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Letters |
1
The Buffalo General Hospital, and,
2
State University of New York, at Buffalo, Buffalo, NY 14203
a Author for correspondence. Fax 716-859-2973; e-mail vladutiu{at}acsu.buffalo.edu
To the Editor:
We studied a 62-year-old man with light chain disease
(multiple myeloma) whose urine protein electrophoresis (UPE;
electrophoresis performed on agarose gel on a Panagel high resolution
electrophoresis system; gel was stained with Amido Black) showed
>15 narrow bands in the ß- and 
-globulin regions; many of the
bands were strongly stained and equidistant (Fig. 1
), suggestive of a
step-ladder pattern originally described for immunofixation
electrophoresis (IFE) of urine (1)(2). The
patient had blood in his stool, and urinalysis showed microhematuria
and a small amount of protein. The abnormal laboratory findings
included 23.5 mmol/L urea nitrogen, 848 µmol/L creatinine, 7.7 mmol/L
glucose, 3.11 mmol/L calcium, 72 g/L hemoglobin, a red blood cell
count of 2.2 x 1012/L, and a hematocrit of
0.20. There was 1.37 g of protein in his urine collected during a
24-h period. UPE showed the pattern just described. Because this was
considered similar to the pattern reported after IFE in urine from
patients with various conditions (1)(2) and this
pattern was reported to be caused by polyclonal light chains
(1)(2), a tubular nephropathy was suspected.
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A bone marrow aspirate showed no abnormalities (4% plasma cells).
Serum protein electrophoresis (Ciba Corning Diagnostic) revealed
decreased -globulins, and serum immunoelectrophoresis (IEP; Ciba
Corning) showed decreased IgG, IgA, and IgM and the presence of a faint
arc with anodal bowing attributable to -Bence Jones protein
(BJP). Quantification of the serum immunoglobulins (rate nephelometry;
QM 300; Beckman) showed 5.17 g/L IgG (reference interval, 7.10–18.58
g/L), 0.62 g/L IgA (reference interval, 0.89–3.87 g/L), and 0.08 g/L
IgM (reference interval, 0.46–2.26 g/L). The concentration of
ß2-microglobulin in his serum was 16.6 mg/L
(reference interval, 1–2.4 mg/L). IEP of his urine (concentrated 50x;
Fig. 1
A) showed albumin, a very faint 
arc, and an intense
arc with bowing at the anodal end (i.e., -BJP). The diagnosis of
multiple myeloma, light-chain type, was considered, and a bone marrow
biopsy performed 1 week later essentially showed replacement of the
marrow cells by plasma cells, as seen in multiple myeloma. The first
bone marrow aspirate was considered to have been inconclusive because
the sample was taken from a region without abnormal plasma cells.
Multiple myeloma is often known to have a "spotty" distribution of
malignant plasma cells in bone marrow.
Microscopic examination of a kidney biopsy showed a "myeloma
kidney" pattern, with tubular casts composed of light chains (as
evidenced by immunofluorescence microscopy). IFE of his urine
(concentrated 20x) with the Panagel system showed narrow,
equidistant bands of light chains. However, it was obvious that no
anti- antibody dilution was optimal for revealing all bands seen in
UPE. The utmost importance of the proper antibody concentration in IFE
is well known; an excess of antigen can occur and precludes
visualization of the intense bands.
The patient underwent routine therapy for multiple myeloma, but his
kidney function decreased rapidly; he became oliguric and required
chronic hemodialysis. Sixteen months after the findings described
above and after therapy, the amount of protein in the patient's
urine decreased substantially and UPE showed fewer (3 to 5) equidistant
bands. His serum IFE no longer showed -BJP.
Many narrow equidistant bands were seen in the slow ß- and
-globulin regions in high resolution agarose gel electrophoresis of
his urine concentrated 10- to 50-fold. Most of the bands in the ß-
and -globulin regions were very intense and sharply defined, others
were faint and not equidistant, but all were detected by UPE without
the need for immunological enhancement by IFE (Fig. 1B
). There also
were less-intense bands representing albumin, 
-globulins, and
transferrin. After incubation of concentrated (20x) urine with 1%
2-mercaptoethanol (2-ME) for 1 h at room temperature, no changes
in the electrophoretic pattern occurred. This procedure has been used
routinely and successfully in our laboratory to determine the type
(light chain) of abnormally appearing IgM in serum IEP. The treatment
with 2-ME breaks the polymeric IgM into monomers, thus overcoming the
"umbrella effect" often seen with monoclonal IgM. We also incubated
concentrated (20x) urine with 2-ME overnight at 37 °C, after which
most of the bands remained the same; however, a few disappeared at the
subsequent UPE, and an intense band was seen in the slow -globulin
region. This suggests that polymers of BJP, which likely were cleaved
into monomers after the treatment with 2-ME, were not the sole
explanation for the electrophoretic pattern. Different electrical
charges of the light chains and their fragments also accounted for the
multiple bands seen in UPE.
To our knowledge, such a pattern of UPE from a patient with light chain
disease has not been reported. These narrow bands were of and not
type, in contrast to reports on IFE of urine (2), in
which light chains were found rarely and only together with
light chains. In two patients with monoclonal gammopathies, the serum
M-component was of type, but the urine had multiple -light chain
bands detected by IFE (3). The finding of both and
light chains in the urine of patients with B-cell malignancies is
surprising in view of the suppression of normal (polyclonal)
immunoglobulins that occurs especially in patients with light chain
disease. It should be mentioned that the UPE pattern seen in the
patient described above is not common, although we have seen several
patients whose UPE had intense, equidistant (albeit fewer) narrow
bands. We can only speculate that the -BJP from this patient is
unusually labile and breaks into fragments and/or forms polymers. The
-BJP also showed a fainter precipitate with commercial anti-
antibodies when compared with most of the chains we have detected
by IEP.
Evenly spaced multiple bands (usually three) were first reported as
"the ladder light chain" pattern in urine IFE by Harrison
(1). The same pattern, named "urinary light-chain
ladder", was later reported by Bailey et al. (3) in IFE of
urine from patients with monoclonal gammopathies or other diseases.
Although Harrison (2) did not see bands in the UPE (Titan
Gel system; Helena Laboratories) because of the stain detection
threshold (i.e., the amount of light chain was low), Bailey et al.
(3) found a faint banding pattern in the stained agarose gel
(in-house system, not commercial reagents). On the basis of
two-dimensional electrophoresis and silver staining, Harrison
(1) concluded that the pattern he described in IFE of urine
can be attributed to polyclonal light chains, even when only light
chains were detected, i.e., when the bands were apparently monotypic.
It is conceivable that the appearance (after protein staining) of
equidistant bands that represent light chains in UPE depends on the
amount of light chains in the urine and the resolution of the
electrophoresis system (e.g., matrix, temperature, and current, as well
as the protein stain) (4). It would be interesting to study
patients with kidney tubular disease (e.g., Balkan nephropathy) as well
as more patients with light chain disease to see whether UPE shows
equidistant, multiple bands in the -globulin region in these
patients.
Multiple bands after electrophoresis of BJP have also been described
(5), but usually there are not more than four bands and they
are not equidistant (6). Our patient's light chains are
uncommon, and we speculate that they may be highly prone to proteolytic
degradation (7). There may be posttranslational changes of
the light chains that give them different isoelectric points. The
clinical relevance of this UPE pattern, if any, is not known. It
might be related to renal tubular cell damage by toxic light chains,
and this hypothesis should be tested in experimental animals.
In conclusion, multiple, equidistant, narrow bands representing light chains may infrequently occur in high resolution UPE, as well as in IFE of urine. To identify whether these bands are attributable to monoclonal or polyclonal immunoglobulins or their fragments, UPE should be followed by IEP or IFE of urine. This pattern depends on the separation technique and the concentration of light chains. It is likely that in patients with light chain disease, multiple narrow bands in UPE represent BJP.
Acknowledgments
We thank the technical personnel of the Immunopathology Laboratory, Buffalo General Hospital, for their help.
References
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