basic cytology techniques.pptx

cytological methods

1. CY T OLOGICAL SAMPLE PREPARATION AND STAINING TECH N IQUES Yona Mbalibulha

2. I NT R ODUC T I O N  The best morpho l og i c presentation obta i ned f r om a ny cyto l o g ic sp e cim e n requ i res an Process that it w ent through during col l ecting and preparation. spec i men. und e rstand i ng of the Princ i ple of cyto t echn i que s  T o reduce the s pec i men to a cell u l a r presentatio n , can be i n te r preted and diag n osed. w h ich

3. C Y T OL O GY S P E CIMENS 1. 2. 3. 4. 5. 6. 7. 8. 9. peritoneal, pericardial C SF Nip p le disc h arge and pleural f l uids B ronc h ial b rushings S putum Gastric w a s hin g s U rine se d iment P rostat i c se c ret i ons / w as h ings C e rvicovaginal ( paps) sme a r

4. E XFOL I A TIVE C Y T OL O GY  It is the study of ce l ls that h ave b e en shed or re m oved organs. f r om the ep i thel i al sur f ace of v a rious sm e ar wash s c ra p ing b r u s h i ng

5. COLLECTI O N OF SA M PL E S S aline moistened cot t on t i p appli c ator A yre s p atula

6. B RON C HIAL W A S H

7. B R U S H ING

8. S C R A P ING

9. F INE N E E D LE A S PIR A TION CY T OL O GY ( F N A C )

10. B ODY FL U IDS P l eural fluid P e ricardial fluid

11. B ODY FL U IDS CSF ascitic fluid

12. C E N T R IFUG A TION  If t oo m uch fluid is o b taine d ,. C e nt r ifuged for 5 mins S e d i ment  If l i t t le amou n t of fluid is aspir a ted (few d r o p s), or if the f l uid is thi c k, the c e n tri f u g e is not r e q u ire d .

13. C Y T OCE N T R IFUG A TION  It is a spec i al machine that perfor m s a ce n t r i f uge and collect i on of s e diment on the center of the slid e s. procedure is useful for hypoc e l l ular spec i men . Th i s

14. M ET H ODS O F S MEAR  s t r e a k ing  spr e ading  p u ll a part P R E P A R A TIO N :  touch or impr e s s ion sm e ar

15. ST R E A KING - Used for preparing mucoid secretions , vag i nal secretions, sputum a nd gastric content - use a spatula, dissecting needle or app l icator stick and st r eak in a zi g zag fashion

16. S P R E A D ING - used for thick mucoid secretions - smears of fresh s p utum and bronch i al asp i rates

17. P U LL A P A R T - for serous flui d s, conce n t r ated sputum, a nd enzymatic lavage fo r m the GI T , smears of u rinary sed i ment, vagin a l pool and breast secreti o ns.

18. TO U CH IM P RESSION I mp res s ion c y tol o g y b eing c ol l e c ted From a p a tient , using a sterile g l a ss sl i d e with pol i shed e dg es.

19. S QUASH SME A R PRE P AR A TION  fairly accurate,  simp l e and r e l i a b l e tool for ra p id i n t r a - op e rative central nervous system l esi o ns.  B a sed on two essential fac t ors: d i ag n osis of A vai l ab i l ity of v e ry small tissue f r agments & good preservation of fi n e cel l u l ar deta i ls. N o t e f fected by e d ema, hemorrha g e, n e cros i s & calc i fication. • •

20. C E L L B L OCK  It is a p r oced u re to con v ert cell sediment into p ar a ff i n block  further pathological procedures can be perfor m ed like i m mun o hist o ch e mistry (IHC).

21. M et h ods o f C e ll B l ock P r e p a r a tion  Dire c t pro c e s si n g of tiss u e fragm e nts pre s e n t in f l uids  Fi x ed s e dim e nt meth o d  Ba c ter i al ag a r method  Sim p li f ied c e ll blo c k tec h niq u e a n d p a ra f fin u s ing al c o h ol, ac e tone  C o mp a ct c e ll blo c k tec h niq u e  Pla s ma throm b in meth o d

22. M et h ods o f C e ll B l ock P r e p a r a tion cont ’  Cell b locks from millipore  Histo g el method  G e latin emb e d d i ng  Celloidin bag  Cell block p repa r ation fr o m scraping of cytolo g y smears  Automated c e ll block p reparation  Alb u min me t hod

23. Example Procedure of cell block prepn C e nt r ifugat i on 2 4 00 rpm P o ur o f f sup e rnatant A d d 5 m l of methanol + f or m a l in ( 9:1) S p in at 2 400 rpm metha n o l +for m a l in for 30 t o 60 min r e move ha r d e ned cell b utton submit in a cass e tte

24. L I Q UID - BASE D C Y T OL O GY  cytology (t h e study of cel l s) through a liquid medium  Cel l s a re collected f r o m cervi x (any o ther s ite) a r e placed d i r e ctly i n t o l iq u id p r e se r vativ e , r a ther than tr a nsf e r r ed to slide.  Samp l e is pr o cess e d and r e sultant thin sme a r e asy to s cre e n

25. L IQUI D - BASE D CY T OL O GY TECHNIQUES

26. P a th sure Th i n prep

27. ( 1 ) C ELL DISP E RSION  S w irl i ng the sa m p l i n g dev i ce in the p reserva t i o n sol u tion  Strong eno u gh to s e parate d e bris a n d d i sp e rse mucus

28. ( 2 ) C ELL C OLLECTION  A ge n tle va c uum i s c r e a t ed w i t hi n the Th i nPrep Pap T est Fi l te r , whi c h c o l l e c ts c e l l s on t h e exter i or s u rfa c e of t he me m br a ne.

29. (3) CE L L T R ANSFER  the Th i nPrep Pap T e s t F i lt e r i s in v erted and g e nt l y pr e s s ed ag a i n st the Th i nPrep M i c ros c ope Sli d e.  Natu r al att r a c ti o n a nd s l i g ht pos i tive a ir pr e s s ure c a u s e the c e l l s to a dh e r e .

30. PA TH SURE 3 4 1 2

31. C o nv e nti o n a l smear Th i n prep sl i de

32. Th i n prep sl i de C o nv e nti o n a l smear

33. Co n v e t i o n a l p a p a n i c o l a o u Th i nPr e p Sur e Path F i x ation Ethanol me t ha n ol Ethan o l C o l l ection Smear on sl i de S a mple rinsed in vial C o l l ection dev i ce l e ft i n vi a l C e ll samp l e Ran d om distribution U n iform d i st r i b ution over 20 mm of s l i d e U n iform d i st r i b ution over 13 mm of s l i d e

34. C o llection device EC bru s h, spatula, Ce r v e x - Bru s h EC bru s h, spatula, Ce r v e x - Bru s h rin s ed in v ial C e rve x - Bru s h most e f fec t i v e, tip dep o sited into v i al Pre s e r va t ion a r ti f a c ts Air dr y ing, blo o d, infla m mation, i r regu l ar distribution of cel l s All p r e s e r va t ion a r ti f a c ts g r eatly r e du c ed All p r e s e r va t ion a r ti f a c ts g r eatly r e du c ed

35. Au t omated pro c essing N o t appli c able V a c uum pre s sure through T r a n s Cyte fi l ter G r avi t y sedimenta t ion p r o c e s s I m aging tech n ology Not applicable A v ai l ab l e A v ai l ab l e Ancilla r y testing N ot appli c able HP V , C h lam y dia, gon o r r hea H P V , C hlamydia, gon o r r hea

36. Staining Methods in cytopathology Common staining methods. Papanicolaou stain (PAP smear) Differential quick method (diff. quick methd ) Acridine orange technique P.A.S Mehtylen blue Perl’s Prusiian Blue Feulgen reaction Heamatoxylin and eosin Florescent dye techniques Others....... Note: Read and make personal notes on these and other specific cytological methods.

37. General concepts in cell examination The overall size and shape of cancer cells are often abnormal. The size and shape of the nucleus of a cancer cell is often abnormal. The nucleus of a cancer cell appears darker when seen under a microscope after being stained with certain dyes.

38. Concepts cont...... Cancer cells do not relate to each other normally. Takes on abnormal functions different from the parent organ. (form glands, secretions, hormones ) cancer cells invade other tissues. cancer cells can metastasize

39. Modes of examination. Ordinary Microscopy Electron Microscopy . Flow Cytometry Image cytometry Molecular Genetic Studies Cytogenetics Note: Read and understand the details of these methods.

40. S T AINING M ET H ODS I N C Y T OL O GY  P a p a n i co l aou Sta i n i ng Method  May - G r un w a l d- Giemsa ( M G G ) Stai n i n g me t hod

41. P A P A N ICOLAOU S T AINING M ET H OD  named af t er D r . George N. P a pa n ic o l a ou  po l ychrome stain i ng reaction  d i sp l ay the many var i ati o ns of ce l l u l a r morph o l o gy sho w i n g de g ree of c e l l u l ar ma t urity a nd me t ab o l i c acti v it y .

42. P R INCIP L ES  H y dration and deh y drati o n: – Hydr a t i on p r e p a r es t h e c e l l s am pl e f o r u p t ak e of t h e n u cl e a r dye; – d e hy d r a t i on p r e p a r es t h e c e l l s am pl e f o r u p t ak e of t he co u nter s tai n s . Deh y drati o n and cl e aring so l uti o ns re s u l t i n c e l l u l a r  tran s pare n cy and p repare the c ell sample for the f i nal st e ps

43. S T AINS  Nu c lear stainin g : Hema t o x y lin  T wo cytoplasmic c o unter stainin g : ( 1 ) Or a nge G - ( O G ) - 6 , OG - 5 and O G- 8 is an a c idic dye, stains ke r at i n a bright, in t ense orang e . ( 2 ) Eosin Azu r e (EA) - EA - 36, EA - 50 and E A - 65 in c luding three stains  –Eosin Y  –Light Gr e en  –Bismar c k b r o w n Y

44. S TEPS OF S T AINING PR O CE D URE (1) Fix a tion - 95% ethyl alcohol or in other - minim u m of 15 min u tes substitutes (2) Nu c lear st a ining  Har r is ha e mat o x y lin - r e gre s si v e st a ining method

45. ( 3 ) Cy t oplasmic staining ( 4 ) D ehydrat i on - R inse t h e smea r s in absolu t e alco h ol for t wo or t h ree removal of water. - Al t ernative to 1 0% et h anol are changes for t h e - 1 0% isopropa n ol a n d 100 % de n atured ( 5 ) Clear i ng - alcohol is bei n g replaced with Xylene - Xylene h a s a refractive in d ex as t h a t of - It p revents c el l ular distor t ion. ( 6 ) Mou n ting - DPX alcohol. glass a n d mounting medium

46. cl e a ri n g d e h y dr a tion h y dration fi x ati o n

47. A g e of dyes T ype of fi x atives N o . Of sl i d es in dye each Moisture a nd y humi d it R e gressiv e or p rogr essive Le n gth of stain i ng tim e Presence or a bse Qual i ty of ll sample ce nce of i nflamma t o ry ce l l chan g es Factors a f fec t i n g P a p stai n i n g

48. Diff-Quik Stain: On - site evaluation of fine - needle a s piration samples requires stain protocols that are quick and easy to perform and allow for assessment of adequacy of the sample, i.e., cell types and amounts. Procedur e : • • • • • • • Place one drop of aspirate on slide Allow slide to air dry and smear Fix in 95% ethanol - 10 dips 1 2 dips 6-8 dips - 20 dips 1- 2 - Place in Place in Distilled solution solution water rinse Review under microscope. • purple nucleus Resul t : • The cells stained exhibit a deep blue for the cytoplasm and different hues of

49. U L T R A F AST P A P ANICOL A OU S T AIN  f ast a s t h e D i f f -Qu ik stain  90 secon d s smeared on a sl i de a l l o w e d to a ir d r y p l aced in nor m al sal i ne f i x e d in a mi x tu r e of % fo rm aldehyde and 65% e t hanol 4 s t ained with Richard Al l an Hema t o x ylin 2 Cytostain and

50. M A Y -G RUN W ALD -G IEMSA (MGG) S T AINING METHOD  M G G stain is perfo r med in a i r –dried asp i rates or flui d s.

51. I MM UNOCY T OCHEM I ST R Y  Det e ction of sur f ace antigens (marke r s) on is o lated cells  The d etecti o n is ba s ed on specific antige n - antib o dy binding (im m unor e actions).  A specific antib o dy that was p r o duced by single B cell cl o ne id e ntifies an epitope with 8 - 15 length amino a cid sequence in protei n . a

52. I H C I N D IAGNOSTIC C Y T OL O GY  Tumor Di a gnosis/Classi f icati o n A PPLIC A TI O NS  Pr o gnostic/Pre d ictor M a rk e rs  T a rget Ther a py

53. FLOWCY T O M ET R Y D e f i ni t ion : An a na l ytic a l t e c hn i que  - in wh i ch c e l l su s pe n s i on /body flu i d, per i pher a l obta i ned f rom any unf i x ed ti s sue b l ood or bo n e marr o w are s ta i ned with f l uo r e s c e nt l y l a be l ed an t i b ody and th e n s ub j e c ted to an a lys i s by a in s trument ca l led as flow cyto m ete r .

54. F LOROC H ROMES  F l orescin is o thiocya n ate ( F ITC ) .  P h ycoeryth r in.  T e x as red.  A l l o phycocy a ni n .  P e rid i n i n chl o rophy l l.  T a n d e m floroc h romes.

55. F LOW C Y T O M ET R Y A P P LIC A TIONS  I m munoph e notyp i ng.  D i ag n osis and prognostic a tion of immunod e ficie n c y .  T o d i a g n o se ca u se of a l l o graft rejection.  D i ag n osis of a uto antibod i es in I TP .  T o measure nuc l e i c acid content.  D N A p l o i dy study in cance r .

56. M OLE C U L AR T E C H N I Q U E S I N C Y T O P A T H OL O GY  F l ourescence in situ hybrid i zation (F I S H )  P o lymerase cha i n reaction (PC R )  Microsatel l ite an a lys i s  L a ser microd i sse c tion  Mu t ation an a lys i s  D N A me t hyl a tion an a lys i s

57. C ONC L U D ING R E MAR K S A s pec i men must b e care f u l ly prepared, well fi x ed, and stai n ed in its j o u rney to the microsc o p e . Less time i s r eq u ired to p repare stain i ng sol u tion, si n ce ready - made products t hat produce rel i ab l e and consistent stai n i n g are av a i l a b le for p u rchas e . L i qu i d - bas e d and automated sys t ems are ent r enc h ed in soph i sticated screeni n g programs. D e vices such as imag i ng flow cyto m et r y and 3 - d i mensio n al cell scann i ng promise f urther a dvanc e s in an a lys i s ca p a b i l it y .    