tipizare hla-a
TRANSCRIPT
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TESTELE DE
HISTOCOMPATIBILITATE
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TRANSPLANTUL = Actul de a transfera celule, tesuturi sau
organe dintr-o parte in alta, fie de la acelasi individ, fie intre indivizi diferiti
Tipuri: Autogrefa= transplantul de tesuturi proprii dintr-o
parte in alta a aceluiasi individ Alogrefa = transplantul de tesuturi de la un donor
genetic diferit de acceptor, dar care apartine aceleiasi specii
Xenogrefa = transplantul de tesuturi de la un donor care apartine unei alte specii
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ISTORIC Secolul II:
India, autogrefa, piele, rinoplastie Secolul III (??):
Inlocuirea membrului inferior gangrenos al imparatului Iustinian cu cel al unui cadavru
1959: Boston, rinichi, gemeni bizigoti
1967: Denver, ficat
1968: Cape Town, inima; Minneapolis,
maduva hematopoetica
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CONDITII TRANSPLANT Compatibilitate AB0 Identitate HLA cat mai mare Absenta in serul pacientului a unor Ac anti-
donor Stimulare slaba la reactia mixta limfocitara
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COMPATIBILITATE AB0 Incompatibilitate minora:
Donorul are Ac (aglutinine) Exemplu: donor 0 si primitor A/B/AB
Incompatibilitate majora: Donorul are Ag Exemplu: donor A/B/AB si primitor 0
Incompatibilitate minora si majora Exemplu: donor A/B si primitor B/A
Nu este esentiala Obiectiv: prevenirea hemolizei post-
transplant
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COMPLEXUL MAJOR DE HISTOCOMPATIBILITATE
= MHC La om – HLA (human leucocyte antigen) Grup inrudit de gene pe cromozomul 6 Roluri:
Imunitate celulara Imunitate umorala Mecanisme de recunoastere intercelulara Discriminare self / nonself
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ORGANIZAREA HLA 3 clase HLA:
Clasa I: Glicoproteine membranare ~ Celule nucleate Prezentarea Ag peptidice endogene LT citotoxice
Clasa II: Glicoproteine membranare CPA Prezentarea Ag exogene LT helper
Clasa III: Proteine cu functie in imunitate
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GENELE HLA
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POLIMORFISM GENIC Clasa I:
HLA A – 451 alele HLA B – 782 alele HLA C – 238 alele
Clasa II: HLA DR – 525 alele HLA DQ – 105 alele HLA DP – 147 alele (HLA DM – 11 alele) (HLA DO – 21 alele)
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CONDITII PENTRU COMPATIBILITATE
Se testeaza cel putin 3 locusuri: HLA-A HLA-B HLA-DRB1
6 Ag ≥4 identice
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CONDITII PENTRU COMPATIBILITATE
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TESTE DE HISTOCOMPATIBILITATE
Moleculare
Serologice
Celulare
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METODE MOLECULARE Leucocite din sange Extractia ADN-ului Amplificarea genelor HLA:
PCR-SSP PCR-SSOP SBT
Interpretarea rezultatelor
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PCR-SSP PCR-SSP (Sequence Specific Primers) Pentru HLA clasa I si II Amplificarea genelor HLA utilizand primeri:
Rezolutie mica – se amplifica grupuri de alele Rezolutie inalta – se amplifica alele specifice
Electroforeza pe gel de agaroza a produsilor de amplificare: Deplasare in functie de dimensiuni Comparare cu marker
Citirea si interpretarea rezultatelor: Apare banda specifica doar daca exista alela
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PCR-SSP
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PCR-SSOP PCR-SSOP (Sequence Specific
Oligonucleotide Probes) Amplificarea intregului exon Hibridizarea produsului de amplificare cu
secventa complementara Vizualizarea strip-urilor cu benzi Interpretarea computerizata Rezolutie scazuta
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PCR-SSOP
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SBT SBT (Sequence Based Typing) Rezolutie maxima Amplificarea genei de interes Secventializare Confruntare cu baza de date – alele
compatibile Detectarea unor alele noi
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SBT
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METODE SEROLOGICE Utilizeaza seruri cu concentratii mari de Ac
anti-HLA specifici Femei multipare Lichid placentar Pacienti cu transplante de organ sau transfuzii
multiple Ac monoclonali
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METODA DE LUCRU
1. Separarea limfocitelor prin centrifugare in gradient de densitate
2. Ser cu Ac anti-HLA in godeuri3. Se adauga suspensie limfocitara in fiecare
godeu (complexe Ag-Ac)4. Se adauga complement (ser de iepure)5. Daca s-au format complexe se activeaza
complementul liza osmotica a celulelor6. Se adauga colorant vital (eozina)7. Se citeste la microscop cu contrast de faza
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INTERPRETAREA REZULTATELOR
Limfocite vii: necolorate, stralucitoare, refractile
Limfocite moarte: umflate, rosu inchis Pentru fiecare godeu: % limfocite moarte Martor negativ: <10% moarte Martor pozitiv: 100% moarte
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INTERPRETAREA REZULTATELOR
Identificarea serurilor care au reactionat cu suspensia limfocitara
Toate serurile cu specificitatea respectiva trebuie sa dea reactie pozitiva (>50% limfocite moarte)
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TIPIZARE HLA-ANumele serului Specificitati ScorA-001 A1 1A-002 A1, Aw36 1A-003 A1, A11 1A-004 A2 1A-005 A2, A28 6A-006 A2, A28, A7 8A-007 A3 8A-008 A3, A4 6A-009 A3, A10, A11, Aw19 8A-010 A11 1A-011 A10, A11 1A-012 A1, A3, A11 4
Interpretare: fenotip HLA A28, A3
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TIPIZARE HLA-BNumele serului Specificitati ScorB-001 B51, Bw52, B35 1B-002 B51, Bw52 1B-003 Bw51, Bw52, Bw53 1B-004 B7, Bw42 8B-005 B7, B27 8B-006 B7, B55 8B-007 B8 1B-008 B8, Bw59 2B-009 B44, B45, B21 6B-010 B44, B45 8B-011 B44 8B-012 B45 1
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SCREENING PENTRU ANTICORPI ANTI-HLA
Detectarea Ac anti-HLA din sangele primitorului
Metoda de lucru:1. Suspensie de leucocite cu HLA cunoscut in
godeuri2. Se adauga ser de la primitor (complexe Ag-Ac)3. Se adauga Ac anti-Fc4. Se adauga complement (ser de iepure)5. Daca s-au format complexe se activeaza
complementul liza osmotica a celulelor6. Se adauga eozina si se citeste la microscop
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INTERPRETAREA REZULTATELOR
Limfocite vii: necolorate, stralucitoare, refractile
Limfocite moarte: umflate, rosu inchis Pentru fiecare godeu: % limfocite moarte Ac anti-HLA probabilitatea de a gasi un
donator compatibil
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REACTIA MIXTA LIMFOCITARA MLR (Mixed Lymphocyte Reaction) LT ale unei persoane (P1) raspund la Ag
HLA de pe limfocitele unei alte persoane (P2) Stimulare Ag proliferare limfocitara
(sinteza crescuta de ADN) Unidirectionala: se blocheaza proliferarea
limfocitelor P2 si se analizeaza proliferarea limfocitelor P1
Bidirectionala: P1 si P2 se stimuleaza reciproc si se insumeaza raspunsurile
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TESTUL DE LIMFOTOXICITATE MEDIATA CELULAR
Se realizeaza o reactie mixta unidirectionala populatie de limfocite citotoxice activate (P1) impotriva limfocitelor P2
P1 se pune in contact cu limfocite P2 marcate cu 51Cr
Se masoara 51Cr eliberat (comparativ cu celule 51Cr nespecifice de la o alta persoana)
~Simulare transplant
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PROBLEME LEGATE DE HLA Rejetul transplantului din cauza LT ale
primitorului care recunosc HLA necompatibile Factori care influenteaza rejetul:
Sarcina, transfuzii Incompatibilitate HLA Terapie pre-transplant mai putin agresiva Terapie imunosupresoare post-transplant
suboptimala Depletia LT din grefele de maduva osoasa Clasa I tolerarea grefei Clasa II GVHD
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REACTIA GREFA CONTRA GAZDA
GVHD (Graft Versus Host Disease) = transplantarea de tesuturi
imunocompetente (celule stem hematopoetice), necompatibile cu tesuturile primitorului in gazde imunodeprimate patologic sau farmacologic (medicatie imunosupresoare)
Celulele imune din grefa recunosc moleculele HLA ale gazdei ca fiind straine raspuns imun impotriva celulelor gazdei
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REACTIA GREFA CONTRA GAZDA
GVHD acut: <100 zile post-transplant Piele, mucoase, plamani, ficat, tract gastro-
intestinal GVHD cronic:
>100 zile post-transplant Piele, mucoase, plamani, ficat, tract gastro-
intestinal, glande exocrine, tesut conjunctiv Corticoterapie
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GVHD ACUT
Eritem palmar
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GVHD ACUT
Ulceratii ale mucoasei bucale
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GVHD ACUT
Degenarea stratului bazal, keratinocite necrozate
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GVHD CRONIC
Papule confluente hiperpigmentate
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GVHD CRONIC
Placi sclerotice hiperpigmentate
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GVHD CRONIC
Modificari lichenoide difuze ale buzelor
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GVHD CRONIC
Hiperkeratoza, hipergranuloza, acantoza, degenerarea stratului bazal