Analysis of Ig heavy chain/light chain pairs (Hevylite™)

From Wikilite

32.1. Introduction: limitations of immunoglobulin measurements

Figure 32.1 Target epitopes (in black) for Hevylite antibodies are on the constant regions (CH1 and CL) between the heavy and light chains of immunoglobulin molecules.

Figure 32.2 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 SPE with scanning densitometry and/or IFE together with sFLC immunoassays. While SPE is a simple, cheap test, it is not particularly sensitive so that 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 it is a non-quantitative assay. Nephelometry is also used for immunoglobulin measurements and is analytically accurate to low concentrations. However, patients’ samples also contain non-tumour polyclonal immunoglobulins of both κ and λ types that are included in the analysis so that results are clinically inaccurate at normal serum concentrations. Furthermore, assessments of monoclonal IgG are unreliable because of variable catabolism as FcRn recycling receptors become saturated or reduced by chemotherapy (Chapter 10).

In contrast, one of the great diagnostic benefits of serum FLC analysis is the κ/λ ratio. This is because:- 1), it provides a quantitative assessment of FLC clonality, 2), it has typical high immunoassay sensitivity, 3), clinical ranges are wide due to immunosuppression of the non-tumour FLCs and 4), there is automatic compensation for variable renal metabolism and changes in blood volume (Chapter 10).

Immunoglobulin heavy chain/light chain immunoassays - “Hevylite” (HLC), have similar analytical advantages. This chapter provides early data on these new immunoglobulin reagents.

32.2. Concept: immunoglobulin heavy chain/light chain assays

Intact immunoglobulin molecules contain unique junctional epitopes between the heavy chain (CH1) and light chain (CL) constant regions (Figure 32.1). These are the target of Hevylite (HLC) antibodies. Hence, they can separately identify the different light chain types of each immunoglobulin class, i.e. IgGκ, IgGλ, IgAκ, IgAλ, IgMκ and IgMλ (Figure 32.2). These molecules are then measured in pairs, e.g., IgGκ/IgGλ, to produce ratios of monoclonal immunoglobulin/background polyclonal immunoglobulin concentrations, in the same manner as sFLC κ/λ ratios.

32.3. Antibody specificity

Inevitably, one of the most demanding aspects of HLC assay production is ensuring good specificity. As for FLC immunoassays, the reagents are polyclonal antibodies produced in sheep. Immunisation and adsorption techniques are designed to ensure no cross-reactivity. For example, IgGκ reagents do not react with free κ or IgGλ, or any other immunoglobulins.

There are 4 HLC epitope regions per 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 per molecule, immune complexes readily form to produce good homogeneous immunoassays that are suitable for nephelometry and turbidimetry. Latex enhancement is not necessary for IgG, A and M HLC assays because of their high concentrations, but may be useful for IgDκ/λ assays and CSF samples.

32.4. Normal ranges of Hevylite assays

Intact immunoglobulin concentrations are normally controlled within narrow limits, as are their HLC κ and λ subsets. The results from testing blood donor panels are shown in Table 32.1 and Figures 32.3 and 32.4. Pearson rank correlations for summation of IgGκ+λ Hevylite samples to total IgG was ~ 0.9 (p<0.01), IgAκ+λ to total IgA was 0.9 (p<0.01) and IgMκ+λ to total IgM was 0.9 (p<0.001).

Ranges that include older individuals, hospital populations and patients with chronic infections and autoimmune diseases are required. Initial studies have indicated that HLC κ/λ ratios in diseases with raised polyclonal immunoglobulins are maintained within the narrow normal limits observed for blood donors (as with sFLC κ/λ ratios).

32.5. Clinical sensitivity of Hevylite assays for monoclonal gammopathies

Figure 32.3 IgG Hevylite tests on 103 blood donor sera (95% range) and 18 IgG MM patients at disease presentation. Immunosuppression of the non-tumour HLC from hypercatabolism is apparent in high concentration samples. One blood donor sample (BTS) had an IgGλ MGUS confirmed by IFE.

Figure 32.4 IgA Hevylite tests on 191 blood donor sera (95% range) and 33 IgA MM patients at disease presentation. All MM samples were abnormal by Hevylite while 6 of the samples could not be quantitated by SPE because of overlying protein bands.

Figure 32.5 SPE gels from 16 patients with IgA monoclonal gammopathies in AL amyloidosis. (Nos 1&16 normal human serum controls, Nos 3 & 12 fixed as IgG Amyloid). Many are difficult to quantify yet can be measured by Hevylite (Figure 32.6).

Figure 32.6 IgA Hevylite tests on 118 unselected blood donor sera (in blue showing 95% range) and 16 AL amyloidosis samples with IgA monoclonal proteins (red). (14/16 had abnormal Hevylite ratios and all could be quantified).

Figure 32.7 Serum IgA Hevylite concentrations in patients with AL amyloidosis that had no detectable monoclonal immunoglobulins by IFE.

Figure 32.8 . Serum IgG Hevylite concentrations in patients with AL amyloidosis that had no detectable monoclonal immunoglobulins by IFE.

Serum samples from patients with MM (courtesy of M Drayson and the UK, MRC MM Trials Centre) and AL amyloidosis (courtesy of P Hawkins and the UK, Amyloidosis Centre) were tested using Hevylite assays. The results are shown as HLC κ/λ log plots (as for sFLC assays) alongside blood donor samples to show normal ranges (Figures 32.3, 32.4 and 32.6 - 32.8).

As with sFLC assays, many MM patients had abnormal concentrations of individual HLC κ or λ molecules in addition to abnormal HLC κ/λ ratios (Table 32.2). Furthermore, ratios were more sensitive for monoclonality because of immunosuppression of the non-tumour HLC immunoglobulin molecules. For IgG, compared with IgA, there was greater suppression of the non-tumour IgG when the monoclonal protein was high because of increased catabolism from FcRn saturation (Chapter 10 and Figures 32.3-32.4). Six of the IgA samples could not be quantified by SPE because of their low concentration and position on other serum proteins. Four of these samples had abnormal HLC concentrations and all were abnormal by HLC ratios.

Table 32.1. Normal concentration ranges of HLC immunoglobulins and HLC ratios in blood donors. (IgG 103 samples; IgA 191 samples IgM 118 samples).

Serum samples were also tested from patients with AL amyloidosis to assess analytical sensitivity compared with IFE. Initially, 16 IgA IFE positive samples were tested of which 6 were quantifiable by SPE and in the remaining 10, IgA was hidden/non-quantifiable (Figure 32.5). The HLC results showed that 14/16 samples could be measured by abnormal IgAκ/λ ratios and 2/16 had normal ratios and results could be quantified (Figure 32.6). Subsequently, 50 IFE negative samples were assessed of which 13 had abnormal IgA HLC ratios (Figure 32.7), 10 had abnormal IgG HLC ratios (Figure 32.8) and one an abnormal IgM HLC ratio – 24/50 in total. Of these, 37/50 had abnormal sFLC ratios.

These results show that HLC assays can quantify monoclonal immunoglobulins as abnormal ratios when they are unmeasureable or undetectable by SPE and IFE. This high sensitivity should help clarify disease status in many patients with subtle monoclonal gammopathies.

Table 32.2. Hevylite concentrations and ratios in 18 IgG and 33 IgA MM patients at disease presentation. (Tum: tumour. N-Tum: non-tumour. Also see Figures 32.3 and 32.4).

32.6. Hevylite assays for monitoring monoclonal gammopathies

Figure 32.9 Monitoring a patient with IgGλ MM using Hevylite ratios (IgGλ/IgGκ), SPE, scanning densitometry (SD) of IgG and nephelometry (N). NR:Normal Range.

Figure 32.10 Monitoring a patient with IgGλ MM using IgGλ Hevylite and IgG Hevylite ratios. Comparison with SPE is shown and see Figure 32.9 for other details. NR:Normal Range.

Figure 32.11 Monitoring a patient with IgGλ MM using IgGλ Hevylite and IgG Hevylite ratios. Comparison with SPE is shown and see Figure 32.9 for other details. NR:Normal Range.

Figure 32.12 Hevylite assays for (A) IgA and (B) IgM in non Hodgkin lymphoma. (Patient identification for A + B are the same).

Figure 32.13 Immunohistochemistry of a lymph node stained with peroxidase labelled IgGλ Hevylite reagent.

Figure 32.14 Immunohistochemistry of a lymph node stained with peroxidase-labelled IgM Kappa Hevylite reagent.

There are several reasons why HLC assays might be useful for monitoring patients with monoclonal gammopathies.

In order to determine these features of HLC assays we assessed 9 patients with IgG MM and 5 with IgA MM who were undergoing treatment in the UK, MRC VII trial. For the IgG patients (4 IgGκ and 5 IgGλ), 25 samples were available. In 4/4 who did not achieve complete response, the ratio remained abnormal throughout. In 3/5 patients achieving complete remission, IgGκ/λ ratios were abnormal at relapse earlier than IFE measurements.

For the IgA patients (4 IgAκ and 1 IgAλ), all of the 26 samples that were positive by IFE had abnormal IgAκ/λ ratios. In 2/5 patients, abnormal ratios indicated residual disease when IFE was negative. For one patient the IgA monoclonal protein was obscured by another protein in the SPE gel but could be monitored by HLC ratios. In a second patient, abnormal HLC ratios indicated a slow relapse more than a year before IFE became positive.

One IgG MM patient was studied in detail during 2 remissions and relapses and illustrates the main features of HLC assays (Figures 32.9 - 32.11). HLC ratios had a greater range of values than IgG quantitation by scanning densitometry or nephelometry and were more sensitive during remissions and indicated relapse earlier. Of particular interest were the HLC results during the second course of chemotherapy. IgG measurements indicated a tumour response but HLC κ/λ ratios did not change indicating no selective tumour cell killing. This was in contrast to the first course of chemotherapy that had huge selective tumour cell killing. Indeed, the patient terminally relapsed after the second chemotherapy. This suggested that HLC ratio provided the correct interpretation of the lack of response to the chemotherapy. The discrepance 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 ratios would be unaffected.

Measurement of HLC IgGλ (Figure 32.10) did not provide more information than total IgG. IgGκ quantitation showed the functional activity of the bone marrow plasma cells, the response to chemotherapy and the subsequent tumour relapse (Figure 32.11). However, it was the HLC IgG κ/λ ratio that provided the most interesting results.

32.7. Hevylite assays in non Hodgkin Lymphoma (NHL)

Quantitative abnormalities of sFLC and/or sHLC were identified in 45/93 (48%) patients with NHL compared with 17/93 (18%) by SPE alone. The frequency of abnormalities varied markedly between disease ie. 8/8 with Waldentröm's Macroglobulinaemia, 13/20 (65%) with diffuse B cell lymphomas, 17/27 (63%) with marginal zone lymphomas and 5/17 (29%) with follicular lymphomas.

The most frequent abnormalities were in sFLC ratios (22/93) and IgDκ/IgDλ ratios (18/93). For both, the abnormalities predominantly indicated an excess production of κ clones (20/22 for κFLC and 17/18 for IgDκ) while only 1 patient had both sFLC and IgD abnormalities. HLC abnormalities were usually present (37/46 positive patients) in only one immunoglobulin class, as would be expected in monoclonal diseases. Abnormally low concentrations of IgM (with normal IgMκ/IgMλ ratios) were found in 28% (26/93) of the sera. This degree of immunoparesis was not seen with the other immunoglobulins or sFLCs. Further studies will determine whether sFLC ratios and sHLC ratios have utility for prognosis or disease monitoring in NHL.

32.8. Hevylite assays for immunohistochemistry

HLC antibodies may also find use in immunohistochemistry (Figures 32.13 and 32.14) for assessing immunoglobulin light chain subsets. Commonly, lymph node, bone marrow and other tissue biopsies contain B cells and plasma cells that need testing for clonality. Measurements of total light chain ratios are not always reliable and in any case, are not specific for each of the IgG, A, M, or D producing cells. These subsets can be evaluated using double staining techniques but such methods are cumbersome. HLC reagents labelled with peroxidase or fluorochromes could be used to provide sensitive analysis of clonality in a variety of immune derived tumours.

32.9 Publications on FcRn receptors and related issues

32.10 Publications on Hevylite assays