Serum Protein Electrophoresis

Dr. Nitin Inamdar Department of BiochemistryTata Memorial Center, Parel,Mumbai.

Phoresis: Separation or migration or movement

Electro: Under influence of electric field

Electrophoresis

Serum

Electrophoresis

Mixture of proteins

Amino acids:◦ Amino group (NH2)◦ Carboxyl group (COOH)

Two amino acids join each other with polypeptide bond to form polypeptide chain & many polypeptide chains make a protein.

Ph 8.6/0.075M Barbitone buffer

1%Agarose

Constant current 5mAM/Slide

Electrophoresis separates proteins based ontheir physical properties.

Serum is placed on a specific medium, and a charge is applied. The net charge (positive or negative) and

the size and shape of the subsets of these proteins are used in interpreting the results.

Serum ElectrophoresisPaper electrophoresis

Starch gel electrophoresisAgarosre gel electrophoresis

Cellulose acetate gel electrophoresisCapillary gel electrophoresis

Serum protein electrophoresis on agarose gel is a type of horizontal gel electrophoresis

Equipment used for the gel electrophoresis

power supply (direct current)

electrophoresis chamber

containers for staining and destaining gel

applicator

Serum/protein electrophoresis: ◦ plasma cell dyscrasia, nephrotic syndrome, etc.

Hemoglobin electrophoresis: ◦ thalassemia, hemoglobinopathies like sickle cell

anemia, HbC, HbD, etc.

Urine electrophoresis: ◦ BJP in plasma cell dyscrasia

Various electrophoresis

Diagnostic: ◦ Diagnosis of plasma cell dyscrasia◦ Diagnosis of Waldenstrom’ macroglbulinemia

Monitoring of disease: ◦ Monitoring of plasma cell dyscrasia

Indication of serum electrophoresis

1. Serum protein electrophoresis2. Quantitative immunoglobulins

(nephelometry)3. Immunofixation, immunoelectrophoresis

and immunodiffusion4. Urine studies5. light chain

Method of detection

Albumin

Globulins:◦ α1, ◦ α2, ◦ β1, ◦ β2, ◦ gamma

A/G Ratio: 2.0

Normal serum electrophoresispH=8.6, 1% agarose gel, sodium barbitone buffer

Anode +ve Cathode -ve

Abnormal serum electrophoresis showing M band

Anode +ve Cathode -ve

Normal serum electrophoresis

Abnormal serum electrophoresis showing M band

Plasma cell myeloma (MM) Plasma cell myeloma variants

Non secretory myelomaIndolent MyelomaSmoldering MyelomaPlasma cell leukemia

PlasmacytomasSolitary plasmacytoma of boneExtramedullary plasmacytoma

Immunoglobulin deposition diseasesPrimary AmyloidosisSystemic light and heavy chain

deposition disease Osteosclerotic myeloma (POEMS

syndrome) Heavy chain disease

Gamma /Mu / Alpha

WHO Classification of Plasma Cell Neoplasms

Symptomatic plasma cell myeloma:BM clonal plasma cells or PlasmacytomaMarrow plasmacytosis (>30%)M-component in serum or urine

Serum: Ig G >3 g/dL, Ig A >2.5 g/dL or Urine: >/= 1g/ day of λ or κ [BJP]

Asymptomatic plasma cell myeloma:M band in serum at myeloma levels > 3 gm/dL and /or 10% or more clonal plasma cell in BM No related organ or tissue impairment (end organ

damage or bony lesion) or myeloma-related symptoms MUGS:

M-Component, present but less than 3 gm/dLMarrow Plasmacytosis < 10%No Lytic bone lesions or related organ impairment etc

Diagnostic criteria for plasma cell dyscrasia

Immunoglobin (Ig) structure and types

Malignant Plasma Cell (Clonal Proliferation) produces one type of Ig either A,M, G, D or E with kappa or lambda light chains in excess amount.

Various patterns

Analbuminemia

Haptoglobin phenotype

Bis-albuminemia

Nephrotic syndrome

Hypergammaglobulinemia

M band in β region

M band in γ region

Nephelometry Antigen-antibody complexes, when formed at a

high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance.

• Nephelometry: is indirect measurement of light scattered by the antigen-antibody complexes.

After separation of serum proteins by electrophoresis separated serum in different lanes is incubated with monospecific anti sera against IgA, M G and κ λ

Immunofixation electrophoresis

Intravascular hemolysis

A type of multiple myeloma in which plasma cell tumors produce only monoclonal light chain proteins. Persons with light chain disease may develop lytic bone lesions, hypercalcemia, impaired kidney function, and amyloidosis.

light chain myeloma

Normal serum mean median 95 %concentration concentration range

Free Kappa 8,36 mg/l 7,30 mg/l 3,30 – 19,40 mg/l

Free Lambda 13,43 mg/l 12,40 mg/l 5,71 – 26,30 mg/l

Kappa/

Lambda ratio

mean median total range

0,63 0,60 0,26 – 1,65

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