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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 .

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serum protein electrophoresis with immunofixation

Serum Protein Electrophoresis with Immunofixation

Dr.AjayPhadke

Centre Head

SRL Diagnostics-Dr.AvinashPhadke’s Lab

what is electrophoresis
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?
slide3

Capillary Electrophoresis in Minicap/Capillarys

Anode +

Cathode -

DETECTION OF

PROTEINS

INJECTION OF

SERUM

Protein migration

EOF

Electro migration

Positive charges of the buffer solution

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.

Negative charges of capillarywall

slide4

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.
slide5

Electrophoretic System in Minicap/Capillarys

Capillarys Electrophoresis Principle

Thermic bridge

Temperature

Controlled by

Peltier device

Migration

Capillary in thermo-conductive resin

Detector

Deuterium lamp

High Voltage

Cathode -

Anode +

when do doctors ask for spe
When do doctors ask for SPE?
  • Unexplained anemia / weakness / fatigue / ↑ ESR
  • Unexplained renal insufficiency
  • Heavy proteinuria in patient >40yrs
  • BenceJones proteinuria
  • Hypercalcaemia
  • Hypergammaglobulinemia
  • Immunoglobulin deficiency
  • Peripheral neuropathy (5% will have MGUS)
  • Recurrent infections
  • Unexplained bone pain / pathologic fracture / lytic lesion-
slide7

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
slide9

Carousel

28 positions

slide10

Reagent Compartment

Easy access to consumables:reagent cups, waste & reagents containers

(2 buffer vials on board)

fraction identification
Fraction identification

Albumin

Beta-1

Alpha-1

Alpha-2

Beta-2

Gamma

a-1 acidglycoprotein

a-1 antitrypsin

TBG, Transcortin

Gammaglobulins

C3 complement

B Lipoprotein

Haptoglobin

Hemopexin

Transferrin

a-2 macroglobulin

Ceruloplasmin

slide12

Normal

Gaussian aspect in gamma

&

No increase or additional deformation/peak

in gamma, beta 1, beta 2 and alpha 2

a2

b2

Alb

g

a1

b1

albumin
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
slide16

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
immunofixation immunotyping
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
slide25

IEP: serum applied to aggelin 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)
slide26

Monoclonal peak or polyclonal increase in gamma?

Pointedpeak

Rounded top

Narrow

basement

Large

basement

Polyclonalincrease

Monoclonal peak

IT

IT

Complete substractionwith the antiserumagainst a heavychain and partial substractionwith the antiseraagainst kappa and lambda

Complete substraction of the peakwith one antiserumagainst a heavychain and a light chain

slide27

The peak disappears in Ig G

Abnormal peak

in gamma

The peak disappears in Kappa

Conclusion: Detection of monoclonal Ig G Kappa

when interpreting it always consider
Wheninterpreting IT, alwaysconsider:

« If removingsomething, whatisremaining? »

In eachwindow, removing one specific class of IgGhighlightswhatishappening with the residualimmunoglobulinsthatremainaftersubstraction

hints and tips for it interpretation
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
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”.
slide38

What history is important?

What would you report?

slide40

Increase in alpha1, alpha 2

Advise renal profile,UPE and IT

slide41

65 year old patient. Weakness,

What do you see on the graph. What will you advise?

slide42

50 year old female

What is your impression?

What would you advise?

slide43

30 Year old Female.

What is your opinion?

What history will you take

slide44

Hb: 8.0

  • RDW: 20.3
  • Retic N
  • Ferritin : 3.0 Normal range(4.6-204)
  • B 12 : 254