Diagnosis of Paraprotein

Diseases

CLS 404
Immunology
Protein Abnormalities

Objectives

Discuss the use of the following

laboratory tests in the diagnosis of
paraprotein diseases:

Protein level determinations

Immunoglobulin level determinations
Electrophoresis
Bone marrow differential

Objectives

Distinguish paraprotein diseases from

these non-paraprotein conditions:

Acute inflammation
Nephrosis
Cirrhosis
Infection

Protein Measurements

Total serum protein

Elevated levels
May be detected before the patient
exhibits symptoms
Also found in non-paraprotein diseases

Additional tests required to distinguish

between diseases

Immunoglobulin Levels

Serum immunoglobulin levels

Detects increased quantities of a specific

immunoglobulin class
Patients serum is mixed with antibodies to IgG,
IgM or IgA and the formation of antigen-antibody
complexes is measured
Cannot distinguish between monoclonal and
polyclonal increase

Serum protein electrophoresis

Demonstrates the monoclonal immunoglobulin

(M protein)

Serum Protein Electrophoresis

Abbreviated SPE
Separation of proteins according to
size and electrical charge
Application point

Anode
(+ electrode)

Patient serum

Cathode
(- electrode)

Protein Fractions
alpha-1-antitrypsin,
alpha-1-glycoprotein,
alpha-1-lipoprotein

alpha-2-macroglobulin,
Haptoglobin, Ceruloplasmin
Transferrin, Complement, betaLipoprotein
IgG, IgA, IgM, IgD, IgE
and C-reactive protein

Serum Protein Electrophoresis

Will differentiate a monoclonal

gammopathy from other causes of
increased protein levels
Will not detect an increase in light
chains, as these are cleared from
circulation too quickly

See slide on Bence Jones proteins

Electrophoresis Pattern of
Normal Individual

anode

cathode

Electrophoresis Pattern of
Monoclonal Gammopathy
Note the
percentage
of the
gamma
globulin
fraction has
doubled
from the
norm.

M protein spike

Electrophoresis Pattern of
Polyclonal Gammopathy
Polyclonal
gammopathy is
typically seen in
infections.
Note the % of the
gamma globulin
fraction is similar to
that seen in
monoclonal
gammopathy, but the
band is wider,
reflecting the
diversity of
antibodies produced.

Electrophoresis Pattern of
Acute Inflammation

Electrophoresis Pattern of

Cirrhosis
The pattern in
cirrhosis shows a
bridging of the
beta and gamma
globulin fractions.

Electrophoresis Pattern of
Nephrosis

Immunoelectrophoresis (IEP)

Semi-quantitative test for determining

specific heavy chain and light chain
components in a monoclonal gammopathy
First, serum or urine proteins are separated
by electrophoresis, usually on an agarose
gel.
Antibodies specific for heavy and light
chains are added to the gel.
The antibodies diffuse through the gel.

IEP

If the antibody encounters its specific

Ig chain, a precipitate forms.
The gel is stained in order to visualize
the precipitates.
The amount of Ig present is indicated
by the thickness and shape of the
precipitate.

Immunofixation Electrophoresis

Abbreviated IFE

More sensitive than IEP

More expensive than IEP

As in IEP, the proteins are separated by

electrophoresis.
Ig specific antibody is applied directly to the gel,
rather than relying on diffusion.
The gel is stained to reveal antigen-antibody
complexes.
Dark bands form when monoclonal antibodies are
present; light diffuse bands indicate polyclonal
antibodies.

IFE Example of an IgG monoclonal

antibody with kappa light chains
= Serum application point

AntiAnti-IgG
Total protein

Anti-IgA

Anti-IgM Anti-Kappa Anti-Lambda

Bence Jones Proteins

Light Ig chains found in the urine of multiple

myeloma patients
NOT detected by routine urine dipstick test
Heat Precipitation non-specific test

Bence Jones proteins remain in solution at room

temperature
Precipitate out of solution at 56oC
Dissolve again at 100oC

IEP and IFE specific test for identification

of particular light chain

Stains

Light chain deposits in tissue, as seen

in amyloidosis, can be detected by
stains:

Congo red show these as apple green

fibers under a polarizing microscope
Antibodies to the light chain tagged with
fluorescent dyes or other chemicals

Immunofluorescence
Tissue that is suspected of
having light chain deposits (pink
dots in the demonstration) is
fixed to a slide.
Fluorescently labeled antibody
specific for kappa or lambda light
chain is added to the slide.
Antibody combines with antigen,
and fluorescence can be
detected microscopically.

Bone Marrow Differential

Used to confirm a diagnosis of

Multiple Myeloma, Waldenstrm's
macroglobulinemia, or MGUS.
An aspirate of bone marrow is
obtained via a large bore needle
inserted into the iliac crest of the hip.

Bone Marrow Differential

Normal marrow typically shows less

than 5% plasma cells.
In Multiple Myeloma, plasma cells will
increase to 10% - 30% or more of all
marrow cells.
In MGUS, plasma cells comprise less
than 10% of marrow constituents.

Plasma Cells in Bone Marrow of

Multiple Myeloma Patient

The differential on this marrow revealed that

over 80% of the cells in the marrow were
plasma cells.

Plasma Cells in Bone Marrow of

Multiple Myeloma Patient

This magnification of the previous slide shows abnormal and very

immature plasma cells (prominent nucleoli at arrows)

Peripheral Blood

Plasma cells may be

seen in Multiple Myeloma
Plasmacytoid
lymphocytes may be
seen in Waldenstrm's
Macroglobulinemia
Red cells may exhibit a
stack of coins
appearance.

Called rouleaux
Caused by excess of
serum proteins

Radiology

X-rays demonstrate lesions in bones

throughout the body

Diagnosis of
paraprotein disease includes:

Detection of high protein levels

Confirming the high protein is due to gamma
globulins
Determining the presence of the M protein &
classifying the Ig present through
electrophoresis
Bone marrow biopsy to verify abnormal
numbers of plasma cells
X-rays to visualize lytic bone lesions

This completes the presentation

on the diagnosis of paraprotein
disease.
You are ready for the self
assessment quiz!