Serum Electrophoresis AND IMMUNOFIXATION june 2013 Dr. Nitin A Inamdar Department of Biochemistry Tata Memorial Center

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

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

Serum 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. Electrophoresis Ph 8.6/0.075M Barbitone buffer 1%Agarose Constant current 5mAM/Slide

 Immunity Immune cell types – T lymphocytes Mature in thymus – B lymphocytes Mature in marrow – Antigen- presenting cells (APCs)

 Immunity Humoral response – Secondary response Memory cells start response to same antigen

 Immunity Antibody classes = Immunoglobulins – IgD, IgM, IgG In blood &/or attached to B lymphocytes – IgA In saliva, sweat, tears, etc. – IgE Bound to basophils & mast cells Causes release of histamine; allergy; parasitic infection

 Immunity Antibodies – Large complex proteins Y-shaped 4 polypeptide chains – 2 “heavy” – 2 “light” Constant region Variable region with antigen binding sites

Programmed cell death is part of normal development Falling of leaves: Apoptosis

In Multiple Myeloma Normal Plasma cell become Abnormal Plasmacell

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

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

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.

 Method of detection 1.Serum protein electrophoresis 2.Quantitative immunoglobulins (nephelometry) 3.Immunofixation, immunoelectrophoresis and immunodiffusion 4.Urine studies 5.Free light chain assay

Equipment used for the gel electrophoresis power supply (direct current) electrophoresis chamber containers for staining and destaining gel applicator

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

Albumin Globulins: – α1, – α2, – β1, – β2, – γ

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

Abnormal serum electrophoresis showing M band Anode +veCathode -ve

M bandNormal study

 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

 Immuno-fixation electrophoresis After separation of serum proteins by electrophoresis fix the separated protein with antiserum of immunoglobulin heavy chain and light chains.

Bis-albuminemia

ABNORMAL?

Intravascular hemolysis

ABNORMAL

Nephrotic syndrome

Hypergammaglobulinemia

M band in β region

M band in γ region