This document provides an overview of squash cytology techniques for diagnosing pediatric central nervous system tumors, describing the smear patterns and key cytological features of common tumor types like medulloblastoma, atypical teratoid/rhabdoid tumors, choroid plexus papilloma, and choroid plexus carcinoma. Squash cytology allows rapid examination of biopsy samples during neurosurgery to aid diagnosis and surgical decision making.

1. Squash Cytology of Paediatric
CNS Tumours
Presented by – Dr. V. Nagesh Kumar (1st Yr PG)
Chair Person: Dr. Kumuda (Prof)
Moderators: Dr. Rama Raju (Assoc Prof)
Dr Shrinivas (Asst Prof)

2. Dr. Harvey Williams Cushing
Dr. Louise Eisenhardt
(1869 – 1939)
(1891 – 1967)

3. Introduction
• Central nervous system (CNS) is one of
the most challenging domains for the
neurosurgeon and Pathologists
• The role of intraoperative pathological
diagnosis is crucial in neurosurgery

4. Introduction
• Making a diagnosis of CNS lesions is
difficult on the basis of clinical and
radiological findings only
• Cytological and/ or histological diagnosis
is required for confirmation and proper
management

5. Introduction
• Besides rapid decision making during
neurosurgical procedures, it is also to be
ensured that minimum injury is caused to
the normal brain structures surrounding
the intracranial neoplasm

6. Introduction
• In this regard, imprint cytology or squash
smear prepared from small biopsy during
surgery seems to be very helpful for the
operating surgeon to make a decision on
further management

7. Introduction
• The application of smear techniques as a
means of obtaining rapid diagnosis for
neurosurgical biopsies was first advocated
by Dr Eisenhardt and Dr Cushing in the
USA in early 1930
• Since then, although the technique has
been modified by various individuals by
changing the fixative or stain, the basic
principle has remained unchanged

8. Introduction
• Though this technique receded to the
background with the advent of CT guided
stereotactic biopsy, it has regained
importance due to its technical simplicity
• Three simple techniques allow rapid
examination of biopsy material during
surgery: touch preparations, squash
preparations, and the smear

9. Squash Preparation
• Tissue is crushed between two slides and
then stained
• Similar to touch preparations, this method
will give excellent cytology, although some
cells may show some crush artifacts

10. Squash Preparation
• As the tissue spreads out under the force
produced by the two glass slides, its
structure partially breaks down, thereby
releasing more cells
• Notably, neoplastic cells that elaborate
their own matrix will retain their
relationship to this milieu on the slide

12. Examination of the smear
• A multilevel process and entails observing
at every level
– Radiology
– gross examination, and
– how the tissue physically separates

13. Examination of the smear
• After it is stained, hold it up to the light
What does it look like?
– Is it pink or blue?
– Lmpy or smooth?
– Easy to spread or resist spreading?

14. Examination of the smear
• Under the microscope
– Intermediate magnification
• the lesion's matrix becomes clear: glial, liquid,
epithelial, or cohesive
• Finally, at high power
– the cells reveal their identity
– Their cytoplasm generally indicates the cell
type, whereas their nuclei tell whether the
process is reactive or neoplastic, benign or
malignant

15. Biopsies of Gray Matter
• Produce smooth, relatively pink smears
• They are hypocellular, which distinguishes
them from tumors and inflammatory
disorders
• A few delicate capillaries often stretch
around the smear, whereas larger vessels
are dragged off to the end of the slide

16. Biopsies of Gray Matter
• Neurons and their synapses make up the
essence of gray matter
• Fine granularity of gray matter or
neuropil depends on the multitude of
axons synapsing on dendrites

17. Normal gray matter

18. Biopsies of White mater
• Normal white matter lacks the neurons
and neuropil of gray matter
• It contains two main populations of cells
that can be difficult to distinguish:
– Oligodendrocytes - a dense, small, round,
blue nucleus
– Astrocytes - larger, slightly round nucleus with
more open chromatin

19. • Unless you see some defining cytoplasm,
you cannot reliably distinguish these
nuclei. Rather, you identify a cell by the
company it keeps, by its context in the
surrounding tissue

20. A. Low magnification view showing the light fluffy appearance of
slightly gliotic white matter.
B. Intermediate power showing a reactive astrocyte (arrow) in a
spattering of small, round, blue cells. Although many of these other
cells are most likely oligodendrocytes (arrowheads), they cannot be
reliably distinguished from a few infiltrating lymphocytes or
nonstimulated macrophages.

21. Cerebellar cortex
• In a smear, identifying cerebellar cortex
requires
– recognizing the monotony of its granular
neurons
– finding a fine neuropil background, and
– looking for the occasional, helpful Purkinje
neuron

22. Normal cerebellar cortex

23. • Distinguishing normal cerebellum from a
small, round, blue cell tumor can be tricky
• However, normal cerebellum smears
evenly, like all other brain, and the
confounding internal granular neurons are
oppressively monotonous at high power

24. Smear Patterns
• Noncohesive smears
• Cohesive smears
• Epithelial tissue smears
• Glial tissue smears
• Necrotic debris smears

25. Noncohesive smear
Noncohesive tissues. In noncohesive processes, cells grow
without significant intercellular attachments. Their matrices are
weak and are insufficient to bind the cells together.

26. Noncohesive smear
This is a classic smear from a pituitary adenoma

27. Cohesive smear
Several different types of tissues, including some tumors and
dura, are tightly bound together by either dense reticulin or
collagen bands. These tissues are unwilling to give up their cells
without a fight.

28. Cohesive smear
Smear of a cohesive schwannoma

29. Epithelial tissue smear
Epithelial tissues come in different forms, but all share two
characteristics: they are bound to each other by cell surface
connections and they have distinct cell membranes. On a
smear, squamous tissues form sheets (upper cells). Tubular
epithelium frequently forms balls (lower cells). Papillary
epithelial tissues form papillary structures on smears.

30. Epithelial tissue smear
Metastatic lung carcinoma

31. Glial tissue smear
A smear prepared from gliotic tissue, be it reactive or neoplastic, will
reflect these highly intermingled processes.
At low magnification, it will look like puffs of cotton seeds clumped
around strands of cotton fibers (center).
On high power, reactive or neoplastic astrocyte nuclei connect
directly to the glial matrix they have elaborated (right).

32. Glial tissue smear
Moderate reactive gliosis near a metastasis

33. Glial tissue smear
High-power view of a reactive astrocyte in this smear

34. Necrotic debris smear
This fragment of tissue from a glioblastoma is necrotic. The pale, dead nuclei
(arrows) are diagnostic evidence of necrosis.
This sample also has some blood bound within it. Remember, erythrocytes
can lyse during the fixation step and leave only ghosts, which can look like
dead nuclei.

39. Paediatric CNS Tumours

40. Paediatric CNS Tumours
• most common solid organ tumor in
children
• are second only to leukemias in children
• 70% of pediatric brain tumors are in the
posterior fossa
– Pilocytic astrocytoma
– Medulloblastoma
– Ependymoma

41. Paediatric CNS Tumours
• Primary
• Secondary
– Metastatic
– Local invasion
• Tumors of the spinal cord

42. Paediatric CNS Tumours
• Most common types
– Astrocytomas
• Grades I-IV
– Medulloblastomas
• primitive neuroectodermal tumor-PNET
– Meningiomas
– Pituitary adenomas

45. Infratentorial or Posterior Fossa
• Astrocytomas
• Medulloblastomas
• Atypical teratoid/rhabdoid tumors
• Ependymomas
• Choroid plexus tumors

46. Parasellar
• Astrocytomas
• Craniopharyngiomas
• Germ cell tumors

47. Medulloblastoma
• Embryonal tumor of the cerebellum and at
sites throughout the neural axis showing
primitive neuroectodermal cells with
neuronal differentiation
• considered malignant, grade IV
neoplasms because of their potentially
rapid growth and to seed distant sites of
the nervous system through the ventricles
and subarachnoid spaces

48. Medulloblastoma
• common solid tumor of children and young
adults
• The classic form has neuroblastic rosettes
(tumor cell nuclei around cytoplasmic
processes)
• Mitoses and apoptoses may be frequent

49. MRI – T1, T2 & gad enhanced images of Medulloblatoma

52. In a smear examined at medium to high microscopic powers, classic
medulloblastomas show cells as if glued end-to-end, small numbers of nuclei stick
together, forming short chains or circles, these represent true rosettes

53. Medulloblastoma smears at medium power.
Few nuclei remain fully isolated from other nuclei (white arrowheads). Instead, many
of the cells seem to form short chains or clusters (black arrowheads) and some form
larger, circular arrangements.

54. Features of a classic medulloblastoma:
A.Cells with scant-to-no cytoplasm having nuclei displaying salt-and-pepper
chromatin but no nucleolus. Nuclear molding (arrows) reflects cellular
cohesion in the presence of minimal cytoplasm.
B.Occasional circles of cells (arrowheads) appear to be more than a
fortuitous arrangement of cells, because many of their nuclei mold onto each
other and lumen they form has a distinct edge.

55. Medulloblastoma showing neuroblastic differentiation

56. Desmoplastic Medulloblastomas
• Like all variants these smear easily
• displays abundant pink neuropil
• Relatively monotonous nuclei dispersed in
a fine neuropil matrix
• Minimal cytoplasm and has more
homogenous nuclei than classic
medulloblastomas but retains the salt-and-
pepper chromatin

57. Desmoplastic medulloblastoma

58. • This tumor displays a prominent nodular
growth pattern on permanent sections.
These nodules are not really desmoplastic
lumps surrounded by fibrous tissue but
rather islands of better-differentiated
neural cells within neuropil that are
demarcated by poorly differentiated bands
of cells

59. Atypical teratoid rhabdoid
tumors (ATRT)
• ATRTs are a tumor of early childhood(<2
years of age) and rare after age 4 years
• These tumors are neither teratomas nor
do they produce muscle
• ATRTs lack neuronal differentiation
• Easily be confused with medulloblastomas

60. • Have predilection for the cerebellum and
posterior fossa, they can occur anywhere
along the neural axis
• These tumors characteristically have lost
one chromosome 22; monosomy 22, in
the right clinical setting, is considered
diagnostic of an ATRT

61. • Unlike medulloblastomas, which can be
eradicated in many patients, these are
highly malignant tumors for which no
satisfactory treatment has been developed

62. Atypical teratoid rhabdoid tumor neuroradiology

63. Atypical teratoid rhabdoid tumor
A.The field view of this microscopic slide shows a choppy or rough spreading of
the tumor.
B.Among the cellular clumps at low magnification, tumor cells shed easily. They
accumulate in large numbers around stiffened or rigid blood vessels (arrow).
Notice the tiny piece of involved cerebellum that was part of the tissue sampled
(cb, demarcated by lines); this piece had been invaded by tumor but still
retained some of its cellular components.

64. Vascular predilection of the tumor. In both preparations, cells cluster around
vessels. In addition, the vessel walls appear thickened and the endothelial cells
enlarged (B).

65. Multinucleated giant tumor cells are helpful, when present. Their nuclei match
those of the surrounding mononuclear tumor cells.

66. ATRTs may have subclones displaying epithelioid features: large cells having
abundant, eosinophilic cytoplasm, sharp cell borders, and eccentric nuclei

67. ATRT (Higher magnification)
A.Collection of primitive cells having scant cytoplasm and a salt-and-pepper
chromatin (arrowheads). Notice how an occasional cell having a similar nucleus
can also have more abundant and distinct cytoplasm (arrow).
B.The volume of cytoplasm ranges from scant to abundant (arrow). As
cytoplasmic volume increases, the nuclei remain eccentric rather than central.

68. C. A true rhabdoid cell, rather than just an epithelioid cell with an eccentric
nucleus, should have condensed cytoplasm and perhaps fine internal
filaments or whorls (arrow).
D. This multinucleated cell could really be a very tight clustering of smaller
epithelioid cells, because some distinct cytoplasmic borders are present
(arrowhead); this is not a cell of a medulloblastoma.

69. Choroid plexus tumors
• Occurs in any portion of choroid plexus,
but usually as papillary neoplasms of
lateral ventricle of children and fourth
ventricle of adults
• Uncommon (0.4 to 0.6% of intracranial
neoplasms)

70. Choroid plexus papilloma
• Grade I of IV - benign
• Rare (<1%), slow growing tumor
• commonly in ventricular system and
associated with hydrocephalus
• Often causes developmental delay,
behavioral problems or epilepsy in
children
• 85% occur at age 10 years or less; often
present at birth

71. • Needle biopsy not recommended since
histologically resembles normal choroid
plexus
• 10-30% become histologically malignant
• High survival unless becomes malignant
(then 5 year survival is 26%), although
histology does not predict behavior

72. Well-demarcated intraventricular (or cerebellopontine angle) mass with
hydrocephalus. Calcification especially frequent in fourth ventricular
tumors. In adult patients the fourth ventricle and in children third ventricle is
more common. The tumor is attached to the choroid plexus.

73. Choroid plexus papilloma (transverse section of the brainstem and the
cerebellum). Pinkish, granular growth with lobular contour in the lateral
recess of the fourth ventricle, at the cerebellopontine angle.

74. A. In the low-power smear, the tumor has grapelike papillary clusters of cells
bulging off vessels. B. A cross-section transforms these clusters into dendritic
arbors or fronds of tumor growing on fibrovascular cores.

75. C. The tumors shed bland epithelial balls or shells of cells. D. Permanent
sections show the labyrinth of tumor cells lining vascular stalks.

76. E. At high magnification, the tumor nuclei in individual shells are bland and
monomorphic. F. Histological sections show the nuclei to be more
hyperchromatic, heaped-up, and longer than those of normal choroid plexus.

77. Choroid plexus papilloma smears.
A.At intermediate magnification, sheets of cells stripped from papillae form a
honeycomb pattern.
B.High magnification shows distinct cytoplasmic borders separating the cells in
these sheets.

78. C. Cells further sheared off the sheets retain their polarized epithelial features;
they have distinct cell membranes and eccentric bits of cytoplasm. D. Ribbons of
epithelial cells mirror the fibrovascular structures on the permanent sections.

79. E. Because they are rigid, thick, and refringent, psammoma bodies often jump
out of the grapelike clusters. F. At high magnification, isolated psammoma
bodies display their diagnostic and aesthetic concentric spheres or laminations.

80. Choroid plexus carcinoma
• WHO grade III of IV
• Extremely rare and resembles metastatic
carcinoma but usually occurs in children
• Associated with germline p53 mutations
• Positive stains: EMA, keratin, S100, INI1;
GFAP (20%)
• DD: atypical rhabdoid tumor (INI1 - ve)

81. Left: Contrast-enhanced T1-weighted MR images showing a choroid plexus
papilloma that developed in the third ventricle. The vascular tumor pedicle
arising from the choroid plexus and the associated hydrocephalus are
typically observed in cases of these lesions.
Right: An MR image showing a choroid plexus carcinoma of the lateral
ventricle without hydrocephalus. In children such appearances can be similar
to those of an ependymoma or an intraventricular meningioma.

82. Gross: well circumscribed, brown-red, cauliflower-like mass; variable
hemorrhage, necrosis and invasiveness

83. Choroid plexus carcinoma. (×200; h&e stain) shows a solid growth of markedly
pleomorphic cells with loss of any papillary growth pattern.

84. Craniopharyngiomas
• Benign - typically very slow growing
tumors and arise from the cells along
the pituitary stalk
• usually suprasellar neoplasm, which may
be cystic, that develops from nests of
epithelium derived from Rathke's pouch
• Rathke's pouch is an embryonic precursor
of the anterior pituitary

87. • Resistant to smearing, reveal cohesive
sheets of squamous cells and keratinous
debris. Background can show cholesterol
crystals, calcified debris, foreign body type
giant cells and histiocytes.
• Microscopically:
– adamantinomatous (pediatric type),
– papillary (adult type)
– mixed

88. Ramified cell cords composed of
Spongy and cystic transformation squamous epithelium bordered by a
of the cell cords and connective layer of cuboidal basal cells. These
tissue. Massive nodule of cell cords are separated by an
compact "wet keratin". edematous, loose connective tissue
that exhibits cystic change in certain
areas.

89. • DD:
– metastatic carcinoma (usually no
calcifications, no squamous epithelium)
– pilocystic astrocytoma (if only gliosis is
sampled, more cellular, has microcysts)
– Rathke cleft cysts (CK8+, CK20+; both
negative in craniopharyngioma)

90. Intracranial Germ Cell Tumors
• 1-3% of primary pediatric CNS tumors
• Multiple tumor types seen:
– Germinomas -55%
– Teratomas and mixed germ cell tumors -33%
– Others: malignant endodermal sinus tumors,
embryonal cell carcinomas,
choriocarcinomas, teratocarcinomas-10%
• In all but germinomas, serum and CSF
alpha-fetoprotein (AFP) and βHCG may
be elevated

91. Germinoma
• MC intracranial germ cell neoplasm
• Seen in teenagers and young adults
• May derive from ectopic rests,
transformation of resident germ cells or
migration of germ cells late in
development
• MC site is pineal region; also anterior or
posterior third ventricle, rarely fourth
ventricle

92. Germinoma
• Relatively good prognosis
• Very sensitive to radiotherapy and
chemotherapy
• Metastases may be due to surgical
displacement of tumor; spinal cord
metastases occur in 10-15% of patients

94. Large pleomorphic cells with vacuolated cytoplasm ,large nuclei and
streaking of lymphoid cells

95. • DD:
– embryonal carcinoma (25% are PLAP+)
– Metastatic carcinoma (keratin+, EMA+, 13%
are PLAP+)

96. Embryonal carcinoma
• Characterized by rapid and bulky growth
and spread to liver and lungs; 60% have
metastases at presentation
• Prognosis poorer than germinoma
• May be associated with precocious
puberty
• Positive stains: alpha-fetoprotein

97. Yolk sac tumor
• Rare intracranial tumor, usually in pineal
or suprasellar regions
• Also called endodermal sinus tumor
• Prognosis poorer than germinoma
(median survival 2 years or less)
• Gross: usually large
• Micro: tubulopapillary structures with
vacuolated cuboidal cells, cystic spaces
with eosinophilic hyaline bodies, and
Schiller-Duval bodies

98. • Tumor occupies dorsal thalamus, cerebral
peduncle, mesencephalic tegmentum and
pons with compression of the third
ventricle and adjacent structures

99. Tumor occupies dorsal thalamus, cerebral peduncle, mesencephalic
tegmentum and pons with compression of the third ventricle and
adjacent structures

100. crushed smears showed loose aggregates of medium sized
hyperchromatic undifferentiated cells with ovoid nuclei, moderate
anisonucleosis, discernible nucleoli, and scant to moderate amounts of
cytoplasm

101. Teratoma
• Tissue derived from ectoderm, endoderm
and mesoderm (at least 2 of 3 germinal
layers)
• Usually well differentiated / grade I of IV
• Congenital cases are usually fatal

102. • Mature teratomas: have well differentiated
tissue from all three germinal layers
• Immature teratomas: have less
differentiated tissue from any of the three
germinal layers
• Poor prognosis: tissue resembling
medulloepithelioma, neuroblastoma,
retinoblastoma or ependymoblastoma

103. • Pineal teratomas are more common in
males, but saccrococcygeal teratomas are
more common in females
• Treatment: newborns - complete surgical
excision (difficult)

104. MRIof the head shows a mass measuring 4.2 cm in diameter with cystic
contents and small areas of nodular and rim enhancement. The mass
causes marked compression of the cortical mantle throughout the inferior
parietal and temporal regions.

107. Choriocarcinoma
• Prognosis poorer than germinoma -
median survival 22 months in cases with
high hCG levels
• Serum levels of hCG are helpful
• Micro: syncytiotrophoblasts (large
multinucleated cells) and cytotrophoblasts
• Positive stains: hCG
• DD: metastatic choriocarcinoma (from
gonads or placenta)

108. References
• Diagnostic Neuropathology Smears, 1st
Edition by Joseph, Jeffrey T
• Intraoperative neurocytlogy of primary
CNS neoplasia, Review article by shama
and Deb
• Internet

109. THANK YOU