+ serum protein electrophoresis with immunofixation dr.ajay phadke centre head srl...
TRANSCRIPT
+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 +Cathode -
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 OFSERUM
DETECTION OFPROTEINS
+ 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 bridgeTemperatureControlled byPeltier 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
a-1 acidglycoprotein
a-1 antitrypsin
TBG, Transcortina-2 macroglobulin
Ceruloplasmin
Hemopexin
Transferrin
Alb
um
i n
Ga
mm
a
Be
ta-2
Be
ta-1
Alp
ha
-2
Alp
ha
-1
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 a1 2a 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 2a b1 b2 g
Monoclonal peak in beta
Alb a1 2a b1 b2 g
Oligoclonal pattern
Alb a1 2a 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?
Monoclonal peak
Narrowbasement
Pointedpeak
Polyclonalincrease
Largebasement
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
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
+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 femaleWhat 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?