Professional Documents
Culture Documents
8/19/2011
NH NGHA:
Phng phap nhuom ac biet Xac nh khang nguyen trong to chc = khang the (nh phan ng mien dch KNKT) va hoa chat
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QUA TRNH PHAT TRIEN phat trien hoa mien dch 1940 : Coons
huynh quang tren mau mo cat lanh thap nien 90: Avrameas va cs phat trien KT men (horseradish peroxidase), quan sat c bang KHV quang hoc Tang o nhay HMMD:
KT men khang men (peroxidase antiperoxidase) Cau noi Biotin- StreptAvidin (BSA) Cau noi Avidin- Biotin (ABC- avidin-biotin conjugate) KT khuech ai (tyramide, )
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Khang nguyen mo Khang the (KT 1, KT2..) Chat tao hp chat mau (chromogen solution) Nhuom nhan (Counterstaining)
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CHAT PHAN NG
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KHANG NGUYEN
Phan ln la protein, 1 so carbohydrate Khang nguyen la dau an cua s biet hoa te bao. Gom nhieu loai:
Cac si trung gian: keratin, vimentin, desmin Thu the hocmon: estrogen, progesteron, androgen
Epitope- v tr tiep xuc : la v tr gan khang the tren be mat khang nguyen. Mang tnh ac hieu cua khang nguyen. La vung quyet nh khang nguyen (antigenic determinant) 1 khang nguyen co nhieu epitope, va ch gan vi 1 khang the ac hieu
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KHANG THE
La cac glycoprotein Gom 1 chuoi nang va hai chuoi nhe Chia thanh 5 loai IgA, IgG, IgM, IgD, IgE da vao chuoi nang Gom vung co nh va vung co the thay oi Phan loai theo phng phap san xuat: Khang the a dong Khang the n dong Phan loai theo v tr phan ng vi khang nguyen: Khang the nguyen phat Khang the th phat
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ong vat bang khang nguyen ac hieu Hon hp nhieu khang the ac hieu va khong ac hieu. Cac khang the khong mong muon se c loai bo sau o u iem:
De san xuat o nhay cao do nhan dien nhieu epitopes KN Phan ng dng gia do cha cac khang the phan ng khong ac hieu Khuynh hng nhuom nen cao
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Khuyet iem:
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Khang the gan trc tiep vi KN. Khong gan ket (khong anh dau) Cau noi gia khang nguyen va he thong quan sat
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Khang the cua mot loai phan ng trc tiep vi khang the cua loai khac. Phan ng vi KT nguyen phat Gan vi biotin hay enzym hoac chat huynh quang Khuech ai phan ng gia KT nguyen phat va KN ch. Cau noi gia KT nguyen phat va he thong quan sat.
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Mau sac: can mau tng phan vi cac mau khac, v du : chromogen khong co mau nau e tng phan vi melanin o hoa tan o nhay phu hp vi quan sat sieu cau truc
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enzyme
Mau
Nau o
peroxidase
Xanh en
+ _
Xanh
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enzyme
Mau
Fast blue BB Alkaline phosphatase Fast red TR New Fuchsin BCIP-NBT Tetrazolium Glucose oxidase Tetranitoblue tetrazolium (TNBT) Bac
Immunogold
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Diaminobenzidine ( DAB )
La chromogen hay dung nhat Cho san pham mau nau, khong tan Lu tr lau Them ion kim loai (cobalt, nikel) cho tng phan mau sac tot Lam lan vi mau melanin, hemosiderine Gay ung th
8/19/2011
AEC (3-amino-9-ethylcarbazole)
Tao ra san pham mau o AEC co hai phan ng mau :bnh thng cho mau o nhng neu lng enzyme qua nhieu th mau se chuyen t mau o sang mau xanh-nau Hay dung trong cac benh da B hoa tan trong cac dung moi cua mo Khong ben, de phai mau Gay ung th nhng t nguy c hn DAB
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Nhuom nhan
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ENZYMES
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HORSERADISH PEROXIDASE
La mot enzym, 40kD Bien oi peroxide hydrogen thanh nc Oxi hoa chromogen nhn thay c Peroxidase noi sinh mot so te bao: neutrophile, eosinophile, basophile, histiocyte nham lan. -> c che peroxidase noi sinh
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ALKALINE PHOSPHATASE
c s dung bi Avrameas, nh la 1 enzyme gan vi khang the th 1. c dung pho bien trong nhuom oi Subtrates dung kem bao gom naphthol salts nh coupling agents, diazonium salts nh la chromogenic capture agents
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GLUCOSE OXIDASE
t c s dung Co the s dung trong nhuom oi do cung cap chat tao mau vi mau sac rieng
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Khong the nhn thay khang the di KHV quang hoc hay KHV ien t Phai gan ket khang the va 1 chat chuyen biet e co the quan sat c Cac chat gan ket khang the la phc hp huynh quang hay cac enzyme -> Do o co hai cach quan sat phc hp KN-KT:
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Ky thuat n gian: hp chat huynh quang + khang the + mo Anh sang kch thch hp chat huynh quang tia sang. Hp chat huynh quang:
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HE x 200
Immunofluorescein x 200
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Mien dch trc tiep (direct procedure) Mien dch gian tiep (indirect sandwich procedure) Ky thuat unlabeled antibody
Cau noi enzyme Mien dch men khang men (peroxidase antiperoxidase method)
Cau noi Biotin-Avidin (Biotin-avidin procedure) Phc hp Biotin-Avidin (Avidin-biotin conjugate procedure) He thong Biotin- streptavidin Polyvalent systems
Alkaline phosphatase- antialkaline phosphatase Protein A Men gan khang nguyen (enzyme-labeled antigen 8/19/2011 method)
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KT TH II GAN BIOTIN
KT TH I
ABC METHOD
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Streptavidin, la tetrameric avidin, 60kD, tach t vi khuan Streptomyces avidinii Gan vi Biotin vi ai lc cao Dung thay the avidin trong phc hp A-B :
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Peroxidase-antiperoxidase
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Giong KT Peroxidaseantiperoxidase (PAP) Dung nhuom mo co peroxidase noi sinh cao nh mo limpho hay tuy xng (cac dd c che peroxidase noi sinh co the lam huy epitope nhng mo nay) Dung nhuom oi Cac chat phan ng mau dung vi pp nay la Fast blue BBN, Fast red TR
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Protein A
Chiet xuat t tu cau khuan ( Staphylococcus aureus), co ai lc cao Co the gan ket vi peroxidase, chat huynh quang fluorescein, gold Gan ket nhanh nhng co the gay nhuom nen
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CO NH MO TRONG HMMD
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HMMD ++ ++
Acetone Formaldehyde
++ ++
0 +
Glutaldehyde Bouins
+ ++
++ ?
0 +/++
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VUI MO
MO HOC Cat lanh Paraffin Methacrylate Lowicryl LR white Epoxy resin + ++ +++ + + +++ SIEU CAU TRUC 0 + 0/+ + + ++ HMMD ++ +/++ +/++ +/++ +/++ 0
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Tieu ban nhuom HMMD phai c nhung trong dung dch Silane (3aminopropyltriethoxy-silane, pha vi acetone) hoac lam trang san silane Tac dung lam gan chat mo vao lam knh khong b troi trong qua trnh boc lo Khang nguyen
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Dung dch A-Dung dch Tris HCL ac: Pha loang 60.55g Tris trong 500ml nc cat trong chai 1lit. Them 35 ml HCL ac, lac eu, ieu chnh pH. Bao quan tu lanh Dung dch B- Dung dch NaCl ac: Hoa tan 90g NaCl vao chai 1 lit roi them nc va u, bao quan tu lanh Dung dch TBS s dung: 1 lit TBS pha 100ml dung dch A va 100 ml dung dch B, kiem tra pH (pH =7.2 -7.6). Bao quan tu lanh
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Ky thuat: un nong Dung Proteinase K Dung Proteinase K (PK): Pha 100 l dd PK trong 50 ml TBS pH=7.6 e c nong o 20 g/ml, bao quan lanh Moi tieu ban c phu 200 l dung dch PK, u 10 phut, niet o phong Ra tieu ban 2 lan bang TBS x5 phut Cac khang nguyen c boc lo bang nhiet vi lo vi song trong dung dch Citrate
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Dung dch citrat: Dung dch A: 21g acid citric/100ml Dung dch B: 14,75 g sodium citrate/ 500ml Dung dch Citrate = 9ml ddA + 41 ml ddB + 450 ml nc cat Cac khang nguyen c boc lo bang nhiet vi lo vi song trong dung dch Citrate Mot so khang nguyen can boc lo vi nhiet o cao hn va dung dch citrate nh CD117, ER, PR Hoac co the boc lo khang nguyen bang dung dch pha san cua DAKO (target retrieval) oi vi cac khang nguyen p53, Ki67, Ki1, CD30 Mot so khang nguyen khong can boc lo nh B cell, T cell, EMA
8/19/2011
Chuan b dung dch H202: 50 ml H202 30% trong 450 ml nc cat, lac eu, bao quan lanh Sau khi tay paraffin, at vao dung dch H202 trong 5 ph nhiet o phong , sau o ra nc cat trong 2 ph
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Pha loang bang dung dch PBS vi 0.2% bovine serum albumin Nong o pha loang: 10ml khang the vi t le pha loang 1/10 , ta can 1ml khang the am ac roi them 9 ml dung dch pha loang Pha loang khang the bao am ket qua chnh xac.
Khang the qua loang -> nhuom nhat, hay khong bat mau Khang the qua ac -> nhuom nen khong ac hieu, hoac dng tnh gia
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5ph 15-20 ph
45 ph
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Neu dung DAB th kh nc tiep bang con va lam trong sang mo bang xylen
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Am tnh: Ch co mau xanh tm cua Hematoxylin nhuom nhan Dng tnh: Neu co hien dien khang nguyen tren be mat te bao, phc hp KN-KT-streptavidinemau se cho mau o ( neu dung EAC) va cho mau vang nau (neu dung DAB)
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HER-2/NEU
AM TNH
DAB
AEC
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DAU AN
NHAN
BAO TNG + + +
Dau an ac biet
+ + + + +
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NHA N
BAO TNG
+ + + +
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Khong bat mau hoac bat yeu Dng tnh gia Am tnh gia Mo b troi Mo b thoai hoa at tien oi luc khong phan biet c lanh ac Can kinh nghiem cua ngi oc
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KT nguyen phat va th Dung KT th phat phu phat khong phu hp hp Khang nguyen khong Boc lo khang nguyen c boc lo
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Dng tnh gia Nhuom nen qua am Hieu ng ra Hieu ng bat mau do nhiet Bat mau do co rut mo
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HIEU NG RA
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AM TNH GIA
Khang nguyen khong c boc lo tot KN b pha huy do co nh Khang the b hong do bao quan khong k Lng khang the qua t Thi gian u qua ngan pH cua dung dch em khong ung Nhiet o qua nong
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Tuan thu nghiem ngat qui trnh nhuom: - nong o khang the - pH cua dung dch em - thi gian
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Lam chng am: giup khang nh tnh ac hieu cua phan ng mien dch Lam chng dng: cho biet khang the van con tac dung tot. Lam chng dng ly tng la mo bnh thng khong benh ly v du nh mo day than kinh ngoai bien lam lam chng cho S-100 Lam chng dng vi mo khong b c che peroxidase noi sinh cho thay chat phan ng tao mau (chromogen) con hoat ong
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Dau an
Khang nguyen Mo chng dng CD3, CD8 CD20, CD43 Bcl-2, CD45 RO LCA, CD68 CD15, CD30 CD 31, CD 34 FVIII CEA EMA Keratin AE1 Amydale, hach limpho
Dau an
Mo chng dng Gan Vu Ruot U hac to U hac to Sarcom Ewing Ruot Ruot ruot
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Dau an bieu mo CK 20 U hac to S-100 HMB-45 CD 99 Dau an Sarcom Muscle-specific actin (SMA) Desmin Vimentin
Dau an
Tuy, si than kinh Mo CTC lanh, mo vu lanh Mo TLT Nhau thai gan
Ki-67 P 53
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CHNG DNG/ CK
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NG DUNG HMMD
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Xac nh nguon goc u khong biet hoa Xac nh yeu to tien lng Chan oan phan biet u lanh hay ac D oan ap ng ieu tr Phat hien nhiem 1 so loai virus va vi khuan
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CYTOKERATIN
EMA
LCA
(+)
SARCOM
(-)
(-) (+)
(+)
LYMPHOM
CARCINOM
U KHAC
DESMIN HMB45 S100 NSE
(+)
C VAN C TRN
(+)
MELANOM
(+)
MO BAO
(+)
THAN KINH
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THYROGLOBULIN
EMA
CEA
CK 7
PSA
(+) (+)
(+)
TUYEN GIAP TE BAO GAI
(+)
(+)
TUYEN
MYOSIN
FIBRONECTIN
CD31
CD34
FVIII
(+) (+)
(+)
C TRN
(+) (+)
(+)
(+)
(+)
C VAN
SI
MACH MAU
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CD30
CD68
MYELOPEROXIDASE
(+) (+)
LYMPHOM T
(+) (+)
(+)
(+)
(+)
TB REED STERNBERG
LYMPHOM B
MO BAO
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Ki67: (+) trong nhan, hien dien trong te bao pha G1, G2, S tr pha G0 PCNA (proliferating cell nuclear antigen, cyclin) Nhng benh nhan co thi gian song them ngan thng co t le Ki67, PCNA cao
Xac nh xam nhap hay gia xam nhap cua TB ung th:
KT khang mang ay- laminin (collagen IV) KT khang actin e xac nh TB c bieu mo(vu) Cytokeratin trong lng phan t cao e xac nh te bao ay (mo tuyen tien liet)
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Xac nh o di can nho tuy xng hay hach Khang nguyen U: Khang nguyen ac hieu u : protein p53 Gen sinh ung, yeu to phat trien, thu the: Her-2/neu EGFR bcl-2 Gen e nen u va san pham gen: Rb 8/19/2011 P53
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Ki67: (+) trong nhan -> dng tnh nhieu ac tnh PCNA
Chan oan phan biet phan ng lanh tnh cua mo limpho : Nhuom ong thi B,T> ca hai dng la lanh tnh, 1 trong hai dng hay ca hai am la limphom CEA hien dien mang te bao tuyen ac tnh ( trong khi te bao bnh thng khong hien dien hoac rat t) -> chan oan carcinom giai oan sm
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ER, PR (+): ap ng ieu tr hocmon trong ung th Vu, ung th noi mac t cung Androgen receptor: giup ieu tr ung th tien liet tuyen bang thuoc c che androgen Phat hien khang thuoc:
D OAN AP NG IEU TR
Her-2/neu (++): Ung th vu khang vi hoa tr N-myc : Ung th phoi te bao nho va u nguyen bao than kinh khang vi hoa tr P-glycoprotein (P-170) : bieu hien nhieu mo ung th, th benh nhan ap ng kem Nguon Anderson vi hoa tr
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Phat hien sm, nhanh chong va chnh xac cac vi sinh vat kho phat hien trong moi trng cay nh Helicobacter Pylori, Histoplasma capsulatum, Pneumocystic carinii, Phat hien khoang hn 60 loai vi khuan va virus
Cytomegalovirus HPV (human papilloma virus) Virus viem gan SV A,B,C Epstein barr virus Toxoplasma gondii HSV (Herpes simplex virus) ,
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HELICOBACTER PYLORI
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Chromoganin, neuron specific enolase ( NSE) Mo than kinh noi tiet HMB45 + S 100 protein LCA Placectal alkalin phosphatase (PLAP) Human chorionic gonadotropin (hCG) Melanocytes Hematolymphoid cells Te bao mam T bo nui
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stt 12 13 14
c tr ng Ung th tuyen Benh Hodgkin Yolk sac tumors, carcinom te bao gan GIST Ung th TLT Lymphom B Lymphom T
15 16 17 18
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DAU AN BIEU MO
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KERATIN
La dau an cua te bao bieu mo Cac keratin trong lng phan t cao thng phan bo te bao bieu mo lat tang (sng hoa va khong sng hoa) Cac keratin trong lng phan t thap thng phan bo cac bieu mo n va bieu mo tuyen Keratin trong te bao la cytokeratin
CK 7: UT bieu mo BT, dau an biet hoa nguyen bao nuoi trong u TB mam CK 10,11: (+) Ung th te bao gai, (-) ung th tuyen CK 18,8 : sarcom te bao hnh thoi
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Adenoid cystic vu
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La carbohydrate Bieu hien bieu mo tuyen vu, nhieu tuyen ngoai tiet va cac loai bieu mo khac nhau Ngoai ra con bieu hien cac u nh sarcom dang bieu mo, sarcom bao hoat dch, u day song
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Desmin : tm thay trong cac te bao c , la dau an cho s biet hoa c cua te bao u -> bieu hien sarcom c van, sarcom c trn Actin, myosin: la dau an cho s biet hoa c
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ACTIN
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DESMIN
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VIMENTIN
Bieu hien cac te bao trung mo Ngoai ra con bieu hien 1 so te bao bieu mo nhat nh ( TB bieu mo cau than)
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NSE , CHROMOGRANIN
NSE: La khang the khang cac neuron, la dau an cho s biet hoa than kinh noi tiet. Chromogranin: La protein co mat trong hat che tiet than kinh noi tiet v the no la dau an co o nhay va o ac hieu cao cho s biet hoa than kinh noi tiet
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S100
La protein, phan bo rong rai trong c the nh mo nao (TB than kinh em, Tb schwann), mo sun, mo m, te bao hac to, TB langerhans Con bieu hien 1 so te bao bieu mo ( TB c bieu mo
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Melanocyte
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FACTOR VIII
Xac nh cac u biet hoa noi mo mach mau nhng khong thc s hu ch trong chan oan u te bao noi mo mach mau biet hoa thap
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LCA( leucocyte common antigen) : La khang nguyen mang, rat nhay vi te bao limpho va u limpho va khoang 10% am tnh vi u limpho te bao ln CD 20: xac nh te bao B CD3, CD43, CD45 RO: xac nh te bao T CD15, CD30: xac nh te bao Reed Sternberg Bcl-2:
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HER-2/NEU
La 1 proto- oncogene tren chromosome 17q21 La thanh vien cua gia nh HER (Human epithelial receptor). Ma hoa 1 glycoprotein xuyen mang te bao 185kD con goi la p185HER2 con co ten la C-erB-2 hay Her 2 S khuech ai gen HER2 (nhan) Bieu hien qua mc protein HER2 (mang bao tng)
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0 (negative)
1+ (negative)
2+ (equivocal)
3+ (positive)
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ESTROGEN
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PROGESTERON
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(+): 1+2 = 3
(++): 2+2 = 4
(+++): 5+3 = 8
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HMB45
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HMB45/ melanoma
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KET LUAN
HMMD la ky thuat hien ai Giup chan oan nguon goc u Xac nh u lanh hay ac Giup tien lng at tien, ky thuat phc tap
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