بسم الله الرحمن الرحيم

Background ( The cell, Nucleus ). Cell cycle ( Mitosis & Mieosis ). Karyotype definition, procedure and importance. Cytogenetics lab. Chromosome analysis methods. Overview of some genetic disorders. Conclusions.

The basic unite of life. The simplest structure capable of independent existence ( By human power ). Consists of: Cytoplasm, bound by a cell membrane and centrally placed nucleus. Cells that have the same general function are often grouped together to form TISSUE (muscle).Tissues combined in larger functional unite to form ORGANS (Kidney).Organs can be grouped by function into ORGAN SYSTEMS (Respiratory System).

Every cell has a nucleus at some stage of its existence in higher organisms. Cells lacking nuclei are severely limited in their metabolic activities. The nucleus: is the site of genetic material ( DNA ), which determines the specific morphologic, biochemical and metabolic characteristics of the cell.

Chromosome: A condense of the threads of chromatin (DNA-protein complex) in the nucleus forming multiple levels of coiled organized structures. DNA: is a double helix made up of two strands, each strand is composed of nucleotides, each consisting of a sugar molecule, a phosphate group and one of four bases: Adenine, Guanine, Thymine and Cytosine.

Mitosis: Each chromosome replicates itself and one copy of each chromosome is delivered to each of two resulting daughter cells. Meiosis: When one diploid cell containing two copies of each chromosome—one from the mother and one from the father— produces four haploid cells containing one copy of each chromosome.

Definition: The transition of interphase to cell division (mitosis) and back to interphase. When the cell is not dividing; it is said to be in interphase. Interphase is the time when the nucleus is the most active in metabolism. This is the time when the cell prepares itself for division. ( copy the DNA, increase the size ). There are four distinct stages in the cell cycle.

Definition: A display of the chromosomes of a cell by lining them up, beginning with the largest arm oriented toward the top of the karyotyping sheet. Each chromosome is paired with its homologous chromosome,its exact match in size and structure, though the homologous chromosomes may carry different alleles of the same gene. During the cell cycle, at the M phase and exactly during the metaphase ( mitosis ), the chromosomes are at their most condensed state, with spindle fibers attaching to the area of the centromere called the kinetochore.

A typical metaphase chromosome consists of two arms separated by a primary constriction or centromere. A chromosome may be characterized by its total length and the position of its centromere. In humans seven (A–G) groups of autosomes are recognized and sex chromosomes (X,Y) are placed last.

Metacentric A chromosome with the centromere at or near the middle. A chromosome has a centromere somewhat displaced from the middle point. A chromosome with a centromere is obviously off center. Chromosomes have their centromeres very near one end. submetacentric very submetacentric Acrocentric

Cytogenetics: Is the study of chromosome structure, pathology, function and behaviour. Chromosomes are best studied at mitotic or meiotic metaphase. Samples suitable for karyotyping: Bone marrow, Lymph nodes, Leukemic blood, Solid tumors, Pleural fluid, Ascetic fluid, Skin, Fibroblasts, PB, Amniotic fluid… etc.

Sample : Peripheral Blood. – Collected in : Green top tube “ Sodium Heparin “. – Fresh, Sterile containers ( Tissues ). – In moist: Isotonic slaine for solid samples, e.g.: solid tumors. – Must never be frozen. – Communication between lab. Specialist and the referral personnel is important. – Labeled with patient name, age, sex and Dx. OR will be rejected !! – After centrifugation: take the “ buffy coat layer “. – Also you can use: whole blood directly.

Culture : RBMI 1640 – Using STRICT aseptic technique. – Media enriched with: Vitamins, Amino acids. – E.g.: Fetal Calf Serum, Newborn Calf Serum, L-Glutamine. – Phytoheamagglutinine ( PHA ): mitogenic agent: Because: in healthy adult lymphocytes are not divided naturally… – Optimum volume of PHA is very important; killing the lymphocytes if increased and insufficient mitotic figures if little amount. – Duration: 72 hrs. ( minimum 48 hrs. for stat samples ). – Incubation: at 37 ̊C with 5 % Co2. – For successful culture you need: suitable T ̊, PH, Co2.

Harvesting: – All the procedures done after 72 hrs of incubation at 37 ̊C; to obtain metaphases. – Colcemid ( Colchicine or Velban ): are examples of mitotic arresting agents. By preventing the synthesis of spindle fiber. Also, causes chromosomal contraction and condensation. For 30 min. – Hypotonic agent ( 0.56 % KCl for 30 min ): TO a- increase cell volume. b- spread the chromosomes. – Fixation : using Methanol + Acetic Acid ( 3 / 1 ) TO: a- Remove water from the cells. b- Kills the cell, lyses RBCs and preserve. c- Hardening the membranes and chromatin. d- Preparing the chromosomes for banding procedure. – 3 times of centrifugation + removing supernatant + adding fixative + centrifugation again at 1000 rpm for 8 min using spinning rotor.

Slide making: – The art of slide making !! – The most critical aspect of good banding preparations. – Immerse slide in methanol and allowed to dry. ( or cold D.water ). – Hold the slide on horizontal edge ( 20 ̊ angle )on paper towels. – Place 2-3 drops of cell suspension across the horizontal axis of the slide. – Label the slide, wait to dry. – Using controlled humidity ( % ) and temperature reading ̊ C. – Too fast or too slow drying can rupture the cells with poor spreading of chromosomes.

Staining: GTG ( G-Bands with Trypsin and Giemsa ) – After keeping the slides at the oven at 80 ̊ C for 2 hrs. Then keep them outside to cool. – Using Trypsin treatment during the staining procedures. – Shows more clear bands. – Trypsin : exact mode of action still unknown. But Wang & Federoff suggested that Trypsin hydrolyzed the protein component of the chromatin, thereby allowing the Giemsa dye to react with the exposed DNA.

DescriptionStep# Whole blood or Buffy coatSample Receiving1 RPMI + PHA / for 72 hrs. / At 37°C / 5 % Co 2 Culturing2 Add Colcimide / Keep for 30 min.Harvesting rpm / for 8 min / then keep pelletCentrifugation4 KCl 0.56 % / keep for 30 minHypotonic Agent5 3 to 1 Methanol to Acetic acid / add 10 drops / Gentle mixFixation6 Keep pellet / add fixative / repeat 3 timesCentrifugation7 Keep the pellet + Add new fixative / Pure lymphocytesDiscard supernatant8 Hold horizontally (20°) / 2-3 drops to the axis of the slide / Let dry with humidity Slide making9 GTG stain / Treated with TrypsinStaining10 After each slide staining, check under microscope..Karyotype11

Chromosomes Abnormality

Aneuploidy: the most common type of clinically significant chromosome abnormality. An abnormal chromosome number due to an extra or missing chromosome. Always associated with physical or mental maldevelopment.

Trisomy 21 Down Syndrome Trisomy can exist for any chromosome of the set. Trisomy 21 : is the most common type in liveborn infants. Kryotype: 47,XX,+21 OR 47.XY,+21 Individuals with Down syndrome have a typical facial appearance. All have some degree of mental retardation, but for most it is mild or moderate. Chromosomes Abnormality Numerical Abnormalities Examples

Trisomy 13 Patau Syndrome Is the presence of 3 chromosomes at chromosome number 13. A very serious condition, only about 5% of babies with this disorder can survive and past their first year. Most pregnancies involving Trisomy 13 end in miscarriage. Children with Trisomy 13 : usually live with severe retardation. Presenting with small head, extra fingers. > 80 % died within the first month. Chromosomes Abnormality Numerical Abnormalities Examples

Monosomy Turner syndrome (Monosomy X) An absence of an entire sex chromosome. all or part of one of the sex chromosomes is absent. Girls with Turner syndrome typically experience gonadal dysfunction which results in amenorrhea and sterility. Chromosomes Abnormality Numerical Abnormalities Examples

Structural abnormalities Balanced Rearrangement e.g. Translocation Burkitt’s Lymphoma 46,XX,t(8;14)(q24;q32) Unbalanced Rearrangement e.g. Deletion Prader-Willi syndrome 46,XY,del(15)(q11.2q13.1)

Understanding the cell cycle is very important in genetics. More diseases can be diagnosed by karyotyping. “ The Art of Cytogenetics “. Cytogenetics methods may vary between labs and even for each sample type. Examining each slide after staining is highly recommended, to evaluate the Trypsin treatment. Analyze the karyotype ( 20 cells ) for any numerical or structural abnormalities.