Long-Term Biological Variation of Serum Protein Electrophoresis M-Spike, Urine M-Spike, and Monoclonal Serum Free Light Chain Quantification: Implications for Monitoring Monoclonal Gammopathies

J.A. Katzmann, M.R. Snyder, S.V. Rajkumar, R.A. Kyle, T.M. Therneau, J.T. Benson, and A. Dispenzieri

www.clinchem.org/cgi/content/article/57/12/1687

December 2011

© Copyright 2011 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry

Introduction:Introduction:

Plasma Cell Proliferative Disease (PCPD)PCPDs are a group of disorders that can be malignant, pre-malignant, or protein diseases whose clinical symptoms are due to the secreted monoclonal protein or peptide. Because the proliferative plasma cell clone usually secretes a monoclonal immunoglobulin, these disorders are also called monoclonal gammopathies.

> Multiple Myeloma (MM)MM is a malignant PCPD that grows in the bone

marrow. The secreted monoclonal protein serves as a serum marker for the plasma cell clone.

© Copyright 2009 by the American Association for Clinical Chemistry

Introduction (cont.):Introduction (cont.):

Monoclonal immunoglobulins – The monoclonal immunoglobulin can often be detected

and quantified by serum protein electrophoresis. The measured serum abnormality is called the serum M-spike.

– Patients with monoclonal light chain diseases such as light chain multiple myeloma often clear the peptide so rapidly from serum that it is only detectable as a urine M-spike.

– Quantification of the serum and/or urine M-spike is a reflection of patient’s tumor cell burden.

– Clinical guidelines require at least a 50% reduction of the serum M-spike and a 90% reduction of the urine M-spike to define a partial response to therapy.

© Copyright 2009 by the American Association for Clinical Chemistry

Introduction (cont.):Introduction (cont.):

Quantitative free light chains (FLC) – Nephelometric immunoassays are now available to

quantify serum kappa or lambda light chains that are not bound to heavy chains (FLC).

– In the absence of a serum or a urine M-spike, quantification of monoclonal FLC can provide a means to monitor the hematologic response to a treatment regimen.

– It has been recommended that a serum FLC partial response be defined as requiring a 50% reduction [analogous to the serum M-spike].

© Copyright 2009 by the American Association for Clinical Chemistry

Introduction (cont.):Introduction (cont.):

Study aim: – We undertook this study to evaluate the variability of

long term serial laboratory results. This allowed the quantification of biologic variation and the calculation of the reductions needed in each assay variable to have confidence that reductions represent a significant partial response following treatment.

© Copyright 2009 by the American Association for Clinical Chemistry

Introduction (cont.):Introduction (cont.):

Question: – Significant changes are usually identified in the

context of the changes in reference (normal) populations. Reference change values are usually calculated in normal populations, within the reference interval, and over a short time span.

– What reference population can be used to define variability of a marker that is only present in diseased individuals?

© Copyright 2009 by the American Association for Clinical Chemistry

Material and methods:Material and methods: Study protocol:

– Patients were included in the study if they had either monoclonal gammopathy of undetermined significance (MGUS) or smoldering multiple myeloma (SMM) or if they had MM in stable partial remission. In addition to having no change in diagnosis, the serum M-spike could not change by >5 g/L during the study interval. An additional requirement for inclusion was at least 3 serial samples within the 5 yr study interval. All laboratory data was retrospectively retrieved from the electronic medical record.

© Copyright 2009 by the American Association for Clinical Chemistry

Material and methods:Material and methods:

Study protocol: – Serum M-spike, urine M-spike, quantitative FLC, and

quantitative immunoglobulins were obtained retrospectively from the clinical practice.

– The total CV for each method was computed as the mean square error using ANOVA on the logarithm of the laboratory value.

– The biologic variability was derived from the total CV of each assay and the analytic CV of each assay.

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 1a: Serum M-spike CVs vs. M-spike mean value. The vertical dashed line at 10 g/L represents the recommendation for minimal values of the serum M-spike to allow for reliable assessment of hematologic response. This figure illustrates why M-spikes must be >10g/L to be considered “measurable”. Small M-spikes have higher variability compared to larger M-spikes.

Figure 1aFigure 1a

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Table 2 Table 2

Measurable Serum M-spike

[M-spike>10g/L]

Serum IgG Measurable Urine M-spike

[M-spike>200 mg/24 hr]

Measurable Serum iFLC

[iFLC>100 mg/L]

CV 8.1% (n=90) 13.0% (n=148) 35.8% (n=25) 28.4 (n=52)

Table 2: Total CVs for patient sample sets. Each cell in the table has the total CV in the patient sample set for each assay variable, and the value in brackets is the number of patients that fulfilled criteria for a measurable results.

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Table 3Table 3

Table 3: Decrease needed in each assay for significance at various probability thresholds. The assays and their total CVs are listed. In addition, the decrease in the laboratory value required for significance at P-values of 0.5, 0.9, and 0.95 are indicated. Reference change values are calculated at a P-value of 0.95.

Test n Total CV

P=0.05 P=0.90 P=0.95

Serum M-spike >10g/L

90 8.1% 7.4% 17.2% 20.1%

IgG 148 13.0% 11.7% 26.1% 30.3%

Urine M-spike >200 mg/24 hr.

25 35.8% 28.8% 56.5% 62.9%

iFLC >100mg/dL

52 28.4% 23.7% 48.3% 54.5%

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Table 4Table 4

Table 4: Total, analytic, and biologic CVs. The total CVs are from Table 2, the analytic CVs are from laboratory validation studies, and the biologic CVs are derived as the square root of the difference of the square of the total CV and the square of the analytic CV.

Serum M-spike(≥10 g/L)

Urine M-spike (≥200 mg/24hr)

Serum iFLC (≥100 mg/L)

Serum IgG

Total CV 8.1% 35.8% 28.4% 13%

Analytic CV 2.1% 4.5% 5.8% 4.2%

Biologic CV 7.8% 35.5% 27.8% 12.3%

© Copyright 2009 by the American Association for Clinical Chemistry

Results (cont.):Results (cont.):

Question: Why does the serum FLC variability more closely

resemble the urine M-spike variability rather than the

serum M-spike variability?

© Copyright 2009 by the American Association for Clinical Chemistry

Author’s Conclusions:Author’s Conclusions:

Stable monoclonal gammopathy patients can be defined, but inherent in the total variability will be pre-analytic

variability, biologic variability, analytic variability, as well as disease variability.

Guidelines have suggested a 25% decrease in serum M-spikes and a 50% decrease in urine M-spikes for a minimal response. These values closely match the decreases needed for confidence at P=0.95.

The clinical guidelines for decreases in serum FLC are currently aligned with guidelines for the serum M-spike but should be altered to reflect the urine M-spike guidelines.

© Copyright 2009 by the American Association for Clinical Chemistry

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