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11.5. Comparison of Hevylite results with other immunoglobulin tests

Chapter 11

11.5.1. Comparison of Hevylite and total immunoglobulin measurements

In general, the agreement between summated Hevylite (e.g. IgGκ + IgGλ) and total immunoglobulin assays (e.g. IgG) is good; indeed, comparison of summation data was used to validate the assays during development and summation checks are used as part of the manufacturing quality control (Sections 9.4.4 and 9.7). Eckold et al. [895] compared summated IgG, IgA and IgM HLC concentrations with total immunoglobulins and reported an excellent linear correlation (Figure 11.3). Theoretically, the more a tumour suppresses the production of polyclonal immunoglobulin (of the same immunoglobulin class), the closer will be the correspondence between iHLC concentrations with both the total immunoglobulin concentrations and the monoclonal immunoglobulin concentrations, as measured by densitometry of SPE gels.

As part of an evaluation of HLC assays for monitoring multiple myeloma (MM) patients, Katzmann et al. [38] compared total IgA quantification and the IgA iHLC for 149 IgA MM samples. Passing–Bablok linear regression gave a slope of 1.124 (95% CI 1.015–1.194), r = 0.969 Figure 11.4A), indicating a good agreement.

Boyle et al. [901] compared total IgM and summated IgM HLC values (IgMκ + IgMλ) for sera from 110 normal donors and 78 Waldenström’s macroglobulinaemia (WM) patients. There was a good correlation between the results (Linear regression R2 = 0.90, Passing-Bablok regression y=-0.32 + 1.17x, Figure 11.5A), although Bland-Altman analysis demonstrated that IgM values measured using IgM Hevylite assays were slightly higher than those measured using total IgM assays (bias = 1.8 g/L, Figure 11.5B). Similar findings were reported by Manier et al. [681] and Sarto et al. [936].

11.5.2. Comparison of Hevylite and immmunoglobulin measurements by SPE

In an in-house study, the IgG iHLC concentration was compared with the monoclonal concentration, determined by SPE scanning densitometry, for 160 IgG MM patients. In the majority of patients, there was good agreement in the concentration determined by the two methods but some samples did show variance (Figure 11.6). Lopez-Anglada et al. [918] reported similar results. Possible explanations for the variance include inaccurate SPE measurements, for example if the monoclonal protein co-migrated with other serum proteins (e.g. transferrin), or dye saturation underestimated the concentration of IgG monoclonal proteins. This is discussed in more detail in Section 17.4. In addition, when a high concentration of polyclonal immunoglobulins is present, Hevylite measurements may be less sensitive than serum electrophoretic techniques for detecting a monoclonal protein (e.g. MGUS; Chapter 13).

Katzmann et al. [38] compared the iHLC concentration with the monoclonal protein concentration determined by SPE for 114 IgG and 41 IgA MM serum samples. Each sample had a band present on SPE (M-spike) and a corresponding abnormal HLC ratio. There was a linear correlation between the iHLC concentrations and the M-spike for both IgG and IgA patients (Figure 11.7 and 11.4B). Other studies have also reported a good correlation of IgA iHLC concentration with the SPE M-spike [918][897][880]. Similar findings were reported by Boyle et al. [917], who compared IgG or IgA M-Spike concentrations with the corresponding dHLC value. Katzmann et al. [38] noted that the correlation of iHLC with total IgA (Figure 11.4A; r=0.97) was better than the correlation of iHLC with the SPE M-spike (Figure 11.4B; r=0.87), and suggested that this was most likely to be due to the difficulty of quantitating β-migrating monoclonal proteins that co-migrate with other serum proteins (Section 17.4). Katzmann et al. [38] study also compared the relative changes in the iHLC and serum M-spike concentration for 13 IgG patients for whom diagnostic and four follow-up samples were available. The relative changes of the mean iHLC and mean M-spike were not statistically different (p>0.75; Figure 11.8).

Boyle et al. [901] compared monoclonal IgM concentrations determined using the involved IgM HLC concentration or SPE densitometry for 66 WM patients with a quantifiable M-spike. The agreement between the two assays was poor, and a higher value was reported by the IgM HLC assays in most cases (Linear regression R2 = 0.49; Passing-Bablok regression y=-4.4 + 1.98x; Bland-Altman systematic bias = 10.1 g/L, Figure 11.9). Similar findings were reported by Manier et al. [681] and Sarto et al. [936]. The lack of agreement between electrophoretic techniques and automated IgM immunoassays has been reported previously [369][902], and was also demonstrated by Boyle et al. [901]. One explanation for these findings is the polymeric nature of IgM molecules, which causes an overread by nephelometric assays [369]. It should be noted that whilst the numerical agreement between IgM HLC and SPE densitometry is poor, there is an excellent correlation between the responses assigned using the two methods (Section 32.4.2).

11.5.3. Comparison of Hevylite and immunofixation electrophoresis

During the follow-up of patients with monoclonal gammopathies, there are occasionally discrepancies between Hevylite and immunofixation results. Two scenarios are discussed below.

1) IFE positive, normal HLC ratio: in IgG patients, recycling of IgG by the FcRn receptor may result in the persistence of small IgG monoclonal proteins in the serum, causing IFE to remain positive long after the tumour has been eradicated and HLC pair suppression has ended (Section 18.4.5). In addition, in a minority of cases, the diagnostic sensitivity of IFE may be superior to that of the HLC ratio. For example, if a small IgG monoclonal protein is present with a large amount of polyclonal IgG [40]. Finally, on rare occasions, Hevylite antigen excess may cause a sample value to be falsely low. If antigen excess is suspected, an additional sample dilution should be performed (Section 11.4).

2) IFE negative, abnormal HLC ratio: in some patients, the HLC ratio may be more sensitive for detecting a monoclonal immunoglobulin than IFE. This is more common for IgA and IgM where there is less polyclonal production so smaller amounts of monoclonal immunoglobulin can produce an abnormal HLC ratio. An abnormal HLC ratio may indicate residual disease in IIMM patients whose electrophoresis results have normalised following therapy (Section 18.4.3).

Questions

  1. When monitoring HLC values in a patient, what percentage change is necessary to be statistically significant (p<0.05)?

Answers

  1. The relative change value (RCV) for HLC measurements has been calculated to be between 30 and 40% (Section 11.2.6).
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