Conventional versus Liquid-based Cytology: "Man versus Machine".

Background: Liquid-based cytology (LBC) can improve adequacy, monolayer quality with a clean background compared to conventional smears (CS). Aims and Objectives: The objective was to compare the quality and diagnostic yield of CS and LBC in routine cytological investigations. Materials and Methods: This retrospective study consisted of 306 samples (255 gynecological, 39 nongynecological, and 12 fine needle aspiration cytology [FNAC]) during a 2-year period (2019-2020). From each patient, two samples were collected in the same manner in the same sitting and processed by CS and LBC (ThinPrep® 2000, Hologic Inc.). Both CS and LBC were compared for adequacy, quality, representativeness, inflammation, hemorrhage, necrosis, preservation, reactive changes, organisms, atypia/dysplasia/malignancy, and preparation/screening time. Statistical analysis was performed. Results: No statistically significant difference was noted for adequacy, representativeness, reactive changes, preservation, and atypia/dysplasia/malignancy. CS was better in cellularity and diagnosis of inflammation and organisms, whereas LBC had a clean background and the difference was statistically significant (P = 0.0005). Conclusions: CS was equivalent to LBC in adequacy, representativeness, reactive changes, and atypia/dysplasia/malignancy. Adequacy comparable to LBC can be achieved in CS by careful sample collection, processing, and screening by trained cytotechnologists. CS was better in detecting organisms and inflammation than LBC. The advantages of LBC were monolayer smear, clean background, and lesser screening time, but the demerit was higher cost and longer processing time. Therefore, LBC is best suited to those laboratories having high sample inadequacy rates, lack of competent cytotechnologists, and no financial constraints. Either man or machine, appropriate and adequate sample collection by trained personnel forms the cornerstone for ensuring adequacy in both CS and LBC.

Heat Induced Processing of Cellblocks with Significant Reduction in Overall Turn Around Time.

Introduction: Cellblock (CB) with immunohistochemistry (IHC) is practically indispensable in the diagnostic workup of serous effusions; however, CB requires a minimum of 15-20 h for routine histopathological processing. A reduction in processing time can expedite a faster diagnosis. Aim: This study was undertaken to evaluate the utility of the heat-induced CB (HICB) technique. Material and Methods: Two sets of agar-embedded CBs were processed from 50 effusion samples. CBs were further processed by conventional and rapid methods. Conventional CBs (CCB) were processed in a histoprocessor, whereas rapid CB was processed in a heated water bath with an agitation facility. For HICB processing, dehydration and clearing were performed at 50°C followed by paraffin wax impregnation at 65°C temperature. From both CBs, sections of 5 um thickness were cut and stained with hematoxylin and eosin (H and E). Cell morphology, cost, and time were compared between the two methods. The feasibility of IHC was attempted in a few cases. Results: HICB was completed within 4.30 h compared with CCB. Diagnoses on both CBs were concordant in all the cases. Incomplete dehydration was noted in six (12%) cases, but the diagnosis was not compromised. No additional cost was involved in HICB. On IHC, both HICB and CCB exhibited equivalent expression. Conclusions: HICB is a rapid, innovative, simple, and cost-effective technique and expedites faster diagnosis. It does not require any advanced equipment.

Study of histopathology reports of loop electrosurgical excision procedure of cervical transformation zone and their correlation with preprocedural cervical biopsy and/or cytology: An audit.

Introduction: Loop electrosurgical procedure of the transformation zone of the cervix (LEEP) is the preferred method for many investigators for early detection and treatment of high grade intraepithelial neoplasia(HGCIN). Histopathology reports of LEEP should contain information about the diagnosis, presence or absence of neoplasia ( with its grade) and comment on excison margins. Aim: Our aim was to study LEEP reports for its contents and to see their correlation with preprocudure histology and/or cytology report. Results: Between 2011 and 2017, 44 LEEP reports were archived and studied for their contents from our records. Slides were not reviewed. Mean age was 47.66 years (median 47 years). Forty two (( 95.45%) reports mentioned that all the tissue was examined. Deep cut examination was mentioned in 17/44 cases (38.64%). The concordance rate between LEEP and preprocudure histology and /or cytology for CIN II plus diagnosis is 65.9%. A strict definition is used. If, however, diagnoses between inflammation and CIN I, ASC-H and inflammation, and ASC-H and CIN I are considered non discordant, then the concordance rate rises to 72.7 %. The breakup of discordant cases is given. Conclusion: Literature shows wide range of concordance due to variable definitions and variety of reasons; possible reasons are discussed.

Incremental value of endoscopic brush cytology in response assessment after chemo-irradiation for Esophageal cancer.

BACKGROUND: Response assessment after chemo-radiotherapy (CTRT) in locally advanced esophageal cancer is usually performed using a PET-CT scan, an upper GI endoscopy (UGIE) and histological correlation with biopsy or cytology. We aim to study the incremental value of brush cytology in addition to PET-CT for response assessment. MATERIALS AND METHODS: In this retrospective analysis, 40 patients with Stage II- IV carcinoma esophagus treated with radical intent between June 2015 and August 2019 were included. Patients were treated with either upfront concurrent CTRT or neo-adjuvant chemotherapy followed by CTRT. All patients underwent PET-CT and UGIE for initial staging and response assessment on follow-up. Patients with esophageal stricture (disease related or treatment induced) had brush cytology done during UGIE. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of brush cytology were calculated considering serial clinical follow-up as gold standard. RESULTS: Twenty-three male (57.5%) and 17 (42.5%) female patients with median age of 57 years (range: 27 - 79 years) were analyzed. Concurrent CTRT was delivered in 52.5%; 75% patients were treated with intensity-modulated radiotherapy (IMRT); median RT dose was 63 Gy (range- 41.4 to 64 Gy). At a median follow-up of 16 months (range 6- 54 months), 20 patients (55.5%) were clinically controlled, 9 (25%) had local recurrence, 5 (13.8%) had loco-regional recurrence and 2 had distant metastasis. Considering clinical follow-up as the gold standard, sensitivity, PPV and NPV of PET-CT combined with brush cytology improved compared to PET-CT alone and was found to be 75%, 90%, 85.7% and 81.8% respectively. CONCLUSION: We found that brush cytology on endoscopy is a simple tool with high specificity which adds value to the findings of response assessment PET-CT scan and thereby can increase the confidence of the treating oncologist in making clinical decisions.

Is Diagnosis of Low-Grade Urothelial Carcinoma Possible in Urine Cytology?

BACKGROUND: Urine cytology is a useful modality, primarily for the diagnosis and follow-up surveillance of high-grade urothelial carcinoma (HGUC). Its utility in diagnosing low-grade urothelial carcinoma (LGUC) remains controversial because of low reported sensitivity compared to cystoscopy. AIM: To study the cytomorphology of LGUC in voided urine samples and analyze its utility in diagnosis. MATERIALS AND METHODS: This is a retrospective study of one year, including 48 voided urine samples in cases which were confirmed as LGUC on subsequent histology. Urine cytology smears of these cases, originally stained with Papanicolaou stain were reviewed, critically analyzed and the specific cytomorphologic and cystoscopic findings were documented. RESULTS: On review 18 samples were re-categorized as LGUC which included 10 samples initially diagnosed as Negative for HGUC, 2 as Atypical Urothelial Cells - Not Otherwise Specified (AUC-NOS) and 6 as Suspicious for Carcinoma. In addition, another 3 samples with initial diagnosis of LGUC remained as LGUC on review. Thus, a total of 21 LGUC samples were identified after the review. 26 (54%) samples with a diagnosis of negative for HGUC remained negative even after review, as the tumor cells were not identified either due to sampling error or unrecognizable morphology. One (2%) samples of AUC-NOS remained the same on review due to very scant atypical cells. In 21 LGUC samples, cytology showed a dual population of benign differentiated urothelial cells and small urothelial cells with subtle nuclear atypia such as irregular and thickened nuclear membrane with increased nuclear cytoplasmic ratio. In 12 false negative LGUC samples, the diagnostic cells were camouflaged by their subtle nuclear atypia coupled with an overwhelming background of differentiated benign urothelial cells as both appeared almost similar in morphology. Papillary fragments were identified only in 2 samples. CONCLUSIONS: Diagnosis of LGUC on cytology is challenging and depends on the presence of diagnostic cells, pick up of diagnostic cells on screening and accurate interpretation. Special attention to papillary fragments and aforementioned nuclear atypia should be paid as tumor cells may resemble normal urothelial cells and can be easily missed.

Evaluation of cell blocks from effusion specimens in Gynecologic Oncopathology: An experience of 220 cases, diagnosed at a Tertiary Cancer Referral Center.

One of the common indications of ascitic fluid examination in gynecological oncopathology is the detection and classification of malignant cells, especially in cases of clinically suspicious tubo-ovarian masses. The present study was undertaken to assess and validate the diagnostic utility of cell blocks (CBs) and compare its results with the corresponding conventional smears, prepared from effusion samples. CBs were prepared by thromboplastin technique in 220 cases. In 208 cases, diagnostic concordance between results obtained from smears and corresponding CBs was evaluated. Various antibody markers were tested, as per individual case. The average age of patients was 52.2 years. Positive immunohistochemical (IHC) staining for various markers was observed in 182 cases (82.7%) The most frequently positive antibody marker was PAX8 (101/134), followed by p53 (85/92) [mutation type (either diffusely positive or completely negative)], WT1 (tumor cells) (80/112), calretinin (2/87) (diffuse), BerEP4 (21/49), CA125 (21/24), CK7 (31/39) and CK20 and CDX2, together (5/16). Various other IHC markers utilized, including their positive expression, were TTF1 (1/10), p40 (3/3), p63 (2/4), ER (21/29), HBME1 (1/7), GATA3 (1/4), and MIC2 (1/1). Complete diagnostic concordance between CBs and smears was observed in 170/208 cases (81.7%). There were 20 major discordances, 10 minor and 8 cases with sampling errors. IHC was useful in classifying 158/182 (86.8%) cases, including serous or Müllerian adenocarcinoma (n = 123), mostly high-grade (121); metastatic squamous carcinoma (3); gastrointestinal-type adenocarcinoma (8); pulmonary adenocarcinoma (1); breast adenocarcinoma (1); Ewing sarcoma (1); and mesothelioma (2). CBs are complementary to smears in the detection of gynecological malignancies, mostly high-grade serous adenocarcinomas. These provide an opportunity for testing several IHC markers, for a precise diagnosis, including in various uncommon case scenarios, associated with significant therapeutic implications.

Reliability of conventional Papanicolaou smear in diagnosing bacterial vaginosis among women with clinical genital infection.

OBJECTIVE: Bacterial vaginosis (BV) is a common reproductive tract infection (RTI) reported among Indian women. BV can influence the persistence of high-risk oncogenic human papillomavirus, a causative factor for cervical cancer. BV and cervical cancer are major public health issues in a developing country like India. It becomes important for a resource-constrained country like India with poor healthcare access to implement control measures to screen and treat RTI in an attempt to prevent the risk for cervical cancer. Papanicolaou (Pap) smear is an established screening tool for cervical cancer and the diagnosis of RTIs, forms a part of its evaluation. The present study explores the validity of conventional Pap smear in diagnosing BV. METHODOLOGY: Pap smear and Gram stain smear were collected for 254 women with clinically evident cervicitis/cervicovaginitis (genital infection). Using the Nugent score on Gram stain as a gold standard, we determined the sensitivity and specificity of Pap smear to diagnose BV. RESULTS: The overall prevalence of BV in the study population was 44% using the Nugent score. Pap smear showed sensitivity and specificity of 70.9%. (CI- 61.5% - 79.2%) and 56.8% (CI - 48.2%-65.2%), respectively. The positive predictive value of Pap smear to diagnose BV was 56.5% (CI - 47.8%-64.9%), and the negative predictive value was 71.2% (CI - 61.8%-79.4%). CONCLUSION: In the present study, conventional Pap smear demonstrates good accuracy to detect BV. Pap testing for cervical cancer screening can additionally serve as an effective screening tool for diagnosing BV among women with genital infection in healthcare settings.

Indian Academy of Cytologists Guidelines for Collection, Preparation, Interpretation, and Reporting of Serous Effusion Fluid Samples.

Cytological examination plays an important role in the initial work-up of the serous cavity effusion fluids to find out the possible etiology as benign or malignant. Among malignant effusions, cytology is helpful in determining the exact type, site, and stage of the tumor. However, for reporting effusion cytology specimens, there is no consistent and reproducible reporting system. AIMS: The aim of these guidelines is to provide a standardized format for effusion cytopathology right from sample receipt to its ultimate report sign-out for implementation in all cytopathology laboratories. The Indian Academy of Cytologists in consultation with experts across the country has prepared guidelines pertaining to collection, preparation, and diagnostic categories of effusion specimens to reduce reporting variability. The guidelines are made keeping in mind the different areas of practices in India, especially low- and medium-resource settings. The guidelines are broadly divided into essential, optimal, and optional categories for best usage and appropriate allocation of the precious specimens. In referral centers or well-established setups, essential ancillary techniques can be done for accurate and final diagnosis. By adhering to and implementing these uniform guidelines, it is hoped that clinical patient care and management in India will improve and be of uniformly good quality by enabling and facilitating good laboratory practices.

Is the Morning Sputum Sample Superior to the Fresh Sputum Sample for the Detection of Malignant Cells?

INTRODUCTION: Sputum cytology is a well-established technique for the detection of lung malignancies. Generally, random or morning samples are used. OBJECTIVES: To evaluate the diagnostic yield of morning sputum and compare it with that of fresh samples. MATERIALS AND METHODS: Patients were instructed to bring a morning sputum sample to the laboratory in a clean plastic container, without any fixative and within 2-3 h of collection. Fresh sputum was then collected in the laboratory from these same patients. Two smears were prepared from each sample by the "pick and smear" technique and then stained by the Papanicolaou method. One hundred samples from each method (total 200 samples; 400 slides) were evaluated by 3 investigators for their adequacy, preservation, and yield of diagnostic cells. The results were analyzed by using the Pearson χ2 test. RESULTS: Cytomorphological details were preserved in 82/84 satisfactory morning samples and in 80/81 satisfactory fresh samples, respectively. Malignancy was detected in 37 morning samples (44%) and 25 fresh samples (30.8%). In the malignant samples, there were more abundant tumor cells in the morning samples than in the fresh samples (65 and 40%, respectively) with a 2+ cellularity in the morning samples. The morning samples showed a better cell yield (25% more), with a 13% increase in the rate of detection of malignancy and an increased sensitivity of 19.68% compared to the fresh samples. CONCLUSIONS: Adequacy and preservation was similar in both sample types. While the morning samples showed a higher sensitivity and a larger number of tumor cells than the fresh samples, the difference was not statistically significant (p < 0.054).

Case report of renal cell carcinoma diagnosed in voided urine confirmed by CD10 immunocytochemistry.

BACKGROUND: Preoperative diagnosis of renal cell carcinoma (RCC) by exfoliative urine cytology is difficult, as infiltration of RCC into the pelvicalyceal system is uncommon. The exfoliation of RCC cells in urine is a rare phenomenon and when it does occur, it is likely to be missed. Cytologic examination of the urine coupled with ancillary immunocytochemistry can clinch the diagnosis leading to appropriate clinical management. CASE: A 50-year-old man presented with complaints of hematuria and abdominal pain of 6 months' duration. Ultrasonographic examination of the abdomen and pelvis showed a well-defined mass lesion in the upper pole of the left kidney, suggestive of neoplastic etiology. In the given clinical context of renal mass, urine cytology was suggestive of RCC and biopsy confirmation was suggested. One cytology smear subjected to immunocytochemistry with anti-CD10 antibody which showed strong diffuse cytoplasmic positivity in these cells confirmed the diagnosis of RCC. Subsequently, fine needle aspiration cytology of the kidney mass was reported as RCC. CONCLUSIONS: RCC has distinct cytologic features that facilitate a diagnosis in urine in an appropriate clinical and radiological context. Their recognition in the urine smear is important to avoid costly and invasive modalities like image-guided needle biopsy.

Papanicolaou stain: Is it economical to switch to rapid, economical, acetic acid, papanicolaou stain?

OBJECTIVE: To standardize an inexpensive and rapid Papanicolaou staining technique with limited ethanol usage. STUDY DESIGN: Smears from 200 patients were collected (2 per patient) and fixed in methanol. Half were subjected to conventional Papanicolaou and half to stain ing with rapid, economical, acetic acid Papanicolaou (REAP) stain. In REAP, pre-OG6 and post-OG6 and post-EA36 ethanol baths were replaced by 1% acetic acid and Scott's tap water with tap water. Hematoxylin was preheated to 60 degrees C. Final dehydration was with methanol. REAP smears were compared with Papanicolaou smears for optimal cytoplasmic and nuclear staining, stain preservation, cost and turnaround time. RESULTS: With the REAP method, cytoplasmic and nuclear staining was optimal in 181 and 192 cases, respectively. The staining time was considerably reduced, to 3 minutes, and the cost per smear was reduced to one fourth. The staining quality remained good in all the smears for > 2 years. CONCLUSION: REAP is a rapid, cost-effective alternative to Papanicolaou stain. Though low stain penetration in large cell clusters is a limitation, final interpretation was not compromised.