Cytology 2 .docx

Objectives - Know how to prepare a site prior to obtaining a cytology sample - Know the different techniques used to obtain samples for cytologic evaluation - Know why one method is chosen over another - Discuss the different slide preparation techniques - Problems encountered in...

Objectives - Know how to prepare a site prior to obtaining a cytology sample - Know the different techniques used to obtain samples for cytologic evaluation - Know why one method is chosen over another - Discuss the different slide preparation techniques - Problems encountered in obtaining and securing diagnostic cytology samples - Know the different slide techniques used to prepare a cytology slide - Commonly used stains Preparation of Collection Site - Depends on site of collection: - Superficial site requires preparation similar to a venipuncture - Surgical preparation is required for aspirates of internal organs, joints, body cavities, bone marrow, and spinal tap - Microbiologic test generally do not require a surgical prep (depends on sample needed) Smear Preparation Tips - Cytologic samples can be collected by: - Imprints - Scraping - Swabbing - Aspirating - Technique used depends on location, characteristics of the tissue, and demeanor of patient - Always make several smears if possible - Leave some smears unstained for samples that are to be sent to an outside lab - prepared for procedure Imprints - External lesions - Living animals best  - But often used on tissue removed during surgery or necropsy - Easy to collect - Results are not always satisfactory - Generally, collect fewer cells than other methods - Contamination-bacteria, blood - For the most part not considered diagnostic - Ulcers - Imprint before cleaning (highest bacterial count) - Then clean with saline and re-imprint or scrape - Cutaneous lesions or tissues collected during surgery/necropsy - Clean blood and tissue before imprinting - Tissue fluid or blood Inhibits cells from sticking to slide - Excessive fluid can cause drying problems - Can cause cells not to spread evenly, therefore cells will not be the correct size and shape A close-up of a cut off Description automatically generated Scrapings - Prepared from tissues collected during necropsy, surgery, or external lesions - Advantage - Collection yields large number of cells especially on firm lesions where it is difficult to obtain cells - Disadvantage - Difficulty in collecting them - Superficial nature of the collection procedure often does not give a true representation of cells. This hinders their use as a diagnostic tool - Clean lesion or tissue - Using a scalpel blade scrape lesion - Blade is perpendicular to lesion, pulling blade towards you - Material sticks to the blade where it is then transferred to a slide - Smear the material - Heat Fix?? Swabs - Generally used only when imprints, scrapings and aspirates are not possible because of location - Fistulous tract - Vaginal cytology - Ears - Lesion should be cleaned if necessary - Using a moistened sterile swab (0.9% Saline) gently swab area (not ears!!) - Gently Roll swab across slide - not rub across slide as this causes cell damage Aspirates - Fine Needle Aspirates (FNA) - Lymph nodes (superficial and deep) - Skin, subcutis - Internal organs - Spleen - Liver - Kidneys, Lungs, Prostate, Thyroid - Masses in thorax or abdomen - The mass, tissue or organ is identified by palpation, radiography, ultrasound, and manually isolated - Preferred method for obtaining samples from masses - Avoids superficial contamination - Allow cells to be collected from various areas within the lesion - More representative of cellular types - Collected either by aspiration or nonaspiration techniques - Method depends on type of lesion and the preference and experience of collector  image (16) Technique - 25 gauge needle (22 ga & 25 ga common) - 6 ml syringe (book states 20cc) - The softer the tissue, the smaller the needle and syringe needed - Larger needles often leads to poor quality slides - Tissue cores tend to be aspirated, which results in poor yield of free cells suitable for diagnosis - Tend to cause greater blood contamination  - Size of syringe is influenced by the consistency of the tissue being aspirated (12ml over all good average) - 3ml good for lymph nodes - Fibrosarcoma requires a larger syringe because of firmness of the tumor  Aspiration Technique - Mass to be aspirated is held firmly to aid: - penetration of mass - control needle - Needle with syringe attached is introduced into the center of the mass - Strong negative pressure is applied by withdrawing the plunger to about 2/3-¾ the volume of the syringe - Multiple areas of the mass should be sampled A syringe in a fured animal Description automatically generated - Aspiration of the surrounding tissue should be avoided - Large mass-redirect needle without releasing negative pressure - sure to release pressure before withdrawing from mass - Small mass -- relieve negative pressure during redirection - Negative pressure is applied only when the needle is static - Often, material is not visible - this point your needle is out of the mass - Remove needle from hub of syringe - Draw air into the syringe - Replace needle onto the syringe - Depress the plunger rapidly - Expelling aspirate onto the middle of a slide - The needle should be close to the slide to allow for a single droplet of material vs a spray - - - Spread aspirate into a monolayer - Techniques will be discussed later A person holding a syringe Description automatically generated Nonaspiration Technique - Technique works well for highly vascular areas - Modification technique of the original nonaspiration method - Using a small gauge needle on a 3 cc syringe - Draw a few cc of air into the syringe barrel prior to collection this allows for rapid expulsion of the material onto the slide - Grasp needle and syringe at the hub for maximum control - Mass stabilized with free hand - Needle is inserted - Move the needle back and forth in a stabbing motion trying to stay on the same tract - This allows cells to be collected by cutting and tissue pressure - careful not to contaminate sample with surrounding tissues - Withdraw the needle without releasing pressure - Quickly expel material onto slide - Smear promptly - Optimal to perform multiple attempt at various sites A close-up of hands holding a needle Description automatically generated Demonstration - Tissue biopsy - Sampling of piece of tissue for cytologic and/or histopathologic examination - Gentle abrasion with a blade - Excision - punch biopsy - Endoscope-guided biopsy - Fix tissue samples for histopathologic examination in 10% neutral phosphate-buffered formalin - ensure adequate fixation, place slabs of tissue no more than 1 cm wide in fluid (10% formalin)- - tight jars containing formalin at approximately 10 times specimen's volume - With large tissues, remove sample to a smaller jar with less formalin once fixed for 24 hours Endoscope Specimens - Gently flush specimens collected by endoscopy from tip of endoscope with sterile saline - Blot specimen gently on a paper towel to remove excess blood; place on a small piece of wooden tongue depressor or cardboard; allow tissue to dry on the "splint" - Immerse or float specimens with attached "splint" specimen side down in fixative Most common Problems - or Few cells seen - One of the most common problems - Lesion does not exfoliate well (mesenchymal neoplasms) - Needle may miss the site of lesion during collection - Timid sample collection - Inadequate negative pressure (aspirate technique) - Slow or shallow needle passes (nonaspiration technique) Blood Contaminate  - Blood - Presence of excessive peripheral blood results in a diluted sample - Two main causes - Use of excessively large needles (\>21ga) - Excessive or prolonged aspiration - Anytime you can visualize material in the hub of the needle you should stop and make smears immediately - Some tissues are highly vascular and blood contamination is difficult to avoid  - use nonaspiration technique - Blood seen on impression smears often results from a failure to blot tissue before making slides Poorly Prepared Slides - Poorly prepared slides - High cell yield is useless when slides are prepared poorly - Smears should be made immediately to prevent drying or clotting - a lot of material is recovered be prepared to make 2 slides or more - Too thick of smears are as useless as not enough cells - Smears should be aired dried immediately - Make as many good quality slides as possible to submit to lab - Cells are often fragile and break apart easily so having more than 1 or 2 slides can be beneficial  Smears for solid masses - There are several methods that can be used - The method used depends on: - The experience of the person - Characteristics of the sample - Combination of methods is also acceptable Squash Preparation - This method takes some experience - inexperience hands the cytologic smear is often unreadable because too many of the cells are ruptured or is insufficiently spread - Method - Expel the aspirate onto the middle of the slide - Place a second spreader slide over the aspirate horizontally - Quickly and smoothly slide across first slide - Important that no downward pressure be applied because this causes rupture of the cells Squash Blood Smear Technique - the aspirate has a large fluid component - Often seen in lymph node aspiates Combination Technique - Place aspirate onto the middle of slide - Draw back spreader slide until it contacts 1/3rd of the sample and push the slide forward (blood smear) - Next place a spreader slide horizontally over the back 1/3rd of the sample, allow sample to spread - Quickly and smoothly slide the slide (squash method) - The middle third of the aspirate is left untouched Benefits of Combination Technique - Leaves the front 1/3rd  of aspirate gently spread - the aspirate is of fragile tissue, the middle 1/3rd area should contain sufficient unruptured cells to evaluate - Also, if the aspirate is of low cellularity the middle 3rd remains more concentrated and is the efficient area to study - The back 3rd area contains cells that are clumped or difficult to spread they should be sufficiently spread to evaluate (squash technique) Combo Other methods - Modification of the squash technique - Starfish - Drag the aspirate with the tip of a needle to make a starfish - Line concentration technique - Blood smear without the feathered edge - Read about in Laboratory Procedures for Veterinary Technicians, Sirois bs00554\_ Preparation of Smears from Fluids - Should be made immediately after collection - Collect in EDTA - Smears can be prepared from fresh or centrifuged samples - Centrifuged if low cellularity is expected - May use the blood smear, squash prep, and line smear technique - Cellularity, viscosity and homogeneity influence selection - Centrifuged samples - Excessive fluid keeps cells from spreading well - Centrifuge at rate similar to urine samples - Supernatant is separated from the sediment - Resuspend sediment in a few drops of supernatant - Blood smear or squash prep technique is used - Line smear is used for samples of low cellularity  Stain Types - Romanowsky type stain - New Methylene Blue - Papanicolaou stain - Excellent nuclear and cytoplasmic detail - Allows viewer to see through layers of cell to evaluate nuclear and nucleolar changes - Requires multiple steps and considerable time Swan Review - Should be able to discuss - Preparation of area and the type of preparation required - Different methods used to obtain samples and when you would choose one method over the other - How each method is performed - Different methods for preparing a cytology slide - Common problems encountered in obtaining cytology samples - Different stains available