Imunologi transfusi

1. Imunologi Transfusi:
Konsep Pemahaman
BUDI MULYONO
Departemen Patologi Klinik & Kedokteran laboratorium
FK-UGM/RSUP Dr. Sardjito
Yogyakarta
E-mail: budimulyono@ugm.ac.id

2. Pengantar :
▪ Transfusion immunology – bagian dari ilmu kedokteran
transfusi yg mempelajari respon imun terhadap elemen
seluler terkait dengan kegiatan transfusi darah
▪ Immunohematology – bidang imunologi yang terkait
studi tentang properti serta aspek keberadaan antigen-
antigen sel darah dan antibodinya
▪ Eritrosit (sel darah merah), lekosit (sel darah putih) dan
trombosit (keping darah) merupakan komponen seluler
darah yg mempunyai kemampuan menginisiasi respon
imun sesudah pemberian transfusi

3. 1. Konsep antigen, antibodi dan
nomenklatur dlm kedokteran transfusi
2. Immunoassay / Tes-tes imunodiagnostik
Sistematika :

4. Konsep antigen, antibodi dan
nomenklatur dalam
kedokteran transfusi

5. ▪ An antigen is a molecule that binds to an antibody or
T-cell receptor.
▪ In chemical terms, antigens are large-molecular-
weight proteins (including conjugated proteins such
as glycoproteins, lipoproteins, and nucleoproteins)
and polysaccharides (including lipopolysac-
charides). Sometimes include nucleic acid
▪ These protein and polysaccharide antigens may be
located on the surfaces of cell membranes or may be
an integral portion of the cell membrane.
▪ Antigens are located on viruses, bacteria, fungi,
protozoa, blood cells, organs, and tissues.
Antigen

6. MV > 3000 Immunogenic
MV < 3000 Hapten
Antigen determinants (epitopes):
the area is typically between about 400
– 1000 À2
Big antigen have more than 1
determinants
( 1 Angstrom = 10-10 meter)
ANTIGEN

7. Antigen dan Imunogen
• Protein: sangat imunogenik
• Pure protein, glycoprotein, lipoprotein
• Polysacharide: imunogen yg bagus
• Nucleic acid: kurang imunogenik
• Lipid: non imunogenik, bisa jadi hapten
Jenis Antigen
• Eksogen
• Endogen
Asal Antigen

8. Antigen: As a substances, that generates a specific
immune response and induces the
formation of a specific antibody or
specially sensitized T cells, can be named
as autoantigen and alloantigen
Autoantigen: a person’s own self
antigens
Alloantigen: antigens found in different
members of the same species
Ex: red blood cell antigens A and B
AUTOANTIGEN vs ALLOANTIGEN

9. Allogeneic & autologous
▪ Transfused red cells contain antigens that may be
recognized as foreign to the individual receiving the
blood.
▪ These antigens are called allogeneic because they
are unfamiliar to the individual being transfused but
are derived from the same species.
▪ The body’s immune system normally recognizes and
tolerates self-antigens.
▪ These antigens are termed autologous because they
originate from the individual.

10. BINDING SITE of Antigen & Antibody :
- the part of antigen which combines with antibody is called
‘epitope’, recognized by the immune system, specifically by
antibodies, B cells, or T cells.
- part of an antibody that recognizes an epitope is called a
‘paratope’.

11. 11
Figure A-10
v
Different Ab bind to distunct epitopes
on Ag
• Antigen valence : maximum
number of antibodies that can be
bound by an antigen molecule
VALENSI ANTIGEN

12. Antibodi
– gamma-globulin
– immunoglobulin
– produk respon imun
– bereaksi dg Ag yang
memicu produksinya

13. Specificity:
- refers to the ability of an individual antibody combining
site to react with only one antigenic determinant (epitope).
- each antibody binds to a specific antigen; an interaction
similar to a lock and key.

14. Variable region of an immunoglobulin. The specificity of an
antibody is determined by the unique variable region that “fits”
antigenic determinants or epitopes.

15. Good fit. A good fit between the antigenic determinant and the
binding site of the antibody molecule results in high attraction.
In a poor fit, the forces of attraction are low.

17. IgM
IgG
Respon Antibodi

20. Naturally occurring vs Immune Antibody
Feature Naturally occurring Immune
Antigen stimulus Obscure, possibly
from microbial origin
Human red cell
antigens
Type of Immunoglobulin IgM IgG
Optimum temperature < 22o C at 37o C
Clinical significance Acute HTR HDN, DHTR
Examples ABO antibodies Rh, Kell, Kidd,
Duffy antibodies

21. Complete and Incomplete Antibody
Complete antibody - IgM
Incomplete antibody -IgG

22. IgM Antibodies (Complete)
• Agglutinate in saline phase
• Pentavalent
• Usually naturally occurring
• Do not cross placenta
• React at temperature
varying from 4 – 20oC
• Example: ABO antibodies

23. IgG Antibodies (Incomplete)
• Agglutinate in IAT phase
• However, may cause
agglutination in saline phase
using albumin / enzymes
• Monovalent
• Usually immune in nature
• Can cross placenta
• React at 37oC
• Example: Rh antibodies

24. Dinamika Reaksi Ag-Ab :
Ikatan tergantung pada struktur dan muatan molekul
• Lock and key
• Kekuatan tarik menarik ditimbulkan oleh muatan Ag dan Ab yg
berlawanan
• Reaksi bersifat REVERSIBLE
Kekuatan ikatan dipengaruhi oleh:
• perubahan pH,
• kekuatan ionik,
• temperatur
• jenis pelarut: salin, albumin, LISS

25. Antigen – Antibody Bonds
1. Coulombic Type ( Electrostatic Model )
-
+
+
+
-
-
+
+
-
-
-
+
+
-
Ag Ab

26. 2. Van der Walls Attraction (Lock and Key
Model)
Ag Ab
Hydrophilic and hydrophobic
perspectives follow those 2
models

28. Kinetics of antigen-antibody reactions: the ratio of the forward and
reverse reaction rates gives the equilibrium constant.
Ab ( antibody); Ag (antigen)

29. K1
Ag + Ab ➔ Ag - Ab
Ag - Ab
Ag Ab
=
K1
K2
= K
K = temperature dependent
Affinity : the speed of interaction to form complex
Avidity : the strenght of complex to avoid dissociation
t Affinity :
Avidity :
t Affinity :
Avidity :
K2
Thermodynamic of
Antibody-antigen
Interactions

30. Binding Force of Ag-Ab (Affinity vs Avidity):
• Closeness between Ag & Ab -> closer=
stronger
• Non – covalent bonds or Intermolecular
forces
• Affinity of antibody -> speed of reaction
between a single epitope & single paratope
• Avidity: multiple interactions between
antigen binding sites and epitopes
The binding of Ag – Ab reaction is due
to 4 factors:

31. Avidity
Avidity is a measure of
the overall strength of
binding of an antigen
with many antigenic
determinants and
multivalent antibodies
Avidity is influenced by
both the valence of the
antibody and the valence
of the antigen.
Avidity is more than the
sum of the individual
affinities.
Dr.T.V.Rao MD 11

32. ANTIBODY :
1. Polyclonal (PCA) : purification of animal sera
polyspecific; various affinity
2. Monoclonal (MCA) : similar clone of B-
lymphocytes
monospecific; similarity in affinity & avidity

33. Immunoassay /
Immuno diagnostic test

34. Immunoassay
34
An immunoassay is a test that
uses antibody and antigen
complexes as a means of
generating a measurable result.
the specific binding of an
antibody to an antigen allows
the detection of analytes by a
variety of immunoassay methods.

35. • Use known antibodies to detect antigens
associated with an infectious agent
• Use antigens to detect specific antibodies in a
patient’s blood to determine exposure to a
specific pathogen
use immunological processes in two
general ways
Immunoassays :

36. 36
Keith chaitoff, 2004 abbot diagnostic division
Antigen
+
Antibody
+
Indicator
system /
detector
Immuno-serology technique

37. Classification of Immunoassay
1. Non amplified IA (Naked IA) :
a. Precipitation tests
b. Agglutination tests
2. Visualized IA :
a. Agar gel IA
b. Latex IA
c. Erythrocyte IA
3. Ligand binding assay :
a. Isotopic IA : RIA (Radio Immunoassay)
b. Non Isotopic IA: EIA, FIA, LIA
The sensitivity of : 10-100 x
The sensitivity of : > 100 x
2
3
1
1

38. Precipitation
/Flocculation
test
Agglutination test
(qualitative
analysis)

39. Visualized Antigen-Antibody
Reaction

40. AGAR GEL IMMUNOASSAY:
• The simplest way to
visualize antigen-
antibody reaction
through agarose
medium. It is because
the property of specific
impermeability of the
precipitate barrier that
dependent to the size
of complex

42. LIGAND BINDING ASSAY
A. Isotopic Method : RIA ( Radio Immuno Assay)
B. Non Isotopic Methods :
a. EIA ( Enzyme Immuno Assay)
b. FIA ( Fluorescence Immuno Assay)
c. LIA ( Luminescence Immuno Assay)

44. Competitive ELISA

46. THE PRINCIPLES OF OTHERS NON-ISOTOPIC
TECHNIQUES
Replacement of radioisotope can be carried out also by :
1. Fluorescent substances FIA (Fluorescence
Immuno Assay)
2. Luminescent substances LIA (Luminescence
Immuno Assay)
Those techniques nowadays have been developed as
the method of choice for hormone assay and
immunological assay.

47. MODIFICATIONS of EIA:
1. The sequence of reaction: using avidin-biotin complex,
paramagnetic separation → to improve the sensitivity of test
2. The method to measure absorbance:
a. Macro or micro ELISA techniques
b. Photometric or Microscopic (Immuno Peroxydase /
Immunohistochemistry Technique - IHC) or Naked eyes / Using
Gel
3. Hybridization with other techniques:
a. Chromatography: ICT (ImmunochromatographicTest) → popular for
infectious diseases detection (Hepatitis B virus, Hepatitis C virus, HIV,
Malaria)
b. FIA or LIA:
F-MEIA (Fluorescence Microparticle EIA)
ELIFA (Enzyme Linked Immunofluorence Assay)
CLEIA (Chemiluminescence EIA)
→ facility to be automated

48. Card Gel test for Blood Group &
Antiglobulin Testing

49. Pengembangan imunokromatografi

50. Keunggulan dan kendala POCT
• Keunggulan:
hasil bisa lebih cepat shg tindakan dpt segera dilakukan
dan masa tinggal di RS dpt lebih pendek
• Kendala:
- linearitas terbatas, cakupan hanya sekitar
normal, tidak mampu mencapai nilai ekstrim
- pengaruh nilai hematokrit pd pemakaian sampel
darah utuh (whole blood)
- kontrol kualitas hasil pengukuran tidak
mudah dilakukan, masa inkubasi reaksi antigen
dg antibodi tidak bisa optimal

51. Automation in
Immunoassay

52. Resume :
• Aplikasi konsep imunologi menjadi keseharian kegiatan
kedokteran transfusi
• Penjelasan tentang fenomena klinis terkait reaksi
antigen-antibodi elemen seluler darah sampai
penerapan dalam teknik-teknik diagnostik yang
berdasar terbentuknya kompleks imun
• Pemanfaatan dlm Bank Darah Rumah Sakit (BD-RS) utk
pelayanan transfusi yg lebih akurat (Card Gel test for
Blood grouping and Antiglobulin testing) & deteksi
cepat dlm praktek klinik penyakit infeksi (Hepatitis,
HIV, Malaria) → peningkatan mutu pelayanan dan
keselamatan pasien