Generality
Immunofixation is a laboratory test that allows the identification and typification of gamma globulins present in a biological sample.
In detail, the analysis allows to study - in the patient's blood or urine - the immunoglobulin classes (IgA, IgG, IgM, IgE or IgD) and the type of light chain kappa or lambda, according to their specific electrophoretic mobility . Subsequently, for the identification of which gamma globulin is involved, the immunofixation involves the inoculation of specific antigens for the fragment or the corresponding antibody ( anti-immunoglobulin antisera ): the presence of the suspected alteration is confirmed by the formation of a precipitate, visible to the naked eye or microscope.
This information is of great importance for establishing, in particular, the diagnosis of plasma cell pathologies (gammopathies or plasma cell dyscrasias). Immunofixation is very useful for the evaluation and monitoring of multiple myeloma, Waldenström's macroglobulinemia and primary amyloidosis (also called light chain amyloidosis).
What's this
Immunofixation (or immunoelectrophoresis, also abbreviated in the acronym IFE ) is a technique able to determine whether and which class of immunoglobulins (IgG, IgM, IgA, IgD, IgE) or type of light chain kappa or lambda, is present in excess or defect in the patient's blood serum and urine.
The kappa and lambda light chains characterize the monoclonal component, ie the antibodies with the same identical chemical structure.
Serum and Urinary Immunofixation: when is it indicated?
Plasmacellular dysplasias or gammopathies are a heterogeneous group of disorders of unknown etiology characterized by:
- Disproportionate proliferation of a B cell clone ;
- Presence in the serum and / or urine of a type of immunoglobulin (or its polypeptide subunit), structurally and electrophoretically homogeneous ( monoclonal ).
Normally, plasma cell dyscrasias are diagnosed using electrophoresis of serum and urinary proteins, followed by serum immunofixation (IFE).
At the same time, a blood test can be prescribed to measure the total concentration of immunoglobulins (IgG + IgM + IgA).
To learn more: Immunoglobulins - What they are, How they are measured and Reference Values »Immunofixation: basic principle
Immunofixation is an examination that involves two phases (first the electrophoresis in agarose gel, then the immunoprecipitation with specific antisera ).
The first step of immunofixation is the same as that of the ELECTROPHORESIS of proteins (or protidogramma), therefore it is necessary to remember some concept:
- Electrophoresis is a laboratory analysis that allows to determine the quantity of proteins present in the blood serum or in other biological samples and, for each fraction, reveals if there are any anomalies in terms of quality. In particular, this exam allows the separation of proteins into five groups: albumin, alpha 1 globulin, alpha 2 globulin, beta globulin and gamma globulin ; the latter are investigated more specifically with immunofixation.
- Electrophoresis is a separation method based on the different migration speed of electrically charged particles, through a solution and an inert support medium, under the influence of an electric field, generated by a direct current. In practice, the exam exploits the electric charge and the molecular mass of the proteins present in the patient's sample. Under the impulse of an electric field, these macromolecules migrate and "group" by type, responding to stress in a characteristic way. The result ( electrophoretic tracing ) consists of various peaks and curves, to which the protein fractions correspond. Normally, the first peak, higher and narrower, is that of albumin ; to follow, we observe the globulin peaks, much lower than albumin. The increase or decrease in amplitude and intensity of the peaks that form in the path indicate a greater or lesser presence of proteins in each category; the more proteins are present in a band, the higher the respective peak. In the case of gamma globulins, if one wants to know the quantity of each of the different classes (IgA, IgM, IgG etc.) it will be necessary to resort to single dosage. In general, the more gamma globulins are present in a band, the higher the respective peak is; the height corresponds to the total quantity of proteins belonging to a specific category.
IMMUNOFISSATION provides:
1) Agarose Gel Electrophoresis : the patient's sample (serum or urine) is deposited on an electrophoretic strip, ie the support for migration (usually, agarose gel). Through the application of an electric field, generated by a direct current, separation in different bands is obtained. In practice, each type of gamma globulin present in the mixture (ie in the patient sample) migrates based on the molecular mass and the electric charge. The different electrophoretic mobility of gamma globulins makes it possible to identify them (each of them coincides with a specific band in the electrophoretic path) and to observe any anomalies.
2) Immunoprecipitation with Specific Antisera (Fixation) : specific antigens for a specific antibody or fragment are added individually to each electrophoretic strip (anti-IgG, anti-IgA, anti-IgM, anti-light kappa or anti-chain) lambda light chain). If a monoclonal protein is present, the interaction of the antigen with the corresponding antibody will produce a narrow band (this means that the result is positive) and the formation of a precipitate, visible to the naked eye or microscope. Finally, the sample is processed (ie washed and colored) to remove proteins that have not precipitated, to obtain the drying of the gel and proceed with the reading of the results.
Why it runs
What is Immunofixation for?
Serum immunofixation is an exam indicated to diagnose or monitor diseases characterized by an alteration of gamma globulins, especially when it concerns the production of the monoclonal component ( monoclonal gammopathy ).
To learn more: Monoclonal Gammopathies - What and which are »Immunofixation is useful in cases of suspected plasma cell dyscrasia and is fundamental for the study of MIELOMA (and related pathologies) and MGUS (an acronym for "monoclonal gammopathies of uncertain meaning").
This test also allows us to evaluate Waldenström's macroglobulinemia and light-chain amyloidosis ( AL ).
Immunofixation: when is the examination prescribed?
The indication for immunofixation is subsequent to clinical or laboratory evidence that raises the suspicion of alterations of monoclonal antibodies or immunoglobulins in serum or urine.
For example, this exam allows to deepen the result of seroprotective or urinary electrophoresis: when in the electrophoretic tracing, the bands that correspond to the gamma globulins deviate from normality, the immunofixation identifies which of these proteins is altered, in terms of quantity and quality.
In summary, serum immunofixation can be prescribed:
- In the case in which other laboratory tests provide an abnormal result, in terms of absence or smaller and greater quantity of plasma proteins with respect to the norm;
- When the presence of diseases characterized by the production of a monoclonal component is suspected, such as:
- Multiple myeloma and its variants;
- Waldenström's macroglobulinemia;
- Amyloidosis;
- When the patient develops symptoms and signs that suggest the presence of a monoclonal gammopathy or multiple myeloma, such as:
- Bone pains;
- Anemia;
- Easy fatigue;
- Recurrent infections;
- Unexplained fractures.
In the case of multiple myeloma, the electrophoresis of proteins and immunofixation are of considerable utility also for monitoring the progress of the disease (follow-up).
Immunofixation: on which samples can be performed?
The biological samples that can be subjected to immunofixation are mainly:
- Serum (serum immunofixation);
- Urine (urinary immunofixation);
- Liquor * (liquor immunofixation).
* Note : CSF or cerebrospinal fluid is the fluid that permeates and protects the brain, spinal cord, cranial nerves and spinal roots.
Normal values
A NEGATIVE result of serum and urinary immunofixation for monoclonal or polyclonal gamma globulins is considered NORMAL .
Gamma globulins
Gamma globulins make up 9-20% of total plasma proteins.
- Reference value Gamma globulin : 0.9-1.4 g / dl
Regarding the total concentration of immunoglobulins ( IgG + IgM + IgA ), it is believed that values between 600 and 2300 mg / dl are normal.
- Reference value Immunogobulins in serum :
- IgA : 90-400 mg / dl
- IgG : 800-1800 mg / dl
- IgM : 60-280 mg / dl
- IgD : 0.3-0.4 mg / dl
- IgE : 20-440 mg / dl
Note : the reference interval of the exam may vary slightly depending on age, sex and instrumentation used in the analysis laboratory. For this reason, it is preferable to consult the ranges listed directly on the report. It should also be remembered that the results of the analyzes must be assessed as a whole by the general practitioner, who knows the patient's medical history.
High Values - Causes
The increase in gamma globulins can be:
- Polyclonal, if the production of immunoglobulins is heterogeneous, that is, all the components of the gamma globulins - produced or deriving from different plasma cells - are affected by the increase;
- Monoclonal, when the gamma globulins are produced or derived from a single type (clone) of plasma cells.
The increase in polyclonal gamma globulins may be associated with:
- Chronic inflammatory diseases;
- Rheumatoid arthritis;
- Systemic lupus erythematosus;
- Chronic liver diseases (such as hepatitis and cirrhosis);
- Acute and chronic infections;
- Recent immunizations;
- Some tumor diseases.
High values of monoclonal gamma globulins may indicate, instead, the presence of:
- Some neoplasms and chronic inflammations;
- Multiple myeloma;
- lymphoma;
- Waldenström's macroglobulinemia.
An excess of gamma globulin is called hypergammaglobulinemia, while a deficiency is known as hypogammaglobulinemia .
Low Values - Causes
The reduction of gamma globulins may indicate the presence of various diseases of the immune system on a genetic basis, including agammaglobulinemia, or a secondary immunodeficiency (eg AIDS, renal failure, acute sepsis and Cushing's syndrome).
The decrease in gamma globulin values may also depend on the use of cytostatic drugs, immunosuppressants and steroids.
How it is performed
Serum immunofixation
Serum immunofixation involves a simple blood draw from the vein of an arm. The serum is obtained by centrifugation of the biological sample; this allows, in fact, to separate the fraction containing the cells (corpuscular part) from the liquid one of the blood (plasma). The blood serum is free of coagulation factors (fibrinogen, factor VIII, factor V and prothrombin).
Urinary Immunofixation
To perform urinary immunofixation it is necessary to collect a small amount of urine in a special sterile container. Based on the indications of the doctor and the laboratory, the sample can be collected without a precise timing ( random ) or within 24 hours .
Immunofixation of the Liquor
The analysis requires the taking of a small amount of cerebrospinal fluid, through the insertion of a needle, inside the spinal cavity ( lumbar puncture ).
Preparation
Immunofixation: how do you prepare for the exam?
- Prior to blood sampling, some laboratories may require you to observe a fast of at least 10-12 hours. During this time a small quantity of water is allowed.
- Immunofixation can be performed on urine collected at a certain time of the day (random) or during the 24 hours, so it is necessary to obtain the appropriate sterile containers beforehand.
- As for the immunofixation of the liquor, instead, no particular preparation is necessary.
What can alter the outcome of the exam?
Some medicines can influence the outcome of immunofixation, so it is advisable to report any ongoing drug therapies to the doctor.
In the six months before the examination, the administration of vaccines (immunization) and the use of drugs, such as phenytoin, procainamide, oral contraceptives, methadone and therapeutic gamma globulin, may increase the concentration of immunoglobulins. Other medicines that can alter the test result are corticosteroids, neomycin, acetylsalicylic acid and chlorpromazine.
Interpretation of Results
The result of the immunofixation is evaluated in combination with the outcome of other analyzes. The interpretation of the finding is the responsibility of the general practitioner or the specialist of reference who can identify and compare the alterations affecting a specific gamma globulin (for example, the band that coincides with the immunoglobulins G) to certain diseases. This evaluation confirms or supports the diagnostic hypothesis: in general, immunofixation can reveal a normal gamma globulin profile or identify a monoclonal protein or a polyclonal model .
Once the disease is defined, immunofixation can be performed for:
- Monitor the disease over time, then follow its course (follow-up);
- Check the effectiveness of the therapeutic protocol .
Pros and Cons of Immunofixation
Compared to protein electrophoresis, immunofixation:
- It is faster : the results of the immunization are usually obtained within three hours;
- It is more sensitive (albeit slightly): immunofixation can reveal an immunoglobulin excluded from protein electrophoresis, especially at low concentrations (less than 1 gram / liter).
Furthermore, the immunofixation:
- It can be partially automated ;
- It is easily readable and interpretable .
However, immunofixation is more expensive than protein electrophoresis and allows only gamma globulin analysis .
Normal result
In immunofixation, a normal path includes:
- A band that coincides with the darker immunoglobulin G ( IgG );
- A band corresponding to the lightest immunoglobulin A ( IgA );
- The absence of the band for immunoglobulin M ( IgM );
- The kappa band is denser than that of lambda (light chain), with a ratio of 2: 1.
In a normal result, the bands are large and there is a gradual and uniform reduction in the color density towards the edges of the immunoelectrophoresis strip.
Polyclonal gamma globulins
In some cases, all the immunofixation bands are evenly obscured to the same extent. This model represents the presence of polyclonal immunoglobulins . Also in this case, the bands are wide and the transition to the edges of the immunoelectrophoretic strip is gradual. The IgM band - normally absent - is wide.
Monoclonal gamma globins
When a narrow band with sharp edges can be identified in the route of the immunofixation, it means that the search for monoclonal components is positive .
The identification of a monoclonal immunoglobulin is useful in the diagnosis of the following conditions:
- Monoclonal gammopathy of undetermined significance (MGUS);
- Multiple myeloma;
- Waldenstrom's macroglobulinemia;
- Amyloidosis.
Furthermore, immunofixation can be used to monitor therapy in plasma cell dyscrasias (ie multiple myeloma and Waldenstrom's macroglobulinemia). If the level of monoclonal protein decreases or is not detectable after chemotherapy, it may indicate a response to treatment . In contrast, a monoclonal protein persisting despite treatment is a sign of refractory disease .
Please note . A negative immunofixation result does not always exclude a plasma cell dyscrasia. For example, a non-secretory multiple myeloma or oligosecretory can give negative results both in urine and in serum. Therefore, if the diagnostic suspicion for gammopathy is still high, the determination of the ratio of the Kappa / Lambda Libere Light Chains ( K / L ratio ) is indicated.
Immunofixation and diagnosis of Multiple Sclerosis
The immunofixation of cerebrospinal fluid (liquor) is prescribed when the doctor suspects the presence of multiple sclerosis . In this case, the path that is determined is characteristic, since it highlights the presence of the so-called oligoclonal bands . The result is reported in the laboratory report as positive . It should be considered that the bands are not normally observed in serum immunofixation, as they correlate to the production of intrathecal gamma globulins .
However, it should be noted that this finding does not necessarily confirm the diagnosis, since other conditions can occur with oligoclonal bands in the cerebrospinal fluid.
In other words, the identification of oligoclonal bands with liquor immunofixation is very useful to support the diagnosis of multiple sclerosis, but must be confirmed by other investigations . Other conditions that can provide this result are inflammations of the central nervous system, including: HIV-related encephalitis, neurosyphilis, neurosarcoidosis, Lyme meningoencephalitis, CNS malignancies, optic neuromyelitis and transverse myelitis.
