+Serum Protein
Electrophoresis with
Immunofixation
Dr.Ajay Phadke
Centre Head
SRL Diagnostics-Dr.Avinash Phadke’s Lab
+
What is electrophoresis?

Electrophoresis is a method of separating proteins based on their
physical properties.

Proteins can be separated using a buffered solid medium (agarose
electrophoresis ) or using only the liquid phase (capillary
electrophoresis)

The net charge (positive or negative) and the size and shape of the
protein commonly are used in differentiating various serum
proteins.

A negatively charged particle usually travels to the positively
charged electrode(Gel EP)

In Capillary EP ? Negatively charged particle travels to the
negatively charged electrode(Cathode)

WHY?
+ Capillary Electrophoresis in
Minicap/Capillarys
EOF
DETECTION OF
PROTEINS
Protein
Anode +
migration
INJECTION OF
SERUM
Electro migration
Positive charges of the buffer
solution
Negative charges of
capillarywall
The Electro-Osmotic Flow (EOF) is a stronger
force than the Electrical Field.
As a result, all proteins are carriedtowards the
cathodic end of the capillary.
+

The complementary positively charged ions in the
surrounding buffer are free to move under the
electromotive force, and they carry with them molecules
of the solvent water.

This buffer flow is termed electro-osmosis or
endosmosis, which also carries the proteins with it to
some extent by mechanical flow, not by charge.

The actual distance traveled by a particular protein
migrating in an electrical field is determined by the electromotive force (a feature of the protein itself and the pH)
and the electro- osmotic force (a function primarily of the
support medium).

When the electro-osmotic force is greater than the
electrophoretic force acting on weakly anionic proteins
(e.g., γ-globulins), those proteins move from the
application point toward the cathode, even though their
charge is slightly negative.
Electrophoretic
System
in
+
Minicap/Capillarys Capillarys Electrophoresis Principle
Thermic bridge
Temperature
Controlled by
Peltier device
Migration
Detector
Capillary in thermoconductive resin
Deuterium lamp
High
Voltage
Cathode -
Anode +
+
When do doctors ask for SPE?

Unexplained anemia / weakness / fatigue / ↑ ESR

Unexplained renal insufficiency

Heavy proteinuria in patient >40yrs

Bence Jones proteinuria

Hypercalcaemia

Hypergammaglobulinemia

Immunoglobulin deficiency

Peripheral neuropathy (5% will have MGUS)

Recurrent infections

Unexplained bone pain / pathologic fracture / lytic lesion-
+

1.Elderly patient with suspicion for MM i.e bone pain, lytic
lesion

2. fever for >1 month

3. ESR increase, persistent anemia, fatigue

4. CRP high

5.Heavy proteinuria in adults

6.persistent increase in calcium

7.Peripheral neuropathy since a percentage have MGUS
Capillarys 2
Minicap
Carousel
28 positions
+
Reagent Compartment
Easy access to consumables:
reagent cups, waste & reagents containers
(2 buffer vials on board)
+
Fraction identification
a-1 acidglycoprotein
a-1 antitrypsin
Haptoglobin
TBG, Transcortin
a-2 macroglobulin
Ceruloplasmin
Gammaglobulins
C3 complement
B Lipoprotein
Hemopexin
Transferrin
Normal
Gaussian aspect in gamma
&
No increase or additional deformation/peak
in gamma, beta 1, beta 2 and alpha 2
Alb
a1 a2 b1 b2 g
+
Albumin

Albumin concentrations are vital to the understanding and
interpretation of calcium and magnesium levels because
these ions are bound to albumin, and so decreases in
albumin are directly responsible for depression of their
concentrations

C3 (and also C4) concentration is a convenient marker for
assessing disease activity in rheumatic disorders such as lupus
erythematosus and rheumatoid arthritis.

C4 is not appreciated on serum protein electrophoresis because
its concentration is normally only about one-fifth that of C3.

Both C3 and C4 are now easily quantitated by nephelometry for
monitoring rheumatic disease activity
Monoclonal peak in Gamma
Alb
a1
a2
b1 b2
g
Monoclonal peak in beta
Alb
a1
a2
b1 b2
g
Oligoclonal pattern
Alb
a1
a2
b1 b2
g
+
Immunofixation & immunotyping

Principle: Apply the patients sample on gel. Separate the
sample. Add antibody . If positive = on washing this sample
remains because of large size of complex.

Immunotyping : similar principle. Automated, not labour
intensive.

BASIC DIFFERENCE: way how sample is processed. WE MIX
sample with antibody before processing.Complex is made
EVEN BEFORE SEPERATION TAKES PLACE. Then injected into
capillary. Monoclonal complex will MIGRATE SLOWLY and will
NOT form a peak.

THEREFORE, in IF you are looking for the band to be PRESENT.
While in IT you are looking for it to be ABSENT!

IFE is Very labour intensive
+

IEP: serum applied to aggel in wells. EP . Antisera added. 24 hr
incubation. ARCS formed

IFE:Sample on solid matrix

IT: NO GEL. Migration in buffered medium. Mono-specific antisera.
REDUCTION technique. Antisera binds to Immunoglobulin. Heavy, large
molecule created. Pulled OUT of viewing area.

If PEAK DETECTEd, just click on immunotyping after selecting dilution.

Hypgogamma : Ig<0.8g/L(1/10)

Std :Ig 0.8- 2.0 g/L(1/20)

Hypergamma: Ig >2.0g/L(1/40)
Monoclonal peak or polyclonal increase in gamma?
Pointedpeak
Narrow
basement
Monoclonal peak
IT
Complete substraction of
the
peakwith
one
antiserumagainst
a
heavychain and a light
chain
Rounded top
Large
basement
Polyclonalincrease
IT
Complete
substractionwith
the
antiserumagainst
a
heavychain
and
partial
substractionwith
the
antiseraagainst kappa and
Abnormal peak
in gamma
The peak disappears in
Ig G
The peak disappears in
Kappa
Conclusion: Detection of monoclonal Ig G
Kappa
IgG lambda
Zoom
IgG kappa
+
When interpreting IT, always consider:
« If removingsomething, whatisremaining? »
In eachwindow, removing one specific class
of IgG highlights what is happening with the
residual immunoglobulins that remain after
substraction
+
Hints and tips for
IT interpretation

Examine carefully all IT curves without a zoom to verify the
correct overlapping on albumin and the zone of interest
between ELP and antisera curves

Verify that the correct sample dilution has been used

Compare the residual heavy and light chains after
substraction and their position to verify additional presence
of other monoclonal Ig

If there is no correspondence between heavy and light
chains, complete the test with an immunofixation to check for
free light chains and/or IgD, IgE
+
+
When does one advise urine EP

The following conditions (to list a few) warrant urine protein electrophoresis:
1) monoclonal protein in serum is >1.5 g/dL, 2) monoclonal free light chains
are detected in serum, 3) hypogammaglobulinemia is present in serum; 4)
serum electrophoresis shows nephrotic pattern.

“In the context of screening, the serum FLC assay in combination with serum
protein electrophoresis (PEL) and immunofixation yields high sensitivity, and
negates the need for 24-h urine studies for diagnoses other than light chain
amyloidosis (AL).”

• “...once diagnosis of a plasma cell disorder is made, 24-h urine studies are
required for all patients.”

• “For AL screening, however, the urine IFE should still be done in addition to
the serum tests including the serum FLC.”

• “The FLC assay cannot replace the 24-h urine protein electrophoresis for
monitoring myeloma patients with measurable urinary M proteins”.
What history is
important?
What would you
report?
Increase in alpha1, alpha 2
Advise renal profile,UPE and IT
65 year old patient.
Weakness,
What do you see on
the graph. What will
you advise?
50 year old female
What is your
impression?
What would you
advise?
30 Year old Female.
What is your opinion?
What history will you take

Hb: 8.0

RDW: 20.3

Retic N

Ferritin : 3.0 Normal range(4.6-204)

B 12 : 254
What is important in
this graph?