Clinical Chemistry 46: 1230-1238, 2000;
(Clinical Chemistry. 2000;46:1230-1238.)
© 2000 American Association for Clinical Chemistry, Inc.
Understanding and Identifying Monoclonal Gammopathies
Mohammed Attaelmannan1 and
Stanley S. Levinson1,2,a
1
Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY 40292.
2
Department of Veteran Affairs Medical Center,
Louisville, KY 40206.
a Address correspondence to this author at: Laboratory Service, VAMC, 800 Zorn Ave., Louisville, KY 40206. Fax 502-894-6265; e-mail levinson{at}louisville.edu
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Abstract
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Monoclonal gammopathies reflect conditions in which abnormal amounts of
immunoglobulins are produced by a clone that developed from a single
pro-B germ cell. The condition may reflect a disease process or be
benign. The primary purpose of this review is to emphasize routine
clinical laboratory techniques that currently are recommended for use
in identifying monoclonal gammopathies from serum and urine. Selection
of the preferred technique and correct interpretation often is
dependent on an understanding of the immunological basis and clinical
sequelae associated with these conditions. For this reason, we first
briefly discuss the structure, production, and nature of
immunoglobulins, and then describe important features of the associated
diseases. Finally, we discuss strengths and weaknesses of the
techniques and make reference to current recommendations to facilitate
optimal testing. We discuss in detail high-resolution electrophoresis,
methods for quantifying immunoglobulins, immunofixation
electrophoresis, problems associated with analysis of urine
immunoglobulins, and identification of cryoglobulins and immune
complexes.
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Introduction
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Monoclonal gammopathies result from an overproduction of a single
abnormal clone of a plasma cell or B lymphocyte. The monoclonal
immunoglobulin is recognized as a band of restricted migration on serum
or urine electrophoresis (M-component).1
When the band represents a monoclonal free light chain, it usually is
called a Bence Jones protein (BJP). In some cases, more than one clone
may produce monoclonal gammopathies (biclonal or, very rarely,
triclonal) (1). Usually, the production of a M-component
does not seem to be a response by the immune system to an offending
immunogen. Still, in some cases, M-components with activity against
immunogens from infectious agents have been identified (2).
The exact meaning of this relationship remains obscure.
If a disease is caused by a monoclonal line of plasma cells, derived
either from a malignant clone(s) or from a nonproliferative population
of cells, the condition is called plasma cell dyscrasia. In some cases,
monoclonal gammopathies may occur as a result of abnormal B cells,
which have not yet developed into plasma cells. This type of gammopathy
is seen in leukemia or lymphoma. It is important to note that many
monoclonal gammopathies identified on serum electrophoresis are benign,
so-called monoclonal gammopathy of undetermined significance (MGUS).
Guidelines for laboratory evaluation of patients with monoclonal
gammopathies recently have been described
(3).
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Structure, Genetic Characteristics, Production of Immunoglobulins,
and Nature of M-Components
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There are five classes of antibodies: IgG, IgA, IgM, IgD, and IgE.
Each basic unit is a monomeric antibody consisting of four chains: two
heavy chains, providing class specificity, and two light chains, 
or
. Each whole antibody consists of a constant, COOH-terminal end
(Fc), and variable, NH2-terminal end (Fab)
(4). Depending on the class, each intact monomeric unit has
a molecular weight varying between ~150 000 and 200 000,
whereas each light chain weighs ~22 000. The variable end of each
antibody contains a unique antigen combining site, whereas the Fc
portion, which contains common determinants, defines class and binds to
plasma proteins and to cell Fc receptors. IgG usually exists as a
monomer, IgA as a dimer in secretions but as a monomer in serum, and
IgM as a pentamer.
The heavy chain variable region is coded by three separate gene
segments, and the light chains by two genes. Before transcription, the
genes are randomly rearranged on the chromosome with a set of variable
region genes being translocated to form a continuous gene product
(4)(5). The constant regions are then spliced.
It appears that there are nearly 200 functional heavy and light chain
gene segments that give rise to combinations of gene products, allowing
typical B-cell lines to produce >5 x
107 antibodies with different unique variable end
antigen combining sites (4)(6)(7)(8). B cells that
contain surface receptors that best fit antigens are encouraged to
multiply (affinity maturation), providing a means whereby more specific
antibodies with greater affinity can be produced (4). This,
together with nucleotide additions or deletions during combinational
joining (junctional diversity), allows for >109
different antibodies (4)(5)(9).
The immunoglobulins formed early in the normal response to an immunogen
are IgM and IgD isotypes; these are located on the B-cell surface as
recognition receptors. Many of the immature cells produce low-affinity
early antibodies that bind to multiple antigens
(10)(11). The activated B cells begin to divide,
and class switching from the IgD and IgM heavy chains produced earlier
to the IgG, IgE, or IgA classes occurs (4)(5).
Much class switching and some somatic mutation occur in geminal centers
of lymphoid organs under the influence of various cytokines
(5). As B cells mature into plasma cells, they home to the
bone marrow.
In contrast to the great diversity of normal immunoglobulins, in
monoclonal gammopathies a single abnormal cell line predominates (or in
the rare case two or three). The abnormal cell(s) may produce an intact
immunoglobulin, free light chains without heavy chains (often both
intact and free), and rarely only heavy chains. The cells may produce
immunoglobulins with peculiar or missing amino acids, or class
switching may occur in some abnormal cells, causing lines that produce
immunoglobulins with two or more heavy chain classes; each abnormal
cell line produces only a chain or chain, never both.
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Plasma Cell Dyscrasias
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multidiscipline diagnosis
Diagnosis of plasma cell dyscrasia requires a multidisciplinary
approach (12)(13). Table 1
outlines the reasons for this.
multiple myeloma
Multiple myeloma is a plasma cell dyscrasia in which an increased
number of monoclonal plasma cells (plasmacytoma) occurs in the bone
marrow. The term multiple is used because plasmacytoma is found in
multiple sites. Usually, there are <4% plasma cells in the bone
marrow. Multiple myeloma is characterized by >30% plasma cells, or
<30% and 
10% plasma cells in bone marrow in the presence of other
evidence, such as that outlined in Table 2
(12)(13)(14). Patients with heavy BJP proteinuria (>1
g/24 h) usually have myeloma (13). In the classical case,
the M-component is abnormally increased in concentration but the normal
immunoglobulins are decreased, giving rise to a profile in which one
heavy chain class is increased but the others decreased. This pattern
is shown in Fig. 1
A. In ~5% of cases, the abnormal plasma cell does not secrete
an M-component.
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Figure 1. Illustration of serum protein electrophoresis showing
M-components.
Both M-components were identified as IgG-. Panel A
shows a dense M-component (arrow) and
densitometer tracing with an almost clear region surrounding the dense
band, indicating reduced synthesis of other (normal) immunoglobulins.
In this case, the best estimate of the M-component concentration can be
made from the densitometer tracing of the  area, which translated
into 38 g/L (reference interval, 0.7–1.6 g/L). Immunonephelometric
assay showed an IgG concentration of 49 g/L (7.2–16.8 g/L), IgA of
0.11 g/L (0.7–3.8 g/L), and IgM of 0.33 g/L (0.6–2.7 g/L), with a
/ ratio of 102 (1.2–2.6). Panel B shows a
low-concentration M-component (large arrow) superimposed
on a surrounding region of diffuse staining, indicating normal or
increased synthesis of other immunoglobulins. Here the densitometer
tracing translated into a -globulin of 22 g/L. IgG = 14 g/L,
IgA = 4.7 g/L, and IgM = 2.5 g/L, with a / ratio of
2.6. In this case, the M-component can only be estimated from the
staining density by visual inspection (the tracing is not shown). The
M-component density is approximately one-third of the total staining in
the region, or 22 g/L ÷ 3 = <10 g/L. A similar
conclusion could be reached by inspection of the densitometer tracing
if it were shown. Protein fractions are indicated in B.
The small arrow indicates the origin. Polarities are
indicated, and direction of electrophoresis is toward the positive
electrode.
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In multiple myeloma, IgG is the most common immunoglobulin, with IgA
being the second most common. IgM is rare. Although in myeloma
M-components typically appear to be low-affinity early antibodies with
unknown antigen specificity, the fact that IgG and IgA are more common
than IgM suggests that maturation of the abnormal cell has proceeded
far enough along for antigen stimulation and class switching to occur
(15)(16)(17)(18)(19).
asymptomatic or smoldering multiple myeloma
Some individuals with multiple myeloma show no symptoms. In this
case, a M-component usually is inadvertently identified by serum
electrophoresis. Various terms have been applied to this syndrome,
including smoldering, indolent, and asymptomatic myeloma. Usually
treatment is withheld until the M-component begins to rise or symptoms
associated with myeloma appear (14)(20).
solitary plasmacytoma
In some cases, only a single site of proliferation exists in bone
(21). Occasionally, the plasmacytoma is in tissue other than
bone, often in the sinuses or nasopharynx. In these cases, the
likelihood of remission or complete cure is higher than for myeloma.
Here, the M-component usually is of low concentration (<25 g/L)
(14).
poems syndrome
This refers to a rare disorder with peripheral neuropathy,
organomegaly, endocrine deficiency, monoclonal gammopathy, and skin
pigmentation (22). The clinical course is more indolent than
multiple myeloma (22).
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Lymphocytic Diseases
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M-Components may be associated with B-cell lymphomas and
leukemias. In these cases, IgM is more common than IgG or IgA,
suggesting that the abnormal clone is more primitive than in myeloma.
Waldenström syndrome is a low-grade small-cell lymphoma
that produces monoclonal IgM (23). Clinical features usually
are related to growth of the tumor. Many patients produce monoclonal
IgM, which may cause hyperviscosity syndrome, or type I or II
cryoglobulinemia, which may lead to Raynaud syndrome, vasculitis, cold
agglutinin hemolytic anemia, peripheral neuropathy, or immune complex
disease (23)(24). M-Components are also seen in
leukemias, especially chronic lymphocytic leukemia where identification
adds little to the diagnosis or predicted outcome.
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Amyloidosis AL
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Amyloidosis is a condition in which an abnormal proteinaceous
material is deposited in tissues (25). In immunoglobulin
light chain-related amyloidosis (AL) the deposits are attributable to
BJPs (25). In amyloidosis, fibrils are formed that appear
arranged in an antiparallel conformation with a ß-pleated sheet
structure when examined by x-ray diffraction. Under the polarizing
microscope, amyloid appears as a Congo red-staining material that
exhibits apple-green birefringence (25). This appearance
under polarized light is the most common method used to identify
amyloid, although the electron microscope is more sensitive and is used
for suspected cases when light microscopy is nondiagnostic
(25). Lambda chains appear to have a greater affinity to
form amyloid than chains (26)(27).
The most common organs affected in amyloidosis are the kidney, heart,
liver, and gastrointestinal tract. Although the central nervous system
usually is not affected in amyloidosis AL, peripheral nervous system
complications are common, especially carpal tunnel syndrome
(25). These diseases may be associated with multiple myeloma
or may occur in the absence of clear plasma cell malignancy. In the
absence of myeloma, biopsy of the bone marrow is not helpful because it
usually shows <5% plasma cells. Amyloid is best identified from
biopsy of an involved organ, but because of the invasiveness associated
with this approach, usually a rectal or more commonly an abdominal
subcutaneous fat pad is biopsied. These are positive under polarized
light in ~85% of cases (25)(28). If the
disease occurs in the absence of myeloma, a low-concentration
monoclonal spike consisting of IgG or IgA, which often is small (<10
g/L), may be seen in serum, and a BJP, whose presence is very helpful
in leading to the diagnosis, usually is seen in urine with or without a
serum M-component.
Approximately 5% of patients with Waldenström macroglobulinemia
develop amyloidosis, and this diagnosis should be suspected in patients
with IgM M-components and appropriate clinical presentation
(23).
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Transplant Monoclonal Gammopathy
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A transient monoclonal or oligoclonal gammopathy has been
associated with bone marrow and solid tumor transplants. Generally,
these gammopathies are of low concentration and short-lived
(14)(29)(30).
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MGUS
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A low-concentration M-component associated with no signs or
symptoms of plasma cell dyscrasia or B-cell abnormality is seen in the
serum of a small number of people under 70 years of age and ~3% of
persons over 70 (14). Usually, the concentration is <20 g/L
IgG or 10 g/L IgA. As shown in Fig. 1B
, on electrophoresis these
low-concentration M-components usually are superimposed on a diffuse
background of normal immunoglobulins. If the concentration is between
20 and 30 g/L or if a BJP is present, it may be difficult to
differentiate from asymptomatic myeloma.
Idiopathic BJP proteinuria has also been described (31).
Some of these patients remain stable, but many develop myeloma. Low
concentrations of BJPs (<0.2 g/24 h) may be seen in the urine of
patients with MGUS and with chronic diseases other than myeloma
(31)(32)(33).
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Identification of M-Components
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Bone marrow biopsy and testing of serum and urine for a
M-component are fundamental in the work-up of monoclonal gammopathies
(1)(12). In some cases, peculiar bands may be
seen in serum that are not M-components. These include transferrin
associated with iron deficiency anemia, highly increased
ß-lipoprotein, fibrinogen, or C-reactive protein (34). For
this reason, suspect bands identified by electrophoresis should be
described as a paraprotein or possible M-component until characterized
by immunoassay.
high-resolution electrophoresis
High-resolution agarose protein electrophoresis (HRE) is a
technique that gives better resolution of serum, urine, and
cerebrospinal fluid proteins than standard electrophoresis
(35).
As illustrated in Fig. 1B
, traditionally agarose electrophoresis of
serum proteins is divided into five fractions: albumin,
1-globulins,
2-globulins, ß-globulins, and -globulins.
HRE systems separate several other fractions, often totaling 10–12.
Importantly, HRE permits better detection of low-concentration
M-components migrating in the 2, ß, or
regions of the gel (36)(37).
On average, the maximal density of IgG is cathodal to the origin, IgM
is coincident with the origin, and IgA is anodal to the origin, closest
to the ß region. Nevertheless, because of their great diversity, some
of each immunoglobulin class exhibits charge characteristics that cause
migration in the , ß, and even pre-ß regions. Likewise, intact
M-components most commonly migrate in the region, but occasionally
they are found in the ß or even the 2
region. Fig. 2
illustrates this effect. In some cases, the smaller BJPs
migrate well into the 2 region.
The most important function of HRE is as a screen for identifying
possible M-components. Visual examination of the gel is the most
sensitive means of detection because low-concentration M-components are
readily missed by densitometry scanning.
quantification of m-components
Quantification of M-components is desirable because it aids in
assessing the tumor load and in determining whether the disease is
progressing. Because of excellent reproducibility and ease of use,
nephelometric and turbidimetric immunoassays of M-components currently
are the most common methods for quantifying immunoglobulins. However,
when assaying M-components, these methods are often inaccurate because
the antibodies and calibrators used with the assay are developed using
the vast variety of diverse normal immunoglobulins, whereas
M-components exhibiting limited or incomplete antigenic determinants
may react incompletely with the antiserum and behave peculiarly
compared with the calibrator. Thus, the concentration of M-components
is often under- or overestimated (38). Quantification of the
normal immunoglobulin classes not involved in the disease may be useful
for determining the functional degree of hypogammaglobulinemia.
Because of these peculiar immunological properties, the best way to
assess the concentration of a high-concentration M-component is by
densitometry. If the M-component is migrating in the region of the
electrophoretic gel and is increased, whereas the other immunoglobulins
are decreased as seen in Fig. 1A
, the concentration of M-component can
be measured accurately by densitometry.
Low-concentration M-components in serum or those migrating in the ß
region are usually superimposed on other proteins. Fig. 1B
shows a
low-concentration M-component superimposed on a diffuse background of
polyclonal immunoglobulins. In such a case, the concentration can only
be qualitatively estimated from the densitometer tracing by subtracting
the approximate density of a small band from the approximate underlying
staining.
immunofixation electrophoresis
In immunofixation electrophoresis (IFE), proteins are fractionated
on electrophoretic strips as with HRE but not stained. Each lane is
overlaid with monospecific antisera, usually with activity against the
three major immunoglobulin classes (IgG, IgA, and IgM), and against
free and bound (intact) and light chains. Immunoglobulins are
precipitated by the antisera in the gel. After a few hours, the gels
are washed to remove unprecipitated proteins and then stained
(39)(40). The results of IFE are illustrated in
Fig. 3
. If a M-component is present, it appears as a band coincident
with the paraprotein seen on HRE. It can be characterized as IgG, IgM,
IgA, and or , depending on the pattern of precipitation.
With a detection limit down to ~0.25 g/L, IFE is more
sensitive for detecting low-concentration M-components than HRE
(41). For this reason, IFE is recommended as the preferred
technique for identification of low-concentration M-components
(3). This feature contributes very little to the
identification of M-components in serum, where tiny components often
are of little clinical significance and their identification adds
expense to an unnecessary additional workup, but with urine greater
sensitivity is important for identifying BJPs present in very low
concentrations.
There is a problem associated with the reaction between antibodies and
very high concentrations of M-components that may affect the
interpretation of IFE patterns. With very high concentrations of
M-component, the antiserum will be relatively dilute and complete
precipitation may not occur. This phenomenon of antigen excess,
illustrated in Fig. 3
, is called the prozone effect. This effect
usually is not a great problem with serum M-components because the
concentration changes over only a limited range of ~10–60 g/L. In
serum, a very dense band may appear as a donut with a clear spot in the
center, but such patterns are still interpretable. In these cases, a
solid band can be demonstrated by diluting the serum 2- to 10-fold and
repeating the assay.
This problem is worse when analyzing BJPs in urine (38). As
described below, in urine much wider ranges of M-component
concentrations are commonly encountered so that the prozone effect
becomes a much greater problem. In addition, compared with intact
immunoglobulins, free light chains more often react poorly with the
antisera, producing indistinct bands (38).
Quality control is an important feature for ensuring accurate testing.
Antisera are produced against polyclonal immunoglobulins and may react
poorly with M-components (42). Antisera of sufficient
quality to perform IFE are now available from several manufacturers.
Each lot of antiserum should be tested against sera containing known
M-components and especially against concentrated urine containing BJPs
and urine containing general proteinuria without BJPs. Many
laboratories keep a stock of at least two antibodies from different
companies to reanalyze incomplete or peculiar precipitation patterns.
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Problems Associated with Analysis of Urine
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monoclonal free light chains
Free light chains usually are Mr
22 000 monomers or Mr 44 000
dimers (43). As a result, BJPs readily pass through the
glomerulus and are more concentrated in urine than in serum. In
addition, because urine is relatively dilute in total protein compared
with serum, urine can be concentrated further by differential
filtration using mechanical devices. To identify BJPs present in very
low concentrations, it is recommended and important that urine samples
be concentrated between 100- and 150-fold, regardless of the amount of
total protein in the urine (27)(44).
It seems to us that excessive concentration of urine above 150-fold is
unwise because BJPs present in very low concentrations (<0.2 g/24 h),
which are MGUS or associated with other chronic diseases, may be seen
(31)(32)(33), which would necessitate an unnecessary workup.
Furthermore, excessive concentrations may cause an increase in
background staining and, as will be discussed below, invariably give
rise to polyclonal patterns that appear as multiple bands that can be
confused with a monoclonal pattern (27).
Because of its greater sensitivity, IFE should be performed along with
HRE on all urine specimens suspected of having a BJP
(27)(45). Specimens should be 24-h collections.
First morning collections may also be adequate, but random collections
are unsuitable (46).
Because of the wide range of protein concentrations achieved by
mechanical concentration, ranging between ~0.1 and 20 g/L,
high-concentration BJPs may appear overloaded on IFE or a prozone
effect may occur, making interpretation more difficult. As shown in
Fig. 3
, more definitive patterns can be obtained by diluting the
specimen and repeating the IFE. In practice, overloading rarely leads
to a mistake in interpretation because the correspondence between the
band seen on urine HRE and a distorted band on IFE provides a means by
which correct interpretation can usually be achieved without repeat
assay (47).
BJPs react very idiosyncratically with antisera. As a result, even BJPs
present in very high concentrations may react poorly, showing little or
no banding. Thus, if the HRE pattern shows a suspect band, it is wise
to test the sample with a second antiserum from a different source
before concluding that a BJP is not present.
Another difficulty with IFE is that BJPs migrating coincidental with
intact M-components cannot be distinguished using free and bound
antiserum. This problem may be more hypothetical than real. As
illustrated in Fig. 3
, a comigrating BJP may be suspected when one
observes a large antigen excess effect with the or free and
bound light chain antiserum compared with a lesser effect with the
heavy chain antiserum. Usually, if a BJP is migrating close to an
intact monoclonal protein, dilution studies will distinguish it,
showing that it is migrating close to but not exactly coincidental with
the intact protein.
Nevertheless, it might be possible to miss a tiny BJP hidden directly
behind a moderately sized intact immunoglobulin. Thus, to ensure that
there is not a comigrating BJP, it may be necessary to fix specimens
with an intact immunoglobulin in urine but without an obvious separate
light chain band with free antisera (antisera that react only with free
light chains). Fig. 3
illustrates IFE with the use of free and bound
(intact), and free antisera. Because of lower activity titers in free
antisera compared with free and bound antisera, the prozone effect
attributable to antigen excess is greater. Thus, if suspect bands stain
strongly with free and bound antisera, but not at all with free
antisera, dilution studies should be performed. In our experience,
because of its weak avidity, free antiserum has little usefulness other
than for this purpose (27)(44).
polyclonal free light chains
Polyclonal free light chains are secreted in excess of heavy
chains (48). Thus, normal urine that is sufficiently
concentrated will exhibit polyclonal free light chains. As shown in
Fig. 4
, polyclonal free light chains may appear as multiple bands
(ladder pattern) after IFE and isoelectric focusing. This pattern,
observed as three to six equally spaced bands, is more often associated
with light chains but may also be found after analysis of light
chains, and can be uncovered in most urine specimens if sufficiently
concentrated (49)(50)(51). MacNamara et al.
(51) demonstrated that this pattern is the
property of normal free light chains by inducing the transient
appearance of multiple bands with arginine infusion in healthy
volunteers. The multiple banding pattern appears to be largely a
product of charge differences (50). The pattern may be
related to homologies in the framework 1 region of the light chain
variable region, which defines four serological subgroups of and
six subgroups of light chains (27)(52).
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Figure 4. Polyclonal free light chain ladder band pattern.
The gel was fixed with antisera against free and bound . Multiple
bands are present but difficult to see. Some electrophoretic systems
show these bands better than others. Notice that the band closest to
the bottom could be mistaken for a BJP. The direction of
migration is toward the bottom (positive electrode).
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As a result of poorer resolving power, IFE devices from some
manufacturers do not appear to have the ability to distinguish this
pattern routinely. With some systems, electrophoresis of polyclonal
free light chains in high concentrations will cause dense, protracted
staining patterns that will evolve into a ladder band pattern with
dilution, whereas patterns on other systems will evolve into diffuse
staining zones. Although a ladder pattern, per se, does not reflect an
abnormal condition, as illustrated in Fig. 4
, the pattern may be
mistakenly interpreted as a BJP or it may obscure a low-concentration
BJP migrating coincidental with the pattern
(50)(51)(53). In the latter case,
the presence of a low-concentration BJP may be overlooked. Because IFE
is not a quantitative technique with a well-defined lower reference
limit for detection, systems with sufficient separation power to
discern these polyclonal patterns may offer little advantage over
otherwise sensitive IFE systems that do not (27).
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Identification of IgD or IgE
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If a or M-component is found in serum in the absence of a
IgG, IgA, or IgM heavy chain, it is necessary to test the sample for
IgD and IgE.
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Identification of Cryoglobulinemia and Immune Complexes
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Cryoglobulins are immunoglobulins that precipitate when cooled
below body temperature. This occurs either because the structure of the
immunoglobulin is such that it precipitates in the cold or because the
immunoglobulin, usually IgM, contains rheumatoid factor activity, in
which case the variable Fab of the rheumatoid factor binds to the Fc
portion of polyclonal immunoglobulins, forming an immune complex that
precipitates in the cold.
Three types of cryoglobulins have been described. Type I consists of a
single monoclonal immunoglobulin; type II is mixed
polyclonal-monoclonal, and type III is mixed polyclonal-polyclonal
(24). Types I and II may be attributable to M-components
associated with multiple myeloma, Waldenström syndrome, or other
lymphoproliferative diseases. As a result of precipitation in blood
vessels close to the surface, these may cause several of the symptoms
associated with monoclonal gammopathies, such as Raynaud syndrome,
peripheral neuropathy, and gangrene in the absence of known vascular
causes.
Thus, it has been recommended that cryoglobulins should be assessed in
all patients with M-components and cold-sensitive complications
(3). Cryoprecipitates appear as gelling or precipitation.
Because of the ambiguity associated with this endpoint, it is important
to collect no less than 10 mL of blood. The blood should be kept at
37 °C during transport, clotting, and serum separation. The warm
serum is placed in a 4 °C refrigerator and examined for up to 7
days. If a cryoprecipitate appears, it can be analyzed by IFE to
confirm that it is a cryoglobulin and to type it (24). To
ensure the removal of adsorbed immunoglobulins before analysis, the
cryoglobulin should be collected by centrifugation and then
reprecipitated in saline by cooling. The pellet should be collected a
second time and washed thoroughly with ice-cold saline. If no albumin
is seen with HRE, it is likely that the precipitate has been adequately
washed.
Soluble immune complex aggregates that may or may not be cryoglobulins
may appear as a paraprotein band on HRE. In IFE, a band is seen in all
lanes or in the IgG, IgM, , and lanes. Treatment for 4 h at
37 °C with a sulfhydryl reagent breaks the aggregates into component
parts (we make a 1:10 dilution of 2-mercaptoethanol with isotonic
saline and add 10 µL of the dilution to 100 µL of the patient’s
serum). After treatment, the IFE is repeated. If the repeat pattern
appears as a monoclonal band in the or and the IgG or IgM
lanes, this indicates a monoclonal IgG or IgM rheumatoid factor,
respectively. If a polyclonal pattern is seen in all lanes, the
rheumatoid factor was not monoclonal. If the pattern is ambiguous after
treatment, the rule of thumb is to repeat the treatment for 12 or
24 h.
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Turbidimetric/Nephelometric Analysis
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Automated immunonephelometric or turbidimetric analysis has two
uses in the workup of monoclonal gammopathies in serum: (a)
They are useful for determining whether the immunoglobulin classes,
other than the monoclonal, are decreased. When decreased, a diagnosis
of myeloma is more certain. (b) Nephelometric and
turbidimetric tests can be used for analyzing the / ratio. In
some cases, this provides a means for identifying whether a paraprotein
component in serum identified by HRE is monoclonal, as well as its
immunoglobulin class, without the need for the more complex IFE. The
typical / ratio is ~2, with a range of ~1–3. If the /
ratio is abnormal and IgG, IgA, or IgM is increased, the class and type
of paraprotein seen on HRE is identified without IFE
(54)(55)(56).
This technique works well when the abnormal immunoglobulin is very
large, such as that shown in Fig. 1A
. (It is clear from the
electrophoretic pattern and immunonephelometric analysis that an IgG
M-component is present.) In such a case, an additional assay is not
needed. But the technique often does not show an abnormal / ratio
when the abnormal immunoglobulin is of low concentration and the normal
immunoglobulins are not decreased (Fig. 1B
). This is attributable to a
masking effect by the and activity of the normal
immunoglobulins. In addition, this technique is not useful when
biclonal or triclonal gammopathies are seen with HRE. In small
laboratories where IFE is performed infrequently, this approach may
effectively reduce the need for the more tedious IFE. On the other
hand, in laboratories where IFE is performed frequently, / ratios
serve little purpose.
Another advantage of having immunonephelometry or turbidimetry
performed in the laboratory is that the various antisera used for the
automated analysis can be used as a second source of antibody for
reanalysis of peculiar IFE patterns.
|
Capillary Electrophoresis
|
|---|
High-performance capillary electrophoresis (CE) is a new technique
in which proteins in a buffer can be separated in a narrow capillary
(usually ~50 µm in diameter). Although no gel is used in the
capillary, samples are relatively small compared with the surface area
of the capillaries so that separation of the proteins into components
is facilitated by adsorption to the walls of the silica capillaries.
Thus, the proteins separate on the basis of chromatographic
partitioning to the capillary walls as well as charge. Very small
sample sizes are made possible by very sensitive monitoring devices. As
a result, similar to the fractions seen with HRE, the proteins are
separated into ~12 identifiable fractions. A detector that records
absorbance as the proteins pass by is located at the cathodal end of
the capillary. CE may be able to detect paraprotein peaks as well as or
even slightly better than HRE (57)(58). The
advantage of CE is that it is both automated and rapid, so that many
samples can be analyzed in only a short time, making it less tedious
than HRE, which usually is manual.
Suspected monoclonal proteins can be identified on CE by
immunosubtraction. Here, CE is performed before and after exposing
samples to solid particles coated with antibodies specific for
immunoglobulin classes and and light chains. Removal of a peak
by the coated beads provides a means for characterizing the nature of
the M-component.
Although this technique has been widely studied for detection of serum
M-components, it has not been perfected for analysis of urine proteins
(58). In addition, its effectiveness is still unclear for
the detection of antigen excess, small M-components, and M-components
migrating in the ß region coincidental with other proteins. For these
reasons, although some large laboratories may have validated its use
and use it routinely, it is premature to recommend its use on a routine
basis, although it may be the choice for the future (58).
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Conclusions
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|---|
Monoclonal gammopathies may be indicative of diseases arising from
B cells or plasma cells. The most common diseases are myeloma and
amyloidosis AL. These diseases may be difficult to diagnose because
they affect many tissues and exhibit nonspecific symptoms. Definitive
diagnoses of these conditions are based on clinical criteria,
laboratory testing, and biopsy. Identification and quantification of a
M-component in serum or urine is helpful in leading to the diagnosis
and evaluation of extent of disease. M-Components usually are
identified and characterized by a combination of HRE and IFE, although
in some cases immunonephelometry or turbidity may be used. The newer
technique of CE may eventually replace these.
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Footnotes
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1 Nonstandard abbreviations: M-component, monoclonal component; BJP, Bence Jones protein; MGUS, monoclonal gammopathy of undetermined significance; HRE, high-resolution electrophoresis; IFE, immunofixation electrophoresis; and CE, capillary electrophoresis. 
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Abstract
Introduction
