Clinical diagnostic Cytology Central Nervous System.pdf

1. www.vut.ac.za
Presented by: I Madonsela
Health Sciences
Learning Unit 4
Central Nervous System (CNS)
1

2. Anatomy of CNS
• The central nervous system consists of the brain, the spinal
cord, and the peripheral nerves.
• Cerebrospinal fluid (CSF) is the liquid that surrounds the
brain & spinal cord.
• The microscopic examination of CSF is essential to the early
detection of many diseases involving the central nervous
system (CNS).
• CSF cytology is used to investigate patients with CNS disease,
especially infections, management of patients with leukemia
or lymphomas, abnormal bleeding and metastatic tumours.

3. Anatomy of CNS

4. Anatomy of CNS
• Brain contains 4 ventricles which are line by cuboidal cell
layer called the ependyma.
• In some areas the ependyma differentiates into a villous
structure called the choroid plexus.
• The brain & spinal cord are surrounded by 3 membranes
known as the meninges that consists:
- Dura mater - outer
- Arachnoid mater - middle and
- Pia mater – inner
• CSF flow between the arachnoid & pia mater in an area
referred to as the subarachnoid space.

5. Anatomy of CNS

7. Anatomy of CNS
• The choroid plexus produces cerebrospinal fluid (CSF) mainly
in the lateral, third and fourth ventricles by filtering plasma
across capillary walls and actively secreting it.
• Some originate from the ependymal cells lining the
ventricles and from the capillaries of the brain and metabolic
water production.
• The total volume of CSF in body is 90 and 150ml.
• CSF is continually produced and absorbed, with complete
turnover every 5 to 7 hours.

8. Functions of CSF
• Cushioning the brain by providing a bath in which the brain
floats.
• Circulation of nutrients .
• Removal of waste.

9. Specimen Collection
• Most CSF specimens are obtained by LP, in which a needle is passed
through the intervertebral space at L3 to L4 or L4 to L5.
• Rarely, because of inflammation at these sites or a bony abnormality,
the specimen must be obtained from the cisterna magna at the base
of the brain.
• CSF is sometimes aspirated directly from a lateral ventricle during a
neurosurgical procedure; such specimens often contain microscopic
fragments of normal brain.
• In patients undergoing chemotherapy for leptomeningeal metastasis,
a silicone pouch (Ommaya reservoir) is implanted in subcutaneous
tissue.
• A cannula leads from the pouch into a lateral ventricle through a 3-
mm burr hole. This is an efficient way to introduce chemotherapeutic
drugs and withdraw CSF periodically for examination.

10. Specimen Collection
• CSF is usually obtained by
lumbar puncture, but can be
obtained from ventricles
(ventricular shunt).

11. Preparatory Techniques
• CSF is normally clear and colourless.
• *Any other appearance is abnormal and indicates disease or
traumatic tap
• CSF specimen should be processed fresh, within 1 hour of
collection, cells undergo rapid degeneration.
• CSF is sparsely cellular even in disease therefore special
concentrating techniques such as :
- Cytocentrifugation (is the method of choice)
- Thinlayer preparation
- Membrane Filtration
• CSF should be processed a.s.a.p, because the cells degenerate
rapidly

12. Preparatory Techniques
• Cytocentrifugation has greater flexibility because both
alcohol-fixed and air-dried slides can be prepared using this
method.
• This method involves spinning of cells directly onto a slide. It
produces a monolayer of cells
• Lymphoid cells are best evaluated using air-dried
preparations, thus it is advisable to prepare an air-dried
Romanowsky-stained slide in addition to the traditional
alcohol-fixed Papanicolaou stained slide.

13. Normal CSF Cells
• LP CSF specimen normally contain a few cells – mainly
mononuclear WBCs (lymphocytes and monocytes)
Lymphocytes
- Small, mature lymphcytes
- Small round nuclei
- Smooth nuclei outline
- Coarse, dense chromatin
- Invisible nucleoli
- Scanty, basophilic cytoplasm
- Reactive lymphocytes have larger nuclei, which may be
cleaved, may have nucleoli and more abundant cytoplasm

14. Normal CSF Cells
Monocytes
- Bone marrow derived.
- Larger than lymphocytes.
- Eccentrically located, oval or folded kidney bean shaped
nuclei.
- Chromatin is bland and has a “salt and pepper appearance,
nucleoli is invisible but when reactive can appear single and
prominent.
- Moderate cytoplasm.

16. CSF Cells
Neutrophils
- Normally sparse , act against bacteria and therefore increased in
acute bacterial meningitis or acute process, surgery, infarcts and early
viral meningitis. Neutrophils are increased when blood is present.
Eosinophils
- Are rare, if present, especially in large numbers, they suggest parasitic
infection
Macrophages
- Associated with destructive disorders, trauma, infarct, foreign bodies
- Derived from transformed monocytes – clean up cells
- Containing fat, blood pigment may be seen

17. CSF Cells
Red Blood Cells:
• RBC’s are not normally present in CSF but a few are
commonly seen in concentrated specimens due to capillary
bleeding when the specimen was obtained.
• When numerous, RBC’s may indicate a pathologic bleeding
or a traumatic tap.
Traumatic Tap
• Common in infants who are difficult to restrain.
• CSF is clear in succeeding tubes
• Show fresh intact, well preserved RBCs

18. CSF Cells
Pathologic Bleeding
• Blood from a pathologic bleed in CSF is evenly distributed
among tubes
• Bleeding is characterized by xanthochromia (yellow) and the
presence of degenerated blood and later haemosiderin-
laden macrophages

19. Normal CNS Cells
• Ventricular shunt samples contain microscopic fragment of brain cells
including: choroid plexus cells, astrocytes, ependymal cells, and
leptomeningeal cells.
Ependymal and choroid plexus cells – line ventricles
- Uniform, small cuboidal/ columnar cells which usually forms loose
clusters and resemble histiocytes.
- Cytoplasm transparent, moderately abundant, basophilic with ill-
defined cell borders and branching processes may be seen
- Terminal bar with cilia or basal bodies characteristic
- Nuclei single, central, round or oval with delicate chromatin and
small nucleoli

20. Normal CNS Cells
Choroid plexus/ependymal cells

21. Normal CNS Cells
Pia Arachnoid (leptomeningeal cells)
- Cells resemble mesothelial cells or monocytes
- Usually sparse and single
- Cytoplasm abundant and delicate with rounded ill defined
borders.
- Nuclei are eccentric, round to oval to bean shape with fine
chromatin.

22. Normal CNS Cells
Neurons or Neuroglia
• Neurons of the CNS are supported by two types of brain
tissue:
• Astrocytes
• Oligodendrocytes
• May be found after a procedure that penetrates the brain
substance or in some disease.
• However, normal neurons or glia are never found in
specimens obtained by LP

23. Normal CNS Cells
Astrocytes
- Are spindle or oval shaped with multiple branches.
- Finely fibrillar cytoplasm, poorly defined borders with multiple
branching processes.
- Nuclei oval, hyperchromatic to pyknotic
Oligodendroglial cells
- Cells are smaller than astrocytes
- Found single or may form small sheets
- Cytoplasm is transparent with elongated fine branching
processes
- Nuclei are small, single, round to elongated & hyperchromatic.

24. CSF of Nonneoplastic conditions
Acute bacterial meningitis
• CSF shows numerous neutrophils, fibrin, macrophages, cell
debris and sometimes bacteria
• Many bacteria can cause meningitis, including Neisseria
meningitidis (meningococcus), Haemophilus influenzae,
Streptococcus pneumoniae (pneumococcus)
• Bacterial meningitis can be fatal if not treated immediately,
prompt diagnosis is crucial.
Tuberculous meningitis
• Lymphocytes and plasma cells are the characteristic
cytological finding, but neutrophils may also be present

25. CSF of Nonneoplastic conditions
Viral meningitis
• CSF shows increase in small, mature lymphocytes, but also
reactive lymphocytes, plasma cells and monocytes
• Early in the course of the disease neutrophils predominate.
Later small mature lymphocytes and degenerated
monocytes become more common
• Diagnostic cellular changes of HSV and CMV not seen in CSF
Cryptococcal meningitis
• Cryptococcus is most common fungal organism seen is CSF,
usually found in immunosuppressed patients such as
patients with transplant, lymphoma/leukemia or AIDS or
occasionally healthy individuals

26. Cryptococcus neoformans
• Round and pale budding yeasts, teardrop shaped, (no pseudohyphae)
that is about 5-15mm in diameter.
• The spherical yeast produce a single bud/spore, attached to the
mother cell by a narrow pinch.
• Yeast have a refractile center and are surrounded by thick
gelatinous/mucoid capsule that does not stain with Pap technique,
thus leaving a characteristic halo around the centrally stained area.
• Seen dispersed extracellularly or engulfed by histiocytes.
• Formation of granulomas may be observed.
• Pink/purple Pap stain
• Special stains : mucicarmine or Gomori-metheneamine silver
positive.

27. Cryptococcus neoformans

28. Malignant Conditions
• Majority of tumors diagnosed with CSF cytology are
secondary lesions, mostly lymphoma/leukemia followed by
metastases
• The most commonly seen primary tumors:
Astrocytoma and glioblastoma multiforme (GM) in adults
and medulloblastoma in children

29. Primary Central Nervous System Tumors
Gliomas: are benign and malignant tumours of supporting
tissue of the CNS.
• Most common primary CNS tumours in children and adults.
• Gliomas difficult to diagnose on CSF are they rarely involve
the leptomeninges and ventricular system.
• Include the following:
- Astrocytomas
- Glioblastoma multiforme
- Oligodendroglioma
- Ependymoma
- Choroid plexus tumours

30. Primary Central Nervous System Tumors
CSF cytology of Astrocytoma:
• Cells form clusters or may be single
• Cells vary in size, shape and nuclear atypia depending on
grade of tumor
• Spindle cells are typical of astrocytoma
• Cytoplasm varies from scanty-abundant
• Nuclei varies from bland( round, oval, pale, fine chromatin)-
clearly malignant (coarse, irregular distributed chromatin
with prominent irregular nucleoli)
• Background may show debris and inflammation cells

31. Primary Central Nervous System Tumors
CSF cytology of Glioblastoma Multiforme:
• Large, pleomorphic, spindle-bizarre shaped cells with dense
cytoplasm are characteristic.
• They have hyperchromatic, highly pleomorphic nuclei with
coarse chromatin, irregular nuclear outlines.
• Cytoplasm is abundant and show cytoplasmic extensions.
• Multinucleated giant cells may be present
• Anaplastic small cell component may also be present
Presence of small and giant malignant cells highly suggestive
of GM

33. Secondary/Metaplastic Central Nervous
System Tumors
Neural crest Tumours
• Neoplasm of children and young adults- includes the
following:
- Medulloblastoma - cerebellum
- Retinoblastoma - Retina
- Neuroblastoma – adrenal medulla or peripheral ganglia
- Pineoblastoma
• Impossible to distinguish the specific type by CSF cytology
alone.
• Clinical history – site of the tumour allows specific diagnosis.

34. Secondary/Metaplstic Central Nervous System
Tumors
Cytomorphology Neural crest
tumours:
• The cells of various neural crest
tumours are all small and similar
in appearance:
• Small, cohesive cell, rosette
formation are characteristic
• High N/C ratio, nuclei molding,
hyperchromasia and
inconspicuous nucleoli
• Cytoplasm is scanty and poorly
defined.

35. Secondary/Metaplastic Central Nervous
System Tumors
Differential diagnosis:
• Other small blue cell tumours
- Small cell carcinoma
- Lymphoma/leukaemia
- Wilm’s tumour
• Glioblastoman Multiforme

36. Neural Crest Tumour: Medulloblastoma

37. Haemato-lymphoid Malignancy
Leukaemia
• Leukaemia are malignant neoplasms of haemopoietic stem
cells, results in high numbers of abnormal cells which are not
fully developed called blasts.
• Acute leukaemia have an abrupt onset.
• Accumulation of blasts in acute leukaemia results from
failure of maturation into functional end cells rather than
rapid proliferation of transformed cells.

38. Haemato-lymphoid Malignancy
Acute lymphoblastic leukaemia
• The peak incidence is between the ages of 2 and 7 years, but
adults are also affected.
• The appearance of ALL cells is variable. According to the
French-American-British (FAB) classification system, ALL is
divided into types L1, L2, and L3 based on the
cytomorphologic appearance of blasts on air-dried
Romanowsky-stained preparations

39. Cytomorphology of acute lymphoblastic
leukaemia
L1
• small blasts
• round nucleus (rare clefts)
• fine chromatin
• inconspicuous nucleolus
• scant, slightly basophilic cytoplasm
L2
• larger blasts
• irregular nucleus
• fine chromatin
• prominent nucleolus
• abundant cytoplasm

40. Cytomorphology of Acute Lymphoblastic
Leukaemia
L3 (leukemic variant of Burkitt lymphoma)
• coarse chromatin
• multiple nucleoli
• dark-blue cytoplasm
• small cytoplasmic vacuoles (lipid)

41. Haemato-lymphoid Malignancy
Acute Myeloid leukaemia
Neoplastic proliferations of the myeloid progenitor cells: immature
granulocytes, monocytes, erythrocytes, and megakaryocytes.

42. Haemato-lymphoid Malignancy
Chronic Lymphocytic leukaemia
• Chronic lymphocytic leukemia (CLL) predominantly affects
adults.
• Chronic Myeloid leukaemia (CML) is a disorder of the elderly
(mean age 60 year)
• The cells of CLL are morphologically indistinguishable from
small, mature lymphocytes.

43. Haemato-lymphoid Malignancy
Lymphoma
• CSF usually cellular
• Monomorphic population of atypical cells, which are usually larger
than small, mature lymphocytes.
• Cells usually occur single with no true tissue aggregates.
• Nuclei enlarged and often have lobulated or irregular nuclear
membranes (grooves and knobs)
• Chromatin varies from fine-coarse and hyperchromatic.

44. Haemato-lymphoid Malignancy
Lymphoma
• Nucleoli may be prominent.
• Mitosis can sometimes be seen.
• Cytoplasm varies from scanty to
abundant depending on type.

45. Other Common Metastasis
• Lung Carcinoma:
Common cancer that commonly metastasize to CNS
-Small cell carcinoma: small cohesive cells with high
N/C ratio and nuclear molding are seen
-Adenocarcinoma: pleomorphic cells with large,
irregular nuclei, irregular chromatin, prominent
nucleoli and foamy cytoplasm can be seen
Glandular features predominate

46. Other Common Metastasis
• Breast Carcinoma
- Common Ductal ca and Lobular carcinoma
Cells usually single/loose clusters
In duct ca. medium-large cells are seen
In lobular ca. small, single, uniform cells are seen

47. Metastatic malignancies (Breast)

48. Other Common Metastasis
• Melanoma
- Large single cells, with large eccentric nuclei,
macronucleoli and cytoplasmic pigment
• Stomach
- Cells are small-medium sized and present
single or in small clusters
Eccentric malignant nuclei with moderate amount
of vacuolated cytoplasm
Signet ring cells may be seen