Liquid-based cytology for risk-adapted cervical screening

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1 Advntges Liquid-bsed cytology for risk-dpted cervicl screening Reinhrd Bollmnn Institute of Pthology Bonn-Duisdorf, Bonn, Germny Reviewers:Wolfgng Kühn, Berlin, Germny nd Jn Dolezil, Hmburg, Germny Summry In Germny, in the erly 70's, two projects deling with the development of computer-ssisted cervicl screening were crried out. It ws soon relized, however, tht mchineredble cytologicl preprtions hd to fulfill certin criteri tht were difficult to meet using conventionl Pp smers: i. e. isolted cells in one opticl plin (monolyer), high contrst between nuclei, cytoplsm nd bckground nd cler bckground.the first issue ws solved by the introduction of single cell preprtion (monodispersion) nd the ltter by cell seprtion nd clensing. This ws the dvent of liquid bsed cytology (LBC). Currently, there re three LBC-systems used in Germny: the ThinPrep, the SurePth nd the PpSpin method. There re only very few differences between the cytomorphology of LBC nd conventionl Pp smers. The min difference is the cler bckground provided by LBC, which mkes it esier to identify nd interpret bnorml cells. So fr, evidence suggests tht liquid-bsed cytologicl methods offer the following dvntges (Tb. 1, 2) compred to trditionl smer techniques: ) reduction in the proportion of indequte specimens, b) n improvement in sensitivity, nd c) possible reduction in specimen interprettion times. In the USA, 98 % of the Pp tests re nowdys liquidbsed. This reflects the normous populrity of the method mongst cytologists, s demonstrtively expressed by the director of cytopthology of the University of Vermont, Tble 1: Advntges nd disdvntges of liquid-bsed cytology Disdvntges who complined tht he ws fed up screening poorly preserved, ir-dried, blood stined conventionl slides of vrying depth of focus, problems which, mongst others, cn be fully eliminted by LBC. Tble 2: Results of the IAC Cervix Cncer Screening Meeting from the 20 th to the 27 th of April 2004, on the relities of liquid-bsed cytology 1. The smple trnsferred from the smpling device to the slide is more representtive in LBC thn in conventionl pp smers 2. Reduced number of unsuitble smers 3. Severl tests cn be crried out on just one specimen 4. Significnt increse in the detection of HSIL ( high-grde squmous intrepithelil lesions ) Introduction! The science of liquid-bsed cytology is still in its infncy. I bet tht George Ppnicolou would be its gretest fn (Leimn 2007).! In 1943, Ppnicolou nd Trut published the fmous monogrphy on vginl cytology s screening method for cervicl cncer. The methods of Aurel Bbes in Rumni (Tsc et l. 2002) were lso documented during the sme period. The technique of extrcting cell smples from the cervix, streking the cells onto slide nd their subsequent fixing, stining, nd microscopic evlution, hs not ltered gretly since then.the method hs proven itself nd hs led to decrese in the number of cervicl crcinom dignosed in Germny. However, the mjor chllenge tht remins re the flse-negtive Pp tests, i. e. when the mlignncy or its precursors remin undetected. Published studies deling with the percentge of flse-negtive results hve become politicl issue in Germny: some clim high flse-negtive rte (Schneider et l. 2000; Petry et l. 2003) while others cn prove low flse-negtive rte (Mrqurdt et l. 2005). The truth probbly lies somewhere in between. Bsed on met-nlyses, n interntionl flse-negtive rte of bout 50 % is ssumed (Nnd et l. 2000).This low sensitivity cn be compensted by regulr prticiption in screening. The possible cuses of flse-negtive cytology re listed in tble 3. Preprtions re stndrdized. Cells re treted gently to void dmge. Almost ll smer test cells cn be exmined. Possibility to crry out other tests on the rest of the liquid (cytometry, immunocytochemistry, moleculr pthology, cell block method). Cells re distributed rndomly on the slide. Greter demnds on equipment nd logistics. Cytologicl interprettion differs from conventionl methods nd users hve got to be trined. The number of cells used in BD SurePth TM Pp Test is not stndrdized; the stining process follows utomticlly. Tble 3: Possible cuses of flse-negtive Pp tests No typicl cells in the preprtion (incorrect smpling) = % Atypicl cells re present but cnnot be identified due to poor fixtion nd/or overlpping Atypicl cells re present, but re being overlooked by the screener ( screening error ) Atypicl cells re present nd re identified but not correctly interpreted, thus they re Schritte, flsely judged wie die Zellentnhme und Die Die vorbereitenden Die vorbereitenden Schritte, wie die Zellentnhme und It is Bollmnn R. Liquid-bsed cytology... Gynkol Geburtsmed Gynkol Endokrinol 2008;4(2): published kdemos Wissenschftsverlg 2008 ISSN

2 It is difficult to stndrdize the preprtory steps, such s smpling the cells nd trnsferring them onto the slides: over 80 % of the specimens tken re discrded together with the smpling device (Hutchinson et l. 1994). The prepred smers demonstrte rtistic vritions (Fig. 1). As described lter on in this rticle, thin-film cytology provides solution to the problem of stndrdizing cell trnsfer to slides. Tble 4: Cytologicl preprtions for use with mchines Requirements Technicl solutions Isolted cell mounting Single cell seprtion (monodispersion) Mounting in one opticl plne (monolyer) High contrst between the nucleus, cytoplsm nd bckground Cell seprtion nd clensing Clen bckground of the preprtion b Figure 1: Problems when trnsferring cells to slides: the conventionl smer technique is limited nd difficult to stndrdize : Wve technique b: Pinting technique Screening errors (typicl cells re overlooked) cn be voided by computer-ssisted screening. The history of liquid-bsed cytology In the 1970s, the Germn stte finnced two reserch projects on computer-ssisted cervicl screening: LEYTAS (Leyden Texture Anlysis System) nd FAZYTAN (utomted erly detection cytologicl nlysis). During the projects, it becme cler tht if they were to be used with mchines, the cytologicl preprtions would hve to fulfill certin criteri (Tb. 4). In Germny, this reliztion led to the development of single cell techniques nd to cell seprtion nd clensing (see Tb. 5) s the min steps in liquid cytology (synonyms: thin-film cytology, monolyer cytology, liquid-bsed cytology). Idelly, the im of the single cell technique (monodispersion) is to produce isolted cells in one opticl lyer (monolyer) nd thus void the formtion of cell clumps. While the term monodisperse cell seprtion focuses on isolted mounting of the individul cells, the expression monolyer preprtion emphsizes the fct tht the cells lie in one plne. However, the liquid-bsed cytology technique does not yet meet these criteri: liquid-bsed cytology preprtions contin more three-dimensionl cell structures thn those of conventionl cytology, requiring the screener to constntly refocus (Fig. 2). It is therefore consistent to replce the term monolyer cytology with thin-film or liquid-bsed cytology. 40 μm Figure 2 -c:three-dimensionl dysplstic cell structures in Thin- Prep preprtion (Pp IVb) hve to be refocused, therefore the term monolyer is not pproprite b c 40 μm 40 μm 69

3 Liquid-bsed cytology systems Three liquid cytology systems re presently vilble in Germny:ThinPrep (Hologic), BD SurePth TM PpTest* (Becton Dickinson) nd PpSpin (ThermoShndon), nd other similr methods (see below). The min differences between the systems re shown in tbles 5 nd 6. Tble 5: Methodologicl requirements of stndrdized liquid-bsed cytology The procedure for cell smpling is lwys the sme: the instrument used for smpling is either thoroughly rinsed in the liquid (ThinPrep ), or the hed of the instrument is removed t the predetermined breking point nd plced in the liquid (SurePth ). The mnufcturers of the equipment provide trining mteril on how to tke the smer. The composition of the liquids is ptented nd is therefore unknown. The liquids probbly consist of buffered cell mediums with reltively low lcohol content (methnol), s they must fulfill both the requirements of cell trnsport medium nd cell fixtive. Single cell isoltion (monodispersion) Mechnicl Vortextion (PpSpin ) Stirring (ThinPrep ) Filtrtion Cnnultion (SurePth ) Homogeniztion nd ultrsound Density grdient centrifugtion (SurePth ) Membrne filter (ThinPrep ) Chemicl Enzymes EDTA Dithiotreitol (PpSpin ) Detergents Single cell isoltion (monodispersion) Cell seprtion 2nd clensing FDA-licensed Cell seprtion nd clensing Density grdient centrifugtion (SurePth ) Membrne filter (ThinPrep ) Tble 6: Differences between different liquid-bsed cytology methods ThinPrep (Hologic) Rottion Yes SurePth PpSpin (Becton Dickinson) (Shndon) Cpillries Membrne Density grdient filter centrifugtion Yes Vortextion No No Aims of liquid cytology To recruit ll exfolited mteril for the cytologicl exmintion Hve method-immnent evidence on the cellulrity s qulity ssurnce mesure Optimize visul morphologicl exmintion through n improved presenttion of the mteril Fulfill the criteri for computer-bsed screening To temporrily document the mteril for further morphologicl nd non-morphologicl exmintions Enble biomedicl ssistnts to work more sfely nd in less tiring mnner, prtly due to the elimintion of disruptive elements (e.g. inflmmtory cells, mucous, fibrin) Production of cytologicl preprtions ThinPrep (Hologic) Figure 3 shows the utomted steps of the ThinPrep method tht tke plce in the T2000 Processor. Firstly, rotting cylinder mechniclly isoltes the cells. Following this dispersion, the cells re filtered. The lower end of the cylinder is covered with micropore filter nd vcuum is mounted t the upper end. The liquid contining the cells is thus sucked through the filter. The pores of the filter re lrge enough to llow neutrophil grnulocytes nd erythrocytes to pss, but too smll to let epithelil cells pss. Grdully, epithelil cells obstruct the pores of the filter. The processor cn detect when ll the pores of the filter re obstructed nd complete the filtrtion process. The filter is then thrown onto n electriclly chrged slide in order to trnsfer the cells onto the slide, which is stined in the following step. *In the following short SurePth Bollmnn R. Liquid-bsed cytology... Gynkol Geburtsmed Gynkol Endokrinol 2008;4(2): published kdemos Wissenschftsverlg 2008 ISSN

4 Cylinder with membrne filter 1. Cells in Cytolyt -solution 2. Dispersion 3. Filtrtion 4. Trnsfer to slide Vcuum Membrne filter Stop 1 Fixtion bth continer 2 Fixtures 3 Rollers 4 Filter components 5 Smple continer 6 Fixtures 7 Slide fixture 8 Slide c Figure 3: Automted steps of the T2000 Processor in the ThinPrep technique : 1. Cells in the liquid; 2. A rotting cylinder cuses cell dispersion (mechnicl single cell isoltion); 3. A vcuum is then mounted on the cylinder nd the cells in the liquid re sucked through the filter; 4.The cylinder with the membrne filter is then thrown onto n electriclly chrged slide nd the cells re thus trnsferred to the slide. b: Step 3 in detil c:thinprep Processor T

5 G 1. Cells in Cytorich solution 2. Dispersion 3. Density grdient centrifugtion (grnulocytes, erythrocytes, debris) Resuspension 5. Trnsfer of pellet nd sedimenttion b Figure 4: Steps of the SurePth technique -b: Density grdient centrifugtion principle with subsequent sedimenttion onto the slide c: Preprtion equipment BD PrepStin TM Slide Processor SurePth (Becton Dickinson) This system is bsed on density grdient centrifugtion (Fig. 4). The smple continers re first vortexed, whereby single cells re isolted. An liquot of the smple is plced into centrifuge vil for density grdient centrifugtion. In this process, the cells re seprted ccording to their specific weight. Grnulocytes, erythrocytes nd debris tht hve lower specific weight thn the epithelil cells ccumulte bove the density grdient nd re then discrded. Menwhile, pellet forms contining the cell popultion relevnt for dignosis; the pellet is resuspended nd trnsferred onto sedimenttion chmber, which is mounted onto slide.the cells sediment on the slide in thin lyers nd the rest of the liquid is discrded. Stining is n integrl prt of the process. The procedure described is not fully utomted. 6. PAP stining (Steps 5 nd 6 Monolyer in BD PrepStin TM (stined) Slide Processor) c PpSpin (ThermoShndon) or similr methods, e. g. Turbitek (Lbornord),Liqui-Prep TM (LGM Interntionl), GluCyteTM (Select Dignostics) With these techniques, the cells re isolted by vortextion nd re subsequently centrifuged. The cells re neither seprted nor clened beforehnd. However, these techniques re not yet uthorized by the FDA (Bollmnn nd Jordn 2005). Pro nd contr: licensed versus unlicensed methods Due to high costs, the methods tht re not uthorized by the FDA (PpSpin, mongst others) do not include the min steps of stndrdized preprtion, i. e. cell seprtion nd clensing (see Tb. 6), so tht the preprtion produced still contins inflmmtory cells, blood, fibrin nd detritus. Bollmnn R. Liquid-bsed cytology... Gynkol Geburtsmed Gynkol Endokrinol 2008;4(2): published kdemos Wissenschftsverlg 2008 ISSN

6 However, if the cells re lso seprted nd clensed, s in the ThinPrep Pp test (Hologic) or the SurePth Pp test (Becton Dickinson), the dignosticlly relevnt cells cn be enriched. The methods tht do not include previous cell seprtion nd clensing with simple centrifugtion deliver preprtions hving coincidentl, questionbly relevnt ccumultion of cells, s the dignosticlly importnt cells re not enriched. Despite this, it ppers tht these less expensive methods provide n lterntive to the estblished methods if the preprtions re crefully processed nd if the cell content of the liquid is determined turbimetriclly. This hs been suggested by study crried out t the John Hopkins Hospitl, Bltimore (Rosenthl et l. 2006). One cn only hope tht the mnufcturers of the methods licensed by the FDA follow different pricing policy in the future so tht in Europe, the equipment becomes vilble t resonble prices, s is the cse in Belgium (ThinPrep ).We hve to consider tht the WHO only pproves of less expensive methods for mss screening. Correspondingly, the Germn Cytologicl Society (DGZ) is politiclly right when rejecting liquid-bsed cytology due to its high costs. However, it is surprising tht scientific society enlists the price of method s n rgument ginst its use. Stining On completion of the utomted ThinPrep process, the so produced unstined preprtion cn be stined using vriety of utomtic stining pprtus. The uthor s own experience shows tht it is dvisble to slightly modify the stining protocol used for conventionl smer preprtions. In the SurePth technique, stining is n integrl prt of the process, whereby the results of stining my differ from stining conventionl preprtions, especilly with regrd to the colortion of cytologicl cornifying processes. Kertinized cells re, however, n importnt mrker both in detecting ltertions cused by HPV s well s kertinized crcinomtous solitry cells. The fct tht kertinized cells do not stin well led to reltively poor rting for the SurePth stining method in n externl technicl qulity ssurnce progrm crried out in Gret Britin (TEQA) (NHS CSP 2004). cn be found in ny position nd re not grouped together in so-clled streets, s observed in conventionl preprtions. The fields must therefore overlp in this type of screening. Additionlly, screening of LBC preprtions is ltogether more intensive compred to conventionl preprtions, s typicl cells cn be hidden in the gps between the norml cells. Additionlly, it is oftentimes necessry to chnge from the 10mm objective (screen enlrgement) to the 40 mm lens to be ble to precisely evlute the solitry cells. Overll, the liquid-bsed cytology screening process requires greter concentrtion of the technicin. Figure 5: Comprison of SurePth (left) nd ThinPrep preprtions (right) Adequte cell number for liquid-bsed cytology preprtions In the Bethesd Clssifiction, 5000 cells re considered s dequte. McQueen nd Duvl (2006) were ble to show tht t lest 87 typicl cells must be present in preprtion in order to detect them with certinty. However, ccording to mthemticl clcultions this cn only be chieved with totl of cells. In Wles, limit of cells hs been estblished (Cervicl Screening Wles 2006). A study presently being crried out by the Ntionl Helth Service (NHS) ims t solving the cell number issue for Gret Britin. The cell number cn only be correctly judged with gret mount of experience, thus hving completed intensive trining nd use of comprtive imges (Solomon nd Nyr 2004). Screening methods for liquid-bsed cytology The screening fields of the slide re round-shped nd much smller thn the fields in conventionl preprtions (Fig. 5). Screening methods therefore differ from those used for conventionl smer preprtions: bnorml cells Cell morphology of liquid-bsed cytologicl preprtions in comprison to conventionl preprtions Liquid-bsed cytology preprtions re very similr to those of the regulr smer method, lthough their morphology cn sometimes differ fundmentlly (Tb. 7). The most importnt difference in comprison to regulr methods is the cler bckground of liquid cytology preprtions, which enbles n esy visul ccess to bnorml cells, thus fcilitting their interprettion (Fig. 6). 169

7 Tble 7: Morphology of liquid-bsed cytology preprtions in comprison to conventionl preprtions More similrities thn differences Lower number of pthologicl cells/slide Esier visul ccess to bnorml cells due to the clerer bckground Shrunk cells (fixtion in lcohol) Cells re more circulr in solution Cytoplsm nd nucler structures re more esily recognized (wet fixtion, lck of ir-drying rtefcts) Thus lower nucler hyperchromsi Aprt from the differing locliztion of typicl cells ( missing streets ), liquid-bsed cytology preprtions lso differ from conventionl preprtions in tht there is less blood, fibrin, nd necrotic debris on the slides due to their specil processing. For mny yers, such rtefcts hve served s evidence for mlignnt processes in conventionl cytology. Although this so-clled tumor dithesis is lso lwys found in preprtions in liquid-bsed cytology, it is much more discreet nd hngs like wllpper on the surfce of the cells nd cell structures (Tb. 9).The squmous epithelil crcinom should rther be recognized due to its chrcteristic morphology nd not due to such debris (Tb. 10, Fig. 7-d). Glndulr neoplsis lso present chrcteristic picture in thin-film cytology (Tb. 11, Fig. 8-d) nd re esily recognized. 170 b 50 μm Figure 6: Comprison of Pp-Smer/ThinPrep : The dignosis of Pp IIID is more difficult using conventionl preprtions due to the poor preservtion of the cells nd the impure bckground of the preprtion b: Monitoring of findings fter three months with the ThinPrep method: clener bckground, well-preserved cells, simple ccess to dysplstic cells In intermedite nd severe dysplsi (HSIL high-grde squmous intrepithelil lesions), isolted dysplstic cells with smller nuclei nd cells with greter nucler-cytoplsmic reltion re found more frequently, without the formtion of so-clled streets (Tb. 8). Tble 8: Morphology of high-grde lesions (HSIL) in liquid-bsed cytology Incresed number of isolted dysplstic cells Smller cells with decresed nucleus re more frequently found Cells with greter nucler-cytoplsmic reltion re frequently found Tble 9: Discreet tumor dithesis in crcinoms using liquid-bsed cytology Dithesis consisting of blood, fine grnules of fibrin nd necrotic debris is lmost lwys found, but is discreet Dithesis hngs like wllpper on the surfce of the cells nd cell structures ( clinging dithesis ) Tble 10: Morphology of the squmous epithelil crcinom shown using liquid-bsed cytology Cornified type Noncornified type There is no difference Tumor cell structures re more threedimensionlly lyered in kertinized cells in comprison to conventionl prepr- nd denser Cytoplsm is more strongly contrcted tions The nucleus ppers smller Chromtin is distributed more evenly (cells pper less mlignnt ) Nucleoli re more prominent At lower mgnifiction, tumor cell structures my be mistken for benign squmous epithelil metplsi Mlignnt nucler chrcteristics re preserved (pleomorphism in size nd shpe plus the irregulr contours of the nucleus) Bollmnn R. Liquid-bsed cytology... Gynkol Geburtsmed Gynkol Endokrinol 2008;4(2): published kdemos Wissenschftsverlg 2008 ISSN

8 Tble 11: Morphology of glndulr neoplsis using liquid-bsed cytology Incresed number of isolted tumor cells Glndulr cell structures re thicker (three-dimensionl), side contours re more difficult to recognize Smller nuclei Chromtin is evenly distributed (cells pper less mlignnt ). Nucleoli re more prominent Mlignnt chrcteristics of the nucleus re preserved (pleomorphism in size nd shpe plus irregulr contours of the nucleus) 90 μm 90 μm 40 μm 40 μm b c d Figure 7: Squmous epithelil crcinom -b: Three-dimensionl cell structures of squmous epithelil crcinom with severely typicl nuclei nd fine grnulr deposits on the mrgins (wllpper-like tumor dithesis) c-d: Individully isolted cells of squmous epithelil crcinom with dignosticlly chrcteristic typicl nuclei nd ccumultions of fine grnulr deposits (discreet tumor dithesis) 40 μm 40 μm 90 μm 40 μm b c d xx Figure 8 -d: Cell structures of vrying size in n denocrcinom of the uterine cervix; glndulr deposition nd conspicuous typicl nuclei re esy to detect Cytologicl detils re well preserved in liquid-bsed cytology, for instnce enbling the cler depiction of the nucler chromtin.this hs led to renissnce of the non- clssicl HPV signs, which hd lredy been described mny decdes go (Schneider et l. 1987).

9 Y Y Y 10 μm 10 μm c 10 μm b Y 10 μm 10 μm f 10 μm 172 d e Figure 9: Non-clssicl HPV signs : Abortive koilocytosis nd micronucleus b: Kertohylin-like grnulr cells; spindle-like nucleus with longitudinl fold c: Fissure-like folds in the cytoplsm nd protrusion of the ectoplsm; two ppendges of the nucleus (protrusion? development of micronuclei?) d: Mesle cells with centrl condenstion of the grnulr cells e: Cells with double nuclei f: Prkertosis Cytomorphology of HPV infection using liquid-bsed cytology An infection with the humn ppillom virus (HPV) dmges the cell s cytoskeleton. A distinct cytoplsmic hlo develops when the cytoskeleton collpses. This so-clled koilocytosis (Greek: koilos = empty) is generlly regrded s clssicl HPV sign. There re, however, lso discreet ( non-clssicl ) HPV signs, which re prticulrly strightforwrd to recognize in LBC preprtions: bortive koilocytosis, mild dyskertosis, prkertosis, mild nucler hyperchromsi, pointed nuclei, grooved nuclei, multinucler cells, mesle cells, kertohylin-like grnule cells, mcrocytes nd condensted cytoplsmic filments (Fig. 9-f). These secondry HPV signs hve negtive predictive vlue of 100 %. If they re missing, it is highly probble tht the womn is HPV negtive (Bollmnn et l. 2005). Bsed on our results, we re convinced tht if these indictions re rigorously exmined, the cytologicl sensitivity increses. We lso believe tht the good results chieved when using liquid-bsed cytology re due to the improved preservtion of such fine cytologicl detils. The ssessment of these criteri combined with the recommended type-specific HPV PCR nlysis is n integrl prt of the Bonn Model of cncer prevention (Bollmnn et l. 2007). Liquid-bsed cytology s pltform for the moleculr Pp possibility for sving helth cre costs? Aprt from possible flse-negtive cytologies, nother difficulty re the flse-positive cytologicl findings, i. e. the low positive predictive vlue of cytology (ppv). The literture on the ppv of typicl cytologies shows results rnging from 11.4 % (Petry et l. 2003; Ronco et l. 2006) to 70.6 % (Schneider 2000), with respect to the histologicl dignosis CIN II. In Germny, the follow-up costs to clrify findings nd to tret nonspecific nd bnorml findings found in cervix cytology mount to 55 million euro (Schneider et l. 2007). A method tht increses the ppv could therefore signify enormous svings: An increse in the ppv of just 1 % in cncer prevention cytology would spre women from unnecessry tretment nd sve helth cre costs (Bk et l. 2005). One of the min mesures tken to improve the ppv is HPV trige of typicl cytologicl findings. In our mteril we chieve ppv of 82 % (Bollmnn et l. 2005).The costeffectiveness of HPV trige ws estblished in decisionnlyticl model (Sheriff et l. 2007). A further improvement in ppv is possible if HPV trige is combined with sttic DNA cytometry. High-Risk HPV infection my led to disruptions in the cell cycle nd to chromosoml neuploidy. Bollmnn R. Liquid-bsed cytology... Gynkol Geburtsmed Gynkol Endokrinol 2008;4(2): published kdemos Wissenschftsverlg 2008 ISSN

10 Aneuploidy is tody regrded s progression mrker nd cn be detected by fluorescent in situ hybridiztion (FISH) (Mehes et l. 2004) or by sttic DNA cytometry in routine dignostics (Grote et l. 2004). The results of DNA cytometric tests support the dul Bethsed clssifiction: low-grde intrepithelil lesions (LSIL) re DNA-diploid, high-grde intrepithelil lesions (HSIL) re DNA-neuploid (Bollmnn et l. 2001). Liquid-bsed cytology therefore provides n optiml pltform for risk-dpted multimodl gynecologicl cncer prevention with high ppv. By simultneous HPV genotypiztion together with DNA imge cytometry, n 88.2 % positive predictive vlue (ppv) for intermedite nd highergrde cervicl dysplsis nd crcinom ( CIN II) cn be chieved (Bollmnn et l.2007). In current literture, no method hs reches higher ppv. This multimodl riskdpted cervicl screening voids unnecessry surgery nd sves costs. In ddition, other moleculr methods cn be crried out with the liquid medium: determintion of oncogenic HPV-E6/E7-mRNA (Vrni et l 2008), p16 ELISA test (Mo et l. 2007), or the simultneous determintion of HPV infection nd 3q26 nd 8q24 neusomi by FISH (Sokolov et l. 2007). Cytologicl correltion (cell block technique) Liquid-bsed cytology llows the production of histologicl preprtions using the so-clled cell block technique (Gupt et l. 2007) (Fig. 10). Liquid-bsed cytologies contin regulr mini-biopsies (Yeoh u. Chn 1999), on which good immunohistochemicl tests cn be performed. Vlidity A pilot study crried out in Englnd by the Deprtment of Helth on the introduction of liquid-bsed cytology showed tht the number of unsuitble preprtions decresed from 9.1 % (conventionl) to 1.6 % (liquid-bsed cytology) (Moss et l. 2002). In ddition, screening hd incresed by 9 %. These results were sufficient resons for including liquid-bsed cytology into Englnd s cncer prevention progrm, even if the results of the pilot study could not prove the superior sensitivity of liquid-bsed cytology when compred to the conventionl smer technique. In the mentime, officil reports (lso from Scotlnd) hve confirmed n increse in the detection rte nd higher ppv (deprtment of helth) (Willims 2006). The FDA hs estblished the following licensing guidelines for recognized methods: For the ThinPrep Test (Hologic): Licensed s n lterntive to the conventionl smer test (1996) Approved s significntly more effective method in the detection of low- nd high-grde lesions in comprison to conventionl techniques (1996) Approved becuse the smple qulity is significntly better in comprison to conventionl preprtions (1996) Approved s being significntly more effective (+59.7 %) in detecting high-grde lesions in comprison to conventionl preprtions (2003) Additionl tests for HPV re pproved using the Preserve- Cyt solution with the Hybrid-Cpture method (2002) The PreserveCyt solution is pproved for PCR screening for chlmydi (2003) Approved due to n incresed rte of detection in bnorml glndulr cells (2005) ThinPrep Imging-System: pproved s computerssisted system for primry screening for cervicl crcinom on ThinPrep slides (2003) 40 μm Figure 10: Histologicl preprtion produced from cell block from the mini biopsy left in the remining solution fter production of the cytologicl slide For the SurePth Test: Approved s n lterntive to the conventionl Pp test (1999) Approved s significntly more effective method (+64.4 %) for detecting high-grde lesions in comprison to the conventionl smer (2003) Focl Point System: pproved for primry screening of cytologicl preprtions. Focl Point identifies 25 % of the successfully screened preprtions s being, with high probbility, norml, thus requiring No Further Review. Licensed for conventionl nd thin-film preprtions (1998, respectively 2001). At present, there is no comprble licensing for PpSpin or other thin-film techniques. 173

11 174 A met-nlysis recently published in the Lncet concludes tht liquid-bsed cytology is no better thn conventionl cytology. The number of unsuitble cytologicl preprtions ws neither lower, nor ws the detection rte of highgrde lesions higher (Dvey et l. 2006). In n investigtion crried out in Medline, the uthors of the met-nlysis found 145 publictions on the vlidity of liquid-bsed cytology. Of these publictions, 116 (80 %!) were discrded, s they did not fulfill the inclusion criteri postulted by the uthors of the met-nlysis. 27 other studies, which were not found in Medline, were dded to the remining 29 studies. Of the remining 56 studies, none were found to be idel, 5 were judged to be of high qulity, 32 intermedite qulity nd 19 were poor qulity. The met-nlysis crried out by Dvey nd coworkers ws bsed on the evlution of 1.25 million slides. In conclusion, the met-nlysis does not support the proposition tht liquid-bsed cytology produce better results nd suggests tht lrge rndomized studies will hve to be crried out. When compring the results of the study by Dvey to those of the British study (Moss et l. 2002), we cn drw the conclusion tht the good English results re bsed on better trining progrm for screeners: liquid-bsed cytology requires experience nd the method cnnot be used without trining. The screener must be wre of the differences between the vrious methods. In Englnd, liquid-bsed cytology hs been vilble to ll women vi the Ntionl Helth Service since the end of In the USA, 98 % of the Pp tests re liquid-bsed (Institute of Mngement nd Administrtion 2007), s in the USA reflex HPV test is required in the cse of ASCUS ( typicl squmous cells of undetermined significnce ). This test is crried out with the preserved liquid. Therefore it is not necessry to recll the ptient in order to collect new smples. Incidentlly, the College of Americn Pthologists (CAP) hs discovered tht in the cytologicl exmintions prescribed by the stte ( proficiency progrm ), cytologists nd cytologicl ssistnts significntly more often detect lesions when using liquid-bsed cytology compred to conventionl preprtions (Renshw et l. 2004). It is regrettble tht liquid-bsed cytology hs led to schism mongst cytologists with regrd to the evlution nd use of the method nd tht the study by Dvey hs been greeted with such vehemence by the prtisns of the ntiliquid legue (Leimn 2007) (Obwegeser nd Schneider 2006). The rigid stnce of the Germn Society for Cytology is eqully incomprehensible; the Society s sttement on liquid-bsed cytology, issued in 2003, hs still not been revised, even if members of the bord of this scientific society, such s the hed of the bord of directors, regulrly mke use of the method (Byer-Pietsch nd Flenker 2004). Above ll, the DGZ considers the high costs of the tests nd the finncing of studies by the mnufcturers s reson for rejecting the method. To be consistent, the DGZ should lso reject HPV vccintion, s it is finnced by the industry nd the vccintion is expensive. In contrst, the Germn HTA study (HTA: Helth Technology Assessment; Siebert et l. 2003) cme to the conclusion tht the use of new screening technologies (thin-film preprtion nd computer-ssisted evlution methods) re cceptble per yer of life sved, if screening tkes plce over period of three or more yers. Computer-ssisted screening Liquid-bsed cytology ws dvnced by the development of computer-ssisted cytology. Nowdys, there re techniclly developed systems tht cn be used in everydy dignostics. Becton-Dickinson Imging ( subsidiry of SurePth) hs developed the Focl Point nd Focl Point GS System, which is now distributed by Becton-Dickinson. Besides preprtions produced by the SurePth technique, the system lso scns conventionl preprtions. A good smer technique is prerequisite, s the computer is not ble to red suboptiml cytologicl smer preprtions. In order to produce optiml preprtions for computerssisted systems, fom-bsed smpling devices cn be used (e. g. Cervisoft, PpCone ) (Bollmnn et l. 2002). The system identifies bnorml cells using n dptive morphometric lgorithm nd sorts them ccording to the probbility of being bnorml. 25 % of the preprtions re clssified s no review nd cn be issued s negtive result in the USA.The procedure is licensed by the FDA. However, in Germny, supplementry informtion hs got to be documented, such s the degree of prolifertion nd microbiology. The preprtions must therefore be evluted by cytologicl ssistnts. In Germny, the system is used together with the Focl Point GS. Pictures of typicl cells re tken nd their coordintes re stored.with the id of motorized microscope tble, the 15 most bnorml cells re presented t the microscope for evlution before the whole of the slide is conventionlly screened. Focl Point GS hs not yet been licensed by the FDA. Bollmnn R. Liquid-bsed cytology... Gynkol Geburtsmed Gynkol Endokrinol 2008;4(2): published kdemos Wissenschftsverlg 2008 ISSN

12 The coordintes of 22 cells with bnorml densitometric vlues re stored nd fter scnning, re presented on the microscope. Both methods re significntly more sensitive thn mnul screening, or t lest s sensitive (Dvey et l. 2007;Wilbur et l. 1998) (Biscotti et l. 2005; Ronco et l. 2003). Slight koilocytic dysplsis my be overlooked by TIS (Zhng et l. 2007). It is possible tht these cells do not differ densometriclly from norml cells nd re therefore not recognized by the system. In this regrd, it should be mentioned tht the percentge of invsive cervicl crcinom tht re DNA-diploid is not low (Horn et l. 2002). Incresed productivity An English pilot study showed 9 % increse in screening when using liquid-bsed cytology preprtions (Moss et l. 2002). This percentge is even higher when combined with computer-ssisted methods (Wilbur et l. 1998; Dvey et l. 2007). Technicl problems ssocited with the processing of liquid-bsed cytology preprtions Liquids contining lot of mucous or blood cn block the pores of the cell filter in the ThinPrep method (Bentz et l. 2002), producing preprtions tht re low in cell content nd pper moth-eten (Figure 11, b). In such cses, second preprtion must be processed fter mucolysis or erythrolysis. The possibility of cell crryover is n dditionl problem with the ThinPrep technique, if the preprtion is not crefully processed. The cylinder with the cell filter nturlly crries cells on its outer wlls tht were immersed in the preprtion liquid. The used cylinder is unscrewed fter preprtion of the smple nd is replced by nother filter. It must be ensured tht no cells re trnsferred from the used to the new cylinder by using disposble gloves. In the SurePth method, cells my be lost (FDA). Costs of liquid-bsed cytology nd billing modes Thin-film preprtion costs euro while conventionl cytology only costs euro (the ltter cost compres with n wrd of only 165 points for the EBM code number 01733!) (EBM = the so-clled Unique Evlution Tble, Einheitlicher Bewertungsmßstb) (Siebert et l. 2003). Following ruling of the Germn Medicl Assocition, liquid-bsed cytology is billed ccording to 6 GOÄ (Gebührenordnung für Ärzte medicl fee schedule) nlogous with code number Fees cn lso be chrged ccording to 10 GOÄ. In the new drft of the GOÄ, one code number, which ws not previously included, hs been dded for smple extrction μm b Figure 11: Problems ssocited with processing in the ThinPrep technique : Cell-free, moth-eten fields on the stined slide b: Microscopic preprtion of filter of such cse shows the obstruction of the comm-shped filter pores by erythrocytes (round components) nd mucous (strongly refrctile tble-like structures) Ptients covered by the sttutory helth insurnce cn only be chrged under the IGeL system (privtely pid helth services). In ny cse, it seems pproprite to use code number 1 GOÄ for consultncy fees. Mny gynecologists

13 nd cytologists re of the opinion tht the IGeL monolyer test should lwys simultneously include conventionl smer, s otherwise the requirements of the services outlined in code number EBM re not fulfilled. Aprt from the fct tht the purpose of liquid-bsed tests is thwrted by such procedure, the bove-mentioned interprettion is, in the uthor s opinion, incorrect. The service legend for EBM code 01733, used for billing cytologicl exmintions ccording to Section B.2 of the cncer prevention guidelines, stipultes the obligtory content s cytologicl exmintion of one or more smers, lso brush smers, of the ecto- nd/or endocervix. Mny gynecologists nd cytologists utomticlly equte the expression smer with the ppliction of the smple mteril to the slide. The uthor cnnot follow this line of rgumenttion: smer is the removl of bodily mteril from the surfce of wound or mucous membrne (mouth, urethr, vgin, nus) using sterile swb, smll brushes, or smll sptul for the purpose of exmintion. CME Prkt Fortbild Gynkol Geburtsmed Gynkol Endokrinol 2008; 4(2): Keywords Liquid cytology, Pp smer, cell block, DNA cytometry, moleculr pp The cncer prevention guidelines (lst ltered on ) stte: Cytologicl exmintion includes the evlution of mteril removed for the purpose of cytologicl exmintion. How to preserve the mteril is not prescribed. Consequently, trnsferring the smple mteril to liquid medium s n lterntive to the regulr processing of slide-bsed preprtions is not excluded. The ltest resolution of the Federl Committee of the lso does not explicitly forbid the use of liquid-bsed cytology: In its meeting on the , the Federl Committee decided tht thin-film cytology nd HPV test methods will not be implemented s cncer prevention screening test for cervicl crcinom. The expression will not be employed does not indicte tht thin-film cytology is not n pproved method; in fct, the committee simply decided not to implement the method t present. It is not expressly forbidden to bill liquidbsed cytologicl exmintions under the code for preventive exmintions. Only the preprtion process hs got to be billed s privtely pid service (IGeL). 176 Bollmnn R. Liquid-bsed cytology... Gynkol Geburtsmed Gynkol Endokrinol 2008;4(2): published kdemos Wissenschftsverlg 2008 ISSN

14 References Bk J, Kruse A, Jnssen E, vn Diermen B. Prediktive testing of erly CIN behviour by moleculr biomrkers. Cell Oncol 2005; 27: Byer-Pietsch E, Flenker H. Gynäkologische Zytologie mit der PpSpin-Technik. Eine Alterntive zum konventionellen Abstrich nch Ppnicolou? Fruenrzt 2004; 45: Bentz JS, Rowe LR, Gopez EV, Mrshll CJ. The unstisfctory ThinPrep Pp Test: missed opportunity for disese detection? Am J Clin Pthol 2002; 117: Bicotti CV, Dwson AE, Dziur B, Glup L, Drrgh T, Rhemtull A, Wills-Frnk L. Assisted primry screening using the utomted ThinPrep Imging System. Am J Clin Pthol 2005; 123: Bollmnn M, Bnkflvi A, Trosic A, Speich N, Schmitt C, Bollmnn R. Cn we detect cervicl humn ppillomvirus (HPV) infection by cytomorphology lone? Dignostic vlue of non-clssic cytologicl signs of HPV effect in minimlly bnorml Pp test. Cytopthology 2005; 16: Bollmnn R, Bollmnn M, Henson DE, Bodo M. DNA Cytometry confirms the Utility of the Bethesd System for the Clssifiction of Ppnicolou Smers. Cncer Cytopthol 2001; 93(3): Bollmnn R, Jordn B. Technische Vorussetzungen einer vlidierten Flüssigkeitszytologie. Reicht die einfche Zytozentrifugtion? Fruenrzt 2005; 46: Bollmnn R, Tork R, Schmitz J, Bollmnn M, Mehes G. Determintion of Ploidy nd Steroid Receptor Sttus in Brest Cncer by Lser Scnning Cytometry. Cytometry 2002; 50: Bollmnn R, Várni AD, Bánkflvi A, Bollmnn M. Risikodptierte multimodle gynäkozytologische Krebsvorsorge PpTest der Zukunft? Pthologe 2007; 5: Bollmnn M, Várni AD, Griefingholt H, Bánkflvi A, Cllenberg H, Speich N, Schmitt C, Bollmnn R. Predicting tretment outcome in cervicl diseses using liquidbsed cytology, dynmic HPV genotyping nd DNA cytometry. Anticncer Res 2006; 26(2B): Bollmnn R, Bánkflvi A, Griefingholt H, Trosic A, Speich N, Schmitt C, Bollmnn M. Vlidity of combined cytology nd humn ppillomvirus (HPV) genotyping with djuvnt DNA-cytometry in routine cervicl screening: Results from women from the Bonn-region in West Germny. Oncol Rep 2005; 13: Cervicl Screening, Wles. Sttisticl Report Crdiff: Cervicl Screening Wles KC 2006; 53/61/65. Dvey E, Brrtt A, Irwig, Chn SF, Mcskill P, Mnnes P, Sville AM. Effect of study design nd qulity on unstisfctory rtes, cytology clssifictions, nd ccurcy in liquid-bsed versus conventionl cervicl cytology: A systemtic review. Lncet 2006; 367: Dvey E, d'assunco J, Irwig L, Mcskill P, Chn SF, Richrds A, Frnsworth A. The Accurcy of Cervicl Cytology: A comprison between the ThinPrep Imging System nd conventionl Methods. BMJ 2007; 31: 335. Epub 2007 Jun 29. Dvey E, Irwig L, Mcsill P, D'Assunco J, Richrds A, Frnsworth A. Accurcy of reding liquid bsed cytology slides using the ThinPrep Imger compred with conventionl cytology: prospective study. BMJ 2007; 335: Deprtment of Helth. Cervicl Screening Progrmme. London: Sttisticl Bulletin 2003/24; October Gupt S, Hlder K, Khn VA, Sodhni. Cell block s n djunt to conventionl Ppnicolou smer or dignosis of cervicl cncer in resource-limited settings. Cytopthology 2007; 18: Horn LC, Rptis G, Nenning H. DNA Cytometric Anlysis of Surgiclly Treted Squmous Cell Cncer of the Uterine Cervix, Stge pt1b1-pt2b. Anl Qunt Cytol Histol 2002; 24(1): Hutchinson ML, Isenstein LM, Goodmn A, Hurley AA, Douglss KL, Mui KK, Ptten FW, Zhniser DJ. Homogenous smpling ccounts for the incresed dignostic ccurcy using the ThinPrep Processor. Am J Clin Pthol 1994; 101(2): Institute of Mngement nd Adminstrtion. Dignostic testing nd technology report: ntomic pthology nd cytology: continued growth driven by Pp testing. Wshington: G2 Reports Jnury Leimn G. Liquid-Bsed Cytology: Under Scrutiny Down- Under. Dign Cytopthology 2007; 35(7): Mo C, Blsubrmin A, Yu M, Kivit N, Ridder R, Reichert A, Herkert M, von Knebel Doeberitz M, Koutsky LA. Evlution of new p16(ink4a) ELISA test nd high-risk HPV DNA test for cervicl cncer screening: results from proof-of-concept study. Int J Cncer 2007; 120(11): Mrqurdt K, Büttner HH, Broschewitz U, Brten M. Die Restinzidenz des Zervixkrzinoms in Deutschlnd. Fruenrzt 2005; 45: McQueen F, Duvll E. Using qulity control pproch to define n «dequtely cellulr«liquid bsed cervicl cytology specimen. Cytopthology 2006; 17: Mehes G, Speich N, Bollmnn M, Bollmnn R. Chromosoml Aberrtion Accumulte in Polyploid cells of High-grde Squmous Intrepithelil Lesions (HSIL). Pthol Oncol Res 2004; 10(3): Moss S, Gry A, Legood R, Henstock E. Evlution of HPV/LBC cervicl screening pilot studies. First report to the Deprtment of Helth on evlution of LBC. London: Deprtment of Helth Nnd K, McCory DC, Myers ER, Bstin LA, Hsselbld V, Hickey JD. Accurcy of the Ppnicolou test in screening for nd follow-up of cervicl cytologic brnormlities: systemtic review. Ann Intern Med 2000; 6(132(10)): NHS CSP publiction. Externl Qulity Assessment Scheme for the Evlution of Ppnicolou Stining in Cervicl Cytology. London: Deprtment of Helth Obwegeser J, Schneider V. Thin-lyer cervicl technology: A new met-nlysis. Lncet 2006; 367: Ppnicolou GN, Trut HF. Dignosis of uterine cncer by the vginl smer. New York: Commonwelth Fund

15 178 Petry KU, Menton S, Loenen-Frosch F, de Crvlho GH, Holz B, Schopp B, Grbrecht-Buettner S, Dvies P, Boehmer G, vn den Akker E, Iftner T. Inclusion of HPV testing in routine cervicl cncer screening for women bove 29 yers in Germny: results for 8466 ptients. Br J Cncer 2003; 88: Renshw AA, Young N, Birdsong GG, Styer PE, Dvey DD, Mody DR, Colgn TJ. Comprison of performnce of conventionl ThinPrep gynecologic preprtions in the College of Americn Pthologists Gynecologic Cytology Progrm. Arch Pthol Lb Med 2004; 128: Ronco G, Segnn N, Giorgi-Rossi P, Zpp M, Csdei GP, Crozzi F, Plm PD, Del Mistro A, Folicldi S, Gillio-Tos A, Nrdo G, Nldoni C, Schincgli P, Zorzi M, Confortini M, Cuszick J. Humn Ppillomvirus Testing nd Liquid-Bsed Cytology: Results t Recruitment From the New Technologies for Cervicl Cncer Rndomized Controlled Tril. J Ntl Cncer Inst 2006; 98: Ronco G, Cuzick J, Pierotti P, Criggi MP, Dll Plm P, Nldoni C, Ghiringhello B, Giorgi-Rossi P, Minucci D, Prisio F, Pojer A, Schiboni ML, Sintoni C, Zorzi M, Segnn N, Confortini M. Accurcy of liquid bsed cytology versus conventionl cytology; overll results of the new technologies for cervicl screening (NTCC) rndomised controlled tril. BMJ 2007; 335: Ronco G, Vineis C, Montnri G, Orlssino R, Prisio F, Arnud S, Berrdengo E, Fbbrini T, Segnn N. Impct of the AutoPp (currently Focl Point) Primry Screening System Loction Guide Use on Interprettion Time nd Dignostic. Cncer 2003; 99: Rosenthl DL, Geddes S, Trimble CL, Crson KA, Alli PM. The PpSpin: A Resonble Alterntive to Other, More Expensive Liquid-Bsed Ppnicolou Tests. Cncer Cytopthology 2006; 108(3): Schenk U, Schwrz G. Probleme der Präprtionstechnik. Micro Act Suppl 1983; 6: Schneider A, Hoyer H, Lotz B, Leistritz S, Kühne-Heid R, Nindl I, Müller B, Herting J, Dürst M. Screening for high-grde cervicl intr-epithelil neoplsi nd cncer by testing for high-risk HPV, routine cytology or colposcopy. Int J Cncer 2000; 89: Schneider A, Meinhrdt G, Villiers EM de, Gissmnn L. Sensitivity of the cytologic dignosis of cervicl condylom in comprison with HPV DNA hybridiztion studies. Dign Cytopthol 1987; 3: Schneider A, Schwrz TF, Hmmerschmidt T, Rsh RM, Siebert U. Vorgehen und Kosten bei der Abklärung und Behndlung unklrer und bnormler zytologischer Befunde des Pp-Abstrichs im Rhmen der Krebsfrüherkennungsuntersuchungen. Geburtsh Fruenheilk 2007; 67: Sheriff SK, Petry KU, Ikenberg H, Crouse G, Mzonson PD, Snts CC. An economic nlysis of humn ppillomvirus trige for the mngement of women with typicl nd bnorml Pp smer results in Germny. Eur J Helth Econ 2007; 8(2): Epub 2007 Feb 17. Siebert U, Muth C, Sroczynski G, Velsco-Grrido M, Gerhrdus A, Gibis B. Dünnschichtpräprtionen und computergestützte Untersuchungen von Zervixbstrichen im Rhmen der Krebsfrüherkennung. Snkt Augustin: Asgrd-Verlg 2003; 35. Sokolov I, Algecirs-Schimnich A, Song M, Sitilo S, Policht F, Kipp BR, Voss JS, Hlling KC, Ruth A, King W, Underwood D, Brinrd J, Morrison L. Chromosoml biomrkers for detection of humn ppillomvirus ssocited genomic instbility in epithelil cells of cervicl cytology specimens. J Mol Dign 2007; 9 (5): Solomon D, Nyr R. The Bethesd System for Reporting Cervicl Cytology. Definitions, Criteri nd Explntory Notes. 2 nd ed. Heidelberg: Springer Tsc L, Östör AG, Bbes V. History of Gynecologic Pthology. Int J Gynecol Pthol 2002; 21: Várni AD, Bollmnn M, Bnkflvi A, Speich N, Schmitt C, Griefingholt H, Kovács K, Klozoris C, Bollmnn R. Predictive testing of erly cervicl pre-cncer by detecting humn ppillomvirus E6/E7 mrna in cervicl cytologies up to high-grde squmous intrepithelil lesions: Dignostic nd Prognostic implictions. Oncol Rep 2008; 19 (2): Várni AD, Bollmnn M, Bánkflvi A, Griefingholt H, Pfening N, Schmitt C, Pjor L, Bollmnn R. The spectrum of cervicl diseses induced by low-risk nd undefined-risk HPVS: impliction for ptient mngement. Anticncer Res 2007; 27 (1B): Wilbur D, Prey M, Miller W, Pwlick G, Colgn T. The AutoPp System for Primry Screening in Cervil Cytology. Compring the Results of Prospective, Intended-Use Study with Routine Mnul Prctice. Act Cytol 1998; 42: Willims A. Liquid bsed cytology nd conventionl smers compred over two 12 month periods. Cytopthology 2006; 17: Yeoh GPS, Chn KW. Cell block preprtion on residul ThinPrep smple. Dign Cytopthol 1999; 21: Zhng FF, Bnks HW, Lngford SM, Dvey DD. Accurcy of ThinPrep Imging System in Detecting Low-Grde Squmous Intrepithelil Lesions. Arch Pthol Lb Med 2007; 131: Bollmnn R. Liquid-bsed cytology... Gynkol Geburtsmed Gynkol Endokrinol 2008;4(2): published kdemos Wissenschftsverlg 2008 ISSN

16 Prof. Dr. med. Reinhrd Bollmnn, MIAC Institute of Pthology Bonn-Duisdorf Heilsbchstrße Bonn Dr. Reinhrd Bollmnn studied in Bonn nd completed his doctorl studies in Freiburg. While working s n ssistnt physicin t the Institute for Pthology t Freiburg University, Dr. Bollmnn ws wrded DFG scholrship to study in London (Deprtment of Histochemistry, Hmmersmith Hospitl). For seven yers he worked s senior physicin in cliniclly oriented hospitl pthology. In 1980, Dr. Bollmnn founded privte institute for pthology tht focuses on the field of cytoptholgy. He is founder of the Leistungserbringungsgemeinschft für Molekulrpthologie (GenOPth GbR compny providing ser vices in the field of moleculr pthology), in which 11 pthologicl institutes re members. Dr. Bollmnn is member of the bord of the Arbeitsgemeinschft zytologisch tätiger Ärzte Deutschlnd (AZÄD work group of Germn physicins ctive in the field of cytology) nd the Berufsverbndes Deutscher Pthologen e.v. (professionl ssocition of Germn pthologists). At present, Dr. Bollmnn is hed of the cytologicl commission of the Kssenärtzliche Vereinigung Nordrhein (Assocition of Physicins North Rhein). Conflict of interest The uthor declres tht this rticle ws written without the influence of ny industril interests. Dr. Bollmnn lso declres tht he hs received consulting fee from Cytc. However, the pyment of this fee hs in no wy influenced the content of this rticle. Mnuscript dt Submitted on: Accepted on: