I M M U N O E L E C T R O P H O R E S I S

1. Serum protein electrophoresis & Immunoelectrophoresis
(Immunofixation
Dr Ajit Kumar Singh
PGT 1st year
Moderator
Dr. Garima Chauhan
MD Biochemistry
Assistant professor
Department of Laboratory medicine
Chittaranjan National Cancer Institute , Kolkata

2. Normal pattern of serum
electrophoresis
 Serum proteins are separated in
to 6 groups:
1. Albumin
2. α1– globulins
3. α2- globulins
4. β1-globulins
5. β2 –globulins
6. γ -globulins

3. Normal pattern of serum
electrophoresis

4.  Suspected cases of multiple myeloma , waldenstrom's macroglobulinemia and
primary amyloidosis.
 New-onset anemia associated with renal failure or insufficiency and back
pain in which multiple myeloma is suspected
 Hypercalcemia attributed to possible malignancy.
 Rouleaux formations noted on peripheral blood smear.
 Unexplained pathologic fracture or lytic lesion identified on
radiograph.
 Bence jones proteinuria.
Indications for serum protein
electrophoresis

5. •Most common plasma cell proliferative disorder reported in approximately 3%
of persons older than 70 years
• MM – preceded by asymptomatic premalignant MGUS stage.
M-Protein
<3g/dl
Clonal Bone marrow
plasma cells
<10%
Absence of organ
damage(CRAB)
All three criteria must
be met
MGUS

6. Smoldering multiple myeloma
Clonal Bone
marrow plasma
cells
10-60%
Absence of organ
damage(CRAB)
• Asymptomatic plasma cell dyscrasia with a higher risk of progression to
multiple myeloma.
M-Protein
>3g/dl
All three criteria must
be met

7. M-Protein
>3g/dl
Clonal Bone
marrow
plasma cells
>10%
Presence of end
damage(CRAB)
Clonal Bone
marrow plasma
cells
>60%
Multiple
myeloma
• Sr. Calcium >11.5mg/dl
• Sr. Creatinine >2mg/dl
• Anemia: NCNC, Hb
<10g/dl
• Bone Lesions
+

8. Abnormal
Immunoglobulins
?Polyclonal
?Monoclonal
Ig Heavy chain
and light chain
typing
Electrophoresis
• Serum
• Urine
Immunofixation
λ
κ
IgM Non-IgM
• Waldenstrom
Macroglobulinemi
•IgG (52%)
•IgA (20%)
•IgD or IgE (2)%
16%
M-Band

9. Electrophoresis
patterns
Polyclonal Gammopathy
• Broad peak
Monoclonal Gammopathy
• “Church Spire” peak

10. Monoclonal gammopathy
 In a monoclonal gammopathy, the pattern will have a large spike in the gamma
region.
 An immunoglobulin (IgG, IgM, IgA) from a single B-cell clone is produced in
excess.
o These are mostly caused by hematological disorders including :
 multiple myeloma,
 Waldenstroms macroglobulinemia,
 heavy chain disease,
 and MGUS.

11. Monoclonal gammopathy
Interpretation :
 Presence of sharp narrow M band monoclonal proteins.
 The M protein is considered to be measurable if it is > 1g/dl in the serum and or >
200 mg/day in the urine.
 It can be quantified – useful in response assessment .
Caveats :
 Does not inform about the type of Ig class/light chain derangement .
 False negative in light chain myeloma

12. Monoclonal gammopathy

13. Biclonal gammopathies

14. Biclonal
Gammopathies
• Discrete monoclonal band may be seen anywhere between the α₂ toϒ globulin
region.
•Oligosecretory or Non secretory myelomas may not be detected by electrophoresis
• Biclonal Gammopathies:

15. Polyclonal Gammopathy
• A broad diffuse increase in the
gamma region is the result of a
polyclonal plasma cell response
to chronic antigen stimulation .
• Seen in the infection /
inflammation / autoimmune
disease .

16. Hypogammaglobulinemia
 In hypogammaglobulinemia, the pattern will have a really low gamma globulin
region.
 Anything that causes a decrease in the gamma globulins (IgG, IgA, IgM) like an
immune deficiency can cause hypogammaglobulinemia.

17. Hypogammaglobulin
emia

18. Immunofixation
electrophoresis (IFE)
 To further diagnose a monoclonal gammopathy an IFE can be performed.
 An IFE will separate the gamma region into IgG, IgA, IgM, kappa chains, and
lambda chains.
 This test can help further differentiate what type of monoclonal gammopathy a
patient has. IgG, IgA, and IgM are termed heavy chains and kappa and lambda,
are termed light chains.

19. Immunofixation

20. Immunofixation studies
 Use to identify the heavy and light chain involved in disease.
 Antisera to the different class IgG, IgA, IgM, k and lemda
Interpretation :
 Sharp band corresponding to antisera added depicts the heavy and light chain.
 In case of only light chain band with no heavy chain band (G/A/M/)- IgD / IgE
myeloma to be ruled out.

21. Interference
 Fibrinogen
 hemolysis

22. α1 antitrypsin deficiency
 α-1antitrypsin is major protein in α-1 region .
 It is a member of a family protease inhibitor called
serpins that inhibits proteolytic enzymes.
 α-1AT deficiency can result in lung and liver disease.

23. Acute inflammatory process
 Immediate response occurs with stress or inflammation cause by infection ,
injury or surgical trauma.
 Acute systemic infection triggers change in the hepatic production of multiple
serum proteins.
 Increased in α1 and α2 and/or (γ)
gammaglobulin pattern is consistent
with an acute inflammatory process.

24. Chronic inflammatory
response
Late response is correlated with chronic inflammation.
 Autoimmune disease
 Chronic liver disease
 Chronic infection

25. Liver damage (cirrhosis)
 Cirrhosis can be caused by chronic alcohol abuse or viral hepatitis.
 There is small bridge between beta 2 and gamma band.
 Anodal slurring of albumin may
occur secondary to the binding of
excess bilirubin and/or other
negatively charged drugs .

26. Anodal albumin slurring and
β-γ bridging

27. Nephrotic syndrome
 Nephrotic syndrome is often
associated with increased
alpha 2 and beta bands.
 Serum albumin and gamma
globulin concentration are
usually reduced in this
condition.

28. Increased transferrin
 Transferrin is a major protein in the B 1 region
 It is increases in iron deficiency anemia
 Some time monoclonal proteins can be seen in B1 region.

29. Q.69 years old non retired farmer come in to our hospital complaining of sever lower
back pain and pain in his right lower chest . He seems confused and look pale . He
cough rusty colored sputum , his calcium level is 12.3 mg/dl and creatinine is high
what is next step ? most likely diagnosis ?

30. DIAGNOSTIC
EVALUATION
History and Physical examination
Complete Blood Count and differential
Biochemical Screening
Serum Protein Electrophoresis
Routine Urine Analysis
24 hour Urine for Electrophoresis
Immunofixation
Serum Free Light Chain Assay
Bone Marrow Aspirate and Trephine Biopsy
Measurement and
characterisation of
Monoclonal protein

31. DIAGNOSTIC
EVALUATION
END ORGAN
DAMAGE
EVALUATION
PROGNOSTIC
EVALUATION
SPECIALISED TEST
FOR SELECTED
PATIENTS
PATIENT
EVALUATION
APPROACH TO LAB
DIAGNOSIS

32. NORMAL VALUES
Blood Urea Nitrogen 8-24 mg/dl
Serum Creatinine 0.8-1.3 mg/dl
Total Protein 6-8.3 gm/dl
Serum Albumin 3.5-5.5 gm/dl
Serum Globulin 2.5-3.5gm/dl
Albumin: Globulin Ratio 0.8-2
Serum Calcium 9-10.5 g/dl
Alkaline Phosphatase 44-147 IU/L
Serum Lactate dehydrogenase 140-240 IU/L

33. SIGNS AND
SYMPTOMS

34. BLOOD
EXAMINATION
•Anemia : Hb <12 gm/dl Normocytic
Normochromic, sometimes macrocytic
•Neutropenia due to replacement of
hematopoietic elements by myeloma cells
•Rouleaux formation of RBC with
background basophillia
• Leukoerythroblastic picture
•Coagulopathy due to anti-protein C
activity of M protein and
thrombocytopenia.

35. ROUTINE URINE ANALYSIS
URINE ELECTROPHORESIS
 Obtain a 24hour- urine sample
 Normal Range:10–140 mg/24 hours
 Methods Of concentration:
 By Ultrafiltration
 By vaccum dialysis
 By centrifugation
 Urine has large amounts of smaller sized lighter chains with only small amount of
intact globulin
 Intact monoclonal immunoglobulin in patients with poor renal function

36. URINE
ELECTROPHORE
SIS URINE ELECTROPHORESIS:
• Obtain a 24hour- urine sample
• Normal Range:10–140 mg/24 hours
• Methods Of concentration: 1. By Ultrafiltration
2. By vaccum dialysis
3. By centrifugation
•Urine has large amounts of smaller sized lighter
chains with only small amount of intact globulin
•Intact monoclonal immunoglobulin in patients with
poor renal function

37. IMMUNOFIXATI
ON

38. SERUM FREE LIGHT
CHAIN ASSAY
•15–20% of patients with myeloma only have
production of free monoclonal light chains (light-
chain myelomas)
• 1–2% of myeloma patients are non-secretory
•May not be detected as a distinct monoclonal
band on serum electrophoresis
•Detect free light chains (κ &λ) in serum by using
anti-FLC antibodies
•Quantifies and determines monoclonality by
using the κ: λ ratio.
•Normal free κ: 3.3 to 19.4mg/l
• Normal free λ: 5.7 to 26.3mg/l
• Normal ratio: 0.26 to 1.65

39. IMMUNOSUBTR
ACTION
•Used in Capillary electrophoresis -analogous to immunofixation on agarose
gels
•Enables the identification of paraprotein found in serum by using
immunoglobulin specific antibodies.
• Antibodies attached to solid supports such as beads
•Component binding to the antibody will become attached to the support and
removed from solution.
•In IgG(kappa) for -binding (and removal) of the paraprotein with IgG and
kappa antibodies cause a diminution of the band and antibodies to IgA, IgM
and lambda components will persist

41.  When electric current is applied to a slide layered with gel, antigen mixture
placed in wells is separated into individual antigen components according to their
charge and size.
 Following electrophoresis, the separated antigens are reacted with specific
antiserum placed in troughs parallel to the electrophoretic migration and diffusion
is allowed to occur.
 Antiserum present in the trough moves toward the antigen components resulting
in formation of separate precipitin lines 18-24hrs, each indicating reaction
between individual proteins with its antibody.

43.  The presence of elliptical precipitin arcs represents antigen-antibody interaction.
 The absence of the formation of precipitate suggests no reaction.
 Different antigens (proteins) can be identified based on the intensity, shape, and
position of the precipitation lines.

44.  The test helps in the identification and approximate quantization of various
proteins present in the serum.
 Immunoelectrophoresis is used in patients with suspected monoclonal and
polyclonal gammopathies.
 This method is useful to monitor antigen and antigen-antibody purity and to
identify a single antigen in a mixture of antigens.
 Immunoelectrophoresis is an older method for qualitative analysis of M-proteins
in serum and urine.
 Immunoelectrophoresis aids in the diagnosis and evaluation of the therapeutic
response in many disease states affecting the immune system.

45. Advantages
• Immunoelectrophoresis is a powerful analytical technique with high
resolving power as it combines the separation of antigens by
electrophoresis with immunodiffusion against an antiserum.
• The main advantage of immunoelectrophoresis is that a number of
antigens can be identified in serum.
Limitations
• Immunoelectrophoresis is slower, less sensitive, and more difficult to interpret than
Immunofixation electrophoresis.
• IEP fails to detect some small monoclonal M-proteins because the most rapidly
migrating immunoglobulins present in the highest concentrations may obscure the
presence of small M-proteins.
• The use of immunoelectrophoresis in food analysis is limited by the availability of
specific antibodies.

46. Definition :
• Hb electrophoresis is used as a screening test to identify variant and
abnormal hemoglobin.
• Hb electrophoresis help in diagnosis of diseases in which abnormal
hemoglobin production occur.
• Electrophoresis uses an electrical current to separate normal and abnormal
types of Hb in the blood Hemoglobin types have different electrical charges
and move at different speeds. The amount of each hemoglobin type in the
current is measured

47.  Abbreviated Hb or Hgb.
 Iron containing oxygen transport metalloprotein in the RBC of almost all vertebrates as
well as the tissues of some invertebrates.
 Hemoglobin has an oxygen binding capacity of 1.34 mL O2 per gram.
 1 molecule carry 4 molecule of O2.

48. Normal
 HbA : 95%-98%
 HbA 2 : 1.5%-3.5%
 HbF : < 2%
Abnormal
 Abnormal form of Hb is also known as varient
 Abnormality in Hb occur mostly due to genetic mutation
 Over 350 types of abnormal Hb have been found
 Hb C, D, E, M, and S are abnormal forms

49. • A part of routine check up ( complete blood test)
• To diagnose blood disorder (thalassemia polycythemia rubra vera and sickle cell anemia)
• To monitor treatment
• To screen for genetic condition
• Help couples find out how likely they are to have a child with certain forms of anemia that
can be passed from a parent to a child (inherited)
• When someone has had a positive Hemoglobin Solubility test

50. It uses the principle of Gel electrophoresis.
Different hemoglobin have different charges and according to those
charges and the amount of hemoglobin, different chains move at
different speed in gel and separates.

51.  Electrophoresis buffer(Tris/EDTA/borate (TEB), pH 8.5)
 Wetting agents ( e . g , Zip Zone Prep solution)
 Fixative stain/solution(Ponceau S 5 g)
 Hemolysing reagents (0.5% Triton in 100 mg potassium cyanide)
 De-staining solution (3% acetic acid)

52.  Specimen: Blood
 Container : green - , or blue-top vacuum tube
Methods
 Cellulose acetate (CA) electrophoresis
 Alkaline electrophoresis
 Citrate agar electrophoresis
 Alkaline and Citrate agar electrophoresis are the commonly used method

53.  Sample collection(blood)
 Centrifuge samples at 1200 g for 5 min
 Prepare the electrophoresis tank with TEB buffer
 Soak the cellulose acetate into buffer for 5 min
 Fill the sample well plate with 5 μl of each diluted sample and cover with glass
slide to prevent evaporation
 Load a second sample well plate with Zip Zone Prep solution

54.  Then Applying them to a blotter
 Blot the cellulose acetate strip twice between two layers of clean blotting paper
 Do not allow the cellulose acetate to dry
 Load the applicator by depressing the tips into the sample wells twice
 Place the cellulose acetate plates across the bridges
 After 25 min of electrophrosis immediately transfer the cellulose acetate to
ponceau S and fix and stain for 5 min
 Remove excess stain by washing for 5 min in the first acetic acid reservoir
 Label the membrane and store in protective envelope

55.  There is very little chance of a problem from having a blood sample taken from a
vein
 Hematoma (can lower the chance by keeping pressure on the site for several
minutes)
 Bleeding
 Infection at the puncture site
 Fainting or feeling lightheaded
 Swelling (also called phlebitis. A warm compress can be used several times a day
to treat this)

56.  Results available after 1-2 days depending on lab
 If normal then no problem
 If abnormal then following conditions can occur depend on type of abnormal Hb
 Higher than normal amounts of both Hb A2 and F may mean a mild form of
thalassemia is present
 High levels of Hb F may be seen in a rare condition called hereditary persistence of
fetal hemoglobin
 Hb S in high amounts means sickle cell disease
 Hb C in high amounts means patients have anemia and an enlarged spleen
 Hb E in high amounts means patients have anemia and RBC size will be smaller then
normal

57.  Reasons you may not be able to have the test or why the results may not be
helpful include
 Having a blood transfusion in the past 3 months
 Having iron deficiency anemia This can cause falsely low results for hemoglobin
A 2

58.  Evaluation of unexplained hemolytic anemia
 Microcytic anemia unrelated to iron deficiency, chronic disease, or lead toxicity
 A peripheral smear with abnormal red cell features (eg, target cells or sickle cells)
 Positive family history of hemoglobinopathy
 Positive neonatal screen results
 Positive results on sickle cell or solubility test

59. Thank you ..