Squash smear cytology - By Anamika dev

Ssquash smear cytology of CNS, ALgorith to aprroach to squash cytology, new WHO CNS Tumor classification, squash smear preparation

1. SQUASH SMEAR CYTOLOGY DR. ANAMIKA DEV

2. introduction Central nervous system (CNS) is one of the most challenging domains for the neurosurgeon and Pathologists 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

3. Simple and rapid methodology for fairly accurate intraoperative diagnosis of CNS space occupying lesions. High accuracy  clinical and radiological picture into consideration. Two procedures : open craniotomy or stereotactic biopsy. Goal of intraoperative cyto -diagnosis in stereotactic biopsies  to confirm the adequacy of the tissue.

4. Use of intraoperative diagnosis in open biopsy: Additional tissue for ancillary techniques (for infections and lymphomas), To decide on extent of resection and makes it possible to institute adjuvant therapy in immediate postoperative period. Last, but not the least, an intraoperative pathological diagnosis is of great value to the anxious family members of the patients .

5. Sample identification and transportation Transportation - sterile container on a saline-moistened lens paper or telfa pad (never gauze) to prevent drying Request form - relevant clinico -radiologic data precise site of biopsy reason for which the intraoperative diagnosis was sought – adequacy or guidance technique used : open biopsy, stereotactic-guided needle aspiration biopsy ; fine-needle aspiration biopsy

6. HOW TO PREPARE A SQUASH SMEAR? The selected fragment is placed on a glass slide and then divided into smaller pieces (1–2 mm 3 ) Sample is inspected with the help of a magnifier or under dissecting microscope to see whether specimen appears necrotic or hemorrhagic or both

7. Each of the smaller pieces is placed on a newer slide A second labelled slide is placed over the first slide on top of the tissue fragment Gently pull both the slides apart to produce two thin well spread smears

8. Fixative of choice : 95% Ethanol, 1-2 min Additional slides are air dried and stained Stain : H&E, Papanicolaou additional slides : WRIGHTs stain Artifacts : crushing, overstretching and dry artifacts When tissue spreads poorly, stain both slides with H & E, as one smear will usually have sufficiently thin areas to make interpretation possible

9. Note : Don’t smear too large a specimen slide too thick for optimal cytological detail Thin specimen  recognition of fine fibrillary process often a/w glial tumors tissue defy smearing  having extensive reticulin, collagen, or glial fibers

10. Comparison of tissue smearing EASILY POORLY NORMAL BRAIN/ CORD REACTIVE GLIOSIS MOST PRIMARY / SECONDARY TUMORS SCHWANNOMA NON-NEOPLASTIC DISORDERS HEMANGIOBLASTOMA SUBEPENDYMOMA DESMOPLASTIC TUMORS VASCULAR MALFORMATIONS

11. ADVANTAGES of smear technique Improved cost-effectiveness More rapid and technically simpler Only small amount of tissue needed Far better preservation of cellular detail Best in demonstrating fibrillary cytoplasmic processes Provides additional information to the permanent sections If the case turns out to be infectious, contamination of the cryostat and subsequent defrosting and sterilization can be avoided Abrogate the risk of distorting valuable diagnostic material during freezing, which compromise permanent sections and immunomarkers

12. LIMITATIONS Relies on tissue soft enough to smear Histologic architecture not apparent Relies on accurate localization by the surgeon Low-magnification architecture is lost Some lesions do not smear well (especially those having extensive reticulin or collagen) Specific training needed (many pathologists are more familiar with frozen section techniques and interpretation)

13. Anaplastic astrocytoma. (a) Cryostat section showing freezing artifact , nuclear distortion, and effacement of the fibrillary background (Methylene blue) (b) Cytologic preparation from the same tumor showing beautifully preserved nuclear and cytoplasmic details. Note the characteristic multipolar astrocytic processes (Smear, H&E)

14. Normal cns cytology

15. NORMAL Brain White matter pattern Will smear easily Felt like background Small, empty vacuoles representing dissolved myelin – scattered in background Cellular population consisting basically of oligodendrocytes and to a lesser extent of astrocytes Delicate capillaries with elongated endothelial cells arranged parallel to the capillary wall Also seen few arterioles with thicker walls, in which cells are arranged parallel (endothelial cells) or perpendicular (muscle cells) to the lumen - bidirectional arrangement

16. Grey matter pattern Smear less evenly than tissue from white matter Accumulate elements of larger size, such as blood vessels and neurons, at the edge of the smear. The background is also eosinophilic and granular (feltlike) No vacuoles of myelin. Numerous neurons seen along with glial cells Neurons appear as large cells with abundant cytoplasm, distinct cell borders, and round, prominent nucleolated nuclei. Capillaries of same pattern as white matter Also seen arterioles and arteries

17. 3. cerebellar cortex pattern More cellular Predominant cells  neurons from the internal granular layer (small, round, hyperchromatic cells without visible cytoplasm) Voluminous Purkinje’s neurons may be observed. These cells show a wide cytoplasm with an expansive dendritic tree and large vesicular nucleus with a visible nucleolus.

18. Choroid plexus pattern A normal choroid plexus may be present in biopsy specimens from the ventricles Vascular-stromal framework  tends to smear poorly and results in a cohesive crush preparation Vessels  complex arched papillary appearance , characteristic of a plexus, lined by a row of cuboidal epithelial cells Cells that appear isolated, due to the squash trauma  wide polygonal “cobblestone-shaped” cytoplasm small, round, basally located nuclei Can have single prominent paranuclear cytoplasmic vacuole

19. 5. Leptomeningeal pattern Large cell clusters The cytoplasm of arachnoid cap cells is wide and delicate Nuclei are round to ovoid with finely granular dispersed chromatin, often giving the appearance of central clearings. As against cells from the choroid plexus, arachnoidal cells have poorly defined boundaries , giving rise to sheets with a syncytial appearance

20. 6. Pineal pattern Infant pineal gland preparations highly cellular uniform, round, single pinealocytes with indistinct cytoplasm. The background is finely granular (neuropil-like) and calcifications are absent Adult pineal gland Less cellular, displaying tissue fibrillary fragments and single cells with ill-defined cytoplasm and moderate nuclear pleomorphism microcalcifications, referred to as corpora arenacea or simply “brain sand”

21. Normal brain tissue pattern and potential misdiagnosis

22. Who 2016 Classification of cns tumors

23. CNS lesions in which the smear technique is an especially useful procedure Astrocytoma Oligodendroglioma Ependymoma Glioblastoma Ganglion cell tumors Meningioma Lymphoma Germinoma Pituitary adenoma Metastases Reactive gliosis Cerebral infarction Inflammatory processes Demyelinating disorders Cranial and spinal bone masses

24. APPROACH TO SMEAR EVALUTAION

25. APPROACH TO SMEAR EVALUTAION A complete evaluation of the smear requires the study of six parameters: Type of smearing Type of background Type of blood vessels Presence of specific cell groups Predominant type of cell Presence of specific cellular elements

26. 1.TYPE OF SMEARING Normal tissue pattern - uniform/smooth Lesional tissue pattern - speckled/granular Eg : Schwannoma smear reveals only tissue fragments without single cells .

27. Smear from metastatic carcinoma showing a mixed pattern with cell sheets, smaller cell clusters, and individual cells. Smear from lymphoma showing single cells without cell aggregates. Note the numerous lymphoglandular bodies in the background

28. 2.TYPE OF BACKGROUND Finely granular (felt like) - normal neuroglial tissue Clear (empty) - In the case of lesions, if intercellular space empty Schwannoma Meningioma Pineocytoma SFT/Hemangiopericytoma Choroid plexus papilloma Pituitary adenoma (if imprints)

29. Fibrillary (threadlike )- if intercellular space occupied by various cell products or components, such as fine cytoplasmic processes Astrocytic tumors Ependymal tumors Mixed glioneural tumors Neuronal tumors (fine neuropil) Necrotic (dirty) Necrotic primary tumors - Necrotizing infections Radiation necrosis - Necrotic metastasis Necrotic CNS lymphoma Cerebral infarction

30. Granular-vacuolated Released intracytoplasmic components of a proteinaceous, lipidic, or glycogenic nature Oligodendroglioma Hemangioblastoma Demyelinating lesions Resolving infarct Pituitary adenoma (if squash/smear) Germinoma (“ tigroid ”) Ewing’s sarcoma (“ tigroid ”) Myxoid/mucoid background Stromal or mucinous matrix Pilocytic/ pilomyxoid astrocytoma Myxopapillary ependymoma Angiocentric glioma DNT Neurofibroma Chordoma Chordoid /myxoid meningioma Chordoid glioma Gliomas with mucinous degeneration Mucinous metastatic carcinoma

31. Mixed background fibrillary-necrotic background (glioblastoma) fibrillary- myxoid background (pilocytic/ pilomyxoid astrocytoma)

32. 3. Type of blood vessels Very important in dealing with gliosis Thin-walled Low-grade diffuse gliomas (astrocytoma, oligodendroglioma) Neurocytoma Dysembryoplastic neuroepithelial tumor . Thick-walled (hyalinized) Tumors with degenerative changes Schwannoma, ganglioglioma, and pilocytic astrocytoma, and in the setting of radiation

33. Endothelial cell proliferation (microvascular hyperplasia/ proliferation [MVP]) Criterion for anaplasia in diffuse astrocytoma and, to a lesser degree, in oligodendroglioma and ependymoma, but not in pilocytic astrocytoma. MVP occasionally seen in: metastatic cancer (mainly small-cell and renal cell carcinomas) nonneoplastic processes (surrounding resolving infarctions and abscesses). Network of vascular channels Intricate network of thin-walled vascular channels  characteristic pattern of hemangioblastoma and hemangiopericytoma .

34. 1. Tumor distribution in relation to blood vessels Perivascular gradient pattern Gliomas – especially astrocytoma Angiocentric and diffuse pattern Lymphoma Randomised clusters with and without vascular affinity Metastatic carcinoma

35. Glioblastoma: Characteristic microvascular hyperplasia displaying a “ glomeruloid ” appearance

36. Diffuse astrocytoma, WHO grade II. Thin-walled, capillary vessels are usually found in low-grade diffuse gliomas

37. Hemangiopericytoma. Network of vascular channels with tumor cell aggregates

38. 4. Specific cell groups Whorls Meningioma Craniopharyngioma Epidermoid carcinoma Schwannoma Transitional meningioma. Typical cell whorls showing tight concentric arrangements

39. Papillae Choroid plexus tumors Papillary/myxopapillary ependymoma Papillary meningioma Papillary craniopharyngioma Papillary tumor of the pineal region Metastatic papillary carcinomas Choroid plexus papilloma. Characteristic papilla with a vascular stromal core

40. True rosettes (Flexner- Wintersteiner ) Ependymoma Subependymoma ETANTR Pineoblastoma Medulloepithelioma Flexner- Wintersteiner rosettes from ependymoma, with prismatic cells arranged around a small luminal structure

41. Pseudorosettes (Homer-Wright) Neuroblastoma Ganglioneuroblastoma Neurocytoma Pineocytoma PPTID Medulloblastoma Rosette forming glioneural tumor Homer- Wright rosettes from neuroblastoma, with central tangles of fibrillary processes

42. Perivascular pseudorosettes Ependymoma Astroblastoma Subependymal giant cell astrocytoma Pilomyxoid astrocytoma Papillary glioneural tumor Rosette- forming glioneural tumor Angiocentric glioma Anaplastic astrocytoma Glioblastoma

43. Perivascular pseudorosettes from ependymoma, with tumor cell processes radiating toward a central vessel

44. 5.Type of cell Glial (fine processes) Ganglion/ganglion-like Round Epithelial/epithelial-like Fusiform Small poorly differentiated Mixed/polymorphic

45. 6.Specific cellular elements Rosenthal fibers Eosinophilic granular bodies Lymphoglandular bodies Keratin Melanin Mucin

46. clump of Rosenthal fibers and eosinophilic granular body - pilocytic astrocytoma

47. Rosenthal fibres Pilocytic astrocytoma Ganglioglioma Chronic piloid gliosis Rosette- forming glioneural tumor Alexander’s disease E-G BODIES Pilocytic astrocytoma Ganglion cell tumors Pleomorphic xanthoastrocytoma Rosette- forming glioneural tumor DNT (occasionally)

48. Keratin -metastatic epidermoid carcinoma show orange hyaline cytoplasm with Papanicolaou stain (a) and sky-blue hyaline aspect with Romanowsky stain (b)

49. KERATIN Craniopharyngioma Teratoma Epidermoid cyst Dermoid cyst Metastatic epidermoid carcinoma LYMPHOGLANDULAR BODIES Lymphomas Non-neoplastic lymphoid infiltrations

50. Smears from a metastatic melanoma show dark-brown cytoplasmic granules with Papanicolaou stain and black granules with Romanowsky stain

51. melanin Melanoma/ Melanocytoma Neurocutaneous melanosis Teratoma Pineal anlage tumor Melanotic schwannoma Melanotic medulloblatoma Melanotic ependymoma Melanotic paraganglioma Melanotic progonoma mucin Mucinous metastatic carcinoma Nonneoplastic cysts of the neuroaxis Glandular component in teratoma Gliomas with mucinous degeneration

52. (a) Metastatic mucinous colonic adenocarcinoma with abundant thick mucoid background. (b) Metastatic lung adenocarcinoma displays numerous intracytoplasmic vacuolar purple granules or “magenta bodies”

53. gliosis Reactive astrocytosis or gliosis is a nonspecific response of the brain tissue Neoplastic and nonneoplastic irritating injuries.

54. Reactive gliosis: (a) Clump of evenly spaced reactive astrocytes and some inflammatory cells (Smear, Romanowsky). (b) Two stellate-shaped reactive astrocytes with tapering cytoplasmic processes and mild nuclear enlargement

55. Binucleation (mirror nuclei) and tapering processes radiating out from all around the cell suggest it is nonneoplastic. Compare this morphology with the surrounding glioblastoma tumor cells

56. Piloid gliosis Chronic reactive gliosis with abundant Rosenthal fibers (RFs ). It is a common process adjacent to cysts and slow-growing tumors (e.g., pineal cyst, craniopharyngioma, hemangioblastoma) Particularly in the midline axis (third ventricle, brainstem, cerebellum, and spinal cord) The long processes forming a compact layer are intermixed with numerous hyaline Rosenthal fibers

57. astrocytic tumors

58. Diffuse astrocytoma Cytologic features- Lumpy tissue fragments mature capillary vessels In comparison with normal brain, smears show: Fibrillary (no felt-like) background Increased cellularity with uneven cellular distribution Mild nuclear atypia Gemistocytic variant ( at least 20% of the tumor cells) Large, plump cells with eccentric nuclei predominate Protoplasmic varian t Small cells with short, cobweb-like processes predominate M yxoid background

59. Fibrillary variant - Cellular tissue fragments have uneven, lumpy appearance, fibrillary background, increased cellularity and mild nuclear atypia. (b) Cytoplasms are not discernible and nuclei show irregular contours and slight hyperchromatism . Vessels are of the capillary type

60. gemistocytic variant  gemistocytic astrocytes with peripherally displaced nuclei and glassy pink cell bodies

61. protoplasmic variant b)Evenly distributed preparation with small cells showing round to oval nuclei and sparse cytoplasmic processes (Smear, Papanicolaou) (c) Metachromatic myxoid material surrounding the tumor cells (Smear, Romanowsky)

62. Diffuse astrocytoma – all 3 variants Absence of mitosis Absence of microvascular proliferation Absence of necrosis, Differential diagnosis and pitfalls Normal brain tissue Reactive gliosis Pilocytic astrocytoma Gemistocytic variant Subependymal giant cell astrocytoma Protoplasmic variant Oligodendroglioma Dysembryoplastic neuroepithelial tumor

63. 2. Anaplastic astrocytoma Cytologic features Fibrillary background Increased cellularity and pleomorphism Distinct nuclear atypia and mitotic activity Perivascular aggregates Microvascular proliferation and necrosis must be absent Differential diagnosis and pitfalls Pleomorphic xanthoastrocytoma Pilocytic astrocytoma with degenerative-type atypia Anaplastic oligodendroglioma Undersampled glioblastoma

64. Dark, elongated and sometimes angulated nuclei without apparent nucleoli are typical of anaplastic astrocytoma and glioblastoma, but in WHO grade III tumors , the vessels are still of capillary type

65. 3.glioblastoma Fibrillary and/or necrotic background Anomalous vessels Microvascular “ glomeruloid ” proliferation, Abnormal fistulous vessels with intraluminal endothelial proliferation Vascular thrombosis Pseudopapillary appearance ( atypical vessels appear surrounded by a large number of tumor cells) Very high cellularity with discohesive pattern Marked cellular pleomorphism with Distinct nuclear atypia (multilobed appearance, coarse chromatin) Often tumor cells conserve fibrillary processes (diagnostic clue)

66. Variants/patterns: Giant cell (bizarre, multinucleated giant cells predominate) Small cell (small, undifferentiated cells predominate) Gliosarcoma (mixed pattern of glial and sarcomatous cells) Epithelioid (large, epithelioid cells predominate) Differential diagnosis and pitfalls Other anaplastic gliomas Pleomorphic xanthoastrocytoma Metastatic carcinoma/melanoma Embryonal tumors CNS lymphomas Sarcomas Nonneoplastic necrotic processes Necrotizing infections Ischemic lesions Radiation necrosis with radiation- induced atypia

67. Atypical asytrocytic cells in fibrillary b/g and giant cells Necrotic granular debris is intermixed with ghost cells and karyorrhectic nuclei

68. 4.PILOCYTIC ASTROCYTOMA Fibrillary-myxoid background Biphasic cellular pattern: Bipolar “ piloid ” cells Multipolar “protoplasmic” cells Oval to elongate ( piloid ) or round (protoplasmic) bland nuclei Rosenthal fibers and eosinophilic granular bodies Regressive changes Hyalinized/ glomeruloid vessels Degenerative-type atypia - including large or giant cells with multiple nuclei circumferentially arranged (“ pennies on a plate ”)

69. Pilomyxoid astrocytoma Enhanced myxoid background Monomorphous cellular pattern of bipolar cells Angiocentric arrangements RFs and EGBs typically absent Differential diagnosis and pitfalls Diffuse astrocytoma (any grade) Ependymoma Piloid gliosis

70. Bipolar “ piloid ” cells exhibiting very long processes. Nuclei are bland with smooth contours Brightly eosinophilic Rosenthal fiber with one blunt pole and one tapered end. It is surrounded by “protoplasmic” cells exhibiting round nuclei and short, cobweb-like processes

71. a)“pennies on a plate” arrangement b) angiomatoid vasculature with complex arborization and hyaline mural fibroplasia, perivascular aggregations of “ piloid ” tumor cells

72. Oligodendroglial tumors

73. oligodendroglioma Uniform single cell pattern without adhering to blood vessels Round nuclei with finely granular (salt and pepper) chromatin and small nucleoli Ill-defined, wispy cytoplasm (no perinuclear halos) Finely granular/ vacoulated or mucoid background No fibrillary background Delicate branching “ chicken-wire” capillary network Microcalcifications

74. Anaplastic oligodendroglioma Increased cellularity and pleomorphism Coarser chromatin Prominent mitotic activity Epithelioid features and minigemistocytes Microvascular proliferation

75. Differential diagnosis and pitfalls Diffuse astrocytoma Subependymoma Dysembryoplastic neuroepithelial tumor Pituitary adenoma Neurocytoma Pineocytoma Lymphoma Macrophage-rich processes Anaplastic oligodendroglioma Anaplastic astrocytoma Glioblastoma Metastatic carcinoma/melanoma

76. a) Uniform population of small, round cells without perinuclear halos. The background is not fibrillary, but finely granular with slim capillaries and tiny vacuoles b)mucoid metachromatic background and a “chicken-wire” capillary network

77. ependymoma

78. Moderately cellular smears Fibrillary background Dual (glial-epithelial) cellular properties Small oval nuclei with stippled chromatin Key diagnostic clues Perivascular pseudorosettes  “arboreal” or “caterpillar” appearance Ependymal rosettes Anaplastic ependymoma Increased cellularity and coarser chromatin Nuclear grooves and indentations Mitotic figures and vascular hyperplasia Perivascular pseudorosettes Fibrillary background Features retained

79. Classic ependymoma – a) The characteristic branching “arboreal” appearance of pseudorosettes . Tumor cells remain tethered to the vessel wall by their glial tails. (b) High-magnification view showing unevenly distributed glial cells in a fibrillary background. Nuclei are small, oval, and uniform

80. Choroid plexus tumor

81. Choroid plexus papilloma: Cellular smears with the joint presence of: Papillae with fibrovascular cores Monolayer fragments Single cells Columnar to cuboidal cells with: Smooth ( nonciliated ) epithelial surfaces Round to oval bland nuclei Clean background Atypical papillomas : Crowded and tall columnar epithelium with the additional presence of mitoses

82. Choroid plexus carcinoma Malignant cellular characteristics Areas of transition papilloma-carcinoma Hemorrhagic or granular (necrotic) background Differential diagnosis and pitfalls Normal choroid plexus Papillary ependymoma Choroid plexus carcinoma Metastatic carcinoma Melanoma Non- germinomatous (malignant) germ cell tumors Atypical teratoid/rhabdoid tumor

83. a)highly cellular preparation has branching papillae, small cell clusters, and isolated epithelial cells (b) Papilla with a central vascular core. c) cell sheets with smooth epithelial surfaces. Nuclei have slightly coarser chromatin than their normal counterparts but still lack significant pleomorphism

84. Papillary tissue fragments composed of cells with little cytoplasm are consistent with choroid plexus carcinoma. (b) This high-power view shows characteristic vesicular nuclei with prominent nucleoli and frequent lobulations

85. Embryonal tumor

86. medulloblastoma Highly cellular Evenly distributed discohesive sheets of small, rounded or wedge- shaped cells with minimal cytoplasm. Nuclei have a slightly coarse “salt-and- pepper” chromatin and lack nucleoli, nuclear membranes show folds and indentations Medulloblastoma nuclei often stick together forming short chains or circles or conform around each other, giving the characteristic nuclear molding

87. Characteristic discohesive pattern of small, round cells with hyperchromatic nuclei and minimal cytoplasm, nuclear molding . Also a small numbers of nuclei stick together, forming short chains or circles

88. meningioma

89. meningioma High cellularity Clear background Uniform, benign cellular aspect with Oval nuclei with delicate chromatin Nuclear pseudoinclusions and clearings Copious “tissue paper “cytoplasm with broad or streaked processes Cell whorls and psammoma bodies Characteristic morphologic variants (fibrous, secretory, chordoid ….)

90. Irregular clusters, small groups, and single cells in a clear background. Tumor cells have copious cytoplasm with broad, borderless processes. Nuclei are slightly oval with frequent intranuclear pseudoinclusions or clearings (arrows) Cellular whorls seen

91. Psammomatous meningioma : calcified psammoma bodies

92. Fibrous meningioma . (a) fusiform cells with tapering cytoplasmic processes. Despite elongation, nuclei show characteristic meningothelial features including nuclear pseudoinclusions (b) In contrast to schwannoma, the fusiform cells of fibrous meningioma tend to appear dispersed with visible cytoplasmic boundaries

93. Secretory meningioma . (a) Histology. Numerous intracellular lumens containing eosinophilic globules are typically (b) Preparation from this tumor displaying target-like intracytoplasmic bodies and cell whorls

94. Microcystic meningioma :cystic spaces of variable sizes within the cell clusters and sheets of an otherwise typical meningioma. The tumor cells exhibit numerous tiny cytoplasmic vacuole

95. Angiomatous meningioma . (a) Histology. Blood vessels constitute most of the mass. The intervening tumor cells are difficult to recognize as meningothelial. (b) Dense network of mature vessels and small clumps of meningothelial cells with characteristic nuclear pseudoinclusions

96. Metaplastic meningioma . (a) Histology. Presence of xanthomatous cell aggregates in this case of metaplastic meningioma. (b) Large, foamy xanthomatous cells admixed with smaller meningothelial cells

97. Pleomorphic meningioma . (a) Some, otherwise, completely benign meningiomas may show conspicuous nuclear pleomorphism, which should not be misinterpreted as signifying a potentially aggressive behavior . (b) Similarly, the presence of bizarre pleomorphic cells in cytological preparations is not an indicator of high-grade tumor

98. Atypical meningioma . (a) Hypercellularity, patternless pattern, and small cell population are characteristic features of this histologic variant. Mitotic figures seen (b) discohesive pattern of small, lymphocyte-like cells and many naked nuclei. Note a nuclear pseudoinclusion

99. Chordoid meningioma . (a) Nests and cords of epithelioid cells in a basophilic, myxoid-rich matrix characterize this variant. (b) Undoubted meningothelial cell group embedded in a metachromatic myxoid matrix . Nuclear pseudoinclusions +

100. Clear cell meningioma . (a) sheets of cells with cleared cytoplasm due to increased glycogen accumulation and strands of collagen scattered throughout. (b) Preparation from this tumor displaying round clear cells with nuclear features of meningioma

101. Anaplastic meningioma . (a) Histology. Sarcomatous-like anaplastic meningioma with spindled morphology, poorly differentiated cytology, and mitosis (b) Preparation from this tumor showing crowded tissue fragments and individual cells with anaplastic features

102. Papillary meningioma . (a) The histologic pattern consists of a perivascular pseudopapillary arrangement of cells on a vascular-fibrous stroma. (b) papillary cluster and single cells

103. Rhabdoid meningioma . (a) Tissue section showing an area of cortical infiltration indicating aggressive tumor behavior . This variant loses the syncytial appearance of most meningiomas. (b) Highly cellular preparation with a discohesive pattern of rhabdoid cells

104. Germ cell tumor

105. germinoma Dual cell population Large, primordial germ cells Large, spherical nuclei with prominent and characteristically angular nucleoli. The cytoplasm is faint, vacuolated, and lacy due to its high glycogen content Small, mature lymphocytes Occasionally admixed with plasma cells and histiocytes. Striped “ tigroid ” background (Romanowsky stains) Release of intracytoplasmic glycogen during the squash better revealed by using Romanowsky stains looks only slightly granular and eosinophilic with H&E or Papanicolaou stains. Frequent granulomatous inflammation

106. a)classic germinoma is made up of sheets of large tumor cells interrupted by fibrous septae sprinkled with small lymphocyte b) large germ cells, small lymphocytes, and striped “ tigroid ” background.

107. Germinoma pitfall : Tight clusters of epithelioid histiocytes admixed with a heavy chronic inflammatory infiltrate are the only feature that appears in this case

108. lymphoma

109. diffuse large B cell lymphoma The vast majority of PCNSLs (almost 98%) are high-grade lymphoma of the diffuse large B-cell type (DLBCL) Highly cellular smears Perivascular cuffing and clearly discohesive cells Large, pleomorphic nuclei with prominent nucleoli Scant cytoplasm without processes Apoptotic bodies and tingible body macrophages Granular-vacuolated or necrotic background with LGBs

110. Diffuse large B-cell lymphoma. A large branching vessel surrounded by atypical cells gives a clue that this is a lymphoma. Characteristic single cell pattern of large, atypical lymphoid cells in a granular-vacuolated background of disintegrating neuropil

111. Discohesive large pleomorphic cells, Numerous lymphoglandular bodies are typically present in the background.

112. Nerve sheath tumor

113. schwannoma Conventional schwannoma (>90%) Very difficult to smear Antoni A areas: Cohesive tissue fragments without single cells Spindle cells with club-shaped nuclei Antoni B areas : Loosely cellular sheets Round to oval nuclei and stellate processes Cellular schwannoma (5-10%) Only highly cellular Antoni A-type groups Mild to moderate hyperchromatism Absence of anaplastic features

114. Antoni A zone

115. Antoni B zone- Loose cluster composed by less regular cells exhibiting round to oval nuclei Cellular schwannoma: highly cellular tissue fragments of spindle cells, cells are more crowded as opposed to those in conventional schwannomas, nuclear uniformity is typical

116. Pituitary adenoma

117. Third most common tumor in the CNS Highly cellular smears Discohesive monolayer pattern Round nuclei with stippled chromatin Small “peppery” nucleoli Granular background with many bare nuclei Plasmacytoid cell appearance When cytoplasm is preserved, looks well-defined with an oval or round morphology without processes along with the frequent nuclear eccentric position

118. Discohesive pattern of small cells and numerous bare nuclei. Plasmacytoid features with oval cytoplasm and eccentric nuclei

119. References César R.  Lacruz , Javier  Saénz  de  Santamaría , Ricardo H.  Bardales , editors. Central Nervous System Intraoperative Cytopathology. 2 nd ed. Switzerland: Springer, 2018. Squash preparation; a reliable diagnostic tool in the intraoperative diagnosis of central nervous system tumors; journal of cytology/ July 2010/volume 27/issue 3 Washington manual of surgical pathology Internet sources

120. Thank you