immunophenotyping leukemia
DESCRIPTION
PPT about immunophenotyping for leukaemiaTRANSCRIPT
![Page 1: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/1.jpg)
FCA pada akut leukemia
![Page 2: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/2.jpg)
spesimen
• BMP, darah tepi, jaringan limfoid• Harus diproses dalam 24 jam ditransport
dalam suhu ruang• Antikoagulan heparin atau EDTA• Populasi Sel berinti RBC dilisiskan dulu• Viabilitas : propidium iodine atau 7-
aminoactinomysin.
![Page 3: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/3.jpg)
spesimen
• Ditambahkan koktail Mo Ab terkonjugasi fluorokrom
• Analisa interseluler perlu proses fiksasi dan permeabilisasi agar antibodo bisa memasuki membran sel.
• Belum ada konsensus standarisasi panel antibodi
![Page 4: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/4.jpg)
Marker yang sering dipakai Lineage markers
Immature CD34CD117Terminal deoxynucleotidyl transferase (tdt)
Granulocytic/monocytic
CD33CD13CD15CD14
Erythroid CD71Glycophorin A
Megakaryocytic CD41CD42CD61
B Lymphocyte CD19CD20CD22k light chainΛ light chain
T Lymphocyte CD2CD3CD4CD5CD7CD8
![Page 5: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/5.jpg)
Marker yang direkomendasikan
![Page 6: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/6.jpg)
Prinsip dan instrumentasi
• Poin utama: – Fluidic– Sumber cahaya (laser)– Sistem deteksi– Computer
![Page 7: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/7.jpg)
Prinsip dan instrumentasi
• Fluidic – Suspensi sel diinjeksikan kedalam suatu aliran
dengan teknik hydrodinamic focusing masuk satu satu
• Laser (488nm)– Menerangi setiap sel yang lewat, yang telah
diwarnai dengan antibodi yang terkonjugasi fluorokrom tertentu
![Page 8: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/8.jpg)
Prinsip dan instrumentasi
• Detektor– FCA selain memiliki FSD,SSD, juga memiliki beberapa
floresence detektor – Sel yang mengabsorbsi cahaya berflouresensi pada
warna dan panjang gelombang ttt– Ditangkap oleh photodetektor yang sesuai dengan
panjang gelombang yang dimiliki flouresens tadi• Computer– Data yang ditangkap detektor dirubah menjadi
bentuk digital (scaterplot)
![Page 9: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/9.jpg)
Flourokrom
• FITC (flourescein isothiocyanate) 519nm (hijau)
• Phycoerythrin (PE) 575nm (oranye)• PerCP 694nm (merah)• APC 660nm (pink)
![Page 10: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/10.jpg)
Direct versus indirect labeling of antigens
B or T Cell marker
B or T cell specific Ab
B or T cell specific Ab
B or T Cell marker
![Page 11: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/11.jpg)
gating
• Gate : batasan elektronik oleh operator yang digunakan untuk menggambarkan suatu kelompok/kluster
• Proses gating : pemilihan suatu populasi berdasarkan parameter FCA menggunakan komputer oleh operator
• Gating : live atau saat analisis
![Page 12: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/12.jpg)
Analisa data FCA
• Analisa scaterplot berdasar ukuran (FCS)dan kompleksitas(SSC)
• Ekspresi CD45 :– Limfosit : paling tinggi – Seri granulosit : sedang– Prekursor : redup-negatif
![Page 13: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/13.jpg)
Identifikasi populasi sel
• Progenitor– CD34– CD117– CD38– HLA-DR– Semakin matur lama-lama hilang
![Page 14: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/14.jpg)
Identifikasi populasi sel• Granulocytic lineage
– Myeloblast : CD34,CD38,HLA-DR,CD117– Pan myeloid marker : CD13,CD33– Promyelosit : CD34 (–) HLA-DR (-), CD 15– Semakin matur : CD11b, CD13 menghilang, penurunan CD33– Band stage : CD13 muncul lagi, CD16(+)
![Page 15: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/15.jpg)
Identifikasi populasi sel• Monocytic lineage
– Monosit muda : peningkatan densitas CD13, CD33,CD11b– Transisi ke promonosit dan monosit matur : CD14 dan CD15– Berlawanan dengan seri granulosit : HLA –DR dan CD64 tetap kuat pada monosit matur
![Page 16: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/16.jpg)
Identifikasi populasi sel• Erythroid lineage– Prekursor eritroid :Tidak mengekspresi CD45– Diferensiasi eritroid : CD71 (pronormoblast- semakin
hilang pada stage retikulosit)– Glycophorin A : pertama muncul pada basophilic
normoblast.
![Page 17: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/17.jpg)
Identifikasi populasi sel• Megakaryocytic lineage
– Maturasinya belum dietahui – CD41 dan CD61 : Gp Iib/IIIa marker diferensiasi pertama– Pada populasi CD34+ dengan CD41/CD61 + megakarioblas– maturasi megakariositplatelet : CD42,CD62P,CD63
![Page 18: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/18.jpg)
Identifikasi populasi sel• B-cell Lymphoid lineage
– Progenitor lymphoid : CD34, terminal deoxynucleotidyl transferase (TdT) dan HLA-DR– B-cell paling muda : CD19, cyCD22, cyCD79– Semakin matur : muncul CD10 B-cell matur : CD20 dan penurunan CD10
• T-cell Lymphoid lineage– Immature T-cell : CD34, TdT– Marker pertama T-cell : CD2, CD7, cyCD3– Cd 2,CD7 NK cell– Pada akhirnya: tinggal CD3 di permukaan sel, CD4 dan CD8 menghlang
![Page 19: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/19.jpg)
![Page 20: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/20.jpg)
![Page 21: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/21.jpg)
![Page 22: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/22.jpg)
![Page 23: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/23.jpg)
Koktail
Bag. PK FK UGMFITC/FL1 PE/FL2 PerCP/FL3
igG1 (kontrol neg)
IgG2(kontrol neg)
CD45(blast)
CD3(T lymp)
CD10(lymp B)
CD45(blast)
CD7(lymph T)
CD33(myelo)
CD45(blast)
cyMPO(myelo)
CD34(progenitor)
CD45(blast)
cyCD3(T lymph)
cyCD79a(B lymph)
cyCD45(blast)
Jurnal • Panel primer :
– CD45 (blast)– CD19 (B lymp)– CD10 (B lymph CALLA(+))– CD3 (T lymp)– CD33 (Myelo)– CD7 (Lymp T)– CD34 (progenitor)– CD13 (Myelo)– Tambahan : CD14 dan CD41
• Panel sekunder :– CD117 (myelo)– CD14 (Mono)
![Page 24: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/24.jpg)
Teori di jurnal
CD34• Immature hematopoietik• Normal BMP hanya <3%• Populasi leukemia :
– 45-65% AML : CD34+– 75%pre B-cell ALL : CD34+– B-cell ALL L3 : dim CD34, TdT -
B-cell ALL• 57,6% >20% CD19+
(earliest B-cell)• B-cell ALL CD19+,HLA-DR+• CD19- rule out B-cell ALL• Aguilera et al : marker B-cell
yang sensi-spesi paling baik : cyCD79a dan cyCD22
• Matur B-cell (ALL L3) CD10 and dim CD34
![Page 25: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/25.jpg)
Teori di Jurnal
T-cell ALL• Aguilera et al : marker T-cell
paling baik sensi-spesi : cyCD3 CD7
• Penelitian lain: CD7 paling sensi-spesi
• CD7 + belum tentu T-cell ALL, diekspresi oleh : – B-cell– Myeloid lineage
• T-cell ALL CD7+, juga disertai marker T-cell lain (CD2,CD3,CD5)
CALLA (common ALL antigen)• CD10 + CALLA +• Biasa ditemukan pada B-cell
ALL pada leukemia anak• Penting pada :
– Dx pre-B cell ALL– Early relapse pre-B cell ALL
• CD 10+ prognosis lebih baik
• Berkorelasi dengan AbN kromosom
![Page 26: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/26.jpg)
Teori di jurnal
AML• Jurnal : Dim CD45
dikombinasi CD34,CD33,CD13
• Aguilera et al : marker paling baik sensi-spesi untuk AML : cyMPO
• Kaleem et al : peling sensi CD33
AML dengan aberant ekspresi Ag
• 25% AML CD7+• Tidak signifikan secara klinis
dan respon thd terapi
![Page 27: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/27.jpg)
Teori di jurnal
• 21,2% ALL CD13,CD33• 5,2% B-ALL CD7• Bradstock et al : – 30-45% B-ALL ekspresi Myeloid marker (My+ALL)
CD33,CD13,CD11b,CD7• Unal et al : ekspresi myeloid antigen pada kasus
ALL tidak signifikan terhadap klinis, angka kekambuhan dan survival rate.
• ALL dengan aberant myeloid antigen
![Page 28: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/28.jpg)
Teori di jurnal
– Lihat skoring – >2 untuk myeloid dan >2 lymphoid biphenotypic
• Mixed lineage leukemia
![Page 29: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/29.jpg)
KLASIFIKASI IMUNOLOGI
![Page 30: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/30.jpg)
KLASIFIKASI IMUNOLOGI
![Page 31: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/31.jpg)
KLASIFIKASI IMUNOLOGI
![Page 32: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/32.jpg)
KLASIFIKASI IMUNOLOGI
![Page 33: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/33.jpg)
KLASIFIKASI IMUNOLOGI
![Page 34: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/34.jpg)
Penelitian di indonesia
![Page 35: Immunophenotyping leukemia](https://reader033.vdocuments.net/reader033/viewer/2022061511/55cf87eb55034664618b8559/html5/thumbnails/35.jpg)
Penelitian di indonesia