Student Internship Programme - Cytology

Hey people, Desmond here. I've been attached to the cytology for 4 weeks now and have been doing non-gynaecological procedures for the first 3. In this posting, I will guide you through what is done from the time a urine specimen is recieved right until it is "reported" to the cytologist.

I will be posting pictures soon and feel free to ask any EASY questions =].

Subject title
Pathology (Cytology)

Aims
Cytology testing of Urine for malignancy

Introduction
The principle of cytology testing of urine is to differentiate the nucleus and cytoplasm of the cells to detect malignancy by comparing the Nucleus: Cytoplasm ratio (N:C ratio). The Papanicolaou staining method is used for this. It is a polychrome staining reaction (staining the cytoplasm of different cells different colours) designed to exhibit differences in cellular morphology, maturity and metabolic activity. Because intact cells in a cytological smear tend to overlap and some appear in 3D configurations, the greatest value of the Pap staining method are the resultant transparency of he cells and clear definition of nuclear detail.

Urine however has small number of cells; Cytocentrifugation is needed to pull the cells into a confined/defined region on the glass slide.

Materials
Glass slides
Depex
Xylene
Autostainer
Centrifuge
Cytocentrifuge
Cytocentrifuge funnel and clamp
10ml centrifuge tube
22mm by 22mm coverlips
Labels/Stickers
Shandon reagent
0.5% sodium hypochloride
Virkon solution

Methods
(a) Receiving Specimens (Urine)

1. Scan the time onto the patient’s form
2. Fill in the type of specimen and initials of the person receiving the specimen (e.g. DH)
3. Label the patient’s form with pre-made stickers which are chronologically numbered and bar-coded.
4. Assign a cytologist to the case and record details like case number and the cytologist assigned to the case.

(b) Processing specimen (urine)

1. Enter Biosafety Cabinet Level 2.
2. Remove the container with the patient’s urine from the biohazard bag
3. Compare IC numbers on patient form and the container
4. Label the container and a 10ml centrifuge tube with the pre-made stickers (e.g. NG1234/07)
5. Indicate on the patient’s form the following:
Volume of urine
Condition of urine (e.g. Colourless, bloody or yellowish)
Initials of the person processing the urine
Date
Number and type of stains required (in this case 2 Pap stains)
6. Gently mix the urine by overturning the container
7. Aliquot about 10ml of urine into the 10ml centrifuge tube
8. Centrifuge the urine for 10 minutes at 2000rpm.
9. Prepare 2 labelled glass slides for cytocentrifugation
10. Decant the supernatant into 0.5% Sodium Hypochloride
11. Add 6 drops of Shandon reagent to the cell pellet.
12. Mix by pipetting up and down
13. Add 3 drops of the mixture of cells and Shandon into the cytocentrifuge funnel.
14. Cytocentrifuge for 6 minutes at 800rpm
15. After cytocentrifugation, fix the glass slides in 95% alcohol.

(c) Staining

1. After fixation for 20 to 30 minutes, load the glass slides onto a staining rack.
2. Load the rack into the autostainer
3. Set the autostainer to the Papanicolaou staining programme.

(d) Mounting

1. After staining, mount the 2 glass slides.
2. Dip the glass slide into xylene
3. Add a drop of depex to the stained side of the glass slide
4. Place a coverlip on the Depex
5. Press out any air bubbles
6. Place the glass slides into an oven (45 degrees Celsius) to let the Depex solidfy (5 to 10 minutes)

(e) Microscopic observation and reporting

1. Observe under microscope to determine if there is an adequate number of cells or staining for the cytologist to detect any abnormalities/malignancy

2. Report the case including the patient’s form and glass slides to the cytologist to detect abnormalities (malignancy)

Results
(Reference)
Nuclei – Blue
Acidophillic cells – Red
Basophilic cells – Blue-green
Erythrocytes – Orange-red

(Urine tested)
Nucleus appears very large when compared to cytoplasm. There are multinucleated cells and the nuclei appear to be intensively stained blue-green.

Discussion

The patient’s Cells are malignant. There is nuclear enlargement without an increase in the overall size of the cell, giving a decreased cytoplasm to nuclear ratio. There is also hyperchromasia due to increased amounts of DNA. The nuclear outline appears irregular and there is a variation in size and shape. Moreover, nucleoli increase in size and number. Abnormal cell division results in multinucleation. Finally, there is uneven distribution and variation in size and chromatin particles.











Desmond Heng Chih Pheng


0503179D


TG02