National Health and Nutrition Examination Survey

Conduct serum protein electrophoresis, immunofixation analyses, and kappa and lambda free light chain (FLC) assays in serum, to determine the age-adjusted prevalence and monoclonal protein size distribution of MGUS by ethnic/racial group. Using our results from applied serum analyses, in combination with available information from the NHANES surveys (such as data from medical exams; routine blood samples; questionnaires covering medical history, drug intake, nutrition, smoking, and demographics), we will be able to dissect etiologic mechanisms involved in MGUS development and transformation to full-blown multiple myeloma.

Because the mucosal immune system is a a major site of extrinsic immune stimuation, and celiac disease is associated with a potent stimulation of the gut immune system, elaborating both T cell and B cell mediated immune responses, typically associated with an increase in expansion of B cells and plasma cells within the gut the assays also include serologic testing for celiac disease. Immune responses in celiac disease include both IgA and IgG and possibly IgM responses. Celiac disease may affect as many as 1% of the population but most remain undiagnosed. Celiac disease has also occasionally been reported in association with multiple myeloma. Using the celiac assays we are evaluating if there is any correlation between seropositivity for celiac disease and markers for plasma protein abnormalities.

Participants ≥ 50 years of age from NHANES III and NHANES 1999-2004.

Measurement of Serum Protein Markers

 A total of 250 microliter of serum was used for each study subject to perform the analyses to detect and quantify M protein and FLC (described below). For those individuals where an abnormal protein band or equivocal pattern with conventional agarose-gel electrophoresis was found, an additional 250 microliter serum was used to perform immunofixation for validation purposes (see below).

Electrophoresis

First, serum samples were analyzed for all identified subjects (described above) by using conventional agarose-gel electrophoresis, which has been described in detail previously. This revealed the occurrence and pattern (including determination of the size) of M proteins in the study cohort. If there was an abnormal band or equivocal pattern, immunofixation was performed to validate and to specify type of M protein.

Free light chains

FLC levels were determined in all positive study samples (described above) using the FLC assay (Freelite; The Binding Site, Birmingham, United Kingdom) performed on a Dade-Behring Nephelometer (Deerfield, IL). It consists of two separate measurements, one to detect free-kappa (normal range, 3.3-19.4 mg/L) and the other to detect free-lambda (normal range, 5.7-26.3 mg/L) light chains. In addition to measuring the absolute levels of FLC, the test also allows the assessment of clonality based on the ratio of kappa-lambda light chain levels (normal reference range, 0.26-1.65). Subjects with a kappa-lambda FLC ratio less than 0.26 are typically defined as having monoclonal lambda FLC and those with ratios greater than 1.65 are defined as having a monoclonal kappa FLC. If the FLC ratio is greater than 1.65, kappa is considered to be the “involved” FLC and lambda the “uninvolved” FLC, and vice versa if the ratio is less than 0.26. The normal reference range of 0.26 to 1.65 for the free-kappa-lambda ratio in the FLC assay reflects a higher serum level of free-lambda light chains than would be expected given the usual kappa-lambda ratio of two intact immuno¬globulins. This occurs because the renal excretion of free kappa (which exists usually in a monomeric state) is much faster than free lambda (which is usually in a dimeric state).

Celiac assays

Testing consists of a sequential test strategy, which has proven both sensitive and specific for establishing a serologic diagnosis of celiac disease. The first level test consists of a tissue transglutaminase IgA ELISA test and, for patients who’s test exceeds the normal range, a confirmatory test with endomysial antibody by immunofluorescence is then carried out.

All protein analyses in this study will be performed at the Protein Immunology Laboratory at Mayo Clinic, Rochester, Minnesota. The Protein Immunology Laboratory is the leading laboratory in the world with regard to protein studies on MGUS and multiple myeloma. Since the early 1960’s Mayo Clinic have published extensively in this field. In order to assess accuracy and precision before study start, we performed quality control (QC) analyses of serum from 2 positive MGUS subjects (known to have MGUS), 2 negative MGUS subject (known not to have MGUS), 2 positive FLC subjects (known to have FLC), and 2 negative FLC subject (known not to have FLC). Briefly, for each of these six QC individuals, we aliquoted serum into 5 tubes at an NCI laboratory and thereafter sent the serum in blinded tubes to the Protein Immunology Laboratory at Mayo Clinic for analyses of M protein and FLC, respectively. The results were very stable.

In terms of sample storage and potential influence on results; the Mayo Clinic lab has analyzed over 1,100 sera collected up to 35 years before testing, without any problems to perform the assays. The practice in the laboratory is to repeat the assay following a variety of storage activities. For example, serum is stored at room temperature for one week and the assays are repeated on days 1, 3, 5, and 7. Also, samples are refrigerated at 0 to 4 degrees Celsius for 7 days and then assays are repeated on days 1, 3, 5, and 7. Further, they freeze serum sample at -20 degrees Celsius for 1 and 2 weeks, then they thaw and run the assays. In addition, they subject samples to 3 cycles of freeze/thaw and then repeat the assays (personal communication, Dr. Kyle, December 2006). Thus, freeze/thaw steps will not have an adverse impact on the methods being used in our proposed study.

Data was received after all the laboratory testing was complete. The data were not edited.

Data Access: All data are publicly available.