Download Coombs test - IMMUNOLOGY

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
2. Uji kuantitatif
hasil + / 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
Non
Labelling
Labelling
1.
2.
3.
4.
5.
Imunopresipitasi
Aglutinasi, flokulasi
Fiksasi komplemen
Radioimmunoassay (RIA)
Enzyme immunoassay (EIA) atau
Enzyme linked immunosorbent 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
Secondary immune phenomena
Kompleks Imun
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
PRESIPITASI ANTIGEN-ANTIBODI
Ekses antibodi
Prozone
Seimbang
KONSENTRASI ANTIGEN
Ekses 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
Tahap I
Tahap II
Ab
K.I
+
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
Ab
tidak hemolisis
Eritrosit
Ag
+
c
C - Komplemen
+
+
+
Hemolisin
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
Ab pd fase
padat
Ag
berlabel
cuci
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, antiRo/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: warm type
AIHA, HDN
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/SemiQuantitative Enzyme-Linked Immunosorbent Assay
• A normal result in an immune complex test is a
negative result.