Cancer Terms PDF

Cancer Terms Neoplasia Cancer Malignant Tumor Neo= New Plasia = Tissues Most common known term Tumor means swelling or Neoplasia may be Is uncontrolled division of mass, it refers to...

Cancer Terms Neoplasia Cancer Malignant Tumor Neo= New Plasia = Tissues Most common known term Tumor means swelling or Neoplasia may be Is uncontrolled division of mass, it refers to a mass of benign (not cancer). abnormal cells in the part non-structured new cells of the body with no known physiological Neoplasm is defined as purpose in the body. new and abnormal growth of tissue  Difference Between Tumors Benign Tumor Malignant Tumor Non-cancerous tumors Cancerous tumors Is composed of cells that won’t invade other unrelated tissues or organs. Is composed of cells that invade the basement membrane and invade or spread other parts of the body. Other terms may be usedin metastasizing to distant sites by means descrption as “mass” and “lump”. of vascular system (blood stream/  Microscopic Features A. Hyperplasia An increase in the number of cells in an organ or tissue. These cells appear normal under a microscope.  B-Metaplasia Metaplasia describes a change where the differentiated cells in a tissue are replaced by differentiated cells normally found in another organ or tissue type. It can occur almost anywhere in the body although it is most commonly seen in the esophagus, stomach,. Metaplasia on its own is a non-cancerous change. However, there are different types of metaplasia, and some types can increase the risk of developing cancer over time. For example, intestinal metaplasia in the esophagus is associated with an increased risk of developing a type of esophagus cancer called adenocarcinoma.  Microscopic Features C. Dysplasia Dysplasia is the abnormal growth or development of cells within tissues or organs. It’s a term used in pathology to describe cells that look abnormal under a microscope but are not cancerous. These abnormal cells can be a sign of early changes that could lead to cancer, but dysplasia itself is not cancer. It indicates a disorderly but non-cancerous cell growth, where the cells have not yet invaded nearby tissues or spread to other body parts.  Feature Hyperplasia Metaplasia Dysplasia reversible if the stimulus is reversible if the stimulus is Reversibility reversible if caught early removed. removed. Chronic irritation, infection, or Cause stimulation (e.g., hormonal,). Chronic irritation or stress inflammation, or progression from hyperplasia or metaplasia. Cells are normal in Normal-appearing cells, but of Cellular Cells show abnormal size, shape, and appearance but increased in a different type than usual for Appearance organization number. that tissue. Barrett's esophagus (columnar Benign prostatic hyperplasia Examples cells replace squamous cells in Cervical dysplasia (BPH) the esophagus). Low risk but can increase the Risk of Low risk, but metaplasia can High risk of progression to cancer, risk in certain contexts Cancer progress to dysplasia, especially if untreated (endometrial hyperplasia). Commonly Glandular or epithelial tissues Epithelial tissues Epithelial tissues Affects Type of Epithelial Tissue Function Stratified Squamous Protection against Simple Squamous Epithelium Diffusion, filtration Epithelium abrasion, pathogens Simple Cuboidal Epithelium Secretion, absorption Stratified Cuboidal Epithelium Protection, secretion Simple Columnar Epithelium Absorption, secretion Stratified Columnar Epithelium Protection, secretion Pseudostratified Columnar Secretion of mucus, movement of mucus by cilia Transitional Epithelium Stretching to accommodate urine storage Different types of tumors Hematologic Tumors Haematologic Lymphoma Leukaemia Multiple myeloma Blood Components Lymphocytes Hematopoietic cells Plasma cells Lymphoid origin Myeloid origin  Carcinoma The epithelium is a type of tissue composed of one or more layers of cells that cover the body’s surfaces, line internal cavities and organs. ‫ث‬ examples Skin: Forms the outer covering of the body, protecting against dehydration, injury, and infections. The surface is made up of specialized squamous cells. Glands: Both endocrine (releasing hormones directly into the blood or lymph) and exocrine (releasing substances through ducts to an epithelial surface) glands are derived from epithelial tissue.  Types of carcinomas include: Squamous cell carcinoma: This is a type of cancer made up of abnormal squamous cells. Adenocarcinoma: This is a type of cancer made up of glandular cells. Neuroendocrine carcinoma: This is a type of cancer made up of cells that have properties of both epithelial cells and endocrine cells. This type of cancer can also arise in almost any location, but it is most commonly found in the lungs, skin, and digestive tract.. As a group sarcomas are rare compared to other types of cancer (such as carcinoma and lymphoma).  What is Histochemistry? Histo = histology: Chemistry = chemical reactions The branch of science concerned with the qualitative and quantitative assessment of chemical compounds in a cell/tissue using stains/dyes and microscopy. It is a marriage between bio/chemistry and cyto/histology 16  Incisional Biopsy It is performed when removal of entire lesion is impossible. Often performed before major surgical procedure.  Excisional Biopsy In this technique, the entire lesion is removed, usually with a rim of normal tissue. It is performed when the lesion is smaller in size. The procedure serves the diagnostic and therapeutic function.  Punch Biopsy It is done by biopsy forceps.  Core Needle Biopsy It is done with a special type of wide-bore biopsy needle. It permits a percutaneous approach to internal structures  Curettage Biopsy Curetting are usually done for diagnosis of endometrial disease.  Handling of Specimen Specimen should be transported in glass, plastic or metal container or in a plastic bag in 10%formalin. If formalin is not available at hand, place the specimen in refrigerator at 4oC to slow down autolysis..  Sampling for Histopathological Examination: Tissue submitted for histopathology must not be more than 3 mm thick and not larger than the diameter of the slides used. Most specimens from solid tissues are cut in the form of pieces measuring 10 to 15 mm on the slides and 2 to 3 mm in thickness. Discrete areas of calcification or ossification should be taken out and should be decalcified in nitric acid.  1. Fixation Definition: This is the process by which the constituents of cells and tissue are fixed in a physical and a chemical state so that they will withstand subsequent treatment with various reagents with minimum loss of architecture.  Properties of an Ideal Fixative: 1. Prevents autolysis and bacterial decomposition. 2. Preserves tissue in their natural state and fix all components. 5. Avoid excessive hardness of tissue.  2. Tissue Processing: A. Dehydration: Tissues are dehydrated by using increasing strength of alcohol; The duration for which tissues are kept in each strength of alcohol depends upon the size of tissue, fixative used and type of tissue. The volume of alcohol should be 50-100 times that of tissue.  B. Clearing: *The next step alcohol should be replaced by paraffin wax. *As paraffin wax is not alcohol soluble, we replace alcohol with a substance in which wax is soluble. This step is calling clearing. *Clearing of tissue is achieved by any of the following reagents: Xylene Chloroform Benzene Xylene is commonly used.  D. Blocking: *Impregnated tissues are placed in a mould with their labels and then fresh melted wax is poured in it and allowed to settle and solidify. Once the block has cooled sufficiently to form a surface skin it should be immersed in cold water to cool it rapidly. *After the block has completely cooled it is cut into individual blocks and each is trimmed. Staining: *Staining is a process by which we give colour to a section. *There are hundreds of stains available. *Classification of Stains: Generally the stains are classified as: A. Acid stains B. Basic stains C. Neutral stains  Classification of Stains: Acid Dyes: *In an acid dye the basic component is coloured and the acid component is colourless. *Acid dyes stain basic components *e.g. eosin stains cytoplasm red. Basic Dyes: *In a basic dye the acid component is coloured and the basic component is colourless. *Basic dyes stain acidic components *e.g. basic fuchsin stains nucleus blue. Neutral Dyes: *When an acid dye is combined with a basic dye a neutral dye is formed. *As it contains both coloured radicals, it gives different colours to cytoplasm and nucleus simultaneously. This is the basis of Leishman stain.  Procedure of staining * *Haematoxylin and Eosin staining: It is the most common used routine stain in histopathology laboratory  Special Stains: 1.PAS (Periodic Acid Schiff) stain: This stain demonstrates glycogen 2.Congo-red: It is used for identification of amyloid. 3.Sudan-Black: It is used for fat staining. 4.Masson’s Trichrome: It is used for differentiation of connective tissue 1. Leukemia: is a cancer that starts in the blood-forming tissues, such as the bone marrow, and leads to the overproduction of abnormal white blood cells. The types of leukemia are based on how quickly the disease progresses (acute vs. chronic) and the type of white blood cell affected (lymphoid vs. myeloid): Acute lymphocytic leukemia (ALL): Affects immature lymphocytes, progresses rapidly. Acute myeloid leukemia (AML): Affects immature myeloid cells, progresses rapidly. Chronic lymphocytic leukemia (CLL): Affects mature lymphocytes, progresses slowly. Chronic myeloid leukemia (CML): Affects mature myeloid cells, progresses slowly  2. Lymphoma Lymphoma is a cancer that starts in the lymphatic system, which is part of the immune system. It primarily affects lymphocytes, a type of white blood cell. A lymphocyte is a type of white blood cell (leukocyte) in the immune system Lymphocytes include T cells (for cell-mediated and cytotoxic adaptive immunity), B cells (for humoral, antibody-driven adaptive immunity) There are two main categories of lymphoma: Hodgkin lymphoma (Hodgkin’s disease): Characterized by the presence of Reed-Sternberg cells, a specific type of abnormal cell. Non-Hodgkin lymphoma (NHL): A more diverse group of lymphomas that do not have Reed-Sternberg cells. It includes various subtypes based on how the lymphoma behaves (aggressive vs. indolent) and the type of lymphocyte affected (B-cell vs. T-cell).  3. Multiple Myeloma Multiple myeloma is a cancer that affects plasma cells, a type of white blood cell responsible for producing antibodies. In multiple myeloma, cancerous plasma cells accumulate in the bone marrow, where they interfere with the production of normal blood cells. This can lead to bone damage, kidney problems, and immune system dysfunction.  1. Complete Blood Count (CBC) The diagnosis of leukemia, including the types mentioned in your image (ALL, AML, CLL, and CML), involves a combination of clinical evaluations, laboratory tests, and imaging studies. Here’s a general outline of how these types of leukemia are diagnosed: 1. Complete Blood Count (CBC) and Blood Smear CBC measures the levels of red blood cells, white blood cells, and platelets in the blood. Leukemia often shows abnormal levels of white blood cells, with immature or abnormal cells appearing in the blood blood smear can reveal abnormal-looking cells under a microscope, which may indicate the presence of immature white blood cells.  Common CBC Findings in Leukemia 1.White Blood Cell (WBC) Count: Elevated or reduced white blood cell counts are common. In acute leukemia (both ALL and AML), the WBC count may be extremely high due to the overproduction of immature cells (blasts). However, in some cases, the WBC count may also be lower than normal. Normal WBC Range: 4,500 - 11,000 cells/µL Leukemia: WBC counts can range from very low to over 100,000 cells/µL. 2. Hemoglobin (HGB) and Hematocrit (HCT): Both hemoglobin and hematocrit levels are often reduced in leukemia due to anemia, which results from the bone marrow's inability to produce sufficient red blood cells. Platelet Count: Platelet counts are typically low in patients with leukemia due to the suppression of normal bone marrow function.. Blasts: In acute leukemia, immature white blood cells called blasts can be found in the peripheral blood. In a healthy individual, blasts are usually confined to the bone marrow and are rarely seen in a CBC. ALL/AML: A high percentage of blasts can be seen (often > 20% of the white blood cells). CLL/CML: Blasts are generally less common, but in blast crisis (a severe phase of CML), blasts can rise significantly  2. Bone Marrow Aspiration and Biopsy A sample of bone marrow is taken, usually from the hip bone, using a needle. This sample is examined under a microscope for the presence of abnormal leukemia cells. The bone marrow biopsy helps confirm the diagnosis and differentiate between the types of leukemia. ALL and AML can be diagnosed by finding immature cells (blasts) in the bone marrow, while CLL and CML show an increase in more mature white blood cells.  3. Immunophenotyping and Flow Cytometry This test identifies specific proteins (antigens) on the surface of leukemia cells, which helps determine the exact type of leukemia. Flow cytometry is commonly used for immunophenotyping. It helps to distinguish between lymphocytic (ALL and CLL) and myeloid (AML and CML) types of leukemia by identifying unique markers on the cells.  4. Cytogenetic Analysis Cytogenetic tests look at the chromosomes of leukemia cells to identify genetic abnormalities, such as translocations or deletions. For example, the Philadelphia chromosome (a translocation between chromosomes 9 and 22) is often found in CML. These tests can help confirm a diagnosis and guide treatment decisions.  5. Molecular Testing (PCR and FISH) Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) are advanced molecular techniques used to detect specific genetic mutations or chromosomal changes in leukemia cells. For instance, BCR-ABL1 gene fusion is often tested in CML, and specific mutations like FLT3 may be tested in AML.  6. Imaging Studies Imaging techniques like CT scans, MRI, or ultrasound may be used to detect leukemia in lymph nodes, the spleen, liver, or other organs. This is especially common in more advanced cases where the cancer has spread. CD- Markers A cluster of differentiation (CD) is a single or group of molecules on the surface of a cell that is highly specific to that cell, allowing one to identify it amongst others. This system is used for the identification and investigation of cell surface molecules providing targets for immunophenotyping of cells.  Flow cytometry Flow cytometry is a technology that rapidly analyses single cells or particles as they flow past single or multiple lasers while suspended in a buffered salt-based solution. Flow cytometry is a powerful tool that has applications in immunology, molecular biology,  Flow Cytometer Mechanism Sample injection 1. The machine uptake the fluid into narrow vessels 2. The sample is mixed with a saline stream in order to form a straight line of the cells 3. The vessels become narrower to cause the cell to be suspended into one cell passing at a time Excitation 1. This Flow (straight line of cells) pass through a laser beam 2. As the single cell pass through the beam of laser the laser scatters into different directions  Flow Cytometer Mechanism 6. The light scatters by 2 manners 1. Side scatter 2. Forward scatter 7. Forward scatter is proportional to the size of the cell 8. Side scatter Side scatter detector are located perpendicular to the path of the laser beam The side scatter is proportional to the sample and internal complexity Flow Cytometer Mechanism Fluorescence detection Fluorophores present in the cell are excited by the correct wavelength of the laser beam The light emitted by the fluorophore is directed along the path with emission filters that allow for the detection multiple fluorophores The light emitted by the fluorophore can be qualitative or relative quantification It also detects different colours emitted from the same cell The data is plotted into a histogram or a dot plot The light filters is the main component for reading fluorophores  Uses of Tumor Markers: Screening: Some markers are used for early detection of cancers, though they are not always specific. Diagnosis: Certain markers help in confirming a diagnosis, especially when combined with other tests. Prognosis: Tumor marker levels can provide information about the aggressiveness of cancer. Monitoring treatment: Changes in levels of tumor markers during treatment can indicate how well the cancer is responding to therapy. Detecting recurrence: After treatment, tumor marker levels can be monitored to check for cancer recurrence.   Tumor markers are defined as a biochemical substance (e.g. hormone, enzymes or proteins) synthesized and released by cancer cells or produced in the host in response to cancerous substance. Types of Tumor Markers Soluble markers – classical tumor markers , various chemical substances Circulating cellular elements – circulating tumor cells, circulating endothelial cells and their precursors Genetic abnormalities – detection of mutations in oncogenes and tumour supressor genes, protein products of oncogenes, further changes  A Good Tumor Maker Should Have Those Properties:  1. A tumor marker should be present in or produced by tumor itself.  2. A tumor marker should not be present in healthy tissues.  3. Plasma level of a tumor marker should be at a minimum level in healthy subjects and in benign conditions. 52  4. A tumor marker should be specific for a tissue, it should have different immunological properties when it is synthesized in other tissues. 5. Plasma level of the tumor marker should be in proportion to the both size of tumor and activity of tumor.  6. A tumor marker should be present in plasma at a detectable level, eventhough tumor size is very small 12/20/2024 53  1- Hormonal Human Chorionic Gonadotropin (hCG): Associated with: Germ cell tumors (testicular and ovarian cancer),  Ferritin Ferritin is a marker for Hodgkin lymphoma, leukemia, liver, cancer. Thyroglobulin It is a useful marker for detection of differentiated thyroid cancer. 12/20/2024 55  2- Oncofetal Antigen S CEA (Carcinoembryonic antigen) Associated with: Colorectal cancer, but also lung, breast,.  Carcinoembryonic antigen (CEA) It is a cell-surface protein and a well defined tumor marker. CEA is a marker for colorectal, gastrointestinal, lung and breast carcinoma. CEA levels are also elevated in smokers and some patients having benign conditions such as cirrhosis, rectal polips, ulcerative colitis and benign breast disease. CEA testing should not be used for screening. Some tumors don’t produce CEA. It is useful for staging and monitoring therapy. 12/20/2024 57  AFP (Alpha-fetoprotein) Associated with: Liver cancer (hepatocellular carcinoma), germ cell tumors (testicular and ovarian cancer).  α-Fetoprotein (AFP) α-fetoprotein is a marker for hepatocellular It is also increased in pregnancy and chronic liver diseases. 12/20/2024 59  3- Lineage specific protein Prostate-Specific Antigen (PSA): Associated with Prostate cancer Use: Primarily for screening and monitoring prostate cancer progression or recurrence after treatment.  Immunoglobulin: Monoclonal immunoglobulin has been used as marker for multiple myeloma for more than 100 years. Monoclonal paraproteins appear as sharp bands in the globulin area of the serum protein electrophoresis. marker for multiple myeloma. Beta-2 Microglobulin (B2M): Associated with: Multiple myeloma When CLL cells replicate, they release B2M into the blood. doctors use the B2M test to assess the number of cancer cells in the body and their growth rate, and it also helps determine the response to treatment.  4- DNA markers RECEPTOR MARKERS Estrogen and progesterone receptors are used in breast cancer as indicators for hormonal therapy. Patients with positive estrogen and progesterone receptors tend to respond to hormonal treatment. Those with negative receptors will be treated by other therapies. 12/20/2024 62  PROTEIN MARKERS β2-microglobulin β2-microglobulin is a marker for multiple myeloma, Hodgkin lymphoma. It also increases in chronic inflammation and viral hepatitis. 12/20/2024 63  GENETIC CHANGES Four classes of genes are implicated in development of cancer: 1) protooncogenes which are responsible for normal cell growth and differentiation 2) tumor suppressor genes Alterations on these genes may lead tumor development. 3)apoptosis-related genes are responsible for regulation of apoptosis 4)DNA repair genes which are involved in recognition and repair of damaged DNA. 12/20/2024 64  5- Mucin and glycoprotein Cancer Antigen 15-3 (CA 15-3) and Cancer Antigen 27.29 (CA 27.29): Associated with: Breast cancer. Cancer Antigen 125 (CA-125): Associated with: Ovarian cancer. Use: Monitoring treatment and recurrence of ovarian cancer. CA 19-9 (Cancer Antigen- 19-9) Associated with: Pancreatic cancer,  6- ENZYMES  Alkaline Phosphatase (ALP)  Increased alkaline phosphatase activities are seen in primary or secondary liver cancer. 12/20/2024 66 Limitations of Tumor Markers: Non-specific: Elevated levels of many tumor markers can occur in non-cancerous conditions. Not all cancers have markers: Many cancers do not produce specific markers, limiting their use. Cannot be used alone for diagnosis: Tumor markers are rarely used as the sole diagnostic tool due to false positives or negatives.  Examples for tumor induction: Cancer Tumor Induction for establishment of animal model: 1- By absolute ethanol - (Induction of oral cancer results in squamous cell carcinoma, given to rats for 16 weeks) (Induce colon cancer , given to rats for 24 weeks, longer period than previous) 2- By Chloroform - ( induce hepatocellular carcinoma with treatment to rats /mice for 104 weeks) (induce gastrointestinal tract tumor in rats, administered for 39 weeks, shorter period than previous) 3- Trichloroethylene, ( Induce liver tumor, By , 4- Diethylnitrosamine DEN: Diethylnitrosamine (DEN)-induced carcinogenic liver injury in mice 5- Ethylene Oxide ethylene oxide was associated with increases in leukemia in female rats and peritoneal mesotheliomas (malignant tumors) in male rats.  The Usage of Animal Cell Culture 1) Model System ▪ Provide a good model system for studying i) basic cell biology and biochemistry; ii) interactions between disease-causing agents and cells; iii) effects of drugs on cells; process and triggers for aging and nutritional studies.  2) Toxicity Testing ▪ Widely used to study the effects of new drugs, cosmetics and chemicals on survival and growth in wide variety of cell types. 3) Cancer Research ▪ To study differences in both normal cells and cancer cells. ▪ To study the mechanism of cancer with the use of use chemicals, viruses and radiation to convert normal cultured cells to cancer causing cells.  Types of continuous culture 1) Cell lines finite life, senesce after approximately thirty cycles of division usually diploid and maintain some degree of differentiation. 2) Continuous cell lines can be propagated indefinitely generally have this ability because they have been transformed – tumor cells. – viral oncogenes – chemical treatments. What is cell culture? Cell culture refers to the removal of cells from a mammal or an animal, and their subsequent growth in a favorable artificial environment.  Why we grow animal cells in culture? TO INVESTIGATE THE NORMAL TO TEST THE EFFECTS OF TO PRODUCE ARTIFICIAL TISSUE TO SYNTHESIZE VALUABLE PHYSIOLOGY OR BIOCHEMISTRY COMPOUNDS ON SPECIFIC CELL BY COMBINING SPECIFIC CELL PRODUCTS (BIOLOGICALS) FROM OF CELLS TYPES. TYPES IN SEQUENCE. LARGE-SCALE CELL CULTURES.  Main types of cell culture `1. Primary cell culture 2. cell line 1. Primary cell culture Derived directly from normal animal tissue following dissociation 1. Explant cultures 1. speeds up the process by adding digesting (proteolytic) enzymes such as trypsin or collagenase to the tissue fragments to breakdown bonds holding the cells together 2. pieces of tissue are immersed in culture medium where they will begin to divide and grow 3. After the first subculture, the primary culture becomes known as a cell line. Environment (CO2, temperature 37oC, humidity): Are the incubation conditions  Advantages Usually retain many of the differentiated characteristics of the cell in vivo. Disadvantages Primary cell culture Such cultures are initially heterogeneous but later become dominated by fibroblasts. The preparation of primary cultures is labor intensive. They can be maintained in vitro only for a limited period of time.  1. Anchorage dependant - Most animal derived cells. - Adhere to bottom of a flask and form a monolayer. - 2. Anchorage independent - Cells associated with body fluid (eg. blood cells). -. 2. Cell lines Continuous cell line Cell lines that can be propagated indefinitely Continuous cell line can be obtained by: Tumor cells Viral oncogene Chemical treatment  Epithelial-like cells Mammalian cell morphology Most mammalian cells in culture can be divided into three basic categories based on their morphology: Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated shapes and grow attached to the surface of growth container. Epithelial-like cells are polygonal in shape with more regular dimensions and grow attached to the surface of growth container in discrete patches. Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface. Fibroblastic cells Cell Culture Media Is a chemically complex liquid mixture suitable for supporting growth of cells in culture for several generations.  The optimal pH for animal cell culture media typically falls within the range of 7.2 to 7.4 Glucose is used to provide an energy Media source as well as a precursor for biosynthesis, such as ribose needed in formulation nucleic acid synthesis. Lactic acid is the major product of glycolysis. ▪ Amino acids are included as a source of precursors for protein synthesis. ▪ Glutamine for the TCA cycle intermediates. ▪ The content of vitamins and hormones varies enormously from one medium formulation to another ▪ Salts are included so that the solution is isotonic and has no imbalances with the intracellular content Media Supplements Serum Serum is normally added to culture media Cow (bovine) or horse (equine) serum is most commonly used while fetal calf serum (FCS) is particularly effective because of its high content of embryonic growth factors..  Disadvantages of serum: Media Chemically undefined and variation Supplements between batches. Expensive, account for 70–80% of the cost of some formulations. Contain high protein content which can compromise (interfere with) the extraction and purification procedures for cell- secreted proteins. Vulnerable to contamination with infectious agents such as viruses and prions.  Antibiotics Antibiotics are often included in media for Media short-term cultures in order to reduce the risk of contamination. Supplements Penicillin G to inhibit the growth of Gram positive bacteria. Streptomycin to inhibit the growth of many Gram-negative and Gram-positive bacteria. Amphotericin B as an antifungal agent. Cryopreservation of cells Transfer to liquid nitrogen storage tank Why cryopreserve cells? Reduced risk of microbial contamination. Reduced risk of cross contamination with other cell lines. Reduced risk of genetic drift and morphological changes. Research conducted using cells at consistent low passage.  Consistency and reproducibility of results Advantages of Reduced statistical analysis of variance Control physico-chemical environment Tissue Culture Control of physiological conditions Change of selective media Cryopreservation  Chemical and bacterial contamination Limitations of Stringent aseptic procedures Disposal of wastes tissue culture Quantity and cost – medium, pipets etc Genetic instability Phenotypic instability - Dedifferentiation