advances in serum protein electrophoresis

ADVANCES IN CLINICAL CHEMISTRY, VOL. 42

ADVANCES IN SERUM PROTEIN ELECTROPHORESIS

Xavier Bossuyt

Laboratory Medicine, Immunology, University Hospitals Leuven,Herestraat 9, B‐3000 Leuven, Belgium

1. A

0065DOI

bstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

43

-2423/06 $35.00 Copyright 200: 10.1016/S0065-2423(06)42002-3 All

6, Elsevierrights rese

44

2. I
ntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3. T
echnique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4. I
nstruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
5. P
recision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
6. C
omparison with Gel‐Based Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
7. R
eference Intervals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
8. E
lectrophoretic Patterns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
9. O
nline Processing of Digital Absorbance Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
10. D
etection of Monoclonal Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
1
0.1. D etection of Monoclonal Proteins by Paragon CZE 2000w. . . . . . . . . . . . . 58
1
0.2. D etection of Monoclonal Proteins by Capillarysw . . . . . . . . . . . . . . . . . . . . . 61
1
0.3. D etection Limit for Monoclonal Proteins by CZE . . . . . . . . . . . . . . . . . . . . 63
1
0.4. D etection of Heavy Chain Disease by CZE . . . . . . . . . . . . . . . . . . . . . . . . . . 65
1
0.5. D etection of Monoclonal Cryoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
11. T
yping of Monoclonal Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
12. U
rine and Body Fluid Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
1
2.1. U rine Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
1
2.2. A nalysis of Pleural and Ascitic Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
13. Q
uality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
14. I
nterferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
1
4.1. D etector Interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
1
4.2. F ibrinogen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
1
4.3. I n Vitro Hemolysis, Bilirubin, and Lipid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
1
4.4. C omplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
1
4.5. G elatin‐Based Plasma Substitutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
15. ‘‘
Open’’ Capillary Electrophoresis Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
16. C
onclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
R
eferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Inc.rved.

44 XAVIER BOSSUYT

1. Abstract

A major advance in serum protein electrophoresis in the last decade has

been the introduction of capillary zone electrophoresis (CZE). Two dedicat-

ed automated multichannel instruments for serum protein separation by

CZE in clinical laboratories are available, the Paragon CZE 2000w (Beckman

Coulter, CA) and the Capillarysw (Sebia, France). This chapter focuses on

the performance of these commercial multichannel CZE systems. Following

topics are addressed: precision, comparison of CZE with gel‐based methods,

dysproteinemia analysis by CZE, detection and identification of mono-

clonal proteins by CZE, quality control, and interferences. Examples of

electrophoretic patterns are given.

2. Introduction

In clinical laboratories serum protein electrophoresis has traditionally

been performed with the use of agarose or cellulose acetate gels. In the last

10 years, classic gel electrophoresis (and immunofixation) has been (partly)

automated. This improved the reproducibility and the hands‐on time of the

techn ique [1] . Besides , capil lary zone electroph oresis (CZE) has e merged as

an attractive alternative to the gel‐based methods, especially for clinical

laboratories that have to deal with a large daily serum protein electrophoresis

workload. The first automated multichannel instrument developed exclusive-

ly for serum protein separation in clinical laboratories was the Beckman

Coulter Paragon 2000w Clinical Capillary Electrophoresis instrument

(Beckman Coulter, Brea, CA). It became commercially available in the late

1990s. A similar dedicated multichannel automated system, the Sebia Capil-

larysw (Lisses, Evry Cedex, France), was introduced in 2001. The features of

these two systems will be overviewed. The chapter partly recapitulates a

previou s review [2] on this topic and further extends on it.

3. Technique

With CZE, separation of the protein fractions occurs in free solution in a

narrow‐bore fused silica capillary that is exposed to a high voltage (Fig. 1).

A borate‐based buVer, pH 10, is used. At alkaline pH, serum proteins are

negatively charged. When voltage is applied, two forces act on the proteins

(Fig. 2). First, the negatively charged proteins are attracted toward the

positive electrode (anode) (force of the electrical field). Second, an electro-

osmotic force is involved. At pH 10, the internal surface of the fused‐silica

FIG. 1. Schematic diagram of the CZE technique.

FIG. 2. Principle of protein separation by CZE. Forces acting on proteins in CZE.

ADVANCES IN SERUM PROTEIN ELECTROPHORESIS 45

46 XAVIER BOSSUYT

capillaries is negatively charged due to ionization of the silanol groups.

Cations in the electrolyte near the capillary wall migrate toward the negative

electrode (cathode), pulling electrolyte solution with them. This flow of

electrolyte solution toward the cathode constitutes the electroosmotic flow.

Thus, the force of the electrical field and the electroosmotic force act in

opposite directions. In CZE, the force of the electroosmotic flow surpasses

the force of the electrical field. As a result, all proteins are carried toward the

cathode. Since prealbumin and albumin (pI 4.7) are more acidic (carry more

negative charges) than the �‐globulins (pI 7.2), prealbumin and albumin are

more resistant to the electroosmotic flow than are the �‐globulins. As a

result, prealbumin and albumin are the last to reach the cathode. At the

cathodal end of the capillary, proteins are detected and quantified real time

by absorbance measurement (200–214 nm) through an optical window. The

absorbance at 200–214 nm is proportional to the number of peptide bonds.

The borate buVer does not interfere with the UV detection.

4. Instruments

The Beckman Coulter Paragon CZE 2000w may contain up to 10 racks of

7 tubes. Ten microliters of the serum sample is 1:20 diluted with the signal

reagent in dilution segments. Thereafter, samples are hydrodynamically

injected for 1 s by vacuum. Separation is achieved by applying a voltage of

10.5 kV for 4.3 min in seven fused‐silica capillaries (total/eVective length:

20/18 cm; 25‐mm i.d.) in a borate buVer (pH 10). The temperature is con-

trolled at 24�C by Peltier eVect. Online detection is at 214 nm in an optical

cell (75‐mm i.d.). The throughput of the system is �42 samples/hour.

The Capillarysw instrument may contain up to 13 racks of 8 sample tubes.

Forty microliters of the serum sample is 1:5 diluted with the migration buVerin dilution segments. Thereafter, samples are hydrodynamically injected for

1 s by anodic depression (injected volume, �1 nl). Separation is achieved by

applying a voltage of 7.8 kV for 3.96 min in eight fused‐silica capillaries

(total/eVective length: 17.5/15.5 cm; 25‐mm i.d.) in a borate buVer (pH 9.9)

containing additives. The temperature is controlled at 35.5�C by Peltier

eVect. Online detection is at 200 nm in an optical cell (100‐mm i.d.). The

throughput of the system is �90 samples/hour.

For both instruments, the manufacturers provide ready‐to‐use buVer andcapillary wash solutions.

With both instruments, serum proteins are separated in five zones: albu-

min, �1‐globulin, �2‐globulin, �‐globulin, and �‐globulin (Fig. 3). Prealbu-

min is visible and in the �‐globulin fraction, transferrin is separated from

complement. With Capillarysw, a high‐resolution kit is available in which

FIG. 3. Protein separation on the Paragon CZE 2000w instrument.


�1‐acid glycoprotein is separated from �2‐antitrypsin and �2‐macroglobulin

from haptoglobin.

5. Precision

Several laboratories have reported coeYcients of variation for protein

fraction ation by Paragon 2000 w [3–8] and by Capi llarys w [9–11] . With bot h

systems, coeYcients of variation (%) for the five classic protein fractions were

consistently <8% in intrarun and <11% in between‐run imprecision studies

(number of aliquots test ed varie d be tween 20 and 120) [3–11] . The impreci-

sion increased as the relative percentage of the fraction decreased. The lowest

coeYcients of variation were found for the albumin fraction (<3.5%), where-

as the highest coeYcients of variations were found for the �1‐globulin frac-

tion (< 8% in intr arun and < 11% in be tween ‐ run studi es; n � 20). Lur aschi

et al . [12] rep orted the long ‐ term precision (205 determinat ions ov er a 1‐ yearperiod) from an internal quality control scheme. The overall imprecision,

expressed as coeYcients of variation, was: 1.2% for albumin, 6.1% for the

�1‐globulin fraction, 3.2% for the �2‐globulin fraction, 3.2% for the �‐globulinfraction, and 4.3% for the �‐globulin fraction. The long‐term imprecision

(n ¼ 498) obtained at our institution (unpublished results, Paragon 2000w,

software 1.6) was 0.92% for the albumin fraction, 5.05% for the �1‐globulinfraction, 4.87% for the �2‐globulin fraction, 5.98% for the �‐globulin fraction,

and 2.43% for the �‐globulin fraction.

48 XAVIER BOSSUYT

Lur aschi et al . [13] also invest igated the varia tion of qua ntification of

monoclonal proteins by CZE and found that the imprecision was negatively

and nonlinearly related to the concentration of the monoclonal protein. The

imprecision (coeYcient of variation) was 2.5%, 5%, and 15% for quantifica-

tion of a monoclonal protein that amounted to, respectively, 40% of total

protein, 14% of total protein, and 5% of total protein.

In conclusion, CZE analysis is a highly reproducible technique, due to

automation and direct measurement of protein absorbance.

6. Comparison with Gel‐Based Techniques

The Paragon 2000 system has been compared to cellulose acetate gel electro-

phoresis [4, 5, 8, 14] and to agarose gel electrophoresis [5, 6, 15]. Capillarysw has

been compared to agarose gel electrophoresis [9–11] and to the Paragon 2000w

CZE system [9]. In all studies, the highest correlation coeYcients were found for

the �‐globulin fraction (r between 0.96 and 0.99 in most studies) and the

albumin fraction (r between 0.94 and 0.99 in most studies), whereas the lowest

correlation coeYcients were obtained for the �1‐globulin fraction (r between

0.73 and 0.92 in most studies) and the �‐globulin fraction (r between 0.52 and

0.91 in most studies). The low‐correlation coeYcient found for the �1‐ and�‐globulin fractions is probably related to the fact that delimitation of these

fractions might be diYcult. With Capillarysw, very low density lipoproteins

(VLDLs) increase the baseline between albumin and the�1‐globulin fraction inhyperlipemic samples and complicate the integration of the �1‐globulin frac-

tion. A computer‐assisted correction (available on Capillarysw with software

version 4.51 or higher), which deletes the area under the curve between albumin

and �1‐globulins bettered the automated integration of �1‐globulins with im-

provement of the analytical precision, and the correlation of Capillarysw and

agarose gel electrophoresis [1 0] .

For both Capillarysw and Paragon 2000w there was a significant diVerencebetween CZE and gel electrophoresis for the �1‐globulin fraction with higher

values given by CZE. For the albumin fraction, lower values were found with

CZE than with gel electrophoresis. The higher values found with CZE for the

�1‐globulin fraction are related to the high‐sialic acid content of �1‐acid glyco-

protein, which interferes with the binding of dyes in gel‐based systems. TheUV

absorption method used in CZE is not aVected by these sugar moieties.

7. Reference Intervals

Table 1 gives an overview of the published reference intervals obtained

with Paragon and with Capillarysw. In general, the reference intervals were

co ncordant [3, 5, 8, 9, 14–17] . Ref erence intervals for ch ildren betw een 1 and

TABLE 1

REFERENCE INTERVALS (%) FOR THE FIVE SERUM PROTEIN FRACTIONS OBTAINED BY CZE IN DIFFERENT LABORATORIES

References Instrument (software version) N Age (year) Albumin �1 �2 � �

[3] Paragon 52–67 3.8–8.3 5.8–12.4 9.8–13.0 9.3–20.4

[5] Paragon 82 18–65 (men) 60–70 3.9–6.4 4.9–9.3 8.4–12.7 7.9–18.3

(1.08) 79 18–65 (women) 56–70 3.6–7.4 5.6–9.7 8.0–12.7 7.9–18.3

[16] Paragon 57–70 3.8–7.8 4.4–10.0 7.8–12.8 9.0–17.4

[8] Paragon (1.16) 63 56–69 3.9–6.3 4.8–8.4 7.9–11.9 9.9–21.5

[14] Paragon 167 54–68 3.7–8.8 5.5–11.7 8.5–13.8 10–20.3

53–70 4.4–9.3 6.4–10.3 6.7–10.6 11–16.8

[18] Paragon (1.2) 33 1–2 55–70 4.2–8.5 7.0–15.6 7.5–11.6 4.7–16.0

44 3–4 54–70 4.8–8.1 7.6–15.2 7.4–11.6 7.1–17.8

70 5–9 53–66 4.2–7.6 7.4–13.5 7.9–11.3 8.5–18.7

48 10–14 54–69 4.4–8.0 6.8–11.4 8.5–12.9 8.8–17.6

[17] Paragon (1.6) 52.3–68.9 4.8–9.8 8.9–12.8 5.9–9.6 10.4–16.2

[9] Capillarys (1.41) 50 56–66 2.0–6.9 5.9–11.4 9.4–14.4 8.0–18.8

[11] Capillarys (4.14) 120 18–70; 60 males,

60 females

50.5–62.7 4.8–8.3 7.1–12.2 10.2–14.9 10.2–18.5

Sebia Capillarys (5.2.1) 55.8–66.1 2.9–4.9 7.1–11.8 8.4–13.1 11.1–18.8

Reproduced and modified from Ref. [2 ] with the permission of the publisher.

TABLE 2

REGRESSION EQUATIONS TO CONVERT REFERENCE VALUES OBTAINED WITH PARAGON CZE 2000W

WITH SOFTWARE 1.5 INTO VALUES EXPECTED WITH SOFTWARE 1.6

Zone

Regression equation Regression equation

Luraschi et al. [17] ( n ¼ 119) Marie n et al. [19] ( n ¼ 174)

Albumin y ¼ 0.55 þ 0.97x y ¼ 1.26 þ 0.95x

�1‐Globulin fraction y ¼ 0.35 þ 1.02x y ¼ 1.65 þ 0.87x

�2‐Globulin fraction y ¼ 2.50 þ 1.00x y ¼ 2.46 þ 0.98x

�‐Globulin fraction y ¼ –0.39 þ 0.94x y ¼ –0.40 þ 0.95x

�‐Globulin fraction y ¼ 0.47 þ 0.99x y ¼ –0.30 þ 0.98x

50 XAVIER BOSSUYT

14 years old are available as well [18] . Sign ificant ag e ‐relat ed chang es werefound for the �2‐ and �‐globulin fraction. The �2‐globulin fraction gradually

decreases with age and the immunoglobulin concentration gradually

increases with age in young children.

On the Paragon 2000w instrument, several software versions have been

released: 0.8, 1.1, 1.2, 1.5, 1.6, and 2. The release of software version 1.6

introduced new analytical conditions, including slightly more alkaline sepa-

ration buVer, increased separation voltage, and a new capillary cover for

improved cooling eYciency. This resulted in changed analytical conditions,

and it is recommendable that reference intervals obtained with earlier soft-

ware versions (most published reference intervals) are interconverted.

Regr essio n eq uations for va lue interc onversio n ha ve be en publ ished [17, 19]

an d are summarize d in Table 2.

8. Electrophoretic Patterns

The typical electrophoretic patterns revealed by classic cellulose acetate

and agarose gel electrophoresis in sera of patients with dysproteinemias such

as monoclonal gammopathy, the inflammatory syndrome (acute or chronic),

hypogammaglobulinemia, the nephrotic syndrome, polyclonal gammo-

pathy, �‐�‐bridging, cirrhosis, and iron deficiency anemia were also revealed

by Paragon 2000 w [3, 5] and by Capi llarys w [9] . The clini cal informat ion

obtained by CZE was comparable to the clinical information obtained by gel

electrophoresis. Examples of electropherograms typical for a variety of

dysproteinemias are illustrated in Figs. 4–13.

Bisalbuminemia is a genetic variant without pathological significance.

It can also be seen in patients with renal insuYciency receiving high doses of

drugs (antibiotics) and in patients with pancreatic disease. Jaeggi‐Groisman


et al. [20] found eight cases of bisalbumine mia with CZE ov er a 1 ‐ year periodin which they an alyzed 6500 specim ens. The pa tients recei ved no antibioti cs

and had no pan creatic disease. In a simila r an nual ana lysis load perfor med by

agarose elect rophoresi s, they detect ed only one case in 4 years. The increa sed

sensi tivity for detection of bisal buminem ia of CZE compared to gel electro-

phor esis was also report ed by Kala mbokis et al . [21] . They descri bed a case

of inheri ted bisal buminem ia that was detect ed by CZE but not by agarose

elect rophoresi s. An exampl e of bisalb uminem ia is shown in Fig. 4. CZE

reveal s slurring albumi n in patients recei ving high dos es of dru gs (Fig. 5).

�1‐ Antitryp sin and � 1‐ acid glycopr otein are the main clinicall y relev ant

protei ns of the �1‐ globuli n fraction. Thes e two protei ns are quan tified forrecogni zing and/or monitoring inflamm ation. Carrier s of �1‐ antitryps in g enemutat ions have a defi ciency of �1‐ antitryps in, whi ch is associ ated with p u l -monary and l iver diseases. The lowest �1 ‐ antitrypsin values are found i nindivi duals with ZZ and SZ phe notypes, intermed iate values in indivi duals

with SS, MZ, and MS phe notypes, and the highest values in ind ividuals with

the MM phen otype.

Luraschi et al . [22] rep orted that CZE measur ement of the �1‐globulinfraction systematically results in higher values than the sum of �1‐antitrypsin

FIG. 4. Bisalbuminemia. Bisalbuminemia is a genetic variant without pathological signifi-

cance. It can also be seen in patients with renal insuYciency receiving high doses of drugs

(antibiotics) and in patients with pancreatic disease.

FIG. 5. Slurring albumin. Slurring albumin (indicated by arrow) can be seen in patients

receiving high doses of drugs.

52 XAVIER BOSSUYT

and �1‐acid glycoprotein and that high‐density lipoprotein (apolipoprotein

A‐1) substantially contributes to such gap. Lipemic samples may have an

increased �1‐globulin fraction with, in some cases, an additional peak close

to albumin.

CZE provides suYcient resolution in the �1‐region to allow distinction of

�1‐ acid glycopr otein from � 1‐a ntitrypsin [23] (Fig. 6). � 1‐ Acid glycopr otein

pro duces a rather plane curve that coinci des with �‐ lipopr otein.Gonzal es ‐ Sagrado et al . [24] de scribed that wi th CZE there was no rela-

tions hip between �1‐ antitryps in phe notype and the concen tration of the�1‐ globuli n fraction and concluded that � 1‐antitrypsin deficiency was masked

by CZE . With gel elect rophoresi s, estimat ion of the �1‐ globuli n fract ionpro vides an estimation of the �1‐ antitryps in concen tration because � 1‐ acidglycoprotein is not e Y ciently stained. A computer‐ suppo r ted algor ithmthat produced a reliable semi quan titative evaluation of �1‐ antitryp sin (onPar agon CZE 2000 w ) reco gnized deficiency varia nts of � 1‐ antitryps in [25] .In pa tients with �1‐ antitryps in deficiency, the specific peak of � 1‐ antitryps inis absent [5] (Fi g. 7). With Capill arys w, a computer ‐ assisted correct ion isavail able, which de letes the area unde r the curve between albumin and �1‐globu lins. Suc h correct ion impro ved the correl ation be tween CZE and aga-

rose elect ropho resis [10] and cou ld allow an e asier screeni ng for �1‐ antitryps inde ficiencies.

FIG. 6. Increased �1‐acid glycoprotein. The arrow indicates the increase at the �1‐acidglycoprotein position. In this sample, the �1‐acid glycoprotein concentration was 3.23 g/liter

(reference interval: 0.51–1.17 g/liter).


Routine CZE with Paragon CZE 2000w and Capillarysw is unable to

clearly diVerentiate �2‐macroglobulin from haptoglobin in the �2‐globulinfraction. Such diVerentiation is achievable with the high‐resolution kit on

Capillarysw. With this high‐resolution kit, �1‐antitrypsin is also clearly dis-

tinguished from �1‐ acid glycopr otein. Larsson and Hansso n [26] co mpared

the qua ntifica tion of �1‐antitrypsin, � 1‐acid glycoprotein, haptoglobin, andfibrinogen in plasma by nephelometry and the corresponding quantification

from the Capillarys electropherogram with the high‐resolution buVer. Theconcentration of albumin was used to assign a value (in g/liter) to individual

peaks in the capillary electropherogram. There was a strong linear correlation

for �1‐antitrypsin, � 1‐acid glycoprotein, and haptoglobin and a weaker corre-lation for fibrinogen. The authors concluded that CZE can be used to monitor

inflammatory responses (acute phase reaction) following tissue injury,

infarction, infection, or immune‐related diseases.CZE allows to distinguish transferrin from complement in the �‐globulin

fraction and can be used to estimate serum levels of these proteins [25, 27].

Figures 8–13 show examples of various dysproteinemias such as acute

phase reaction, chronic inflammatory syndrome, polyclonal gammopathy,

nephritic syndrome, iron deficiency anemia, and �‐� bridging.

FIG. 7. �1‐Antitrypsin deficiency. The arrow indicates the absent �1‐antitrypsin peak. In this

sample the �1‐antitrypsin concentration was 0.24 g/liter (reference interval: 0.88–1.74 g/liter).

54 XAVIER BOSSUYT

9. Online Processing of Digital Absorbance Data

As CZE produces digital absorbance data, online computerized interpre-

tatio n of the patte rns is possibl e. Jon sson and Carlson [25] an d Jonsson et al.

[28] were success ful in de velopi ng a mathe matica l algori thm ‐ based expertsyst em for the interpreta tion of pro tein profi les, the de tection of monoclonal

imm unoglob ulins, and the evaluat ion of imm unog lobulins afte r CZE . The

algori thm detected 94 of 95 mono clonal pr oteins (98. 9%) an d 100% of those

visi ble after CZE. Of 607 sampl es lacki ng a monoclon al protein , only 3 were

identi fied by the algori thm (specifi city 99%). The sensitiv ity of the algorithm

for recogni zing oligoc lonality, as a whol e, was low (37% ) compared with the

sub jective evaluat ion [28] .

An artifi cial neu ral ne twork ‐ based algorithm has also been suggest ed as a

power ful too l capab le of providin g de cision supp ort for rap id and standar -

dized inter pretation of serum pro tein elect rophoresi s. Ogni bene et al . [29]

repo rted that the artifi cial ne ural network ‐ba sed algori thm was able to

correctly classify 4009/4971 normal samples and 493/503 pathological sam-

ples (monoclonal proteins and oligoclonal patterns), with a sensitivity of

98.4% and a specificity of 80.6%.

FIG. 8. Acute phase reaction (inflammatory syndrome). The characteristic electrophoretic

changes for the acute phase reaction are depressed albumin, and increased �1‐globulin and

�2‐globulin fractions. In the �‐fraction, a depressed tranferrin and increased C3c is observed.

FIG. 9. Chronic inflammatory syndrome. The characteristic electrophoretic changes for the

chronic inflammatory syndrome are similar to those of the acute phase reaction but with

increased �‐globulin fraction.


FIG. 10. Polyclonal gammopathy. Increased �‐globulin fraction with decreased albumin

fraction.

FIG. 11. Nephrotic syndrome. Nephrotic syndrome gives rise to a high �2‐macroglobulin

fraction (elevation of the �2‐globulin fraction) associated with hypoproteinemia (i.e., decreased

albumin and decreased �‐globulin fraction).

56 XAVIER BOSSUYT

FIG. 12. Iron deficiency anemia. In iron deficiency anemia, increased transferrin is observed.

FIG. 13. �‐� Bridging. �‐� Bridging is caused by increased polyclonal IgA as is seen in liver

cirrhosis.


58 XAVIER BOSSUYT

10. Detection of Monoclonal Proteins

Detection and quantification of monoclonal proteins is the main indica-

tion to perform serum protein electrophoresis. Several investigators have

evaluated CZE for its potential to recognize monoclonal proteins.

10.1. DETECTION OF MONOCLONAL PROTEINS BY PARAGON CZE 2000W

Joli V a nd Blessum [3] reported a 1 00% conco rdance be tween Paragon CZE

2000 w an d agarose gel elect rophoresi s for detect ing monoclonal gamm opa-

thy in 100 sampl es wi th a mon oclonal protei n that did not migr ate in the

�‐ globulin fract ion, an d with a concentra tion >0.5 g/liter for IgG an d

> 0.75 g/lite r for IgM and IgA.

Bos suyt et al . [30] found that Paragon CZE 2000 w detected a monoclonal

pro tein in 54 (93% ) of 58 selec ted sera in whi ch immu nofixati on and/or

imm unoelect ropho resis revealed a mono clonal protei n. The sensi tivity of

agaro se gel electro phoresi s and cellulose acetate electroph oresis in the same

co hort of sampl es was 86% an d 74%, respect ively.

Litwin et al . [31] an alyzed 617 sampl es with Paragon CZE 2000 w an d

agaro se gel elect rophoresi s to de termine sensitiv ity and sp ecificity for detect-

ing immun ofixati on‐ confirmed monoclon al protein s ( n ¼ 6 3). The sensitiv ity

was 100% for CZE and 95% for agarose elect rophoresi s. Agaro se electro-

phor esis mis sed three IgA monoclon al protei ns with a concen tration

< 0.65 g/lite r. In this study the specificity of CZE was 100%.

A study by Clark et al . [32] of serum samples problem atic by agarose gel

elect rophoresi s illu strated that CZE was superi or to agarose electropho resis

for evaluat ing: (1) serum sampl es con taining mon oclonal protei ns within a

polyc lonal increa se, (2) sampl es ch aracterize d by point of applic ation arti-

fact s with agarose gel elect ropho resis, (3) abno rmalities in the �‐ region, and(4) the presence of free light chains. Some free light chains, howev er, remai ned

unr ecognized by CZE an d requir ed immun ofixati on for detection.

Meu nier [33] selec ted 105 di Yc ult mono clonal proteins an d perfor med

agaro se gel e lectropho resis and CZE (Paragon CZE 2000) on these samples.

Sixty ‐ five of the mon oclonal protei ns migr ated in the �‐ an d 10 in the �2‐globu lin region. For ty‐ one and 80 of the 105 monoclon al pro teins wer e

reveal ed by agarose gel elect rophoresi s an d CZE, respect ively. These data

obv iously exemplified that Par agon CZE 2 000 w was superi or to agarose gel

elect rophoresi s for reveal ing monoclonal pro teins in the �‐ globulin region.The sensi tivity and specifici ty of Parago n CZE 2000 w for detect ion of

mon oclonal protei ns ha s been evaluate d in two large pr ospective studies.

Katz mann et al . [15] evaluate d 1518 patien ts. Imm unofix ation and /or immu-

noelectrophoresis were regarded as the gold standard. A monoclonal protein


was identified in 215 patients. An electrophoretic abnormality was found in

195 (91%) and 204 (95%) samples with agarose gel electrophoresis and CZE,

respectively. CZE failed to detect 2 IgM monoclonal proteins, 3 IgA mono-

clonal protei ns, and 6 out of 13 light ch ains. The specifici ty was 99% for bot h

CZE an d agarose electrop horesis. Bossuy t and Marie n [34] evaluat ed serum

sampl es from 1692 diV erent indivi duals sub mitted to the laborato ry to screen

for the presence of a monoclonal protein or to reevaluate a known monoclo-

nal protei n by CZE [Paragon CZE 2000 w system (sof tware versi on 1.5) ] an d

by immun ofixati on. A mono clonal protei n was found in 481 of the 1692

patie nts. CZE failed to detect an ab normality in 24 (5%) of these samples.

The 95% sensi tivity agreed with the findings of Katzmann et al . [15] . The

mono clonal proteins that wer e missed typic ally included monoc lonal light

chains and low ‐ concen tration monoclonal pr oteins that could only be

detect ed by immun ofixatio n.

Three cases of false‐negative results in the detection of a large IgM mono-

clonal protein by Paragon CZE 2000w have been reported. The first report

was from Keren et al. [35]. They described an IgM monoclonal protein that

was detected by Paragon CZE 2000w, but at a much lower concentration

than indicated by nephelometry or agarose electrophoresis. Treatment with

2‐mercaptoethanol produced a monoclonal protein that was more consistent

with the other results. The authors recommended that laboratories using CZE

should consider the use of 2‐mercaptoethanol to evaluate monoclonal proteins

when the nephelometric concentrations or clinical features are inconsistent

with the CZE electropherogram measurement. Marie n and Bossuyt described

a similar case of an IgM� m ono cl on al pr ot ei n f or wh ic h P ar ag on CZ E g av e a n

error code [36]. Analysis of the sample after 1:2 dilution resulted in an electro-

pherogram in which no obvious peak was present, whereas nephelometry

and agarose electrophoresis indicated a large monoclonal protein. Treatment

with 2‐mercaptoethanol revealed the abnormal protein on CZE [36]. Finally,

Zetterberg and Nilsson‐Ehle [37] reported a comparable case of an IgM

monoclonal protein that was missed by CZE but not by agarose gel electro-

phoresis and by nephelometry. Cleaving the disulfide bonds in the IgM

pentamer by penicillamine treatment revealed the monoclonal protein.

Paragon 2000 w CZE (softw are versi on 1.5) has also been report ed to fail to

prop erly separate some occasio nal large non ‐ IgM mon oclonal comp onents

[34] . Thes e monoclon al‐ protei ns charact eristic ally had a high ‐ pI value ( >8.5)

and migr ated in the slow �‐ region on agarose gel elect rophoresi s. Suchinaccur ate separat ion of monoclonal protei ns on CZE due to high ‐ isoelec tricpoint had already been descri bed in early studi es using the singl e ‐ capillaryBec kman Coul ter P/ACE resear ch instrument [38, 39 ]. By increa sing the

ionic strength and the pH of the buVer, the authors were able to overcome

the problem. Thus, the pH of the buVer is an important aspect in CZE.

60 XAVIER BOSSUYT

Take n toget her, CZE is a sensi tive method to detect monoclonal proteins.

Dis crepanci es be tween CZE and agarose electro phoresi s and /or immu nofixa-

tion for detection of mono clonal protein s have been rep orted and attribut ed

to the small size of the monoclona l protei n, to migr ation in the �‐ region withmaski ng by trans ferrin or C3, to pentam erization of IgM monoclonal pro-

teins , or to incorr ect separat ion with CZE due to high ‐ isoelectr ic point of themon oclonal protei n. Additional ly, a case has been repo rted in which CZE

failed to detect a high ‐ concentra tion IgG l monoclonal co mponent with a pI�= 7 that migr ated in the mid ‐�‐ region [34, 4 0, 41] . The reason for this failurewas unknow n. It was ne ither due to the presence of cryogl obulins nor to

lipi d–protei n complex es.

To overcome the pro blem that so me low ‐ concen tration and /or unus ualmon oclonal imm unoglobu lins (e.g., unusu al isoelectr ic poin t, agg regation)

may be mis sed, the manufa cturer relea sed new an alytical conditio ns for serum

pro tein electrop horesis with the Bec kman Coulter Par agon CZE 2000 w sys-

tem. This included the use of a modified bu Ver, applic ation of a higher vo ltage(10. 3 kV), more e Y cient co oling of the capil laries, and relea se of an adaptedne w softwar e (vers ion 1.6 or higher) . Under these cond itions, �‐ lipopr oteinan d fibr inogen (heparin ized sampl es or incomp letely clott ed serum) are

resol ved. Fibrinog en migrates in the anodic part of the �‐glob ulin fractionclose to C3, wher eas �‐ lipopr otein migr ates in the � 2‐ globulin fraction.

Marie n et al. [41] ev aluated wheth er the upg rade increa sed the resol ution of

previou sly unresol ved monoclonal protein s. Five sera with a mo noclonal

pro tein that could be separat ed by agarose gel electrop horesis but not by

CZE wer e an alyzed. Of the four post ‐�‐ migrati ng monoclonal pr oteins, one

mon oclonal pro tein was detected wi th the new buV er syst em, one generat ed

an error co de, and two wer e missed. A high ‐ concentra tion IgG l monoclonal

pro tein with a pI �= 7 migrati ng in the mid‐�‐ region on agaro se elect rophore-sis was not separated under softwar e 1.5 and gave an error code unde r

soft ware 1.6 [41] . Thus , the adap ted syst em did sho w fairly improv ed resol u-

tion of certa in (but not all) rarel y occurri ng monoclonal pro teins that wer e

previou sly missed by CZE . The overal l sensi tivity for detect ion of monoclo-

nal proteins with the new analytical conditions was also determined on 1106

consecutive samples and was found to be 95%, which was comparable to

previous analytical conditions. The specificity was 78%, which was lower than

the specificity reported for the previous operating conditions (98%). The

most frequently observed abnormalities in immunofixation‐negative samples

were slight changes in the anodal part of the �‐globulin fraction. Monoclonal

proteins that are undetected by CZE most frequently hide in normal bands.

Such hidden monoclonal proteins can only be detected by immunofixation

and are missed by CZE (and gel electrophoresis). Figures 14–17 illustrate the

electropherograms of various hidden monoclonal proteins. Small monoclonal

FIG. 14. Hidden monoclonal protein (IgGl). CZE electrophorogram of a sample that con-

tains a hidden IgGl monoclonal protein (in the �‐globulin region). Inset shows the

corresponding immunofixation results obtained with Hydragel/hydrasisw system (Sebia). Lanes:

ELP: total protein; G, A, and M: �‐, �‐, and �‐heavy chains, respectively; K and L: �‐ and�‐light chains, respectively. The arrow indicates the position of the monoclonal protein.


proteins are frequently part of monoclonal gammopathy of undetermined

significance. They can also be found in patients with infectious disease,

autoimmune disease, or immunosuppression but also in patients with B‐celllymphoma, leukemia, light chain disease, neuropathy, and amyloidosis.

10.2. DETECTION OF MONOCLONAL PROTEINS BY CAPILLARYSW

At present, only two studies are available that deal with the performance of

Capillarysw to detect monoclonal proteins.

Bossuyt et al. [9] an alyzed 23 8 sampl es with agarose gel electroph oresis,

Capillarysw (software version 1.4.1), Paragon (software version 1.5), and

immunofixation. Sample selection was based on the presence of a disturbed

morphology (e.g., spike) of the protein profile or hypogammaglobulinemia

with agarose electrophoresis and/or Capillarysw. Immunofixation revealed

the presence of amonoclonal protein, oligoclonal bands, and polyclonal and a

normal profile in, respectively, 89, 66, 19, and 64 samples. With Capillarysw,

Paragon and AGE, a spike and/or disturbed morphology of the profile was

FIG. 15. Hidden monoclonal protein (IgAl). CZE electrophorogram of a sample that

contains a hidden IgAl monoclonal protein (in the �‐globulin region). Inset shows the



62 XAVIER BOSSUYT

found in 222, 182, and 180 samples, respectively. In these samples, immuno-

fixation was negative in 73 (33%), 46 (25%), and 39 (22%) samples, respec-

tively. These data indicated that for the detection of monoclonal proteins

Capillarysw had a lower specificity than agarose electrophoresis. Of the

89 samples with a monoclonal protein, both Capillarysw and Paragon failed

to detect 3 monoclonal proteins. Two of these three monoclonal proteins

were missed by both CZE systems. The third monoclonal protein was a

�‐migrating monoclonal protein that was detected by Paragon CZE 2000w

but not by Capillarysw and a monoclonal protein that migrated in the slow

�‐region that was missed by Paragon but not by Capillarysw.

Gay ‐ Bellile et al . [10] an alyzed 265 selec ted sera coveri ng a wid e range ofelectrophoretic patterns with agarose electrophoresis and with Capillarysw

(software version 4.51). They found a detectable abnormal peak in the �2‐ or�‐globulin zone in 135 samples with Capillarysw and in 130 samples with

agarose electrophoresis. There was a 100% agreement between Capillarysw

and agarose gel electrophoresis in a subgroup of samples with a single

well‐defined monoclonal protein by agarose electrophoresis (n ¼ 64). In

oligoclonal, hypo‐ and hyper‐�‐globulinemic subgroups, the Capillarysw dis-

played higher sensitivity than agarose electrophoresis for detection of weak

FIG. 16. Hidden monoclonal protein (�‐light chain). CZE electrophorogram of a sample that

contains a hidden �‐light chain monoclonal protein (in the �‐globulin region). Inset shows the




monoclonal bands. Additional peaks detected by CZE but not by agarose gel

electrophoresis corresponded to small or faint bands identified asmonoclonal

by immunofixation in 70% of cases. In 30% of discordant results, abnormal-

ities detected by CZE were not confirmed by immunofixation, indicating that

CZE was less specific than agarose gel electrophoresis. Fewer peaks could be

quantified by CZE than by agarose gel electrophoresis because 15 (11.5%) of

them shifted from the �‐zone to the �‐zone (mostly IgM and IgG).

Supplementary prospective studies are needed to evaluate the sensitivity

and specificity of Capillarysw in a routine clinical setting.

10.3. DETECTION LIMIT FOR MONOCLONAL PROTEINS BY CZE

Several authors have tried to find out the detection limit for recognizing a

monoclonal protein with Paragon CZE 2000w. These studies were typically

done by mixing a sample containing a monoclonal protein with a normal

sampl e. Joli V and Bl essum [3] report ed 0.5 g/l iter for IgG, and 0.75 g/lite r

for IgM and IgA as the de tection limits, provided the mono clonal protei ns

wer e not co ‐ migr ating with other pro teins. Bienven u et al . [4] and Katz man

et al. [15] report ed, respect ively, <0.5 g/liter and 0.14 g/l iter as the limit for

FIG. 17. Hidden monoclonal protein (�‐light chain in �2‐globulin region). CZE electrophoro-

gram of a sample that contains a hidden �‐light chain monoclonal protein (�2‐globulin region).

Inset shows the corresponding immunofixation results obtained with Hydragel/hydrasisw system

(Sebia). Lanes: ELP: total protein; G, A, and M: �‐, �‐, and �‐heavy chains, respectively; K and

L: �‐ and �‐light chains, respectively. The arrow indicates the position of the monoclonal

protein.

64 XAVIER BOSSUYT

de tection for monoclonal IgG by CZE. God ey et al . [6] report ed a sensitiv ity

of 4.25, 0.23, and 1.25 g/l iter for a monoclonal protei n migrati ng in the

�2‐g lobulin fraction, be tween the �‐ and the �‐ globuli n fract ion, and inthe �‐region (polyclonal), respectively. Gay‐Bellile et al. [10] checked the sensi-tivity for monoclonal protein detection by Capillarysw by mixing samples

containing either a cathodic or anodic �‐monoclonal peak with a normal

serum. The detection limit was 0.5 g/liter for the anodic �‐migrating monoclo-

nal protein and 0.7 g/liter for the more cathodic �‐migrating monoclonal

protein.

Keula rts et al. [42] performed a study in which 49 di Verent CZE plots werejudge d by 1 6 investiga tors from 12 clini cal chemistry laborat ories in Dutch

Hospitals. The plots included mixed sera with a low concentration (0.4, 1, or

4 g/liter) of a monoclonal protein. At 0.4 g/liter, 9 out of 12 diVerentmonoclonal proteins were hard to detect. At 1 g/liter, 6 out of 12 monoclonal

proteins were detected by >85% of the participants and at 4 g/liter 10/12

monoclonal proteins were detected by all participants. Normal sera were

always judged correctly. The authors concluded that at concentrations below

4 g/liter, M proteins become increasingly hard to detect.


10.4. DETECTION OF HEAVY CHAIN DISEASE BY CZE

Heavy chain diseases are rare B‐cell malignancies that are marked by the

production of a monoclonal heavy chain without associated light chains.

�‐Heavy chain disease is most common and occurs generally as intestinal

malabsorption in young adults from the Mediterranean Sea area. �‐Heavy

chain and �‐heavy chain disease are rare and largely found in patients with a

lymphoplasma cell proliferative disorder.

Luraschi et al . [43] describe d that CZE coupled with immu nosubtra ction

was ab le to detect and ch aracterize low ‐ concen tration free �‐ heavy chains inserum . By contras t, M arie n et al . [44] report ed a case in whi ch CZE failed to

detect and charact erize �‐ heavy ch ain disease.

10.5. D ETECTI ON OF M ONOCLONAL CRYOGL OBULINS

As sampl es are incubat ed and run at 24 � C with Parago n CZE 2000 w ,

mono clonal cryogl obulins that precipi tate at tempe ratures >24 � C might be

mis sed. With Capillarys w , the tempe ratur e of the capillari es is controlled at

35.5 � C. Thi s prevent s cryogl obulins from pr ecipitat ing during electrop ho-

retic migr ation, thus allowi ng their detect ion an d a ccurate quan tification.

Gay ‐ Bellile et al . [10] report ed that afte r cryogl obulin storage at 4 � C, no pe akwas detect ed in the �‐ zone of the Capillarys electroph erogram in seven of

nine sampl es. At 37 � C, a pea k was detected in 100% of the cases that

corres ponde d to the measur ed cryopreci pitate.

11. Typing of Mon ocl onal Protein s

Mon oclonal protei ns may be classified by immuno subtra ction. This feat ure

is available on Par agon CZE 2000 w an d also on Capill arysw . The method

involv es perfor ming CZE be fore an d after incubat ion of the serum sampl e

with seph arose beads coated with an tibodies agains t specific immun oglobuli n

isot ypes (IgG , IgA, IgM, kappa, lambda ). Identi fication is based upon whi ch

of the imm unosorbants remove s the mono clonal ba nd. No reagent s are

avail able for the identifi cation of the rare IgD and IgE mon oclonal protei ns

by immu nosubtra ction. An examp le of the immun osubtra ction proced ure is

given in Fig. 18 .

Joli V and Ble ssum [3] report ed that the results of immu nosubtra ction

wer e identi cal to immuno fixation if a monoclonal protein was de tectable by

CZE ( >0.5 g/liter for IgG an d > 0.75 g/liter for IgA or IgM). Bienvenu et al .

[4] a ssessed mono clonal pr oteins by imm unosu btraction on 40 3 samples.

The monoclonal type was correctly identified in all samples with peak

FIG. 18. Immunosubtraction. The serum shows a monoclonal peak in the �‐fraction. Theserum is treated with beads coated with the various antibodies (IgG, IgM, IgA, kappa, lambda).

The monoclonal peak is removed using beads with anti‐IgG and anti‐�. The monoclonal peak is

thus an IgG�.

66 XAVIER BOSSUYT

concentrations >10 g/liter, whereas only 50% of monoclonal proteins that

could not be quantified by densitometric scan were typed.

Bos suyt et al . [30] evaluat ed the Parago n CZE 2000 w imm unosub traction

method on 58 selec ted sampl es in whi ch a mon oclonal protein had been

identi fied by immun ofixati on and /or imm unoelect rophoresi s. CZE detect ed

93% of the monoclonal s an d immu nosubtra ction was able to immu notype

91% of these mon oclonal s. A full iden tification of the mono clonal protei n by

imm unosub traction was possible in 100% of the sampl es with a monoclonal

pro tein conce ntration > 30 g/l iter and in 80% of the sample s with a monoclo-

na l pr otein concentra tion < 5 g/liter.

Litwin et al . [31] evaluat ed 78 serum sampl es, 48 of whi ch ha d a monoclo-

na l gamm opathy, by immun osubtra ction. Only 60– 75% of the monoclonal

gammopathies were correctly immunotyped with immunosubtraction by

four readers blinded for the immunofixation immunotype. The authors con-

cluded that immunosubtraction was less accurate than IF in determining the


immunotype of the monoclonal gammopathy. Serum samples containing

missed monoclonal gammopathies had small quantities of a monoclonal

protein or the monoclonal spike was present in the background of a

polyclonal increase.

Katzmann et al. [15] evaluated 1518 serum samples. Immunofixation was

positive in 215 samples and immunsubtraction in 208 samples. Of the 208

samples that contained a monoclonal protein detected by immunosubtraction,

there were 16 that were initially believed to be polyconal by immunofixation.

Because of the monoclonal protein detected by immunosubtraction, these

samples were retested at diVerent dilutions, and the immunofixation results

confirmed the presence of a monoclonal protein. These cases illustrated the

technical diYculties of immunofixation. Katzmann et al. [15] reported that

when an electrophoretic abnormality was detected by CZE, the immunosub-

traction method was usually able to immunotype the monoclonal protein. They

commented that the immunosubtraction method seemed to have comparable

sensitivity to immunofixation. Immunofixation was still required as a comple-

mentary method for detection of free light chain and detection of a second

small monoclonal in a sample with bi‐ or multiclonal gammopathy.

Meun ier [33] foun d that CZE immun osubtra ction was able to co rrectly

identi fy 7 out of 17 mult iclona l gamm opathie s, 32 out of 4 3 �‐ migrati ng

mono clonal protein s, an d 5 out of 6 �2‐ migratin g monoclonal pro teins.

Smalley et al . [45] report ed that 6% of seque ntial CZE imm unosubtra ction

analyses needed to be tested by immunofixation for definite immunotyping.

Those needing further assessment mainly were cases in which there was a

polyclonal gammopathy present in addition to a suspected monoclonal

gammopathy. The authors reported immunosubtraction as an accurate and

eYcient method to identify the monoclonal type. In a panel of 50 selected

specimens, 3 readers correctly identified 29 monoclonal proteins and 21

nonmonoclonal proteins.

Collectively, immunosubtraction is a dependable technique for typing (large)

monoclonal proteins.However, being a subtractive technique, it cannot achieve

the sensitivity of immunofixation. Immunofixation continues to be the ‘‘gold

standard’’ for detection and characterization of monoclonal proteins.

12. Urine and Body Fluid Analysis

12.1. URINE ANALYSIS

A problem with analyzing urine proteins by CZE is that urine may contain

many low‐molecular weight components that absorb at 200–214 nm. These

interfering substances may comigrate with the proteins of interest and

68 XAVIER BOSSUYT

hamper interpretation. Therefore, urine samples need to be cleared from

interfering substances by adsorptive filtration or cold ethanol precipitation

[46] . Ano ther prob lem is that the protei n concen tration in urine samples may

be low, necessi tating co ncentra tion of the sampl e.

After co ncentra ting and de salting the urine, Jolli V et al . [47] found agree-ment betwee n CZE (Paragon 2000 w ) a nd AGE in the detect ion of free light

ch ains in patie nts. Beckman Coul ter sup ported evaluation of urine on the

Par agon 2000 clini cal CZE Instrument with soft ware version 1.5 or 1.6

afte r concentra ting (to a minimum of 25 g/lite r total protein ) and desalting

the samples (e.g ., us ing Vivaspi n 20 concentra tors and Diafiltr ation cups

[V ivascience, W estford, M A]). Overridi ng the program med dilution step of

the Paragon 2000 instrument, Kolios et al. [48] were able to distinguish diVerenttypes of proteinurias without concentration of specimens with a total protein

content of 150–200 mg/liter. More recently (2004), a kit for urine protein

electrophoresis and for immunosubtraction on unconcentrated urine samples

became available for the Paragon CZE 2000w with software version 2.0.

Desalting the samples prior to electrophoresis remains necessary. The analyti-

cal range claimed by the manufacturer is 5–46,000 mg/liter for a single protein

component and 0.2–48 g/liter for total protein. At the moment, no studies are

available that evaluated this newly released kit.

At present , Se bia oVers no procedu re to analyze urine sampl es.

12.2. A NALYSIS OF PLEURA L AND ASCIT IC F LUID

CZE has be en used to an alyze pleural and ascitic body flui ds, using

method s developed for serum pro tein separation. Claeys et al . [49] and Porcel

et al. [50] demon strated that CZE can be used for the fractionat ion of

pro teins in pleural an d ascit ic fluids. The results co rrelate wi th those obtaine d

by AGE. By calcul ating the �2‐ macrog lobuli n/albu min ratio, Claeys et al.

[49] cou ld identify exud ates with a sensi tivity of 81% an d a sp ecificity of 91%.

13. Quality Control

To guarantee the quality of results obtained with CZE it is important to

monitor the well‐functioning of the instrument. This implies the regular use

of an internal quality control material and participation in a quality assur-

ance program. Commercial quality control material is available (e.g., Biorad,

Hele na Laborator ies, Sebia) but does not co ntain a mo noclonal pro tein.

Jen kins [51] suggeste d that a quan titative commer cial co ntrol mate rial con-

taining a monoclonal protein at decision level for treating myeloma patients

would be beneficial as a serum protein quality control.


14. Interferences

14.1. DETECTOR INTERFERENCE

With CZE, detection of proteins is based on the absorbance of peptide

bonds at 200–214 nm, in contrast to gel‐based methods, which use dye

binding. Substances that absorb at 200–214 nm can cause interference in

CZE.

14.1.1. Radio‐Opaque Agents

Radio‐opaque agents interfere by producing an abnormal spike, which

may be mistaken for a monoclonal protein. The first report described inter-

ference by intravenously administered Urografinw (sodium‐meglumine ami-

dotri zoate), Tel ebrix w (ioxitalami c acid) , and Omnipaqu e w (iohex ol) [52] .

These substances produce an interfering peak in the �‐globulin fraction

(Figs. 19–21). The elimination half‐life is 60–120 min for sodium‐meglumine

amidotrizoate, 120 min for ioxitalamic acid, and 121 min for iohexol.

Arr anz ‐ Pena et al. [53] described interfer ence of megl umine iotr oxate in the

prealbumin region, iobitridol in the �2‐globulin fraction, and sodium‐meglumine ioxaglate, ioversol, iomeprol in the �‐globulin fraction. Finally,

FIG. 19. EVect of Telebrixw on CZE analysis. The interfering substance Telebrixw (meglu-

mine ioxitalamate) is indicated by an arrow. Adapted from Bossuyt et al. [52] with permission of

the publisher.

FIG. 20. EVect of Omnipaquew on CZE analysis. The interfering substance Omnipaquew

(iohexol) is indicated by an arrow. Adapted from Bossuyt et al. [52] with permission of the

publisher.

FIG. 21. EVect of Urografinw on CZE analysis. The interfering substance Urografinw

(meglumine amindotrizoate) is indicated by an arrow. Adapted from Bossuyt et al. [52] with

permission of the publisher.

70 XAVIER BOSSUYT

FIG. 22. Monoclonal protein (IgA�) migrating in the �2‐globulin region. Inset shows the


ELP: total protein; G, A, andM: �‐, �‐, and �‐heavy chains, respectively; K and L: �‐ and �‐lightchains, respectively. The arrow indicates the position of the monoclonal protein.


Van der Watt an d Berman [54] rep orted a case of ps eudoparap roteinemi a at

the cathod al end of the �2‐g lobulin fraction afte r iopami dol infus ion for

coron ary an giogra phy in a pa tient with renal failure. Desal ting the sampl e

[55] or treatment with activated charcoal [53] can remove the interfer ing

substance. To avoid interference, instructions should be given not to collect

blood for protein electrophoresis shortly after a patient received an iodinated

contrast agent. Figures 22 and 23 illustrate examples of monoclonal proteins

that migrate in the �2‐globulin fraction.

14.1.2. Antibiotics

In addition to radio‐opaque agents also antibiotics can give additional

spikes in CZE analysis of serum proteins. The sulfamide sulfamethoxazole

prod uces a spike at the anodal site of the albumin fraction [56] ( Fig. 24 ).

The half‐life of sulfamethoxazole is 9 hours. Intravenously administered

piperacillin–tazobactam gives a peak at the anodal side of the �‐globulinfraction [57] . The eliminat ion half ‐ life of this compoun d is 0.7–1.2 hour s.

FIG. 23. Monoclonal protein (l monoclonal protein) migrating in the �2‐globulin region.

Inset shows the corresponding immunofixation results obtained with Hydragel/hydrasisw system

(Sebia). Lanes: ELP: total protein; G, A, and M: �‐, �‐, and �‐heavy chains, respectively; K and

L: �‐ and �‐light chains, respectively. The arrow indicates the position of the monoclonal

protein.

FIG. 24. EVect of sulfamethoxazole on CZE analysis. The interfering substance sulfamethox-

azole is indicated by an arrow. Adapted from Bossuyt et al. [57] with permission of the publisher.

72 XAVIER BOSSUYT


14.2. FIBRINOGEN

Fibrinog en is revealed with Capillarys w [9] an d with Paragon CZE 2000 w

with soft ware versi on 1.6 or higher ( Fig. 2 5) [4, 5, 9] . The fibr inogen ban d

may interfer e wi th the interpreta tion as it obscures part of the �‐�‐ region inincompl etely clotted serum or in hepa rinized sampl es. Ethano l precipitat ion

is not reliab le for selec tive ly removi ng nonmono clonal peaks seen in the

fibrinog en region on CZE [58] . Fig ures 26 and 27 illustr ate exampl es of

mono clonal protein s that migr ate in the fibrinoge n posit ion.

14.3. IN VITRO HEMOLYSIS, BILIRUBIN, AND LIPID

In vitro hemolysis results in an increased and disturbed peak at the trans-

ferr in position with both Capill arysw and Par agon CZE 2000 w [4, 5, 9]

(Fig. 28). Figure 29 illustrates a monoclonal protein that migrates in the

transferrin position. Interference by bilirubin is not seen upon visual inspec-

tion of the electroph erogram [4, 5]. A minor e Vect of biliru bin on the albumi n

wid th, howeve r, was foun d by a comp uter algori thm that calcul ates the

ano dal width of the albu min peak [25] . �‐Lip oprotei n is reveal ed withCapi llarys w and with Paragon CZE 200 0w using the most recent buV ersyst em [9] . El evated levels of triglyce rides may disturb the (pre) albumin

region, the �1‐ globuli n region, and/or the � 2‐ globuli n region [9] ( Fig. 30 ).

FIG. 25. Plasma sample. The fibrinogen peak is indicated by an arrow.

FIG. 26. Monoclonal protein (IgM�) migrating in the fibrinogen region. Inset shows the



FIG. 27. Monoclonal protein (IgA�) migrating in the fibrinogen region. Inset shows the



74 XAVIER BOSSUYT

FIG. 28. Hemolytic sample. Hemolysis results in an increased and disturbed peak at the

transferrin position (see arrow).

FIG. 29. Monoclonal protein (IgM�) migrating at the transferrin position. Inset shows the




FIG. 30. Lipemic sample. Example of a lipemic sample. Triglycerides: 966 mg/dl (reference

interval: �180 mg/dl). The electropherogram shows an elevation between the albumin and the

�1‐globulin fraction.

76 XAVIER BOSSUYT

14.4. COMPLEMENT

Complement degradation products (aging samples) may produce a small

pe ak at the ano dal side of the �‐ globulin region [9] .

14.5. GELATIN‐BASED PLASMA SUBSTITUTES

Gelatin‐based plasma substitutes are denatured collagens and thus contain

complex mixtures of proteins. They produce an increase in the �‐/�‐globulinregion in a polycl onal like way [10, 59] . They are rapidl y eliminat ed (half ‐ life:2.5 hours) and only pro duce interfer ence in the first hour s after infusion.

Dext ran ‐ and starch ‐ ba sed plasm a substitut es do not interfer e [59] .

15. ‘‘Open’’ Capillary Electrophoresis Systems

This chapter was focused on the performance of dedicated CZE systems

for serum protein electrophoresis. It should be mentioned, however, that

additional clinically useful information may be obtained by ‘‘open’’ capillary


electrophoresis systems. For example, capillary electrophoresis can be ap-

plied for the determination of carbohydrate‐deficient transferring in patient

sera [60, 61] .

16. Conclusions

Separation of serum proteins by automated CZE is labor saving and

reproducible. It is an eVective method for detecting monoclonal proteins in

serum with a sensitivity of about 95%. Immunosubtraction is reliable for

typing large monoclonal proteins but is less sensitive than immunofixation.

With CZE, radio‐opaque agents and some antibiotics interfere because they

absorb at 200–214 nm.

ACKNOWLEDGMENT

The author thanks Dr. G. Marien for helpful discussion, for critically reading the manuscript,

and for helping with the selection of the cases.

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advances in serum protein electrophoresis

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