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					                      Effusion cytology
             Dr Alpha Tsui Royal Melbourne Hospital 2008
General points:
-large unilateral effusion (>1 litre) in the elderly is highly suspicious for malignancy
-effusions associated with malignancies may be just a reactive response without
presence of any tumour cells
-number of neoplastic cells in recurrent effusions often increases. Ask for re-tap if
first tap shows no malignant cells or inconclusive
-in large suspicious effusions, if initial 4 slides show no malignant cells, re-prep
further slides and cell blocks

Gross appearance of effusion fluids:
-note volume, colour, clarity, malodour, opalescence, high viscosity
-fluid containing many cancer cells, if allowed to stand, may form a thick, whitish
yellow layer at bottom of container
-pleural fluid from rheumatoid pleuritis may contain a heavy, whitish, flocculent
sediment and the supernatant may have appearance of fruit juice
-fluids containing numerous pigmented melanoma cells may be chocolate brown
-fluids containing many haemosiderin-laden macrophages are lighter brown
-effusions caused by mesothelioma often contain a high concentration of hyaluronic
acid which increases the viscosity (may be a “dry-tap” clinically)

-low protein content, usually <3g/dL
-low specific gravity, <1.015
-lower cellular content
-lower fibrin content
-implies systemic problems e.g. cardiac, liver, renal failure

-higher protein content, usually >3g/dL
-higher specific gravity, >1.015
-high cellular content
-contains many inflammatory cells or neoplastic cells
-implies local problems e.g. infection, tumour

ADEQUACY: No quantitative rule for number of mesothelial cells on a slide. But at
least 50ml of effusion fluid is required for optimal assessment.

Normal cells in effusion fluid:
-RBCs, leukocytes, mesothelial cells

Normal mesothelial cells:
-cohesive, monolayered sheets of cells
-mesothelial cells forming small cell balls; may have central collagen core (especially
when forcibly removed)
-collagen cores are more commonly seen (but not diagnostic) in mesothelial cell
groups (benign or malignant). They are rare in carcinomatous effusions.
-round or oval nucleus centrally or slightly eccentrically located
-nucleus may be single or multiple
-most often show 1 or multiple small nucleoli
-nucleus rarely appears to actually form outer boundary of a cell
-abundant dense cytoplasm
-lacy collar (due to degeneration of microvilli), forming a vacuolated rim around the
entire circumference of the cell
-cytoplasmic blebbing (larger vacuole with coalescence of microvilli)
-mesothelial window, formed by interdigitating long and slender surface microvilli

Reactive mesothelial cells:
-the cytoplasm becomes dense with 2-tone endoplasmic-ectoplasmic margin
-endoplasm: paler area around the nucleus. Ectoplasm: darker area at the periphery of
the cytoplasm
-may form cell balls, containing generally fewer than 20-25 cells per cluster
-may also form small papillary structures
-mild nuclear enlargement, smooth nuclear membrane
-chromatin fine, open, vesicular (not coarse, not hyperchromatic)
-nucleoli usually present and centrally located
-immunostains generally not helpful: desmin positive; weak or absent membranous
staining for EMA; p53 largely negative

Vacuolated mesothelial cells:
-vacuolation commonly due to degeneration; may be therapy-induced
-cytoplasmic vacuoles, initially present at the endo-ectoplasmic border
-degenerative mesothelial vacuoles often overlap the nucleus without pushing it aside
or distorting it (i.e. not forming a signet-ring cell with compressed nucleus)
-vacuoles do not contain mucin
-the nuclear morphology of the vacuolated cells is exactly the same as the
neighbouring non-vacuolated mesothelial cells
-no malignant nuclear features
-as effusion becomes chronic, the vacuoles become larger

Numerous neutrophils in fluid:
-infarction and rupture of organ
-beware broncho or oesophageal pleural fistula by squamous cell carcinoma. Look for
malignant cells in necrotic cell debris

Numerous lymphocytes in fluid:
-chronic pleurisy
-viral pneumonia
-lymphoma / leukaemia
-other malignancies
Numerous eosinophils in fluid:
Note: eosinophilic cytoplasmic granules not seen on Pap stain
-repeated taps, chronic drain tube placement
-autoimmune disorders
-pulmonary infarct
-fungal infection
-parasitic infection
-pneumothorax and haemothorax
-peritoneal dialysis

Differential diagnosis of second benign population of cells in peritoneal fluid:
-fallopian tube epithelium
-ruptured cysts
-pelvic inflammatory disease
-ectopic pregnancy
-ovarian torsion
-tubal abscess

Specific entities:

Rheumatoid arthritis:
-spindle cell macrophages: tapering ends, moderately dense cytoplasm, often has a
finely granular ground-glass appearance, nuclei frequently multiple
-multinucleated giant cells: may contain 20 or more nuclei
-necrotic granular background material
-ragocytes: neutrophils containing degenerated nuclear debris, appearing as spheric
cytoplasmic inclusions
-neutrophils may also contain fat droplets
-eosinophils, neutrophils, lymphocytes may be present
-mesothelial cells are noticeably absent
-cholesterol crystals may be present

-LE cell: when a haematoxylin body is phagocytosed by a cell
-haematoxylin bodies in the background
-tart cell: macrophage which has phagocytosed a bare nucleus of a cell that underwent
-reactive mesothelial cells
Pulmonary infarct:
-cellularity ranges from slight to profuse
-mesothelial cells, macrophages including haemosiderophages, lymphocytes,
neutrophils are present
-eosinophils may be seen

-high proportion of lymphocytes (80-100%)
-very few or no mesothelial cells
-numerous mesothelial cells should be regarded as strong evidence that the effusion is
not caused by TB
-may also contain neutrophils, plasma cells, macrophages, RBCs
-multinucleated giant cells and epithelioid cells are very rare

-2 steps in the diagnosis of mesothelioma:
    -cells must be recognised as being mesothelial in origin
    -they must be neoplastic
-hypercellular smear with large cell clusters, most clusters >50 cells
-cell clusters have scalloped contours (mulberry appearance), may be elongated or
papillary. Acinar structures can be seen (more so in FNA samples).
-large cell clusters often accompanied by smaller clusters and single cells
-single population of cells and all cells are mesothelial-like
-individual cells are enlarged with increased N/C ratio
-nuclei smoothly round or oval, nucleoli prominent
-nuclear chromatin more coarsely granular
-dense 2-tone cytoplasm
-may see large, crescent-shaped lakes of glycogen in perinuclear position (in Pap
stain: yellow). This is not a feature of adenocarcinoma.
-lacy collar maintained
-cytoplasmic blebbing is seen in degenerate cells with coalescing microvilli
-cell-in-cell engulfment seen more often in mesothelioma but also present in reactive
mesothelial cells
-cytoplasmic vacuolation is non-specific (present in both mesothelioma and
adenocarcinoma). However, demonstration of hyaluronic acid in the cytoplasmic
vacuoles is characteristic for mesothelioma. Hyaluronic acid presents as magenta-
coloured intra / extracytoplasmic material with Giemsa stain and grey streaks with
Pap stain (needs to do Alcian blue and hyaluronidase to confirm)
-cytoplasm is elongated and tapered in sarcomatoid mesotheliomas. Immunostaining
is required to confirm the diagnosis.
-occasional apoptotic cells with pyknotic nuclei and orangeophilic cytoplasm
(squamous-like cells) are present and they are quite specific for mesothelioma (due to
abundant keratin filaments in the cytoplasm). They are not generally seen in benign
effusions or in adenocarcinomas.
-may have psammoma bodies (non-specific: seen in both benign and malignant cases)
-always perform immunostains on the cell block (initial panel: Calretinin, Mesothelin,
CK5/6, WT1, CEA, TTF-1, BerEP4)
          Helpful features favouring mesothelioma over adenocarcinoma: One-cell
   population, windows, lacy collar, dense 2-tone cytoplasm, collagen cores, presence of
   hyaluronic acid, squamous-like cells.

            Helpful features favouring mesothelioma over reactive change: Marked
   cellularity with large cell groups and papillae, high N/C ratio, hyperchromasia, irregular
   nuclear contour, prominent irregular nucleoli, coarse chromatin, squamous-like cells,

-tendency to form smoothly contoured cohesive groups
-'proliferation spheres' frequently present with breast and to lesser extent with ovarian
and lung carcinomas (proliferation spheres not seen in recently developed malignant
-may also be single cells or papillary fragments
-large cells with eccentric nuclei, forming edge of cytoplasm
-prominent nucleoli
-less well-defined, delicate, foamy and vacuolated cytoplasm, containing mucin
-signet-ring cells
-crowded vacuolated tumour cells may produce irregular intercellular spaces but these
are not true windows
-metastatic breast, gastric, renal and pancreatic carcinomas can be very difficult to
pick out as the tumour cells are often single with only mild atypia. Must do
immunostains if there is a strong clinical suspicion or for staging purposes e.g.
peritoneal washings. Panel: ER, PR, CDX-2, CK20, CD10.

          Features suggestive of adenocarcinoma from the breast: Cannonballs
   with smooth borders; targetoid inclusions; linear files.

Serous cystadenoma of the ovary:
-usually of low cellularity
-clusters, sheets and occasionally, papillary groups
-cells have cuboidal or columnar shape
-round to oval nuclei containing finely granular chromatin
-inconspicuous nucleoli
-scant to moderate cytoplasm
-cell borders well-defined within sheets
-overlapping cells not present
-ciliated columnar cells may be found

Mucinous cystadenoma:
-honeycomb sheets may be seen
-round uniform basal nuclei
-uniform columnar cells with cytoplasmic vacuolisation
-lack cilia
-mucin-filled macrophages and mucin in the background
Borderline mucinous cystadenoma:
-replacement of honeycomb pattern by crowded and palisaded cells which form
-dissociated cells may be seen

Adenocarcinoma of ovary:
-usually very cellular
-cells in large acinar or papillary clusters mixed with numerous single cells
-nuclear pleomorphism with coarse irregularly distributed chromatin, prominent
-many vacuolated cells
-tumour giant cells
-necrotic debris
-psammoma bodies

Ovarian borderline tumour vs carcinoma:
Features favouring carcinoma:
-more cellular fragments with irregular borders
-frequent single cells and mitoses
-cells larger, more pleomorphic

Pseudomyxoma peritonei:
-most from tumours of the appendix (adenoma / adenocarcinoma). Some cases from
ovary, pancreas
-abundant mucin +/- epithelium
-epithelium usually at least dysplastic

Squamous cell carcinoma:
-SCC in serous fluid is uncommon, most from lung, larynx, female genital tract
-malignant cells are usually few
-more flat, 2-D appearance of fragments
-tadpole and fibre cells are rare
-well-defined cell boundaries within fragments
-nuclei centrally located
-coarse chromatin
-single malignant cells with 'ink black' nuclei and abundant densely staining
cytoplasm characteristic

Small cell carcinoma:
-cohesive tiny groups, frequently in the form of chains
-tumour cells are small but at least 2x the size of lymphocytes
-nuclear angulation, nuclear moulding
-scant cytoplasm

Urothelial carcinoma:
-morphology non-specific
-cells are epithelioid or spindled
-eccentric angulated and hyperchromatic nuclei
-some may show squamous differentiation
-large nucleoli
-bi or multinucleation
-nuclei eccentric and oval
-intranuclear pseudoinclusions
-abundant cytoplasm
-melanin pigment

-post radiation or chemotherapy - large bizarre multinucleated mesothelial cells
-extramedullary haemopoiesis including megakaryocytes
-reactive changes from various causes
-degenerate benign vacuolated mesothelial cells, mimicking adenocarcinoma

-single cell presentation in some carcinomas e.g. breast, stomach, pancreas
-bland looking carcinoma e.g. renal cell carcinoma, looking like histiocytes
-small cell melanoma with bland morphology
-single cell presentation in mesothelioma with only mild nuclear atypia
-tumour cells (e.g. small cell carcinoma) obscured by inflammation, reactive
mesothelial cells
-hypocellular tumours e.g. squamous cell carcinoma, sarcoma
-low grade lymphoma
-difficult to distinguish borderline ovarian tumours from low grade carcinomas


1. If a foreign population of atypical epithelial cells can be seen, it is malignant until
   proven otherwise.

2. Should not see many cell balls in normal pleural fluid, some in pericardial fluid,
   common in ascitic fluid.

3. Large papillary aggregates are significant in pleural effusions. The greater the
   number and the size of the aggregates, the greater the concern. Large cell balls
   >50 cells, even with only mild atypia → beware neoplastic process.

4. Necrosis and apoptosis are abnormal, often associated with malignancy.
5. Presence of thick 3D groups is a warning sign for malignancy, as compared with
   benign 2-D flat monolayered honeycomb sheets.

6. If there are too many mesothelial cells (markedly cellular on low-power), even if
   single or in small groups, beware that it may be mesothelioma. Beware single cell
   presentation in mesothelioma.

7. Even if it is poorly differentiated mesothelioma, the cells are still mesothelial-like.
   If the tumour cells are bizarre and markedly pleomorphic, think about carcinoma

8. Pure population of carcinoma cells with no mesothelial cells can be seen but the
   tumour cells are not mesothelial-like.

9. Look carefully at the inflammatory cells in the background (reactive lymphocytes
   are predominantly T-cells). Beware haematopoietic malignancy esp. if the
   lymphocytes are mainly B-cells.

10. Always do immunostains on a cell block to confirm mesothelioma, even if the
    cytology is diagnostic. All cases of mesotheliomas (esp. first presentation) should
    be confirmed by histology.
        Approach to effusion cytology:
                        1 cell or 2 cell population

                one cell population             2 cell population
            (all cells mesothelial-like) (some cells not mesothelial-like)
                                                   (see below)

                     nuclear atypia
            yes                            no

  inflammatory background                  Normal
        yes                               no

Reactive mesothelial cells                Mesothelioma
    (small cell ball)                    (large cell ball)

                             2 cell population
                            Groups or single cell pattern

               Groups                                Single cell pattern
  acinar arrangement / linear files
          Adenocarcinoma                   small cell                      large cell

                                                            pleomorphic nuclei
 nuclear moulding
                         targetoid inclusions                 eccentric nuclei
granular chromatin
                        cytoplasmic vacuoles                     nucleoli
    no nucleoli
                         Lobular carcinoma                  moderate cytoplasm
   Small cell ca                                               Poorly diff. ca

        no nuclear moulding                 binucleation               prominent nucleoli
        scant cytoplasm                  prominent nucleoli             scant cytoplasm
        lymphoglandular bodies         intranuclear inclusions       lymphoglandular bodies
        Lymphoma                        cytoplasmic pigment               Lymphoma
          Fig 1. Normal mesothelial cells. Note lacy collar (Arrow)

Fig 2. Mesothelial cells showing a window: interdigitating microvilli (Arrow)
          Fig 3. Reactive mesothelial cells with inflammatory background.

Fig 4. Reactive mesothelial cells with low N/C ratio, round nuclei, smooth nuclear
membrane and central nucleoli.
                          Fig 5. Reactive mesothelial cells.

Fig 6. Reactive mesothelial cells. Mild nuclear enlargement with open chromatin.
Small cell groups. Inflammatory background.
Fig 7. Benign mesothelial cells with degenerate vacuoles in the cytoplasm (vacuoles
not indenting the nuclei; no nuclear features of malignancy).

   Fig 8. Rheumatoid arthritis. Note ragocytes: Debris within neutrophils (Arrows)
Fig 9. Mesothelioma. Markedly cellular with large cell balls.

        Fig 10. Mesothelioma with papillary groups.
Fig 11. Mesothelioma. Nuclear atypia present (red arrow). Compare with normal
mesothelial cells (green arrow).

                Fig 12. Mesothelioma. Note collagen core (Arrow)
                              Fig 13. Mesothelioma.

Fig 14. Mesothelioma. Tumour cells with cytoplasmic vacuoles containing magenta-
coloured hyaluronic acid.
Fig 15. Mesothelioma. Note presence of squamous-like cells (Arrow) which is a
helpful clue to the diagnosis.

                         Fig 16. Cell block: Mesothelioma.
                         Fig 17. Sarcomatoid mesothelioma.

Fig 18. Pericardial effusion. Adenocarcinoma from the breast. Often with large cell
groups showing smooth border.
    Fig 19. Adenocarcinoma from the breast. Nuclei forming edge of cell group.

Fig 20. Adenocarcinoma from the breast. Single cell presentation. Small tumour cells
with targetoid inclusions (Arrows).
Fig 21. Adenocarcinoma from the breast (invasive lobular carcinoma). Note Indian
file (Arrow).

                      Fig 22. Adenocarcinoma from the lung.
          Fig 23. Adenocarcinoma from the stomach with signet-ring cells.

Fig 24. Adenocarcinoma from the ovary. Note papillary group with straight edge
Fig 25. Adenocarcinoma from the ovary. Note papillary structure with fibrovascular

                         Fig 26. Pseudomyxoma peritonei.
Fig 27. Metastatic renal cell carcinoma. Single tumour cells with mild nuclear atypia
and vacuolated cytoplasm, mimicking foamy macrophages.

Fig 28. Metastatic renal cell carcinoma (cell block). Tumour cells CD10 positive.
             Fig 29. Small cell carcinoma. Note nuclear moulding.

Fig 30. Leukaemia. Note myeloid precursors with eosinophilic granules (Arrow).
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recognition approach. Adv Anat Pathol 2006;13:174-184

Bhatti TR et al. Malignant mesothelioma. Fluid cytology and differential diagnostic
features. Pathology Case Reviews 2006;11:67-73

Boerner S. Mimicry and pitfalls in effusion cytology. Pathology Case Reviews

Shield P. Peritoneal washing cytology. Cytopathology 2004;15:131-141

Shidham VB, Atkinson BF. Cytopathologic diagnosis of serous fluids. Saunders
Elsevier. 2007

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