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Cytological tests
This document provides information and instructions for various cytological tests including Pap smear, sputum/bronchoalveolar lavage, urine examination, fine needle aspirate collections, and gastric lavage/esophageal brush smears. It describes how specimens are collected, transported, prepared as smears or cell blocks, and fixed prior to staining and examination under a microscope. Proper collection and handling procedures are outlined to ensure optimal specimen quality and diagnostic accuracy. Common fixation methods, staining techniques including Papanicolaou, hematoxylin and eosin, and May-Grunwald Giemsa are also summarized.
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Cytological tests
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- 2. Pap smear: • PAPsmear test is screening test to detect malignant cells of cervical/ vaginal cancers as well as cells, which represent a precursor of malignancy through microscopic evaluation of representative cells. • Certain infections (candida, trichomonas, etc.) and inflammatory conditions may also be detected in this process. • The smears are scraped from the cervix / vagina and spread on a slide. • Specimens are collected by physician at clinic or at nursing home. Specimens are received either in the form of specific solution vials or as previously fixed smears (bio-fix spray). • Smears can also be received in couplin jars containing ether alcohol as fixative.
- 3. Sputum/Broncho-Alveolar Lavage/ bronchial Brushing: •Study fluids such as pleural fluid, peritoneal fluid, pericardial fluid, synovial fluid, cerebrospinal fluid etc. are commonly evaluated for presence of malignant cells. • Effusions can be neoplastic or non-neoplastic. • Cells suspension are received from body cavity fluids. Fluids should be collected in clean, dry container and should be submitted to the laboratory as early as possible. In case of delay in transport, fluid should be preserved at 4◦C. • Cells can remain viable for 4 days at 4◦C. Heparin can be used as an anticoagulant to preserve cellular details.
- 4. Urine: • Urine examinationin cytology is performed for detection of inflammatory lesion including specific infections, diseases related urinary calculi and neoplasm arising from urinary tract. • Proper collection of urine is necessary for correct diagnosis. • A fully voided fresh specimen of urine is the most suitable sample for cytological investigations. • It should not be the first morning sample or a 24 hour collection, since cells deteriorate quickly in urine
- 5. Following are theinstructions for the collection urine: • Allow patient to drink one glass of water every fifteen minutes for two to three hours. • At the end of two to thres hours, patient should collect urine in a clean and dry container( about 50ml capacity). • Urine sample should be dispatched to the laboratory without delay. • Repeat the urine cytological tests for three successive days.
- 6. Gastric lavage /Esophageal-Gasstric Brush Smears/ Scraping from Oral- Laryngeal Lesions: • Specimens from gastrointestinal tract are assessed to rule out malignancy. • Evolution of flexible fiberoptic endoscopy enables to view and sample the lesion from GI tract. • These endoscopes are provided with accessories like brush, a plastic tubes or forceps for small biopsy. • For gastric/ esophageal collections, patient is instructed to have soft meal and water any time one hour before the procedure. • After completion of brushing, brush smears are preoaraed by rotating brush on a glass slide.
- 7. • The smearsare fixed immediately. For collection of lavage a tube is passed through mouth. 50 to 100ml of normal saline is passed through the tube and reaspirated back and collected in a tube. • This aspirate is centrifuged and smears are prepared from deposit and fixed immediately. • For collection of colonic lavage, patient is instructed to have soft meal and laxative at night. Next day, in the morning, multiple normal saline enemas are given to the patient. • Fluid is collected and submitted for cytology examination. Cytological sample is collected before the biopsy.
- 8. Fine Needle Aspirate Collections(FNAC) •Fine needle aspirates are usually obtained to diagnose or rule out malignancy. Infection or inflammatory condition can also be diagnosed by FNAC. • It is necessary to indicate, whenever palpable mass or lesion is visualized within any organ. • FNAC of deep-seated lesions are perormed under radiological guidance using a similar technique that is used for superficial masses. • FNAC can be performed in out patient department(OPD), clinics or in specific room in nursing homes. The sample is received in the form of smears.
- 9. Following are therequirements for FNACs: 1. Examination couch with following facilities: • Pillow • Disposable bed sheet • Blanket • Screen for privacy 2. Solution and equipments: • Fixative: 8%(v/v) isopropanol • Formal alcohol • 10ml syringes • Needle size 22g and 23g • Sterile gauge and plaster • Gloves • Glass slides with frostedd ends • Consent form staining solution
- 10. Procedure • Palpitation ofsuspected lump: The lump is localized and checked by palpating between the tips of the index and middle fingers. The selected area is clearned with an alcohol swab. • Aspiration: The needle is inserted through skin rapidly and then carefully into the lump; plunger is pulled back and needle is moved in straight line with back and forth motion and plunger is released before withdrawing needle. • After withdrawing the needle a gauze piece is applied over the puncture site with pressure. • After removing the needle the collected material is placed on the glass slides. • Samear preparation: Smears are preparaed by using glass slides. • Smears are immediately fixed in 95% alcohol • Smears are immediately stained with toluidine blue.
- 13. Fixation of cytologicalsmears • The specimen should be immediately fixed while the smears is moist. • This prevents drying of the smear and the distortion of the cells. • Following fixatives are generally used in the laboratory.
- 14. Alcohol-Ether Fixative • Mixedand placed in a jar with a tight stopper. Fixation is carried out for about 30 minutes, following by a rinse in alcohol and then the section is taken to water. • It is specially used with the Papanicolaou staininng method. Absolute ethyl alcohol 50ml Ether 50ml
- 15. Schaudinn’s fluid • Fixationis carried out for about 2 minutes. • After washing in distilled water, the mercuric chloride black clumps are removed by addinng a few drops of saturated alcoholic iodine solution. • After rinsing in water the smear is taken for staining. • This reagent is rapidly in penetrating one and preserves the smears which are to be stained by hematoxylin and eosin.
- 16. Schaudinn’s fluid Saturated mercuric chloridesolution 60ml Absolute alcohol 33ml Glacial acetic acid 1ml
- 17. Carnoy’s fluid: • Itis prepared as follows: • The fixation is performed from 30minutes to 3hrs Absolute alcohol 60ml chloroform 30ml Glacial acetic acid 10ml
- 18. Fixation procedure: • Fixthe pre-labeled smear while moist. • Fix adequately ( at least for our hour). • Fixed slides can be kept aside for staining or can be left in the fixative until ready for staining . • Prolonged fixation does not affect the smear
- 19. Fixatives used forspecimens other than smears • 10% neutral buffered formalin • Bouin’s fluid • Methanol acetic acid fixative: It is prepared by mixing 1 part of methanol and 19 parts of glacial acetic acid. This fixative is recommended for cytology and flow cytometry. • Ethanol formalin fixative:It is used as mixture of 1 part of 40%(v/v) formaldehyde and 9 partsof absolute alcohol. • Formal vapor: These are recommended for demonstration of lipids.
- 20. Preparation of smears: •Smears are prepared according to the type of the specimen. By using wire loop or an applicator stick, fixed specimen or sediment is transferred on a cleanand pre- labeled slide. Following methods can be used to prepare smears: • Streaking: This method is used for the mucoid specimens such as vaginal secretion, sputum or gastric contents. About 2/3 area of the slide is covered by spreading the specimen uniformly. The smears are placed in the fixative immediately.
- 21. Spreading: • A moderatelythick flim is prepared by this method which maintains the cellular interrelationship. This method is used for the speimens such as sputum and bronchial aspirates. • Direct smears: Cervical smears, breast secretions, FNAC are directly smeared on a slide. The slides are then paced in a jar containing fixative or can be coated with a spray fixative.
- 22. • Pick andsmear method: It is used for mucinous specimens such as sputum, bronchial lavage and gastric lavage. Pick and smear method is performed as follows: • Specimen should be poured in to a petri dish under aspetic conditions. • Gross description of the sputum sample is documented. • The sputum can be teased out using forceps and blood stained; discoloured and purulent areas can be selected and placed onto the glass slides. • About three smears are prepared. • Once prepared the smear should be immersed in the fixative-containing jar immediately. • Smears should be fixed in fixative for at least 30 minutes and then proceed for staining.
- 24. Saccomanno’s technique • Itis performed for mucinous specimen by using saccomanno’s fixative. It is prepared as follows: Carbowax 2g is mixed in 100ml od 50%(v/v) alcohol. Following procedure is used for fixation. • Collect specimen in saccomanno’s fixative. • Pour in a blender (for 5-10sec). • Pour in the 50ml tube. • Centerifuge for 10 minutes. • Decant and agitate well . • Aspirate sediment and place a drop on a slide and smear it.
- 26. Cell block technique •Cell- block technique is a meethod of preparing material for microscopic examination using a paraffin wax embeddig method. • This supports in providing more definitive cytopathology diagnosis. • Cell blocks can be prepared from fluid specimens of all types, particularly from effusions, edometrial aspirates and brush samples as well as fine needle washing.
- 28. Liquid based cytology(LBC): •Liquid based cytology technique has increased sensitivity and specificity of Pap smear technique. • Sample is collected using a brush and is rinsed in the fluid. • This ensures complete transfer of material collected for the testing. • The smears preapred using LBC minimizes obscuring inflammatory cells, red blood cells, necrosis and displays monolayer of well- preserved cells.
- 31. Papanicolaou stain(PAP stain) •Reagents : • Harris alum hematoxylin • 0.5% (v/v) hydrochloric acid in 70% ethanol( acid – alcohol reagent) • 0.5%(v/v)hydrochloric acid in distilled water. • 0.25%(v/v)hydrochloric acid in distilled water • Orange G6 (OG6) and EA36 and 50 (eosin-azure): This reagents are avaliable commercially.
- 32. Procedure : • Fixthe smears in equal parts of 95% ethanol and ether. • Take the smears to water ( hydration): 6 dips in each of the following solutions of ethanol • 80% (v/v) • 70% (v/v) and • 50% (v/v). • Rinse gently in distilled water. • Stain in diluted Harris’ hematoxylin for 2 to 3 minutes. • Rinse in distilled water • Dip in 0.25% (v/v) hydrochloric acid 6 times.( or in 0.5%(v/v) hydrochloric acid 3 times) • Place under running tap water for 5 minutes. • Check up the slides under the microscope to see if the nuclei are stained properly. (if over stained, decolorize in acid- alcohol and if pale, return to hematoxylin stain)
- 33. • Dehydrate throughdistilled water and alcohol solutions • 50% (v/v) • 70% (v/v) • 80% (v/v)and • 95% (v/v) by dipping 6 times in each solution. • Stain in OG6 for 2 minutes • Rinse in 95% (v/v) alcohol(3 separate changes) and 6 dips each. • Satin in EA36 (or EA50) for 2 minutes. • Rinse in 95%(v/v) alcohol (3 separate changes) • Dehydration in absolute alcohol(2 changes) • Clear in alcohol xylene (6 dips), following by 3 changes in xylene (6dips) • Mount in any satisfactory neutral medium
- 34. Precautions: • This slidesshould be handled carefully during rinsing and washig. Rough handling may wash off the specimen. • Results : • Nuclei: Blue • Cytoplasm: Varying shades of pink, blue, yellow green, grey.
- 35. The method ishighly reproducible. The method is used for the smears such as • Vaginal • Cervical • Prostatic • Urinary as well as for the smears made from the specimen such as • CSF • Pleural fluid and • Other fluids.
- 37. Rapid papanicolaou staining: •Stock reagents: • Eosin Y 20% aqueous • Orange G-6 3% aqueous • Light Green SF 3% aqueous. Working reagent is prepared as follows. Eosin 20ml Orange G-6 6ml Light green SF 20ml Isopropyl alcohol 760ml Methanol 240ml Glacial acetic acid 10ml Phosphotungstic 5g
- 38. • Mix reagentsand light green slowly to avoid precipitation. • Procedure : Nuclear staining can be performed as routine PAP procedure followed by using modified EA regent. • OG- EA reagent treatment: 2-4min. • 95% alcohol: 10 dips, with 2 changes • Xylene: 10 dips with 2 changes • Blot dry and mount with DPX
- 39. Hematoxylin and eosinstaining • Reagents: • Harris’ hematoxylin • 1.0% (v/v) hydrochloric acid in 70% ethanol( acid- alcohol reagent) • Eosin (Y): 1% (w/v) aqueous
- 40. Procedure: • Treat with95% alcohol for 2 minutes. • Wash in running tap water for 2-3 minutes • Stain in harris hematoxylin for 5-10 minutes • Wash in running tap water for 5 minutes. • Differentiate in acid alcohol solution for 10-15 seconds • Wash in running tap water for 5-10 minutes • Treat with eosin solution using 2 dips • Blot and dehydration in absolute alcohol. • Clear in xylene and fix coverslip by using a drop of DPX.
- 41. May grunwald giemsastain • It is useful technique for FNAC smears, and body fluids. • May-Grunwald Giemsa stain is Romanowsky staining technique containing two dyes, methylene blue and eosin Y. • During staining procedure, methylene blue on oxidation produces colored compound known as azure that has ability to combine with eosin. • Methylene blue azure is blue violet and as follow stains mainly acidic cell comonents. • Eosin is red in color and stains more basic structures. Combination of acidic and basic dye optimizes staining reaction.
- 42. Reagents: • May-Grunwald stain(stock): It is prepared as follow: • Mix and store at room temperature (25◦ C ± 5◦C). May-Grunwald power 2.5g Methanol 1000ml
- 43. May –Grunwald workingsolution: It is prepared as follows: • This working solution should be prepared fresh everyday. Stock solution 2ml Methanol 8ml
- 44. Giemsa stock solution:It is prepared as follows: Giemsa powder 1gm Glycerin 54ml Methanol 84ml
- 45. Stock buffer solution( pH 6.8): It is prepared as follows: • Mix 50ml of K2HPO4 with 236ml of NaOH. K2HPO4 2.7% (w/v)in distilled water NaOH 0.8g% (w/v) in distilled water
- 46. • Working buffersolution: It is prepared as follows: • Mix 40ml of stock buffer solution with 60ml of distilled water. • Working Giemsa solution: It is prepared as follows: It is prepared fresh everyday by mixing 1ml of Giemsa solution and 9ml working buffer solution.
- 47. Procedure: • Fix smearsin methanol for 15 minutes. • Stain the snears with working May-Grunwald solution for 25 minutes • Wash in tap water. • Stain the smears with working Giemsa stain for 20 minutes. • Wash in running tap water, dry and mount with DPX.
- 48. Rapid stains: • Thesestain are used mainly to check adequacy of fine needle aspirates. Rapid stains can also be used for immediate evaluation to control cross contamination. The dyes used in rapid staining procedures are, Methylene blue, Thionin blue, and Toluidine blue. • Toluidine bue stain is prepared as follows: Toluidine blue 0.5g 95% ethyl alcohol 20ml Distilled water 80ml
- 49. Toluidine blue stainingprocedure: It is performed as follows: • Fix the smear for 10 to 15 seconds in 95% alcohol • Blot dry. • Treat with toluidine blue, use 10 dips. • Rinse in water to remove excess of stain. • Put a coverslip on wet smears and submit for microscopic examination
- 50. Maintainence of thestains • Following precautions are taken while using the stains. • Stain used must be certified by biological stains commission. • Dye content on the certified label of dye must be considered in stain preparation. • Contamination control: Gynec and Non- gynec slides are stained separately. Cells are detached from the slides during staining. These cells can cause cross contamination which results in female in false diagnosis. • Filter stains and reagents daily. • Non- Gynec specimens have high potential for cross contminationduring staining procedures • Daily microscopic checks are documented for the quality control of stains.
- 51.
- 52. • The sexchromatin or Barr body is a condensation of chromatin present at the nucleus of cells in female individuals. • Their observation is possible in different cell types and is used for the rapid diagnosis of biological sex. • Sample: • Scrapings from buccal mucosa, or in some women from the vaginal wall. • Buccal mucosa is scrapped and smear the slide. • While the Drumstick is done on the white cells (WBC) to see the presence of drumstick.
- 54. Normal • Male =No barr bodies are seen. • Female = Barr body is positive. • Interpretation • This Barr body test is advised for the screening of ambiguous or doubtful sex characters. • Turner’s syndrome female (XO) will have no barr body. • Male with Klinefelter syndrome (XXY) will have a barr body. • Female with XXX chromosome will have two barr bodies. – The female cell with four X chromosomes has three barr bodies.