STUDY OF CYTOLOGY & CYTOLOGY STAINING

 

 

                              CYTOLOGY

Cytology:- Microscopic Exanination of indivisual cell in smear is known as cytology.

→ The purpose of cytology:-

1. Early diagnosis of cener.

2. Hormonal study of cytology of vaginal smear.

3. Two identify nature of lesion such as exujudate or tranjudate.

4. Sex determination in buccle smear.

5. Anti partum tuptare of membrane in pregnant ledies.

Type of specimen for cytological examination

1. Urine:- urine sediment, bladder washing, prostatic massage.

2. Sputum:- Bronchial washing, brushing, brushing, expectorated sputum.

3. Gastric lavage often through gastri expiration.

4. Cervical descharges:- Lateral vaginal smear, vaginal pull smear, cervical smear, endocervical smear.

5. Breast secreation.

6. Body cavity fluid.

7. FNAC (Fine Needle Axpiration Cytology)

Prepration of Smear:-

1. Prepration of smear with use of swab→ the swab is soked it exuiudate or fluid cervical canal or other cavity fluid.soked swab is rolled over the glass slide. The material on the swab is transferred on the glass slide. Swab can also be used for collecting material for mucosal surface.

2. Liquid Exujudate → Transffer a drop of on the glass slide at one conner and draw the smear like blood flim.

3. Streking → This is done material from vaginal, stomach and thick exujudate streking is done with fine needle.

4. Spreading → This is done for bronchial expiration with the help of platinum loop.

5. Pullapart → this is done for sticky materials. Material is covered with a clean glass slide. It is firmly applear on the slido over material the two slides are pulled a part smear appears on both slide. Gastric washing sputam and unine specimen first and hence they most be fixed soon after. Collection. This is done with the help of 70% alcohol in ratio 1:1 the sample is then centrifuse and smear prepared from the deposit.

6. Body fluid rich in protein such as pleural fluid, pericardial fluid, peritoneal fluid and CSF make clot. This fluid are mix with citrate fluid (2ml/100ml of body fluid). The citrate fluid contain sodium citrate and citric acid. Body fluid mix with citrate fluid centrifase and smear prepared from the deposit.

Ø  Use of Adhesive on slide on which smear to be prepared:-

1. For vaginal corical and thick exujugate rich is protein adhesive before smear is drawn. There protein contain work as adhesive.

2. For specimen not rich in protein, adhumin is use at adhesive as case of histology. Either egg albumin pulled human serum is use add adhesive.

3. For specimen form body lineing the buckle the smear is fixed in a jar containing fixative before the smear gets ready this is done to prevent shrinkage of the cell. One of the following fixative can be used for this purpose →

1. Alcohol ether mixture is 1:1 → slide is kept in the fixative for one hours.

2. Achaudinn’s fluid composition→

1. Mercuric chloride → 66ml

2. Absolute alcohol →  33ml

3. Glacial acitic acid →  1ml

→  The smear is kept in the fixative for two minutes only.

3. Caroy’s fluid:-

Composition:-

1. Absolute alcohol →  60ml

2. Chloroform →  10ml

3. Glacial acitic acid →  1oml

→  keep the smear in the fixative 1-2 hours.

4. Alcohol – ether, Alcohol Polyethlene Glycod Mixture:-

Composition→

1. Ethyle Alcohol → 50ml

2. Ether → 50ml

3. Polyethlene Glycol →  5ml

→  This fixative is used if the smear is to be sent to other laboratory. The smear kept in this fixative for six hours.

 

                Staining for cytology

The most commen stain is papaniculau stain. Other stains may also be used such as H.E stain, cresyl violet stain, pas stain, silver stain, muelgen stain, nile blue sulphale stain, and may grounwld stain.

Papanicolau stain:-

Test procedure:-

1. Fix the smear in alcohol mixture.

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2. Pas the smear through 80%, 70%, 50% alcohol 6 dips in each alcohol.

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3. Rinse gently in DLW.

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4. Deep in haematoxylene stain for 8-10 mit.

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5. Rinse in DLW.

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6. Dip in 0.25% Hcl 6 dips.

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7. Wash the smear in running tap water.

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8. Dehydrate the smear through 50%, 70%, 80% & 95% alcohol 6 diups in each.

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9. Place the smear in orange G6 stain for 2 minutes.

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10. 95% Alcohol 3 changes 6 dips in each.

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11. EA 36 (Eosin Azure 36 stain) for 2 minute.

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12. 95% Alcohol 3 changes.

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13. Clearing in Alcohol xylene mixture 6 dips.

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14. Xylene 3 changes 6 dips in each.

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15. Maunt in DPX (Di putyl xylal.)

Result:-

1. Nucleus → blue

2. Acidophilic cell → Red colour

3. Basophilic cell → blue – red colour

4. Rpc → red colour

5. Cytoplasm → varying (blue, green, yellow or red)

Rapid pap staining (hiteture)

1. Stain and reagent preparation:-

→  prepare working cytoplasm stain by mixing equal volume of cytoplasm stain 2A & 2B.

→  Working cytoplasm stain is stable in an air tight bottle and if protected from water contamination for atleast 90 days.

→  Discard the stain that the smear poorly.

Procedure:-

1. Dip fixed smear for 3 minutes in tap water and blot out excess water from the slide.

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2. Dip for 60 sec in rapid pap Tm nuclear stain.

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3. Add 3 drop of scotte’s tap water buffer and wash after 10 sec. blot out excess water from the slide.

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4. Dip with two change in rapid- pap dehydrant for 30 seconds each.

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5. Dip for 45 seconds in working cytoplasm stain.

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6. Wash in tap water and blot out excess water from the slide.

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7. Repeat dehydration in a second both of rapid pap Tm dehydrant for 30 sec and dry at air.

Requirement:-

1. Coplin jars 8 pis

2. Slides & cover slip

3. Filter & tissue paper

Precaution:-

1. Dip the slide with slight agitation.

2. Ensure maximum draining of stain from smear on each both by subsequent dip in tap water.

3. Blot out excess water with filter paper or tissue paper.

4. Guard the stains stain from dilution and contamination of suspended specimen from the smears. Filter the stain if necessary.

5. Rapid-pap reagents and stain are for “invitro” professional use only. Biolab accept no liability for accidents arising in handing or using the contect for any other purpose.

6. use protective hard gloves.

7. Rapid pap dehydrant is highly inflammable. Keep away from fire/heat.

8. Store all reagent at 25-35˚ c.

9. Dpx can be diluted with xylene if require.

Papnicolaou Stain:-Normal finding of vaginal cytology. The fououring type of cells may be sound.

1. Epithelial cells (Normal):- The cells are sound in groups closely attached to each other. Cytoplasmic area is more than nuclear area. Nucleus is without nucleolus and chromatin matter is uniform.

These epithelial cells can be of fouauring cells:-

a. Basal cells → Raund or oval, large nucleus, cytoplasm takes blue stain.

b. Pora basal cells → Same as basal cells but wruth smaller nucleus. If cytoplasm can accumulate anather nucleus it is a para basal cells.

c. intermediate cells → Polygonal cells, nucleus, smaller than para basal cells, cytoplasm is pale blue (cynophilic).

d. Superficial cells → polygonal cells with pyknotic nucleus, cytoplasm is eosinophilc.

2. Endocervical cella → they are longitudinal cells in sheets. Nuclei are of different sizes which reach upto suryace of cytoplasm.

3. Endometrial stromal cells → Endametrium is inner lining of uterine cavity. It has endometrial glands and endometrial stroma. Endometrial stroma are found in vaginal secreations only during mensturation. This may also come from tumour of uterues. Endometrial stromal cells are large lymphocyte type of cells. Nucles is small and darkly stain cytoplasm is eosinophilic.

4. Malignant cells (cancer cells) →

These cells have the follouring characteristic →

1. Larger than normal in size.

2. Irreguar out line in cells.

3. Pleo morphic cells or giant cells.

4. Loss of cross striations, cilia, mucus, keratine.

5. Retrograde chnges such as cytoplasmic vaeules.

6. Anaplasia and loss of polarity.

7. Nuclear changes such as naked nuclear matter, Nuclear area more than cytoplasmic area. Nucleolus coarse chromatin dots, hypes chromasia.

8. Presence of abnormal cells such tad pole cells. It is a cell with elongated cytoplasm. Cytoplasm is keratinized. It takes yellow staining nucleus is hyper chromatin. Tad pole cells is an indication of differentiated malignant cells.

Ø  Study of infection in vaginal:-

→ Epithelium → infection by trichamonas and pracess. Trichomonas infection is characterized by vaginal cells with grey-blue cytoplasm and only visible nucleus. Epithelial cells may show perinuclear halo. Epithelial cell show variation in size, shape and orientation. Nuclear cytoplasmic ratio is change. There is no hyper chromasia.

Candida infection is characterized by presence of bundles of pink stain and unloranched hyphae along with this spores are also found. Spores are bright red in colour. Which have small round or ovel out line. If stained by Gram’s Method, they are gram positive.

Ø  FNAC (Fine Needle Aspiration Cytology) :-

This is examination of deep seated lesion in the body with the help of line needle this is done as an alternative to excesuional biopsy for diagnosis of tumour super licial tumour mass can be othained under direct vision deep seated tumour in intra-abdominal situration or in pelvic organs are aspirated by taking help of ultra sound technique or computed tomography.

FNAC Technique:-

1. 5ml plastic disposable syringe with 23 gauze line needle is used for as piration.

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2. Tumour mask is fixed with one hand and needle is introduced in the mass with the other hand when needle enters tumor plungers of syringe is retracted to create a vaccum in the barrel.

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3. The needle is moved to an for several times in different direction.

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4. If tumour substance appear in the barrel of syringe.

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5. Syringe is withdrawn needle is disconnected and contens of the barrel is poured over a slide.

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6. Smear is drawn by apply another slide over it and by drying the slide acress the smear.

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7. The smear is immediately fixed with in ether-alcohol mixture after hay an hour.

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8. Smear can be stained by H.E staining or papanicolaou stain and other stain.

Such as MGG stain (May- Grunwald Geimsa Stain) or Leishman stain can be used. The man purpose of FNAC is diagnosis of tumour and granuloma. Such as tu berculosis and syphilis.

Advantage of this technique is diagnosis without operating, procedure limitation of this test is failure toobtain the tissue mass and small amount of material obtained.