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Cytopreparation The act of preparing & staining diagnostic slides
Diagnostic cytology Examination of cellular material for disease, esp. cancer
Gynecological cytology specimens Pap smears, endocervical brushings, collected & smeared on slide, fixed while still wet, then dried & sent for staining
Non-gynecological cytology specimens Urine, sputum, CSF, bronchial/gastric/esophageal washings or brushings, pleural/peritoneal/ascites/pericardial fluids, FNAs, best collect fresh w/ no fixative/additive, most can be refrigerated 24-72 hrs, don't freeze
Pap smears Liquid-based cytology rather than direct smear - better for automated systems - ThinPrep system & SurePath system, smeared on slide, fixed while wet, dry & stain
ThinPrep System Specimens w/o brush head in vial w/ weak methanol preservative, homogenize & filter, filter pressed on slide to deposit cells, slide can be stained traditionally, nongynecological specimens need extra suspension & centrifugation
SurePath System Specimens w/ brush head in vial w/ very weak ethanol preservative, cell enrichment - centrifuge conc. abnormal cells, remove blood/mucus, specimen aliquot sediments on slides, nongynecological req. alt. suspension & centrifugation, higher yield cells
Preferred cytology fixative: 95% ethanol, rapid fixation important to prevent distortion of cell & loss of morphology,
Alcohol vs. formalin fixation Alcohol better b/c crisp fixation of nuclear chromatin, avoid formalin - causes different nuclear chromatin patterns which may impact diagnosis
Other cytology fixatives: Commerical cytology sprays - usu. ethanol w/ acetone & polyethylene glycol (remove latter w/ alcohol before staining), 95% denatured alcohol, 80% isopropanol (b/c more shrinkage than ethanol), 100% methanol (b/c less shrinkage than ethanol)
Sparsely cellular specimens Sample w/ not many cells in suspension can be made more conc'd by Cytospin or centrifugation preparation: 1mL properly prepared cell suspension in funnel of cytospin, centrifuge, cells will be deposited on slide & fluid absorbed by filter paper
Problem: bloody specimens: Can obscure morphology or outnumber diagnostic cells, crosshatch can help separate cells, dip in Carnoy's or Clark to lyse rbc, commercial products can lyse rbc (maybe diagnostic too)
Problem: poorly adhesive specimens Often urine & breast fluid, formalin-fixed may wash, coated, positive-charged slides, specimens prepared by centrifugation less likely to shed cells, spray fixing (rather than liquid fixing) decr. slide cell loss
Problem: cross contamination Ongoing concern, cells may float into reagents or stick to tools, change reagents between samples & use disposable tools
Cell block Can process, section, stain, as histo section, easier special stain, incl. IHC, made of material left after smear, make from tissue fragments, clots, heavy mucus, "block" together w/ thrombin-prothrombin clot, albumin, or agar
Fixation time: Smears fixed w/i 1-2 seconds of application to slide, thick smears/smears w/ mucus may take 2-4 seconds, liquid suspensions longer, air-drying causes nuclear swelling, distortion, loss of cytoplasmic density, cytoplasm becomes eosinophilic
EA stains metabolically inactive cells? Various shades of pink
EA stains metabolically active cells? Various shades of blue-green
Nickel smear method Smear material from scraper/brush in circular motion onto slide in area the size of a nickel, fix immediately w/ 95% ethanol or spray fixative, avoid cotton swabs, avoid feathered-edge smears
Pull-apart smear method 2-3 drops of sediment on slide, place another slide on top upside-down, pull slides apart endways, fix immediately w/ 95% ethanol or spray fixative, good general-purpose for fluids w/o mucus
Crush/mash smear method Mash material between two slides to give thin, even smear, may use back & forth "slap" motion to break chunks, fix w/ 95% ethanol or spray-fixative
Cross-hatch smear method 2-3 loopfuls of sediment on slide, spread diagonally, back & forth, corner to corner, fix immediately w/ 95% ethanol or spray fixative, if sediment beads form, spread again for monolayer of cells, good for cellular body fluids, esp. w/ lots of blood
Cytocentrifuge smear method Specimen placed into funnel apparatus, centrifugation forces specimen thru tunnel, cells onto slide, filter absorbs fluid
To evaluate cellularity of specimen to determine adequacy: Toluidine blue wet film, or air-dry & stain w/ Diff-Quik
Problem: Pap-counterstained smears look dirty & not optimal: Ensure clean alcohol rinses after counterstains, overused alcohol rinses may cause "dirty" colors
Bodys fluids pleural, peritoneal, pericardial, ascites treatment: Send entire amount collected to lab, good for cell block
Breast/nipple discharges treatment: Prepare smear at time of collection, nickel smear, spray-fix, don't let air-dry
CSF treatment: Deteriorates rapidly, if can't take to lab immediately use pre-fixative holding solution - alcoholic saline or Saccomanno fluid = vol. of specimen
Direct scrapings for viral lesions (Tzank smears) treatment: Prepare smear at time of collection, nickel smear, spray-fix, don't let air-dry
Washings - bronchial, esophageal, & gastric treatment: Don't fix, refrigerate immediately
Urine treatment: V. fragile, doesn't hold pH, first morning urine not recommended b/c cells degenerated, add pre-fixative if can't process or send to lab right away, equal volume of alcoholic saline or Sacomanno fluid
Brusings - bronchial, esophageal, & gastric treatment: Prepare smear at time of collection, nickel smear, spray-fix, don't let air-dry, or submit brush in physiological saline for lab processing, never fix
FNAs include: Breast, thyroid, lung, liver, pancreas, cell-laden fluid removed from site, initially use pull-apart method & fix, never feathered-edge, rinse needle w/ physiologic saline (don't rinse w/ fixative) to recover remaining specimen
Alcoholic saline pre-fixative reagents 1 part saline 1 part 50% alcohol
Saccomanno Fluid pre-fixative reagents 980 mL ethanol 20 mL melted Carbowax (remove Carbowax after slide prep, before staining)
Cytology smears: Fresh, unfixed specimens, wet-fixed on slide, better cell adhesion, causes cells to flatten - better visualization, pre-fixed specimens - round cells
Direct smears include: Scrapings, brushings, viral inclusion smears, use nickel method
Fluids include: Pleural, peritoneal, pericardial, ascites, effusions, urine, cyst fluids, washings, first centrifuge to concentrate cells, use pull-apart, or cross-hatch method
Best centrifugation for cytology: 2,000 rpm for ~10 min, high speed packs cells too tightly, lower speed won't force cells to bottom of tube, 50 mL conical tube best (15 mL usu. not adequate specimen), swinging-arm centrifuge best concentrates cells @ bottom
Mucoid specimens include: Sputum & thick bronchial washings, use crush method
Sparsely cellular specimens include: CSF, sparsely cellular urines or body fluids, filter w/ cellulose or polycarbonate filter (5 ┬Ám pore) to capture all cells, stain & mount filter (old method), more common - cytocentrifugation
Cytopreparation for sample with mucus? Crush method w/ spray fixative
Cytopreparation for sample with good cellularity & adhesiveness? Pull apart or crosshatch w/ spray fixative or 95% alcohol
Cytopreparation for sample with good cellularity & poor adhesivenss? Pos slide or cytocentrifuge w/ spray fixative
Cytopreparation for sample with poor cellularity? Cytocentrifuge
Cell block for specimen w/ spontaneous clots: Squeeze out excess liquid until solid mass, place mass in labeled cassette, may wrap w/ lens paper, if too large submit portion, formalin process routinely
Cell block for specimen w/ cellular material entrapped in mucus: To remaining sediment add 95% ethanol, denatured alcohol, or 10% NBF, mix by inversion, fix 15-30 min, centrifuge 10 min, 2,000 rpm, pour off supernatant, mass in cassette, may wrap w/ lens paper, if large submit portion, formalin process routinely
Cell block for specimen w/ loose cellular material - albumin method: To remaining sediment add 30% BSA, mix w/ stick, add 95% alcohol, place mass in cassette, may wrap w/ lens paper, if too large submit portion, formalin process routinely
Cell block for specimen w/ loose cellular material - agar method: To remaining sediment add melted 4% agar, mix gently, centrifuge 10 min, 2,000 rpm, refrigerate until agar solid, remove from tube, cut away cellular material, bisect, place in cassette w/ common cut side down, formalin process routinely
Papanicolaou technique stains: Hematoxylin nuclear stain, Harris or Gill, OG-6 counterstains tonofilaments in keratinized cells, EA differentially counterstains cytoplasm of cells, store all in light-blocking containers
OG-6 reagents Orange G dH2O phosphotungstic acid (mordant) 95%ethanol glacial acetic acid (opt.) (enhance specificity, decr. stain time)
EA reagents eosin Y 20% light green SF 3% phosphotungstic acid 95% ethanol abs. methanol glacial acetic acid Bismarck brown (may be excl'd makes stain weaker)
Pap stain purpose To distinguish cellular components by obtaining highly detailed chromatin, differential counterstaining, & cytoplasmic transparency
Pap stain reagents hematoxylin ammonia water, or Scott tap water OG-6 EA
Pap stain results chromatin - blue keratin - orange superficial squamous cells, nucleoli, cilia, rbc - variable pinks all metabolic cell cytoplasm - variable blue-greens
Toluidine blue wet film purpose Rapid determination of cellularity, esp. FNAs, & eval. potential cross-contamination
Toluidine blue wet film facts Gives metachromatic staining in unfixed cells, use unfixed cytological specimen
Toluidine blue wet film reagents Toluidine blue in 95% alcohol w/ dH2O, filtered
Cross-contamination in cytology specimens: Significant chance of loose cells floating during staining, to prevent stain separately specimens w/ higher potential to float, filter solutions between batches
What is a LEEP biopsy? Loop electrosurgical excision procedure, cervix biopsy
Created by: CCF



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