Study of cap pap versus conventional pap in suspicious cervical lesions

BACKGROUND: CAP Pap test is a new test developed as an adjunct to routine PAP to improve its sensitivity. This test combines a simple biochemical test of enzyme labelling with conventional Pap Test in which abnormal squamous cells of the cervix are labelled for the presence of the Lysosomal enzyme, cervical acid phosphatase (CAP). Objectives: 1. To establish the sensitivity of new test CAP PAP in cervical carcinoma screening. 2. To assess the utility of CAP PAP as an adjunct to routine PAP. MATERIAL AND METHODS: The study comprised of 100 females who were randomly selected. Two smears of each patient were examined by panel of four experts independently and reported. The study was a single centric, random assignment, blinded, 2-group (test and control), and split-sample design to assess safety and efficacy of the new test in comparison with the control for cervical cancer screening in standard Pap test environment. Cervical biopsy was the Gold standard test for the present study. This data was compared and the sensitivity and specificity of the test was noted. RESULT: CAP PAP showed greater sensitivity(100% )as compared to Pap test(75%), while specificity(97.2% )is little lesser than Pap(100%). CONCLUSION: The CAP-PAP Test is a simple diagnostic tool which can be used with routine Pap staining on the same slide adding to the accuracy of routine Pap .This Test could be the future of cervical carcinoma screening. Keyword: Cervical Carcinoma, Screening, sensitivity Study of cap pap versus conventional pap in suspicious cervical lesions 103 Int J Res Med. 2015; 4(1);102-108 e ISSN:2320-2742 p ISSN: 2320-2734 squamous cells of the cervix are labelled for the presence of the Lysosomal enzyme, cervical acid phosphatase (CAP). 8,9,10 Demonstration of acid phosphatase in tissues and cells is based on enzyme catalysis of organophosphate substrate, capture of phosphate by a metallic ion (i.e. lead), or an organic radical (aromatic ring) by a diazonium salt, formation of a product which is insoluble at acid pH range (pH<5.0), and precipitation of a colourful, granular deposit at sites of enzyme activity which was available for microscopic examination. The amount is measurable and it is proportional to acid phosphatase activity. 11,12,13 Acid phosphatase is abundant in metabolically active cells in inflammation and malignancy. The alteration of synthesis, processing and trafficking of lysosomal enzymes in malignancy has been demonstrated. 14,15,16,17 A consistent increase of lysosomal enzymes (i.e., prostatic acid phosphatase) has been found in tumor cells in comparison with their normal counterparts .18 This property contributes to "aggressiveness" of malignant cells. In blood cells, an increase of acid phosphatase activity was found in connection with infection and inflammation (i.e., PMN, monocytes) 19,20 In 1986-88, Markovic et al investigated a number of human tissue specimens versus many cytochemical techniques to select candidates for quantitative image analysis. In one of series, cervical smears were exposed to cytochemical techniques for demonstration of lysosomal enzymes. Surprisingly, acid phosphatase activity was found in atypical squamous epithelial cells while normal-looking cells did not present this type of activity. Acid phosphatase activity was inversely proportional to maturity of epithelial cells, and abnormal-looking cells possessed most active acid phosphatase. 8 The double-staining, single-slide procedure in which acid phosphatase could be stained inside abnormal cervical cells, and counterstained by a modified Papanicolaou (for visualization of classical cytological criteria) was named the Cervical Acid PhosphatasePapanicolaou (CAP-PAP) Test. Cervical acid phosphatase(CAP) test 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 ( will serve as internal quality control for adequacy of sampling and staining) 8 .(Fig 2 ) CAP activity increases with the degree of cervical dysplasia .CAP activity is also present in cervical cancer cells, and in HeLa cell line cells derived from human cervical cancer . Control slides made of HeLa cell line cells and buccal cells (COMBO controls) serve as external QC/QA. 9,10 (Fig.1) MATERIAL AND METHODS The study comprised of 100 females who were randomly selected according to inclusion and exclusion criteria of study and these were the females whose follow up was available. The study was approved by Institutional ethical committee. Inclusion criteria 1. All women aged 21-70 yrs who have ever had sex & who attended gynaecological O.P.D of tertiary health centre. 2. Females with gynaecological complaints like bleeding/discharge per vaginum were included. 3. All females who attended Pap smear screening camps. (Those without positive history were considered as control) 4. Women who have been attending ART centre. Exclusion criteria 1. Females in menstruation. 2. Those who had a normal Pap smear less than a year ago. 3. Less than 21 years of age. 4. The cervix showing obvious inflammation The two smears collected from the female The conventional cervical smears were fixed by 100% methanol whereas CAP PAP smear was fixed by fixative made by  25 ml Citrate Solution  65 ml acetone, and  8 ml 37% formaldehyde. Placed in glass bottle and capped tightly. It Study of cap pap versus conventional pap in suspicious cervical lesions 104 Int J Res Med. 2015; 4(1);102-108 e ISSN:2320-2742 p ISSN: 2320-2734 was stored under refrigeration at 2 to 8 degree Celsius. Staining First smear was stained as the conventional Pap staining procedure. Second smear labelled as CAP PAP was stained by following technique. Staining procedure 9 was same as Pap staining plus steps for staining of enzyme (Cervical Acid Phosphatase).This includes the following steps: marker visualization and counterstaining.(by conventional Pap stain). Slides were fixed at room temp for 50 sec, followed by rinsing in two changes of distilled water. Slides are then air dried and stored in dust free container at 4-6degree Celsius. At the time of staining, incubation solution is freshly prepared by mixing a.10 drops sodium nitrate b.10 drops of Fast Garnet GBC (incubated at room temp for three minutes) in an Erlenmeyer flask containing 46ml prewarmed distilled water and 2.5ml acetate solution. c.Ten drops of substrate solution (containing alpha napthyl ASBI phosphate) was finally added to this mixture. Slides are to be rehydrated and incubated for 45min at 37 degree Celsius in the dark , followed by serial rinsing in running tap water , distilled water ,and finally in phosphate buffered saline for 5min each . Slides are then counterstained using a modified pap smear with different incubation time in Gill haematoxylin (4min) OG-6(3min) EA -65 solutions (5 sec) Slides were mounted using crystal mount. Eventually evaluated at magnification b/w 100x and 400x Interpretation of CAP PAP smear CAP is a brown-red deposit scattered throughout the cytoplasm of "abnormal" cervical cells. PAP staining produces a colour that is combination of original Papanicolao recommendation: Hematoxylin stains cervical cells nuclei blue, and adds a bluish coloration to the color of cytoplasm. Orange G (OG-6) stains cervical cell cytoplasm orange (red + yellow). Eosin Alcohol (EA-65) stains cervical cell cytoplasm red. However, Light Green added to the solution of EA, causes green color of cytoplasm. The "ideal" staining will produce the following results: CAP -red-brown individual granules scattered through cytoplasm. Other cytological features (nucleus, nuclei, vacuoles, other granulation) easy distinguishable. CAP staining produces a red-brown granular precipitate at intracellular sites of enzyme activity. Counterstaining assists presenting cell morphology, cell identification and classification. Two smears of each patient were examined by panel of four experts independently and reported. STUDY DESIGN (Statistics) The study was a single centric, random assignment, blinded, 2-group (test and control), and split-sample design to assess safety and efficacy of the new test in comparison with the control for cervical cancer screening in standard Pap test environment. Cervical biopsy was the Gold standard test for the present study. Data was collected as a. The smears positive by Pap, b. The smears positives by CAP PAP. This data was compared and the sensitivity and specificity of the tests was noted .Appropriate statistical tests were applied. 1) Efficacy was measured with primary end points (portion of positive/abnormal specimens detected, and the false negative rate). 2) Accuracy was measured by a. Sensitivity & b. Specificity.