Protocol for the Examination of Specimens From Patients With Tumors of the Brain/Spinal Cord: A Basis for Checklists

• I. Cytologic Material

  • A. Clinical information

    • 1. Patient identification

      • a. Name

      • b. Identification number

      • c. Age (birth date)

      • d. Gender

    • 2. Responsible physician(s)

    • 3. Date of procedure

    • 4. Other clinical information

      • a. Relevant history (note A)

      • b. Relevant findings (note B)

      • c. Clinical/imaging differential diagnosis

      • d. Procedure (eg, percutaneous fine-needle aspiration)

      • e. Anatomic site of specimen (note C)

  • B. Macroscopic examination

    • 1. Specimen

      • a. Unfixed/fixed (specify fixative) (note D)

      • b. Number of slides received, if appropriate

      • c. Cytologic preparation of tissue specimen (touch or squash preparation)

    • 2. Material submitted for microscopic evaluation (eg, smear of fluid, cell block) (note E)

    • 3. Special studies (eg, cytochemistry, immunocytochemistry, microbiology, flow cytometry) (note F)

  • C. Microscopic evaluation

    • 1. Adequacy of specimen for diagnostic evaluation (if unsatisfactory or limited, specify reason)

    • 2. Tumor

      • a. Histologic type, if possible (note G)

    • 3. Other pathologic findings

    • 4. Results/status of special studies (specify)

    • 5. Comments

      • a. Correlation with intraoperative consultation

      • b. Correlation with other specimens

      • c. Correlation with clinical information (note H)

• II. Biopsy

  • A. Clinical information

    • 1. Patient identification

      • a. Name

      • b. Identification number

      • c. Age (birth date)

      • d. Gender

    • 2. Responsible physician(s)

    • 3. Date of procedure

    • 4. Other clinical information

      • a. Relevant history (note A)

      • b. Relevant findings (note B)

      • c. Clinical/imaging differential diagnosis

      • d. Procedure (eg, stereotactic needle core biopsy, open biopsy)

      • e. Anatomic site of specimen (note C)

  • B. Macroscopic examination

    • 1. Specimen

      • a. Unfixed/fixed (specify fixative) (note D)

      • b. Size (number of cores or size of biopsy in dimensions or approximate volume)

      • c. Descriptive features (grossly obvious meninges, gray matter or white matter, color, texture, cut surface, mucinous, fibrous, bloody, necrotic, gritty)

      • d. Recognition of gross and microscopic correlates is helpful in correct interpretation of microscopic findings and is also helpful in selecting cores for frozen section analysis

    • 2. Special studies (note F)

      • a. Frozen sections, if requested

      • b. Squash, touch, or scrape preparations

      • c. Histochemistry

      • d. Immunohistochemistry

      • e. Electron microscopy

      • f. Other (microbiology, flow cytometry, cytogenetics, molecular diagnostics)

      • g. Was a portion of tissue frozen for later potential studies?

    • 3. Tissue submitted for microscopic evaluation: The specimen is usually totally submitted after removing tissue for frozen sections, electron microscopy, or other special studies as indicated in note F. Try to orient at right angles to surface.

  • C. Microscopic evaluation

    • 1. Tumor

      • a. Histologic type (note I)

      • b. Histologic grade (note J)

      • c. Additional features, if present

        • (1) Hemosiderin deposition

        • (2) Calcification

        • (3) Microcyst formation

        • (4) Mitotic activity

        • (5) Pleomorphism

        • (6) Presence of gemistocytes

        • (7) Vascular proliferation

        • (8) Necrosis

      • d. Findings in squash, touch, or scrape preparations (note K)

    • 2. Status/results of special studies (specify)

    • 3. Comments

      • a. Correlation with intraoperative consultation

      • b. Correlation with previous specimens

      • c. Correlation with clinical information (note H)

• III. Resection

  • A. Clinical information

    • 1. Patient identification

      • a. Name

      • b. Identification number

      • c. Age (birth date)

      • d. Gender

    • 2. Responsible physician(s)

    • 3. Date of procedure

    • 4. Other clinical information

      • a. Relevant history (note A)

      • b. Relevant findings (note B)

      • c. Clinical/imaging differential diagnosis

      • d. Procedure (total, subtotal, or partial resection)

      • e. Operative findings

      • f. Anatomic site of specimen (note C)

  • B. Macroscopic examination

    • 1. Specimen

      • a. Unfixed/fixed (specify fixative) (note D)

      • b. Number of pieces with combined aggregate dimensions (the extent of resection can have prognostic significance) (note A)

      • c. Descriptive features (grossly obvious meninges, gray matter or white matter, color, texture, cut surface, mucinous, fibrous, bloody, necrotic, gritty)

      • d. Recognition of gross and microscopic correlates is helpful in correct interpretation of microscopic findings and is also helpful in selecting cores for frozen section analysis

      • e. Margins, as appropriate. For the majority of central nervous system neoplasms, margins are not evaluated because specimens are fragmented. Exceptions would be some meningeal or metastatic tumors.

      • f. Results of intraoperative consultation

    • 2. Tissue submitted for microscopic evaluation. The specimen is usually totally submitted after removing tissue for frozen sections, electron microscopy, or special studies, as suggested in note F.

    • 3. Special studies (note F)

      • a. Frozen sections, if requested

      • b. Squash, touch, or scrape preparations

      • c. Histochemistry

      • d. Immunohistochemistry

      • e. Electron microscopy

      • f. Receptor analysis

      • g. Other (microbiology, flow cytometry, cytogenetics, molecular diagnostics)

      • h. Was a portion of tissue frozen for later potential studies?

  • C. Microscopic evaluation

    • 1. Tumor

      • a. Histologic type (note I)

      • b. Histologic grade (note J)

      • c. Local extension (eg, bony or soft tissue invasion, subarachnoid spread; note K)

      • d. Additional features, if present

        • (1) Hemosiderin deposition

        • (2) Calcification

        • (3) Microcyst formation

        • (4) Mitotic activity

        • (5) Pleomorphism

        • (6) Presence of gemistocytes

        • (7) Vascular proliferation

        • (8) Necrosis

      • e. Findings in squash, touch, or scrape preparations (note K)

    • 2. Status/results of special studies (specify)

    • 3. Comments

      • a. Correlation with intraoperative consultation

      • b. Correlation with previous specimens

      • c. Correlation with clinical information (note H)

A: Relevant History.—Patient Age.—Most central nervous system (CNS) tumors show an age predilection, and patient age has been shown to predict survival in many malignant CNS neoplasms. With diffusely infiltrating astrocytic tumors, age followed by histologic grade represent the 2 strongest prognostic indicators for patient outcome, with patient age older than 50 years and high-grade tumors serving as negative indicators.1–4 

Duration of Symptoms (Acute or Chronic).—A long clinical history of CNS symptoms or seizures prior to the diagnosis of a CNS tumor favors a slowly growing neoplasm that is more likely to be benign. A rapidly progressive neurological deficit of sudden onset is more consistent with, but not always indicative of, a high-grade malignant tumor.5 

Extent of Resection.—For most CNS tumors, the amount of tumor removed (total, subtotal, or partial resection) is an important predictor of patient outcome.3,4,6 

The extent of resection can be estimated by recording the gross dimensions of the aggregate pieces. In most operating rooms, a suction device is frequently used in conjunction with gross debulking to remove tumors. We recommend that the surgical team be encouraged to submit the suction specimen to surgical pathology. This will serve to better estimate the extent of resection, and the tissue present in the suction specimen might be critical in making the correct diagnosis.

Tumor Location and Size.—The extent of surgical resection possible is determined by tumor location and size.

Previous Diagnoses.—Knowledge of the presence or absence of extracranial disease, that is, a history of immunosuppression or a history of a primary malignant neoplasm outside the CNS, can be critical in the correct interpretation of biopsy material.5 If a metastatic tumor is included in the differential diagnosis, it is helpful to have slides of the primary tumor available.

Previous CNS Biopsies.—History of radiation or radiosurgery.—Knowledge of prior radiation therapy or radiosurgery can help in interpreting specimens in which there are large areas of radiation change (eg, coagulative necrosis, gliosis, vascular hyalinization).^7(pp125,126) Central nervous system tumors noted to arise in a field of prior irradiation include meningiomas, meningeal sarcomas, astrocytomas, primitive neuroectodermal tumors, and gliosarcomas.^{7(pp586–587)} Radiation therapy of diffusely infiltrating astrocytomas has been shown to increase survival.3,8 

Family History of Cancer or Primary CNS Tumors.

B: Relevant Findings.—Imaging Features (density; enhancement pattern; well-circumscribed or infiltrative borders; cyst formation; calcification; location [intraventricular]; white matter, gray matter, or both).—Recognition of characteristic imaging patterns and locations of CNS tumors is important in correct interpretation of biopsy specimens, for example, low-grade infiltrating astrocytomas usually do not enhance, whereas high-grade ones do.5 Tumor enhancement and peritumoral edema in infiltrating astrocytomas are associated with a worse prognosis, and diffuse tumors have been shown to have a poorer prognosis than focal ones.9,10 

C: Anatomic Site of Specimen.

Cytologic Material

Cerebrospinal fluid (ventricular, lumbar, cisternal)

Cyst fluid

Fine-needle aspiration

Percutaneous (specify site)

Stereotactic computed tomography–guided

Other

Biopsy or Resection

Dura (convexity, falx, tentorium, sphenoid wing, skull base)

Leptomeninges

Cerebrum (specify lobe: frontal, parietal, temporal, occipital)

Basal ganglia

Thalamus

Hypothalamus

Pituitary

Suprasellar area

Pineal

Cerebellum (specify lobe: right or left hemisphere, midline or lateral)

Cerebellopontine angle

Ventricle (third, lateral, fourth)

Brain stem (midbrain, pons, or medulla)

Spine (extradural, intradural/extramedullary, intradural/intramedullary, conus medullaris, filum terminale)

Nerve root(s)/canal (extradural, intradural, anterior root or posterior root)

D: Specimen Unfixed/Fixed.—Cytologic Material.—Cytologic preservation in cerebrospinal fluid depends on the time interval before processing, especially for hematopoietic and some neuroepithelial cells. Refrigerate if delayed more than 30 to 45 minutes. Record the time interval to aid in interpretation.

Biopsy or Resection.—Cellular detail is very important for interpreting CNS neoplasms, and previously frozen tissue is suboptimal, especially for grading and subclassifying gliomas. Recommendations for optimally freezing and cutting frozen sections from tissue from the brain and spinal cord have been made in a previously published article.5 Make every attempt to retain tissue that has not been previously frozen for permanent sections. Avoid using sponges in cassettes because they produce angular defects, which resemble vascular/luminal spaces in the final sections. Wrapping small biopsies in lens paper prior to placing them in cassettes is recommended.5 

E: Cytologic Material Submitted for Microscopic Evaluation.—Cytospin slides or liquid-based monolayer cytology, both air-dried Romanowsky-stained and fixed Papanicolaou-stained slides, as well as unstained slides, should be prepared from fluid specimens, especially cerebrospinal fluid, meningeal fluid, and tumoral cyst fluid.

F: Special Studies.—It may be necessary to divide biopsy/resection tissue into portions for the following procedures:

1. Squash, touch, or scrape preparations

2. Unfrozen permanent paraffin sections

3. Frozen sections, if requested

4. Electron microscopy (retain a small portion in 3% glutaraldehyde or “embed and hold” for electron microscopy if necessary)

5. Other (microbiology, flow cytometry, cytogenetics, molecular diagnostics)

6. Frozen tissue, if requested (freeze fresh tissue as soon as possible and store at −70°C), especially for possible future molecular diagnostic studies

When the tissue is a biopsy and the tissue sample is small, the order of priority for processing tissue for the procedures outlined above is as listed. Recommendations for optimally freezing and cutting frozen sections from tissue from the brain and spinal cord have been made in a previously published article.5 If biopsy frozen and permanent sections are nondiagnostic, tissue that was retained in 3% glutaraldehyde could be submitted for electron microscopy or for additional paraffin sections, depending on the amount of tissue available, with the hope of making a diagnosis. Some pathologists may choose to examine semithin or 1-μm-thick stained sections with toluidine blue instead.

Squash preparations are prepared by placing a tiny (1–2 mm) fragment of tissue onto a glass slide, placing another glass slide over it, pressing the slides together, squashing the tissue between them, then sliding the 2 slides past each other, dragging squashed tissue across each slide. Slides are then rapidly placed into fixative in the same rack used for frozen sections and stained as for frozen sections.5 

Squash preparations are recommended for most CNS lesions. Touch preparations are recommended for pituitary adenomas, oligodendrogliomas, and lymphomas. Scrape preparations, in which tissue is scraped with a scalpel blade and scrapings are applied to glass slides and stained similar to squash and touch preparations, are recommended for desmoplastic tumors, such as dural metastases that cannot be squashed or do not shed well on touch preparations.

If infectious etiologies are suspected, a portion of fresh tissue can be sent to the microbiology laboratory in a sterile container to be processed for bacterial, fungal, or viral cultures. Tissue from patients with symptoms suggestive of spongiform encephalopathy (Creutzfeldt-Jakob disease) requires special handling. The infectious agent of Creutzfeldt-Jakob disease may be inactivated by immersing formalin-fixed tissue in 50 to 100 mL of pure formic acid for 1 hour, followed by reimmersion in fresh formalin.11 While the clinical diagnosis of spongiform encephalopathy encompasses a spectrum of neurologic dysfunction, rapidly progressive dementia and myoclonus are especially suggestive of this diagnosis.12 

If a lymphoproliferative disorder is suspected, a portion of fresh tissue can be sent to the surgical pathology laboratory, where it will be placed in appropriate holding media (RPMI) for flow cytometry. Refer to a previously published protocol for processing specimens from patients with non-Hodgkin lymphoma.13 

Molecular diagnostic testing is playing an increasingly important role in the diagnosis, staging, and treatment of tumors.14 Tissue that has been frozen shortly after arrival in the laboratory and stored at −70°C will be suitable for these studies. Paraffin-embedded tissue can also occasionally be used.

G: Cytopathology: Histologic Type.—Tumor cells, especially those of glial lineage, are often altered by time in fluid/cerebrospinal fluid and are difficult to interpret unless cell clusters of tissue fragments are available. Choroid plexus and ependymal cells are quite similar, with the latter showing more “degenerative” cytologic features and fewer cellular clusters. Therefore, the designation choroid-ependymal cells is appropriate. Ependymomas and choroid plexus papillomas generally appear cytologically benign or bland. It is helpful to prepare squash preparations routinely during intraoperative consultations to develop or keep a sharp cytologic eye for CNS neoplasms.

H: Comments.—Correlation of clinical and radiographic information should be critically reviewed before final sign-out of the biopsy diagnosis.15 

I: Histologic Type.—The World Health Organization (WHO) classification of tumors of the central nervous system is shown below.16 

WHO Histologic Typing of Tumors of the Nervous System

• TUMORS OF NEUROEPITHELIAL TISSUE

• Astrocytic Tumors

• Diffuse astrocytoma

  • • Fibrillary astrocytoma

  •   Protoplasmic astrocytoma

  •   Gemistocytic astrocytoma

• Anaplastic astrocytoma

• Glioblastoma

  • • Giant cell glioblastoma

  •   Gliosarcoma

• Pilocytic astrocytoma

• Pleomorphic astrocytoma

• Pleomorphic xanthoastrocytoma

• Subependymal giant cell astrocytoma

• Oligodendroglial Tumors

• Oligodendroglioma

•    Anaplastic oligodendroglioma

• Mixed gliomas

•    Oligoastrocytoma

•    Anaplastic oligoastrocytoma

• Ependymal Tumors

• Ependymoma

  • • Cellular

  •   Papillary

  •   Clear cell

  •   Tanycytic

• Anaplastic ependymoma

• Myxopapillary ependymoma

• Subependymoma

• Choroid Plexus Tumors

• Choroid plexus papilloma

• Choroid plexus carcinoma

• Glial Tumors of Uncertain Origin

• Astroblastoma

• Gliomatosis cerebri

• Chordoid glioma of the third ventricle

• Neuronal and Mixed Neuronal-Glial Tumors

• Gangliocytoma

• Dysplastic gangliocytoma of cerebellum (Lhermitte-Duclos)

• Desmoplastic infantile astrocytoma/ganglioglioma

• Dysembryoplastic neuroepithelial tumor

• Ganglioglioma

• Anaplastic ganglioglioma

• Central neurocytoma

• Cerebellar liponeurocytoma

• Paraganglioma of the filum terminale

• Neuroblastic Tumors

• Olfactory neuroblastoma (esthesioneuroblastoma)

• Olfactory neuroepithelioma

• Neuroblastomas of the adrenal gland and sympathetic nervous system

• Pineal Parenchymal Tumors

• Pineocytoma

• Pineoblastoma

• Pineal parenchymal tumor of intermediate differentiation

• Embryonal Tumors

• Medulloepithelioma

• Ependymoblastoma

• Medulloblastoma

  • Desmoplastic medulloblastoma

  • Large cell medulloblastoma

  • Medullomyoblastoma

  • Melanotic medulloblastoma

• Supratentorial primitive neuroectodermal tumor

  • Neuroblastoma

  • Ganglioneuroblastoma

• Atypical teratoid/rhabdoid tumor

• TUMORS OF PERIPHERAL NERVES

• Schwannoma (neurilemmoma, neurinoma)

  • • Cellular

  •   Plexiform

  •   Melanotic

• Neurofibroma

  • • Plexiform

• Perineurioma

  • Intraneural perineurioma

  • Soft tissue perineurioma

• Malignant Peripheral Nerve Sheath Tumor

  • • Epithelioid

  • Malignant peripheral nerve sheath tumor with divergent mesenchymal and/or epithelial differentiation

  • Melanotic

  • Melanotic psammomatous

• TUMORS OF THE MENINGES

• Tumors of Meningothelial Cells

• Meningioma

  • • Meningothelial

  •   Fibrous (fibroblastic)

  •   Transitional (mixed)

  •   Psammomatous

  •   Angiomatous

  •   Microcystic

  •   Secretory

  •   Lymphoplasmacyte-rich

  •   Metaplastic

  •   Clear cell

  •   Chordoid

    • Atypical

    • Papillary

    • Rhabdoid

    • Anaplastic meningioma

• Mesenchymal, Nonmeningothelial Tumors

• Lipoma

• Angiolipoma

• Hibernoma

• Liposarcoma (intracranial)

• Solitary fibrous tumor

• Fibrosarcoma

• Malignant fibrous histiocytoma

• Leiomyoma

• Leiomyosarcoma

• Rhabdomyoma

• Rhabdomyosarcoma

• Chondroma

• Chondrosarcoma

• Osteoma

• Osteosarcoma

• Osteochondroma

• Hemangioma

• Epithelioid hemangioendothelioma

• Hemangiopericytoma

• Angiosarcoma

• Kaposi sarcoma

• Primary Melanocytic Lesions

• Diffuse melanocytosis

• Melanocytoma

• Malignant melanoma

• Meningeal melanomatosis

• Tumors of Uncertain Histogenesis

• Hemangioblastoma

• LYMPHOMAS AND HEMOPOIETIC NEOPLASMS

• Malignant lymphomas

• Plasmacytoma

• Granulocytic sarcoma

• GERM CELL TUMORS

• Germinoma

• Embryonal carcinoma

• Yolk sac tumor (endodermal sinus tumor)

• Choriocarcinoma

• Teratoma

  • • Mature

    • Immature

    • Teratoma with malignant transformation

• Mixed germ cell tumors

• TUMORS OF THE SELLAR REGION

• Craniopharyngioma

  • • Adamantinomatous

  •   Papillary

• Granular cell tumor

METASTATIC TUMORS

J: Histologic Grade.—The WHO grading system (malignancy scale) of CNS tumors is shown below.16 There is no formal TNM-based classification and staging system for the CNS at this time.17 

After patient age, tumor histology and grade have been shown to be the strongest predictors of clinical course in selected CNS astrocytomas.2,18 Several grading systems for diffusely infiltrating astrocytomas have been proposed, based on their ability to define distinct patient groups with significantly different survival curves. Both 3-tiered and 4-tiered systems are currently in use and have been reviewed.19 Two examples of popular grading systems are shown below.16 For a complete review and comparison of these systems, including the 3-tiered system such as the Ringertz system and modifications thereof, the reader is referred to the review by McClendon et al.19 

Comparison of the WHO and St. Anne/Mayo Grading Systems for Astrocytomas.16 

K: Other Pathologic Features.—Hemosiderin deposition, calcification, and microcyst formation are nonspecific findings that occur in both malignant and benign CNS neoplasms. However, in general, calcification usually favors a slowly growing neoplasm, which is more likely to be benign. The presence of gemistocytes, vascular proliferation, and necrosis represent negative prognostic indicators, and the latter 2 histologic changes are diagnostic of high-grade astrocytomas.^9,20–22

Findings in Touch, Squash, or Scrape Preparations.—The presence of process-forming cells is suggestive of a primary CNS neoplasm. Extreme fibrillarity may represent reactive astrocytosis.^4(p39) Touch or squash preparations are also of value in evaluating specimens for the presence of macrophages. A macrophage-rich lesion is more consistent with a subacute infarct or demyelinative lesion, rather than a tumor.

Local extension, rapid growth, invasion of adjacent structures, and CNS spread via the ventricular system or subarachnoid space are often suggestive of, but not always diagnostic of, malignancy. Low-grade neoplasms, such as meningioma and pilocytic astrocytoma, may also exhibit this type of spread, but at a slower rate of growth than most malignant tumors. Malignant and atypical meningeal tumors often invade brain substance.²⁴

Members of the College of American Pathologists Neuropathology Committee contributed to the development of this protocol.