Algorithm for procuring fine needle aspirations with rapid on-site evaluation (ROSE): The algorithm for optimizing FNAs with ROSE is divided into 3 main categories: (1) specimen procurement and triage, (2) slide preparation, and (3) tissue evaluation for diagnoses and assessment of adequacy for ancillary studies (AS), if necessary. First, select diagnostic tissue for slide preparation. This is best accomplished by expelling the material in the needle and hub onto a slide and identifying tissue particles. These are usually tan white but may vary depending on the nature of the lesion (e.g., mucoid material in cases of a mucinous carcinoma). The sample may be scant and allow for preparation of only smear(s). Alternatively, the sample may be bloody and contain clots. In samples with clots, gently pressing the clot between two slides distinguishes lesional tissue from blood. Second, prepare two slides from the tissue particles selected. Perform ROSE to determine if there is diagnostic tissue and if additional material is needed for ancillary studies. Repeat these steps in cases of insufficient tissue. Meanwhile, to capture all possible cells, rinse the needle within a liquid media (e.g., alcohol, saline, RPMI). In cases of smaller length needles, like the ones used for CT-guided FNAs, the media/preservative in the transport vial can be drawn into the syringe and then flushed back and rinsed into the vial multiple times. For longer needles, like the ones used for EBUS-FNAs, pass approximately 0.5 to 1 ml of sterile saline through the proximal end of the needle and into the vial containing the media/preservative. In instances where only blood is seen under microscopic examination, an additional smear can be prepared from the unused material. Otherwise, the remainder of expelled and unused specimen is placed in the appropriate fixative for ancillary studies, if necessary and indicated
In case the material cannot be expelled due to clotting, use a stylet to dislodge the specimen.
Prepare two smears from the selected tissue particles for each pass  (Fig. 4.2).
Smear preparation: With the corner of a slide, select a tissue particle(s). Place the tissue on a clean slide and smear holding the second slide perpendicularly
Air-dry one smear and stain with Diff-Quik, or similar method, for ROSE.
Fix one smear in alcohol for Papanicolaou staining in the laboratory.
Flush the needle and/or syringe to remove any remaining cells.
In cases of CT-guided aspirations, the needle and syringe are rinsed in CytoLyt (or other preservatives used by the laboratory).
In cases of EBUS and/or EUS FNAs, approximately 0.5 to 1 ml of saline is passed through the needle into CytoLyt (or other preservatives used by the laboratory).
Place the remaining specimen in media appropriate for ancillary studies and/or cell block preparation.
One technique for making cell blocks involves allowing the specimen to clot on the expelled slide for a few minutes and then placing it into formalin. (Partial) Clotting simplifies cell block processing.
Separating diagnostic material/more cellular passes from non-diagnostic ones (e.g., containing mostly blood) prevents specimen dilution and improves cellular yield of cell blocks.
Is there sufficient material for diagnosis?
If not, repeat steps 1–5.
If so, and there is a need for ancillary studies, determine whether there is sufficient material.
If no, perform dedicated passes for ancillary studies.
If yes, the procedure is terminated.
Maximizing Efficiency of ROSE
Some of the time challenges of ROSE can be overcome by implementing the following steps that increase efficiency: (1) arrangement with the interventionist to notify pathology staff with the appropriate lead time (i.e., after the patient has been consented and prepped and the lesion has been identified/localized) necessary to travel to the procedure site and set up in advance of when the specimen will be procured—a mutual coordination effort increases efficiency and minimizes perpetuation of late arrivals of the cytologist and unnecessary advance requests for ROSE by the interventionists, (2) completed requisition with pertinent clinical history and labels prepared for identifying the specimen vial(s), (3) microscopes deployed in all procedure rooms and clinics that routinely request ROSE, (4) fully stocked FNA baskets and cart that are restocked following each FNA to ensure all necessary items, such as needles, syringes, CytoLyt, 95 % alcohol, Diff-Quik stain, RPMI, sterile saline syringes, etc., are readily available, and (5) ROSE forms in all FNA baskets and FNA cart.
FNA Without ROSE
An experienced cytotechnologist or pathologist is qualified to render an accurate, immediate interpretation . Yet, due to workload, time and cost constraints, as well as limited diagnostic accuracy [14, 15], yield [14, 16–18], or adequacy [17, 19] at a facility, some forgo ROSE.
There are several steps that can be undertaken in the absence of ROSE to ensure that diagnostic material is sampled and optimally processed. First, even the best preparations cannot overcome inadequate sampling. In some instances, as described for EUS FNAs, utilizing the fanning technique, which involves sampling different areas with multiple back and forth motions , increases cellular yield and samples from different areas. This is advantageous in cases of heterogeneity of lesional tissue. Second, performing three aspirates per site  and a dedicated pass(es) for cell block preparation  and/or ancillary studies can enhance the cellular yield.
Even in the presence of adequate tissue, slides of suboptimal technical quality can compromise diagnosis. Though an experienced cytologist makes smear preparation appear effortless, smearing requires skill. Liquid-based preparations, like ThinPrep® and SurePath™, offer a standardized alternative for slide preparation. For the interventionist, the process involves placing the aspirate in an alcohol-based solution (e.g., CytoLyt or SurePath™ vial), which serves as a fixative and transport medium. Slides are prepared in the laboratory with an automated processor. This eliminates the need for non-experienced personnel to prepare smears, possibly poorly, and reduces the number of slides for diagnosis. These factors in turn decrease the amount of time spent examining slide(s) for each case and result in fewer issues with fixation/preservation . Also, a combination of a liquid-based slide and cell block preparation from the fixative standardizes processing. Similar adequacy rates between conventionally processed aspirates  and those placed directly into CytoLyt—especially with an experienced aspirator—and high-quality specimens without on-site support at the time of the procedure  have been reported. For hemorrhagic thyroid aspirates, preparing only cell blocks correlates with slide reduction . These methods neither ensure nor are substitutes for sample adequacy, however.
Optimization and Triage
Optimizing a specimen requires appropriate handling and triaging. Even in the presence of abundant tissue, poor preparation and allocation can preclude a definitive diagnosis and ancillary studies. When performing ROSE, particularly for aspirations or core biopsies of lung and difficult-to-access lesions, it is of utmost importance to approach the triage of each pass as though that is the only specimen that will be procured. Complications, such as pneumothorax and obscuring hemorrhage, may preclude the opportunity to obtain additional material necessary for ancillary tests, resulting in the need for a repeat procedure.
Optimizing Slide Preparation: Fine Needle Aspirations
There are several explanations for not having sufficient tissue for ancillary studies. One of the more common reasons is “wasting” tissue by making excessive smears—those beyond what is required to make a diagnosis  (Fig. 4.3). At the time of ROSE, whenever possible, only two smears should be prepared—one air-dried for Diff-Quik staining and the second fixed in alcohol for Papanicolaou staining—with the remaining material, if any, allocated for cell block or other ancillary studies. Also, avoiding thick smears serves two purposes: first, it allows for easier visualization of diagnostic cells, and second, an even and thin preparation minimizes the tissue expended on smears. Placing excessive material and clots in solution reduces the likelihood of additional passes for ancillary studies. Lastly, smearing by placing slides perpendicular, rather than parallel, to each other creates smears with concentration of specimen at the top and thin distribution below for easy visualization. Application of excessive pressure, which can create artifacts, may hinder interpretation and/or lead to misdiagnosis.
Optimal and suboptimal utilization of tissue: Optimal utilization involves preparation of smears (stained with Diff-Quik and Papanicolaou stains) from selected tissue particles with excess placed in media for potential ancillary studies. Suboptimally prepared specimens consist of numerous slides that are thick and bloody, contain clots, and encompass almost the entire surface of the slides. This obscures cellular detail, hinders accurate interpretation of slides, and risks having inadequate material for ancillary studies
Optimizing Slide Preparation: Touch Preparations
There is debate about performing touch preparations of core biopsies. If performed, touch preparations require handling with care . Provided that core biopsies are typically thin and delicate, they dry rapidly, and significant manipulation can cause them to fragment. Touching the core once or twice to a slide by lifting it with a needle from the sheath or while it is still in the needle sheath and then placing it into formalin (or the appropriate media) minimizes excessive handling and yields optimal results (Fig. 4.4). If the touch preparation has abundant material, it can be smeared with a second slide to yield a thin and even distribution of cells.
Optimal and suboptimal touch preparations: Optimal touch preparations involve gently touching the core to the slide once or twice. If it adheres to the slide, lift the core with a needle and place it in the appropriate media for fixation or transport. A smear can be prepared from a touch preparation when there is excessive tissue or liquid component. Suboptimal touch preparations: smearing or rubbing the core onto a slide can result in crush artifact and transfer of a significant portion of lesional cells onto the slide that can hinder final interpretation and compromise the core
Preparing a Papanicolaou-stained touch preparation can be challenging. Even if touched rapidly and placed in alcohol immediately, the slide may have air-drying artifact. One approach to minimizing this is to hydrate the slide with a few drops of normal saline, which is available in syringes, for a few minutes. Subsequently, placing a slide hydrated in this manner in alcohol salvages the nuclear features. This technique can also be applied to FNA smears when there is a delay in alcohol fixation.
At times, core biopsies consist of minute and thin fragments. If the laboratory is proficient at making cell blocks, the possibility of processing the cores using the same methodology as that employed for FNAs may prevent inadvertent loss of tissue during multiple transfer steps involved in routine core biopsy handling in histology.
For carcinomas, smears should have material to render a diagnosis of small cell carcinoma or NSCLC; the remainder of the specimen, if any, should be allocated for cell block preparation for subtyping (Fig. 4.5). It has been noted, especially in instances of poorly differentiated NSCLCs, that IHC and/or special stains (e.g., mucicarmine) are necessary to establish the histologic subtype [26, 27]. For NSCLCs, notably those presenting at advanced stages, an effort should be made to preserve as much tissue as possible for molecular analysis  when any proportion of adenocarcinoma is present or cannot entirely be excluded.
Algorithm for triage of small specimens. NSCLC, non-small cell lung carcinoma; IHC, immunohistochemistry; (asterisk) Perform immunohistochemistry to confirm diagnosis, if necessary; (double asterisk) Perform molecular testing on carcinomas with adenocarcinoma or when a component of adenocarcinoma cannot entirely be excluded
Optimizing: Processing Core Biopsies and Cell Blocks for Carcinomas
There are several measures that can be undertaken to ensure that there is sufficient material in core biopsies and cell blocks and that the tissue is not exhausted (Table 4.1). The clinician obtaining the biopsy can play a significant role by performing a gross examination of the biopsy. It is important to determine if the core represents a solid piece of tan white tissue, which is typical of carcinomas, or not. Sometimes the “cores” consist of mostly red blood cell clot, mucus, liquefied necrotic inflammatory tissue, or bronchial cell contamination . Similar findings can be observed in FNA samples. Both situations necessitate additional tissue, and typically, the greater the number of cores obtained, the greater the likelihood of having sufficient tissue for ancillary testing. Currently, there are no guidelines dictating the minimum number. However, if feasible and accessible without significant risk to the patient, 1 to 6 cores (from 18- to 20-gauge core needles) have yielded sufficient tissue for mutational analysis [7, 29–31] By placing fewer fragments and/or cores  (1 to 3) in a single histology cassette, inadvertent trimming of one block leaves the other(s) available for ancillary testing, if necessary. The consequences of excessive facing of the block should also be communicated to the histotechnologists cutting the blocks.
Core biopsy and cell block management in the laboratory
Optimizing: Immunohistochemistry for Carcinomas
When using immunohistochemistry in the distinction between adenocarcinoma and squamous cell carcinoma, a limited panel should be performed in a stepwise fashion to ensure that tissue remains for molecular diagnosis in case of lung adenocarcinoma. If sufficient tumor tissue/cells are available, then a minimal set of two IHC stains, i.e., TTF-1 and p63, can be utilized; however, if the amount of tumor is limited, then the initial work-up can begin with a TTF-1 stain alone  (Fig. 4.6).
Triage of NSCLC: The most important step in optimizing a specimen involves appropriate triage. Especially in a case of an advanced, poorly differentiated NSCLC, selecting tissue particles for smear preparation permits allocation of the majority of specimen for ancillary studies. A limited panel of immunohistochemical stains leaves adequate tissue for molecular testing, when indicated
Optimizing: Molecular Testing for Carcinomas
For patients presenting with advanced stage (IV) lung adenocarcinomas who are suitable for therapy, testing for epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) is standard of care and should be ordered at the time of diagnosis . Even in the presence of an EGFR mutation or ALK rearrangement, patients have relatively short life expectancies , so obtaining sufficient tissue and appropriately triaging it without delay or having to perform a repeat procedure is important. Similarly, when EBUS is being performed for simultaneous staging and diagnosis of NSCLC, the sample has to be managed especially carefully. Dedicated passes improve the yield of the specimen . For various reasons, including the inability of the patient to tolerate the procedure, risk of a pneumothorax, difficulty reaching the target, or lack of time or willingness, obtaining an additional pass may be difficult .