CYTOPATHOLOGY (HDD 30203) LABORATORY REPORT

UNIVERSITI KUALA LUMPUR

DIPLOMA IN MEDICAL LABORATORY TECHNOLOGY

CYTOPATHOLOGY
HDD 30203
LABORATORY
REPORT

SEMESTER: OCTOBER 2023

EXFOLIATIVE CYTOLOGY: BUCCAL SMEAR PREPARATION, STAINING &

EXAMINATION

LECTURER NAME: MDM. AZLINA MUHSIN

GROUP NO: L01-B02

STUDENTS NAME ID NUMBER CONTRIBUTION

1. NURSHAMIMI AUFIYAH BINTI SHAZLEAN 12105121069 - MATERIALS

- CONCLUSION

2. WAN IZZATI NADHIRAH BINTI AZMAN 12105121066 - OBJECTIVES

- DISCUSSION

4. NURUL ALIA NAFISSA BINTI YUSRI 12105121078 - INTRODUCTION

- RESULTS
INTRODUCTION

Exfoliative cytology is the examination of specimens that contain cells exfoliated from body
cavities and surface such as the cervix, abdominal fluid, pleural fluid, oral mucosa, etc. It is
useful for the detection of precancerous or cancerous lesion in the body.

For staining of cytological preparations, Papanicolaou staining is used as a universal staining.

It is a polychrome staining which enables to differentiate cells according to their maturity and
metabolic activity. When performed properly, the stained specimen would display hues from
the entire spectrum of red, orange, yellow, green, blue, and violet. The staining results in very
transparent cells, so even thicker specimens with overlapping cells can be interpreted.

OBJECTIVES
1. To prepare buccal smear
2. To perform Papanicolaou staining on prepared buccal smear
3. To examine the morphology of cells in the prepared smear

METHODOLOGY

Equipment and material

1. Wooden applicator
2. Glass slide
3. 95 % alcohol (fixative)
4. DPX (mounting medium) and applicator
5. Coverslip
6. Gloves
7. Staining rack
8. Pap stain:
a. 70% ethanol, 80% ethanol, 95% ethanol, 100% ethanol
b. Harris’ hematoxylin
c. 1% acid alcohol
d. Scotts tap water (bluing solution)
e. Tap water
f. Orange G-6
g. Eosin azure-50
h. Xylene
 Specimen
Buccal swab

Procedure

(a) Buccal swab collection and smear preparation

1. The glass slide was labeled using a pencil with the following: Name; Date of collection;
Type of specimen.
2. Clean water was used to rinse the mouth prior to sample collection.
3. By using a wooden applicator, it was placed at one side of the mouth.
4. The applicator was pressed firmly and twirled against the inside of the inner cheek
using an up and down motion.
5. The wooden applicator was avoid being saturated with excess saliva. The wooden
applicator was removed from the mouth.
6. The collected sample was smeared onto the labeled glass slide by spreading over a
large area, preventing clumping of cells.
7. The smear was fixed immediately into 95% ethanol for 30 minutes.

(b) Papanicolaou (Pap) staining procedure

1. Stain the prepared buccal smear according to the following Pap staining protocol:

Step Duration
80% ethanol 5 sec - 20 dips
Running tap water 1 min
Hematoxylin 5 min
Running tap water 1 min
1 % acid alcohol 2 dips
Running tap water 1 min
Scotts tap water 1 min
Running tap water 1 min
70% ethanol 5 sec – 20 dips
Orange G-6 2 min 30 sec
95% ethanol 5 sec – 20 dips
Eosin azure-50 2 min 30 sec
95% ethanol 5 sec - 20 dips
100% ethanol 5 sec – 20 dips
100% ethanol 5 sec – 20 dips
Xylene 5 sec – 20 dips
Xylene 5 sec – 20 dips
(c) Mounting and cover slipping
1. 2 to 3 drops of DPX were dropped onto the slide.
2. At an angle (approx. 45°), the coverslip was placed against one side of the slide
contacting the outer edge of the mounting medium.
3. The coverslip was lowered slowly (avoiding air bubbles) over the slide.
4. The mounting medium was allowed to spread, covering the whole area beneath the
coverslip.
5. The slides were air dried.

Safety and precaution

Avoid drying of smear before fixation as dryness leads to alterations in the cellular
morphology.
RESULTS

Under 10x magnification lens:

Under 100x magnification lens:

Intermediate squamous cell

 Oval nucleus
Superficial squamous cell
Under 40x magnification lens:  Open vesicular
 Small and dense chromatin
pyknotic nuclei
 Polygonal cells, folded
 Eosinophilic cytoplasm edges
DISCUSSION

In this experiment, we focused on the Papanicolaou staining (PAP Stain) by using a buccal
smear as a sample. The basal mucosa is the tissue that lines the interior of the mouth and is
comprised up of squamous epithelial cells. These structures are also known as cheek cells.
Therefore, mucin is the main component of mucous which is secreted continuously by cheek
cells. The mucin in combination with the salivary glands keeps the mouth cavity wet. A suitable
moisture level must be maintained on a continuous basis to help enzymes in softening food,
facilitating swallowing and initiating the digestive process.

Moreover, the result above indicates that intermediate squamous cells appear blue-green color
when it stains with Pap stain. Additionally, superficial squamous cells appear pink color when
it stains with Pap stain. To conclude it, the result of this experiment showed that the cheek
cells are normal.

In addition, both squamous cells have visible borders due to improper staining. A few errors
occurred throughout the staining process that gave effect to the cell. Also, the result shows
superficial squamous cells have eosinophilic cytoplasm with small and dense pyknotic. Thus,
intermediate squamous cells have oval nucleus, open vesicular chromatin and polygonal
cells with folded edges.

CONCLUSION

To summarize, the cells that were observed in a buccal smear test that are taken by scraping
the cheek can be used for genetic testing as it can evaluate for the presence of Barr bodies
that were often found in a large amount in a normal female sex chromosome. Cells found in
buccal swabs test under the microscope we observe are majorly homogenous tissue and only
contain two major cell types, buccal epithelial cells and leukocytes. Moreover, cheek cells were
used because they have the same cell found in vagina. A buccal smear tests also an
inexpensive diagnostic method, noninvasive and fast for collecting genetic material. It can also
be used as aneusomy, microdeletion syndromes, and a variety of polymerase-chain reaction
based molecular genetic tests.

REFERENCE

Buccal smear. (n.d.). Mount Sinai Health System.

https://www.mountsinai.org/health-library/tests/buccal-
smear#:~:text=Cells%20are%20collected%20by%20scraping,is%20a%20male%20or%20fe
male