Unit 10 Flashcards
- Peritoneal, pericardial and pleural fluids;
- CSF;
- Nipple discharge;
- Bronchial brushings/washings;
- Sputum;
- *Gastric washings;
- Urine sediment;
- Prostatic secretions/fluid;
- Cervicovaginal (paps) smear
SPECIMEN FOR EXAMINATION
● Volume;
○ Measure using the graduated cylinder or syringe (if the volume is small).
○ mL is used.
● Color;
● Consistency.
○ To check whether the specimen is watery,
mucoid, purulent, etc.
LIQUID FORM
● Number of slides received;
○ Usually in a fixed state.
○ Document how many slides are submitted
which are part of the documentation.
● Labeled specimen.
PREPARED SMEARS
● Urinary Sediment;
● Bronchial Lavage Specimen;
● Specimens that utilize proteolytic enzymes during processing.
○ Examples
■ Trypsin
■ Conc. Sputum
■ Enzymatic lavage sample from the GIT
SPECIMENS THAT REQUIRE ADHESIVE AGENT
The more watery the specimen is, the more difficult the adherence of the specimen to the slide.
TRUE
● Must be permeable to both fixative and the stain;
● Must not retain the stain.
CHARACTERISTICS OF A GOOD ADHESIVE
● Pooled human serum or plasma;
● Celloidin Ether-Alcohol;
● Leukonostoc culture.
GOOD ADHESIVES FOR CYTOLOGIC METHOD
● Smears should be from fresh material;
● Complete requisition data;
○ Patient’s ID: name, age, gender, LMP, PMP,
date, and type of specimen
● Label the slide.
SMEAR PREPARATION
- Streaking;
- Spreading;
- Pull apart;
- Touch or impression smear (Imprint).
- All have to be fixed immediately in the fixative of choice.
METHODS OF SMEAR PREPARATION
- Cellblock Preparation
A histologic preparation that is a sediment
that would be processed just like a tissue
and will be used as a complement to the
smears.
CELLBLOCK PREPARATION
Used for preparing mucoid secretions, vaginal secretions/pap smear preparation, sputum, and gastric contents.
Use a spatula, dissecting needle or applicator stick and streak in a zigzag fashion.
A spatula or a stainless wire comb loop needle which is streaked in a zigzag fashion.
STREAKING
Used for thick mucoid secretions (smears of fresh sputum and bronchial aspirates).
The material is placed at the center of the slide and gently spread out in a centrifugal fashion where it is thin and evenly distributed.
Star pattern: another spreading technique that may also be done wherein the material is gently teased in a centrifugal fashion forming a star-like pattern.
Twist method can also be done.
SPREADING
We have two slides wherein the material is sandwiched and macerated in between and gently pressed/squashed in opposite directions using both fingers.
PULL-APART
Advantage: Final results consist of two smear slides. The material is more thin and evenly.
Thinner material = preferred = better cytologic evaluation.
pull-apart method
For preparation of direct impression from the cut surface of tissue like the lymph nodes, mucosa, and other surgical /autopsy material.
TOUCH OR IMPRESSION SLIDE
Impression cytology being collected from a patient with herpes simplex keratitis, using a sterile glass slide with polished edges.
A sterile glass slide is used and gently imprints/ touches the cornea surface → fragments are transferred/adhered to the slide.
The touch imprint complements a frozen section.
TOUCH OR IMPRESSION SLIDE
- Using standard paracentesis technique,
obtain a MINIMUM 10ML fluid specimen from
body cavity. HEPARIN may be added to the
specimen to reduce clotting. - Place three (3) units of heparin per mL
capacity of the collection container. Gently
agitate to thoroughly mix the specimen with
heparin. Submit the specimen along with the
completed cytology request form. - The specimen should be refrigerated or
kept on wet ice until transported to the lab.
● Aside from the liquid specimen, smears can also be used. In most instances, the pull-apart method is the most popular and easiest technique to do.
● For specimens that will be delayed: all liquid
specimens must kept in the refrigerator to avoid the generation of the cytologic material.
○ Exception: CSF–considered as stab material,
meaning should be collected and processed
immediately.
COLLECTION OF SPECIMEN: BODY FLUIDS
Fluid that accumulates in the lungs and is aspirated using the aspiration technique.
The liquid material collected is called the pleural fluid/pleural effusion.
The fluid can be smeared onto the slide using the pull-apart method or cell block technique.
PLEURAL FLUID: THORACENTESIS
Another method of collecting specimens that are abnormally seen/accumulates in the peritoneal cavity.
Fluid is referred to as Ascitic fluid/Peritoneal fluid
ASCITIC FLUID: PARACENTESIS
Fluid that is found on the pericardial sac/pericardium.
Fluid can be directly placed onto the slide and processed to form a cell block.
PERICARDIAL EFFUSION: PERICARDIOCENTESIS
L3 and L4 of the spinal column
Manner of collection: Lumbar tap/puncture.
Should be collected and processed immediately.
CSF: LUMBAR TAP
Help diagnose breast cancer early in the disease process.
Used to aspirate material from palpable masses as seen in the image.
Used to diagnose breast cancer in the early phase of its development.
A hypodermic syringe is used, and the fine needle gauge is between 22-25.
The aspirated material is gently placed onto a slide and a smear is prepared.
FINE NEEDLE ASPIRATION BIOPSY (FNAB)
- Gently strip the sub-areolar area and nipple with the thumb and forefinger.
- Place the slide upon the nipple and draw it quickly across the nipple.
-> If 1 drop is obtained, get another slide and do the pull-apart technique. - Immediately immerse the slide into a bottle of 95% Ethyl or Isopropyl ROH or use a spray fixative.
- Label the secretions obtained indicating laterality (left or right breast)
Advantage: Immunohistochemistry can be done directly from the sample as seen in D.
BREAST: NIPPLE DISCHARGE - TOUCH (IMPRINT CYTOLOGY)
Bronchial aspirate;
Bronchial brushings;
Bronchial lavage (BAL).
BRONCHOSCOPY
Specimen: Bronchoscopically-directed brushing of the identified lesion.
Collection Procedure:
1. Using standard bronchoscopy technique, identify the lesion in question and obtain a brushing sample of the lesion.
2. Gently apply the sample on to glass slide and immediately immerse slide into 3% GLACIAL ACETIC ACID ALCOHOL FIXATIVE
3. The brush tip should also be cut off into the solution.
BRONCHIAL BRUSHINGS
Specimen: Bronchoscopically-obtained washing (preferable at least 10 mL) of the bronchi in the region of the suspected lesion.
Collection Procedure:
1. Using standard bronchoscopy technique, lavage the distribution of the bronchus to be sampled.
2. Collect the wash in a clean container. Label the container with correct patient information.
BRONCHIAL WASHINGS
Obtained at three consecutive morning sputum specimens by deep cough method.
Collect the sample in a wide-mouth container containing Saccomano fluid (50% EtOH and 2% carbowax)
SPUTUM CYTOLOGY: NORMAL
Inhalation of aerosol solution for 20 mins to produce a deep cough sample.
Collect the sample in a wide-mouth container containing Saccomano fluid (50% EtOH and 2% carbowax)
SPUTUM CYTOLOGY: INDUCED
Exfoliated cells decompose/autolyze rapidly (whether mechanically or physiologically from the body), which may destroy cellular and nuclear details and will give inadequate results for diagnosis.
Why should FIXATION be done immediately?
All are liquid fixatives = Wet fixation
- Equal parts of 95% EtOh and Ether
-> Ether: highly flammable and ignite spontaneously, also a mild anesthetic (continued inhalation may cause drowsiness). - 95% EtOH
- Carnoy’s fluid
- Equal parts of tertiary butyl alcohol and 1 part 95% EtOH
- SCHAUDINN’S FLUID – sat. aq. Hg2Cl, absolute HoAc
- Methanol – for dried films, bone marrow smears, blood smears, etc.
- Conventional hairspray
Common Fixatives
A cell block is a method of preparing cytologic material so that it can be processed, sectioned, stained, and viewed as a histology section.
Pass through the paraffin processing technique
CELL BLOCK
It can provide diagnostic information in addition to that obtained from cytology slides.
Advantage: complements the smears.
CELL BLOCK
It is easier to do special stains, and immunohistochemistry on cell block slides rather than on regular cytology slides because the slides often require adaptations of the staining protocols and different controls.
TRUE
‘_____’ in this context is not a reference to placing the cells in a paraffin block but refers to the technique of ‘______’ something to hold it together and maintain its shape.
Block; Blocking
Cell blocks are micro biopsies embedded in paraffin that broaden the diagnostic value of cytology specimens and are complementary to cytology preparations.
Cell block is fixed in a 10% neutral buffered formalin.
It employs retrieval of small tissue fragments from FNA specimens which are processed to form a paraffin block.
TRUE
The supernatant is decanted and the sediment is directly placed on the slide and uses proper methods such as pull-apart or streaking.
Plasma Thrombin Method: can also be used wherein the plasma + thrombin is added until the preparation forms a semi-solid clot fixed in formalin.
Another option: Adding agar to the sediment and is fixed with formalin and embedded in paraffin, hence, cell block.
CELL BLOCK TECHNIQUE
__: can also be used wherein the plasma + thrombin is added until the preparation forms a semi-solid clot fixed in formalin.
Plasma Thrombin Method
Histogel is used wherein the material is mixed with a special gel material and similarly prepared into a block.
HISTOGEL METHOD
____ is collected into a conical tube.
A double embedding processing step is performed as well.
COLLODION BAG TECHNIQUE
A branch of General Cytology which deals with the microscopic study of cells that have been desquamated from the epithelial surfaces.
BASIC EXFOLIATIVE CYTOLOGY OF THE FEMALE GENITAL TRACT
Cytologic detection of malignant cells or precancerous lesions in the body.
Detection of asymptomatic or precancerous cervical lesions in women.
Assessment of female hormonal status in case of sterility and endocrine disorders.
Determination of genetic (phenotypic) sex.
Detection of the presence of infectious microorganisms.
BASIC EXFOLIATIVE CYTOLOGY OF THE FEMALE GENITAL TRACT recommended for:
Natural collection - exfoliation
Forceful collection - scraping of mucosa
TRUE
Use an unlubricated speculum (saline or warm water may be used).
After visualization of the cervix is accomplished, insert the sampling device (e.g. cytobrush®) into the endocervical canal and rotate one complete turn.
Withdraw the cytobrush and spread the collected material quickly and evenly onto the slide opposite the frosted end.
ROUTINE CERVICAL/ENDOCERVICAL (PAP) SMEAR
The endocervical mucus will prevent air-drying during collection of the subsequent cervical component.
Mucus may be absent or scant, especially in elderly patients wherein the mucosa is atrophic.
TRUE FOR PAP SMEAR
Using the extended-tip spatula (e.g. Ayerst®), scrape material with the spatula from the whole circumference of the cervix.
Withdraw the spatula and spread the collected material quickly and evenly onto the slide adjacent to the frosted end.
TRUE FOR PAP SMEAR
Fix Immediately (drop slide into fixative or spray with fixative, holding the spray bottle approximately 8 to 12 inches from the slide).
95% ethyl alcohol (fixative of choice)
Since we prepare the smears using the wet fixation technique.
TRUE FOR PAP SMEAR
- Cervix;
-> Ectocervix
-> Endocervix - Vaginal mucosa.
Pap smear has two sites:
Cells are scraped from the cervix and examined under a microscope to check for disease or other problems.
Cervix viewed through speculum with patient in LITHOMY position.
TRUE
Shiny mucosal portion - Stratified squamous non-keratinizing epithelium.
Orifice - Opening of the endocervix containing endocervical glands and mucus.
Transition zone - Transition point between ectocervix and ectocervix.
Ectocervix - Stratified squamous epithelium
Endocervix - Simple columnar/glandular epithelium.
Dynamic location where pathologic processes occur (e.g. hyperplasia, metaplasia, dysplasia, and cancer).
TRUE
Special wooden fish-tail spatula
Used to collect material from the ectocervix.
Ayres spatula
● Suspending cells in a fixative solution and centrifuged in the machine.
● Sediment is prepared onto the slide.
● SurePath
○ ThinPrep
○ MonoPrep
Advantage
○ Removes non-essential debris (e.g. rbc, bacteria).
○ Suspension of cells which can be preserved in a fixative solution.
○ Small aliquot only is used.
LIQUID-BASED CYTOLOGY-CANCER (LBC) SCREENING
ADVANTAGE
- Transparent blue or pink staining of cytoplasm is observed.
- Excellent nuclear staining.
- Color range is predictable and of great value in identification of cells.
PAPANICOLAOU OR PAP’S STAIN
DISADVANTAGE
- Procedure is lengthy and complicated.
- Does not give an accurate acidophilic index.
PAPANICOLAOU OR PAP’S STAIN
- Harris Hematoxylin
- OG 6 Stain
- EA 50 / EA 65
STAINS FOR PAP’S SMEAR
PAPS SMEAR STAIN: Demonstrates keratin in cytoplasm.
OG 6 Stain
a polychromatic cytoplasmic stain comparable to EA 36. It differentiates cellular components with pink, green and blue-green hues.
EA 50
has a lower concentration of light green that improves expression of eosin-stained components.
Brings out more of the orange color of the cytoplasm.
EA 65
- Fix in ether-ROH and pass through 80% ROH, 40% ROH and distilled H2O.
- Stain in Harris Hematoxylin for 4-5 minutes.
- Wash with H2O.
- Pass thru 0.25% HCl in 50% ROH (Acid alcohol)*.
-> AKA differentiation step
-> Purpose: improve cell clarity by
removing excess hematoxylin - Immerse in 1.5% NH4OH in 70% ROH for 1 minute.
- Rinse in 70% ROH and pass through 80% and 95% ROH.
-> Differentiation step: acid alcohol enhances staining by removing excess Haematoxylin from the slide improving cell clarity.
-> It will also clear the residual - Stain with OG 6 for 1.5 minutes. (1st Counterstain)
- Pass through 3 changes of 95% ROH.
AKA differentiation step.
Excess OG 6 is removed. - Stain with EA 65 or EA 50 for 3 minutes. (2nd counterstain)
- Pass through 3 changes of 95% ROH.
Excess EA is removed. - Dehydrate and clear in:
Absolute ROH,
Equal parts of ether and absolute ROH,
2 changes of xylol - Mount in resinous media (e.g. Canada Balsam)
PROCEDURE FOR PAP’S SMEAR
Result: Cytoplasm
Either bright red/pink or greenish to blue-gray
Result: Vesicular Nucleus
Blue
Result: Pyknotic Nucleus
Dark blue to black
Result: Bacteria
Dark blue
Result: Mycelia
Violet
Result: Trichomonas vaginalis
Pale greenish blue blob of cytoplasm
Blue cytoplasmic cells - Intermediate cells
-> Younger cells
-> Granular nucleus
Pink cytoplasmic cells - Superficial cells
-> Older variants/more mature cells
-> Pyknotic nucleus (dark blue to black; ink drop)
Vesicular Nucleus - blue
TRUE
- Evaluation of specimens
- Preparation of smears
- Fixation
- Staining and coverslipping
STEPS IN CYTOPREPARATORY TECHNIQUES
PROB: Red nuclei
Remove time in bluing reagent
PROB: Nuclear staining too weak
Increase staining time. Use
higher strength Haematoxylin.
Reduce concentration (not
time) of acid alcohol
PROB: Background Hematoxylin
staining
PROB: Overall color too blue
Increase time in acid alcohol
PROB: Cytoplasm too pink
Cytoplasm too pink
PROB: Cytoplasm too orange
Increase time in alcohol after
OG-6
PROB: Cytoplasm too blue
Increase time in EA-50
PROB: Cytoplasm too green
Increase time in alcohol after
EA-50
Pap Stained Smear
Stained bluish green with eccentric pale black nuclei.
Pear-shaped morphology.
Trichomonas vaginalis
Pap Stained Smear
Malignancy of the cervix.
Dark staining chromatin and atypia of the nuclear membrane.
Background consisting of neutrophils indicate necrosis of the stroma.
Keratinizing squamous cell carcinoma
Pap Stained Smear
Herpes-simplex virus type II
Ground glass chromatin and molding of the membrane contour.
Multinucleated giant cell
Nuclear Change
Due to the enlargement of nuclei and the most important criteria of malignancy.
However, this is benign in certain tissues, e.g. endocervical and renal pelvic cells.
Normal: cytoplasm is larger than nuclei
ALTERED NUCLEAR-CYTOPLASMIC RATIOS
Nuclear Change
Although common in malignant cells, one must be careful to exclude overstaining and not to mistake pyknosis for hyperchromasia.
Pyknosis ≠ hyperchromasia
Polyploidy
HYPERCHROMASIA
Nuclear Change
Irregular hyperchromatic or bizarre nuclei should be suspicious, but caution is warranted in certain situations, e.g. after abortion, or expulsion of H-mole,
Herpes complex infection.
Herpes-simplex virus type II
MULTINUCLEATED GIANT CELLS
Nuclear Change
Frequently seen in reactive pleural and peritoneal mesothelial cells, in histiocytes and in any tissue undergoing active repair and benign growths.
Increased proliferative activity
INCREASED MITOTIC ACTIVITY
Nuclear Change
Triple or quantiple spindles are highly suspicious and important signs of malignancy.
ATYPICAL MITOSES
Nuclear Change
Considerable variation in nuclear size and shape is common in malignant.
Pleiomporphism
ANISOKARYOSIS
Keratinizing change is seen in increased eosinophilia.
Cells of epidermoid carcinomas frequently show a tendency to cytoplasmic eosinophila.
Adenocarcinoma cells may enclose PMNs, (eg. endometrial andcolonic cancers)
Glandular formation
—-> Polymorphonuclear leukocytes
—-> Rosette formation
—-> Acinar formation
—-> Trabecular formation
Cytoplasmic vacuolation is common in adenocarcinoma cells, but may be also be seen in endometrial cells following cutterage.
Adenocarcinoma cells secrete mucin and produce glycogen and appear as vacuolated-looking cytoplasm.
CYTOPLASMIC CHANGES
Nuclear Change
Often seen in malignant cells, polypoidic cells may also occur in benign conditions, especially in thyroids of older women and less in adrenal cortex and hyperactive islets of Langerhans.
In vaginal smears, they may result from pregnancy as well as malignancy.
POLYPLOIDY
