QFR 3

[Grab your reader’s attention with a

1) Cytology and indications great quote from the document or use
this space to emphasize a key point. To
Diagnostic Cytology place this text box anywhere on the
o microscopic exam’n of page, just drag it.] cells from diff. body sites for
diagnostic purposes
o Cells are from secretions or body fluids
o Secretions must not allow to dry before Pap’s staining
*Pap Smear: from female genital tract (cervicovaginal secretions) or breast
But we used cheek lining as our specimen also with squamous cells
o Body fluids obtained from hollow cavities
 Lungs
 Abdominal
 Pleural
 Pericardial fluid from Pericardium
 Ascitic fluid from GIT
o CELL BLOCKING TECHNIQUE is employed not Tissue Processing
-concentrate to come up with sediments by Cytocentrifugation

Recall Tissue Processing

- One 1st slide we passed to ma’am Abdominal and Pleural Cavity
1. Centrifuge the body fluid
2. Obtain sediment secured in a filter paper ( not wash off they’re unfixed might loss sediment)
3. Enclose cassette with label (abdominal or pleural)
4. Perform tissue processing but omit 1st step w/c is Fixation. Thus, start with Dehydration
5. Obtain serial sections
6. Stain with H& E

H & S Staining Technique

1. Xylene: 2.5 minutes
2. Xylene: -do-
3. Absolute alcohol: -do-
4. Absolute alcohol: -do-
5. 95% Ethyl alcohol: few dips (5-10)
6. Tap water: several rinsing
7. Hematoxylin: 5-10 minutes
8. Tap water: few changes
9. 0.5% acid alcohol: 1-3 rapid dips
10. Tap water(running): 1 minute
11. LiCO3: 30-60 seconds
12. Tap water(running): several changes
Note: microscopy
Nuclei: bright clear blue
Background: light/ colorless

13. Eosin: Few dips to 1 min

14. 95% Ethyl alcohol:
15. 95% Ethyl alcohol:
16. Absolute alcohol:
17. Absolute alcohol:
18. Xylene:
19. Xylene:
20. Xylene:
several dips
several dips
several dips to 1 min
several dips to 1 min
several minutes (5mins)
several minutes (5mins)
several minutes (5mins)

A. Exfoliative Cytology
(ma’am)
-sample: shed/ exfoliated/ desquamated cells from superficial surfaces like the skin
Causes of Normal Desquamation
 Constant growth and development
 Replacement with new cells
Indications/ Use of Exfoliative (5)
o Cancer staging
o Detection of cervical lesion
o Assessment of female hormonal status like endocrine d/o
o Genetic Sex determination
-drum stick shape : neutrophilic segmenters
o Detection of infectious indication
 (Book)
-m’c study of cells that have been desquamated from epithelial surfaces.
-found in smears that are shed or physically removed from epithelial and mucous membranes
(vagina, buccal mucosa and from body fluids, sputum, urine, pleural fluid, peritoneal <ascitic> fluid,
gastric juice and cerebrospinal fluid <CSF>)
-spontaneous exfoliation is due to constant growth and replacement w/ new cells seen in malignant
tumor cells.
Recommended to:
1. Detect malignant cells in body fluids, esp. Staging cancer
2. Detect precancerous cervical lesions in women(cervicovaginal smear/Pap smear)
3. Assess female hormonal status in cases of sterility and endocrine d/o. Through Evaluation for
determination of maturation index (MI), from lateral vaginal walls.
4. Determine Genetic Sex- women nuclei w/ conglomeration of chromatin, XX chromosomes(Barr
Body), demo from buccal or vaginal mucosa.
5. Detect infectious agents.

FIXATION
50% Alcohol - for all types of effusions not yet ready for smear. Or replaced by
Saccomano preservative (50% Alcohol and Carbowax)
10-15 - minutes air dry after fixation
2000 RPM for 2minutes – for specimens >few drops
Common fixatives:
1. Equal parts of 95% ethyl alcohol and ether -flammable
2. 95% ethyl alcohol –use in routine prep’n

B. FNA (Fine Needle Aspiration) CYTOLOGY

-simple, safe and rapid
-commonly used for Dx for cancer in last 30 yrs
-FNA superficial masses: breast, thyroid, and peripheral lymph nodes- done by clinicians or
pathologists
-Deeply Lesions: lung, mediastinum, abdominal organs like liver & pancreas, retroperitoneal organs like
kidney, adrenals and lymph nodes- performed under LAPAROSCOPY, COMPUTERIZED
TOMOGRAPHY (CT scan) or ultrasound (SONOGRAPHY)

2) Indications of FNA (Fine Needle Aspiration Cytology)

(ma’am)
-no shedding
-through aspiration technique taken from a Lesion
-from solid organs/ tissue masses
 Thyroid
 Lymph nodes
 Soft tissuesu
 Nodules
-23 gauge needle is used
-secretions :for smear preparation

(Book)
 FNA Technique for Palpable Masses
 Common sites: breast, thyroid, soft tissues & lymph nodes
 22-23 gauge needle
 FNA Technique for Non-Palpable Masses
 Aspirated under fluoroscopy, computed tomography, or ultrasound
 For non-cystic masses material remains in the needle
Fixed with 95% alcohol or spray fixative
 For cystic Lesions the fluid is submitted fresh in the syringe
No fixative and w/o needle attached
*Easy collection of FNA by SIMPLE SCRAPING : direct smear-slide-pull technique-routine fixative-H&E
staining

SLIDE PREP’N
o Solid lesion aspiration- usually few drops fr the tip of the needle has the most diagnostic mat’l
o Bloody specimen- diagnostic cells are diluted hard to find on a direct smear
o Maximum of 4 slides
o Slide-pull technique(like technique for PBS)
o Rinse needle in a preservative sol’n: Saccomano Fluid

SLIDE FIXATION
o Most crucial
o Immediate fixation is important
o 95% alcohol or spray fixative (can be alcohol-acetone or formalin if papanicolau staining is used)
o Air-dried
3) Describe different methods of collection for Gynecologic specimens. Note indications of each.
(ma’am)
1. Smear Preparation
-secretions/effusions
Wet fixation: 95% ethyl alcohol or 50% ethyl alcohol
2. Cell Blocking technique
-need to concentrate first
-use of Cytocentrifuge : 100-2000 RPM for 1-2 minutes
solutions:
a. 95% ethyl alcohol
-can coagulate protein
b. Picric acid ( optional )
-can precipitate proteins
Body fluids classification
1.SEROUS
-normal substance that accumulate into body cavities and passages
-peritoneal, synovial, pleural

2.EFFUSIONS
-can accumulate more than of its amount
-with physicochemical changes
-indication of malignancy
2 Types
i. Transudate – low specific gravity , low proteins
ii. Exudate – high specific gravity , high proteins

Collection for Diagnostic Cytology

1. Scraping
 Volume: 20-30 mL of effusion / 50 mL of urine
 3 smears for sputum consecutive mornings with alveolar macrophage
 Body fluids are not fixed (?)
 Only method for preservation: Refrigeration @ 4C for
24-48 hours(with protein content) or
12-24 hours (with mucous content) or
1-2 hours (with protein and mucous content)
 For low pH sample collected on ice processed w/n minutes to prevent from HCl damage
 Fixatives for wet specimens
 50% ethyl alcohol
 Saccomano’s fixative
 50% ethyl alcohol
 2% carbowax
 alcoholic formaldehyde
-substitute for NAFS/ AFF used for cell blocks (?)
AAF components:
 34 mL 95% ethyl alcohol
 4 mL formalin
 2 mL glacial HAc
 95% ethyl alcohol
-gives nuclear detail
 Ether + Alcohol
 Coating fixatives
-seen in spray fixatives
-with alcohol based (fixes cells) and wax-like substance like carbowax (protective film on
surface)
- 10-12 inches away from slide using spray fixatives to prevent pressure from washing out the
specimen
-DisAdvantage: Carbowax as coating material if not remove will inhibit staining. Submerge in
95% ethyl alcohol overnight prior to staining to eliminate carbowax.

4) Describe the cells found in a normal cervicovaginal smear. Note significance.

NORMAL CELLS IN CERVICOVAGINAL SMEAR (3)

 MATURE SUPERFICIAL CELLS
-polygonal squamous cells
-45-50 um in diameter
-pale, pink-staining cytoplasm and dark pyknotic nuclei (<6u in diameter)
-True acidophilia under estrogen influence(but not reliable index of maturation
-Pseudo-acidophilia may be observed due to the drying of smears esp before fix’n, prolapse and
drying of vaginal epith’m, infxn and chem’ls.
 INTERMEDIATE CELLS
-medium sized polyhedral or elongated calls
 -basophilic vacuolated cytoplasm
 PARABASAL CELLS
-round to oval cells
-small dense basophilic cytoplasm
-15-30 um
-smaller than intermediate cells
-larger vesicular nucleus
-found fr 2 weeks of age to puberty, after childbirth, w/ abortions and after menopause
OTHERS: (4)
 NAVICULAR CELLS
-boat-shaped intermediate cells
-has strong tendency to fold or curl on edges
-presence suggests Estrogen-Progesterone effect
-found in latter half of the menstrual cycle, pregnancy and menopause
 PREGNANCY CELLS
-round, oval, or boat- shaped cells
-translucent basophilic cytoplasm in center of the cell due to glycogen accumulation pushing the
nucleus towards the side or to the cell membrane
-appearance is due to deeper blue stain of the cytoplasm at the periphery
 ENDOMETRIAL CELLS
-small cells and slightly cylindrical
-less basophilic cytoplasm
-occur in tightly packed group of 3 or more
-found during and 1-10 days after menstruation and shed in response to ovarian hormones
 ENDOCERVICAL GLANDULAR CELLS
-occur in large groups or small sheets
-pale blue/gray cytoplasm and finely vacuolated
-often indistinct cell borders and nuclei w/ finely granular chromatin
-HONEYCOMB appearance when viewed on end

5) Discuss the principle of PAP SMEAR. Identify solution and use of each. Give advantages.
(ma’am)
PAP SMEAR/Papanicolau Method
-diagnosis of malignancy by microscopic exam’n
-sample specimen :cervicovaginal secretions
-staining method of choice for exfoliative cytology or gynecological specimens
Advantages
a.Transparent blue staining of cytoplasm due to high alcoholic content of cytoplasmic counterstain
for overlapping cells to be seen and identified.
b.Excellent nuclear detail
c.Predictable color range and great value in ID and class’n of cells, good differential coloring of
basophilic and acidophilic cells.
d.Valuable in comparing cellular appearances in smears w/ their counterpart in similarly stained
sections.

PAPANICOLAU STAINING

1. 80% Alcohol 6 DIPS

2. 70% Alcohol 6 DIPS
3. 50% Alcohol 6 DIPS
4. Distilled water 3-5 DIPS
5. HEMATOXYLIN 3 minutes
6. Distilled water 3-5 DIPS
7. 0.5% HCl 3 DIPS
8. Running water RINSE
9. 50% Alcohol 6 DIPS
10. 70% Alcohol 6 DIPS
11. 80% Alcohol 6 DIPS
12. 95% Alcohol 6 DIPS
13. OG-6 1.5 mins
14. 95% Alcohol 6 DIPS
15. 95% Alcohol 6 DIPS
16. 95% Alcohol 6 DIPS
17. EA-50 1.5 min
18. 95% Alcohol 6 DIPS
19. 95% Alcohol 6 DIPS
20. 95% Alcohol 6 DIPS
21. Absolute Alcohol 5 DIPS
22. Absolute Alcohol 5 DIPS
23. Alcohol + Xylene 5 DIPS
 24. Xylene 6 DIPS
25. Xylene 6 DIPS
26. Xylene 6 DIPS

PATIENT PREPARATION

1. Avoid DOUCHING AGENTS (eg.feminine wash) for 24-48 hrs prior to collection
*Effects:
 Alters pH of surrounding env’t thus affecting microorganisms
 Predisposed to disease like amoebiasis and UTI
*Remedy/Alternative:
 2-3 drops of white vinegar
2. Avoid INTRAVAGINAL DRUGS (eg.suppositories) for a week prior to collection
3. Avoid coitus for 24 hrs/ a day prior to collection
4. Avoid collection during menstrual period

*Materials for collection should be:

 Non-lubricated and powder-free gloves
 Dry & sterile mat’ls for collxn and smear prep’n

SAMPLING DEVICES
1) AYRE’S SPATULA
- both ends are rounded
- a speculum with different sizes
- use for opening vagina
- like tongue depressor
Types:
a. Wooden Spatula – most preferred bec. w/ rough surface so more is collected
b. Plastic
- DisAdvantage: painful and traumatic
2) AYLESBURY VERSION OF AYRE’S
- one end is rounded and the other end is pointed
3) CERVIX BRUSH
- flexible plastic brush to follow the shape of ectocervix, endocervix, and T-zone
- suitable for all cervical shape
4) ENDOCERVICAL BRUSH
- like bottle brush
- one end w/ nylon bristly
- one rotation and press the up and down
5) CYTOBRUSH
- use for the whole cervix
- similar w/ #4 but w/o nylon brush

*Better Sampling – spatula & endocervical brush

*Triple Smear – vaginal, cervical & endocervical
*considerations for post menopausal women – spatula & endocervical brush
*Prolapsed uterus – cytobrush

TECHNIQUES FOR COLLECTION

 SCRAPING
 SWABBING
 ASPIRATION (can’t be in cervical secretion)
 WASHING

FAULTY TECHNIQUES (results to Artifacts)

1. Delay in processing or smear preparation
-must not allow to dry
-fix in 95% alcohol
EFFECT:
 Loss of cellular morphology
 Bacterial contamination

2. Delay in Fixation
-Formation of air-drying artifact characterized by a pale stained nuclei
EFFECT:
 Lack of differential cytoplasmic staining
 Cytoplasmic and nuclear eosinophilia

*Remedy for air-drying artifact (dehydrated)

 REDYDRATE unfixed air dry smears by placing smears in:
 a.50% aqueous sol’n of Glycerine for 3 minutes
b.95% Ethyl alcohol at least 2 rinses then proceed to staining

3. Using of unfiltered stains

-stains must changed weekly
EFFECT:
 Contamination
Manner of reporting in Bethesda’s System
-basis: nuclei
 Maturation Index
 Acidophilic Index

*minimum Fixation Time : 15minutes w/ Ethyl Alcohol

*maximum Fixation Time : 1 hour
*combination of OG-6 and EA 50 gives subtle range of Green, blue and pink hues to cytoplasm

6) Note special considerations and guidelines in handling, collection and preserving of gynecologic
specimens.
1-Gynecologic Specimens : Conventional/ Liquid-Based
 T-Zone /Transformation Zone
- junction b/n Endocervix and Ectocervix
–where majority of cervical carcinomas and precancerous lesions of the cervix arise.
-important for detection of dysplasia and carcinomas of the cervix
 adequacy of sampling is indicated by the ff.-Types of specimens w/c shows mitotic div’n
o Squamous cells
o Columnar cells
o Metaplastic cells
 Other Samples to be collected:
o Conventional Pap Smear
o Endocervical brush- samples of endocervial canal
o Vaginal scrape- patients w/ hysterectomy
o Lateral vaginal scrape- hormonal evaluation
o Four Quadrant Vaginal Scrape- localization of vaginal adenosis
o Vulvar scrape- herpetic lesions or carcinoma

 Vaginal Hormonal Cytology

-inexpensive
-performed w/o undue risk even in pregnant women
-vaginal smears taken fr UPPER LATERAL THIRD OF THE VAGINAL WALL, more accessible
and less likely to be contaminated cellular debris or vaginal discharges
-examined first under low magnification to assess quality of smear and staining, to detect RBC
and Leukocytes and type of Exfoliated cells
-give rough assessment of the proportion of mature superficial pyknotic acidophilic cells
-quantitative evaluation of the smear under 40 x objective

7) What is the Bethesda’s system of reporting in diagnostic cytology. Describe.

*Best time of Collection: 2 weeks after 1st day of last menstrual period
*5 purposes of Exfoliative Cytology
i. Detect malignant cells in body fluids, esp. used in Staging cancer
ii. Detect precancerous cervical lesions in women(cervicovaginal smear/Pap smear)
iii. Assess female hormonal status in cases of sterility and endocrine d/o. Through Evaluation for
determination of maturation index (MI), from lateral vaginal walls.
iv. Determine Genetic Sex- women nuclei w/ conglomeration of chromatin, XX chromosomes(Barr Body),
demo from buccal or vaginal mucosa.
v. Detect infectious agents.
*Stains used:
 OG-6
 EA-50

EXAM coverage:

CELL BLOCKING
note
 Paraffin Tissue Processing
 Ethyl alcohol
 How to prepare Xylene and Paraffin
Special Stains
1,2,3 QFRs
**GOOD LUCK AND GOD BLESS SA ATIN! WE CAN DO THIS! IN GOD WE TRUST 🙏🙏**