Pap Stain Lab Lecture questions answered 100% correct

Pap Stain Lab Lecture questions answered 100% correct Remove coating fixative in 95% alcohol for 15 min Coating Fixative ingredients 95% Ethanol and 2% CarboWax ( water soluble) Running in Tap water For excess dye off Harris' Hematoxylin To demonstrate nucleus Acid alcohol Clarifier Running tap water after acid alcohol To stop the reaction Bluing reagent Permanent Nuclei dye : acidic to alkaline After microscopic check, not enough nuclei dye , Go back to Hematoxylin dye OG solution. To demonstrate Keratin Eosin Polychrome stain Purpose of rinsing in Ethanol in fume hood To dehydrate for cover slipping and mounting Mounting aid Entellan Alcohol doesn't react with Mounting agent Papanicolaou Stain 1. Most common stain used in cytology 2. Polychrome staining reaction 3. Designed to display variations of cell morphology and show varying degrees of cell maturity and metabolic activity 4. Indirect staining method 5. Dysplastic : bad formation - precancerous change 3 main objectives to the Pap Stain 1. Crisp nuclear staining 2. Differential Cytoplasmic Counterstaining 3. Cytoplasmic Transparency Crisp nuclear staining 1. Most important 2. Cytologic diagnosis is based on the evaluation and analysis of nuclear detail within cells 3. Recognition of malignancy requires assessment of chromatin pattern Differential Cytoplasmic Counterstaining 1. Serve as a sharp color contrast to the nuclear stain 2. Supplies info related to cell maturity and functional differentiation 3. Polychromatic Counterstaining provides a range of colours. ( green- blue-pink) 4. Depends on the metabolic activity of the cells 5. Orange G- bright orange color to Keratin Floaters Contamination from one specimen to another Small nuclei Dying Older cells of cytoplasm Pink Abundant keratin Well differentiated cancer Cytoplasmic Transparency 1. Allows visualization and evaluation of nuclear detail 2. Cytotechnologist and cytopathology still must be able to see through cluster of cells 3. Maintained by using Counterstaining with a high concentration SOP 1. Standardization produces consistent results, consistent intensity of the nuclear stain, consistent range of colours in the cytoplasm Preparation and staining techniques must be consistent , why? Cytodiagnosis is a comparative analysis Factors that can affect the pap stain 1. Type of fixative 2. Type of hematoxylin( haffis, Mayers, Gills) 3. Formula of counterstains 4. Staining time 5. Slide volume 6. pH and chemical content of water use ) neutral pH is best. Deionized water is preferred b/c the chlorine in tap water can bleach dyes) 7. Water temperature 8. pH of stain solutions ( Eosin is best at pH of 4-5) 9. Age/shelf-line of stain 10. Presence of dye particles in unfiltered solutions 11. Inconsistency of the staining technique 12. Type of staining method employed ( regressive or progressive) 13. Quality of the prepared cell sample 14. Contaminated dehydrating solutions 15. Environmental factors 16. pH of the specimen 17. Presence or absence of inflammation Progressive Stain 1. Staining to desired intensity 2. Intensity of color of cell components become greater with time 3. Timing must be carefully controlled 4. 2 step nuclear staining 5. Hematoxylin ( nuclear stain) 6. Bluing agent : increase in pH 7. Use of acidic alcohol Regressive Pap Stain 1. Cells are overstrained and differentiated - pullout 8-10 times in Acidic Alcohol 2. Differentiation must be carefully controlled 3. More common than progressive 4. 3-step nuclear staining 5. Hematoxylin 6. Acidic Alcohol- clarifier: pullout Hematoxylin 7. Bluing- Hematoxylin 1. Natural dye powder 2. Derived from Hematoxylon compechianum 3. Dye precursor - b/c it has not stating properties in its natural form 4. Requires Oxidation and Mordanting Oxidation Hematoxylin to hematein ( weak acid dye) Two ways of Oxidation 1. Synthetic or chemical : oxidizing agent is added Ex) potassium permanganate , sodium iodate, mercuric oxide 2. Natural : exposure to air and light , Slow ( 6-8 weeks)