Techniques in Experimental Pathology (3 credits)

Sample Course Schedule
PTR 538, SPRING 2004
Mondays 9:30 am TO 12:30 pm

Week	Topic	Lecturer
1	Course Organization	Leong
	Autopsy Video	Broccolo
2	Martin Luther King Holiday	No Class
3	Tissue Culture	Musser
4	Photodynamic Therapy	Dougherty
5	Pathology	Cheney
6	President’s Holiday	No Class
7	Nuclear Medicine	LaMonica
8	Cytogenetics and Fish	Block
9	Animal Resources	Jackson
10	Spring Recess	No Class
11	PCR	Starostik
12	Image Analysis	Kolega
13	Microarray	Nowak (Conroy)
14	Flow Cytometry	Wallace
15	Immunohistochemistry	Vaughan
16	DNA Sequencing	Kazim

Course Coordinator: Susan S. Leong PhD E-mail: sleong@adelphia.net

Basic Policies and Course Details

• This course should be registered for 3 credit hours.
• There are no make up classes available for this course
• Punctuality in reporting to scheduled classes are required
• A weekly written one page report on what was presented at the class and what the student learned about the technique must be sent to Dr. Leong via e-mail, or fax within two weeks of the scheduled class to satisfy the requirements of this course.
• The semester grade will be PASS or FAIL based on attendance and quality of the written report.

Week 1
Topic: Course Organization and Autopsy Video
Lecturer: Leong & Broccolo
Place: RPCI Department of Pathology Small Conference Room K280

Students will be shown an autopsy on video. Actual autopsy observation will be shown by arrangement.

Week 3
Topic: Tissue Culture
Lecturer: Musser
Place: RPCI MRC 231

Cell culture techniques will be outlined. Definitions and applications; laminar flow hood and sterile techniques; labware; culture environment; incubators, cell cycle and growth (normal vs transformed); types of cultures: suspension, adherent and spheroids; culture maintenance, cryopreservation, contamination; establishment of a primary cell line; cloning and isolation of particular cell types; enumeration and viability; specific applications: 1) cytotoxicity assays; 2) hybridoma production; 3) transfection.

Week 4
Topic: Photodynamic Therapy
Lecturer: Dougherty
Place: RPCI MRC 1st floor PDT Conference Room 140

Lecture will describe basic photosensitizer; method of activation; uptake in tissues; basic clinical application of PDT; mechanism of action of PDT; future applications; demonstration of lasers and treatment therapies.

Week 5
Topic: Techniques in Pathology
Lecturer: Cheney
Place: RPCI Department of Pathology Conference Room K240

Techniques (frozen section, paraffin embedding) used in routine processing of pathological specimens will be discussed and demonstrated. Special stains and methods used for identification of specific structures/markers will also be discussed.

Week 7
Topic: Nuclear Medicine
Lecturer: LaMonica
Place: RPCI 2nd floor Radiology Conference Room

Basic and technical considerations; Radiation protection; pharmaceuticals - preparation and usage; Radiation detection (film badges, survey meter, dose calibrator, gamma cameras, uptake probe); quality assurance; nuclear medicine studies. Clinical application and patient studies will also be discussed.

Week 8
Topic: Cytogenetics and FISH
Lecturer: Block
Place: RPCI Science Building Room S517

Students will receive instructions in the basic concepts of clinical cancer cytogenetics. Recurrent chromosome abnormalities associated with hematologic malignancies will be discussed using actual patient clinical material. Students will have the opportunity to cut and construct representative karyotypes.

The use of fluorescence in situ hybridization (FISH) technology will be discussed in the setting of diagnosis, minimal residual disease detection and bone marrow transplantation. A FISH demonstration using centromeric and translocation probes will be presented.

Week 9
Topic: Animal Resources
Lecturer: Jackson
Place: RPCI MRC South Room M262

After reviewing Federal, State, and Institute regulations for animal care and use, the students will learn of the resources available for laboratory animals. A tour of the Animal Facilities will be concluded with a discussion of the various caging systems and requirements for sanitary and safe cage processing. Some technical procedures used routinely with laboratory mice will be demonstrated.

Week 11
Topic: Polymerase Chain Reaction
Lecturer: Starostik
Place: Science Building Room 520

Polymerase chain reaction (PCR) is a powerful tool for molecular diagnostics. PCR is an in vitro method of nucleic acid synthesis by which a particular region of DNA can be specifically amplified. It involves two primers that flank the DNA fragment to be amplified and repeated cycles of heat denaturation of the DNA, annealing of the primers and extension of the annealed primers with DNA polymerase. This results in an exponential accumulation of the specific DNA fragment, such that one cancer cell amongst 10 normal cells can be detected.

Week 12
Topic: Image Analysis
Lecturer: Kolega
Place: UB Main Street Campus Farber Hall Room 306

Students will be introduced to the concepts of digital image enhancement and analysis through hands-on experience with various image manipulations concurrently with discussion of the underlying principles, results and limitation of each procedure. Examples of types of image processing to be covered include contrast enhancement, histogram equalization, convolutions, and image segmentation. An image grabber/processing board installed in a PC will be available to the students for this session.

Week 13
Topic: Microarray Analysis
Lecturer: Nowak & Conroy
Place: Cell and Virus Building

Comprehensive molecular analysis, where individual gene function is studied within the context of all other genes in organisms= cells or tissue, will be the keystone for transforming research in this century. Previously, gene expression analyses were done in a sequential fashion making genome wide comprehensive analysis untenable. The DNA Microarray Facility at RPCI is generating high-density arrays with a GMS arraying robot of cDNAs, as spotted PCR products. These arrays are then hybridized to fluorescently labeled test and control samples with the results read on a multicolor fluorescence scanner. The results reflect the expression of the arrayed set of genes in the test sample as compared to the control allowing the investigator to survey hundreds to thousands of genes at a time in a single experiment.

Week 14
Topic: Flow Cytometry
Lecturer: Wallace
Place: RPCI, Flow Cytometry Lab, Science Building Room 617

A lecture/demonstration of immunophenotyping by flow Cytometry will be presented. Methods for staining cells with specific antibodies to membrane proteins will be described. There will be a brief overview of how a flow cytometer and cell sorter work. The strategies used to analyze and interpret flow Cytometry data will also be discussed.

Week 15
Topic: Immunohistochemistry
Lecturer: Vaughan
Place: RPCI CCC Room 413

The following topics will be discussed:
I. Characteristics of Antibodies
1. Polyclonal versus monoclonal
2. Specificity of antibody
3. Method of increase specificity/quench Anon-specific@ staining
II. Labeling antibodies for detection of antigen
1. Directly conjugated antibodies versus multi-step staining
2. Enzyme conjugation to primary or secondary antibodies
3. Fluorochrome labeling of antibodies
4. Avidin/Biotin systems
5. Colloidal gold
III. Sample preparation
1. Single cell suspensions
2. Frozen sectioning
3. Paraffin embedding
4. Immunoelectron microscopy
IV Methodologies for staining tissues/cells with antibodies
1. Sample protocols
2. Practical laboratory demonstration: immunoglobulin distribution on the surface/interior of splenic B cells

Week 16
Topic: DNA Sequencing
Lecturer: Kazim
Place: Biophysics Lab, Science Building, Room 4932

The lecture will be an overview of the theory and applications of analytical and synthetic techniques for proteins and DNA that are performed in the Biopolymer Facility. The demonstration will focus on DNA synthesis and capillary electrophoresis of synthetic oligonucleotide. These techniques can be demonstrated within the time allotted for this session.