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Technical Briefs |
Departments of
1
Pathology and
2
Medicine, The University of Texas Southwestern Medical Center, Dallas, TX 75390
3 Department of Pathology, University of California Davis Medical Center, 4635 2nd Ave., Room 3000, Sacramento, CA 95817
aauthor for correspondence: fax 916-734-6593, e-mail ishwarlal.jialal{at}ucdmc.ucdavis.edu
High-resolution serum protein electrophoresis (SPE) provides a clearer separation of ß1- and ß2-globulins than does low-resolution SPE. Currently, immunofixation electrophoresis (IFE) is performed in most laboratories based on high clinical suspicion of a disease associated with a monoclonal gammopathy or an abnormality in the pattern displayed on the electrophoretogram. The aim of this study was to prospectively determine the utility of performing IFE on specimens with increased ß1- or ß2-globulins on high-resolution SPE but with a normal electrophoretic pattern on visual inspection.
SPE was performed with a Sebia-Hydrasys automated electrophoresis system (Hydragel ß-1 + ß-2 gels; Sebia). The same system was used for IFE with polyclonal anti-human serum for identifying immunoglobulin heavy and light chains. The reference intervals for ß1 and ß2 concentrations, determined from 100 fresh samples from healthy volunteers in this laboratory, were 4–8 and 1–5 g/L (mean ± 2 SD), respectively.
This study received approval from our Institutional Review Board. During the period from March to December 2001, 3179 samples were submitted for SPE. Of these, 963 had IFE performed because of clinical suspicion or abnormalities in the SPE. All serum specimens submitted for SPE with increased ß1 or ß2 (n = 51) were prospectively evaluated by IFE. Fifteen of the samples had an obvious M-spike that warranted an IFE and were excluded from the analyses. As a control group, 50 samples with protein concentrations within the reference intervals and a visually normal electrophoretogram were also subjected to IFE. Criteria for inclusion were (a) no distinct protein band evident outside of the usual six bands (albumin, 
1, 2, ß1, ß2, and 
) and (b) an increased ß1 or ß2 concentration. LDL-cholesterol, iron, and total iron binding capacity (TIBC) were determined on all samples with increased ß1- or ß2-globulins in our clinical laboratory. The specimens were segregated according to the presence of a M-spike detected by IFE. Student t-tests were used to compare the ages in the two groups. The distribution of the two groups by gender was compared using a binomial distribution. A one-tailed Mann–Whitney–Wilcoxon nonparametric test was used to compare concentrations of the ß-globulins in the two groups.
During the study period, the ß-globulin (ß1 or ß2) concentrations by densitometry were increased in 36 specimens subjected to SPE. In 12 of the 36 specimens, a M-spike was identified by IFE, yielding a prevalence of 33%. In addition, the abnormally high ß in each sample matched the location of the M-spike. In 50 patients [mean (SD) age, 60 (15) years; 27 males and 23 females] with protein concentrations within the reference interval and visually normal electrophoretograms, IFE did not identify a M-spike in any sample.
The patients with a M-spike were significantly older (mean age, 64 years; range, 45–83 years) than the patients without a M-spike (mean, 55 years; range, 28–81 years; P <0.05). There was no difference in the gender distribution. We detected no significant differences in the ß1- or ß2-globulin concentrations between the two populations (M-spike present and absent). Ten of the 12 M-spikes (83%) migrated in the ß2 region, whereas 2 appeared in the ß1 region. The majority of the M-spikes in the ß2 region were IgA-
(6 of 10). The two M-spikes in the ß1 region were of the IgA-
class. M-Spikes in the ß2 region included six IgA- and one each of IgA-, IgG-, IgG-, and IgM-, respectively. -Globulin concentrations were decreased in 5 of the 12 samples and increased in 3.
Shown in Fig. 1A
are the SPE patterns for the 12 samples with a subsequently identified M-spike and 12 specimens from the group (n = 24) with increased ß1- or ß2-globulin with no M-spike on IFE. IFE gels for the M-spike-positive samples are shown in Fig. 1B
. Subtle abnormalities in the SPE, when present, are difficult to visualize; thus it is difficult to predict which specimen should have IFE performed.
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In an attempt to explain the increase in either ß1- or ß2-globulins in the 24 patients who did not have a M-spike on electrophoresis, we measured transferrin saturation, TIBC, and LDL-cholesterol. Ten of the 24 patients had evidence of iron deficiency as indicated by decreased transferrin saturation (<15%) and/or an increase in the TIBC. ß-Lipoprotein (LDL-cholesterol >1300 mg/L) was increased in an additional three patients. This accounted for 
54% of the patients who had increased ß1- or ß2-globulin and did not have a M-spike. Complement (C3) was not measured because all measurements were performed on frozen samples at the end of the study. In addition, none of the samples displayed a reactive pattern.
We suggest that IFE should be performed routinely on specimens with increased ß1 or ß2 concentrations, especially if there is no obvious reason to account for the increase, e.g., iron deficiency or increased ß-lipoprotein or C3, even if there are no abnormalities in the SPE pattern. A monoclonal gammopathy of undetermined significance (MGUS) is marked by the presence of monoclonal immunoglobulin without evidence of plasma cell dyscrasia (1)(2)(3). The temporal pattern of risk of multiple myeloma after detection of MGUS has no plateau, and multiple myeloma can supervene more than 30 years after the initial detection of gammopathy (1). Because it is not possible to predict malignant conversion, patients with MGUS need long-term follow-up to avoid any delay in the diagnosis of overt disease (1)(2)(3). This gives enough justification to closely explore all the possible diagnostics available for identifying monoclonal gammopathies. Our study adds to the existing evidence that it is possible to miss some M-spikes present in the ß1 or ß2 region when high-resolution electrophoresis is used for detection of monoclonal gammopathies and that if densitometry reveals increased ß1- or ß2-globulins, these samples should be subjected to IFE.
This study was conducted with 36 consecutive patient samples with increased ß1- or ß2-globulins. Twelve (33%) of the 36 had monoclonal gammopathies that would have been missed if they had been evaluated with the existing criteria used by most clinical laboratories (IFE is prompted by clinical suspicion, a M-spike, or altered mobility in the 2-, ß1-, ß2-, or -globulin regions). The SPE patterns observed in this series suggest that it is not always possible to identify a M-spike visually or densitometrically. Because there was no difference in the ß-globulin concentrations between the two populations, no threshold other than the reference interval could be established for performing an IFE. Thus, the protein concentrations in the ß1 and ß2 fractions, although increased, are of no value in predicting the presence of a monoclonal band.
Our experience shows that performing IFE on all samples that have high ß1- or ß2-globulin concentrations on densitometry is more reliable than visual comparison alone. As there appears to be a high prevalence of a monoclonal component in the ß-globulin region without any obvious anomaly in the gel pattern, it could be worth pursuing cases with high ß-globulin concentrations. Of the 12 samples that were identified as M-spike-positive, 2 patients have been diagnosed as having multiple myeloma. This further underscores our recommendation. In addition, it does not add significantly to the overall number of IFE procedures that will need to be performed because over a 10-month period, only 36 samples met our inclusion criteria. Considering the costs per test of SPE and IFE together, which are approximately $18.00 and $23.00 (SEBIA System), respectively, it should not be a financial burden and would clearly be advantageous in providing valuable information and facilitating management.
In conclusion, it appears that samples with M-spikes may still be missed with high-resolution SPE, and it is imperative that any sample with a high ß1 or ß2 band on densitometry be subjected to IFE because this will help confirm the presence of an underlying M-spike, even in the absence of any obvious visual abnormality.
Acknowledgments
This work was supported by NIH Grant K24 AT 00596.
References
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