**3.1 Albumin**

Albumin is a 69 kDa protein. It is the most abundant protein in serum. Albumin is synthesized in the liver and functions as a transport protein of various substances like bilirubin, enzymes, hormones, drugs etc. It also maintains fluid volume within the vascular space. Albumin is the first protein fraction to appear near the anode in

**3**

(**Figure 3**).

**Figure 2.**

*Serum protein family with fractions.*

*Serum Protein Electrophoresis and Its Clinical Applications*

SPE. Altered levels of serum albumin are associated with various clinical conditions. Low levels of albumin are clinically significant and are termed as hypoalbuminemia. Decreased concentration of serum albumin (hypoalbuminemia) indicates either a poor dietary intake (malnutrition) or a decreased production or an increased loss. Chronic liver disease is a common clinical condition associated with decreased albumin production, and chronic kidney disease (CKD) is the most common disease associated with an increased loss of albumin in urine (proteinuria). This clinical condition is otherwise known as nephropathy. Other causes of hypoalbuminemia

Apart from hypoalbuminemia which is commonly observed in an electrophoretogram, there are a few variations which can be observed in the in albumin peak including:

a.Bilirubin, Triglycerides if present in high levels in serum may appear as a blunt

b.Prealbumin (transthyretin)—increased levels of pre albumin, if present due to various clinical conditions including several inflammatory diseases is seen as a

c.A rare variant observed in the albumin peak is bisalbuminemia which is a rare condition, with no clinical features, in which the serum contains two albumin variants of different electrophoretic mobilities, usually in equal concentrations, though the total concentration of albumin is normal. Bisalbuminemia may be hereditary or acquired. The acquired type has been more frequently reported in chronic renal disease and pancreatitis and in patient with chronic renal disease. Two (rather than one) albumin bands may represent bisalbuminemia. Hereditary condition is a rare anomaly caused by a genetic lesion in

d.Analbuminemia (absence of albumin) is another genetically inherited metabolic disorder and was first described in 1954. This disorder is rare and affects

The most important aspect of such albumin variants lies in quantification of an albumin peak in such scenarios followed by interpretation and clinical correlation

include acute and chronic inflammation, critical illness, pregnancy etc.

peak which is seen adjacent to the cathode near the albumin peak.

blunt anodal peak distinctly separated from the peak of albumin.

the albumin gene usually a point mutation.

less than 1 in 1 million births.

*DOI: http://dx.doi.org/10.5772/intechopen.88367*

**Figure 2.** *Serum protein family with fractions.*

*Biochemical Testing - Clinical correlation and Diagnosis*

*Sebia Minicap flex piercing capillary electrophoresis.*

fractions and interpreted accordingly [5]. There are several support mediums available for separation of serum proteins including agarose, cellulose acetate, capillary medium etc. [6]; when a capillary medium is used, the technique is known as capillary zone electrophoresis (CZE). Capillary electrophoresis is the preferred method when compared to its competitors including agarose gel electrophoresis due to the following reasons. CZE provides an improved resolution due to the following factors:

a.The use of "electroendosmosis" principle which improves the resolution of

b.Employing a "high-voltage" electric current which aids in improving the throughput (the processing time) and the resolution of protein separation.

(**Figure 1**). Sebia Minicap Flex Piercing capillary electrophoresis works on the principle of capillary electroendosmosis under high-voltage electric current. The Flex Piercing model of Sebia CZE aids in testing of human blood with capped tubes which in turn eliminates the biohazard xassociated with handling of uncapped samples.

**3. Revisiting the basics: an insight into the protein family**

includes Serum Albumin: 3.5–4.5 g/dl and Globulins: 2.5–3.5 g/dl.

Below is an illustration of capillary electrophoresis (Sebia Minicap Flex Piercing)

Serum proteins are a family of albumin and globulins. Albumin is the major fraction synthesized from human liver endogenously and available through various dietary sources exogenously including egg, meat, pulses, milk etc. Globulins are a group of proteins subclassified into alpha-1, alpha-2, beta-1, beta-2, and gamma globulins based on the electrophoretic mobility (**Figure 2**). The normal biological interval of serum total proteins in a healthy adult ranges between 6 and 8 g/dl which

Albumin is a 69 kDa protein. It is the most abundant protein in serum. Albumin is synthesized in the liver and functions as a transport protein of various substances like bilirubin, enzymes, hormones, drugs etc. It also maintains fluid volume within the vascular space. Albumin is the first protein fraction to appear near the anode in

**2**

**3.1 Albumin**

separation

**Figure 1.**

SPE. Altered levels of serum albumin are associated with various clinical conditions. Low levels of albumin are clinically significant and are termed as hypoalbuminemia.

Decreased concentration of serum albumin (hypoalbuminemia) indicates either a poor dietary intake (malnutrition) or a decreased production or an increased loss. Chronic liver disease is a common clinical condition associated with decreased albumin production, and chronic kidney disease (CKD) is the most common disease associated with an increased loss of albumin in urine (proteinuria). This clinical condition is otherwise known as nephropathy. Other causes of hypoalbuminemia include acute and chronic inflammation, critical illness, pregnancy etc.

Apart from hypoalbuminemia which is commonly observed in an electrophoretogram, there are a few variations which can be observed in the in albumin peak including:


The most important aspect of such albumin variants lies in quantification of an albumin peak in such scenarios followed by interpretation and clinical correlation (**Figure 3**).

**Figure 3.**

#### **3.2 Alpha fraction**

As electrophoresis proceeds toward the negative portion of the gel (cathode), the alpha zone is the next band after albumin. The alpha zone is subdivided into two zones: the alpha-1 peak and alpha-2 peak.

The alpha-1 peak consists of alpha-1 antitrypsin (AT), alpha-1-chymotrypsin, and thyroid-binding globulin. Alpha-1 antitrypsin is an acute-phase reactant. The concentration of alpha-1 antitrypsin increases in conditions of inflammation and is usually decreased in patients with alpha-1 antitrypsin deficiency or decreased production of globulin in patients with severe liver disease. A rare variant of alpha-1 antitrypsin is encountered occasionally characterized by a split peak pattern of alpha-1 globulins.

The alpha-2 peak consists of alpha-2 macroglobulin, haptoglobin, and ceruloplasmin. Alpha-2 macroglobulin accounts for about 3% of the total protein in the serum. Because of the variable migration of the haptoglobin types, a2-macroglobulin is often adjacent to, or co migrating with, haptoglobin and is therefore not seen as a discrete band.

A distorted pattern of alpha-2 region in electrophoresis is seen commonly in conditions of hemolysis, including in vivo and in vitro. The pathophysiology behind this pattern is the formation of hemoglobin-haptoglobin complexes in these conditions. This is a physiological adaptive response by human physiology to conserve hemoglobin released as a result of RBC breakdown into circulation and hemoglobin being a smaller globular protein is bound to be lost in urine. Hence to preserve it, haptoglobin is consumed to form complex with hemoglobin which results in the formation of a macromolecular protein which is retained in circulation making hemoglobin available for the production of RBCs and prevention of anemia.

Haptoglobin and ceruloplasmin are acute-phase reactants, and hence increased in acute inflammatory states.

Alpha-2-macroglobulin is increased in nephrotic syndrome and cirrhosis of the liver. The elevation of alpha-2 macroglobulin is distinctly evident in nephritic syndrome, since it is a bulky molecule, and hence retained in circulation to compensate

**5**

including:

*Serum Protein Electrophoresis and Its Clinical Applications*

not aid in the detection of a decreased ceruloplasmin.

for the loss of other proteins in urine which is evident in form of proteinuria in

The beta zone usually is subdivided into two peaks, beta-1 and beta-2 in CZE. Beta-1 zone comprises proteins like transferrin and low-density lipoprotein

Transferrin functions to transport non-heme ferric iron from the gastrointestinal tract. Each Transferrin molecule can bind two molecules of free iron. An increased beta-1 band is observed in iron deficiency anemia due to an increased level of free transferrin and also in pregnancy. Determinations of the transferrin levels are useful in distinguishing between iron deficiency anemia (inadequate intake or chronic hemorrhage with loss of iron stores) and hemolytic anemia, in which transferrin levels are low resulting in a beta-1 peak of low amplitude. Transferrin is usually decreased in alcoholic cirrhosis. Transferrin is also decreased

The beta-2 band is mostly composed of complement proteins, C3 and C4. Elevated beta-2 zone can be caused in inflammatory states due to activation of

A reduced beta-2 peak intensity can be encountered in an aged sample, since the immune complexes are used up and low serum levels of complements are

Fibrinogen is a protein with molecular weight of 340 kDa protein. Sometimes a small fibrinogen band can be seen in serum protein electrophoresis due to the insufficient clotting or failure to remove the serum from the clot. This fibrinogen band is seen between beta-1 and beta-2 regions. This band is also seen in patients who are receiving heparin therapy. It is also an important indicator of the sample type being analyzed. When plasma is used in the place of serum for protein electrophoresis, fibrinogen present in plasma appears in the beta-2 region, and this has the potentiality to interfere

One of the main clinical implications of serum protein electrophoresis is to aid diagnosis of disorders associated with alterations of gamma globulins. Gamma region comprises mainly of serum immunoglobulins. The five major classes of immunoglobulins are IgG, IgA, IgM, IgD, and IgE. The immunoglobulins are characterized by the presence of two protein moieties named as heavy chain and light chain. The classification of immunoglobulin had been made based on the composition of heavy chains, while the light chains are of two types including kappa or lambda. Physiologically,

Various clinical conditions are associated with alteration of gamma globulins

a.Hypergammaglobinemia (increased serum gamma globulin levels)

b.Hypogammaglobinemia (decreased serum gamma globulin levels)

with the detection of monoclonal gammopathies in such patients (**Figure 4**).

kappa forms the major light chain fraction among the two.

Ceruloplasmin is an important copper-binding transport protein produced by the liver. Ceruloplasmin concentrations are markedly decreased in conditions of Wilson's disease. The disadvantage of serum protein electrophoresis is that it will

*DOI: http://dx.doi.org/10.5772/intechopen.88367*

during renal disease and thermal injuries.

complement cascade which include C3 and C4 too.

urine microscopic examination.

**3.3 Beta fraction**

(LDL).

evidenced.

**3.4 Gamma fraction**

for the loss of other proteins in urine which is evident in form of proteinuria in urine microscopic examination.

Ceruloplasmin is an important copper-binding transport protein produced by the liver. Ceruloplasmin concentrations are markedly decreased in conditions of Wilson's disease. The disadvantage of serum protein electrophoresis is that it will not aid in the detection of a decreased ceruloplasmin.

#### **3.3 Beta fraction**

*Biochemical Testing - Clinical correlation and Diagnosis*

As electrophoresis proceeds toward the negative portion of the gel (cathode), the alpha zone is the next band after albumin. The alpha zone is subdivided into two

The alpha-1 peak consists of alpha-1 antitrypsin (AT), alpha-1-chymotrypsin, and thyroid-binding globulin. Alpha-1 antitrypsin is an acute-phase reactant. The concentration of alpha-1 antitrypsin increases in conditions of inflammation and is usually decreased in patients with alpha-1 antitrypsin deficiency or decreased production of globulin in patients with severe liver disease. A rare variant of alpha-1 antitrypsin is encountered occasionally characterized by a split peak pattern of

The alpha-2 peak consists of alpha-2 macroglobulin, haptoglobin, and ceruloplasmin. Alpha-2 macroglobulin accounts for about 3% of the total protein in the serum. Because of the variable migration of the haptoglobin types, a2-macroglobulin is often adjacent to, or co migrating with, haptoglobin and is therefore not seen

A distorted pattern of alpha-2 region in electrophoresis is seen commonly in conditions of hemolysis, including in vivo and in vitro. The pathophysiology behind this pattern is the formation of hemoglobin-haptoglobin complexes in these conditions. This is a physiological adaptive response by human physiology to conserve hemoglobin released as a result of RBC breakdown into circulation and hemoglobin being a smaller globular protein is bound to be lost in urine. Hence to preserve it, haptoglobin is consumed to form complex with hemoglobin which results in the formation of a macromolecular protein which is retained in circulation making hemoglobin available for the production of RBCs and prevention of anemia.

Haptoglobin and ceruloplasmin are acute-phase reactants, and hence increased

Alpha-2-macroglobulin is increased in nephrotic syndrome and cirrhosis of the liver. The elevation of alpha-2 macroglobulin is distinctly evident in nephritic syndrome, since it is a bulky molecule, and hence retained in circulation to compensate

**3.2 Alpha fraction**

**Figure 3.**

alpha-1 globulins.

as a discrete band.

in acute inflammatory states.

zones: the alpha-1 peak and alpha-2 peak.

*Abnormal electrophoretic patterns of albumin zone.*

**4**

The beta zone usually is subdivided into two peaks, beta-1 and beta-2 in CZE. Beta-1 zone comprises proteins like transferrin and low-density lipoprotein (LDL).

Transferrin functions to transport non-heme ferric iron from the gastrointestinal tract. Each Transferrin molecule can bind two molecules of free iron. An increased beta-1 band is observed in iron deficiency anemia due to an increased level of free transferrin and also in pregnancy. Determinations of the transferrin levels are useful in distinguishing between iron deficiency anemia (inadequate intake or chronic hemorrhage with loss of iron stores) and hemolytic anemia, in which transferrin levels are low resulting in a beta-1 peak of low amplitude. Transferrin is usually decreased in alcoholic cirrhosis. Transferrin is also decreased during renal disease and thermal injuries.

The beta-2 band is mostly composed of complement proteins, C3 and C4. Elevated beta-2 zone can be caused in inflammatory states due to activation of complement cascade which include C3 and C4 too.

A reduced beta-2 peak intensity can be encountered in an aged sample, since the immune complexes are used up and low serum levels of complements are evidenced.

Fibrinogen is a protein with molecular weight of 340 kDa protein. Sometimes a small fibrinogen band can be seen in serum protein electrophoresis due to the insufficient clotting or failure to remove the serum from the clot. This fibrinogen band is seen between beta-1 and beta-2 regions. This band is also seen in patients who are receiving heparin therapy. It is also an important indicator of the sample type being analyzed. When plasma is used in the place of serum for protein electrophoresis, fibrinogen present in plasma appears in the beta-2 region, and this has the potentiality to interfere with the detection of monoclonal gammopathies in such patients (**Figure 4**).

#### **3.4 Gamma fraction**

One of the main clinical implications of serum protein electrophoresis is to aid diagnosis of disorders associated with alterations of gamma globulins. Gamma region comprises mainly of serum immunoglobulins. The five major classes of immunoglobulins are IgG, IgA, IgM, IgD, and IgE. The immunoglobulins are characterized by the presence of two protein moieties named as heavy chain and light chain. The classification of immunoglobulin had been made based on the composition of heavy chains, while the light chains are of two types including kappa or lambda. Physiologically, kappa forms the major light chain fraction among the two.

Various clinical conditions are associated with alteration of gamma globulins including:

a.Hypergammaglobinemia (increased serum gamma globulin levels)

b.Hypogammaglobinemia (decreased serum gamma globulin levels)

**5**

#### **Figure 4.**

*Fibrinogen producing a peak in beta 2 region (from a plasma sample).*

Hypergammaglobinemia (gammopathies):

Gammopathy is defined as abnormal proliferation of the lymphoid cells producing immunoglobulins. There are four types of gammopathies: polyclonal, monoclonal, biclonal, and oligoclonal.

Polyclonal gammopathies are defined as heterogeneous increase in immunoglobulins involving more than one cell line, commonly caused by a variety of inflammatory conditions (chronic inflammation), infections, chronic liver diseases (cirrhosis), chronic kidney diseases, etc.

Monoclonal gammopathies are characterized by a homogenous increase produced by clonal population of mature B cells, most commonly plasma cells. Monoclonal immunoglobulins seen in these conditions are also known as Para proteins. The classic interpretative terminology used in clinical laboratory medicine for describing a monoclonal immunoglobulin in SPE is "M" band where M stands for monoclonal. Common clinical disorders producing "M" Band in SPE include multiple myeloma and plasmacytoma in usually 60% of cases and Waldenströms Macroglobulinemia, lymphomas, and leukemia in approximately 10% of cases. Certain monoclonal gammopathies produce "M" band in electrophoretic regions other than in gamma regions, commonly being beta region especially in case of IgA and IgG myeloma.

Biclonal gammopathies are characterized by a double peak in the gamma region. This electrophoretic pattern is seen when there is a biclonal proliferation of immunoglobulins encountered in multiple myeloma. A biclonal pattern is also seen in monoclonal gammopathies associated with IgA and IgG. In such scenarios, these immunoglobulins appear as polymerized and monomerized forms which elute as biclonal peaks in gamma region or in beta region, respectively (**Figure 5**).

The oligoclonal pattern of gamma region is characterized by more than two peaks evident in the gamma region. This pattern is commonly seen in autoimmune disorders, light chain myelomas (characterized by clonal proliferation of light chains), amyloidosis, etc. (**Figure 5**).

Apart from serum immunoglobulin, C-reactive protein (CRP) also is evident in the gamma region. C-reactive proteins levels usually increase during inflammatory responses.

Apart from the common causes of altered electrophoresis picture specific to the particular zones, a sharp distinct peak when evident especially in beta or alpha region

**7**

*Serum Protein Electrophoresis and Its Clinical Applications*

should raise a high index of diagnostic suspicion of multiple myeloma since a few monoclonal immunoglobulins shall migrate in these zones too, in contrary to the classical gamma zone M protein pattern, which is commonly reported in these conditions.

According to the International Myeloma Foundation, plasma cell dyscrasias are group of plasma cell disorders involving a wide spectrum of pathologies including:

4.Multiple myeloma (which includes various subtypes including nonsecretory myeloma (NSMM), light chain myeloma, secretory multiple myeloma)

Criteria for diagnosis and differentiation of the plasma cell disorders based on

1.**Monoclonal gammopathy of undetermined significance (MGUS)**

1.MGUS—monoclonal gammopathy of undetermined significance

2.MGRS—monoclonal gammopathy of renal significance

International Myeloma Working Group (IMWG) criteria:

• M protein (Monoclonal band)—<3 g/dl

• Bone marrow biopsy—<10% plasma cells seen

**4. Role of SPE in multiple myeloma work-up**

*Abnormal electrophoretic patterns of gamma zone.*

3.Smoldering myeloma

**Figure 5.**

*DOI: http://dx.doi.org/10.5772/intechopen.88367*

*Serum Protein Electrophoresis and Its Clinical Applications DOI: http://dx.doi.org/10.5772/intechopen.88367*

*Biochemical Testing - Clinical correlation and Diagnosis*

Hypergammaglobinemia (gammopathies):

*Fibrinogen producing a peak in beta 2 region (from a plasma sample).*

nal, biclonal, and oligoclonal.

**Figure 4.**

and IgG myeloma.

(cirrhosis), chronic kidney diseases, etc.

chains), amyloidosis, etc. (**Figure 5**).

Gammopathy is defined as abnormal proliferation of the lymphoid cells producing immunoglobulins. There are four types of gammopathies: polyclonal, monoclo-

Biclonal gammopathies are characterized by a double peak in the gamma region. This electrophoretic pattern is seen when there is a biclonal proliferation of immunoglobulins encountered in multiple myeloma. A biclonal pattern is also seen in monoclonal gammopathies associated with IgA and IgG. In such scenarios, these immunoglobulins appear as polymerized and monomerized forms which elute as

biclonal peaks in gamma region or in beta region, respectively (**Figure 5**).

The oligoclonal pattern of gamma region is characterized by more than two peaks evident in the gamma region. This pattern is commonly seen in autoimmune disorders, light chain myelomas (characterized by clonal proliferation of light

Apart from serum immunoglobulin, C-reactive protein (CRP) also is evident in the gamma region. C-reactive proteins levels usually increase during inflammatory

Apart from the common causes of altered electrophoresis picture specific to the particular zones, a sharp distinct peak when evident especially in beta or alpha region

Polyclonal gammopathies are defined as heterogeneous increase in immunoglobulins involving more than one cell line, commonly caused by a variety of inflammatory conditions (chronic inflammation), infections, chronic liver diseases

Monoclonal gammopathies are characterized by a homogenous increase produced by clonal population of mature B cells, most commonly plasma cells. Monoclonal immunoglobulins seen in these conditions are also known as Para proteins. The classic interpretative terminology used in clinical laboratory medicine for describing a monoclonal immunoglobulin in SPE is "M" band where M stands for monoclonal. Common clinical disorders producing "M" Band in SPE include multiple myeloma and plasmacytoma in usually 60% of cases and Waldenströms Macroglobulinemia, lymphomas, and leukemia in approximately 10% of cases. Certain monoclonal gammopathies produce "M" band in electrophoretic regions other than in gamma regions, commonly being beta region especially in case of IgA

**6**

responses.

**Figure 5.** *Abnormal electrophoretic patterns of gamma zone.*

should raise a high index of diagnostic suspicion of multiple myeloma since a few monoclonal immunoglobulins shall migrate in these zones too, in contrary to the classical gamma zone M protein pattern, which is commonly reported in these conditions.
