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Chapter 11

Summary:

  • Hevylite® assays are validated for the quantification of immunoglobulins in human serum.
  • Hevylite results should be interpreted alongside a patient’s clinical details and other laboratory tests including serum electrophoresis and total immunoglobulin measurements.
  • Summated Hevylite values for a particular immunoglobulin class (Ig’κ + Ig’λ) should equate to the corresponding total immunoglobulin value.

IgG, IgA and IgM Hevylite (HLC) assays are polyclonal antisera-based immunoassays, and can be performed on automated laboratory turbidimeters/nephelometers. For each immunoglobulin class, the Ig’κ and Ig’λ (e.g. IgGκ and IgGλ) are measured separately, then results can be expressed as an Ig’κ/Ig’λ HLC ratio (e.g. IgGκ/IgGλ), or as the difference between the involved (iHLC) and uninvolved (uHLC) HLC concentrations (dHLC), in a similar manner to Freelite® serum free light chain (sFLC) assays.

This chapter discusses both the practical aspects of implementing Hevylite assays (including choice of instrument, sample types and biological variation) and interpretation of results.

11.1.1. Choice of instrument

Hevylite immunoassays are available for the Binding Site SPAPLUS®, Optilite® and Siemens BN™II instruments (Chapter 38). Factors that may influence a laboratory’s choice of instrument include assay time, workload, complete testing menu offered and existing platforms etc.

It is recommended that all laboratories performing Hevylite assays participate in external quality assurance (EQA) schemes. These are discussed in Chapter 39.

11.1.2. Reporting units

It is important to ensure that IgG, IgA and IgM HLC concentrations are reported in consistent units. In the UK the preferred reporting units are g/L. Within the USA, results may be either in g/L or g/dL.

11.1.3. Choice of sample

IgG, IgA and IgM Hevylite assays are validated for the quantification of immunoglobulins in human serum. Hevylite assays are not validated for use with any other sample types.

Samples that are grossly haemolysed, lipaemic, or with highly elevated billirubin may impact upon the performance of the assay. The maximum concentration of interfering substances that do not cause significant assay interference is stated in the product insert. An example of interference testing for IgGκ and IgGλ HLC assays is shown for the Binding Site SPAPLUS in Table 11.1.

Interfering substance Concentrations Deviation from target value
IgGκ HLC IgGλ HLC
Haemoglobin 4.56 g/L -1.3% 2.2%
Chyle 1540 formazin turbidity units 1.6% 3.8%
Bilirubin 200 mg/L -2.0% -1.6%

Table 11.1. Interference testing for IgGκ and IgGλ HLC assays on the Binding Site SPAPLUS. Interference was tested using a control serum containing 0.71 g/L of IgGκ or 0.74 g/L of IgGλ, tested at the minimum sample dilution (1/1).

11.1.4. Sample and reagent stability

An in-house study was conducted to assess the stability of HLC measurements in unpreserved serum samples (n=10) stored first at 22 °C for 2 days, then at 2 - 8 °C for 28 days (in order to mimic shipping followed by refrigerated storage) [236]. For each sample, IgGκ, IgGλ, IgAκ, IgAλ, IgMκ and IgMλ HLCs were measured in triplicate on the day of collection and at regular intervals during storage. There was no significant change observed in HLC values over 28 days (Table 11.2). However, the HLC product inserts recommend that samples are stored for a maximum of 21 days at 2 to 8 °C prior to analysis, to match the guidance in the Freelite product inserts (as sFLCs may be measured in the same sample; Section 7.1.4).

Specificty Mean concentration
at collection (g/L)
Change in concentration at day 28
(% difference from collection value)
Mean Range of values
IgAκ 1.316 4.3 +1 to +5
IgAλ 1.212 1.6 0 to +5
IgGκ 8.400 2.6 0 to +6
IgGλ 3.643 -2.8 -7 to 0
IgMκ 0.503 -1 0 to -11
IgMλ 0.378 -7 -2 to -12

Table 11.2. HLC concentrations in unpreserved sera measured after storage at 2 - 8 °C.

A second in-house study assessed the stability of HLCs in unpreserved serum samples (n=10) stored at 22 °C for 2 days, and then at -20 °C for 28 days [236]. For each sample, IgGκ, IgGλ, IgAκ, IgAλ, IgMκ and IgMλ HLC were measured in triplicate on the day of collection and after 0 and 28 days storage at -20 °C. There was no significant change in HLC values over the time course (Table 11.3, similar to the reported stability of FLCs; Section 7.1.4). Therefore, for long term storage of serum samples prior to HLC analysis, it is recommended that samples are stored frozen at ≤-20 °C.

Specificity Mean concentration
at collection (g/L)
Change in concentration at day 28
(% difference from collection value)
Mean
IgAκ 1.31 4.0%
IgAλ 1.21 0.8%
IgGκ 8.40 0.4%
IgGλ 3.64 -2.7%
IgMκ 0.50 -5%
IgMλ 0.38 -3%

Table 11.3. Change in HLC values measured after storage of unpreserved sera at -20 °C.

Katzmann et al. [38] studied the stability of all six HLC specificities in 10 serum samples. Sample aliquots were assayed after storage for 7 days at room temperature, storage for 7 days at 4 °C or after 3 freeze/thaw cycles. The observed percentage change in HLC values was minimal (Table 11.4), and the authors concluded that the stability was acceptable for routine use in a clinical laboratory.

Storage Mean change in HLC concentration from day 0 Maximum decrease in HLC concentration from day 0
7 days at room temperature -2.5% 4.7%
7 days at 4 °C -2.2% 4.9%
3 freeze/thaw cycles -3.4% 6.7%

Table 11.4. Change in HLC values measured after storage of sera under different conditions [38].

Stability of the Hevylite reagents is also an important issue. “Open-vial stability” refers to the shelf-life of the reagents after their first use. Open-vial stability of 3 months has been validated for all HLC specificities.

11.1.5. Changing batch of reagent or instrument

During Hevylite assay manufacture batch-to-batch consistency is maintained (Section 9.7), so that changing from one batch to the next should not present the laboratory with any issues. However, it is still recommended that identical internal quality control samples are measured with both the new batch and the existing batch to confirm consistency. Similar checks should be employed when changing instruments.

References