+ 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?

+

Anode +

EOF

Electro migration

Protein migration

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.

Positive charges of the buffer

solution

Negative charges of

capillarywall

Capillary Electrophoresis in

Minicap/Capillarys

INJECTION OF

SERUM

DETECTION OF

PROTEINS

+ � 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 elec- tromotive 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.

+ Capillarys Electrophoresis Principle

Thermic bridge

Temperature Controlled by

Peltier device

Anode + Cathode -

Detector Deuterium lamp

Migration

High

Voltage

Electrophoretic System in

Minicap/Capillarys

Capillary in thermo-

conductive resin

+ 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

28 positions

Carousel

+

Easy access to consumables:

reagent cups, waste & reagents containers

(2 buffer vials on board)

Reagent Compartment

+ Fraction identification

Haptoglobin

αααα -1 acidglycoprotein

αααα -1 antitrypsin

TBG, Transcortin αααα -2 macroglobulin

Ceruloplasmin

Hemopexin

Transferrin

C3 complement

B Lipoprotein

Gammaglobulins

Normal

Gaussian aspect in gamma &

No increase or additional deformation/peak in gamma, beta 1, beta 2 and alpha 2

Alb α1 α2 β1 β2 γ

Bis albuminemia

Causes: genetic, lipoproteins, biliary pigments, antibiotics, products of contraste

+ � In diabetes mellitus, patients with Type 2-2 haptoglobin have

shown higher risk for vascular complications, perhaps owing

to different ability to clear hemoglobin, thus leading to

altered iron handling and heightened oxidative load

� Serum haptoglobin rises in response to stress, infection,

acute inflam- mation, or tissue necrosis,

+ � Fibrinogen levels become elevated with the other acute

phase reactants, occasionally to over 1.0 g/L.

� In such instances, the erythrocyte sedimentation rate

(ESR) is also markedly elevated owing to fibrinogen

content directly.

� Fibrinogen levels also rise with pregnancy and use of

contraceptive medications.

� Low levels generally indicate extensive activation of

coagulation with consumption of fibrinogen.

Congenital deficiency of A1AT

A1AT = 0.17g/L (normal range: 0.97 – 1.93 g/L)

Alb α1 α2 β1 β2 γ

+

� Because of the rapid dynamics of its synthesis and

clearance, prealbumin is considered to be a better early

indicator of change in nutritional status

� Presence of a distinct band of prealbumin is used only as a

landmark to confirm that the specimen was likely CSF.

� A protein band frequently appears in the pre-albumin

position of of serum from patients who have had heparin

therapy. In the circulation, heparin activates and releases

lipoprotein lipase activity, which attacks triglycerides in

lipoprotein fractions.

+ 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

Phenotype I - II

Haptoglobin phenotypes in alpha 2 zone

Phenotype I - I

Phenotype II - II

Monoclonal peak in Gamma

Alb α1 α2 β1 β2 γ

Monoclonal peak in beta

Alb α1 α2 β1 β2 γ

Oligoclonal pattern

Alb α1 α2 β1 β2 γ

+ 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?

Monoclonal peak

Narrow

basement

Pointedpeak

Polyclonalincrease

Large

basement

Rounded top

Complete substraction of

the peakwith one

antiserumagainst a

heavychain and a light

chain

Complete substractionwith

the antiserumagainst a

heavychain and partial

substractionwith the

antiseraagainst kappa and

lambda

IT IT

+ Normal sample

Zoom

~ 80% IgG ~ 15% IgA

~ 5% IgM 2/3 Kappa 1/3 Lambda

ELP G A

M K L

The peak disappears in Ig G

The peak disappears in Kappa

Conclusion: Detection of monoclonal Ig G Kappa

Abnormal peak in gamma

IgG lambda

Zoom

IgG kappa

Fusion between beta 1& 2

IgA lambda

IgG lambda + free light chains lambda

+ 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

When removing the IgM, the whole peak disappears

When removing the Kappa → a slight peak in the residual Lambda remains

When removing the Lambda → a peak in the residual Kappa remains

There are 2 monoclonal peaks, one Kappa and one Lambda.

Both disappear when removing the IgM…

Ig M Kappa + Ig M Lambda

Zoom

IgM kappa

+

IgM L IgM K

IgM Kappa+ IgM Lambda + oligoclonal profile in IgG

+

Before BME treatment

IgM IgG

+ Treatment with Beta Mercaptoethanol (BME) to depolymerize a monoclonal Ig :

* Prepare a 1% BME reducing solution : 1) 90µL H2O + 10µL BME 2) 10µL 1/10 diluted BME + 90µL Fluidil (ref 4587) * 100µL of 1% BME reducing solution + 300µL serum * Incubate 10 mn at room temperature • Analyze immediately the treated sample in Capillarys or Minicap without any delay ⇒ To separate co-migrating Igs of different types (Ig M vs Ig G)

+ IgG kappa + IgM Lambda

After BME treatment

IgM L IgG K

+

One or Two Ig G kappa?

+ Smoothing 0

Smoothing 2

2 IgG kappa

Change of the

smoothing option

Polyclonal increase or monoclonal Ig in gamma?

Zoom

Polyclonal increase or monoclonal Ig in gamma?

+

+ Polyclonal increase of Ig G

+

Oligoclonal profile

Oligoclonal: Presence of multiple faint peaks or

distorsions

This profile is observed in: •Autoimmune diseases (Rheumatoid arthritis, Gougerot Sjögren

syndrome, Lupus erythematosus)

•Infectious (viral, bacterial, parasitic) diseases

•Autoimmune reactions (Patients with transplants or patients under

immuno- suppressive treatments)

Oligoclonal profile is linked with a dysregulation of immune system

+ Oligoclonal profile on Hydragel IF

Zoom

Oligoclonal profile in Ig G (with major Ig G lambda to recontrol)

+ 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

+

� Once a monoclonal gammopathy is identified by serum

protein electrophoresis, multiple myeloma must be

differentiated from other causes of this type of gammopathy.

(Waldenström’smacroglobulinemia, smoldering multiple

myeloma, monoclonal gammopathy of undetermined

significance, plasma cell leukemia, heavy chain disease)

+

� sizeof the M-protein spike. Although this spike is usually greater than 3 g per dL in patients with multiple myeloma, up to one fifth of patients with this tumor may have an M-protein spike of less than 1 g per dL.10Hypogammaglobulinemia on serum protein electrophoresis occurs in about 10 percent of patients with multiple myeloma who do not have a serum M-protein spike.11 Most of these patients have a large amount of Bence Jones protein (monoclonal free kappa or lambda chain) in their urine.11 Thus, the size of the M-protein spike is not helpful in excluding multiple myeloma.

� If multiple myeloma still is considered clinically in a patient who does not have an M-protein spike on serum protein electrophoresis, urine protein electrophoresis should be performed.

+

� With the addition of sodium sulfate, sodium sulfite,

ammonium sulfate, or methanol, the globulins tend to

precipitate, leaving albumin in solution. By measuring total

protein in the original serum and protein in the precipitate or

the supernatant, values for albumin and globulin can be

derived.

+

� 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

+

+ 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?

+