Clin Path Unit 3 (Cytology)

What is an imprint in cytology and how is it prepared?

• Cleaned lesions/tissues collected during surgery/necropsy

• Remove excess blood + tissue w/ clean absorbent material to help cells stick to slide

• Blot slide to site of concern

What is a scraping in cytology and how is it prepared?

• Obtained from necropsy, surgery, or external lesions

• Scalpel held perpendicular to blotted site + scraped against surface of site several time

• Be careful to not destroy cells by being too aggressive (pro = lots of cells, con = difficult to collect, can only obtain superficial samples + not helpful in neoplasm DX

What is a swab concerning cytology and how is it prepared? Pros/cons?

• Useful when imprint, scraping, and aspirates are not available

• Wet sites can use sterile swab to collect, dry site must use 0.9% saline to dampen site (water will lyse cells)

• Roll swab on clean slide (wiping will destroy)

How is cytology performed on an ulcer?

• Initial imprint of unclean site, imprint after cleaning

• FNA of underlying tissue

• Imprint under scab

How is an aspiration performed in cytology?

• Can do aspirations on skin, subcutis, deep + superficial nodes, spleen, liver, kidneys, lungs, thyroid, prostate/intracavitary sites

• 21-25g needle (*softer tissue = smaller needle; yeilds single cell suspension vs. core of tissue*)

• 3-20mL syringe (*softer tissue = small syringe*)

• Don’t throw away hubs of needle! May have good sample!

• Aspirate mass w/ ¾ volume of syringe of negative pressure, release before removing to avoid drawing blood*

• Cytology collected in EDTA*

What medium should an aspiration/most cytologies be collected in? What type of collection might you use something different, and what would it be?

• Collected in EDTA

• Cavity centesis may use red top tube (clot tube) depending on testing (exudates), but definitely EDTA

What are 4 differences between a transudate and an exudate?

Transudate:

1. Clear, serous, light yellow

2. Protein/SPG = <3.0, <1.017

3. Cells = few mesothelial, small lymphs, and RBC

4. Bacteria = absent

Exudate:

1. Clear/cloudy

2. Protein/SPG = >3.0, >1.017

3. Cells = neutrophils, lymphs, RBC

4. Bacteria = usually present

What is a complication of trying to make a smear on a sample that has extra fluid (transudate)?

Cells will shrink when slide dries (exudates have enough protein to protect the cells from drying + when transudate dries, environment of cells becomes hypertonic and water rushes out of the cell); can add albumin to try to remedy this issue by buffering cells

How are smears from a cavity centesis made? What are the 3 types?

Smears made with traditional blood smear method or use cytocentrifugation (easier with transudates)

1. Thoracocentesis

2. Pericardiocentesis

3. Abdominocentesis

What are the 4 types of smear techniques in cytology?

1. Squash prep - good for bone marrow when done correctly; lay spreader flat on aspirate (spicule is flat portion on slide), light pressure and drag down (can lay horizontal), con = cells very concentrated + hard to read

2. Combination prep - good for unknown fluid (1/3 of aspirate to squash prep, 1/3 to blood smear, 1/3 is untouched)

3. Needle spread - spread aspirate in starfish shape w/ point of needle, pro = less ruptured cells, con = more fluid around cells which changes cell size (natural shape not determinable)

4. Line concentration - same as blood smear, but stop halfway

What is a method of fixing a sample to a slide?

Can alcohol fix hemo/micro slides (do not use heat!)

When should each of the 4 cytology techniques be used?

Technique determined by viscosity + cellularity of fluid

• Increased viscosity = squash prep

• Small sample = line smear concentration

If you have a low cellularity fluid, what can be done to prepare an accurate smear? What about high cellularity fluid?

• Low cellularity = sediment (prepare smear from sediment)

• High cellularity/homogenous = blood smear method

What are 3 things you should scan for on a cytology slide before reading?

Scan on 4x and/or 10x

1. Abnormal clumps

2. Quality of staining

3. Foreign objects (crystals, parasites, fungal hyphae)

What are the most commonly used stains in cytology?

1. Romanowsky (Wright’s, Giemmsa, Diff Quik)

2. Papanicolaou stain (looks for mitotic cells, pap smears)

3. New methylene blue

• Smears that are thinner and have lower TP should have lower staining time

What are the advantages/disadvantages of Romanowsky stains?

1. Pros = easier, more available, good overall stain

2. Cons = Diff Quik does not stain granules of some mast cells (often called macrophages), doesn’t stain mitotic cells

What are the advantages/disadvantages of papanicolaou stain?

1. Pros = stains nuclear + nucleolar detail better

2. Cons = doesn’t stain well (cytoplasm, bacteria, other organisms), multiple steps, increased time, wet fixed, reagents difficult to maintain

What are the advantages/disadvantages of new methylene blue stain?

1. Pros = stains nuclear/nucleolar areas well, can be used in conjunction w/ Romanowsky

2. Cons = stains cytoplasm weakly

What are the steps to evaluating a cytology slide?

1. Cells normal/abnormal?

2. Inflammatory/non-inflammatory?

• Non-inflammatory = decreased cells & TP

• Inflammatory = increased cells & TP

3. Evidence of neoplasm?

4. If malignant carcinoma or sarcoma?

5. Cell type (difficult)

What are 4 general characteristics of malignant cells?

1. Multiple, pleomorphic bizarre nucleoli (mitotic)

2. Divide often

3. Basophilic cytoplasm

4. Often in clumps (don’t mistake for mesothelial cells coming off in sheets; lining cells), unusual homogenous

Benign cells may be obtained from a malignant tumor

What are some defining characteristics of epithelial cells?

1. Tend to form clusters

2. Nuclei + cytoplasm well-differentiated

3. Blue cytoplasm + round nuceli

4. Form normal tissue

5. When this cell goes malignant = carcinoma**

What are some defining characteristics of mesenchymal cells?

1. Spindle shaped** + irregular nuclei

2. Cytoplasmic boundaries indistinct, cell clumps occur

3. Difficult to obtain w/ FNA/scraping due to intracellular matrix

4. From mesenchymal tissue + form normal tissue (lining cell)

5. When this cell goes malignant = sarcoma**

6. Name for mesothelial/epithelial cell

What is hematopoietic tissue?

Bone marrow

• All cell maturation series of circulating blood cells

• Blasts to differentiated circulating cells

What cells are really the only cells that will be ID’d in cytology samples?

• Segmented neutrophils

• Mesothelial cells

• Macrophages

• Eosinophils

• Basophils

• Lymphocytes

T/F

Hyperplasia in some organs (prostate) may be suggestive of malignancy, but cells are still normal

True

What characterizes an inflammatory process?

1. Cells present = neutrophils + eosinophils

2. Acute = granulocytes (neutrophils, eosinophils, basophils)

3. Chronic = macrophages + lymphocytes

What are some defining characteristics of carcinoma cells?

Adenocarcinoma

• Round/polygonal cells in clusters

• Deep blue

• Vacuoles

Squamous cell carcinoma

• Individual cells

• Deep blue cytoplasm

• No vacuoles

Overall**

• DARK cells

• Clumped

• Massive cells

What are some defining characteristics of sarcoma cells?

• Spindle shaped to oval cells

• Reddish-blue cytoplasm

• Irregular shaped nuclei

• Cells often form tails + nuclei protrude from cytoplasm

• General rule = sarcomas do not exfoliate well; aspirates may not yield any cells

What are characteristic of round cell tumors?

1. Round cells

2. Common in dogs/cats

3. Include lymphoma, mast cell tumor, malignant melanomas

What is normal within a lymph node? What does reactive lymphadenopathy cause? Lymphadenitis? Neoplastic lymph node?

1. 75-90% small lymphs

Reactive lymphadenopathy

1. Mix of small lymphs, prolymphocyte, lymphoblasts, plasma cells, and macrophages

2. May see eosinophils

Lymphadenitis

1. Similar to reactive, but see neutrophils + pyknosis (nucleus becomes small and leads to cell death)

Neoplastic lymph node

1. Monomorphous population w/ large immature lymphoid cells, increased nucleus to cytoplasm ratio, coarse chromatin & nucleoli

What is the advantage of doing a touch prep of an excised mass?

Ability to determine what a mass is before excising it gives greater knowledge of how aggressive to be in surgery

What is a specific area of the body you may find mesenchymal cells?

1. Nerves

2. Connective tissue

What comprises acute inflammation? What are common causes?

1. >70% neutrophils

2. Most common cause = bacterial but could be other organisms (Sporothricosis) or noninfectious disorders (necrotic areas in tumors, immune mediated disorders)

What comprises pyrogranulomatous (chronic active) inflammation? What are some causes?

1. Neutrophils

2. Macrophages (15-50%)

3. Chronic active inflammation

4. Cause other than bacterial infection includes: fungal infections, mycobacterium, protozoa, noninfectious disorders (foreign bodies, necrosis)

How would you prepare a transudate vs. exudate smear?

Transudate

• Cytocentriguation/centrifuge and resuspend sediment

• Make at least 2 smears, one air dried and one fixed immediately

• Do squash prep or line concentration (squash prep ideal to not damage cells)

Exudate

• Can do traditional 45 degree angle blood smear technique due to higher protein content

What comprises granulomatous (chronic) inflammation? What are some causes?

1. >50% macrophages

2. Causes = fungal, mycobacterium, protozoa, foreign bodies, necrosis

What comprises eosinophilic inflammation? What are some causes?

1. >10-20% eosinophils

2. Mixture of neutrophils, macrophages, mast cells, and lymphocytes

3. Causes = immune/allergic reactions, parasitic infections/disorders, fungal

What comprises lymphocytic or lymphocytic/plasmacytic inflammation? What are some causes?

1. Nonlymphoid tissue (abnormal to see increased lymphs outside of lymphoid tissue)

2. Increased lymphocytes (small + mature)

• Lymphosarcoma = large lymphoblasts

3. Causes = injection site reactions, feline stomatitis/gingivitis, lymphocytic/plasmacytic gastroenteritis

What is the purpose of a tracheal wash?

Detection of pulmonary disease, only done in dry disease since wet disease can be extracted and prepped

• Infuse saline into lungs via catheter through ET tube (tip in tracheal/bronchial/bronchoalveolar site), move around, withdraw fluid, prep

• Cell count not performed (depends on amount of fluid placed by DVM, doesn’t matter since body is not producing that fluid, presence matters)**

What cells are seen in a normal BAL (bronchoalveolar lavage)? What is abnormal?

1. Few cells (epithelial cells/macrophages), small amount of mucus

2. Abnormal = exudate

• Chronic bronchiolar problem = granulocytes (neutrophils, eso, basos) + macrophages

• TP obtained via refractometer

• Acute inflammation = granulocytes

• Inflammatory RXN = eos

• RBC rare, neoplastic cells

What is the appearance of neutrophil degeneration? What are some causes?

1. Appearance = cytoplasmic breakdown

2. Suggests bacterial causes

3. Induced by autolysis (rot)

What is the appearance of non degenerative neutrophil damage? What are some causes?

1. Appearance = pyknotic/hypersegmented

2. Aging changes

What are 3 examples of common pathogenic bacteria?

1. Coccoid bacteria

• Gram + (staph/strep)

2. Bacterial rods

• E. Coli

• Clostridium spp (spore forming)

3. Oral contaminant

• Simonssiella - extracellular on squamous epithelial cells

What is the predominant cells in spinal fluid? How many are common?

• Small, mature lymphs

• Typically less than 5 cells

What are common pathogenic fungi in cytology?

1. Fungi that primarily form hyphae

• Do not stain well with hem (H & E) stain

• Make sure that stains are not contaminated if finding fungi

What are some common dermatophytes in cytology?

Microsporum + Trichophyta spp

• Commonly cause ringworm

• Seen as mall spores on or w/in hair shafts

What are 6 examples of noninfectious inflammatory lesions in cytology and what cells they consist of?

Cysts

• Usually contain macrophages

• May contain neutrophils induced by pressure of cyst

• RBC’s in intracystic hemorrhage

Neoplasia

• Tumors that outgrow blood supply create necrotic areas containing neutrophils w/wout macrophages

Eosinophilic Granuloma

• Scraping/aspirate often containing increased eosinophils

Steatitis (fat necrosis)

• Contain inflammatory cells + variable size fat vacuoles

• Macrophages contain lipid vacuoles + multinucleated

Immune Mediated Skin Lesions

• Nondegenerated neutrophils + necrotic debris + lymphocytes

• Squamous epithelial cells

Insect Bites

• May be neutrophilic to eosinophilic to mixed to all basophils

What 2 main hormones have an influence on estrous cycle changes and effects on cells?

1. Estrogen

2. Progesterone

What cells are seen in vaginal cytology?

1. Neutrophils

2. RBC’s

3. Morphology of epithelial cells

What is vaginal cytology used in conjunction with?

Used in conjunction w/ history and clinical examination in determining the stage of estrous cycle in females

What are parabasal cells? What phases of estrous are they most common in? What are some characteristics?

1. Basal epithelial cells seen on typical vaginal smear

2. Round/nearly round + have high nuclear to cytoplasmic ratio

3. Prevalent during diestrus + an estrus

4. Absent during estrus

What are intermediate cells? What stages of estrous are they common in? What are some characteristics?

1. Diameter is 2-3x of that of a parabasal cell

2. May contain prominent nuclei

3. Prevalent during all stages of cycle except estrus

4. Vary in size

5. Some are rounded shape and others have polygonal shape

What are superficial cells? What phases of estrous are they prevalent in? What are some characteristics?

1. Largest cells seen on vaginal smear

2. Polygonal + distinctly flat, may appear folded**

3. Often seen in large sheets/strings

4. Nuclei are absent/pyknotic

5. Referred to as being “fully cornified”

6. Not normally seen during anestrus; large numbers of only superficial cells are defining characteristic of cytologic estrus

What other cells can be seen in vaginal cytologies?

1. Neutrophils

2. RBCs

3. Bacteria

4. Usually no pathologic significance

What are some characteristics of anestrus?

1. No vulvar swelling

2. Predominately non-cornified squamous epithelial cells

3. Primarily parabasal + intermediate cells

4. No RBCs, some neutrophils

5. Lasts <4.5 months

What are some characteristics of proestrus?

1. Vulvar swelling

2. Slightly bloody discharge (RBCs)

3. Lasts 4-13 days

4. Cells are non-cornified w/ nucleus to large superficial squamous epithelial cells (usually later)

5. Early = high RBCs, basal + parabasal epithelial cells

6. Late = cornified epithelial cells + pyknotic nuclei

7. Small number of neutrophils especially in early stage

What are some characteristics of estrus?

1. Lasts about 4-13 days

2. Vulvar swelling

3. Discharge is pinkish to straw colored

4. All cornified squamous epithelial cells, usually anuclear

5. Small # of RBCs present, neutrophils absent

What are some characteristics of diestrus?

1. Lasts 2-3 months, coming out of heat

2. Decreased cornified cells, increased non-cornified cells

3. Neutrophils increase until about day 3, then decrease

4. Often hard to differentiate from anestrus

What will you see cell wise in vaginitis/metritis?

Reveals non-cornified squamous epithelial cells + large number of neutrophils w/ free phagocytized bacteria

(Inflammation of vagina/uterus results in pinkish-white discharge)

What are some things to avoid to preserve semen samples?

1. Avoid exposing to marked change in temperatures (especially cold)

2. Avoid water

3. Avoid disinfectants

4. Avoid variations in pH

5. Tools used to examine should be warmed to 98.6 degrees F (37 degrees C)

6. Stains/diluents should be warmed

What are some things viewed for a semen sample being evaluated?

1. Volume

2. Gross appearance

3. Microscopic motility

4. Spermatozoal concentration

5. Ratio of live/dead spermatozoal concentration (dead will pick up stain, live will not)

6. Assessment of morphologic features

7. Presence of foreign cells/material

(T/F)

Method of collection greatly influences volume obtained, gross appearance, and spermatozoal concentration

True

(T/F)

There are 3 fractions to sperm (sperm free, sperm rich, and sperm poor). Bucks, bulls, and toms have all three portions collected while boars, dogs, and stallions will have sperm rich and poor separated

True

What is assessed for sperm?

1. Motility (wave motion (swirling activity in a drop of semen at low mag), motility (dilute sample examined at 100x mag w/ warm isotonic saline, look at % of motile sperm (60% moderately active motility is ideal**)

What are the basic differences between sperm concentration and live/dead sperm ratio?

Sperm Concentration

• Diluted + counted w/ hemocytometer

• Sample settles for a few minutes and checked for homogenous distribution of sperm

Live/Dead Sperm Ratio

• Stained w/ vital dye (eosin/nigrosin mix)

• After a few seconds of contact, make blood smear

How many cells are counted in sperm morphology?

• Observe 100-500 cells and not percentage of abnormal sperm

• Abnormalities = head, mid-piece, tail

• Other cells = normal - contains few, if any WBCs, RBCs, or epithelial cells and no bacteria/fungi

What are some reasons for sperm collection?

1. Breeding soundness exams

2. Examine prostatic fluid for culture or cytology in case of prostatic enlargement

What tests are done on sperm?

1. Concentration

2. Motility

3. Morphology (referred to as viability in lecture)