Year : 2008 | Volume
: 25 | Issue : 1 | Page : 1--5
Cervical acid phosphatase detection: A guide to abnormal cells in cytology smear screening for cervical cancer
Prabal Deb1, Venkateswaran K Iyer1, Neerja Bhatla2, O Markovic3, Kusum Verma1,
1 Department of Pathology, All India Institute of Medical Sciences (AIIMS), New Delhi, India
2 Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences (AIIMS), New Delhi, India
3 BioSciCon Inc, Rockville, Maryland, USA
Venkateswaran K Iyer
Department of Pathology, All India Institute of Medical Sciences, New Delhi - 110 029
Background: Cervical acid phosphatase-Papanicolaou (CAP-PAP) test has recently been described for detection of acid phosphatase enzyme in abnormal squamous cells, and has been proposed as a biomarker-based technology for the screening of cervical cancer.
Materials and Methods: Eighty-one consecutive cervical smears were subjected to routine Papanicolaou (Pap) staining as well as CAP-PAP, which combined cytochemical staining for acid phosphatase with modified Pap stain. Statistical evaluation of its utility was examined.
Results: Of 81 smears, 16 (19.75%) showed the presence of mature squamous cells with acid phosphatase by CAP-PAP technique and were considered positive. Of these 16, atypical squamous cells of undetermined significance (ASCUS) or above were initially diagnosed in five of the corresponding routine Pap smears. After re-evaluation with CAP-PAP, eight of the routine Pap smears were considered to have ASCUS or above. Of these eight, three were reported as low-grade squamous intraepithelial lesions and five as ASCUS on conventional Pap smears. The remaining 8/16 CAP-PAP-positive cases were negative for atypical squamous cells on the corresponding Pap smears. None of the CAP-PAP-negative smears were positive on routine Pap smear screening.
Conclusions: This study highlights the efficacy of CAP-PAP in quality assurance of cervical smear screening. It is also an inexpensive method for segregating smears for subsequent re-screening. In the absence of trained cytologists in peripheral laboratories, this technique can be adopted for identifying smears that would require proper evaluation.
|How to cite this article:|
Deb P, Iyer VK, Bhatla N, Markovic O, Verma K. Cervical acid phosphatase detection: A guide to abnormal cells in cytology smear screening for cervical cancer.J Cytol 2008;25:1-5
|How to cite this URL:|
Deb P, Iyer VK, Bhatla N, Markovic O, Verma K. Cervical acid phosphatase detection: A guide to abnormal cells in cytology smear screening for cervical cancer. J Cytol [serial online] 2008 [cited 2018 Dec 17 ];25:1-5
Available from: http://www.jcytol.org/text.asp?2008/25/1/1/40649
Cervical smear cytology screening by Papanicolaou (Pap)-stained smears is the most efficacious and cost-effective method of cancer screening in developed countries, decreasing the incidence and mortality from cervical cancer.  However, cervical smear screening has significant rates of false-positive and false-negative results. , To improve the detection of precancerous cervical lesions using Pap smear screening, a number of adjunctive tests have been developed including thin layer cytology;  use of magnified chemiluminescent screening examination (speculoscopy) combined with Pap smear (Papsure);  combined cytology and Cervicogram;  automated rescreening methods;  detection of human papilloma virus DNA (HPV DNA) by the Hybrid Capture 2 (HC2) or polymerase chain reaction tests;  evaluation of telomerase repeat activity by the telomeric repeat amplification protocol (TRAP),  Fourier-transform infrared (FTIR) spectroscopy  and immunocytochemical detection of p16INK4a protein.  However, limited by their prohibitive cost factor and unavailability beyond few tertiary care referral centers, these newer technologies have no role in large scale screening programs of developing countries.
Recently, an inexpensive modification of the conventional Pap test, viz ., cervical acid phosphatase-Papanicolaou test (CAP-PAP Test; MarkPap® Test) has been described. In this test, abnormal squamous cells of the cervix are labeled for the presence of the lysosomal enzyme, cervical acid phosphatase (CAP). ,,, Cytochemistry for CAP results in red, granular deposits against a modified Papanicolaou background. CAP is not present in the squamous cells of the normal female genital tract. Endocervical cells and monocytes however, contain CAP.  Preneoplastic lesions of the cervix arise in the transformation zone, which contains CAP. Hence, abnormal cells of both low- and high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively) are positive for CAP. Other lesions like ASC-H (atypical squamous cells can not rule out high-grade SIL) and other cervical intraepithelial neoplasia (CIN) lesions also express CAP. Studies conducted decades ago have described acid phosphatase in cervical cancer, preneoplastic lesions of the cervix and in vaginal secretions of patients suffering from cervical and uterine cancer. ,, The present study was undertaken to assess the utility of CAP-PAP in addition to routine Pap smears as an aid to visualization of abnormal squamous cells and in screening for cervical carcinoma and its precursors.
Materials and Methods
The study was conducted on healthy women undergoing routine cervical smear screening in the months of January and February 2005. Two smears were collected from each subject. One was alcohol-fixed and stained by the conventional Pap stain and evaluated using the Bethesda System (2001) for reporting cervical/endocervical/vaginal cytology (TBS).  Slides were subjected to routine reporting and were also re-evaluated with the CAP-PAP slide as part of this study.
Procedure for CAP-PAP
The second smear of each patient was processed by the CAP-PAP technique using the BioSciCon's MarkPap® Research kit (M/S Ricca Chemical Company, Arlington, TX, USA). Immediately after the collection of a routine cervical smear, slides for CAP-PAP were immersed in a special fixative instead of alcohol. The fixative solution was prepared by mixing 15 ml citrate solution, 15 ml distilled water, 60 ml acetone and 10 ml 4% formaldehyde. Slides were fixed at room temperature for 50 seconds, followed by rinsing in two changes of distilled water (DW). Slides were then air-dried and stored in a dust-free container at 4-6°C.
At the time of staining, the incubation solution was freshly prepared by mixing ten drops of Fast Garnet GBC with ten drops of sodium nitrate (incubated at room temperature for three minutes) in an Erlenmeyer flask containing 46 ml prewarmed DW and 2.5 ml acetate solution. Ten drops of substrate solution (containing alpha-naphthyl AS-BI-phosphate) was finally added to this mixture. Slides were then rehydrated and incubated for 45 minutes at 37°C in the dark, followed by serial rinsing in running tap water, DW and finally in phosphate buffered saline (PBS) for five minutes each. Subsequently, slides were counterstained using a modified Pap technique with different incubation times in Gill hematoxylin (four minutes), OG-6 solution (three minutes), and EA-65 solution (five seconds), as compared to the conventional Pap stain. Finally, slides were mounted using Crystal Mount (M/S Biomeda Corp, Foster City, CA, USA), and evaluated at magnifications varying between 100× and 400×.
COMBO slides (combination of HeLa cell lines with buccal cell lines) (M/S Ricca Chemical Company, Arlington, TX, USA) were used as external controls. HeLa cells displayed red, granular cytoplasmic deposits indicating CAP activity, while squamous cells of buccal origin served as a negative control. Inflammatory and endocervical cells in cervical smears served as internal positive controls as they are always positive and can easily be distinguished from squamous cells. Staining was adjusted to achieve optimal staining of these cells, avoiding overstaining.
Evaluation of CAP-PAP smears
Positivity in CAP-PAP smears was visualized as red, granular deposits at the sites of enzyme activity inside cells, while other cellular structures and CAP-negative cells were counterstained by the modified Pap technique. Cytomorphology of all CAP-positive cells was carefully evaluated to distinguish abnormal squamous cells (as per TBS).  For analysis, smears showing CAP positivity in endocervical or metaplastic squamous cells were considered negative by the CAP-PAP test. Smears having CAP-positive mature squamous cells or CAP-positive squamous cells with nuclear enlargement or atypia were considered positive by the CAP-PAP test.
The present study consisted of cervical smears from 81 consecutive subjects attending the Gynecology OPD for routine screening. The mean age of the subjects was 35.4 years (range 25-55 years).
Cytological features on routine Pap stain
On routine reporting, out of a total of 81 smears, 73 were reported 'negative for intraepithelial lesion or malignancy', which included one case each of an atrophic smear, candidiasis, and Trichomonas vaginalis [Table 1]. Three smears were unsatisfactory. Of the remaining smears, one was LSIL and four were atypical squamous cells of undetermined significance (ASCUS), of which one was combined with atypical glandular cells of undetermined significance.
On re-evaluation of routine smears with CAP-PAP smears, among the 73 routine smears initially considered negative, two were re-evaluated as LSIL while another was re-evaluated as ASCUS. In each of these three instances, strong CAP-positive, large squamous cells were identified in CAP-PAP smears. Careful searching of the corresponding Pap smears located a few cells, evaluated finally as LSIL (two cases) and ASCUS (one case). For the purpose of analysis, the diagnosis after re-evaluation was taken as the final diagnosis on routine cytology. Thus, a total of 8/81 smears had abnormality comprising of three with LSIL and five with ASCUS [Table 2].
Cytological profile on CAP-PAP stain
The CAP-PAP stain was easy to perform following the manufacturer's instructions and gave uniform staining results. Endocervical cells always showed marked cytoplasmic granular red deposits. There was no extracellular diffusion [Figure 1] although in many instances, the positivity was intense and obscured nuclear details. Squamous metaplastic cells were always CAP-positive [Figure 1]. Red granules were seen mostly as small foci of positive staining in the cytoplasm of metaplastic cells, although many cells showed quite intense staining of most of the cytoplasm. There was no deposition on the nucleus whose morphology could be evaluated.
Parabasal, intermediate, and superficial cells were uniformly negative, except in smears considered positive with CAP-PAP. Inflammatory cells showed CAP-positivity, with monocytes and neutrophils having the most intense cellular reaction with CAP-PAP. Trichomonas vaginalis and fungal profiles of Candida were CAP-negative. Fungal contamination occurred in a few slides; however, this did not interfere with evaluation.
Smears having CAP-positive mature squamous cells or CAP-positive squamous cells with nuclear enlargement or nuclear atypia were considered positive in the CAP-PAP test. In such smears, CAP-positive cells were seen focally, either as isolated cells or cell groups. Staining intensity was variable but usually quite intense and unmistakable [Figure 1]E, F. There was no deposition on the nucleus, permitting evaluation of nuclear details. A total of 16 smears (19.75%) showed CAP positivity [Table 1]. In 8/16 smears, the positivity was in mature squamous cells in which the nuclei appeared enlarged. This included all eight cases evaluated as LSIL or ASCUS on routine Pap staining. Of the remaining eight, six showed mature squamous cells without nuclear enlargement to be focally CAP-positive (possibly mature squamous metaplasia) while in two smears, metaplastic cells with nuclear enlargement (sufficient to arouse the anxiety of an ASCUS diagnosis) showed CAP positivity. No abnormal cells were identified on the corresponding routine Pap smears of these eight cases. The remaining 65 smears were negative for CAP.
Comparison of Pap stain with CAP-PAP stain
Overall, the CAP-PAP screening demonstrated a sensitivity of 100% and specificity of 89%, as compared to the conventional Pap stain. The predictive value of a positive CAP-PAP test was 50%, while it was 100% for a negative test. Overall, there were no false negatives in the study, while false positives were 11%.
Benign and malignant lesions of the uterine cervix have been evaluated for a number of histochemical reactions including ribonucleic acid, glycogen, acid phosphatase, nonspecific esterase, glucuronidase, and phosphamidase. , ,,,, Of these, acid phosphatase evoked considerable interest due to its easy intracellular localization using cytochemical techniques. , High acid phosphatase activity was seen in normal basal cells, 'atypical' basal cells, histiocytes, and endocervical cells as well as in malignant squamous cells. , Subsequently, acid phosphatase remained unnoticed until 1997, when it was first proposed for its role in detecting atypical squamous cells on routine Pap smears. 
The present study utilized the CAP-PAP technique of Markovic ,,, and selectively detected CAP in endocervical cells, metaplastic cells, monocytes, leukocytes, and atypical squamous cells. Parabasal, intermediate, and superficial cells were always negative. This study was designed to compare the efficacy of the CAP-PAP test with the conventional Pap test for screening of cervical smears. CAP-PAP had a sensitivity of 100% for detecting atypical squamous cells with no false negative cases. This is similar to earlier observations.  The most important aspect of the present study is the re-evaluation prompted by CAP-PAP positivity. Of 16 cases found to be positive by CAP-PAP, five were initially positive in routine Pap smears (considering ASCUS and above as positive). After the CAP-PAP test, three more smears were upgraded to being positive. In two instances, occasional squamous cells with LSIL had been overlooked while in one instance; focal atypical cells with features pointing toward the suspicion of LSIL (ASCUS) were identified. Earlier studies  also observed a significantly improved rate of detection of abnormal cells, which helped to reduce the false negative rate.
Although technical simplicity and ability to detect SIL lesions appears to be advantageous for the CAP-PAP test, this technique does have many interpretation problems. In comparison to the conventional Pap stain, the modification adopted for this test appears to be technically inferior in evaluating nuclear features. Nuclear details are the most essential criteria for detecting atypical squamous cells. In some instances, endocervical cells with very high cervical acid phosphatase activity where the red granules tend to shroud the nucleus are difficult to evaluate. Overstaining needs to be scrupulously avoided. The presence of enzyme activity in squamous metaplastic cells implies that only a person well-versed with routine cytopathology can evaluate a CAP-PAP smear. Otherwise, all smears with metaplastic squamous cells would be considered positive, which in the absence of clear nuclear details, can mimic an atypical cell and lead to an incorrect diagnosis. Due to these reasons, we have taken CAP to be positive when found in a mature squamous cell regardless of the nuclear features as the features of LSIL may be difficult to evaluate on CAP-PAP. Similarly, any CAP-positive cell with nuclear enlargement or atypia, regardless of its size or presumed nature, needs to be considered as being a positive result with CAP-PAP. Metaplastic squamous cells can show nuclear enlargement, as seen in two smears in the present study. Such cells would also be CAP-positive and would be considered positive with the CAP-PAP test. In all these instances, a conventional Pap smear is essential for proper evaluation of nuclear features. Despite this caveat, the smears considered as being false positive in this study showed sufficient nuclear details in the CAP-PAP smear to be categorized as being negative in the hands of a competent cytopathologist. The issue of false positives is not a concern if a two-tier screening procedure is followed with a pathologist reviewing the positive slides. Other limitations to this technique were encountered. There was fungal contamination of some smears despite adequate precautions. The special fixative needs filtration and refrigeration, hence, it may not be practical in peripheral labs.
The role of the CAP-PAP test in screening for cervical cancer needs greater evaluation. A screening technique requires high sensitivity so that the disease is not missed if present. The CAP-PAP test fulfils this criterion. We feel that its greatest utility would be for quality assurance and selecting cervical smears for re-screening. CAP positivity helps to focus attention on only the significant cells. Ignoring the CAP-negative cells greatly speeds up the screening process. The role of CAP-PAP in resource-poor settings also needs appraisal. It would be far easier to train cytoscreeners to recognize CAP positivity, ignoring CAP-positive metaplastic cells, than to train them to accurately pick up LSIL and HSIL on screening. All patients with CAP-positive smears can be re-called for re-evaluation. In the present study, around 20% of smears were CAP-PAP positive. Hence, the load on regular cytology services can be cut to one-fifth the normal level. Similarly, use of CAP-PAP with HPV detection using HC2 is an option. As HC2 is expensive, an initial screening with CAP-PAP can be done with collection of specimen for HC2. CAP-PAP-positive cases alone need to undergo HC2, while specimens of CAP-PAP-negative patients can be discarded or stored. In addition, this method can be easily adapted for automated, computer-assisted screening including telepathology-assisted screening.
In the absence of HSIL cases in the present study, the diagnostic ability of the CAP-PAP test in such cases remains hypothetical and needs to be evaluated in future studies. As endocervical groups are intensely CAP-PAP-positive, it may be difficult to detect small cells of CIN III using this technique, which would be a major limitation. However, other reports have not encountered such a problem. ,,,
Thus, the main role of the CAP-PAP test may be for initial screening or as an adjunct for quality assurance surveys in cervical smears. Furthermore, this technique appears promising in low-resource settings lacking trained cytologists. When used in conjunction with routine Pap smears, it has the potential to improve the sensitivity of the latter, translating into an improved cervical smear-screening program.
|1||Bristow RE, Montz FJ. Workup of the abnormal Pap test. Clin Cornerstone 2000;3:12-24.|
|2||Markovic O, Markovic N. Can acid phosphatase reduce pap test false-negative readings? J Natl Cancer Inst 1997;89:1459.|
|3||Volgareva G, Zavalishina L, Andreeva Y, Frank G, Krutikova E, Golovina D, et al. Protein p16 as a marker of dysplastic and neoplastic alterations in cervical epithelial cells. BMC Cancer 2004;4:58.|
|4||O'Meara AT. Present standards for cervical cancer screening. Curr Opin Oncol 2002;14:505-11.|
|5||Chang JI, Ou CH, Wu KM, Chen CC, Cheng KC. The evaluation of cervical cancer screening by combining speculoscopy with Papanicolaou smear examination in Taiwan. Zhonghua Yi Xue Za Zhi 2002;65:430-4.|
|6||Moscicki AB, Durako SJ, Ma Y, Darragh T, Vermund SH. Utility of cervicography in HIV-infected and uninfected adolescents. J Adolesc Health 2003;32:204-13.|
|7||Nieminen P, Vuorma S, Viikki M, Hakama M, Anttila A. Comparison of HPV test versus conventional and automation-assisted Pap screening as potential screening tools for preventing cervical cancer. BJOG 2004;111:842-8.|
|8||Kumar K, Iyer VK, Bhatla N, Kriplani A, Verma K. Comparative evaluation of smear cytology and hybrid capture II for the diagnosis of cervical cancer. Indian J Med Res 2007;126:39-44.|
|9||Jarboe EA, Thompson LC, Heinz D, McGregor JA, Shroyer KR. Telomerase and human papillomavirus as diagnostic adjuncts for cervical dysplasia and carcinoma. Hum Pathol 2004;35:396-402.|
|10||Fung Kee Fung M, Senterman M, Eid P, Faught W, Mikhael NZ, Wong PT. Comparison of Fourier-transform infrared spectroscopic screening of exfoliated cervical cells with standard Papanicolaou screening. Gynecol Oncol 1997;66:10-5.|
|11||Markovic O, Markovic N. Cervical acid phosphatase: a biomarker of cervical dysplasia and a potential surrogate endpoint for colposcopy. Dis Markers 2003-2004;19:279-86.|
|12||Markovic O, Markovic N, Belledone M. Cervical acid phosphatase - Papanicolaou (CAP-PAP) test. J Histotechnology 1999;22: 43-7.|
|13||Markovic O, Markovic N. Acid phosphatase in cervical smears (CAP-PAP test). Arch Oncol 1998;6:137-9.|
|14||Malvi SG, Sirsat SM. A cytochemical study of acid phosphatase in carcinoma of the cervix uteri. Indian J Cancer 1974;11:81-7.|
|15||Gross SJ, Kinzie G. Cytochemistry of benign and malignant squamous epithelium of the cervix uteri. Acid phosphatase, nonspecific esterase, and alkaline phosphatase. Obstet Gynecol 1960;15:261-79.|
|16||Panazzolo A, Bergantino L, Arrotta S, Napoli F, Pacilli L. Lysosomal enzymes in neoplastic pathology of the cervix uteri. Study of acid phosphatase, as a diagnostic test, in vaginal secretions of patients with pre-invasive and invasive carcinoma of the cervix. Minerva Ginecol 1978;30:1123-45.|
|17||Smith JH. Bethesda 2001.Cytopathology 2002;13:4-10.|
|18||Danziger S, Gross SJ. Histochemical techniques applied to the study of benign and malignant squamous epithelium of the cervix uteri. Am J Obstet Gynecol 1957;73:94-119.|
|19||Walker BS, Lemon HM, Davison MM, Schwartz MK. Acid phosphatases; a review. Am J Clin Pathol 1954;24:807-37.|
|20||Blonk DI, Schaberg A, Willighagen RG. Enzyme cytochemistry of benign and malignant cells in pleural and peritoneal fluid. Acta Cytol 1967;11:460-5.|
|21||Fishman WH, Mitchell GW Jr, Dimitrakis H, Hayashi M. Enzymorphology of adenocarcinoma of the endometrium; beta-glucuronidase, acid phosphatase, reduced diphosphopyridine nucleotide (DPNH) diaphorase, and alpha-naphthyl esterase. Cancer. 1963;16:126-32.|
|22||Santis H, Shklar G, Chauncey HH. Histochemistry of experimentally induced leukoplakia and carcinoma of the hamster buccal pouch. Oral Surg Oral Med Oral Pathol 1964;17:207-18.|
|23||Burton JF. Histochemical demonstration of acid phosphatase by an improved azo-dye method. J Histochem 1954; 2: 88.|
|24||Pearse AGE. Histochemistry, theoretical and applied. 3rd ed. London: Churchill Livingstone; 1972. p. 728.|