Methods for serum & urine electrophoresis & immunofixation: pros and cons

Samarina Musaad Chemical Pathologist, Labtests Workshop Wellington February 2015 Outline: All in context of the clinical chemistry laboratory and protein separation in serum and urine . Amino acids & proteins are charged . The basic concept of electrophoresis: definition, instrumentation & technique . Methods . Clinical indications in serum & urine . Differences: technical & interpretive . Advantages and disadvantages of common methods: a marriage between technology, practicality & clinical need . Summary

11/03/2015 S Musaad 2 Amino acids & proteins are charged

Amino acid = RCH(NH2)COOH An amino group & a carboxyl group with a side chain specific to the amino acid Glycine is the simplest amino acid . Hydrogen is it’s side chain

Protein= polymerised amino acids

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Ampholyte = a molecule that can be negatively or positively charged depending on pH

A zwitterion is one with net zero charge

Isoelectric point ( PI)= the pH at which zwitterions predominate and remaining +ve. and –ve. charges balance each other (ions in a solution are in dynamic equilibrium)

At PI, proteins (& amino acid) have zero mobility in electrophoresis

Electrophoresis utilises these properties

11/03/2015 S Musaad 4 What is electrophoresis ? “The migration of charged solutes or particles in a liquid medium under the influence of an electrical field” Tietz

Related mathematics F=(EMF)(Q)/d

As d decreases , F increases resulting in an accelerating force This acceleration is counteracted by F′ F′= 6∏rɳv

There comes a point when F= F′ when solute moves at a constant velocity

11/03/2015 S Musaad 5 Instrumentation & technique

• Instrumentation Power source Medium: solid or fluid Buffers- carry the current and fix the pH Dyes/stains- choice depends on type of protein under investigation and it’s denaturation commonest are amido black for serum prtein electrophoresis (SPEP) , acid violet for immunofixation (IFIX) & coomassie blue for isoelectric focussing (IEF) Detector: densitometer, photodetector ( e.g. capillary electrophoresis)

• Technique Separation Detection & quantitation- by densitometry, UV photometry, fluorescence Interpretation

11/03/2015 S Musaad 6 Methods

1. electrophoresis (AGE) 2. Capillary zone electrophoresis (CZE) 3. 4. Cellulose acetate electrophoresis (CAE) 5. Polyacrylamide (PAGE)

11/03/2015 S Musaad 7 1-Agarose gel electrophoresis (AGE)- high resolution

Medium- neutral “agarose” fraction of agar Sample volume≈ 10 µL

11/03/2015 S Musaad 8 Preparation and Procedure

GEL

- Prepare Stain

- Prepare Destain

- Prepare Wash Solution

- Apply samples to applicator

- Insert buffer strips

- Dry gel with filter paper

-Place gel and insert applicators

- Migration (automatic, managed by Hydrasys)

- Stain and dry gel after migration

2-Capillary zone electrophoresis (CZE)

Medium- buffer in a small bore capillary tube 35.5oC The high surface-to-volume ratio improves heat dissipation allowing higher voltages. The narrow capillary chamber minimises flow convection ( a combination of diffusion and movement of heat or matter via large-scale mass flow of currents) Both factors improve resolution

High surface-to-volume ratio may increase chances of adsorption of solute onto the inner surface of the tubing by hydrophobic and ionic interactions. This is minimised by chemically conditioning the inner wall

11/03/2015 S Musaad 10 Electro-osmotic flow opposes electrophoretic forces (-vely charged silanol groups on surface attracts +vely charged hydrated ions which flow towards cathode when electricity is applied. This movement is against the overall anodal flow)

The order of migration of proteins past the detector reflects the balance between them. (Jenkins 2009)

Detection is at 200nm (peptide bonds)

11/03/2015 S Musaad 11 Preparation and Procedure

CAPILLARY FLEX PIERCING: Waste Wash Buffer - Prepare Wash Solution

- Insert Buffer, Water and Wash into reagent bay

- Load dilution segments and samples tubes onto racks

- Load racks onto Capillarys and walk away!

- Migration automatic (managed by Phoresis) and no staining required

-Results are stored in Phoresis

3-Isoelecric focusing (IEF) Medium- agarose gel, cellulose acetate, sephadex

A mix of “carrier ampholytes” form a pH gradient during electrophoresis depending on their PIs. Simultaneously proteins in question migrate during electrophoresis but their migration stops when & where pH = PIs

Resolution is 5x better than immunofixation (Jenkins 2009) because PI manifests as a narrow pH range hence a discrete protein peak , and change in charge counteracts movement from the PI position

Used commonly for CSF and α1 antitrypsin phenotyping

11/03/2015 S Musaad 14 4-Cellulose acetate electrophoresis (CAE)

Obsolete. Need to pre-soak the strips and render them transparent before densitometry. Replaced by AGE

5- Polyacrylamide gel electrophoresis (PAGE) Not used in clinical chemistry laboratory for proteins because the are thin and fragile, the glass plates can be cumbersome to work with and acrylamide can be a lung irritant and neurotoxin.

11/03/2015 S Musaad 15 Complementary methods : Immunofixation and immunosubtraction

Antigen- binding is utilised in immunofixation (IFIX) & immunosubtraction (IT)

• IFIX After SPEP or IEF, to particular proteins in question are added to the gel Washing removes all unbound antiserum and un-precipitated (unbound) antigen followed by staining, detection & quantitation

• IT Used in capillary electrophoresis Antibodies to the defined immunoglobulin are bound to a solid phase → removing it from solution and removing the “band” from the electrophoretogram

11/03/2015 S Musaad 16 11/03/2015 S Musaad 17 Clinical indications

• Serum Detection of monoclonal proteins in case of suspected MM, unexplained peripheral neuropathy, Waldenström’s macroglobulinemia, primary amyloidosis & cryoglobulinaemia

Other potentially clinically relevant findings

A rise in acute phase proteins, α1-antitrypsin deficiency, increased transferrin levels in iron deficiency anaemia, polyclonal increase in liver disease

• Urine Monoclonal proteins in case of suspected MM (Bence Jones protein) Glomerular proteinuria ( selective and non-selective) e.g. glomerulonephritis Tubular proteinuria e.g. heavy metal poisoning, Fanconi’s syndrome….

Urine protein electrophoresis for monoclonal proteins is being superseded by serum free light chains

11/03/2015 S Musaad 18 Differences between AGE & CZE- technical Sample loading

GEL CAPILLARY

• 10ul of sample is pipette from •Primary tubes loaded onto racks primary tube into applicator well

• Initial load: 13 racks, 104 samples •Applicator placed in wet storage chamber for 5 mins before •Continuous loading during process application to gel

• More samples can be loaded only after migration is complete

Differences in procedure as discussed…………

AGE • Prepare Stain • Prepare Destain • Prepare Wash Solution • Apply samples to applicator CZE • Insert buffer strips • Prepare Wash Solution • Dry gel with filter paper • Insert Buffer, Water and Wash into • Place gel and insert applicators reagent bay • Migration (automatic, managed by • Load dilution segments and Hydrasys) samples tubes onto racks • Stain and dry gel after migration • Load racks onto Capillarys and walk away!

• Migration automatic (managed by

Phoresis) and no staining required • Results are stored in Phoresis

11/03/2015 S Musaad 20 Differences in migration

GEL MIGRATION CAPILLARY MIGRATION

- Automatic migration managed by -Automatic migration managed by Hydrasys software Phoresis software

-Through a solid gel matrix -Through a liquid medium

- Migration carried out under 240 -Migration carried out under 10,000 volts volts

- Drying and staining of the gel is required - Fractions detected by OD detector, to visualise bands no staining required

Differences in resolution

CZE AGE Differences in sensitivity

0.2 – 0.6 g/L

0.18 g/L Differences in throughput

Zhaohai et al 2007

24 Differences between AGE & CZE- interpretive

“Five reviewers interpreted 149 consecutively ordered serum specimens following agarose gel electrophoresis, capillary electrophoresis, immunofixation electrophoresis and subtraction immunotyping” .

11/03/2015 S Musaad 25 11/03/2015 S Musaad 26 78 samples classified as abnormal by IT then compared to IFIX: inter- interpreter agreement

Samples had multiple bands (therefore > 78 results) Overall 86% agreement 12 bands (2.8%) missed by IFIX- already had a large monoclonal protein identified 31 bands (7.2%) missed by IT In the presence of other monoclonal proteins, free light chain bands were missed by IT

11/03/2015 S Musaad 27 Samples missed by IT

• 5/91 abnormal samples were missed by CZE & IT • 2 have a known monoclonal protein • Clinical significance of the others is questionable

11/03/2015 S Musaad 28 Quantitation

42 specimens: slight +ve bias for CE at monoclonal protein conc. <10g/L & slight –ve bias at conc. > 30g/L. overall not clinically significant.

11/03/2015 S Musaad 29 11/03/2015 S Musaad 30 11/03/2015 S Musaad 31 11/03/2015 S Musaad 32 11/03/2015 S Musaad 33 11/03/2015 S Musaad 34 11/03/2015 S Musaad 35 11/03/2015 S Musaad 36 11/03/2015 S Musaad 37 Similar to McCudden et al…..

11/03/2015 S Musaad 38 11/03/2015 S Musaad 39 11/03/2015 S Musaad 40 11/03/2015 S Musaad 41 11/03/2015 S Musaad 42 International staging system for MM (ISS): stage 1 have albumin >=35g/L as a criterion (amongst others)

11/03/2015 S Musaad 43 11/03/2015 S Musaad 44 11/03/2015 S Musaad 45 11/03/2015 S Musaad 46 Therefore…… Advantages and disadvantages of primary methods-AGE & CZE

Method AGE CZE IEF Advantages .Most labs have .↑ separation .Very fine more experience efficiency separation with it .ease of automation .IFIX is 5x more .↑ throughput sensitive than .can be interfaced SPEP IFIX (e.g. to MS) .UV detection eliminates variability improving imprecision

11/03/2015 S Musaad 47 Method AGE CZE IEF Disadvantages .Staining can be .It can be difficult to .Time variable depending detect small consuming on protein monoclonal bands .Needs concentration esp. in cases of expertise (&type of stain) ↓gammaglobulinemia .Over estimates .More sensitive esp. in albumin with high β region concentration of .Less specific monoclonal proteins . Overestimates .Time consuming albumin with high .Prone to concentration of misinterpretation monoclonal proteins due to disulphide .Compounds that bonds and Ig. absorb at 200nm can complexes interfere

11/03/2015 S Musaad 48 Advantages and disadvantages of complementary methods

Method 1o versus IFIX IT complementary methods Advantages .Sensitivity .More sensitive .Automated .Qualification esp. for low .Better resolution e.g. level IgA and total or complete IgM bands band subtraction Disadvantages .IFIX-time .Semi manual .Can miss subtle consuming subtraction .IT-may need confirmation with IFIX

11/03/2015 S Musaad 49 Urine PEP and IFIX complement serum testing

Urine PEP .May need concentrating .Shows monoclonal bands and types of proteinuria CE .Not widely used IFIX .Needed to exclude amyloidosis

11/03/2015 S Musaad 50 In summary

• Different separation techniques with different detection technologies

• Experience plays a big role in interpretation

• Preferred method depends on experience but mostly on laboratory requirements

• The availability of both is advantageous: one can compensate for interferences in another

• Know your method’s limitations….

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• IT has not totally replaced IFIX

• Follow up monoclonal bands using the same technique and preferably the same laboratory

• Acknowledging limitations in both, both methods, AGE and CZE, fulfil requirements for detection and qualification of monoclonal bands as instructed by AACB guidelines for the standardisation of reporting of monoclonal bands (Tate 2012)

11/03/2015 S Musaad 52 Acknowledgments

for slides

11/03/2015 S Musaad 53 Thank you

[email protected]

11/03/2015 S Musaad 54 References

1. Jenkins MA. Serum and Urine Electrophoresis for Detection and Identification of Monoclonal Proteins. Clin Biochem Rev 2009;30:119-122

2. Tate J, Caldwell G, Daly J et al. Recommendations for Standardised Reporting of Protein Electrophoresis in Australia and New Zealand . Ann Clin Biochem 2012 ;1-5

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