13. Cytologic Techniques

Diagnostic Cytology

- Microscopic examination of cells from different
body sites for diagnostic purposes.
- Includes Exfoliative cytology and Fine Needle
Aspiration (FNA)

Exfoliative Cytology

It deals with the study of
cells that have been desquamated or shed off
from the epithelial surfaces.

Fine Needle Aspiration

It is the study of
the cellular sample obtained from the organ that
does not shed cells spontaneously such as
breast, thyroid, liver, lymph nodes, lungs, skin,
soft tissue, and even the bones.

Methods of Smear Preparation

Streaking
Spreading
Pull apart
Touch, Impression, Imprint, Abraded Smear

Streaking

application or doing direct zigzag lines
throughout the slides, mainly for sputum
samples and bronchial aspirates

Spreading

preparation of thick mucoid secretions.
- The specimen is applied and is spread by
teasing using an applicator

Pull Apart

- Use for secretion such as serous fluid,
concentrated sputum, enzymatic lavage sample
from gastrointestinal tract.
- The specimen is spread evenly on the
surface of two slides

Touch/ Impression/ Imprint/ Abraded smear

the slide is made to touch the cut surface of an
organ or tissue such as tumors (esp. lymph
nodes) and other surgical or autopsy specimens

EXFOLIATIVE CYTOLOGY

- Deals with the microscopic study of cells that
have been desquamated from epithelial surfaces.
- harvested by rubbing or brushing a lesional tissue
from epithelial surface
- proven to be of great value in the
early detection of cervical cancer

Exfoliated cells either came from:

Physical removal or shed off spontaneously from the epithelial and mucous membranes.

Purpose of exfoliative cytology

1. Detection of malignant cells in body fluids,
mainly used for staging cancer
2. Detection of precancerous cervical lesions
among women.
3. Assessment of female hormonal status in case of
sterility and endocrine disorder.
4. Determination of genetic sex
5. Detection of infectious agents

Techniques in Exfoliative Cytology

A. Smear Technique
B. Cell Block Technique
C. Membrane Filter Mode
D. Concentration Technique
E. Aspiration Technology

Smear Technique

Smear is the product of a diagnostic technique in
which cells and other components are spread
out thinly in a glass slide.

Air Drying

- to give optimal results and optimal diagnosis whenever using slides for checking blood or bone marrow smears
- Should be avoided if you are going to
check the cytological appearances if
NEOPLASIA, because it can change the
appearance of the cell

Cell Block Technique

- A cell block is a paraffin embedded specimen derived from different fluids and fine needle aspirates.
- Considered as a more definitive method for
cytopathologic diagnosis

Membrane Filter Method

- Testing Fluid Samples
- It is a technique for collecting cells using a filter
with a specialized pore size depending on the
sample that will be processed.
- Filter is made up of polycarbonate and a wide
range of synthetic materials such as
polycarbonate, cellulose acetate, cellulose
nitrate, polyamide, polypropylene, and
polytetrafluoroethylene (Teflon).

Concentration Technique

- Uses Cytospin
- Centrifuge: 1000 rpm for 1 minute
- glass slide coated with adhesive (thin layer of egg albumin)
- Outcome: concentrated suspension.
- The cells are isolated via a series of centrifugation steps to concentrate the cells into a small suspension.

Cytospin Preparation:

best method to collect cells from body fluids

Edges are dried while the center is moist:

the smear should be fixed at 95% alcohol
- If smears are not prepared immediately,
cover it with absolute alcohol, then
refrigerate

Aspiration Technology

- It is used to obtain specimens (organs) that do
not shed cells
- It can be carried out in diagnosing any palpable
lesions (FNAB/cytology) and deep-seated/ nonpalpable
lesions (NLBB or Needle localization
breast biopsy)

TYPE OF SPECIMEN

GYNECOLOGIC SPECIMENS
NON-GYNECOLOGIC SPECIMENS

GYNECOLOGICAL SPECIMENS

a. Transformation Zone (T-zone)
b. Vaginal Scrape
c. Upper lateral 3rd of the vaginal wall
scrape
d. Four Quadrant vaginal scrape:
e. Vulvar scrape

Transformational Zone (T-zone):

composed of endocervix and ectocervix

Cytologic Collection and Preparation

Endocervical brush: collecting samples
from the endocervical canal.

Vaginal scrape

collecting samples from
the hysterectomy (surgical removal of
the uterus)

Upper lateral 3rd of the vaginal wall
scrape:

used for hormonal study

Four Quadrant vaginal scrape

to check the localization of vaginal adenosis
(presence of metaplastic cervical or
endometrial epithelium within the
vaginal wall)

Vulvar scrape

used for herpetic lesions or carcinoma

Other collection device:

a. Wooden Spatula
b. Plastic Spatula
c. Brome type plastic samplers

How to collect the Specimen:

1. The patient should not have been douched or should not have undergone vaginal exam for at least 1-2 days
2. The gloves used by the examiner should not contain lubricant or any powder
3. The smear should be prepared thinly at a rotating motion instead of doing pull apart

Position of the patient:

Lithotomy position

Lithotomy position

- used during childbirth and surgery in the pelvic area.
- legs are flexed to 90° and knees are slightly bent at 70-90°
- The feet of the patient are rested on the padded foot rest attached to the table which will support the legs of the patient

Gynecological Specimen Staining Procedure:
Modified Papanicolaou Method

- provides optimum nuclear detail information
a. Harry Hematoxylin
b. Eosin Azure 50 (EA-50)
c. Orange Green 6 (OG-6)

Harry Hematoxylin

primary nuclear stain

Eosin Azure 50 (EA-50)

counter stain that will stain the cytoplasm of the parabasal and intermediate cells

Orange Green 6 (OG-6)

counter stain that will stain the cytoplasm of the parabasal and superficial cells

Vaginal Hormonal Cytology

Advantages:
- relatively expensive
- may be regularly performed with minimal risk (even for pregnant work)

Vaginal Hormonal Cytology

- Specimen is taken from the upper lateral third of the vaginal well
a. prone to hormonal changes
b. more accessible and it's less likely to be contaminated by cellular debris pr vaginal discharges

Vaginal Hormonal Cytology

- this area is prone to hormonal changes
- It is more accessible and it's less likely to be contaminated by cellular debris or vaginal discharges.

Cells found in Cervicovaginal smears are divided
into the following:

a. Mature Superficial Cells
b. Intermediate Cells
c. Parabasal Cells
d. Basal Cells

Mature Superficial Cells:

- top layer, mature, and largest
- 45-50 um
- polyhedral flat cells
- cytoplasm can be acidophilic or basophilic

Intermediate cells

- 20-30 um
- polyhedral, elongated cells
- basophilic cytoplasm, that often shows vacuoles and vesicular nuclei.

Parabasal Cells

- 15-25 um
- Round to oval cells
- Fried egg with sunny side up appearance.
- Strongly basophilic cytoplasm and vesicular nuclei that are similar to intermediate cells.

Exhibits 4 As:

Normally seen from 2 weeks of
Age to puberty, After childbirth,
with Abortions, and After
menopause

Basal Cells

- 13-20 um
- small, round to oval cells.
- relatively large nuclei that occupy
half or more than half of the cell volume
- Seen in vaginal smear only after puberty
and after menopause

Other cells that may be found in cervicovaginal
smears includes:

a. Navicular cells
b. Pregnancy cells
c. Endometrial cells
d. Endocervical Glandular cells

Navicular cells

- Boat-shaped, intermediate cell with
strong tendency to fold or curl on the
edges.
- combined estrogen-progesterone effect
- latter half of menstrual cycle, during pregnancy and during menopause

Pregnancy cells

- Also characterized as being "boatshaped"
- greatest translucency at the center due to glycogen accumulation
- More round to oval shape

Endometrial cells

- Groups of 3 or more cells
- Seen 1-10 days after menstruation

Endocervical Glandular Cells

- large group or small sheets
- honeycomb appearance
- similar to parabasal cells

Doderlein Bacillus

- Gram positive bacillus
- First described in 1892 by Doderlain
- thick, non-motile gram-positive bacillus
- Most common organism of the normal vaginal
secretion.

Pap's stain

blue to lavender

Numerous naked nuclei with many Doderlein
bacilli may indicate: [LIED]

Last trimester of pregnancy
Infection
Estrogen deficiency
DM

Ferning

- "palm-leaf" pattern
- high estrogen level
- detection of early pregnancy
- Arborization of mucus due to the formation of
salt crystals in high NaCl concentration.

Criteria for Cytologic Diagnosis of Normal
Pregnancy:

1. Marked progesterone effect (presence of
ferning)
2. At least 50% of intermediate cells in clusters
3. At least some typical pregnancy cells are present
(average of 3-7 cells)
4. Less than 30% superficial cells
5. Doderlein filled "dirty" background

Quantitation in Vaginal Cytology

a. Acidophilic Index:
b. Pyknotic index (karyopyknotic index)
c. Maturation index

Acidophilic Index

assessment of percentage of cells appearing pink, orange, and red after pap smear

Pyknotic index (karyopyknotic index)

Checking of percentage of cells having shrunken, dark, and structureless nucleus

Maturation index

consists of proportions of cells from the 3 layers of the vaginal epithelium (superficial, intermediate, deep)

Maturation index

- Provides pyknotic nuclei of <6 um in
diameter.
- criteria for mature superficial
cells (Index is based on careful
differential count)
- most accurate index available

Two Systems of Reporting of Cytologic Smears

a. Class System
b. Bethesda System

Class System

- developed in 1959
- malignant cells
- No longer used, last time it was used: December 1988
Why?
- incomplete (has no classes for non cancerous entities)
- Not reproducible
- Obsolete

Class 1

absence of atypical or abnormal cells

Class 2

Atypical cytologic picture but no evidence of
malignancy

Class 3

Cytologic picture suggestive but not conclusive of malignancy

Class 4

Cytologic picture strongly suggestive of malignancy

Class V

Cytologic picture conclusive of malignancy

Bethesda System:

- National Cancer Institute in 1988
- 3rd reworking of Pap Smear Classification System

Report format:

Specimen Adequacy
General Categorization (optional)
Descriptive Diagnosis

LMP

date of the Last Menstrual Period of the
patient

OB history

G (Gravida)
P (Parity)
TPAL:
a. T (Term)
b. P (Premature)
c. A (Abortion)
d. L (Live Birth)

G (Gravida)

it pertains to the number of
pregnancy that the patient already
encountered

P (Parity):

number of complete delivery

T (Term):

complete 9 months of pregnancy

P (Premature):

Incomplete 9 months of pregnancy

A (Abortion):

termination of pregnancy, induced or
spontaneous

L (Live Birth)

number of babies delivered alive

Descriptive Diagnosis:

WNL
ASCUS/AGUS
LSIL/HSIL

WNL:

Within Normal Limits

ASCUS/AGUS

Atypical Squamous Cells
of Undetermined Significance (ASCUS) /
Atypical Glandular cells of Undetermined
Significance (AGUS)

LSIL

Low Grade Squamous Intraepithelial Lesions
(LSIL): a common abnormal result found on a
Pap Test or Pap Smear

(HSIL):

High Grade Squamous Intraepithelial Lesions
(HSIL): there are more defined changes in the
size and also the shape of the cervical cells and
it indicates that there is lightly moderate to
severe cervical dysplasia

NON GYNECOLOGIC SPECIMEN

Respiratory Tract Specimens

Respiratory Tract Specimens:

exclude the possibility of malignancy
or infectious agents

Sputum Smear

- Specimen: Morning Sputum Specimen
- Collection and Preparation: Obtain at least 3
consecutive morning sputum specimens.
- Saccomano's fixative (composed of 50% alcohol with 2% carbowax 1540)

Carbowax 1540

solid at RT and melting point is at 43°C to 46°C

Sputum Induction:

- Extensive Studies: requires 2-4 slides, one is air dried for Giemsa staining.
- At least 2 slides should be stained with Pap's method.
- Sputum is examined first for bloodflecked
or solid particles Bronchial brushing

Bronchoscopy specimens

Bronchial Brushing
Bronchial Washing
Bronchoalveolar Lavage
Bronchoalveolar Lavage

Bronchial Brushing

specimen is directly smeared onto two labeled slides by pull technique

Bronchial Washing

specimens are freshly
collected in the bronchoscopy collection
container and hand delivered to the laboratory

Bronchoalveolar Lavage

Used for the detection
of Pneumocystis jirovecci in AIDS patient

Bronchial Aspirates

The secretions obtained at
bronchoscopy are collected either by aspiration
into a glass suction apparatus or by washing the
bronchi with 1-2 cc. of saline, into a cup.

Cell Suspensions

- direct tap of pleural/peritoneal
effusions, CSF, and synovial fluid.
- The optimum amount: 20-30 mL
- Cells can remain viable for days if the specimen
is kept refrigerated at 4°C.
- sample should not be frozen

Staining technique:
Modified Papanicolaou Method

Harry Hematoxylin:
Eosin Azure 50 (EA-50):
Orange Green 6 (OG-6):

Harry Hematoxylin:

primary nuclear stain

Eosin Azure 50 (EA-50):

counter stain that will
stain the cytoplasm of the parabasal and
intermediate cell

Orange Green 6 (OG-6):

counter stain that will
stain the cytoplasm of superficial cells

Gastrointestinal Specimens

Specimens: Gastric Lavage, Gastric Brush, FNA

Gastrointestinal Specimens

- Gastric smear preparations are quite difficult to
make because of inaccessibility of the specimen
and the presence of the gastric juices
- can cause deleterious effect on the
morphology of the exfoliated cell

Specimen should be ASAP:

since a delay of 30 minutes will digest the cell and make it unsatisfactory for evaluation.