- 19 KRAJ str. 257

Acta Haematologica Polonica 2011, 42, Nr 2, str. 257–271
PRACA ORYGINALNA – Original Article
MARIA KRAJ, BARBARA KRUK, RYSZARD POGŁÓD, KRZYSZTOF WARZOCHA
Evaluation of IgG, IgA and IgM monoclonal and biclonal gammopathies
by nephelometric measurement of individual immunoglobulin κ/λ ratios –
Hevylite assay versus immunofixation
Ocena IgG, IgA i IgM monoklonalnej i biklonalnej gammapatii metodą nefelometrycznego oznaczania stosunku κ/λ indywidualnych immunoglobulin – Test Hevylite versus immunofiksacja
Institute of Hematology and Transfusion Medicine, Warsaw, Poland
Head: Prof. Krzysztof Warzocha
SUMMARY
Immunofixation is a standard method for detecting monoclonal immunoglobulins and characterizing its isotype. Recently clonality can also be determined by using immunoglobulin heavy chain /light chain immunoassays – “Hevylite” HLC. Availability of antibodies which bind to conformational epitopes spanning the junctional regions between
bound κ or λ light chains and their respective heavy chain partners has allowed the specific measurement of serum
IgGκ, IgGλ, IgAκ, IgAλ IgMκ and IgMλ concentrations. In turn, this has enabled the calculation of IgGκ/IgGλ,
IgAκ/IgAλ and IgMκ /IgMλ ratios (heavy/light chain or HLC ratios) for individual patients. In this study, the diagnostic value of HLC ratios was compared with the serum protein immunofixation (IFE) results. Sera from 51 patients
with monoclonal and biclonal gammopathy including 34 with multiple myeloma and 11 with Waldenström’s macroglobulinemia were assayed. IFE and HLC ratio results were concordant for 44 of the 51 IFE – monoclonal protein positive samples. IFE detected 2 “minor” IgGκ bands (1oligoclonal in multiple myeloma patient in CR after ASCT, 1 in
Hodgkin lymphoma patient in 10 year CR), and 1 IgAλ with polyclonal background (in a patient with multiple
myeloma and liver cirrhosis) that were not detected by HLC assay.
In 6 cases with biclonal IgGκ + IgGλ gammopathy revealed in IFE, the HLC ratios were normal in 3, also in the three
cases the HLC ratios revealed only one component IgGκ or IgGλ. In 4 cases with biclonal IgG + IgA gammopathy,
including 3- IgGκ + IgAκ cases and one IgGκ + IgAλ, the HLC ratios were concordant with IFE. In one patient with
Waldenström’s macroglobulinemia and 3 homogenous bands IgG+IgA+ IgM in serum IFE analysis, the HLC assay
correctly revealed only IgMκ/IgMλ abnormal ratio while IgGκ/IgGλ and IgAκ/IgAλ ratios were normal, as has been
also proved in quantitative studies of total immunoglobulin concentration. Our results indicate that HLC and IFE are
complementary methods. HLC assay provides numerical results. There is raised the question of HLC predictive
value. A carried out in our study evaluation of survival of 21 patients with IgM monoclonal gammopathy diagnosed
with multiple myeloma or Waldenström macroglobulinemia whose average time of observation was 6 years showed a
median survival time of 7 years in patients with the values of HLC ratios < the median and 5.5 years in patients with
values of HLC ratios> the median.
KEY WORDS: Hevylite – Monoclonal gammopathy – Multiple myeloma – Waldenström’s macroglobulinemia.
STRESZCZENIE
Standardową metodą wykrywania i identyfikacji immunologicznej izotypu immunoglobuliny monoklonalnej jest
immunofiksacja (IF). Klonalność immunoglobuliny moŜe takŜe być określona przy zastosowaniu ostatnio opracowanych testów „Hevylite” HLC (immunoglobulin heavy chain /light chain immunoassays). Dostępność przeciwciał
wiąŜących się z epitopami łączącymi przyległe regiony między związanymi lekkimi łańcuchami κ lub λ i ich odpowiednimi partnerami cięŜkich łańcuchów pozwoliła na swoiste mierzenie w surowicy IgGκ, IgGλ, IgAκ, IgAλ, IgMκ,
IgMλ, a to z kolei umoŜliwiło wyliczanie stosunku IgGκ/IgGλ, IgAκ/IgAλ, IgMκ/IgMλ (HLC ratio- HLCR) dla indywidualnych chorych. W pracy oceniono skuteczność diagnostyczną testu HLC w porównaniu do IF białek surowicy. Badaniom poddano surowice 51 chorych z gammapatią monoklonalną i biklonalną w tym 34 chorych na szpiczaka plazmocytowego i 11 na makroglobulinemię Waldenströma. Spośród 51 przypadków z białkiem monoklonalnym
wykrywalnym w IF u 44 wyniki badań HLCR były zgodne z wynikami IF. IF wykazała w 2 przypadkach „małe”
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M. KRAJ et al.
prąŜki IgGκ (w jednym przypadku u chorej na szpiczaka w całkowitej remisji po autologicznym przeszczepie
a w drugim u chorego na chłoniaka Hadgkina w 10-letniej całkowitej remisji) oraz w 1 przypadku IgAλ na tle obrazu
poliklonalnego (u chorego na szpiczaka i marskość wątroby), które nie były wykrywalne w testach HLC. Spośród 6
przypadków z biklonalną gammapatią IgGκ + IgGλ ujawnioną w IF, HLCR były prawidłowe w 3, takŜe w 3 HLCR
ujawniły tylko 1 komponent IgGκ lub IgGλ natomiast w 4 przypadkach z biklonalną gammapatią IgG + IgA, włączając 3 przypadki IgGκ + IgAκ i 1 przypadek IgGκ + IgAλ, wyniki HLCR były zgodne z IF. U 1 chorego z makroglobulinemią Waldenströma i trzema jednorodnymi prąŜkami IgG + IgA + IgM w IF surowicy, testy HLC słusznie
ujawniły nieprawidłowy HLCR IgMκ/IgMλ, podczas gdy HLCR IgGκ/IgGλ oraz IgAκ/IgAλ były prawidłowe, co
udowodniono takŜe w badaniach ilościowych stęŜenia całkowitych immunoglobulin. Uzyskane przez nas wyniki
wskazują, Ŝe HLC i IF są metodami uzupełniającymi się. HLC dostarcza wyników ilościowych. Podnoszona jest wartość prognostyczna HLCR. Przeprowadzona w pracy ocena rzeczywistego czasu przeŜycia 21 chorych z gammapatią
IgM z rozpoznaniem szpiczaka lub makroglobulinemii Waldenströma i średnią czasu obserwacji 6 lat wykazała medianę czasu przeŜycia 7 lat chorych z wartościami HLCR < mediany i 5,5 lat chorych z wartościami HLCR > mediany.
SŁOWA KLUCZOWE: Hevylite – Gammapatia monoklonalna – Szpiczak plazmocytowy – Makroglobulinemia Waldenströma.
INTRODUCTION
Basic goals of the methods used to investigate monoclonal proteins (M proteins) are detection, immunochemical characterization and quantification. Serum protein electrophoresis (SPE) techniques
have undergone considerable improvements in the past few decades and now provided crisp resolution
(especially in the β-globulin region) to detect many subtle electrophoretic differences in M proteins not
readily distinguished by older 5-band gel techniques [1, 2]. However, problems in detecting and especially in quantifying M proteins still exist. By SPE, M proteins can migrate anywhere from the α to the
γ region. They can be obscured by large proteins, such as haptoglobin in the α2 region or more commonly by transferrin and complement component 3 (C3) in the β region [3]. This is particularly apparent for monoclonal IgA. Occasionally, IgM M proteins self-aggregate, making problems with detection
and measurement.
Immunofixation (IFE) is the current gold standard for detecting and immunochemically characterizing M proteins. Unfortunately, IFE is not a quantitative technique.
At present, the M protein spike is commonly measured by densitometric scanning. As noted above,
when a relatively small M protein overlays a typical serum component, it can be difficult to obtain a
precise measurement with this technique. Nephelometry is also used for immunoglobulin measurements
and is analytically accurate at their low concentrations. However, patients’ samples also contain nontumor origin 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 [4–6]. In addition, in multiple myeloma, changes in hematocrit and blood
volume usually occur, causing serum concentrations of immunoglobulins to vary by 50% or more, independently of changes in immunoglobulin production by tumor [7].
In 2009 Bradwell et al. [8] developed immunoglobulin heavy/light chain immunoassays – “Hevylite” (HLC). Availability of antibodies which bind to conformational epitopes spanning the junctional
regions between bound κ or λ light chains and their respective heavy chain partners has allowed the
specific measurement of serum IgGκ, IgGλ, IgAκ, IgAλ, IgMκ and IgMλ concentrations. In turn, this
has enabled the calculation of IgGκ/IgGλ, IgAκ/IgAλ and IgMκ/IgMλ ratios (heavy /light chain or HLC
ratios) for individual patients. Separate measurements of the κ and λ light chain types of IgG, IgA and
IgM allow evaluation of individual tumor clones and give quantitative information about the immunosuppression of each non-tumor immunoglobulin. Measurement of molecule pairs, such as IgGκ/IgGλ,
IgAκ/IgAλ, IgMκ/IgMλ, would indicate clonality in the same manner as serum free light chain (FLC)
κ/λ ratios [9].
Evaluation of IgG, IgA and IgM
259
Bradwell et al. [8] proved that use of anti – HLC reagents could help in the vexing issue of detecting and providing reproducible measurements of M proteins that comigrate with other major serum
components, such as transferrin or C3. They had also reason that the M protein type will be similarly
affected by changes in blood volume and the half-life of the affected heavy-chain class. Thus, the HLC
ratio will theoretically provide more dynamic information than measurement of total M protein alone
[2].
Until now, apart from a key Bradwell’s et al. [8] publication, there appeared congress reports on the
results of the use of nephelometric measurement of individual immunoglobulin κ/λ ratios in assessment
of monoclonal gammopathies [10–16] including evaluation of prognostic value of Hevylite assays in
these diseases [17–21]. The aim of the present study was to compare the value of the HLC and the immunofixation test in the detection of monoclonal and biclonal gammopathy.
MATERIAL AND METHODS
The study included 51 patients with monoclonal or biclonal gammopathy hospitalized at the Institute of Hematology and Transfusion Medicine in Warsaw: 28 women and 23 men aged 25 to 82 years
(median: 64 years). Thirty five patients were diagnosed with multiple myeloma (including 10 with IgMM protein), 11 with Waldenström’s macroglobulinemia, 4 with other lymphoma and one with polycythemia vera.
Serum protein electrophoresis and immunofixation were performed on agarose media with densitometric scanning using HydrasysTM 2 apparatus (Sebia, France) and antisera from the same company and
also using Beckman Paragon Immunofixation Electrophoresis Kit.
Nephelometric immunoglobulin assays, performed using a Siemens BNTM II nephelometer, were
used to measure IgGκ/IgGλ, IgAκ/IgAλ, IgMκ/IgMλ (HLC) and also to quantify FLC in the fresh and
archived frozen sera of assessed patients. In this method there were applied antibodies (HevyliteTM Human IgG Kappa Kit; IgG lambda Kit, HevyliteTM Human IgA Kappa Kit; IgA Lambda Kit, HevyliteTM
Human IgM Kappa Kit; IgM Lambda Kit; The Binding Site, Ltd, Birmingham, UK) specific for IgG κ,
IgG λ, IgA κ, IgA λ, IgM κ, IgM λ and antibodies (FreeliteR ; The Binding Site, Ltd, Birmingham, UK)
specific for κ and λ light chains in free form, not bound to the heavy chain.
In patients with IgM monoclonal gammopathy who underwent testing at diagnosis, there was
determined the actual survival time. The observation period of patients ranged from several months to
16 years. For the statistical analysis of differences in survival time there was used Student's t –test.
RESULTS
Tumor and non – tumor HLC concentrations and HLC IgGκ/IgGλ, IgAκ/IgAλ ratios in sera of patients with IgG and IgA multiple myeloma are presented in Table 1 while tumor and non-tumor HLC
concentrations and HLC IgMκ/IgMλ ratio in sera of patients with IgM monoclonal gammopathy are
presented in Table 2.
Table 3 summarizes results of HLC immunoglobulins concentrations in patients with biclonal
gammopathy and in one patient with multiple myeloma evaluated at diagnosis (M-IgAλ present), in
complete remission (CR) after autologous stem cell transplantation (ASCT) (oligoclonal IgGκ in IFE
but normal HLC), and at relapse (M-IgAλ present, oligoclonal IgGκ band absent) (Figure 1).
IFE and HLC ratio results were concordant for 44 of 51 IFE – M – protein positive samples. IFE detected 2 “minor” IgGκ bands: 1 oligoclonal in CR after ASCT, 1 in patient with Hodgkin lymphoma in
10-year CR (Table 4 sample 1, 2, Figures 1, 2) and one IgAλ with polyclonal background (Table 4
sample 9, Figure 3C) that were not detected by HLC assay.
M. KRAJ et al.
260
Table 1. Tumor and non-tumor HLC concentrations and HLC IgGκ/IgGλ, IgAκ/IgAλ ratios in sera of patients with IgG and
IgA multiple myeloma
Tabela 1. StęŜenia nowotworowej i nie nowotworowej HLC i wartości stosunku IgGκ/IgGλ, IgAκ/IgAλ HLC w surowicach
chorych na szpiczaka plazmocytowego IgG i IgA
Tumor sera
with
M-protein
isotype acc.
to IFE
IgG κ
IgG λ
IgAκ
HLC immunoglobulin g/L
n
3
1
IgGκ
Median,
range
11.70
8.52-38.30
23.40
IgGλ
Median,
range
0.91
0.519-1.32
0.43
HLC ratios
IgGκ /IgGλ
n
HLC immunoglobulin g/L
IgAκ
Median,
range
IgAλ
Median,
range
Median,
range
5.13
3.56-7.17
0.62
0.08-1.20
1.27
0.43-2.36
0.55
0.07-1.48
17.20
3.72-21.30
0.87
0.4-1.73
9.31
4.84-47.46
0.02
0.01-0.06
1.4
0.58-2.52
Median,
range
40.12
6.45-73.79
0.01
3
IgAλ
4
Blood donor
109
7.76
4.00
sera (acc. to
4.23-12.18
2.37-5.91
Bradwell et al.
[8])
Abbreviations: HLC – heavy chain /light chain
HLC ratios
IgAκ /IgAλ
1.96
1.26-3.2
191
Table 2. Tumor and non-tumor HLC concentrations and HLC IgMκ/IgMλ ratios in sera of patients with IgM
monoclonal gammopathy
Tabela 2. StęŜenia nowotworowej i nie nowotworowej HLC i wartości stosunku IgMκ/IgMλ HLC w surowicach
chorych z gammapatią monoklonalną IgM
HLC ratios
IgMκ /IgMλ
HLC immunoglobulin g/L
Tumor sera with
M-protein isotype acc.to IFE
n
IgM κ
16
IgM λ
5
Blood donor sera Bradwell et al. [8]
118
IgMκ
Median,
range
IgMλ
Median,
range
Median
range
70.3
2.09-353.00
0.77
0.26-1.55
0.77
0.33-1.54
0.27
0.008-2.72
29.40
16.70-37.20
0.50
0.20-1.10
257
4.22-2680
0.04
0.01-0.05
1.6
0.81-2.52
Table 3. HLC immunoglobulins concentrations in patients with biclonal gammopathy (sample 1–9) and one patient with
multiple myeloma evaluated at diagnosis (sample 11), in CR after ASCT (sample 12) and at relapse (sample 13)
Tabela 3. StęŜenie HLC w surowicach chorych z biklonalną gammapatią (przypadki 1–9) i chorej na szpiczaka plazmocytowego ocenianej w chwili rozpoznania (próbka 11), w czasie remisji całkowitej po ASCT (próbka 12) i w nawrocie choroby
(próbka 13)
Sample
IFE
IgGκ
10.20
3.30
HLC immunoglobulins, g/L
IgGλ
IgAκ
IgAλ
0.53
83.90
0.11
0.97
29.90
0.15
HLC ratios
IgGκ/IgGλ
IgAκ/IgAλ
18.95
742.47
3.38
189.00
1.
2.
IgAκ + IgGκ
IgAκ + IgGκ
3.
4.
5.
6.
IgGκ + IgAλ
IgAλ + IgGκ
IgGλ + IgGκ
IgGλ +IgGκ
36.80
0.85
2.84
5.61
30.70
24.00
0.09
0.23
7.
IgGκ + IgGλ
19.30
9.00
2.14
0.37
1.43
5.02
6.25
43.29
0.07
0.22
Evaluation of IgG, IgA and IgM
8.
9.
10.
11.
12.
13.
IgAκ + IgAλ
IgGκ + IgGλ
IgGκ + IgAκ + IgMκ
IgAλ
IgGκ
IgAλ
14.40
3.60
3.69
6.38
5.57
6.19
0.95
2.20
3.68
2.54
10.50
1.01
0.30
0.91
0.93
0.60
0.10
0.76
0.18
40.70
0.94
6.14
261
2.32
3.78
1.67
1.73
2.19
102.94
1.32
1.65
0.02
0.98
0.09
Abbreviations: IFE – immunofixation electrophoresis; HLC –heavy chain/light chain; ASCT – autologous stem cell transplantation; CR – complete remission
Table 4. Patients and theirs SPE samples that were discordant between IFE and HLC ratios
Tabela 4. Przypadki z podejrzeniem białka M w elektroforezie surowicy, w których wykazano niezgodności
w testach IFE I HLC
Patient
Sample
Disease
diagnosis
SPE
IFE
HLC
ratios
1.
Multiple myeloma
(CR after ASCT)
Suspicious
Minor IgGκ
Normal
2.
Suspicious
Minor IgGκ
Normal
3.
Hodgkin lymphoma
in 10 years CR
Polycythemia
Positive
IgGκ + IgGλ
Normal
4.
Non-Hodgkin lymphoma
IgGκ + IgGλ
Normal
5.
Multiple myeloma
IgGκ + IgGλ
Normal
6.
Multiple myeloma
IgGκ + IgGλ
IgGλ 0.09
7.
8.
Multiple myeloma
Follicular lymphoma
Suspicious/
polyclonal
Suspicious/
polyclonal
Positive
2 M-spikes
Positive
Suspicious/
polyclonal
Suspicious/
polyclonal
Positive
IgGκ + IgGλ
IgGκ + IgGλ
IgGλ 0.23
IgGκ 4.61
9.
sFLC ratios
(κ : λ, mg/L)
Normal (12:18)
Normal (40:42)
FLC λ 0.08
(22 : 280)
Liver cirrhosis,
IgAλ
Normal
Multiple myeloma
(polyclonal background)
10.
Waldenström’s macrogloIgGκ + IgAκ + IgMκ
IgMκ
bulinemia
Abbreviations: SPE – serum protein electrophoresis; IFE – immunofixation electrophoresis; HLC – heavy chain/ light chain;
FLC – free light chains; ASCT – autologous stem cell transplantation; CR – complete remission.
In 6 cases with biclonal IgGκ + IgGλ gammopathy revealed in IFE, the HLC ratios were normal in
3 and also in 3 revealed only 1 component IgGκ or IgGλ (Fig. 4). In 4 cases with biclonal IgG + IgA
gammopathy, including 3 – IgGκ + IgAκ cases and one IgGκ + IgAλ, the HLC ratios were concordant
with IFE results (Fig. 5AB).
In one patient with Waldenström’s macroglobulinemia and 3 homogeneous bands of IgG + IgA +
IgM in serum IFE analysis, HLC assay correctly revealed only IgMκ/IgMλ abnormal ratio (IgMκ-93.1
g/L, IgMλ 0.29 g/L, IgMκ/IgMλ 322.0) while IgGκ/IgGλ and IgAκ/IgAλ ratios were normal (Table 3,
sample 10, Fig. 5C). In this case quantitative determination of total immunoglobulins performed on
ARRAY 360 analyzer revealed following results: IgG – 0.51 g/L (normal range 0.69-1.6 g/L), IgA –
0.03 g/L (n. range 0.06-0.37 g/L), IgM – 7.96 g/L (n. range 0.06-0.23 g/L).
Serum FLC and HLC immunoglobulins concentrations in 2 patients with Bence Jones myeloma and
one healthy individual are presented in Table 5.
Median overall survival in the whole group of patients with IgM monoclonal gammopathy (Table 2)
was 6 years, in the group of patients with IgM HLC ratio < median it was 7 years and in the group of
patients with IgM HLC ratio > median it was 5.5 years (p= 0.2738).
M. KRAJ et al.
262
Table 5. Serum FLC and HLC immunoglobulins concentrations in 2 patients with Bence Jones myeloma
and healthy individual
Tabela 5. StęŜenia FLC i HLC w surowicy 2 chorych z chorobą łańcucha lekkiego i osoby zdrowej
Bence Jones κ myeloma
SPE
UPE
SIFE
UIFE
HLC immunoglobulins, g/L
IgGκ
IgGλ
IgAκ
IgAλ
HLC ratios
IgG κ/ IgG λ
IgA κ/ IgA λ
κ FLC, mg/L
λ FLC, mg/L
FLC ratios (κ : λ, mg/L)
hipo - γ
M-spike
FLC κ band
FLC κ band
Bence Jones λ
myeloma
hipo - γ
Healthy person
FLC λ band
FLC λ band
2.66
1.80
0.35
0.14
11.80
5.84
8.48
15.70
7.030
2.970
1.230
0.752
1.47
2.39
3980.00
10.30
387
2.02
0.54
2.08
194.00
0.01
2.36
1.63
8.91
8.60
1.03
Abbreviations: HLC –heavy chain/light chain; FLC –free light chains; SPE – serum protein electrophoresis; UPE – urine
protein electrophoresis; SIFE – serum immunofixation electrophoresis; UIFE – urine immunofixation electrophoresis
DISCUSSION
Currently, monoclonal immunoglobulins are identified and quantified from bands on electrophoretic
gels. As an alternative, clonality can be determined by measuring the separate light chain types of each
immunoglobulin class to allow numerical assessment of IgGκ/IgGλ, IgAκ /IgAλ, IgMκ/IgMλ ratios,
analogous to FLC κ/λ ratios [8, 22].
Bradwell et al. (8 defined the normal range of HLC immunoglobulins serum concentrations: – Using 109 blood donor sera the 95% reference interval for HLC IgGκ was 4.23–12.18 (median 7.76) g/L
and that for HLC IgGλ was 2.37–5.91 (median 4.00) g/L. The normal diagnostic range for HLC
IgGκ/IgGλ ratio was 1.26–3.2 (median 1.96). Patients with ratios greater than 3.2 have excess of IgGκ
and are presumed to be producing clonal HLC IgG. In turn, patients with ratios less than 1.26 contain
excess of HLC IgGλ and are presumed to be producing clonal HLC IgGλ.
Using 191 blood donor sera, the 95% reference interval for HCL IgAκ was 0.43–2.36 (median 1.27)
g/L and that for HLC IgAλ was 0.4–1.73 (median 0.87) g/L. The normal diagnostic range for HLC
IgAκ/ IgAλ ratio was 0.58–2.52 (median 1.4). Patients with ratios greater than 2.58 have excess of IgAκ
and are presumed to be producing clonal HLC IgAκ. In turn, patients with ratios less than 0.58 contain
excess of HLC IgAλ and are presumed to be producing clonal HLC IgAλ.
Using 118 blood donor sera the 95% reference interval for HLC IgMκ was 0.33-1.54 (median 0.77)
g/L and that for HLC IgMλ was 0.20–1.10 (median 0.50) g/L. The normal diagnostic range for HLC
IgMκ /IgMλ ratio was 0.81–2.52 (median 1.6). Patients with ratios greater than 2.52 are presumed to be
producing clonal HLC IgMκ. In turn, patients with ratios less than 0.81 are presumed to be producing
clonal IgMλ.
Our results obtained from typing of M proteins into IgG, IgA or IgM classes, using immunoassays
for intact immunoglobulin κ /λ pairs, were mostly comparable with results obtained by immunofixation
– electrophoresis methods. IFE and HLC results were concordant for 41 of the 51 SPE- positive or
suspicious samples and discordant in 10 cases (Table 4). Two cases with Bence Jones myeloma and of
Evaluation of IgG, IgA and IgM
263
Fig. 1. SPE and IFE of serum proteins in a multiple myeloma patient in CR after ASCT in whom these methods revealed an
oligoclonal IgGκ band while HLCR tests were normal
Ryc. 1. Elektroforeza i immunofiksacja białek surowicy u chorej na szpiczaka plazmocytowego w całkowitej remisji po przeszczepieniu autologicznych krwiotwórczych komórek macierzystych, u której tymi metodami ujawniono oligoklonalną frakcję
IgGκ podczas gdy testy HLCR były w normie
264
M. KRAJ et al.
Fig. 2. Serum SPE and IFE: (A) in a patient with polycythemia vera: IgGκ + IgGλ present in IFE while HLCR is normal; (B)
in a patient with Hodgkin lymphoma in CR: present IgGκ in IFE while HLCR is normal; (C) in a non-Hodgkin lymphoma
patient: IgGκ+ IgGλ present in IFE while HLCR is normal
Ryc. 2. Elektroforeza i immunofiksacja (IF) surowicy: (A) u chorego z czerwienicą prawdziwą: w IF obecne IgGκ + IgGλ, a
HLCR w normie; (B) u chorego z chłoniakiem Hodgkina w remisji: w IF obecna IgGκ, a HLCR w normie; (C) u chorego z
chłoniakiem nieziarniczym: w IF obecne IgGκ + IgGλ, a HLCR w normie
Evaluation of IgG, IgA and IgM
265
Fig. 3. Serum SPE and IFE: (A) in a patient with multiple myeloma: two IgGκ + IgGλ components present in IFE while
HLCR is normal; (B) in a patient with non-Hodgkin lymphoma: present IgAκ in IFE, HLCR is consistent with IFE; (C) in a
patient with liver cirrhosis and multiple myeloma: present IgAλ on polyclonal background in IFE, while HLCR is normal.
Ryc. 3. Elektroforeza i immunofiksacja: (A) u chorego na szpiczaka: w IF obecne dwa komponenty IgGκ + IgGλ, a HLCR
w normie; (B) u chorego z chłoniakiem nieziarniczym: w IF obecna IgAκ; HLCR zgodny z IF; (C) u chorego z marskością
wątroby i szpiczakiem: w IF na tle poliklonalnym obecna IgAλ, a HLCR w normie.
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M. KRAJ et al.
Fig. 4. Serum SPE and IFE of multiple myeloma patients (AB) and non-Hodgkin lymphoma patient (C) in whom two IgGκ +
IgGλ components are present in IFE while only one IgGλ (AB) or IgGκ (C) in HLCR test
Ryc. 4. Elektroforeza i immunofiksacja (IF) u chorych na szpiczaka (AB) i chłoniaka nieziarniczego (C), u których w IF obecne są dwa komponenty IgGκ + IgGλ, podczas gdy test HLCR świadczy o obecności tylko jednego komponentu IgGλ (AB),
albo IgGκ (C)
Evaluation of IgG, IgA and IgM
267
Fig. 5. Serum SPE and IFE : (AB) in patients with multiple myeloma - consistence of IFE and HLCR outcomes; (C) in a patient with Waldenström’s macroglobulinemia the result of HLCR, and not IFE, is correct
Ryc. 5. Elektroforeza i immunofiksacja (IF) surowicy: (AB) u chorych na szpiczaka – zgodność wyników IF i HLCR; (C)
u chorego z makroglobulinemią Waldenströma – wynik HLCR, a nie wynik IF jest zgodny z prawdą
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FLC M-protein according to IFE were correctly negative by HLC assay (and positive for an increased
serum FLC ratio) (Table 5).
In one IgA multiple myeloma patient, in CR after ASCT, oligoclonal IgGκ according to IFE was
negative by HLC assay (Table 3 sample 11, 12, 13, Fig.1; Table 4, sample 1). Oligoclonal bands often
appear after chemotherapy and stem cell transplantation [1, 23, 24]. An oligoclonal band is defined as
the presence of a serum and/or urine IFE M-spike different either in heavy and/or light chain component from the orginal myeloma protein. Larrea et al. [25] assessed 33 patients with multiple myeloma in
CR after different induction regimens. In the group treated with cytotoxic agents, the prevalence of
oligoclonal bands was observed in 2 out of 18 patients (11.1%). In contrast, induction with novel drugs
resulted in developing oligoclonal bands in serum and/or urine in 9 of 15(60%) patients (p=0.003). This
difference is likely due to greater anti-myeloma effect, and stronger immune reconstitution resulting
from the effect of novel drugs, or both. The mechanism associated with the emergence of the oligoclonal bands as well as their prognostic impact are unknown. It was suggested that these atypical serum IFE
patterns are associated with good prognosis, likely due to durable immune reconstitution.
Among 6 cases with biclonal IgGκ +IgGλ gammopathy revealed in IFE, the HLC ratios were normal in 3 (Table 4, sample 3,4, Fig. 4) while in 3 other cases only one M protein IgGκ or IgGλ was
shown (Table 4 sample 6, 7, 8). Biclonal gammopathies represent about 5% of clonal gammopathies.
Decaux et al. [26] identified 203 cases with biclonal gammopathy. The most frequent diagnosis in their
study was biclonal gammopathy of undetermined significance (MGUS) (60,6%), while 21% of patients
had lymphoproliferative diseases, including Waldenström’s macroglobulinemia, non-Hodgkin lymphoma and chronic lymphocytic leukemia; in 8.87% of patients multiple myeloma was diagnosed and
in 7.4% biclonal gammopathy was associated with a non-lymphoid hemopathy or with systemic disease. Clinical outcomes of persons with multiple M-spikes at initial diagnosis in multiple myeloma
were evaluated by Mark et al. [27]. Patients with single M-spike versus patients with multiple M-spikes
were compared by treatment received, disease response, PFS, event free survival and overall survival.
Overall 26 of 161 patients had multiple M-spikes (16%). The outcomes were similar for patients with
single M-spike and those with multiple M-spikes. It means that the method of adding M-spikes and
following the aggregate throught out the treatment course is appropriate. Conventionally, multiple Mspikes are summed and the total M-protein is used to assess response.
In one patient with multiple myeloma and liver cirrhoris IFE revealed IgAλ and a polyclonal background but HLC assay was normal (Table 4, sample 9, Fig. 3C). On the other hand, in one case with
Waldenström’s macroglobulinemia and three bands IgGκ + IgAλ + IgMκ in IFE analysis, HLC assays
correctly revealed only IgMκ M protein (Table 4 sample 10, Fig. 5C).
Our results indicate that HLC assay is not a replacement method for the highly sensitive IFE technique. IFE does not exhibit any interference resulting from comigration of M-proteins with serum components and readily detects biclonal gammopathies. The HLC assays are an addition to our armamentarium for detecting and measuring M proteins [28]. They bring quantifiable number to the evaluation of
M-proteins together with the ability to visualize M-protein HLC type and provide numerical results for
patients who are only IFE positive.
Suvannasankha et al. [16] evaluated the correlation of serum HLC assays with FLC assays, serum
monoclonal protein measurement by electrophoresis (M spike), and total immunoglobulin G or A in
serial samples of patients with newly diagnosed multiple myeloma undergoing a lenalidomide containing induction regimen. They found that HLC kinetics showed differences compared to FLC and correlated better with the monoclonal spike; FLC normalized faster but, once normalized, seemed to correlate worse than HLC with M spike and ongoing clinical response. Increased HLC of the uninvolved
chain was observed in a subset of patients, after therapy. The HLC κ/λ ratio, which takes into account
the changes of the uninvolved chain, is an appealing tool for disease monitoring once HLC of the involved chain normalized.
Evaluation of IgG, IgA and IgM
269
Some congress publications provide data on prognostic value of HLC analysis in multiple myeloma
and MGUS [18, 19, 20, 21].
Avet –Loiseau et al. [18, 19] reported that heavy/light chain specific immunoglobulin ratios at presentation were prognostic for progression free survival (PFS) in the IFM 2005-01 myeloma trial. In that
study, Kaplan Meier analysis indicated that more abnormal HLC ratios were associated with reduced
PFS (> median for IgGκ and IgAκ patients, < median for IgGλ and IgAλ patients; P = 0.007). When
using more extreme ratios (>200 or < 0.01), the significance level was higher increased (P= 0.002). Cox
regression analysis confirmed the association of the latter HLC ratios with reduced PFS (P < 0.001) and
indicated that the association was independent of and more significant than that of β2M or albumin. The
combined use of the extreme HLC ratios and β2M > 3.5 mg/L, in a risk stratification model, showed
significant differences in PFS for patients with 0, 1, or 2 adverse risk factors (P=0.000013). A more
complex risk stratification model combining HLC ratios with the International Staging System also
showed significant differences in PFS according to the number of risk factors (P= 0.0001). The use of
HLC ratios provides a measure of both tumor immunoglobulin production and immunoparesis. Probably, the combination of these two factors has a prognostic value. HLC measurements may be a useful
addition to the current ISS assessments.
Also Ludwig et al. [20] found that the ratio of monoclonal to polyclonal immunoglobulins assessed
with the Hevylite test predicts prognosis, is superior for monitoring the course of the disease and allows
detection of monoclonal immunoglobulin in multiple myeloma patients with normal or subnormal involved immunoglobulin isotype. In a study of 103 multiple myeloma patients median overall survival of
the entire group was 37.9 months. In multivariate analysis, β2M, and HLC ratio were found as the only
parameters correlating with survival. A three tiered risk stratification model utilizing β2M > 3.5 mg/L,
and HLC > median value had a grater prognostic value than ISS (p= 0.001 vs p=0.09). Patients with 0
risk factors (β2M < 3.5 mg/L, HLC ratio < median) had a 50% survival time of 118 months, patients
with 1 risk factor (either β2M > 3.5 mg/L or HLC ratio > median ) had a 50% survival of 53 months and
those with both risk factors (β2M > 3.5 mg/L and HLC ratio > median) had a 50% survival of 29
months (p= 0.001).
The serum FLC ratio at presentation has been shown to be an independent prognostic marker in
multiple myeloma [29]. Harding et al. [17] reported the use of serum FLC and HLC ratios to predict
survival in multiple myeloma patients. Archived presentation sera (n=186) from British Medical Research Council multiple myeloma trials were analyzed retrospectively using sFLC and HLC assays.
Kaplan-Meier survival curves were constructed to compare patients above or below the median value
for FLC and HLC ratio and M-spike concentration. There was no significant difference in survival
when analyzing intact M protein concentrations, serum FLC concentrations, FLC ratios or HLC ratios
individually. Adding FLC and HLC ratios together there was a significantly shorter survival for those
patients with values greater then the median (50% survival 949 days versus 1592 days; P = 0.02). The
serum FLC ratio is likely to be more predictive of outcome than the concentration of the tumor sFLC
because it includes a measure of immunoparesis (the denominator) as well as tumor production (the
numerator). Use of the HLC ratio also incorporates a measure of immunoparesis but additionally, it will
compensate for any reduction in the concentration of monoclonal immunoglobulin due to increased
catabolism (IgG) and / or increased plasma volume. It is probable that the use of the summated sFLC
and HLC ratios was more predictive of outcome because there were some patients with very low monoclonal intact immunoglobulin production for whom the FLC ratio was the most appropriate prognostic marker and vice versa for patients with low FLC production [17].
A carried out in our study evaluation of actual survival time of 21 IgM gammopathy patients
diagnosed with multiple myeloma or Waldenström’s macroglobulinemia and mean follow –up time of
six years showed no statistically significant difference in median survival depending on the results of
the HLC ratios tested at diagnosis amounting 7 years in patients with HLC ratio values < the median
and 5.5 years for patients with values of HLC ratio > the median (p = 0.2738).
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However, Koulieris et al. [14] evaluated the prognostic value of IgMκ /IgMλ HLC ratios at diagnosis and the role of HLC ratio in disease monitoring in 31 patients with Waldenström’s macroglobulinemia. Median IgM HLC ratio was significantly higher in patients requiring treatment at presentation than
in those not requiring treatment. IgM HLC ratio correlated with bone marrow infiltration and time to
first treatment. A simple risk stratification model utilizing IgM HLC ratio > median, β2M > 5 mg/L and
abnormal LDH identified 3 prognostic groups with respect to survival (p< 0.001) in this series with a
median follow – up of 59 months. Authors suggest that HLC IgM and IgM HLC ratio seem to separate
patients with a more aggressive disease.
IgG but not IgA HLC ratios have been shown to predict malignant transformation in MGUS patients [21].
In our study HLC measurements were readily made on an automated nephelometer. It is worth to
stress that in many patients, an applicable result can be obtained only having repeated the measurements
at different dilution levels; therefore, it is not possible to predict the number of examinations for performing of which can serve one kit.
Acknowledgement
The authors thank The Binding Site Company Ltd. Birmingham, UK, Dr. B. Olszewska and Dr. D.
Rutkowski from Biokom company for providing the reagents for Hevylite tests.
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Praca wpłynęła do Redakcji 20.06.2011 r. i została zakwalifikowana do druku 29.06.2011 r.
Correspondence:
Prof. Maria Kraj
Institute of Hematology and Transfusion Medicine,
Indiry Gandhi 14
02-776 Warsaw, Poland
e-mail: [email protected]