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 starting 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 (not a heterogenous sample)

what are the components in Saccomanno’s fixative?

• 50% ethanol and 2% carbowax

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

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

how do you concentrate a Saccomanno’s specimen after blending?

centrifugation

pros and cons to Saccomanno’s fixative

pros

• representative sample (homogenous sample)

• can be done at home

cons

• due to fixative, cells can round up

• oral contamination not decreased

what are 3 respiratory samples and their prep methods discussed in the prep types in cytology lecture?

sputum

• can use plain or frosted slides, pick and smear, mucolexx technique (mucolexx helps break up the mucus)

brochial washings

• 2-4 slides, plain or frosted slides

• thinprep and surepath can also be used

BAL

• special kind of washing that goes into alveolar spaces

• most common pulmonary sample

• shared with microbio

• for IUHPL:

  • 1 wet prep (surepath)

  • 3 air dried surepath (one for iron stain, one for lipid stain AKA Oil Red O, one for rapid stain, one for GMS fungal stain —> look for pneumocystis)

are bronchial brushings high or low cellularity? what method is used to prep this specimen?

low cellularity and methods done:

• cytospins or a monolayer prep (use a stick to knock off stuff from the brush into the vial/tube)

• directly brush the sample onto the slide

what method for cell blocks shouldn’t be performed anymore and why should we not do it? (Eskenazi still does though :\)

plasma thrombin method —> add thrombin and expired human plasma and creates a pellet

problem: plasma cells contain human DNA which could lead to false reports

*IUHPL uses histogel which is kind of like an agar that can be melted and then hardened with the cell spot inside it

what are cell blocks fixed in? what are they stained with and what can they be tested for?

formalin and stained with H&E stain

used for genetic and molecular testing

blood lysing options?

• saponin: added before centrifuge (ex: instead of adding 20 mL of specimen, do 10 mL of specimen and 10 mL of saponin)

• Carnoy’s: added after slide prep but before final fixation; slide is dipped in

• cytorich red: added to cell pellet after centrifugation; add 30 mL of this to cell pellet

• glacial acetic acid: added to specimen container before centrifugation

• cytolite: add to specimen after 1st centrifugation and centrifuge a 2nd time or until a pellet forms

what suspected condition do we never use blood lysing techniques on?

lymphoma and/or leukemia

what test is run when lymphomas and/or leukemias are suspected?

flow cytometry —> collected in an RPMI

*avoid frosted slides

types of slides

plain

frosted —> NEVER use for CSF or lymphoma/leukemia specimens

*if using frosted slides for smear, use a frosted slide and one plain

what can air drying do to the cells?

• swelling

• loss of nuclear details

• inability to take up counterstains

what is a fixative?

“permanent preservative”

effectively freezes the sample in time

needed for accurate cytologic interpretation

preservatives prevent…

• autolysis

• evaporation

• osmotic swelling

• shrinking

• bacterial growth

fill in the blank: fixatives act on ____ side chains

protein

types of fixatives

heat fixative: act on entire protein structure

chemical fixative: act selectively on protein side chains

what are the 4 characteristics of chemical fixatives?

coagulants

• removes water from proteins

• causes shrinkage and rounding

non-coagulants

• water is left behind

• cellular swelling

additive

• leaves a portion of fixative onto protein

non-additive

• fixative is washed away after doing work

is ethanol a coagulant/non coagulant? Additive/non additive?

coagulant and non additive (doesn’t leave any ethanol and water behind)

is formalin a coagulant/non coagulant? Additive/non additive?

non coagulant and additive. (leaves a portion of fixative behind and water is also left behind)

what is the fixative used for histology slides?

formalin —> used for tissue and cell blocks

how is formalin made? is it a carcinogen?

• methanol is heated and vaporized —> produced formalin

• gas is added to water to make aqueous formaldehyde (40%)

• one part of aqueous formaldehyde is mixed with 9 parts water —> 10% formalin

• buffer is added

• NOT a carcinogen (even though it contains formaldehyde)

what is the fixative used in cytology?

95% ethanol —> immersion fixation

polyethylene glycol mixed w/ 95% ethanol —> spray fixation (mainly used for FNA procedures)

what was the original “pap” fixative? why was it problematic?

95% ethanol and ether 1:1 —> faster than just 95% ethanol

problematic —> ether explodes 😐

spray fixatives

• acts as a pre-fixative

• protects cells during transport

• slides must be placed in 95% ethanol after procedure to remove waxy film from slide

  • ~ 10 min.

• hold pump 5-7 in. from slide

what can be used as a substitute for carbowax?

cheap hairspray (Aqua Net) —> has polyethylene glycol

why do we have prefixatives?

prevents degeneration, especially if specimen was mailed in or brought over the weekend (during off hours)

types of prefixatives?

• 50% ethanol

• Saccomanno fixative —> 2% polyethylene glycol in 50% ethanol

• mucolexx —> 3% polyethylene glycol, buffers, formaldehyde

final fixation?

95% ethanol for 10 minutes before staining

necessary for specimens that have been —> spray fixed, pre-fixed, treated with coating fixatives

is air drying a type of fixative?

yep :]
can be intentional for rapid stains or unintentional (which causes staining artifacts)

type of rapid stain we use for air drying prep?

Romanowsky style rapid stain —> done during FNA procedures

*air drying is also used for Oil Red O (meant for lipids —> discussed in the staining lecture)

pros and cons to air drying

pros

• allows for rapid staining

• ideal for observing cytoplasm

• used for adequecy assessments and rapid interpretations —> FNAs

cons

• causes nuclear swelling and distortion —> loss of nuclear detail

• loss of cytoplasmic density

• loss of “counter stainability”

• so air drying effect :)

what is done during an FNA procedure?

• each pass produces 2 slides —> one air dried and one wet fixed

• needle rinse in saline or formalin

  • used to make a cell block

  • all needle rinses from the same site (multiple passes) can be combined

spray fixation: what appearance does it create when sprayed onto slide?

produces a “beaded” effect on the slide

at IUHPL, both slides are wet or air dried?

• both are air dried

  • allows for easy and safe transport

  • prevents the fixation artifact that is produced by spray

• one is used for adequacy assessment, other for pap staining

• second one is rehydrated using saline and then final fixed in 95% ethanol before pap stain

types of effusions

• exudate

• transudate

• chylous