LN status is a key prognostic factor in the decision-making process of cancer management. For a long time, sectioning and H&E staining technique has been the gold standard for the evaluation of LN metastases. Even though it remains an adequate tool, some limitations have been described, especially low sensitivity in detecting the accurate tumour burden, mainly as a consequence of sampling bias , as well as a subjective and time-consuming result. To overcome these limitations, OSNA assay has been developed as a fast, objective, automated, and reproducible way to examine the whole LN, raising a general interest to explore its utility for lymphatic metastases identification in different tumours.
OSNA gives a quantitative result of CK19 mRNA copies, which is present in several simple epithelia but is not expressed in healthy lymphatic tissue . CK19 was initially proposed as a marker for the detection of LN metastases in breast cancer, where it is found in up to 98% of cases . In 2007, Tsujimoto et al.  determined 250 copies/µl as the optimum cut-off point to define a positive axillary LN in breast cancer population. Nonetheless, it is known that the number of positive LN and the size of metastases are significant prognostic factors in most tumours. Therefore, it was also established a second cut-off point of 5,000 copies/µl to distinguish between micro and macrometastases . Subsequent studies have confirmed these values and all the results reflected in this review are based on them.
In 2013 V. Peg et al.  defined the concept of total tumour load (TTL) as the total CK19 mRNA copies of all positive SLNs. TTL serves as a predictive and prognosis value, providing more accurate staging than pathological findings. Accordingly, different OSNA studies in breast SLN have set cut-off values in order to predict the axillary LN status; some of which (10,000–15,000 copies) are already included in clinical guidelines [52, 53]. In 2017, Rakislova et al.  explored its utility to predict recurrences in colorectal carcinoma, and a recent study confirmed that a TTL ≥ 6,000 copies/µl was associated with worse disease free survival in those patients .
The analysis of SLN in breast cancer patients is still its main clinical application, but over the years OSNA has raised interest of the pathology community for a more accurate LN staging in other cancer entities. In the last decade, several reports comparing OSNA with histopathological examination have been published, but after a systematic review of the available literature, to date only two studies related to PCa have been found [23, 24].
All the articles included in this review compare OSNA assay with postoperative H&E staining in the same LN. There is a general concordance between OSNA and standard H&E of over 85%. No full information about discordant cases is available, but we have found not only different explanations for them but also heterogeneity in its analysis. Main justifications for the discordant cases are tumour allocation bias (TAB), and more rarely low or no tumour CK19 expression and contamination by other epithelial cells .
As CK19 is the single molecular marker used in OSNA assay, low tumour CK19 expression may result in a false-negative OSNA case. Different CK19 expression levels have been described for other malignancies such as colorectal (94.1%) , gastric (98.6%) , gynecological (98%) , lung (96%) , HNSCC (91.1%)  or PCa (100%) . Moreover, certain tumour subtypes are more likely to the lack of CK19 expression, as observed in metaplastic and lobular breast carcinomas . Interestingly, Goda et al.  performed a CK19 IHC in primary HNSCC as a first step when analyzing discordant results, detecting no expression of CK19 in 75% of those cases. Aiming to reduce false-negative cases in breast, thyroid and lung carcinomas, Vegué et al. , del Carmen et al. , and Escalante Pérez et al.  verified the presence of CK19 in primary tumour by IHC before LN analysis. For a more accurate interpretation of the results, we encourage future researchers to include CK19 expression in primary tumours as patient inclusion criteria.
One of the main advantages of OSNA is its capacity to analyse the whole LN, leading to a more precise diagnosis than conventional histology. However, to properly compare both techniques on the same node, it is mandatory to split it, leading to a possible misdetection of metastasis by one of the methods, which is called TAB. Trying to justify the discrepancies by the need for sectioning, 17 studies have reported different strategies: second-round OSNA analysis, CK19 IHC, exhaustion of the paraffin-embedded SLN slices, CK19 Western blot, or CK19 qRT-PCR. Most of the metastases from the discrepancies were confirmed thanks to these strategies.
Regarding PCa, CK19 has been found not only in neoplastic tissue but also in basal and luminal cells of normal, dysplastic and benign hyperplastic tissues, although complete data concerning CK19-RNA levels is still missing . In 2018, Winter et al.  published the first OSNA assay in PCa. A total of 20 primary PCa tumours from intermediate-high risk PCa patients (Gleason ≥ 7) were analysed. A central slice was analysed by OSNA, while surrounding slices were sent for both conventional H&E staining and CK19 IHC. PCa was confirmed by H&E in all 20 samples; OSNA was able to detect CK19 mRNA in 100% of cases, ranging from 320 to 250.000copies/µl while IHC did not detect CK19 in one specimen. Given the small sample size and the high tumour burden of the selected patients, we cannot fully extrapolate these findings for all PCa patients.
Recently, Engels et al.  published a second OSNA assay in PCa, including a total of 574 SLNs from 64 PCa patients. SLNs were assessed by conventional H&E staining and OSNA assay. The comparison between both techniques showed a sensitivity, specificity and concordance of 84.2%, 96.1% and 94.4%, respectively.
To date, the ePLND remains the most accurate staging procedure , but individual assessment of all dissected LNs is laborious and time consuming. Using OSNA, LNs can be pooled together and analysed in a few samples as already done in colorectal cancer . In fact, Engels et al.  suggested that such approach could be feasible to analysed PCa LNs as a faster and economic alternative. Moreover, it could be interesting to define a TTL value to predict the risk of early biochemical recurrence and the need of adjuvant treatment.
When compared to histopathological examination, OSNA offers the advantage to obtain objective and quantitative data about tumour load of the whole LN in a fast and effortless way, avoiding interobserver variability. Multicentric prospective studies with large cohorts of patients may investigate the the benefits of OSNA and its impact as a potential prognostic factor for PCa. In contrast, a potential disadvantage of examining the whole LN with OSNA is that there is no tissue left for subsequent histopathological examination following complete homogenization. Nonetheless, RNA-based molecular tests are possible using OSNA lysate, thus allowing any follow-up molecular testing. Moreover, fresh lymphatic tissue requires a thorough dissection to avoid missing nodes, which must be completely separated from fat tissue by a trained pathologist. It is also important to note that in the case of a coexisting neoplasm, OSNA cannot define which primary tumour the metastases come from and is unable to distinguish LN tumour cells from other benign epithelial inclusions. Therefore, it is imperative to exclude other cancers with the same lymphatic drainage and avoid contaminations.
Despite these concerns, the results reported show a high specificity, concordance rate, and NPV of OSNA assay when compared with the H&E method. Specifically, in breast cancer patients, that high NPV provides enough evidence to become the gold standard for SLN evaluation. As slicing is required for the analysis per node, a concordance of 100% cannot be achieved due to TAB. A high concordance rate of over 85% suggests that OSNA could be an alternative technique to histopathological examination in terms of its ability to detect lymph node metastases. Future investigations may bring light to the clinical impact of OSNA in PCa as well as to its potential predictive and prognostic roles.