Non Gynecological Cytologic Specimens

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Non Gynecological Cytologic Specimens

• Collection cytopreparatory
• technique

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Goals of Standardizationspecimen collection
processing

• To obtain
• Well distributed
• Well preserved
• Well stained Cells
• Minimize unwanted artifacts

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• Detection of
• Malignancy
• Inflamatory disease
• Infectious disease

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Non gynecological cytology Samples

• Cerebrospinal tract
• GI tract
• Joint space
• Ocular area
• Pericardium
• Peritoneum
• Pleura
• Respiratory tract
• Skin Mucosal membrane
• Urinary tract
• Breast /Nipple

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Methods of specimen collection

• Brushing Washing
• Direct puncture Drainge
• Touch preparation Scraping
• Voiding
• Expectoration

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Cytologic Samples

• Hypercellular or Hypocellular
• Containing
• Blood
• Mucus
• Inflammatory cells
• Microbial agent
• Crystals
• proteinaceous material
• Other debries

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Cell integrity

• 4 hours in room temp. (22-25 c )
• 72 hours in refrigerator with anticoagulant
• (4 c )
• Further delay specimen should be fixed

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• Exception
• CSF Urine
• Degenerate within 1 hour
• Even with refrigeration

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Body fluids

• Separate / clearly labeled / leak proof container
• Fresh specimen is prefered
• Anticoagulant heparin
• Keep body fluid uniformly suspended
• Fixation 50 ethanol equal to specimen volume

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Specimen volume

• minimum of 1-2 ml
• 20-50 ml is preferred
• for pleural or peritoneal tap
• 50-200 ml is favored

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Washing of body cavity

• Collected in balanced salt solution(Ringer)
• Optimally fresh
• If not possible refrigerated or
• fixed in equal volume of 50 ethanol

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Brushing of body cavity

• Slide preparation
• roll the brush across the glass slide ,
• pressing firmly in a small area in a
  circular motion (prevent air drying )
• Fixation fixative spray or immersion in 95
  ethanol for 15 minutes
• papanicolaou staining
• Alternatively
• Clip the end of the brush send it in a
  container with balanced salt solution or
  fixative

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C S F

• Minimum of 1 ml is needed
• 2-3 ml is preferred
• If immediate evaluation is not possible
  refrigeration is recommended
• Delay beyond 72 hours fixation with an equal
  volume of 50 ethanol

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Gastric washing

• Blind or at endoscopy
• Retrieve swallowed acid fast bacilli or
  hemosiderin-laden macrophages (IPH),.
• Balanced salt solution is introduced in to the
  stomach to flush gastric mucosa in different
  position
• Specimen is send to lab. immediately

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Abrasive baloon collection

• Recovery of the cells by rolling the baloon
  surface on to glass slides
• Immediate fixation in 95 ethanol
• Rinsing the baloon in balanced salt solution or
  50 ethanol
• Preparation of cytocentrifuged or cell block
  material

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Respiratory specimensSputum

• Separate specimens should be collected for each
  analysis. Shared specimens are not recommended.
• Early morning sputum yields the greatest number
  of diagnostic cells
• 3-5 consecutive daily sputum or 3-day-pooled
  sputum collection
• patient instruction clearing postnasal
  discharge
• gargle rinse
  the mouth
• opening the
  airway (bronchodilators , mucolytics..)
• deep coughing
• Adequacy presence of macrophages dust cells
  on microscopy

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Sputum.

• Expectorated material collected in a wide-mouth
  labeled container
• Fresh specimen is preferred
• Delay more than 4 hours fixation with
• 70 ethanol or 50 ethanol with 2 PEG
• (saccomanno,s fixative)
• Refrigeration more than 24 hours is not
  recommended (multiplying of microorganisms)

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Respiratory specimenswashing / brushing

• Via fiberoptic broncoscopy
• Washing
• with balanced salt solution
• Bronchioalveolar lavage(BAL)
• Rinsing distal airways with a balanced
  salt solution suction of fluid cytologic
  analysis

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Breast secretion

• Gently express the nipple subareolar area only
  using thumb forefinger
• Allow pea-size drop of fluid to collect upon the
  nipple tip
• Smear the material across a glass slide
• Immediately drop the slides in to the fixative
• (95 ethanol or spray fixative)
• Repeat the procedure until all available
  secretion is used .
• If no secretion appears at the nipple with this
  gentle compression, do not manipulate further.

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Lesion Speciemens

• Touch preparation (Touch Imprint)
• to obtain information about a lesion on an
  urgent basis.
• Surgical Specimen
• Lymph nodes
• Bone Marrow Biopsy
• Wound
• Vesicles

Touching a wet tissue with a glass slide
Adherence of the cells to glass slide in the
same orientation enable cells to be examined
apart from their connective tissue
matrix. Slides may be Air dryed Wet fixed by
immersion Spray fixed
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Lesion Speciemen

• Brush Preparation
• Direct transfer to glass slide
• Immersion the brush into balanced salt solution
  or fixative

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Tzanck Smear Preparation

• Direct Smear of Lesion for viral screening
• (CMV, HSV, HPV, RSV, Measles,.)
• Premoisten the lesion (fresh non-ruptured
  vesicle) with saline
• Vesicle is opened or crust from a ruptured lesion
  is removed
• Margin of the lesion is scraped
• Spread the obtained material on an alcohol
  moistened slide
• Fix immediately after the smearing
• Scraping tool may be rinsed in the preservative
  and then concentration procedure is done

Note Cotton swab should not be used
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Ocular Sample

• Ocular discharge and secretions , conjunctival or
  corneal lesions handled similar to Tzanck
  preparation
• Local anesthetic administered by drops will
  usually facilitate the acquisition of ocular
  samples.
• - Intraocular aspiration of small amount of fluid
  treated like a FNA specimen

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Urinary Specimen

• Urine
• Sample First morning specimen is discarded
• subsequent midstream clean catch
  specimen is collected
• in a wide mouth labeled container
• Sample Condition
• 
  Refrigerated
• 
  Fixed(Equal volume of 50 ethanol or
• 
  Saccomannos fixative)

FRESH
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• Catheter-Collected Specimen
• the catheter should be passed with only just
  enough lubricant to effect placement.
• If too much lubricant is used it will accumulate
  in the cytologic sample and obscure cellular
  features.
• Any instrumentation should be noted on the
  requisition

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• Bladder Washing
• 1-Introduce a balanced salt solution via a
  large syringe connected to the port of a
  cystoscopic device or catheter
• 2-Withdraw and re-inject with a moderate force
  to dislodge epithelial cells
• 3- Send the resultant fluid for evaluation

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Specimen handling and transport
Record Number
Patients full name
Labeling the Specimen
Specimen Source, Date of Collection, Name of
Physician
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• Date time that sample was received
• Condition of sample
• amount
• color
• turbidity
• other gross
  characteristics
• If the specimen is fixed or unfixed
• If anticoagulant is added

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Transport of fluid specimen

• Separate , clearly labeled , leakproof container
• Placed inside a sealable plasstic bag
• Standard precautions in transporting biohazardous
  material should be followed

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Turnaround time

• It is recommended that results be reported
• within two working days
• (unless special studies are requested)

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Requisition Form

• Full name of the patient
• Identification number
• Date of birth
• Date time of collection
• Source of specimen
• Number type of specimen submitted

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Requisition form

• Type of examination requested
• Ordering physician's full name
• Relevant clinical history
• Physical, radiological endoscopic findings
• Collection method(washing, brushing,)
• Differential diagnosis

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Rejection criteria

• improper documentation
• Discrepant or lack of patient demographics
• on container and requisition form
• Incorrect or lack of requisition form
• Requisition form lacking the essential
  information
• No specimen submitted with cytology form

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Rejection Criteria

• Improper Specimen
• Insufficient amount of specimen
• Standard safety precautions are not followed
• container is leaking
• Slides are broken
• Specimen deterioration
• Delay in transport of fresh specimen
• Improper fixation
• Improper storage

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Cytopreparationof Specimen

• anticoagulation

Regardless of source or cellularity,
anticoagulation of the specimen may be
preferred.
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Cytopreparationof Specimen

• anticoagulation
• Active components of coagulation system in
  some cell
  suspension
• formation of fibrin polymers
• entrapment of the cells in a clot

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• Common laboratory Anticoagulant
• Heparin / EDTA / Citrate / Oxalate
• To keep body cavity fluids
  uniformly suspended, 3-5 IU of heparin per mL is
  added to the collection vessel before the
  specimen is introduced.

Heparin
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• some anticoagulants interfere with ancillary
  studies
• Lithium Heparin interfere with cell culture
• EDTA interfere with Flow cytometry

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Cytopreparation of Specimen

• Specimen concentration
• Most of cell suspension (fresh or fixed)
• need to be concentrated before study

Conventional centrifugation Cytocentrifugation Fi
ltration Gravity sedimentation
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Conventional centrifugation

• Time 5 15 minutes
• Centrifugal force 400 1600 RCF
• Low RCF for urine
• High RCF for proteinaceous
  material

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• Conventional centrifugation .
• Packed sediment is produced at the bottom of
  the tube
• Bulk of supernate is removed
• Sediment is resuspended either in a portion of
  supernate or another solution for further
  processing
• Concentrated sediment may be directly applied to
  glass slide or transferred to electrolyte ,
  nutrient , or fixative solution

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Cytocentrifugation

• Produce a cell monolayer on a glass slide
• applying a constant centrifugal force
• at right angles to the surface of glass slide
• causing cells to deposit on the slide

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Cell filter

• Comprise of
• nitrocellulose, polycarbonate,
• Two broad categories
• 1- whole mount filters upon which cells are
  collected, processed, stained, and examined,
• (hypocellular, unfixed specimens)
• 2- Touch transfer filters from which cells are
  transferred to glass slides by touch imprint
• (fixed specimens)

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Cell blocks

• Embedding centrifuged cell samples
• in Agar, Thrombin and other gels
• or directly in paraffin and processed as a
  histologic sample

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Cytopreparationof Specimen

• Adhesion
• Degree of cellular adhesion to glass slide
  depends on
• Body site (source of sample)
• condition of the cells

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Adhesion

• Plain glass slides are suitable for specimens
• which naturally adhere well to glass
• In specimens that adhere weakly
• frosted slides
  or
• pre-coated slides with
  an adhesive
• Adhesion is not absolutely predictable
  therefore,
• it can never hurt to use adhesive-coated glass
  slides.

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Adhesion

• Frosted slides
• -problem of distracting, refracting granularity
• -should be mounted in a medium with similar
  refractive index

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Adhesion

• Coated slides
• With Albumin(mixture of egg white glycerin)
• With water soluble glue
• With chrome alum (mixture of gelatin and
  chromium potassium sulfate)
• 4. poly-L-lysine
• Be sure of cleanliness of slides , before coating
  them with adhesive
• detergent wash ,thoroughly rinse, and then
  dip them into a weak (0.01N) ammonium hydroxide
  solution to ensure their cleanliness
  immediately before coating them with adhesive.

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Adhesion

• Activated slides
• is preferred for in situ hybridization
  studies

1- Clean slides are dipped in clean acetone,
2- dried, 3-soaked for two minutes in a 2
solution of 3- aminopropyltriethoxysilane
in acetone, 4- rinsed in distilled water, 5-
dried at 60 C for 30 minutes, and stored.
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Cytopreparationof SpecimenFixation

• advantage of fresh (unfixed ) specimen
• Ease of handling
• Greater cell recovery
• Better cell flattening
• Crisp nuclear morphology
• Facillity of special staining studies
• (flow cytometry, cell culture, EM..)

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Wet-mounted fresh specimen

• Rapid examination of unfixed cellular fluid
• After conventional centrifugation
• a drop of sediment is mixed with a supravital
  stain (toluidine blue) and examined
  microscopically
• useful for highly cellular malignant specimens

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Fixation

• Alter the cell morphology
• Diagnostic criteria of cell health and disease
  ,is based on morphologic features in alcoholic
  wet-fixed specimen

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Fixation.

• Wet-fixed smears,
• stained with Papanicolaaou stain or HE
• ensure maximum resemblance between cells in
  cytology specimen
• corresponding tissue section

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Fixation of the specimen

• Fixation must be completed within seconds of
  smearing
• Air-drying alters cellular features
• result in autolysis

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• Air-dried specimens
• desirable for
• Wright
• Romanowsky
• ultrafast pap staining

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Characteristcs of a good cytology fixative

• Penetrate cell rapidly
• Minimize cell shrinkage
• Maintain cell morphology
• Replace cell water
• Inactivate autolytic enzyme activity
• Allow stain permeability across cell boundaries
• Permit cell adhesion to glass surfaces
• Kill pathogens
• Afford a permanent cellular record

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Immersion Fixative

• Spreading the fresh cells as a thin layer on a
  glass slide
• Immersion the slide in immersion fixatives
• 95 Ethanol
• Absolute Methanol
• 80 Isopropyl
  Alcohol
• 80 n-propanol
• 90 Acetone

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Coating fixative

• More popular than immersion fixatives
• Comprise
• an Alcohol that fixes cells
• a Wax-like substance(PEG)
• that forms a protective coat over the cells

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Coating fixative

• Can be sprayed or dripped over slides
• Slides may be dipped into fixative

Coated slides need to be soaked in 95
ethanol to remove their coating
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Only fixatives prepared specifically for
cytology specimen should be Used, Not Alcohol
based hair spray
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Fixing the specimens of body fluid

• Cell preparation conventionally centrifuged
• Its supernate is decanted
• Sediments are resuspended in at least 10X of
  balanced electrolyte solution to which an equal
  volume of 50 ethanol or fixative is added
• This specimen can be stored for further
  processing

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• Urine can be treated in a similar fashion
• if it is to be transported off site
• or
• if its processing is to be delayed

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Cytopreparationof SpecimenErythrocyte lysis

• Hemolytic agents are used to reduce the effect
  of blood contamination

• Acid Elution of air dried prep.
• Carnoys Fixative
• Acidic Alcohols
• Saponin

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Erythrocyte Lysis in blood contaminated freshly
produced cell spreads
1- Immerse cell spreads in Carnoys fixative for
3-30 min ( fixation occurs with greater
shrinkage/ darker staining) 2- Transfer the
slides to 95 ethanol
1-Place the slides in 50-70 ethanol 2- Then
transfer it to 95 ethanol
1- Place the slide in 95 ethanol for 5
minutes 2-Then place it in 12 aqueous urea for
20-30 minutes 3- Place again in 95 ethanol
1- Place the slide in acidified 95 ethanol
(1 drop of HCL per 500 ml alcohol or
Clarkes solution) 2- Then place in 95 ethanol
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Erythrocyte Lysis in fresh cell sediment
1- Cell sediment re-suspend in at least 10X
electrolyte solution containing 1 ml glacial
acetic acid per100 ml suspension 2- Suspension is
mixed by several inversions 3- Held for 10
minutes 4- Centrifuged 5- Process is repeated
until cell sediment no longer contains
colored erytrocytes
1- For excessively bloody samples saponin is
used 2-Time is critical Saponons action must
be stopped after exactly 1 minute by addition
of Ca ions 3- Excessive exposure may destroy
non-erythrocyte component of cell
sample Saponin solution should be prepared under
a hood to prevent inhalation of saponin powder
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Cytopreparationof SpecimenMucolysis

• Saccomannos method
• Make it possible to achieve concentrating cells
  dispersed in mucus without ruining their
  morphology!

Note Homogenization using chemical methods is
not generally favored. although commercial
mucolytic solutions are available.
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