Metode Pemeriksaan

Sistem Imun
Agustin
Iskandar,dr,MKes,SpPK
FKUB

Indications for Laboratory testing

for immune competance
Clinical diagnosis, Tx monitoring or Prognosis of :
Congenital or acquired immunodeficiency
disease
Immune reconstitution following bone marrow
or other lymphoid tissue graft
Immunosupression induced by drug, radiation
or other
Autoimmune disorders
Immunization to monitor efficacy or immune
status
Clinical or basic research

Innate and Adaptive

Immunity

Clinical clues to the presence of

an immunodeficiency syndrome
Increased frequency of infections
Failure to clear infection rapidly
dispite adequate Tx
Dissemination of local infections to
distant sites
Occurrence of opportunistic infections
Development of certain types of
cancer (Kaposis sarcoma in AIDS
patients)

The evaluation the four

component of the immune system
B cell (humoral immunity)
T cell (cellular immunity)
Phagocyte and granulocyte
function
Complement component

VARIABLES & LIMITATIONS IN

IMMUNOLOGIC TESTING
Result of test of components immunity can
vary between different laboratory are
caused by :
Technical reasons
Biologic variation : genetic, gender, age,
environment
Intercurrent infections
Exacerbation of autoimmunity
Medications

Must be interpreted with other result

(clinical examination and history
taking)

Evaluation of Non-spesific Immunity

Roled by macrophage, monocyte and
PMN
Closely related with spesific immune
response.
Defect and evaluation:
Quantitative: the number of PMN,
macrophage
Qualitative: leukocyte function test

Evaluation of Specific
Immunity
Indication:
defficiency/ dysfunction of
lymphocyte
autoimmune disease
donor selection organ transplant
Evaluation:
Cellular Immunity
Humoral Immunity

Evaluation of Cellular
Immunity

Quantitative Enumeration of T cell

number and subtype (FC)
T cell deficiency : AIDS
CD4 T cells : used to monitor disease
progression in AIDS ang the response to
Tx
Qualitative Functional assay
DTH testing : T cell defect
T cell function proliferation induced
with nonspecific mitogenic (PHA, ConA)
Cytolytic function
Cytokine responses

Evaluation of Humoral
Immunity
Immunoglobulin level :
Selective IgA deficiency
Hypo IgG (< 100mg/dL) : XLA
Hyper IgM syndrome : congenital deficiency of
CD40L
Hyper IgE : allergy, hypersensitivity syndromes
Para protein (monoclonal Ig) : malignancy
(lymphoma or MM)
Level antibodies specific particular Ag--- ability
individual to make a humoral IR ---- Dx
Antibody function --- naturally occurring
isohemagglutinins (IgM) --- stil usefull in infant
Invitro proliferation and Ig production by B cell
in response to mitogenic stimulation

Evaluation of Phagocyte
Function
Indication
Chronic bacterial infectious
Repeated pneumonia
Abnormal blood count
Morphologic examination
MDS, granule deficiency, Chediac Higashi
Syndrome
Defects in cytokine production
Kostmanns syndrome
Functional test
Superoxide production --- CGD
Microbicidal function

Evaluation of Complement
deficiencies
Indication
Breakdown in host resistance to certain
bacteria and autoimmune diseases
Examination
CH 50
C3 level
C4 level

Laboratory Methods for Detection

of Antigens - Antibodies

Immunoassay
Molecular Genetic Techniques

IMMUNOASSAY
Sistem pemeriksaan yang
mempergunakan satu atau lebih
produk atau reagen imunologik
Prinsip dasar : ikatan antara molekul
imunoglobulin (Ab) dengan antigen
(Ag)
Hasil interaksi Ag Ab (kompleks
imun) harus terlihat dan dapat diukur

MACAM IMMUNOASSAY
Non
Labelli
ng

Labelling

1.
2.
3.
4.
5.

Imunopresipitasi
Aglutinasi
Fiksasi Komplemen
Radioimmunoassay ( RIA )
Enzyme immuno assay
( EIA )
6. Immunofluorecent assay
( IFA )
7. Immuochromatografi (ICT)

NON LABELLING
IMMUNOASSAY

Aglutinasi
Dasar : reaksi antara Ag ( tidak larut
atau partikel ) dgn Ab spesifik
cross linked antara kompleks imun
aglutinat
Uji kualitatif / semi kuantitatif
Fenomena imun sekunder
Dipengaruhi :

Klas Ab ( Ig G, Ig M )
Aviditas Ab
Jumlah binding site partikel
Prozone postzone effect

AGLUTINASI ANTIGEN-ANTIBODI

Ekses antibodi
Seimbang

KONSENTRASI

Ekses
antigen

PRIMARY IMMUNE PHENOMENA

+
Ag
Imun

Ab

Kompleks

SECONDARY IMMUNE PHENOMENA

a. Aglutinasi langsung
- Deteksi Ag / Ab

Ag berupa partikel / sel + Ab -------> Aglutinasi

b. Aglutinasi tak langsung / Pasif

Deteksi Ab
- Ag yg larut diikatkan lebih dahulu dengan
karier yaitu
?
eritrosit, lateks,
bentonit, dll
-

K
K
Ag

K
K
K

c. Aglutinasi pasif terbalik

Deteksi Ag larut
karier

Ab spesifik dilekatkan pd

( Eritrosit )

Ag
Ag
ERI

ERI

Ag

Ag

Reverse Passive Hemaglutination - RPHA

Kepraktisan Tes Aglutinasi

Sederhana
Cepat
Tidak membutuhkan ketrampilan khusus
Tidak memerlukan peralatan canggih
Murah

Kelemahannya
Sensitifitas dan spesifisitasnya rendah

LABELLING IMMUNOASSAY

Prinsip Pemeriksaan
- Deteksi

Ag/Ab dengan konsentrasi rendah

- Menggunakan Ab / protein sebagai bahan pengikat

- Memerlukan label:
- Radioisotop : I 125, C 14, H 3
- Enzyme : ALP, HRP
- Fluoresen : Fluorescein, Rhodamine
-Pengukuran Ag/Ab melalui pengukuran radioaktif,
produk enzim, intensitas fluoresen

Enzyme linked immunosorbent assay

(ELISA)

Deteksi Ag/Ab cukup sensitif spesifik

Half life reagen panjang
Tidak bahaya
Memerlukan peralatan spektrofotometer
dapat diautomatisasi
Metoda : - Sandwich
- Kompetitif

Sandwich - ELISA
E

Ab - E

Optical density

substrat

Perubahan
warna

[ ] Ag
E = enzym

Kompetitif - ELISA
?
+

-E
-E
-E

-E
-E
-E

Substrat

Ag - E

Optical density

Perubahan warna

Diukur dengan
spektrofotometer
[ ] Ag

S : substrat TMB, NADPH

Indirect - ELISA

Y Y

Y Y

Radioaktifitas

Y Y

Diukur
[ ] Ab

Ab II E

Immunoflouresence Assay /
IFA
Merupakan metoda untuk deteksi Ag/Ab
pada jaringan/sel atau dalam cairan tubuh
Menggunakan label :
fluorescein warna hijau
Rhodamin warna merah
Memerlukan mikroskop
Fluoresen/fluorometer
Ada dua metode : - metode direk
- metode indirek

IFA UNTUK DETEKSI Ag/Ab

JARINGAN

Ab II - F

Y
Deteksi Ag

Indirek IF

Direk IF

Deteksi Ag/Ab

Sandwich - IFA

Intensitas fluoresen

Ab - F

[ ] Ag

F = Fluoresent

Indirect - IFA

Y Y

Y Y

Intensitas fluoresen

Y Y

[ ] Ab

Ab II F

Microparticle enzyme
immunoassay MEIA

Ab IIE- ALP

S : MUP

+
MU

Substrat fluorogenic
MUP : Methylumbelliferyl phosphate
MU : Methylumbelliferon

Diukur dengan
fluorometer

IMMUNOCHROMATOGARFI / ICT
Deteksi Ag/Ab
Macam :
Langsung
Kompetitif

Keuntungan :

Format lebih disukai

Cepat
Stabil
Relatif tidak mahal
Sensitifitas dan spesifisitas baik

PRINSIP ICT Langsung

Deteksi Antigen
Ag
?

Capture : Ab

Garis tes

Tes Positif

Garis Kontrol

Ab
conjugated
colloidal
gold

Garis tes

Garis Kontrol

Tes Negatif

PRINSIP ICT Langsung

Deteksi Antibodi
Ab
?

Capture : Ag

Garis tes

Tes Positif

Garis Kontrol

Ab
conjugated
colloidal
gold

Garis tes

Ab
conjugated

Garis Kontrol

Tes Negatif

Molecular Genetic
Techniques

Dasar: spesifisitas pasangan basa atau

DNA komplementer serta enzim-enzim
bakteri yang dapat mengiris molekul
DNA pada bagian tertentu.
Tehnik DNA cloning probe
hibridisasi dilabel dibaca secara
kolorimetrik atau fluoresensi.
Metode: Southern blotting
Northern blotting
PCR

Southern Blotting
Tujuan: menentukan karakteristik
susunan DNA pada gen tertentu.
DNA di-isolasi/ diekstraksi Di-irisiris/ didigesti menggunakan enzim
fragmen DNA yang dituju/ restriction
sites dilabel
Indikasi : umumnya digunakan untuk
menentukan jenis limfosit T atau B
pada kasus limfoma atau leukemia
yang meragukan.

Northern Blotting
Tujuan: menganalisis molekul mRNA.
Tehnik: elektroforesis asam nukleat,
blotting dan hibridisasi dengan
probe DNA.
Single strain mRNA dihibridisasi
dengan probe RNA berlabel
dibubuhkan RNAse untuk memotong
untaian mRNA tsb dsRNA yang
berlabel dipisahkan dengan PAGE
dianalisis dengan autoradiografi.

Northern Blot
Cells
Rupture in
detergents

Isolate RNA

Location of Gene Specific Gene

Transcript (Identify by Size)

Size
Markers

Hybridize to
single
Stranded 32 p
labeled Probe

Blot to

Large

Electrophoretically
separate on the basis of
size on a denaturing
agarose gel

nitrocellulose

Filter

Small
Gel

RNA

Filter
Northern Blot

Autoradiogram

Western blotting
A method to identify specific protein
in biologic sample
Protein are denatured with an ionic
detergent ( SDS)
Followed by EP in polyacrylamide gels
(SDS-PAGE)
Transferred to nylon or nitrocellulose
filter paper then incubated with
antisera to reveal the reactive protein

Polymerase Chain Reaction

Adalah cara cepat dan sederhana
untuk membuat multiple copies
sekuen DNA spesifik yang di-inginkan
dengan meniru replikasi DNA in vivo.
Beberapa tahapan: isolasi
denaturasi annealing
elongation multiple copies

DNA Isolation

Salting Out

E L Buffer
darah EDTA

Inkubasi 40C 1 jam, putar cuci dgn

PBS

Proteinase K + SDS
10%
pelet

NaCl 6 M

Ethanol abs
supernatan

koco
k

Inkubasi
560C

putar

Benang
DNA

supernatan
endapan
protein

Polymerase Chain Reaction

A technique to amplify a small DNA segment beginning with
as little as 1 microgram
PCR condition
Pair of primers
Taq polymerase
Mg Cl2

dNTPs
aquadest

PCR condition
95 970C --- 1-2 --denaturation
50 550C --- 1-2 --- annealing
72 750C --- 1-2 --- clongatin

20 cycles + 220.000 copies

Polymerase Chain Reaction Sequencing Specific Primers

Sample of cell or tissue

Extract DNA

Combine DNA with

sequence-specific primer
mix for each allele

DR1
primers

DR15
primers

DR4
primers

Thermal
cycler

Amplify by PCR
Gel electrophoresis of each
sample

DR1

DR15 DR16

DR3

1. Stain ethidium bromide

2. Photograph under UV

Amplified
HLA allele(s)

DR3

DR4

DR11

DR12

Internal
amplification
control

Terima Kasih