Pemeriksaan

Laboratorium
Imunologi
Immunological laboratory diagnostic methods
can be classified from several aspects:

I. Based on a group of diseases that facilitate diagnosis

• Immunological profile tests for the detection of
immunodefi ciency
• Hypersensitivity tests
• Autoimmunity tests
II. Based on availability of the methods
– Methods performed in a surgery
– Methods included in haematological or
– biochemical analysis, and histological
– or visualisation methods that provide valuable
information for immunological diagnosis
– A group of basic methods conducted in a
specialised immunological laboratory
– Advanced immunological methods above all,
used in clinical research
 Basic laboratory examinations

• Leukocyte count and differential leukocyte count, i.e.

standard haematological parameters should be included in
basic laboratory examinations,
• Cytology and histology of various samples obtained from
biopsies are also of great value for immunological diagnosis.
• Preliminary methods selected for humoural immunity
testing involve the assessment of total immunoglobulins
using a simple precipitation method or serum
electrophoresis.
• Inflammatory parameters: the C-reactive protein test
A non-specific immunological profile testing
 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

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• Dasar
Reaksi Ag dengan Ab spesifik
• Tujuan
Mendeteksi keberadaan Ag dalam serum
memakai Ab spesifik
Mendeteksi keberadaan Ab dalam serum
memakai Ag yang sesuai
• Manfaat
1. Menentukan status imunitas
2. Memperkirakan prevalensi
penyakit
3. Mengetahui adanya invasi
mikroorganisme, jika isolasi
kuman tidak dapat dilakukan
4. Menunjang diagnosis penyakit
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Macam :
1. Uji kualitatif hasil + / -
2. Uji kuantitatif hasil kadar

Pengenceran tertinggi hasil +

(uji semikuantitatif)
Contoh :
 pengenceran 1 dalam 8: 1 vol serum +7
vol pengencer: titer 1/ 8 sampel
diencerkan 8 X
 Bahan pemeriksaan

Serum, plasma, urin

Plasma hanya untuk pemeriksaan tertentu saja.
Puasa: untuk metode aglutinasi.
Serum harus dihindarkan dari hemolisis, lipemik &
kontaminasi bakteri (pengiriman < 2 jam)
Disimpan dalam
Suhu 2 – 8oC : 48 jam
-20oC s/d - 70oC: > 48 jam
Diberi label
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 Teknik Pemeriksaan Imunoserologi

1. Imunopresipitasi
Non
Labellin 2. Aglutinasi, flokulasi
g 3. Fiksasi komplemen
4. Radioimmunoassay (RIA)
5. Enzyme immunoassay (EIA) atau
Enzyme linked immunosorbent assay
Labelling (ELISA)
6. Immunofluorescent (IF)
7. Immunochromatographic technique
(ICT)
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 Interaksi Antigen-Antibodi

Interaksi primer:
• Pengikatan Ag-Ab tingkat molekuler
• Memerlukan indikator/label (isotop, enzim, floresen)
• Sesuai utk pengukuran Ag/Ab dgn kadar yg rendah

Interaksi sekunder:
• Reaksi Ag-Ab bisa secara langsung atau dgn bantuan
komplemen
• Prinsip dasar : reaksi presipitasi/ aglutinasi
• Bila partikel Ag terikat latex atau eritrosit → aglutinasi
Primary immune phenomena

Ag Ab Kompleks Imun

Secondary immune phenomena

IMUNOASSAI
NON LABEL
 1. Imunopresipitasi

• Interaksi sekunder → Ag-Ab komplek tdk larut

(presipitat)
• Media : cair atau semisolid (gel)
• Faktor yg mempengaruhi :
 Aviditas Ab → stabilitas komplek Ag-Ab
 Suhu (optimal 0-37o C)
 pH (netral = 6-7,5), pH < 6 ; >7,5 → mudah
disosiasi
 Molaritas (molaritas < 0,15 M) ; >0,15 M →
men-cegah presipitasi
 Imunopresipitasi…

Pembentukkan presipitat terjadi apabila

konsentrasi Ag dan Ab seimbang (zona ekivalen =
ZE)
Konsentrasi Ag berlebih → Komplek Ag-Ab yg
terbentuk larut kembali disebut postzone effect
Konsentrasi Ab berlebih → Komplek Ag-Ab yg
terbentuk tetap larut disebut prozone effect
ZE sempit → Ag bersifat mudah larut
ZE lebar → Ag bersifat tdk mudah larut, BM
besar, & multikomponen Ag
 Ekses antibodi Seimbang Ekses antigen
PRESIPITASI ANTIGEN-ANTIBODI

Prozone KONSENTRASI ANTIGEN Postzone

 2. Aglutinasi

• Umumnya : Ag bentuk partikel + Ab spesifik →

Aglutinasi
• Reaksi 2 tahap :
1. Ab dgn salah satu antigen binding site (Fab) bereaksi dgn
Ag
2. Fab yg lainnya berikatan dgn Ag lain yg sudah berikatan
dgn Ab gumpalan (lattice)
• Aglutinasi lebih mudah terjadi pada IgM o/k
pentamer dibanding IgA dan IgG
 Ag Ab K.I

Tahap I
+

Tahap II Aglutinasi
Contoh-contoh pemeriksaan aglutinasi
1. Aglutinasi direk

2. Aglutinasi indirek (pasif)

3. Aglutinasi pasif terbalik

4. Hambatan aglutinasi
 3. Fiksasi komplemen

 Tahap :
1. Pengikatan sejumlah komplemen oleh kompleks Ag –
Ab
2. Penghancuran Eritrosit yg telah dilapisi hemolisin oleh
komplemen
Interpretasi :
 + tidak hemolisis
 _ hemolisis
Contoh : Deteksi tripanosoma, virus
 Tes Fiksasi Komplemen
Fiksasi komplemen Sistem hemolitik

Eritrosit
Ag

+
c ++
+ Hemolisin tidak hemolisis
? Ab

Eritrosit

Ag
++
+ +
c Hemolisin

C - Komplemen
hemolisis
 Imunoasai berlabel

 Imunoassai yang menggunakan indikator utk melacak Ag

atau Ab dengan konsentrasi rendah
 Mampu melacak interaksi primer Ag-Ab (initial binding)
 Sensitifitas analitik lebih tinggi dibanding imunoassai non
label
 Label yg digunakan :
 isotop : I125, H3, C14
 non isotop : enzim (ALP, HRP), floresen
(fluorescein, rhodamine), kemiluminesen
 Selain uji kuantitatif, dpt digunakan pada uji kualitatif (ANA
tes, antitiroid Ab)
 Imunoassai Berlabel

Imunoassai berlabel homogen

Sinyal kadar analit diperoleh langsung dari reaksi ikatan
label dgn analit
Tidak memerlukan separasi Ag terikat dan Ag bebas
(B/F)

Imunoassai berlabel heterogen

Sinyal kadar analit diperoleh secara tdk langsung
Memerlukan seperasi B/F
Lebih sensitif dibandingkan imunoassai homogen
Imunoasai kompetitif
● Ab label dan analit direaksikan sekaligus terhadap Ag

Imunoasai non kompetitif

● Ag analit yg diukur terikat antara Ab phase solid &
label Ab
● Lebih sensitif dibandingkan metode kompetitif
Imunoasai Berlabel

1. Radioimunoassai (RIA/IRMA)

2. Imunofloresen (IF)

3. Enzyme Imunoassay (EIA/ELISA)

4. Imunokromatografi (ICT)
 1. Radioimunoasai

● Immunoassay berlabel radioisotop  membedakan Ag

yang terikat Ab dengan Ag bebas.
● Sensitif
& spesifik untuk ttk kadar bahan yang amat
rendah dalam serum
● Yang diukur : - γ-ray → I125
- β particle → H3

Radiolabel pada imunoassai dibagi 2 kelompok:

a.Radioimmunoassay (RIA): radiolabelisasi pada Ag
b.Immunoradiometric Assay (IRMA): radiolabelisasi pada
Ab
●Kerugian:
 Bahaya efek radiasi bahan radioaktif
 Waktu paruh reagen singkat, γ dan β counter
mahal

●Keuntungan:
 Presisi baik and high sensitivity
 Isotope conjugation lebih mudah
 Signal detection tanpa optimalisasi
 Lebih stabil terhadap interferensi environment
(pH, suhu)
 Prinsip dasar RIA

E
+
E
E + E
E
E
E
E

cuci
Ab pd fase Ag
padat berlabel Ag

Radiaton
counter
 2. Imunofloresen assay (IFA)

 Merupakan teknik untuk deteksi Ag/Ab pada

cairan tubuh atau jaringan/sel
 Prinsip :
Molekul yg mampu menyerap energi radiasi
dan memancarkannya kembali dlm btk
cahaya (floresensi)
 Memerlukan alat fluorometer/mikroskop
 Menggunakan label:
• Fluorescein → warna hijau
• Rhodamin → warna merah
Imunofloresen assay
 Enzyme immunoassay

• Immunoassay dengan menggunakan label

enzim
• Relatif murah, banyak tersedia, reagen
bertahan lama, mudah diotomatisasi,
peralatan yang relatif murah
• Enzim yang digunakan dipilih berdasakan
jumlah molekul substrat yang dapat
dirubah per satu molekul enzim, mudah
dan cepat mendeteksi serta stabil.
• Dibaca dengan alat :
• Spektrofotometer ( λ = 492 m) →
microELISA reader
 Kerugian:
 Reaksi enzim lebih kompleks dari pada label
isotop
 Masih dipengaruhi faktor environment (plasma
constituents)

 Keuntungan:
 Mudah dikerjakan
 Relatif murah, simultan dgn pemeriksaan yg
lain
 Bahaya radioaktif (-)
One-step sandwich EIA
 Imunokromatografi

Imunokromatografi
 Lateral flow test
 Membacanya cukup dgn mata saja
 Tidak membutuhkan substrat
 Penggunaan colloidal gold waktu inkubasi pendek (<15 menit)

Kerugian :
 Nitrocelulose membrane tdk stabil pada suhu ↑

Keuntungan :
 Prosedur cepat (<15 menit) dan praktis
 Nilai diagnostik baik
 Stabil untuk jangka panjang
Prinsip dasar ICT

A. Melacak Analit (Ag)

a. Reaksi langsung (Double Antibody Sandwich)/Asai
Imunometrik  untuk melacak analit yang besar dan
memiliki > 1 epitop (LH,hCG dan HIV)
b. Reaksi kompetitif/Hambatan kompetitif (competitive
Inhibition)  untuk melacak molekul kecil dengan
epitop tunggal

B. Melacak Ab  Indirect Assay

The Tuberculin test
• The Tuberculin test has been the traditional method for detection of
infection with tubercle bacilli.
• In clinical practice, it is used to find out the presence or absence of
tuberculous infection. This aids in the differential diagnosis of TB
among children and to decide about administration of
chemoprophylaxis.
• The TST is performed by injecting 0.1 ml of tuberculin purified protein
derivative (PPD) into the inner surface of the forearm. The injection
should be made with a tuberculin syringe, with the needle bevel
facing upward. The TST is an intradermal injection. When placed
correctly, the injection should produce a pale elevation of the skin (a
wheal) 6 to 10 mm in diameter.
• The skin test reaction should be read between 48 and
72 hours after administration. A patient who does not
return within 72 hours will need to be rescheduled for
another skin test.
• The reaction should be measured in millimeters of the
induration (palpable, raised, hardened area or
swelling). The reader should not measure erythema
(redness). The diameter of the indurated area should
be measured across the forearm (perpendicular to the
long axis).
An induration of 5 or more millimeters is considered
positive in:
• HIV-infected persons
• A recent contact of a person with TB disease
• Persons with fibrotic changes on chest radiograph
consistent with prior TB
• Patients with organ transplants
• Persons who are immunosuppressed for other reasons
(e.g., taking the equivalent of >15 mg/day of prednisone
for 1 month or longer, taking TNF-a antagonists)
An induration of ≥ 10 mm is considered positive in:
• Recent immigrants (< 5 years) from high-prevalence countries
• Injection drug users
• Residents and employees of high-risk congregate settings
• Mycobacteriology laboratory personnel
• Persons with clinical conditions that place them at high risk
• Children < 4 years of age
• Infants, children, and adolescents exposed to adults in high-risk
categories
• An induration of 15 or more millimeters is considered positive in any
person, including persons with no known risk factors for TB. However,
targeted skin testing programs should only be conducted among high-
risk groups.
 Skin testing for allergies

• Skin testing for allergies is used to identify the substances that are
causing your allergy symptoms.
• It is often performed by applying an extract of an allergen to your
skin, scratching or pricking the skin to allow exposure, and then
evaluating the skin's reaction.
• It may also be done by injecting the allergen under the skin, or by
applying it to a patch that is worn on the skin for a specified period of
time.
 The three main types of skin tests are

• Scratch test (also known as a puncture or prick

test). Areas on your skin are then marked with
a pen to identify each allergen that will be
tested. A drop of extract for each potential
allergen is placed on the corresponding mark.
A small disposable pricking device is then used
so the extract can enter into the epidermis.
• Intradermal test
• Patch test. Another method is to apply an
allergen to a patch which is then placed on the
skin.
Autoantibodies assay

• Detection of autoantibodies is an important

diagnostic tool for diagnosis of autoimmune diseases.
Despite the fact that their occurrence is not quite
specific for a respective disease, it may considerably
facilitate the diagnosis.
Serologic Tests Useful in the
Diagnosis of Systemic Lupus
Erythematosus
1. Fluorescent antinuclear antibody (ANA)
2. ANA panel: anti-ds DNA*, anti-Sm, anti-U1RNP,
anti-Ro/SSA, anti-La/SSB
3. Serum complement level
4. VDRL**
5. Anticardiolipin antibodies
6. Lupus anticoagulant
7. Coombs test
*dsDNA, double-stranded DNA
**VDRL, Venereal Disease Research Laboratories
Anti Nuclear Antibodies

• Antibodies directed against cell nuclei

• Screening for Rheumatic Autoimmune
Diseases, especially for SLE (a diagnostic
criteria)
• Very sensitive but less specific
• 2 detection methods : IF and EIA
• Normal value: < 1/100 (IF), < 20 units (EIA)
• Positive result should be evaluated for
more specific auto ab (anti-dsDNA, anti-
ENA, etc)
Anti-dsDNA antibodies

• Autoantibodies which target the ribose

phosphate backbone of native DNA
• Appear in approximately 75% of SLE
patients
• SLE diagnostic test and disease activity
monitoring (specificity 94%)
• Play an important role in the pathogenesis
of SLE and strongly correlated with lupus
nephritis
• Normal Value : < 25 IU/ml
Anti Phospholipid Antibodies

• Antibodies directed against anionic

phospholipids membrane cell
• 2 laboratory test : ACA and LA
• Diagnosis of SLE and APS
• Strongly associated with
thrombocytopenia, thrombosis and
reccurrent abortion
• Normal value : ACA IgM / IgG < 12
U/ml
 Complement

• Plasma and cells surface protein

• 15 % globulin fraction, proenzyme or
zymogen
• Measurement Methods
• Total Hemolytic Complement Assay
• Immunoassay
• Normal Value :
C3 : 85 – 185 mg/L ; C4 : 10 - 50 mg/L
• Indication: Monitoring the disease activity
Rheumatoid Factor

• Autoantibodies reactive with epitopes

in the Fc portion of IgG

• Diagnosis and prognosis of RA

• Sensitivity is 60- 90% and specificity

50-60%

• Correlation with disease activity and

disease progression ???

• Normal Value : < 1/8 (<8 IU/ml)

Rheumatoid
Factor
 Coombs test

• Agglutination of red blood cells is used in

the Coombs test.
• Coombs test (also known as Coombs' test,
antiglobulin test or AGT) refers to two
clinical blood tests used in
immunohematology and immunology.
• The two Coombs tests are:
Direct Coombs test (also known as direct
antiglobulin test or DAT).
Indirect Coombs test (also known as indirect
antiglobulin test or IAT).
 The direct Coombs test

• Used to detect red blood cells sensitized

with IgG alloantibody, IgG autoantibody,
and complement proteins.
• It detects antibodies bound to the surface
of red blood cells in vivo. The red blood
cells (RBCs) are washed (removing the
patient's own plasma) and then
incubated with antihuman globulin (also
known as "Coombs reagent").
• If this produces agglutination of the RBCs,
the direct Coombs test is positive. Ex:
 The indirect Coombs test

• Used in prenatal testing of pregnant women, and in

testing blood prior to a blood transfusion.
• It detects antibodies against RBCs that are present
unbound in the patient's serum. In this case, serum
is extracted from the blood, and the serum is
incubated with RBCs of known antigenicity.
• If agglutination occurs, the indirect Coombs test is
positive
The immune complex test
• The immune complex test is a test designed to evaluate the
status or proper functioning of the immune system.
• An immune complex is an association formed between
large numbers of antigens and the corresponding
antibodies.
• Normally, immune complexes are removed from the
bloodstream by macrophage of the spleen and by other
specialized cells located in the liver. if this clearance does
not occur, the immune complexes will continue to circulate,
and will become trapped in the kidneys, lung, skin, joints,
or blood vessels.
• Immune complexes can be detected by the application of special
stains to tissue that has been obtained from a patient. The stains
contain antibodies that bind to the complexes and this binding is
highlighted by the presence of the staining agent. This test is useful
because it directly detects the presence of the immune complexes.
However, for routine clinical use, this method is cumbersome and
invasive. This has stimulated the development of blood tests that
indirectly detect the complexes in the blood serum.
• There are several methods available. Immune complex tests include
the Raji cell, C1q binding, conglutinin, and anti-C3 assays. The Raji cell
assay, for example, detects the immune complexes following the
binding of the complexes with complement.
• C1q binding assy dan Raji cell immune complex assay: Quantitative
Flow Cytometry/Semi-Quantitative Enzyme-Linked Immunosorbent
Assay
• A normal result in an immune complex test is a negative result.
Thank You