Secondary monoclonal gammopathy may be further subdivided into diseases associated with neoplasia and those associated with nonneoplastic disorders. In the neoplasm group, monoclonal proteins are most often found with malignant lymphoma and chronic lymphocytic leukemia. Among carcinomas, those of the rectosigmoid are most frequent, followed by carcinomas of the prostate, breast, and lung. The incidence in one large cancer hospital ranged from 0.2% in the fourth decade of life to 5.7% in the ninth. In three large series of patients with monoclonal gammopathy, 6%-8% of cases were associated with lymphoma or lymphocytic leukemia and 4%-8% with other types of neoplasms. Nonneoplastic diseases that have been associated with monoclonal proteins are many and varied, but the greatest number appear in the rheumatoid-collagen-autoimmune group, cirrhosis, chronic infection (particularly tuberculosis and chronic infection of the biliary tract, urinary tract, and lung), Gaucher’s disease, osteitis deformans (Paget’s disease of bone), and sarcoidosis. One study detected monoclonal or oligoclonal serum proteins in 9% of patients who had HIV-1 infection without the criteria for acquired immunodeficiency syndrome (AIDS). There is also increased incidence in AIDS. Most of these nonneoplastic conditions are ordinarily associated with polyclonal hyperglobulinemia rather than monoclonal gammopathy. The incidence of nonneoplastic monoclonal protein in the three series mentioned varied from 4%-10%. Monoclonal protein type in the secondary paraproteinemias may be IgG, IgA, or IgM. Occasionally patients in either the neoplastic or nonneoplastic group excrete Bence Jones protein in the urine, usually in small amounts. In some cases the heat test gives false positive results due to an increase in normal light chains associated with polyclonal gammopathy.

Diagnostic techniques

In many instances the diagnosis of myeloma or Waldenstrцm’s macroglobulinemia can be made by serum protein electrophoresis, followed by bone marrow aspiration. In patients without a monoclonal-type serum peak, urine electrophoresis on a concentrated specimen is essential to detect cases in which the only protein abnormality is urinary excretion of Bence Jones protein. In problem cases, it is necessary to resort to serum and urine immunoelectrophoresis. Many authorities advocate immunoelectrophoresis in all patients since this is the only way to classify monoclonal immunoglobulin disorders with certainty (even subgrouping of IgG and IgA is now possible.) Although subclassification of myeloma into immunoglobulin categories at present has more academic than practical application from the standpoint of therapy, such classification may become important in the future, and in any event, it provides additional confirmation of the diagnosis. About 1%-2% of myeloma patients fail to secrete abnormal proteins that are detectable in either serum or urine by immunoelectrophoresis (“non-secretory” myeloma).

Immunoelectrophoresis consists of three steps. First, the unknown (patient’s) serum is subjected to ordinary electrophoresis in a substance such as agar gel; this separates the immunoglobulins from one another to some extent. Second, antiserum against a specific type of human globulin (or a polyvalent antiserum against several types) is added to a trench cut nearby and parallel to the electrophoretic separation bands. Then the immunoglobulins and antiimmunoglobulin antibodies diffuse toward each other. Finally, the reaction between the patient’s immunoglobulin fractions and any specific antibodies against one or more of those immunoglobulin fractions forms visual precipitin lines. The combination of electrophoresis and agar diffusion antigen-antibody reaction produces better separation of the immunoglobulin components and demonstrates abnormal quantities of any type present. Immunofixation is a modification of the immunoelectrophoresis technique that takes a little longer to perform but is easier to interpret and may be a little more sensitive. The test sample (usually diluted serum or concentrated urine) is spotted into each of 6 side-by-side slots on a cellulose acetate or agar gel plate at the same end of each slot. The proteins are then separated by electrophoresis. After that, antiserum against IgG, IgA, IgM, kappa light chain, and lambda light chain are placed into different slots. One slot does not receive antiserum. Incubation permits antigen-antibody reaction, if any. A protein stain is applied to visualize any antigen-antibody reaction. Monoclonal proteins are seen as a sharp narrow band; polyclonal proteins appear to be a wider, more diffuse band. Immunoelectrophoresis and immunofixation are most useful to demonstrate monoclonal proteins, differentiate monoclonal from polyclonal proteins, and to identify any monoclonal or polyclonal proteins. Therefore, these methods differentiate between macroglobulinemia (IgM) and other categories of monoclonal gammopathy. It should be noted that antisera against IgD and IgE are ordinarily not used, because these monoclonal gammopathies are uncommon, so that no reaction with the antisera used does not exclude the possibility of IgD or IgE. To exclude them would require repeating the procedure with these antisera. The same is true for immunoelectrophoresis.

If a monoclonal peak is shown to be IgM, this is evidence against myeloma, since only a few cases of IgM myeloma have been reported. On the other hand, if the peak is not IgM, this rules out Waldenstrцm’s macroglobulinemia. The idiopathic or secondary paraproteinemias can be of the IgG, IgA, or IgM class.

There are two types of immunoglobulin light chains: kappa and lambda. Normally about twice as much kappa is produced as lambda. Immunoelectrophoresis can detect these light chains, differentiate kappa from lambda, demonstrate whether one is increased or decreased in relation to the other, and afford a rough visual estimate whether either one is increased or decreased in quantity. Malignant monoclonal gammopathies, such as myeloma or Waldenstrцm’s macroglobulinemia, usually have an abnormal predominance of either kappa or lambda, with the other markedly decreased or absent. Unfortunately, commercial companies have had problems in producing consistently good antisera to kappa and lambda light chains. Controls must be run with every lot of antiserum to guard against false results.

Differentiation of benign and malignant monoclonal gammopathies

Patients with monoclonal gammopathies are usually detected in one of two ways. Either clinical symptoms suggest myeloma and electrophoretic studies are performed, or the studies are ordered for some other reason and monoclonal abnormality is discovered by accident. In 80%-90% of patients with myeloma, bone marrow aspiration findings make the correct diagnosis relatively easy. In those patients with monoclonal gammopathy but normal or equivocal results of bone marrow aspirate, diagnosis becomes a problem. As listed in Table 22-1, nonmyelomatous monoclonal gammopathies include Waldenstrцm’s macroglobulinemia, leukemia and lymphoma (usually the lymphocytic B-cell types), secondary monoclonal gammopathies (carcinomas and inflammatory disease), and idiopathic cases without known associated disease. Some investigators divide the monoclonal gammopathies into two categories, malignant (myeloma, Waldenstrцm’s macroglobulinemia, and lymphoproliferative disorders) and benign (secondary and idiopathic types). The majority of diagnostic problems involve the secondary and idiopathic monoclonal gammopathies, and the usual difficulty consists of excluding a plasma cell or lymphocytic malignancy when bone marrow results are normal and other studies are negative.

In general, if the monoclonal serum protein is greater in quantity than 3.0 gm/100 ml (30 g/L), if the bone marrow contains more than 20% plasma cells, or if the patient excretes more than 60 mg/L of Bence Jones protein, the disorder is probably malignant. On the other hand, if serum monoclonal protein does not exceed 2.0 gm/100 ml, marrow plasma cells do not exceed 5%, and Bence Jones protein does not exceed 60 mg/L, the condition is more likely to be benign. However, up to 5% of those with benign monoclonal gammopathies are reported to exceed at least one of the three criteria for malignancy, although usually not more than one criterion. About 15% of myeloma patients are reported to have less than 10% plasma cells in their initial bone marrow aspirate, with about 5% of myeloma patients having less than 5%. About 30% of myeloma patients have monoclonal protein in serum less than 3.0 gm/100 ml, and about 20% (literature range, 17%-22%) have less than 2.0 gm/100 ml. About 20%-30% have no Bence Jones protein in the urine, and of those who do, about 10% excrete less than 60 mg/L. Thus, the criteria for malignancy are more reliable than those for benign monoclonal etiology. In addition, in one study about 10% of patients thought to have benign monoclonal gammopathy developed myeloma or macroglobulinemia within 5 years.

Some patients require quantitation of one or more immunoglobulin groups. The most frequent reasons include serial quantitation of monoclonal protein to monitor the effects of therapy and immunoglobulin quantitation to detect deficiency of one or more specific immunoglobulin groups. The current standard method to quantitate immunoglobulins is some type of immunologic technique (radial immunodiffusion, immunonephelometry, immunoassay, and others). It must be emphasized that immunoelectrophoresis is not suitable for immunoglobulin quantitation. Immunoelectrophoresis detects and classifies immunoglobulins, but the quantity of any detectable immunoglobulin can only be very roughly estimated as normal, increased, or decreased rather than measured. Serum protein electrophoresis is sometimes used to monitor myeloma protein response to therapy. However, protein electrophoresis is not ideal for this purpose, since standard methods depend on measuring total protein, finding the percentage that each protein fraction contributes to total protein, and then multiplying the total protein numerical value by the percentage representing each protein fraction to derive the numerical quantity of each protein fraction. This means that abnormality in one fraction (e.g., albumin) can change total protein and secondarily change the percentage of all the protein fractions relative to total protein. This can produce some degree of artifactual change in quantity of protein fractions calculated from percent of total protein. Finally, if serial determinations of a specific protein fraction are necessary, the same laboratory should perform the assays, since different techniques can yield somewhat different results on the same specimen.


Cryoglobulins are immunoglobulins that precipitate reversibly in serum or at least partially gel at cold temperature. The most common symptoms are purpura (60%-100% of cases), arthralgias (60%-90%), or Raynaud’s phenomenon (about 50% of cases). The symptoms are usually referable to cryoglobulin precipitation in blood vessels. Cryoglobulins can be primary (“idiopathic,” “essential”) or secondary (associated with some disease). In either case the cryoglobulins can be monoclonal or mixed. In type I cryoglobulins (about 25% of cryoglobulinemia), there is monoclonal IgM or IgG (rarely IgA) only; in type II, there is monoclonal IgM rheumatoid factor (RF) plus polyclonal IgG; and in type III, there is polyclonal IgM RF and IgG.

Cryoglobulins most often do not appear as discrete peaks in serum protein electrophoresis but are incorporated into areas occupied by other globulins. Although the classic cryoglobulin test takes place at 4°C, some cryoglobulins agglutinate to some degree at higher temperatures, with reports even as high as 35°C. Cryofibrinogens exist as well as cryoglobulins. The most common conditions associated with cryoglobulins are vasculitis, rheumatoid-collagen diseases, leukemias and lymphomas, myeloma and Waldenstrцm’s macroglobulinemia, infections, and liver diseases.

Diagnosis consists of drawing a blood specimen and maintaining it at 37°C until clotting is completed. After that, the serum is incubated at 4°C. There is disagreement over the maximum time needed to terminate the test if no agglutination or gelation occurs. Some use a cutoff point of 3 days and others propose 7 days. If cryoglobulins become visible, they should be analyzed to determine which immunoglobulins are present. For this, it would often be necessary to send the specimen to a reference laboratory. Other applicable tests are a screening test for RF and for antinuclear antibodies (ANA). To detect cryofibrinogenemia, a plasma specimen collected in ethylenediamine tetraacetic acid (EDTA) or citrate plus a serum specimen obtained at the same time are incubated together at 4°C. A precipitate in the plasma but not serum would indicate cryofibrinogens.