Serum Proteins Capillary Zone Electrophoresis: A Comparison of Two Systems

Science Received 9.1.05 | Revisions Received 11.8.05 | Accepted 11.9.05

Serum Proteins Capillary Zone Electrophoresis: A Comparison of Two Systems Alessandro Terreni, Anna Caldini, Agostino Ognibene, Benedetta Salvadori, Gianni Messeri (Central Clinical Biochemistry Laboratory, Hospital of Careggi, Florence, Italy) DOI: 10.1309/E8P5GXDWAKDP3B0Q

We compared the analytical performance of the CE instrument Paragon CZE (Beckman) and the new instrument Capillarys recently developed by Sebia. Within- and between-day imprecision, variability among capillaries, interference from hemoglobin and fibrinogen, linearity, and sensitivity for monoclonal components (MC) detection have been estimated. For comparison studies, 313 unselected sera were simultaneously processed with both instruments.

Within-day CV’s were 1.07% and 0.62% for albumin, 3.98% and 3.90% for α1, 2.41% and 4.17% for α2-, 1.58% and 3.69% for β-, and 1.20% and 1.78% for γ- globulins; betweenday CV’s were 0.90% and 0.78% for albumin, 6.43% and 3.80% for α1, 5.70% and 4.53% for α2-, 3.72% and 2.66% for β-, and 2.19% and 2.32% for γ- globulins for Capillarys and Paragon CZE, respectively. Bland-Altman (n=273) analysis showed low mean differences between the electrophoretic fractions measured by the 2 instruments, but all the

Over the last years capillary zone electrophoresis has emerged as a powerful tool for the separation of proteins and other biopolymers,1 including serum protein fractions.2 In clinical use, it allows the detection of monoclonal immunoglobulins (MCs). In comparison to traditional electrophoretic methods, Paragon CZE (Beckman Coulter, Fullerton, CA) offers several advantages: automation, high resolution,3,4 and faster turnaround time.5 More recently, a new capillary electrophoresis instrument has been developed by Sebia (Capillarys, Sebia, Issyles-Moulineaux, France). Considering the importance of automation and reproducibility of serum electrophoresis, we compared the analytical performance of the 2 instruments: Paragon CZE and Capillarys. As a further goal, we evaluated the ability of MCs (n=175) detection of both instruments.

Materials and Methods Instruments The Paragon CZE system basically consists of a sample unit which accommodates the primary tubes and a phoresis chamber equipped with 7 fused silica capillaries (20 cm in length and 25 µm inner diameter). Proteins are measured by direct absorption at 214 nm through a small optical window in the capillary. The sample carousel consists of 10 sectors each containing 7 primary tubes and 1 segment with 7 traps for dilutions.4 The protein separation occurs in 4.3 min, at 10.3 kV and 24°C.5 The software version used was 1.6.02, with modified buffer and more efficient cooling, with respect to the previous versions. labmedicine.com

fractions demonstrated a systematic difference, possibly due to a difference in electrophoretic mobility. The 2 systems showed a substantially superimposable ability in revealing MCs (n=175). The detection limit of MCs was 0.4 g/L for both systems. The analytical performance of the new instrument Capillarys is substantially comparable to that of Paragon CZE.

Capillarys instrument includes a flowing tape for sample handling, a system of 8 fused silica (17 cm in length and 25 µm inner diameter) capillaries arranged in parallel for the analysis, and a computer with software (4.41 version) for the management of operation and results. Ultraviolet detection at 200 nm is used for protein quantification. The system is provided with sample racks for the primary tubes and segment for dilutions, each consisting of eight traps. The electrophoretic migration occurs in 2.30 min at 9KV and 35°C. The temperature is controlled by Peltier’s effect. Both instruments were used as indicated by the manufacturer. Patient Samples The specimens used for our study were patient samples submitted to the laboratory for serum protein analysis. For the comparison study, a total of 313 serum specimens were examined, but only 273 specimens were considered for comparison studies (the criteria was that there be no suspicion for MC and an albumin/globulin ratio >0.8).4 Serum protein electrophoresis was performed by Cellulose Acetate Electrophoresis (CAE; CTE8000 JOKOH), and by capillary zone electrophoresis with both instruments used by 2 different operators in a blind mode. In our laboratory, when the presence of MC is suspected, the sample is then run by Immuno Fixation Electrophoresis (IFE) (Hydrasys System, Sebia). Monoclonal component evaluation was performed on the other 175 MC samples, selected on the basis of positive IFE results, that revealed the presence of 24 β-fraction MCs, 129 γ-fractions MCs, 13 oligoclonal patterns, and 9 biclonal profiles. For the evaluation of MC linearity, we used a sample with a γ-fraction MC immunotyped as IgGk (12.8g/L), spiked with April 2006 䊏 Volume 37 Number 4 䊏 LABMEDICINE

233

Downloaded from http://labmed.oxfordjournals.org/ by guest on November 16, 2016

Abstract

Science Table 1_Imprecision of Capillary Electrophoresis Serum Proteins (n=20) Albumin Sebia Control

Capillarys Mean CV% SD Paragon CZE Mean CV% SD

Alpha 1

Alpha 2

Gamma

Within Day

Between Day Within Day

Between Day Within Day

Between Day Within Day

Between Day Within Day Between Day

56.59 1.07 0.60

60.04 0.90 0.54

5.77 3.98 0.23

4.81 6.43 0.31

9.83 2.41 0.23

8.64 5.70 0.49

12.35 1.58 0.19

11.96 3.72 0.44

15.44 1.20 0.18

14.53 2.19 0.31

57.38 0.62 0.35

61.26 0.78 0.48

6.75 3.90 0.26

5.76 3.80 0.21

11.22 4.17 0.46

9.58 4.53 0.43

10.46 3.69 0.38

9.87 2.66 0.26

14.20 1.78 0.25

13.27 2.32 0.30

Table 2_Regression Analysis (Capillarys vs. Paragon CZE) Coefficient of Correlation (Pearson)

P-Value (t test)

Albumin α1-globulin α2-globulin β-globulin γ-globulin

0.93 0.97 0.91 0.91 1.00

0.95 0.76 0.95 0.91 0.99