cyto patho tech (2).pptx presentation ppt
- 1. CYTOPATHOLOGICAL TECHNIQUES AND THEIR APPLICATIONS PRESENTER- DR ASHA GHIDOLE MODERATOR- DR MILI PATEL
- 2. CONTENT • INTRODUCTION • SAMPLING TECHNIQUE IN CYTOLOGY • CYTOLOGICAL TEST • PAP SMEAR • FNAC • SMEAR PREPERATION • STAINING OF SMEAR • FNAC OF DEEP SITED LESIONS • ANCILLIARY TECHNIQUES
- 3. TERM: Cytology: branch of life science, which deals with study of cells in terms of structure, function and chemistry. Cytopathology: frequently called cytology means “ the study of cells”. • It is a branch of pathology that studies and diagnoses diseases on cellular level. • It was founded by George Nicholas Papanicolaou in 1928
- 4. USES OF CYTOPATHOLOGY: • Cancer • Infectious diseases • Inflammatory condition • Pap smear • Thyroid lesion • Disease involving body cavities( pleural, peritoneal, csf) Cytopathology is used on free cells or tissue fragments, in contrast to histopathology which studies whole tissues.
- 5. • SPECIMEN REQUIRED FOR CYTOLOGICAL STUDIES : Discharges collected from( nipples, sinuses, vagina) Scrapping from buccal mucosal surface Aspirates from palpable lumps(abscesses, growth)
- 6. SAMPLING TECHNIQUES IN CYTOLOGY 1) EXFOLIATIVE CYTOLOGY/Exfoliation of cell by nonabrasive method:- spontaneous shedding of cells derived from lining of an organ into body cavity. • Ex- vaginal smear, sputum, urine, and other body fluids 2) INTERVENTION CYTOLOGY/Collection of cells by brushing or similar abrasive technique:- direct sampling of target such as uterine cervix or bronchus. • Ex- flexible fibre optic instrument used for respiratory and GI tract.
- 7. 3) WASHING OR LAVAGE:- In this method normal saline is installed in target organ under visual control.These solution are aspirated and collected in small container. • Ex- Bronchoalveolar lavage, peritoneal lavage 4) FINE NEEDLE ASPIRATION CYTOLOGY(FNAC):- Any palpable or non palpable lesions in the body can be sampled using either palpation or imaging techniques
- 8. CYTOLOGICAL TESTS 1) PAP SMEAR • Pap smear test is screening test to detect malignant cells of cervical/vaginal cancer. • Certain infection like candida, trichomonas and other inflammatory condition can also be detected. PROCEDURE: sample scrapped from cervix or vagina and spread on slide by physician at clinic or nursing home. • Specimen is fixed by bio-fix spray or received in couplin jar containing ether alcohol as fixative.
- 9. STEPS OF PAP STAIN: • Rehydration of smear- gradual dip of smear in graded concentration of alcohol • Nuclear staining by hematoxylin- dipped into harris hematoxylin. Excess hematoxylin removed by 0.05% aqueous solution of HCL.To make more stable smear treated with weak alkaline solution. • Cytoplasmic staining by orange G- smear again dip in alcohol and stained with OG stain which stain keratin into orange colour • Cytoplasmic staining by eosin azure- synthetic dye and stains cytoplasm as blue green color • Dehydration by absolute alcohol • Clearing by xylene • Mounting by DPX
- 10. PAP TEST
- 11. 2) SPUTUM/BRONCHO-ALVEOLAR LAVAGE/BRONCHIAL BRUSHINGS • Study of cells from sputum/brocho alveolar lavage used for detecting tuberculosis, fungal infection, pneumocystic jirovecii pneumonia, and malignat lesion of respiratory tract. 3) BODY FLUIDS/EFFUSIONS • Pleural fluid, peritoneal fluid, cerebrospinal fluid, pericardial fluid, synovial fluid are evaluated for malignant cells. • Effusion can be neoplastic or non neoplastic. • Fluid collected in clean, dry container and submitted to lab as early as possible.
- 12. 4) URINE • A fully voided fresh urine at the end of 2 to 3 hr after drinking 1 glass of water at every fifteen minutes is best sample for detecting urinary calculi, neoplasm from urinary tract.Also repeat the test for 3 successive days. 5) GASTRIC LAVAGE/ESOPHAGEAL-GASTRIC BRUSH SMEARS/SCRAPPING FROM ORAL-LARYNGEAL LESIONS • Flexible fibre optic endoscopy enable to view the lesion from GI endoscopy.These endoscope are provided with brush, plastic tubes, forceps for small biopsy. • For gastric/esophageal collection, pt told to eat soft meal with water one hr before procedure. For collection of lavage tube passed trough mouth and 50-100 ml normal saline passed through tube reaspirated back in test tube. • This aspirate is centrifuged and smear prepared and fixed immediately.
- 13. 6) NIPPLE DISCHARGE • Any discharge which not related to lactation or pregnancy is abnormal. • Procedure; wipe and clean nipple surface with saline. • Gently press nipple and sub areolar region for any secretion in ducts. • Smear secretion on clean slide directly if scanty or between two slide if thick. • Immediately put in fixative.
- 14. FINE NEEDLE ASPIRATE COLLECTIONS (FNAC) • Fine-needle aspiration, or fine-needle aspiration cytology (FNAC) • Involve needle attached to syringe to collect cells from lesions or masses in various body organs. • Microcoring with application of negative pressure or suction to increase yield. • Technique: • By palpation guidance • By ultrasound guidance • CAT scan
- 15. Palpation guidance • Clinician feel the lesion on a mass in superficial region like neck, thyroid or breast. Ultrasound guidance • Used for deep seated lessions within the body, as the can not be localized via palpation
- 16. ADVANTAGE OF FNAC • Simple office technique • Rapid diagnosis • Economical • Sampling from multiple site in same sitting • High sensitivity and specificity • Avoid further biopsy in inoperable malignancy and infective condition LIMITATION OF FNAC • Loss of tissue architecture • Capsular invasion and lymphovascular invasion cannot be detected • Difficult to differentiate insitu versus invasive carcinoma • Training needed for accurate interpretation
- 17. FNAC CLINIC • Location preferably hospital outdoor • Well ventilated and lighted • Examination bed • Writing table and chair • Working table • Basic equipment for FNAC • Rapid staining kit • Small binocular microscope • Sink with water connection • cytotechnologist CLINICAL HISTORY • Chief complaint • Swelling site,size,consistency • History of any bleeding diathesis • Past history of illness • Relevant biochemical investigation • Radiological investigation
- 18. FNAC CLINIC
- 19. COMPLICATION OF FNAC • Minor hematoma • Surgical emphysema • Accidental rupture of aneurysmal vessel or spleen • Anaphylaxis in hydatid cyst • Needle tract seedling of cancer cells, specially if thick bore needle used. CONTRAINDICATION OF FNAC • Bleeding diathesis • Hydatid cyst • Aneurysmal artery
- 20. PATIENT PREPERATION FOR FNAC • Talk with patient, it makes him or her relax • Ask patient’s version of swelling • Explain procedure, purpose, complication, advantages • Take written consent
- 21. S T E P S O F F I N E N E E D L E A S P I R AT E C O L L E C T I O N
- 22. F I N E N E E D L E B I O P S Y W I T H A S P I R AT I O N
- 24. F I N E N E E D L E B I O P S Y W I T H O U T A S P I R A T I O N
- 25. SAFETY PRECAUTION • Well lighted and well ventilated room • Barrier precaution like apron, gloves, mask • Waste disposable • Needle and syringe cutter • No needle resheathing
- 26. SUBOPTIMAL MATERIAL • Various cause of suboptimal material in FNAC are: • Faulty technique • Vigrous suction in vascular organ like thyroid during FNAC may yield blood.best is to get intermittent suction during to and fro movement of needle • Always check weather blood is in needle hub or not and immediately stopped if so happen. • Sufficient to and fro action to dissociate hard sclerotic needle, also use thick bore cannula. • Too much crushing of smear during preparation will cause crush artifact • Too delay in smear making cause clotted smear
- 27. CAUSE OF UNSATISFACTORY YEILD
- 28. P R E P E R AT I O N O F S M E A R • Smear are prepared according to type of sample specimen. • Different method of smear preparation are: 1) STREAKING: 2-3rd slide is covered by spreading specimen and immediately placed in fixatives. Ex- vaginal secretion, sputum and gastric content. 2) SPREADING: thick film prepared to maintain cellular interrelationship. Ex- sputum, bronchial aspirtes 3) DIRECT SMEAR: cervical smear, breast secretions, FNAC are directly smeared on slide.The slides are then placed in fixative jar. 4) PICK AND SMEAR METHOD: specimen poured in petri-dish under aseptic condition and musinous specimen like sputum are teased out using forcep. Discolored, purulent, blood stained ares selected and placed on slide. 3 smear prepared and put in fixative jar. 5) SACCOMANNO’S TECHNIQUE: saccomanno’s fixative used.
- 29. DRY SMEAR • A optimal sample from cell rich tissue has creamy consistency due to high cellularity with little or no blood or fluid called DRY Smear. • A dry smear best smeared withflat of second slide exerting light pressure as it is move along the specimen slide. • Dry smear dry quicklynd result into milky, finely granular film on the slide. • A wet aspirate mainly consist of blood or fluid containing small number of cells. • The cells can be concentrated and separated from fluid using two step smear technique. WET SMEAR DIRECT SMEARING
- 31. A RT I FAC T • The pressure must be carefully adjusted to achieve thin, even spread without causing disruption of tissue fragments with loss of micro- architecture or smudging artifacts.
- 32. • Thick smear and bloody smear is uneven and dry slowly and cause clumping and shrinkage of cell .
- 33. S M E A R P R E P E R AT I O N
- 35. • Some time best part of sample is found in hub of needle which is not expelled out properly for which another needle also.
- 36. MISCELLANEOUS TECHNIQUE LIKE LIQUID BASED CYTOLOGY(LCB) • Liquid based cytology technique has increased sensitivity and specificity of pap smear technique. • Principle: its basic principle is to collect cell sample into a liquid fixative solution and then create a monolayer of cell, ready for microscopic examination after staining. • LBC minimize obscuring inflammatory cell, red blood cells, necrosis.
- 37. THIN PREP ANALYSER Equipment require homogenization of specimen before filtering onto slide. Poly carbonate filter used. Thin prep analyser work on 3 principle: 1) Dispersion- sample homogenized by filter and separate debries and disperse mucus. 2)Cell collection- negative pressure draws fluid and cells are collected on exterior surface of filter. 3)Cell transfer- filter inverted and with slight positive pressure cells are transferred to the slides.The slide are then fixed and stained. Advantage- Ancilliary tech like IHC, moleculat test can be performed on LCB sample. • Number of slide per patient decreased • Improve cell preservation, and prevent cell drying during preparation. • Improve background for microscopy and eliminate preparation artifact
- 39. CELL BLOCK TECHNIQUE It is a method of preparing material for microscopic examination using a paraffin wax embedding method. Advantage:This support by providing more definitive cytopathology diagnosis. Cell block give better idea of tissue architecture and allow multiple section for panel of immune markers with controls It can be prepared from fluid specimens of all type like effusions, endometrial aspirates, brush samples as well as fine needle washings. Disadvantage:Time consuming and coastly process Cell block mainly used if need of immunocytochemistry is anticipated. CELL BUTTON Recently its simplified version called cell button is also developed which bare mainly applicable for cell rich tissue like lymph node and cellular neoplasm.
- 40. CELL BUTTON Recently a simplified version of cell block called cell button is also developed which mainly applicable for cell rich tissue like lymph node and cellular neoplasm. Procedure: A thick. Creamy material obtained from 27-25 gauge needle without aspirate is expelled onto glass slide but not spread or smeared. • After few second letting the drop to adhere to slide, put it into 90% ethanol.The drop struck like drop or button over slide. • The button gently detached from scalpel blade and processed like small biopsy. Advantage: Cell material is more concentrated whereas multiple section are required to find scanty tissue fragment in cell block.
- 42. FIXATION OF CYTOLOGICAL SMEAR • Specimen are immediately fixed when moist, this prevent it from drying and cell distortion. Types of fixatives: 1)ALCOHOL-ETHER FIXATIVE- mostly used for Papanicolaou staining. • Absolute-Ethyl alcohol(50 ml)+Ether(50 ml) for 30 minutes. 2) SCHAUDINN’S FLUID- This reagent is rapidly penetrating one and preserve the smear which are to be stained for hematoxyline and eosin. • Saturated mercuric chloride solution(60 ml)+Absolute alcohol(33 ml) +Glacial acetic acid(1 ml) for 2 minutes. • After washing in distill water the mercuric chloride clumps are removed by adding few drops of saturated alcoholic iodide and then rining in water.
- 43. 3)CARNOY’S FLUID- used for fixing small tissue pieces in urgent biopsy cases. • Absolute alcohol(60 ml)+Chloroform(30 ml)+Glacial acetic acid(10 ml) for 30 minutes to 3 hrs. 4) AIR DRYING- used for mostly Romanowsky staining.
- 44. FIXATION OF CYTOLOGICAL SMEAR
- 45. S TA I N I N G O F S M E A R • May Grunwald Giemsa (MGG) or Romanowsky stain and Diff-Quik stain all are done in Air dried smear. ROMANOWSKY STAIN: • Mixture of Methylene blue and Azure A,B,C. • It is metachromatic stain and therefor stain epithelial cell different from stromal cells • Also good for mucin and extra-cellular matrix.
- 46. May Grunwald Giemsa • Most convenient stain for FNAC • Give excellent detail of cytoplasm. • Steps: • May Grunwald solution:1 mint • Running water:1 mint • Giemsa stain:15 mint • Running water:1 mint • Air drying Diff-Quick Stain • Modified Wright Giemsa stain. • Much faster(2-3 mints) and give equal detail of cells and stromal tissue. • Contains 2 solution: • Sol 1- contain buffered eosinY • Sol 2- contain methylene blue+ azure A.
- 47. WET FIXATION • Alcohol fixation smear: 1) Papanicolaou stain- it is a metachromatic transparent stain showing cells overlapping nuclei. • Help in displaying cell maturation, cytoplasmic detail, nuclear details. • Orange G component of Papanicolaou stain demonstrate keratin and help in identification of squamous cell. 2) Hematoxylin and eosin stain-this is not cytological stain and mostly used for histopathological section. Hand E stained smear give resemblance of histology section.
- 48. COMPARISION OF AIR DRIED AND WET FIXED SMEAR ACCORDING TO GENERAL PROPERTIES
- 49. COMPARISION OF AIR DRIED AND WET FIXED SMEAR ACCORDING TO TISSUE SPECIFIC PROPERTIES
- 50. S P E C I A L S TA I N S • PAS/DIASTASE OR ALCIAN BLUE for Mucin. • PRUSSIAN BLUE for Iron. • MASSON-FONTANA for Melanin • GRIMELIUS for Argyrophilic granules • CONGO RED forAmyloid • MICRO-ORGANISMS by Gram, PAS, Z-N or Gomori silver stain • GLYCOGEN stained by PAS • FAT by Oil-Red-O
- 51. RAPID STAIN Uses: to check adequacy of FNAC and also immediate evaluation to control cross contamination. • Dye used- Methylene blue,Thionin blue, toluidine blue. Procedure- Fix smear for 10-15 second in 95% alcohol • Blot dry • Treat with toluidine blue 10 dips. • Rinse in water to remove excess stain. • Put cover slip on wet smear and see under microscope
- 52. F N AC O F D E E P S E AT E D L E S S I O N S • FNAC are widely used for various deep seated lesion of body. Advantages: • Area of interest or more representative part of tissue can be sampled. • Injury of major structure is avoided • Necrotic tissue or cystic area is avoided • Small lesion can be sampled • Nonpalpable and deep seated lesion can be accessed
- 53. INDICATION OF IMAGE GUIDED FNAC • To diagnose the character and type of the primary lesion • To know staging of the malignant lesion • To collect material for ancilliary studies such as flow cytometery, microbial culture. • Clinical history • Simple X-RAY, USG, OR CT scan • Size of the lesion • Routine blood test • Coagulation parameter PRIOR INFORMATION NEEDED FOR GUIDED FNAC
- 54. COMPLICATION OF FNAC GUIDED • Bleeding • Pneumothorax • Infection • Fluoroscopy guided FNAC • USG guided FNAC • ComputedTomographic scan guided FNAC • Endoscopy guided FNAC • Magnetic Resonance Image guided FNAC • Mammography guided FNAC • Transrectal FNAC of prostate. EXAMPLE OF FNAC GUIDED
- 55. FLUROSCOPY GUIDED FNAC • Easiest method. • Used for cortical bony lesion • Disadvantage; high radiation • Adequate shield necessary to prevent radiation
- 56. USG GUIDED FNAC Principle: high frequency sound wave is applied and the reflection of the wave from tissue interface is recorded to construct image. Advantage: • Economic, portable, rapid, easy to do, real time. Disadvantage: • Significant obscuration due to air or bone may affect the image. • Operator dependent • Not of high resolution. Application: almost all the body lesion except bony lesion.
- 58. • CT GUIDED FNAC • Principle: multiple images are generated in different tissue plane depending on the difference of physical density of the tissue. Advantage: • High resolution • Exact localization of needle possible • Operator independent • Deep lesion near vital structure need CT guidance Disadvantage: • Costly, time taking, radiation exposure Application: small lesion near vital structure.
- 59. • MAGNETIC RESONANCE IMAGE GUIDANCE Principle: radiofrequency energy used. Advantage: no radiation exposure Disadvantage: costliest, time consuming, special equipment required Application: breast, lung, soft tissue of head and neck region
- 60. DIFFERENCE BETWEEN DIFFERENT GUIDED PROCEDURE
- 61. ANCILLIARY TECHNIQUES IN ASPIRATION CYTOLOGY Various ancilliary technique are essential part of fine needle aspiration cytology.These help in diagnosis, prognosis, and management of disease: • Immunocytochemistry • Flow cytometry • Molecular techniques • Electron microscopy
- 62. IMMUNOCYTOCHEMISTRY: • Use of monoclonal antibodies provide valuable information regarding poorly differentiated malignant tumor, their exact categorization etc. • IC can be done on FNAC material by different ways like: 1) Direct smear preparation 2) Cytospin preparation 3) Liquid based cytology 4) Cell block
- 63. INDICATIONS OF IMMUNOCYTOCHEMISTRY • Typing of poorly differentiated malignancy into carcinoma, lymphoma, sarcoma, etc • Subtyping of lymphoma • Exact typing og carcinoma • Diagnosis of exact typing of malignant round cell tumor • Hormone or receptor expression, such as estrogen, progesterone receptors, her-2 receptor in breast • Studying cell proliferation marker
- 64. PROCEDURE OF CYTOLOGY SMEAR FOR IHC • Fix smear in fixative for 30 mints • Stain smear with routein pap and HE stain • Scan the slide and mark suitable area for staining • Remove cover slip by dipping slide in xylene.This will remove mounting medium • Rehydrate slides by passing through xylene and alcohol • Do not destain the slides • Block endogenous peroxidase activity using 0.3%(v/v) hydrogen peroxidase/methanol for 15 mints • Rinse slide in distill water and wash in PBS for 5 mint • Proceed for enzyme retrieval • Wash in PBS for 5 mints • Continue with desired staining
- 67. FLOW CYTOMETERY • Flow cytometery help to study the different characteristic of the single cell in fluid steam. • The single cell suspension is made from FNAC material and the cell are stained with a fluorescent dye that binds with the specific component of the cells surface antigen or receptors. • The cell are flowed in front of the laser beam and the emitted fluorescence is recorded in photomultiplier tube and is converted into digital signal. • FCM of FNAC material is used for immunophenotyping, DNA analysis, and cell cycle analysis.
- 68. MOLECULAR GENETICS • FLURORESCENT IN SITU HYBRIDIZATION
- 69. • POLYMERASE CHAIN REACTION
- 72. REFERANCES • FINE NEEDLE ASPIRATION CYTOLOGY BY SVANTE R. ORELL AND GREGORY F. STERRETT • FINE NEEDLE ASPIRATION CYTOLOGY BY PRANAB DEY • TEXTBOOK OF MEDICAL LABORATORY TECHNOLOGY BY PRAFUL B. GODKAR AND DARSHAN P. GODKAR
- 73. THANK YOU
Editor's Notes
- #34: Needle should be 45 degree angle with slide and initial placement of material should be 1 inch away from margin to label it.