Electrophoresis and its Application for diagnosis Ratchada Cressey, PhD Clin. Chem 202.

Electrophoresis and its Application for

diagnosis

Ratchada Cressey, PhD

Clin. Chem 202

Principal of electrophoresis

• Electrophoresis is the process of moving charged molecule in solution by applying an electric field across the mixture

- - -+ - - +

-+

+ + - + +

- +

Electrode

Serum Protein Electrophoresis(SPE)

• Electrophoresis is refer to the migration of all charged solutes or particle in liquid medium under influence of an electric field

Serum Protein Electrophoresis

SPE procedure

• Sample preparation• Gel loading• Gel running• Protein staining• quantification

Procedure (i) Membrane and Sample preparation

Procedure (ii) sample application and electroph

ores

Separated protein bands can be quantified using

Densitometer

Densitometer is one of the application of

spectrophotometer

• Absorbance measurement is made through a solid material,using support medium is the cuvet

• Accuracy and precision are limited by variations in path length and scatter from the solid medium

Normal SPE

Albumin 47-71% (3.63-4.91 g/dL)Alpha-1 globulin 2.7-5.8% (0.11-0.35 g/dL)Alpha-2 globulin 5.1-12.0% (0.65-1.17 g/dL)Beta- globulin 4.5-15.7% (0.74-1.26 g/dL)Gamma globulin 11.3-24.0% (0.58-1.74 g/dL)

Clinical Significance of Albumin

• Reference range 3.5-5.5 g/dL• Hyperalbuminemia dehydration• Hypoalbuminemia

• Low intake, synthesis• Increase loss:

• kidney; nephrotic syndrome, wound, burn• GI tract; protein-losing enteropathy

• Increase catabolism

Clinical Significance of 1-globulin

•Increased alpha 1 globulin due to increased alpha 1 antitrypsin: inflammatory/acute phase

•Decreased/absent alpha1 globulin due to alpha1antitrypsin deficiency: infant with neonatal cirrhosis or child with emphysema

Clinical Significance of 2-globulin

•Increased alpha 2 globulin due to alpha 2 macroglobulin: nephrotic sx with peripheral edema, membranous glomerulonephritis, hyperlipidemia

•Decreased alpha 2 globulin due to haptoglobin: hemolytic anemia with spherocytes in PB

Clinical Significance of -globulin

•Increased beta globulin due to increased transferrin in iron deficiency anemia: microcytic hypochromic RBCs; third trimester of pregnancy

•Increased beta-gamma with bridging: cirrhosis

Clinical Significance of -globulin

MONOCLONAL GAMMOPATHY

• Monoclonal peak: narrow needle-shaped “spike”

• With plasma cell myeloma (IgG,IgA) or IgM type in LPL lymphoplasmacytic lymphoma

• In gamma region usually IgG • In gamma, beta or even alpha 2 globulin

region, usually IgA • If at application point between beta & gamma

regions, IgM type

POLYCLONAL HYPERGAMMAGLOBULINEMIA

• Caused by reactive processes• Broad asymmetric peak• Chronic infection including HIV+/AIDS • Autoimmune/collagen vascular disease:

systemic lupus erythematosus (SLE), rheumatoid arthritis (RA)

• Liver disease

• The nephrotic pattern • illustrates the long term loss of lower molecular weight proteins

(Examples: albumin, IgG) and retention of higher molecular weight proteins (example alpha-2-macroglobulin)

• This patient was a 39 year old female with SLE. Her daily total urinary protein loss exceeded 3700 mg. (Normal loss is less that 150 mg/day.)

http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE

Normal

• Polyclonal gammopathy • usually occurs secondary to many chronic diseases. This patient was a 3

9 year old male with sarcordosis. The sequential increase of the globulin fractions illustrated "sarcoid stepping." IFE excluded the possibility of a monoclonal protein.

http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE

Normal

• Liver disease:• Patient was a 46 year old male with end stage liver disease se

condary to chronic alcohol abuse. He died 5 months after this sample was obtained.

• In the cirrhotic pattern, the distinction between beta and gamma globulin is blurred and is sometimes referred to as the "beta-gamma bridge" pattern.

Normal

http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE

• Acute inflammatory pattern• This patient was a 42 year old female who presented with a temp

erature of 40 degrees oC and was diagnosed with both pneumonia and pyelonephritis.

• In the acute inflammatory pattern albumin and gamma globulin are decreased and alpha-2-globulin becomes very prominent. In both the strip and scan note that the intensity of the alpha-2 fraction is greater that the gamma globulin fraction.

Normal

http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE

• Alpha-1-anti-trypsin deficiency • can be congenital or acquired, typically secondary to liver or pulmo

nary diseases. • Congenital alpha-1-anti-trypsin deficiency is most commonly assoc

iated with early onset emphysema, pancreatic insufficiency, or cirrhosis.

Normal

http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE

• Immunofixation (IFIX) is a powerful enhancement of immunoelectrophoresis in which a series of post-electrophoretic gel slabs are layered with cellulose-acetate gels saturated with specific antibodies.

• The resulting antigen-antibody complexes fixed on the second gel may then be stained, allowing sensitive and specific qualitative visual identification of paraproteins by electrophoretic position.

Immunofixation Electrophoresis (IFE)

1. Diluted patient serum is applied to each track of the gel using an application mask.

2. The gel is electrophoresed, separating proteins according to their charge.

3. Protein fixative and monospecific antisera to IgG, IgA, IgM, Kappa and Lambda are applied to the gel.

4. During incubation, insoluble immune complexes are formed.

5. The gel is now washed, pressed, dried, and stained.

6. Samples are examined for paraprotein bands in the TSP and Ig tracks

IFE of serum

IFE of Urine

Samples required for IFE

• 100 mL aliquot of 24 hour urine • No preservative required.

Random urine will be accepted, but not a recommended specimen due to low protein content.

• 0.5 mL serum

• Monoclonalprotein present• The patient was a 72 year old male

who presented with lower back pain.

• Quantitative immunoglobulin measurements showed a large increase in serum IgG, but decreased IgA and IgM. Bone marrow exam revealed a large increase in plasma cells that were frequently aggregated.

• IFE on this patient's serum showed the M protein was IgG kappa. A diagnosis of multiple myeloma was made.

http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE

• Biclonal gammopathy• This sample is from a 62 yea

r old male who presented with weight loss and fatigue. He was found to have multiple myeloma.

• In this disease, biclonal gammopathies are rare, occurring in about 1.7 % of patients. IFE showed the 2 M proteins to be IgG-k and IgA-lambda

http://erl.pathology.iupui.edu/labmed/Generator.cfm?Image=SPE

Electrophoresis of Urine• a minimum of 10 ml of urine for

electrophoresis. • Most urine samples require

concentration to obtain this minimum amount and for optimal concentration,

• Electrophoresis on urine is the preferred technique for identification of Bence Jones proteins in patients with suspected multiple myeloma.

• It should always be done in conjunction with serum, so the results can be compared directly.

Electrophoresis of Urine

Normal RangeUrine Total Protein ( < 140 mg/24hr )

Urine Albumin ( < 30 mg/24hr )