lab materials chemicals storage centrifugeworkstation
TRANSCRIPT
![Page 1: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/1.jpg)
Lab Materials
Chemicals Storage
Centrifuge Workstation
![Page 2: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/2.jpg)
Procedure for Isolating Polymorphonuclear Cells
• Blood (Cancer patient)
• “Buffy Coat”
• Storage
![Page 3: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/3.jpg)
Lab Materials
![Page 4: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/4.jpg)
Isolation of RNA Using Immunomagnetic Beads
![Page 5: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/5.jpg)
Immunomagnetic Isolation of mRNA
![Page 6: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/6.jpg)
Images of Immunomagnetic Isolation of CTC
Immunomagnetic Beads Target Ber-Ep4 Epithelial Cell Antigen (ECA)
![Page 7: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/7.jpg)
Reverse Transcriptase Polymerase Chain Reaction
Animation
![Page 8: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/8.jpg)
![Page 9: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/9.jpg)
Polymerase Chain Reaction (PCR)
![Page 10: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/10.jpg)
Lab Materials
PCR Machines Incubator
PCR Workstation Cold Room
![Page 11: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/11.jpg)
Identifying Type of Cancer Cell
Most cells have three types of genes:
1. Housekeeping Genes (Actin)
2. Tissue-specific Genes (Cytokeratin-19)
3. Organ Specific Genes (Mammaglobin)
4. Cancer Specific Genes (BRCA1 & BRCA 2)
![Page 12: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/12.jpg)
Gel Electrophoresis
Molecular Markers:
1. Actin (~350 bp)
2. Cytokeratin-19 (~300 bp)
3. Mammaglobin (~280 bp)
![Page 13: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/13.jpg)
RT-PCR Results in the Detection of Breast CTC
Positive Result: +Negative Result: -
Sample # Actin Cytokeratin 19 Mammaglobin Overall
1 + + - +
2 + + + +
3 + - - -
4 + - - -
5 + - - -
6 + + + +
7 + + + +
8 + + - +
9 + + - +
10 + - - -
11 + + + +
12 + - - -
13 + + - +
14 + + - +
15 + + - +
Percentage 100% 67% 27% 67%
![Page 14: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/14.jpg)
Conclusion
Use of multiple markers enhances sensitivity
Cancer cells can be detected at early stages
Less radiation therapy, chemotherapy, and immunotherapy
![Page 15: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/15.jpg)
Future Plans
Use additional molecular markers to enhance sensitivity (Telomerase)
Associate risk factors genes (BRCA1 & BRCA2)
![Page 16: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/16.jpg)
References
Cotran, Kumar, Collins: The Pathological Basis of Disease 6th Edition (W.B. Saunders Company)
Hospital Practice (http://www.hosppract.com/)
Foss AJ, Guille MJ, Occleston NL, Hykin PG, Hungerford JL, and LightmanS: The detection of melanoma cells in peripheral blood by reverse transcriptase polymerase chain reaction. Br J Cancer 72: 155 – 159, 1995
Ghossein RA, Bhattacharya S, Coit DG. Reverse transcriptase polymerase chain reaction (RT-PCR) detection of melanoma-related transcripts in the peripheral blood and bone marrow of patients with malignant melanoma. What have we learned? Recent Results Cancer Res. 2001;158:63-77.
Ghossein RA, Bhattacharya S.: Molecular detection and characterization of circulating tumour cells and micrometastases in solid tumours. Eur J Cancer. 2000 Aug 36 (13 Spec No):1681-94.
NATIONAL BREAST CANCER FOUNDATION (http://www.nationalbreastcancer.org/)
Pelkey TJ, Frierson HF and Bruns DE: Molecular and immunological detection of circulating tumor cells and micrometastases from solid tumors. Clin Chem 42:1369 – 1381, 1996
Ronald A. Ghossien, Leo Carusone, and Satyajit Bhattacharya: Molecular detection of micrometastases and circulating tumor cells in melanoma prostatic and breast carcinomas. In vivo 14: 237 – 250, 2000
![Page 17: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/17.jpg)
Acknowledgements
Dr. Sat Bhattacharya Memorial Sloan-
Kettering Cancer Research Center
Harlem Children Society
Minimal Residual Lab (MRD Lab)
![Page 18: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/18.jpg)
![Page 19: Lab Materials Chemicals Storage CentrifugeWorkstation](https://reader036.vdocuments.site/reader036/viewer/2022062315/56649e435503460f94b3647d/html5/thumbnails/19.jpg)
Thank You for Your Cooperation!Don’t be afraid to ask questions
Rami
Pankaj