Chapter 2: Introduction to Cytology PDF

# Chapter 2: Introduction to Cytology ## Cytogenetics - A branch of genetics that studies the function of the cell, specifically the chromosomes, in the process of inheritance. - Relatively a new field compared to systematics and evolution, given that chromosomes were not properly explained until...

# Chapter 2: Introduction to Cytology ## Cytogenetics - A branch of genetics that studies the function of the cell, specifically the chromosomes, in the process of inheritance. - Relatively a new field compared to systematics and evolution, given that chromosomes were not properly explained until its discovery in plant cells in 1842 by Karl Wilhelm von Nägeli. - Among animal cells, Walther Fleming described the chromosomes using salamander. - The number of human chromosomes was mistakenly identified to 48. - Only in 1956, it became generally accepted that the human karyotype included only 46. - Relies heavily on the techniques it employs. - Karyotyping - Fluorescent in-situ hybridization (FISH) - DNA microarray technology ## Karyotyping - One of the most basic techniques in genetics. - Refers to the routine analysis of chromosomes at the metaphase stage which have been banded using trypsin followed by stains such as Giemsa, Leishman, or a combination of the two. ## Giemsa Banding (G-banding) - Especially useful in creating a karyogram. - Stain specifically binds to phosphate groups of the DNA and where there is high adenine-thymine bonding. - Can identify chromosomal aberrations such as translocations and rearrangements. ## Q banding - Much earlier fluorescent staining technique. - Q stands for Quinacrine. - Developed by Toborson Caspersson in the late 1960s. - Less used today. ## C banding - Stains the heterochromatin near the centromere. ## Nucleolar Organizing Region Stains (NOR stains) - Highlights the satellites and stalks of acrocentric chromosomes. ## Fluorescent In-situ hybridization (FISH) - Developed in the 1980s. - Refers to a cytogenetic technique that uses fluorescent probes that attaches to specific areas in the chromosome with high degree of sequence complementarity. - Used to detect and localize the presence or absence of specific DNA sequences in the chromosomes. - Can be used to determine the possible cause of a child’s developmental disability, identify pathogens that do not grow well in laboratory conditions and infer evolutionary techniques. ## DNA Microarray Analysis - Makes use of a DNA microarray or a DNA chip. - Is a collection of microscopic DNA spots attached to a solid surface. - Types of microarray: - Traditional solid-phase array with spots attached to a surface. - Alternative bead array using polyesterene beads, each with a specific probe and a ratio of two or more dyes. ## Constitutional Versus Cancer Cytogenetics ### Constitutional Cytogenetics - Diagnosis of heritable genetic abnormalities in children, adults, pregnancy, and fetal loss. - Abnormalities may be inherited or *de novo*. - Examples of abnormalities: - Robertsonian translocation - Trisomy 21 - Klinefelter’s syndrome - DiGeorge Syndrome - XYY ### Cancer Cytogenetics - Detection of acquired, or somatic (versus germline/constitutional) genetic abnormalities, for the diagnosis, prognosis, therapy, and/or monitoring of many types of cancer, esp. hematologic. ## Indications of Cytogenetics ### Constitutional - **Postnatal, childhood growth and development**: - Birth defects, malformations, dysmorphisms, ambiguous genitalia. - Growth: failure to thrive, growth delay, short stature. - Developmental delay: fine and gross motor, speech. - Cognitive: intellectual disability, learning disability. - Neurological: hypotonia, seizures, ataxia. - Behavioral: autism, OCD, psychiatric illness. - **Tissues studied**: peripheral blood, buccal swab, skin biopsy. - **Adolescent, adult sexual development and fertility**: - Amenorrhea, primary or secondary ovarian failure, premature menopause. - Azoospermia, oligospermia, hypogonadism. - History of infertility or spontaneous abortions. - Birth of a child with a chromosomal abnormality. - **Tissues studied**: peripheral blood. - **Prenatal**: - Abnormal maternal serum screening (first or second trimester). - Abnormal cell-free DNA testing (cfDNA), non-invasive prenatal testing (NIPT)/screening (NIPS) - Abnormal ultrasound findings: cystic hygromas/hydrops, cardiac defects, other malformations, IUGR, etc. - Advanced maternal age (AMA), generally ≥ 35 yrs. - Parental or familial chromosome/genomic abnormality. - Tissues studied: amniotic fluid, chorionic villus sampling, fetal tissues. - **Fetal or neonatal demise (products of conception, POC)**: - Tissues studied: amniotic fluid, chorionic villus sampling, fetal tissues. ### Cancer - **Hematologic oncology**: - Myeloid diseases: AML, CML, MDS, MPNs. - Lymphoid diseases: ALL, CLL, NHL, PCNs/MM. - **Bone marrow transplant** - **Other areas of oncology (solid tumors)** - **Tissues studied**: bone marrow, peripheral blood, lymph nodes, solid tumor, pleural fluid, spinal fluid. ## The Chromosomes - Are organized structures containing the DNA of an organism associated with structural proteins called histones and some other factors that help maintain its integrity and facilitate its formation and replication. - Associated with structural proteins (negatively charged), provides structural support. | Organism | Body Cell (2n) | Gamete (n) | |:---------------|:--------------:|:-----------:| | Fruit Fly | 8 | 4 | | Garden Pea | 14 | 7 | | Corn | 20 | 10 | | Tomato | 24 | 12 | | Leopard Frog | 26 | 13 | | Apple | 34 | 17 | | Human | 46 | 23 | | Chimpanzee | 48 | 24 | | Dog | 78 | 39 | | Adder's Tongue | 1260 | 630 | | Fern | | | *** *** - **Linear chromosome**: - Found among eukaryotes (animals). - Supercoiled DNA structure. - Typically 0.2-20 um in length. - Found in the nucleus of a cell. - May exist as unduplicated or duplicated: - Unduplicated appear as lines. - Duplicated contains two identical copies called as chromatids or sister chromatids held together by a centromere - referred to as chromatin in its diffused state. - Chromatids from different chromosomes are referred to as non-sister chromatids. - **Centromere**: Chromatin in its diffused state. - **Chromatids**: - **Sister**: Same/identical. - **Non-sister**: From diff. chromosomes ## Human Linear Chromosome Contains Varied Number of Genes - The x-axis represents the chromosome number - The y-axis represents the number of genes and base pairs ## The Cell Theory - All living organisms are composed of cells. (They may be unicellular or multicellular). - The cell is the basic unit of life. - Cells arise from pre-existing cells. (They are *not* derived from spontaneous generation) ## Cell Cycle - The cells undergo the interphase prior to the M phase. - The dividing cell spends most of its time in interphase, in which they increase in mass and replicate DNA in preparation for cell division. - To aid this process, cells employ cell cycle check points between substages. - Checkpoints acts as both police and quality control agents to ensure that the cell has achieved the right size, replicated the DNA properly and has the proteins and other chemicals needed for cell division. ### Mitotic Phase - The mitotic phase involves the separation of nuclear chromosomes, followed by cytokinesis (division of cytoplasm forming two distinct cells). - At the end of the mitotic cycle, two distinct daughter cells are produced, each cell contains identical genetic material. - Also occurs in the replication of sex cells, or meiosis, upon completion of the cell cycle in meiosis, four daughter cells are produced. ### Interphase - The nucleolus and the nuclear envelope are distinct and the chromosomes are in the form of threadlike chromatin. ### Prophase - The chromosomes appear condensed, and the nuclear envelope is *not* apparent. ### Metaphase - Thick, coiled chromosomes, each with two chromatids, are lined up on the metaphase plate. ### Anaphase - The chromatids of each chromosome have separated and are moving towards the poles. ### Telophase - Spindle fibers are at the poles, and are becoming more diffuse. The nuclear envelope is reforming. The cytoplasm may be dividing. ### Cytokinesis - Division into two daughter cells is completed. ## The Cell Cycle | Phase | Sub-phase | Processes | |:------------------------|:-------------------|:----------------------------------------------------------------------------------| | Resting or Quiescent phase | Gap O | Resting stage; cell is nondividing | | Interphase | Gap 1 (G1) | Growth phase; synthesis of amino acids and other biochemicals needed for the S phase | | | Synthesis (S) | DNA in chromosomes are replicated | | | Gap 2 (G2) | Synthesis of chemicals needed for the production of microtubules | | M phase | Karyokinesis | The cell nucleus divides | | | Cytokinesis | The cell itself divides into daughter cells | ## Mitosis - Nuclear division among somatic cells; cells divide or separate the chromosomes into two identical nuclei. - Relatively a short phase in the cell cycle. - Has four phases: prophase, metaphase, anaphase and telophase. - Ensures that the number of chromosomes of each of the daughter cells is equal to that of the parent cell. | Sub stage | Key events in mitosis | |:----------|:-----------------------| | Prophase | - Nuclear membrane begin to disintegrate | - Nucleoli disappear | - DNA begin to supercoil and appear as chromosomes | | Metaphase | - Chromosomes appear a sister chromatids attached together at the centromere containing the kinetochore | - Centrioles appear on both poles of the cell | - Spindle fibers appear and attach to the kinetochore | | Anaphase | - Chromosomes align at the center of the cell | - Sister chromatids are pulled towards the opposite poles | | Telophase | - Nuclear membrane reappears | - DNA begin to diffuse into the nucleus | - Nucleoli reappears | ## Meiosis - Nuclear division undergone by germ cells (specialized cells). - Involves a course of double division producing four daughter cells with half the number of chromosomes as that of the parent cell (2n). - This process is required to produce egg and sperm cells for sexual reproduction. - During reproduction, when the sperm and egg unite to form a single cell, the number of chromosomes is restored in the offspring. - Begins with a parent cell that is diploid, meaning it has two copies of each chromosome. - The parent cell undergoes one round of DNA replication followed by two separate cycles of nuclear division, thus resulting in four daughter cells that are haploid (contain half of the number of chromosomes of the diploid parent cell (2n)). - Process is split into: - Meiosis I - reductional phase (haploid cells are made) - Meiosis II - similar to mitosis - Follows a long interphase or preparation stage before entering Prophase I. - Chromosomes begins to become visible. - There is a maternal and paternal version of chromosomes of exact length and centromere position. - In this stage, each chromosome searches for its homologous pair in a process called homology search. - Once done they are referred to as tetrads or bivalents. - Then, *synapsis* or intimate pairing leading to crossing over happens - exchange of genetic material between non-sister chromatids of homologous pairs. - This crossing over process is key feature of meiosis and guarantee genetic diversity among offspring. - Site of exchange is known as chiasmata. | Sub-stage | Key events | |:----------|:-----------| | Leptotene | Individual chromosomes condense to form visible strands within the nucleus. | | Zygotene | Homologous chromosomes pair up during synapsis. | | Pachytene | Crossing over happens. | | Diplotene | Chromosomes separate slightly. | | Diakinesis | - Nucleolus disappear | - Nuclear envelope disintegrate completely | - Spindle fibers begin to form | - **Metaphase I**: Chromosomes line up at the metaphase plate and spindle fibers attach to the centromeres via the kinetochores. - **Anaphase I**: Chromosomes migrate to the poles. - **Telophase I**: - Completed when chromosomes arrived at the poles. - The number of chromosomes is halved in each of the daughter cells, thus meiosis I is referred to as reductional phase. ## Meiosis II - Similar to mitosis. - Prior to meiosis II, cell may undergo an interphase II but no DNA replication will happen. - **Prophase II**: - Begins with the disappearance of the nucleoli and nuclear envelope and the thickening and shortening of chromosomes. - **Metaphase II**: - The chromosomes migrate to the center of the cell and spindle fibers attach to their kinetochores. - **Anaphase II**: - Sister chromatids are pulled apart from one another. - **Telophase II**: - Reorganization of the nucleoli and the cell membrane - Chromosomes again diffuse into the nucleus - At the end of meiosis II, four daughter cells are formed with a haploid number of chromosomes - equational division. ## Meiosis Differs From Male and Female Division - **Spermatogenesis for males**: - Produces four sperms of generally equal potency. - **Oogenesis for females**: - Produces one large ovum and three polar bodies. - These polar bodies serve as depository of chromosomes to comply with the haploid requirement of meiosis and they are then reabsorbed by the body of the female. ## Mitosis vs. Meiosis | | Mitosis | Meiosis | |:--|:------------------------------------------------------------------------------|:-------------------------------------------------------------------------------------------| | | Produces diploid cells (2n) | Produces haploid cells (n) | | | Includes one nuclear division | Includes two nuclear divisions | | | The product is somatic cell | The product is gamete cell | | | Responsible for asexual reproduction | Responsible for sexual reproduction | | | No crossing over | Crossing over takes place | | | Two cells are produced | Four cells are produced | ## Differences Between Mitosis and Meiosis *Mitosis* - **Parent Cells**: 2n (diploid) - **Replication**: DNA is replicated. - **Daughter cells**: 2n (diploid), two daughter cells are produced. - **Key features**: - nuclear division, chromosomes are separated, daughter cells are genetically identical to the parent cell. - Growth and repair of body tissues. *Meiosis* - **Parent Cells**: 2n (diploid) - **Replication**: DNA is replicated. - **Daughter cells**: n (haploid), four daughter cells are produced. - **Key features**: - Produces four daughter cells from one parent cell. - Daughter cells only have half the number of chromosomes. - Crossing over occurs. - Produces gametes (sperm, egg). - Sexual reproduction