Maimun Z Arthamin

Lab Patologi Klinik FKUB/RSSA

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
6
• 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
8
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

10
 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
Labelling assay (ELISA)
6. Immunofluorescent (IF)
7. Immunochromatographic
technique (ICT)
11
 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
– Fluorometer/Luminometer
 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
 Relatif tidak mahal
 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.
 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.