1. Serum Protein Electrophoresis (SPEP)
Lab 3
Serum Protein Electrophoresis (SPEP)

2. What does it mean ?
Serum protein electrophoresis is an easy method of separating serum proteins based on their net charge, size, and shape.
A small amount of serum is placed on a specific medium (such as agarose) and an electrical charge is applied.
The proteins then migrate across the medium in a characteristic manner, due to the net charge and size and shape of the protein.
In routine serum protein electrophoresis, the protein will separate into five main components, identified as albumin and the globulins (alpha1, alpha2, beta and gamma) .
Protein electrophoresis is used to identify the presence of abnormal proteins, to identify the absence of normal proteins, and to determine when different groups of proteins are present in unusually high or low amounts in blood or other body fluids.

3. Direction of protein migration

4. Electrophoresis fractions
Approximately 60% of the total protein in the serum is albumin, and represents the largest peak that lies closest to the positive electrode, while the remaining fractions are composed mainly of globulins
Globulins comprise a much smaller fraction of the total serum protein but represent the primary focus of interpretation of serum protein electrophoresis.
The γ region contains the largest portion of globulins, therefore monoclonal gammopathies are most frequently encountered in the electrophoresis
An increase in gamma globulin is referred to as a gammopathy.
Serum proteins are negative charged at pH 8.6 (a buffer helps to maintain a constant pH) and they move toward the anode at the rate dependent on their net charge

5. Overview for the pattern of SPEP
electrode
-ve
electrode

6. 1. Albumin
Albumin represents the highest peak in serum protein electrophoresis, usually seen as a single, tall band.
Occasionally, two equally staining bands, referred to as bisalbuminemia or a widely staining band may be seen, which both represent normal variations.
 Common conditions associated with decreased albumin include malnutrition, cirrhosis and nephrotic syndrome.
Dehydration, on the other hand, causes a high albumin.
Absence of albumin, known as analbuminaemia, is rare
An area between albumin and the alpha-1 band is called the albumin-alpha-1 interzone. Even staining in this zone is caused by alpha-1 lipoprotein (High-density lipoprotein or HDL). An increase is usually seen in alcoholic liver disease, pregnancy and during puberty. A sharp band may also be seen in those with hepatocellular carcinoma as a result of elevated alpha-fetoprotein (AFP)
Heterozygous individuals may produce bisalbuminemia - two equally staining bands, the product of two genes. Some variants give rise to a wide band or two bands of unequal intensity but none of these variants is associated with disease

7. 2. Alpha zone
The alpha-1 fraction includes α-1 antitrypsin, transcortin, and thyroid-binding globulin.
α-2 fraction is comprised of ceruloplasmin,
α-2macroglobulin, and haptoglobin.
Both alpha-1 and 2 represent the acute phase reactants; hence, malignancy, infection or any inflammatory condition can cause their elevation.

8. A relative increase in α-2 fraction may be seen in nephrotic syndrome due to the relative larger size of the proteins and the inability to pass through the glomeruli.
A decrease in α-1 component may be seen in α-1 antitrypsin deficiency
A decrease in α -2 component may be seen in hemolytic anemia due to decreased haptoglobin levels.

9. 3. Beta zone
The beta zone consist of β-1 and β-2 but is often represented a graphically as a single band.
β-1 consists mostly of transferrin, and is increased in conditions such as iron-deficiency anemia, pregnancy and estrogen therapy. B-lipoprotein and C3 complement are included in the β -2 component.
IgA has an anodal mobility and migrates in the region between the beta and gamma zones also causing a beta/gamma fusion in patients with cirrhosis, respiratory infection, skin disease, or rheumatoid arthritis (increased IgA).

10. 4. Gamma zone
Immunoglobulins mainly comprise this area including IgG, IgA, IgM, IgD and IgE, but note that Igs may be found in the α and β zones.
If the γ zone shows an increase (or spike), the first step in interpretation is to establish if the region is narrow or wide.
If it is elevated in a single narrow "spike-like" manner it could indicate monoclonal production of a single immunoglobulin (monoclonal gammopathy), while a broad "swell-like" manner (wide) indicates polyclonal immunoglobulin production.
However, a homogenous, spike-like increase in the gamma region is of special interest as it may represent an abnormal expansion of immunoglobulin-producing plasma cells.

11. Various inflammatory, autoimmune and hematologic, and non-hematologic diseases are associated with an increased gamma peak
Agammaglobulinemia and hypogammaglobulinemia syndromes such as IgA deficiency are associated with a decrease in this area.

12. Summary

13. Are there another fractions ?!
Standard SPE separates the protein into five distinct bands but, by modifying the electrophoretic parameters, proteins can be further separated into as many as 12 bands.
The modification, known as HRE, uses a higher voltage coupled with a cooling system in the electrophoretic apparatus and a more concentrated buffer.

15. Indications for requesting SPEP
1ST : Indications based on clinical findings:
Suspected multiple myeloma, Waldenström’s macroglobulinemia, primary amyloidosis or other related disorders
Unexplained bone pain or fracture
Recurrent infections
Unexplained peripheral neuropathy (not able to be attributed to another condition, e.g. type 2 diabetes, chemotherapy)
Unexplained proteinuria
When a health practitioner is investigating symptoms that suggest multiple myeloma
To monitor treatment of multiple myeloma to see if the monoclonal band is reduced in quantity or disappears completely with treatment

16. 2ND :Indications based on laboratory findings
High (or low) total serum globulin or immunoglobulin
Extremely high percentage of lymphocytes
Unexplained anaemia (multiple myeloma is a recognised cause of non-iron deficiency anaemia) or other persisting cytopaenias for which there is no other explanation
Unexplained high ESR (>50) with a normal CRP
Unexplained hypercalcaemia or renal impairment
Red cell rouleaux formations noted on the peripheral blood smear
Presence of urine free light chains (Bence-Jones proteinuria)
As a follow up to abnormal findings on other laboratory tests, such as total protein and/or albumin level, elevated urine protein levels, elevated calcium levels, or low white or red blood cell counts

17. Specimen
The specimen most often used to determine the total protein is serum rather than plasma.
Container : Red-top tube, serum separator tube (SST)
A fasting specimen is not needed.
Interferences in some of the methods occur in the presence of lipemia; hemolysis falsely elevates the total protein result because of the release of RBC proteins into the serum.

18. Run the test
When an abnormality is found in the total protein or albumin, an electrophoresis is usually performed.
In the standard method for serum protein electrophoresis (SPE) :
1. serum samples are applied close to the cathode end of a support medium that is saturated with an alkaline buffer (pH 8.6).
2. The support medium is connected to two electrodes and a current is passed through the medium to separate the proteins.
3. All major serum proteins carry a net negative charge at pH 8.6
and migrate toward the anode.
If an abnormality is seen on the electrophoretic pattern, an analysis of the individual proteins within the area of abnormality is made

20. For quantitative investegations
Many scanning densitometers compute the area under the absorbance curve for each band and the percentage of total dye that appears in each fraction.
The concentration is then calculated as a percentage of the total protein that was determined by one of the protein methods, such as the biuret procedure.
For example: n Spike is 60% of total protein n Total protein=12 grams/deciliter (g/dL) n Spike level=7.2 g/dL (60% of 12)
The crucial information comes from SPEP by calculating the size of the “spike”, which depicts the amount of abnormal protein (done by measuring the area between the top of the spike and the baseline of the graph)

21. Reference serum control
A reference serum control is processed with each electrophoretic run
Reference values for each fraction are as follows: 1. Albumin : –65% of the total protein (3.5–5.0 g/dL) 2. α : –5% (0.1–0.3 g/dL) 3. α : 7–13% (0.6–1.0 g/dL) 4. β : 8–14% (0.7–1.1 g/dL) 5. γ : –22% (0.8–1.6 g/dL)

22. Note that :
The great advantage of electrophoresis compared with the quantitation of specific proteins is the overview it provides.
The electrophoretic pattern can give information about the relative increases and decreases within the protein population, as well as information about the homogeneity of a fraction.

23. Let’s assess some electrophoresis patterns

24. In α1-antitrypsin deficiency , there is marked decrease in α1-fraction

25. In nephrotic syndrome, the patient loses serum albumin and low-molecular-weight proteins in the urine. Some IgG is also lost.
At the same time, an increase occurs in 2-macroglobulin, -lipoprotein, complement components, and haptoglobin.
These lead to a dramatic decrease in the relative amount of albumin and a significant increase in the relative amounts of 2-globulin fractions

26. An inflammatory pattern indicating an inflammatory condition is seen when there is a decrease in albumin and an increase in the 1-globulins (1-acid glycoprotein, 1-antitrypsin), 2-globulins (ceruloplasmin and haptoglobin), and -globulin band (C-reactive protein) (Fig. 10-9E). This pattern, also called an acute-phase reactant pattern, is seen in trauma, burns, infarction, malignancy, and liver disease. Acute-phase reactants are so named because they are increased in the serum within days following trauma or exposure to inflammatory agents. Fibrinogen, haptoglobin, ceruloplasmin, and serum amyloid A increase severalfold, whereas CRP and 2-macrofetoprotein are increased several hundredfold. Chronic infections also produce a decrease in the albumin, but the globulin increase is found in the fraction as well as the 1, 2, and fractions.
An inflammatory pattern indicating an inflammatory condition is seen when there is a decrease in albumin and an increase in the α1-globulins (α 1-acid glycoprotein,
α 1-antitrypsin), α 2-globulins (ceruloplasmin and haptoglobin), and -globulin band
(C-reactive protein).
This pattern, also called an acute-phase reactant pattern, is seen in trauma, burns, infarction, malignancy, and liver disease

27. The electrophoretic pattern of serum proteins in liver disease shows the decrease in serum albumin concentration and the increase in -globulin. In the pattern in cirrhosis of the liver, there are some fast-moving -globulins that prevent resolution of the - and -globulin bands. This is known as the – bridge of cirrhosis (Fig. 10-9F). In infectious hepatitis, the -globulin fraction rises with increasing hepatocellular damage. In obstructive jaundice, there is an increase in the 2- and -globulins. Also noted in obstructive jaundice is an increased concentration of lipoproteins, which is an indicator of its biliary origin. This is especially the case when there is little or no decrease of the serum albumin
The electrophoretic pattern of serum proteins in liver disease shows the decrease in serum albumin concentration and the increase in β-globulin.
In the pattern in cirrhosis of the liver, there is fast-moving globulin prevent resolution of the β - and γ-globulin bands. This is known as the β- γ bridge of cirrhosis

28. Probably the most significant finding from an electrophoretic pattern
A dense narrow band that is composed of a single class of immunoglobulins secreted by an abnormally expanded clone of plasma cells is known as M-protein (paraprotein, monoclonal protein or M-component).[2] An M-protein usually presents as a single narrow peak, resembling a "church spire," in the gamma, beta, or alpha-2 region of the densitometer tracing, or as a dense, discrete band on the agarose gel
Probably the most significant finding from an electrophoretic pattern

29. Interpretation
A dense narrow band that is composed of a single class of immunoglobulins secreted by an abnormally expanded clone of plasma cells is known as M-protein (paraprotein, monoclonal protein or M-component).
An M protein is characterized by the presence of a sharp, well-defined band with a single heavy chain and a similar band with a kappa or lambda light chain.
Diseases that produce an increase in the γ-globulin level include Hodgkin’s disease, malignant lymphoma, chronic lymphocytic leukemia, connective tissue diseases, liver diseases, multiple myeloma, Waldenström’s macroglobulinemia, and amyloidosis
Once a monoclonal gammopathy is identified by serum protein electrophoresis, multiple myeloma must be differentiated from other causes of this type of gammopathy. Among these other causes are Waldenström’s macroglobulinemia,, plasma cell leukemia, heavy chain disease, and amyloidosis

30. Polyclonal Immunoglobulin disease
It is extremely important to differentiate monoclonal from polyclonal gammopathies. Monoclonal gammopathies are associated with a clonal process that is malignant or potentially malignant. In contrast, polyclonal gammopathies may be caused by any reactive or inflammatory process, and they usually are associated with nonmalignant conditions.
A polyclonal gammopathy is characterized by a broad diffuse band
Infectious, Inflammatory or various reactive processes may be associated with a broad-based peak or band in the gamma region .
This pattern suggests a polyclonal increase in immunoglobulins. Liver disease, autoimmune disease, chronic viral or bacterial infections

32. The End