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

Patient ID
Select the dilution (according
with the total concentration of
Igs in g/l)
- Hypogamma
(Ig<8g/l )
- Standard (Ig 8-20g/l)
- Hypergamma
(Ig>20g/l)

Require an
IT

Verify your IT request in

Immunotyping 6

Verify the presence of your

sample in the IT list

Require an IT without a barcode for a

sample
Select the mode tube without barcode
in the IT list
No samples must be already selected in the IT
list

Require an IT without a barcode for a

sample

Add the sample to analyze in IT by entering

a rack number for Capillarys or a position
on the carrousel for Minicap

Require an IT in Immunotyping

Click on standard to change the dilution

Select the dilution (according

with the total concentration of
Igs in g/l)
- Hypogamma (Ig<8g/l)
- Standard (Ig 8-20g/l)
- Hypergamma
(Ig>20g/l)

Immunotyping on Capillarys
Sample : Position 1

Antiserum segment

Diluent (380 L)

L K M A G ELP
Antisera/Fixative solution (20 L)

Capillarys Immunotyping:
Principle

Serum dilution in the double wellof the IT segment

with a specific diluent:

- Hypogamma (for Ig<8g/l ) : 1/10 - Collect 40 L of serum
- Standard (for Ig 8-20g/l) : 1/20 - Collect 20 L of serum
- Hypergamma (for Ig>20g/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)

Capillarys Immunotyping:
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.

ELP
Ig G
Ig A
Ig M
K

ELP

IgGX IgAX IgMX Ig

Ig

L
DILUENT

Capillarys Immunotyping:
* Delay to obtain the first result: 12
min after starting
* Cadence: 8 IT / hour

Immunotyping:
Interpretation
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

INTERPRETATION

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

INTERPRETATION

Ig G
Lambda

Ig A

Ig M

Kappa

Normal sample

ELP

~ 80% IgG

~ 5% IgM

~ 15% IgA

2/3 Kappa

1/3 Lambda

Monoclonal peak or polyclonal increase in gamma

Pointed peak

Narrow
basement
Monoclonal peak
IT
Complete substraction of
the
peak
with
one
antiserum
against
a
heavy chain and a light
chain

Rounded top

Large
basement
Polyclonal increase
IT
Complete substraction with
the antiserum against a
heavy chain and partial
substraction
with
the
antisera against kappa and

Polyclonal increase or monoclonal Ig

in gamma?

Polyclonal increase or monoclonal Ig

in gamma?

Polyclonal increase of Ig G

2/3 polyclonal Ig G kappa

1/3 polyclonal Ig G Lambda

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 no bridge aspect between Beta 2 and Gamma zone
Usually,

(valley)
Elevation of the Beta 2 occurs in a narrow width of the
fraction
Beta 2 peak decreases more significantly on one of the 2 light chains

IgG lambda

Zoom

IgG kappa

IgG lambda

Weak IgM lambda

IgA lambda

IgG lambda + free light chains

lambda

Polyclonal increase of Ig G

Zoom

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

Zoom

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

Zoom

IgA lambda

Zoom

IgM kappa and IgM lambda

IgM L

IgM K

Weak IgG lambda + very weak IgG kappa

IgG L

IgG K

IgG lambda + weak IgM kappa

IgM kappa

IgM kappa

IgG lambda

IgG

IgM

Before treatment with BME

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 BME
2) 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

IgG K

After treatment with BME

IgM L

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
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