DNA Replication and Cellular Processes | Lecture notes Health sciences

Histotechnology

Fixation

 Most important step in histopathology

 Alteration in tissues by stabilizing protein so it

is resistant to further changes

Fixative

 Changes the soluble contents of the cell into

insoluble substances so that they are not lost

during subsequent processing steps (maybe

chemical-fixative solutions or physical-

heat/desiccation).

Fixative Functions

 Prevents decay, putrefaction and autolysis.

 Maintain proper relationship between cells

and extracellular substance.

 Firms tissue for ease of gross dissection and

taking of thin sections for processing.

 Brings out differences in refractive index to

Increase contrast between different tissue

elements.

 Enhance staining.

Fixative Actions

 Kill bacteria and molds (prevents

putrefaction).

 Mordants (links dye to tissue).

 Inactivates enzymes (prevents autolysis).

 Stabilize tissue elements (proteins)

FACTORS AFFECTING FIXATION

 Volume Ratio

 Fixative volume should be 20X greater

than the tissue volume.

 Time

 Place tissue in fixative immediately after

removal.

 Temperature

 ideally, around 45 degrees

 room temperature fixation is ideal for

Electron microscopy

 the rate of penetration is affected by heat

but not by the concentration of fixation

 Increase in temperature will increase the

rate of fixation, rate of autolysis, and

diffusion of cellular elements

 Size of tissues

 ideally, large specimens need to be

opened up upon receipt like intestines,

uterus etc.

 3 mm thickness ideal for sufficient fixation

American Society of Clinical Oncology (ASCO) & College

of American Pathologists (CAP) – released guidelines to

improve accuracy of Her-2 testing for invasive breast

cancer

 Tissue be fixed in 10% neutral buffered

formalin (6-48 hours)

Fixatives in order of decreasing speed of penetration:

1. Formaldehyde

2. Acetic acid

3. Mercuric chloride

4. Ethyl/Methyl alcohol

5. Osmium tetroxide

6. Picric acid

Reagent penetration of Gross specimens

 Large specimens (uteri, bowel resections,

mastectomies) should be opened or sectioned

thinly on removal to prevent autolysis.

 Lymph nodes should also be cut open as the

capsule prevents quick penetration of fixative.

 At ph 7.0 & temperature of 25°C,

Formalin penetrates:

3.6 mm in 1 hour

3.7 7.2 mm in 4 hours

 Cross-linking (complete fixation) is complete in

48 hours

Choice of Fixative

10% Neutral Buffered Formalin

 Components:

Formaldehyde, 37% to 40% 100mL

Distilled water 900mL

Sodium phosphate, monobasic 4g

Sodium phosphate, dibasic 6.5g

 Optimal choice for many reasons:

o fast penetration

o non-coagulant and additive

o prevents alterations during processing

o less shrinkage than other fixatives

o not osmotically active

o hardens tissue better (except alcohol &

acetone)

o tissue can be stored in formalin indefinitely

(except for IHC)

o fixative of choice for

Immunohistochemistry & molecular tests

o cheap & stable

Formaldehyde

 colorless gas commonly obtained in

histopathology laboratory as a 37% to 40%

solution in water

 Commercial solutions are 37% to 40%

formaldehyde but are considered to be 100%

formalin.

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Histotechnology Fixation  Most important step in histopathology  Alteration in tissues by stabilizing protein so it is resistant to further changes Fixative  Changes the soluble contents of the cell into insoluble substances so that they are not lost during subsequent processing steps (maybe chemical-fixative solutions or physical- heat/desiccation). Fixative Functions  Prevents decay, putrefaction and autolysis.  Maintain proper relationship between cells and extracellular substance.  Firms tissue for ease of gross dissection and taking of thin sections for processing.  Brings out differences in refractive index to Increase contrast between different tissue elements.  Enhance staining. Fixative Actions  Kill bacteria and molds (prevents putrefaction).  Mordants (links dye to tissue).  Inactivates enzymes (prevents autolysis).  Stabilize tissue elements (proteins) FACTORS AFFECTING FIXATION  Volume Ratio  Fixative volume should be 20X greater than the tissue volume.  Time  Place tissue in fixative immediately after removal.  Temperature  ideally, around 45 degrees  room temperature fixation is ideal for Electron microscopy  the rate of penetration is affected by heat but not by the concentration of fixation  Increase in temperature will increase the rate of fixation, rate of autolysis, and diffusion of cellular elements  Size of tissues  ideally, large specimens need to be opened up upon receipt like intestines, uterus etc.  3 mm thickness ideal for sufficient fixation American Society of Clinical Oncology (ASCO) & College of American Pathologists (CAP) – released guidelines to improve accuracy of Her-2 testing for invasive breast cancer  Tissue be fixed in 10% neutral buffered formalin (6-48 hours) Fixatives in order of decreasing speed of penetration:

Formaldehyde
Acetic acid
Mercuric chloride
Ethyl/Methyl alcohol
Osmium tetroxide
Picric acid Reagent penetration of Gross specimens  Large specimens (uteri, bowel resections, mastectomies) should be opened or sectioned thinly on removal to prevent autolysis.  Lymph nodes should also be cut open as the capsule prevents quick penetration of fixative.  At ph 7.0 & temperature of 25°C, Formalin penetrates: 3.6 mm in 1 hour 3.7 7.2 mm in 4 hours  Cross-linking (complete fixation) is complete in 48 hours Choice of Fixative 10% Neutral Buffered Formalin  Components: Formaldehyde, 37% to 40% 100mL Distilled water 900mL Sodium phosphate, monobasic 4g Sodium phosphate, dibasic 6.5g  Optimal choice for many reasons: o fast penetration o non-coagulant and additive o prevents alterations during processing o less shrinkage than other fixatives o not osmotically active o hardens tissue better (except alcohol & acetone) o tissue can be stored in formalin indefinitely (except for IHC) o fixative of choice for Immunohistochemistry & molecular tests o cheap & stable Formaldehyde  colorless gas commonly obtained in histopathology laboratory as a 37% to 40% solution in water  Commercial solutions are 37% to 40% formaldehyde but are considered to be 100% formalin.

 Most widely used solution for routine formalin fixation  pH of approximately 6. Specimen identification  Assign each case a unique ID number  1 case = all samples derived from same patient and performed on same day  Identify and verify all components of sample  Label of sample container: patient name, birth date, hospital number Specimen rejection  Discrepancies between requisition and specimen label  Unlabeled or mislabeled specimens  Contaminated specimen/leaking container  No clinical data/ history  Inappropriately identified specimen Gross study  Tissue must be fixed for 6-48 hours and sectioned (5mm) for proper fixative penetration  Gross worksheet o Accession no., number of sections and blocks taken per case, manner of embedding  Samples for gross description only o Specimen submitted for documentation purposes o No microscopic exam; no histologic diagnosis  Special requests: o ex. abortus or calculi to be returned to patient Tissues for gross description only  Prepuce/foreskin  Vaginal mucosa  Scars/cicatrix  Foreign bodies (e.g., bullets, ortho/ medical devices, silicone implants)  Hair, fingernails and toenails removed for cosmetic reasons  Lens cataracts  Nasal septum (rhinoplasty)  Calculi, stones  Eyelid  Placenta from normal spontaneous delivery  Teeth  Fetuses Gross examination and dissection  Each specimen approached with clear goals in mind based on type of specimen and reason/s for surgery  Identify all anatomic structures present (may use diagrams)  Orientation markers o Inking (1 or more colors, liquid paper) o Nicking (usually for laterality) o Sutures: long – lateral (LL), short- superior (SS)  Measurements o Weights taken on intact specimens and in nearest 0.1 grams o 3 dimensions in nearest 0.1cm  Inking margins o Inking small samples may ensure that the entire face of specimen fragment is present on glass slide o When in doubt, ink!  Artifactual ink in non-marginal areas o Blot specimens dry after inking, or o Allow specimen to air dry before cutting to avoid such artifacts  Dissect specimen thoroughly  Identify pathologic processes  Take judicious, representative histologic sections Selection of tissue for microscopic exam  All lesions  Lesional tissue placed in special fixatives for histologic exam o ex. Decalcify, EM  Representative sections of all normal structures not included in other sections  Lymph nodes  All margins, when appropriate  Frozen section remnants Special issues in processing  Lymph nodes: sample all!  Margins o En face: lesion 0.5cm away from margin o Perpendicular: lesion close/abutting margin  Multiple lesions: sample all o if multiple similar lesions, tissue between lesions is sampled to know whether separate or connected Other gross exam issues  Tissue thickness o <3mm, especially fatty tissues o Tissue cassette must not bulge when closed (allow space)  Paper tags: o Use pencil or computer print (prefer dot matrix), not pen or marker  Dissect fat away from lymph nodes before loading

notched lightly or can be marked with India ink or tattoo ink. o Inking-used to indicate margins; different colors may be used  Very important the light pressure be applied over the entire specimen during the orientation and initial chilling so that the tissue will be embedded flat; otherwise complete section cannot be obtained  Orientation of hard tissues such as bones: o embedded diagonally in the mold and not parallel to the mold edges.  Tissues with a wall, such as cysts, gallbladder and gastrointestinal tract, must be embedded on edge so that all layers are visible.  Tubular structures, such as fallopian tubes or the appendix, are embedded in cross-section so that the lumen and all layers of the mucosa, submucosa, and external muscle layers are obvious microscopically.  Skin: o Should be embedded so that the epidermis, or epithelium is facing 1 side of the mold. o If more than 1 piece of skin is to be embedded in the same block, the epidermis of all pieces should be faced to the same side of the mold. o arranged in a line parallel to the longer axis of the mold and never just randomly placed in the mold. Staining  Hematoxylin-most widely used nuclear stain is extracted from logwood (Hematoxylon campechianum) which when freshly cut is colorless but becomes dark reddish-brown when exposed to atmospheric oxidation. The oxidized dye is hematein, a weak anionic dye. Oxidation of Hematoxylin (ripening)  Necessary to convert it to a dye (hematein)  May be achieved by:

Exposing solution to atmospheric oxygen (Erlich solution).
Using oxidizing agents such as sodium iodate, mercuric oxide & potassium permanganate (Harris hematoxylin).  Harris hematoxylin o Hematoxylin 5 g o Absolute ethyl alcohol 50 ml o Ammonium aluminum sulfate (mordant) 100 g o Distilled water 1,000 ml o Sodium iodate (oxidizer) 2.5 g Eosin Counterstain  Most widely used counterstain in the routine staining of sections.  Eosin is the sodium salt of a color acid; the chromophore is in the anionic (-) part of the molecule.  Eosin is fully charged at pH of 7 but because IEP of protein is 6 we must stain below pH 6.  Below pH 4, eosin is converted to free acid , may remain in solution and nonspecifically bind to sections making it appear muddy.  The best staining with Eosin will occur at pH of approximately 4.6 to 5.  Used properly, at least 3 shades of pink can be obtained with eosin alone; o erythocytes should be the deepest of pink; o collagen or muscle/epithelial cells are the intermediate shade of pink and is determined by choice of fixative, duration of fixation, heat during processing, stain formulation, differentiation step and nature of collagen itself.  Nuclei : Blue  Erythrocytes and eosinophilic granules : bright pink to red  Cytoplasm and other tissue elements : various shades of pink  Usual problem: stain unable to bind completely Mounting/ Coverslipping  Tissue has an average refractive index of 1. to 1.54 while most synthetic resins have a refractive index ranging from 1.51 to 1.55.  Cause: o sections are not completely dehydrated before clearing in xylene or a xylene substitute.  Prevention/Correction: o Change all dehydrating and clearing solutions before staining any more sections. o Remove coverslip and mounting medium on mounted sections with xylene, and return to fresh absolute alcohol; after sections are dehydrated, clear with fresh xylene and mount with synthetic resin.  Cause: o Mounting Medium on top of the cover glass.

 Correction /Prevention: o Avoid Mounting Medium on top of the cover glass; if necessary, modify or change technique for applying the cover glass. o I f section is mounted, remove cover glass and remount section with clean cover glass.  Cause: o When sections are allowed to air-dry before mounting ; a drying artifact is created in the tissue This may manifest as granular brown stippling resembling pigment or as glossy black nuclei.  Correction/Prevention: o If sections are mounted, remove the coverslip and mounting medium with xylene ; return to water to rehydrate, and then rehydrate and clear; keep the slide wet with xylene before mounting. Hazard = WEAR PPE Disposal of tissue  Schedule disposal of surgical tissues and body fluids.  Only those samples with official report are to be discarded.  Samples for disposal are removed from formalin and placed in a doubled yellow plastic bag with a newspaper underneath to absorb the formalin.  The plastic bag should be labeled “for disposal” and must be endorsed to the assigned housekeeper.

replication, each of which initiate a replication bubble.  Replication at the Telomere o Eukaryotic chromosomes end in distinctive sequences called telomeres that help preserve the integrity and stability of the chromosome. The telomeres consist of simple sequence repeats. Fidelity of DNA Replication  Proofreading Mechanism  DNA replication takes place once each generation in each cell. As such, it is essential that the fidelity of the replication process be as high as possible to prevent mutations, which are errors in replication and can lead to diseases.  Eukaryotic DNA replication is likely to be at least this accurate. Typically, errors in hydrogen bonding lead to the incorporation of an incorrect nucleotide into a growing DNA chain once in every 105 base pair. At this rate, replication would introduce errors into a significant percentage of genes every generation in E. coli, whose genome contains over four million base pairs.  Fortunately, the 3’-5’ exonuclease activity can remove the incorrect nucleotide, and replication resumes when the correct nucleotide is added.  Errors in replication occur approximately once in every 10 9–10 10 base pairs after the combination results of polymerase activity and proofreading activity.  As such, the proofreading activity can improve the fidelity of replication significantly. DNA Mutation  Point Mutation  Changes can occur in the nucleotide sequence of a DNA molecule if the changes escape the proofreading and repair. Such a genetic change is called a mutation.  Mutations are classified by the kind of change in the DNA molecule.  Point mutations arise when a base pairs with an inappropriate partner during DNA replication.  A change of one nucleotide of a triplet within a protein coding region of a gene may result in the creation of a new triplet that codes for a different amino acid in the protein product. If this occurs, the mutation is known as missense mutation.  A second possible outcome is that the triplet is changed into a stop codon, resulting in the early termination of the protein synthesis. This is known as the nonsense mutation.  The third possibility is that the point mutation changes the nucleotide, but the triplet still codes for the same amino acid; this is known as the silent mutation, because here, there is no effect on the final protein product.  Insertion and Deletion o In addition to point mutation, DNA replication can lead to the introduction of small insertions or deletions. o The addition or removal of one or more base pairs leads to insertion or deletion mutations, respectively.  The loss or addition of a single nucleotide causes all of the subsequent three-letter codons to be changed. These are called frameshift mutations because the frame of the triplet reading during translation is altered. DNA DAMAGE  In addition to experiencing those spontaneous mutations caused by misreading the genetic code, organisms are frequently exposed to mutagens that cause damage to DNA.  DNA damage is a chemical alteration to DNA which can be introduced by many different ways. If the DNA damage is left unrepaired, it can lead to mutation.  If a particular kind of DNA damage is likely to lead to a mutation, it is deemed genotoxic. Some common examples of DNA damage are base modifications caused by base analogs and alkylating agents, pyrimidine dimers caused by ultraviolet (UV) radiation, and free radicals caused by ionizing radiation. DNA Mutation  Point mutation  Insertion and deletion  Tautomeric shifts—bases can occur in many forms called Tautomers and can have mispairing in replication

 Mismatch repair-Exonuclease I removes the section of DNA containing the mistake. DNA Damage  Chemical alteration of the DNA due to mutagens  Base analogs-can substitute purines and pyrimidines  Alkylating agents-attack the negatively charged DNA and add an alkyl group and alter or halt replication. This kills the cell and or the replication may continue but not the defective DNA is not repaired (e.g. ethylmethane sulfonate) and is prone to mutation.  Ultraviolet radiation—it cross link adjacent pyrimidines and distort the DNA affecting transcription  Ionizing radiation-(gamma and xrays)-ionizes water near the DNA molecule and forms free radicals which are extremely reactive molecules causing breaks in the DNA molecules  By-products damage: these products are produced after a normal cellular processes:

Superoxides
Hydroxyl radicals
Hydrogen peroxides : also called free radicals with reactive oxygen species Genome  Cell Cycle o Before a cell can divide, it must replicate its DNA and segregate the duplicated chromosomes equally to what will become the two daughter cells. o This highly coordinated sequence of events that occurs during the division process is called the cell cycle. o The cell cycle of a somatic cell has four phases that occurs in sequence: G1, S, G2, and M o In the first phase, G1 (gap phase I), cell growth occurs until cells attain a minimum size that is required to progress to the next phase. o During the S phase, the DNA is replicated, thereby duplicating all of the chromosomes. o In the G2 phase, the cell prepares for mitosis o M phase, sister chromatids are separated and a complete set of chromosomes is delivered to separate pole of the cell. o Cytokinesis-completes the division of the mother cell into two daughter cells o Not all cells in the body enter mitosis o Once the organ is fully developed, all cells in that organ are in the G0 phase, which means, the cells are not dividing but 100% functional. o In injury and repair, the stem cells enter into S phase and finally mitosis. o Some organs remain in G0 forever: Brain, kidney, lung, heart Versus: Liver with increase capacity for regeneration Mb = megabase 1 megabase = 1 million base

DNA Replication and Cellular Processes, Lecture notes of Health sciences

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