Microscopy methods
Microscopy methodsShanley
Overview
Body fluids are generally lumped together even though they are
completely different sources, sites and reasons for collecting
them. We treat sterile sites the same but we have body fluids the
same even though they are different sources.
In most cases we collect and analyze body fluids for 2
reasons:To determine the etiology of the fluid accumulationTo
distinguish between benign and malignant processes
Several steps must be covered:Specimen collectionSpecimen
transportSpecimen recieptWhat kind of test we are doingRoutine
testingGross examMicroscopic exam for detrmining infectious
processCell count to determine why the fluid is accumlating. Most
stools dont need microbio studies.Chemical analysis includes flow
cyto, cytogenetics that must be handled differently from
normal.Microbiology studies - Specialized testingFlow
cytometryImmunohistochemical stainsCytogeneticsMolecular
testing
2
General PrinciplesCollection more complex than phlebotomy: Since
collection is generally more complex than phlebotomy, these
specimens are considered more valuable. Every step along the way
should be carefully monitored from transport to reporting to ensure
accurate results.Transport more closely watched than blood:
Transport for most routine tests is at room temp, and STAT.
Pneumatic tubes is ill advised because of the shaking/agitation
that can degrade components (esp. cellular)Labeling issues remain:
All labeling issues must still be followed institutions must have a
policy for irretrievable specimens.Collection containers: Must be
made from materials that wont affect the test results ex: glass can
cause cellular adherence and inaccurate cell or diff countsSpeed of
analysis: Analysis must be done as soon as possible upon receipt
into labMost body fluids stable for 2-4 hours at RTMost body fluids
are stable for up to 24 hrs if refrigeratedCrystals may remain
stable for up to 48-72 hours in refrigerated specIdeally body
fluids for culture should be inoculated into commercial blood
culture bottlesCertainly collecting the appropriate amount is vital
to get all the tests doneClotting invalidates cell counts. Synovial
fluids must be placed in an EDTA tube to ensure that a cell count
can be done. Freezing invalidates cell counts since the cells lyse
when frozenIf gns for all the tests ordered it is the physician who
should decide the priority. They will determine what tests they
want to run. You cannot make the decision to run a certain test.
While the minimum required is 1cc through practice we dont usually
use that much. If it is sent out however you must send the specific
amount of fluid they require for testing.Remaining specimen should
always be saved in the freezer in case further testing is needed
and each lab needs a procedure to spell out how to store left over
BF and for how long. Cell count cannot be done after
refrigeration.
Preanalytic, analytic & Postanalytic variables affecting
body fluid analysis and result
reportingPreanalyticAnalyticPostanalyticCollection methodSpecimen
viability/integrityResult reportingSpecimen handlingInsufficient
specimenReport deliverySpecimen labelingInstrument failureSpecimen
storageCollection vesselQCAnticoagulant usedTransport time &
temperature
Since collection is generally more complex than phlebotomy,
these specimens are considered more valuableEvery step along the
way should be carefully monitored from transport to reporting to
ensure accurate results
Transport for most routine tests is at room temp, and
STATPneumatic tubes is ill advised because of the shaking/agitation
that can degrade components (esp. cellular)
All labeling issues must still be followed institutions must
have a policy for irretrievable specimens
Collection containers:Must be made from materials that wont
affect the test results ex: glass can cause cellular adherence and
inaccurate cell or diff counts
Analysis must be done as soon as possible upon receipt into
labMost body fluids stable for 2-4 hours at RTMost stable for up to
24 hrs is refrigeratedCrystals may remain stable for up to 48-72
hours in refrigerated spec
Ideally body fluids for culture should be inoculated into
commercial blood culture bottles
Certainly collecting the appropriate amount is vital to get all
the tests doneClotting invalidates cell countsFreezing invalidates
cell counts since the cells lyse when frozen
If qns for all the tests ordered it is the physician who should
decide the priority. They will determine what tests they want to
run. You cannot make the decision to run a certain test. While the
minimum required is 1cc through practice we dont usually use that
much. If it is sent out however you must send the specific amount
of fluid they require for testing.
Remaining specimen should always be saved in case further
testing is needed and each lab needs a procedure to spell out how
to store left over BF and for how long
3
Suggestions for various fluidsSpecimen requirements for amniotic
fluidTestAnticoagulantVolumeCommentsFetal lung maturityNone6-7
mLRefrigerate to prevent digredation of phospholipidsChemical
analysisNone3-5 mLProtect from light for bilirubin
testCytogenetics/karyotypingNone6-7 mLRTMicrobiology studieNone3-5
mLBlood culture bottle collection recommended
Specimen requirements & suggested tube collection sequence
for CSFTestAnticoagulantVolume Collection sequenceChemical analysis
& immunologic studiesNone1-3 mLTube 1Microbiology
studiesNone3-5 mLTube 2Cell count and diffNone1-3 mLTube 3Cytologic
exam and other studiesNone5-10 mLTube 4
Note with the csf that tube order is important although
physicians often change the order based on their success at
collecting the specimens.Always go with the physicians order. This
is only if the order doesnt include tube numbers. It is done in
this order because the initial stick will affect the results of
both microbioogy and cell count from contamination. First chemical
analysisSecond microbiologyThird cell countForth cytology or other
exams
Amniotic fluid has specific requirements such as fetal lung
maturity and you are looking for spingophospholipid maturity. The
phospholipid will disintegrate when left. Cytogenetics/karyotyping:
done before birth for genetic studiesMicrobiology studies:
collected and placed in blood culture
4
Suggestions for various fluidsSpecimen requirements for serous
fluidsTestAnticoagulantVolumeCommentsCell count and diffEDTA:
prevents clotting. 3-5 mLChemical analysis Sodium heparin or
none5-8 mLMicrobiology studiesNone8-10 mLBlood culture bottles
recommendedCytologic exam and other studiesNone, because it will
interfere with testing15-100 mLSpecimens in heparin or EDTA are
acceptable
Specimen requirements for synovial
fluidTestAnticoagulantVolumeCommentsCell count and diff, crystal
id, wet mountsLiquid EDTA, sodium heparin3-5 mLOxalate, powdered
EDTA and powdered lithium heparin are unacceptable as they may form
crystalsChemical analysisNone3-5 mLMicrobiology
studiesNone>5mLBlood culture bottles recommended
Chemical analysis for these can include total protein, LDH,
amylase, glucose and CH50 (total complement activity). This looks
for signs of infection.
Synovial fluid can be viscous because of hyloronidase. 5
Gross ExamAll are generally noted before centrifugation (except
where noted)VolumeAmniotic fluidColor: Should be colorless in 3rd
trimester looking for bleeds that may be seen as red color.
Meconium: fetal poop that should be noted as +/-CSFColor: should be
colorless, and thinXanthochromia is read after
centrifugationClarity: clearClots: free of clotsSerous fluids
(pericardial, pleural, peritoneal fluids)Color & clarity:
Should be clear to pale yellow but these can normally have some rbc
which may have broken down and then give you a straw or yellowish
color. Synovial fluid this is the most viscous of normal fluids
because of the hyaluronidaseColor: colorless to pale yellow
color.Clarity: clear but can be slightly cloudy.
All are generally noted before centrifugation (except where
noted)
Amniotic fluidShould be colorless in 3rd trimester looking for
bleeds that may be seen as red colorMeconium fetal poop
CSF: should be colorless and thin
Serous fluids (pericardial, pleural, peritoneal fluids)Should be
clear to pale yellow but these can normally have some rbc which may
have broken down and then give you a straw or yellowish color
Synovial fluid most viscous of normal fluidsNormally colorless
to pale yellow6
Microscopic examManual Count: Can be either Manual or automated
and you are looking for cells and/or crystalsMix sample: We must
thoroughly mixed with a recommended 10X inversion. It should not be
rocking on mechanical rocker to exceed 5 minutes so it wont damage
cells except for viscous synovial fluids which can go to 10 min,
which will protect the cell a bit longer. Extremely viscous
synovial fluids may need to be treated w/ hyaluronidase first
before analysisDilution may be necessary:Clear sample dilution may
not be necessarySlightly to moderately turbid specimen dilute 1:3,
1:5, 1:10 or 1:20Very turbid to grossly bloody start dilution at
1:20 (a higher dilution)Serially dilute up to 1:100 for TNCs (total
nucleated cells) and up to 1:200 for RBCCount TNC and RBC together:
Both RBC and TNC can be counted on same chamber.We Scan chamber 10X
for the presence of cell clumps if they are present note count may
be affected by presence of clumpsWe Scan at least 10 fields at 10X
and 40X for debris if present note count may be affected by
presence of debrisBased on TNC and RBC present determine
appropriate area of chamber to count200 cells in entire chamber
count 4 corner squares (4mm2)>200 cells in 1 large square count
5 small squares w/in center large square (0.2mm2)If the cells are
crowded and overlapping, consider a higher dilutionOnce counted use
the formula:Total cells/L = #cells counted x dilution factor#
squares counted x volume each squareKnow what to include and not to
include:Dont count cells touching the right or bottom boundary
lines but count the oppositeIf the boundary line is a triple line
count all cells w/in square and those touching the middle line
inwardInclude in RBC count:RBC w/ distinct outlines w/ halos &
clear centersCrenated RBCGhost RBCInclude in the TNC
countLymphocytes, monocytes/macrophages, mesothelial cells,
synovial lining cells, PMN, blasts, lymphoma cells, nucleated
RBCsDO NOT INCLUDE IN COUNT:Broken cells, sheets, clumps of tumor
cellsTissue cellsDistinguishable tumor cells Automated count: these
are fine but must be validated to match manual counts. Most
difficulty is w/ CSF since their normal cell counts are so low.
Can be either Manual or automated and you are looking for cells
and/or crystals
Manual count:We must thoroughly mixed with a recommended 10X
inversion. It should not be rocking on mechanical rocker to exceed
5 minutes so it wont damage cells except for viscous synovial
fluids which can go to 10 min, which will protect the cell a bit
longer. Extremely viscous synovial fluids may need to be treated w/
hyaluronidase first before analysis
Clear sample dilution may not be necessarySlightly to moderately
turbid specimen dilute 1:3, 1:5, 1:10 or 1:20Very turbid to grossly
bloody start dilution at 1:20 (a higher dilution)Serially dilute up
to 1:100 for TNCs (total nucleated cells) and up to 1:200 for
RBCBoth RBC and TNC can be counted on same chamberWe Scan chamber
10X for the presence of cell clumps if they are present note count
may be affected by presence of clumpsWe Scan at least 10 fields at
10X and 40X for debris if present note count may be affected by
presence of debrisBased on TNC and RBC present determine
appropriate area of chamber to count200 cells in entire chamber
cound 4 corner squares (4mm2)>200 cells in 1 large square count
5 small squares w/in center large square (0.2mm2)If the cells are
crowded and overlapping, consider a higher dilutionOnce counted use
the formula:
Total cells/uL = # cells counted x dilution factor # squares
counted x volume of each square
Dont count cells touching the right or bottom boundary lines but
count the oppositeIf the boundary line is a triple line count all
cells w/in square and those touching the middle line inwardInclude
in RBC count:RBC w/ distinct outlines w/ halos & clear
centersCrenated RBCGhost RBCInclude in the TNC countLyphocytes,
monocytes/macrophages, mesothelial cells, synovial lining cells,
PMN, blasts, lymphoma cells, nucleated RBCsDO NOT INCLUDE IN
COUNT:Broken cells, sheets, clumps of tumor cellsTissue
cellsDistinguishable tumor cells
Automated counts: fine but must be validated to match manual
countsMost difficulty w/ CSF since their normal cell counts are so
low
7
Cytospin & cell concentrationCytospin is another means of
preparing for microscopic exam. It is a slow speed centrifuge with
low spin. You gently move cells into specialized space. Fluid will
be absorbed by the filter and wil be concentrated on the center.
They can get distorted because of the spinning. If you have enough
cells use the regular drying method. Low cell counts should be used
with a cytospin.DO NOT USE WEDGE OR PUSH SMEARS SINCE THEY WILL
DAMAGE CELLS. Cytocentrifuge: is low speed, low acceleration
centrifuge that makes concentrated thin layer cell prepsRecommended
method by ASCPLow speedLow accelerationSpecial assemblies for each
specimen20 fold concentrationCan cause cellular distortionIdeal for
low cell count fluids
Another means of preparing for microscopic examDO NOT USE WEDGE
OR PUSH SMEARS SINCE THEY WILL DAMAGE CELLS
Cytocentrifuge is low speed, low acceleration centrifuge that
makes concentrated thin layer cell preps
8
StainingRomanowskyPapanicolaou
Romanowsky stains - several variations including
Wright-GiemsaProvides information about the cytoplasm and
background (stroma). It can be troublesome in fluids. Ex:
wright-giemsa, may-grunwald-giemsa (MGG)
Papanicolaou stains provide great information about the nucleus
and are used in assessing malignancyOriginally developed for
vaginal and cervical preps
Then you still have gram stains and afb stains for
microorganisms9
Differentials of body fluidsSteps in differential:We scan the
smears at 10X and 40XIf cells overlap or are distorted on cytospin
we must repeat cytospin w/ a fewer drops or higher dilution. We can
also scan at 50-100x to verify any abnormal findings.We must
Classify 100 cells by counting them then classify them to their
type. If
