Practice gap. Currently, there is no standardized algorithm for optimal procurement, processing, and triaging of small specimens
Since diagnoses are based more frequently on small biopsy and/or cytology specimens, which sometimes represent the only sample, it is of utmost importance to obtain and manage specimens in a manner that provides all of the pertinent information necessary for appropriate therapy. This chapter outlines methods to optimize small biopsy and cytology acquisition and preservation.
Cytology Specimens and Core Biopsies
There is ongoing debate centering on which specimen type, an aspirate or a core biopsy, is the better method for providing sufficient tissue. The method, FNA or needle core biopsy, chosen is influenced by several issues, including the preference and comfort of the radiologist and/or pathologist , the procedure being performed (e.g., only FNAs are performed with EBUS), availability of on-site evaluation by a cytologist, risk to the patient (e.g., underlying lung pathology, such as emphysema/bullous disease), and nature of the lesion (e.g., size, location, consistency).
There are various types of cytology specimens available for diagnosis, including FNAs and exfoliative specimens such as pleural effusions, bronchoalveolar lavages, and bronchial brushings. Of these, FNAs offer the highest sensitivity (80–95 %) and specificity (98–100 %) in diagnosing malignancies [15–17]. Data strongly support that subtyping of NSCLC on cytological specimens is possible and accurate  particularly when cytomorphology is combined with IHC stains . When compared, FNAs are equivalent to core biopsies at definitively and specifically classifying NSCLC  and have similar rates of yielding sufficient material for molecular testing [19, 21, 22]. Recent studies have reported that not only are cytology specimens, including FNAs, effusions, and exfoliative specimens, suitable for EGFR and Kirsten rat sarcoma (KRAS) testing [19, 21], but there is also complete concordance of molecular testing results between FNA specimens and their histological counterparts, including resections and cores . For heterogeneous lesions, FNAs may outperform cores because of their strength to sample different aspects of the lesion with a single pass .
Core biopsies are an alternative source of diagnostic tissue. Recently, the performance of cores has been advocated by pathologists who are unavailable for rapid on-site assessment of FNAs and/or who sign-out mostly surgical pathology . Some have reported that optimal results are obtained when the two modalities are performed concurrently . Others follow a FNA with a core, if necessary , or commonly irrespective of a diagnostic result at the time of ROSE due to clinicians’ requests . In scenarios where both FNA and core biopsies are performed, comparison of the two is recommended because the two modalities may provide complementary information to render a specific and concordant diagnosis .
Overall, there is no consensus on the method of choice—FNA and/or core biopsy—and ultimate determination may rest upon institutional establishment of adequacy protocols and rates of success with each modality.
Rapid On-Site Evaluation: Fine-Needle Aspirations
Rapid on-site evaluation (ROSE) involves a cytology staff member, either a (cyto)-pathologist or cytotechnologist, providing real-time evaluation of the specimen during the procedure and communicating the findings to the clinician (e.g., pulmonologist, interventional radiologist, surgeon, or endosonographer). Correlation with the history and imaging provides an integrated approach to the diagnosis (Table 3.1).
Advantages of ROSE
Ensures sufficient tissue for diagnosis
Specimen procured for ancillary studies
Expedites clinical decisions
Enables scheduling of additional imaging/appointment(s) during single visit
Improves diagnostic accuracy
Positive predictive value
Enhances efficiency and cost-effectiveness of patient care
Reduces repeat procedures
Minimizes complication rate of procedure
Traditionally, providing a preliminary assessment was sufficient. Now, in light of the added importance and responsibility for the cytologist to properly triage the specimen, it is intuitive that the individual performing ROSE has experience in cytomorphology interpretation, be knowledgeable about ancillary tests necessary for various diagnoses, ensures that sufficient tissue is allocated for potential ancillary tests (e.g., immunohistochemistry and/or molecular testing for carcinomas, microbiological cultures in cases of inflammation or granulomas, or flow cytometry for lymphoma assessment), and be informed about methods of specimen fixation/preservation required for each test. Proper decision-making and specimen submission at the time of the procedure can directly affect patient care. Recent data strongly support that aspirates performed with ROSE optimize the use of the aspiration procedure [27, 28] by improving diagnostic yield  and aiding in the procurement of additional material for potential necessary special studies [29–40].
There is also value added to a well-prepared specimen—one without poor preservation or preparation—which expedites final examination. When the technical quality of a specimen is maximized, final interpretation is not compromised (Fig. 3.2). High-quality preparations limit intradepartmental consults and time consumed interpreting slides with limited cellularity, poor preparation, or artifacts and shift focus on diagnostic dilemmas. In cases where the same pathologist performs ROSE and issues the final report, time may not be “wasted”  as on-site assessment provides an opportunity to obtain the clinical history and preview of a fraction of the slides.
Role of specimen preparation in diagnostic interpretation. Technical quality, which includes smear preparation and staining, plays a significant role in the final interpretation. When performed appropriately, the morphology is well preserved, and interpretations at time of ROSE and final diagnosis are not compromised
ROSE of lung nodule aspirates is effective at discriminating malignant versus nonmalignant lesions, such as sarcoidosis , and having a cytopathologist present is associated with a significantly greater accuracy in diagnosis of malignancy [32–34, 37, 39]. ROSE increases sensitivity and specificity  and the predictive value of a negative test. Moreover, it increases the likelihood of attaining a higher rate of positive diagnoses , when present, and decreases unsatisfactory results . While the average reported non-diagnostic rate of FNAs is 20 % when on-site evaluation is not utilized , a diagnostic rate of 98 % has been achieved in a study of over 5,600 FNAs with ROSE . Diagnoses rendered at time of ROSE are highly accurate and usually concordant with the final interpretation [43, 44]. In the same study with >5,600 FNAs, the on-site interpretation was in agreement with the final diagnosis in 85 % of cases; there was disagreement in 2.7 % of cases and deferment to the final diagnosis in 12 %.
The high concordance of on-site immediate interpretation and final diagnosis allows for reliable decision-making that can enhance efficiency and cost-effectiveness of patient care . With a preliminary diagnosis, clinical decisions can be expedited [27, 45–49] and scheduling for additional imaging, appointment with a specialist, or surgical procedure can be initiated. The procedure can be terminated as soon as diagnostic material is obtained. By doing so, fewer needle passes [34, 50, 51] than those predetermined in the absence of ROSE  are performed at fewer sites, thereby decreasing the time a patient is under sedation, reducing the complication rate , and increasing the safety of the procedure. Next, ROSE results in a decrease in the number of repeat  or additional procedures necessary for a diagnosis , which has downstream effects. Assuming non-diagnostic FNAs are repeated, direct institutional charges can be significant. The diagnostic value and cost-effectiveness of ROSE was evaluated in 5,688 FNAs . In the absence of ROSE, the estimated calculated cost of performing repeat FNAs of non-diagnostic specimens resulted in additional direct institutional charges of $2,022,626 over 5 years. These savings are significant despite the professional service fee of on-site interpretation . Also, this does not take into account indirect costs to the patient, such as time off work and prolonged hospital stays. ROSE has downstream effects on the facility as well. Specifically, with shorter procedures times and fewer re-biopsies, utilization of procedure rooms and imaging facilities  is optimized.
ROSE is not always feasible, either by a pathologist or cytotechnologist, due to limited resources of the facility. For a pathologist, ROSE results in time consumption, workflow disruption, and little reimbursement, i.e., high cost for time spent at the procedure [29, 53, 54] (Table 3.2). From a pathologist’s economic standpoint, time is spent better signing out cases in the office than performing immediate, on-site evaluation of FNA specimens . Compared with routine surgical pathology, compensation schedules for intraprocedural FNA consultations by pathologists are insufficient [40, 53]. Moreover, relative value units for signing out cases in the office outweigh those generated during a single ROSE. The time and overall benefit are not calculated in the measurement of productivity. In light of this suboptimal compensation and often prolonged procedure times , some institutions defer ROSE to cytotechnologists or forego ROSE and place the entire specimen in a liquid-based cytological container . Despite the extra expense for the on-site cytology service , reports document that on-site analysis, specifically for transbronchial FNA, is cost-effective [56, 57].
Disadvantages of ROSE
Prolonged procedure time
Relative value units disproportionate to time and effort
Possibility of diagnostic misinterpretation
Need for consultation or ancillary studies
Though infrequent , another drawback of ROSE includes diagnostic misinterpretation, both false positive and false negative [58, 59]. In the majority of cases with differing on-site immediate diagnosis and final diagnosis, the reason is sampling error, i.e., only normal-appearing tissue evident on Diff-Quik stained smears and diagnostic cells present in the remaining specimen. In one report, the diagnostic yield (determining tissue adequacy) of ROSE in cases with high suspicion for lung cancer was 77.1 % (101 of 131) but increased to 93.9 % upon final cytological analysis following review of the remaining material processed in the laboratory . This suggests that the lesional tissue was sampled but not identified during ROSE; training in procurement and triage can improve the on-site yield in such instances. Additional reasons for discrepancy include scant cellularity, need for intradepartmental consultation or ancillary studies, intraobserver variability, and challenging diagnosis .
ROSE: Lymph Node Assessment with Endobronchial Ultrasound
When sampling a lymph node for staging, it is important to determine if the sample is adequate or not, especially when it lacks metastatic tumor or granulomatous inflammation [29, 31]. In the absence of lesional cells, the presence or absence of lymphocytes and/or pigmented (anthracotic) macrophages to confirm localization of the needle within the target and assessment of the quantity of lymphocytes impacts the negative predictive value, which is reportedly variable .
Currently, there are no strict guidelines to determine adequacy of a negative lymph node sample , and evaluation of the amount of lymphocytes can be subjective . For instance, a focally dense collection may be deemed adequate, but a similar number of lymphocytes scattered throughout the slide and diluted by blood and bronchial cells may be considered scant .
Criteria for objective evaluation have been proposed (Table 3.3) [29, 61, 62]. While some have suggested that a certain number of lymphocytes per high-power field and/or the presence of clusters of pigmented macrophages are good predictors of final adequacy assessment of a benign lymph node [29, 62], others report that the occurrence of moderate to abundant numbers of lymphocytes and/or pigmented macrophages is indicative of adequate lymph node sampling in most cases [31, 59]. One proposed system uses a scale (unsatisfactory = 0 lymphocytes; less than optimal = <40 lymphocytes/high power field (HPF); satisfactory= > 40 lymphocytes/HPF in most cellular areas) , and a high false-negative rate was associated with unsatisfactory and less than optimal lymphocytes, demonstrating that adequacy impacts the false-negative rate. Another semiquantitative scoring system for evaluating lymphocytes (0 = <40 lymphocytes; 1 = 41–200 lymphocytes; 2 = >200 non-confluent lymphocytes; 3 = confluent sheets of lymphocytes or germinal centers;  true negatives had scores of 2 or 3) also confirmed that true negatives were associated with greater numbers of lymphocytes. These criteria require time to count lymphocytes, which can be cumbersome, can delay the procedure, and can thus be a drawback. Also, presence of pigmented macrophages has to be interpreted with caution, as they can be derived inadvertently from non-lesional adjacent lung.
Proposed systems for evaluation of lymph nodes in the absence of pathology
Karunamurthy et al. 
Less than optimal
Number of lymphocytes/high-power field (HPF)
Higher false negativity in unsatisfactory and less than optimal cases.
Fibrosis/hyalinization due to Hodgkin lymphoma or treatment related changes have lower cellular yield
Alsharif et al. 
Number of lymphocytes
Confluent sheets or germinal centers
True negatives have scores of 2 or 3
Nayak et al. 
Number of lymphocytes/low power field (LFP) (×100)
Either “adequate” category is not satisfied
Germinal center fragments
>5 fields with > 100/LPF and <2 groups of bronchial cells/LPF
If not adequate, re-aspirate lymph node or aspirate another lymph node
The presence of bronchial cells in transbronchial aspirations of lymph nodes has no bearing on adequacy. However, excess incidental ciliated bronchial cells, reactive bronchial cells lacking cilia, or dysplastic bronchial cells may pose difficult differential diagnostic problems; they may obscure the presence of scant malignant cells and/or mimic carcinoma [29, 58, 62]. Air-drying artifact and poor fixation/preservation may also cause difficulty with interpretation since atypical bronchial epithelium and reserve cell hyperplasia may be present as tight cohesive clusters with apparent nuclear molding and without apparent cilia, thus mimicking small cell carcinoma . Performing IHC on cell block material may be helpful in such cases . Additionally, presence of granulomas in a sampled lymph node does not necessarily exclude malignancy since they may coexist with metastatic carcinoma.
ROSE: Touch Preparations of Core Biopsies
While ROSE is typically utilized for FNAs, touch preparations of core biopsies for on-site assessment of adequacy and preliminary diagnoses are also frequently employed. As with ROSE of FNAs, radiologists prefer ROSE on touch preparations of core biopsies to ensure adequate sampling of the lesion and triage of the specimen .
Performing on-site touch preparations of CT-guided core biopsies has been associated with greater diagnostic accuracy  and is comparable to that of frozen section diagnosis [64–66]. Touch preparations are sensitive (96 %) in detecting malignant thoracic lesions and predicting malignancy (98 % of cases) . Diagnostic accuracies of touch preparations and core biopsies are both high (92.3–94 % and 93–94 %, respectively), but the combination of touch preparation with core biopsy improves the diagnostic accuracy to 96.4–98 % [67, 68]. Similarly, it has been observed in a study of touch preparations of thoracic lesions that the combined evaluation of touch preparations and core biopsies has a higher NPV (90 %) than that of touch preparations (83 %) or core biopsies (79 %) alone . This stresses the value in a collective interpretation of both preparations.
The number of biopsies procured has been reported to be higher in patients with negative or inadequate touch preparation diagnoses compared to those with positive or suspicious touch preparation diagnoses (2.9 versus 3.7, respectively) . These data suggest that positive touch preparations indicate a true positive, and no further biopsy attempts are necessary, but further biopsy or diagnostic evaluation should be considered if the touch preparation is negative. Negative results may represent true negatives, as is evident in cases of organizing pneumonia in which the fibroblastic tissue is not readily evident on touch preparations , or
false negatives, stemming from suboptimal on-site specimen processing , sampling of adjacent normal tissue , scant cellularity, suboptimal needle position, surrounding or superimposed inflammation, severe necrosis, mucinous nature of the tumor, or coexisting tuberculosis .
There is debate about the role of touch preparations. There is concern of lesional cell transfer onto the touch preparation, leaving behind non-lesional tissue on the core biopsy . This may deem a specimen adequate on-site but inadequate for ancillary studies. Assessment of a touch preparation differs from that of a FNA. The transfer (touch) onto a slide is unlike that of a smear of a FNA . This may result in examination of fewer cells, cellular distortion, and air-drying artifact . Clues to diagnosis, such as dyscohesion of cells associated with lymphocytes, may be muted on a touch preparation, and excessive manipulation of the core may cause crush artifact raising concern for possible small cell carcinoma. Care in processing can minimize these artifacts.
Alternatives to ROSE
Laboratories may find it challenging to provide ROSE due to high volume, insufficient staffing, time constraints, and distance issues. Telecytology (TC), a relatively recent means to view slides remotely in real time, addresses these issues while allowing the pathologist to be involved in the evaluation process. Telepathology (TP) has been shown to be a valuable tool for frozen section diagnosis with high diagnostic accuracy [71–74]. While TC still lags behind TP in validation and use , it has value in off-site assessment of multiple organs [36, 76–83] and lymph nodes sampled under EUS, EBUS, and CT guidance [36, 78, 82] without significant difference in agreement between TC and ROSE . Concordance between TC and conventional ROSE has also been reported in adequacy assessment, assignment of preliminary diagnostic categories (non-diagnostic, benign, atypical, suspicious, malignant), correlation with final diagnoses [36, 77–81], and average number of passes performed . Although sample size was small, one study showed an 89 % sensitivity of both ROSE and TC; however, ROSE showed a higher specificity compared to TC (93 % versus 87 %, respectively) .
There are three main available methods of image transmission for TC and TP including (1) static image capture, (2) dynamic live video (real time), and (3) whole slide image (WSI) [75, 84]. For both the static and dynamic systems, a team approach that involves a skilled on-site operator experienced in cytology is essential. Briefly, static image capture, consisting of a microscope with an attached digital camera and internet access for file transmission, offers low cost, wide applicability, and access. Limitations include selective and narrow scope of image review and inability to view on multifocal planes [75, 84]. With the dynamic system, comprising a microscope with an attached camera capable of generating video, the operator at the originating site controls the microscope, and the pathologist situated remotely views the slide passively . Robotic systems are also available with capability of complete and unbiased slide review as well changes in magnification and focusing by the interpreting pathologist. WSI captures the entire slide content and permits slide review in a manner similar to that of a glass slide under a microscope [75, 84]. Disadvantages include increased time for entire slide scan and suboptimal depth of field views [75, 84].
TC has been shown to be a more effective use of a pathologist’s time on the clinical service, with a reported average time of 30 min saved per procedure . Although the actual time spent reviewing a case via TC versus conventional on-site assessment may be similar and not statistically significant [36, 78], the potential time saved in a given day for a pathologist working on a busy FNA service can be significantly reduced by eliminating time spent traveling to and from the procedure site and downtime either during prepping or in between passes. In a high-volume FNA service, the time saved for cytopathologists could potentially be measured in hours.
Loss of personal contact and communication between the cytopathologist and clinician/radiologist performing the FNA is a noteworthy concern. Although this issue is not addressed in much of the TC literature, one study noted that clinician satisfaction has not been negatively impacted by using TC over in-person assessment and that no issues related to TC have been identified by them . Others specifically report that even with TC, interaction with the endoscopist is not sacrificed and that TC allows for mass communication/teaching beyond any distance and advances communication so that pathologists can reach out to more clinicians, thereby improving patient care .
TC also allows the cytopathologist to actively participate in making decisions about proper triage for ancillary studies , including IHC, flow cytometry, molecular testing, and cultures; however, successful triaging requires that the on-site operator, whether a cytotechnologist, cytology fellow, or pathology resident, be knowledgeable of various preservation media and technically skilled in the preparation of slides that allows for necessary material to be saved for such ancillary tests. Comparison of rates of adequate material for ancillary tests, specifically IHC and molecular testing for lung cancer, in FNAs with in-person ROSE versus TC ROSE has not yet been addressed and is an issue that is worth investigating.
Several fixatives and transport media are available for cytology specimens. For EGFR molecular testing, guidelines provided by the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology recommend using formalin-fixed (10 % neutral buffered), alcohol-fixed (70 % ethanol), fresh, or frozen specimens following validation . Cytology specimens initially placed in other solutions (i.e., saline, RPMI) can be transferred to formalin or CytoLyt subsequently. Fixation for 6 to 12 hours is recommended for small specimens .
As stated above and discussed in the following section on cell blocks, formalin-fixed paraffin-embedded (FFPE) tissue is currently recommended for ancillary tests such as IHC stains and molecular assays [2, 11, 85, 86]. Typically, formalin is the fixative in which IHC stains and most molecular assays are optimized and validated, and FFPE material has potential to yield multiple tissue sections that are suitable for these ancillary studies [38, 87–90].
Liquid-based preparations (LBPs) offer a standardized alternative to conventional smear preparation and fixation for FNAs and exfoliative specimens, including respiratory specimens and effusions. The current FDA-approved liquid-based preparations (LBPs) in use are ThinPrep®/CytoLyt (Cytyc Corp, Marlborough, MA) and SurePath™ (Becton Dickinson) . The specimen is placed in an alcohol-based solution (e.g., CytoLyt), which serves as a fixative and transport medium. An automated system in the laboratory creates slides, which are stained with Papanicolaou stain, with an even distribution of cells and without loss of cells. Additionally, the process and solution minimize contamination by blood, inflammation, and debris.
There are several additional advantages of LBPs (Table 3.4). They preserve cells at room temperature in the fixative solution for extended periods of time (3 weeks in CytoRich and 3 months in PreservCyt) . CytoLyt solution appears to be superior to CytoRich Red in terms of the yield of suitable DNA . Cell blocks for ancillary tests  can be prepared from the unused cells in solution. Even without cell blocks, ancillary tests, such as IHC [93–95] and molecular analysis [92, 96–100], can be performed on LBPs. In fact, the percentage of malignant cells on FNA specimens assessed on ThinPrep® has been found to be significantly high . EGFR and KRAS mutation analyses have been successfully carried on lung carcinomas preserved in CytoLyt [96–98] and have shown comparable results between FNA samples and histological tissues . Potential advantages of utilizing LBP for molecular techniques include (1) homogeneous cell distribution, which is helpful in determining percentage of malignant cells in a specimen dominated by benign or normal cells, and (2) preparation of multiple slides, which spares the original routinely stained slides/smears .