Quoting Dr. Frost from the 2nd edition of The Cell in Health and Disease published in 1986: “For practical purposes, however, only a few stains usually are needed to permit good identification of types and degrees of the most useful cellular changes.” 1. As professionals in cytopathology, our practices often reflect the foundational training we received early in our careers. I was trained as a cytopathology fellow by Dr. Prabodh Gupta at the University of Pennsylvania Medical Center in Philadelphia, PA, where the laboratory routinely employs to date Diff-Quik stain for air-dried smears during rapid onsite evaluation (ROSE), Papanicolaou stain for alcohol-fixed smears and cytospins, and hematoxylin and eosin (H&E) for cell block sections. It is important to recognize that we often remain creatures of habit, practicing pathology in ways that align with the methods we were taught during training. These preferences are not only shaped by tradition but also by the comfort and confidence we develop with specific techniques over time. Over the course of my 30-years career in cytology, I have occasionally encountered cytologic smears and cytospins stained with H&E, which I have consistently regarded as suboptimal. Recently, however, I was involved in a discussion with a well-known senior cytopathologist who highlighted the continued use of H&E in many countries for both ROSE and permanent cytology smear preparations. Dr. John K. Frost also noted the distinction between staining methods, stating that two techniques dominate routine practice: H&E for histopathology and Papanicolaou's multichromatic stain for cytopathology. While the latter offers clear advantages for cytologic evaluation, the two methods share enough similarities that expertise developed with one can be readily applied to the other 1. To explore global staining practices among cytopathologists, I conducted an informal survey via LinkedIn (Baloch Zubair | LinkedIn) and X (Zubair Baloch (@aakasharmand)/X) by posting the question: “Please let me know how many of you use H&E stain for your cytology preparations.” Our star pathology trainees assisted with data compilation and analysis, ensuring accuracy and clarity in summarizing these findings. This post (Figure 1A,B) generated 108 responses, summarized in Table 1. Respondents represented a wide range of experience levels, from residents to attending pathologists, and worked in diverse institutional settings. Most were affiliated with academic institutions (n = 56, 51.9%), followed by non-academic institutions (n = 39, 36.1%), government facilities (n = 9, 8.3%), and unknown settings (n = 4, 3.7%). Participants were geographically diverse, with the largest proportion from Europe (n = 39, 36.1%), followed by North America (n = 22, 20.4%) and South Asia (n = 22, 20.3%). Additional respondents were from the Middle East (n = 9, 8.3%), Central/South America and the Caribbean (n = 8, 7.4%), Southeast/East Asia (n = 4, 3.7%), and Africa (n = 4, 3.7%). Overall, 63 respondents (58.3%) reported using H&E in some capacity for cytology, while 45 (41.7%) reported no use. Among H&E users, practice patterns varied widely. For ROSE, 12 respondents (11%) used H&E alone, and one (0.9%) combined it with Diff-Quik. Routine use in permanent cytology preparations was uncommon, reported by two respondents (1.9%) alone and two (1.9%) in combination with Papanicolaou and/or Giemsa stains. Marked heterogeneity was observed across regions. In North America, 59.1% (n = 13) reported H&E use, primarily during ROSE (36.3%, n = 8), while routine use was rare (4.5%, n = 1). In Europe, only 17.9% (n = 7) reported H&E use, mostly unspecified. In contrast, South Asia showed the highest prevalence (72.7%, n = 16), though most did not specify context. Respondents from the Middle East, Latin America, East/Southeast Asia, and Africa also demonstrated variable practices, though smaller sample sizes limited detailed comparisons. Variation was also evident across institutional types. Among academic respondents, 57.7% (n = 30) reported H&E use, compared to 22.2% (n = 2) in government institutions and 53.8% (n = 21) in non-academic settings. Context of use—ROSE, permanent evaluation, or unspecified varied within each group. Qualitative responses indicated that staining preferences were largely influenced by familiarity and training rather than perceived diagnostic superiority. Many respondents emphasized reliance on techniques learned during residency, reflecting a broader tendency toward tradition and resistance to change. Representative comments included: “You should work with the stain that allows you to make a high-quality diagnosis and that you are trained in” and “There are a few pathologists who insist on H&E in their reporting.” When H&E was preferred, common reasons included lower cost, limited access to Papanicolaou or Romanowsky stains, simplified workflow, and workforce constraints, such as insufficient technologists trained in Papanicolaou-preparation or interpretation. In several cases, H&E was described as a pragmatic compromise rather than an optimal choice, as illustrated by the comment: “I know it's not the optimal stain for cytology, but I don't have a choice if that particular lab uses only H&E for cost reasons.” Conversely, objections to H&E focused primarily on its perceived inferiority in nuclear detail compared with Papanicolaou staining. Overall, these findings underscore substantial variability in cytology staining practices, with H&E use shaped by regional training norms, institutional resources, and operational constraints rather than adherence to a uniform standard. As international collaboration, digital consultation, and multi-institutional research expand, such heterogeneity may impact diagnostic concordance, shared case review, and cytology education. Familiarity with morphologic features across different staining methods will therefore be increasingly important to ensure diagnostic consistency and accuracy in collaborative settings. Responses were collected via LinkedIn and X, enabling rapid dissemination and broad international participation. However, this approach was subject to selection bias, as participation was voluntary and limited to active users of these platforms. The informal nature of the survey also precluded control over respondent demographics and independent verification of responses. Nonetheless, we appreciate the engagement and insights provided by all participants and thank those who contributed to the discussion. This experience highlights how much one can learn simply by asking. The willingness of colleagues worldwide to share their practices made this an enriching learning opportunity and reinforces that, despite geographic distance, we can learn from each other to better serve our ultimate client, “the patient”. We thank the following individuals, listed in no particular order, for their responses and contributions to the discussion on Linkedin and X: Mitadru De Sarkar, Sule Canberk Schmitt, Carla Ferreira Walecka, Valeriia Vyostskaia, Fernando Schmitt, Katja Magali Marwedel, George Stoyanov, Mehroosh Shakeel, Charles Sturgis, Severino Rey, Samia Nawaz, Nelson Fuentes Martinez, Apoorva N, Rene Gerhard, Jovana Radakovic, Istvan Kovacs, Annette Salillas, Pawel Piotrowsky, Jovana Jovanovic Andersson, Ewe Seng Ch'ng, Kari Beaudry, Helen Trihia, Shazia Khan, Ricardo Guillermo Sanchez Marull, Zafar Ali, Khalid Nasser Khalfan al-Housni, Miranda van Hulzen, Sophia Taylor, Eva Darai Ramqvist, Nicole Samijo-Theunissen, Dietmar Schmidt, Devika Gupta, Sana Afroze, Nazeel A., Lia van Zuylen, Sunil Kumar Katari, Antoine Washington, Nazila Rastegar rad, Patricia Gregg, Sepi Mahooti, Fatima Shamsuddin, Shakeel Akhtar, Fouzia Kauser Vali, Danjiela Vrdoljak Mozetic, Kristen N., Suzana Harabajsa, Ciolly Rivero Colmenarez, Claudio Bellevicine, Shagufta Iram, Rafay A., Leonie Wheeldon, Claudia Silva, Ana Yuil Valdes, Panagiota Mikou, Pembe Oltulu, Khan Ummiya, Pragya Katoch, Mary Dorsey, Anita Flynn, Laetitia Collin, Sangeeta Palaskar, Sean McNair, Emilian O., Hector-Enrique Torres-Rivas, Maria Sevastiadou, Aung Phyo, Eulenda Shabangu, Durre aden, Aparna Gangoli, Mohammed S. I. Mansour, Gopal Parikh, Priyangie Amarabandu, Dauda E. Suleiman, Israh Akhtar Khan, Elba De Oliveira Andrade, Rashida Saleem, Maria del Mar Rivera Rolon, Dina Zenezan, Kalyani Bambal, Eduardo Alcaraz, Ivana Kholová, Pepe Jiménez Heffernan (José A Jiménez-Heffernan), Laila Farid, Ana Yuil, Jose Candido, Frida Rosenblum, Esther Diana Rossi, Philippe Stephenson, Mark Chien-Chin Chen, Maria Luisa Cabanas, Tania Labiano, Michael Nweke, Cioly Rivero Colmenarez, Ruth Estrada, Mohsin Sayed, Vidya Monappa, Swikrity U Baskota, Roxana Chávez, Demis Abate, Mario Gámez, Shubhada Kanhere, Pinakin Kaushik, and Clemencia Bencosme. The authors declare no conflicts of interest.
Leung et al. (Sun,) studied this question.