Specimen types and fixation: A462 quiz 3

prep type depends on what factors?

• specimen types

• clinical suspicions

• laboratory/clinician preference

cytopathologic sample

cell suspension: direct smear or cellular concentration —> smear or filter

why are two slides made from one pass?

• 2 reportable tests

• one is for gross description

• one is for rendering diagnosis (also for billing patient)

what is the first thing we should do when receiving the specimen?

verify patient name and the location of the specimen

what is accessioning?

assigning a pathology case number to the specimen

what is done after accessioning?

centrifugation

what are the volumes of the vials we use in centrifugation?

• 10 mL vials (1-10 mL specimens go in here)

• 50 mL vials (any specimen over 10 mL)

what should you do before staring centrifugation?

• make sure the centrifuge is balanced by adding salt solution (saline)

what is thinpreps version of a saline solution?

cytolite

what are the two things seen in a vial after centrifugation?

• supernatant (liquid)

• pellet or sediment —> cells

what are the major body cavity fluids?

• pleural fluid

• peritoneal fluid —> ascites (excess peritoneal fluid)

• pelvic washings

• pericardial fluid

what are the minor body cavity fluids?

• CSF

• synovial fluid

• cysts

• eye fluid

What are the steps to centrifugation of a major body cavity fluid?

• centrifuge 2-4 50mL tubes of sample

• decant the supernatant

• make direct smears of sediment

  • perform a rapid assessment if necessary

if the major body cavity fluid has RBCs, what should you do?

• lyse the RBCs first

• do a rapid assessment if neccessary

• then centrifuge

why do we perform a rapid assessment for high cellularity specimens?

to see cellularity of fluid

Are major body cavity fluids high or low cellularity?

high cellularity

Are minor body cavity fluids high or low cellularity?

low cellularity

what do we do if the volume of a low cellularity specimen is greater than 1 mL?

centrifuge and then cytospin (cytocentrifugation) or monolayer prep

what do we do if the volume of a low cellularity specimen is less than 1 mL?

skip centrifugation and prep for analysis

what do we do with urine specimens?

• check patient history

• process immediately —> centrifuge

what specimen do we reject?

a 24 hr urine —> extremely degenerated —> can’t make diagnosis

other than a 24 hr urine, what other specimens can we reject?

• improperly labeled specimens

  • names don’t match

  • improperly labeled fluid (ex: on paper it says a pleural fluid but on the bottle it says a peritoneal fluid)

what do we do if the urine specimen has sediment visible?

• 2-4 pull smears

• immediately fix slides

what do we do if the urine specimen has no sediment?

• 2-4 cytospins or other monolayer prep

• only do wet fixed preps

• commonly done compared to ones that have sediment

what is the fixation for wet preps?

95% ethanol

what can we use instead of ethanol as our fixative for wet preps?

• 80% isopropanol

• 100% methanol

what is the fixative for an air dried prep?

air

what are the liquid based monolayer preps?

• thinprep (hologic)

• surepath (BD)

which liquid based monolayer is used nationwide?

thinprep

thinprep vs. surepath

thinprep

• methanol based

• larger cell spot

• used at eskenazi and VA (not exclusively)

surepath

• ethanol based

• smaller cell spot

• bidirectional screening (move horizontal and vertically)

• used at IUHPL for almost everything —> except FNAs

processing differences between thinprep and surepath

• thinprep has a machine that will lower a vial with a filter at the bottom into the thinprep bottle and then apply vacuum to separate cells from liquid, resulting in a thicker layer of cells

• surepath involves centrifugation until larger particles are seen and then filtered

True or false: surepath slides are charged

true —> charged slides help the cells adhere more to slide

cytospin is used for what cellularity specimens?

low cellularity specimens

• especially CSF

• urines too

• maybe BALS, washings, brushings

what are the steps to load a specimen into the cytospin?

• place a slide into the plastic chamber

• then place blotter/filter paper above slide

• then close chamber

what can you add to the cytospin chamber to prevent air drying of wet prep specimen?

add a few drops of carbowax (ex: 3 drops of specimen = 3 drops of carbowax)

what can you add to the air dried specimen’s cytospin chamber to balance the cytospin?

albumin —> doesn’t fix air dried cells or hydrate them

True or false: you ALWAYS do an air dried and a wet prep for a CSF specimen

true

other low celllularity fluids have how many preps?

4 wet preps or 1 monolayer prep

relationship between cellularity of specimen and drops added to cytospin chambers

• high cellularity —> less drops

• low cellularity —> more drops

what effect is seen when slides aren’t quickly dropped into 95% ethanol?

ribbon effect —> happens when slide is stopped in the ethanol, creating thick lines of specimen

what methods are used when processing sputums?

• thinpreps

• surepath

• pick and smear

• Saccomanno’s fixative

pick and smear

• pick at mucus and pick something that has some tissue and maybe some blood

• then place on slide and use sticks to thin out the mucus

• then smear onto another slide

  • do this until the mucus is thin (usually 4-8 slides per specimen 😐 )

pros and cons to pick and smear method

pros

• fresh specimen

• no degeneration

• direct sampling (ex: if squamous cells are next to respiratory epithelial glandular cells on the slide, those cells were likely next to each other in the body)

cons

• not a good representative of the specimen collected

what are the components in Saccomanno’s fixative?

• 50% ethanol and 2% carbowax

True or false: cytospins were used for Saccomanno’s specimens

false —> blenders were used to blend the specimens :]