Immunoglobulins and

Waldenstroms
B cell malignancy macroglobulinaemia
Common ALL Secretory B Lympho
Pro -B plasmacytoid
Immature B cell
Stem cell
Pre-B
CLL,
Non-Hodgkin's Plasma cell Making immunoglobulin
lymphoma • Heavy chain rearrangement on Chromosome 14
• Randomly join 1V, 1D and1J region
• Splice this VDJ onto 
• Reactivity check
Memory B • If high or autoreactive delete and try to
Multiple rearrange on other Chromosome 14
Mature B Myeloma • Rearrange  (chromosome 2) and join to 
• Reactivity check
• If  -  has high or autoreactivity, delete  and
try to rearrange other . Reactivity check and
if high go on to rearrange s (chromosome 22)
• B cell now goes out into circulation, lymph nodes,
lymphoid tissue, looking for antigen to react with
• Activation and proliferation with T cell help
• Class switching to make IgG, IgA, IgE
• Back to bone marrow as a plasma cell for long
term production of specific antibody
 • IgA has the “fastest” mobility running in the
Immunoglobulins and •
beta-fast gamma
IgM is next in the fast gamma - gamma
Disorders with monoclonal Ig
Malignant
o Multiple myeloma
o Solitary plasmacytoma
B cell malignancy •
•
IgG runs in the gamma
Monoclonal proteins may not obey these
o Waldenstroms
macroglobulinaemia
rules – paraproteins can run from just after o Non-Hodgkins lymphoma
the albumin to the post gamma o Chronic lymphocytic leukaemia
• Amino acids Clonal B cells – not necessarily
• Huge variation in the sequence for the malignant
hypervariable region of HC and LC o Monoclonal gammopathy of
• Represents all classes, subclasses  and  unknown significance (MGUS)
o Peripheral neuropathy (MGUS)
• Glycosylated
o AL amyloidosis
• Estimated 1010 specificities therefore 1010 o Cryoglobulinaemia
slightly different variable amino acid sequences, o Primary cold agglutinin disease
different HC, LC, protein charge o Dermatological disorders e.g.
• Product of billions of B cell clones lichen myxedematosis,
• POLYCLONAL immunoglobulins pyoderma gangrenous,
Transient
o Post infection
• 1 B cell becomes “malignant” and develops out
IgA IgM IgG o During immune reconstitution
of balance with the other B cells e.g. post PBSCT
• Lots of cells producing identical Ig with identical • Some B cells may be stimulated to
heavy chains, identical light chains, identical produce extra immunoglobulins e.g. as
variable region etc. and identical charge a result of infection or inflammation
• MONOCLONAL immunoglobulin • This can appear as small “zones” or
even obvious bands e.g.
OLIGOCLONAL banding
 Haemoglobin
Fibrinogen

Lymphoid tissue • If you are losing protein, low or falling

and bone marrow
• Igs and albumin +  +  proteins are under
different controls
• Protein malnutrition – all proteins low
Liver • Low Igs due to BM failure – albumin normal
• Liver failure – Igs normal/raised
albumin will be the most obvious followed
by low or falling IgG (due to high-ish
concentrations and/or low-ish mwt).
• Can’t lose IgG without losing albumin
• IgM has high mwt (like alpha-2
macroglobulin) so often relatively normal
or raised in protein loss
• IgA produced at mucosal surfaces so can
also be normal in protein loss

Proteins, Basic Pathological processes

VITAMIN CDEF
V: vascular
Concentrations •
•
Genetic
Tumour
I: infective/Inflammatory
T: traumatic
• Trauma A: autoimmune
and processes •
•
Infection
Infarction
M: metabolic
I: iatrogenic/idiopathic
• Inflammation N: neoplastic
• iatrogenic C: congenital
D: degenerative/developmental
E: endocrine/environmental
F: functional
How low can they go? – interpret with age related reference ranges
• IgG - <1g/L - unexplained low IgG needs urgent action
• IgG replacement may be indicated at concentrations <5g/L in secondary
Antibody deficiencies
• Can be primary
Immunoglobulins
antibody deficiency
• Can be secondary
• IgA - <0.06 g/L consistent with IgA deficiency (~1/700 of population)
• IgG or any of the subclasses or IgA or IgM
• Also seen in secondary immune suppression
• IgA and IgG subclasses
• IgM - <0.1g/L
• Pan hypogamma
• Slightly low IgM seen in the elderly
• Also seen in secondary immune suppression

IgG IgA IgM IgD IgE

Usual form Monomer Monomer, dimer, Pentamer Monomer Monomer
trimer and (monomer)
secretory
Subclasses 4 2 - - -

Mwt (kDa) 150 (IgG3 170) 160 970 175 190

HC domains 4 4 5 4 5

~ 21 ~5 ~5 ~3 ~3 Approx. magnitude of increase

Half life
(days) Depends on serum Polyclonal Monoclonal
concentration (g/L) (g/L)
Adult serum 6-16 0.8-4.0 0.5-2.0 0.04-0.4 0.0003 IgG 100 140
concn (g/L) (<81 kU/L)
IgA 15 100
Function • MOST important • Mucosal • Primary • Marker of • Active when bound IgM 10 100
• MAIN secondary secretions antibody mature B cells to mast cells
antibody response response • Important • Defence against IgD - 25
• Antibody “memory” • Mainly against some parasite IgE <1 20
• Bathes all tissues intravascular respiratory • Allergic reactions
pathogens BJP - 20

Traceable ERM Da 470k/IFCC No 3rd WHO International Free HC - 10

Standard 11/234
 Know the principles of  Immunofix
o What proteins are made where –
liver vs bone marrow
Reading serum protein o new monoclonals or bands
o unexplained high IgA and IgM
o What is “normal” – for various age o unexplained or new low Igs in adults (and ask for
groups
o Children have raised alpha-2
electrophoresis urine)
o disappeared monoclonals – for minimal residual
o Young children have low disease
gammas  Read the EP separation – look systematically at each o don’t forget D and E for monoclonal light chains
o How much can the concentrations “zone” in serum
change in disease o Note high, low, odd shape - monoclonals can run  Immunofixation increases SPECIFICITY and
o Acute phase proteins anywhere from alpha-1 to slow gamma SENSITIVITY
o immunoglobulins  Compare patterns to a normal control o It enhances the signal by binding an antibody to
 Compare patterns to each other the protein so increasing the amount of protein
 Know and understand the concepts o Do lots of samples seem to have raised alpha-1 and fixed into the gel
of polyclonality, moncolonality, alpha-2 globulins o It is NOT quantitative – it relies on the
oligoclonality o Is there a shoulder on most beta-2 zones characteristics of the Ag-Ab affinity and avidity
o Does everyone seem to have polyclonally raised  Immunotyping cannot enhance a signal it only
 Know the EP patterns for broad gamma “removes” signal
groups of conditions  Interpret and report the EP pattern in the context of: o Immunotyping cannot detect bands that are not
o B cell malignancy o Immunoglobulin concentrations seen on the CZE trace
o Persistent infection or inflammation o Patients age o will miss small bands e.g. of retained BJP
o Acute infection or inflammation o Previous serum and urine protein patterns and  Know what you are expecting on the immunofixation
o Severe infection results – but be prepared to be surprised
o Immune deficiency o Clinical details o I think this will be polyclonally raised IgA and I
o Immune suppression  Add further tests as indicated don’t think it will be a monoclonal
o Protein loss o AAT for an “unknown” low alpha-1 o I think this will be a large IgG paraprotein
 Repeat tests if the pattern and the quantifications are o I think this will show polyclonally raised gamma
inconsistent and there may be some “zones”
 Rules and
Oddities
Rules
• Look at every zone on the electrophoresis
• Interpret the electrophoresis WITH the serum
immunoglobulin concentrations
• Interpret the Igs and EP with the patients age
• Low Igs in an adult is a worrying finding
o Could be immune deficiency e.g. common
variable immune deficiency
o Could be secondary to another process e.g.
malignancy
• ALWAYS immunofix low Igs in an adult (1st time)
• ALWAYS check D and E if you have light chains
only in a serum
• Myeloma does not mean paraproteinaemia and
paraproteinaemia does not mean myeloma
• Make sense of the results – does it all fit together
consistently
• The same paraprotein band will always have the
same electrophoretic mobility – look at the X axis
on the CZE
• A band on an immunofixation is the same protein
in e.g. the IgG and the kappa land, just identified
using a different antibody
Expectations of Serum Immunofixation
• No paraprotein detected IS a possibility!
• A serum paraprotein will most often have a heavy chain and a
Oddities
• Immunoglobulins have a complex
folded three dimensional structure
• The can aggregate into high
light chain….but you can have retained Bence Jones protein
with or without an intact immunoglobulin
molecular weight immune complexes
• These can get stuck on the origin
• Light chains on their own in the serum without an intact of a gel electrophoresis or
immunoglobulin must be checked for D and E immunofixation
• Heavy chains can occur – often with post synthetic degradation • Some Igs can become
but can be with malignancy inappropriately folded which “hides”
• Often diffuse “zones” rather than bands their light chains (especially IgA)
• Di-thio-threitol is a mild sulphydryl
• Can be confirmed by Laurell Rockets immunoselection
reducing agent that can dis-associate
• -chain disease (used to be called Mediterranean these complexes and is very good at
lymphoma) - seen in young adult males in the revealing immunoglobulin with
Mediterranean. Environmental factors important in immune complexes and hidden light
pathogenesis. Antibiotic treatment can be effective. chains
• -chain disease – heterogeneous clinical picture but more
often “lymphomatous” rather than “myelomatous”
• -chain disease – very rare and presents more like CLL
Other things that we see on electrophoresis
 Fibrinogen – runs in the 2 region often giving a shoulder on
the complement band
 Patients are increasingly anti-coagulated e.g. with low mwt.
heparin so even a sample collected into a serum tube with
not clot – you can check by fixing with anti-fibrinogen
 Haemoglobin in the 1 – look at the sample! (serum or urine)
 Drugs
 UV absorbing e.g some antibiotics in the 2- on CZE
 Contrast media
 Monoclonal antibodies e.g Daratumumab
Rules - electrophoresis
● Locate the albumin
● Locate the transferrin – this
should be much fainter than the
albumin, if not, could be BJP
● If not previously known,
immunofix ALL samples that
have bands additional to the
albumin
● Glomerular pattern –  Look for “leaks” of intact monoclonal Ig from the
predominant bands of albumin
and transferrin e.g. track 11
● Tubular pattern – albumin,
transferrin and smudge of low
molecular weight protein
fragments e.g. track 4
Things that we see on urine
electrophoresis
 Haemoglobin – big band in the 1 that
does not immunofix for Ig – look at
the sample!
 Myoglobin – similar mobility to
haemoglobin
 Things people add e.g. egg albumin
 Seminal plasma particularly in elderly
men
Urine patterns Expectations - Urine Immunofix
 No obvious band – you may want to fix with
(GAM) 
 Immunofixation improves specificity AND
sensitivity so you may see bands on the
immunofixation that are not visible on the urine
protein electrophoresis
 Use good antibodies – anti total- and  are
better than anti free- and 
serum AND monoclonal free light chains (Bence
Jones protein) - most often, these will have
different electrophoretic mobility
 Urines will usually have some polyclonal  and ,
usually more  than 
 Patient who are elderly or who have underling
infection or inflammation will often show “light
chain banding”
o 2-5 tiny “zones” of usually  but can be  or 
and  - the distinction between this and BJP
can be hard and needs experience
 Smelly and degraded urines give indistinct
patterns – get fresh!
 2 microglobulin can be a very distinct band in the
2
 Immunofixation
Serum Immunofixation
 Read every track – is there a band in the SPE, IgG, IgA etc.
 Is there polyclonal IgG, IgA etc.
 Look at the whole length of the track – bands can appear right up
to the alpha-1
 No obvious band on EP – you may still find a monoclonal protein
– do a fix if there is a high index of suspicion
 The IFIX should be consistent with the electrophoresis
 You will only detect the protein if the antibody is added
 When you are detecting an intact immunoglobulin e.g. an IgG
kappa, you are detecting the SAME protein using different
antisera in the different tracks
 Always expect more polyclonal kappa than lambda – this may
mean that tiny “zones” are more apparent in the lambda and
hidden in the IgG and kappa lanes by background staining
 You can have retained BJP in the serum
 Optimum length for electrophoresis and immunofixation is
approx. 4cm
 IgA paraproteins often appear as 1, 2, 3 or even 4 bands
 Use good antibodies
 Immunofixation is NOT quantitative – the strength of staining will
depend on the affinity and avidity of the antibody for the antigen
 Have a mechanism to check for IgD and IgE paraproteins
 Don’t forget that haemoglobin, fibrinogen and drugs will appear
on the electrophoresis but not on the IF
Immunofixation is NOT
quantitative – this sample is
run at the SAME dilution in
the IgM and lambda lanes
Band vs Zone
• “zoning” is a useful term when there is no distinct
monoclonal but there is a tiny “restriction” on the EP
and IFIX – serum “zoning” is of intact Ig, tiny
“zones” of BJP in the serum must be followed-up
• There may be multiple “zones” - oligoclonal - but
these may also be distinct oligoclonal bands
• “zoning” can be an incidental finding in the elderly
• “zoning” can be seen with ongoing or recent
infection or inflammation,
• Polyclonally raised IgG and IgM often have some
“zoning” visible
• Always fix “zones” in case they are BJP but be
prepared to call them “zones” and report as likely
incidental findings or secondary to recent infection