Brain, Spinal Cord, and Meninges
Paul E. McKeever
TERMS AND ABBREVIATIONS USED IN THIS CHAPTER
α-ACT: α-antichymotrypsin macrophage marker; AFP: α-fetoprotein; CADASIL: cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; CD45RB: LCA: leukocyte common antigen; CD3: T-cell-specific marker; CD20: L26, B-cell-specific marker; CD68: KP-1, macrophage marker; CK: cytokeratin; EBV: Epstein-Barr virus; ETANTR: embryonal tumor with abundant neuropil and true rosettes; EMA: epithelial membrane antigen; epithelioid: cells with distinct cytoplasm and nonfibrillar cellular margins that resemble epithelial cells; ECM: extracellular matrix; fibrillar: cells that extend cytoplasmic processes bound by plasma membranes (unipolar, bipolar, or multipolar); FISH: fluorescent in situ hybridization; GFAP: glial fibrillary acidic protein constituent of glial fibrils (intermediate filament); gliosis: brain reaction to injury remotely similar to systemic fibrosis; HV: herpesvirus; HSV: herpes simplex virus; HCG: human chorionic gonadotropin; Ig: immunoglobulin; IDH: isocitrate dehydrogenase; IHC: immunohistochemistry; κ and λ: kappa and lambda light chain subclasses of immunoglobulin molecules; Ki-67: epitope for Kiel-67 antibody clone, detected in paraffin by MIB-1 and other antibodies, and preferred nuclear marker of proliferation; NF: neurofilament; PCR: polymerase chain reaction; PLAP: placental alkaline phosphatase; PNET: primitive neuroectodermal tumor; PML: progressive multifocal leukoencephalopathy; S-100: highly acidic small protein soluble in 100% ammonium sulfate; syncytial: cells that have no cytoplasmic borders by light microscopy; TTF-1: thyroid transcription factor 1, a protein present in thyroid and large cell lung neoplasms.
This chapter focuses on the diagnosis of neurosurgical biopsy and resection specimens from the central nervous system (CNS) and within the cranial-spinal vault. First, it presents ways to identify and manage various surgical specimens and tools that help with this process. Then, it provides a reference guide first to reactive changes and then to major etiologic categories of disease processes: infectious, inflammatory, demyelinating, cerebrovascular, neoplastic, degenerative, and developmental diseases. Toxic, metabolic, and traumatic diseases are noted where appropriate. The focus is on diseases seen by the surgical pathologist. Extensive recent comprehensive reviews of different fields of neuropathology are also available (1,2,3,4,5,6,7 and 8). The evaluation of lesions of the pituitary gland and sellar region are reviewed in Chapter 12.
The approach to diagnosis employed in this chapter relies principally on histologic evaluation of hematoxylin and eosin (H&E)-stained sections, with incorporation of smear preparations, histochemical stains, electron microscopy, and immunohistochemical preparations as supplemental aids in diagnosis.
The first table lists reasons for intraoperative biopsies. Tables 10.210.3,10.4,10.5,10.6,10.7,10.8,10.9,10.10,10.11,10.12,10.13,10.14,10.15,10.16,10.17,10.18,10.19,10.20 and 10.21 provide information about pathologic entities to assist in reading the text and diagnosing cases. Suspected specific disease can be checked directly on the individual table where its key structural, chemical, and topographic features are listed. The text and figures elaborate upon the information in the tables. Features of unknown cases may be found in the tables, which should be helpful to the diagnostician. For example, if the lesion is a mass, specific tables summarize differential features depending on whether the mass is composed of fibrillar cells (Table 10.10; see Fig. 10.39), epithelioid cells (Table 10.11; see Fig. 10.125), more than one type of cell (Table 10.12; see Fig. 10.110), small anaplastic cells (Table 10.13; see Fig. 10.81), or syncytial cells (Table 10.14; see Fig. 10.88).
ALGORITHMIC APPROACH TO DIAGNOSIS EMPLOYING TABLES AND TEXT
An example of a case interpreted from start to finish employing an algorithmic approach and the tables and text is presented in Figure 10.1. As in any other organ system, an algorithmic approach to specimens, at the time of frozen section and subsequently, is valuable. It prevents overlooking key etiologic considerations including secondary pathologic processes.
Neoplasm or not neoplasm: This hypercellular lesion is composed of pleomorphic cells with atypical nuclei, which
indicate neoplasm. If the lesion were not neoplastic, other etiologic categories, including infectious, inflammatory, toxic-metabolic, traumatic, vascular, developmental, and degenerative, would be considered.
TABLE 10.1 Indications for Intraoperative Consultation at Biopsy
Lesion requires proper surgical sampling of tissue for diagnosis and grading
Lesion may require special tissue processing
Culture for microorganisms
Fixation for electron microscopy
Fixation for immunohistochemistry
Touch preparation
Other special processing
Diagnosis affects immediate surgical procedure
TABLE 10.2 Surgery Directed Toward a Neurologic Symptom or Specific Disease
CONFIRMATORY FEATURES OF SUSPECTED DISEASE
Symptom/Suspected Disease
Structures
Reactant
Locationsa
Herpes simplex encephalitis
Encephalitis (Table 10.3), Cowdry A amphophilic nuclear inclusions of 90- to 100-nm “target” capsids
HSV antigen
Temporal or basilar frontal lobe, CNS; frequently bilateral
Toxoplasmosis
Necrosis containing 3- to 5-nm tachyzoites; (cysts); (inflammation)b
Toxoplasma antigen
CNS, frequent multiple lesions
Progressive multifocal leukoencephalopathy
Demyelination, bizarre, glial, amphophilic nuclear inclusions of 15- to 25-nm or 30- to 40-nm diameter
JC/SV40 antigen, myelin, neurofilament
Cerebral white matter, CNS
Dementia/Creutzfeldt-Jakob disease
Cytoplasmic vacuoles indenting nuclei, gliosis (Table 10.3)
GFAP, prion (DANGER)
Bilateral cerebral cortex, gray matter
Small vessel disease
Vasculitis or arterial sclerosis or congophilic angiopathy
Amyloid, iron
Cerebrum, CNS; frequent multiple lesions
Dementia/Alzheimer disease
Argyrophilic plaques; neurofibrillary tangles of bihelical filaments
Neurofilament, tau
Bilateral cerebral cortex
Demyelination
Loss of myelin, gitter cells, with or without axonal preservation
Myelin, neurofilament
Cerebral white matter, CNS
Epilepsy
Low-grade glioma or ganglioglioma (Table 10.10), or gliosis (Tables 10.3 and 10.18), or vascular malformation
GFAP, neurofilament, synaptophysin, iron
Temporal lobe, cerebral cortex
a Most common or most specific location is listed first.
b Parentheses around a differential feature indicate an uncommon feature very useful in differential diagnosis when found.
CNS, central nervous system; GFAP, glial fibrillary acidic protein; HSV, herpes simplex virus; JC, JC virus; SV, simian virus.
TABLE 10.3 Differential Features of Cells Infiltrating Central Nervous Parenchyma
DIFFERENTIAL FEATURES
Diagnosis
Structures
Reactant
Locationsa
Gliosisb
Cells are fibrillar, uncrowded; round or oval nuclei
GFAP in glial filament
CNS
Macrophages
Cells and nuclei are round to elongated; cell content reflects injury
CD68; α-ACT
CNS, meninges
Encephalitis and/or cerebritis
Perivascular mixture of inflammatory cells
CD3, CD20, LCA, κ and λ Ig, α-ACT; CD68, microorganism
CNS gray matter, CNS
Hemorrhage
Red blood cells or macrophages with hemosiderin
Fibrin, iron
Deep cerebrum, cerebellum, CNS
Margin of gliomasc
Cells are fibrillar; angular nuclei indent each other; (mitoses)c,d
GFAP
CNS
Lymphoma
Perivascular, noncohesive small, round cells
CD3, CD20, LCA, κ and λ Ig
Deep cerebrum, CNS; meninges
a Most common or most specific location is listed first.
b Nonspecific reaction to injury.
c Suspicion of margin of glioma on frozen section should be followed by a request for another more central biopsy. Mitoses suggest margin of a high-grade glioma.
d Parentheses around a differential feature indicate an uncommon feature that is very useful in differential diagnosis when it is found.
α-ACT, α-antichymotrypsin macrophage marker; CNS, central nervous system; GFAP, glial fibrillary acidic protein; Ig, immunoglobulin; LCA, leukocyte common antigen (CD45/CD45RB).
TABLE 10.4 Viral Inclusions in the Central Nervous System
Nucleus
Cytoplasm
Neurons
Herpes simplex and zoster
+
−
Rabies
−
+
SSPE (measles)
+
−
Oligodendrocytes
PML (JC virus)
+
−
SSPE (measles)
+
−
Various cells (endothelial, ependymal, glial)
Cytomegalovirus
+
+
PML, progressive multifocal leukoencephalopathy; SSPE, subacute sclerosing panencephalitis.
TABLE 10.5 Causes to Consider in Intracranial Hematoma
Traumatic
Cerebral contusion
Epidural or subdural hematoma
Infectious and inflammatory
Vasculitis
Fungus (mycotic aneurysm)
Developmental vascular
Vascular malformation (arteriovenous malformation, cavernous angioma)
Aneurysm
Hematologic
Thrombocytopenia
Sickle cell anemia
Neoplastic
Primary (high-grade glioma)
Metastatic (e.g., melanoma, renal cell carcinoma)
Leukemia (chloroma in acute myelogenous leukemia)
Vascular
Amyloid angiopathy
Hypertension
Conversion of ischemic infarct
Toxic-metabolic: anticoagulation related
TABLE 10.6 Vascular Malformations
Malformationa
Vessels
Neuropil
Gliosis and Hemosiderin-Laden Macrophages
Arteriovenous malformation
Arteries, veins, arterialized veins
Absent, except around feeding vessels
Presentb
Cavernous angioma
Compact cluster thin-walled to thickwalled vessels, mineralization
Absent usually
Presentb
Capillary telangiectasia
Capillaries, thin-walled
Admixed
Absent
Venous malformation
Veins, often dilated
Admixed
Absent
Varix
Vein, dilated (vein of Galen)
Absent
Absent unless ruptured
a Location: Each can be found anywhere in the central nervous system; capillary telangiectasias often in brainstem and venous malformations in the spinal leptomeninges.
b Gliosis and hemosiderin-laden macrophages are consistent with leakage; they serve as a seizure focus, and they signal a tendency for spontaneous hemorrhage.
TABLE 10.7 Relative Frequency of Most Common Pediatric and Adult Brain Neoplasms
CHILDREN
ADULTS
Location
Diagnosis and Frequency
Location
Diagnosis and Frequency
Anterior fossa
Miscellaneous
33%
Anterior fossa
Gliomas (cerebrum)a
33%
Meningiomas (dura)
13%
Metastases (cerebrum)
12%
Pituitary adenomas (sella)
5%
Miscellaneous
4%
Total
67%
Posterior fossa
Astrocytomas (cerebellum)a
26%
Medulloblastomas (cerebellum)
24%
Ependymomas (fourth ventricle)
14%
Posterior fossa
Schwannomas (eighth cranial nerve)
8%
Miscellaneous
3%
Miscellaneous
25%
Total
67%
Total
33%
a Most common site in parentheses. See entry in text for other locations.
TABLE 10.8 World Health Organization Criteria for Grading of Astrocytomas
Grade
Nomenclature
Histologic Features
1
Pilocytic
Circumscribed; biphasic: bipolar piloid cells and multipolar cells; microcysts, Rosenthal fibers, and granular bodies; may or may not have rare mitotic figures, vascular proliferation, or focal necrosis
2
Diffuse
Moderate hypercellularity of monotonous cells; mild nuclear atypia; no or minimal mitotic activity
3
Anaplastic
Increased cellularity and diffuse infiltration; increased nuclear atypia; increased mitotic activity
4
Glioblastoma
Vascular proliferation or necrosis; crowded anaplastic cells; marked nuclear atypia; brisk mitotic activity
From Louis DN, Ohgaki H, Wiestler OD, Cavenee WK. World Health Organization Classification of Tumours: Pathology and Genetics of Tumors of the Nervous System. Geneva: IARC Press, 2007, with permission.
Primary or metastatic: These cells are fibrillar. They are plump and large but not spindled or vacuolated. The neoplastic cell nuclei are moderately pleomorphic; the cells have small processes emanating from them. The lesion lacks rosettes. There is a vessel cuffed by many small, reactive-appearing mononuclear cells. No epithelial elements are recognized. The findings are those of a primary CNS neoplasm rather than a metastatic lesion (Tables 10.10 and 10.11).
Glial, neuronal, and other primary considerations: Possible primary CNS neoplasm diagnoses in Table 10.10 include giant cell astrocytoma, gemistocytic astrocytoma, ganglion cell tumor, or histiocytosis (Fig. 10.1). Although nuclei of the lesional cells have a slight resemblance to neuronal nuclei, their chromatin is more condensed. Their cytoplasm is pink without purple Nissl substance of neurons. Bielschowsky stain shows no silver staining of neoplastic cells (Fig. 10.1); neoplastic cells were also negative for synaptophysin, a neuronal marker (not shown). Aggregate features do not support a ganglion cell tumor. The neoplastic cells are positive for the intermediate filament glial fibrillary acidic protein (GFAP), a marker that accentuates their fibrillarity and confirms their astrocytic nature (Fig. 10.1). The related text in this chapter describes differences between gemistocytic astrocytoma and giant cell astrocytoma. Gemistocytic astrocytomas resemble reactive astrocytes and have abundant, plump, hyaline cytoplasm and peripheralized nuclei. Macrophages (histiocytes) are GFAP-negative and lack the fibrillary structure highlighted by GFAP, arguing against the diagnosis histiocytosis. Neoplastic cells are negative for the T-lymphocyte marker CD45RO (Fig. 10.1D) and the B-lymphocyte marker CD20 (Fig. 10.1E), whereas polyclonal perivascular lymphocytes are positive. Therefore, the aggregate histologic, histochemical, and immunohistochemistry (IHC) findings support the diagnosis of gemistocytic astrocytoma.
TABLE 10.9 World Health Organization Criteria for Grading of Oligodendrogliomas
Grade
Nomenclature
Histologic Features
2
Oligodendroglioma
Moderate cellularity; homogeneously round nuclei, “fried egg” halo (paraffin); fine capillary network; mineralization (microcalcifications)
3
Anaplastic oligodendroglioma
Increased cellularity; high mitotic rate; marked cytologic atypia; microvascular proliferation; necrosis
From Louis DN, Ohgaki H, Wiestler OD, Cavenee WK. World Health Organization Classification of Tumours: Pathology and Genetics of Tumors of the Nervous System. Geneva: IARC Press, 2007, with permission.
Grade of astrocytoma: Bielschowsky stain reveals passing axons of brain tissue infiltrated by this neoplasm (Fig. 10.1). Infiltration of brain tissue is seen in astrocytomas of grade II to IV. Grade III astrocytomas have mitotic activity, but there is no evidence of mitotic activity. Features of grade IV, microvascular proliferation and necrosis, are absent. This astrocytoma is grade II. Most gemistocytic astrocytomas are grade II, although they tend to progress to a higher grade. Grading is further discussed in the “Gemistocytic Astrocytoma,” “Diffuse Astrocytoma,” and “Anaplastic Astrocytoma” sections. Other algorithmic approaches to the brain biopsy are available (9,10).
CLINICAL AND RADIOGRAPHIC PERSPECTIVE OF LESIONS
Biopsies should be examined with knowledge of the clinical history. If a specific diagnosis or differential is not suspected preoperatively, at the least, a major neurologic symptom (e.g., weakness or visual loss) or category of neurologic disease (e.g., refractory epilepsy) is usually available to focus the search for a diagnosis (Table 10.2). The pathologist should always know, at a minimum, the age and sex of the patient, the precise location of the targeted lesion, and imaging characteristics.
Knowledge about past medical history (e.g., previous CNS or primary neoplasms, connective tissue disease, immunosuppressive disease) is critical to interpretation. Likewise, knowledge about preoperative therapy (e.g., corticosteroids, chemotherapy, radiation therapy, radiosurgery) is also critical to interpretation of findings (e.g., necrosis, vascular fibrosis).
Knowledge about past medical history (e.g., previous CNS or primary neoplasms, connective tissue disease, immunosuppressive disease) is critical to interpretation. Likewise, knowledge about preoperative therapy (e.g., corticosteroids, chemotherapy, radiation therapy, radiosurgery) is also critical to interpretation of findings (e.g., necrosis, vascular fibrosis).
TABLE 10.10 Differential Diagnosis of a Mass of Fibrillar Cells | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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TABLE 10.11 Differential Diagnosis of a Mass of Epithelioid Cells | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Certain pathologic entities predominate in pediatric, adult, or geriatric age categories as described in the text. A communication system between the operating rooms and the pathology diagnostic rooms is especially important to share relevant clinical and pathologic observations on individual cases examined by frozen section (Table 10.1).
Radiology provides extremely valuable gross pathologic information—most importantly, computed axial tomography (CT) scan and magnetic resonance imaging (MRI); use of contrast and special imaging techniques (e.g., diffusion- and perfusion-weighted imaging) adds additional data in characterizing normal and abnormal structures. It is now possible to map sensitive regions such as corticospinal tracts and perform MRI during the surgical interval to aid localization and removal of a lesion. Emerging imaging techniques including magnetic resonance (MR) spectroscopy (evaluation of chemical components in a lesion) offer additional information about specific lesions including the likelihood that a lesion is neoplastic (6,11).
Major categories of lesions of the brain, spinal cord, and meninges, such as solitary or multiple masses, cysts, vascular malformations, or abscesses, are likely to be recognized by imaging or upon
viewing a gross specimen. The neurosurgical lesions summarized in Tables 10.610.7,10.8,10.9,10.10,10.11,10.12,10.13,10.14,10.15,10.16,10.17,10.18 and 10.19 are usually focal, whereas the lesions in the biopsies directed toward a neurologic disease tend to be more diffuse (Table 10.2). One particularly problematic diagnosis is vasculitis, which can be focal, multifocal, or diffuse. If you are lucky enough to be consulted on any multifocal case, advise the surgeon to target a new or subacute radiographic lesion.
viewing a gross specimen. The neurosurgical lesions summarized in Tables 10.610.7,10.8,10.9,10.10,10.11,10.12,10.13,10.14,10.15,10.16,10.17,10.18 and 10.19 are usually focal, whereas the lesions in the biopsies directed toward a neurologic disease tend to be more diffuse (Table 10.2). One particularly problematic diagnosis is vasculitis, which can be focal, multifocal, or diffuse. If you are lucky enough to be consulted on any multifocal case, advise the surgeon to target a new or subacute radiographic lesion.
TABLE 10.12 Differential Diagnosis of a Mass of Conspicuously Different Cells | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Multiple lesions can be produced by neoplasms or by inflammatory, vascular, and infectious diseases. If inflammation or infection is suspected prior to or seen at biopsy, cultures for appropriate microorganisms should be sent in sterile containers directly from the operating room to microbiology laboratory. The “M rule” for common multiple CNS neoplasms includes metastases, malignant lymphoma, melanoma, and (late stages only) medulloblastoma.
In some settings (e.g., in a patient with neurologic deficits), a biopsy is performed in a desperate attempt to find a diagnosis. In such a setting, to optimize the probability of a pathologic diagnosis, the biopsy should be directed at a region of recent radiologic abnormality such as contrast enhancement and optimally should include (a) dura, (b) arachnoid, (c) gray matter, and (d) white matter. In the setting of radiologic lesions, biopsy of a normal radiologic region or a so-called unguided or undirected (“blind”) biopsy is, in our experience, a useless waste
of everyone’s time. It is a ritual that helps the clinician say that everything was tried on a moribund patient.
of everyone’s time. It is a ritual that helps the clinician say that everything was tried on a moribund patient.
TABLE 10.13 Differential Diagnosis of a Mass of Small Blue Cell Tumor | ||||||||||||||||||||||||||||||||
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TABLE 10.14 Differential Diagnosis of a Mass That Includes Syncytial Cells | ||||||||||||||||||||||||||||
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TABLE 10.15 World Health Organization Criteria for Grading of Meningiomasa | ||||||||||||||||||||||||||||
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TABLE 10.16 Histiocytoses Affecting the Central Nervous System Compared with Macrophages | ||||||||||||||||||||||||||||||||||||
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TABLE 10.17 Carcinomas and Melanoma Metastatic to Brain | ||||||||||||||||||||||||
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TABLE 10.18 Differential Diagnosis of Cyst with Wall of Fibrillar Cells | ||||||||||||||||||||||||||||||||||||||||
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TABLE 10.19 Differential Diagnosis of a Cyst with Wall Lined by Epithelium | ||||||||||||||||||||||||||||||||||||
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The tomographic density of hemorrhage is sufficiently unique at certain stages of organization to identify preoperatively hemorrhage as a major component of a lesion. In general, calcifications and relationships with the skull are resolved well by CT. Gray and white matter, edema, and melanin are resolved well by MRI. Vascular abnormalities are frequently appreciated clinically and angiographically. CT or MRI angiography of flow voids is sometimes sufficient and is less invasive than classic angiography.
INTRAOPERATIVE CONSULTATION
Certain entities that may be either suspected clinically or suggested on cytologic preparation and frozen section can affect the immediate surgical procedure (see “Primary Open Biopsy” section and Table 10.1). Most significantly, attempts at total resection are often made for these neoplasms: meningiomas, schwannomas, solitary metastases, cysts, ependymomas, hemangioblastomas, cerebellar pilocytic astrocytomas, and craniopharyngiomas. Therefore, assessment of such specimens requires careful clinicopathologic correlation at the time of the primary operation, and intraoperative consultation guides the procedure.
Any undefined lesion should be biopsied and inspected by both cytologic preparation and frozen sections. Cytologic preparations add fine nuclear detail and the presence or absence of (a) glial-type processes; (b) discohesiveness in pituitary adenomas, oligodendrogliomas, medulloblastomas, and lymphomas; and (c) “epithelioid” features, with cellular cohesion (suggesting junctions) in carcinomas. The value of such preparations has been demonstrated (12,13) and, in some centers, intraoperative diagnosis is based only on evaluation of cytologic preparations. Although smear and crush preparations may be done, touch preparations minimize effort and artifact. Touch a glass slide to the wet tissue and then immediately fix it in ethanol before it dries. Stain with H&E or with a cytologic stain.
Difficult access to, and fragility of, central nervous tissue places a high premium on obtaining tissue suitable for diagnosis during the first surgical procedure and avoiding secondary biopsy after a primary nondiagnostic biopsy (4). The goal of the pathologist in the intraoperative setting is not to diagnose
definitively and grade every case but, rather, (a) to ensure that lesional tissue has been obtained for subsequent diagnosis and grading, (b) to ensure the lesional tissue has been appropriately sampled (e.g., that high-grade features are noted on a suspected glioblastoma based on imaging features), (c) to provide sufficient preliminary diagnostic information to optimize surgery, and (d) to perform appropriate special tissue processing (Table 10.1). Optimizing surgery depends on the individual case and on whether open biopsy or stereotactic needle biopsy is being done (see later sections).
definitively and grade every case but, rather, (a) to ensure that lesional tissue has been obtained for subsequent diagnosis and grading, (b) to ensure the lesional tissue has been appropriately sampled (e.g., that high-grade features are noted on a suspected glioblastoma based on imaging features), (c) to provide sufficient preliminary diagnostic information to optimize surgery, and (d) to perform appropriate special tissue processing (Table 10.1). Optimizing surgery depends on the individual case and on whether open biopsy or stereotactic needle biopsy is being done (see later sections).
TABLE 10.20 Inclusion Immunophenotype | ||||||||||||||||||||||||||||||||||||||||
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TABLE 10.21 Findings in Surgically Resected Temporal Lobes in Setting of Seizure Disorder | ||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Given the constraints of intraoperative evaluation of tissue, including sampling limitations and freezing artifact, precise diagnosis is often neither possible nor necessary for a successful intraoperative consultation. The surgical pathologist need only go as far as necessary with his or her diagnosis (e.g., highgrade glioma, metastatic neoplasm, atypical lymphoid infiltrate suspicious for lymphoma, abundant neutrophils suggestive of abscess, granulomatous inflammation) to guide the surgery without attempting to make complex diagnoses that optimally employ paraffin sections. The pathologist’s microscopic impression, even if rendered as a differential diagnosis, guides the surgical procedure.
In the setting of a glial neoplasm, it is not wise to offer a grade during intraoperative frozen section or touch preparation interpretation. Because gliomas are quite heterogeneous, diagnostic features that affect the classification and grade may be revealed only in the “permanent” specimens and not on frozen section. For instance, oligodendroglial elements in a mixed glioma may not be well represented or recognized on the frozen material; the characteristic halo is a paraffin-embedding artifact. A diagnosis of “primary glial neoplasm with abundant mitotic activity, at least World Health Organization grade III,” or “highgrade glioma,” effectively characterizes a biopsy when malignant glial cells are unequivocally identified on cytologic and frozen section preparations and allows for the later identification of oligodendroglial and other elements not evident on the fresh sample.
The intraoperative consultation provides the opportunity to obtain tissue for microbiologic culture, special fixation (e.g., lymphoma), and special processing (e.g., flow cytometry, cytogenetics, molecular evaluation, and electron microscopy) when preliminary assessment deems it necessary or pragmatic (see “Tissue Processing” later in this chapter).
SUMMARY POINTS ABOUT INTRAOPERATIVE CONSULTATION
I. Be prepared:
History: Patient age, duration of symptoms, and surgical location are the minimum.
Radiography: View images and/or read reports. On reports, objective findings trump interpretation. Are there one or more masses? Infiltrative? Enhancing?
Check for prior specimens: Slides and reports.
II. New specimen: Both cytologic and frozen sections preferred.
III. Observations:
Cell type: Fibrillar, epithelioid, mixed, small and crowded, or syncytial (e.g., fibrillar*)
Major features: Such as mass; marked cellular and nuclear pleomorphism*
Differential (Dif): Such as glioblastoma (GbM), giant cell astrocytoma (GCA), pleomorphic xanthoastrocytoma (PXA), sarcoma (Src), or melanoma (Mel)*
IV. Narrow the differential: Check excluding and corroborating features (e.g., for earlier Dif*).
Location: Entirely extramedullary (e.g., outside of CNS [excludes GbM, GCA]), skull invasion (favors Src, Mel), subependymal (favors GCA)
Radiography: Diffuse margin with CNS (favors GbM)
Duration of symptoms: For a new mass, less than 3 months favors GbM, Src, Mel
Microscopic features:
No mitoses (favors GCA, PXA)
Granular bodies (favors PXA)
Necrosis (favors GbM, Src, Mel)
Microvascular proliferation (favors GbM)
Intercellular collagen (favors PXA, Src)
*The differential (set in parentheses) of a mass with fibrillar cells of marked cellular and nuclear pleomorphism is a single algorithmic example of how to use the summary points.
CATEGORIES OF SURGICAL SPECIMENS
Optimal management of a specimen requires knowledge of the aim of the procedure. Three major types of neurosurgical procedures routinely yield tissue: (a) primary biopsies, (b) secondary biopsies, and (c) therapeutic resections. A primary biopsy seeks a diagnosis. Secondary biopsies may try again to establish
a diagnosis or monitor consequences of therapy. Biopsies can be performed using CT- or MRI-guided stereotactic needle biopsy or using an open technique. Therapeutic resections attempt gross total excision of lesional tissue. Individual cases may combine more than one procedure at a single operation. For example, open biopsy for diagnosis of an intramedullary spinal cord tumor may proceed directly to a therapeutic resection if the intraoperative evaluation suggests ependymoma.
a diagnosis or monitor consequences of therapy. Biopsies can be performed using CT- or MRI-guided stereotactic needle biopsy or using an open technique. Therapeutic resections attempt gross total excision of lesional tissue. Individual cases may combine more than one procedure at a single operation. For example, open biopsy for diagnosis of an intramedullary spinal cord tumor may proceed directly to a therapeutic resection if the intraoperative evaluation suggests ependymoma.
 FIGURE 10.1 A solitary mass from the right frontotemporal cerebral cortex of a 38-year-old woman. This case and the first paragraphs of the text illustrate how the tables and text assist in the interpretation from the initial impression to the final diagnosis. (A) Hematoxylin and eosin stain. (B) Anti-glial fibrillary acidic protein with hematoxylin counterstain. (C) Bielschowsky silver stain for axons and neurofilaments. (D) CD45RO anti-T lymphocyte with hematoxylin counterstain. (E) CD20 anti-B lymphocyte with hematoxylin counterstain. |
PRIMARY OPEN BIOPSY
The primary biopsy for diagnosis is critical. The craniotomy and open biopsy, where the neurosurgeon “turns a flap” by sawing through, and temporarily opening, a portion of the calvarium, affords a macroscopic view of the lesion. In some centers, localization of the lesion is aided by intraoperative imaging, including MRI. The pathologist’s database should include the preoperative radiologic data as well as macroscopic findings in situ, either by direct observation or by conversation with the neurosurgeon. Descriptive observations of the margin, color, consistency, precise point of attachment, and immediate surroundings of a lesion are frequently helpful. Because an open biopsy provides surgical latitude from biopsy to total resection, it is important to recognize resectable tumors whenever possible during surgery. Resectable tumors include meningiomas, carcinomas, adenomas, schwannomas, pilocytic astrocytomas, and many ependymomas.
STEREOTACTIC NEEDLE BIOPSY
Radiographically guided stereotactic needle biopsies of vital and sensitive locations through a small hole in the skull are increasing in frequency (14). Needle biopsy specimens have requirements for tissue handling similar to open biopsy specimens, with the following exceptions.
Radiographs substitute for direct visual inspection of the lesion in situ. This and the small size of the specimen confound even the most careful attempts at proper sampling of the lesion. Movement of a centimeter or less may result in totally necrotic or gliotic nondiagnostic tissue. This so-called black box effect requires that sampling of needle biopsies for diagnosis be monitored with frozen sections. However, the intraoperative interpretation does not affect the immediate surgical procedure with regard to therapy because excision of the lesion is not intended.
Better results come from examining both whole cell cytology and frozen section histology than from either alone, but special care is needed to avoid harming tiny stereotactic biopsies by touching and drying. One approach is to freeze the entire tissue and touch only the gauze pad it came on to a slide for cytology. When the pathologist indicates that a diagnostic region has been sampled, the neurosurgeon should take a second sample for permanent section from the same region, placing it directly into fixative. Those who excessively ponder the macroscopic appearance of this tiny tissue fragment under powerful lights risk a dried, nondiagnostic sample.
The most important task for the pathologist during stereotactic surgery is to advise the surgeon of the likelihood that he or she is sampling the lesion itself, rather than normal, gliotic, or otherwise nondiagnostic tissue (Fig. 10.2). Frequently, initial biopsies yield minimally abnormal tissue and lesional tissue is identified on subsequent biopsies, or an initial biopsy may yield low-grade features when high-grade features are suspected radiologically. The features to look for are addressed in the “Tumor and Tumor Margin” section later in this chapter.
The most frequent lesions to be approached by stereotactic biopsy are suspected gliomas in critically sensitive regions of the brain, including speech and motor cortex, thalamus, basal ganglia, and brainstem. Lesions in less sensitive regions should be approached by open biopsy to provide a better tissue sample than needle biopsy. Lesions firmer than gliomas, particularly common in the pineal region, tend to resist the needle. The relative difficulty of capture of the stereotactic specimen is an important clue to its diagnosis because gliomas and lymphomas tend to be easily obtained, whereas others are difficult. The presence of a large vessel in a needle biopsy specimen is a matter of concern that should be reported to the neurosurgeon.
 FIGURE 10.2 Stereotactic biopsy of pineal tumor. The first biopsy from this patient revealed epithelium on a fibrous base resembling choroid plexus epithelium (A). Another biopsy revealed germinoma (B). Still another specimen from this second biopsy was α-fetoprotein-positive (C), and it contained Schiller-Duval bodies (not shown) in a focus of yolk sac tumor. |
Stereotactic biopsies of cystic lesions yield a lower percentage of positive diagnoses than do those of solid tumors because of the difficulty of obtaining diagnostic tissue from a mural nodule or the wall of a lesion. Stereotactic biopsies of tumors with architectural and cytologic appearances similar to normal tissue (e.g., choroid plexus and choroid plexus papilloma, pineal and pineocytoma) can be reported as the appropriate type
of tissue, with a comment about the need for neuroanatomical and clinical correlation to rule out the possibility of normal structure. Stereotactic biopsies of pineal tumors present problems analogous to the problems the fabled elephant presented to the six blind men (Fig. 10.2).
of tissue, with a comment about the need for neuroanatomical and clinical correlation to rule out the possibility of normal structure. Stereotactic biopsies of pineal tumors present problems analogous to the problems the fabled elephant presented to the six blind men (Fig. 10.2).
SECONDARY BIOPSY
Secondary biopsies are repeat biopsies of the lesion or its immediate vicinity. The need for a secondary biopsy following a nondiagnostic primary biopsy can usually be avoided by adequate sampling of the tissue at the primary biopsy. Most helpful in this regard is monitoring the primary biopsy procedure with frozen sections (Table 10.1). The proper time to render the judgment that a specimen is nondiagnostic is during the biopsy procedure, allowing the surgeon the opportunity to obtain another specimen.
BIOPSY AFTER THERAPY
Accurate diagnosis is enhanced when the patient’s clinical status and the type of neurosurgical procedure are understood. Moreover, ancillary microscopic findings may be helpful. One of many examples is the significance of necrosis in a cerebral astrocytoma. Spontaneous coagulation necrosis in a diffuse cerebral astrocytoma is a feature of a glioblastoma multiforme. On the other hand, if necrosis is found in an astrocytoma biopsied after cytotoxic or radiotherapy, it may simply represent a good therapeutic response (Fig. 10.3A,B). Geographic necrosis is less reliable than necrosis with pseudopalisades found in cellular tumors. “Pseudopalisades” are rings of increased cellular density around the necrosis. Geographic necrosis alone has been seen in white matter infiltrated by glioma as part of the therapeutic response to radiotherapy.
 FIGURE 10.3 Treatment effects. Largely necrotic lesion in a young man a year after radiation therapy to a total dose of 81 Gy along with concurrent temozolomide chemotherapy for his glioblastoma shows (A) radiation effects, including fibrinoid necrosis of vascular walls and extensive tissue coagulation necrosis with beginning cavitation; (B) vessel has cavitation, macrophages, and fibrinoid material (FM) in its wall. Small vessels within the FM resemble revascularization in a thrombus. (C,D). Recently treated glioblastoma. One month after the end of 2 months of radiation and chemotherapy, there are (C) regions with a few remaining glioma cells (short arrow), macrophages with bubbly cytoplasmic lipids, gliosis, and attenuated vascular wall (long arrow); (D) necrosis with tiny flecks of calcium and nuclear debris; and remnants of abnormal vascular walls (arrow). This case was interpreted as treatment effects more prominent than glioma cells. (continues) |
 FIGURE 10.3 (Continued) Chronic radiation necrosis: (E) This man received radiation to his right temporal lobe. Years later, fibrinoid extravasation from a vessel and brain necrosis are evident. (F) Myelin basic protein from damaged white matter accumulated in macrophages. (G) Tumor progression: The original oligodendroglioma of this man was grade II. Years after radiation and chemotherapy, high cell density and numerous mitoses plus MVP lacking fibrinoid change show progression to anaplastic oligodendroglioma, grade III. |
Exceptionally problematic are biopsies after radiotherapy, chemotherapy, or vascular embolization of tumors. Even the previous surgical procedure can radically alter the appearance of a neoplasm (Fig. 10.4). Previous treatment information should be obtained prior to rendering an opinion. Biopsies for diagnosis before the initiation of such therapy should be encouraged.
Glioma Regrowth or Progression versus Treatment Effect
Some biopsies, often prompted by brain swelling, intentionally monitor the effects of therapy. Determining status of a glioma during or soon after therapy is often critical. Procedures including choline to N-acetyl aspartate (NAA) ratios on MR spectroscopy have improved radiographic estimation of glioma versus necrosis in a glioma treated with radiation and/or chemotherapy. However, glioma regrowth and treatment effects (TEs) are more complex than glioma versus necrosis. When radiographic procedures do not work, biopsy is needed to distinguish TE from tumor regrowth (Fig. 10.3C,D).
Tumor progression and TE can both involve necrosis, vascular changes, and cellular pleomorphism. The necrosis of TE often shows fibrinoid change (Fig. 10.3E,F). Either tumor progression or TE can show vascular proliferation, but only TE shows fibrinoid necrosis or extravasation affecting vascular
walls. Radiation necrosis is not pseudopalisading, and during its maturation, it shows tiny flecks of calcification. Both the tumor and white matter are particularly sensitive to radiation, grey matter being more resistant. Mitoses and tumor cell density are decreased with TE.
walls. Radiation necrosis is not pseudopalisading, and during its maturation, it shows tiny flecks of calcification. Both the tumor and white matter are particularly sensitive to radiation, grey matter being more resistant. Mitoses and tumor cell density are decreased with TE.
 FIGURE 10.4 This frontal lobe specimen resected 24 days after an initial biopsy simulates an organizing infarct (A). The original biopsy revealed an obvious glioma (B). |
A few scattered glioma cells are usually present in biopsies done to monitor TE versus glioma regrowth and/or progression. Scattered glioma cells do not dissect the differential. Instead, look for significant glioma cell density and mitoses as signs of regrowth. Tumor cell density and mitoses also are usually increased with tumor progression (Fig. 10.3G). The character of necrosis in tumor progression is often oddly shaped (geographic) and may include adjacent increased cell density (pseudopalisades). Pseudopalisades are more reliable indicators of progression, in my experience. Microvascular proliferation in tumor progression lacks fibrinoid changes.
Months or years following tissue diagnosis and radiotherapy of a glioma, a radiologic abnormality or declining clinical status may prompt another operation to distinguish between recurrence of tumor and radiation necrosis (Fig. 10.3E-G).
THERAPEUTIC RESECTION
If the initial operation for diagnosis was incomplete, a second operation may be done to achieve a therapeutic effect. Other operations are primary resections of brain, often the temporal lobe, to treat medically refractory seizure foci. Categories of therapeutic resection include gross total (100% removal of visible mass), radical subtotal (95% to 99% removal), subtotal (75% to 95% removal), and partial (10% to 75% removal) resection.
If the lesion was removed with a Cavitron ultrasonic aspirator (CUSA), tissue obtained from the suction collection device retains diagnostic features and immunoreactivity. However, tissue artifact is common and easy to confuse with necrosis in aspirated specimens. Blue-tinged necrosis and linear streaks of nuclear material suggest artifact.
The pathologist should compare prior diagnostic findings to the remaining lesion. Familiarity with the histologic appearance of a lesion on previous biopsy is critical for accurate interpretation (Fig. 10.4). If the resection is performed months or years following the primary biopsy, the pathologist may be expected to render an opinion about the effect of therapy. Evidence of treatment response, such as necrosis and vascular fibrosis, should be noted (Fig. 10.3).
Alternatively, the second procedure is performed with therapeutic intent, such as to place a wafer impregnated with a chemotherapeutic agent. In such cases, specific histologic features (e.g., definitive features of glioblastoma) may be necessary to qualify for specific treatment protocols for placement of the wafer.
MACROSCOPIC SPECIMEN INTERPRETATION AND PROCESSING
All specimens should be examined for arachnoid, gray matter, and white matter. To optimize evaluation, sectioning of specimens with identifiable architecture should be performed perpendicular to the meninges, from arachnoid through gray matter to white matter.
Many lesions are macroscopically evident in biopsy specimens. Yellow or firm gray or white matter typically corresponds to gliosis. Solid tissue that is not discernible as either gray matter or white matter typically represents an abnormal mass. Some tumors are often semiliquid and gelatinous in nature (oligodendrogliomas, lymphomas, pituitary adenomas). See Tables 10.1110.12 and 10.13 for diagnosis by distinguishing features. Multiplicity of masses is usually impossible to establish from the neurosurgical specimen alone; it requires radiographic correlation.
Flat tissue no thicker than 3 mm that is neither dura nor arachnoid may represent the wall of a cyst (see Tables 10.18 and 10.19). This can be compared and verified with the in situ or radiographic impressions of the lesion. Intracranial and intraspinal cysts are often fragmented in the course of their removal.
A vascular conglomerate larger than the usual aggregates of arachnoidal vessels may represent a vascular malformation (Table 10.6). Vascular malformations involve meninges and/or parenchyma and are often associated with recent or chronic hemorrhage.
Necrosis of the CNS parenchyma or of tumors is initially very soft and friable. It cavitates with age more rapidly in CNS parenchyma than in tumors. It can be clear, white, or yellow depending on the state of microcavitation and macrophage reaction. The arachnoid is typically preserved despite underlying parenchymal necrosis.
Although clinical diagnosis is variable, radiographic and macroscopic examination often do not distinguish inflammatory from other lesions. This must be done microscopically, as described in Tables 10.210.3,10.4,10.5,10.6,10.7,10.8,10.9,10.10,10.11,10.12,10.13,10.14,10.15,10.16,10.17,10.18 and 10.19 and the accompanying text and figures.
Some lesions have many of the features described earlier. A mass may contain regions of necrosis or hemorrhage. Arteriovenous malformations and cavernous angiomas are commonly linked with hemorrhage. Macroscopic cysts may occur within a mass, especially within cystic astrocytomas, oligodendrogliomas, craniopharyngiomas, and teratomas. There fore, all tissue regions should be adequately sampled, particularly any interface between lesion and recognizable brain.
MARGINS
Assessment of margins in the resection of many CNS lesions, particularly diffuse gliomas, is an unrealistic goal. Due to limitations of access, CNS lesions are frequently resected piecemeal and with the use of combined ultrasound and suction. Although a gross total resection is attempted in some cases, guided by imaging studies, most adult primary gliomas manifest a diffusely infiltrative growth pattern that defies even radical resections attempted in the past and are considered nonresectable. Some exceptions exist, with, for example, a pilocytic astrocytoma from the cerebellum of a child or a dysembryoplastic neuroepithelial tumor from the cerebrum. See further considerations later in the “Gliomas” section. In rare cases, an en bloc excision of an entire neoplasm (e.g., a meningioma) is obtained. It is best to mark the margin of tissue around such an intact specimen with ink, unless the surgeon requests otherwise.
TISSUE PROCESSING
General Considerations
Squash/smear/touch preparations
Unfrozen routine permanent paraffin sections
Frozen sections
Electron microscopy (EM) (retain a small portion in a fixative for EM, if necessary)
Frozen tissue, for possible future molecular diagnostic studies (freeze fresh tissue as soon as possible and store)
Other (microbiology, flow cytometry, cytogenetics, molecular diagnostics)
Routine Fixation
In most cases, with the critical exception of suspected hematopoietic neoplasms, fixation of biopsy and resection specimens in formalin and routine processing are sufficient to meet diagnostic needs.
Electron Microscopic Fixation
Glutaraldehyde fixation of a small portion of a lesion can be done any time after formalin fixation, making it easy to save tiny pieces of wet tissue in formalin in case EM is needed. It may be needed for (a) poorly differentiated or unusual tumors, (b) differential that includes a lesion best confirmed by EM (like ependymoma), (c) toxic-metabolic disease, and (d) infection. Saving a bit of tissue for possible future EM evaluation until completion of a challenging case is smart. Keeping it in formalin in its original jar is easy.
Hematopoietic Specimens
When a hematopoietic lesion is identified intraoperatively or suspected clinically, the specimen should be treated like a lymph node biopsy. Nuclear morphology is a key element of hematopoietic diagnoses. As such, a portion of the specimen should be fixed in a mercuric chloride-formalin fixative (e.g., B5) or, alternatively, zinc-containing fixative. The cassette must be marked to indicate that removal of the metallic fixative is required after the appropriate fixation interval. If enough tissue is available, a suspected hematopoietic lesion can also be placed in cell culture medium and sent for flow cytometric evaluation for phenotypic characterization. Touch preparations are often useful, especially intraoperatively. A portion can also be flash frozen to accommodate molecular evaluation and labile antigen evaluation by IHC; retaining a frozen section specimen in the frozen state can accomplish this goal simply and effectively.
Microbiologic Workup Recommendations
Cultures usually are more sensitive than other methods (including molecular methods) to detect organisms that grow in vitro. If an infectious etiology is suspected intraoperatively, it is critical that the surgeon be notified of the possibility immediately so that optimal samples for bacterial (aerobic and anaerobic), mycobacterial, fungal, and viral cultures can be obtained by the surgeon from the sterile operating field and sent directly to the microbiology laboratory. Saving a small, fixed portion of the lesion for possible EM evaluation and freezing another small piece are wise.
Molecular Diagnosis
Incorporation of genetic data into the assessment of a CNS neoplasm is now well established for glial neoplasms and is emerging for other tumors and lesions including infections. Formalin-fixed, paraffin-embedded tissue sections can be used for most of the techniques, including IHC, fluorescence in situ hybridization (FISH), in situ hybridization (ISH), and polymerase chain reaction (PCR) techniques. Other techniques require unfixed tissue for evaluation. When abundant lesional tissue is present, the pathologist is wise to snap-freeze a portion and store it at -80°C. Retaining the frozen specimen in a frozen state can accomplish this, assuming adequate tissue is available for permanent sections.
Toxic-Metabolic Processes
When a biopsy from a patient with suspected toxic-metabolic disorder is performed (e.g., for a suspected lysosomal storage disease, mitochondrial cytopathy, or cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy [CADASIL]), representative gray and white matter should be fixed in formalin for routine processing, retained in formalin or glutaraldehyde for possible electron microscopic evaluation, and frozen for possible biochemical studies if enough tissue is available to meet histologic needs.
HIGH-RISK INFECTIOUS DISEASE CASE CONSIDERATIONS
Universal precautions prevent transmission of most infectious diseases from central nervous specimens to pathologists and assistants. However, biopsies obtained from (a) patients with known HIV infection/AIDS and (b) patients with a clinical differential diagnosis that includes Creutzfeldt-Jakob disease (CJD) require special consideration and handling (4,15).
Minimization of risk to laboratory personnel includes (a) advance warning by neurosurgeons when operations are performed on high-risk patients, (b) cytologic preparations instead of frozen sections on specimens from these patients and only if needed to confirm lesional tissue, (c) declining frozen sections from patients with a clinical differential diagnosis that includes CJD, and (d) proper handling of specimens once received in the laboratory.
Human Immunodeficiency Virus
Brain biopsies obtained from patients with known HIV infection or acquired immunodeficiency syndrome (AIDS) are usually tested for infectious processes (e.g., toxoplasmosis) and neoplasm (e.g., lymphoma or other neoplasms). HIV is inactivated by formalin, so, after fixation, there is minimal risk of handling specimens from such patients. Cytologic preparations often provide assurance of lesional tissue. However, if a frozen section is necessary from such a specimen, decontamination of the cryostat should be carried out by using and replacing a disposable blade, removing all freezing medium debris, and wiping surfaces that came into contact with the specimen with a solution of 10% bleach in water. Use of a separate cryostat for such specimens, when available, can minimize downtime in a busy frozen section laboratory.
Prion Diseases, Including Creutzfeldt-Jakob Disease
Biopsy for possible CJD requires a compelling clinical reason, such as a treatable disease being in the clinical differential. A neurologist with expertise in prion diseases should certify all biopsies for dementia: (a) Is there a possibility of prion disease or not? (b) Could the patient have another treatable cause of the dementia? Without an affirmative answer to both questions, biopsy is not appropriate because of the concern for contamination of surgical and pathology personnel.
If biopsy is undertaken, frozen sections should not be done. Specimens must include (a) tissue for formalin fixation and (b) snap-frozen tissue for Western blot and gene sequencing evaluation. It is also desirable to collect blood for germline comparison. Prior to biopsy, the neurosurgeon should confer with the neurologist to sample an appropriate portion of brain, depending on symptoms. In addition to gray matter, white matter and meninges should be sampled.
The National Prion Disease Pathology Surveillance Center (NPDPSC) at Case Western Reserve University is funded and equipped for molecular diagnosis of prion diseases. For Western blot, the NPDPSC requests at least 10 mg of tissue (pea-sized or 0.5 cm diameter) for biopsies. Put the tissue into a small plastic vial and screw on the cap. Put the vial into a biohazard bag and close the bag. Store initially in a -70°C freezer until it can be sent by overnight mail to the NPDPSC. Further instructions and contact information can be obtained by contacting the NPDPSC directly.
In the rare biopsied case in which CJD is in the differential, decontamination of tissue for histologic processing, sectioning, and examination can be obtained as follows. Specimens should be sectioned to fit into cassettes (see special notes in the following section); if desired, a small portion of cortex can be placed into glutaraldehyde for possible electron microscopic evaluation. The cassettes are (a) placed into 10% buffered formalin for fixation for at least 24 hours; (b) transferred to undiluted, 95% to 100% formic acid for 1 hour; and (c) then returned to fresh 10% formalin for another 48 hours or more before routine processing. Although of very low risk, embedded tissue should be handled as if infectious—sectioned by an experienced technologist using disposable blades, with incineration of all section waste and wiping of microtome and cutting station with bleach or 2N NaOH. Histologic and many IHC evaluations are minimally impeded by the formic acid processing. Disposable towels, gloves, and equipment should be used in initial processing and subsequently incinerated. Contaminated surfaces can be cleaned using 5% bleach (corrosive for steel instruments) or 2N NaOH (incompatible with aluminum) with prolonged exposure (15 minutes up to 2 hours) for equipment (4,15). More information is available online at http://www.pathology .med.umich.edu/Safety%20Manual/CJD%20Final.pdf.
Special Notes
Cassettes should be labeled with pencil as formic acid will dissolve away even the ink specially formulated for histologic processing.
Normal plastic cassettes are resistant to formic acid, but the thin plastic netting of cassettes designed for small biopsies will dissolve in formic acid, so a small biopsy should be wrapped in lens paper to keep it in the cassette. Metal tops should not be used.
HISTOCHEMICAL AND IMMUNOHISTOCHEMICAL EVALUATION
Special stains (16), IHC stains, and cytologic techniques that aid the diagnosis of specific entities at biopsy are described in Tables 10.2 and 10.10,10.11,10.12,10.13,10.14,10.15,10.16,10.17,10.18,10.19 and 10.20. Routinely use a standard tissue control that contains regions both positive and negative for the IHC marker under evaluation. If possible, choose a block with the lesion of interest that also contains tissue with known positive and negative regions to use as an internal standard tissue control (i.e., the edge of a tumor with gliotic brain and vessels for GFAP IHC).
Polarization microscopy quickly reveals mature collagen fibers and hair shafts (see Fig. 10.133 in the “Dermoid Cyst” section later in this chapter). On the Congo red stain, polarization distinguishes light yellow-green amyloid from whiter collagen (17).
Individual neoplasms may lack a marker generally representative of its category. Others may lose expression of markers as they progress to higher grades of malignancy. Because these factors or technical reasons may cause individual neoplasms to fail to label for a marker, a positive immunostaining result is more meaningful than a negative result.
Based on initial histologic findings, one can construct a differential diagnosis for which a group of appropriate special stains can be assembled from information in the text and tables here. To minimize redundancy, positive rather than negative
features are emphasized in the following sections, under each entity. Depending on difficulty, the differential can be solved on H&E or IHC (18).
features are emphasized in the following sections, under each entity. Depending on difficulty, the differential can be solved on H&E or IHC (18).
There are many Web sites for locating IHC expertise, reagents, and suppliers. www.biocompare.com searches for very specific reagents and their suppliers. www.histonet.org provides numerous comments, recipes, and images from histology and histochemistry professionals. Our University of Michigan (UM) IHC laboratory professionals often use the antibody name in Google searches to find new antibodies.
ELECTRON MICROSCOPY
The importance of EM in cranial and spinal diagnostic neuropathology has been diminished by faster IHC. However, EM is valuable in defined settings in which IHC cannot distinguish among specific subtypes of tumor (19). Unlike largely confirmatory IHC, EM shows many structures not well seen on light microscopy that may point to a new direction in interpretation. At times, it is the only path to a definitive answer. For example, among various clear cell tumors, basal bodies, cilia, elongated junctions, and microvillous lumens distinguish clear cell ependymoma from the other tumors, including oligodendroglioma. EM can be a valuable adjunct to diagnosis in specific settings; see the “Tissue Processing” section earlier in this chapter.
EM is needed to confirm certain metabolic diseases. Specimens from sites other than brain are frequently biopsied for evaluation of various metabolic diseases that affect the CNS, such as neuronal ceroid lipofuscinosis, the various lysosomal storage diseases, leukodystrophies, mitochondrial encephalopathies, and vascular diseases including CADASIL (19).
REACTIVE CHANGES
GLIOSIS
Gliosis is a reaction of the CNS to injury of the brain or spinal cord (see Fig. 10.5A). It identifies abnormal CNS tissue. Gliosis is defined as either (a) increased number of astrocytes or (b) increased number and length of processes or increased cytoplasmic synthesis of GFAP representing gemistocytic astrocytes. Although subtle changes such as astrocyte swelling with fluid occur earlier, gliosis is usually appreciated on H&E-stained slides within 2 weeks after an injury. Because nearly any injury of the CNS can cause gliosis, it is not diagnostic of a specific pathologic entity.
Anti-GFAP IHC differentiates subtle forms of gliosis (Table 10.3). When this differentiation is critical, a control slide from the same region of normal CNS can help. Compared to control, look for a greater number of astrocytes, a greater number and density of cell processes, and the presence of GFAP-positive gemistocytes with reactive nuclei, cells spaced apart from one another.
The problem of distinguishing gliosis from low-grade glioma is challenging and is described later (see “Gliomas”). Gliosis does not expand, although gliotic parenchyma may surround an expanding tumor. “Pure” gliosis, which lacks noticeable CNS parenchyma, is associated with long-standing cysts (Table 10.18). In contrast to microcysts of gliomas (see Fig. 10.32 in the “Gliomas” section), these cysts are macroscopic, confluent, and often have numerous residual macrophages. However, in the absence of evidence of another origin, the possibility of neoplastic origin of such cysts must still be considered. In gliosis, astrocytes respect each other’s space and show more uniform spacing between individual glial cells than in the margin of a glioma (see Fig. 10.5A; also see Figs. 10.23, 10.24, and 10.35 later in this chapter). The nuclear-to-cytoplasmic ratio of gliosis is less than that of a glioma. IDH1 mutation and p53 overexpression are not found in gliosis. This distinguishes gliomas with IDH1 mutation and p53 overexpression from gliosis (20). Gliosis has a less than 5% Ki-67 proliferation index, assessed on hot spots within 100 cells (see “Assays of Proliferative Capacity” section). Excessive fibrillarity and Rosenthal fibers do not distinguish gliosis from glioma (see Figs. 10.35 and 10.40 in the “Gliomas” section).
 FIGURE 10.5 Reactive changes. Common reactions to injury in the central nervous system include gliosis (A) and “gitter cell” reaction of macrophages filled with neutral fat (B). The gliosis occurred in edematous brain near a metastatic carcinoma. The macrophages are in a subacute infarct clinically suspected to be a glioma. |
MACROPHAGES, MICROGLIAL CELLS
Macrophages present as (a) granular or foamy macrophages, (b) microglial cells with rod-shaped nuclei, or (c) epithelioid macrophages with or without multinucleated giant cells. They may be seen, in variable and sometimes striking abundance, in any condition that involves tissue disruption, including infectious,
inflammatory, necrotic, traumatic, degenerative, and neoplastic processes (21,22 and 23). The origin of these cells is variable among lesions and consists of some combination of (a) “native” parenchymal microglial cells, which can proliferate along with (b) blood-derived macrophages entering the brain parenchyma in response to damage (22). The fate of the latter cells may be to remain at the site of injury or migrate to the perivascular space (23). Because distinction among these origins is not useful diagnostically, the inclusive term macrophages can be employed.
inflammatory, necrotic, traumatic, degenerative, and neoplastic processes (21,22 and 23). The origin of these cells is variable among lesions and consists of some combination of (a) “native” parenchymal microglial cells, which can proliferate along with (b) blood-derived macrophages entering the brain parenchyma in response to damage (22). The fate of the latter cells may be to remain at the site of injury or migrate to the perivascular space (23). Because distinction among these origins is not useful diagnostically, the inclusive term macrophages can be employed.
In the brain, a useful marker for macrophages is CD68 (KP-1) IHC (see Fig. 10.25 in the “Demyelination” section later in this chapter), and it frequently identifies considerably more macrophages than are anticipated by H&E evaluation. A special stain for myelin (e.g., Luxol fast blue-hematoxylin and eosin [LFB-H&E]) (see Fig. 10.19 in the “Progressive Multifocal Leukoencephalopathy” section later in this chapter) identifies engulfed myelin debris, and a stain for axons (see Fig. 10.26 in the “Demyelination” section later in this chapter) helps to distinguish between necrotic CNS tissue (axons disintegrate and leave axonal debris in macrophages) and demyelination (foamy macrophages contain myelin but not axonal debris). GFAP IHC is helpful to discern gemistocytic reactive astrocytes from macrophages. Reactive macrophages must be distinguished from the histiocytoses; see the “Histiocytoses” section later in this chapter.
Foamy macrophages are common in infarcts and demyelination; their lysosomes are loaded with cellular debris and may contain yeast forms and other organisms. Macrophages swollen by phagocytosis within the CNS are called endocytic or gitter cells (Figs. 10.4A and 10.5B). Hemosiderin-laden macrophages can be noted around organizing hemorrhages, traumatic lesions, and some vascular malformations. Large, rounded cells with foamy cytoplasm and eccentric nuclei, having the appearance of macrophages, present occasionally as a part of such rare lesions as xanthogranulomas of choroid plexus. They are occasionally evident in various brain tumors, along with lymphocytes. Lipid-laden macrophages can be distinguished from amebae in the rare amebic brain abscess by their larger and more distinct nuclei and less chromatin clumping than amebae and by their CD68 positivity (see the “Parasites” section under “Infectious Diseases” later in this chapter).
Epithelioid macrophages are activated but nonfoamy macrophages are identified in granulomatous inflammation in infectious (e.g., fungus, acid-fast bacillus [AFB]) and inflammatory (e.g., sarcoid) diseases of the CNS.
Giant cell macrophages are usually seen in granulomatous inflammation. Individual multinucleated giant cells in brain tissue are evidence of HIV encephalitis.
Microglial cells are macrophages that closely resemble antigen-presenting dendritic cells seen elsewhere in the body and play a key role in response to a variety of etiologic insults (22,23). They have a spindle-shaped nucleus and little discernible cytoplasm and must be distinguished from the so-called naked nuclei of infiltrating glioma and from endothelial cell nuclei. They are CD68-positive (see Fig. 10.25). They are notably seen diffusely in brain tissue or as small aggregates, so-called microglial nodules; in viral and rickettsial diseases; and around dying neurons (neuronophagocytosis; see Fig. 10.7).
GRANULOMATOUS INFLAMMATION
Granulomatous inflammation is defined by both cell types and architecture. Cells include infiltrates of (a) activated, epithelioid macrophages with or without multinucleated giant cells and (b) lymphoid cells. Foamy macrophages may or may not be seen. Architecturally, the infiltrate may form nodules, or granulomas, with or without central necrosis. Identification of granulomatous inflammation raises the differential of infectious or autoimmune inflammatory conditions. Granulomatous inflammation is common in the setting of bacterial (tuberculosis, syphilis, Whipple disease), fungal, and parasitic infections. These entities must be distinguished from rare, noninfectious granulomatous processes that can affect the CNS, including systemic lupus erythematosus (SLE), Wegener granulomatosis, sarcoidosis, rheumatoid meningitis, and Crohn disease. If a histiocytic process is in the differential diagnosis, IHC for S-100 and CD1a can be performed (see the “Histiocytoses” section later in this chapter).
 FIGURE 10.6 (A) Perivascular mixed inflammatory infiltrate is composed of lymphocytes, plasma cells, and macrophages around but not within vessel walls. (B) Chronic encephalitis stained with CD68 shows elongated microglial macrophages within brain tissue and around vessels but not within the vascular wall. Note the normal thin endothelial lining (compare with vasculitis, Fig. 10.25C,D). |
PERIVASCULAR INFLAMMATION
Virtually, any irritation of the CNS can stimulate accumulation of cells around blood vessels (Fig. 10.6), including surgical
wounds and implants (24). Macrophages ingest the irritant or injured cellular constituents and move them to the perivascular space (23). In the absence of classic lymph nodes in the brain, this perivascular region is where cells that respond to antigens intermingle. Depending on severity and duration of illness, the perivascular inflammation varies substantially. Old hemorrhage is characterized mainly by perivascular macrophages laden with hemosiderin. Viral or autoimmune encephalitis produces a maximal response, with abundant perivascular macrophages and lymphocytes along with parenchymal infiltrates of microglial cells and microglial nodules.
wounds and implants (24). Macrophages ingest the irritant or injured cellular constituents and move them to the perivascular space (23). In the absence of classic lymph nodes in the brain, this perivascular region is where cells that respond to antigens intermingle. Depending on severity and duration of illness, the perivascular inflammation varies substantially. Old hemorrhage is characterized mainly by perivascular macrophages laden with hemosiderin. Viral or autoimmune encephalitis produces a maximal response, with abundant perivascular macrophages and lymphocytes along with parenchymal infiltrates of microglial cells and microglial nodules.
MENINGEAL FIBROSIS
Meningeal fibrosis develops following traumatic injuries, meningitis, vasculitis involving meningeal vessels, radiation therapy, or as a desmoplastic response to a tumor, especially if the tumor triggers massive vascular proliferation. Multifocal fibrosclerosis with hypertrophic intracranial pachymeningitis is a firm, fibrous lesion with dural thickening, frequently found in conjunction with an intracranial pseudotumor (25). Arachnoiditis ossificans deposits bone within the chronic inflammatory response.
INFECTIOUS DISEASES
In a symptomatic patient with imaging findings suggesting an infectious or inflammatory disease, when evaluation of cerebrospinal fluid (CSF) is unrevealing, biopsy is undertaken to attempt establish a definitive diagnosis and rule out other processes (see Fig. 10.9). Optimal sampling by biopsy that (a) includes dura, arachnoid, and gray and white matter and (b) is performed in the site of a radiologically enhancing lesion markedly improves the chance of reaching a diagnosis.
When biopsy is performed, the principal goal of the surgical pathologist in this setting is to evaluate features of infectious and inflammatory processes and rule out ischemic damage and neoplasm. Check the history; the immunocompromised patient is particularly susceptible to CNS infection. Specific susceptibilities are found in the following discussions.
MICROBIAL CULTURE
If an infectious etiology is suggested at the time of biopsy, the surgeon must be notified to send cultures (see “Tissue Processing”).
SPECIAL STAINS
A combination of histochemical microbial stains is employed in the setting of inflammation to evaluate for organisms and assess for specific cell types. IHC or ISH may help to identify organisms, particularly toxoplasma, viruses (including herpes, cytomegalovirus [CMV], and JC virus), fungi, and Rickettsia (26). Available molecular biologic approaches for identification of microorganisms vary among institutions. Some require frozen section, but PCR usually can be performed on DNA extracted from paraffin-embedded sections of tissue following deparaffinization steps. Most useful is the ability to detect mycobacteria, in the setting of necrotizing granulomatous inflammation, and assess tuberculosis (TB) or not TB. Cultures for TB are more sensitive but they take much longer.
EM has a role in the identification of microorganisms that do not grow in culture and are hard to stain, including viruses, Whipple bacillus, amebae, and spirochetes.
MENINGITIS, CEREBRITIS (FOCAL ENCEPHALITIS), MYELITIS
The terms meningitis, cerebritis, and myelitis literally mean inflammation of the meninges (dura or leptomeninges), cerebral parenchyma, or spinal cord parenchyma, respectively (27). Acute (neutrophils), chronic (lymphocytes and macrophages), and granulomatous (round, fibrous clusters of lymphocytes and epithelioid macrophages) inflammation can be identified. These forms of inflammation can be seen in infectious processes and in autoimmune inflammatory processes. In general, inflammation is not a prominent feature in neoplastic processes, and caution must be exercised in a hypercellular lesion with a large number of macrophages and other inflammatory cells. However, inflammatory infiltrates can be seen in neoplastic processes, especially perivascular lymphocytic infiltrates (particularly common in lymphomas, gangliogliomas, gemistocytic astrocytomas, and oligodendrogliomas) and macrophages mixed with neoplastic cells, but they are a secondary feature.
The term cerebritis has been used to describe a focal cerebral encephalitis of bacterial origin that is not sufficiently encapsulated to be an abcess (27,28). Unfortunately, cerebritis is confusing because it excludes CNS structures posterior to the cerebrum, leaving cumbersome alternatives like cerebellitis, mesencephalitis, or brainstemitis. I prefer the term focal encephalitis to cerebritis. Focal encephalitis (FE) precedes frank abscess formation but requires early biopsy to be seen. FE may be sterile or may contain an infectious agent. Its inflammatory infiltrate is composed of neutrophils, macrophages, lymphocytes, and plasma cells, with or without parenchymal necrosis (Figs. 10.6 and 10.7). FE is found around septic thromboemboli, neoplasms, ruptured vascular malformations, infarcts, or traumatic lesions. Meningitis may precede FE. The aforementioned features also apply to focal myelitis. Septic FE is usually caused by bacterial agents, most often streptococci or staphylococci and less frequently by Gram-negative organisms such as Escherichia coli, Pseudomonas, or Haemophilus influenzae.
ENCEPHALITIS
The term encephalitis literally means brain inflammation. Viruses and Rickettsia usually cause inflammation of brain tissue that is more diffuse than that caused by bacteria and may involve more than the cerebrum. By convention, this brain inflammation is usually called encephalitis (27). Most viral infections are self-limited and cause inflammation that resolves without permanent brain damage. Other infections, emphasized in the section “Viruses” later in this chapter, can result in a serious necrotizing process.
MYELITIS AND TRANSVERSE MYELITIS
Myelitis and so-called transverse myelitis (specifically inflammation of the spinal cord parenchyma) present clinically with symptoms can involve sensory, motor, and autonomic
modalities of variable severity. The etiology is often elusive, but etiologies include infectious (herpes simplex virus [HSV], CMV, etc.), inflammatory (multiple sclerosis [MS], connective tissue disease, etc.), vascular (infarct due to thrombosis), developmental (vascular malformation), traumatic, and iatrogenic processes (29,30). Workup is based on imaging and CSF and serum evaluation; biopsy is occasionally performed to search for an etiology.
modalities of variable severity. The etiology is often elusive, but etiologies include infectious (herpes simplex virus [HSV], CMV, etc.), inflammatory (multiple sclerosis [MS], connective tissue disease, etc.), vascular (infarct due to thrombosis), developmental (vascular malformation), traumatic, and iatrogenic processes (29,30). Workup is based on imaging and CSF and serum evaluation; biopsy is occasionally performed to search for an etiology.
 FIGURE 10.7 Chronic encephalitis. The hallmark of chronic encephalitis is neuronophagia, the “eating of neurons.” Lymphocytes recognize infected neurons, even ones such as these that show no inclusions, and direct microglial macrophages to eat them (arrows). Notice that these infected neurons are not hypoxic; they have not lost their purple cytoplasmic Nissl substance nor have they turned orange. When there is no encephalitis, the most common cause of neuronophagia is hypoxic damage to neurons. |
ABSCESS
An abscess, typically preceded by FE or cerebritis, combines features of inflammation, gliosis, and fibrosis. A mixture of polymorphonuclear leukocytes, lymphocytes, macrophages, and plasma cells on H&E-stained sections confirms inflammation (Fig. 10.8). Polymorphism of inflammatory components can be verified by finding mixed B and T lymphocytes admixed with CD68-positive macrophages.
The wall of a brain abscess consists of collagen and reactive gliosis. The collagen layer is formed by fibroblasts that migrate out from the surrounding blood vessels. Thus, the thickness of this layer varies depending on the number of viable vessels and their distance from the abscess as well as the age of the abscess. Because collagen is rare within the CNS, its presence is an important diagnostic feature of an abscess (Table 10.18). Collagen may be difficult to distinguish from fibrillary gliosis on a slide stained with H&E. Points to remember are the extracellular and often wavy appearance of collagen compared with the intracellular fibrillar character of gliosis. Collagen will polarize yellow-white on H&E- or Congo red-stained sections, whereas gliosis will not. If there is any doubt, it is best to stain histochemically for collagen or reticulin or stain immunohistochemically for collagen type IV.
Other entities that produce collagen within the CNS may be confused with the wall of an abscess (Tables 10.10, 10.11 and 10.12, 10.18, 10.19). Sarcomatous and desmoplastic neoplasms, and various cysts with collagenous walls, generally lack an inflammatory component. Notable exceptions are cysts that have ruptured and extruded material foreign to the CNS, such as colloid or squamous epithelial cells (see Fig. 10.132 in the “Common Metastatic Tumors” section later in this chapter and Table 10.19). If this material is not detectable within the inflammatory reaction to the cyst, clinical and pathologic correlation assists the interpretation. These other lesions that combine collagen and inflammation are sterile in situ and do not contain microorganisms.
Nocardial abscesses of the cerebrum and the spinal cord are rising in frequency in drug addicts. Multiple microabscesses should be considered in the differential diagnosis of encephalopathy in hospitalized patients with chronic disease, immunosuppression, and sepsis (27).
MYCOBACTERIA, SPIROCHETES, AND WHIPPLE DISEASE
Suppurative bacterial infections have been discussed earlier under meningitis, cerebritis (focal encephalitis), myelitis; and shown in Fig. 10.8B. Here, consideration will be given to several special diseases, including TB, syphilis, and Whipple disease (31,32).
TB and atypical mycobacteria can involve any region of the CNS, principally manifesting as meningitis and meningoencephalitis (32). AFB stains are useful, but slower cultures are more sensitive. A role for PCR in identifying and speciating mycobacteria has been established (33). In human immunodeficiency virus (HIV)-infected patients, newly acquired TB can spread readily and progress rapidly to active disease. TB can be the first clinical manifestation of immunodeficiency in these patients.
The frequency of neurosyphilis continues to increase in AIDS patients (31). Microscopic examination reveals focal lymphoplasmacytic inflammatory infiltrates in a predominantly perivascular arrangement. Syphilis contributes to the list of the differential diagnoses of granulomatous inflammations. Silver stains for spirochetes do not work well in brain due to background staining. Special identification procedures are available at the Centers for Disease Control and Prevention (CDC).
Whipple disease rarely presents as a primary brain disease and even then usually with concomitant gastrointestinal symptoms. The diagnosis can be made by brain biopsy. Histologic features include periodic acid-Schiff (PAS)-positive, diastaseresistant macrophages; microgranulomas; giant multinucleated cells; perivascular lymphocytes; and reactive microglia (Fig. 10.9). Supplemental data come from immunohistochemical evaluation for group B streptococci and EM (34). Biopsy of the central cingulate gyrus, mediobasal temporal region, and insular cortex yields the most diagnostic tissue. EM can confirm the presence of a characteristic bacillus that has been named Tropheryma whippeli.
FUNGI
Fungal organisms that involve the CNS include those presenting as (a) yeast forms (Fig. 10.10), including Cryptococcus neoformans, Coccidioides, histoplasmosis, Candida, and blastomycosis; and (b) hyphal forms (Fig. 10.11), including the Zygomycetes (Mucor and Rhizopus, Rhizomucor), Aspergillus, and Pseudallescheria. Involvement of the CNS can be via a hematogenous route
(e.g., from a primary pulmonary source) or via direct extension from an infected sinus.
(e.g., from a primary pulmonary source) or via direct extension from an infected sinus.
 FIGURE 10.8 Abscess in a 49-year-old man who had a first seizure after a dental procedure. (A) Imaging revealed an enhancing cystic lesion. (B) Suppurative inflammation was noted, including neutrophils with admixed macrophages and lymphocytes. (C) Gram stain demonstrates filamentous bacteria. (D) Grocott methenamine silver stain of filamentous bacteria. Actinomycosis was cultured. |
Fungal organisms tend to involve the meninges, in some cases presumably passing from subarachnoid space along perivascular spaces to involve parenchyma. Vascular wall invasion is common in hyphal forms, which may lead to catastrophic rupture and hemorrhage.
Variable inflammatory infiltrates are elicited by fungi, most often chronic and granulomatous. When fungi are suspected, use of both Grocott methenamine silver (GMS) and PAS is recommended because some forms stain better with one than the other stain (Fig. 10.11). Mucicarmine is of value in evaluating the capsule of Cryptococcus. This relatively nonantigenic capsule delays the inflammatory response to Cryptococcus.
Cryptococcal meningitis is the most common form of fungal meningitis, with a variable inflammatory response, often minimal in acute infections and in the setting of AIDS (Fig. 10.10). Chronic infections can produce granulomatous inflammation. Histoplasmosis, blastomycosis, and coccidioidomycosis can present as CNS parenchymal lesions, after presumed dissemination from lung, with variable suppurative to granulomatous inflammatory reactions; characteristic yeast forms can be identified by H&E, GMS, and PAS (Fig. 10.10). Candida can also involve the CNS as intraparenchymal masses; inflammation is typically acute (28). Zygomycetes organisms tend to be angiocentric and angioinvasive (Fig. 10.11). Zygomycetes may involve brain by direct extension from a nasal sinus infection; diabetic patients appear to be at particular risk for such infections (35).
 FIGURE 10.9 Whipple disease of the central nervous system. Typical cluster of reactive cells stained with periodic acid-Schiff stain (A) and by immunoperoxidase for anti-group B Streptococcus (B). (B, Courtesy of Dr. M. E. Velasco, Medical College of Ohio, Toledo, OH.) |
Assessment includes the identification of hyphal or yeast forms and an attempt at identification of the organism. Ideally, a portion of the specimen is sent for culture and specific identification. The report of hyphal organisms as suspicious for aspergillosis (based on dichotomous branching and septa formation) or Zygomycetes (based on ribbonlike hyphae of varying width, absence of definite septa, and rightangle branching) should be confirmed by culture, IHC, or ISH. Pseudallescheria can appear histologically as hyphae with acute branches and septa, and treatment is different (36). Histologic consultation with a microbiologist prior to reporting is wise.
 FIGURE 10.10 (A) Cryptococcosis in the subarachnoid space with a capsule (Grocott methenamine silver). (B) Coccidioidomycosis spherule and free endospore (inset) from a necrotizing brain lesion (hematoxylin and eosin). (C) Histoplasmosis in macrophages and free in brain tissue in a necrotizing brain lesion (Grocott methenamine silver). |
 FIGURE 10.11 Zygomycetes. (A) Vessel wall is diffusely invaded by hyphal organisms with associated chronic inflammatory cell infiltrate. (B) Periodic acid-Schiff and (C) Grocott methenamine silver stains highlight the fungal morphology. |
PARASITES
A variety of parasitic organisms can involve the CNS, including endemic organisms or exotic organisms most frequently seen in immigrants or travelers (37,38,39 and 40). Important parasites include (a) protozoa, including toxoplasma and amebae; (b) the pork tapeworm Taenia solium; and (c) schistosoma, a trematode. The organisms can be seen at various stages of their development and involve the leptomeninges and neuropil of the brain and spinal cord.
Opportunistic infections are common in AIDS and other immunosuppressed patients. Toxoplasmosis is the most frequent of these infections, and clinically, the differential between toxoplasmosis and lymphoma can be difficult, requiring biopsy if antimicrobial therapy fails (40). A TORCH (toxoplasmosis, other infections, rubella, CMV, and HSV) organism that affects the intrauterine developing brain, toxoplasmosis in an adult likely represents a reactivation of dormant parasites introduced by previous exposure. It presents with a necrotizing encephalomyelitis, typically multifocal by imaging, which is characterized by ill-defined necrotic lesions containing thin-walled cysts filled with bradyzoites (“slow [-growing] animal”), dark-staining dotlike structures representing nuclei, and individual tachyzoites (“fast [-growing] animal”) (Figs. 10.12 and 10.13). IHC or immunofluorescence pinpoints the individual organism not easily found on routine H&E sections. Toxoplasma may coexist with Candida in the same patient as well as with Cryptococcus, CMV, and other organisms. Rarely, toxoplasmosis is complicated by cerebral hemorrhage.
Neurocysticercosis, the result of migration to the CNS of larval forms of the tapeworm Taenia solium, is a common parasitic infection of the CNS found occasionally in people who have been in developing countries. About 50% to 70% of patients with neurocysticercosis present with seizures (37). Some resected specimens are tan, firm nodules containing a cavity with either a cystic region (Fig. 10.14A) or grumous, gray-yellow material. Microscopically, pale eosinophilic membranes separate the cavity from gliotic brain parenchyma with chronic perivascular inflammation. The characteristic invaginated scolex may be seen.
 FIGURE 10.12 Toxoplasmosis in an immunodeficient patient. Amphophilic pseudocyst full of a cluster of bradyzoites is irregular and larger than surrounding cells. |
 FIGURE 10.13 Cyst and free tachyzoites immunostained for Toxoplasma antigen (immunoperoxidase with hematoxylin counterstain). |
 FIGURE 10.14 (A) Cysticercosis cyst resected from the cerebral hemisphere of a young South American man with seizures. (B) Degenerating schistosome egg at the center surrounded by heavy mixed inflammatory infiltrate. (A, Courtesy of Dr. D. Burns, University of Texas Southwestern, Dallas, TX.) |
Schistosomiasis in the CNS is the result of migration of eggs of the organisms. The eggs characteristically elicit an intense, granulomatous inflammatory response and can be misinterpreted radiologically as a high-grade neoplasm (Fig. 10.14B).
Amebae including Naegleria and, less commonly, Acanthamoeba, Sappinia diploidea, and Balamuthia (seen mostly in the setting of immunocompromise) can affect the CNS (38,39). The organisms, containing a nucleus (some with a distinct karyosome), have vacuolated cytoplasm and can easily be mistaken for foamy macrophages. They may contain ingested red cells. Macrophages can be distinguished from amebae in the rare amebic brain abscess by their larger and more distinct nuclei and less chromatin clumping than amebae; the organisms are negative for CD68.
Exotic CNS infections often occur in AIDS patients. They include trypanosomiasis and strongyloidiasis.
VIRUSES
A large number of viruses affect the CNS, with or without systemic (especially respiratory system) involvement, with variability among seasons and geographical regions (e.g., presence of specific vectors), and with considerable variation in clinical presentation (41,42). It is highly likely that viral infections have been significantly underreported as the result of difficulty in identifying the etiologic agent.
Most diagnostic tools are clinical and include serologic (serum or CSF, usually with a rise between acute and convalescent specimens), PCR (serum, CSF, or brain), culture on mosquito or mammalian cell line or by intracranial inoculation into suckling mice (blood, CSF, brain tissue), or immunocytochemical (brain and other organ tissues) evaluations. CSF or postmortem brain specimens can be evaluated at reference laboratories or the CDC.
Histologically, most viral encephalitides induce perivascular lymphocytic cuffing. This (an increased number of parenchymal microglial cells) and neuronophagia (“neuron eating”; Fig. 10.7) should raise suspicion for a viral infection. Some viruses produce recognizable inclusions (Table 10.4), many of which can be identified with IHC or ISH. Neuronal inclusion bodies are (a) in nuclei with herpesviruses and measles virus of subacute sclerosing panencephalitis (SSPE) and (b) in cytoplasm in rabies. Oligodendrocyte nuclear inclusions are seen in progressive multifocal leukoencephalopathy (PML) and SSPE. CMV induces inclusions in the nucleus and cytoplasm of endothelial cells, ependymal cells, and glial cells. Many viruses do not manifest inclusions, including HIV, poliovirus, and arboviruses including West Nile virus.
Enteroviruses and Arboviruses
In acute viral meningitis and meningoencephalitis, it is estimated that enterovirus infections (e.g., coxsackievirus, echovirus) cause between 60% and 90% of cases and that arbovirus infections (e.g., Eastern equine, St. Louis, West Nile, etc.) comprise many of the remaining cases (42). Pathologic findings in cases described to date are nonspecific, without characteristic inclusions.
West Nile Virus
This arbovirus, a member of the Flaviviridae family, has recently emerged in the United States and Canada, with mosquito and bird vectors and hosts and involvement of humans (41). Transmission by blood transfusion and organ transplantation has been documented recently. Symptoms are variable, multisystemic, and nonspecific and include fever, myalgia, lymphadenopathy, rash, and headache. Neurologic symptoms include headache, encephalopathy, and diffuse weakness, including acute flaccid paralysis (41,43,44). A proportion of cases are fatal, and fatalities occur principally in the elderly—most with underlying systemic diseases.
The findings are nonspecific. They include a patchy meningitis, encephalitis, and polio-like myelitis with variable but, in some cases, severe involvement of the substantia nigra, brainstem (particularly the medulla), and cerebellum. Perivascular inflammation with evidence of meningoencephalitis and, in some cases, microglial nodules and neuronophagia can be seen (43,44). Anterior horn cells appear to be targeted in some patients, providing a substrate for the weakness (43). Of the involved regions, only the cerebrum and cerebellum are reasonable biopsy candidates. Laboratory diagnosis is based predominantly on serology (45). Additional information on this and other arboviruses is available from the Arbovirus Diagnostic Laboratory in Fort Collins, Colorado.
 FIGURE 10.15 Inflammatory infiltrate in herpes simplex virus encephalitis. (Courtesy of Dr. F. Stephen Vogel, Duke University Medical Center, Durham, NC.) |
Herpes Simplex Virus
The most common cause of nonepidemic encephalitis is HSV (Tables 10.2 and 10.3). The process is usually localized to the temporal and frontal lobes. When suspected by clinical and imaging findings, the diagnosis of HSV can be made by CSF culture, serology, and PCR evaluation rather than biopsy. Biopsy of the medial portion of a temporal lobe is still carried out in some cases. In the acute and subacute phase, perivascular inflammation, composed predominantly of lymphocytes mixed with macrophages, is accompanied by varying degrees of necrosis and hemorrhage (Fig. 10.15). Inclusion bodies are not easy to find in small brain biopsies (Fig. 10.16). Sensitive and specific methods of identification include ISH and IHC. EM also may demonstrate viral particles but is less sensitive and less specific.
Cytomegalovirus
CMV infection in an adult likely represents reactivation of dormant disease (28,46). Histologic findings vary from cells with typical CMV inclusions with virtually no associated inflammation to severe necrotizing ependymitis and meningoencephalitis. Other lesions may be complicated by CMV infection. IHC, ISH, and PCR are useful techniques for detecting the virus in paraffin-embedded tissue.
 FIGURE 10.16 Herpes simplex virus encephalitis. Cowdry type A inclusion body at the center. (Courtesy of Dr. F. Stephen Vogel, Duke University Medical Center, Durham, NC.) |
Progressive Multifocal Leukoencephalopathy
PML is the result of activation of the DNA papovavirus, predominantly JC virus (rarely SV-40 virus), in the brain. The disease is typically seen in the setting of severe immunodeficiency (e.g., AIDS) and lymphoproliferative disorders and associated treatment; occasional cases occur in the setting of immunosuppression after solid organ transplant and in connective tissue diseases (e.g., lupus) treated with immunosuppression (47).
PML manifests as multiple discrete foci of demyelination. Radiographically, it may simulate MS or a mass (Fig. 10.17). Brain biopsy frequently shows an active to subacute process, (a) typically with abundant foamy macrophages, many of which, on LFB-H&E stain, contain myelin; (b) marked reactive gliosis with many astrocytes manifesting bizarre, anaplastic nuclei that can be confused with glioma; and (c) glassy nuclear inclusions in oligodendrocytes that enlarge the nucleus and marginate the chromatin (Figs. 10.18 and 10.19). Perivascular infiltrates of mature lymphocytes are prominent in some cases. The histologic findings of PML are similar in patients with and without AIDS, although bizarre astrocytes are less frequent, and perivascular inflammatory cells more frequent, in AIDS patients. Diagnosis of PML can be confirmed by IHC, ISH (Fig. 10.19), or EM (1,28,48).
 FIGURE 10.17 Progressive multifocal leukoencephalopathy simulates an edematous mass in this T2-weighted magnetic resonance image from a patient with severe immunodeficiency. (Courtesy of Dr. Herbert Ichinose and Dr. Joseph Epps, Jr., Pendleton Memorial Methodist Hospital, New Orleans, LA.) |
Subacute Sclerosing Panencephalitis/Measles
SSPE is a rare disease, with a relatively high incidence in Asia and the Middle East. This slowly progressive disease is caused by a persistent infection of immune-resistant measles virus, usually in unvaccinated or late-vaccinated children. Optimal testing involves identification of antimeasles antibodies, both
IgG and IgM in both the CSF and serum. Brain biopsy is rarely performed. When undertaken, viral encephalitis-type changes predominate and there is loss of myelin in subcortical white matter (28). Nuclear inclusions may be detected in neurons and oligodendroglial cells by H&E. IHC or ISH evaluation detects measles virus antigen.
IgG and IgM in both the CSF and serum. Brain biopsy is rarely performed. When undertaken, viral encephalitis-type changes predominate and there is loss of myelin in subcortical white matter (28). Nuclear inclusions may be detected in neurons and oligodendroglial cells by H&E. IHC or ISH evaluation detects measles virus antigen.
 FIGURE 10.18 Progressive multifocal leukoencephalopathy. An infected oligodendroglial nucleus is enlarged by a pink-purple (amphophilic) accumulation of virions and peripheralization of chromatin. |
HIV/AIDS
The CNS is significantly affected by HIV- and AIDS-related diseases. Such diseases can be classified as primary (due directly to HIV) and secondary, with opportunistic diseases resulting from the immunodeficiency. Primary and secondary CNS disease were very common in the early era of AIDS, with neurologic symptoms at clinical presentation and CNS abnormalities noted in more than half of patients (3,49). Highly active antiretroviral therapy (HAART) has markedly altered the disease course but not eliminated CNS diseases (50,51).
 FIGURE 10.19 Deep white matter lesion from a 54-year-old woman treated with immunosuppressives for lupus. Astrocytes manifest strikingly atypical nuclei. Abundant foamy macrophages contain blue balls of myelin, which is consistent with demyelination. |
The primary and secondary disease processes in HIV infection and AIDS produce a spectrum of neuropathology, beginning with cerebritis, meningitis, encephalitis, and vascular pathology and ending with degenerative-metabolic changes and neoplasia.
Primary Manifestations of Human Immunodeficiency Virus
HIV causes several primary CNS diseases, including (a) HIV encephalitis, (b) leukoencephalopathy and vacuolar myelopathy, and (c) lymphocytic meningitis (51). These changes can be seen in biopsies of secondary lesions (e.g., toxoplasmosis or lymphoma) or in cerebral biopsies carried out to assess the etiology of unanticipated cognitive decline and detect a treatable cause of disease (see previous section “High-Risk Infectious Disease Case Considerations” for tissue handling).
The hallmarks of HIV encephalitis, considered pathognomonic of the disease, are multinucleated giant cells in the setting of microglial nodules, which form multiple foci of various sizes within white and gray matter (Fig. 10.20). Nonspecific viral encephalitis changes are also noted, including microglial nodules without giant cells. By IHC, the giant cells can contain abundant HIV markers, including p23.
HIV leukoencephalopathy is characterized by diffuse damage to the white matter with loss of myelin, reactive astrogliosis, multinucleated cells, and macrophage infiltrates with scant lymphocytic infiltrates.
Lymphocytic meningitis is yet another primary manifestation of HIV infection and is remarkable for heavy lymphocytic infiltrates within the leptomeninges and perivascular spaces. HIV cerebral vasculitis and granulomatous angiitis may occur with lymphocytic or lymphoplasmahistiocytic multinucleated giant cell infiltration of cerebral vessel walls, occasionally accompanied by necrosis.
Since the onset of HAART, the severity and incidence of primary HIV encephalitis and leukoencephalopathy have decreased (50,51). However, a new form of HIV encephalitis, with severe leukoencephalopathy and intense perivascular macrophage and lymphocyte infiltrates, has been described— postulated to be a response of the revived immune system (50).
 FIGURE 10.20 Human immunodeficiency virus encephalitis. Multinucleated giant cells (arrows) in the setting of a loosely formed microglial nodule accompanied by gliosis. (Courtesy of Dr. Javad Towfighi, Penn State University, Hershey, PA.) |
Secondary Manifestations of AIDS and Other Immunosuppression: Infections and Neoplastic Processes
A wide range of opportunistic diseases occur secondary to the immunosuppression caused by AIDS as well as in the context of therapy-related immunosuppression (3). The organisms of note include (a) fungi, including C. neoformans (Fig. 10.10) and Candida; (b) viruses, including the JC virus (Figs. 10.17,10.18,10.19,10.20 and 10.21) and CMV; (c) parasites, including toxoplasmosis (Figs. 10.12 and 10.13); and (d) bacteria, including Listeria monocytogenes, H. influenzae, Streptococcus pneumonia, Staphylococcus epidermidis, and Pseudomonas (28). Many viral and parasitic diseases likely represent reactivation of the infectious agent, which had been encountered in the past and held in check by the normal immune system.
Primary CNS lymphoma is a well-recognized neoplasm in AIDS (see “Lymphoma” later in this chapter). Other neoplasms can (rarely) involve the CNS in the setting of immunosuppression, including Kaposi sarcoma and leiomyosarcoma.
HAART allows recovery of the immune system in many patients with AIDS and prevents the immunodeficiency with early treatment. This has markedly decreased the incidence of some secondary, opportunistic disease processes. However, the incidence of PML and lymphoma has not been clearly reduced, and it is possible that PML may shift toward a more chronic disease in treated patients (50).
PRION DISEASES
“PRION” was coined from PROteinaceous INfectious particle with the vowels transposed (4). The counterpart of the pathogenic prion protein is a normal constituent of neurons, white blood cells, and other cell membranes with a still-undefined function (4). When an abnormal tertiary structure is acquired, the protein both (a) becomes resistant to normal protein degradation and (b) has the autocatalytic capacity to convert the tertiary structure of other prion proteins to the pathogenic conformation. As this cycle continues, an increasing percentage of normal protein is converted into the pathogenic prion configuration.
 FIGURE 10.21 Progressive multifocal leukoencephalopathy. Cytochemical detection of JC viral sequences by in situ hybridization in larger nuclei stained blue by alkaline phosphatase reaction product. Negative nuclei counterstain pink, and perivascular macrophages contain light brown cytoplasmic granules. (Courtesy of Dr. Ricardo Lloyd, Mayo Clinic, Rochester, MN.) |
Several forms of prion disease affect man, including (a) the most common sporadic form, CJD, in which no germline mutation is present; (b) familial forms, with germline mutations, including Gerstmann-Sträussler-Scheinker, familial CJD, and fatal familial insomnia; and (c) acquired forms, including kuru (passed from person to person by the eating of the human brain of dead relatives), variant CJD (see the following texts), and iatrogenic CJD (patients receiving growth hormone derived from pooled cadaver pituitaries, contaminated neurosurgical and ophthalmologic surgical equipment, depth electrodes, or contaminated transplanted tissue [dura, cornea]) (28). Sporadic CJD is the most common of these. Prion diseases, known as “transmissible spongiform encephalopathies,” occur sporadically in humans, cows, sheep (scrapie), goats, mink, cats, deer, and elk. Variant CJD appears to have arisen in cows in the setting of use of sheep tissue, including scrapiecontaining brain as cattle feed, and was perpetuated by inclusion of affected cow nervous tissue in feed. In man and animals, these diseases are uniformly fatal.
Clinical diagnosis of CJD is difficult and imprecise, especially at early stages. Detection of the 14-3-3 protein in CSF is a valuable but imperfect tool (52). Because many CNS diseases can increase 14-3-3, its utility for CJD may tilt toward a negative 14-3-3 ruling out CJD, rather than a positive 14-3-3 confirming CJD. Ultimately, histopathologic and molecular evaluations of brain tissue are the gold standard for diagnosis (53).
Key considerations for biopsy of a “possible CJD” case include a reasonable chance to find a treatable cause of the cognitive deficits and safety of operating room and pathology staff. Biopsy for the identification of a prion-related disease is not warranted if the clinical suspicion is very high, based on clinical features (rapid progression of the disease), brain imaging studies, electrodiagnostic studies (electroencephalogram), and confirmatory serologic and CSF studies that exclude other explanations (e.g., stroke, vasculitis, meningoencephalitis).
Histologic Appearance
Several histologic changes are evidence of prion disease, including a combination of neuropil vacuolation and gliosis (54). Histologic changes in CJD include (a) vacuolation of the neuropil, with involvement of all six cortical layers in cerebrum or with diffuse involvement of the cerebellar molecular layer (Figs. 10.22 and 10.23); (b) reactive gliosis that is particularly prominent in late stages of the disease (Fig. 10.24); and (c) occasional plaques of amyloid-like material in the neocortex in new-variant CJD. By H&E, a few vacuoles in the neuropil are glassy or lightly eosinophilic rather than clear. Microglial cells are increased in number, but perivascular inflammatory infiltrates are negligible.
Lack of identification of diagnostic histologic changes from a single biopsy site does not rule out CJD because (a) different subtypes of the disease exist, variable with the mutation in the prion protein, with variably cortical-predominant, basal ganglia-predominant, thalamus-predominant, and hippocampalpredominant disease and (b) considerable variation can be seen from gyrus to gyrus in CJD and at different stages of the disease (55,56). A diagnostic pitfall—diffuse cortical vacuolization—is a common finding in advanced Alzheimer disease. Superficial cortical vacuolization occurs in ischemia and in frontotemporal dementia; the vacuoles in these cases are clear, punched out, and different from those in CJD. Processing artifacts can
produce vacuoles that encircle neurons, vessels, and glia; these are larger than CJD vacuoles. Nonetheless, because many vacuoles are not related to prions, molecular diagnosis of prions is needed to confirm suspicions based on vacuoles.
produce vacuoles that encircle neurons, vessels, and glia; these are larger than CJD vacuoles. Nonetheless, because many vacuoles are not related to prions, molecular diagnosis of prions is needed to confirm suspicions based on vacuoles.
 FIGURE 10.22 Creutzfeldt-Jakob disease. Biopsy of the cerebral frontal lobe from a 57-year-old man with suspected Alzheimer disease. Coalescing round vacuoles and gliosis are present in the neuropil. Some of the vacuoles have a glassy appearance rather than a punched out look. These involved all six cortical layers (cresyl violet with eosin). |
 FIGURE 10.23 Variant Creutzfeldt-Jakob disease. This variant is from the cerebral cortex of a 29-year-old woman in the United Kingdom who experienced 17 months of progressive dementia, ataxia, and chorea. Fibrillary plaques composed of prion protein resemble skyrocket bursts, and they are surrounded by very coarse spongiform changes in the neuropil. (Courtesy of Dr. Jeanne E. Bell, National CJD Surveillance Unit, Western General Hospital, Edinburgh, United Kingdom.) |
 FIGURE 10.24 Familial Creutzfeldt-Jakob disease. Most of the neurons have been lost and replaced by gliosis in this case. Phosphotungstic acid hematoxylin stain highlights gliotic fibrils and vacuolated neuropils. |
Immunohistochemistry
IHC after protease predigestion of normal prion proteins helps identify protease-resistant prion protein. However, there is a fairly high background labeling among the IHC techniques, challenging their sensitivity and specificity (54,55). Specifically, most available antibodies manifest some degree of background labeling of neurons in control cases, and they can stain neuritic plaques. These should be interpreted only by an experienced observer.
Molecular Diagnosis
The gold standard for identification of the prion protein is molecular evaluation for the protease digestion-resistant protein by Western blot, performed in specialized medical centers and at the NPDPSC in Cleveland, Ohio (53) (see “High-Risk Infectious Disease Case Considerations” section earlier in this chapter). Sporadic CJD lacks a germline mutation, which can be confirmed by sequencing of the prion protein gene to rule out known familial CJD variants.
Variant Creutzfeldt-Jakob Disease/“Mad Cow Disease”
In the mid-1990s, “new-variant” CJD (vCJD) emerged in Great Britain and France, originally called “mad cow disease.” It has been linked by molecular analysis to the eating of meat from cows with bovine spongiform encephalopathy (BSE), in most cases through contamination of the meat with brain and other highly infected tissue during slaughter and butchering. The previously postulated “species barrier” to transmission was broached (54). By early 2003, approximately 135 cases of vCJD had been reported, most cases diagnosed in the United Kingdom.
In the United Kingdom and Europe, the BSE epidemic is now ending and the number of cases is decreasing, thanks to the strict control of animal foodstuff that was the main source of prion contamination (57). BSE in North America (two cases to date) may be at its beginning. Significant measures have been adopted to reduce the risk from recycling of infection via feed, but it remains to be seen whether they are watertight (58).
Despite vCJD being a small fraction of prion diseases, it is a major public health and economic concern. After halting all imports of U.S. beef in 2003, the Japanese government decided in December 2005 to allow importation of beef from the United States derived from young cattle from which specified risk materials are removed (59). Rapid tests for misfolded prion protein in central nervous tissue of slaughter cattle now allows hope for better monitoring (58), provided our hands are on our wallets and our heads out of the sand.
The histologic appearance of vCJD differs from sporadic CJD. In the cerebral and cerebellar cortex, (a) striking stellate, “florid” plaques composed of prion protein, easily seen by H&E, labeled by PAS and anti-prion protein antibodies; (b) coarse vacuolization rather than the fine vacuolation of CJD; and (c) marked gliosis are the most consistent neuropathologic features (Fig. 10.23) (54). These cerebral cortical plaques and coarse spongiform features have not been seen among typical sporadic CJD cases. Similar plaques can be noted in the cerebellum of sporadic CJD (4,56).
Hereditary Prion Diseases
The vacuolization, neuron loss, and gliosis in hereditary prion disorders such as Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia, and familial CJD (Fig. 10.24) are similar to sporadic CJD and kuru (28). These diseases are rarely biopsied.
INFLAMMATORY DISEASES
Causes of inflammatory diseases of the CNS are elusive because there is considerable overlap in their clinical and histologic findings. A range of inflammatory diseases affects the brain, including connective tissue diseases, sarcoid, and rheumatoid disease. They variably affect leptomeninges, blood vessels, and brain tissue. Altered mental status and meningeal changes invite biopsy to evaluate etiology and to rule out infections. Optimal sampling that includes dura, arachnoid, and gray matter in the area of a radiologic enhancement improves the chance of reaching a lesion.
GRANULOMATOUS INFLAMMATION
Inflammatory diseases can present a granulomatous appearance, with or without distinct granuloma formation, with or without regions of necrosis. See “Reactive Changes” and “Infectious Diseases” sections earlier in this chapter.
Granulomatous inflammation can also be seen in the setting of dural, leptomeningeal, or parenchymal involvement of the CNS by Wegener granulomatosis and rheumatoid arthritis. Granulomatous meningitis has a predilection for the base of the brain. Neurosarcoidosis is always a diagnosis of exclusion in cases of granulomatous inflammation. Infection must be ruled out first. Sarcoid typically affects the leptomeninges and blood vessels of the brain or spinal cord. Lesions contain lymphocytic leptomeningeal infiltration, slight lymphocytic cuffing of intraparenchymal vessels, scattered small leptomeningeal and parenchymal noncaseating granulomas, and mild reactive astrocytosis, especially in the white matter, with only moderate loss of myelin.
SMALL VESSEL DISEASE AND VASCULITIS
Brain biopsies performed in search of small vessel disease, particularly vasculitis, frequently do not provide a satisfactory correlation between the pathologic specimen and the radiographic data, although step sectioning through the entire block of tissue may occasionally yield diagnostic material. Small vessels are primarily affected in isolated angiitis of the CNS, and both angiography and leptomeningeal plus parenchymal biopsy of a radiographic lesion are essential to the diagnosis (60,61 and 62).
Criteria for diagnosis of vasculitis include both (a) vessel wall inflammation and (b) vessel wall damage and destruction. Chronic, healed, or resolving vasculitis manifests irregular vessel wall fibrosis, often with partial damage of elastica. H&E, trichrome, type IV collagen, and elastic stains are keys to confirming vasculitis.
Temporal arteritis is a common disorder that affects small to medium-sized arteries of elderly women. It is often biopsied bilaterally (61). The disease particularly affects the intima, with giant cells a prominent part of the infiltrate. Although uncommon, SLE affects the CNS more frequently than other collagen diseases (62). Rarely, SLE produces microinfarcts and necrotizing vasculitis with thrombosis. Isolated angiitis and arteritis associated with collagen vascular disease, infection, toxins, and granulomatous disease can cause strokes in young people (63).
A multitude of diseases are responsible for individual cases of vasculitis. Cerebral angiitis can be a part of systemic vasculitis in mixed cryoglobulinemia and Wegener disease. Isolated, primary CNS angiitis may include inflammation of very small vessels (60). Angiitis also occurs in association with herpes varicella-zoster virus (64), relapsing polychondritis, and Behçet disease (65). Lymphomatoid granulomatosis is a process that spans inflammatory and neoplastic entities. It is discussed under “Lymphoma.”
Intrathecal methotrexate therapy can cause mineralizing microangiopathy (66). Meningovascular syphilis and associated vasculitis is rising in frequency associated with AIDS. Illicit drugs, such as cocaine, heroin, and amphetamines, affect not only the cardiac vessels but also cerebral vessels and can cause cerebral vasculitis associated with intraparenchymal or subarachnoid hemorrhage (67).
DEMYELINATION
Demyelination results principally from (a) presumed autoimmune etiologies such as MS or acute disseminated encephalomyelitis (ADEM; perivenous demyelination) (68); (b) infectious etiologies, most notably PML (see “Infectious Disease” section earlier in this chapter); and (c) toxic metabolic etiologies, including the leukodystrophies (e.g., Gaucher disease, adrenoleukodystrophy). Survivors of carbon monoxide poisoning and patients with long-standing hypertension and atherosclerosis can have diffuse cerebral hemisphere white matter demyelinating lesions that can histologically mimic the demyelination of MS in surgical specimens. The absence of a margin with less affected brain may help distinguish this from MS plaques, which typically have a sharp margin.
Demyelinating diseases are usually investigated clinically without biopsy (Table 10.2). Exceptions occur with unusual clinical presentations where the clinical diagnosis is unknown or unclear. For instance, active MS lesions can be mistaken radiologically for neoplasm (69). Demyelination due to a primary viral process (PML, HIV leukoencephalitis, CMV, varicella-zoster) is also occasionally seen at biopsy; these entities are considered in the “Viruses” section earlier in this chapter. The leukodystrophies are very rarely seen by the surgical pathologist.
Histology
Primary, acute demyelinating lesions histologically show destruction of myelin and abundant foamy macrophages containing myelin debris and lipid droplets (Fig. 10.25). There is relative sparing of axons in demyelinating disease, although a
fraction of axons are lost in chronic MS. In contrast, axonal loss precedes myelin loss in an infarct (Fig. 10.26). The amount of gliosis increases with the chronicity of the lesion.
fraction of axons are lost in chronic MS. In contrast, axonal loss precedes myelin loss in an infarct (Fig. 10.26). The amount of gliosis increases with the chronicity of the lesion.
 FIGURE 10.25 Vascular involvement in multiple sclerosis (A,B) compared with vasculitis (C,D). Active MS lesion presented on MRI as ring-enhancing lesion in a 24-year-old woman. (A) Abundant plump cells and scant lymphocytes around blood vessel but not in its wall. Initial impression could mistake this lesion for vasculitis or glioma. (B) CD68 immunostain identifies the plump cells as macrophages. Active vasculitis presented on MRI as severe white matter disease with patchy enhancement in a 32-year-old woman. (C) Lymphocytes and macrophages penetrate the pink necrotic wall of the vessel and insinuate between collagen fibers. Endothelium is swollen. (D) Trichrome stain reveals vascular wall penetration and plump endothelium. |
Histochemically, special stains for myelin (LFB-H&E) and axons (Bodian or Bielschowsky silver stain or neurofilament IHC) help to distinguish between (a) demyelination (foamy macrophages containing LFB-positive myelin debris present with axons mostly retained intact in brain tissue) and (b) necrosis, including infarct (axons lost), and Wallerian degeneration (myelin loss is secondary to axonal loss) (Fig. 10.26). GFAP stain helps discern gemistocytic reactive astrocytes from macrophages; CD68 and CD45/CD45RB identify macrophages and lymphocytes. If the lesion was induced by a virus, neuronal or oligodendroglial intranuclear inclusions may be found, particularly at the periphery of the lesion.
Multiple Sclerosis
The major demyelinating disorder of adults is MS. MS is likely an autoimmune disease (70). Despite an immense amount of sophisticated research spanning the past three decades, much remains unknown about this disease. It remains uncertain why some areas of myelin are attacked and others remain spared.
MS manifests acute (or “active”), subacute, and chronic lesions. The former are those that are typically seen at biopsy from a patient suspected of having something other than MS (Fig. 10.25). Due to loss of integrity of the blood-brain barrier, they can have imaging characteristics that mimic a malignant neoplasm (69). MS should be differentiated from other disorders that have similar lesions, including Behçet disease and late-onset forms of leukodystrophies.
Acute Disseminated Encephalomyelitis
Perivenous demyelination is a prominent pathologic finding in postinfectious and postvaccination encephalomyelitis, which is thought to be an autoimmune phenomenon initiated by a viral pathogen, most commonly varicella-zoster and influenza (68,71,72 and 73). ADEM is in some cases associated with hemorrhage. It is rarely biopsied. If biopsied, identification of its perivascular pattern of demyelination is important. The demyelination of MS extends beyond vessels to a confluent patch or plaque.
 FIGURE 10.26 Two different white matter lesions (A,B) are each stained for neurofilaments, with hematoxylin counterstain for nuclei. (A) Demyelination is hard to see except for some pale, lipid-filled macrophages (arrow) between straight brown axons. Axons are spared in demyelination. (B) Focal white matter necrosis produced the pale side of this image in this small infarct. Necrotic axons become very pale and finally disintegrate into small beads that are eaten by macrophages. Some damaged axons swell (arrow). (C) Vacuolar myelopathy as seen in vitamin deficiency, metabolic disorders, toxins, and HIV where vacuolation and demyelation with naked axons (arrow) and blue balls of myelin, some in macrophages (arrowhead), precede axonal damage. Neurofilaments/Luxol fast blue/hematoxylin. |
Differential Diagnosis
A demyelinating lesion may be confused with neoplastic disease. Gliosis in the subacute demyelinating lesion can be robust, with large astrocytes with chromosomes spread apart in their cytoplasm (so-called Creutzfeldt cells), mimicking glioma cells (69). In addition, macrophages can accumulate in large numbers in an active region of demyelination to the extent that they mimic glioma, especially oligodendroglioma, or they may mimic hemangioblastoma. Leu-7- and S-100-positive oligodendrogliomas have fewer cytoplasmic vacuoles and more central nuclei than lipid-laden CD68-positive macrophages. Hemangioblastomas are more vascular than oligodendrogliomas (see Fig. 10.5B and Figs. 10.54 and 10.120 in the “Tumors” section later in this chapter). LFB-H&E stain will reveal phagocytosis of myelin in a demyelinating lesion.
CEREBROVASCULAR DISEASES
INFARCT
Infarction of CNS parenchyma is a common pathologic process often associated with clinical stroke. Thrombosis of an atherosclerotic artery or emboli of blood clot or of atherosclerotic plaque commonly causes infarcts. Other etiologies include coagulopathy, amyloid angiopathy, local vasospasm, major changes in blood pressure, CADASIL, mitochondrial encephalopathy with lactic acidosis and strokes (MELAS), and infectious and inflammatory processes. Infarcts can occur in young adults with normal arteries as a result of a severe episode of hypotension or in association with coagulopathy or cerebral vasospasm (74).
Representative portions of the specimens should be submitted for histologic evaluation to optimize evaluation of blood vessels by sectioning perpendicular to the surface of the brain and by including arachnoid, gray matter, and white matter. Vessels are carefully examined for evidence of embolism, arteriosclerosis, atherosclerosis, amyloid angiopathy (see “Amyloid Angiopathy” section later in this chapter), and an infectious-inflammatory process (see “Small Vessel Diseases” later in this chapter). If mitochondrial disease and CADASIL have not been ruled out, then portions of gray matter and gray-white matter junction should be saved for possible EM examination.
In CNS tissue, the age of the infarct is evaluated for active changes and evidence of organization and classified as acute, subacute, or remote. Special stains for axons (Bodian or Bielschowsky silver stain, neurofilament IHC) help to distinguish between necrotic tissue (axons missing or disintegrated) and demyelination (axons retained).
INTRACRANIAL HEMORRHAGE
Clotted blood or hematoma removed from an intracranial location represents a common surgical neuropathology specimen. Such a specimen can be removed from the epidural, subdural, subarachnoid, or intraparenchymal regions. Possible causes of intracranial hemorrhage are summarized in Table 10.5 (75).
The contribution of the surgical pathologist to such specimens lies in careful macroscopic and microscopic inspection of the material and clinicopathologic correlation with (a) evaluation of potential etiologies for intracranial hemorrhage, including clinically unsuspected causes, such as a cryptic vascular malformation, vascular disease, or neoplasm; and (b) approximate dating of the hematoma as acute, subacute, or chronic.
Close macroscopic examination of aspiration collection device contents and resected specimens should be done, submitting for histologic evaluation representative brain parenchyma and other solid regions when present. At least one cassette of hematoma, or more as indicated by gross findings, should be submitted for processing. Bone fragments may represent bone dust generated during the surgery or may be secondary to traumatic injury.
Microscopic examination of the hematoma can roughly distinguish among (a) acute hemorrhage (minutes to hours old, in which white cell nuclei are intact and no evidence of organization is noted), (b) subacute hemorrhage (days old, in which white cell nuclei are karyorrhectic and macrophages ingesting red cells are noted), and (c) remote or chronic hemorrhage (weeks to years old, with hemosiderin-containing macrophages and ingrowth of blood vessels and fibroblasts in the meninges) and reactive astrocytes surrounding the lesion (in parenchymal lesions). Hemosiderin is evident 4 days after hemorrhage and reactive astrocytes 2 weeks after hemorrhage. Both persist indefinitely.
Epidural and Subdural Hemorrhage
Epidural hemorrhage typically results from trauma, most frequently with temporal bone fracture and rupture of the middle meningeal artery. Most such evacuated specimens contain acute hematoma.
Subdural hemorrhages may or may not follow known trauma (76). They are most common in elderly patients with some degree of cerebral atrophy (and bridging vein susceptibility to tear) as well as in patients with systemic cancer (77) and CNS tumors in postoperative settings (76). Acute subdural hematoma is, as a rule, removed by aspiration; contents may come for evaluation in a suction collection device. Subacute or chronic subdural hematomas require excision and histologic evaluation of the subdural membranes to rule out a neoplasm.
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