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 Patient’s 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

Anode Patient serum Cathode

(+ electrode) (- electrode)

Application point

Protein Fractions

IgG, IgA, IgM, IgD, IgE and C-reactive protein

Transferrin, Complement, beta-Lipoprotein

alpha-2-macroglobulin, Haptoglobin, Ceruloplasmin

alpha-1-antitrypsin, alpha-1-glycoprotein, alpha-1-lipoprotein

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 ofMonoclonal Gammopathy

M protein spike

Note the percentage of the gamma globulin fraction has doubled from the norm.

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

Anti- Anti-IgG Anti-IgA Anti-IgM Anti-Kappa Anti-LambdaTotal protein

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, Waldenström'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 Waldenström'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!