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HP
LAB - 8/5
| Question | Answer |
|---|---|
| rooms in histopathology lab should have? | proper ventilation and good lighting |
| equipment & apparatus GROSS TABLE | ● Forceps ● Scalpel ● Chopping board ● Weighing balance ● Ruler ● Orientation markers ● Watch glass/petri dish ● Tissue cassettes |
| Tissue cassettes ○ Wax is heated at ? in the embedding machine | 60C |
| Automated Tissue Processor FDCI - | ● Paraffin oven ● Embedding center ● Refrigerator ● Microtome ● Flotation Bath ● Slide Dryers |
| Processes that an automated machine can perform | FDCI (Fixation, Dehydration, Clearing, Infiltration): |
| sections produced by a microtome | Ribbons: |
| tissue cassettes function by color; ● Yellow: | Liver/renal |
| tissue cassettes function by color;● Green | Routine (e.g. placenta/uterus) |
| tissue cassettes function by color;● White: | Bones |
| tissue cassettes function by color;● Gray | Skin |
| tissue cassettes function by color; ● Pink: | Lymph nodes |
| fresh tissue examination ADVANTAGE: | visualizing cell processes such as motion, mitosis, phagocytosis, and pinocytosis |
| fresh tissue examination DISADVANTAGE: | not permanent; liable to develop the changes that have usually been observed after death |
| fresh tissue examination - procedure | A. Teasing or Dissociation B. Squash Preparation C. Smear Preparation ➢ Streaking (zigzag manner) ➢ Spreading (spreading in a circular motion) ➢ Pull-apart ➢ Touch Preparation |
| A. Teasing or Dissociation | 1. Cut small and thin slices of tissue from the main organ/tissue 2. The selected slices are immersed in a watch glass/petri dish containing isotonic salt solution (NSS) 3. dissect/separate the tissue using dissecting needles or applicator sticks |
| B. Squash Preparation | 1. Transfer a small tissue slice (diameter: <1 mm) in a glass slide 2. Add 2 drops of methylene blue stain directly to the slide 3. Using another glass slide, cover the tissue and forcibly compressed it with the right amount of pressure |
| C. Smear Preparation ➢ Streaking (zigzag manner) | - |
| C. Smear Preparation SPREADING | 1. Transfer a selected portion of the secretion into a clean glass slide 2. Using an applicator stick, gently spread the specimen into a moderately thick film by teasing the mucous strands 3. Label the slides correctly |
| C. Smear Preparation PULL-APART | 1. Place a drop of secretion/sediment into a clean glass slide 2. Using a different glass slide, cover the slide containing the sample and spread evenly the secretion 3. With an even and continuous pressure, the two slides are then pulled apart |
| C. Smear Preparation TOUCH PREPARATION | 1. Obtain a freshly cut piece of tissue 2. The selected tissue is then brought into contact and pressed on the surface of a clean glass slide 3. Apply pressure gently |
| Accomplished by preserving and carefully processing solid structures and tissues in the following order: | 1. Fixation 2. Decalcification (OPTIONAL) 3. Dehydration 4. Clearing 5. Impregnation (INFILTRATION) 6. Embedding 7. Trimming 8. Microtomy 9. Staining 10. Mounting 11. Labeling |
| The specimen is placed in a liquid fixing agent (fixative) such as formaldehyde solution (formalin) | Fixation |
| Tissue-to-Fixative Ratio | 1:10 or 1:20 |
| Gas produced by oxidation of methanol | formaldehyde (Formalin) |
| gas form ● Concentrations | 100% |
| stock concentration (causes overhardening of the external surfaces of tissues) ● Concentrations | 37-40% |
| Neutral Buffered Formalin– working solution; MOST COMMONLY USED IN PH ● Concentrations | 10% |
| ● Formaldehyde + Na Dihydrogen Phosphate + Disodium hydrogen Phosphate ● pH 7 | 10% Neutral Buffered Formalin– |
| BEST GENERAL TISSUE FIXATIVE | 10% Neutral Buffered Formalin– |
| 10% Neutral Buffered Formalin DISADVANTAGE: | longer to prepare, inert to phospholipids and neutral fats |
| 10% Neutral Buffered Formalin Fixation Time: | 4-24 hours |
| factors affecting fixation | ● Fixative of choice ● Time (20-30 minutes) ● Tissue-to-Fixative Ratio |
| Osmic Acid ratio | 1:5 |
| The thicker the specimen, the longer the ? | time |
| Light Microscope: | 2 cm2 x 0.4 cm2 |
| Electron Microscope | 1-2 mm2 |
| takes longer to fix | Fibrous, mucus, fatty (cut to pieces), blood (wash with NSS) specimen: |
| ? pH of fixative if NBF has a pH of 7, so it will not disturb the pH | 6-8 |
| Room Temperature ROUTINE | (22C–28C)–40C |
| ● Temperature Tissue Processors: | 45C |
| ● Temperature Microwave: | 65C |
| ● Temperature Electron Microscope: | 0C–40C |
| ● Temperature Tuberculosed Tissues: | 100C |
| ● Temperature Rapid Biopsy: | 60C |
| ● Osmolality ○ MOST OPTIMAL: | 400–450 mOsm (milliosmole) |
| Removal of fixative and water from the tissue | dehydration |
| dehydration Carried out by passing the tissues through a series of ascending grades of alcohol: | 70%, 90%, and absolute alcohol |
| MOST COMMONLY USED in dehydration | Ethanol |
| Tissue-to-Dehydrating Agent Ratio | 1:10 |
| most commonly employed medium in histopathology | Paraffin Wax: |
| SOLUTION 1. 70% ethanol 2. 90% ethanol 3. 100% ethanol 4. 100% ethanol 5. 100% ethanol 6. 100% ethanol | DURATION 15 min 15 min 15 min 15 min 30 min 45 min |
| decalcification of calcified tissues Done for | 48 hours |
| Tissue-to-Decalcifying Agent Ratio– | 1:120 |
| factors influencing the rate of decalcification | 1. CONCENTRATION 2. FLUID ACCESS 3. SIZE AND CONSISTENCY 4. AGITATION 5. TEMPERATURE |
| A fresh decalcifier should have ready access to all surfaces of the specimen | FLUID ACCESS |
| Concentration of active ingredient and volume of decalcification agent is depleted as it combines with calcium | CONCENTRATIO |
| SIZE AND CONSISTENCY ● Ideal: | 24-48 hours |
| 2 weeks or longer to decalcify | Dense tissues |
| Slowly dislodge precipitates from tissue | AGITATION |
| Heat hastens decalcification, lower temperature decreases reaction rate, directly proportional | TEMPERATURE |
| AKA dealcoholization | CLEARING |
| CLEARING MAIN GOAL: | Make tissues transparent/translucent |
| Commonly employed clearing agent; cheapest | ● Xylene |
| can use glycerine and gum syrup as dehydrating agent | FROZEN SECTIONS: |
| Xylene 1 Xylene 2 Xylene 3 | 20 minutes 20 minutes 45 minutes |
| INFILTRATION AND EMBEDDING Duration per Section: | 1 hour–1 hour–3 hours |
| Wax used should be pure ○ Wax/Paraffin Oven: | >2C–5C than melting point |
| Wax can only be reused | twice |
| soft and crumbly tissues, making sections difficult | INSUFFICIENT: |
| excessive shrinkage and produces dry, brittle tissues which are equally difficult to section | TOO MUCH |
| Most commonly used: IN INFILTRATION | paraffin wax |
| 1st change paraffin wax bath: | 30 minutes |
| 2nd change paraffin wax bath: | 30 minutes |
| 3rd change paraffin wax bath: | : 45 minutes |
| Temperature of embedding paraffin wax: | 2-4C above its melting point |
| OVERHEATING OF WAX cause ○ Hardening and distortion of the tissue ○ Breakdown of the paraffinic additive (e..g resin polymer) ■ Results in poor performance | - |
| TRIMMING & MICROTOMY - One of the most directly correlated factors is the thickness in which a specimen is cut | - |
| Removal of excess wax from the tissue block | TRIMMING |
| Performed to expose the tissue surface and allow the block to fit into the block holder of the microtome | TRIMMING |
| Usual thickness of tissue: | 10 microns |
| Floatation Bath: | 5-10C lower than temperature of paraffin wax |
| SAFEST TYPE OF MICROTOME: | knife if stable and not moving |
| ESSENTIAL PARTS IN MICROTOME | ● Block Holder ● Knife Carrier and Knife ○ Xylene/Wax Paper/Brush: used to clean the knife ● Pawl, Ratchet Feed Wheel, and Adjustment Screws |
| AFTER MICROTOMY Heat the slide gently on the slide warmer | (40-45C) |
| basic, attracted to acidic part (nucleus) of the cell | Hematoxylin Stain |
| Stains purple-blue | Hematoxylin Stain |
| acidic, attracted to basic part (cytoplasm) of the cell | Eosin Stain: |
| Stains red-orange | Eosin Stain: |
| descending grades of alcohol (100%–90%–70%) | Rehydration |
| Process of removing excess hydrogen ion from the stain | BLUING: |
Created by:
blues1234