A brief overview of imprint cytology in thoracic cytopathology

INTRODUCTION

Touch imprint cytology is a conventional cytological technique known for its simplicity, cost-effectiveness, and broad applicability. Although histopathology remains the gold standard for pathological diagnosis,^[1] touch imprint cytology can support cytopathologists by providing a highly accurate intraoperative preliminary diagnosis and by assisting in the evaluation of specimen adequacy during rapid on-site evaluation (ROSE). Despite the procedural simplicity of the method, the cytological interpretation of touch imprint specimens is complex and requires substantial training and expertise on the part of the cytopathologist.^[2]

IMPRINT CYTOLOGY IN ASSISTANCE TO FROZEN SECTION

With the global expansion of lung cancer screening programs, an increasing number of small pulmonary lesions are being detected. These lesions are often challenging to sample using transthoracic or endoscopic techniques, rendering intraoperative diagnosis crucial in many cases.^[3]

At our institution, intraoperative touch imprint cytology is routinely performed using the same sampling technique described by Nakagiri et al.,^[3] followed by rapid staining with the MayGrünwald-Giemsa quick stain (Bio-Optica, Milan).

In the intraoperative setting, imprint cytology can facilitate the subtyping of non-small cell lung carcinoma (NSCLC), especially when frozen sections demonstrate solid cellular arrangements. In addition to conventional cytomorphological features of NSCLC, our experience suggests that imprint cytology is particularly useful in identifying cells with distinct blue cytoplasm, which may reveal intracytoplasmic keratinization. This feature can sometimes be more apparent than in frozen hematoxylin and eosin sections, especially in the absence of extracellular keratin.

Neuroendocrine morphology is identifiable in imprint smears with a frequency and clarity comparable to that seen in frozen sections. Naked nuclei are frequently present in cytologic preparations, which may pose a diagnostic challenge when distinguishing between large cell neuroendocrine carcinoma (LCNEC) and small cell lung carcinoma (SCLC). However, a subset of LCNEC cells typically displays clear cytoplasm, providing a useful discriminating feature.

Rosette formation, although commonly observed in neuroendocrine tumors, may also suggest glandular differentiation. Therefore, it should not be regarded as a definitive diagnostic criterion when considered in isolation.

In the context of metastatic disease, imprint cytology does not play a central role during frozen section analysis, as distinguishing a metastatic tumor from a primary lung carcinoma typically requires evaluation of features such as lepidic growth and overall architectural patterns, along with confirmation by immunohistochemistry. Nevertheless, we present two illustrative cases of metastases with characteristic cytological features [Figure 1a and b].

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Figure 1:

Touch imprint cytology of lung masses obtained intraoperatively. (a) Clusters of pleomorphic cells with large, atypical nuclei, prominent nucleoli, and cytoplasmic pigment, consistent with metastatic melanoma (scale bar: 50 µm; ×400, May-Grünwald-Giemsa [MGG]); (b) Clusters of atypical tumor cells with clear cytoplasm and enlarged nuclei with prominent nucleoli, indicative of metastatic clear cell renal cell carcinoma (scale bar: 100 µm; ×200, MGG); (c) Imprint cytology sample from a patient with granulomatous inflammation and necrosis, showing a granuloma composed of epithelioid cells, lymphocytes, histiocytes, and a Langhans-type multinucleated giant cell (upper right) (scale bar: 100 µm; ×200, MGG).

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We reviewed two reputable studies that reported sensitivity ranging from 75.9% to 87.7%, and specificity between 97.9% and 100% for intraoperative touch imprint cytology.^[4,5] According to Sugiyama et al., discrepancies between cytological and histological diagnoses were observed in cases involving precancerous lesions, minimally invasive adenocarcinomas, and tumors with clear cell or mucinous morphology. In that study, 10.6% of smears were considered inadequate for analysis.^[5] In our experience, extensive necrosis can also compromise the adequacy of touch imprint samples. One study demonstrated that combining cytological and histological evaluation during intraoperative diagnosis improves overall sensitivity.^[4]

Although fewer studies have assessed the utility of imprint cytology in benign lesions, one investigation reported poor correlation between cytological and histopathological findings.^[5] In contrast, Elmas et al. demonstrated excellent diagnostic performance, accurately identifying 248 out of 260 benign cases using imprint cytology.^[4] Our department has had a similarly favorable experience, particularly in the diagnosis of granulomatous diseases [Figure 1c].

IMPRINT CYTOLOGY IN BRONCHOSCOPY AND TRANSTHORACIC CORE NEEDLE BIOPSY

ROSE during bronchoscopy is a valuable, real-time technique that supports clinical decision-making. Small peripheral pulmonary lesions often present a broad differential diagnosis and are particularly challenging to sample and diagnose using cytological smears alone. In such cases, transbronchial and transthoracic core needle biopsies are essential diagnostic tools.

Numerous studies have demonstrated the effectiveness of touch imprint cytology as a ROSE technique, highlighting its role in enhancing the diagnostic yield of the primary biopsy method it supports.^[6,7] In our experience, imprint cytology has proven highly effective for assessing sample adequacy in bronchial biopsies, particularly for centrally located lesions. In many cases, not only could the presence of tumor cells be confirmed but also tumor subtyping was also achievable [Figure 2].

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Figure 2:

Touch imprint cytology of bronchoscopic samples. (a) Squamous cell carcinoma – Three-dimensional clusters of tumor cells (microbiopsy) with peripheral cells visible in the upper right (×400, May-Grünwald-Giemsa [MGG]), displaying mildly spindle-shaped morphology, enlarged nuclei, and moderately abundant “robin’s egg” blue cytoplasm, indicative of intracytoplasmic keratinization (scale bar: 200 µm; ×100, MGG); (b) large cell neuroendocrine carcinoma – Cluster of tumor cells exhibiting large nuclei with finely dispersed “salt-and-pepper” chromatin and a moderate amount of cytoplasm (scale bar: 100 µm; ×200, MGG); (c) small cell lung carcinoma – Groups of atypical cells characterized by small nuclei with finely dispersed chromatin and very scant cytoplasm, displaying nuclear molding (scale bar: 100 µm; ×200, MGG).

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One limitation of the imprint technique is the frequent presence of large, three-dimensional cell clusters [Figure 2a], which can be difficult to interpret. These may mimic squamous metaplastic reactive epithelium and complicate the differential diagnosis. However, malignant cells are often identifiable at the periphery of these clusters. This challenge is especially evident in well-differentiated squamous cell carcinomas, where strong desmosomal attachments inhibit dissociation into smaller, more readily analyzable cellular groups.

Reactive changes in ciliated epithelial cells present a diagnostic challenge in touch imprint cytology, as they may lead to false-positive interpretations and, consequently, unnecessary repeat bronchoscopic procedures. Such reactive alterations are commonly observed in the context of inflammatory conditions, toxic exposures, and as sequelae of therapeutic interventions such as radiotherapy or chemotherapy.^[8]

Another limitation inherent to the imprint technique is the occurrence of crush artifacts, including chromatin smearing, which can simulate the cytological features of SCLC. Nevertheless, given that the primary intraoperative aim is to ascertain malignancy rather than to precisely subtype the tumor, this artifact is typically regarded as a minor concern. Notably, as demonstrated in Figure 2b and c, neoplastic cells of SCLC and LCNEC in our samples exhibited good preservation with minimal crush distortion.

While most published reports describe sampling using forceps, we included Figure 3 to depict the touch imprint sampling approach routinely employed in our department.

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Figure 3:

Touch imprint cytology using the “two-slide” technique performed rapid on-site evaluation in bronchoscopy. (a) Bronchial biopsy specimen positioned between two glass slides, with multiple imprints created by applying gentle pressure; (b) Black arrows indicate visible imprint areas on the slide; (c) The same imprint areas (black arrows) following staining with May-Grünwald-Giemsa.

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Several studies evaluating the diagnostic performance of touch imprint cytology in bronchoscopy for malignancy detection have reported sensitivity ranging from 77% to 91.1% and specificity from 90.4% to 95%.^[9,10] The largest cohort study to date demonstrated that transbronchial biopsy combined with ROSE touch imprint cytology achieved a molecular testing success rate between 87.3% and 96.6%, depending on the specific molecular marker analyzed. In addition, Atheetha et al. reported successful histologic subtyping in approximately 70% of cases.^[9] Furthermore, the use of imprint cytology as an adjunct to transthoracic needle biopsy has been shown to enhance diagnostic accuracy compared to histopathological evaluation alone.^[7]

IMPRINT CYTOLOGY OF LYMPH NODES

Intraoperative pathologic diagnosis of mediastinal lymph nodes in patients with NSCLC, especially because of inaccurate evaluation of computed tomography (CT) and even positron emission tomography (PET), is essential in adopting the further intraoperative protocol for the most effective treatment.

Intraoperative pathological assessment of mediastinal lymph nodes in patients with NSCLC is critical for guiding surgical decision-making, particularly given the limitations in accuracy associated with pre-operative imaging modalities such as CT and PET. Touch imprint cytology has demonstrated utility as a rapid intraoperative diagnostic tool for lymph node evaluation. However, comparative analyses have indicated that frozen section examination offers superior diagnostic performance. Notably, touch imprint cytology is less time-consuming and technically simpler than frozen section analysis. In one study, the sensitivity of touch imprint cytology was reported at 75.68%, compared to 97.30% for frozen sections, while both methods exhibited a specificity of 100%.^[11] Interestingly, one study reported superior diagnostic performance of touch imprint cytology compared to frozen section analysis; however, this observation was based on a limited patient cohort and should be interpreted with caution.^[12]

DISCUSSION

Findings from our experience, supported by a review of the literature, reaffirm the continued relevance of touch imprint cytology in thoracic pathology. This technique offers rapid and reliable intraoperative and bronchoscopic support, particularly in the assessment of specimen adequacy. Nonetheless, its limitations – including the potential for false-positive results due to reactive epithelial changes in the setting of necrosis, and reduced sensitivity in detecting certain metastatic lesions – underscore the importance of interpretation by experienced cytopathologists.

Importantly, touch imprint cytology should not be regarded as a standalone diagnostic modality. Rather, it functions best as a complementary technique alongside frozen section analysis, providing preliminary diagnostic orientation intraoperatively and contributing to the reduction of repeat procedures and diagnostic delays.

Most studies describe imprint smear preparation using fine-needle aspiration needles or forceps to roll, drag, or press biopsy material onto glass slides, as well as through the “squash technique.” These methods, however, often result in excessive blood contamination and mechanical artifacts, including cytoplasmic and nuclear distortion – particularly problematic in fragile neoplasms such as SCLC and lymphomas. In contrast, the two-slide touch imprint technique enables the acquisition of multiple cytological impressions from a single biopsy fragment while preserving tissue integrity for downstream ancillary testing, including immunohistochemistry and molecular profiling. Moreover, this method aligns with the principles of ROSE, offering immediate assessment of sample adequacy without compromising histopathological yield.

SUMMARY

Touch imprint cytology represents a valuable and cost-effective adjunct to frozen section analysis. Its effectiveness is particularly evident in the setting of ROSE, where it aids in determining specimen adequacy and directing clinical decision-making. This, in turn, enhances the diagnostic yield and reduces the frequency of repeat procedures. It is hoped that this concise review will encourage broader adoption of this technique in routine clinical practice.

AVAILABILITY OF DATA AND MATERIALS

The materials from this article are available from the corresponding author on reasonable request.

ABBREVIATIONS

H&E: Hematoxylin and Eosin

LCNEC: Large cell neuroendocrine carcinoma

MGG: May-Grünwald-Giemsa

NSCLC: Non-small cell lung carcinoma

ROSE: Rapid on-site evaluation

SCLC: Small cell lung carcinoma

AUTHOR CONTRIBUTIONS

NG: Concept of the study, acquisition of data, analysis and interpretation of data, literature searching, writing and editing original draft, writing the review; DM: Analysis and interpretation of data, preparing the figures and technical support; VS: Acquisition of data, critically review the article, supervision; AL: Concept of the study, supervision, analysis and interpretation of findings, writing the review. All authors have been involved in revising it critically for important intellectual content. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work. All authors are eligible for ICMJE authorship.