Serum Protein Electrophoresis Dr. Nitin Inamdar Department of Biochemistry Tata Memorial Center, Parel, Mumbai.

Electrophoresis Phoresis: Separation or migration or movement Electro: Under influence of electric field

Constant current 5mAM/Slide 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 on their 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 Electrophoresis Paper electrophoresis Starch gel electrophoresis Agarosre gel electrophoresis Cellulose acetate gel electrophoresis Capillary 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) containers for staining and destaining gel electrophoresis chamber applicator

Various electrophoresis 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

Indication of serum electrophoresis Diagnostic: Diagnosis of plasma cell dyscrasia Diagnosis of Waldenstrom’ macroglbulinemia Monitoring of disease: Monitoring of plasma cell dyscrasia

Method of detection Serum protein electrophoresis Quantitative immunoglobulins (nephelometry) Immunofixation, immunoelectrophoresis and immunodiffusion Urine studies light chain

Albumin Globulins: α1, α2, β1, β2, gamma A/G Ratio: 2.0

Normal serum electrophoresis Anode +ve Cathode -ve Normal serum electrophoresis pH=8.6, 1% agarose gel, sodium barbitone buffer

Abnormal serum electrophoresis showing M band Anode +ve Cathode -ve Abnormal serum electrophoresis showing M band

Normal serum electrophoresis

Abnormal serum electrophoresis showing M band

WHO Classification of Plasma Cell Neoplasms Plasma cell myeloma (MM) Plasma cell myeloma variants Non secretory myeloma Indolent Myeloma Smoldering Myeloma Plasma cell leukemia Plasmacytomas Solitary plasmacytoma of bone Extramedullary plasmacytoma Immunoglobulin deposition diseases Primary Amyloidosis Systemic light and heavy chain deposition disease Osteosclerotic myeloma (POEMS syndrome) Heavy chain disease Gamma /Mu / Alpha

Diagnostic criteria for plasma cell dyscrasia Symptomatic plasma cell myeloma: BM clonal plasma cells or Plasmacytoma Marrow 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/dL Marrow Plasmacytosis < 10% No Lytic bone lesions or related organ impairment etc

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.

Immunofixation electrophoresis 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

Intravascular hemolysis

light chain myeloma 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.

Freelite test Free Kappa Free Lambda Kappa/ Lambda ratio 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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