This month, i'm scheduled to the Cytology department in which the bulk of the specimens it deals with are mainly PAP smears, urine and body fluids (such as peritoneal, pleural and pericardial fluids, CSF).
Scope of Cytology:
- Diagnosis of malignancy
- Identify premalignant conditions
- Identify infections and associated organisms
- Evaluation of hormonal effects of estrogen and progesterone
PAP (Papanicolaou) Smearing and Staining
Purpose: Diagnosis of cervix cancer (causative agent: Human Papillomavirus).
Procedures:
- After smearing on slides, specimems will be sent to the Cytology lab.
- Check to make sure the patients' data on the request log tallies with that on the slides.
- Load them into a rack then into the automated staining machine which has been programmed to perform PAP staining.
- After staining, the slides are mounted for microscopic examination.
PAP Staining (The Regressive Method- Nuclei Overstained)
Principle: Exhibit differences in cellular morphology, maturity and metabolic activity. As intact cells in a cytologic smear tend to appear in 3-D configurations and overlap, PAP allows resultant transparency and clear definition of nuclear details.
Procedures:
- Fix the cells in 95% ethanol for 15 minutes to allow the cells to absorb the dyes in the subsequent staining procedures which are fully automated.
- Unacidified haematoxylin
- Blue in water (nuclear staining)
- Dilute hydrochloric acid (remove background staining caused by excess haematoxyin)
- Rinse in water(3 rounds to set the haematoxylin)
- 95% alcohol (3 rounds to remove all water as subsequent staining reagents are alcohol-based and water cannot mix with alcohol)
- OG (Orange-G) stain (stains keratin orange)
- 95% alcohol (2 rounds)
- EA Polychrome stain (stains mature cells- mostly cancerous cells red and immature cells green)
- 95% alcohol (2 rounds to provide clearer view of overlapping cells)
- 100% alcohol (2 rounds for final dehydration)
- Xylene (2 rounds for clearing to allow microscopic examination)
Results: Nuclei- blue or black
Cytoplasm (non-keratinised)- green or blue
Cytoplasm (keratinised)- pink or orange
Red blood cells- orange
Quality Control of PAP Staining
- Checking of nuclear staining
- Staining is crisp and sharp
- Parachromatin is clear (not muddy)
2. Cytoplasmic counterstains
- Sharp and clean
- 3 distinct colours: red (Eosin-Y); orange (OG); green (EA)
Troubleshooting Guidelines for PAP Staining
- Problems with absorption of haematoxylin due to inadequate removal of carbowax resulting in irregular and spotty staining (poor quality).
- Soak in 95% ethanol for complete fixation to enable the cells to have better absorption of haematoxylin
2. Lack of contrast in nuclear staining due to poor quality of haematoxylin.
- Replace water rinses used for blueing purposes
- Tap water should be slightly alkaline. Acidic tap water will result in nuclear fading while heavily chlorinated tap water will bleach out the haematoxylin.
3. Problems with cytoplasmic stains due to improper rinsing of stains after OG and EA
- Do not allow the slides to sit in alcohol solution following OG and EA stains as it can wash the stains away
- Rotate ethanol rinses as ethanol nearest the dye becomes discoloured
4. Water droplets seen on slides due to water in xylene (xylene will appear milky)
- Change the xylene
- Add Silica-Gel pellets to absolute alcohol to minimize the possibility of water contamination of xylene
That's all! Enjoy your SIP! Take care! ^_^
June Tham
TG02
0505073G
7 comments:
hey,
generally i understand what you are posting but i am curious to know if you get to read the slides after staining?
Sally
hey
just curious, for EA Polychrome stain, what does the 'EA' stand for?
Wing Fat
tg01
Hey
What is Carbowax? Why is it found on the smear?
Adrian TG01
hi
May i know what is the difference between a keratinised and a non-keratinised cytoplasm? Thanks
Ci Liang
TG01
Hello June.
Trichomoniasis and Candidiasis are common occurences in Pap smears. Could you elaborate on their clinical significance in women?
Desmond Heng
TG02
0503179D
To Sally: We don't screen the slides after staining. That's the cytotechnologists' job.
To Wingfat: It stands for Eosin Azure.
To Adrian: Carbowax is a coating fixative composing of polyethylene glycol in an alcohol base and are usually applied as a spray. It protects cells from damage.
To Desmond: Candidiasis, commonly called yeast infection or thrush, is a fungal infection of any of the Candida species, of which Candida albicans is the most common. Symptoms include severe itching, burning, and soreness, irritation of the vagina and/or vulva, and a whitish or whitish-gray discharge, often with a curd-like appearance.
Trichomoniasis, sometimes referred to as "trich", is a common sexually transmitted disease. It is caused by a single-celled protozoan parasite Trichomonas vaginalis. Trichomoniasis is primarily an infection of the genitourinary tract and the vagina is the most common site of infection in women. The symptoms in women include a heavy, yellow-green or gray vaginal discharge, discomfort during intercourse, unpleasant vaginal odor, and painful urination. Irritation and itching of the female genital area, and on rare occasions, lower abdominal pain also can be present. In about two-thirds of infected females, there is edema, inflammation, cell hypertrophy and metaplasia.
hey,
will there be problems reading the slides? esp pale nuclear or cytoplasm? any corrective actions?
elaine
Post a Comment