CSF Cytology

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CSF/brain smear cytologyDr Alpha Tsui Royal Melbourne Hospital 2008 CSFADEQUACY IN CSF: Sufficient diagnostic cells. If the background is heavily bloodstained from a traumatic tap, it is considered unsatisfactory. At least 5ml of CSF should be submitted if malignancy is suspected clinically. Lymphoid cells in normal CSF: -normal CSF is pauci-cellular -CSF contains just a few mature lymphocytes (mainly T-cells) and monocytes -ratio of lymphocytes to monocytes is approx. 2:1 -normal cell count in CSF is 90% of the cytospin covered by overlapping red blood cells Increase in neutrophil count: -bacterial meningitis -early stage of viral, TB or fungal meningitis -ischaemia -recent surgery -neoplasia Increase in lymphocyte count: -nonbacterial infections -multiple sclerosis -Guillain-Barr -syringomyelia -AIDS Increase in eosinophil count: -drug reaction

-foreign material e.g. shunt -parasitic infection -some tumours e.g. lymphoma, Langerhans cell histiocytosis Increase in macrophage count: -trauma -cerebral infarction -haemorrhage -sarcoidosis -demyelination -Whipples disease -vasculitis -treated lymphoma Specific entities in the CSF: Reaction after subarachnoid haemorrhage: -proliferation of macrophages -phagocytosis of erythrocytes -enzymatic destruction of haemoglobin and ingested RBCs appear as empty cytoplasmic vacuoles in the macrophages -appearance of haemosiderin (after 4 days) -haemosiderin-laden macrophages may persist for more than 6 months Infectious conditions: -increased number of inflammatory cells, esp. polymorphs or activated lymphocytes, suggest infections -acute bacterial meningitis: infiltrate composed predominantly of polymorphs -viral meningoencephalitis: predominantly lymphocytic response -look for organisms (quantity, morphology, intra or extracellular) -needs microbiological culture for definitive diagnosis Reactive lymphocytosis: -reactive and atypical lymphocytosis may be seen in multiple sclerosis, Guillain-Barr syndrome, cryptococcal meningitis -activated lymphocytes and monocytes may become larger with multiple nuclei and prominent nucleoli, mimicking malignant cells -a heterogeneous population of lymphocytes may be difficult to appreciate if there are not that many cells -clues: -heterogeneous population with lymphocytes of varying sizes; majority small and mature -intermixed with monocytes and plasma cells or neutrophils -cells do not have significant nuclear pleomorphism, coarsely clumped chromatin or macronucleoli -look at the Pap stain (Diff-Quik exaggerates cell size) -immunostaining: most of the lymphocytes are T-cells -repeat sample and send fluid off for flow cytometry Cryptococcus:

-yeast itself 7-10m, surrounded by mucoid capsule up to 20m -look for the capsule and budding yeasts -when intracellular, may look like intracytoplasmic vacuole -capsule-deficient form may be difficult to recognise (in AIDS patients). Yeasts much smaller (2.5m) -DDx: erythrocytes; talc powder - polarise it with Maltese-cross pattern; corpora amylacea Chronic asceptic meningitis (Mollaret meningitis): -culture negative -aetiology unclear -characterised by initial neutrophilic response that progresses to a lymphocytic infiltrate -Mollaret cells are large monocytes with abundant vacuolated cytoplasm Lymphoma / leukaemia: -cannot distinguish lymphoma from leukaemia on cytology alone -some leukaemic cells tend to have convoluted, horseshoe type nuclei -leukaemic cells may have more cytoplasm -myeloid cells have cytoplasmic granules, Auer rods -small numbers of malignant cells problematic (e.g. during chemotherapy) How to deal with small numbers of malignant lymphoid cells: -be sure that the cells have malignant criteria (nuclear pleomorphism beyond that seen in reactive cells, hyperchromasia, coarse chromatin, prominent irregular nucleoli) -look for specific differentiation e.g. cytoplasmic granules, Auer rods -compare and contrast with adjacent normal lymphoid cells -compare and contrast with previous cytology -immunostaining -flow cytometry Metastatic carcinoma: -tumours need to extend into the ventricular system or subarachnoid space +/leptomeningeal involvement before shedding the cells into the CSF -this usually implies late stage disease and a diagnosis would have almost certainly been made in the past -commonly from lung, breast, stomach -greater tendency for cells to shed singly or in small loose clusters rather than in cohesive tissue fragments (i.e. the tumour cells may look like melanoma or lymphoma) -in adenocarcinomas, glandular structures are rarely identified -can confirm mucinous vacuoles by mucin stains -keratinisation is also difficult to demonstrate. Look for dense cytoplasm, tadpole cells in squamous cell carcinomaIn a fluid milieu, any cell may become vacuolated and this does not necessarily indicate adenocarcinoma.

Metastatic melanoma: -cells are large -coarse nuclear chromatin -prominent macronucleoli, binucleation, intranuclear pseudoinclusions -moderate amounts of cytoplasm, containing melanin Astrocytoma: -single cells or forming aggregates -fibrillary matrix made from the astrocytic cytoplasmic processes not shed in the CSF -cells vary from small to large in size -high N/C ratio with irregular hyperchromatic nuclei and scanty cytoplasm -may have gemistocytic form with moderate amounts of eosinophilic cytoplasm and eccentric nuclei Medulloblastoma: -shows a tendency for cell cohesion -cells show nuclear moulding, scant cytoplasm, hyperchromatic nuclei, mimicking a small cell carcinoma Choroid plexus papilloma: -the tumour cells may be as bland as the normal choroid plexus cells -the smears are much more cellular with numerous papillary clusters -the diagnosis is based on the finding of an intraventricular mass

FALSE POSITIVES: -degenerate cells -normal components: chondrocytes, ependymal / choroidal cells, bone marrow elements: megakaryocytes, immature myeloid elements -traumatic tap containing circulating tumour cells from peripheral blood -reactive lymphocytosis from various causes -foamy and vacuolated macrophages mimicking adenocarcinoma -haemosiderin-laden macrophages mimicking melanoma cells -viral inclusions -post-intrathecal treatment effect

FALSE NEGATIVES: -often only small numbers of tumour cells are seen -necrotic tumours -low grade astrocytic tumours e.g. pilocytic astrocytoma -AML with monocytic differentiation mimicking benign monocytes

PRACTICAL TIPS:

1. Know the cellularity of normal CSF. Any derivation is abnormal. Cells from CSF often become degenerate (looking atypical) if not processed quickly. Only assess well-preserved cells. 2. Beware starch granules or erythrocytes mimicking cryptococcus. Use polarised light. 3. Bloody background with entrapped inflammatory cells is unsatisfactory. It should not be assumed to represent true inflammation. Also beware traumatic tap containing circulating tumour cells. Need to repeat specimen in these instances. 4. Reactive lymphocytosis with activated lymphocytes may mimic lymphoma. Look for heterogeneity of the cells. May need flow cytometry in difficult cases. Check the serum white cell count. 5. Carcinomas often present as single cells, mimicking a melanoma or lymphoma. Need to do immunostains on a cell block to confirm unless there is a known past history.

Fig 1. Normal CSF. Note hypocellularity.

Fig 2. Normal chondrocytes.

Fig 3. Normal ependymal/choroidal cells. May be mistaken for a carcinoma.

Fig 4. Normal ependymal/choroidal cells with columnar shaped cytoplasm.

Fig 5. Unsatisfactory. Degenerate cells with smudged nuclear detail.

Fig 6. Bacterial meningitis with neutrophils and bacterial cocci.

Fig 7. Viral meningitis with lymphocytes and monocytes.

Fig 8. Chronic asceptic meningitis with lymphocytes and vacuolated monocytes (Mollaret cell Arrow).

Fig 9. Cryptococcus.

Fig 10. Erythrophagocytosis, which may mimic cryptococcus.

Fig 11. Corpora amylacea, which resembles cryptococcus.

Fig 12. Subarachnoid haemorrhage.

Fig 13. Metastatic carcinoma with single cell presentation.

Fig 14. Metastatic squamous cell carcinoma, which often elicits prominent acute inflammation.

Fig 15. Metastatic small cell carcinoma from the lung. Note nuclear moulding.

Fig 16. Metastatic melanoma.

Fig 17. Non-Hodgkin lymphoma.

Fig 18. Leukaemia (Acute myeloid leukaemia).

Fig 19. Leukaemia. Tumour cells usually show marked nuclear irregularity.

Fig 20. Glioblastoma multiforme.

Fig 21. Brain fluid cytology: Glioblastoma multiforme.

Brain smears:Normal: Normal cerebral cortex: -abundant neurons that are uniformly distributed -oligodendroglial cells with small round nuclei -microglial cells with small elongated nuclei -fibrillary background, branching meshwork of thin-walled capillaries Normal deep cerebral grey matter: -larger neurons often prominent -calcospherites may be seen in older patients Normal white matter: -background neuropil is typically coarser and more fibrillary than that of grey matter because of greater proportion of myelinated fibres -mixture of oligodendroglial, microglial and astrocytic nuclei -less vascular -normal glial cell nuclei are small with no perinuclear cytoplasm Normal cerebellar cortex: -the granular layer of the normal cerebellar cortex is one of the most cellular regions of the CNS -smears show hypercellular zones in a fibrillary background -most numerous cells are granule cells: round nuclei with central chromatin aggregate (targetoid appearance). Size of the nucleus is about that of a normal lymphocyte. -Purkinje cells: very large cell with pyramidal or bulbous shape cell body, 1-2 prominent nucleoli, abundant granular cytoplasm, thick dendritic processes extending from the cell bodySmears from normal cerebellar cortex may mimic a small round cell tumour, esp. medulloblastoma.