PLENARY LECTURE

36. INTRAEPITHELIAL LESIONS OF UTERINE CERVIX

Silvana Audy-Jurković

Clinics of Gynecology and Obstetrics KBC and

Medical Faculty in Zagreb

Since 1993, in Croatia the classification of cytological uterine cervix findings, "Zagreb 1990" (Audy, Singer, Pajtler, Dražančić, Grizelj)  has been applied which presents the modification of "The Bethesda System 1988" (TBS). The key distinctivness relates to medium heavy dysplasia in which, in the "Zagreb 1990", a clinician is left the possibility of conservative or active approach.

According to the assessment of diagnostical accuracy of differential cytological diagnosis of squamous intrepithelial lesions (SIL), the CIN (cervical intraepithelial neoplasia) III is a higly reliable (88%) diagnosis, and in glandular intraepithelial lesions (GIL) the correspondence with  histological diagnosis is higher for the lesion intensity (86%) than for the  epithelal type (55% in glandular lesions and 44% in glandulo-squamous ones). In the defferential cytodiagnostics,  uterine cervical lesions ASCUS and AGCUS (atypic squamous and glandular cells of undetermined significance), the  group introduced by TBS,  present a particular problem because of unprecise criteria, low reproducibility and frequently too high prevalences, as well as high percentage of unidentified intraepithelial, but also invasive lesions. That problem is present in Croatia as well, but we should also add  distinctive understanding  of the groups ASCUS, AGCUS (TBS) and Abnormal cell of undetermined significance ("Zagreb 1990"), which is necessary to  equalize.

In the further diagnostical procedure, a colposcopic examination is common, and in  cases of adenocarcinoma in situ also the classical conization. Lately, the HPV DNK typing has most frequently been  recommended as an addition to  cytological findings of CIN I,II, ASCUS, and AGCUS.

Nowadays,  excisive and destructive methods are mainly applied in the treatment, and immunotherapy application is tested. However,  in the next century, the prophylactic serum is the mostly expected from.

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37. PREVENTION OF CERVICAL CANCER

BY ORGANIZED SCREENING IN FINLAND

^1,2 Kari J. Syrjanen, M.D.,Ph.D, FIAC

¹ MediCity Research Laboratory, Faculty of Medicine,

University of Turku, Finland;   ² International Cytopathology

Services, ICS Ltd, UK.

The organized national mass-screening for cervical cancer in Finland has been implemented since the early 1960s, when this disease in our country was the third most frequent malignancy in women (with the incidence of 16/105). The Finnish programme is a collaboration between the National Board of Health, municipal health authorities, and the Finnish Cancer Society, fulfilling the UICC definition for an organized screening programme. The programme is based on routine PAP smears taken according to standard procedures. The slides are screened by well trained cytotechnologists (majority of whom with the IAC certificate), and the abnormal findings are controlled by cytopathologists. Age groups from 30 to 60 years are screened at 5-year intervals.

In the country with a population of 5 million, around 150.000 women are smeared every year as a part of this organized screening programme. The nation-wide attendance rate has been constantly around 75% of the women invited. Reliable age-adjusted incidence and mortality rates of cervical cancer (and carcinoma in situ CIS) have been available in the Finnish Cancer Registry since 1953. The reduction in the incidence of invasive cervical cancer due to the screening has been about 60-70% being most pronounced (80%) in women aged 35-50 years. Parallel changes have been observed in cancer mortality as well. By the early 1990s, the annual incidence of invasive cervical cancer in Finland reached the level of 2.7/105 women, being the lowest in the whole world. Since 1992, however, a minor increase in these figures has been observed, the reasons of which are no clear yet.

A few years ago, the Finnish Cancer Society made a comprehensive approach to evaluate the quality of this mass-screening programme, run by its 11 cytopathology laboratories (Quality Assurance Project). The targets were the 148,147 annual PAP smears taken during the normal mass-screening programme, of which, 11.641 (7,9%) samples were subjected to rescreening. Out of the smears originally classified normal (Papanicolaou Class I), 19 cases were reclassified as Class II+and 8 cases into Class III. On histopathological confirmation, however, significant histology was found in few cases only, as follows; 6 cases of CIN I, and of CIN II, and 3 CIN III lesions. This resulted in the incredibly law false negative rate of 0.12%. Not a single case of invasive cancer was detected. As a part of this programme, a number of other QA issues also rendered as discussed in detail. According to the Finnish experience, improving the attendance, the quality of  smear and the validity of cytological diagnosis are likely to cause a reduction in the risk of cervical cancer more than increasing the frequency of screening and by starting the screening at very young ages. Clearly, cervical cancer ia a preventable disease, and the only effective means to do accomplish this is based on an organized screening programme with a high coverage.

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38. CYTOLOGY OF SEXUALLY TRANSMITTED

DISEASE AGENTS IN ADOLESCENTS AND

YOUNG WOMEN IN THREE AGES

V. Mahovlić, S. Audy-Jurković, A.Ovanin-Rakić

Institute of Gynecological Cytology, Department of

Obstetrics and Gynecology, Medical School, University of Zagreb

Background: To determine the prevalence of cytologically identified agents of sexually transmitted diseases (STDs) in vaginal-cervical-endocervical (VCE) smears in the population of adolescents (14-19y) and young women (20-25y) during three periods.

Patients and methods: VCE smears were analysed in 20000, 6984 and 6089 adolescents and young women  during three periods (A,B,C) with regard to cytologically diagnosed agents of STDs. XZ test was applied to determine the differences among the groups, and statistical significance was assessed at P=0.05 level.

Results: The prevalence of cytologically diagnosed microorganisms increases with regard to the period studied (A-~B-.C), as for the whole group so too for adolescents and young women separately (P>0.05). This primarily refers to the incidence of HPV and yeast infections (P>0.05), while the prevalence of Gardnerella vaginalis and Trichomonas vaginalis (TV) shows a decreasing tendency (A~-BBC). HPV and TV are cytologically more frequently identified in adolescents than in young women of A-period (P<0.05), while in the later periods (B and C), STDs agents are equally present in both age groups (P>0.05), except for TV (C-period) which is also more frequently diagnosed cytologically in adolescents (P<0.05). The frequency of abnormal cytological findings increases with the period studid (A->B-~C) in adolescents, as well as in young women, with cytologically most frequent diagnosed HPV infection, particularly in C-period (P<0.05):

Conclusion: Sexually active adolescents should be by all means under gynecological surveillance, including the cervical screening programme on carcinoma and its precursors, the infection treatment and prevention of its further complications.

Cytopathology, 2000.; 5 (11): 461, (Abstract No. 138.)

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40. CYTOLOGICAL MONITORING AFTER

BIOPSIES AND CONIZATIONS

Škopljanac-Mačina L, Ovanin-Rakić A,  Audy-Jurković S,   Barišić A

Clinic of Gynecology and Obstetrics KBC and

Faculty of Medicine Zagreb

The aim of the work is to estimate the adequacy of monitored patients after biopsy and/or conization, and the monitoring results in relation to abnormal cytological findings before a surgical operation.

In 169 patients who were subjected to a biopsy and/or conization during 1996, we analysed  cytological observations retrospectively until the end of 1999. 

A biopsy was performed in 74 examinees, conization in 28, and  biopsy with subsequent conization in 67 of them.

During the cytological monitoring period we lost 37 (21,9%) examinees, and one or more cytological findings were detected in 132 (78,1%) examinees. They were classified according to the duration of monitoring: up to 6 months 43 (32,6%), from 6 months to 1 year 22 (16,7%) examinees, from 1 to 2 years 30 (22,7%), from 2 to 3 years also 30 (22,7%), and more than 3 years 7 (5,3%) examinees.

The monitoring results were assessed as negativisation or persistence of abnormal cytological findings. The criteria for negativisation were 3 negative cytological findings at a row, and for persistency   at least 1 abnormal cytological finding. We could assess the outcome in 84 (63,6%) examinees, and in the other 48 (36,4%) we found only 1 or 2 negative cytological findings at a row, which was not sufficient for the assessment of monitoring findings.

After the biopsy, 57 examinees were followed and assessed. Only 1 (1,8%) of them fulfilled the critera for negativisation, and the other 56 (98,2%) had persistent abnormal cytological finding. After the conization, 46 examinees were followed and assessed. Negativisaton was found in 31 (67,4%), and persistence in 15 (32,6%) examinees.

We consider the monitoring procedure after biopsy and/or conizaiton inadequate and needing  to be significantly improved. We also think the follow-up evidence of cytological monitoring should be improved by the electron data  processing and connecting of laboratories to the central data base.

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40. CYTOLOGICAL  MONITORING OF PATIENTS

AFTER DIATHERMIC CONIZATION (LETZ)

Vukosavić-Cimić B, Grubišić G, Pirkić A, Vignjević D, Matković-Bilin M

Clinic of  Gynecology and Obstetrics,

KB "Sestre milosrdnice", Zagreb

BACKGROUND AND AIM:  In the period between 1995-2000 in the Clinic Gynecology and Obstetrics of Clinical Hospital "Sestre milosrdnice", we citologycally monitored 159 patients out of  248 who had been submitted to the diathermic conization.

PATIENTS AND METHODS: The patients were classified in two groups according to the modern cytological cervical classification.  In the first group were the patients with normal cytological finding after the operation (N-130-81,80%), and in another the patients with abnormal finding (N-29-18,20%). In both groups, the affection of conus edges and top  with coagulational necrosis was examined followed by control cytological finding after the operation performed.

 RESULTS: In the group without cytological abnormalities after LETZ,the PHD was: Cerviatis 5 (3,80%), CIN I 16 (12,30%), CIN II 46 (35,30%), CIN III 62 (47,69%), MIC 1 (0,76%), and in the group with cytological abnormality: Cervicitis 1 (3,45%), CIN I 1 (3,45%), CIN II 9 (31,00%) and CIN III 18 (62,00%). Concerning the affection of the edges of diathermic conus with coagulation necrosis 43 patients (71,70%) out of 60 had valid cytological result after the operation, and in 17 (28,30%) it was abnormal. In the group of 43 women with normal cytological findings after the operation, the PHD was CIN I in one patient (2,32%), CIN II in 12 (27,90%), and CIN III in 30 (69,76%), while in the group of 17 women with abnormal cytological results, CIN I was found in 1 patient (5,80%), CIN II in 4 patients (23,50%), and CIN III in 12 (70,50%).

CONCLUSION: Based on the number of cytological controls and insights into the mentioned relations, we want to emphasize the importance of strict appreciation of preoperational processing, precise approach to operation under the colposcopic control, and right timing for the first cytological control after the operation. The diathermic conization is technically easy to perform, therefore, we have to avoid the risk of its non-critic application. This operation has its position and significance in the treatment of squamous intrepithelial lesions of high degree in young nulliparous and parturient women.

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41. FACTORS PREDICTING DISEASE OUTCOME

IN ADENOCARCINOMA OF   UTERINE CERVIX

Mojca Erzen, M.D.,Ph.D., MIAC

Dept. of Obstetrics and Gynecology, SIZE Diagnostic

Centre of Gynecological Cytopathology, Ljubljana, Slovenija

OBJECT OF  STUDY: Adenocarcinomas (ACs) and adenosquamous carcinomas (ASC) of the uterine cervix were analysed for the determinants of disease progression.

MATERIALS, METHODS: Series of 94 women with cervical ACs diagnosed at our Department during 1995-1999 and subsequently followed- up for a mean of 26,4 (SD 14,8) months were analysed for the clinical data, screening histories, colposcopy, as well as tumor histology, stage of the disease, treatment and disease outcome. Out of the 24 patiens, a total of 72 cytological smears collected upon screening, 1-6 years prior to diagnosis, were reexamined and analysed for the sampling-, screening- and interpretation errors. The mean age of the patients was 44,2 years (range 24-81), the peak incidence rate (32%) being in the age group of 30-41 years. For statistical calculations, univariate and multivariate analysis were made using SPSS.

RESULTS. Symptoms: vaginal bleeding and discharge were present in 37 patients (39,4%), whereas fifty-seven (60.6%) patients were completely asymptomatic. Screening history:Fifty (53.2°fo)   patients were regularly screened at 1-2 years intervals. In 6 cases, the PAP test was performed at 3-4 year intervals, while 8 patients had their last smear 5 to 10 years before the detection of AC. No screening history was available for 28 (29,8%) women. Interpretation errors were established in 17 (23,6%) and sampling errors in 6 (8,3%) of the 72 reviewed smears. Colposcopy: On clinical examination, 55 patients (58,5%) had visible lesions, including exophytic tumor, ulcer, ATZ, polyp or diffusely enlarged cervix. Importantly, no gross cervical lesions were visible in 28 (29,8%) women, 11 of them after conisation. FIGO stage: Adenocarcinoma in situ (AIS) was found in 9 (6,2%) cones, microinvasive AC, stage IA1 in 9 (8,6%), stage IA2 in 8 (22,2%), IB in 62 (56,8%), IIA in 5 (6,5%) and IIB in 1 of the cases. Treatment: conisation was the conclusive diagnostical mean and the only therapeutical procedure in 12 (12,8%) cases. Radical hysterectomy was performed in 63 cases (67%), total hysterectomy in 13 (13.8  10) and radiotherapy in 5 (5.3) patients. Follow-up data: were available from 72 patients, of whom the disease progressed in 4 (one died), whereas 68 patients are alive and well at the moment. Cox multivariate survival analysisdisclosed FIGO stage (p=0.001) and screening (p=0.03) as significant independent predictors of the overall survival (OS), whereas age, age groups, histological type, tumor grade, lymph node status and clinical symptoms were not significant predictors. Patient´s age, screening, stage, final therapy, and presence of concomitant squamous cell lesions associated with AC were significant prognostic indicators in univariate (Kaplan-Meier) analysis (p=0.01 - 0.001).

CONCLUSIONS. In the present series, statistically significant predictors of disease outcome emphasize the importance of early detection of cervical AC and its precursors by routine PAP smear. This can only be achieved by increasing the sensitivity for the PAP smear detection of abnormal glandular cells in asymptomatic women.

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42. DIFFERENTIAL LESION CYTOLOGY OF ENDOCERVICAL

CYLINDRICAL EPITHELIUM OF UTERINE CERVIX

Pajtler M, Milojković M

KB Osijek, Unit of Clinical Cytology

Aim: To determine clinical significance and pathohistological background of atypias of endocervical cylindrical uterine cervix epithelium identified by cytological screening of VCE smear. Methods: The number of patients with finding of "abnormal cylindrical cell of undetermined significance (AGUS) -  endocervical one and "adenocarcinoma -endocervix" (Bethesda 1988) is determined in relation to the number of VCE smears in the period between 1993 - 1999, as well as the results of control procedures. For the same period, in the patients with pathohistologically diagnosed in situ (AIS) or invasive endocervical adenocarcinoma, these detections were compared to cytological findings, and in the patients with the AIS, cytologic findings were revised.

Results: On 182180 examined VCE smears "abnormal cylindrical cells of undetermined significance - endocervical ones" were found in 152 (0,08%), and "endocervical adenocarcinoma" in 24 patients (o,013%). The mean age of the AGUS patients was 41,4, and the adenocarcinoma patients 54,8 years. Out of 152 patients with the AGUS, 76 (50%) did not have control procedures, 56 (36%) had only cytological results (in 48 it was negative). 13 (9%) had cytological and pathohistological results, and 7 (5%) had only pathohistological results. In the 20 AGUS patients pathohistologically  normal epithelium was found in 20%, and CIN in 40%, and only in 30% lesions of endocervical cylindrical epithelium (10% AIS and 20% adenocarcinoma).   In 22 out of 24 patients with endocervical adenocarcinoma, the pathohistological results  revealed  endocervical adenocarcinoma in 18 cases (82%), and in 1 (4,5%) AIS+CIS, in 2 cases (9%) squamous carcinoma and in 1 (4,5%) metastatic adenocarcinoma. During the analysed period in 12 patients the AIS was pathohistologically diagnosed, and in 30 invasive endocervical adenocarcinoma. Out of 12 patients with the AIS, cytological findings was in 3 cases (25%) endocervical adenocarcinoma, in 1 (8,3%) squamous carcinoma, in 6 (50%) CIN3, and in 2 (16,7) negative. After the revision, we found, in 6 cases, cytomorphological changes in accordance with  AIS, in 4 only CIN 3 was found, while 2 remained negative. Out of 30 invasive adenocarcinomas, the cytological finding was endocervical carcinoma in 23 cases (76,2%), and in 4 (13,1%) AGUS, in 2 (6,5%) minute cellular type of squamous carcinoma, and in 1 case (3,2%) only carcinoma.  Conclusion: The cytological finding of "abnormal cylindrical cell of undetermined significance - endocervical", as it has been applied here, has extremely low frequency and high rate of spontaneous regression. Pathohistologically speaking, they are considered more  intrapeithelial lesions, with the prevalence of squamous (40%) in relation to  cylindrical (10%) ones,   than invasive changes (20%) that, as a rule,  have cylindrical origin.  The cytological finding of "endocervical adenocarcinoma" is clinically a very respectible finding since its pathohistologic background in 95,5% cases is an invasive carcinoma (in 82% endocervical carcinoma, in 9% squamous carcinoma, and 4,5% metastatic adenocarcinoma). The accurate cytological diagnosis AIS is limited by inadequate samples.

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43. RELIABILITY OF  DETECTING METHODS OF    CARCINOMA IN SITU OF UTERINE CERVICAL GLANDULAR EPITHELIUM  - COMPARISON TO SQUAMOUS LESIONS.    PERIOD BETWEEN 1990-1999

Milojković M,  Pajtler M

Clinical Hospital Osijek

 AIM - To evaluate the detection reliability  carcinoma in situ of glandular uterine cervical epithelium (ACIS) by screening methods in Croatia and its effect on the success of secondary prevention and early diagnosis of this type of cancer.

EXAMINEES - Patients (N=678) treated because of carcinoma in situ or invasive uterine cervical carcinoma at the Unit of Gynecology and Obstetrics of Clinical Hospital in Osijek in the period between 1990-1999.

RESULTS - Out of totally   678 patients, carcinoma in situ was detected in 387 (57,1%) and invasive cervial carcinoma in 291 (42,9%). Squamous carcinoma was found in 244 (83,3%) patients , adenocarcinoma in 35 (12,0%), adenosquamous 5 (1,7%) and non-differentiated in 7 (2,4%). SCIC was found in 375 (96,9%) patients and ACIS in 12 (3,1%). Out of totally 619 patients with SCIC or invasive carcinoma of squamous cervical epithelium, SCIS was revealed in 375 (60,6%), and invasive carcinoma in 244 (39,4%). AQCIS or invasive carcinoma of glandular epithelium was detected in totally 47 patients; ACIS 12 (25,5%) and invasive carcinoma 35 (74,5%)- The ratio between invasive carcinoma of glandular epithelium and ACIS was 3:1, and between invasive carcinoma of squamous epithelium and SCIS was 1:1,5. It was observed that the number of ACIS was significantly increased in the monitored period, while the number of other lesions was not significantly changed. All 12 histologically proved atypias were accompained by precancerosis or carcinoma of squamous epithelium. Only in 3 examinees (25,0%)  with ACIS  atypia of glandular epithelium ( in situ or invasive carcinoma) was discovered in the cytological finding. ACIS did not display characteristic colposcopic abnormalities but it is interesting to mention that a lesion in 4 (33,3%) patients was located within a large ectopia, not in the transformational zone.

CONCLUSION - Modern screening methods of uterine cervical carcinoma in Croatia (cytology, colposcopy) are not reliable enough for detection of the pure ACIS form. Mostly detected are the changes that, apart form the glandular, contain squamous component or precancerosis,  whilst pure lesions of glandular epithelium are overlooked in detection, and in that way, they keep an unfavourable ratio between precancerosis and carcinoma of glandular cervical epithelium which is different from the ratio precancerosis/carcinoma of squamous epithelium which has, in a last few decades, been significantly improved due to the methods of secondary prevention.

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44. AUTOMATIZATION IN CERVICAL CYTOLOGY -

PRACTICAL EXPERIENCE IN AUTOPAP SYSTEM

A. Trošić, G.Brungs, J.Schmitz, M.Bollmann, R.Bollmann

The  screening equipment presents the most complicated application of automatization in cytology. Its purpose is to, followed by the same or increased sensitivity and specifity,  ease the screening by shortening its time or by excluding possibly negative slides. Since June 1, 199? to December 15, 1999, 21525 cervicovaginal smears were analysed by AutoPap. 8435 (39,1%) slides were selected for the archive (No Further Review). 10390 (48,26%) slides needed   manual screening  (Review), and the analysis of 2700 (12,54%) smears could not be completed (Process Review).

Two LSILs were determined by careful manual screening of all the "No Further Review" slides. In the "Review" group, majority of SILs (68,5% LSIL and 81% HSIL) were detected in the first two out of 5 groups (Rank) with greater possibility of abnormal cell prevalence. The AutoPap is capable to classify a big number of slides to be  normal, but false negative results are not excluded. In the "Review" group there is a correlation between a probable  detection of abnormal cells and subgroup (Rank) of slides. Statements about (non)-presence of cylindrical endocervical cells are not reliable.

POSTER SECTION

45. EOSINOPHILIC SYNCYTIAL CHANGE OF

ENDOMETRIA - DIAGNOSTICAL TRAP IN PAP SMEAR

Marija Pajtler, Milanka Mrčela

KB Osijek, Unit of Clinical Cytology and Clinical

Department of Pathology and Forensic Medicine

An eosinophilic syncytial change, (previously called a superficial syncytial change, papillary eosinophilic change or papillary metaplasia) in which cells of superficial endometrium show papillary proliferation,  is usually associated with the breakage of endometrium or inflammation, and it is regarded as a degenerative or reparatory process. However, it can also be observed in different  types of endometrial hyperplasia, and recently  its combination  with endometrial carcinoma has been described.

The case presentation:

A patient K.M:, 50-year old, suffering hypertonia in the postmenopause for a long time, the gynecological  finding b.o. The cytological finding in  the VCE smear: Endometrial adenocarcinoma ( there are numerous papillary sheets  of atypic endometrial cells revealed in the smears accompained by granulocytes and hemolized erythrocytes in the background)   The pathohistological results of fractioned abrasion: an eosinophilic syncytial change of endometria. (Superficial cells of eosinophilic cytoplasma are amalgamated into syncytium forming papillary extentions. The quantity of cytoplasma is variable, and partly vacuolated. Papillary extentions have no connective-vascular stroma, and cystic spaces fulfilled with polimorphoneclear leukocytes can be occasionally observed inside them .  Nuclei are distributed without order in the syncytium; usually they are small, round or vesicular with changes in the chromatine structure. Vesicular nuclei with visible nucleolus and smooth nuclear membrane seem to be reactive (No mitosis.)

Although the pathohistologic eosinophilic syncytial change of endometria has certain similarities with papillary adenocarcinoma, it should not be mistaken for it, because it is a clinically insignificant change. Since it can be followed by cellular desquamation, found in VCE smear, it can cytologically be mistaken for cervical dysplasia and endometrial adenocarcinoma.

POSTER SECTION

46. STRUMA OVARIA IN PREGNANT WOMEN

- CASE PRESENTATION

Šentija K¹,  Krivak-Bolanča I¹ ,    Kardum-Skelin I² ,   Ljubanović D³

Clinic of Gynecology and Obstetrics KB"Merkur", Unit of

Citology and Cytogenetics¹ , Clinic of  Internal Diseases KB "Merkur"²,

Clinical Pathology and Cytology KB "Merkur"³ , Zagreb

Struma ovaria is a type of ovarial monodermal teratoma occured extremely rarely, consisting mainly of the thyroid tissue. Most frequently it is unilateral, and it can be combined with other types of teratoma or epithelial ovarial tumours.

A 38-week pregnant  woman, who had been detected a right ovarium cyst by ultrasound review in the second month of pregnancy, was admitted to the Clinic of Gynecology and Obstetrics KB "Merkur". Immediately, she was examined by ultrasound vaginal tube which revealed irregular multilocular cytic tumor of 10×9 in size with fluid cysts of different thickness. In the solid tumor parts there was no neo-vascularization found. The finding CA -125 was within borders of reference values. During the Caesarian section, a right-sided adnexetomy was performed.The cystic formation was, intraoperatively, aspirated and the content sent to the cytologic analysis. The rest of tumorous formation was sent to pathohistological verification. The cytological intraoperative finding, based on the cellular morphology suggested a benign process. Epithelial  cell sheets were found partly of follicular appearance, stroma cells, blood, numerous pigmentophages and erythrophages, and low cubic epithelium. The detected cellular elements indicated the thyroid tissue. The suspected rare tumor, struma ovarii,  was confirmed by   staining immunocytochemical reaction on thyroglobulin. The intraoperative biopsy findings confirmed   the struma diagnosis, and the final pathohistological diagnosis was the serous cystadenoma (in prevailing part) with teratoma, for which there was not enough  elements in the cytological finding of the aspirated fragment.