JOURNAL PRESENTATION

Dr. Md. Raiq Raihan Chowdhury

Resident

Department of Haematology

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Serum Free Light Chain Assay: Clinical Utility and Limitations

Malini V Bhole , Ross Sadler and Karthik Ramasamy

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Annals of Clinical Biochemistry

2014, Vol. 51(5) 528–542

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Background

• Approximately 15–20% of patients with myeloma only have production of free monoclonal light chains (light-chain myelomas)

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• These may not be detected as a distinct monoclonal band on serum electrophoresis due to limited sensitivity of this method.

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• An additional 1–2% of myeloma patients are non-secretory, thus without a detectable band on routine electrophoresis.

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Background

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• Monoclonal light-chain diseases were diagnosed by measurement of free light chains excreted in urine also known as Bence-Jones proteins (BJP);

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• a more sensitive method than serum electrophoresis for the detection of light chains but limited by renal threshold and function

Background

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In 2001, the diagnosis of light-chain diseases was revolutionised by the introduction of a new serum assay.

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This assay was originally set up using polyclonal antisera obtained from sheep immunised with human BJP.

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Background

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This method provided an improved sensitivity for the determination of serum-free light-chain (sFLC) compared to previous methods.

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In addition to quantification, the method also allowed indirect determination of monoclonality by using the κ : λ ratio

Background

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Once purified, the antisera were digested with pepsin to produce F(ab)2 fragments, these fragments were then adsorbed onto polystyrene latex particles to enhance stability and sensitivity

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All plasma cells produce both intact immunoglobulin and relevant free light-chain molecules (up to 40% excess). Free light chains can either be or with -free light chains pre-dominantly existing as dimers. Epitopes used for specific detection of free light chains are to be found on the ‘hidden’ region on the constant and variable domain of the free light chain. These are hidden when the light chain is integrated into an intact immunoglobulin molecule but exposed when the light is produced as a free molecule.

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Free light chains purified from the urine of patients with light-chain myeloma are injected into sheep in addition to adjuvants by the manufacturer. The sheep is then bled weeks later to retrieve serum samples containing a polyclonal response to the free light chains.

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These serum samples are further affinity purified and digested to produce (Fab’)2 and bound to latex particles to enhance stability and sensitivity.

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Signal is generated when the latex particle-bound antibody fragments bind to free light chains from test serum samples leading to cross linking and immune complex formation. A laser beam aimed at these immune complexes leads to laser scatter. Measurement of this laser scatter (nephelometry) or of the unscattered laser light (turbidimetry) can be used to quantify free light-chain amount.

The detection of intact immunoglobulins and immunoglobulin-free light chains in serum and urine is of paramount importance in the diagnosis, prognosis and management of patients with plasma dyscrasias.

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In addition, the short physiological half life of sFLC in blood (κ : 2–4 h and λ : 3–6 h) potentially allows this to be used as a serum marker for real-time monitoring of response to treatment as well as disease progression.

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In addition, the short physiological half life of sFLC in blood (κ : 2–4 h and λ : 3–6 h) potentially allows this to be used as a serum marker for real-time monitoring of response to treatment as well as disease progression.

• Immunoglobulin free kappa (κ) and lambda (λ) light chain concentration in the serum is dependent on the rate of production from plasma cells and renal clearance.

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• This results in a defined serum concentration and ratio.

• In clonal plasma cell disorders, there is an excess production of only one of the light chain types, resulting in higher levels, with suppression of the uninvolved light chain, leading to an abnormal κ/λ ratio

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• The levels and ratio however may be affected by renal failure, as the light chains are cleared by the kidneys.

• Immunoglobulin free kappa (κ) and lambda (λ) light chain concentration in the serum is dependent on the rate of production from plasma cells and renal clearance.
• This results in a defined serum concentration and ratio.
• In clonal plasma cell disorders, there is an excess production of only one of the light chain types, resulting in higher levels, with suppression of the uninvolved light chain, leading to an abnormal κ/λ ratio
• The levels and ratio however may be affected by renal failure, as the light chains are cleared by the kidneys.

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• The normal serum free κ level is 3.3 to 19.4 mg/L

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• the normal free λ level is 5.7 to 26.3 mg/L.

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• The normal ratio of κ and λ FLC is 0.26 to 1.65.

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• The normal reference range in the FLC assay reflects a higher serum level of λ FLCs than would be expected, given the usual κ to λ ratio of 2 for intact immunoglobulins.

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• This occurs because the renal excretion of free κ (which exists usually in a monomeric state) is faster than free λ (which is usually in a dimeric state).

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• Patients with a κ to λ FLC ratio 1.65 are defined as having a monoclonal κ FLC.

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• If the FLC ratio is >1.65, κ is referred to as the “involved” FLC and λ the “uninvolved” FLC, and vice versa if the ratio is <0.26

• Polyclonal production of free light chains can increase markedly during infections or in inflammatory states resulting in an increase in the absolute values of both and chains with a relatively unchanged κ/λ ratio.

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• In contrast, haematological malignancies and plasma cell dyscrasias producing monoclonal or FLC with suppression of the alternate light chain display a skewed distribution with altered κ/λ ratio

• The serum FLC assay can be used in place of urine protein electrophoresis and immunofixation in the initial screening algorithm for M-proteins.

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• However, if a monoclonal plasma cell disorder is identified on screening, a 24-hour urine collection followed by electrophoresis and immunofixation should always be done to aid in the assessment of disease progression and response to therapy over time

• An interesting relationship exists between sFLC concentrations and renal function

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• excessive light-chain production and subsequent filtration through renal glomeruli can damage renal tubules with resultant tubular dysfunction

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• renal failure due to any cause can independently raise sFLC concentrations secondary to reduced filtration rate.

• Patients with renal failure were noted to have elevated and concentrations with ratios falling just outside the established ranges.

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• a new ‘normal’ sFLC ratio range for patients with renal failure (0.37– 3.1).

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• This new renal range has been subsequently validated and found to be useful in diagnosing monoclonal gammopathies in patients with pre-existing renal disease.

sFLC in Myeloma

The role of sFLC assay in the diagnosis of monoclonal gammopathies gained recognition in 2006

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the IMWG incorporated sFLC assays into the diagnostic criteria stating that in patients with no detectable M component, an abnormal sFLC ratio on the sFLC assay can substitute and satisfy this criterion.

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The Working Group have since published further updates and specific guidelines for sFLC analysis in MM and related disorders recommending its use in all newly diagnosed patients with plasma cell dyscrasias.

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The recommendation also includes the use of sFLC assay in patients with solitary plasmacytoma, smoldering (asymptomatic) myeloma and MGUS as in these patients an abnormal result is associated with higher risk of progression to symptomatic myeloma

• The presence of an abnormal / ratio at baseline has been shown to be an independent risk factor for progression to symptomatic myeloma in MGUS, SMM and solitary plasmacytoma

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• risk stratification models have been described incorporating sFLC assay into multivariate parameters

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• Similarly, sFLC concentrations have been identified as an independent risk factor for poor prognosis in a retrospective analysis of patients with primary systemic amyloidosis (AL).

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The ability to measure sFLC presents a significant advance in the management of patients with AL amyloidosis.

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The combination of sFLC and SEP with IFE will identify up to 98% of patients with AL amyloidosis.

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The majority of patients with AL amyloidosis do not have an intact monoclonal protein in serum or urine.

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Therefore, serial use of sFLC during monitoring has been recommended by the IMWG.

Pitfalls and Limitation

Availability

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Cost

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Antigen excess

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Polymerisation of free light chain

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A common technical problem faced by users of the sFLC assay has been that of antigen excess and nonlinearity resulting in over or under-estimation of the monoclonal protein.

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Due to the prerequisite of immune complex formation to produce a signal, both nephelometric and turbidimetric assays are vulnerable to antigen excess, otherwise known as the hook effect

Conclusion

• Free Light Chain Assay is an important investigation in diagnosis and follow up of patients of myeloma and other plasma cell dyscrasia

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• prognostic value in case of MGUS and SMM cannot be overestimated

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• Careful integration in evaluation and management plan for these patients may help to achieve better outcome.

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Thank You