Freelite

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Freelite is the brand name of an immunoassay for the determination of free light chains , manufactured and sold by the English diagnostics company The Binding Site , based in Birmingham . The determination of free light chains plays an important role in the diagnosis and monitoring of monoclonal gammopathies such as monoclonal gammopathy of unclear significance (MGUS), AL amyloidosis or multiple myeloma . In addition, the test is also of importance in diseases with a polyclonal increase in free light chains, such as Sjogren's syndrome and rheumatoid arthritis, as well as in multiple sclerosis .

Biological background

Light chains are small molecules ( proteins ) that form intact immunoglobulins ( antibodies ) in covalent connection with the so-called heavy chains (two heavy chains and two light chains each). A distinction is made here between the light chain type kappa (κ) and lambda (λ), with only one type of these two always being present in an intact immunoglobulin. In healthy people, in addition to intact immunoglobulins, small amounts of free (i.e. not bound to heavy chains) light chains are formed by the so-called plasma cells (approx. 40% excess compared to the light chains bound in intact immunoglobulins; a total of approx. 0.5 g - 1 g per day; about twice as much κ as λ; production ratio 2 κ: 1 λ) and released into the blood . The free light chain κ is typically present in the serum as a monomeric molecule with a molecular weight of approx. 25  kDa . The free light chain λ, however, forms i. d. Usually a covalently linked dimeric molecule with a molecular weight of approx. 50 kDa. Due to their small size or low molecular weight, the free light chains can leave the blood via the filter of the kidney ( glomerulus ), reach the so-called tubule , where they are almost completely reabsorbed and broken down in the tubular cells. In people with normal kidney function and normal production of free light chains, the ratio of the two molecules in the blood or serum of 2 κ: 1 is due to the different filtration speed of the kidneys (κ is filtered about twice as fast as λ due to its smaller size) λ (κ / λ ratio; averages 0.63; the normal range in serum is between 0.26 and 1.65). A pathological change in the production of free light chains by monoclonal plasma cells, as occurs in monoclonal gammopathies, can lead to an increase in the concentration of typically one type of free light chain in the serum (like intact immunoglobulins, such abnormal monoclonal free light chains are referred to as monoclonal proteins ). This increase is also reflected in an increased (with κ overproduction) or reduced (with λ overproduction) κ / λ ratio in the serum. It should be noted that, due to the kidney's reabsorbability capacity of 10-30 g of protein per day, significant amounts of free light chains only enter the urine when there is severe overproduction or loss of the kidney's reabsorbability.

Overproduction of monoclonal free light chains can be found in almost all monoclonal gammopathies (e.g. in about 90% of all patients with multiple myeloma). This fact forms the basis for the clinical significance of the determination of free light chains.

Chronic inflammatory processes, such as in multiple sclerosis, are usually characterized by intrathecal antibody production in the central nervous system . This can also lead to an increased concentration of the free light chain κ in the liquor . A determination of free light chains in such samples is also possible.

Measuring principle

The Freelite test is based on the principle of an antigen- antibody reaction in solution . In this case, the antigen is a specific epitope of the free light chain that is otherwise inaccessible in intact immunoglobulins. This makes this measurement method highly specific for this particular parameter. The antibody, which reacts with an epitope of the free light chains by binding to it, is part of the test (the so-called detection antibodies; monospecific polyclonal sheep antibodies). The detection antibodies form high-molecular, insoluble complexes with the epitopes of the free light chains. In order to enhance the sensitivity of measurement, the detection antibody of the Freelite tests are polystyrene - latex particles coupled to a suitable size.

The actual measurement is based on a turbidimetric or nephelometric measuring principle, depending on the device used. In turbidimetry, light is radiated through a cuvette in which the antigen-antibody reaction takes place. The amount of light that can pass through the solution is focused and measured with the help of an optical lens system. The concentration of free light chains is inversely proportional to the amount of incident light. With nephelometric measurements, however, the light scattering is determined by measuring the intensity of the light at a defined angle to the incident light. After the measurement, the concentration of the free light chains is automatically calculated and output using a calibration curve previously created and stored in the corresponding analysis device.

It should be noted that the two free light chains κ and λ must each be measured separately. Due to the complexity of the free light chains as analytes, sample-specific dilution phenomena are possible, which can be expressed in non-linear measurement results in different dilutions or an antigen excess. Modern analysis devices have z. T. the property to recognize such anomalies and to automatically carry out further measurements. In case of doubt, a more precise assessment of the measurement results by the user is recommended. However, these anomalies are rare and if a consistent and rigorous approach is taken (e.g. by following the device-specific dilution scheme) i. d. Usually easy to identify, which guarantees reliable measurement results. The lower detection limit of Freelite is approx. 1 mg / l and thus below the normal physiological range.

Clinical significance

The determination of free light chains is a central laboratory parameter in the clarification of monoclonal gammopathy, in particular multiple myeloma but also similar diseases and precursors such as monoclonal gammopathy of unclear significance, smoldering multiple myeloma (SMM) or AL amyloidosis. Even in the course of the disease, values ​​of the free light chains are used to assess the response to therapy, the prognosis of the further course of the disease and the early detection of an impending progression or relapse .

The concentration of the free light chains or the κ / λ ratio formed from them usually allows - except for so-called nonsecretory forms of the disease in which no free light chains or intact immunoglobulins are excreted by the cell - a conclusion about the presence of a monoclonal accumulation of malignant plasma cells Reference ranges: κ = 3.30 - 19.40 mg / l, λ = 5.71 - 26.30 mg / l, κ / λ ratio = 0.26 - 1.65). The κ / λ ratio therefore reflects the clonality of the disease. Using the example of a multiple myeloma with κ overproduction, this is expressed, for example, in an increase in the concentration of the free light chain κ (e.g. 350 mg / l), a free light chain λ within or below the reference range (e.g. 10 mg / l) and a resulting increased κ / λ ratio (based on the exemplary concentrations mentioned above here: 35).

Risk stratification MGUS and SMM

Studies have shown that various risk factors can promote the progression of the previous illnesses MGUS and SMM, can be consulted for a prognosis and therefore their determination is part of hematological guidelines. The risk stratification model for MGUS patients includes the following parameters:

  • abnormal κ / λ ratio
  • monoclonal protein in serum (quantification of a peak in serum protein electrophoresis) ≥ 15 g / l
  • Immunoglobulin (of type IgA , IgM , IgD or IgE )

For SMM patients, however, the following factors are considered to be the risk of progression (if at least two of these factors are met, we speak of high-risk SMM patients):

  • Abnormal ratio of the free light chain produced by the monoclonal plasma cell (involved free light chain, iFLC) to the free light chains produced by polyclonal plasma cells (non-involved free light chain, uFLC) - with a λ-SMM i.e. λ / κ ratio - of> 20
  • monoclonal protein in serum (quantification of a peak in serum protein electrophoresis)> 20 g / l
  • > 20% plasma cells in the bone marrow

Significance in multiple myeloma

The free light chains are central parameters when examining patients with suspected multiple myeloma or when monitoring the course of the disease or the success of a therapy:

  • Diagnosis: Part of the so-called SLiM criteria . a κ / λ ratio of ≥ 100 or ≤ 0.01 (with simultaneous iFLC concentration of ≥ 100 mg / l) together with at least 10% monoclonal plasma cells in the bone marrow or a proven plasmacytoma ( confirmed by biopsy ) defines a myeloma Event.
  • Response: In the case of an exclusive production of free light chains, the response of patients to a specific therapy is assessed on the basis of the change in the concentration of free light chains. The following limit values ​​apply to the various remission criteria :
    • partial remission (PR): Reduction of the difference between iFLC and uFLC concentration (dFLC) ≥ 50%
    • very good partial remission (VGPR): reduction of dFLC ≥ 90%
    • Stringent complete remission (sCR): normalization of the κ / λ ratio
  • Progress: With a 25% increase in dFLC (with an absolute increase of> 100 mg / L)

Significance in AL amyloidosis

Patients with MGUS and an abnormal κ / λ ratio are at increased risk of developing AL amyloidosis. In addition, a dFLC of ≥ 180 mg / l is a prognostic marker and a risk factor for an aggressive disease. To control the success of therapy for AL amyloidosis, the free light chains serve as important parameters, as in multiple myeloma:

  • PR: With a reduction of the dFLC> 50%
  • VGPR: With a dFLC <40mg / l
  • CR: If the κ / λ ratio is normalized

Current research focus

The determination of the free light chains is currently being investigated as an alternative to the test for oligoclonal bands in CSF and has shown promising results in previous studies.

A large population study in Iceland is currently investigating the possible benefit of a general examination of the entire population over the age of 40 for the presence of MGUS and the subsequent regular follow-up examination, regardless of possible risk factors.

Differences from other tests

Comparison with electrophoretic methods

With Freelite, a much higher sensitivity can be achieved in direct comparison to electrophoretic analysis methods (such as serum protein electrophoresis and immunofixation electrophoresis). The lower detection limit for monoclonal proteins is a minimum of about 500 mg / l for serum protein electrophoresis and about 100 mg / l for immunofixation electrophoresis in serum. The lower limit with Freelite is 1 mg / l. Due to strong fluctuations in concentrations below 10 g / l, serum protein electrophoresis is only recommended for quantifications of monoclonal proteins from a concentration of more than 10 g / l (i.e. ≥ 10,000 mg / l).

Comparison of sample materials serum with urine

From a clinical point of view, a determination of the free light chains from serum samples is far more meaningful due to the described influence of metabolism . In addition, serum samples have proven to be more stable and their results more reproducible than urine samples. Urine samples can be obtained in different ways (e.g. 24-hour urine, spontaneous urine, first morning urine, etc.), which can have an influence on the measurement results. Urine samples are also more susceptible to contamination, which further reduces the reliability of the measurement results for this sample material. A turbidimetric or nephelometric determination of free light chains in urine samples is still possible, although the guidelines only recommend such a determination only in the serum if a monoclonal gammoapthy is suspected or during controls during therapy. Under certain circumstances, the concentration of free light chains in the urine can allow conclusions to be drawn about kidney function or the degree of overproduction of free light chains compared to the reabsorption capacity of the kidney. In principle, a low to very low concentration of free light chains in the urine is the rule in people with healthy kidney function, as long as the reabsorption capacity of the kidneys is not exceeded by overproduced free light chains.

Comparison to the determination of the total light chain concentration

The test to determine the total light chain differs fundamentally from Freelite. Not only the free light chains are quantified, but also the light chains bound in intact immunoglobulins. The proportion that is caused by polyclonal intact immunoglobulins has a major effect on the measurement results (the ratio of free light chains to bound light chains is approx. 1: 250, depending on the amount of tumor production). When determining total light chains , the results are in the gram range , whereas Freelite measures in the milligram range. This corresponds to the typical and physiologically normal as well as the (in the vast majority of samples) pathological concentration range. When determining the total light chains, a measuring range with far lower sensitivity results, in particular for patients with an exclusive production of free light chains, who under certain circumstances cannot be identified as patients with monoclonal gammopathy. The use of this test is therefore not recommended for the clarification of monoclonal gammopathy or in its follow-up.

Compared to other FLC assays

A unique selling point of Freelite compared to tests from other providers is the use of highly specific polyclonal detection antibodies in a turbidimetric or nephelometric test procedure. Previous studies have shown that the results of the various other Freelite tests (and from each other) differ, which means that there is no comparability. These differences can affect the clinical assessment of patients. The guidelines also refer exclusively to Freelite in their recommendations and not to tests by other manufacturers.

Web links

Individual evidence

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