Strengths and weaknesses of methods for identifying monoclonal free light chains of Ig: examples from two cases with renal disease.

BACKGROUND: Serum free light chain (FLC) analysis with ratio and urine immunofixation electrophoresis (IFE) are both available for routine use in helping to detect plasma cell dyscrasia and related diseases. CASES: Case reports showing one serum positive for serum FLC but that showed a hook effect and overestimated the amount of monoclonal FLC while urine IFE was negative for Bence Jones protein, and a second serum that showed elevated FLC κ and λ but a normal κ/λ ratio, while urine IFE was positive for Bence Jones protein. CONCLUSIONS: These two techniques complement one another. Neither of the techniques is truly quantitative, and both exhibit methodological defects.

[A case of renal Fanconi syndrome due to Bence Jones' protein K-type multiple myeloma].

A 60-year-old man, who had been treated for chronic kidney disease and chronic hepatitis B infection, was referred to our hospital following presentation with thoracic bone pain and exacerbation of proteinuria and hematu- ria. On admission, laboratory test results showed evidence of hypophosphatemia, glucosuria and elevated levels of both urinary NAG and 62MG.The patient was diagnosed with Fanconi syndrome based on findings indicating the presence of pan-aminoaciduria, elevated urinary excretion of uric acid and an increased phosphorus reabsorption rate. Furthermore, bone scintigraphy showed increased multiple symmetric uptake of radiotracer in both sides of the ribs, leading to the diagnosis"of hypoposphatemia-related osteomalacia with renal Fanconi syndrome. Urinary immunoelectrophoresis indicated the presence of K Bence Jones' protein (BJP). A bone marrow biopsy examina- tion showed that the plasma-to-cell ratio was less than 10%. However, the patient had over lg/day of proteinuria and suppression of serum IgM (18mg/dL) and was, therefore, diagnosed with multiple myeloma based on SWOG criteria. Light microscopic examination showed evidence of glomerulosclerosis, intimal thickness of interlobular arteries and acidophilic granular deposits in the cytoplasm of the proximal epithelial tubular cells. Immunofluores- cence indicated positive anti-K staining in these regions. Electron microscopic examination of the proximal tubular epithelial cells revealed the presence of numerous diamond-shaped and oval crystals, thought to be the K light chain of BJP. In general, cast nephropathy, light chain deposition disease (LCDD) and AL amyloidosis are recog- nized renal injuries caused by myeloma. However, there have been few clinical reports of Fanconi syndrome with multiple myeloma, such as the case study we have described here. In addition, histological examination of a biopsy sample provided further evidence of K BJP in the proximal epithelial tubular cells.

[Secondary monoclonal gammopathy after bone marrow autotransplantation as a cause of worse renal function in light chain immunoglobulin deposition disease].

The paper describes a clinical case of a female woman with nephropathy due to light chain deposition disease caused by secretion of κ Bence-Jones protein. Complete immunochemical remission was achieved after induction therapy using a bortezomib + cyclophosphamide + dexamethasone regimen. Renal function remained unchanged (glomerular filtration rate 16 ml/min), there was a reduction in proteinuria from 5.8 to 2.6 g/day. High-dose melphalan (200 mg/m2) chemotherapy with peripheral blood stem cell autotransplantation was performed as consolidation of remission. A year posttransplantation, there was no secretion of κ light chains; however, monoclonal IgG lambda emerged in a quantity of 3.2 g/l. At the same period, nephrotic syndrome became progressive (daily proteinuria 12 g) and dialysis-dependent renal failure developed. A repeat renal biopsy specimen revealed changes, suggesting that there was a decrease in renal deposits of κ light chains. Simultaneously with this, the obvious negative trend as progressive nephrosclerosis and fixation of IgG and λ light chains in the glomeruli (in the sclerotic areas) cause IgGλ monoclonal protein to be involved in the genesis of further kidney injury. Attention is also paid to different characteristics of capillary wall deposits by density (according to the electron microscopic findings), which may point to their different qualitative composition and possibly different formation duration. Papaprotein Gλ disappeared after a year without therapy, suggesting its reactivity. The findings confirm that worse renal function is caused by the action of paraprotein Gλ due to secondary (after autologous hematopoietic stem cells transplantation) monoclonal gammopathy.

[Improvement of hepatomegaly after treatment with autologous hematopoietic stem cell transplantation followed by bortezomib and dexamethasone in Bence-Jones protein κ-type of multiple myeloma with systemic amyloidosis].

A 51-year-old man was admitted to our hospital complaining of right upper quadrant abdominal and back pain. Severe hepatomegaly (six fingerbreadths) was detected by liver palpation. Blood test results showed cholestatic liver disease. He was diagnosed with amyloidosis by liver biopsy. Bone marrow aspiration revealed 15% of contents to be plasma cells. BJPκ was detected by urine electrophoresis. Therefore, he was diagnosed with the BJPκ type of multiple myeloma with systemic amyloidosis. The patient was treated with bortezomib, dexamethasone and high-dose melphalan with autologous peripheral blood stem cell transplantation. He achieved VGPR (very good partial response) after transplantation. Hepatomegaly improved but swelling persisted, and he was therefore treated with 1.3 mg/m(2)/day of bortezomib and 20 mg/day of dexamethasone on day 1 and day 15 in 28-day cycles. Upon finishing 22 cycles in June 2014, his liver had returned to normal size. Restoration of normal liver size after treatment is rare in cases with severe hepatomegaly due to systemic amyloidosis. We thus report our case with a review of the relevant literature.

Acute kidney injury due to tubular intraluminal monoclonal light chain crystals mimicking acute pyelonephritis.

Tubular intraluminal inflammatory cells may be seen in kidney biopsies of patients with pyelonephritis, cell-mediated transplant rejection, autoimmune tubulointerstitial nephritis, allergic reactions, or in association with monoclonal light chain casts. When casts in a native kidney are primarily composed of granulocytes, the cause is most commonly acute pyelonephritis due to an ascending bacterial urinary tract infection. We report a 57-year-old man with acute kidney injury and an intense intraluminal neutrophil response to monoclonal lambda light chain crystal containing casts.

History of multiple myeloma.

Multiple Myeloma has been recognized since Ancient Times. The first well-documented case was reported in 1844 by Samuel Solly. The most commonly recognized case is that of Thomas Alexander McBean, a highly respectable tradesman from London in 1850. Mr. McBean excreted a large amount of protein that was described by Henry Bence Jones in the middle of the 19th century. Jones was a well-known physician and made many contributions to medicine. One of the best known cases of multiple myeloma was that of Dr. Loos that was reported by Otto Kahler. The recognition of plasma cells and subsequently their product, a monoclonal protein has been described in detail. The authors have reviewed the treatment of multiple myeloma including the novel agents, thalidomide, bortezomib and lenalidomide.

Monoclonal free light chain detection and quantification: Performances and limits of available laboratory assays.

The detection and quantification of immunoglobulin free light chains in serum and urine is recommended for the diagnosis and monitoring of monoclonal gammopathies according to the guidelines of the International Myeloma Working Group (IMWG). Several tests are currently available in the clinical laboratory to detect and quantify free light chains but although quality, efficiency, and effectiveness have been improved, the results are still variable and poorly harmonized and standardized. The present review article wants to analyze these aspects, with a keen eye on techniques, such as mass spectrometry, that could replace in the practical clinical laboratory the current methods including Bence-Jones protein assay and free light chain immunoassays.

A study of immunoglobulin structure. II. The comparison of Bence Jones proteins by peptide mapping.

Urinary proteins of patients with myeloma, prepared by precipitation with ammonium sulphate, have been separated by gel filtration on Sephadex G-100 after reduction and aminoethylation. Many specimens separated into a major peak of Bence Jones protein and into minor peaks of albumin, a protein tentatively identified with heavy chain and a smaller molecular weight protein corresponding to the variable portion of the corresponding Bence Jones protein. The Bence Jones protein purified by gel filtration was analyzed by electrophoresis and by peptide mapping after tryptic digestion. The peptide maps of 24 type K and 20 type L Bence Jones proteins were compared. A set of common peptides was identified in the peptide maps of the Bence Jones proteins of the same type; the common peptides of type K proteins were completely different from the common peptides of type L proteins. The patterns of distinctive peptides was compared; no similarities were found between distinctive peptides of type K and of type L proteins. Some similarities were observed in the distinctive peptides of proteins of the same type. The similarities involved in many cases peptides containing cysteine, whereas similarities in other peptides were limited. This observation suggested that the amino acid sequence around the cysteines of the variable NH(2)-terminal half of the Bence Jones proteins may show less variability than other sequences. A few proteins of the same type differed in all their distinctive peptides, an indication that multiple amino acid differences exist between individual Bence Jones proteins. The genetic mechanisms responsible for the variability in the amino acid sequence of the NH(2)-terminal half of the light chains of immunoglobulins are discussed in view of the results of the comparison by peptide mapping of the Bence Jones proteins.

Bence Jones proteins and light chains of immunoglobulins. II. Immunochemical differentiation and classification of kappa-chains.

Three distinct classes of kappa light polypeptide chains have been detected immunochemically by an antiserum (R185) prepared against a kappa Bence Jones protein with a glutamyl amino terminal residue. This antiserum had specificity for kappa light chains with glutamyl amino terminal residues and differentiated kappa-chains with aspartyl amino terminal residues into two classes: the three kappa-chain classes have been designated as kappa(glu), kappa(aspII), and kappa(aspI). The ability of antiserum R185 to detect these antigenic differences on the intact immunoglobulin molecule, as well as on the isolated light chain or Bence Jones protein, made feasible the direct classification of type K myeloma proteins and M-macroglobulins (Waldenström). The multispecificity of the antiserum permitted the quantitation of type kappa(glu) light chains in normal, hypergammaglobulinemic, and hypogammaglobulinemic sera. Whereas the distribution of myeloma proteins and Bence Jones proteins in the kappa(glu) class correlated with the distribution of kappa(glu) chains in normal and hypergammaglobulinemic sera, the M-macroglobulins in the kappa(glu) class represented 90% of the total M-macroglobulins tested and revealed a marked divergence from the range of 24-31% of kappa(glu) immunoglobulins in normal sera. A preponderance of kappa(glu) chains was detected in the sera from patients with non-sex-linked hypogammaglobulinemia and represented 60-77% of the total type K light chain content. The controlled cleavage of a Bence Jones protein representative of each kappa-chain class into its variant half and constant half made possible the localization on the light polypeptide chain, the reactive sites for which antiserum R185 had specificity. The correlations between immunochemical and structural classification of kappa light chains are discussed.

A study of immunoglobulin structure. 3. An estimate of the variability of human light chains.

102 human Bence Jones proteins have been purified by gel filtration, digested with trypsin, and analyzed by peptide mapping. In several cases Bence Jones "fragments", corresponding to the variable half of the corresponding proteins, were observed. The peptide maps of the proteins were compared to establish whether any identical proteins were present in the sample analyzed. No Bence Jones protein showed a peptide map identical to that of any other protein, although remarkable similarities in the peptide maps were observed for some proteins. Two proteins that gave very similar peptide maps were then examined in detail, by purifying and analyzing the tryptic peptides. It was then found that these two proteins differ in amino acid sequence in at least six positions. The probability of not finding two identical sequences by examining a sample extracted from populations of light chains of different sizes has been calculated. This has led to an estimate of the minimal size of the population of light chain sequences in humans. The number of light chain sequences appears to be at least a few thousand. Information on the frequency of Inv and Oz antigenic determinants and on the relative frequency of subtypes of K chains has been obtained. Proteins of KI subtype are found most frequently. The possibility that different subtypes may be predominant in different species is discussed in relation to the evolutionary arguments used in favor of the somatic theories on the origin of variability of immunoglobulin chains.

An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity.

In an attempt to account for antibody specificity and complementarity in terms of structure, human kappa-, human lambda-, and mouse kappa-Bence Jones proteins and light chains are considered as a single population and the variable and constant regions are compared using the sequence data available. Statistical criteria are used in evaluating each position in the sequence as to whether it is essentially invariant or group-specific, subgroup-specific, species-specific, etc. Examination of the invariant residues of the variable and constant regions confirms the existence of a large number of invariant glycines, no invariant valine, lysine, and histidine, and only one invariant leucine and alanine in the variable region, as compared with the absence of invariant glycines and presence of three each of invariant alanine, leucine, and valine and two each of invariant lysine and histidine in the constant region. The unique role of glycine in the variable region is emphasized. Hydrophobicity of the invariant residues of the two regions is also evaluated. A parameter termed variability is defined and plotted against the position for the 107 residues of the variable region. Three stretches of unusually high variability are noted at residues 24-34, 50-56, and 89-97; variations in length have been found in the first and third of these. It is hypothesized that positions 24-34 and 89-97 contain the complementarity-determining residues of the light chain-those which make contact with the antigenic determinant. The heavy chain also has been reported to have a similar region of very high variability which would also participate in forming the antibody-combining site. It is postulated that the information for site complementarity is contained in some extrachromosomal DNA such as an episome and is incorporated by insertion into the DNA of the structural genes for the variable region of short linear sequences of nucleotides. The advantages and disadvantages of this hypothesis are discussed.

[Current status regarding detection of monoclonal component in Japan].

Monoclonal immunoglobulin component (M-component) presence is suspected based on serum protein analysis using cellulose acetate membrane electrophoresis, and finally clarified by determining its immunoglobulin class using immunoelectrophoresis (IEP) or immunofixation (IFE). M-component presence is essential for diagnosing multiple myeloma (MM) and primary macroglobulinemia; however, since it is also found in non-malignant conditions, called MGUS (monoclonal gammopathy of undetermined significance), the differentiation of MGUS from malignant diseases is often important. Bence Jones protein (BJP), once detected, can support the diagnoses of MM and primary AL-amyloidosis. In the latter condition, which is often difficult to diagnose, BJP is very helpful. The newly developed method measuring free immunoglobulin light chains can effectively indicate the presence of BJP in serum. The detection of BJP in urine is still important. Capillary electrophoresis combined with immunoabsorption can detect BJP in non-concentrated urine. It may be time to take such new methods into consideration in Japan.

Antibody variability. Somatic recombination between the elements of "antibody gene pairs" may explain antibody variability.

I have analyzed the available amino acid sequence data from 30 myelomatosis-derived proteins. Several types of variation are apparent. I conclude that a major and genetically predetermined contribution to the variability of these proteins and of antibodies could be provided by chromosomal rearrangements resulting from somatic recombination between similar but not identical genes in antibody gene pairs. My hypothesis suggests many new types of experiment and can be tested (31).

Evolution of immunoglobulins: structural homology of kappa and lambda Bence Jones proteins.

The amino acid sequence of a human lamba chain has been determined. There are many identities in sequence with human kappa chains, but this intraspecies homology is less than the interspecies homology of kappa light chains of man and mouse. Structural relationships suggest a common evolutionary origin and early differentiation of light- and heavy-chain genes.

Genetic implications of common region sequence comparisons of lambda immunoglobulin chains differing at position 190.

The common regions of two lambda chains (amino acid residues 109 to 213) have been partially sequenced. These two human immunoglobulin chains have lysine at position 190, but are otherwise identical in their common-region sequence to four reported lambda chains that have arginine at position 190. The single amino acid interchange at position 190 may be explained either by an ambiguous codon at this position or by a gene duplication so recent that only a single mutational event has occured.

Deletions in immunoglobulin polypeptide chains as evidence for breakage and repair in DNA.

The partial sequence of the light chain of the myeloma-like immunoglobulin Sac shows a large deletion in its variable region. The sequence provides evidence that the corresponding gene was formed by the repair of DNA broken at nonhomologous positions. Data from other immunoglobulin (heavy) chains containing large deletions are compatible with their genes also being the result of DNA breakage and nonhomologous repair. Single homologous reciprocal exchanges in DNA networks at immunoglobulin loci could be the cause of the nonhomologous breaks. The relevance of these events to the generation of normal antibody variability remains to be determined.

Immunoglobulin structure: variation in amino acid sequence and length of human lambda light chains.

Variation and conservation in the primary structure of human lambda light chains is revealed by complete amino acid sequence of three Bence Jones proteins. These proteins differ in amino acid sequence in from 38 to 48 positions; they are of unequal length in the amino-terminal half of the chain but have identical sequence in the last 105 amino acids.