Hämatologie
   

Freelite® serum free light chain analysis

Freelite® is a major breakthrough for the detection and monitoring of Multiple Myeloma (MM) and other B-cell dyscrasias. Freelite assays were developed by Binding Site to measure free lambda and free kappa immunoglobulin light chains. Our expertise in the manufacture of antibodies has enabled us to provide a quantifiable, highly specific, automatable free light chain assay for serum.

Significant clinical evidence indicates the benefit of Freelite® serum free light chain assays in initial screening for monoclonal gammopathies, identifying AL amyloidosis and Nonsecretory MM patients missed by conventional electrophoretic methods, as a prognostic indicator for progression in myeloma, for risk stratification of MGUS patients and rapid evaluation of treatment efficacy.

Freelite® is a sensitive, specific marker of kappa and lambda free light chains (FLC) in serum and provides quantitative measurement of:

  • Free Kappa in serum
  • Free Lambda in serum
  • The serum free Kappa/free Lambda ratio

The Freelite® serum free light chain ratio is a strong indicator of monoclonality and is valuable for distinguishing monoclonal from polyclonal diseases.

 

 

In 2009 the International Myeloma Working Group published guidelines recommending the measurement of serum free light chain concentrations as an aid in the diagnosis, prognosis and monitoring of multiple myeloma patients.
The advice was given based upon results obtained in extensive clinical trials using the polyclonal Freelite® assays.1

Key recommendations are:-

  • Recommended for the use of the serum FLC assay in screening
  • Recommended for the use of the serum FLC assay in prognosis
  • Recommended for the use of the serum FLC assay in response assessment

A recent report by Katzmann et al2 recommends a simplification of the plasma cell dyscrasia diagnostic algorithm mentioned in current International Myeloma Working Group guidelines indicating that only serum protein electrophoresis (SPE) and Freelite® free light chain (FLC) tests may be needed in many cases.

"...because of the small incremental sensitivity provided by urine studies and serum IFE, the use of PEL [SPE] plus FLC provides a simple and efficient initial diagnostic screen for the high-tumour-burden monoclonal gammopathies such as MM [multiple myeloma], WM and SMM [smouldering multiple myeloma]. Urine studies and serum IFE can be ordered more selectively."

Ordering information for Freelite® assays

 

Hevylite® immunoglobulin heavy chain/light chain analysis

Hevylite®* (HLC), a new assay now available from Binding Site, promises to transform the way patients with B cell dyscrasia are managed, just as the Freelite® assay has done.

In summary Hevylite® assays offer:

  • Higher sensitivity than serum protein electrophoresis for quantifying monoclonal immunoglobulins
  • Numerical results for patients at a sensitivity as high as or better than immunofixation electrophoresis
  • Clinical value when monitoring patients with monoclonal gammopathies
  • HLC ratios which have a greater range of changes than monoclonal immunoglobulin measurements because the non-tumour immunoglobulin allows assessment of immunosuppression
  • HLC ratios which are not affected by changes in blood volume, haematocrit and variable metabolism (via FcRn receptors for IgG) that affect current assays for serum immunoglobulins
  • HLC ratios that provide information about the tumour selective killing rate versus non-tumour plasma cell kill rates. The assessment of selective tumour killing rates may help with decision making regarding effective chemotherapies


Ordering information for Hevylite® assays 

Intact immunoglobulin molecules contain unique junctional epitopes between the heavy chain and light chain constant regions. These are the target of the Hevylite® antibodies. They can identify separately the different light chain types of each immunoglobulin class ie IgGκ, IgGλ, IgAκ, IgAλ, IgMκ, IgMλ. The molecules are then measured in pairs e.g. IgGκ/IgGλ to produce ratios of involved monoclonal immunoglobulin/background uninvolved immunoglobulin concentrations, in the same manner as serum free light chain κ and λ ratios when using Freelite®.

Target epitopes (in black) for Hevylite® antibodies are on the constant regions between the heavy and light chains of immunoglobulin molecules

 

Six Hevylite® kits are available now

As with Freelite® assays, the reagents used to make the Hevylite® tests are polyclonal antibodies produced in sheep. Immunisation and adsorption techniques are designed to ensure no cross-reactivity. So, for example, IgGκ reagents do not react with free κ or IgGλ, or any other immunoglobulins.
 

There are 4 Hevylite® epitope regions per immunoglobulin molecule - one on each side of the heavy chain / light chain contact regions and the same on the other arm of the molecule. Because there are 4 epitope regions per molecule, immune complexes readily form with immunoglobulins in the patient's serum. This is needed to produce good homogeneous immunoassays that are suitable for nephelometric and turbidimetric automated laboratory analysers.

Heavy chain / light chain pairs of IgG, IgA and IgM molecules showing the target epitopes for Hevylite® immunoassays in black.

Typical analytical tests for monoclonal gammopathies are serum protein electrophoresis (SPE or SPEP) with scanning densitometry and/or immunofixation electrophoresis (IFE) together with serum free light chain immunoassays. While SPE is a simple, low cost test, it is not very sensitive and quantification of proteins at low concentrations (1-3g/L) is inaccurate.
This is particularly apparent for monoclonal IgA since its anodal electrophoretic migration positions it over other bands such as transferrin. Improved sensitivity is achieved with IFE but IFE is a non-quantitative assay. Nephelometry is also used for immunoglobulin measurements and is analytically accurate down to low concentrations. However, patients' samples also contain non-tumour polyclonal immunoglobulins of both κ and λ types that are included in the analysis. This means results are clinically inaccurate at normal serum concentrations.
Furthermore, assessments of monoclonal IgG are unreliable because of variable catabolism as FcRn recycling receptors that bind albumin and IgG become saturated or reduced by chemotherapy. 

Normal ranges for Hevylite® 

The ranges below were obtained by measuring the IgA kappa and IgA lambda concentrations of 138 normal (blood donor) sera and are intended for guidance purposes only. Wherever possible it is recommended that local ranges are generated. 

Normal Adult Serum
Mean
Median
95 Percentile Range

IgA kappa (g/L)

1.24

1.19

0.48 - 2.82

IgA lambda (g/L)

1.00

0.98

0.36 - 1.98

IgA kappa/IgA lambda ratio

1.28

1.27

0.80 - 2.04

Ranges that include older individuals, hospital patients and patients with chronic infections and autoimmune diseases are required. Initial studies have indicated that Hevylite® κ/λ ratios in diseases with raised polyclonal immunoglobulins are maintained within the narrow limits observed for blood donors in the same way as has been seen with Freelite® serum free light chain κ/λ ratios.

 

 

Monitor with Hevylite® to obtain rapid and reliable information

 

Case Study 1


 

Hevylite® ratios (IgAκ/IgAλ) (pink), SPE scanning densitometry of IgA (red) and total IgA by nephelometry (blue). NR:shows the upper limit of the Normal Range


At presentation this Multiple Myeloma patient had 66g/L IgA kappa. The patient initially responded well to treatment and received an autologous cell transplant. Both monoclonal IgA (quantified by densitometry) and heavy light chain (HLC) ratio clearly showed the reduction in paraprotein, although the ratio did not become normal until 194 days after the monoclonal protein became negative by IFE.
Residual disease was identified 596 days earlier using IgA kappa/IgA lambda ratios than detection by either SPE or IFE.

Case Study 2


 

Hevylite® ratios (IgGλ/IgGκ) (pink), SPE scanning densitometry of IgG (red) and total Ig by nephelometry (blue). NR: shows the upper limit of the Normal Range.


One IgG lambda Multiple Myeloma patient was studied in detail during 2 remissions and relapses and illustrates the main features of HLC assays. The HLC ratio had a greater range of values than IgG quantitation by scanning densitometry or nephelometry and was more sensitive during remissions and indicated relapse earlier.

During the first course of chemotherapy (Cyclophosphamide) there was significant selective tumour cell kill, shown by the reductions of IgG (by either scanning densitometry or nephelometry) and Hevylite® ratio. Following disease relapse the patient received a second course of therapy (C-VAMP), during which IgG measurements indicated a tumour response but the HLC κ/λ ratio remained unchanged, indicating no selective tumour cell kill was taking place. Unfortunately the patient subsequently died.

This indicates that the HLC ratio provided the correct interpretation of the lack of response to C-VAMP. The discrepancy between total IgG measurements and IgG HLC κ/λ ratios may, in part, be due to inhibition of the FcRn receptor by the chemotherapy. This would cause a fall in total IgG (because of faster turnover) but IgG HLC κ/λ ratio would be unaffected. 

  1. A Dispenzieri, R Kyle, G Merlini, JS Miguel, H Ludwig et al. International Myeloma Working Group guidelines for serum-free light chain analysis in multiple myeloma and related disorders Leukemia 2009; 23:215-224
     
  2. Katzmann et al.  Screening Panels for Detection of Monoclonal Gammopathies. Clinical Chemistry 2009;55:8:1517-1522
5th Edition Serum Free Light Chain Analysis

AR Bradwell
Serum Free Light Chain Analysis (Plus Hevylite®) 5th Edition 2008, Chapter 32, p275-285
The book is available to order US $75.
Please contact your local office or distributor