L7 - Serous Effusion Cytology

Question 1

Serous effusion

Answer 1

• Accumulation of fluid is an abnormal finding
• medical history of neoplasia-confirm metastasis
• Recurrent effusion –determine cause
• Benign disease-confirm cause

Question 2

Serous Effusion Cytology

Answer 2

• Valuable for diagnosis of spectrum of benign and neoplastic conditions

Diagnosis dependent on:

• Collection
• Preparation
• Application of adjunct tests
• Approach to reporting

Question 3

Accuracy and limitations

Answer 3

• More sensitive than biopsy
• Allows adaptation of adjunct tests
• Sensitivity 58-64% (true positives)
• Specificity-97-100% (true negatives)
• PPV 99.3-100% (positive test : all positives)
• NPV 56-80%(negative test : all negatives

Question 4

Effusion cytology

Answer 4

How useful is the test:

• Sensitivity 58% (Range 22-81%)
• Specificity 97% (Range 91-100%)
• Individual reports show improvement using adjunct testing
• Clinicians still nervous

Question 5

Influences on Accuracy

Answer 5

• Sample quality
• Laboratory preparation
• Extent of laboratory investigation
• Diagnostic experience
• Number of cases seen by the laboratory

Question 6

Specimen Collection

Answer 6

• Volume: 180-200ml
• Advantage of added anticoagulant
• Fresh /refrigerated

Provision for dealing with clotted specimens:

• Mechanical manipulation to release trapped cells
• Fix tenacious clots in NBF and process

Question 7

Anticoagulant

Answer 7

Formula:

• Sodium Citrate - 4gms
• Glucose - 6gms
• Distilled water - 240ml
• Sodium Azide - 0.24gms

• Use at 10% v/v

Question 8

Effusion Specimen

Answer 8

• Volume: 180-200ml
• Advantage of added anticoagulant
• Fresh /refrigerated

Provision for dealing with clotted specimens:

• Mechanical manipulation to release trapped cells
• Fix tenacious clots in NBF and process

Question 9

Serous Effusion Cytology

Answer 9

Clues for laboratory preparation initiatives:

• Clinical information
• Macroscopic appearances (colour, viscosity)

Question 10

Transudate

Answer 10

• less than 3g/100ml serum
• Poorly cellular
• Associated with alteration in vascular hydrostatic and osmotic pressure
• Develops in cardiac failure, hypoproteinaemia

Question 11

Exudate

Answer 11

• more than 3g/100ml
• Highly cellular
• Develops in bengin infection or neoplasia

Question 12

Changing the cell concentration

Answer 12

• Centrifugation, Cytocentrifugation

* Liquid based preparation devices (ThinPrep, SurePath)

Question 13

Blood contamination reduction

Answer 13

• Gradient centrifugation
• Mixing specimen with lysing agent
• Fixing smears in haemolytic fixative

Question 14

Smears and staining

Answer 14

Smear technique:

• Squash
• Blood film
• Cytospin

Minimum 2 smears:

• Air dried - Giemsa
• Wet fixed - Papanicolaou

Question 15

Purpose of a cell block

Answer 15

• Improves diagnostic accuracy:
• May contain material not seen in smears
• May show morphological details not seen in smears
• Platform for special stains and adjunct tests
• Provides a permanent reserve of cells
• Method of preparation –trap cells in an inert support medium, fix in NBF, process as a biopsy

Question 16

Special Stains and IHC

Answer 16

• Smears useful for some histochemical stains e.g. PAS+/-Diastase
• IHC on smears (preservation issues for some markers, require standardisation and controls)
• Histochemical and IHC usually performed on cell block sections (NBF fixation, serial section tracking)
• Special staining allows for differential diagnosis of reactive / mesothelioma /adenocarcinoma.

Question 17

Ultrastructural studies

Answer 17

• May be of assistance when morphology and IHC are equivocal
• Cell button fixed in 2.5% glutaraldehyde

Question 18

Flow cytometry

Answer 18

Serous effusion applications:

• Lymphoma studies
• DNA ploidy analysis
• Immunophenotyping using monoclonal antibodies against epithelial markers

Question 19

“Advanced” Adjunct Tests

Answer 19

• Targeted therapies-EGFR and HER2/Neu using FISH, CISH
• Therapeutic response markers-NAC1-ovarian carcinoma, Inhibitor of Apoptosis Protein IAPmalignant mesothelioma
• RT-PCR assay of human cancer gene MN/CA9
• TRAP assay: telomeric repeat amplification protocol – in situ test to distinguish benign/malignant effusions.

Question 20

Malignant effusions- Adenocarcinnoma

Answer 20

• Highly cellular
• Tendency for neoplastic cells to form microglandular structures-balls, papillae
• Nuclear criteria usually satisfied
• Secretory product can often be identified
• Often with clinical history

Question 21

Malignant Mesothelioma

Answer 21

• Monotonous population
• Retains some mesothelial characteristics
• Overlap of morphology with adenocarcinoma demands the use of specific cell markers to distinguish the tumour type