Sip sharing..cytology!

Hey people! I’m now attached to a routine cytology laboratory. My tasks assigned are: mount stained slides manually, sort slides, receiving/collection of specimens (both gynae & non-gynae), processed urine and sputum samples. So this time, I will blog on the procedure for sputum processing and Papanicolaou staining.

Sputum testing is to differentiate the nucleus and cytoplasm of the cells to detect malignancy by comparing the Nucleus: Cytoplasm ratio (N:C ratio).

Procedure for sputum processing
1. Receive sputum sample
2. heck particulars of patient and nature/number of specimen
3. Assign a doctor to the patient and stamp date/time received and initialized by technician
4. Label all containers containing sample with pre-printed labels
5. Initial of technician performing sputum sample and the condition of sputum sample
6. Label the specimen lab number on the frosted end of the four clean glass slides.
7. Label on one of the slide the name of the special stain, if requested example Ziehl Neelsen
8. Pick the selected material with a Pasteur pipette and flush them onto the surface of the labeled glass slide
9. Holding a slide on one hand, pick up another slide to pull the material evenly over their surface to make smear. Avoid leaving rough or raised areas that may cause cover-slipping problems.
10. Used Pasteur pipette are being disinfected in sodium hypochlorite.
11. Place slide immediately into container containing 95% ethanol. 12.Separate out slides for special staining.
13. Additional slides are prepared if future request for special stains are made.
14. Load slides into autostainer using slide hold (rack)
15. Stain with Papnicolaou stain
16. Mount with 24 by 50mm cover slips and Depex manually
17. Place in oven for 10 to 15 minutes to let Depex dried up
18. Slides are ready to be observed under microscope

Label with specimen lab number and name of specimen
A drop of sputum sample

Making a smear using the ‘touch and pull’ method’

A smear done

0.5% Sodium Hypochlorite (prepare and use only fresh solution) is a disinfectant. It is active against bacteria, spores, fungi, viruses including HIV and HB. Concentration is 0.50%.
Used for specimen disposal, contaminated waste, materials spillage and non-metal equipments. The contact time must be at least 30 minutes for optimal effectiveness.

Routine Papanicolaou Staining
Papanicolaou method is a polychrome staining reaction (staining the cytoplasm of different cell types different colors) designed to exhibit differences in cellular morphology, maturity and metabolic activity
Intact cells in a cytologic smear tend to overlap and some appear in three dimensional configurations, the greatest value of papnicolaou staining method are the resultant transparency of the cells and clear definition of nuclear detail
Used for gynaecologic and non-gynaecologic specimens

Principles
1.Fixation
2.Nuclear Staining
3.Cytoplasmic staining
4.Clearing

A series of graded percentage of alcohol (80%-70%-50%) to hydrate the cells gradually before immersion in the aqueous haematoxylin solution
After approximately two minutes in haematoxylin the cells are then dehydrated (70%-80%-95%) prior to immersing in the alcoholic counter stains (Orange G and Eosin Azure)
Following the two cytoplasmic stains, the slides are then rinsed in alcohol

Fixation
To fix/preserve the morphological details of the cell in as perfect a condition as possible

Nuclear staining
Regressive method: The nuclei are overstained with unacidified haematoxylin; excess stain is then removed with dilute hydrochloric acid. The hydrochloric acid must be removed by a bath of running water.
Progressive method: To stain for the desired color intensity. This method eliminates decolorizing with hydrochorite acid and the need for subsequent running in a water bath. Recommend for non-gyanecological cell samples because they do not adhere to slides as well as those from the female genital tract. To cause the colour of the stain to change from red to blue, the slides may be ‘blued’ with dilute solutions of ammonium hydroxide (NH4OH, Ph 8.0-8.5), lithium carbonate ( Li2CO3, pH 8.0-8.5) or Scott’s tap water substitute (pH8.2).

Cytoplasmic staining:
After the nuclear staining, the cells are dehydrated through rinses of 95% alcohol. This dehydration prepares the cells for the two alcohol stains. First, the OG-6 stain, the slides will be in the alcohol for 1 minute. If there is any keratin in the cytoplasm of these cells, the OG-6 will stain it a brilliant orange. Following the OG-6 stain, the cells are rinsed in two 95% alcohol baths. They are then immersed in the modified EA stain for 2 minutes. The modified EA is a combination of these two stains : First is the eosin, which stains the cytoplasm of mature squamous cells, nucleoli and cilia. Second, is light green, which stains the cytoplasm of parabasal and immediate squamous and columnar cells.

Clearing:
Following the EA stain, the cells are then taken through two 95% alcohol rinses. These high concentrations of alcohol help to provide a clearer view through areas of overlapping cells
Next, the cells are taken through 10% alcohol rinses for final dehydration
Upon complete dehydration, the cells are placed in the two to four rinses of xylene where they remain until the coverslipped. The xylene will carry light rays from the microscope in the same way that the cell will, thus, making the cells transparent.

Note: Pictures are taken with the permission from my supervisor.

Yup so thats all :) do feel free to ask any questions..

Sharon Ang

0503219H

Tg02