Clinical Chemistry / Comparison of ElECtrophorEtiC mEthods

Performance Comparison of Capillary and Agarose Gel Electrophoresis for the Identification and Characterization of Monoclonal Immunoglobulins
Christopher R. McCudden, PhD,1 Stephanie P. Mathews, MD,1 Shirley A. Hainsworth,2 John F. Chapman, DrPH,1 Catherine A. Hammett-Stabler, PhD,1 Monte S. Willis, MD, PhD,1 and David G. Grenache, PhD1
Key Words: Serum protein electrophoresis; Agarose gel electrophoresis; Immunofixation electrophoresis; Capillary electrophoresis; Immunotyping electrophoresis; Immunosubtraction electrophoresis; Monoclonal gammopathy; Multiple myeloma
DOI: 10.1309/6KT8N49BRNVVVBT1

Abstract
The objective of this study was to compare geland capillary-based serum protein electrophoresis methods to identify and characterize monoclonal immunoglobulins (M proteins). Five reviewers interpreted 149 consecutively ordered serum specimens following agarose gel electrophoresis (AGE), capillary electrophoresis (CE), immunofixation electrophoresis (IFE), and subtraction immunotyping (IT). As a screening test for detecting M proteins, AGE and CE displayed similar sensitivity (91% and 92%, respectively). CE was less specific (74%) than AGE (81%). An analysis of interinterpreter agreement revealed that interpretations were more consistent using gel-based methods than capillary-based methods, with 80% of the gel interpretations being in complete (5/5) agreement compared with 67% of the capillary interpretations. After implementing the capillary-based methods, the number of tests per reportable result increased (from 1.58 to 1.73). CE is an analytically suitable alternative to AGE, but laboratories implementing it will need to continue IFE testing to characterize all M proteins detected by CE.

Serum protein electrophoresis is frequently used to identify monoclonal gammopathies such as multiple myeloma, Waldenstrm macroglobulinemia, and other plasma cell dyscrasias. Protein electrophoresis most often involves the separation of serum proteins by passing an electric current through an appropriate electrolytic solution and a solid support medium such as agarose. Currently, agarose gel electrophoresis (AGE) is the most commonly used clinical method for protein electrophoresis. Within the last decade, capillary electrophoresis (CE) has been adapted for use in clinical laboratories. CE is a liquid-based system that uses small bore (10-100 m) fused silica tubes rather than agarose gels and has several advantages over AGE, including rapid separation and automation, resulting in quicker turnaround of patient results. CE is generally accepted as a sensitive and specific means to detect qualitative serum protein abnormalities.1-10 When qualitative abnormalities that suggest a monoclonal immunoglobulin are observed following AGE or CE, immunofixation electrophoresis (IFE) is used to confirm the presence of monoclonal bands and to identify the type of paraprotein. IFE incorporates the use of specific antisera to heavy and light chains to precipitate immunoglobulins following electrophoresis and is a highly sensitive and specific method to detect and classify monoclonal immunoglobulins.11 Most laboratories routinely include antisera for , , and heavy chains and and light chains. In parallel with the development of the CE format, an alternative method for identifying monoclonal immunoglobulins has been developed. Subtraction immunotyping electrophoresis (IT) separates serum proteins following brief incubation of serum in the presence of antisera for each of the heavy and light chains, thereby removing them and enabling the qualitative detection
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of monoclonal immunoglobulins based on their absence in comparison with the serum protein electrophoresis performed without antisera. Although capillary-based methods have been used for several years, only a handful of reports have compared capillary-based methods (CE and IT) with the traditional AGE and IFE.1,4-7,10-13 Existing reports support greater sensitivity for the CE method in detecting serum protein abnormalities with an associated lower specificity.1,6 In contrast, IT is less sensitive than IFE for the identification of monoclonal immunoglobulins in some studies.1,6 Specifically, IT is reported to be more prone to missing free light chain monoclonal immunoglobulins and monoclonal immunoglobulins present at low concentrations.8,10,12 Although these previous studies have focused on comparing methods, several aspects regarding the practical and clinical use of this new technology have not been previously investigated. The objectives of this study were to compare interindividual interpretations of AGE and IFE with CE and IT with respect to sensitivity and specificity, determine the clinical relevance of method sensitivity, compare techniques for the quantitation of monoclonal immunoglobulins, and determine the consistency of interpretations using the different techniques.

capillary at constant high voltage (7700 V). For each patient specimen, diluted serum was automatically injected into the capillaries and electrophoresed at 35.5C. Proteins were directly detected by monitoring the absorbance at 200 nm to generate an electrophoretogram. Immunotyping was performed as for CE except that specimens were incubated with specific antisera against , , and heavy chains, as well as and light chains. The patterns were interpreted by superimposing the resulting electrophoretogram on top of one obtained without the use of antisera. Examples of all 4 techniques are shown in Figure 1. Method Performance Characteristics To determine the sensitivity and specificity of each method, results from each were interpreted by 5 reviewers (C.R.M., S.P.M., J.F.C., C.A.H.-S., and M.S.W.) blinded to clinical information, previous results, and each others interpretations. The mean performance characteristics of each method were determined by comparing the individual interpretations with a consensus interpretation of the IFE gel used as the gold standard. The consensus interpretations were based on agreement among all 5 interpreters. When discrepancies between interpretations arose, a 4 of 5 majority was used as the final interpretation. If fewer than 4 of 5 of the interpretations agreed, the group reexamined the specimens together to reach a consensus. The performance data were analyzed using a nonparametric analysis of variance (Kruskal-Wallis test). Clinical Relevance of Method Sensitivity To estimate the impact that the use of capillary techniques may have on follow-up procedures, additional clinical data for patients with monoclonal proteins observed with IFE but not IT were obtained from the medical records. Guidelines from the International Myeloma Working Group were used and included a calcium level of more than 11.0 mg/dL (2.75 mmol/L), a creatinine level of more than 2.0 mg/dL (176.8 mol/L), a hemoglobin concentration of less than 10.0 g/dL (100 g/L) for women or less than 11.1 g/dL (111 g/L) for men, and evidence of bone lesions.14 Quantitation of Monoclonal Immunoglobulins To establish the correlation between AGE and CE methods for quantifying monoclonal proteins, specimens with monoclonal immunoglobulins that exceeded the limit of quantification (0.3 g/dL [3 g/L] determined in-house during method validation) were analyzed by Deming regression and Spearman correlation. Monoclonal Immunoglobulin Characterization in Hypogammaglobulinemic Specimens Because it is a subtractive method, the ability of IT to correctly characterize monoclonal immunoglobulins in specimens
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Materials and Methods

Study Design This was a retrospective study using data from 149 consecutively received serum specimens sent to the laboratory for physician-ordered serum protein electrophoresis tests. These specimens included patients being evaluated or treated for B-cell dyscrasias and patients with a history of bone marrow transplantation for multiple myeloma or leukemia. There were no a priori exclusions. The study was reviewed and approved by the University of North Carolina (Chapel Hill) Human Research and Ethics Board (IRB No. 06-0740). Electrophoretic Techniques AGE and IFE were performed on the Hydrasys (Sebia, Norcross, GA) according to the manufacturers directions. For AGE, 10 L of serum was applied to 0.8% agarose gel in a proprietary Tris-barbital buffer (pH 8.6) (Sebia) and subjected to electrophoresis for 7 minutes at 10 W (36 volt hours; Vh). Proteins were stained with amido black and destained, and an electrophoretogram was generated by scanning densitometry. For IFE, serum proteins were separated in an alkaline buffer (pH 9.1) for 9 minutes at 20 W (42 Vh) and then precipitated in the presence of specific antisera to , , and heavy chains as well as and light chains. Proteins were stained with acid violet following the removal of unprecipitated protein. CE and IT were performed on the Capillarys II (Sebia) according to the manufacturers directions. For both methods, proteins were separated using a proprietary borate buffer with additives (pH 9.9) (Sebia) through a narrow-bore fused silica
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B
IFE

AGE

CE

IgG

IgG IgA IgM

IgA

IgM

Figure 1 Examples of the electrophoretic techniques used in the study. A serum specimen containing an IgG monoclonal immunoglobulin was analyzed by agarose gel electrophoresis (AGE) (A), immunofixation electrophoresis (IFE) (B), capillary electrophoresis (CE) (C), and capillary immunotyping (D-H). The shaded area in panels D through H represents the electrophoretogram following removal of the indicated heavy or light chain. Note the removal of the IgG monoclonal protein in panels D and H.

with hypogammaglobulinemia (-globulin concentration, <0.5 g/dL [5 g/L]) was determined by comparing IT interpretations with those derived from IFE.

Results
Method Performance Characteristics We analyzed 149 serum specimens by AGE, CE, IFE, and IT with results interpreted by 5 reviewers. A consensus interpretation of the IFE results was considered the gold standard and was obtained for 146 (98.0%) of the specimens. Because no consensus interpretation could be reached for 3 specimens, they were excluded from further analysis.

Interpretations of these specimens ranged from no abnormality detected to the presence of a faint restriction suggestive of a monoclonal protein with or without an oligoclonal or polyclonal background. The performance characteristics of AGE, CE, and IT were determined by comparing each with IFE and are shown in Table 1 as the mean and 95% confidence interval derived from the interpretations by the 5 interpreters. Of the 146 specimens included in the study, 91 (62.3%) contained a monoclonal immunoglobulin as identified by IFE. The sensitivity of CE for detecting monoclonal immunoglobulins was 91.5% and was slightly higher than that for AGE (90.9%), which had a higher specificity (Table 1).

Table 1 Method Performance Characteristics for the Detection of Monoclonal Immunoglobulins Compared With Immunofixation Electrophoresis as the Gold Standard*
Electrophoresis Method Agarose Gel Sensitivity Specificity Positive predictive value Negative predictive value
*

Capillary 91.5 (85.9-97.1) 73.7 (60.1-87.3) 87.4 (80.8-93.9) 84.3 (78.8-89.8)

Immunotyping 84.9 (81.7-88.1) 93.8 (91.0-96.6) 95.9 (94.2-97.7) 78.2 (74.4-82.1)

90.9 (86.2-95.6) 81.0 (71.7-90.4) 89.4 (84.4-94.3) 84.1 (78.8-89.3)

Data are given as mean (95% confidence interval). 91 specimens with monoclonal immunoglobulins. 55 specimens without monoclonal immunoglobulins. Disease prevalence was 62%.

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The sensitivity of IT as a screening test was 84.9%, which was lower than either screening method, yet it demonstrated the highest specificity (93.8%). The observed differences in performance characteristics were not statistically significant (P = .443). Table 2 shows the interinterpreter agreement between IFE and IT interpretations among 78 specimens identified as abnormal by IT. Table 2 shows the combined interpretations for each of the 5 interpreters of the 78 specimens (390 total interpretations; 430 results are presented because multiple bands were identified in some specimens). Overall, the IFE and IT methods showed 86% agreement for characterizing the isotype of monoclonal immunoglobulin(s) present in these specimens. Using IT, the 5 interpreters failed to correctly characterize 31 (7.2%) monoclonal immunoglobulins that were observed by IFE. Conversely, there were 12 (2.8%) cases in which monoclonal immunoglobulins were characterized by IT but were not evident by IFE. Last, interpreters using the IT method were prone to missing monoclonal free

light chains in the presence of other monoclonal proteins, and none of the 7 free light chain interpretations identified by IFE were reported by interpreters using IT (Table 2). Clinical Relevance of Method Sensitivity Of the 91 specimens that contained monoclonal immunoglobulins following IFE, 5 (5%) were not identified as abnormal by interpreters using CE or IT. The medical records of these patients were examined for clinical information and other biochemical indicators of possible multiple myeloma using diagnostic criteria defined by the International Myeloma Working Group.14 Data are shown in Table 3. All patients had biochemical evidence of impairment to at least one organ. In addition, 2 patients had a history of a plasma cell dyscrasia (plasmacytoma or multiple myeloma) and 4 had a history of chronic renal insufficiency. Only the patient with a history of plasmacytoma underwent bone marrow biopsy at the time of specimen collection for electrophoresis testing. The biopsy specimen was notable for hypocellularity with no evidence

Table 2 Characterization of Monoclonal Immunoglobulins by IT Compared With IFE*

IFE IT G G A A M M Free Free Other ND Total G 178 G 6 105 1 1 4 182 11 124 7 33 2 27 A A 1 26 23 1 M 1 1 19 1 1 23 M 2 Free Free 1 Other ND 5 4 1 1 0 0 6 7 1 0 12 Total 194 110 27 23 21 16 1 2 5 31 430

15

5 5

17

IFE, immunofixation electrophoresis (gel); IT, immunotyping electrophoresis (capillary); ND, not detected. * Numbers represent the combined interpretations for each of 5 interpreters of 78 specimens (390 total interpretations). A total of 430 results are shown because multiple bands were identified in some specimens. The bold numbers represent interpretations in agreement. G heavy-chain disease.

Table 3 Clinical Biochemical Results and History of Patients With a Monoclonal Immunoglobulin Identified by Immunofixation Electrophoresis Only*
Monoclonal Immunoglobulin IgG IgM Free IgG IgG Hemoglobin, g/dL (13.5-17.5) 10.2 11.8 14.2 10.8 15.3 Creatinine, mg/dL (0.8-1.4) 4.6 2.5 2.1 1.4 1.5 Calcium, mg/dL (8.5-10.2) 8.1 8.7 9.6 8.7 8.8 Clinical History Plasmacytoma; postkidney and pancreas transplant lymphoproliferative disorder Chronic renal insufficiency; MGUS Stage III chronic kidney disease; HIV and HCV infections Cardiomyopathy; renal insufficiency; hairy cell leukemia Multiple myeloma; postautologous stem cell transplantation; renal insufficiency

HCV, hepatitis C virus; MGUS, monoclonal gammopathy of undetermined significance. * Numbers in bold indicate results outside the reference interval. All patients were men aged 45-89 years. Laboratory values are given in conventional units. Conversions to Systme International units are as follows: hemoglobin concentration (g/L), multiply by 10.0; creatinine (mol/L), multiply by 88.4; calcium (mmol/L), multiply by 0.25. All monoclonal immunoglobulins were too low to quantify. Reference interval.

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Quantitation of Monoclonal Immunoglobulins The ability of CE to accurately quantify the concentration of monoclonal immunoglobulins that exceeded the limit of quantitation (0.3 g/dL [3 g/L]) was compared with that derived from scanning densitometry of AGE (n = 42). Deming regression produced a slope of 0.93 and a Y-intercept of 0.09. The Spearman correlation coefficient was r = 0.98. A Bland-Altman plot of these data is shown in Figure 2. Monoclonal Immunoglobulin Characterization in Hypogammaglobulinemic Specimens Before this study, it was observed that results from the subtractive IT method were difficult to interpret in specimens with hypogammaglobulinemia (-globulin concentration, <0.5 g/dL [5 g/L]). Subsequently, we hypothesized that monoclonal immunoglobulins present in such specimens might be missed or misidentified. To test this hypothesis, the IT interpretations from 17 hypogammaglobulinemic specimens were compared with those obtained by IFE Table 4. Of these 17 specimens, 9 contained monoclonal immunoglobulins by IFE. Of the 9, 4 were correctly characterized by IT by all 5 interpreters and 5 were mischaracterized by some interpreters. In these specimens, the isotypes of the monoclonal immunoglobulins were mischaracterized by IT or the interpreter noted an abnormality by IT when none was observed with IFE. Also, some interpreters failed to note monoclonal immunoglobulins by IT that were observed with IFE. Comparison of Interpreters To assess the consistency of interpretations using each of the electrophoretic methods (AGE, IFE, CE, and IT), the agreement among the 5 interpreters was evaluated for all 146 specimens. Agreement between interpretations for monoclonal immunoglobulin detection Figure 3A and characterization Figure 3B was determined. The number of specimens that were not in complete agreement varied widely depending on method. Overall, interpretations from gel-based techniques demonstrated greater agreement than did those from capillary-based methods. When using AGE methods to identify a monoclonal immunoglobulin, 75.3% (110/146) of interpretations were in complete agreement compared with only 63.0% (92/146) of the CE interpretations. Similarly, when using IFE for characterizing monoclonal proteins, the agreement between interpreters was higher. IFE interpretations were in complete agreement for 78.1% (114/146) of the specimens, whereas IT interpretations agreed completely in 62.3% (91/146).
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CE AGE (g/dL)

of a lymphoproliferative disorder. None of the other patients underwent bone marrow biopsies, nor was there radiologic evidence of lytic bone lesions or hypercalcemia in any of the patients at the time of specimen collection.

0.9 0.6 0.3 0.0 0.3 0.6 0.9 0 1 2 3 4 5 6 7

Mean Monoclonal Protein (g/dL)

Figure 2 Bland-Altman plot of the quantitation of monoclonal immunoglobulins by agarose gel electrophoresis (AGE) and capillary electrophoresis (CE). Dotted lines represent the 95% confidence intervals. Table 4 Characterization of Monoclonal Immunoglobulins in Hypogammaglobulinemic Specimens*
IT Interpretation by Interpreter No. Specimen No./IFE Interpretation 1 1 2 3 4 5 6 7 8 9 A G M A G A G A A G G A G M G A G A G A A G G 2 A Free M Free A G A G A A G G 3 A Free M A G A Unable to interpret A A None G 4 A M G A G A G A A None G 5 A M None G A None A A None G

IFE, immunofixation electrophoresis; IT, immunotyping electrophoresis. * Of 146 specimens, 17 were hypogammaglobulinemic (gamma globulin, <0.5 g/dL [5 g/L]) by scanning densitometry; 8 of 17 hypogammaglobulinemic specimens did not contain monoclonal proteins by any interpretation (not shown). Interpretations in bold represent mischaracterizations compared with IFE. None indicates an interpretation of no monoclonal immunoglobulin.

Tests per Reportable Result The lower sensitivity of IT suggested that IFE would still need to be performed to correctly characterize all monoclonal immunoglobulins. To determine the number of tests required to yield a reportable result, the number of tests
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A
100 % of Total Samples Gel Capillary

Table 5 Number of Tests Required to Produce a Reportable Result Before and After Implementing Capillary Electrophoresis
Before Implementation (December 1-31, 2005) After Implementation (April 1-30, 2007)

75 Test(s) Performed AGE AGE and IFE CE CE and IT CE and IFE CE, IT, and IFE Total No. of tests per reportable result

50

No. (%) of Specimens 95 (42) 133 (58) NA NA NA NA 228 1.58

No. (%) of Specimens NA NA 72 (30) 47 (19) 119 (49) 6 (2) 244 1.73

25

0 AGE
5/5 Agreement

IFE

CE
3/5 Agreement

IT
2/5 Agreement

4/5 Agreement

AGE, agarose gel electrophoresis; CE, capillary electrophoresis; IFE, immunofixation electrophoresis; IT, immunotyping electrophoresis; NA, test not available.

B
100 % of Total Samples Gel Capillary

Discussion
The primary objective of this study was to compare the performance of agarose gelbased and capillary-based protein electrophoresis methods for the detection and characterization of monoclonal immunoglobulins. A consensus interpretation from IFE was used as the gold standard. The performance of the 2 screening methods (AGE and CE) was comparable to that published in other studies1,6,8,10,12 in which CE was demonstrated to be slightly more sensitive than AGE at detecting monoclonal immunoglobulins. This modest increase in the sensitivity of CE was associated with a lower specificity, which is also consistent with other reports.1,6 The higher sensitivity of CE compared with AGE has been attributed to the avoidance of application artifacts and superior detection of -region migrating monoclonal proteins.1,6 In our experience, the lower specificity of CE is attributed to the direct spectrophotometric detection of proteins during electrophoresis, which can result in minor abnormalities in the electrophoretogram not typically observed in those derived from scanning densitometry. These subtle alterations, which were not confirmed by IFE, were more likely to be identified as abnormal by the interpreters in this study. The IT method was less sensitive than AGE or CE for the detection of monoclonal immunoglobulins, a finding that is also consistent with other reports.1,6 In particular, interpreters using IT were unable to detect monoclonal free light chains and low concentrations of monoclonal immunoglobulins, such as those often observed in patients with monoclonal gammopathy of undetermined significance.8,10,12 Following the detection of a monoclonal immunoglobulin, it is necessary to characterize its isotype. With combined characterization data from all 5 interpreters, the 78 IT-positive specimens showed 86% agreement with IFE
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75

50

25

0 IFE
5/5 Agreement 4/5 Agreement

IT
3/5 Agreement 2/5 Agreement

Figure 3 Agreement in 5 individual interpretations using different electrophoretic methods for monoclonal immunoglobulin detection (A) and characterization (B). AGE, agarose gel electrophoresis; CE, capillary electrophoresis; IFE, immunofixation electrophoresis; IT, immunotyping electrophoresis.

performed during a 30-day period before implementing capillary electrophoresis (December 1-31, 2005) was compared with a 30-day period 8 months after its implementation (April 1-30, 2007). As shown in Table 5, the number of tests per reportable result increased from 1.58 to 1.73 after implementing the Capillarys II system. IFE tests continued to be used frequently after implementing the new system because some interpreters relied on IFE more often than IT for making interpretations. This was attributed to the necessity of the IFE method when interpreting specimens that were hypogammaglobulinemic or contained a low concentration of monoclonal proteins.
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isotype characterization. Other studies have reported a wide range of agreement between gel- and capillary-based methods, with agreement ranging from 44% to 100% depending on the concentration of the monoclonal immunoglobulin.2,3,6,13 Of the 78 IT-positive specimens in this study, 42 contained a monoclonal immunoglobulin at a quantifiable concentration. Each of these specimens was correctly detected as abnormal by CE and IT and characterized correctly by IT. Conversely, specimens that contained monoclonal immunoglobulins at low concentrations (<0.3 g/dL [3 g/L]), monoclonal free light chains, or multiple (oligoclonal) bands were often incorrectly typed by interpreters using IT. It is also noteworthy that interpreters identified additional monoclonal immunoglobulins in 4 specimens following IT that were not evident by IFE (Table 2, ND column). These apparent false-positive characterizations were always in specimens that already contained a substantial quantity of monoclonal immunoglobulin that was correctly characterized. Given that these patients would be monitored and/or treated for the predominant paraprotein, it is unclear what, if any, clinical significance can be attributed to this observation. Before this study, it was hypothesized that interpreters might experience difficulties using the IT method to detect low-concentration monoclonal immunoglobulins. We observed that more than half (5/9) of the specimens that were interpreted to contain monoclonal immunoglobulins by IFE were mischaracterized by some interpreters when using the IT method. All of these samples contained monoclonal immunoglobulins at a concentration below the limits of quantitation (<0.3 g/dL [3 g/L]). These observations highlight the difficulty in using the subtractive IT method to characterize low-concentration monoclonal immunoglobulins. Although several studies have reported acceptable agreement between AGE and CE for quantifying the major serum protein fractions, there are fewer reports on the ability of CE to accurately quantify monoclonal immunoglobulins.8-10 In this study, 42 specimens contained a quantifiable paraprotein. At concentrations of less than 1.0 g/dL (10 g/L), CE had a slight positive bias, whereas a slight negative bias was observed at concentrations of more than 3.0 g/dL (30 g/L). The overall bias was 0.09 g/dL (0.9 g/L) for CE in comparison with AGE and is clinically insignificant. Other reports have indicated that quantitation by CE may be slightly higher or lower in comparison with analysis by scanning densitometry, with the variation attributed to differences in the detection methods.1,3 Whereas AGE uses protein staining and scanning densitometry, CE uses direct UV spectrophotometric detection of the proteins as they are separated during electrophoresis. Scanning densitometry is influenced by the protein concentration and the type of stain used and is associated with wide ranges of imprecision, particularly when used to quantify the -globulins.15 Conversely, the direct detection
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used by capillary electrophoresis is unaffected by staining and has a much lower level of imprecision.9 Regardless of these methodological differences, the data presented herein support the use of AGE or CE to accurately quantify monoclonal proteins. Because the presence of a monoclonal immunoglobulin by itself is not diagnostic of multiple myeloma, other criteria are also used to identify and diagnose the disease. These include biochemical evidence of end-organ damage, such as an increase in the serum level of calcium and/or creatinine, a decrease in hemoglobin concentration, and the presence of lytic bone lesions,14 which, collectively, are used for decision making about follow-up tests such as a bone marrow biopsy. To our knowledge, there are no studies to date that have addressed the clinical significance that the electrophoresis method may have on patients. In this study, we used diagnostic criteria defined by the International Myeloma Working Group14 to determine if the patients would be monitored or treated differently based on electrophoresis results. In this study population, 5% of specimens (5/91) contained a monoclonal immunoglobulin as determined by IFE interpretations that were not observed with CE or IT. Review of each patients medical record using the aforementioned diagnostic criteria for multiple myeloma revealed limited information. However, it is noteworthy that 2 of these patients had a history of a plasma cell dyscrasia. This history alone would warrant consistent monitoring of the patients and suggests that the lower sensitivity of the capillary techniques was not a liability. In all cases, there was biochemical evidence of organ damage (increased creatinine level, hypercalcemia, and/or decreased hemoglobin concentration), and, while it is possible that these patients had an underlying plasma cell dyscrasia, no definitive conclusions can be drawn without long-term follow-up studies. When the capillary-based methods were first introduced into our laboratory, an 11% increase in the number of tests performed per specimen was noted (data not shown). This increase was attributed to testing of specimens by both IT and IFE that occurred as the interpreters adjusted to a new electrophoretic method. To minimize this duplicate testing, a strategy was implemented whereby specimens containing a subtle abnormality by CE were characterized exclusively by IFE. This strategy effectively limited performing IT tests that could not be adequately interpreted. However, it should be noted that using this approach may result in a high number of IFE tests (49% of specimens in this study had IFE tests performed after implementation of the Capillarys II system). Another unique aspect of this study was the use of 5 experienced interpreters to examine the electrophoretic results of all specimens tested. Following independent review of the data, the interpretations were quantitatively evaluated for agreement. Generally, the interpretations of CE and IT
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agreed less often than those of AGE and IFE (Figure 3). The IT method was particularly prone to interpreter disagreement, in which only 62% of interpretations were in complete agreement in comparison with 78% of the IFE interpretations. This observation highlights the importance of the method on the consistency of interpretations. Laboratories planning to implement capillary-based techniques should take this into consideration to ensure consistency between multiple interpreters. The Capillarys II is a rapid alternative to traditional AGE and IFE for the identification and characterization of monoclonal immunoglobulins. Although the overall performance of both systems is effective, there are some limitations to CE and IT that necessitate immunofixation testing for some specimens. IFE was the most consistently interpretable technique for the detection and characterization of monoclonal immunoglobulins. Additional studies are required to establish if methodological differences are clinically significant for patient care and monitoring.
From the 1Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, and 2W.W. McLendon Clinical Laboratories, UNC Hospitals, Chapel Hill. Address reprint requests to Dr Grenache: Dept of Pathology, University of Utah School of Medicine and ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108.

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4. Claeys R, Groven C, Gorus FK. Capillary zone electrophoresis of proteins in body fluids: comparison of capillary and agarose gel electrophoresis. Clin Chem. 2001;47:967-970. 5. Fernandez FJ, Rivas G, Salgado J, et al. Comparative evaluation of serum protein analysis by capillary zone electrophoresis and agarose gel electrophoresis. Am Clin Lab. 2002;21:26-29. 6. Katzmann JA, Clark R, Sanders E, et al. Prospective study of serum protein capillary zone electrophoresis and immunotyping of monoclonal proteins by immunosubtraction. Am J Clin Pathol. 1998;110:503-509. 7. Lissoir B, Wallemacq P, Maisin D. Serum protein electrophoresis: comparison of capillary zone electrophoresis Capillarys (Sebia) and agarose gel electrophoresis Hydrasys (Sebia) [in French]. Ann Biol Clin (Paris). 2003;61:557-562. 8. Mussap M, Ponchia S, Zaninotto M, et al. Evaluation of a new capillary zone electrophoresis system for the identification and typing of Bence Jones protein. Clin Biochem. 2006;39:152-159. 9. Bossuyt X, Lissoir B, Marien G, et al. Automated serum protein electrophoresis by Capillarys. Clin Chem Lab Med. 2003;41:704-710. 10. Jolliff CR, Blessum CR. Comparison of serum protein electrophoresis by agarose gel and capillary zone electrophoresis in a clinical setting. Electrophoresis. 1997;18:1781-1784. 11. Salkie ML. A retrospective study of the relative utility of electrophoresis, immunoelectrophoresis, immunofixation, and nephelometry in the investigation of serum proteins. Clin Biochem. 1996;29:39-42. 12. Bakshi NA, Gulbranson R, Garstka D, et al. Serum free light chain (FLC) measurement can aid capillary zone electrophoresis in detecting subtle FLC-producing M proteins. Am J Clin Pathol. 2005;124:214-218. 13. Smalley DL, Mayer RP, Bugg MF. Capillary zone electrophoresis compared with agarose gel and immunofixation electrophoresis. Am J Clin Pathol. 2000;114:487-488. 14. Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group. Br J Haematol. 2003;121:749-757. 15. Keren DF. Protein Electrophoresis in Clinical Diagnosis. New York, NY: Oxford University Press; 2003.

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