Frozen sections CNS - Rcpa.tv

Frozen sections of the CNS
Dr Alpha Tsui Royal Melbourne Hospital 2008
*Note: This study guide should be used in conjunction with the brain smear
cytology module.
General aspects of frozen sections:
-specimen should be fresh and free of excess moisture
-wrapping specimen in gauze with saline solution may produce severe ice crystal
artefact
-liquid nitrogen (-190 °C) is used to freeze the tissue. Alternatively for brain tissue,
isopentane cooled in liquid nitrogen (-150 °C) may be used.
-cryostat is kept at -20 °C
-optimal cutting temperature solution (OCT), a mixture of glycols and resins, is watersoluble
and will rinse away during fixation and staining
-number of ice crystals in the tissue depends on the speed of tissue freezing. Slowfreezing
causes larger numbers of ice crystals and should be avoided.
-thinner specimens will freeze faster
-ideal thickness of tissue submitted for frozen section is 1-2mm
-tissue to be frozen should never be thicker than 3mm (not usually a problem in
stereotactic brain biopsies)
-most tissues are cut well at -10 to -20 °C
-extremely soft tissue e.g. brain, spleen, adrenal gland, lymph node, is best cut at -7 to
-10°C
-very low temperature of -20 to -40°C is required to cut fatty tissue e.g. breast
(alternatively, cut thicker sections)
-most important in the adherence of tissue to the slide is presence of proteins in the
tissue section. Since fixation denatures proteins, frozen sections cut from fixed tissue
will not adhere to the slides. To correct this, slides may be dipped in a solution of
gelatin, albumin, chrome alum and distilled water.
Frozen section procedure:
1. Check the request slip and specimen container to ensure correct patient
identification
2. Examine the tissue under safety cabinet. Describe macroscopic appearance.
3. Select appropriate tissue (Avoid freezing the entire specimen if there is a chance
that the surgeon will not give you more tissue)
4. If the tissue is wet, blot the outer surface of the tissue dry with paper towel to
minimize ice-crystal artefact
5. Tissue Freezing
-OCT is first applied to the circular Reichert-Jung chuck
-place the chuck into liquid nitrogen or using the cooling bar inside the cryostat
-place the specimen onto the frozen chuck, add more OCT and then freeze the
chuck again. Do not overfreeze the tissue as this causes difficult sectioning and
chattering artefact

-the tissue is rapidly frozen to minimize ice-crystal artefact
6. Tissue sectioning (5µm thick for brain tissue) using rotary microtome. Section
pressed onto a glass slide.
7. Slides must be immediately placed in fixative (about 30secs), not allowing airdrying
to occur. For brain tissue: 95% ethanol is used. For non-neural tissue: 10%
formalin / 95% ethanol (1:1 ratio) is used. A delay in fixation, even for several
seconds, can significantly affect the cellular detail.
8. Rapid H&E stain
9. Dehydrate, clear, mount and coverslip
10. Report to the surgeon and write down the diagnosis on the request slip
11. Special studies (send remaining fresh tissue for culture, flow cytometry if
indicated)
12. The piece of frozen tissue should be then fixed in formalin and processed for
paraffin section
Rapid H&E stain for frozen section:
-progressive staining
1. fix sections in ethanol / formalin for 30 seconds
2. rinse in water
3. stain in Harris' haematoxylin for 30 sec
4. rinse in water
5. rinse in Scott's tap water
6. rinse in water
7. stain in eosin for 10 seconds
8. dehydrate (in alcohol), clear, mount in DPX and coverslip
Indications for CNS frozen sections:
-to obtain adequate abnormal material for a diagnosis (including tissue for special
studies e.g. microbiology)
-to know the nature of the lesion: neoplastic or not; benign vs malignant; tumour
typing (esp. lymphoma; primary vs metastatic); low grade vs high grade glioma (in
some cases, the surgeon is trying to biopsy the contrast enhancing component of the
lesion which may be high grade)
-status of resection margins (not usually an issue with CNS tumours)
Advantages of frozen section:
-architecture of the tumours can be better assessed as compared to cytology smears
-not a problem with firm tumours that are difficult to smear
-can do multiple levels
Disadvantages of frozen section:
-prone to artefacts
-some tumours are altered by the freezing procedure: oligodendroglioma, pilocytic
astrocytoma, ganglion cell tumour, lymphoma
-some tumours are difficult to cut e.g. bony / calcified lesions, cysts, fatty tissue
-takes more time to prepare the slides
-technically more difficult to have satisfactory sections
-more expensive
-requires maintenance of the cryostat

-decontamination procedure takes a longer time and a spare cryostat is required
Use of cytology smears:
Advantages:
-faster than doing a frozen section
-ease of preparation
-technically simple
-nuclear detail is better seen with brain smears e.g. round nuclei of oligodendroglioma
much better visualized; lymphoma; differentiation features of a carcinoma e.g.
keratinisation
-when infectious process is suspected, smears can be used to avoid contamination of
the cryostat
-only small amount of tissue is required
-less artefacts as compared to frozen section
Disadvantages:
-some tumours are hard to smear e.g. schwannoma, meningioma, haemangioblastoma,
neurofibroma, subependymoma, etc
-cysts are better assessed with frozen section
-some features are not seen e.g. vascular pattern of haemangioblastoma; perinuclear
haloes of oligodendroglioma
Technical problems and artefacts during frozen section:
-ice crystal artefact (damage usually most prominent in the centre of the tissue):
freezing too slow; tissue too wet; tissue too large to permit rapid freezing
-chattering artefact: tissue too cold and too hard (e.g. calcified); specimen or blade
poorly supported or adjusted; damaged blade; section too thick
-folding artefact: section not straightened out when pressed onto the glass slide
-pale staining: section dried out before putting the slide into fixative
-section falling off the slide: fatty tissue; formalin-fixed tissue; small cauterized
specimens; cartilage; necrotic tissue
-tissue section adherence to knife edge: high block temperature, incorrectly positioned
antiroll plate
-contamination e.g. in OCT medium; fixative; staining solutions
Decontamination procedure for the cryostat:
-use formalin bomb: put concentrated formalin (40%) and potassium permanganate in
the cryostat with lid closed overnight
-the cryotome is removed the following day. The components are cleaned up with
70% ethanol
-the cryotome is placed into the 37°C incubator to allow thorough drying and then
lubricated
Approach to frozen sections of the CNS:
1. check the radiology (site, size, single or multiple, solid or cystic, calcification,
contrast enhancement or not), clinical history (esp. age, symptoms), previous biopsies,
past treatment
2. pick abnormal tissue for smear + frozen section – often has different consistency
and colour from normal cortex/white matter

3. avoid freezing entire specimen (unless surgeon is happy to take more tissue later
on)
4. decide normal or abnormal (cellularity, architecture, cellular detail)
5. if abnormal, is it benign or malignant
6. if benign, is it specific ? e.g. infarct, abscess, benign neoplasm
7. if possibly infectious, must ask surgeon for more tissue for microbiology (or submit
unused tissue)
8. if malignant, is it primary or metastatic
9. if primary tumour, may need to give grading (low or high grade, look for mitoses,
endothelial cell proliferation, necrosis)
10. if secondary, is it carcinoma, melanoma, lymphoma or sarcoma
11. if suspected lymphoma, ask for more tissue for flow cytometry
12. communicate clearly to the surgeon; ask for more tissue if there is insufficient
material for a diagnosis or if the lesion is entirely necrotic
Normal:
Normal cerebral cortex and white matter:
-uniformly distributed neurons maintaining the normal architecture
-astrocytes evenly spaced showing no nuclear atypia
-oligodendrocytes may form linear rows in white matter tracts
-deep layers of temporal or parietal cortex often show normal perineuronal satellitosis
by oligodendrocytes. In oligodendroglioma, the number of these cells is increased and
they have nuclear enlargement.
Normal cerebellar cortex:
-beware normal granule cells mimicking a small round blue tumour such as
medulloblastoma or metastatic small cell carcinoma
-normal granule cells are small and they have round nuclei. They don’t show nuclear
moulding.
-look for adjacent evenly spaced Purkinje cells
Normal anterior pituitary gland:
-uniformly packeted arrangement
-cells show different size and staining properties
-it is important to advise the surgeon if normal tissue has been removed during
adenoma surgery as too much normal tissue resected will cause hypopituitism postoperatively
Non-neoplastic lesions:
Reactive gliosis:
-characterised by evenly spaced astrocytes of uniform size with abundant eosinophilic
cytoplasm and radiating long processes (neoplastic astrocytes have truncated or absent
processes)
-mitoses are morphologically normal and usually sparse
-with time, reactive astrocytes become more densely fibrillated
-do not form secondary structures: dense subpial, subependymal, perivascular and
perineuronal aggregation not seen
-microcysts are not a feature of reactive gliosis
-if there are foamy macrophages or perivascular lymphocytes, a reactive process
should be first considered

Infarct:
-acute infarcts are not usually biopsied. Organising infarcts with ring enhancement,
mimicking glioblastoma radiologically, are the ones for biopsy.
-infarcts always involve the cerebral cortex
-predominant cells are ovoid to polygonal lipid-rich histiocytes (gitter cells) with
round nuclei and foamy cytoplasm
-mitoses may be seen but are not atypical
-aggregation of histiocytes in cuffs about the vasculature is common and is often
accompanied by small numbers of lymphocytes or polymorphs
-reactive astrocytes are large with prominent cell bodies and possess long processes
-there is a proliferation of blood vessels and they can show endothelial cell
hyperplasia. Thrombi may be present.
-look for ischaemic neurons (pyknotic nuclei, dense hypereosinophilic cytoplasm)
Demyelination:
-the lesion is centered in the white matter
-sheets of foamy histiocytes (may be difficult to see in some cases)
-perivascular cuffs of lymphocytes a clue
-neurons and their processes are intact
-no ischaemic red neurons
-necrosis is rare
-no cavitation
-final diagnosis requires clinical correlation and special stains for myelin and axons
Do not misinterpret macrophages as gemistocytes. Foamy
macrophages are seen in infarcts, demyelination disorders, vasculitis,
Whipple’s disease, post-chemoradiotherapy and lymphomas esp. when
steroids are given pre-operatively.
Organising abscess:
-circumscribed lesion with a liquefied centre
-there are polymorphs, lymphocytes, plasma cells, histiocytes, broad areas of necrosis,
plump reactive astrocytes and fibrocapillary proliferation
-the lesion is characteristically zonated: centre of polymorphs; layers of necrotic
tissue; foamy macrophages among reactive capillaries, monocytes, proliferating
fibroblasts; intense reactive gliosis in surrounding brain
-finding an organising collagenous capsule is a clue
-beware reactive gliosis at the periphery, mimicking a glioma
-endothelial cell hyperplasia can be prominent, forming a rim. This does not equate
high grade glioma.
If the features are suggestive of an abscess during frozen section, fresh tissue
must be sent for microbiology. Paraffin sections may not show the organisms.
Progressive multifocal leukoencephalopathy:

-histiocytes and reactive astrocytes may be numerous. The astrocytes may appear
pleomorphic and multinucleated. Again, presence of foamy macrophages gives a clue
that the lesion is probably not a glioma.
-the pleomorphic astrocytes are usually single and they do not form cellular sheets
-overall cellularity falls short of that seen in high grade astrocytoma
-presence of diagnostic homogeneous glassy intranuclear inclusions in
oligodendroglial cells
-absence of microvascular proliferation, palisaded necrosis, secondary structures
Prolapsed intervertebral disc:
-degenerate granular amorphous acellular material (may mimic necrosis)
-fibrosis may be seen
-inflammation usually sparse
Astrocytic tumours:
Diffuse (low grade) astrocytoma:
-Macro: grey with a hint of yellow; vary from soft, gelatinous to firm; cysts may be
seen
-increased cellularity
-astrocytes are irregularly distributed, forming small clusters of a few cells
-nuclear atypia seen: nuclear enlargement, hyperchromasia, irregular nuclear
membrane
-perineuronal satellitosis is a valuable clue: Atypical cells cluster around cortical
neurons (not seen in reactive gliosis)
-microcystic change seen with glioma rather than gliosis
The freezing process may cause artefactual nuclear angulation and
hyperchromasia. It can cause normal or gliotic tissue to look neoplastic; a
grade II lesion appearing as a grade III.
Anaplastic astrocytoma:
-use the term “high grade astrocytoma” is sufficient at the time of frozen section
-usually more cellular than diffuse astrocytoma
-look for mitoses
-may see endothelial cell hyperplasia
-no necrosis
Glioblastoma multiforme:
-Macro: soft, grey to tan, may show increased vascularity or white-yellow necrotic
areas
-beware gliomas may be multifocal up to 10% of cases
-cellular tumour with poorly defined cell borders in fibrillary background
-look for mitoses, endothelial cell proliferation and necrosis
-necrosis with pseudopalisading a characteristic feature
-necrotic blood vessel also a good clue to GBM (more indicative of a neoplastic
process, esp. when inflammatory cells are sparse)
-abundant foamy macrophages or neutrophils are not seen in GBM and should suspect
alternative diagnosis (non-neoplastic entities) if present

-giant cell variant may be deceptively well-circumscribed, mimicking a metastasis
radiologically and surgically. It may also have perivascular lymphocytes.
If the lesion is strongly suspicious for a GBM radiologically and the
frozen section only shows low grade tumour or reactive gliosis, the tissue is
most likely not representative of the lesion. Should request deeper tissue from
the surgeon.
Radionecrosis:
-the issue with frozen section is to decide whether there is residual/recurrent glioma or
not. The radiology usually shows new contrast enhancement, suspicious for tumour.
-vascular damage is a prominent feature
-fibrinoid necrosis is often widespread and accompanied by thrombosis
-chronic changes include recanalisation and mural hyalinisation in addition to
telangiectasia. Subendothelial foam cells are often present.
-the degree of macrophage response is much less as compared with that in ischaemic
infarct and demyelinating disease
-granulation tissue often seen with chronic inflammation and haemosiderin deposits
-reactive astrocytes are abundant and may show radiation atypia: nuclear and
cytoplasmic enlargement, multinucleation, cytoplasmic vacuolisation
Features favouring radionecrosis: areas of coagulative necrosis with no
pseudopalisading by tumour cells; all the usual features of reactive gliosis;
typical vascular and inflammatory changes.
Features favouring tumour: cellular sheets of atypical cells; astrocytes
have high N/C ratio and hyperchromasia; high mitotic rate; palisaded necrosis.
Gliosarcoma:
-Macro: focally firm and discrete (mimicking a metastasis or even a meningioma
surgically)
-biphasic pattern of more typical GBM and malignant spindled tumour cells, forming
fascicles
Pilocytic astrocytoma:
-Macro: tough and rubbery; cysts containing clear fluid
-typical biphasic pattern with dense spindled areas and microcysts
-presence of multinucleated cells is a helpful clue
-tumour looks less conspicuously “pilocytic” in frozen section than in paraffin section
-some cells may have degenerative type change with marked nuclear pleomorphism,
mimicking a high grade glioma
-look for microcysts, hyalinised vessels, Rosenthal fibres, eosinophilic granular
bodies
-beware presence of glomeruloid microvascular proliferation which is common in
pilocytic astrocytoma and should not overcall it as a high grade astrocytoma
-correlate with the radiology (discrete, contrast-enhancing mural nodule in a cyst), site
and age of the patient

Rosenthal fibres may be seen in the cyst wall of a haemangioblastoma or
gliotic periphery of a craniopharyngioma, which may be potentially misdiagnosed
as a pilocytic astrocytoma.
Pleomorphic xanthoastrocytoma:
-Macro: sharp interface with adjacent brain; yellow hue from lipids in the cytoplasm
-despite the pleomorphic cells, mitoses, endothelial proliferation and necrosis are not
present
-the tumour cells may be elongated and may form vague fascicles
-look for xanthomatous change in the cytoplasm
-look for eosinophilic granular bodies
-presence of perivascular lymphocytes is a clue
-correlate with the radiology (superficial location, involving meninges, mural nodule
in a cyst in 50% of the cases)
Subependymal giant cell astrocytoma:
-Macro: well-circumscribed, generally firm, may be calcified; grey to pink
-usually protruding into the lateral ventricle near the foramen of Munro
-large tumour cells with abundant cytoplasm (much larger than a gemistocyte)
-some of the cells are more spindled
-enlarged vesicular nuclei with prominent nucleoli, resembling neurons
-the cells may arrange themselves around blood vessels forming perivascular
pseudorosettes
-no mitoses, endothelial cell hyperplasia or necrosis
Oligodendroglial tumours:
Oligodendroglioma:
-Macro: gelatinous, soft; grey to pink; may have cystic change; some are gritty
-oligodendrogliomas or oligoastrocytomas involve the cortex more commonly as
compared to pure astrocytomas. Think oligodendroglioma if there is rather extensive
cortical involvement.
-perinuclear haloes (“fried egg” appearance) are not seen in frozen section (artefact of
delayed fixation)
-secondary structuring (perineuronal satellitosis, perivascular aggregation, subpial
accumulation), mucin-filled microcysts, calcifications, chicken-wire vasculature are
valuable clues
-aggregation of tumour cells to form palisaded clusters may be seen
-nuclei may be distorted by the freezing process but the round nuclear contour can
still be appreciated in some cells
-cell processes and gliofibrillary matrix are absent
-correlate with the smears (round nuclei better appreciated in cytology preparation;
look for absence of background fibrillary matrix)
-in difficult cases, a diagnosis of ‘low grade glioma’ is reasonable. It makes no
difference to the surgeon during surgery whether the tumour is astrocytoma or
oligodendroglioma.
-beware minigemistocytes / gliofibrillary oligodendrocytes. Should not immediately
suggest mixed glioma.

Anaplastic oligodendroglioma:
-Macro: yellow-white soft necrotic areas and haemorrhage
-increased cellularity; mitoses; endothelial cell hyperplasia; necrosis
-tumour cells may be more pleomorphic and losing the nuclear roundness. It may be
difficult to distinguish from a high grade astrocytoma.
Frozen section process produces nuclear angulation and
hyperchromasia, making an oligodendroglioma appearing like an astrocytoma.
In Grade II oligodendroglioma or oligoastrocytoma, the tumour may be densely
cellular, easily mistaken for a high grade glioma. It is important to ask the
surgeon for more tissue for paraffin section if the entire specimen has already
been submitted for frozen.
Ependymal tumours:
Ependymoma:
-Macro: sharply demarcated, soft and fleshy; may be gritty
-tumour may look like a fibrillary astrocytoma on frozen section as the fibrillarity and
gemistocytic-like quality of the cells are emphasized
-perivascular pseudorosettes a clue (slender tapering cytoplasmic processes of the
tumour cells oriented perpendicular to the blood vessels)
-ependymal canal-like structures and true rosettes with lumina may be seen
Myxopapillary ependymoma:
-Macro: well-defined, soft, saccular mass
-mucin is characteristically located within the walls of blood vessels and in turn,
surrounded by collar of tumour cells
Subependymoma:
-Macro: bosselated (cauliflower-like), firm, white to tan; may be gritty; may show
cysts or haemorrhage
-look for clustering of nuclei, separated by dense fibrillary matrix
-microcysts are common
-hyalinised vessels frequently present
-perivascular pseudorosettes are not seen in subependymomas
Choroid plexus tumours:
Choroid plexus papilloma:
-Macro: very vascular; pink; tufted or fronded surface; friable, granular texture; may
be gritty
-increased cellularity, exceeding that of normal choroid plexus
-papillary structures lined by multi-layered epithelial cells
-apical surface is usually flat and lacks the typical “cobblestone” appearance of
normal choroid plexus tissue
-oncocytic change is a helpful clue to indicate choroid plexus origin of the lesion
Choroid plexus carcinoma:
-Macro: very haemorrhagic, friable; surface less obviously fronded; may have
necrosis; calcification uncommon
-marked architectural complexity with partially solid areas

-papillary structures with surface layer of columnar cells may be lost in poorly
differentiated tumours
-tumour cells are more pleomorphic
-mitoses, vascular proliferation and necrosis present
Neuronal and mixed neuronal-glial tumours:
Ganglioglioma:
-Macro: well-circumscribed, firm, gritty, grey
-look for atypical ganglion cells: bi or multinucleation; cell clustering; loss of normal
orientation
-ganglion cells become smudged and distorted during freezing and are easily missed
-the pleomorphic glial component may be more conspicuous and mistaken for a high
grade astrocytoma
-correlate with radiology (mural nodule in a cyst) to avoid potential misdiagnosis
-eosinophilic granular bodies and perivascular lymphocytes are clues to the diagnosis
-adjacent cortical dysplasia may be seen (losing normal architecture of the cortical
layers)
Beware entrapped residual neurons in astrocytomas, mimicking a
ganglion cell tumour. The entrapped neurons show no cytologic atypia and
they are evenly distributed.
Central neurocytoma:
-Macro: well-circumscribed; soft to gritty; tan; may be partly cystic
-beware of the diagnosis in a typical location (intraventricular, near foramen of
Munro)
-columns or rosette-like arrangement of uniform tumour cells
-areas of eosinophilic fibrillar matrix without nuclei a useful clue
-the fibrillar zones are altered by the freezing process and may mimic necrosis
-most cells have uniform round nuclei which may be distorted during the freezing
process. Some cells show nuclear angulation and hyperchromasia.
-perinuclear haloes not seen
The main differential diagnosis is oligodendroglioma, which has an almost
identical histological appearance. Intraventricular location and anuclear fibrillar areas
favour a neurocytoma.
Dysembryoplastic neuroepithelial tumour (DNET):
-Macro: soft with mucoid areas
-intracortical nodules may be seen
-cords of oligodendroglial-like cells with interspersed mature neurons floating in pale
mucoid matrix
-calcification and Rosenthal fibres may be present
-know the clinical history (long history of seizures) and the typical intracortical
location
Pineal region tumours:
Pineocytoma:

-adequate to use the term “pineal parenchymal tumour” at the time of frozen section
-may see pineocytomatous rosettes
Pineoblastoma:
-appears as undifferentiated tumour, forming diffuse sheets
-rosettes of the pineocytic type not seen
-tumour cells have high N/C ratio
-high mitotic rate
Embryonal tumours:
Medulloblastoma and PNET:
-Macro: highly vascular; soft (firmness indicates desmoplasia); grey-pink
-highly cellular, mitotically active tumour forming sheets
-Homer-Wright rosettes may be present
-tumour cells have elongated nuclei which often show moulding
Cranial and paraspinal nerve tumours:
Schwannoma:
-Macro: very firm, tan; may have yellow areas, cysts, haemorrhage
-look for fascicles with nuclear palisading and Verocay bodies (Antoni A areas) and
microcystic change (Antoni B areas)
-secondary changes common: foamy macrophages, haemosiderin deposits
-hyalinised, thick-walled blood vessels a clue (uncommon in fibrous meningioma)
-beware ancient change (no mitoses)
Neurofibroma:
-Macro: firm, white
-collagen bundles in-between cells with wavy nuclei
-loose stroma with frequent mast cells
Tumours of meningothelial cells:
Meningioma:
-Macro: white to tan, usually firm but can be soft; may be gritty
-look for whorls, fascicles, psammoma bodies
-tumour cells have intranuclear pseudoinclusions, uniform nuclear features
-fibrous meningioma may be difficult to distinguish from schwannoma. Secondary
change (foamy macrophages, haemosiderin deposits) is more commonly seen in
schwannoma.
Mesenchymal tumours:
Haemangiopericytoma:
-Macro: soft and red (highly vascular) as compared to pale and firm in meningioma
-staghorn blood vessels on low-power a clue
-densely cellular
-high N/C ratio, ovoid nuclei
-variable mitotic rate
Primary melanocytic lesions:
Malignant melanoma:
-Macro: soft brown tissue with foci of haemorrhage and necrosis

-often form sheets with areas of haemorrhage in the background
-tumour cells are pleomorphic with prominent nucleoli, intranuclear pseudoinclusions
and pigmented cytoplasm
Other tumours related to the meninges:
Haemangioblastoma:
-Macro: firm, dark red/yellow nodule; may be associated with a cyst containing clear
fluid
-the intricate vascular network of thin-walled small vessels is a clue
-the foaminess of the cytoplasm in the stromal cells may not be obvious
-presence of pleomorphic and hyperchromatic nuclei of the stromal cells may mimic a
glioma
-presence of fat supports the diagnosis of haemangioblastoma
-correlate with the radiology (discrete, contrast-enhancing nodule in a cerebellar cyst)
Haematopoietic tumours:
Lymphoma:
-Macro: soft, fleshy grey-tan with haemorrhage and necrosis
-it is very important to make an accurate diagnosis of lymphoma. The surgeon will
not need to further excise the tumour, once the lymphoma diagnosis is given.
-tumour has distinct tendency to form perivascular cuffs – angiocentric invasion (not
usually seen in gliomas)
-histiocytes, reactive astrocytes, inflammatory cells may be numerous. The
background reactive astrocytes may suggest a glioma.
-the cytology of the tumour cells may be distorted by the freezing process, mimicking
a glioma or metastatic carcinoma
-crush artefact and prominent apoptotic debris are clues
-the tumour cells often are large with scanty cytoplasm
-correlate with the smears (nuclear detail much better seen)
-in patients receiving pre-op steroids, the tumour cells may shrink
Myeloma:
-Macro: fleshy grey-purple
-solid sheets of tumour cells
-look for classic features of plasma cell differentiation: perinuclear hof, “clock-face”
chromatin
-Dutcher and Russell bodies
Germ cell tumours:
Germinoma:
-Macro: soft to firm (depends on the amount of collagenous septa), grey-pink
-large tumour cells with vesicular nuclei, prominent nucleoli and abundant cytoplasm
-abundant lymphocytes may obscure the tumour cells (making a misdiagnosis of
lymphocytic hypophysitis)
-tumour cells may be crushed and difficult to see
-granulomas may be present
Lesions of the sellar region:
Pituitary adenoma:
-Macro: soft, tan (normal pituitary gland tissue is more rubbery)

-sheets, trabeculae, rosettes with vascularised stroma
-monotonous population of cells with uniform nuclei and granular eosinophilic
cytoplasm
-some functioning adenomas may have pleomorphic tumour cells
-in prolactinomas showing Bromocriptine effect, the tumour cells have high N/C
ratio, resembling small cell carcinoma, lymphocytic hypophysitis or lymphoma
-tumour cells are necrotic in pituitary apoplexy
Pituitary hyperplasia:
-Macro: slightly softer than normal tissue
-acini expanded and becoming irregular in shape
-the cells becoming more monotonous as compared to adjacent normal acini with a
mixed population of cell types
-better demonstrated with reticulin stain on paraffin sections
Lymphocytic hypophysitis:
-Macro: tough, rubbery, pale
-abundant lymphocytes and plasma cells
-background of residual normal acini and fibrosis
-look carefully for hidden tumour e.g. germinoma, spindle cell oncocytoma
Craniopharyngioma:
-Macro: solid, partly grey-white, firm, focally calcified; cystic part containing
“machine oil” viscous brown fluid, glistened with minute cholesterol droplets
-the tumour forms prominent multinodular epithelial lobules
-cells at the periphery of the lobules are palisaded whereas internally situated cells
show a stellate myxoid appearance
-a diagnostic finding is wet keratin: nodules of plump, eosinophilic, keratinised cells
-calcium is frequently seen in nodules of wet keratin
-chronic inflammation and cholesterol clefts present
-specimens obtained from the immediate periphery of a craniopharyngioma show
Rosenthal fibre-rich gliosis which may mimic a pilocytic astrocytoma
-wet preparation of cyst fluid may show characteristic birefringent layered polygonal
cholesterol crystals
Chordoma:
-Macro: soft, translucent grey
-cords of cells in myxomatous stroma
-cytoplasmic vacuolation may be seen but not as striking as in paraffin sections
Rathke’s cleft cyst:
-cyst lined by single-layered cuboidal or columnar ciliated epithelium
Metastatic tumours:
Metastatic carcinoma:
-Macro: soft (may be firm due to desmoplasia), granular; may be necrotic; mucoid if
adenocarcinoma
-characteristically circumscribed, multiple
-abrupt, sharply demarcated tumour margins that push into rather than wildly infiltrate
the parenchyma

-endothelial cell hyperplasia not a common feature (usually at the periphery of the
lesion if present). In GBM, it is seen throughout the tumour.
-palisaded necrosis may be seen but not as frequent as in GBMs
-if necrotic, residual viable tumour often forms cuffs around blood vessels
-tumour cells have well-defined cell borders
-collagenous stroma is seen, instead of fibrillary background
-look for specific features of differentiation: glands, mucin, keratinisation,
intercellular bridges
Common secondary tumours of the CNS: carcinomas of the lung, breast,
GIT, kidney; melanoma; lymphoma / leukaemia.
Typically haemorrhagic metastatic tumours to the CNS: melanoma,
choriocarcinoma, renal cell carcinoma, vascular neoplasms e.g. angiosarcoma.
Metastatic small cell carcinoma:
-Macro: soft, tan, necrotic
-extensive necrosis present
-lack desmoplastic growth pattern of medulloblastoma
-show less tendency to rosette formation
-high N/C ratio, nuclear moulding, granular chromatin, inconspicuous nucleoli
-tumour cells often show crush artefact
-high mitotic activity
-Azzopardi effect
Metastatic melanoma:
-Macro: soft, typically haemorrhagic; may be brown or black
-sheets and loosely cohesive tumour cells
-cells with pleomorphic nuclei, intranuclear pseudoinclusions, prominent nucleoli, binucleation,
abundant cytoplasm
-cytoplasmic brown melanin pigment may be seen
Cysts of the CNS:
Colloid cyst:
-lined by ciliated columnar epithelium with goblet cells
Epidermoid cyst:
-lined by multi-layered squamous epithelium with keratinisation
Dermoid cyst:
-lined by keratinised squamous epithelium
-sebaceous glands, sweat glands, hair follicles in the cyst wall
Endodermal (enterogenous, neuroenteric) cyst:
-lined by respiratory or intestinal type epithelium with goblet cells
Arachnoid cyst:
-lined by meningothelial cells
Ependymal (glioependymal) cyst:

-lined by ciliated ependymal cells
-cilia often lost
FALSE POSITIVES:
-granular cell layer of the cerebellum mistaken for small round blue cell tumour
-normal architecture of anterior pituitary gland not appreciated, overcalling as
adenoma
-calling normal arachnoid cell clusters in leptomeninges as a meningioma
-reactive astrocytosis mistaken for astrocytoma; gliosis with Rosenthal fibres,
overcalling as pilocytic astrocytoma
-reactive lymphocytes, perivascular lymphocytes mimicking a lymphoma
-infarct, demyelination plaque: foamy macrophages mistaken for gemistocytes
FALSE NEGATIVES:
-sampling error: by surgeon or pathologist picking non-tumour tissue for freezing
-tumour obscured by frozen section artefacts (esp. too many ice crystals make tumour
appearing as less cellular)
-low grade astrocytoma
-necrotic tumour
-lymphocytes and granulomas obscuring a germinoma
PRACTICAL TIPS:
1. Know the clinical history, radiology and previous biopsies. Correlate with the brain
smears.
2. The most important indication for frozen section is to get enough tissue for
histology rather than making a specific diagnosis on the spot.
3. Microcysts have round circular profile. Ice crystal artefact has cleft-like, elongated
appearance (may mimic microcystic change, overcalling normal / reactive lesion as
low grade astrocytoma).
4. If there are abundant neutrophils, the lesion is probably infectious rather than
neoplastic. Must submit fresh tissue for culture.
5. If there are prominent perivascular lymphocytes, think about vasculitis, viral
infection, infarct, demyelination, lymphoma. Primary tumours with perivascular
lymphocytes include ganglioglioma, pleomorphic xanthoastrocytoma and sometimes
GBM (gemistocyte-rich or giant cell variant).

6. Features favouring low grade astrocytoma over reactive gliosis: clustering,
crowding of astrocytes; definite nuclear atypia (nuclear irregularity, hyperchromasia,
nuclear enlargement); absence of radiating long cytoplasmic processes; microcysts;
absence of red neurons, foamy macrophages, perivascular lymphocytes.
7. Vascular proliferation with endothelial cell hyperplasia is not specific to high grade
astrocytomas. It may be seen at the edge of an organizing infarct / abscess, low grade
tumours such as pilocytic astrocytoma, metastasis e.g. renal cell carcinoma.
8. Palisaded necrosis is seen in tumours (typically GBMs, some metastases) and it is
not present in an infarct or abscess. Radionecrosis is also unaccompanied by
pseudopalisading.

Fig 1. Calcified tissue difficult to cut and severely fragmented during sectioning.
Fig 2. Air-drying artefact. The cells show pale staining due to excessive air-drying
prior to putting the slide into formalin fixation.

Fig 3. Chattering artefact. Parallel torn spaces.
Fig 4. Ice crystal artefact with elongated spaces (Arrow).

Fig 5. Folding artefact. Section not straightened out before putting onto the slide
(Arrow).
Fig 6. Normal white matter with evenly distributed astrocytes.

Fig 7. Normal cerebral cortex with neurons and glial cells.
Fig 8. Normal cerebral cortex, containing neurons.

Fig 9. Normal cerebellar cortex. Small granule cells with round nuclei. Note: evenly
spaced Purkinje cells.
Fig 10. Reactive gliosis with increased numbers of evenly distributed astrocytes.

Fig 11. Reactive gliosis. Evenly distributed astrocytes with mild nuclear enlargement.
Fig 12. Reactive astrocytes with radiating cytoplasmic processes (Arrows).

Fig 13. Cerebral infarct with ischaemic neurons (Arrows).
Fig 14. Cerebral abscess. Necrotic debris and neutrophils. Abundant neutrophils are
usually not seen in GBM. Be sure to submit tissue for microbiology.

Fig 15. Cerebral abscess. Central necrosis (Yellow arrow), surrounded by reactive
gliosis (Blue arrow).
Fig 16. Cerebral abscess with reactive gliosis. There is no palisaded necrosis.

Fig 17. Cryptococcus.
Fig 18. Demyelination.

Fig 19. Demyelination. Reactive astrocytes with processes (Blue arrows) and
perivascular lymphocytes (Yellow arrow).
Fig 20. Progressive multifocal leukoencephalopathy.

Fig 21. Progressive multifocal leukoencephalopathy. Oligodendroglial cells with
round glassy intranuclear inclusions.
Fig 22. Progressive multifocal leukoencephalopathy. Scattered reactive astrocytes are
seen, which may show prominent nuclear pleomorphism.

Fig 23. Low grade astrocytoma.
Fig 24. Low grade astrocytoma. Neoplastic astrocytes unevenly distributed.

Fig 25. Low grade astrocytoma. Neoplastic astrocytes show focal crowding.
Fig 26. Pilocytic astrocytoma. Rosenthal fibres (Yellow arrow) and hyalinised blood
vessels (Orange arrow).

Fig 27. Pilocytic astrocytoma.
Fig 28. Pleomorphic xanthoastrocytoma. Can quite easily overcall this as a high grade
astrocytoma on low-power.

Fig 29. Pleomorphic xanthoastrocytoma. Note perivascular lymphocytes.
Fig 30. Pleomorphic xanthoastrocytoma. Eosinophilic granular body (Arrow).

Fig 31. High grade astrocytoma. Astrocytes with prominent nuclear atypia.
Fig 32. High grade astrocytoma. Endothelial cell hyperplasia (Arrows).

Fig 33. Glioblastoma multiforme with palisaded necrosis.
Fig 34. Radionecrosis. Note fibrinoid necrosis of the blood vessels.

Fig 35. Radiotherapy effect. The neuropil is often fibrous, containing chronic
inflammatory cells, blood vessels and haemosiderin deposits.
Fig 36. Residual/recurrent glioma post radiotherapy. The tumour cells often appear
small with high N/C ratio.

Fig 37. Gliosarcoma containing spindled tumour cells.
Fig 38. Oligodendroglioma. Scattered tumour cells have round nuclei.

Fig 39. Oligodendroglioma with subpial accumulation.
Fig 40. Oligodendroglioma with perineuronal satellitosis.

Fig 41. Oligodendroglioma. Tumour nuclei are altered by the freezing process
(becoming angulated and hyperchromatic).
Fig 42. Ependymoma. Perivascular pseudorosettes (Arrows).

Fig 43. Myxopapillary ependymoma. Mucoid material in and surrounding central
blood vessels.
Fig 44. Subependymoma. Clustering of nuclei.

Fig 45. Subependymoma.
Fig 46. Choroid plexus papilloma.

Fig 47. Ganglioglioma with abnormally clustered ganglion cells (Arrows).
Fig 48. Central neurocytoma. The typical intraventricular location is a helpful clue to
the diagnosis.

Fig 49. Central neurocytoma. The nuclear contour may become irregular during the
freezing process. Perinuclear haloes are not seen.
Fig 50. Central neurocytoma. Anuclear fibrillary zones present.

Fig 51. Pineal parenchymal tumour. Paraffin sections show intermediate
differentiation.
Fig 52. Medulloblastoma.

Fig 53. Medulloblastoma. Tumour cells with nuclear moulding.
Fig 54. Schwannoma. Spindled cells with palisading (Antoni A area). Thick-walled
blood vessels are commonly seen.

Fig 55. Schwannoma with haemosiderin deposits and foamy macrophages.
Fig 56. Neurofibroma. Tumour cells have wavy nuclei.

Fig 57. Meningioma.
Fig 58. Meningioma. Intranuclear pseudoinclusions appearing as clear holes.

Fig 59. Meningioma with brain invasion. Rosenthal fibres present (Arrow).
Fig 60. Malignant meningioma, mimicking a sarcoma.

Fig 61. Haemangiopericytoma with dense cellularity.
Fig 62. Haemangioblastoma. Prominent vascular network.

Fig 63. Haemangioblastoma. Stromal cells with foamy cytoplasm.
Fig 64. Normal anterior pituitary gland tissue.

Fig 65. Normal anterior pituitary gland tissue with packeted arrangement. The cells
show different staining properties.
Fig 66. Pituitary adenoma with vascularised stroma.

Fig 67. Pituitary adenoma. Uniform population of tumour cells with abundant
granular cytoplasm.
Fig 68. Prolactinoma with Bromocriptine effect. The tumour cells may look like a
small cell carcinoma or lymphoma.

Fig 69. Craniopharyngioma.
Fig 70. Craniopharyngioma with wet keratin.

Fig 71. Lymphocytic hypophysitis with many mature lymphocytes.
Fig 72. Chordoma.

Fig 73. Non-Hodgkin lymphoma. Cuffs of lymphoid cells around blood vessels.
Fig 74. Non-Hodgkin lymphoma.

Fig 75. Myeloma with neoplastic plasma cells.
Fig 76. Metastatic carcinoma (breast). Cribriform structures present. Tumour nests
have well-defined edge.

Fig 77. Metastatic small cell carcinoma to the cerebellum. The tumour aggregates
have well-defined borders and do not merge into the adjacent neuropil.
Fig 78. Metastatic small cell carcinoma to the cerebellum.

Fig 79. Metastatic carcinoma. Viable tumour cells often show perivascular cuffing in
areas of necrosis. This pattern is less commonly seen in gliomas.
Fig 80. Metastatic melanoma, associated with prominent areas of haemorrhage.

Fig 81. Metastatic melanoma. Tumour cells with intranuclear pseudoinclusions
(Arrows) and abundant eosinophilic cytoplasm.

References
Yachnis AT. Intraoperative consultation for nervous system lesions. Sem Diag Pathol
2002;19(4):192-206.
Moss TH et al. Intra-operative diagnosis of CNS tumours. Arnold publishers. 1997.
Burger PC et al. Surgical pathology of the nervous system and its coverings. ed.
2002.
Burger PC, Scheithauer BW. Tumors of the central nervous system. AFIP Series 4.
2007.