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Chapter 8 : Clinical Indications and Applications of Serum and Urine Protein Electrophoresis

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Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, Page 1 of 2

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Abstract:

Protein electrophoresis is performed on serum (SPEP) and urine (UPEP) to detect and quantify monoclonal gammopathies (M proteins). In effect, serum and urine protein electrophoresis provides an immunochemical biopsy of the humoral immune system. However, other clinically relevant information is also available from examination of the proteins demonstrated from these studies. This chapter reviews basic principles of electrophoresis, the types of apparatus that are available, quality control and quality assurance procedures, costs, and illustrative patterns with recommended interpretations.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Figures

Image of FIGURE 1
FIGURE 1

General structure of an amino acid. The R group determines the specific amino acid.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 2
FIGURE 2

The peptide bond formation between two amino acids creates a dipeptide.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 3
FIGURE 3

The serum has sharp separation of major protein bands on the electropherogram (Sebia Capillarys). Ref., reference; T.P., total protein; A/G, albumin-to-globulin ratio.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 4
FIGURE 4

Heterozygous α antitrypsin. The capillary zone electropherogram has two α antitrypsin bands (arrows). Insert shows (top) a normal serum on a gel and (bottom) a heterozygous pattern with two α antitrypsin bands. (B) The electropherogram pattern for a normal serum sample displays the single peak of the normal α antitrypsin band (arrow). Ref., reference; T.P., total protein; A/G ratio, albumin-to-globulin ratio; L, low value.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 5
FIGURE 5

(A) This Levey-Jennings chart monitors the daily variation of albumin in the normal control sample performed on capillary zone electrophoresis for 3 months. No control sample is beyond 2SD (2 standard deviations) and no trend of high or low values is evident, indicating consistent precision. (B) This Levey-Jennings chart monitors the daily variation of the α fraction in the normal control sample performed on capillary zone electrophoresis for 3 months. Wider swings of the daily value are present in the α fraction than in albumin (panel A) due to the smaller percentage of proteins present in this fraction. Nonetheless, no sample is beyond 2SD and no trend of high or low values is evident on either chart, indicating consistent precision. V.M., median value.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 6
FIGURE 6

The capillary zone electropherogram pattern has a relatively flat α fraction with a markedly blunted α antitrypsin peak (arrow). This indicates the lack of the α antitrypsin band which is consistent with the quantitative decrease of that fraction. However, the protein that remains in the α fraction is orosomucoid (α acid glycoprotein). This protein is measured by capillary zone electrophoresis, but not usually stained by protein dye on gel electrophoresis. Insert above shows two serum samples performed on gel electrophoresis. The top pattern shows a normal serum with a prominent α antitrypsin band while the bottom pattern has a clear α region because the α antitrypsin band is not seen and the orosomucoid does not stain well with the protein dye. Ref., reference; T.P., total protein; A/G ratio, albumin-to-globulin ratio; L, low value.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 7
FIGURE 7

(A) Results of agarose gel electrophoresis for four samples are shown. The bottom sample has two unusual findings. The α region shows two light staining bands that almost merge. This reflects the presence of an α antitrypsin variant. In this case, it would be consistent with a faster-migrating variant (F) but needs to be confirmed by molecular studies. However, the second unusual feature is a densely staining band in the slow β region (β). This band could be an M protein; however, fibrinogen also migrates in this region. (B) IFE results for a sample similar to that shown in panel A are shown. Here the band reacts with anti-fibrinogen but not to anti-IgG, -IgM, -κ (K), or -λ (L) antisera.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 8
FIGURE 8

A capillary zone electropherogram is shown with a prominent M protein spike (shaded) and suppression of the normal gamma globulin. The spike measures 1.9 g/dl, and the total gamma globulin is 2.0 g/dl. Only 0.1 g/dl is accounted for by the gamma globulin not measured in the M protein spike. Ref., reference; T.P., total protein; A/G ratio, albumin-to-globulin ratio; H, high value.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 9
FIGURE 9

On the left, a capillary zone electropherogram demonstrates a slight decrease in albumin but a broad polyclonal increase in gamma globulins for a serum sample. On the right, three serum samples are shown on agarose gels. The top serum sample has a broad polyclonal increase in gamma globulins. The increased gamma globulins extend across the entire γ region. Ref., reference; T.P., total protein; A/G, albumin-to-globulin ratio; H, high value; L, low value.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 10
FIGURE 10

On the left, a capillary zone electropherogram is shown from a serum sample with an acute-phase reaction and a polyclonal increase in the IgG4 subclass. The acute-phase reaction is confirmed by the combination of low albumin (2.0 g/dl), increased α and α fractions, and a low transferrin (β) band. The γ region has relatively broad restriction, but it is limited to the anodal half of the γ region. This is a typical finding with a polyclonal increase in IgG4 subclass. As shown, the IgG4 subclass was increased 8-fold over the upper end of the normal range. On the right, four gel electrophoresis patterns are shown. The top one has a polyclonal increase in the IgG4 subclass. It shows a broad increase similar to that shown in Fig. 9 , but this one extends from the slow β region to midway in the γ region. Polyclonal increases in the IgG4 subclass can be mistaken for monoclonal proteins unless an immunofixation electrophoresis is performed. Ref., reference; T.P., total protein; A/G, albumin-to-globulin ratio; H, high value; L, low value.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 11
FIGURE 11

This capillary zone electropherogram demonstrates a cirrhosis pattern. Ref., reference; T.P., total protein; A/G, albumin-to-globulin ratio; H, high value; L, low value.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 12
FIGURE 12

This capillary zone electropherogram demonstrates a nephrotic pattern. The α region is exaggerated by comparison with the dramatic decrease in albumin. Ref., reference; T.P., total protein; A/G, albumin-to-globulin ratio; H, high value; L, low value.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 13
FIGURE 13

This agarose gel electrophoresis demonstrates 30 serum patterns. Samples 6, 13, 14, 25, and 30 demonstrate monoclonal restrictions in the γ region. Sample 24 shows an M protein overlying the usual C3 (β region) band. When the usual β region bands appear fuzzy or enlarged, it often indicates the presence of an M protein. IFE is indicated.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 14
FIGURE 14

This capillary zone electropherogram shows a prominent b region band due to the presence of an IgM M protein in this case of Waldenström's macroglobulinemia. Ref., reference; T.P., total protein; A/G, albumin-to-globulin ratio; H, high value; L, low value.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 15
FIGURE 15

This capillary zone electropherogram shows an increase in the C3 band due to an IgA M protein overlying it. IFE is indicated. Ref., reference; T.P., total protein; A/G ratio, albumin-to-globulin ratio; H, high value; L, low value.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 16
FIGURE 16

Two capillary zone electropherogram patterns are shown. On the left, the C3 band appears enlarged and distorted. In a normal serum sample, it is usually equal to or less than the transferrin (β) band. Here, it is not only larger but shifted anodally compared to a sample from another patient (on the right). It is difficult to determine how much of this area is due to the M protein. However, if one can immunosubtract this band, one may be able to improve such measurements (see chapters 9 and 11). The serum sample for which results are shown on the right also has a distinct, though small, M protein in the γ region. It should be characterized by IFE, but it is too small to measure due to the relatively large amount of underlying polyclonal immunoglobulins. Ref., reference; T.P., total protein; A/G, albumin-to-globulin ratio; L, low value.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 17
FIGURE 17

Several urine protein electrophoretic patterns are shown from concentrated urine samples. The arrow indicates the M protein in the third sample.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 18
FIGURE 18

A densitometric scan of an agarose gel is shown from a urine sample with an MFLC measuring almost 2,000 mg/24 h. A/G ratio, albumin-to-globulin ratio; T.P., total protein.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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Image of FIGURE 19
FIGURE 19

Two capillary zone electropherograms are shown. The one on the right is a control. The one on the left demonstrates the radiocontrast dye Omnipaque as a sharp band in the slow α fraction (arrow). These samples require IFE or immunosubtraction electrophoresis to rule out a monoclonal protein. Ref., reference; T.P., total protein; A/G, albumin-to-globulin ratio; H, high value; L, low value.

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
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References

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Tables

Generic image for table
TABLE 1

Location of radiocontrast and antibiotic spikes on capillary electrophoresis

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
Generic image for table
TABLE 2

Comparison of serum protein intervals by agarose and capillary zone electrophoresis

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8
Generic image for table
TABLE 3

Reflex testing for suspicious bands

Citation: Keren D, Humphrey R. 2016. Clinical Indications and Applications of Serum and Urine Protein Electrophoresis, p 74-88. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch8

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