| Question | Answer |
| 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 µm 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 |
