The Value of the Cytobrush for Obtaining Cells From the Uterine Cervix Keiko Kawaguchi, M.D., Masao Nogi, M.D., Mikako Ohya, C.T. (I.A.C.), Yuko Nishikawa, C.T. (I.A.C.), and Tadao K. Kobayashi, M.T., C.F.I.A.C.

A study was undertaken to evaluate the efficacy of the Cyto- brush compared with the cotton-tipped applicator f o r obtaining cells from the uterine cervix. Four consecutive Papanicolaou smears were obtained in 1,000 patients during a single clinic visit. In every patient, a total of four smears on two slides were submitted: one slide contained endocervical material obtained with a cotton-tipped applicator and with the Cytobrush and the other slide contained ectocervical material obtained with both a cotton-tipped applicator and the Cytobrush. In endocervical smears, the Cytobrush technique produced numerous groups and clusters of cervical columnar cells in contrast to the use of the cotton swab. This was particularly true in older patients and pregnant women. In ectocervical smears, the detection of the squamous component was also higher in the Cytobrush smear than in cotton swab smears. However, the cell yield was not influenced by the cell sampling sequence in either endocer- viral or ectocervical specimens. It is the conclusion of this study that the utility of the Cytobrush technique for collecting specimens for endocervical and ectocervical smears is greater and provides a higher yield of cells than the cotton swab technique. Wider use of this simple procedure is encour- aged. Diagn Cytopathol 1987;3:262-267.

Key Words: Cytobrush technique; Endocervical sampling; Chla- mydia; Cervical smear

Since the introduction of cervical cytology screening by George Papanicolaou in 1940, much has been written about the specificity of the Papanicolaou smear as a procedure in the detection of abnormalities of the cervical epithelium. There is evidence that such screening is associated with reduction of mortality from carcinoma of the uterine cervix. Based on such observations, a rationale

Received November 18, 1986. Accepted April 28, 1987. From the Departments of Obstetrics and Gynecology and Cytopa-

thology, Kobe Shinko Hospital, Kobe, Hyogo, and the Department of Cytopathology, Saiseikai Shiga Hospital, Imperial Gift Foundation, Inc., Ritto, Shiga, Japan.

Presented in part at the 9th International Congress of Cytology, Brussels, May 26-29, 1986.

Address reprint requests to Tadao K. Kobayashi, M.T., C.F.I.A.C., Department of Cytopathology, Saiseikai Shiga Hospital, 280 Oh-hashi, Ritto, Shiga 520-30, Japan.

for population screening can be developed.’ However, the published estimates of false-negative rates from the Papanicolaou smear range from 6% to 56%.24 One expla- nation for the high false-negative rate might be sampling error.

In 1977, Rubio’ suggested that both cotton swab applicators and wooden spatulas retain large numbers of atypical cells that never reach the slides. It is now established that the diagnostic value of gynecological cellular samples in the detection of cervical malignancies depends on the collection of an optimal specimen from appropriate areas. Moreover, the cellular composition of cervical smears is also influenced by the sample taker.6 Wied,’ who studied 50 cases of early invasive cancer including carcinoma in situ, found significantly more atypical cells in the endocervical specimen than in the cervical smear. This is in agreement with modern con- cepts of the topography of cervical carcinogenesis, which localize the highest degree of precancerous lesions more proximally in the endocervical canal. For the successful collection of endocervical cells, it is important that the smears are adeq~ate .~s* ,~ In recent years, therefore, endo- cervical smears have been used extensively, and the literature suggests that a combined sample of endocervi- cal and cervical cells is the most accurate way of identify- ing cervical rnalignancie~.**~*~*~-”

Use of a cotton swab technique in both the endocervix and ectocervix has found wide acceptance in Japan and in Western countries12 because of its ease and convenience. Since the introduction of the cervical brush, however, several papers have shown the efficacy of brush cytology in the endocervical ~ a n a l . l ~ - ’ ~ These reports emphasized that the use of the cervical brush to collect material for cervical smears is more effective and provides a higher yield of cells than the use of the conventional technique.

Recently, these devices have become available in Japan. The current study adds further documentation to the usefulness of the Cytobrush compared with the cotton

262 Diagnostic Cytopathology, Vol3, No 3, September 1987 0 1987 IGAKU-SHOIN MEDICAL PUBLISHERS, INC.

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0

Fig. 1. Sampling devices used for this study. (A) Cotton-tipped applica- tor for both endocervical and ectocervical cell sampling. (B) Cytobrush for endocervical cell sampling. (C) Cytobrush for ectocervical cell sampling.

swab in routine gynecological practice in the outpatient clinic.

Materials and Methods A total of 1,000 patients seen from July 1985 through May 1986 in the gynecologic outpatient clinic at the Department of Obstetrics and Gynecology, Kobe Shinko Hospital, were used in the study. No patient had a history of abnormal cytology. The cellular samples were obtained using two different sampling devices: a cotton-tip applica-

Group A ECTOCERVIX

tor (Sasaki Bandage Co., Tokyo, Japan) and a cervical brush (Cytobrush, Medscand AB, Malmo, Sweden; Tei- koku Hormone MFG, Tokyo) (Fig. 1). The tip of the Cytobrush was curved, suitable for ectocervical cell sam- pling, as shown in Fig. 1. All cellular samples were obtained by one of the authors (K.K.). In every patient, a total of four smears on two slides were submitted. The cell sampling sequence shown in Fig. 2 was used because we thought at first that the smears tended to contain many accumulated cells. Thus, we wished to eliminate any possible bias favoring the brushing technique.

The cellular samples were divided into two groups. In group A, the cellular sample was initially obtained from the endocervix with a cotton swab, smeared on half of the glass slide and spray fixed, with the other half of the slide covered. Then another sample was obtained by the Cyto- brush. In group B, the cellular sample was initially obtained from the endocervix with the Cytobrush, then smeared and fixed as in group A. Then another sample was obtained by cotton swab (Fig. 1). All smears were routinely stained by the Papanicolaou method. The smears were interpreted consecutively in a blind study design by three well-qualified cytotechnologists, with special attention directed to the presence of squamous components and endocervical cell clusters. If such factors were found, they were semiquantitatively classified using arbitrary scores. All cases showing any abnormality more severe than inflammatory atypia were reviewed by an experienced cytotechnologist or cytopathologist. The smears were reported as “no malignancy or dysplastic changes” and “benign atypias.” “Mild dysplasia” was classified as cervical intraepithelial neoplasia (CIN) I, moderate dysplasia was designated CIN 11, and severe

ENDOCERVIX

Group 6 ECTOCERVIX ENDOCERVIX

Fig. 2. Schematic drawing of the smear preparation in both group A and group B.

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Table I. Age Distribution of Patients in the Study

Number of patients

Age (YrJ Group A Group B

10-19 8 5 20-29 107 (61) 113 (48) 30-39 160 (38) 176 (40) 40-49 130 I15 50-59 56 52 60-69 24 20 z 70 15 19 Total 500 (99) 500 (88)

Numbers in parentheses represent pregnant women.

dysplasia and carcinoma in situ were grouped under CIN 111. The statistical analysis was carried out with a chi- square (x’) test.

Results The age distribution of the patients is listed in Table I. Their age ranged from 18 to 80. Of 1,000 patients, 187 (19%) were pregnant. Table I1 shows the relative frequen- cies of smears containing squamous cells. The appearance of squamous cells in ectocervical smears was graded using arbitrary scores. An inadequate smear was defined as scored of 1 with scanty cellular yield, not adequate for making a cytologic interpretation. The incidence of inade- quate smears was 1% by the cotton swab and 0.4% with the Cytobrush technique. The percentage of smears con- taining squamous cells was higher in the Cytobrush smears than in the cotton swab smears (Table 11). Figure 3 shows the occurrence of columnar cell clusters in endocervical smears by two techniques according to patient age. The frequency of columnar cell clusters in each slide was graded using an arbitrary score (1, no columnar cell clusters; 2, one to two columnar cell clus- ters; 3, three to four columnar cell clusters; 4, five to nine columnar cell clusters; 5, 10 to 19 columnar cell clusters; 6,20 to 49 columnar cell clusters, 7, over 50 columnar cell clusters). The smears prepared by the brush always contained more columnar cells than the smears prepared by the cotton swab technique. The overall score of colum- nar cell clusters was significantly higher ( P < 0.001) in Cytobrush smears than in cotton swab smears in any age group. No difference in the cell yield was found between

6 .1 Cytobrush

=Cotton swab

5 m 8 4 In

3

2

1

Group A B A B A B A B A B A B

Age -29 30-39 40-49 50-59 60-69 70- n= 54 70 122 136 130 115 56 52 24 20 15 19

Fig. 3. Occurrence of columnar cells in endocervical smears of nonpreg- nant women.

the two groups in either endocervical or ectocervical smears. The proportion of smears containing endocervical columnar cells increased with age in both the Cytobrush and cotton swab specimens; however, the Cytobrush score was higher than that of cotton swab. The relative frequen- cies of columnar cell clusters in pregnant women is compared for the two techniques in Fig. 4. This compari- son reveals that the smears prepared by brush contained many more columnar cell clusters than those prepared by cotton swab ( P < 0.001).

The frequency of cellular samples without columnar cells in the endocervical smears is shown in Fig. 5. In both groups, the frequency of smears without columnar cells was higher in cotton swab samples than in the Cytobrush smears. These frequencies were particularly low in sam- ples from women over the age of 50. Table 111 shows the relationship between the presence of endocervical colum- nar cells and abnormal epithelial changes in both sam- pling techniques. There tended to be differences in the quality of cellular material in the two smears (endocervi- cal and ectocervical) prepared by both techniques. Smears of the cotton swab contained relatively less mate- rial. The presence of abnormal epithelial changes is frequently associated with the presence of endocervical columnar cells as compared with the absence of columnar cells. In our series, 21 cases were reported as atypical epithelial changes, and 18 cases were histologically veri-

Table 11. Occurrence of Squarnous-Cell Components in Ectocervical Smears

Group B Group A

Cotton swab Cytohrush Cotton swab Cytohrush Score (arbitrary) Number Percentage Number Percentage Number Percentage Number Percentage

1 6 1.2 4 0.8 4 0.8 0 2 186 37.2 12 14.4 284 49.6 78 15.6 3 284 56.8 319 63.8 24 1 48.2 355 71.0 4 24 4.8 105 21.0 7 L .4 61 13.4

Score: 1, none; 2, few; 3, moderate; 4, abundant.

264 Diagnostic Cytopathology. Vol3, No 3, September 1987

VALUE OF CYTOBRUSH SAMPLING

7-

6-

5-

100 %1

Cytobrush

0 Cotton swab

Grow A B A B

Age -29 30-39 n= 61 48 38 40

Fig. 4. Occurrence of columnar cells in endocervical smears of pregnant women.

fied; there was false-positive cytology in three cases. Among 18 cases with a histologic confirmation more severe than CIN I, four cases were detected in endocervi- cal sampkes. False-negative Papanicolaou smears for cot- ton swab samples were found twice in endocervical speci- mens and once in an ectocervical specimen. Because of the small number of abnormal epithelial changes in our study, the difference in false negativity between the two

1 n n Cytobrush

=Cotton swab

G r o u p A B A B A B A B A B A B

Age -29 30-39 40-49 3-59 60-69 70- n=. 54 70 122 136 130 115 56 52 24 20 15 19

Fig. 5. Frequency of cellular findings without columnar cells in end* cervical sniears.

groups was not significant. One case of endocervical adenocarcinoma was diagnosed with both the Cytobrush and cotton swab technique, and both smears contained numerous columnar cells. Our experience confirms the conclusions of most authors that the Cytobrush technique is practical on an outpatient basis with no pain or discom- fort for the patient.

Discussion The Cytobrush technique has been reported by several investigator^'^-^^ as an ideal technique for use with the Papanicolaou smear. In 1983, Ros et al.14 reported the use

Table 111; Cytologic-Diagnoses Using Two Sampling Techniques in Cases With Histologically Proven Malignant or Premalignant Changes

Endocervix Ectocervix

Presence of atypical cells Presence of Patient Cytolonic endocervical Presence of atypical cells

number diagnosis cells Cytobrush Cotton swab Cytobrush Cotton swab Histology

A1 I c - +++ +++ + + + +++ IC A2 IC - +++ +++ + + + + + + IC A3 IC + +++ +++ +++ +++ IC A4 IC - +++ +++ +++ +++ IC A5 CIN 111 +++ +++ - + + + +++ CIN I11 A6 CIN 111 + - - + + + + CIN 111 A? ClN I1 + + - - +++ + CIN 111 A8 CIN I11 + +++ +++ +++ +++ CIN Il l A9 CIN I1 + + - - + + + + CIN I A10 CIN I - - - + + i CIN I1 A1 1 CIN I11 + + + + - + + - CIN Ill A1 2 CIN I1 - - - + + + + + CIN I1 BI IC +++ +++ +++ +++ +++ IC B2 AC +++ +++ +++ +++ + + + AC B3 IC - + + + + + + + + + + + + IC B4 €IN I1 + + + + + +++ + CIN 1 B5 CIN I1 + + + + + + + + CIN I1 B6 CIN I1 + + - - + + + CIN I1

Key: the presence of endocervical cells and atypical cells in each smear was categorized as numerous (+ + +), moderate (+ +), rare (+), or absent ( -). IC, invasive squamous-cell carcinoma; AC, cervical adenocarcinoma; CIN, cervical intraepithelial neoplasia.

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KAWAGUCHI ET AL.

of a brushing technique to obtain cells from the cervical canal. They found the number of usable endocervical smears obtained with the Cytobrush to be three times greater than obtained with the cotton swab. Moreover, the material was better preserved and more plentiful, with a statistically significant superior specificity. Two Dutch studies13*15 subsequently demonstrated that Cytobrush smears provide a higher yield of cells than the conven- tional cotton swab technique. Boon et al.I3 advocated the use of a combined spatula-Cytobrush method because they found a higher positive cytology rate in the combined smear as compared with the spatula-alone smears. Trim- bos and Arentz” recently explained that the reason for the greater effectiveness of the Cytobrush must be sought in the characteristics of the device itself the Cytobrush has a thinner tip than does the cotton swab; it is easier to introduce into the narrow cervical canal since the hairs fold down along the shaft; and it is less likely to retain cells than do other devices such as the cotton swab and the wooden spatula.

Our results also show that the use of a cotton swab for taking endocervical and ectocervical cells resulted in a higher incidence of inadequate cellular samples (Table 11; Fig. 3). Although our study included a relatively small number of patients with abnormal epithelial changes, false-negative smears with the cotton swab included two endocervical specimens and one ectocervical specimen (Table 111). Fortunately, abnormal lesions were detected in their counterpart smears. In patients with abnormal cellular changes, Cytobrush samples always gave more numerous cells than the cotton swab. As pointed out by Rubio,16 the abnormal cells from these lesions are proba- bly more adherent and thus more difficult to dislodge from the lesion with a cotton swab. It is well accepted that all the dynamic, physiological, and pathological processes in the human cervix occur in the squamocolumnar junc- tion. Therefore, the presence of endocervical cells depends on the patient’s age; the squamocolumnar junction is situated higher in postmenopausal women. It has been shown by Takahashi et al.17 that the brushing smear is also valuable, especially in cases of unsatisfactory colpos- copic findings (squamocolumnar junction not visible). Gondos et al.’ described an incidence of cervical smears without endocervical cells of 8% in women younger than 45 vears and 36%-48% in women older than 45. Our

Although the technique of colposcopy is established as safe, accurate, and effective in determining the presence or absence of cervical cancer in pregnant patients’ cer- vices, the evaluation of abnormal cytology in pregnant patients plays an important role in proper clinical man- agement. In 1979, Ostergard’’ noted that the location of the squamocolumnar junction during pregnancy was pre- dominantly in the endocervical canal. This information further supports the need for routine endocervical smears in addition to ectocervical smears for cytologic screening in pregnant women. Thus, it seems likely that brush cytology can also be applied in such clinical situations.

Many centers report the absence of endocervical cells as an important factor in false-negative smears; neverthe- less, Kivlahan and Ingram” observed that women with Papanicolaou smears lacking endocervical cells did not show an increased incidence of cervical atypias on subse- quent smears.

Cytologic screening also facilitates the diagnosis of infections such as herpes, Trichomonas, Chlamydia, and Candida. Of particular interest is the possibility of cyto- logic detection of chlamydial infection using the Cyto- brush sampling. We recently succeeded in detecting cervical chlamydial infection aided by the immunoperoxi- dase technique in Cytobrush specimens and showed that the brush smears could have produced adequate cellular components of chlamydial infection?’ since Chlamydia infects mainly endocervical or metaplastic cells. There- fore, we emphasize that endocervical cell sampling by the brush method can be extremely suited for the cytologic detection of such infections.

In conclusion, this study confirms that the use of the Cytobrush technique significantly increases the yield of cells, especially in postmenopausal and pregnant patients. Likewise, detection of Chlamydia by the Cytobrush technique would seem to be suitable for use with immuno- cytochemical methods. Routine use of the Cytobrush technique is recommended in both endocervical and ecto- cervical cell sampling.

References 1. Carke EA. Does screening by “pap” smears help prevent cervical cancer? Lancet 1979;2:1-2. 2. Richart RM, Vailant HW. Influence of cell collection techniques

study, however, showed improved yield of endocervical columnar cells by both techniques, particularly in older patients (Figs. 3 and 5). Abudul-Karim et al.,” in a morphometric study Of CIN Of the Uterine cervix, found most lesions (87%) to be associated with the transforma-

upon cytological diagnosis. Cancer 19653 8:1474-8. 3. Sedlis A, Waters AT, Balig H, et al. Evaluation of two simulta- neously obtained cervical cytological smears: a comparison study. Acta Cytol 1974;18:291-6, 4. Berkowitz RS, Eharmann RL, Lavizzo-Mourey R, et al. Invasive cervical carcinoma in young women. Gynecol Oncol 1979;8:311-8.

, I

tion zone. Thus, the presence of endocervical cells is Of the smear’ and the

absence Of these be considered a warning

5. Rubio CA. The false negative smear. 11: The trapping effect of collecting instruments. Obstet Gynecol 1977;49:576-80. 6. Vooijs PG, Elias A, van der Graaf Y, et al. Relationship between the diagnosis of epithelial abnormalities and the composition of cervical

as an indicator Of the

signal for a potential false-negative diagnosis. smiars. Acta Cytol 1985;29:323-8.

266 Diagnostic Cyropathology, Vol3, No 3, September 1987

VALUE OF CYTOBRUSH SAMPLING

7. Wied GL. Importance of the site from which vaginal cytology smears are taken. Am J Clin Pathol 1955;25:742-50. 8. Gondos B, Marchall D, Ostergard DR. Endocervical cells in cervical smears. Am J Obstet Gynecol 1972;114:833-4. 9. Elias A, Linthorst G, Bekker B, et al. The significance of endocervi- cal cells in the diagnosis of cervical epithelial changes. Acta Cytol 1983;27:225-9. 10. Johansen P, Arffmann E, Pallesen G. Evaluation of smears obtained by the cervical scraping and an endocervical swab in the diagnosis of neoplastic disease of the uterine cervix. Acta Obstet Gynecol Scand 1979;58:265-70. 11. Garite TJ, Feldman MJ. An evaluation of cytologic sampling techniques: a comparative study. Acta Cytol 1978;22:83-5. 12. Shen JT, Nalick RH, Schlaerth JB, et al. Efficacy of cotton-tipped applicators for obtaining cells from the uterine cervix for Papanicolaou smears. Acta Cytol 1984;28:541-5. 13. Boon ME, Alons-van Kordelaar JJM, Rietveld-Scheffers PEM. Consequences of the introduction of combined spatula and cytobrush sampling for cervical cytology: improvements in smear quality and detection rates. Acta Cytol 1986;30:264-70. 14. Ros E, Jimenez Ayala M, Vilaplana E, et al. New technique for

endocervical cytological sampling with Stormby’s brush: preliminary results. Cytologia 1983;3:9-20 (in Spanish). 15. Trimbos JB, Arentz NPW. The efficacy of the Cytobrush versus the cotton swab in the collection of endocervical cells in cervical smears. Acta Cytol 1986;30:261-3. 16. Rubio CA. A trap for atypical cells. Am J Obstet Gynecol 1977;128:687-8. 17. Takahashi M, Kiguchi K, Hasegawa T, et al. Endocervical smears using Cytobrush in cases of unsatisfactory colposcopic findings. J Jpn Clin Cytol 1986;25:6&4 (in Japanese). 18. Abdul-Karim FW, Fu YS, Reagan JW, et al. Morphometric study of intraepithelial neoplasia of the uterine cervix. Obstet Gynecol 1982;60:21&4. 19. Ostergard DR. The effect of pregnancy on the cervical squamoco- lumnar junction in patients with abnormal cervical cytology. Am J Obstet Gynecol 1979;134:759-60. 20. Kivlahan C, Ingram E. Papanicolaou smears without endocervical cells: are they inadequate? Acta Cytol 1986;30:258-60. 21. Kobayashi TK, Ueda M, Araki H, et al. Immunocytochemical demonstration of chlamydia1 infection in the urogenital tract. Diag Cytdpathol (in press).

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