305 cytology

exudate

effusion - associated with damage to the vasculature, higher rate of malignancy than transudate

cytology

study of cells exfoliated from the body, looking to differentiate between benign and malignant conditions, material is prepared stained and examined under light microscope, use nuclear and cytoplasmic criteria to reach a diagnosis

squamous epithelium

lines the vagina, exocervix and endocervix to the transformation zone, mature non keratinizing stratified squamous epithelium comprising of 3 zones, basal intermediate and superficial zone

basal layer

basal and parabasal cells present here, layer undergoes epithelial regeneration

intermediate layer

cells undergo maturation, gradual increase in cytoplasmic volume, intermediate cells present here

superficial layer

full differentiation, cells have large cytoplasmic area with small pyknotic nuclei, superficial cells

endocervical epithelium

columnar, lines the cervical canal and underlying glands, single mucus secreting layer, nuclei are basally located, cytoplasm is elongate with a fine granular cytoplasm, non secretory cells with cilia to propel mucus

squamo-columnar junction

border between stratified squamous epithelium and columanr epithelium of the endocervix, site at the time of birth at the exocervix, physical of function TZ columnar epithelium replaced by metaplastic epithelium (movement continues throughout the reproductive life)

Transformation zone

region between the neonatal S-C junction and the post pubertal functional SC junction, histologically recognised by the presence of metaplastic epithelium, virtually all cervical squamous neoplasia begins at the transformation xone

squamous metaplasia

metaplasia replacement of one type of tissue by another type of tissue, involves proliferation of sub columnar reserve cells of the endocervical epithelium that differentiate into squamous epithelium, protective function, initiated by the external environment

smear collection

cervix must be well visualised, transformation zone must be sampled, cervix broom is recommended, fixation must be immediate, smears should be taken preferable, preferably at mid-cycle

collection method

insert longer central bristles into cervical os, begin turning in clockwise direction, push towards the cervix while still rotating clockwise, lateral bristles will splay out over ectocervix and the central bristles will penetrate the endocervical canal, complete 5 full rotations, remove and pop off broom head into surepath vial

fixation

maintain cells in as life like state as possible, acts to stabilise proteins producing cross links between them, ideal fixative penetrates the cell quickly replacing water content without distorting the cellular features, encourages adhesion of cells to the slide and allows various dyes to cross into the cells

95% ethanol

fixative of choice as presence of salts from alcohol is needed for max precipitation of nucleic acids, water content acts to rehydrate denatured DNA following its collapse with alcohol, allows for maximum cellular staining with minimum distortion because of its rapid penetrating capacity, cell structures coarsened, nuclear detail sharpened resulting in nuclear chromatin pattern, formalin not suitable as DNA and RNA do not react with these at normal temp

coating form

type of fixative that contains carbowax for extra protection

fixation timing

should be immediate, wither by submersion or by direct spraying of coating fixative, slide should be submerged for at least 10 mins (preferably 20-30 mins), after this it can be sent for staining or stored for later use

Papanicolaou stain

devised for optimal visualisation of cells exfoliated from epithelial surfaces of the body, not specific for cancer, polychromatic stain which shows variations of cellular morphology including degrees of cellular maturity and metabolic activity

Pap stain steps

fixation, nuclear staining, cytoplasmic staining, clearing

nuclear pap stain

haematoxylin (extracted from tree, requires mordant to bind haematin to nuclear proteins, aluminium sulphate), need to differentiate nuclear stain - can be progressive staining with bluing or regressive staining with non acidic dye removed by acid solution

pap cytoplasmic stain

display the variations of cellular morphology, contain a high alcohol content providing clear visualisation through overlapping cells, 2 cytoplasmic stains used

Orange G (OG-6)

monochromatic stain, intensely stains keratin brilliant orange, keratin not normally found in cervical/vaginal epithelium but is found in keratinising carcinoma, keratin staining is therefore significant

EA 50

polychromatic stain, composed of eosin light green and bismark brown, eosin stains the cytoplasm of mature squamous cells nucleoli cilia and some nuclear proteins, light green stains the cytoplasm of metabolically active cells (parabasals, intermediate and columanr)

clearing

final pap stain step, provides cellular transparency, allows the transmission of light from the microscope through the cells, use xylene as this has a similar refractive index as the mountant and glass used

normal endocervical cells

honeycomb arrangment, uniform nuclei

degeneration

normal process present in any smear, metabolic degeneration (programmed cell death), inflammatory conditions (infections), trauma (cervical biopsy, iud removal), insufficient blood supply

benign cytoplasm changes

vacuolisation, swelling, cytoplasmic inclusions due to secretory products building up in cells that have lost the ability to eliminate and absorb, perinuclear halo due to fixation when degenerate cells have enlarged due to excess fluid and nucleus shrinks back, loss of organelles e.g. cilia, polychromasia, hyperkeratosis, cytolysis

polychromasia

multiple staining present in one cell

hyperkeratosis

thickening of cervical epithelia

benign nuclear changes

multinucleation due to merging of adjacent cells (particularly evident in viral infections), nuclear enlargement, chromatin clumping, karyopyknosis (nuclear shrinkage), karyolysis (nucleus breaks up), nuclear vacuolisation, bare nuclei

inflammatory changes

common to see in cervical smears, minor changes not reported, major changes reported as may be clinically significant, often group inflammatory/repair/degenerative changes together

Non infectious cervicitis

can be due to chemical or mechanical changes e.g. IUDs that irritate cervical cells, can occur following changes, follicular cervicitis, erosion, clinically cervix can look friable and may have discharge

Erosion

histologically occurs when thin layer of endocervical cells extends pas the transformation zone, looks red due to vessels beneath, commonly seen post partum, usually followed by squamous metaplasia, cytologically see metaplastic type cells, immature and reactive endocervical cells in sheets, vacuolated cytoplasm common, sheets of repair, more commonly seen in teens pregnant women and those on the oral contraceptive

bacterial infectious cervicitis

acute and chronic inflammation, associated with a non specific inflammatory response, can be commensal and of low virulence, e.g. chlamydia

Bacterial vaginosis

most common bacterial infection, includes a number of bacteria including haemophilus vaginalis and gardnerella vaginalis, small gram negative bacilli adhere to cells (clue cells), can have a mixture of bacteria in background (don’t need to culture), asymptomatic but can have smelly discharge

actinomyces

often grow on strings of IUD, fluffy appearance with filamentous structures originating from its core, red sulphur granules, not to be confused with bacterial infiltrate (differentiate with characteristic filaments), may see IUD associated inflammatory changes, endometrial cells may be evident out of phase

Candida albicans

common fungal infection, can be symptomatic or asymptomatic, cellular changes may be non existent to marked, vacuoles, perinuclear halos, nuclear enlargement, hyperchromasia, characteristic pseudo hyphae with or without spores, usually see candida in smear

herpes viral infection

ulcero-necrotic process, HSV type II, fevers and blisters in severe cases, large multinucleated cells with intranuclear ground glass viral inclusions that mould together, squamous and endocervical appear multinucleate, dense cytoplasm, chromatin washed out with dark ring around outer nucleus

trichomonas vaginalis

oval shaped protozoan parasite with flagella, stains grey/blue/green with characteristic eosinophilic inclusions, may see nucleus, smears often have a dirty background comprised of cellular debris, often marked inflammatory response with marked reactive changes, itch and offensive green yellow discharge common, organism often seen

mild inflammation changes

cytoplasmic eosinophilia, nuclear enlargement, nucleolus more evident, peri nuclear halo, cytomegaly

marked inflammation changes

cytomegaly, nuclear variation including multinucleation, prominent nucleoli, multinucleoli, cytoplasmic vacuolation, inflammatory exudate

repair

part of tissue response to inflammation, sheets of immature squamous metaplastic cells with prominent nucleoli, sheets tend to be flat and monolayered with nuclei streaming in one direction (polarity), sheets tend to be organised and cohesive, minor nuclear variation, normal to increased N:C ratio, hypochromatic, mitoses, cytoplasmic vacuolation

atypical repair

can be difficult to distinguish from dysplasia and in some cases cancer, have repair features but hyperchromatic polymorphic nuclei, tend to retain cohesiveness but can look disorganised within the cell sheet/group

hyperkeratosis

protective mechanism, superficial cells nucleated or anucleated, thickening of epithelium, can be due to trauma infection of cancer, superficial cells >> intermediate cells

parakeratosis

similar to hyperkeratosis but keratinised cells are nucleated, cells are small, also seen in atrophic vaginitis

atrophic vaginitis

associated with atrophy and low oestrogen levels so occurs in older post-menopausal patients, irritation leads to inflammatory reaction, parakeratosis, inflammatory exudate, discharge common and sometimes blood stained, can be mistake for SCC, lots of neutrophils

radiation and chemo changes

marked inflammation and repair changes quite atypical, large sheets of repair, orangephilic cytoplasm common, large nuclei, prominent nucleoli, multinucleation, commonly low N:C ration

Liquid based cytology

used since 2010, ThinPrep and SurePath in NZ, produce a well fixed thin layer specimen free of unwanted background elements that can obscure cells resulting in a prep that is less difficult to screen, the samples are designed to allow for automated screening and ancillary testing, removes unwanted background elements

LBC ThinPrep steps

sample is collected using cervical sampling device, device is rinsed into thin prep vial containing PreservCyt solution, device discarded, sample vial is capped labelled and sent to lab, vial is placed into Thin Prep 5000, cells are dispersed via vortexing breaking up blood and mucous, negative pressure draws the fluid through a thin prep filter, cellular material is then transferred to glass slide, stained with pap stain

processing of LBC

vials vortexed to disaggregate cells (remove from brush and bottom of vial), put on prepmate which begins cell transfer process, transfers cell solution from the vial to a centrifuge tube containing density gradient solution, centrifuge tube is pre filled with density gradient solution, cell suspension layered on top of density reagent, centrifugation for 2 mins, supernatant removed and second centrifugation for 10 mins, concentrated enriched cell pellet remains, density reagent is a polysaccharid solution with sodium azide as preservative, elements caught based on shape weigh size and density, reduces obscuring artefacts such as mucus protein blood and WBCs

PrepStain processor

primary function is to transfer cell solution from centrifuge tubes to the corresponding slide, can process and stain batches of 48 samples, cell pellets are resuspended and transferred to settling chamber on top of the slide

LBC slides

modified poly-L-ysine, positive charge to aid in adherence of neg cells, cells distributed evenly in 13mm area, approx 50-70,000 cells per slide, 70 slides per day max and standard rescreening of all cases when manual, automation allows 140 slides per day max and full targeted rescreen only

adequacy

patient and specimen ID, pertinent clinical info, technical interpretability, cellular composition and sampling of the TZ

NSCP guidelines for abnormal cervical smeats

HSIL to colposcopy, ASCUS/LSIL x 2 to colposcopy, women with LSIL can revert to 3 yearly screening after treatment and clear results, inflammatory smear should not indicate increased follow up

respiratory cell anatomy

bronchus has pseudostratified columnar epithelium - goblet cells and cartilage also seen, bronchioles have simple cuboidal epithelium, alveolar epithelium have type 1 pneumocytes for gas exchange and type 2 pneumocytes to secrete surfactant

benign respiratory conditions

vacular, congestion or oedema have no cyto evidence, diffuse alveolar damage, adult respiratory distress syndrome, pulmonary embolism, hemorrhage, COPD, asthma, dilation of bronchi with wall destruction (bronchiectasis), infections, diffuse interstitial lung disease, non caseous granulomas

respiratory specimen types

sputum (deep cough sample), bronchial brushings and washing, bronchoalveolar lavage, FNA (used rarely in outer areas to reduce risk of needle puncture)

Primary lung cancers

benign papillomas and adenomas, squamous cell carcinoma, small cell neuroendocrine, adenocarcinoma (acinar, papillary, bronchioloalveolar), large cell undifferentiated, adenosquamous carcinoma, carcinoid tumour

primary lung cancer distributions

35-50% SCC, 15-35% adenocarcinoma, 20-25% small cell carcinoma, 10-15% large cell undiff

general cancer features

most primary lung cancers are central arising from first second and third order bronchi, remainder are peripheral arising in terminal bronchioles alveoli and are usually adenocarcinomas

respiratory tumour development

local effects are bronchial ulceration (haemoptysis) and bronchial obstruction (unresollving pneumonias, absecess, bronchiectasis, tumour necrosis), local spread to pleura local hilar medistinal lymph nodes pericardium and chest wall, distant metastases due to spread in blood or lymph

lung cancer growth patterns

exophytic into bronchus, endophytic into surrounding tissues

respiratory SCC

most common in males, high correlation with smoking, centrally located (tends to affect main airway), fast growing, expansive, infiltrating, may undergo central cystic degeneration, microscopically keratinisation seen, keratinised cells tend to occur at the surface of tumours in bronchi and centrally in cavitating tumours

respiratory adenocarcinoma

often peripheral, smaller than SCC and slower growing, similar incidence in both male/female, less association with smoking, has associations with asbestosis and pulmonary scars, microscopically vary from well differentiated glandular or papillary tumours to solid poorly differentiated tumours with only occasional glands or intracellular mucin vacuoles, scalloping of cell borders, visible nucleoli, delicate and bubbly cytoplasm

bronchioloalveolar carcinoma

in deeper part of lungs (smaller airways and alveoli), difficult to distinguish from normal adenocarcinoma, similar incidence in male/female, often single lesion but may have multiple nodules or extensive diffuse areas in one or both lungs, 25% survival rate at 5 years

microscopic bronchioloalveolar carcinoma

well differentiated mucin secreting columnar epithelium growing over alveolar architecture, solitary lesions are surgically resectable

lung small cell carcinoma

most aggressive primary lung cancer, highly malignant, distinctive cell type, strong association with smoking, commonly central, often with widespread metastasis at presentation, insidious onset (cough, weight loss, chest pain, lymphadenopathy), without treatment death in 6-17 weeks, with treatment 1% survival at 5 years

microscopic small cell carcinoma

small to intermediate sized epithelial cells with little cytoplasm, cellular moulding, coarse chromatin, no macronucleoli, salt and pepper chromatin, can be misinterpreted as bare nuclei

lung cancer diagnosis

require cyto and/or histo diagnosis, sputum cytology can be used as a prelim test, radiology, bronchoscopy, CT guided FNA, mediastinal lymph node biopsy, biopsy of distant metastasis, radiology often done first

non small cell lung cancer treatment

surgery for localised disease, radiotherapy for non resectable disease, chemo of limited value

small cell carcinoma treatment

chemo is the main line of treatment

indications for urinary cytology in symptomatic patient

clinical suspicion of urothelial tumour, previous treat urothelial tumour (follow ups), symptoms are haematuria, obstruction, urinary frequency urgency and pain

indications for urinary cytology in asymptomatic patient

follow up of treated urothelial tumour, screening of known high risk industrial workers (e.g. working with chemicals), screening of persons with shistosomiasis (parasitic infection that can cause SCC)

voided urine

3 specimens on consecutive days, 2nd catch of morning or 3 hours after last void preferred, 30-50 mL, unless processed immediately should be fixed with an equal or greater volume of 50% alcohol, non invasive, sensitivity increases with the no. of specimens submitted, specimens often degenerate as instructions not followed

catheterised urine

fresh specimen, less risk of contamination, not commonly used, problems associated with instrumentation effect (irritation can cause cells that mimic SCC)

cytoscopic urine

commonly bladder washings, usually when malignancy suspected, saline wash withdrawing a volume of the irrigant, fixed immediately in alcohol, generally very cellular (due to physical exfoliation, less contamination due to more direct sample, high sensitivity

Ileal conduit urine

very cellular, difficult to screen due to crystals, degenerate intestinal epithelium and inflammatory cells

superficial (umbrella) cells in urine

large cells with polygonal shaped cytoplasm, multiple nuclei, low N:C ratio

deep transitional cells in urine

urathelial cells, single cells or in clusters, much smaller than umbrella cells, single nuclei

squamous cells in urine

common contaminate in females, usually from urethra but can originate from trigone of the bladder

columnar transitional cells in urine

not common in voided urine, usually present due to instrumentation

renal tubular cells

small cells often degenerate but rarely seen, presence can indicate kidney disease, can be distinguished from deep transitional cells by their granular cytoplasm

urothelial tumour pathology

most primary tumours in UT are of transitional/urothelial cell type, most occur in bladder but can arise in the ureter renal pelvis or posterior urethra

Non papillary urothelial carcinoma

flat nodular lesions, obvious malignant features evident, cellular preparations, single cells and occasional small groups, large hyperchromatic nuclei, high N:C ratio, shape and size variation in nuclei, prominent nucleoli, may be diagnosed as in-situ or invasive (histology diagnosis), development through dysplasia without significant hyperplasia, high risk of invasion, high risk of metastasis, poor prognosis

papillary urothelial carcinoma

most common, thought to evolve through stages of hyperplasia and dysplasia, recurrence is frequent, less likely to develop invasion, rarely metastasise, longer patient survival, graded 1-4

low grade UC

grade 1 and papillomas, finger like projections growing into bladder lumen, do not shed cells as easily as HG tumours, cells may have minimal/no cytological abnormality, nuclei may show slight enlargement and chromatin clumping but can be very subtle, main features are papillary fragments palisading and rosette arrangements, architectural features should only be interpreted in voided urine with no history of recent instrumentation and no stones, cytology not diagnostic as features can be caused by irritation

high grade papillary UC

features similar to non papillary with the added feature of large clusters and papillary arrangements, prominent nucleoli, may see some squamous/glandular differentiation, dirty background, inflammatory with blood and necrotic debris, very cellular, can show squamous/glandular differentiation

carcinoma in situ UC

similar features to high grade except usually clean background and fewer number of cell clusters, differentiation relies on histology

urinary squamous cell carcinoma

can result from chronic infection (cystitis) from shistosomiasis, prolonged cyclophosphamide therapy, extensive squamous differentiation in UC, poor prognosis

bladder adenocarcinoma

0\.5-2.5% of bladder tumours, usually mucin producing, wide range of gland differentiation, poor prognosis

Renal cell carcinoma

cells from RCC tend to shed late in the course of the cancer, cells are usually non-diagnostic due to marked cellular degeneration, malignant cells in tumour can travel down into the urine

advantages of urinary cytology

accurate and specific for detection and diagnosis of invasive cancer and non invasive HG papillary cancer of the bladder, important to detect non papillary carcinoma in situ, valuable for co exisiting LG/HG lesions, tumours of the renal pelvis and ureters may be diagnosed

Limitations of urinary cytology

papillomas and LG tumours are not diagnosed with certainty

lithiasis

can produce marked atypia, exfoliation of large fragments DD LG tumour, nuclear hyperchromasia, mitotic figures

drugs and radiotherapy

cyclophosphamide and busulphan, can produce atypia and a granulomatous response

instrumentation

any procedure can dislodge large urothelial fragments, changes due to procedure/regeneration can persist for a few weeks

urinary cytology reporting categories

NMCS, inconclusive, suspicious for LG, suspicious for malignancy, positive for malignancy, unsatisfactory

body cavities

pleural peritoneal and pericardium, lined by serous membranes composed of mesothelial cells, gap between the serosal membranes contains fluid, delicate layer of connective tissue, capillaries venules arterioles and lymphatics all present, undergo physiological responses to pathologic processes

pathophysiology

as long as pressure are equilibrated fluid remains in the intraluminal compartment of the microvasculature, a break in this delicate balance leads to effusion, increase in hydrostatic pressure can lead to effusion (accumulation of fluid), circulatory disease infections and neoplasms are the main causes of effusions, cavities are commonly the medium for metastasis

mesothelial hyperplasia

chronic response to injury, mechanical trauma or sustained pressure can lead to thickening of the mesothelial membrane, causes: infection infarction liver disease radiation chemo chronic inflammation neoplasms

transudate

effusions - filtrates of blood across a physiologically intact vasculature