Abnormal immunoglobulins and immunoglobulin specificities (1)

1. Abnormal immunoglobulins and
immunoglobulin specificities
Dr. Dinesh Jain
SMS MC Jaipur

2. IMMUNOGLOBULINS
• Immunoglobulins are glycoproteins
secreted by plasma cells that recognise a
particular epitope on antigen binds
specifically to it and facilitates its
clearance.
• Immunoglobulins constitute 20-25% of
total serum proteins.
• There are 5 classes of immunoglobulins
• IgG,IgA,IgM,IgD,IgE.

3.  2 Light chains
 2 Heavy chains
 Chains are
linked together
by disulfide
bonds
 Amino terminal
variable region
 Carboxy
terminal
constant

5. IMMUNOGLOBULIN G
Most abundant class in serum
constituting about 80% of total serum
immunoglobulins.
Present in blood ‘plasma and tissue
fluids.
It has four subclasses that differ in
constant region of their heavy chain.
It is the only maternal
immunoglobulin that can cross the
placenta.

6. It participates in most immunological
reactions such as complement fixation ,
precipitation and neutralisation of toxins
and viruses.

7. IMMUNOGLOBULIN A
It constitutes 10-15% of total
immunoglobulins.
It is present in milk, saliva ,tears,mucous
of respiratory, digestive and genitourinary
tract.
In serum it exists as monomer
In secretions it exists as a dimer where
the two monomeric units are joined
together by J chain.
Secretory IgA also has secretory piece.

8. IMMUNOGLOBULIN M
It constitutes 5-10% of total serum
iimunoglobulins
A heavy molecule and are polymers of five four
peptide subunits each bearing an extra Ch
domain.
Presence of IgM in newborn indicates
intrauterine infection.
They also act as a sign for recent infection.
Largely confined to intravascular space they
are responsible for protection against blood

9. IMMUNOGLOBULIN E
Structurally similar to igG.
Heat labile inactivated at 56degrees
in 1 hour.
Mostly present extracellularly .
It is chiefly produced in the lining of
respiratory and intestinal tracts.
It is responsible for anaphylactic type
of hypersensitivity.

10. IMMUNOGLOBULIN D
Its hinge region is particularly
extended making it uniquely
susceptible to proteolytic degradation.
Nearly all IgD is present together with
igM on the surface of blood
lymphocytes where they operate as
mutually interacting antigen receptors
for the control of lymphocyte
activation and suppression

11. IMMUNOGLOBULIN
SPECIFICITIES

12. Immunoglobulin specificities are of greatest
biological importance in immunology.
There are antigenic determinants present on
specific sites on immunoglobulin molecules
which have immunogenic potential.
Based on the location of these determinants
immunoglobulin molecules are divided into
ISOTYPES
ALLOTYPES
IDIOTYPES

14. ISOTYPES
Isotypic determinants refer to the
genetic variations in constant region
of heavy chain of Ig classes and
subclasses within a species.
Different species inherit different
constant region genes and therefore
express different isotypes.
Within a species each normal
individual will express all isotypes in
serum.

15. When an antibody from one species gets
injected into another then the isotypic
determinants are recognised as foreign
inducing as antibody response to isotypic
determinants.
The term isotype switching is a
biological mechanism that changes a B
cell’s production of immunoglobulins from
one type to another such as isotype IgM to
IgG ,during this the constant region portion
change but the variable region of heavy
Isotypes...

16. IMPORTANCE OF ISOTYPES
To measure immunoglobulin levels
To check for immunodeficiency
In detection of B cell tumours.

17. ALLOTYPES
Allotypic determinants refer to multiple alleles
that exist for some of the genes which lead to
subtle amino acid differences that occur in
some of members of species.
Allotypes are characterised for all four IgG
subclasses ,for one IgA subclass and kappa
light chains.
No allotypic markers are found for gamma
light chains or epsilon ,delta heavy chains.
Allotypic determinants can arise during blood
transfusions.

18. Allotypic determinants are sometimes
produced by mother during pregnancy in
response to paternal allotypic
determinants on fetal immunoglobulins.
 Importance of allotypes seen in
 Monitoring bone marrow grafts
 Paternity testing
 Forensics.
Allotypes...

19. ALLOTYPE SYSTEMS
3 system of allotypic markers seen in
humans-
1. For kappa light chains-Km system
(3 Km allotypes)
2. For gamma heavy chains-Gm system
(25 Gm allotypes)
3. For alpha heavy chains-Am system.

20. IDIOTYPES
Idiotypic determinants arise from the sequence of
heavy and light chain variable regions.
An idiotope may be the actual antigen binding site
or variable region sequences outside of antigen
binding site.
Immunisation with fab fragments may produce anti
idiotypic antibodies.
Idiotypes arise due to mutations in genes of
variable region.
Importance –
 Treatment of B cell tumours
 Vaccines

21. ABNORMAL
IMMUNOGLOBULIN

22. PARAPROTEINEMIA
• Paraproteinemia (also known as "monoclonal
gammopathy") is the presence of excessive
amounts of a single monoclonal
gammaglobulin in the blood.
• It denotes an underlying immunoproliferative
disorder or hematologic neoplasms,
sometimes considered equivalent to plasma
cell dyscrasia.
• Exhibit either:
 Excessive amounts of normal
immunoglobulin proteins (Igs)
 Accumulation of Igs in an abnormal
location
 Structurally abnormal Igs

23. Normal Lymphocytes (left) &
Plasma Cell (right) in Peripheral Blood
Note: Plasma cells are not normally seen
in peripheral blood

24. TYPES
Paraproteinaemias categorised according to
the type of monoclonal protein found in blood:
• Light chains ("Bence Jones protein")
only; associated with multiple myeloma or AL
amyloidosis
• Heavy chains only; (also known as "heavy
chain disease")
• Whole immunoglobulins In this case, the
paraprotein goes under the name of "M-
protein" ("M" for monoclonal).

25. • Sometimes paraproteins (usually whole
immunoglobulins) form polymers by
aggregating with each other
k/a macroglobulinemia .
• Certain macroglobulins tend to precipitate
within blood vessel with cold, known as
cryoglobulinemia.
• Others may make blood too viscous to flow
smoothly (usually with IgM pentamer
macroglobulins), known as Waldenström
Types…

26. Serum Protein Electrophoresis
Normally in our body two large classes of
blood protein are found
1.Albumin
2.Globulin
Albumin is much smaller and slightly negatively
charge which leads to an accumulation at one
end of the electrophoretic gel

27. Globulin separate out into three regions on
electrophoretic gel that are
1: α band: two componant
α 1: α 1 antitrypsin , α 1 acid glycoprotein
α 2: heptoglobin, α 2 macroglobin, α 2
antiplasmin, ceruloplasmin
2: b band: transferring low density lipoprotein,
complement system protein
3: y band: here immunoglobulin appear so c/a
Gammaglobulin
Majority of paraprotein appear here

29. Causes
1. Leukemia ,Lymphoma
2. Multiple Myeloma
3. Bence Jones Proteinuria
4. Plasmacytoma
5. Waldenstrom’s macroglobulinemia(IgM only)
6. Amyloidosis (either light or heavy chain)
7. Heavy chain disease
If no cause is identified then c/a monoclonal
gammopathy of undetermined significance

30. Multiple Myeloma

31. MULTIPLE MYELOMA
• Malignancy of a single clone of plasma cells
in bone marrow
• This results in the overproduction of
abnormal Ig mostly IgG.
• When only one mass k/a plasmacytoma
• When more than one mass k/a multiple
myeloma
Symptoms CRAB C: Calcium elevated
R: Renal failure
A: Anaemia
B: Bone lesion

32. Pathophysiology
Bone marrow  B Lymphocyte Lymph
nodes
and mature here and display different
protein on their cell surface
When they get activated they secrete
antibodies k/a Plasma cell.
Multiple myeloma develops from
1. Activated Memory B-cell
2. Precursor to plasma cell k/a Plasmablast

33. Main reason..
A chromosomal translocation between
immunoglobulin heavy chain gene (Ch 14)
and an oncogene (11q13, 6p21,20q11)
frequently results in formation of MM
protein, ie an abnormal immunoglobulin.

34. Ch 14
Translocation
Cloning of marrow
plasma cell
MGUS
(asymptomatic )
SMOLDERING
MM
Increased Myeloma Protein IG
in Bone Marrow & circulation
More aggressive
clone of plasma
cells
One / More specific
sets of CRAB
symptoms
Basis of diag. of
MM
Further genetic
changes
Abnormal plasma
cell clones into
circulation &
distant tissue
Plasma cell
Dyscrasia/
Leukemia

35. Diagnosis
A. Presence of
1. Unexplained anemia
2. Renal dysfunction
3. High ESR
4. Lytic Bone lesions.
5. Elevated 2 microglobulin.
6. High serum protein (especially Globulin)
B. Blood Tests
1. Protein Electophoresis of blood and
urine.
Sharp and distinct band called M band with
narrow spike (M band or monoclonal band)

37. cntd…
This M band almost replace  globulin band
due to diminished synthesis of normal 
globulins.
C. Laboratory Tests: Quantitative measures
of
1. IgA, IgG, IgM for immune paresis.
2. 2 microglobulin – prognostic
information
3. Peripheral blood smear – RBC rouleaux
formation

38. Histo-Pathology
1. Bone Marrow Biopsy : Estimate % of bone
marrow occupied by plasma cells.
2. Immuno - histochemistry : Staining using
antibody against surface protein detect
plasma cell which express immunoglobulin
in cytosol and on the surface.
Myeloma cells typically
CD56,CD38,CD138,CD319 +ve
CD 19,CD45 –ve
3. Cytogenetic: Myeloma specific FISH and
virtual karyotyping

39. Diagnostic Criteria
According to International Myeloma Working
Group
A. Symptomatic myeloma (all three criteria must
be met):
1. Clonal plasma cells >10% on bone
marrow biopsy.
2. A monoclonal protein (Myeloma protein) in
either serum or urine.
3. Evidence of end-organ damage (CRAB):
HyperCalcemia (corrected calcium >11 mg/dl)
Renal insufficiency attributable to myeloma
Anemia (hemoglobin <10 g/dl)

40. Diagnostic Criteria…
B. Asymptomatic /smoldering myeloma:
1. Serum M protein >30 g/l or
2. Clonal plasma cells >10% on bone marrow
biopsy
3. No myeloma-related organ or tissue
impairment
C. Monoclonal gammopathy of undetermined
significance (MGUS):
1. Serum paraprotein <30 g/l and
2. Clonal plasma cells <10% on bone marrow
biopsy and

42. Bence – Jones Proteinuria

43. Bence – Jones Proteinuria
• Mostly heavy chains and whole
immunoglobulin remains within blood
vessels.
• Light chains frequently escape and
excreted by the kidney into urine k/a
Bence jones protein
• These protein found in urine due to
decresed kidney filtration capability due
to renal failure.
Bence jones protein presents in:-

44. Diagnosis
1. Heating method– These proteins
precipitate when urine is heated between
40-50C and re-dissolve on further
heating at higher temperature of 80C;
precipitate reappear again on cooling the
urine
2. Electrophoresis of concentrated urine
3. Serum free light chain assay
4. Bradshaw test :- When urine is layered
on few mL of concentrated HCl a white
ring of ppt

45. PLASMACYTOMA
It is a plasma cell dyscrasia in which a
plasma cell tumour grows within soft
tissue / axial skeleton.
According to IMWG, 3 types:
1. Solitary Plasmacytoma of bone (m/c)
2. Extramedullary Plasmacytoma
3. Multiple Plasmacytoma
a. Primary b. Recurrant

46. Diagnosis
1. CBC
2. Plain Radiography
3. Serum Protein Electrophoresis.
4. Bone Marrow Biopsy.
5. Urine Analysis
6. CT/MRI/PET

47. WALDENSTROM’S
MACROGLOBULINEMIA
It is type of cancer affecting 2 types of B-
cells,
1. Lymphoplasmocytoid cells
2. Plasma cells.
High level of circulating IgM.
Pathogenesis:
1. m/c associated with somatic mutations in
MYD88 and CXCR4 locates on
chromosome6.
2. Protein Src tyrosine kinase is
overexpressed that cause uncontrolled

48. Diagnosis
1. Serum Protein Electrophoresis-
Significant monoclonal IgM spike.
2. Bone Marrow Biopsy- Show malignant
cells.
3. Flowcytometry- Examines markers on the
cell surface or inside the lymphocytes.
4. CBC – Anemia, Low WBC and low
Platelets.
5. SIA test – Due to hyperviscosity, serum
forms globular precipitates in water

49. MGUS
Monoclonal Gammopathy of
Undetermined Significance

50. Characteristics
• Also called benign monoclonal
gammopathy
• Precancerous condition
• Monoclonal protein present without the
invasive symptoms of multiple myeloma
• MGUS resembles multiple myeloma and
similar diseases, but the levels of antibody
are lower, the number of plasma cells in
the bone marrow is lower, and it has no
symptoms or major problems.
• Usually seen in people over age 70

51. Pathophysiology
• Pathologically, the lesion in MGUS similar to that
in multiple myeloma.
• Predominance of clonal plasma cells in the bone
marrow with an
abnormal immunophenotype (CD38+ CD56+ CD1
9−) mixed in with cells of a normal phenotype
(CD38+ CD56− CD19+);
• More than 3% of the clonal plasma cells have the
normal phenotypein MGUS, whereas in multiple
myeloma, less than 3% of the cells have the
normal phenotype.

52. Prognosis
• Good – patient often remains stable for
years.
• May progress to multiple myeloma,
Waldenström's macroglobulinemia, or
amyloidosis,Bcell lymphoma or chronic
lymphocytic leukemia in some patients.

54. Diagnosis
• Patients may be diagnosed with MGUS if
they fulfill the following four criteria:
• A monoclonal paraprotein band less than
30 g/l (< 3g/dl);
• Plasma cells less than 10% on bone
marrow examination;
• No evidence of bone
lesions, anemia, hypercalcemia, or renal
insufficiency related to the paraprotein,
and
• No evidence of another B-cell proliferative

55. Treatment
• As there are no symptoms, there is no
need for treatment
• Patient will be monitored for an increase in
monoclonal protein level and physical
symptoms of more serious paraprotein
disease.

56. Heavy Chain
Disease

57. • There is excessive production of short and
truncated heavy chains.
• There are deletions in heavy chains, mainly in
their amino-terminal part, leading to loss of
their ability to form disulfide bonds with
the light chains.
• Deletion of the N-terminal part leads to
aggregation and signaling of the B cell
receptor, presumably due to the loss of the
anti-aggregating properties of the light chain.

58. Classification
There are three forms:
1. Alpha chain disease (Seligmann's
disease)
2. Gamma chain disease (Franklin's
disease)
3. Mu chain disease

59. Alpha Heavy Chain Disease
Mediterranean Lymphoma

60. Characteristics
• Affects young adults.
• More common in those of Mediterranean
or Middle Eastern decent.
• Lymphoid tissue in the GI tract becomes
infiltrated with lymphocytes and plasma
cells.
• Cells may be normal to extremely bizarre
in appearance.
• Alpha chain may have abnormal structure.

61. Prognosis
• Guarded – some patients experience
complete remission with appropriate
therapy while others die despite intensive
therapy
• When treatment fails, disease progression
is rapid
–Death within 1 year

62. Treatment
• Antibiotics
• Anti-lymphoma therapy
• Corticosteroids

63. Other Heavy Chain
Diseases
• Gamma Heavy Chain Disease – seen in elderly
– Characterised by excess of mostly y1 –paraprotein.
– Symptoms –enlarged liver and spleen, recurrent infections,
and anemia
– Some patients experience no symptoms
– Treatment with anti-lymphoma drugs and corticosteroids
• Mu Heavy Chain Disease – rare
– Produce mu chains that don’t appear in urine and also k
chains that appear in urine
– Presence of vacuoles in malignanat b lymphocyte
– Symptoms include enlarged spleen, liver and abdominal
lymph nodes
– Survival and response to treatment varies

64. Amyloidosis
Accumulation of abnormal protein
i.e. amyloid (a waxy, stringy
protein) in patients with persistent
infection or plasma cell disorders

65. • Amyloid light chain (AL) amyloidosis, primary
systemic amyloidosis (PSA) or just primary
amyloidosis -
• Antibody-producing cells do not function properly
and produce abnormal protein fibers made of
components of antibodies called light chains.
• These light chains come together to form amyloid
deposit.
• Abnormal light chains in urine are sometimes
referred to as "Bence Jones protein".

66. Characteristics
• Usually occurs in the elderly
• More common in men
–Protein comprised of immunoglobulin
fragments
• Variable region
• All or part of the constant domain
• Protein deposits in a variety of tissues
• Other forms of amyloidosis exist that do
not have an immune basis

67. Prognosis
• Poor in many cases – Median survival for
patients diagnosed with AL amyloidosis-
40 months
Treatment
• No specific treatment
• Treat underlying infection or plasma cell
disorder to limit disease progression
• Damage done from protein deposits
cannot be reversed
–Limited use of organ transplants to
“stall” the disease

70. REFERENCES
• Ivan M Roitt Essential Immunology.
• Harrison’s Principles of Medicine
• Robbins and cotrans pathological bais of
diseases.
• ncbi post graduate medical journal