Microscopy methods

Shanley

Overview
Result
reporting

Specimen
collection

Specimen
transport

Specimen
processing

Specimen
receipt

Routine testing
Gross exam
Microscopic exam
Cell count
Chemical analysis
Microbiology
studies

Specialized testing
Flow cytometry
Immunohistochemica
l stains

Cytogenetics
Molecular testing

General Principles

Collection 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 counts
Speed of analysis: Analysis must be done as soon as possible upon receipt
into lab

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 done

Most body fluids stable for 2-4 hours at RT

Most body fluids are stable for up to 24 hrs if refrigerated
Crystals may remain stable for up to 48-72 hours in refrigerated spec

Clotting 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 frozen

If 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
reporting
Preanalytic

Analytic

Postanalytic

Collection
method

Specimen
viability/integrit
y

Result reporting

Specimen
handling

Insufficient
specimen

Report delivery

Specimen
labeling

Instrument
failure

Specimen
storage

Collection
vessel

QC

Anticoagulant
used
Transport time
& temperature

Suggestions for various fluids

Specimen requirements for amniotic
fluid

Specimen requirements & suggested

tube collection sequence for CSF

Test

Anticoagula
nt

Volume

Comments

Test

Anticoagulan
t

Volume

Collection
sequence

Fetal lung
maturity

None

6-7 mL

Refrigerate to
prevent
digredation of
phospholipids

Chemical
analysis &
immunologic
studies

None

1-3 mL

Tube 1

Chemical
analysis

None

3-5 mL

Protect from light

for bilirubin test

Microbiology
studies

None

3-5 mL

Tube 2

Cytogenetic
s/karyotypin
g

None

6-7 mL

RT

Cell count and

diff

None

1-3 mL

Tube 3

Microbiology
studie

None

3-5 mL

Blood culture
bottle collection
recommended

Cytologic
exam and
other studies

None

5-10 mL

Tube 4

Suggestions for various fluids

Specimen requirements for serous fluids
Test

Anticoagula
nt

Volume

Comments

Cell count and

diff

EDTA:
prevents
clotting.

3-5 mL

Chemical
analysis

Sodium
heparin
or none

5-8 mL

Microbiology
studies

None

8-10 mL

Blood culture bottles

recommended

Cytologic
exam and
other studies

None,
because
it will
interfere
with
testing

15-100
mL

Specimens in
heparin or EDTA are
acceptable

Specimen requirements for synovial

fluid
Test

Anticoagula
nt

Volume

Comments

Cell count
and diff,
crystal id, wet
mounts

Liquid
EDTA,
sodium
heparin

3-5 mL

Oxalate,
powdered EDTA
and powdered
lithium heparin
are unacceptable
as they may form
crystals

Chemical
analysis

None

3-5 mL

Microbiology
studies

None

>5mL

Blood culture
bottles
recommended

Gross Exam

All are generally noted before centrifugation (except where

noted)

Volume

Amniotic fluid
Color: Should be colorless in 3 rd trimester looking for bleeds that may be
seen as red color.
Meconium: fetal poop that should be noted as +/-

CSF
Color: should be colorless, and thin

Xanthochromia is read after centrifugation

Clarity: clear
Clots: free of clots

Serous 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
hyaluronidase
Color: colorless to pale yellow color.
Clarity: clear but can be slightly cloudy.

Microscopic exam
Manual Count: Can be either Manual or automated and you are looking for cells and/or crystals

Mix 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 analysis

Dilution may be necessary:

Clear sample dilution may not be necessary
Slightly to moderately turbid specimen dilute 1:3, 1:5, 1:10 or 1:20
Very 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 RBC

Count 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 clumps
We Scan at least 10 fields at 10X and 40X for debris if present note count may be affected by presence of debris
Based on TNC and RBC present determine appropriate area of chamber to count
<200 cells in entire chamber count all 9 squares (9mm 2)
>200 cells in entire chamber count 4 corner squares (4mm 2)
>200 cells in 1 large square count 5 small squares w/in center large square (0.2mm 2)
If the cells are crowded and overlapping, consider a higher dilution
Once counted use the formula:

Total cells/L = #cells counted x dilution factor

# squares counted x volume each square

Know what to include and not to include:

Dont count cells touching the right or bottom boundary lines but count the opposite
If the boundary line is a triple line count all cells w/in square and those touching the middle line inward
Include in RBC count:
RBC w/ distinct outlines w/ halos & clear centers
Crenated RBC
Ghost RBC
Include in the TNC count
Lymphocytes, monocytes/macrophages, mesothelial cells, synovial lining cells, PMN, blasts, lymphoma cells, nucleated
RBCs
DO NOT INCLUDE IN COUNT:
Broken cells, sheets, clumps of tumor cells
Tissue cells
Distinguishable 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.

Cytospin & cell concentration

Cytospin 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 preps

Recommended method by ASCP

Low speed

Low acceleration

Special assemblies for each specimen

20 fold concentration

Can cause cellular distortion

Ideal for low cell count fluids

Staining
Romanowsky

Papanicolaou

Differentials of body fluids

Steps in differential:

We scan the smears at 10X and 40X

If 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 <100 cells record number counted and convert to

absolute number or percentage.

Each lab will define the subclasses of nucleated cells to

report.

Image shows smear of csf w/ lymphs and activated

lymphs and a few macrophages. Note no rbc so it is a
good stick. This was a case of viral meningitis (lymphs
mean viral, segs mean bacteria usually).

Crystals

Synovial fluid are checked for crystals.

Identification of crystals in synovial fluid is
very useful in evaluation of arthritis.

Common synovial fluid crystals

Crystal

Disorder

Lab findings

Monosodium urate

Urate gout

Negatively birefringent
needlelike (intra or
extracellular)

Calcium pyrophosphate
dehydrate (CPPD)

CPPD deposition disease;

pseudogout, pyrophosphate
gout, chondrocalcinosis

Positively birefringent,
rhombohedral but may have
other shapes

Basic calcium phosphates

Hydroxyapatite
Octacalcium
Tricalcium (whitlockite)
Hydroxyl phosphate
dehydrate (brushite)

Apatite gout

Usually not identified with

conventional polarized
microscopy. Alizarin red S
stain

Calcium oxalate
Monohydrate (whewellite)
Calcium oxalate (weddelite)

Primary oxalosis, chronic

renal failure (renal dialysis pt)

Small, variably irefringent,

bypyramidal

Cholesterol

Cholesterol gout (chronic

effusions, rheumatoid
atrthritis, osteoarthiritis)

Large, rectangular notched

plates or occasionally
needles

Lipid crystals

Rheumatoid arthritis, trauma,

atraumatic acute arthritis

Maltese cross, needles,

positive birefrigent

Charcot-Leyden crystals

Hypereosinophilic syndromes
with synovitis

Bipyramidal hexagons

Hematoidin

Hemarthrosis, sickle cell

arthropathy

Golden rhomboids in bright

field light

Must include:

Wet prep

Unstained cytospin

Stained cytospin

Alizarin red S

Stains Calcium

Under bright field light crystals containing

calcium are orange

Under polarized light crystals w/ calcium

appear bright red

KNOW CHART

Crystals

CPPD

MSU

Liquid lipid
crystals

Cholesterol

Corticosteroid
crystals

Lipid crystals

Hematin
crystals

Chemistr
y

Only test standard body fluids regularly. Testing analytes in nonstandard body fluids
has to be seriously considered before attempting we do not necessarily have normal
ranges, and instruments dont have validation for every fluid

Ex: usefulness of measuring glucose in a CSF is well established, but in an amniotic fluid its
meaning would be questionable

Matrix effect: Must also consider the matrix of the fluid (what else is in there besides
the analyte) because it can affect the measurement of the analyte in question:
matrix effect which can have significant influence on method accuracy.
Validation studies need to include at least:

Precision
Accuracy
Recovery experiments: known quantity of analyte is added to body fluid and percent recovered is
calculated from the concentration of the measured analyte
Percent recovery must be evaluated against acceptance criteria that meets medical requirements of
test

Analytical measuring range

Linearity experiments demonstrate the concentration range over which the analyte can be accurately
measured
Combine body fluid samples w/ both low and high concentrations of the analyte in different ratios in
order to obtain several samples that bracket the intended range

Analytic sensitivity
Lowest analyte concentration that can be measured
Repeat measurements on a sample containing a low quantity of the analyte; then analyze the
coefficient of variation for acceptability

Analytic specificity
Help identify possible interferences and their influences on measurement accuracy
Common sources of interference are blood, bilirubin, lipid

Determination of a reference range

This is very difficult to do since we dont normally obtain BF from healthy individuals
Correlate findings with clinical outcomes and use that to help establish a normal range

Reproducibility

The chart shows routine tests for certain specimen.

Microbiology

Body fluids are generally considered sterile. Seminal fluid can have
skin flora this is because the collection methods may not be sterile.
Each hospital has a different procedure for reporting results out.

Gram stain does not differentiate WBC, so it is reported as WBCs

seen. We need a wright or giemsa stain for this.

Blood culture bottles 8 -10 mL/bottle: cannot do gram stain from a

bottle it can only be done from the source itself.

We must isolate for both Aerobic and anaerobic coverage

Depending on the source the doctor might order Acid fast bacilli with
Kinyon stain

Fungus using KOH prep mostly done on specimen with a lot of cellular
material like skin. But India ink is more acceptable. KOH remove
epithelial cells so that you can see the hyphae. If it was a turbid fluid
or viscous the KOH would work because it would clear the cellular
debris.

Image shows GNDC in CSF (18 yr old meningitis pt)

Immunophenotyping

Identify specific antigens: this Identifies and localizes

specific protein antigens in tissues or cells based on
recognition of specific epitopes by antibodies. Generally
the fluid is CSF.
Uses both polyclonal and monoclonal ab to ID specific
antigen

Done in cytospin preps or unfixed cells directly from fluid

or on cell blocks. Cell block prep of fluid in a paraffin gel
which is then processed for histology
Modern techniques are mostly colormetric because of
colored tags that enables you to ID the color of the
antigen. Because most methods are colormetric
conventional microscopy can be used. Either it is there or
not.

Antibodies useful in workup of malignancie

Disease

Antibodies

Hodgkin lymphoma

CD30, CD15, CD20

Non-Hodgkin lymphoma B cell

CD20, CD79a, CD19, PAX-5,

chains

Non-Hodgkin lymphoma T cell

CD2, CD3, CD4, CD5, CD7, C

CD43

Other antibodies useful in

non-Hodgkin lymphoma

ALK, Cyclin D1, Bcl2, CD1a,

CD56/CD57, TIA1, Perforin/g
TdT, EBV-LMP

Myeloid/monocytic neoplasms

CD34, CD117, CD15, CD33,

lysozyme

Plasma cells

CD138, CD79a, / light cha

Erythroid

Glycophorin, Hemoglobin A

Breast tumors

Calponin, P63, E-cadherin, G

disease fluid protein 15, Estr
receptor, Progesterone recep

Gastrointestinal tumors

CD117, HepPar1, Synapophy

Chromogranin A, Helicobact

Skin tumors

S100, HMB45, Melan A

Flow cytometry

Fluorochrome conjugated ab: Fluorochrome conjugated antibodies

will bind w/ specific cellular antigen when incubated, unbound stuff
washed away and individual cells are evaluated as they pass single
file through a laser. Cells w/ bound antibody will emit specific
wavelengths as they pass through which can be detected, processed
and analyzed by computer

This is through a Single file analysis

It is Displayed in a dotplot or histogram.

1-3 mL fluid (small amount of fluid) adequate in cellular fluids, more

for low cell fluids

We can evaluate 5-6 antigen at the same time.

EDTA, sodium heparin or ACD if contaminated w/ blood

KNOW MARKERS ASSOCIATED WITH CELL LINES.

Common antigens evaluated by flow cytometry

Lymphoid markers

CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD30, CD38, CD56/CD57 CD103,
/, /, TdT

Myelomonocytic markers

CD13, CD15, CD33, CD34, CD117, myeloperoxidase

Plasma cell markers

CD38, CD138, / light chains

Erythroid markers

Glycophorin

Megakarocytic markers

CD42, CD61

Molecular testing

We have a lot of MT for TB in the lab. There are also MT

for viral infections. It is used for Infectious disease testing.

Image shows direct molecular testing for TB from a

sputum. The same technology can be applied to fluids.
Fluid does not require pretreatment. This method helps to
reduce contamination.

There are 3 general applications:

Hybridization/probe based signal amplification

Direct nucleic acid amplification

Nucleic acid sequencing