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Technical Briefs |
1
Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY 40292, and
2
Laboratory Service, Department of Veterans Affairs Medical Center, 800 Zorn Avenue, Louisville, KY 40206;
a author for correspondence: fax 502-894-6265, e-mail levinson{at}louisville.edu
Usually urine protein electrophoresis (UPE) gives rise to distinctive patterns that indicate the source of proteinuria. However, abnormalities of endogenous protein synthesis and peculiarities attributable to mechanical concentration of the sample before analysis can cause misinterpretation. Here we describe the analysis of urine from a patient with hypogammaglobulinemia in which a probable mixed pattern appeared as pure glomerular proteinuria as a result of altered immunoglobulin synthesis.
A 72-year-old, Caucasian male patient with a 1-year history of chronic lymphocytic leukemia and a 20-year history of insulin-requiring type 2 diabetes with diabetic retinopathy and neuropathy was admitted to the hospital because of syncope. Selected test results at the time of admission were as follows (reference intervals in parentheses): urea nitrogen, 0.22 g/L (0.07–0.22 g/L); creatinine, 0.01 g/L (0.006–0.014 g/L); total protein, 55 g/L (62–82 g/L); albumin, 33 g/L (35–50 g/L); IgG, 1.7 g/L (7.2–16.8 g/L); IgA,0.2 g/L (0.69–3.8 g/L); IgM, 0.1 g/L (0.63–2.7 g/L); phosphorous, 0.022 g/L (0.025–0.048 g/L); potassium, 3.7 mmol/L (3.5–5.1 mmol/L); and glucose, 2.35 g/L (0.65–1.1 g/L, fasting). Sodium, bicarbonate, and chloride were within the health-related reference intervals. Urinalysis was remarkable for occasional squamous epithelial cells and hyaline casts, a glucose >2.5 g/L, and 6.4 g protein/24 h, with a volume of 1.5 L (reference interval, <200 mg/24 h). Hematologic studies were unremarkable except for a white blood cell count of 115 000/mm3 with 86% lymphocytes.
A 24-h urine specimen was obtained without the use of preservatives. Urinary total protein was measured using pyrogallol red-molybdate with a kit (Biotrol urine proteins, Biotrol USA) on a Cobas Fara II (Roche Diagnostics), which was modified by use of a linear interpolation calibration mode with calibrators down to 50 mg/L to optimize accuracy in the low range. Before electrophoresis, urine samples were concentrated mechanically using Minicon-B15 concentrators (Amicon).
Protein electrophoresis was performed with the PEP SPE-8-Template procedure (Helena Laboratories), according to the manufacturer's directions. Immunofixation electrophoresis (IFE) was performed using the Titan Gel Immunofix procedure and antibodies (Helena).
The patient's serum SPE pattern (Fig. 1
A) showed little staining in the 
-globulin region, which
indicates hypogammaglobulinemia, consistent with the quantitative
immunoglobulin results. As in the glomerular proteinuria profile (Fig. 1A
), the patient's 20-fold urine concentrate shows little staining in
the -globulin region after UPE, but a small diffuse band of
staining appears with the 100-fold concentrate (Fig. 1A
). Of note, the
2 region of the 100-fold concentrate in Fig. 1A
, dual
bands (indicated by brackets) indicate the presence of
2-microglobulins (1)(2); however,
these bands are not seen in the 20-fold concentrate. IFE (Fig. 1B
) of
the 100-fold concentrate revealed that the diffuse band seen after UPE
of the 100-fold concentrate (Fig. 1A
) was a 
-Bence Jones protein
(BJP). For comparison, Fig. 1C
shows the UPE and urine IFE from another
patient in whom multiple myeloma was ultimately diagnosed. In this
case, even a 100-fold concentrate of UPE showed no banding, whereas IFE
showed a band of restricted migration, stained for 
, which indicated
a BJP.
|
The findings in the patient in Fig. 1A
can be compared with typical
patterns (also Fig. 1A
). In glomerular disease (Fig. 1A
, third lane
from top), larger proteins pass more freely than usual
through the leaky glomerulus so that >3 g of protein in
24 h passes. This excreted protein is largely albumin (the most
abundant protein in plasma), without small globulins, which are
reabsorbed by the tubules, but with larger proteins other than albumin
seen when the threshold for tubular reabsorption is exceeded. In pure
tubular proteinuria, the small proteins, usually found in the
glomerular filtrate (e.g., polyclonal free light chains and
2-microglobulins), are not reabsorbed because
of renal tubular failure; they appear in the urine, giving the tubular
pattern seen in Fig. 1A
. The mixed pattern (Fig. 1A
, top lane) is seen
with combined glomerular and tubular damage. The overflow pattern (Fig. 1A
, fourth lane from top) occurs when the BJP concentration exceeds the
tubular capacity for reabsorption, and does not necessarily imply
major renal damage.
Although the physiology and pathophysiology that produce the typical
patterns shown in Fig. 1A
are well characterized, peculiarities in the
synthesis of serum proteins and methodological manipulations that
effect interpretation have not been as well defined and can lead to
misinterpretations. Both of these features are illustrated in the
present case.
Hypogammaglobulinemia is not uncommon as chronic lymphocytic leukemia
progresses. In many cases, BJP is also associated with this syndrome.
In the present case, it was assumed by the clinical staff that the
nephropathy was most likely of diabetic origin, which most commonly
leads to increased glomerular permeability and the development of
nephrotic syndrome (3). The initial UPE with a 20-fold
concentrate (Fig. 1A
) along with 6.4 g protein/day seemed to
justify this view because the UPE pattern resembled a pure glomerular
pattern. However, because the serum electrophoresis showed
hypogammaglobulinemia (Fig. 1A
), the urine was reexamined at 100-fold
concentrate, and double bands were seen in the
2-globulin region, indicating a tubular
component (1)(2). Other evidence suggesting a tubular
component included the low serum potassium and phosphorous, and urea
nitrogen and creatinine within the health-related reference
intervals. These are all generally increased in pure
glomerular failure, but serum potassium and phosphorous are low in
cases of failure of tubular reabsorption (4). Epithelial
cells with hyaline casts are also seen in the urinalysis with tubular
damage. Most likely, the correct interpretation for this case is mixed
proteinuria because of both glomerular and tubular disease.
These patterns highlight the effect of mechanical concentration on the
interpretation. With a mixed UPE pattern containing a substantial
glomerular component, it may be difficult to see all tubular
constituents in a more dilute specimen. Usually, the intense
contribution from polyclonal free light chains is sufficient to enable
the correct interpretation in the absence of visible
2-microglobulins; however, in the present case
this was not true because of a lack of polyclonal immunoglobulin
synthesis. This case illustrates the importance of comparing serum and
urine results whenever possible.
A second misinterpretation that may emerge from inadequate
concentration of urine is failure to identify a BJP. In the case
described here, although nothing was observed in the region of the
20-fold concentrate with UPE, a diffuse region of staining was seen in
the 100-fold concentrate. After IFE, this diffuse staining clearly
appears as a band of restricted migration, which stained as a -BJP
(Fig. 1B
). The problem of screening for BJPs with UPE alone is further
accented by the patterns shown in Fig. 1C
, from a second patient, where
nothing is seen after UPE of a 100-fold concentrate, but a -BJP is
seen clearly after IFE of the same sample. In the case shown in Fig. 1C
, the total protein was found to be 150 mg/24 h, an amount that is
within the reference interval. Most laboratories quantify urine protein
with a precipitation method, such as trichloroacetic or
sulfosalicylic acid, or with a protein binding dye such as Coomassie
blue or Ponceau S. The degree to which these methods measure free light
chains is unclear. Very few laboratories use the biuret method, which
reacts with the peptide bonds in proteins, measuring all proteins more
equally. As a result, except for biuret method, these methods cannot
always be relied on to provide useful information regarding
quantification of free light chains. Furthermore, the detection limit
of all of these methods, including the biuret, are higher than that of
IFE (5). Although disagreement remains as to which
combination of UPE and IFE provides the most efficient and
cost-effective screening approach to detecting BJP (6), the
cases presented here show that even with a 100-fold
concentration, UPE in the absence of IFE is an inadequate screen for
low concentrations of BJP in samples.
Some commercial manufacturers of kits indicate that the urine sample
should be concentrated to a defined protein concentration, which in our
experience often turns out to be only a 10- to 20-fold concentration
for UPE or IFE. The importance of greater mechanical concentration is
emphasized in this study. In our opinion, the best solution to these
problems is to concentrate specimens 80- to 160-fold and to perform IFE
as well as UPE on all specimens for which identification of BJPs is
important. An alternative method for screening urine samples by using
/ ratios in conjunction with UPE has been described
(7). We have found that this alternative approach may reduce
the more tedious IFE by ~30%.
References
/ ratios for decreasing volume of urinary immunoelectrophoresis and improving accuracy of protein electrophoresis for Bence Jones proteins. Clin Chim Acta 1997;262:121-130.
[ISI][Medline]
[Order article via Infotrieve]
The following articles in journals at HighWire Press have cited this article:
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  M. Attaelmannan and S. S. Levinson Understanding and Identifying Monoclonal Gammopathies Clin. Chem., August 1, 2000; 46(8): 1230 - 1238. [Abstract] [Full Text] [PDF]
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