1

Cytologic Evaluation of CellPrep® Liquid-based Cytology in Cervicovaginal, Body Fluid, and Urine Specimens

- Comparison with ThinPrep® –

Jae Soo Koh

Department of Pathology, Korea Cancer Center Hospital, KIRAMS.

Cytologic Evaluation of CellPrep® Liquid-based Cytology in Cervicovaginal, Body Fluid, and Urine Specimens -Comparison with ThinPrep® –

Department of Pathology, Korea Cancer Center Hospital, KIRAMS.

Jae Soo Koh, M.D., Soo Yeon Cho, M.D., Hwa Jeong Ha, C.M.I.A.C.,

Jung Soon Kim, C.M.I.A.C., Myung Soon Shin, C.T.I.A.C. Thesis character: Title: Cytologic Evaluation of CellPrep® Liquid-based Cytology Chief author: Ko, Jae-soo Zip code:139-706 Address: 215-4 Gongneung-dong, Nowon-ku, Seoul,Korea Department of Pathology, Korea Cancer Center Hospital, KIRAMS. Position Tel:02-970-2545 Fax:02-970-2430 E-mail:jskoh@kcch.re.kr Note: This research was funded by CellPrep Manufacturer(MEDIMEX Co.LTD, Korea)

Cytologic Evaluation of CellPrep® Liquid-based Cytology in Cervicovaginal. Body Fluid, and Urine Specimens – Comparison with ThinPrep® -

Abstract

This study purposed to evaluate a CellPrep® (CP) of liquid-based cytology (LBC) to search for a less expensive and automated alternative cytologic preparation technique applicable to usually encountered cytologic specimens. Cervicovaginal direct-to-vial split samples from 457 gynecologic patients, 40 body fluid samples, and 34 urine samples were processed with the CP technique and the results were compared with those of the currently used ThinPrep® (TP) method. Both CP and TP methods provide evenly distributed thin layers of cells with little cellular overlaps or significant obscuring elements in most cases . Staining quality of both preparations showed little difference due to the difference of fixative solutions without significant distractions in cytologic interpretation. On the supposition that TP was a gold standard, sensitivity, specificity, positive predictive value, and negative predictive value of CP cytology were 89%, 98%, 86%, and 99% in the cervicovaginal smear, 89%, 82%, 80%, and 90% in body fluid, and all of these values were 100% in urine samples. To testify the availability of immunohistochemistry on CP preparations, cytokeratin, vimentin, and Ki-67 were applied on body fluid specimens, and all of these antibodies were specifically stained on targeted cells. Conclusively, the CP method gave comparable results to those of TP in terms of smear quality and cytologic diagnostic evaluation, and was available on immunohistochemistry. The CP method could offer a cost-effective and automated alternative to the current expensive techniques of liquid-based cytology on popular cytologic materials including cervicovaginal, body fluid, and urine specimens. Key Words: Liquid-based cytology, cytology, gynecological, exfoliative, immunohistochemistry

Introduction ecent liquid-based cytologic examination development is being used by Korean research institutes for its

improvement of specimen quality and simplifying of screening. Gradually this trend has been replacing existing evaluation methods, resulting in health care coverage for cervicovaginal CellPrep evaluations from 2006.

As opposed to the traditional cell smearing method, liquid-based cytology arranges cells on one side of the slide, so that not only is it easier to view cells at high magnification, but it also makes a clean background by removing red blood cells and exudate which made viewing difficult with current evaluation methods due to the

2

smeared backgrounds. In addition, current methods must screen the entire slide in smeared cell examinations, but the liquid-based

cytologic examination can decrease time and mental labor of the screening remarkably because the cells are fixed forms on a confined surface. Because existing tests generally do not have a preservation system for remaining specimens after the cell smear, there is no retrieval method when, in the pathologist’s judgment additional examinations are necessary after the initial cytological test. Due to liquid-based cytology manufacturing procedures it has the advantage that additional tests are possible for examinations for immunohistochemical staining because remaining specimens can be preserved for a given period after the initial testing.

CellPrep (CP)Liquid-based cytology (MEDIMEX, Korea) is a thin, smear method, slide manufacturing system developed in Korea with a liquid base which uses automatic vacuum filter equipment. This method collects samples from patients and floats the samples on a fixed liquid-base. This sample can then be installed in examination equipment by attaching it to the filter using sound pressure at proper levels to adhere the sample to the slide. This automatic system can produce a slide for a thin cellular layer with a diameter of 20mm. This process is similar to ThinPrep® (below, TP, Cytyc, USA).

The author’s institute introduced TP in 2000 for cytologic examination of exfoliative cervicovaginal cells and this method has been used up until now in almost all cytology such as urine, other body fluids and phlegm examinations, except in fine needle aspiration cytology. The usefulness of TP in cytologic examinations was presented in many studies and supported by the evaluations preformed by the author’s institute; therefore, it has been the standard method.

The purpose of this research is to compare TP with CP liquid-based cytology, one of the current standard liquid -based cytology, that had been recently developed in Korea, including examining the quality of slides for cytologic tests on exfoliative cervicovaginal cells and non-gynecological body fluids; the sensitivity and specificity of cytological testing; an assessment of the possible application of immunohistochemical staining. The structure of the equipment and the merits, demerits and efficiency of the technological problems of slide manufacturing due to the structure, are irrelevant to the study.

Material & Methodology

The material for this report is from research conducted at the obstetrics and gynecology department of the Cancer Center Hospital on patients from March to May 2006 and consisted of specimens collected at the same time for a TP examination for the diagnosis of the patients’ cytology of exfoliative cervicovaginal cells. After collecting the specimens for the TP examination, with the patients’ permission, the specimens that were acquired by using a swab supplied by the CP manufacturing company, were placed in a specimen storage solution that was supplied by the same company. The gynecological specimens totaled 463 samples.

Other body fluids and urine specimens were among the remaining specimen which had cellular components after being used for the diagnosis of the cytological specimen as requested by the pathology department. Forty body fluid samples and 34 urine samples were used. The specimens collected by the same method as above, were stained with ordinary Papanicolaou staining, with CP equipment to produce slides in cooperation with MEDIMEX laboratory. This was followed by a screening at the Cancer Center Hospital pathology department by three clinical pathologists and then two pathologists gave the final analysis so that CP exam and the TP exam could be done independently. The results of the CP analysis did not include the patients’ diagnosis.

After the analysis of the CP slide, the TP results and the CP diagnosis which were analyzed by the pathology department of the main hospital were compared. The general evaluation of each slide, specifically, the cellular density of a slide sample, and the morphologic preservation and staining of the cells adhered to the slide were evaluated qualitatively. These did not include a measured evaluation. Conversely, immunohistochemical staining was applied to each of the five samples with relatively many cells from the body fluid samples for cytokeratin (Zymed, 1 : 100), vimentin (Zymed, 1 : 200), and Ki-67(Zymed, 1 : 100) to judge the possibility of application to immunohistochemical staining in the sample tested with the CP method.

Immunohistochemical staining was applied in the same method as the ordinary ABC method except when applying heat in a citrate buffer to expose the antigen. Statistical significance verification of the analysis of the diagnostic sequences between the two methods was conducted by the McNemar test using SPSS 12.0.

Resuts

In order to compare the two cytologic examination methods of CP and TP for cervicovaginal cell samples, a total of 463 samples were collected; however, six samples that were processed by the TP method were inadequate specimens, leaving 457 samples for comparison between the two methods. Because these specimens consisted of so few cells, the number of cells of the six inadequate samples that were processed by TP adhered to the slide inadequately in the CP test according to cytologic evaluation.

3

The caliber of the TP slides which cells can be adhered to is 22mm and for the CP slides 20mm. It was observed that the cytological findings on the clean smear background were interfered with by red blood cells or albuminous suspended matter. The advantage that the surface around the area of the original attached cells in the screening can be kept clear was also observed. Many inflammatory cells would get entangled with epithelial cells, which produced a large mass of cells that could not adhere to the slide. This tendency was observed in both the TP and CP methods. Neither method was found to be able to remove inflammatory cells except for red blood cells. According to the difference of the cell attachment dimensions the difference of screening times was impossible to be measured exactly. Therefore it was not accepted subjectively.

The collective cellular exfoliation phenomenon which causes clear areas within the caliber of the cell smear is found often in the liquid-based cytologic examination. Specific differences of the clear areas were not found in either method. The degree of cell adherence to the slide had a tendency to depend on the sample’s condition. That is, when there were excessive epithelial cells in the liquid of the collected samples both tests showed high quality findings of uniform cell smearing (Fig. 1). According to cytology the merits and demerits of the difference of the ways of observation between the two methods of the nucleus protoplasm ratio, the distribution aspect of over staining of chromatin, and the shape and size of the cytoplasm, was not significant. In both methods regardless of the type of specimen the observation was adequate in the cytological findings.

In TP tests alone, the nucleus had a slight tendency to be over stained but the interpretation of the results was satisfactory. In the case of CP, the observation of the aspects of chromatin or of cytoplasm was easy. However, although the observation of over staining was insufficient, it did not deter the interpretation. When such subjective judgment factors were applied and evaluated equally to all of the body fluids or urine specimens, a difference was not significant.

Suitable specimens were processed in a cytological test that used a TP examination of the cervix. When TP was the standard analysis for the 457 samples, three inadequate specimens of CP that had poor cellular adherence were observed (0.7%). When the cytological sequence of the results of both methods was checked according to cytological analysis classification by the Bethesda System, TBS, there was no difference found between the two methods statistically according to the McNemar test (Table 1, p=0.048).

When the results of the TP analysis were the standard, the cases of the cytological negative observations, the positive reaction observations, and the ASCUS/AGC observations, etc. were divided into two parts according to the positive results. Consequently, CP examination's responsiveness, specificity, positive predictive value, and negative predictive value were each 89%, 98%, 86%, 99% (Table 4). The test using body fluids was done on 25 abdominal cavity exudate samples, 13 thoracic cavity exudate samples, one pericardium sample, and one bile sample, totaling 40 samples. In this case the samples of high density cells of TP were chosen for CP. As summarized in Table 2, the results of the cytology show that no differences between the two methods were identified statistically (p=0.215). According to the standard of TP, CP examination's responsiveness, specificity, positive predictive value, and negative predictive value were each 89%, 82%, 80%, 90% (Table 4). The test using urine totaled 34 samples and the results, as summarized in Table 3, show that the differences between the two methods was below p=0.01 which was not significant. According to TP, CP examination's responsiveness, specificity, positive predictive value, and negative predictive value totaled 100% (Table 4).

In using CP body fluid samples it was possible to interpret cytokeratin (Zymed, 1:100) and vimentin (Zymed, 1:200) through immunohistochemical staining. The nucleus was stained to identify peculiarities and had slight benign findings (Fig. 4A). This phenomenon is often found in immunohistochemical staining using cytological samples as well as in TP samples. In immunohistochemical staining using Ki-67 (Zymed, 1:100) positive cells were identified only in the nucleus (Fig. 4B).

Investigation

An ideal exam method in a hospital laboratory requires high sensitivity and specificity and must be attained at low material and labor costs. Cytological testing methods are not an exception. A cytological smearing examination of the cervix is a useful testing method which has greatly decreased fatality from uterine cancer. This is being used widely as a cancer screening test for women.

Recently, the so called “thin layer smearing test” using Liquid-based cytology(LBC) was introduced in place of existing smearing examinations. It has the advantage that it can overcome the differentiation of the quality of slide smeared according to the proficiency of clinicians who do the smearing. It also has a cytologic sensitivity and specificity in spite of the narrowness of the surface area of slides for screening. 1,2,4-11 In Korea many various kinds of LBC are being compared to current smearing methods and in some laboratories LBC is replacing current methods. 7-9,13-1 At the author’s hospital this method was recently introduced in body fluid, urine and phlegm, etc. cytologic specimens after the TP method in gynecological examinations was introduced in 2000. Especially this TP method showed remarkable results in urine samples compared to existing methods.

In spite of these advantages, the reason it is difficult to immediately introduce LBC in a medical situation in

4

Korea is the expensive equipment needed for manufacturing the slides and the material cost required to perform each test individually, which are far more expensive than existing methods. 15 To solve this problem MonoPrepTM, which does not require special equipment and can be done relatively cheaply, was introduced in an attempt to find a way to improve the quality of testing methods. 14,15 The CP used for this study is the LBC testing method which was developed in Korea to compensate for this problem considering the high cost of TP and the manual labor required for MonoPrepTM. The principles of manufacturing slides are, first, cervical cells and the swab are put in a vial with a fixative. Then the vial is shaken to make cellular suspension. By using sound pressure it must then be filtered and finally adhered to the slide. This is very similar to TP in many aspects.

In this research the result of TP was used as a gold standard, which showed satisfactory results as a cytologic examination method in existing studies. The purpose of this study is to prove the efficiency of CP compared to similar existing testing methods rather than the absolute efficiency of the CP testing method. However, to have more exact examinations it must be the standard to check the entire condition of the cervix like a cone biopsy. But in reality it is difficult to apply an invasive exam to all patients.

In this research the cervicovaginal cytologic testing had histological results which conflicted with the cervical

examinations. The number of histological results was small and was not enough to be a standard in the case of discord between TP and CP method results. For statistic processing the McNemar test excluded inadequate specimens in the cervical cytodiagnosis test to verify the similarities of the test results of both methods. In the urine test, TP excluded two samples diagnosed as atypical. It was proved statistically that there was no difference in the test results between two methods in the cervical cytodiagnosis test and urine test. However, a difference in the test for body fluids was not recognized.

However, in TP some "suspicious" aspects were diagnosed but the discord of the diagnosis did not have an influence on the clinical process except for one sample diagnosed as negative in CP. One sample, which was 100% in sensitivity, specificity, positive predictive value, and negative predictive value in the urine was positive. Therefore exaggerating the meaning of statistical results must be avoided. In cervical cytodiagnosis , body fluid and urine specimens the increasing frequency of “atypical” was observed. This was understood as a temporary phenomenon appearing in the process of reading CP in the condition of using TP as the standard testing method.

In this research the samples were picked from the cervix of patients two times sequently and a swab was used for another picking of specimens. The first picking was used for TP. The latter picking was used for CP. To have an objective comparison the order of each procedure had to be changed; however, the picking of the specimens for diagnosis must not be interfered with so the specimens for CP was always processed after the picking of the specimens for TP. As a result of this method the number of exfoliative cells was small. At this time cell density in the CP sample was low compared to the TP sample. Six inadequate TP samples were barely observed in CP.

There can be a "collection bias" in this research before the analysis is shown in the testing to compare both examination methods. However there was no statistical similarity between both methods in the diagnostic results. Therefore the second picking after the first picking had a slight influence on the test results. To decrease expenses for a collection bias and specimen picking, remaining specimens, that is, cell suspension which was left over in this research after the TP test, could have been used. But because the difference of fixative between TP and CP can have an influence on the whole evaluation including cellular preservation conditions, the stainability, etc, cell suspension was not used.

Even though there is a similar method to the one already being used in the laboratory, it takes a lot of effort and trial-error until other methods are introduced and become commonly used. In the early steps the Cancer Center Hospital installed CP equipment in the pathology department and then the MEDIMEX personnel did the process to adhere cells to the slides. The pathologist did the staining according to the common method as described in this study which was the same as the CP slides. However, the condition of cellular adherence was not good and the stainability was not clear. In the condition of cellular adherence, cytoplasm was folded and curled over when aspiration pressure was too strong on suspended cells. This phenomenon was observed often. Stainability occurs when the difference of preservation fluids is combined with a lack of quality control. This did not have a fundamental influence on the diagnoses but on subjective judgment of the quality of slides. To solve this problem the study could have been done again after undergoing certain quality control measures in the MEDIMEX laboratory.

An advantage of LBC is that additional tests are possible by using the remaining specimens in the sample vial when the diagnoses in the first cytologic test is not made and needs additional testing or immunohistochemical staining, etc. There are few cases which require immunologic testing in cervix cytodiagnosis , but are required in the body fluid samples. To test the application of immunohistochemical staining in the CP sample, the subjects were cytoplasm and antigen. In all the results which did immunohistochemical staining by using cytokeratin, vimentin, and Ki-67 the application was possible. It was concluded that the reason cytokeratin and vimentin appeared slightly positive is that the cytologic sample was not a cross-section of cells so some cytoplasm

5

overlapped in the part that looked like the morphological nucleus. In the cervical cytodiagnosis , supplementary HPV detection was not done. HPV, a DNA virus which is undetectable in few cases, can be used when additional testing is needed.

Conclusion

When CP was developed as LBC in Korea it had no clear defect in cervicovaginal cytologic testing, body fluid testing and urine testing by the TP standard. The sensitivity of diagnosis, specificity, positive predictive value, and negative predictive value were over 80%. This was confirmed by using immunohistochemical staining. Therefore CellPrep® is considered to be the suitable testing method for LBC in the cervix cytodiagnosis test, body fluid test, and urine cytologic test. Also when CP is used each laboratory needs quality control procedures .

Literature Cited 1. Payne N, Chilcott J, McGoogan E. Liquid -based cytology in cervical screening: a rapid and systematic

review. Health Technol Assess 2000; 4: 1– 73. 2. Linder J. Recent advances in thin-layer cytology. Diagn Cytopathol 1998; 18: 24–32. 3. Sahebali S, Depuydt CE, Segers K, et al. p16INK4a as an adjunct marker in liquid -based cervical

cytology. Int J Cancer 2004; 108: 871-6. 4. Lee KR, Ashfaq R, Birdsong GG, Corkill ME, McIntosh KM, Inhorn SL. Comparison of conventional

Papanicolaou smears and a fluid-based, thin-layer system for cervical cancer screening. Obstet Gynecol 1997; 90: 278-84.

5. Abulafia O, Pezzullo JC, Sherer DM. Performance of ThinPrep liquid-based cervical cytology in comparison with conventionally prepared Papanicolaou smears: a quantitative survey. Gynecol Oncol 2003; 90: 137-44.

6. Schledermann D, Ejersbo D, Hoelund B. Improvement of diagnostic accuracy and screening conditions with liquid -based cytology. Diagn Cytopathol 2006; 34: 780-5.

7. Lee KC, Choi HS, Kim HJ, et al. The Comparison of Conventional Pap Smear with ThinPrep Pap Smear in detecting HSIL and Cervix cancer. Korean J Obstet Gynecol 2002; 45: 753-8.

8. Kim YR, Kim YT, Kim SH, Kim JH, Kim JW, Park YW. Comparative analysis of conventional Papanicolaou smear, fluid-based thin-layer method and cervicography. Korean J Obstet Gynecol 2005; 48: 2932-40.

9. Park YW , Chung JH, Lee HM. A Comparison of the Availability of the Urine ThinPrep test and Urine Cytology in the Diagnosis of Bladder Cancer. Korean J Urol 2003; 44: 734-8.

10. Ylagan LR, Zhai J. The value of ThinPrep and cytospin preparation in pleural effusion cytological diagnosis of mesothelioma and adenocarcinoma . Diagn Cytopathol 2005; 32: 137-44.

11. Gabriel C, Achten R, Drijkoningen M. Use of liquid-based cytology in serous fluids: a comparison with conventional cytopreparatory techniques. Acta Cytol 2004; 48: 825-35.

12. Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 2002; 287: 2114-9.

13. Jang J, Kim J, Cho KJ, Khang SK, Nam JH, Gong G. A comparison of AutoCyte PREP with Matched Conventional Smear in Cervicovaginal Cytology. Korean J Cytopathol 2002; 13: 8-13.

14. Park JM, Lee JG, Suh IS. Clinical Efficacy of Manual Liquid -Based Cervicovaginal Cytology Preparation: Comparative Study with Conventional Papanicolaou Test. Korean J Cytopathol 2005; 16: 10-7.

15. Nam JH, Kim HS, Lee JS, Choi HS, Min KW, Park CS. A comparison of modified MonoPrep2 of liquid -based cytology with ThinPrep Pap test. Gynecol Oncol 2004; 94: 693-8.

16. Maksem JA, Dhanwada V, Trueblood JE, et al. Testing automated liquid-based cytology samples with a manual liquid-based cytology method using residual cell suspensions from 500 ThinPrep cases. Diagn Cytopathol 2006; 34: 391-6.

17. Mullink H, Walboomers JM, Tadema TM, Jansen DJ, Meijer CJ. Combined immuno- and non-radioactive hybridocytochemistry on cells and tissue sections: influence of fixation, enzyme pre -treatment, and choice of chromogen on detection of antigen and DNA sequences. J Histochem Cytochem 1989; 37: 603-9.

Legends for Figures Figure 1. The gross photograph of slides show evenly distributed cell preparation in both methods. There is little difference in cell density between them (from right to left; ThinPrep® of cervicovaginal specimen, CellPrep® of cervicovaginal specimen, ThinPrep® of urine, and CellPrep® of urine)

6

Figure 2. Representative sample of cervicovaginal specimen shows low-grade squamous intraepithelial lesion prepared by CellPrep®. Thin-layer preparation and clean background are appreciated. Figure 3. Peritoneal fluid prepared by CellPrep® demonstrates metastatic adenocarcinoma Figure 4. Immunohistochemical staining of vimentin (A) and Ki-67 (B) successfully demonstrates their specific expression in targeted cells. Table 1. Comparison of the cytologic diagnosis between ThinPrep® and CellPrep® Pap test in cervicovaginal smear CellPrep® ThinPrep® Negative RCC ASCUS/AGC LSIL HSIL SCC/Adc Inadequate Total Negative 379 1 2 0 0 0 2 384 RCC 10 23 3 0 0 0 0 36 ASCUS/AGC 3 1 14 3 0 0 0 21 LSIL 0 0 1 8 0 0 1 10 HSIL 0 0 0 0 2 0 0 2 SCC/Adc 0 0 0 0 0 4 0 4 Inadequate 0 0 0 0 0 0 0 0 Total 392 25 20 11 2 4 3 457 RCC: reactive cellular change; ASCUS/AGC: atypical squamous cells of undetermined significance/atypical glandular cells; LSIL: low grade squamous intraepithelial lesion; HSIL: high grade squamous intraepithelial lesion; SCC/Adc: squamous cell carcinoma/adenocarcinoma. Table 2. Comparison of the cytologic diagnosis between ThinPrep® and CellPrep® test in body fluid CellPrep® ThinPrep® Negative Atypical Suspicious Malignancy Total Negative 17 1 0 0 18 Atypical 4 0 0 0 4 Suspicious 1 1 0 0 2 Malignancy 0 0 2 14 16 Total 22 2 2 14 40 Table 3. Comparison of the cytologic diagnosis between ThinPrep® and CellPrep® test in urine CellPrep® ThinPrep® Negative Atypical Suspicious Malignancy Total Negative 31 0 0 0 31 Atypical 2 0 0 0 2 Suspicious 0 0 0 0 0 Malignancy 0 0 0 1 1 Total 33 0 0 1 34 Table 4. The relationship between results of ThinPrep® and CellPrep® test

Cervicovaginal smear Body fluid Urine

CellPrep® CellPrep® CellPrep®

ThinPrep® Negative* Positive† Total ThinPrep® Negative‡ Positive§ Total ThinPrep® Negative‡ Positive§ Total

Negative* 413 5 418 Negative‡ 18 4 22 Negative‡ 33 0 33

Positive† 4 32 36 Positive§ 2 16 18 Positive§ 0 1 1

Total 417 37 454 Total 20 20 40 Total 33 1 34

Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 89%, 98%, 86%, and 99% in cervicovaginal smear, 89%, 82%, 80%, and 90% in body fluid, respectively. In urine sample, all of these values were 100%.

7

Negative*: Negative or reactive cellular change Positive†: Atypical squamous cells of undetermined significance/atypical glandular cells or higher lesion Negative‡: Negative or atypical cells Positive§: Suspicious malignancy or higher lesion

Figure 1

Figure 2

8

Figure 3

Figure 4

A B