Conventional Smear vs. Liquid-Based Preparation in Fine Needle Aspirations Cytology

Introduction 

Fine Needle Aspirations Cytology (FNAC) serves as a critical modality regarding the diagnosis and management of deep-seated and superficial malignant and benign lesions. FNAC provides the primary investigative method for diagnosing and managing cancerous lesions in different body parts, such as the head & neck, breast, and cervix. The assessment method is considered non-invasive, pain-free, fast, simple, and can be practised safely. FNAC is accomplished through conventional smears and liquid-based preparations. The procedure involves the insertion of a thin needle into the area with abnormally appearing fluid or tissue for sample collection to enable examination under a microscope. The samples collected can be analyzed through conventional smears or liquid base preparations. While liquid-based preparation has been identified as more efficient in diagnosing gynecological cases than conventional smears, data on its efficacy in non-gynecological diagnosis remain inconsistent. Conventional smear is widely accepted as a diagnostic tool for developing diagnosis and treatment plans for malignant and benign lesions in different body parts, given its cost-effectiveness, repetition possibilities, safety, and simplicity. Liquid-based cytology (LBC) was developed for improved accuracy and reliability of Papanicolaou smears. The comparative efficiency and effectiveness of the two techniques have been the subject of extensive studies, with results conflicting between different researchers; the review suggests the lack of evidence for recommending one approach over the other.

Conventional Smears

Conventional smear has been in use for the last few decades as the preferred technique for diagnosing and developing treatment plans for malignant and benign lesions. The technique gained wide acceptance in FNAC due to its cost-effectiveness, possibility of repetition, safety, and simplicity. Despite the potential presented by the technique, it presents certain limitations such as poor cellularity, background obscuring, and smearing technique variation, resulting in dry artifacts.¹ These limitations may lead to sample inadequacy, negatively affecting the overall effectiveness of diagnosis. According to Chong et al., sample inadequacy is observed in approximately 50.5% of conventional smear tests due to the limitations identified.¹ As a result, the technique presents high chances of misdiagnosis of malignant and benign lesions.

Liquid-Based Cytology

LBC was first introduced in gynecological cervical cytology as an alternative for conventional smears. The technique involves collecting aspirates liquid fixative. The next step is the removal of inflammatory cells, red blood cells, and cell debris, followed by vortexing for homogenization.² The final step of sample preparation is slide preparation achieved through sedimentation or vacuum application techniques. Liquid-based preparations can be realized through two systems: SurePath and ThinPrep.² As an alternative to conventional smears, the two systems minimize artifacts and variations, standardizing smears through automation.² Even though originally developed specifically for gynecological cervical cytology; the technique has proven more effective in terms of outcome reproducibility, duration of interpretation, and visualization clarity, with its application extending to non-gynecological specimens. The efficacies provided by LBC derive from its association with homogenous cellular smears and well-preserved cell morphology.³ Developed to address the limitations of conventional smear, liquid-based preparation demonstrates less obscuring by rinsing materials such as inflammatory cells, mucus, and blood, enables more interpretation accuracy, serves as a faster and more efficient alternative, has comparatively fewer unsatisfactory smears.³ The comparatively fewer unsatisfactory specimen in liquid-based preparations is attributed to a lack of obscuring material, enabling better visualization under a microscope. Most importantly, the technique enables the testing of human papillomavirus DNA using residual cell suspension.

Similarities between Conventional Smear and Liquid-Based Cytology 

The conventional smear and liquid-based preparations techniques share a few similarities despite serving a similar role in diagnosing malignant and benign lesions. First, the techniques are deemed simple, fast, and non-invasive; both involve obtaining sample cells from abnormal or suspicious body fluid or tissue by inserting a thin needle into the affected area for specimen collection to be examined under a microscope.¹ The processes involved in both FNAC techniques are relatively pain-free and safe.

Slide preparation procedures between LBC and conventional smear are significantly similar, with slight variations. Both techniques involve collecting sample cells from suspected lesions using a thin needle, which is smeared on glass slides.¹ Despite the differences between the techniques regarding the presence of surrounding cellular matrix, cell distribution is considerably similar. LBC may involve the use of monolayer smears or thin layers of sample cells distributed in either small or large patches. While the monolayer distribution of sample cells is not characteristic of conventional smears, the technique shares the other characteristics.³ For instance, some parts of conventional smears are thinner than the thin layers used in liquid-based preparations. Lastly, smears in both approaches are stained with antibodies linked to either fluorescent dyes or enzymes that become activated upon the binding of the antibodies to the cell sample antigen to enable visualization of the antigen under a microscope.⁴ Nevertheless, researchers need to pay particular attention to smear thickness, given that the slide cellularity may not always indicate the original specimen cellularity.

Difference between Conventional Smear and Liquid-Based Cytology

Sample Preparation

Studies assessing the morphology and the accuracy of diagnosis between conventional smears and liquid-based preparations in gynecological and non-gynecological specimens have established critical differences between the two techniques. The conventional smear technique involves the collection of sample cells from a suspicious or abnormally appearing fluid or tissue which are transferred to a glass slide for viewing under a microscope.¹ Give the direct transfer of specimen to glass slides, blood, mucus, and inflammatory cells are often transferred along with the sample cells, potentially complicating the interpretation process due to background obscuring. Liquid-based preparation offers a solution to obscuring by blood, mucus, or inflammable cells transferred to glass slides for observation under a microscope. The process involves the collection of sample cells from abnormally appearing fluid or tissue; the collected sample is first rinsed in liquid to remove blood samples, among other forms of impurities that may compromise the clarity of viewing.³ The rinsed sample cells are isolated by processing the liquid, after which they are observed under a microscope spread on a glass slide in a thin layer or monolayer spreading.

Procedures

Unlike the single-step procedure in the conventional smear, liquid-based preparation involves two steps; fixation in a preservative liquid/solution and automated processing of the prepared slide. Fixation in a preservative solution results in certain critical advantages, including viewing size area and cell allocation consistency, better cellular preservation, randomization of sample cells and complete capture of sample cells. Furthermore, automated screening is vital for enhanced accuracy. Computerized processing eliminates or minimizes human error; it ensures that scanning covers the entire field of view, unlike manual scanning in conventional smears, which is subject to attention lapses. While conventional smears are affected by human biases, non-objectivity, and variability, computerized scanning allows standardization of assessment and uniform application throughout the slide. Effectiveness in human beings is subject to factors such as preoccupation with diagnosis and the immediate surroundings of the sample cells. The lack of human biases often translates to improved specificity in detecting malignant or benign lesions, improving the likelihood of truly positive or truly negative outcomes. Computerized scanning also has the potential for increased sensitivity, hence improved lesion detection potential. Human beings are more likely to ignore other areas of the slide, concentrating on areas identified to present the greatest probability, increasing the likelihood of false negatives or false positives.

Technique Sensitivity

Sensitivity provides a vital area of distinction between conventional smears and LBC. Sensitivity in FNAC refers to the level of effectiveness with respect to detecting tissue or fluid abnormalities, such as cervical malignancy. Some studies recognize the LBC as superior to the conventional approach. A study by Chong et al. suggests that liquid-based preparation is associated with detecting more malignant and benign abnormalities compared to the conventional smear technique, which produces fewer unsatisfactory samples.¹ The researchers identify conventional smear as limited in terms of inter-individual variation, sample adequacy, accuracy, and clinical utility; however, the liquid-based preparation technique is identified as capable of overcoming such limitations, hence the relatively superior approach to conventional preparations.¹ Nevertheless, the debate about sensitivity between the FNAC techniques varies depending on the location of the suspicious tissue, making it a contested issue among researchers.

Sensitivity between conventional smears and LBC differ based on the site of the malignant or benign lesion under examination. Conventional smear has served as the primary test for different types of carcinogenic cells in various body parts. On the other hand, liquid-based was originally developed for gynecological cervical cytology, where it was deemed to provide improved effectiveness over the conventional Papanicolaou smear. Nishio et al. identify LBC as more sensitive than conventional smear with respect to gynecological applications.⁴ Compared to a sensitivity rate of 98.8% in conventional smears, liquid-based preparation achieved a sensitivity rate of 100%, making it the best-suited technique for cervical cytological examinations.⁴ Liquid-based preparations also proved more sensitive at 87.7% than conventional preparation at 83.2% concerning fine-needle aspiration of pancreatic masses.⁵ Contrarily, the sensitivity of conventional smears proved higher compared to LBC in the diagnosis of thyroid lesions. A study by Chong et al. examining thyroid lesion cells showed a sensitivity rate of 100% for the conventional smear technique against a 71.4% sensitivity rate for LBC.¹ Based on the Study findings, the sensitivity of each technique varied based on the types of lesions under assessment.

Technique Specificity

Another area of distinction between the two FNAC techniques is specificity. Specificity measures how well a technique excludes malignant and benign abnormalities. Various studies suggest that specificity, similar to sensitivity, varied based on the types of benign and malignant cells examined. Nishio et al. identify liquid-based preparation as having a relatively higher sensitivity rate at 23.8% than 17.2% realized with conventional smear in cervical cytology.⁴ Regarding the application of conventional smear and liquid-based preparations in diagnosing thyroid lesions, Chong et al. suggest no significant differences in terms of specificity, with both techniques realizing approximately equal rates.¹ Similar outcome is demonstrated in the examination of solid pancreatic neoplasm, with both conventional smear and LBC demonstrating specificity rates of 100%.3 The findings demonstrate that the two techniques present different specificity rates based on the types of lesions being diagnosed.

Technique Accuracy

Accuracy provides one of the most critical areas of malignant and benign lesion examinations. While both conventional smear and LBC are critical techniques for diagnosing and managing different types of lesions, various research findings associate the latter technique with better accuracy rates across different types of malignancies. Applying the two techniques in diagnosing pancreatic neoplasm demonstrated significant accuracy differences; conventional smear achieved an accuracy level of 83.8% compared to an accuracy rate of 88.0% realized with liquid-based smear in diagnosing cervical cancer.³ In addition, the rate of unsatisfactory smears is 7.1% and 1.61%, with specimen inadequacy rates of 1.3% and 1.2% in conventional smears and LBC, respectively, regarding cervical cancer diagnoses.^6,7 Accurate detection of recurrence of cervical malignancy was realized at rates of 79.16% and 97.6% with conventional smear and liquid-based preparation, respectively.⁸ The relatively higher accuracy rates associated with liquid-based is attributed to the absence of background obscuring. On the contrary, Yeon et al. suggest that conventional smears proved more effective in diagnosing biliary and pancreatic lesions at an accuracy rate of 78.1% and 91.7% compared to an accuracy rate of 54.5% and 77.1% realized with LBC.⁹ The lower accuracy realized observed in LBC results due to low cellularity in relation to conventional smear. The study findings referenced suggest that accuracy levels in diagnosing malignant and benign lesions are based on the types of cancers examined. In addition, the accuracy rate between the two may also be influenced by the system of liquid-based preparation employed.

Positive Predictive Value and Negative Predictive Value

Positive and negative predictive values are critical measures of the effectiveness of FNAC examination of malignant and benign lesions. Positive predictive value refers to the percentage of patients whose results are actually positive among those whose test results indicated positive, hence prediction of the likelihood of a patient truly being sick in case of a positive test result.¹⁰ Negative predictive value refers to the ratio of cases that are actually negative to all patients whose test results turned out negative, indicating the likelihood of a patient being truly free from cancer in the event of a negative result.¹⁰ Positive and negative predictive values serve as measures of accuracy, hence differ between the conventional smear and LBC and between the types of malignancies examined. In cervical cytology, conventional smear demonstrated a higher rate of positive predictive value at 57.9% compared to 56.1% in LBC.⁴ On the other hand, the negative predictive value was higher in LBC than in conventional smear at 100% and 94.7%, respectively.⁴ While the positive predictive value is nearly equal between the two techniques, the difference in terms of negative predictive value suggests that conventional smear in cervical cytology is comparatively more prone to false negatives. A comparative study involving the diagnosis of salivary gland neoplasm indicates that both the conventional smear and liquid-based preparation approaches have equal predictive potentials; the positive predictive potential for both techniques is 100%, while the negative predictive potential varied slightly at 97.6% for conventional smear and 97.7% for liquid-based preparation.¹¹ Overall, there are no significant predictive differences between the two FNAC techniques.

Cost-Effectiveness

Conventional smear and LBC differ significantly in terms of the costs involved. In comparison to conventional smear, liquid-based preparation is considered the low-cost option. The cost-effectiveness associated with liquid-based preparations derives from the simplicity and efficiency afforded by the technique. It allows mono-layered smears characterized by clearly visible morphological details. This leads to a higher pick-up rate of low-grade lesions and infections, enabling the process to be achieved by low-cost equipment.¹² The associated efficiencies and effectiveness also translate into reduced screening time. Other than improved detection rates, the liquid-based preparations demonstrate better potential for detecting the possibility of recurrence of malignancy and reducing disease progression at comparatively lower costs.13 On the contrary, obscuring material makes conventional smear more complex, requiring comparatively higher skill levels and advanced examination equipment to guarantee accurate and reliable outcomes.

Immunostaining in Conventional Smear and Liquid-Based Cytology

Staining in FNAC involves immunocytochemistry and immunohistochemistry. Immunocytochemistry is a method used in the laboratory to realize anatomical visualization of specific antigens or proteins recognized as markers. It involves visualizing the antigens or protein in sample cells using antibodies. The antibodies used are linked to fluorescent dyes or enzymes activated when the antibodies bind with the cell sample antigen allowing an observer to see the antigen under a microscope.¹⁴ Researchers can visualize and determine whether the cells in a given specimen express the antigen of interest, suggesting the presence of malignant or benign cells. Immunohistochemistry entails the selective identification of antigens or proteins in tissue cells by exploiting the superficial binding of a particular antibody to antigens in the cells contained in a specimen.¹⁴ The two immunostaining techniques differ in their application, hence the distinction between conventional smear and liquid-based preparation.

Immunocytochemistry and immunohistochemistry have different applications in cytology. Immunostaining in LBC corresponds to immunocytochemistry, applied to sample cells without an extracellular matrix. The technique is used to stain sample cells that have been stripped of the matrices, such as blood and mucus or separated from solid tissue blocks.¹⁵ Liquid-based preparations involve rinsing sample cells in a preservative liquid to remove the surrounding materials deemed to obscure visualization. In addition, the preparation of the slides involves the development of a monolayer created by isolating cells from a solid block of tissue, hence the application of immunocytochemical staining.¹ On the other hand, conventional smear relies on immunohistochemical staining. The technique applies to sample cells surrounded by their extracellular matrix, which is characteristic of conventional smears.¹⁵ Conventional smears do not involve isolating sample cells from the other cells and materials found in intact tissue, including blood, mucus, and inflammatory cells.

Liquid-Based Cytology Methods

The difference between conventional smear and LBC is dependent on the type of liquid-based preparation method employed. LBC relies on several preparation methods, influencing various elements of malignant lesions diagnosis and management, such as sensitivity and accuracy. A study by Chong et al. focusing on the diagnosis of thyroid lesions suggested no significant differences between conventional smear and LBC, with both techniques indicating a sensitivity rate of 100% with diagnoses of thyroid lesions.¹ Chun et al. expressed similar sentiments, suggesting slight differences in sensitivity rates between the two techniques with diagnoses of pancreatic neoplasm.³ However, Zhao et al. discovered that diagnostic parameters for LBC varied with the preparation method used.¹⁶ The application of the membrane-based method demonstrates a sensitivity rate of 87.0% regarding the diagnosis of thyroid lesions and an overall sensitivity rate of 86.4%.16 Diagnosis of thyroid lesion using the sedimentation method demonstrated a sensitivity rate of 91.4%.16 Statistical analysis of the outcomes presented by the different LBC methods demonstrated insignificant differences between the postoperative histopathological examination and cytological interpretation. Further differences between LBC and conventional smears were realized when combined with other patient assessment technologies, such as ultrasound. According to Zhao et al., differences between the two techniques in ultrasound-guided FNA are largely influenced by operator proficiency and level of experience.¹⁶ The different outcomes observed imply that the various liquid-based preparation methods have distinct effects on FNAC.

Conclusion

The last few decades have seen the increasing application of FNAC in the diagnosis and management of deep-seated and superficial malignant and benign lesions. It is dependent on two techniques; conventional smear and LBC. Conventional smear constitutes the primary testing technique used to identify cancerous cells. However, it is associated with false negatives resulting from interpretation errors, laboratory detection errors, inadequate slide preparation, and sampling inadequacy. LBC was introduced to improve the diagnostic accuracy of Papanicolaou smears. The approach entails rinsing of samples collected from lesions in a preservative liquid to eliminate potentially obscuring elements such as blood, mucus, and inflammatory cells for a clearer view. The efficiency and efficacy of each FNAC technique have been subject to extensive studies, yielding contradictory outcomes based on a range of factors, such as the types of malignancies examined and styles of liquid-based preparation used. A systematic review of various studies indicates no solid evidence to suggest the superiority of one technique over the other; each technique performs better than the other in one area, with both presenting significant limitations.

References

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