Capillarys Immunotyping

IMMUNOTYPING (IT)

LIKE IMMUNOFIXATION, THE PURPOSE OF AN IMMUNOTYPING (IT) IS TO CONFIRM THE PRESENCE OF A MONOCLONAL IMMUNOGLOBULIN AND DEFINE ITS TYPE

Request an Immunotyping in Protein 6

Verify your IT request in Immunotyping 6

Select the mode tube without barcode in the IT listNo samples must be already selected in the IT listRequire an IT without a barcode for a sample

Require an IT without a barcode for a sample

Require an IT in ImmunotypingClick on standard to change the dilution

Immunotyping on CapillarysSample : Position 1Diluent (380 L) L K M A G ELP Antisera/Fixative solution (20 L)Antiserum segment

Capillarys Immunotyping: PrincipleSerum dilution in the double wellof the IT segment with a specific diluent:- Hypogamma (for Ig20g/l) : 1/40 - Collect 20 L of serum

The diluted serum is mixed inside 5 other wells with the 5 antisera (IgG, IgA, IgM, Kappa and Lambda). The reaction between AS and Ag is very fast in liquid medium, without any incubation or sedimentation step. One well without AS will be used to obtain the reference pattern (ELP)

Injection of the mixture AS-serum in 6 capillaries at the anodic side

Migration at 7000 V (4 min)

Visual analysis of the patterns by comparison with the reference one.

Capillarys Immunotyping:

ELPGAMklDILUENT

* Delay to obtain the first result: 12 min after starting

* Cadence: 8 IT / hour

Capillarys Immunotyping:

An immune complex is formed which migrates in anodic position: more anodic than albumin or between albumin and alpha 1 fraction

The monoclonal peak is identified when the peak disappears with a given antibody against an heavy chain (G, A or M) and with an antibody against kappa or lambda light chain

Immunotyping: Interpretation

INTERPRETATIONAll Ag-Ac complexes migrate in the zone albumin - 1

INTERPRETATION Ig G Ig A Ig M Kappa Lambda

Normal sample

~ 80% IgG~ 15% IgA~ 5% IgM2/3 Kappa1/3 LambdaELPGAMKL

Monoclonal peak or polyclonal increase in gamma?Monoclonal peakNarrow basementPointed peakPolyclonal increaseLarge basementRounded topComplete substraction of the peak with one antiserum against a heavy chain and a light chainComplete substraction with the antiserum against a heavy chain and partial substraction with the antisera against kappa and lambdaITIT

Polyclonal increase or monoclonal Ig in gamma?

Polyclonal increase or monoclonal Ig in gamma?

Polyclonal increase of Ig G

Distinguishing features of polyclonal Ig A increase Bridge aspect between Beta 2 and Gamma zone Elevation of the Beta 2 occurs in a broad width of the curve Beta 2 peak decreases equally with both light chains removal

How to identify a monoclonal IgA migrating in the beta2 zone? Isolated elevation of Beta 2 (Beta 2 > Beta 1) without any increase of Alpha 1 & 2 Elevation of the Beta 2 occurs in a narrow width of the fraction Usually, no bridge aspect between Beta 2 and Gamma zone (valley) Beta 2 peak decreases more significantly on one of the 2 light chains

IgG lambda

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IgG kappa

IgG lambda

Weak IgM lambda

IgA lambda

IgG lambda + free light chains lambda

Polyclonal increase of Ig G

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Polyclonal increase of Ig G + underlying IgG lambda(more visible on IT than IF)

Polyclonal IgA: bloc beta-gamma

Distinguishing features of polyclonal Ig A increase Bridge aspect between Beta 2 and Gamma zone Elevation of the Beta 2 occurs in a broad width of the curve Beta 2 peak decreases equally with both light chains removal

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Oligoclonal profil in Ig G

IgG lambda associated with oligoclonal profil in Ig G

When interpreting IT, always consider: If removing something, what is remaining? In each window, removing one specific class of Ig highlights what is happening with the residual immunoglobulins that remain after substraction

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IgA lambda

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IgM L IgM KIgM kappa and IgM lambda

Weak IgG lambda + very weak IgG kappaIgG LIgG K

IgG lambda + weak IgM kappa IgM kappaIgM kappaIgG lambda

Before treatment with BMEIgM IgG

Treatment with Beta Mercaptoethanol (BME) to depolymerize a monoclonal Ig or separate co-migrating Igs :* Prepare a 1% BME reducing solution :1) 90L H2O + 10L BME2) 10L 1/10 diluted BME + 90L Fluidil (ref 4587)

* 100L of 1% BME reducing solution + 300L serum

* Incubate 10 mn at room temperature

Analyze immediately the treated sample in Capillarys or Minicap without any delay

IgG kappa + IgM Lambda After treatment with BMEIgM LIgG K

Hints and tips for IT interpretationExamine 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 subtraction 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