imunofenotipizacija in diagnostika imunskih pomankljivosti · cs521 cd nomencl ature cd (cluster...
TRANSCRIPT
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Imunofenotipizacija in
diagnostika imunskih
pomankljivosti
A. Ihan
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Imunski sistem
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Ekspresija receptorjev, adhezijskih in drugih
molekul je značilna za posamezen tip celice
cs895f
Mikroskopsko imajo limfociti enak izgled...
...vendar vezava s določenimi, dobro opredeljenimi
monoklonskimi protitelesi pokaže razlike v ekspresiji molekul
med posameznimi vrstami limfocitov. Vezavo
fluorescenčno označenih monoklonskih protiteles na celice
ugotavljamo z metodo fluorescenčne mikroskopije ali z
metodo pretočne citometrije.
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Imunofluorescenca
Specifična vezava
Nespecifična vezava
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Direktno označevanje
Fluorescenčno barvilo vezano na protitelo
Specifični signal razmeroma majhen
Nespecifični signal zelo majhen
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Avidin-Biotin
biotinilirano
primarno
protitelo
biotin
avidin
Biotinilirano
barvilo
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Do sedaj je opredeljenih 350 različnih površinskih molekul
na celicah, ki jih lahko določimo z monoklonskimi
protitelesi, uvrščenimi v sistem"Clusters of Differentiation" (CD).
gStainin
Mrtva c.
CD3
T
CD14
Mo
aktivirani
T
CD69
CD3
Ekspresija receptorjev, adhezijskih in drugih
molekul je značilna za posamezen tip celice, ker vsak tip
celice za svoje delovanje potrebuje svoji funkciji ustrezne
signale, metabolne poti, encime, efektorske mehanizme
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cs22
origin of
blood
cells
Kako z imunofenotipizacijo opredelimo funkcioniranje
imunskega sistema ?Kostni mozek
pluripotentna matična celica
Kostni mozek
Mieloidna
progenitorna celica
Kri
limfoidna
Progenitorna celica
granulocitno/makrofagna
progenitorna celica
megakariocitna/
eritrocitna
Progenitorna celica
Efektorji Bezgavke
megakariocit eritroblast
Zela dendritična celica
Beritrocit
plazmatka
nevtrofilci eozinofilci bazofilci monocitiNezrela
dendritična celica
Granulociti
trombocitiT
Aktivirani
limfocit Tl
Tkiva
mastocit makrofag
Nezrela dendritična celica
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cs521
CD
nomencl
ature
CD (Cluster Designations) Nomenklatura
HLDA Workshops (Human Leukocyte Differentiation Antigens)
)
CD350
CD1
Alternative
Name
Ligand/
receptor/
substrate/
associated
molecule
Description and Function
CD195 CCR5 HIV-1
Regulates lymphocyte
chemotoxis, activation
and transendothelial
migration during in-
flammation.Neutralizes
HIV infection
T +
B -
DC (+)
NK -
Mono +
Granulo +
platelet -
erythr -
EC -
gene
locus
kDa
? 37.0
www.bdbiosciences.com/pharmingen/
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Dozorevanje B limfocitov
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Deficiti protiteles
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Zaščitni nivoji protiteles
Cepivo Zaščitni nivo IgG
Difterija 0.1 IU/mL11
Haemophilus influenzae B 0.15 g/mL29
Hepatitis A 10 mIU/mL30
Hepatitis B s –Ab 10 mIU/mL31
Ošpice (rubeola) 120 mIU/mL
Polio (inactiviran) nevtralizacijski titer 1:8
Steklina 0.5 IU/mL
Rubella 10 IU/mL
Tetanus 0.1 IU/mL
Rumena mrzlica 0.7 IU/mL
IU – mednarodna enota; mIU – mili-mednarodna enota
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Cepljenje • Protitelesni odziv
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Razvoj limfocitov B –do plazmatk
Naivni limfociti B imajo antigenski receptor IgM. Ko se med kroženjem po telesu na njihov IgM veže ustrezen antigen, izpostavijo naivni limfociti B kemokinski receptor CCR7, ki jih privede v področje limfocitov T sekundarnih limfatičnih organov. Tam se limfociti B srečajo z nedavno aktiviranimi (manj kot 24 ur) dendritičnimi celicami in celicami T pomagalkami. Ob nadaljnji navzočnosti antigena se ob pomoči limfocitov T hitro zgodi diferenciacija limfocitov B v plazmatke, ki tvorijo nizkoafinitetna protitelesa – proces imenujemo zunajfolikularni protitelesni odziv. Poglavitna protitelesa zunajfolikularnega protitelesnega odziva so IgM, spremlja jih tudi nekaj protiteles IgG1.
Dovolj velika aktivacija limfocitov Th pri zunajfolikularnem odzivu proizvede dovolj citokinov, da nekateri z antigenom aktivirani limfociti B razvijejo kemokinski receptor CXCD5 in migrirajo v folikle, kamor jih s kemokini privabijo folikularne dendritične celice (FDC) – folikularni odziv.
Limfociti B, ki se aktivirajo na FDC, postanejo germinalne celice in preidejo v masivno klonsko razmnoževanje. Hkrati z razmnoževanjem poteka tudi preklop med imunoglobulinskimi razredi in afinitetno zorenje limfocitov B. To omogoči razmnoževanje tistih klonov limfocitov B, ki imajo večjo afiniteto za antigen (pozitivna selekcija), medtem ko kloni z manjšo afiniteto za antigen propadejo.
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Normalno
MBO
MB1
CVID Warnatz et al 2002,
Piqueras et al 2003
MB2
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HSC
T
NKCLP
Mieloid
Pro B Pre B
Plazmacit
HIGM Preklopljeni BIgG
IgA
IgECD40L
CD40
AICDA UNG HIGM4
AICDAlC
CVID
ICOS
Immunoglobulin IgM
Mutiran IgM
IgG
IgA
XLA
BTK
0
0
0
0
0-low
0-low
0-low
0-low
Low
Low
Low
Low
N-hi
Low
0
0
N-hi
0
0
0
N-hi
biased
0-low
0
N-hi
N
0-low
0-low
zreli B
Iz: A Fischer Nature Immunology 2004
L5
V preB
m
Kostni mozek
IgM
IgD
Avtosomna recesivna agamaglob.
l5 Ig a/b m BLNK LRRC8
Izražanje molekul na celicah
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Pretočni citometer
RačunalnikDetektor &
Mehanika Pretočni sistem
Epruveta s celicami, ki so bile predhodno
“obarvane” z označenimi monoklonskimi
protitelesi. Celično suspenzijo posrka
pretočni sistem, nato celice potujejo skozi
snop laserske svetlobe. Hitrost analize je
nekaj 100 celic / sekundo.
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Celični sorterji (FACS – Fluorescence Activated Cell Sorter)
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Optika
PMT 1
PMT 2
PMT 5
PMT 4
Filtri
Laser
Pretočna
celica
PMT 3
Scatter
Senzor
Vzorec
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Optika za FSC (forward scatter)
detektor
reža
blok
Laser
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0 200 400 600 800 1000
0
20
0 4
00
60
0 8
00
10
00
Side Scatter (SSc)
Velikost /
granuliranost
Forward Scatter (FSc)
Nevtrofilci
Limfociti
Monociti
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Velikost celic
Velikost delcev
Šte
vil
o d
og
odk
ov
mali veliki
0.1 1 10 100 1000
0.920
90
200
700
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“B”
“T”
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3 Parameterska analiza
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4 barvna analiza
Emisija (nm)530 580 630 680 730 780
FITC PE PE-TR
PE-CY5
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10 1 10 2 10 3 10 4
CD56 -->
10
11
02
10
31
04
CD
4 -
->
10 1 10 2 10 3 10 4
CD3 -->
10
11
02
10
31
04
CD
4 -
->
CD3CD3 CD3
10 1 10 2 10 3 10 4
CD3 -->
10
11
02
10
31
04
CD
56 -
->
10 1 10 2 10 3 10 4
CD3 -->
10
11
02
10
31
04
CD
8 -
->
CD56
10 1 10 2 10 3 10 4
CD56 -->
10
11
02
10
31
04
CD
8 -
->
CD56 CD8
10 1 10 2 10 3 10 4
CD8 -->
10
11
02
10
31
04
CD
4 -
->
4 barvna analiza
CD
4
CD
8
CD
56 -
NK
CD
8
CD
4
CD
4
CD56 – NK
CD3 – T
CD4 – Th
CD8 – TcThis is a subset of cells
It is CD3+ CD56+This is a subset of cells
It is CD3+ CD4+
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Večbarvna analiza
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Sortiranje celic
Fizično ločevanje celic na osnovi razlik v
parametrih, izmerjenih s pretočnim
citometrom
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HidrodinamikaVzorec
Tekočinski ovoj
Laser
Piezoelektrični
cristalni oscilator
Fluorescenčni
Senzor
Scatter Senzor
jedroOvoj
Smer
svetlobe
Pretočna celica
++ --
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Celične funkcije
Viabilnost
Fagocitoza
Funkcije organel mitohondriji, ER
endosomi, Golgi
Oksidativne reakcije Superoksid
Vodikov peroksid
NO
Glutation
• Koncentracija ionov
–Znotrajcelični kalcij
– znotrajcelični pH
• Membranski el. potencial
• Membranska polarizacija
• Peroksidacija lipidov
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FluorescencaE
NE
RG
Y
S0
S1
S2
T2
T1
ABS FL I.C.
ABS - Absorbance S 0.1.2 - Singlet Electronic Energy Levels
FL - Fluorescence T 1,2 - Corresponding Triplet States
I.C.- Nonradiative Internal Conversion IsC - Intersystem Crossing PH - Phosphorescence
IsC
IsC
PH
[Vibrational sublevels]
Vibrational energy levels
Rotational energy levelsElectronic energy levels
Singlet States Triplet States
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Ethidium
PE
cis-Parinarična kisl.
Texas Red
PE-TR Conj.
PI
FITC
600 nm300 nm 500 nm 700 nm400 nm
457350 514 610 632488 Linije najpogostejših
laserjev
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Viri svetlobe - laserji
Argon Ar 353-361, 488, 514 nm
Krypton-Ar Kr-Ar 488, 568, 647 nm
Helium-Neon He-Ne 543 nm, 633 nm
He-Cadmium He-Cd 325 - 441 nm
Laser Okrajš. Vzbujevalna svetloba
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Sipana svetloba Molekule in zelo mali delci
svetlobe ne absorbirajo
ampak jo razpršijo, pri tem
valovna dolžina ostane enaka.
Nebo je modro, ker molekule plinov bolj pršijo svetlobo manjših
valovnih dolžin (modro) kot večjih valovnih dolžin (rdečo)
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Vrhovi ekscitacije in emisije
Fluorofor EX EM
% Max ekcitacije pri
488 568 647 nm
FITC 496 518 87 0 0
Bodipy 503 511 58 1 1
Tetra-M-Rho 554 576 10 61 0
L-Rhodamine 572 590 5 92 0
Texas Red 592 610 3 45 1
CY5 649 666 1 11 98
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Fluorescenčni mikroskop s 35 mm kamero
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Hoechst 33342 (AT rich) (uv) 346 460
DAPI (uv) 359 461
POPO-1 434 456
YOYO-1 491 509
Acridine Orange (RNA) 460 650
Acridine Orange (DNA) 502 536
Thiazole Orange (vis) 509 525
TOTO-1 514 533
Ethidium Bromide 526 604
PI (uv/vis) 536 620
7-Aminoactinomycin D (7AAD) 555 655
Barvila za nukleinske kisline
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DNA barvila AO
Metakromatsko barvilo
• Emisija odvisna od koncentracije
• Dvojna vijačnica - zeleno
• Enojna vijačnica - rdeče
AT/GC vezavna barvila
AT : DAPI, Hoechst, quinacrine
GC : antibiotics bleomycin, chromamycin A3, mithramycin, olivomycin, rhodamine 800
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Celični ciklus
G1
MG2
S G0
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G0G0- G1
s G2M
Količina DNA2N 4N
G2M G0
G1
s
0 200 400 600 800 10000
75
150
225
300
Šte
vilo
ce
lic
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DNA Histogram
G0-G1
S
G2-M
Fluorescenca
Šte
vil
o d
ogodkov
2n 4n
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Analiza DNA Histograma
DNA Content0 50 100 150 200
Num
ber
02
00
40
06
00
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Kromosomska analiza
Večina humanih kromosomov se loči s pretočno citometrijo
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DNA fragmentacija DNA barvilo – Acridine orange
Metakromatsko barvilo
• Dvojno vijačna DNA/ enovijačna (fragmentirana) DNA
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Annexin V - PI
Annexin V
PI
Annexin V
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Barvila za oksidacijsko stanje
DCFH-DA (H2O2) 488 525
HE (O2-) 488 590
DHR 123 (H2O2) 488 525
Barvilo Oksidant Ekscitacija Emisija
DCFH-DA - dichlorofluorescin diacetate
HE - hydroethidine
DHR-123 - dihydrorhodamine 123
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RKZ - spermiji
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Čas (sekunde)
0 240018001200600
Log D
CF
.1
1000
100
1
0 1
Skala
345
115
38
12
4
Nevtrofilni oksidativni izbruh
PMA-Stimulacija
Kontrola
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FITC-Označene bakterije
Fagocitoza
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Oksidativne reakcije
Superoksid: hidroetidin
Peroksid: DCFH-DA prehaja skozi
membrano celic, znotrajcelično postane
polaren (hidroliza z esterazo). H2O2 oksidira
DCFH v fluorescenten DCF
Glutation: 40 mM monobromobiman
reagira z glutathion-S-transferazo
Dušikov nitrit (NO): DAF
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DCFH-DA DCFH DCF
COOHH
Cl
O
O-C-CH3
O
CH3-C-O
Cl
O
COOHH
Cl
OHHO
Cl
O
COOHH
Cl
OHO
Cl
O
Fluorescenca
Hidroliza
Oksidacija
2’,7’-dichlorofluorescin
2’,7’-dichlorofluorescin diacetate
2’,7’-dichlorofluorescein
Cellular Esterases
H2O2
DCFH-DA
DCFH-DA
DCFH
DCF
H O2 2Lymphocytes
Monocytes
Neutrophils
log FITC fluorescenca.
1 1000100101
0
20
40
60
co
un
ts
PMA-stimulacija PMNkontrola
80
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Fagosom
O2
O2-
H2O2
NADPH + H+
NADP+
HMP
NADPH
Oksidaza
GSSG
GSH
GRGP
H2O2SOD
O2-
H+
H2O
katalaza
H2O + O2
PCB
SOD
PCB
Stimulator
PKC
PCB
(PMA)
Nevtrofilna celica
?
?
+O2
-
OH.
Lipidna Peroksidacija
Fosfolipaza A2Levkotrieni
H2O2
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Barvila za organele
BODIPY Golgi 505 511
NBD Golgi 488 525
DPH Lipidi 350 420
TMA-DPH Lipidi 350 420
Rhodamine 123 Mitohondr. 488 525
DiO Lipidi 488 500
diI-Cn-(5) Lipidi 550 565
diO-Cn-(3) Lipidi 488 500
Barvilo Mesto Ekscitacija Emisija
BODIPY - borate-dipyrromethene complexes
NBD - nitrobenzoxadiazole
DPH - diphenylhexatriene
TMA - trimethylammonium
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Funkcije organel
Mitohondriji Rhodamine 123
Endosomi Ceramidi
Golgi BODIPY-Ceramid
ER DiOC6(3)
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Methods: MMP (mitochondrial
membrane potential)
DiOC6(3)
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Parametri spermatocitov
Zdravih spermijev (več kot 60 %)*
Nekrotičnih spermijev (manj kot 40 %)*
Apoptotičnih spermijev (manj kot 25
%)*
Spermijev s fragment. DNA (manj kot 10 %)*
Indeks (N + Ap) / Z (manj kot 1 )*
Celični drobir (delež) (manj kot 30%)
Povp. PMA indeks RKZ/ celico: (manj kot 1,2
)*
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Apoptosis
Initiator phase
(mitochondrial changes)
Effector phase
(membrane and
cytoplasmic changes)
Cell degradation phase
(DNA fragmentation)
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Barvila za proteine
FITC 488 525
PE 488 575
APC 630 650
PerCP™ 488 680
Cascade Blue 360 450
Coumerin-phalloidin 350 450
Texas Red™ 610 630
Tetramethylrhodamine-amines 550 575
CY3 (indotrimethinecyanines) 540 575
CY5 (indopentamethinecyanines) 640 670
Barvilo Ekcitacija Emisija
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Barvila za ione
INDO-1 Ex350 Em405/480
QUIN-2 Ex350 Em490
Fluo-3 Ex488 Em525
Fura -2 Ex330/360 Em510
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pH indikatorska barvila
SNARF-1 488 575
BCECF 488 525/620
440/488 525[2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein]
Barvilo Ekcitacija Emisija
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Področja uporabe pretočne citometrije
•Imunologija
•Hematologija
•Patologija
•Mikrobiologija
•Genetika
•Odkrivanje zdravil
•Testiranje toksičnosti
• Gojenje celic
•Funkcijske študije celic
Klinika in raziskovanje
•Biotehnologija
•Agronomija
•Veterina
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Specifični T limfociti – označeni polimeri MHC
za spremljanje imunskega odziva
Polimeri MHC I za detekcijo specifičnih citotoksičnih limfocitov T (CD8)
Polimeri MHC II za detekcijo specifičnih limfocitov T –celic pomagalk T (CD4)
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Pentameri MHC I vezani s peptidi HCV –
spremljanje anti-HCV odziva
Peptidi HCV:
CINGVCWTV
DLMGYIPAV
KLVALGINAV
RVCEKMALY
AYSQQTRGL
Ostali virusi: CMV, EBV, HBV, InfluA, HIV, HPV, HSV, ...
Onkoantigeni
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Bazofilni aktivacijski test (BAT)
In vitro (kri)
specifična aktivacija
bazofilcev z
alergenom in
detekcija ekspresije
CD63 na bazofilcih
(CD123)