonal antibodies used for the treatment of plasma cell dyscrasias (eg, daratumumab, isatuximab, elotuzumab) are human IgG kappa monoclonal antibodies that can be detected on immunofixation assays. As such, they may obfuscate the response assessment in patients with IgG kappa myeloma protein.

Indications — Serum immunofixation is more sensitive than SPEP and also determines the heavy and light chain type of the monoclonal protein. However, unlike SPEP, immunofixation does not give an estimate of the size of the M protein (ie, its serum concentration), and thus should be done in conjunction with electrophoresis. Serum immunofixation should be performed when a sharp band or peak is found in the agarose gel (ie, a monoclonal protein on SPEP) or when multiple myeloma, macroglobulinemia, primary amyloidosis, solitary or extramedullary plasmacytoma, or a related disorder is suspected, despite a normal SPEP pattern [2].

Immunofixation will detect a serum M protein at a concentration of at least 0.02 g/dL (0.2 g/L) and a urine M protein at a concentration of ≥0.04 g/L [15]. It should always be performed in the presence of otherwise unexplained sensory motor peripheral neuropathy, nephrotic syndrome, refractory heart failure, orthostatic hypotension, carpal tunnel syndrome, malabsorption, or whenever the clinical situation suggests the possibility of AL amyloidosis. (See "Clinical presentation, laboratory manifestations, and diagnosis of immunoglobulin light chain (AL) amyloidosis".)

There are also a variety of other indications for immunofixation:

●Detection of a small M protein in the presence of normal or increased background immunoglobulins.

●In patients with multiple myeloma or macroglobulinemia in whom treatment has resulted in disappearance of the band on SPEP.

●Recognition and distinction of biclonal (two M proteins) (figure 3) or triclonal (three M proteins) (figure 5) gammopathies [36]. Such multiple M proteins may have only a single band or spike on SPEP and may otherwise be missed. (See 'Biclonal gammopathy' above.)

In addition, the possibility of IgD and IgE monoclonal proteins must be excluded by immunofixation using IgD and IgE antisera in all patients with a monoclonal light chain in the serum but no reactivity to anti-G, anti-M, or anti-A.

When following patients with multiple myeloma, MGUS, or a related disorder, once the presence of a monoclonal protein and its type are initially confirmed by immunofixation, it is not necessary to repeat immunofixation unless needed to document complete response to therapy. Patients can usually be followed with electrophoresis of serum (SPEP) or urine (UPEP) proteins. (See "Multiple myeloma: Evaluating response to treatment".)

Serum free light chains — The serum free light chain (FLC) assay is a sensitive antibody-based system that can detect low concentrations of monoclonal FLCs (ie, kappa or lambda) in the serum. This assay is of particular importance in the approximately 16 percent of patients with multiple myeloma that produce only a Bence Jones protein (FLC that is not bound to a heavy chain), often in concentrations too low to be detected by routine serum immunofixation techniques [22]. Under such circumstances, or when AL amyloidosis or light chain deposition disease are suspected, it is routine to analyze a 24-hour urine collection for the presence of FLCs. The serum FLC assay complements and potentially obviates the need for a 24-hour UPEP when screening for multiple myeloma and related plasma cell disorders. (See 'Indications and uses' below and '24-hour urine protein electrophoresis (UPEP)' below.)

The serum FLC assay can detect low concentrations of monoclonal FLCs in serum. For the serum FLC assay developed by The Binding Site, normal levels are as follows (95 percent confidence intervals) [37]:

●Free serum kappa light chains – 3.3 to 19.4 mg/L

●Free serum lambda light chains – 5.7 to 26.3 mg/L

●Ratio of kappa to lambda FLCs – 0.26 to 1.65

The serum FLC assay is now developed and marketed commercially by several manufacturer. Although the overall correlations between these tests have been demonstrated, measured kappa and lambda values from different manufacturers can vary significantly for individual patients [38]. It is important to take note of the specific FLC assay used when interpreting serial measurements of quantitative FLCs as a change in the assay used may aid the interpretation of what appear to be discrepant results for an individual. Reference ranges are also dependent on the instrument used for measurement [39]. When interpreting the FLC assay in clinical practice, clinicians should refer to the normal reference range specified in the laboratory reporting the result. It is also imperative that laboratories verify reference ranges on a cohort that reflects their patient population. Whenever possible, serial measurements for an individual should use an assay from the same manufacturer.

FLC assays are more sensitive for the detection of monoclonal FLCs than urine immunofixation. This was illustrated in a study of paired serum and urine samples from patients with multiple myeloma who tested positive for either kappa or lambda monoclonal FLCs in their serum; only 51 and 35 percent, respectively, of their paired urine samples were positive for Bence Jones protein on urine immunofixation [40].

When this assay was applied to sera from patients previously diagnosed as having nonsecretory myeloma, the improved sensitivity allowed 19 of 28 to be reclassified as secretory light chain myeloma (figure 6) [41,42]. (See "Multiple myeloma: Clinical features, laboratory manifestations, and diagnosis", section on 'Light chain myeloma'.)

Indications and uses — Measurement of serum FLCs is useful in a number of settings, such as [43]:

●Diagnosis and monitoring progress of patients with non-secretory myeloma and oligosecretory (<1 g/dL [10 g/L] monoclonal protein in the serum and <200 mg/day monoclonal protein in the urine) myeloma.

●Diagnosis and monitoring progress of patients with light chain myeloma as well as AL amyloidosis, in whom the underlying clonal plasma cell disorder may otherwise be difficult to detect and monitor [44,45].

●Predicting risk of progression of MGUS. (See "Clinical course and management of monoclonal gammopathy of undetermined significance", section on 'Risk stratification to estimate risk of progression'.)

●Predicting risk of progression of smoldering multiple myeloma. (See "Multiple myeloma: Clinical features, laboratory manifestations, and diagnosis", section on 'Smoldering multiple myeloma'.)

●Predicting risk of progression of solitary plasmacytoma of bone. (See "Solitary plasmacytoma of bone", section on 'Prognosis'.)

●Diagnosis, monitoring during and after treatment, and perhaps predicting prognosis of patients with multiple myeloma and an intact immunoglobulin [46,47].

●Potentially obviating the initial need for urine protein studies in the initial evaluation for monoclonal gammopathies when serum FLC analysis is performed along with serum protein electrophoresis and serum immunoelectrophoresis [48]. If a monoclonal protein is found, electrophoresis and immunofixation of an aliquot from a 24-hour urine collection should then be performed. (See "Diagnosis of monoclonal gammopathy of undetermined significance", section on 'Evaluation'.)

Use in patients with kidney impairment — The normally rapid renal clearance of serum FLCs is reduced in the presence of kidney impairment (eg, creatinine clearance <60 mL/min). As a result, serum FLC concentrations rise as the glomerular filtration rate falls, and may be 20 to 30 times normal in end-stage kidney failure [49].

Kidney impairment may also impact the kappa/lambda FLC ratio. Data from a large screening study (iStopMM) were used to develop new FLC ratio reference intervals adjusted for the estimated glomerular filtration rate (eGFR) [50]:

●eGFR 45 to 59 mL/min/1.73 m² – FLC ratio reference interval: 0.46 to 2.62

●eGFR 30 to 44 mL/min/1.73 m² – FLC ratio reference interval: 0.48 to 3.38

●eGFR <30 mL/min/1.73 m² – FLC ratio reference interval: 0.54 to 3.30

Other studies evaluating the kappa/lambda ratio in patients with kidney impairment have had conflicting results. While some studies have suggested that the ratio is elevated in patients with severely reduced kidney function [51], others have not [37,52,53]. As an example, one study demonstrated that the kappa/lambda ratio, which is normally in the range of 0.26 to 1.65, may rise to as high as 3.1 in the presence of dialysis-dependent kidney failure [51]. The change in kappa/lambda ratios with increasing kidney impairment is of potential clinical relevance in the following settings:

●Patients might be misclassified as having a kappa monoclonal gammopathy because of an increased kappa/lambda ratio due solely to kidney impairment.

●Patients with a lambda monoclonal gammopathy along with kidney impairment might have a relatively normal kappa/lambda ratio and be missed because of the relative increase in kappa chains due to the kidney impairment.

Kappa/lambda ratios >3.0 are unlikely to be due to kidney impairment alone. It is difficult to interpret kappa/lambda ratios between 1.65 and 3.0 in the context of kidney impairment. In such cases, further investigation with a 24-hour urine protein electrophoresis and urine immunofixation helps to guide interpretation. If both of these subsequent studies are normal and the patient has no other symptoms suggestive of a plasma cell dyscrasia, then the increased ratio is likely due to the kidney impairment.

Mass spectrometry — Mass spectrometry (MS) uses high-resolution molecular mass measurements to accurately identify and classify M proteins in the serum. At the Mayo Clinic, immunofixation has been replaced by MS for identification of M proteins. However, few hospitals or medical centers have clinical laboratories that are able to conduct MS testing. At present, MS is not a standard part of the evaluation of M proteins in most centers.

In addition to being a more accurate potential replacement for immunofixation, initial reports suggest that, unlike other methods, MS is also able to accurately discriminate between therapeutic monoclonal antibodies (eg, daratumumab, elotuzumab, isatuximab) and endogenous M proteins [35,54-57]. MS measurements have revealed that approximately 5 percent of patients with an M protein have Ig light chain N-linked glycosylation. These patients were found to be at higher risk of developing AL amyloidosis, multiple myeloma, and other plasma cell dyscrasias [58]. Patients with a monoclonal IgM with light chain glycosylation were shown to be associated with cold agglutinin disease [59].

A 2022 study reported a higher prevalence of monoclonal protein abnormalities using mass spectrometry, but we feel that most of the small abnormalities reported in this study are likely artifacts or reactive changes that appear in the IgM region where there is less polyclonal background [60]. Most samples studied were from a serum bank in a large referral hospital, and reactive changes in populations seeking hospital care are expected. In our laboratory where mass spectrometry is used as a clinical test, we do not see this level of prevalence. Additional confirmatory studies from other laboratories and longer follow-up are needed before we can determine if these changes are of clinical importance.

Blood viscosity — Viscosity is a measure of a fluid's resistance to flow, commonly perceived as the fluid's "thickness." Blood viscosity can be elevated in persons with monoclonal or polyclonal increases in immunoglobulin and/or other plasma protein fractions, such as fibrinogen. Waldenström macroglobulinemia, with increased concentrations of IgM, is the most common cause of elevated serum viscosity, but hyperviscosity can also occur in patients with multiple myeloma and high concentrations of monoclonal IgA or IgG. Rarely, hyperviscosity has been noted in patients with severe infections, including HIV [61] and coronavirus disease 2019 (COVID-19) [62].

Blood, plasma, or serum viscosity should be performed in any patient with a monoclonal gammopathy and signs and symptoms suggesting the hyperviscosity syndrome. These symptoms include [63]:

●Oronasal bleeding

●Blurred vision, dilatation of retinal veins, flame-shaped retinal hemorrhages

●Unexplained heart failure

●Neurologic symptoms such as headaches, vertigo, nystagmus, deafness, ataxia, diplopia, paresthesias, stupor, or somnolence

Blood, plasma or serum viscosity should also be determined whenever the monoclonal IgM protein spike is >4 g/dL (40 g/L) or the IgA or IgG protein spike is >6 g/dL (60 g/L), regardless of signs/symptoms. (See "Epidemiology, pathogenesis, clinical manifestations, and diagnosis of Waldenström macroglobulinemia", section on 'Hyperviscosity syndrome'.)

The normal value for serum viscosity is 1.5 centipoise (CP), but hyperviscosity symptoms are rarely present unless the viscosity is >4 CP. However, many laboratories report viscosity in relative terms (eg, relative to distilled water or saline). The normal relative viscosity in our laboratory is 1.8. Because of the often poor correlation between viscosity and symptoms, and the fact that relative and absolute (in centipoise, see below) viscosities of plasma are similar, these two units can be used interchangeably.

The relationship between serum viscosity and IgM protein concentration is nonlinear. Thus, with low serum IgM concentrations, an increase of 1 to 2 g/dL (10 to 20 g/L) produces only a small increase in serum viscosity, but with IgM levels of 4 to 5 g/dL (40 to 50 g/L), an increment of 1 to 2 g/dL (10 to 20 g/L) greatly increases viscosity. In addition, the relationship between serum viscosity and symptoms of hyperviscosity is not precise. Although many patients have symptoms when the viscosity is >4 CP, most have symptoms when the viscosity reaches 6 to 7 CP. However, we have also seen higher viscosities in the absence of symptoms or physical findings of the hyperviscosity syndrome.

The specific viscosity level at which clinical symptoms occur is affected not only by the serum protein concentration but also by molecular characteristics of the protein, aggregation of protein molecules, and the simultaneous presence of diseases involving the microvasculature, hematocrit, and cardiac status. Consequently, clinical evaluation of the patient is important. The decision to perform plasmapheresis should be made on the basis of signs and symptoms of hyperviscosity rather than the viscosity value per se. (See "Epidemiology, pathogenesis, clinical manifestations, and diagnosis of Waldenström macroglobulinemia", section on 'Hyperviscosity syndrome' and "Treatment and prognosis of Waldenström macroglobulinemia", section on 'Emergency management of hyperviscosity'.)

It is useful to measure the serum viscosity and to perform SPEP before and after each plasmapheresis to determine effectiveness. Such patients should also be monitored by periodic SPEP. If the serum M protein values increase or if symptoms or signs of hyperviscosity recur, the serum viscosity should be repeated.

Quantitation of immunoglobulins — Quantitation of immunoglobulins is the most useful technique for the detection of hypogammaglobulinemia. Nephelometry and turbidimetry are commonly used methods. Levels measured can be impacted by the specifics of the testing technique so levels measured at one laboratory should not be compared with levels from a different laboratory. The degree of turbidity produced by antigen-antibody interaction is measured by nephelometry in the near ultraviolet regions. Because the method is not affected by the molecular size of the antigen, the nephelometric technique accurately measures 7S IgM, polymers of IgA, or aggregates of IgG.

Estimation of quantitative immunoglobulins by nephelometry does not allow an assessment of monoclonality. Increased levels can be due to polyclonal or monoclonal elevations; clonality needs to be established using immunofixation. (See 'Serum immunofixation' above.)

Although the reason is not well understood, nephelometric levels of IgM are often 1000 to 2000 mg/dL greater than expected on the basis of the SPEP densitometry tracing [64]. Because of the overestimation of IgM by nephelometry, it has been recommended that SPEP followed by densitometry is preferable, the results of which usually more closely agree with the clinical presentation [65]. IgG and IgA may also be spuriously increased using nephelometry.

Quantitation of immunoglobulins using nephelometry is a useful adjunct to the SPEP and UPEP in following patients with multiple myeloma specifically to assess response to therapy. However, when assessing response, SPEP values should only be compared with SPEP values, and quantitative immunoglobulin values only with quantitative immunoglobulin values.

In certain situations, quantitative immunoglobulin values may be more reliable than the SPEP [66]. As examples:

●Small beta-migrating M proteins (usually IgA M proteins) are contaminated by normal immunoglobulins that may be greater in quantity than the M spike itself.

●When the M spike is so large (>4 g/dL [>40 g/L]) and narrow on agarose that the SPEP underestimates the actual immunoglobulin level (by more than 1.5 g/dL [15 g/L]), due to technical staining properties of the agarose gel.

ANALYSIS OF URINE — The presence of an M protein in the urine is estimated by urine protein electrophoresis (UPEP) and urine protein immunofixation. Quantitation of the urine M protein also requires a simultaneous measurement of 24-hour total urine protein. Although dipsticks are used in many laboratories to screen for the presence of protein in the urine, these tests are often unable to detect Bence Jones protein (free kappa or lambda light chains). (See "Assessment of urinary protein excretion and evaluation of isolated non-nephrotic proteinuria in adults", section on 'Standard urine dipstick'.)

24-hour urine protein electrophoresis (UPEP) — The UPEP is analogous to the serum protein electrophoresis (SPEP) and is used to detect M proteins in the urine by an electrophoretic method. Once detected, the monoclonality must be confirmed using urine immunofixation. (See 'Urine immunofixation' below.)

A 24-hour urine collection is necessary for determination of the total amount of protein excreted in the urine per day. The quantity of M protein excreted is determined by measuring the size (percent) of the M spike in the densitometer tracing and multiplying it by the total 24-hour urinary protein excretion. The amount of protein can be expressed as mg/dL or mg/L but it is much more useful to report the M protein in g/24 hours because of wide variability in the daily urinary volume. The 24-hour urine specimen requires no preservative and may be kept at room temperature during collection.

On UPEP, a urinary M protein is seen as a dense localized band on agarose or a tall narrow peak on the densitometer tracing (figure 7). Generally, the amount of urinary monoclonal protein correlates directly with the size of the plasma cell burden, as long as kidney function is relatively normal. Consequently, urinary M protein excretion is useful in determining the response to therapy or progression of disease.

Indications — All patients with a diagnosis of a plasma cell dyscrasia should have a baseline UPEP (and immunofixation) of an aliquot from a 24-hour urine collection. This test is essential to detect the presence of potentially nephrotoxic concentrations of urinary light chains.

UPEP testing is subsequently required to detect progression and to monitor response to therapy in patients who have urinary M proteins at baseline.

UPEP (and immunofixation) has also been used as a standard screening test for patients in whom there is clinical suspicion for a monoclonal plasma cell proliferative disorder such as multiple myeloma or AL amyloidosis. The serum free light chain assay can be used as an alternative method. (See 'Serum free light chains' above.)

Urine immunofixation — Urine immunofixation is the preferred method for identification of a monoclonal protein in the urine. As in the case of serum immunofixation it is more sensitive than UPEP and allows determination of the heavy and light chain type of the urinary monoclonal protein. It does not estimate the size of the monoclonal protein and hence is done in conjunction with UPEP. (See '24-hour urine protein electrophoresis (UPEP)' above.)

The presence of a urinary monoclonal light chain on immunofixation is characterized by a discrete band with reactivity to either kappa or lambda antisera, but not both (figure 8). Two discrete bands with either kappa or lambda antisera (but not both) may be found; this is usually due to the presence of monomers and dimers of the monoclonal light chain protein. However, a discrete band with heavy chain antisera reactivity corresponding to the type of heavy chain present in the patient's serum and coinciding with one of the two light chain bands indicates the presence of a fragment of the intact immunoglobulin (figure 9). This is of no particular clinical relevance.

A biclonal gammopathy with one monoclonal protein having a kappa chain and the other having a lambda chain rarely occurs. Occasionally, regularly spaced faint but discrete multiple bands are seen on immunofixation. These restricted bands represent related polyclonal free light chains and are not to be confused with monoclonal light chains. This phenomenon has been described as "ladder light chain" or a "pseudo-oligoclonal pattern" [67]. (See 'Biclonal gammopathy' above.)

If the patient has nephrotic syndrome, the presence of a monoclonal light chain strongly suggests either AL amyloidosis or light chain deposition disease in almost all instances. If UPEP reveals a localized globulin band and immunofixation does not demonstrate a monoclonal light chain, one should suspect the possibility of heavy chain deposition disease. Immunofixation should then be performed with antisera to IgG (gamma heavy chains). (See "Clinical presentation, laboratory manifestations, and diagnosis of immunoglobulin light chain (AL) amyloidosis" and "Monoclonal immunoglobulin deposition disease".)

Theoretically, antisera that recognize only free kappa or free lambda light chains should be used rather than antisera that recognize both free light chains as well as light chains that are part of an intact immunoglobulin. However, such antisera are often either nonspecific or insufficiently potent. In addition, a patient may have an immunoglobulin fragment that free kappa or free lambda antisera do not recognize. Thus, it is advisable to use kappa and lambda antisera that are monospecific and potent and able to recognize both free and combined light chains when performing immunofixation. This is especially important, as some patients have isolated Bence Jones proteinuria with no M spike in the serum [31,41].

Indications — Urine immunofixation should be performed at baseline in all patients with a diagnosis of a plasma cell dyscrasia. Immunofixation should be performed in these patients even if the routine urine analysis is negative for protein, 24-hour urine protein concentration is within normal limits, and UPEP of a concentrated urine specimen shows no globulin peak. Immunofixation is sufficiently sensitive to detect a urine M protein of ≥0.004 g/dL (0.04 g/L) [15]. Urine immunofixation is subsequently required to assess and document complete response in patients receiving therapy.

Urine immunofixation has also been used as a standard test for patients in whom there is clinical suspicion for a monoclonal plasma cell proliferative disorder such as myeloma or primary amyloidosis. The serum free light chain assay can be used as an alternative method. (See 'Serum free light chains' above.)

SUMMARY

●Definition and clinical implications – An M protein is a monoclonal immunoglobulin present in an amount that can be detected by immunofixation of serum or urine, or by a serum free light chain (FLC) assay. The M protein can be an intact immunoglobulin (ie, containing both heavy and light chains) or can be composed of only light chains or only heavy chains. (See 'Definition of an M protein' above.)

The presence of an M protein indicates an underlying clonal plasma cell or lymphoproliferative disorder (table 1). This may represent an asymptomatic expansion of clonal plasma cells, a premalignant clonal expansion that may lead to life-threatening complications from the secreted immunoglobulin, or a malignancy. (See 'Causes' above.)

The clinical history influences the choice of tests performed and their interpretation. Studies for monoclonal proteins are indicated in the evaluation of all patients in whom multiple myeloma, Waldenström macroglobulinemia, AL amyloidosis, or a related disorder is suspected (algorithm 1). They should also be considered in any patient with an elevated total serum protein or otherwise unexplained signs and symptoms suggestive of the presence of a plasma cell disorder. (See 'Importance of clinical history in testing' above and 'Indications' above.)

Testing classically uses electrophoretic techniques, supplemented with additional tests for protein quantification and methodologies to determine whether the protein arises from a single clone (ie, monoclonal) (table 4). The initial evaluation usually includes a serum protein electrophoresis (SPEP), serum immunofixation, routine urinalysis, 24-hour urine protein electrophoresis (UPEP), and urine immunofixation. Alternatively, a serum FLC assay may be used instead of the 24-hour UPEP as a screening test.

●Serum studies – SPEP separates serum proteins and allows for the detection and quantification of an M protein in the blood. The M protein usually presents as a single narrow peak in the gamma, beta, or alpha-2 region of the densitometer tracing (figure 2A-B). In contrast, a broad-based peak or band suggests a polyclonal increase in immunoglobulins, most often due to an infectious, inflammatory, or reactive process (figure 2B, 2D). (See 'Serum protein electrophoresis (SPEP)' above.)

Serum immunofixation uses antibodies directed against heavy and light chain components to differentiate a monoclonal from a polyclonal increase in immunoglobulins and to determine the type of immunoglobulin involved (eg, IgG kappa). An M protein is characterized on immunofixation by the combined presence of a sharp, well-defined band associated with a single heavy chain class and a sharp and well-defined band with similar mobility characteristics which reacts with either kappa or lambda light chain antisera (figure 4). These tests do not quantify the M protein. (See 'Serum immunofixation' above.)

The serum FLC assay is an antibody-based system that can detect low concentrations of monoclonal free light chains (ie, kappa or lambda) in the serum. This assay is more sensitive for the detection of light chains than urine immunofixation; however, results may be affected by the presence of kidney impairment. (See 'Serum free light chains' above.)

Quantitative immunoglobulins can detect hypogammaglobulinemia and hypergammaglobulinemia. Increased levels can be due to polyclonal or monoclonal elevations; clonality needs to be established using SPEP and immunofixation. (See 'Quantitation of immunoglobulins' above.)

Serum viscosity should be performed in any patient with a monoclonal gammopathy and signs and symptoms suggesting the hyperviscosity syndrome and for all patients with a monoclonal IgM protein spike >4 g/dL (40 g/L) or an IgA or IgG protein spike >6 g/dL (60 g/L). (See 'Blood viscosity' above.)

●Urine studies – Dipsticks used to screen for the presence of protein in the urine are often unable to detect Bence Jones protein (free kappa or lambda light chains). (See "Assessment of urinary protein excretion and evaluation of isolated non-nephrotic proteinuria in adults", section on 'Standard urine dipstick'.)

A 24-hour UPEP separates urine proteins and allows for the detection and quantification of an M protein in the urine. Urine immunofixation must be performed in conjunction with the UPEP in order to differentiate a monoclonal from a polyclonal increase in immunoglobulins and to determine the type of light chain involved. (See '24-hour urine protein electrophoresis (UPEP)' above and 'Urine immunofixation' above.)

ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges extensive contributions of Robert A Kyle, MD to earlier versions of this topic review.