Cytological Variation Research Articles

Evaluation of Morphological and Cytological Variations in Colchicine-Induced M1 and M2 Generations of Bambara Groundnut (Vigna subterranea L.)

Kacang bambara (Vigna subterranea L.) memiliki potensi dalam mengamankan pasokan pangan dalam menghadapi perubahan iklim karena memiliki manfaat dari aspek kandungan gizi, kesehatan, dan agronominya. Keterbatasan sumberdaya genetik menghambat pengembangan varietas unggul kacang bambara di Indonesia. Pemuliaan mutasi menggunakan kolkisin merupakan salah satu metode untuk meningkatkan keragaman kacang bambara. Penelitian ini bertujuan untuk mengevaluasi keragaman morfologi dan sitologi pada dua generasi kacang bambara yang diinduksi oleh kolkisin pada konsentrasi yang berbeda. Eksperimen dilakukan di dua waktu dan lokasi yang berbeda, yaitu Maret-Juli 2022 di lapangan percobaan Fakultas Pertanian Universitas Brawijaya dan Maret-Juli 2023 di rumah kaca di wilayah Joyo Agung, Lowokwaru, Kota Malang, Jawa Timur. Perlakuan kolkisin dilakukan dengan metode tetes pada pucuk apikal bibit dengan konsentrasi yang berbeda (0, 0,1, 0,2, 0,3, dan 0,4%). Pengamatan karakteristik morfologi dan sitologi dilakukan pada generasi M1 dan M2. Perlakuan kolkisin mempengaruhi perubahan morfologi dan sitologi tanaman kacang bambara pada generasi M1 dan M2. Pada generasi M2, perlakuan kolkisin konsentrasi 0,4% meningkatkan lebar daun sebesar 31%, panjang daun sebesar 32%, waktu berbunga sebesar 28%, lebar biji sebesar 29%, panjang biji sebesar 47%, bobot 100 biji sebesar 3%, lebar stomata sebesar 20%, dan panjang stomata sebesar 25% dibandingkan dengan kontrol. Namun, perlakuan kolkisin menurunkan jumlah daun sebesar 30%, tinggi tanaman sebesar 1%, jumlah biji pertanaman sebesar 58%, bobot biji pertanaman sebesar 56%, dan kerapatan stomata sebesar 15% dibandingkan dengan kontrol. Perlakuan induksi kolkisin menyebabkan peningkatan jumlah kromosom pada generasi M2 sehingga dapat digunakan untuk meningkatkan variasi dan mengatasi keterbatasan sumber daya genetik kacang Bambara.

New diploid populations of the Chrysanthemum zawadskii complex (Asteraceae) in natural habitats of Korea

Chrysanthemum zawadskii Herbich is widely distributed from northern Europe to East Asia. It has been reported that various ploidy levels ranging from diploid (2n = 2x = 18) to decaploid (2n = 10x = 90) are distributed in Korea. The diploid populations of C. zawadskii, however, have limited habitats, with only four populations from two locations compared to polyploids that are widely distributed in Korea. During recent biodiversity research on the C. zawadskii complex, new habitats of diploid populations of C. zawadskii not reported earlier in Korea were identified. To understand the speciation mechanism accompanying polyploidization of the C. zawadskii complex, it was necessary to investigate the features of these new populations as to whether they identical to or different from previously reported diploid populations. For this purpose, we identified the number and karyotypes of somatic chromosomes in four new diploid populations and compared the morphological features. It was found that the new diploid populations differed slightly in terms of the size and color of the flowers and in the shape of the leaves, with continuous variations. Representative karyotypes of the new populations were included in previously reported diploid karyotypes, but the numbers and locations of satellite chromosomes differed. In addition, we found that one of them is a sympatric population with different ploidy levels, including diploid, tetraploid, and hexaploid. From these results, it was confirmed that morphological and cytological variations exist among diploid populations in the C. zawadskii complex.

The cervical microbiota of Hispanics living in Puerto Rico is nonoptimal regardless of HPV status.

The cervicovaginal microbiota is influenced by host physiology, immunology, lifestyle, and ethnicity. We hypothesized that there would be differences in the cervicovaginal microbiota among pregnant, nonpregnant, and menopausal women living in Puerto Rico (PR) with and without human papillomavirus (HPV) infection and cervical cancer. We specifically wanted to determine if the microbiota is associated with variations in cervical cytology. A total of 294 women, including reproductive-age nonpregnant (N = 196), pregnant (N = 37), and menopausal (N = 61) women, were enrolled. The cervicovaginal bacteria were characterized by 16S rRNA amplicon sequencing, the HPV was genotyped with SPF10-LiPA, and cervical cytology was quantified. High-risk HPV (HR-HPV, 67.3%) was prevalent, including genotypes not covered by the 9vt HPV vaccine. Cervical lesions (34%) were also common. The cervical microbiota was dominated by Lactobacillus iners. Pregnant women in the second and third trimesters exhibited a decrease in diversity and abundance of microbes associated with bacterial vaginosis. Women in menopause had greater alpha diversity, a greater proportion of facultative and strictly anaerobic bacteria, and higher cervicovaginal pH than premenopausal women. Cervical lesions were associated with greater alpha diversity. However, no significant associations between the microbiota and HPV infection (HR or LR-HPV types) were found. The cervicovaginal microbiota of women living in Puerto Rican were either dominated by L. iners or diverse microbial communities regardless of a woman's physiological stage. We postulate that the microbiota and the high prevalence of HR-HPV increase the risk of cervical lesions among women living in PR. IMPORTANCE In the enclosed manuscript, we provide the first in-depth characterization of the cervicovaginal microbiota of Hispanic women living in Puerto Rico (PR), using a 16S rRNA approach, and include women of different physiological stages. Surprisingly we found that high-risk HPV was ubiquitous with a prevalence of 67.3%, including types not covered by the 9vt HPV vaccine. We also found highly diverse microbial communities across women groups-with a reduction in pregnant women, but dominated by nonoptimal Lactobacillus iners. Additionally, we found vaginosis-associated bacteria as Dialister spp., Gardnerella spp., Clostridium, or Prevotella among most women. We believe this is a relevant and timely article expanding knowledge on the cervicovaginal microbiome of PR women, where we postulate that these highly diverse communities are conducive to cervical disease.

Induced polyploidization in buckwheat (Fagopyrum esculentum Moench)

The significance of polyploidy in the evolutionary process of plants history is well documented. In this concern, seed priming with colchicine as well as the cotton-swab method for polyploidy induction in plants becomes much acquainted in cytogenetic study. The aim of present study was to achieve chromosomal doubling by the application of colchicine to apical meristem of young seedlings. The young seedlings were treated with three different concentrations of colchicine (0.2, 0.4 and 0.6%) for durations of 12, 24 and 36 h each. The colchiploidy with highest proportion approximately 50% with 2n=4x=32 was found at 0.2% concentration for 24 and 36 h. Autotetraploid enduring plants were identified with morphological and cytological variations such as thickness of leaf, larger stomata with low density, larger pollen, large flower etc. Buds were selected from these plants for the cytological study. Cytological analysis including chromosome counting demonstrated that the chromosome number was doubled. The confirmation of autotetraploid plants was achieved by this technique. Tetraploid plants were grown up to maturity and the harvested seeds were sown to establish the second generation which may be used in future breeding programme

Two shades of grey: effect of temperature on seed germination of the escaping ornamental species Lychnis coronaria and Stachys byzantina

This paper describes germination and cytological variability in two popular ornamental and potentially invasive species, lamb’s ear Stachys byzantina and rose campion Lychnis coronaria. Both xerophytic species have the potential to invade natural habitats across Europe and create viable naturalised populations, with subsequent impacts on native vegetation. To assess the species’ invasiveness, seeds were collected from naturalised populations and germination was recorded under different temperature regimes. Flow cytometry, used to record cytological variability, indicated that all populations of both species were cytologically homogeneous. Germination success, an important means of distribution factor in both species, was significantly influenced by temperature, with final germination of L. coronaria being extremely high at temperatures > 15/10 °C (98.5%) and extremely low at temperatures < 10/5 °C (2.9%). The final germination in S. byzantina is the highest at 22/15 °C (55.6% germinated seeds), reduced to 40.3% at 15/10 °C and to 0.3% at temperatures lower than 10/5 °C. No significant differences in final germination were observed between escaping and non-escaping populations, although there were some differences between particular populations. Our results indicate germination temperature limits of two species whose non-native areas include Central Europe. The observed germination allows for successful generative reproduction of both species over their non-native ranges, suggesting that these species are likely to become invasive species of European grasslands soon.

Histological Study of Glandular Variability in the Skin of the Natterjack Toad-Epidalea calamita (Laurenti, 1768)-Used in Spanish Historical Ethnoveterinary Medicine and Ethnomedicine.

Simple SummaryCommon toads, including the natterjack toad (Epidalea calamita), have been used since ancient times for remedies, and thus constitute excellent biological material for pharmacological and natural product research. After a previous analysis of the historical-folk therapeutic use of amphibians in Spain, a histological study was carried out to provide a complementary ethnopharmacological view through the analysis of the integumentary heterogeneity of the serous (venom) and mucous glands from two adult specimens. Plastic-embedded semi-thin sections showed that serous/venom glands are cytologically homogeneous in spite of their genetic and biochemical complexity, leading to a cocktail that remains stored until extrusion. On the contrary, mucous glands, working continuously, show a more complex cytological variation and regional heterogeneity, which suggests an adaptive variability, leading to an invisible topographic map of skin toxicity. Natterjack toad-based folk remedies are usually extracted from the whole animal as a therapeutic unit in ethnoveterinary practice. However, a new ethnopharmacological vision could emerge from the study of tegumentary regional variation.Common toads have been used since ancient times for remedies and thus constitute excellent biological material for pharmacological and natural product research. According to the results of a previous analysis of the therapeutic use of amphibians in Spain, we decided to carry out a histological study that provides a complementary view of their ethnopharmacology, through the natterjack toad (Epidalea calamita). This species possesses a characteristic integument, where the parotoid glands stand out, and it has been used in different ethnoveterinary and ethnomedical practices. This histological study of their glandular variability allow us to understand the stages through which the animal synthesises and stores a heterogeneous glandular content according to the areas of the body and the functional moment of the glands. To study tegumentary cytology, a high-resolution, plastic embedding, semi-thin (1 micron) section method was applied. Up to 20 skin patches sampled from the dorsal and ventral sides were processed from the two adult specimens collected, which were roadkill. Serous/venom glands display a genetic and biochemical complexity, leading to a cocktail that remains stored (and perhaps changes over time) until extrusion, but mucous glands, working continuously to produce a surface protection layer, also produce a set of active protein (and other) substances that dissolve into mucous material, making a biologically active covering. This study provides a better understanding of the use of traditional remedies in ethnoveterinary medicine.

First Glimpse on Spring Starflower Domestication.

The cultivation and domestication of plants are human-driven processes that change the biology and attributes of a plant. Ipheion uniflorum is a bulbous geophyte known as Spring Starflower whose cultivation dates back to the first half of the 19th century. At least seven cultivars have been developed from natural stands. However, comparative analyses of wild and cultivated materials are largely missing. In the present study, we provide a morphological evaluation and analyses of the cytological and genetic variability of I. uniflorum that reveal significant levels of differentiation and evidence of artificial selection in the Spring Starflower. Distinctive phenotypic characters in cultivated materials that are rarely found or lacking in wild plants and natural populations, such as pink or violet flowers, together with its reduced heterozygosity and starting genetic differentiation support the view of early mechanisms of domestication acting upon Spring Starflower plants. The probable geographic origin of the cultivated forms is discussed together with perspectives for plant breeding.

Ploidy Level, Karyotype, and Genome Size of Bletilla Species (Orchidaceae) From China

Bletilla is an Orchidaceae genus with high medical value, including detumescence, antibacterial, and hemostasis. In this study, detailed estimates of ploidy level, karyotype, and genome size were first obtained, and a comprehensive cytological analysis was carried out to better understand the evolution of the genus. The karyotypes of Bletilla were mainly composed of metacentric and submetacentric chromosomes with lengths ranging from 1.25 to 4.93 μm. There was moderate cytological variation in Bletilla (chromosome number 2n = 32 to 76). Diploid with 2n = 34 and 2n = 36 was detected in Bletilla ochracea and Bletilla formosana, respectively, whereas diploid (2n = 32) was dominant in Bletilla striata, dysploidy (2n = 34, 2n = 36) and polyploid (2n = 48, 51, 64, 76) variations were also observed. Three species had a relatively symmetric karyotype, and which of B. ochracea was more asymmetry. The genome size (1C-values) varied from 2.94 pg (B. striata) to 3.33 pg (B. ochracea), of which B. ochracea was significantly larger than the others (P &lt; 0.05). A positive correlation (P &lt; 0.01) between 1Cx vs. haploid chromosome length (HCL) and asymmetry coefficient of karyotypes (AsK%) was observed.

Follicular dendritic cell sarcoma.

SummaryFollicular dendritic cells (FDC) are mesenchymal-derived dendritic cells located in B-follicles where they play a pivotal role in triggering and maintaining B-cell adaptive immune response. In 1986 Dr. Juan Rosai first reported a series of neoplasms showing features of FDC and defined it as Follicular Dendritic Cell Tumor, subsequently renamed as “sarcoma” (FDCS). In its seminal and subsequent articles Rosai and colleagues highlighted the heterogeneous microscopic appearance of FDCS and its immunohistochemical and ultrastructural features.FDCS mostly occurs in extranodal sites (79.4% of cases) and lymph nodes (15.1%); in about 7%-10% of cases it is associated with hyaline-vascular Castleman disease. Given its significant growth pattern and cytological variability, FDCS can be confused with various neoplasms and even inflammatory processes. The diagnosis requires the use of a broad spectrum of FDC markers (e.g. CD21, CD23, CD35, clusterin, CXCL13, podoplanin), particularly considering that tumor antigen-loss is frequent. The inflammatory-pseudotumor-like (IPT-like) variant of FDCS, in addition to its peculiar histopathological and clinical features, is characterized by positivity of tumor cells for Epstein-Barr virus, representing a diagnostic requisite.No distinctive genetic and molecular anomalies have been identified in FDCS. It often carries an aberrant clonal karyotype and chromosomal structural alterations, frequently involving onco-suppressor genes. Direct or next generation sequencing showed alterations on genes belonging to the NF-κB regulatory pathway and cell-cycle regulators. In contrast to hematopoietic-derived histiocytic and dendritic cells tumors, FDCS typically lacks mutations in genes related to the MAPK pathway.FDCS recurs locally in 28% and metastasizes in 27% of cases. Extent of the disease, surgical resectability and histopathological features are significantly associated with the outcome. IPT-like FDCS behaves as an indolent tumor, even if it often recurs locally over years.Complete surgical excision is the gold standard of treatment. Data on targeted therapies (e.g.: tyrosine kinase inhibitors) or immune checkpoint inhibitors are very limited and responses are variable. A better understanding of the molecular drivers of this tumor may lead to potential new therapeutic strategies.

Aplicación de la Hematoxilina férrica de M. Heidenhain para el estudio de cortes semifinos (adenohipófisis humana)

Heidenhain´s iron hematoxylin has been one of the most useful methods of classical cytology. Its ability to stain simultaneously many cell and extracellular structures makes it a general purpose histological technique. This current work describes its application to semithin sections, using human adenohypophysis, discussing the staining mechanism and showing his usefulness to evidence cytological variability and to perform image analysis.

Correction: Morphological, cytological, and molecular variations induced by gamma rays in ground-grown chrysanthemum ‘Pinkling’

In this study, we analyzed the morphological, cytological, and molecular changes induced by different doses of 60Co-γ radiation on root cuttings of the ground-grown chrysanthemum ‘Pinkling’ plants ...

Pediatric Sinonasal Rhabdomyosarcoma: Clinical Characteristics and Surgical Role

Introduction: Rhabdomyosarcoma is the most common pediatric soft tissue sarcoma. It represents 5-8% of pediatric tumors. Head and neck is considered the most common site of RMS origin (40%). Parameningeal, orbital and non parameningeal RMS approximately represent 50%, 25% and 25% respectively. Histopathological evaluation demonstrates small round cells with high cytological variability which stain positive with desmin, myogenin and MyoD1. The mainstay treatment of RMS depends on chemotherapy and radiation therapy with a limited role of surgery. Method: A retrospective chart review for pediatric sinonasal rhabdomyosarcoma diagnosed and managed at King Fahad Specialist Hospital, Dammam, Saudi Arabia, and a literature review of pediatric sinonasal rhabdomyosarcoma was conducted. Results: A total of four cases were identified for the period (2011-2017), and a thorough review of their medical records and radiological imaging were done. Conclusion: Pediatric sinonasal rhabdomyosarcoma may initially present with symptoms mimicking rhinosinusitis. Biopsy and histological evaluation are the most essential steps to exclude malignancy. Intergroup Rhabdomyosarcoma Studies (IRS) established a staging system based on tumor extension and resectability. The role of surgery in RMS may be limited for obtaining biopsies for diagnosis and for palliative purposes. Popular antineoplastic agents used to treat RMS include vincristine, cyclophosphamide, actinomycin D, and adriamycin. Chemotherapy with alkylating agents has achieved a relapsed free survival 90% for nonparameningeal tumors and 65% for parameningeal tumors.

Cytological variations and long terminal repeat (LTR) retrotransposon diversities among diploids and B-chromosome aneuploids in Lilium amabile Palibin.

B chromosomes are supernumerary chromosomes found in numerous plant species, including in the genus Lilium. Lilium amabile, an endemic Korean Lilium species, carries B chromosomes which are highly variable in terms of numbers and shape among the accessions collected throughout the Korea. Class 1 retrotransposons are highly abundant in the genome of Lilium species, but their biological functions are still obscure. Lilium species were known to hold high diversities derived from retrotransposons. In this study, genetic diversities among the L. amabile accessions were analyzed to better understand relationships between genetic variations and cytological diversities. Chromosomes were prepared from 95 L. amabile accessions for cytological identification. Genetic variations were analyzed by inter-retrotransposon amplified polymorphism (IRAP), and genetic differentiation was evaluated via Tajima's D neutrality and FST analyses. Population structure and phylogenetic analyses were also carried out. The L. amabile accessions were classified into 11 cytotypes by the chromosome constitutions. Genetic diversity measured by IRAP analysis revealed high genetic diversity among the accessions. In the joint analysis of cytological variation with genetical variation, IRAP diversity was not related to the cytological diversities of diploid and aneuploids among L. amabile accessions, and genetic differentiation was not obvious. Moreover, the geographical distribution of L. amabile was not related to either IRAP diversity or cytological diversity. The B chromosome-carrying aneuploids occurred randomly among diploids throughout Korea, and IRAP diversification predated L. amabile dispersion in Korea without genetic differentiation.

Expression of Cytological Variability in Some Miscellaneous Legume Accessions

Expression of Cytological Variability in Some Miscellaneous Legume Accessions

High frequency regeneration from leaf derived callus and its histological studies in Lepianthes umbellata

ABSTRACTA protocol was standardized for callus initiation and shoots regeneration from leaf explants of Lepianthes umbellata. The explant was cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 2,4-Dichlorophenoxyacetic acid (2,4-D), 6-Benzylaminopurine (BAP), and 1-Naphthalene acetic acid (NAA) for shoot bud initiation and multiplication. High frequency of shoot regeneration and maximum number of shoots per explant were achieved on MS medium fortified with 0.5 mg L−1 NAA and 1.0 mg L−1 BAP. The elongations of shoots were achieved on the same medium supplemented with 1.0 mg L−1 Gibberelic acid (GA3), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA) at different concentrations on half strength MS medium were used for rooting of the micro shorts. Highest efficiency for rooting was observed on MS with 1.5 mg L−1 IBA. Regenerated plantlets were successfully transferred for acclimatization (70%). Histological studies showed that embryoids and shoot buds initiating from the callus masses, that is, indirect organogenesis. Cytological variations were observed in callus cultures.

Diagnostic utility of molecular and imaging biomarkers in cytological indeterminate thyroid nodules

Indeterminate thyroid cytology (Bethesda III and IV) corresponds to follicular-patterned benign and malignant lesions, which are particularly difficult to differentiate on cytology alone. As similar to 25% of these nodules harbor malignancy, diagnostic hemithyroidectomy is still custom. However, advanced preoperative diagnostics are rapidly evolving. This review provides an overview of additional molecular and imaging diagnostics for indeterminate thyroid nodules in a preoperative clinical setting, including considerations regarding cost-effectiveness, availability, and feasibility of combining techniques. Addressed diagnostics include gene mutation analysis, microRNA, immunocytochemistry, ultrasonography, elastosonography, computed tomography, sestamibi scintigraphy, [F-18]-2-fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET), and diffusion-weighted magnetic resonance imaging. The best rule-out tests for malignancy were the Afirma (R) gene expression classifier and FDG-PET. The most accurate rule-in test was sole BRAF mutation analysis. No diagnostic had both near-perfect sensitivity and specificity, and estimated cost-effectiveness. Molecular techniques are rapidly advancing. However, given the currently available techniques, a multimodality stepwise approach likely offers the most accurate diagnosis, sequentially applying one sensitive rule-out test and one specific rule-in test. Geographical variations in cytology (e.g., Hurthle cell neoplasms) and tumor genetics strongly influence local test performance and clinical utility. Multidisciplinary collaboration and implementation studies can aid the local decision for one or more eligible diagnostics.

Seasonal Variation in Tracheal Mucous and Bronchoalveolar Lavage Cytology for Adult Clinically Healthy Stabled Horses

Seasonal changes affect the concentration of dust particles and aeroallergens in the equine stable environment and are hypothesized to change airway cytology in healthy stabled horses. The objective of the study was to evaluate if seasonal changes from May to November influenced the lower airway health cytological findings in clinically healthy stabled horses. Endoscopic examination, including tracheal aspiration (TA) and bronchoalveolar lavage (BAL) cytology was performed in November and May on 63 clinically healthy stabled Danish Warmblood geldings, aged 12.9 (±4.6) years. Tracheal aspiration and BAL cytology were evaluated via May-Grünwald-Giemsa stain after cytocentrifugation of samples. Five-hundred cells were identified microscopically as macrophages, lymphocytes, neutrophils, eosinophils, or mast cells. A significant difference in climatic conditions with lower temperatures (P < .001), fewer sunlight hours (P = .004), less precipitation (P = .006), and higher humidity (P < .001) was found in November compared to May. A significantly higher percentage of neutrophils (Median 6.10%, Quartiles 4.00–8.80, P < .001) and a higher tracheal mucus score (Median 3.50%, Quartiles 1.95–5.20, P = .010) were found during November compared to May period. Furthermore, a positive correlation between TA and BAL neutrophils was found for both sampling times. Mild winter weather conditions with lower temperatures, fewer minutes of sunlight, and a higher percent humidity were associated with significantly higher BAL neutrophil percentage and significantly higher mucus score in stabled clinical healthy horses. The clear seasonal influence on BAL neutrophilia and tracheal mucous findings in clinically normal horse needs to be included in the working diagnosis of asthma in horses.

A study of association between cervical cytology and period of co-habitation with husbands in the wives of serving soldiers

BackgroundThe wives of serving soldiers constitute a special demographic cohort, as they spend variable amount of time in co-habitation with their husbands. The present study aims to find if any independent association, adjusted for age, exists between the time spent living together with the husband and findings on cervical smear. MethodsOver a period of one year, cervical smears were taken from subjects at two different hospitals. The approximate length of co-habitation with husbands in the last 10 years was elicited through a questionnaire. ResultsAmong 262 subjects, majority of smears showed normal findings (82.06%). 17.93% of the smears displayed abnormality, the maximum proportion of cases with abnormal findings were seen in the group who have spent 61–90 months, in the last ten years. Age adjusted chi square statistics failed to show statistically significant association between period of co-habitation and abnormal Pap smear. Odds’ ratio (OR) for each age stratum varied from each other and was also different from the overall (crude) OR, suggesting that age is an effect modifier. Variation in epithelial cytology did not appear to be an effect of duration of cohabitation but was because of the increasing age. ConclusionsCervical cytology does not show association with length of cohabitation with husbands in this study. However, age is seen to be an effect modifier.

English

English

Diagnostic Utility of Molecular and Imaging Biomarkers in Cytological Indeterminate Thyroid Nodules

Indeterminate thyroid cytology (Bethesda III and IV) corresponds to follicular-patterned benign and malignant lesions, which are particularly difficult to differentiate on cytology alone. As ~25% of these nodules harbor malignancy, diagnostic hemithyroidectomy is still custom. However, advanced preoperative diagnostics are rapidly evolving.This review provides an overview of additional molecular and imaging diagnostics for indeterminate thyroid nodules in a preoperative clinical setting, including considerations regarding cost-effectiveness, availability, and feasibility of combining techniques. Addressed diagnostics include gene mutation analysis, microRNA, immunocytochemistry, ultrasonography, elastosonography, computed tomography, sestamibi scintigraphy, [18F]-2-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET), and diffusion-weighted magnetic resonance imaging.The best rule-out tests for malignancy were the Afirma® gene expression classifier and FDG-PET. The most accurate rule-in test was sole BRAF mutation analysis. No diagnostic had both near-perfect sensitivity and specificity, and estimated cost-effectiveness. Molecular techniques are rapidly advancing. However, given the currently available techniques, a multimodality stepwise approach likely offers the most accurate diagnosis, sequentially applying one sensitive rule-out test and one specific rule-in test. Geographical variations in cytology (e.g., Hürthle cell neoplasms) and tumor genetics strongly influence local test performance and clinical utility. Multidisciplinary collaboration and implementation studies can aid the local decision for one or more eligible diagnostics.