GenPath Lab

QFR 3

1) Exfoliative Cytology and indications

Diagnostic Cytology- microscopic exam’n of cells from diff. body sites for diagnostic purposes.

A. Exfoliative Cytology
(ma’am)
-specimen 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 tissues
 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 : 1000-2000 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
-fr 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
2. Aspiration
3. Lavage
4. Brush technique

**Non-Gynecologic
 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.

(Book)
**Gynecologic
1. Conventional Pap Smear
2. Endocervical brush- samples of endocervical canal
3. Vaginal scrape- patients w/ hysterectomy
4. Lateral vaginal scrape- hormonal evaluation
5. Four Quadrant Vaginal Scrape- localization of vaginal adenosis
6. Vulvar scrape- herpetic lesions or carcinoma

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

 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:
 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.

PAP SMEAR/Papanicolau Method

-diagnosis of malignancy by microscopic exam’n
-vaginal smears to detect human uterine and cervical cancers
-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.
5 dyes in 3 solutions
(internet source)
1. HEMATOXYLIN
-stain nuclei blue
- affinity for chromatin attaching sulfate groups on the DNA
-most common is Harris’ hematoxylin
2. ORANGE GREEN 6 (OG-6)
-first acidic counterstain (cytoplasmic stain) stains matured and keratinized cells
-target structure is stained orange in diff intensities
3. EOSIN AZURE
-second counterstain, a polychrome mixture of eosin Y, light green SF and Bismarck brown
EOSIN Y gives pink color to cytoplasm of mature sq. cells, nuclei, cilia and Rbc. Commonly used are
EA 31 and EA 50, EA 65.
LIGHT GREEN SF
BIRMARCK BROWN Y

*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
–where majority of cervical carcinomas and precancerous lesions of the cervix arise.
-important for detection of dysplasias and carcinomas of the cervix
 Types of specimens
o Squamous cells
o Columnar cells
o Metaplastic cells

 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 Betheda’s system of reporting in diagnostic cytology. Describe.

*Techniques of collecting
 Scraping
 Aspiration
 swabbing
 Brushing
*Patient considerations
o No collection during menstrual period
o No sexual intercourse before collection
* Best are of collection: T-zone / Transformation Zone
*Materials for Collection
 Spatula
 Brush
 Swab
*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