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

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

3. 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

4. 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.

5. Serum Electrophoresis
Paper electrophoresis
Starch gel electrophoresis
Agarosre gel electrophoresis
Cellulose acetate gel electrophoresis
Capillary gel electrophoresis

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

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

8. 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

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

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

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

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

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

14. Normal serum electrophoresis

15. Abnormal serum electrophoresis showing M band

16. 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

17. 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

18. 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.

19. Various patterns

22. Analbuminemia

25. Haptoglobin phenotype

31. Bis-albuminemia

33. Nephrotic syndrome

34. Hypergammaglobulinemia

35. M band in β region

36. M band in γ region

39. 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.

40. 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

51. Intravascular hemolysis

53. 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.

54. Freelite test Free Kappa Free Lambda Kappa/ Lambda ratio Normal serum
mean median %
concentration concentration range
Free Kappa
8,36 mg/l ,30 mg/l ,30 – 19,40 mg/l
Free Lambda
13,43 mg/l ,40 mg/l ,71 – 26,30 mg/l
Kappa/
Lambda ratio
mean median total range
0, , ,26 – 1,65

55. THANK YOU