Immunocytology, or immunocytochemistry (ICC), involves the use of ICC in diagnostic cytopathology. The application of immunostains in cytology cases has expanded and improved our ability to make definitive and accurate diagnoses beyond prior special stains. As seen in surgical pathology, a great deal of progress and expansion has occurred over the years in immunocytology, which has led to an increased number of available antibodies and subsequently an increased use of antibodies in cytopathology.
Use of Immunocytology
The many roles of immunostains in cytopathology are summarized in Box 21.1 . These include the ability to determine the cell type, such as differentiating lymphoid and epithelial cells by using CD45 (leukocyte common antigen [LCA]) and cytokeratin, respectively. In addition, immunostains help provide site-specific markers to determine the tissue of origin in a metastatic carcinoma and help to subclassify poorly differentiated malignancies. Immunostains for infectious etiologies also exist and can help determine the type of bacterial, fungal, viral, or other infection. Immunostains such as Ki-67 also help to grade certain malignancies, particularly neuroendocrine and mesenchymal tumors. New antibodies are also being used to screen cases for potential molecular abnormalities, as a quicker and cheaper alternative to upfront molecular testing. This increasing role and interest in using immunostains to provide predictive and prognostic information for patients continues to grow, and these roles will be addressed in greater detail in this chapter.
Determine cell type (e.g., lymphocyte vs. epithelial cell).
Determine tissue of origin by using site-specific markers, such as use of thyroid transcription factor 1 in metastatic carcinoma to support a pulmonary or thyroid origin.
Subclassify the type of infection by using immunostains for viruses, such as herpes simplex virus and cytomegalovirus, and so on.
Subclassify or subtype tumors (e.g., subclassify non-small cell carcinoma as adenocarcinoma or squamous cell carcinoma).
Grade malignancies, such as with Ki-67 in neuroendocrine and mesenchymal neoplasms.
Provide prognostic and predictive information (e.g., ERBB2 in breast carcinoma).
Diagnostic Immunocytochemistry: Techniques
Both aspiration and exfoliative specimens can be used for ICC by using a variety of cell preparations that include cytospins, smears or imprints, liquid-based cytology (LBC) specimens (e.g., ThinPrep [Hologic, Bedford, Massachusetts] specimens), and cell blocks ( Figs. 21.1 and 21.2 ). The initial cytomorphologic evaluation helps to determine the need for ICC and other ancillary studies to reach a definitive diagnosis and exclude other entities in the differential diagnosis. Once the conventional Romanowsky or Papanicolaou stains have been examined, a differential diagnosis is generated, and the specimen is appropriately triaged; this procedure includes selection of the optimal cell preparation for immunostains and other ancillary studies, which can help answer important questions and support the diagnosis.
Cytospins, smears, and imprint cytology slides may be air-dried or alcohol-fixed before staining (see Fig. 21.2 ). The most important feature is a thin, even layer of cells with minimal obscuring factors to avoid problems interpreting the stain results. Direct smears are helpful for ICC when no other material is available; however, the background artifacts can obscure interpretation of the immunostains, and limited material is available for a panel of markers. Cytospins are also helpful when there is no cell block or other material available, and given the limited material needed, several cytospin slides could be generated from a small sample for a panel of immunostains. In all of these specimens, prior staining or alcohol fixation may affect the results. A separate validation study should be done for these alcohol-based methods to ensure that results are comparable to formalin-fixed surgical specimens.
LBC or thin-layer techniques can also be used for ICC and have achieved good immunostaining results by using the proprietary solutions CytoLyt and PreservCyt (Hologic, Marlboro, MA) with or without processing on the ThinPrep processor. Even with long-term storage in PreservCyt, immunostaining is crisp, the background is cleaner than with direct smears, and immunoreactivity is stable. It is crucial that the immunostaining pattern is validated to the standard formalin protocol to ensure optimum results, and appropriate PreservCyt fixed controls should be used. A recent study showed that immunostaining for hormone receptors and ERBB2 on alcohol-fixed ThinPrep specimens was not affected by the fixation and correlated well with the results on formalin-fixed paraffin-embedded (FFPE) tissue. However, some antibodies may be affected by the alcohol fixation.
Cell blocks are all-purpose cellular material that can be used for special stains and for ICC (see Figs. 21.1 and 21.2 ). Similar to their paraffin-embedded histology specimen counterparts, cell blocks can withstand the processing protocols. In addition to the obvious advantage in studying the tissue architecture, additional tissue sections can be cut for ICC panels, and the paraffin blocks are robust ways to preserve tissue for up to about 25 years. This provides long-term storage option if additional theranostic studies or research is needed years after the biopsy. Ten percent neutral phosphate-buffered formalin (NBF) is used for fixation of tissue fragments. Interpretation of the results and storage of the specimen are easier, and antigen preservation is unlimited; for these reasons, cell blocks are the superior method for immunohistochemistry (IHC) for cytologic specimens. Cell block specimens can be collected directly into formalin or an alternative solution (e.g., RPMI salt solution, CytoLyt, other preservative), treated with a commercial thrombin-plasma agent to organize a clot or treated with HistoGel to create a pellet, and then fixed in 10% formalin and processed in a manner similar to that of small tissue biopsies. The main disadvantage of using cell blocks for ICC is that there is no way to assess cell block cellularity on-site, and thus, there may be a lack of sufficient cells of interest within the block for immunostaining.
The first fully automated rapid cell block (RCB) system introduced was Cellient (Hologic, Marlborough, MA), which has been shown to increase overall cellularity in the resulting sections with decreased time and a necessity for fewer reagents to make a cell block. A proprietary tissue cassette and filter assembly designed to capture tissue fragments also permits them to be positioned in a plane for microtomy ( Fig. 21.3 ). Small aliquots of xylene, alcohol, and paraffin are rapidly drawn through the sample to produce a broad, uniform layer of cells embedded in paraffin. The RCB produces a cell block in 15 minutes from residual ThinPrep vials or other specimens and can be used for a variety of gynecologic and respiratory tissues, fine needle aspiration (FNA) biopsies, body fluids, and other materials. If formalin is not used as the primary fixative, the alternative fixative must be validated against the formalin-fixed specimen, and controls must be used that have been fixed in the alternative material. In addition, the cost of the equipment can be a limiting factor for some institutions.
The use of cytoscrape cell blocks (SCBs) is another technique to prepare cell blocks, especially from stained FNA smears. This technique is useful when cell groups are obscured by clotted blood or when overlapping cell clusters interfere with the cytologic details and make interpretation difficult. The method involves previously stained smears that contain thick material in which the coverslip is removed with xylene. The slides are passed through two changes of absolute alcohol and water. Papanicolaou-stained smears are destained by 1% acid alcohol, whereas Romanowsky-stained smears are destained by 2% glacial acetic acid. The smears are then thoroughly rinsed in running tap water for 2 hours. Slides are carefully scraped with a scalpel blade, and the scraped material is meticulously transferred with forceps in 3% molten agar to form a small button. After the agar solidifies, it is wrapped in Whatman filter paper No. 1 and put in a tissue cassette. The scraped material is refixed in a histologic fixative, such as Bouin fluid or formal saline, for 5 to 6 hours and is routinely processed to make a paraffin wax block. Sections of 5 µm are cut and stained with hematoxylin and eosin. A study that compared SCBs with conventional cell blocks found that cytomorphologic details are equally superior in both types of specimen samples. An added advantage with this method is that additional panels of immunostains can be performed on SCBs, particularly when no cell block is available and repeat FNA is not feasible.
Although cell blocks are commonly and routinely used in nongynecologic cytopathology, they can also be helpful in cervicovaginal cytology. The utility of cell blocks prepared from residual material from Pap tests has been reported to be helpful for morphologic evaluation and ICC and for molecular studies. Cell blocks can be useful for identifying a high-grade squamous intraepithelial lesion (HSIL) and glandular abnormalities in addition to differentiating between atrophy and metaplasia. The diagnostic sensitivity and specificity of cell blocks from the residual samples have been shown to range from 86% to 100%. Another important use of cell blocks is for performing ICC when the Pap test interpretation is equivocal. A few papers have demonstrated the performance of IHC stains on cell blocks. A study that looked at the utility of p16 on cell blocks obtained from the residual samples found that the sensitivity of this immunostain was as high as 85% in Pap tests with a diagnosis of atypical squamous cells, cannot exclude HSIL (ASC-H); another similar study that used p16 and MIB-1 (Ki-67) found the concordance of Pap test and cell block interpretation to be as high as 85% ; thus it is important to recognize the growing importance of cell block preparations in cervicovaginal cytology.
Some cytologic preparations are not suitable for ICC. This category includes filter preparations, because the filter can detach from the slide and absorb the immunologic reagents and the chromogen, leading to high background staining. In addition, fluids with abundant mucoid material, necrosis, blood, or high protein content may have a precipitate that prevents penetration of reagents; thus these specimens may benefit from an additional washing step or from the use of an alternative preparation for ICC, such as a ThinPrep specimen that can help to minimize obscuring blood.
Some of the important prerequisites for optimal ICC results include well-prepared material, that is, a thin and uniform spread of cells with adequate fixation; minimal obscuring factors (e.g., necrosis, blood, mucus, and proteinaceous material); and a reproducible quality-controlled method of ICC. Wet fixation in alcohol (WFA) must be performed without delay, because air drying may result in distortion and false-positive results. An air-dried smear (ADS) is often more cellular than an alcohol-fixed slide, because some material often floats off the slide when it is alcohol fixed. Such air-dried slides must be fixed immediately before performing ICC, and the types of preferred fixatives vary among cytopathologists and different institutions. Cold acetone, formalin, CytoLyt, and 95% alcohol are commonly used fixatives. Of all the specimens and fixatives, FFPE cell blocks using 10% NBF at a pH of 5 to 7 provides an optimal specimen for ICC because of better specimen standardization, and thus they are commonly used in most institutions. A list of common fixatives and quality of the resultant specimen is provided in Table 21.1 .
|Fixative||Description of Fixative||DNA Quality||RNA Quality|
|Ethanol (includes BD SurePath solution)||70%–100% ethanol (ethyl alcohol)||Good||Good|
|ThinPrep (with CytoLyt and PreservCyt)||Methanol-based buffered solution||Good||Good|
|CytoRich Red/Blue||Ethanol-based solution |
CytoRich Blue preserves the red blood cells
|RPMI medium||Cell culture media with sodium bicarbonate||Good||Good|
|Neutral buffered formalin||Formaldehyde, phosphate buffers||Good-Fair||Good-Fair|
|Bouin’s||Picric acid, formaldehyde, and glacial acetic acid||Poor||Poor|
|Decalcifying agents||Weak acids (picric, acetic, and formic acid), strong acids (nitric and hydrochloric acid), or chelating agents (EDTA) a||Poor||Poor|
|B-5||Mercuric chloride, sodium acetate anhydrous||Poor||Poor|
a EDTA may yield higher quality DNA suitable for molecular studies, and less DNA degradation than samples decalcified with formic acid agents.
Suthipintawong and Leong and colleagues advocated postfixation of ADSs after testing different fixatives. Although physiologic saline and 96% alcohol with rehydration in normal saline for 30 minutes are the best fixatives, the cytomorphology is crisper, and less background staining is seen with the latter. A study by Fulciniti and associates has shown that formalin postfixed ADSs are reliable and better than the standard wet-fixed smears. The study proposed that the slow dehydration and short rehydration might contribute to a superior interpretation of the results. Our experience showed similar results with preparations that were relatively free of background blood and mucus.
An important point to keep in mind is that certain antibodies—for example, S100 protein, HepPar-1, estrogen receptor (ER), and gross cystic disease fluid protein 15 (GCDFP-15)—may be leached from alcohol fixatives and render false-negative results. For instance, ER staining on FNA smears by using different methods has shown that destaining the slides with alcohol before immunostaining significantly reduces the number of cells with positive nuclear staining. The available anti-ER antibodies (SP1, ID5, 6F11) perform best with 10% NBF for hormone fixation, as do other immunostains. In addition, S100 protein can show false-negative or decreased staining with alcohol fixation, as shown in the case of malignant melanoma involving the biliary tract in Fig. 21.4 .
An example that illustrates the importance of fixation is the evaluation of breast carcinoma. ERs and progesterone receptors (PRs) and overexpression of ERBB2 are important prognostic and predictive markers of breast carcinoma. The 2010 and 2013 American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) guideline recommendations for hormone receptor testing highlighted the importance of an accurate and reproducible assay with optimal tissue handling. The need for standardizing preanalytical variables is now well recognized, particularly tissue fixation, including the time to fixation (i.e., cold ischemic time), type of fixative, and duration of tissue fixation. Underfixation of tissue cannot be repaired and is the least desirable result of handling tissues in the anatomic pathology laboratory. No amount of antigen retrieval can resurrect a tissue that is underfixed. Antigens will be lost and false negatives will abound in such situations, regardless of the quality of instrumentation. Overfixation, on the other hand, may result in a specimen needing changes in antigen retrieval, antibody titer, and detection systems. The optimal result is to have a standard fixation time, ideally one that is unique for every antigen the pathologist is attempting to detect. For example, per the ASCO/CAP guidelines, the recommended minimum fixation in 10% NBF for hormone receptors is no less than 6 hours and no more than 72 hours, and usually approximately 24 hours is optimal. Routinely processed cytologic specimens fixed in formalin can also be used to assess the receptor status and HER2 protein by IHC.
One of the advantages of using cytologic specimens for immunostaining is that there is limited prefixation time in most cases, especially if on-site evaluation is used for FNAs, given that needles can be rinsed directly into formalin or other fixative. This is helpful given that the ASCO/CAP guidelines recommend limiting prefixation time to under 1 hour to minimize the degradation of nucleic acids and proteins in the tissue. Some of the effects of delayed fixation would include loss of immunostaining from degraded proteins, in addition to the loss of mitotic figures resulting in a falsely low proliferation index and inaccurate grading of tumors. In addition, although a fixation time of approximately 24 hours is preferred (range, 6 to 72 hours), efforts to reduce fixation time include the use of heated fixation. Another important aspect of fixation that is usually not an issue in cytology is fixative penetration given that the amount of cellular material is usually drastically less than the amount of fixative present. This penetration is more of a concern in surgical pathology where a 1 : 10 (range, 1 : 1 to 1 : 20) ratio of tissue to fixative is preferred for surgical specimens to optimize fixation.
The history of standardization attempts is long, and it has been well discussed by Clive Taylor, beginning with his transactions on the Biological Stain Commission and continuing into detailed discussions about the “total test concept” of the IHC tests (see also Chapter 1 ). The Biologic Stain Commission was one of the original agencies to oversee the special stains used in the anatomic pathology laboratory. The agencies that followed include the Clinical Laboratory Standards Institute (CLSI, previously NCCLS), the Food and Drug Administration (FDA), and commissions set up by professional organizations, including CAP. The emphasis of all these organizations has been on consistent, quality assay components for IHC. The consequence of all this work has resulted in the package inserts that accompany various IHC reagents. Despite all this work, there are still many preanalytical variables that have not been adequately addressed. In fact, in one paper from 2011, only 44% of the 61 preanalytical variables had published results, whereas the remaining did not have clear-cut evidence from the literature.
Furthermore, most of the established articles on preanalytical standardization focus on surgical pathology specimens, not cytology specimens, and thus, there is a paucity of data on all the preanalytical variables in cytopathology.
A study by members of the Ad Hoc Committee on Immunohistochemistry Standardization recommended formalin fixative as the standard for IHC testing, along with a minimum of 6 hours of fixative time for prognostic/predictive markers, including ER, PR, and HER2. These guidelines have been incorporated by the ASCO/CAP guidelines for breast cancer hormone receptor testing and follow the simple fact that all clinically validated studies on ER, PR, and HER2 antibodies have been performed on FFPE tissue. Alcohol fixatives may result in spurious results for certain antibodies, such as S100, HepPar-1, hormone receptors, and Her2/neu, as noted earlier; thus alcohol fixative is not recommended for evaluation of these markers. Although alcohol fixatives can be used for other antibodies, it is imperative for the laboratory director to validate protocols and use appropriate alcohol-based controls if alcohol fixation of cytologic specimens is used. FFPE cell blocks are the preferred samples for IHC in most laboratories for the reasons discussed, and with the FFPE cytology cell blocks, standardization protocols for surgical specimens can be easily applied to cytology specimens.
Rehydration and Storage
Air-dried slides and partially fixed air-dried slides can be rehydrated in normal saline to optimize immunoreactivity as well as cytomorphology. Chan and Kung found that the optimal time for rehydration is less than 1 minute, provided that the air-drying time did not exceed 30 minutes. This procedure may be used when cytomorphology is critically important, because air-dried slides can sit for up to 1 week at room temperature and still be used for ICC, provided they are fixed immediately before use, as already described. Slides for ICC, whether air dried or fixed, can be stored at −70°C for at least 1 month and still maintain immunoreactivity.
One of the important methods through which standardization of IHC can be achieved is antigen retrieval. An ideal antigen-retrieval technique is considered to maintain formalin as a standard fixative for both morphology and IHC. High-temperature heating is the most crucial step in this methodology to retrieve the antigens masked by formalin fixation. However, simple methods such as immersing in water or an NaOH-methanol solution will yield dramatic retrieval results. Although use of metal ions with zinc and lead in the antigen retrieval solutions has shown improved results in many studies, their environmental toxicity has given way to alternatives such as citrate, Tris, urea, and ethylenediaminetetraacetic acid (EDTA). Antigen retrieval has been widely used for detection of ER, PR, MIB-1, p53, Bcl-2, retinoblastoma gene (pRB) , and some cluster designation (CD) markers. Although the literature on the subject of use of antigen retrieval on cytology specimens is still evolving, some important studies have successfully shown that antigen retrieval can be applied to these specimens for a wide range of antibodies. Results are satisfactory and are comparable to their tissue specimen counterparts. Sherman and colleagues have shown a method of “cell transfer” that can facilitate immunostaining on small samples. In a study that compared the effects of heat (with a pressure cooker) on air-dried versus alcohol-fixed smears for cytokeratins AE1/AE3, 7, and 20; neurofibrillary protein (NFP); synaptophysin; ER (clone ID5) and PR (clone 1294); and vimentin, it was found that ADS results improved with antigen retrieval. Furthermore, alcohol-fixed smears can be successfully immunostained without antigen retrieval or with mild heat-induced antigen retrieval by citrate buffer. Miller and associates found that FNA biopsy material studied through tissue transfer to adhesive slides followed by antigen retrieval using the same conditions as for routine paraffin sections substantially improved the ease of interpretation, especially in the ADSs.
Positive and negative controls must be performed with each test sample. Tissue controls are typically used in most laboratories for convenience, but the ideal control should be a comparably fixed cytology sample. If tissue controls are used, the pathologist must exercise caution in interpretation, because a different set of artifacts is present in tissue compared with cytology samples. Cytology controls can be obtained daily from the surgical pathology bench with the use of aspirates or direct imprints. It is more practical to use cells on the cytology slide as a positive or negative internal control depending on the antibody and cells present. In a meta-analysis of the use of controls in cytopathology articles, only 13% of articles described the use of positive and negative controls run on identically prepared samples. This finding suggests that there should be more stringent documentation of appropriate controls in cytopathology on par with that seen in surgical pathology.
Specimens of Limited Quality
ICC can be hampered by a limited quantity of the specimen, which happens more often in cytopathology than surgical pathology. Some of the ways to overcome limited specimens include appropriately triaging the material for the studies needed, cutting blank slides from a cell block up front to avoid trimming the block, using alternative material if available, appropriately selecting the important studies needed, and performing multiplex staining for ICC. For example, in scenarios of scant cellularity on a cell block, aspirate smears or other material may be used (see Fig. 21.2 ). In addition, dual immunolabeling may be performed on the same slide instead of on separate slides, or even more antibodies may be multiplexed on the same slide, which will maximize the immunomarkers that can be used on smaller amounts of material. Some of the ways to maximize the cytologic material available for ICC are summarized in Box 21.2 .
Educate those who obtain the biopsies about the importance of procuring sufficient tissue.
Select the optimal type of specimen (exfoliative or aspiration cytology).
Ensure rapid on-site assessment by cytologists for adequacy assessment and appropriate triage.
Select appropriate fixative and processing by avoiding certain fixatives for certain antibodies.
Acquire additional dedicated material for ancillary studies, such as dedicated passes for cell block.
Establish, validate, and standardize laboratory protocol for processing and evaluation of samples.
Cut extra blank slides up front in an effort to conserve tissue and triage the blank slides for the appropriate stains.
Limit immunoperoxidase staining panels.
Use alternative material in cases with insufficient tissue in the cell block (e.g., aspirate smear material).
Use alternative procedures in cases of scant material, such as dual immunostaining or multiplex immunostaining.
Optimize and assess specimen processing with appropriate quality assurance.
In one study, Abendroth and Dabbs described a double-labeling method to address the problem of limited material when more than one antibody is required to make a diagnosis. Immunostaining was performed on these slides with and without a preceding decolorization step, and the results were similar, with good immunostain results. Background staining was more of a problem on the air-dried, unfixed cases. Thus recently stained or archived cytology slides can be used for ICC studies. Dual staining has also been shown in other studies in which more than one antibody, typically a nuclear and cytoplasmic or membranous stain, is used on the same slide as opposed to separate slides. Cytologic material can also be used in a variation of the dual immunolabeling technique. Cytology slides that were subjected to an immunoperoxidase stain and produced a negative result can also be subjected to another immunoperoxidase test using a different antibody. It is imperative to use positive and negative controls with both test runs. For example, a poorly differentiated tumor found to be negative for LCA can then be tested with cytokeratin to determine whether it is an epithelial tumor. Antibodies that have been used with this method include CAM5.2, AE1/AE3, 34βE12, GCDFP-15, vimentin, CD20, CD45RO, muscle-specific actin (HHF-35), desmin, CEA, and S100. Sherman and associates described the cell-transfer technique for use with limited cytologic samples. In this technique, cells are lifted off the slide by redissolving them in a new mounting medium. The medium can be removed from the slide, cut into pieces, and reapplied to slides for individual antibody studies. The results of immunostains are generally good, although Hunt and colleagues described some decreased staining with this method. In the future, multiplex staining with multiple antibodies on a single slide may help to minimize the tissue needed for ICC and maximize the results.
Small specimens can also be difficult to interpret due to equivocal or nonspecific staining, false-negative staining due to different fixatives, folded tissue, and other problems, which are discussed more in detail in the “Pitfalls in Immunocytochemistry” section (see Figs. 21.4 and 21.5 ).
Diagnostic Immunocytochemistry: Applications
Effusion cytology is one of the most challenging areas of cytopathology, wherein ICC serves as a valuable adjunct tool in definitive interpretation. Immunostains are critical to distinguish reactive mesothelial cells from carcinomas that involve body cavity fluids, because mesothelial cells can show significant atypia that mimics adenocarcinomas and other malignancies. In the absence of these studies, the sensitivity for the detection of malignant cells has been reported to be as low as 40%.
Normal fluid specimens contain mesothelial cells, which can exhibit a spectrum of cytomorphologic features that include changes that can mimic malignancy and features that can mimic bland-appearing neoplasms; this makes it difficult to recognize the foreign (i.e., neoplastic) population; thus ICC plays an important role in the identification of reactive mesothelial cells from adenocarcinoma. Several studies have highlighted and proposed panels of antibodies to resolve this issue. These studies indicate that combining the cytomorphologic evaluation with the results of a panel of antibodies that comprises both mesothelial and adenocarcinoma markers can significantly improve diagnostic accuracy.
ICC can be performed on a variety of preparations from fluid specimens, such as cell blocks, direct smears, cytospins, and LBC preparations (see Fig. 21.2 ). Storage of effusions at 4°C gives a satisfactory IHC outcome when a delay in processing of the samples is anticipated. In our experience, optimal results are achieved when ICC is performed on 10% NBF cell-block material, because the immunoreactivity pattern may be changed by different fixation methods.
A 29-kDa calcium-binding protein that is a member of the elongation factor (EF) family of proteins, calretinin, is thought to play a role in the cell cycle and is helpful in confirming cells of mesothelial origin, but it does not distinguish benign or reactive mesothelial cells from malignant mesothelial cells (i.e., mesothelioma; Fig. 21.6 ). Gotzos and colleagues were the first to report its expression by epithelioid mesotheliomas and epithelioid components of mixed mesotheliomas, and they reported negative results in adenocarcinoma. These results were later confirmed by studies that showed only rare focal positivity, primarily as a cytoplasmic blush, in a variety of carcinomas that included 10% to 30% of adenocarcinomas. The sensitivity of calretinin to distinguish reactive mesothelial cells from adenocarcinoma cells approaches 100%, and the specificity is as high as 80%. Interpretation of this marker is based on finding strong nuclear and cytoplasmic positivity in the cells of interest (see Fig. 21.6C ).