STAINING OF BONE MARROW AND BLOOD
ELEMENTS to reduce storage artifact.
OUTLINE
I.INTRODUCTION
II.BONE MARROW PREPARATIONS
III.BONE MARROW
IV. BONE MARROW TREPHINE BIOPSY
V. STAINS INVOLVED
A. Romanowsky Stains
B. Wright Stains
C. Giemsa Stain
D. Wright-Giemsa or Jenner-Giemsa Stain
E. May-Grun-Giemsa Stain
F. Perl’s Prussian Blue Iron Stain
G. Myeloperoxidase (MPO) Stain
H. Masson’s Trichrome Stain
VI. ANCILLARY PROCEDURES
VII. LYMPH NODE BIOPSIES
VIII. SPECIAL STUDIES
I. INTRODUCTION
● Bone marrow may be processed as a touch or smear
preparation, or in the form of biopsy.
● Usually indicated when material cannot be obtained by
aspiration (“dry tap”) as in aplastic anemia and
myelofibrosis or when visualization of bone marrow is
required, as in lipid storage diseases, malignancy or
metastatic disease
● Bone marrow biopsy demonstrates the topographic
distribution of cell types which cannot be obtained by
bone marrow aspiration.
→ Romanowsky technique:
▪ Where bone marrow aspirates stain
▪ Main diagnostic tool used by the
hematopathologist.
II. BONE MARROW PREPARATIONS
● Examination of the bone marrow aspirate and trephine
biopsy is essential for the diagnosis of bone marrow and
malignant hematologic disorders…
→ including acute and chronic leukemias,
myelodysplastic syndromes, chronic
myeloproliferative disorders, lymphomas, and
plasma cell myeloma.
● Additional investigations including flow cytometric
immunophenotyping, cytochemistry, FISH, and
molecular genetics can be performed on the bone
marrow aspirates.
III. BONE MARROW ASPIRATE
● Bone marrow smears should be prepared immediately
following aspiration.
● EDTA samples should be prepared as soon as possible
● To prepare smears:
→ Aspirate should be expelled into a small plastic
or siliconized glass dish
→ A Pasteur pipette is used to draw up particles,
which are placed on glass slides and then smeared
→ Alternatively, a drop of aspirate can be placed on
each glass slide and excess blood drained off by a
Pasteur pipette or plastic syringe before making
the smear.
● Six smears and two particle squash preparations
should be made, and particle clot preparations may
provide additional information.
● Bone marrow smears and touch preparations are fixed
and stained with standard methods used for blood smear
like Wright's or Giemsa's stain.
● Romanowsky stains used in hematology only work on
air-dried material that is relatively fresh, less than one
month old.
● It's important not to use fixatives like hairspray, alcohol,
or formalin on bone marrow smears or touch
preparations.
● For bone marrow biopsies, Zenker's solution is
recommended for fixing and decalcifying small bone
pieces mixed with the clot.
● Zenker's fluid and acetic acid are used to fix a thin slice
of bone marrow for 14-24 hours.
● The bone marrow section is then washed in running
water for 3-6 hours and processed like a bone marrow
smear.
● Usually, bone marrow smears are made at the bedside,
directly after the aspiration method.
→ How to prepare:
− At the bedside, 1ml of the aspirate is added to
a snap cap vial with 1.8% EDTA, then mixed
well, and sent to the laboratory.
− Using a Pasteur pipette that has been rinsed
previously with EDTA solution, transfer an
aliquot of the aspirate to a watch glass.
− Tilt the watch glass gently to let the blood run
to one side so that marrow particles will be
visible.
− Any excess blood can be gently blotted away
with filter paper, taking care not to touch the
marrow particles or contaminate one’s hands
with blood.
− Particles are then transferred to glass slides,
from which smears can be made
● Squash smear - often causes considerable cellular
artifacts and is useful for the assessment of cellularity,
megakaryocyte numbers, focal disease (e.g. lymphoma,
plasma cell myeloma, mast cells, metastatic carcinoma,
storage histiocytes, and granulomas) and fibrotic
marrows.
→ How to prepare squash smear : squash the marrow
particles with another slide and pull to the end of
the
● Spread smear - allows excellent detail of individual
cell morphology
 → How to prepare spread smear: place marrow
particles at one end of the slide with another slide
as for a blood smear.
● Two air-dried smears and one squash slide should be
fixed with fresh acetone-free absolute methanol and
stained with a Romanowsky stain (May-Grunwald
Giemsa or Wright Giemsa stain)
● A methanol-fixed smear and a squash slide should be
stained with Prussian blue (Perls’ reaction) and
counterstained with nuclear fast red.
● All bone marrow smears should be cover-slipped
using a mounting medium that hardens and dries
rapidly.
● Additional slides may be used for Cytochemistry,
Immunohistochemistry, Fluorescent in situ
hybridization, or archived as unfixed, unstained smears,
as required.
→ Unfixed and unstained aspirate smears stored at RT
for long periods may give variable results on
retrospective Giemsa staining.
● Aspirate slides fixed in absolute methanol preserve
DNA (and possibly many antigens) for future FISH, or
DNA extraction and subsequent PCR amplification.
● Cases with suspected acute leukemia - cytochemical
stains for myeloperoxidase and nonspecific esterases are
recommended.
IV. BONE MARROW TREPHINE (CORE) BIOPSY
● Bone Marrow Trephine (Core) Biopsy may be
performed either before or after bone marrow aspiration.
● Trephine sections are useful for the assessment of
overall marrow architecture, cellularity, provide greater
sensitivity for the assessment of of focal lesions and
patchy infiltrates
● Biopsy specimen shrinks by approx. 20% after
processing
→ Length of the core from an adult - should be at least
2cm
→ Shorter core (e.g 1 cm) = sometimes contain
sufficient diagnostic information
→ Larger the amount of tissue biopsied = greater the
likelihood of a focal lesion (e.g. lymphoma,
metastatic tumor, granulomas) detected
→ Fixation time varies from 1 to 24 hours depending
on the fixative used.
− Standard fixative for trephine core biopsies -
Neutral buffered formalin
− Other commonly used fixatives:
o Zinc formaldehyde
o B5 - gives good morphology with a short
turnaround time (TAT) but causes safety
and environmental concerns (mercuric
chloride, sodium acetate and formalin)
o Acetic acid-zinc-formalin
o Isotonic buffered formalin
o Bouin’s fixative (picric acid, acetic-acid
& formaldehyde)
o Formaldehyde and glutaraldehyde
● Touch imprints should be made prior to placing the core
tissue in fixative.
→ Imprints are made by gently touching the fresh
unfixed core on the slide or the slide on the core.
These are fixed and stained using the same method
for aspirate smear and squash preparations.
● To study a bone:
− A small piece of bone marrow attached to a thin
hard bone is decalcified for 24-48 hours.
− Decalcification time varies depending on the type
of decalcifying agent and the size of the biopsy
specimen.
● Organic and mineral acids are commonly used for
decalcifying bone marrow specimens.
● Solutions used for decalcification:
− EDTA = better preservation of nucleic acids but
slower compared to other acid reagents.
− Formic acid
− Acetic acid
− Picric
− Nitric acid
● Organic acids (e.g., formic & arctic) decalcify slower
than mineral acids (e.g., hydrochloric & nitric).
● Tissues in acid decalcifiers should not be left unchecked
for an extended period like over a weekend.
● Over-decalcification with acids, especially mineral
acids, results in tissue destruction.
● Sections should not be left in solution for a longer
period of time since this may injure, if not destroy, the
cellular detail of the bone marrow.
● To protect the cellular and fibrous elements of bone
from damage caused by the decalcifying acids, it is
important to thoroughly fix these specimens prior to
decalcification.
→ Poorly fixed = macerated and poorly stained
● To ensure even distribution of decalcifying acid:
− Tissue suspension, mild agitation, gentle mixing or
air bubble percolation.
● Section must be washed thoroughly after
decalcification, cleared, embedded in paraffin and
stained (hematoxylin and eosin, and Van Gieson).
● Recommended thickness of sections are 2-3 microns.
● Decalcification results in the leaching out of some
storage iron from the core biopsy.
→ It may affect morphology and cytological detail
and the ability to perform histochemistry and
immunohistochemistry.
→ Nitric acid and hydrochloric acid diminish the acid
fastness of mycobacteria resulting in false negative
results.
→ Acid fastness is retained when decalcification is in
formic acid, sodium citrate or citric acid buffer
● Bone marrow core biopsy specimens can also be
embedded in plastic.
→ Results with good cytological details.
→ Does not require decalcification.
→ Useful for evaluation of metabolic bone diseases.
→ Histochemical reactions removed by the
decalcification process.
● Undecalcified resin-embedded trephines have replaced Preparation/s:
the older techniques that employ decalcification and
paraffin embedding. ● Weight out 0.3 g of the
● For iron and reticulin demonstration, hematoxylin and powdered dye and
eosin (H&E) stains are used. transfer to a conical flask
→ Cytological details necessary for diagnosis of of 200-250 ml capacity.
hematopoietic disorders can be obtained from ● Add 100 ml of methanol
marrow biopsies stained by H&E technique. May-Grunwald Stain and warm the mixture to
● For identifying plasma cells, mast cells, lymphoid cells, 50°C. Allow the flask to
eosinophils, and for distinguishing between myeloblasts cool to c 20°C and shake
and proerythroblasts, use Giemsa stain. several times during the
● For glycol methacrylate sections, use toluidine blue and day.
eosin. ● After letting it stand for
→ Toluidine is the most useful and informative stain 24 hours, filter the
for plastic-embedded sections. solution
o When heated and used at high alkaline pH, it
easily penetrates the plastic and stains a blue Preparation/s:
color, with no appreciable staining of the
embedding medium. ● Prepare a 5gl solution in
● Myelodysplastic syndromes is best defined in bone Jenner’s Stain methanol in exactly the
marrow aspirate samples with May-Grünwald Giemsa same way as described
stain. earlier for the May-
→ Other stains used are: Grunwald stain
o Giemsa
o Gomori’s silver Preparation/s:
= Silver impregnation staining methods are
fixation dependent. ● Weigh 1 g of the
o Perls powdered dye and
● Reticulin stain is useful for the detection of transfer to a conical flask
myelofibrosis. of 200-250 ml capacity.
→ Reticulin is greatly increased in this disease and Giemsa’s Stain
● Add 100 ml of methanol
slightly increased with increasing cellularity of the and warm the mixture to
marrow like conditions of leukemia and lymphoma. 50°C; keep at this
● Trichrome stain may be used to identify collagen temperature for 15 mins
fibrosis. with occasional shaking,
then filter the solution.
V. STAINS INVOLVED:
Preparation/s:

A. Romanowsky Stains ● The stock includes azure

Romanowsky Stains
● Consist of methylene
blue/azure B and eosin
dissolved in acetone-free
methanol
● Include Jenner, Giemsa,
Description/Details
May Grunwald, and
Leishman stains.
● Romanowsky found that
better staining resulted
when old (ripened and
therefore “polychromed”)
Preparations of Solutions of Romanowsky Dyes:
Azure B–Eosin Y Stock B, tetrafluoroborate or
Solution thiocyanate (Color index
52010), >80% pure, and
eosin Y (Color index
45380), >80% pure.
Disease/s involved with Romanowsky Stains:
● All are used to examine blood or bone marrow
samples.
● They are preferred over H&E for inspection of blood
cells because different types of leukocytes (white
blood cells) can be readily distinguished.
● All are also suited to examination of blood to detect
blood-borne parasites like malaria.
B. Wright’s Stain
Wright’s Stain
● Methylene blue is
polychromed by heating
with sodium
bicarbonate.
● It may be purchased in
Detail/s:
solution ready for use,
or as a powder, 1.0 gm.
which is carefully
dissolved in 600 ml. of
methyl alcohol.
● The eosinates of
polychromed methylene
blue are dissolved in
absolute methyl
alcohol.
● When this solution is
placed on a dried blood
smear, the methyl
alcohol acts as the
fixative, and the
dissolved dye begins
the staining process.
● After one to three
Principle:
minutes, the stain is
diluted with an equal
volume of distilled
water. This
differentially stains the
cytoplasmic granules
and is allowed to act for
about three minutes.
● It is then poured off
and the preparation is
washed briefly in tap
water and allowed to
dry.
● Streak thin
(approximately one cell
thick) smears across a
Fixation:
sterile slide by means of
a second slide or cover
glass. Air dry quickly.
● Bacterial Infections = ↑ White Blood Cells = Increase
1. Place the air-dried
is called Leukocytosis
smear, film side up,
→ Diseases that cause ↑ WBC
on a staining rack.
2. Cover the smear with
● Decreases called Leukopenia
undiluted Wright stain
→ Disease that cause ↓ WBC
Method: and leave for 1
minute. The methyl
alcohol fixes the
● Bloodborne Parasites
smear.
3. Dilute with distilled
water (approximately
equal volume) until a
metallic scum
appears. Allow this
diluted stain to act for
2 1/2 to 5 minutes.
4. Without disturbing the
slide, flood with
distilled water and
wash until the thinner
parts of the film are
pinkish red.
5. If distilled water does
not give adequate
differentiation, a
phosphate buffer (pH
6.4 to 6.5) should be
used, at least for
washing, but possibly
also for diluting the
stain (step 3).
Results:
Erythrocytes ● Yellowish-red
● Nucleus - dark blue
Polymorphonuclears ● Granules - reddish-lilac
● Cytoplasm - pale-pink
● Nuclei - blue
● Granules - red to
Eosinophils
orange-red
● Cytoplasm - blue
● Nucleus - purple to dark
Basophils
blue
● Granules - very dark
purple
● Nuclei - dark purple
Lymphocytes
● Cytoplasm- sky blue
● Granules - violet to
Platelets
purple
Disease/s involved with Wright Stains:
▪ Appendicitis, Meningitis, Bacterial Pneumonia
▪ Typhoid Fever and Tuberculosis,
▪ Measles and Influenza (Viral)
 → African Trypanosomiasis, Babesiosis, Chagas Microorganisms fungi, ● Purplish-blue
Disease, and Toxoplasmosis = often best parasites
accomplished with Wright-Giemsa Stain (specific)
● Sky blue
● Pigments
C.Giemsa Stain
→ Native color is
Giemsa Stain Starch granules, cellulose yellow/brown
→ Green if fixed in
● A member of the dichromate
Romanowsky group of containing fixative
stains - being the black
precipitate formed from Nuclei ● Dark blue to violet
the addition of aqueous
solutions of methylene Erythrocytes ● Salmon pink
blue and eosin
dissolved in methanol. ● Varying light blue
Cytoplasm
Detail/s: ● This stain employs shades
various azure
compounds (thionine Notes:
and its methyl
derivatives) with eosin 1) It is usually performed at RT overnight.
and methylene blue a) Increase stain temperature = short staining time
● Best purchased 2) The stain stains red cells and neutrophil poorly, but
commercially in azurophil granules (red) are well stained.
solution 3) It is not commonly used alone in Hematology
4) An excellent stain for inclusion bodies if the smears
● Giemsas\’s stain, stock are allowed to stain in dilute Giemsa stain for 12 to
solution - obtain from 18 hours
commercial sources 5) Differentiation with acetic acid will vary according
→ Giemsa reagent to the staining time and temperature (Generally
improves with age achieved within 30s)
Solutions: ● Giemsa stain, working
solution Disease/s involved with Giemsa Stain:
→ Giemsa stock
solution 40 drops ● Book: binds to some pathogens
→ Distilled water 40mL → Spirochetes = Syphilis
● Acetic acid 0.5% → Tryapanosomes = Sleeping Sickness and Chagas
Disease
1. Bring sections to → Plasmodium = Malarial
distilled water → Parasites
2. Stain with diluted ● Additional references: makes it possible to
Giemsa’s stain made demonstrate the presence of microorganisms in all
up fresh types of tissues.
3. Rinse in distilled → Helicobacter Pylori = Gastric Ulcers or Chronic
Method: Gastritis
water
4. Differentiate with → Parasite Leishmaniasis (Detect)
0.5% aqueous acetic → Fungus Aspergillus niger = Pulmonary Mycosis
acid (Detect)
5. Dehydrate rapidly
6. Clear and mount

Results:

Bile pigments ● Green

Collagen, muscle, bone ● Pale pink

D. Wright-Giemsa or Jenner-Giemsa Stain
Wright-Giemsa or Jenner-Giemsa Stain
● Combination of
Romanowsky and
Giemsa.
● Improves staining of
cytoplasmic granules.
→ When properly
performed, it gives
Detail/s: sharp and clear
cytoplasmic, nuclear,
and granular details.
● Keep a separate stock
for bone marrow
staining at least 6
months prior to use to
ripen.
● Eosin Y = stains
cytoplasm; orang-pink
Components: ● Methylene Blue &
Azure Blue = stains
nucleus; blue-purple
1. Dip smears in
methanol to fix the
specimen.
2. Place in Wright's or
Wright-Giemsa stain
for 10-15 minutes.
3. Move the smear to a
mixture of stain 6.8
Method: pH phosphate buffer
for 20-30 minutes.
(One part stain and 2-
3 parts buffer).
4. After staining, give a
quick rinse in distilled
water and air dry
before mounting or
cover-slipping.
Results:
Nuclei ● Purple/blue
Cytoplasm ● Pink/blue
Eosinophils ● Pink/red
Disease/s involved with Wright-Giemsa or Jenner-
Giemsa Stain:
● Testing for bloodborne parasites, including African
trypanosomiasis, babesiosis, Chagas disease, and
toxoplasmosis, is often best accomplished with a
Wright-Giemsa stain.
E. May-Grunwald-Giemsa Stain
May-Grunwald-Giemsa Stain
● Contains alkaline
methylene blue (basic
Detail/s: dye), related azures
(basic dye) and acidic
eosin dye.
1. Fix air-dried smears
in methyl alcohol for
5 minutes.
2. Transfer to May-
Grunwald stain
(another
Romanowsky dye)
freshly diluted with 1
or 2 volumes of
buffered distilled
water (pH 6.8). Leave
for 3 to 5 minutes.
3. Transfer, without
washing, to fresh
diluted Giemsa and
allow to stain for 7 to
Method:
15 minutes.
4. Differentiate by
washing quickly in
three changes of
buffered (pH 6.8)
distilled water and
place in a fourth
change of buffered
water for 3 to 12
minutes. (Monitor
degree of
differentiation under
low power
microscope).
5. Stand the slide on end
to dry.
Notes
1. The use of Coplin jars for staining will minimize
the possibility of precipitation of stain on the glass
slide. If this occurs, the slide can be re-immersed in
methanol and in preferred Romanowsky stain.
2. Differentiation in phosphate buffers can be adjusted
to suit personal preference in the balance between
blue and the eosin staining.
3. It is essential that the slide is completely dry before
mounting.
F. Perl’s Prussian Blue Iron Stain
Perl’s Prussian Blue Iron Stain
● Iron staining on bone
marrow aspirate smears
is commonly part of the
standard order protocol
for bone marrows
aspirates.
● Bone marrow aspirates
are more sensitive than
trephine biopsy sections
for the detection of
hemosiderin when the
biopsy specimens are
decalcified in formic
acid.
● The detection of
hemosiderin in a Perls'
stained bone marrow
G. Myeloperoxidase (MPO) Stain
aspirate is regarded as
the gold standard by
which other tests for
iron deficiency or
depletion are assessed.
● In the adult setting, it is
commonly performed
on the bone marrow
Detail/s: biopsy, but can be
requested on the
aspirates as well.
● Mercurial fixatives
seem to do a better job
of preserving iron in
bone marrow than
formalin.
● A Prussian blue stain
should be performed on
a bone marrow smear
for the evaluation of
storage iron and
sideroblasts.
● A bone marrow smear
with increased iron
stores should be
included as a positive
control.
● Core biopsy sections
are less reliable than the
aspirate for the
assessment of storage
iron, since
decalcification removes
storage iron.
● The section is treated
with dilute hydrochloric
Principle
acid to release ferric
ions from binding
proteins. These ions
then react with
potassium ferrocyanide
to produce an insoluble
blue compound (the
Prussian blue reaction).
This procedure is
particularly helpful
when evaluating
patients with anemia,
iron overload, and
myelodysplasia., etc.
Disease/s involved with Perl’s Prussian Blue Iron Stain
● Hemochromatosis, esp., liver. excess iron deposition
is stained as blue granules
● Gastric ulcer 2nd to iron overdose
Myeloperoxidase (MPO) Stain
● Helpful in identifying
cytoplasmic granules
characteristic of
myeloid cells.
● Useful when there are
large, immature WBCs
in the peripheral blood,
to differentiate myeloid
Detail/s: leukemia cells from
those of lymphoid
origin.
● Useful in differentiating
the blasts of acute
myeloid leukemia
(AML) from those
acute lymphoblastic
leukemia (ALL)
● 40% formaldehyde 1
Fixative: part
● 95% ethanol 9 parts
● 90% Ethanol 100 mL
● Benzidine
hydrochloride 0.3gm
● 3.8% Zinc sulfate 1.0
mL
Staining Solution:
● Sodium acetate 1 gm
● 1N Sodium hydroxide
1.5 mL
● 3% Hydrogen peroxide
(10 volumes) 1.7 mL
● Giemsa stain diluted 1
Counterstain: in 10 with buffered
distilled water at pH 6.8
 ● 30% ethyl alcohol 100
mL
● Benzidine
dihydrochloride 0.3 gm
● 0.132M (3.8 % w/v)
Incubation Mixture: ZnSO4 7 H20 1.0 mL
● 3% Hydrogen peroxide
0.7mL
● 1.0N Sodium hydroxide
1.5mL
● Safranin 0 0.2 gm
● Positive
● Stained most intensely
Eosinophils ● Color: often tinged
brown black or green-
black
● Positive
Cytoplasm of neutrophils
● Filled with blue dye
Basophils, lymphocytes ● Negative
and erythroblasts

1. Use fresh smears of ● Positive

blood or bone marrow ● Shows slight peroxidase
Monocytes
imprints. These may activity
be preserved for as ● Cytoplasm
long as 3 weeks if
preparations are Disease/s involved with Myeloperoxidase (MPO) Stain
stored in the dark.
2. Fix slides for 60 ● Myeloid Leukemias and Granulocytic Sarcoma
seconds at room temp. ● Congenital Deficiency of Myeloperoxidase
In 10% formal ethanol deficiency
( 10ml of 37%
formaldehyde and 90 H. Masson’s Trichrome Stain
ml of absolute ethyl-
alcohol) Masson’s Trichrome Stain
3. Wash the slides for
15-30s with gently ● For GMA Plastic Bone
Detail/s:
running tap water. Marrow Sections
Shake off excess
Method: water. ● Undecalcified glycol
4. Place wet slides in an Sections: methacrylate embedded bone
incubation mixture in marrow sections cut at 4 μm.
a Coplin jar for 30s at
room temp. ● Modified Weigert’s Iron
Solutions:
5. Wash briefly for 5- Hematoxylin
10s in running tap
water, dry and Solution ● Hematoxylin 2.0 gm
examine. A: ● Alcohol, 90% 100.0 ml
6. If greater nuclear
detail is desired, the ● Ferric chloride, FeCl3.6H20,
stained preparations Solution 62% aqueous 4.0 ml
may be re- B: ● Distilled water 95.0 ml
counterstained in 1% ● HCL concentrated 1.0 ml
aqueous cresyl violet
acetate for 1min, or in ● Working Modified Weigert’s
freshly prepared Iron Hematoxylin
Giemsa stain for 10 → Equal parts of Solution A
minutes. and Solution B
● Biebrich Scarlet-Acid Fuchsin
Results: Solution
Counterstain: → Biebrich scarlet, C.I. 26905
● Positive 0.45 gm
● Peroxidase activity = → Acid fuchsin, C.I. 42685
Myeloid cells (except 0.05 gm
presence of green to
basophils) → Acetic acid, glacial 0.50 ml
dark blue granules in
the cytoplasm ● Phosphomolybdic-
Phosphotungstic Acid Solution
 → Phosphomolybdic acid 2.5
gm
→ Phosphotungstic acid 2.5
gm
→ Distilled water 100.0 ml
● Aniline Blue Solution
→ Aniline blue, C.I. 42755 1.0
gm
→ Distilled water 40.0 ml
→ Acetic acid, glacial 0.8 ml
● 1% Acetic Acid Solution
→ Acetic acid, glacial 1.0 ml
→ Distilled water 99.0 ml
1. Place in modified Weigert’s
iron hematoxylin for 5
minutes.
2. Wash briefly in running tap
water and rinse in two
changes of distilled water.
3. Decolorize with 0.5%
hydrochloric acid in 70%
alcohol for 5 seconds.
4. Wash in running tap water
for 30 seconds and rinse in
two changes of distilled
water.
5. Place in a Biebrich scarlet-
acid fuchsin solution for 30
minutes.
Method: 6. Rinse in three changes of
distilled water
7. Place in phosphomolybdic-
phosphotungstic acid for 10
minutes.
8. Rinse in two changes of
distilled water.
9. Place in aniline blue solution
for 7 minutes.
10. Rinse in two changes of
distilled water.
11. Place in 1% acetic acid for 1
minute.
12. Rinse in three changes of
distilled water and air dry.
13. Dip in xylene and mount
with synthetic resin.
Results:
Osteoid ● Red
Mineralized ● Blue
Nuclei ● Dark gray
Disease/s involved with Masson’s Trichrome Stain
● Diagnosis of fibrotic changes, such as those that
occur in liver cirrhosis, renal disease (glomerular
fibrosis) .
VI. ANCILLARY PROCEDURES
● Immunochemistry — performed by
immunoperoxidase/immune alkaline phosphatase
methods.
● Immunohistochemistry — required for the
determination of the lineage and differentiation stage of
normal/abnormal cells.
→ For specimens: prolonged fixation in 10% NBF may
be detrimental
→ Reduces fixation-related effects — transfer the tissue
to 70% ethanol after 12-hour fixation
▪ In-situ hybridization — performed on bone
marrow biopsies
o Formalin fixation: superior antigen and DNA
preservation
o Metallic fixatives (Zenker’s and B-5):
degrade DNA and impair subsequent
molecular diagnostic testing.
VII. LYMPH NODE BIOPSIES
● Lymph node - small round of organs that are part of the
lymphatic system that contain a fluid (lymph) that
carries infection-fighting white blood cells and fluid and
waste products from the body’s cells and tissue.
● Removes all or part of the lymph node to be examined
under the microscope for signs of infection or a disease
(e.g. cancer)
● Other tests like culture, genetic tests, or immunological
tests may also be used to check lymph tissue samples.
● There are several ways to do a lymph node biopsy:
→ Fine needle aspiration
− A long thin needle is inserted into a lymph node
to remove a sample of cells or tissue.
→ Open (excisional) biopsy
− A small cut is made in the skin and one or more
lymph nodes are taken.
→ Sentinel lymph node (SLN) biopsy
− New technique that is increasingly used in
cancer patients
− The first one to receive lymphatic fluid draining
from a cancer site, and this can predict whether
cancer is likely to be found in the rest of the
lymphatic system without removing all of the
nearby nodes.
− Most important aspect of sentinel node
examination
= Slicing the SLN no thicker than 2.0 mm
= Correct embedding of the slices - all
macro-metastases larger than 2.0mm are
identified
 − Most reliable method to assess metastatic
melanoma
= In Immunohistochemistry - performed
with both antibodies (S-100 and HMB-45)
combined with careful conventional
histopathology based on multiple sections.
− Allows more accurate diagnostic and prognostic
staging
Results in Sentinel Lymph Node (SLN) Biopsy
● Sentinel node contains no
cancer cells
● Cancer has not yet spread to
Negative nearby lymph nodes or organs
A complete lymph node
dissection may be unnecessary
● Cancer is present and may
have spread to other nearby
lymph nodes (aka regional
lymph nodes) or possibly to
Positive
other organs
All nearby nodes must be
removed
Reference:
● https://www.cancer.gov/about-cancer/diagnosis-
staging/staging/sentinel-node-biopsy-fact-
sheet#:~:text=nearby%20lymph%20nodes.-
,What%20is%20a%20sentinel%20lymph%20node
%3F,than%20one%20sentinel%20lymph%20node
Processing Lymph Node Biopsies
● Samples need to be processed such that the following
investigations can be carried out if required:
→ Microscopy on appropriately fixed and stained
tissue samples
→ Immunological investigation by
immunohistochemistry and/or FC
→ Cytogenetic analysis by Giemsa-banding (G-
banding)
→ FISH on cell suspensions, films, imprints or
paraffin sections
→ Molecular genetic analysis by polymerase chain
reaction (PCR), real-time PCR (RT-PCR) or gene
sequencing
● Lymph nodes may be received as complete nodes or as
needle core biopsy specimens.
● In the absence of gross tumor, the entire node is cut into
3- to 4-mm slices in the longitudinal or transverse plane,
taking care to process different surfaces for microscopic
examination.
● If the node is so small that it cannot be sliced in this
manner, it may be totally submitted as one piece.
● In the presence of visible tumor within a single lymph
node, one or several routine sections to demonstrate the
tumor and its possible extra-nodal extension. Every
node must be submitted for processing.
● Lymph node dissections are best processed fresh,
although the specimen may be fixed for paraffin or
plastic embedding and morphological examination.
● If a frozen section is performed, the results of frozen
section will guide the subsequent approach.
● If a lymphoproliferative disorder is suspected at the
time of frozen section, it is important to ensure that
sufficient fresh unfrozen tissue is kept for routine
histopathology.
● If macroscopic examination of a larger node leads to the
detection of a possible infective focus, a small piece of
the fresh tissue should be sent for microbiological
culture.
● If tuberculosis or fungal infection is suspected, the fixed
sample should be placed in formalin for 48 hours.
● Also, if HIV is suspected most laboratories would
expect the node to be fixed for 48 hours prior to
processing.
● Cytological diagnosis based on morphology and
confirmation by a positive Ziehl-Neelsen stain for acid-
fast bacilli is a time-tested method for the diagnosis of
tuberculous lymphadenitis, it has low sensitivity.
● Auramine-rhodamine stain is considered better than
routine Ziehl-Neelsen stain in sensitivity, but is less
specific.
● During paraffin sectioning, the tissue is cut at several
levels. At least 10 intervening sections from the ribbon
should be prepared, using coated or charged glass slides,
and stored unstained.
● These are invaluable if a small infiltrate is noted in any
one level, or unexpected features suspicious of
lymphoma are found, requiring subsequent
immunohistochemical analysis.
● Lymph node sections are stained using the standard
H&E stain.
● Reticulin staining may be helpful in assessing the
follicular architecture. Immunohistochemical staining
and other specialized techniques may be used but are
not mandatory unless indicated.
● The specimen may be fixed in formalin to allow
paraffin or plastic embedded sections for morphological
analysis.
● Small samples such as needle core biopsies may be
insufficient material for any additional investigations.
● However, if large enough, a whole fresh lymph node
can be divided so that part of the specimen can be fixed
for histological sections and part can be used fresh for
other investigations.
● Tissue sampled for paraffin blocks news to be 3 mm
thick and fixed for 24-48 hrs.
→ Prolonged fixation = immunochemistry more
difficult and recovery of DNA from paraffin blocks
unreliable
● Specimen preparation with hematoxylin and eosin
(H&E) staining = remains gold standard for histologic
interpretation of lymph node material
● Both serial sectioning and immunohistochemical
staining for breast cancer or melanoma-associated tumor
markers = facilitate detection of metastatic tumor
→ Ex. high sensitive S-100 & high-specific MART-1)
● Rapid immunostaining of frozen sections may be IX. REFERENCES
possible using the enhanced polymer one-step staining
(EPOS) system (Dako) with antibodies against ● Bruce- Gregorios, J. H. (2016) Histopathologic
leucocyte common antigen (LCA), cytokeratin (CK), Techniques. (2nd Revised Edition). Makati, PH: Katha
and antimelanoma (MEL) after fixing samples in 100% Publising (611.0812/883)
acetone for 20 seconds (CK, LCA) or two minutes ● Clinisciences. (n.d.). CliniSciences.
(MEL), followed by incubation of the primary antibody https://www.clinisciences.com/en/buy/cat-giemsa-stain-
and development of the chromogen reaction with 3 3,' 3967.html
diaminobenzidine (DAB) for three and five minutes at ● Ethos BioSciences, Inc. (2021a, December 21).
37°C, respectively. Choosing Between Wright's Stains Or Wright-Giemsa
● For rapid intraoperative diagnosis of lymph node Stains • Ethos Biosciences. Ethos Biosciences.
micrometastasis, combining anti-cytokeratin https://www.ethosbiosciences.com/choosing-wrights-or-
antibodylabeled nano-crystal beads with rapid wright-giemsa-stains
hematoxylin and eosin (H&E) staining on the same ● Ethos BioSciences, Inc. (2021b, December 21).
section, referred to as the rapid double staining (RDS) Choosing Between Wright's Stains Or Wright-Giemsa
technique has also been reported. Stains • Ethos Biosciences. Ethos Biosciences.
https://www.ethosbiosciences.com/choosing-wrights-or-
wright-giemsa-
VIII. SPECIAL STUDIES
stains#:~:text=Testing%20for%20bloodborne%20parasi
tes%2C%20including,with%20a%20Wright%2DGiems
● Optional cytogenetic analysis - fresh sample of
a%20stain.
specimen in tissue culture medium.
● Method of the Histochemical Stains and Diagnostic
→ Cells left more than a few hours without being
Application - Department of Pathology and Laboratory
placed in culture medium are unlikely to yield
Medicine - University of Rochester Medical Center.
useful cytogenetic information.
(n.d.). https://www.urmc.rochester.edu/urmc-
→ Cytogenetic analysis may not be necessary if H & E
labs/pathology/stainsmanual/index.html?stain5
stained sections show reactive changes or evidence
● Myeloperoxidase. (n.d.).
of metastatic disease.
https://www.pathologyoutlines.com/topic/stainsmyelope
● Fluorescent in situ hybridization (FISH) -examines
roxidase.html
tissue imprints made from fresh biopsies.
● University of Wyoming. (n.d.). WRIGHT BLOOD
→ If the lymph node is fresh, make up to five imprints
SMEARS. UW Navigation. Retrieved April 22, 2023,
from the lymph node and preserve for FISH
from http://www.uwyo.edu/molb2021/virtual-
analysis
edge/lab06/exp_6c.html#:~:text=Wright%20stained%20
→ Imprints are initially air-dried.
blood%20smears%3A&text=Diseases%20that%20can%
o Fixed in chilled methanol-acetic acid (3:1) for
20cause%20the,typhoid%20fever%2C%20tuberculosis
15– 30 minutes and stored at –20ºC in a
%2C%20etc
desiccated chamber.
● Cytogenetic or FISH analysis — used as adjuncts for the
diagnosis of Burkitt’s lymphoma, but should not be
requested as a routine investigation on all samples.
→ Sections for FISH should be thinly sliced (2-4
microns).
→ Protease digestion times will vary depending on the
thickness of the section.
● The use of PCR for specific translocations:
→ t(14:18)
→ for detection of T cell receptor clonality and B cell
clonality,
→ based on T-cell receptor
→ immunoglobulin heavy
→ light chain gene rearrangement
о All important them are parts of the diagnostic
study.
● No diagnostic material should be discarded until all
investigations are complete.