The Bence-Jones protein (BJP) was described in 1962 as “free monoclonal light chains”, synthesized by a
single clone of B cells. Normal plasma cells appear to produce a slight excess of light chains, but B cell neoplasms may produce a much greater excess.
Once the mechanism of tubular reabsorption becomes saturated, BJPs are excreted in urine.
The molecular mass of BJP is quite variable; BJPs appear in urine as monomers (22 kDa), dimers (44 kDa), or low-molecular mass fragments (5–18 kDa), or show a high degree of polymerization.
The most frequent associations are with the following:
- multiple myeloma,
- Waldenström’s macroglobulinemia,
- monoclonal light chain-related amyloidosis (AL),and
- light chain deposition disease
BJP determinations are useful in the following (3):
- Subjects with serum monoclonal component (MC): at diagnosis and during follow-up.
- Patients suspected of having a monoclonal gammopathy, clinically or from laboratory findings as follows:
– bone pain, fatigue, recurrent infections, purpura, edema,
– unexpected hypogammaglobulinemia in adults, unexplained increased erythrocyte sedimentation rate, anemia, leukopenia, and thrombocytopenia, proteinuria
- The College of American Pathologists issued guidelines defining the methods for BJP detection and quantification based on a 24-hour urine specimen.
- However, 24-hour collections are cumbersome and er-ratic, and BJP is particularly prone to bacterial degradation.
- The latter can be minimized by adding an antibacterial agent.
- Considering these drawbacks, we recommend the use of the second morning void and expressing the concentration of BJP relative to urinary creatinine.
- If the method in use is sensitive enough, the urine can be used unconcentrated; when the greatest sensitivity of BJP detection is clinically needed, urine should be concentrated.
- In such case, the membrane used in the concentrating devices should have a cut-off preferably of 5 kDa and, in any case, less than 10 kDa.
- The chosen method should verify that the two fundamental characteristics of the LCs are present: e., that they are free and monoclonal.
- Immunofixation combines an electrophoretic step to verify the molecular homogeneity of the protein with immunologic typing,it is the recommended method for BJP detection.
- Antisera to KIEκ and LAMBDA LCs together with antiserum to the heavy chain (HC) of the serum monoclonal immunoglobulin should be used.
- Antisera to free light chains (FLCs) are expensive; in addition, they are often nonspecific and can have low avidity.
- They can be useful only if it is suspected that a BJP is co-migrating with an intact immunoglobulin.
- This suspicion is raised when there is a discrepancy between the HC and the LC signal grossly in favor of the latter
- The indication of a detection limit for BJP can only be approximate since there is no way of obtaining an accurate quantification of the protein.
- However, since the indicated amount for polyclonal LC excretion is approximately 10 mg/l, a method with a sensitivity down to this limit is suggested.
- Among the sensitive stains, colloidal gold provides the highest sensitivity (1–2 mg/l); colloidal Coomassie stain can detect BJP at 10 mg/l or less
- Using methods of high resolution and adequate sensitivity, the appearance of so-called LC ladder patterns is common.
- These multiple, evenly-spaced bands have been well described and are the consequence of the excretion
of polyclonal LCs in individuals with impaired tubular reabsorption.
- The pattern is typical, and an experienced eye can distinguish them from BJP; however, it may sometimes be difficult to identify a BJP band co-migrating with one of the multiple bands
- Immunochemical methods (nephelometry, turbidimetry) for the quantification of FLCs in urine can be used for BJP detection as an initial screening to exclude BJP, thus reducing the number of samples to process further.However, the amount of BJP can range from a few milligrams to grams per liter, so that the assay can eas-ily fall into the antigen excess zone.
- Therefore, it is mandatory to control and avoid antigen excess and to document a detection limit below 10 mg/l.
- A positive test should be followed by immunofixation (IF) for the following reasons:
- If the antisera used in the immunochemical method are against LCs (free and bound) it is necessary to document that the LCs are free;
- Since FLC antiserum is incapable of distinguishing monoclonal from polyclonal LCs it is necessary to define the clonality. Although in LC multiple myeloma the synthesis of polyclonal LCs is usually depressed, in several other clinically important instances (AL, LCDDs) the concentration of polyclonal FLCs in urine can be significant and variable in the course of the disease;
- IF is presently recommended to define the response to high-dose chemotherapy in multiple myeloma. It has been shown that patients achieving a negative IF have the best prognosis; accordingly, therapeutic strategies are presently designed to achieve negative IF.
- The cost-benefit ratio of screening samples for BJP using quantitative immunochemical methods should be carefully evaluated depending on the type of population to be examined and the analytical performance of the immunochemical method used.
Tests to be discouraged
- Methods for measuring total proteins in urine (both precipitating and dye-binding) are insensitive and not accurate for detecting BJP.
- Dipsticks used to screen for protein in the standard urine examination are impregnated with a buffered dye which is sensitive mainly to albumin and can completely miss the presence of BJP.
- The unreliable heat test is mentioned only because of its historical value since not all BJP precipitate upon heating.
- Several staging systems, definitions of indolent disease and treatment guidelines for multiple myeloma and related conditions, are based on decision levels of BJP 24-hour excretion.
- However, none of these studies specifies how to identify and measure “something called BJP”.
- The clinical value of BJP quantification is limited by metabolic and analytical problems.
- The excretion of BJP is influenced by its degree of polymerization, by renal function, and by the deposition rate of the protein in different tissues, so the amount of BJP in urine is not directly related to the tumor cell mass.
- Again, an accurate measurement of BJP cannot be easily achieved with present laboratory techniques.
- The guidelines of the College of American Pathologists suggest the following procedure:
- Measurement of total protein in a 24-hour specimen;
- Electrophoresis and IF of concentrated urine to detect BJP;
- Densitometric scan of the BJP peak; and
- Determination of the ratio of the BJP peak percentageto the total protein.
- This procedure has some drawbacks:
– Inaccuracy of the methods in use to measure total protein in urine: these methods are often insensitive to microproteins in general and to BJP in particular.However, if the urine electrophoresis shows that BJP constitutes almost all urinary protein excretion, the determination of total urinary protein performed in the same laboratory by the same method at two points in time may provide useful indications regarding the efficacy of therapy;
– Different proteins can have different affinities for the dyes used to stain electrophoretic strips, and thus a lack of linearity of the densitometric response can be observed;
– Quite often multiple bands of BJP are present in the urine or the BJP co-migrates with other proteins, so that it is difficult to delimit the BJP peak correctly by densitometry.
It is suggested that follow-up of patients be performed in the same laboratory in order to minimize analytical variability.
- Immunochemical methods using antisera against FLCs have the drawbacks listed in the “Alternative approach”
– Antisera raised against a polyclonal mixture of LCs do not necessarily react in the same way with the monoclonal LCs of the sample;
– The molecular mass of BJP is quite variable; in urine, they appear as monomers (22 kDa), dimers (44 kDa), low molecular mass fragments, or can show a high degree of polymerization. The state of aggregation/ fragmentation of FLCs in urine is highly variable and unpredictable, depending on many. In addition, the antisera used for the quantification of FLCs are directed against epitopesthat are hidden in whole immunoglobulin molecules. In some severe conditions, such as AL, LC fragments of 5-18 kDa, comprising the amino-terminal region, are present in serum and urine and are the main constituents of amyloid fibrils. These pathogenic LC fragments can lack some or all relevant epitopes and be poorly recognized, or missed, by the antisera. All these factors can influence the immune reaction and may invalidate the calibration making the quantification of urinary monoclonal LC unreliable;
– The precision of the quantitative methods at the extremes of the dynamic range is poorly defined;
– There is no reference material for monoclonal LCs,and accuracy therefore remains an open problem;
– There is no standardization of the several methods available for the quantification of urine FLCs.
- Results could differ significantly between methods; this represents a serious problem in consideration of the present extreme mobility of patients.
- Despite all these drawbacks, the immunochemical estimation of BJP may be of clinical value to monitor the clone during treatment, but it is necessary to utilize the same antisera and calibrators throughout the followup and to keep in mind all the caveats listed above.
- Recently, it has been reported that in LC myeloma the quantification of FLCs in serum by nephelometry
- correlates with changes in urinary FLC excretion.
- The authors suggest that serum measurements may be an alternative to the cumbersome 24-hour urine collections in monitoring patients with LC myeloma. However, more data are needed before considering this alternative.