SurePathTM Liquid-based Cytology Research Articles

Cervical Cytology-Histopathology Concordance and Role of Dual Immunomarkers in Biopsy Samples of Uterine Cervix: A Cross-sectional Study from a Tertiary Care Hospital of West Bengal, India

Introduction: Cervical cancer is one of the leading causes of cancer deaths. Liquid-based cervical cytology enables the detection and diagnosis of the disease at an early stage. p40 is a specific immunomarker that distinguishes squamous cell carcinomas from other cervical carcinomas with glandular and neuroendocrine differentiation. Aim: To determine the role of Liquid-based Cytology (LBC) in the early detection of premalignant and malignant lesions of the uterine cervix and its concordance with histopathological findings. Materials and Methods: This cross-sectional study was conducted at the Institute of Post Graduate Medical Education and Research (IPGME&R), Kolkata, West Bengal, India, over a period of one year and six months (from October 2020 to March 2022). Cervical samples were collected from a total of 200 females, and the materials were processed using the BD SurePath™ LBC method. The cytologically confirmed cases were biopsied, and histopathological concordance was established. Immunohistochemical (IHC) staining for p40 and p63 was performed using the peroxidase-antiperoxidase method to differentiate between premalignant, malignant squamous, and glandular lesions of the uterine cervix. For quantitative analysis, all cells were counted in 10 random fields at 400x magnification, and p63 and p40 were expressed as a percentage of positive cells per the total number of counted cells. Cases were considered positive if 5% or more of the tumour cell nuclei showed brown nuclear staining. The mean percentage positivity of p63 and p40 nuclear staining of all the cases in each category was calculated for non neoplastic and precursor/neoplastic lesions of the cervix. Results: Among the 200 cases evaluated by LBC, 169 (84.5%) were non neoplastic, and the remaining 31 (15.5%) were precursor/neoplastic lesions. The vast majority (126 cases, 63%) were inflammatory smears, followed by 20 cases (10%) that were Negative for Intraepithelial Lesion or Malignancy (NILM). Total of 40 cases were followed-up with biopsy and histopathology, and concordance with the cytological diagnosis was evaluated. Among 40 cases, 23 were precursor/ neoplastic on histopathology, and seven cases that were non neoplastic on Histopathological Examination (HPE) had been correctly diagnosed on LBC, giving a concordance rate of 75%. The overall sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) of LBC were 92%, 46.6%, 74.1%, and 77.7%, respectively, considering histological diagnosis as the gold standard. Regarding IHC expression on histopathology, the mean positivity of p40 and p63 for non neoplastic lesions was 9.40% and 10.06%, and for precursor/neoplastic lesions, 47.44% and 46.4%, respectively. Adenocarcinoma-in-situ and adenocarcinoma were negative for both p40 and p63. Conclusion: Cervical cytology is a less invasive, cost-effective, and simple procedure to diagnose cervical epithelial cell abnormalities. Cytological-histopathological concordance revealed that LBC is a sensitive diagnostic method. The comparison between non neoplastic and neoplastic lesions of the cervix revealed a statistically significant difference with respect to the mean percent positivity of p40 and p63 IHC staining. Both of these markers can be used to differentiate squamous cell carcinoma from adenocarcinoma of the cervix.

Cervical Cytology-Histopathology Concordance and Role of Dual Immunomarkers in Biopsy Samples of Uterine Cervix: A Cross-sectional Study from a Tertiary Care Hospital of West Bengal, India

Introduction: Cervical cancer is one of the leading causes of cancer deaths. Liquid-based cervical cytology enables the detection and diagnosis of the disease at an early stage. p40 is a specific immunomarker that distinguishes squamous cell carcinomas from other cervical carcinomas with glandular and neuroendocrine differentiation. Aim: To determine the role of Liquid-based Cytology (LBC) in the early detection of premalignant and malignant lesions of the uterine cervix and its concordance with histopathological findings. Materials and Methods: This cross-sectional study was conducted at the Institute of Post Graduate Medical Education and Research (IPGME&R), Kolkata, West Bengal, India, over a period of one year and six months (from October 2020 to March 2022). Cervical samples were collected from a total of 200 females, and the materials were processed using the BD SurePath™ LBC method. The cytologically confirmed cases were biopsied, and histopathological concordance was established. Immunohistochemical (IHC) staining for p40 and p63 was performed using the peroxidase-antiperoxidase method to differentiate between premalignant, malignant squamous, and glandular lesions of the uterine cervix. For quantitative analysis, all cells were counted in 10 random fields at 400x magnification, and p63 and p40 were expressed as a percentage of positive cells per the total number of counted cells. Cases were considered positive if 5% or more of the tumour cell nuclei showed brown nuclear staining. The mean percentage positivity of p63 and p40 nuclear staining of all the cases in each category was calculated for non neoplastic and precursor/neoplastic lesions of the cervix. Results: Among the 200 cases evaluated by LBC, 169 (84.5%) were non neoplastic, and the remaining 31 (15.5%) were precursor/neoplastic lesions. The vast majority (126 cases, 63%) were inflammatory smears, followed by 20 cases (10%) that were Negative for Intraepithelial Lesion or Malignancy (NILM). Total of 40 cases were followed-up with biopsy and histopathology, and concordance with the cytological diagnosis was evaluated. Among 40 cases, 23 were precursor/ neoplastic on histopathology, and seven cases that were non neoplastic on Histopathological Examination (HPE) had been correctly diagnosed on LBC, giving a concordance rate of 75%. The overall sensitivity, specificity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) of LBC were 92%, 46.6%, 74.1%, and 77.7%, respectively, considering histological diagnosis as the gold standard. Regarding IHC expression on histopathology, the mean positivity of p40 and p63 for non neoplastic lesions was 9.40% and 10.06%, and for precursor/neoplastic lesions, 47.44% and 46.4%, respectively. Adenocarcinoma-in-situ and adenocarcinoma were negative for both p40 and p63. Conclusion: Cervical cytology is a less invasive, cost-effective, and simple procedure to diagnose cervical epithelial cell abnormalities. Cytological-histopathological concordance revealed that LBC is a sensitive diagnostic method. The comparison between non neoplastic and neoplastic lesions of the cervix revealed a statistically significant difference with respect to the mean percent positivity of p40 and p63 IHC staining. Both of these markers can be used to differentiate squamous cell carcinoma from adenocarcinoma of the cervix.

Optimal Papanicolaou Smear Conditions for Manual Microdissection of Single Target Cells.

This study aimed to investigate the optimal conditions for Papanicolaou (Pap) smear to increase the success rate of target cell isolation through manual microdissection (MMD) and prevent cell spread. Pap smears were prepared using an HPV42-positive SurePath™ liquid-based cytology case, and 46 and 50 koilocytes were used in wet and dried Pap smears, respectively, to verify the success rate of target cell isolation using MMD based on the HPV detection rate. During MMD, the microscopic examination of both specimens revealed that cells in dried smears could be easily identified; however, cell debris remained in the surrounding area after MMD. Although it was difficult to observe cells in wet smears, there was no cell debris. When the needle tip was immersed in DNA lysate after cell isolation through MMD, a difference in cell solubility was found between dry and wet smears. HPV42 was detected in 94.7% and 97.4% of dried and wet Pap smears, respectively, via polymerase chain reaction genotyping using lysed cell solution; the detection rates were not significantly different. The isolation of target cells from wet Pap smears using MMD reduced the risk of contamination and increased the success rate of HPV detection. This study might facilitate the identification of new CIN-derived HPV-infected cells using MMD with wet Pap smears.

ThinPrep liquid-based cytologic diagnosis of hepatic entomophthoromycosis (basidiobolomycosis) with Splendore-Hoeppli phenomenon.

Zygomycotic fungal infections are increasingly recognized in immunocompetent and immunocompromised patients in nonendemic countries because of the increased global travels. One of the emerging fungal infections is Entomophthorales which cause deep visceral infections that clinically and radiologically mimic malignancies because they form invasive obstructing masses. A quick diagnosis is life-saving. The current standard diagnosis relies on histopathologic examination and microbiology culture which are time consuming. Cytologic examination is a rapid reliable alternative diagnostic tool. There are a few reports of a primary cytologic diagnosis of zygomycotic and Aspergillus fungi of the lungs based on conventional smear and SurePath™ liquid-based cytology. We report a case of an immunocompetent adult female who presented with a liver mass that was clinically and radiologically diagnosed as cholangiocarcinoma. Fine-needle aspiration cytology revealed the cytopathologic features of basidiobolomycosis in ThinPrep® cytology. The ThinPrep® slide revealed broad thin-walled widely branching septate hyphae with sporangiophores and zygospores. The background revealed bile-stained granular necrotic debris intermixed with inflammatory cells, degenerate hepatocytes, and highly eosinophilic glassy crystal-like structures suggestive of Splendore-Hoeppli phenomenon. The cytologic diagnosis was hepatic entomophthoromycosis (basidiobolomycosis) with Splendore-Hoeppli phenomenon. This was confirmed by subsequent tissue biopsy and microbiology culture. The detailed cytomorphologic features in the ThinPrep® slide allowed the correct cytologic diagnosis and proper classification of the filamentous fungi. The ThinPrep® cytology was superior to tissue biopsy because it was faster and revealed the characteristic pauciseptate fungal hyphae, sporangiophores and zygospores. Liquid-based cytology is a valuable quick tool in the diagnosis of fungal mass-forming lesions.

A study on preserving endometrial glandular architecture during preparation using BD SurePath™ liquid‐based cytology reagents: Cellular fixation with preservative fluid requires at least 18 h

AbstractIntroductionThis study aimed to determine the causes of disruption of the three‐dimensional architecture of endometrial glands prepared using BD SurePath™ liquid‐based cytology (SP‐LBC) reagents. One sample preparation method for endometrial cytology is presented in which this three‐dimensional architecture can be retained.MethodsSP‐LBC specimens were prepared by the following three methods: (1) using the BD PrepMateTM (PrepMate) System after cellular fixation for 1‐6 h (method A); (2) without using the PrepMate System after cellular fixation for 1‐6 h (method B); and (3) using the PrepMate System after cellular fixation for at least 18 h (method C). Size and numbers of endometrial cell clusters and numbers of solitary scattered cells were then evaluated.ResultsSignificantly higher numbers of cell clusters with a major axis of 200 μm or more were yielded by method C (71.3 ± 57.2) than methods A (9.3 ± 5.9, P < 0.001) or B (44.3 ± 28.8, P < 0.05). Method B yielded significantly higher numbers of cell clusters than method A (P < 0.001). Method A (132.2 ± 107.7, p < 0.001) yielded significantly higher numbers of solitary scattered cells than methods B (29.1 ± 14.8) and C (35.7 ± 23.3). No significant difference in solitary cell numbers was found between methods B and C.ConclusionsRetention of endometrial glandular architecture is rendered possible by allowing sample fixation times of 18 h or more when preparing specimens using the PrepMate System.

Impact of SurePath® liquid-based preparation in cytological analysis of peritoneal washing in practice of gynecologic oncology

Context:Peritoneal washing is performed for staging of gynecologic tumors to detect subclinical intraperitoneal metastases.Aim:The aim of the present study was to assess the impact of SurePath™ liquid-based cytology (LBC) in peritoneal washing in various gynecological malignancies.Settings and Design:An audit of peritoneal-fluid/washing (January 2012 to July 2013) was performed with corresponding gynecologic specimens. All peritoneal washings were processed using both conventional and LBC technique. Suspicious cases on cytology were reported along with gynecologic specimens.Results:There were a total of 393 peritoneal fluids. Eighty-three (21.1%) were positive for malignancy, and the corresponding histology was available in 352 (89.6%) cases. Sixty-nine positive samples had ovarian malignancies and 5 had uterine causes. There were 9 cases of peritoneal washings in which no histopathology was available. The most common cause of positive peritoneal cytology was ovarian serous carcinoma in 55/84 (65.5%) cases. Other causes included mucinous cystadenocarcinoma, dysgerminoma, squamous cell carcinoma in teratoma, yolk sac tumor, and granulosa cell tumor. Uterine causes included 2/45 (4.4%) cases of endometrioid adenocarcinoma, ¼ (25%) cases of clear cell carcinoma, ½ (50%) cases of carcinosarcoma, and ¼ (25%) cervix carcinoma. On review of positive cases (n = 83), 10 cases were identified, which had nil (n = 4) to low cellularity (<3 tumor clusters/smear; n = 6) on conventional smears, and were confirmed malignant on LBC.Conclusions:The most common ovarian malignancy causing positive peritoneal cytology is papillary serous carcinoma. Endometrioid adenocarcinoma rarely leads to positive peritoneal cytology. LBC technique leads to concentration of tumor cells causing reduction in false negative cases, especially in hemorrhagic and low-cellular cases.

液状処理法と従来法での採取器具による子宮頸部細胞像の比較検討

目的 : 液状処理法と従来法において採取器具をかえて得られる子宮頸部細胞像について比較検討する.方法 : 705 例からそれぞれ 4 つの子宮頸部細胞診標本を作製した. 綿棒+従来法 (サンプル A) ・ブルーム型ブラシ+従来法 (同 B) ・綿棒+液状処理法 (同 C) ・ブラシ+液状処理法 (同 D). ベセスダシステム 2001 に基づき, 細胞像を中心に比較検討した.成績 : 頸管腺細胞の出現はサンプル A で 7%, B で 87%, C で 4%, D で 75%とブラシで有意に多かった. 背景が清明な標本はサンプル A で 76%, B で 64%, C で 90%, D で 81%と液状処理法で有意に多かった. 従来法での細胞少数例はサンプル A で 10%, B で 3%であった. 液状処理法で細胞数 5,000 個以下はサンプル C で 25%, D で 9%であった. 従来法で 705 例中 44 例, 液状処理法で 42 例に細胞診断の不一致がみられたが, ブラシにおいてそれぞれ 36 例と 37 例にハイグレイドの病変と診断された. HSIL の検出についてはサンプル B と D で差はなかった.結論 : ブラシ+液状処理法で最も異型細胞を検出する機会が多く, ブラシによる細胞採取法が推奨される.

BD シュアパス&lt;sup&gt;TM&lt;/sup&gt;液状処理細胞診システムへの粘液添加実験

目的 : BD シュアパスTM液状処理細胞診システムは, 粘液, 血液, debris などを除去する前処理工程を標準化した標本作製システムである. BD シュアパスTMバイアルに粘液 (喀痰) を添加し, 前処理工程の性能を明らかにする.方法 : 表層型扁平上皮細胞数を 40 倍で 1 視野約 10 個に調整した BD シュアパスTMバイアルに 2000∼5000μl までの粘液を添加し, シュアパス標本を作製した. 標本は 40 倍で表層型扁平上皮細胞を 10 視野カウントし, 平均値を算出した. 粘液 5000μl 添加を行った前処理群と未処理群の各 100 データを用いて, マンホイットニーの U 検定を実施した.成績 : 表層型扁平上皮細胞の平均カウント数は, 粘液の添加なし (0μl) で 10.6 個, 2000μl 添加で 42.3 個, 3000μl 添加で 40.3 個, 4000μl 添加で 35.9 個, 5000μl 添加で 27.0 個, 前処理工程なしの 5000μl 添加で 5.0 個であった. 5000μl までは, 粘液が除去され, 扁平上皮細胞数が増加した. 5000μl 未処理群は, 処理群に比較し扁平上皮細胞数は統計学的に有意に減少した (p<0.001).結論 : BD シュアパスTM法に標準化された前処理工程は, 粘液による不適正標本の排除と診断上重要な細胞を回収し, 子宮頸部病変の検出向上に寄与する.

Eight cases of malignant mixed müllerian tumor (carcinosarcoma) of the uterus: Findings in surepath™ cervical cytology

Malignant mixed Müllerian tumor (MMMT)/carcinosarcoma is a rare neoplasm of the female genital tract characterized by a mixture of epithelial and mesenchymal components. There are published reports of conventional cervical smear findings in uterine MMMT, but to the best of our knowledge the cytomorphology of MMMT in SurePath™ liquid-based cytology samples has not been described. We present a series of eight cases of uterine MMMT in SurePath™ cervical samples. The mean age of the women was 65.5 years (range, 60-82 years) and the most common presenting symptom was postmenopausal bleeding. Three out of the eight cases had both epithelial and sarcomatous elements in highly cellular cervical samples. In two cases, abnormal glandular cell clusters were closely associated with the mesenchymal component. The glandular component was usually represented by three-dimensional clusters of malignant glandular cells and a dispersed population of large, round to oval malignant epithelial cells with single prominent nucleoli. Necrosis or tumor diathesis was not seen in any cases. Six out of the eight cases were initially reported as adenocarcinoma, endometrial type, and two cases as normal. Five cases had an atrophic background and tiny atypical glandular clusters that could easily be overlooked. Although cervical sample is not the method of choice for primary uterine carcinosarcoma screening, the possibility of the same should be kept in mind, when reporting a cervical sample from a woman with postmenopausal vaginal bleeding.

Analytical performance of RNA isolated from BD SurePath™ cervical cytology specimens by the PreTect™ HPV-Proofer assay

Several commercial HPV ancillary tests are available for detection of E6/E7 RNA. It is not clear how storage of a cervical Pap affects the analytical and clinical performance of the PreTect™ HPV-Proofer assay. To investigate the qualitative performance of RNA extracted from BD SurePath™ liquid-based cytology (LBC) specimens for the detection of human papillomavirus (HPV) E6/E7 mRNA using the PreTect™ HPV-Proofer assay, studies including stability, reproducibility, residual specimen analysis, and storage medium comparison assays were performed. Cervical cytology specimens were collected and stored in BD SurePath™ LBC preservative fluid and/or PreTect™ Transport Media. RNA was isolated using the RecoverAll™ Total Nucleic Acid Isolation kit and RNA integrity was evaluated in the PreTect™ HPV-Proofer assay. The performance of RNA isolated from cervical cells collected and stored in BD SurePath™ LBC preservative fluid or PreTect™ Transport Media was also evaluated through a storage medium comparison study. The RNA was found to be stable for a minimum of 21 days when stored at ambient temperature and displayed high reproducibility with the mean percentage reproducibility ranging from 90.5% to 100% for the HPV types detected by the PreTect™ HPV-Proofer assay. The prevalence rate of HPV types in this study cohort was consistent with published reports. A 93.7% first pass acceptance rate was demonstrated across all cytology grades. The positive human U1 snRNP specific A protein (U1A) and HPV rate for BD SurePath™ LBC and PreTect™ Transport Media specimens was statistically equivalent for both normal and abnormal specimens. This data support the use of RNA isolated from BD SurePath™ LBC for ancillary HPV testing and demonstrates the feasibility of using BD SurePath™ preservative fluid as a specimen type with the PreTect™ HPV-Proofer assay.

Diagnostic value of urine erythrocyte morphology in the detection of glomerular disease in SurePath™ liquid‐based cytology compared with fresh urine sediment examination

To assess whether the morphology of urine erythrocytes can be an effective tool for distinguishing glomerular disease from lower urinary tract disease in SurePath™ liquid-based cytology (SP-LBC). We examined four morphological parameters of erythrocytes: (1) irregular erythrocytes (of all types including fragmented forms) comprising greater than or equal to 20% of erythrocytes; (2) uniform erythrocytes (>80%); (3) doughnut or target-like shaped (D/T) erythrocytes (≥1%); and (4) acanthocytes (≥1%) in glomerular disease (n = 32) and lower urinary tract disease (n = 20) with SP-LBC slides in cases that had also been assessed by fresh urine sediment examination. Sensitivity of D/T erythrocytes and acanthocytes (dysmorphic erythrocytes) for glomerular disease were 100% and 87.5%, respectively, with urine sediment examination, and 81.3% and 46.9%, respectively, in SP-LBC slides. Specificity was 100% for D/T erythrocytes and acanthocytes using either procedure. While irregular erythrocytes were specific for glomerular disease using urine sediment examination, they were seen in 70% of those with lower urinary tract disease using SP-LBC slides as a result of the deformation of erythrocytes by the fixative. Although the sensitivity of D/T erythrocytes and acanthocytes for glomerular disease was lower in SP-LBC slides than fresh urine sediment examination, their specificity was equally high. Therefore, urine erythrocyte morphology is useful in the detection of glomerular disease with the SP-LBC slides. However, morphological features apart from D/T erythrocytes and acanthocytes are not useful in SP-LBC slides.

Clinical Usefulness of SurePath™ Liquid-based Cytology in Thyroid Fine Needle Aspiration: Comparison with the Conventional Smear in Diagnostic Efficacy and Applicability ofBRAFMutation Test

Clinical Usefulness of SurePath™ Liquid-based Cytology in Thyroid Fine Needle Aspiration: Comparison with the Conventional Smear in Diagnostic Efficacy and Applicability of<i>BRAF</i>Mutation Test

Glandular neoplasia and borderline endocervical reporting rates before and after conversion to the SurePathTM liquid‐based cytology (LBC) system

Reporting rates for glandular neoplasia in 464,754 cervical samples reported at six laboratories in 12-month periods before and after the implementation of Surepath™ LBC processing are compared. The introduction of LBC processing is seen to have resulted in a significant (P = 0.001) increase in the detection rate for endocervical glandular neoplasia (from 2.2 per 10,000 tests to 3.9 per 10,000) while maintaining high levels of reporting specificity. An observed fall in the number of samples reported as showing borderline glandular neoplasia falls short of statistical significance, and the reporting of possible endometrial and 'other' glandular abnormalities appears to be unaffected. The underlying reasons for the observed improvement in detection of endocervical glandular neoplasia are discussed.

Improved sensitivity over time with rapid prescreening in gynecologic cytology

Rapid prescreening (RPS) is a powerful tool to measure and improve performance in the cytology laboratory. Long-term use of RPS has been shown to result in improved sensitivity and precision in routine screening. The effect of long-term RPS on RPS itself is not known. We compared the sensitivity of 100% RPS of Surepath™ liquid-based cytology over a 4-year period in a laboratory of 11 cytotechnologists (CTs). In comparison with the first 2 years, RPS for the laboratory showed a significant increase in sensitivity for all abnormalities (72.2% vs. 67.3%, P < 0.001) and ASCUS + LSIL (68.5% vs. 62.1%, P < 0.001). For individual CTs s, the lowest sensitivity for all abnormalities increased from 59.0 to 63.2%, whereas, for HSIL, it increased from 71.4% to 75.0%. We conclude that long-term use of RPS leads to significant increase in the sensitivity of RPS.

Isolation of RNA from residual BD SurePath™ liquid-based cytology specimens and detection of HPV E6/E7 mRNA using the PreTect™ HPV-Proofer assay

Infection with high-risk human papillomavirus (HPV) is known to be associated directly with the development of cervical cancer. Recent data suggests that the detection of E6/E7 mRNA from high-risk HPV types may serve as a better diagnostic method for detecting the presence of cervical pre-cancer than HPV DNA testing. This report details a commercially available nucleic acid isolation protocol which can be used to isolate reproducibly RNA from residual BD SurePath™ liquid-based cytology specimens stored for up to 28 days, and have demonstrated the quality and quantity of mRNA is sufficient for detection with the NorChip PreTect™ HPV-Proofer assay. Of the 242 specimens tested in this study, 236 (97.5%) tested positive for U1A internal control gene expression. HPV type 16, 18, 31, 33 or 45 mRNA was detected in 16/20 (80%) of the analyzed high-grade squamous intraepithelial lesion (HSIL) specimens, with a low frequency of HPV mRNA detected in the normal lesions (3%). The presence of HPV E6 expression in a subset of HPV positive specimens was also detected by real-time RT-PCR. These findings confirm that RNA of sufficient quality can be isolated from residual BD SurePath™ cervical cytology specimens for use in downstream NASBA and RT-PCR-based assays.

P‐11 THE IMPACT OF INTRODUCING LIQUID BASED CYTOLOGY INTO A ROUTINE SCREENING LABORATORY

With the exception of information from the liquid based cytology (LBC) pilot site there has been limited data to date on the impact of the introduction of SurePath™ LBC in the NHSCSP. We will present data to show the impact on a medium sized laboratory (42 000 requests per annum) over the first phases of rollout. Data from before, during and post conversion, a period of 28 months, shows the following trends: (1) A significant fall in the inadequate rate (2) A slight decrease in the borderline / mild dyskaryosis rate (3) A small increase in the high‐grade squamous dyskaryosis reporting rate (4) An increase in both the number and specificity of glandular lesions reported (5) A largely unchanged positive predictive value for high‐grade abnormalities (6) A significant increase in laboratory productivity, unrelated to inadequate rate but through increased speed of screening. Although our experience is not directly comparable to the experience of the LBC pilot, our observations may well reflect that we were fortunate to be trained by staff from one of the LBC sites and thereby benefited directly from their experience.