Immunohistochemistry Practices of Cytopathology Laboratories: A Survey of Participants in the College of American Pathologists Nongynecologic Cytopathology Education Program

A supplemental questionnaire was sent out to 1899 laboratories participating in the CAP Nongynecologic Cytopathology Education Program in the final quarter of 2009. The questionnaire was developed by members of the CAP Cytopathology Committee to reflect potential issues and problems with cytology immunohistochemical processes. The questionnaire included demographic data about laboratory size and complexity, current practices in cytology IHC performance, and types of platforms used by laboratories for IHC. By April 2010, 818 laboratories (43%) had voluntarily responded. The data collected did not distinguish fine-needle aspiration specimens from fluids or other types of cytology samples.

Less than half of respondents (345 of 791; 43.6%) performed IHC on cytology specimens. The survey listed 12 possible preanalytic conditions or platforms for performing IHC. As shown in Table 1, each of the 12 possible platforms offered as options in the survey was used by multiple laboratories. The least commonly used platform (cell blocks fixed in SurePath media without additional formalin exposure) was used by 11 of the 345 laboratories. Laboratories were allowed to select more than one platform for this part of the survey. Restricting attention to the single “principal” platform, Table 1 shows overall, 48.8% of laboratories used paraffin-embedded cell blocks. This included 25.5% of laboratories that used FFPE cell blocks, 21.2% that used alcohol-fixed material postfixed in formalin, and 2.1% that used alcohol-fixed material embedded in paraffin without any formalin. In addition to the 12 platforms cited in the survey, laboratories were given the chance to report specific fixative combinations other than those on the survey list that they used for direct smears. Participants submitted 5 additional fixatives used for direct smears prior to IHC. In total, the survey identified 17 discrete platforms or preanalytic conditions, with most of the platforms used independently by many laboratories (Table 1).

Table 2 shows the volume of testing and the number of different antibodies offered for use on cytology specimens. The median monthly volume of IHC testing was 15. When integrated for all 307 respondents, the total annual IHC test volume for cytology specimens in the survey sample was estimated to be 146 376. The median number of antibodies available per laboratory was 50. A total of 82.6% of laboratories used an automated immunostainer, 10.1% used both automated and manual immunostaining, and 7.3% of laboratories used only manual immunostaining.

A total of 311 laboratories responded to the question asking if the preanalytic conditions or platform for clinical cytology specimens differed from those of their positive IHC controls (Table 3). A total of 44.7% of laboratories indicated that the platform for positive controls was different from the platform for cytology specimens for all antibodies, whereas 14.5% indicated that the platform differed between positive controls and the cytology specimens for some of the antibodies. Thus, for 59.2% of laboratories, the platform for clinical and control specimens sometimes differed. A total of 40.8% of laboratories responded that the platform for cytology specimens was the same as the platform for the positive controls for all antibodies. Only a minority of laboratories (37.5%) that used the same platform for clinical cytology specimens as their positive controls reported that their primary platform for IHC was FFPE tissue.

A total of 184 laboratories responded to the question of whether antigen retrieval or antibody titer differed between cytology and surgical pathology specimens. A total of 26 (14%) reported that the antigen-retrieval process differed at least sometimes for cytology specimens compared with surgical pathology specimens for the same antigen. A total of 3 of 184 respondents (1.6%) reported that the antibody dilution used for any of the cytology IHC stains differed from that used for the surgical pathology specimens for the same antigen. Of the laboratories that indicated an alteration in antigen retrieval (n = 26) or an alteration of antibody titers (n = 3) for cytology specimens, 11 laboratories responded to a request to list the 2 most commonly used antibodies that required an altered protocol for cytology specimens compared with surgical specimens (Table 4). These 11 laboratories listed 14 different antibodies for which they altered either antigen retrieval or antibody concentrations for cytology specimens, and at least 6 different platforms were employed. The most frequently altered antibodies were pankeratin or cytokeratin AE1/AE3, and all 3 laboratories that altered the process for these antibodies used alcohol-fixed direct smears as their principal platform for testing.

Validation of nonformalin fixation conditions for HER2 IHC has become an established guideline promulgated by a joint publication of CAP and the American Society of Clinical Oncologists (ASCO) in 2007.¹⁷  Laboratories that currently offer cytology IHC testing were surveyed to examine whether the institution of the CAP/ASCO guidelines had changed practice patterns. As shown in Table 5, 30.6% of respondents offered HER2 testing prior to publication of the CAP/ASCO HER2 validation guidelines, and 33.6% offered HER2 testing after publication of the guidelines. The percentage of laboratories that sent cytology specimens for HER2 testing to a reference laboratory increased from 9.5% to 12.5% following publication of the guidelines (χ²  test; P < .001). Four laboratories attempted validation of a non-FFPE platform. Three laboratories successfully validated an alcohol-fixed, nonembedded platform for HER2 testing. One laboratory found air-dried smears could not be successfully validated.

The data for this survey were collected between the fall of 2009 and April 2010, but they remain relevant to efforts to understand problems and offer solutions for validating IHC testing on cytology specimens. Although most surgical pathology laboratories use FFPE material as their platform for IHC, this survey found that only 25.5% of laboratories used FFPE as the principal platform for cytology IHC. Only 48.8% of laboratories used paraffin-embedded material (fixed by any means) as their principal platform, and only about half of laboratories included any use of formalin in their principal platform. This estimate included SurePath media as a formalin-containing platform because it contains a small concentration of formalin in addition to alcohol. A survey of European cytology laboratories¹⁸  found a lower proportion using cell blocks as a platform (13 of 66; 20%), and fewer respondents reported formalin use for fixation (7 of 24; 30%).

A total of 40.8% of responding laboratories used control specimens that were always processed in the same manner as their cytology specimens. For these laboratories, only 37.5% reported that FFPE was their principal platform. The remaining 62.5% of laboratories (about 25% of total respondents) apparently use cytology specimens—or they make cytologic preparations from surgical specimens—for their controls. Our finding that only about 41% of laboratories use controls that are fixed or prepared in the same manner as their cytology specimens mirrors that of other published academic studies. In a meta-analysis of 100 journal articles, only 46% (11 of 24) of these studies specified that the controls for cytology were processed in the same manner as their clinical cytology specimens.¹⁹  Thus, in our study the platforms for cytology IHC differed from that of IHC controls in more than half of surveyed laboratories, similar to the proportion found in published academic studies.

The CAP accreditation checklist (relevant parts unchanged since 2009) stresses the importance of using control specimens processed identically to clinical specimens, but it acknowledges the difficulty in providing control specimens for all types of platforms and fixatives. According to the current checklist,¹⁶  differences between the platform for clinical patient specimens and controls are acceptable, provided “the [individual] laboratory can show that these patient specimens exhibit equivalent immunoreactivity.” Our survey did not specifically ask whether laboratories actually validated equivalent immunoreactivity, but our survey shows that this checklist question pertained to most laboratories (about 60%) that performed IHC on cytology samples. Validations are expensive and time-consuming for laboratories, and their cost would be expected to disproportionately discourage low-volume tests. Our survey showed that cytology IHC is a low-volume test. Our respondents performed only a median of 180 tests per year per laboratory and offered a median of 50 antibodies. It therefore appears that many antibodies are used less often than a few times per year per laboratory, and yet verifications could have been needed to ensure that clinical samples exhibited equivalent immunoreactivity if positive controls were fixed differently. The CAP checklist item ANP.22550 acknowledges the burden of validating low-volume tests by stating: “this can be accomplished by parallel testing a small panel of common markers to show…equivalent immunoreactivity to routinely processed, formalin-fixed tissue.”¹⁶ 

Appropriate IHC results are dependent on preanalytic platform conditions, analytic conditions (eg, antigen retrieval, antibody titer, and staining technique), and postanalytic interpretation. The relative importance of these 3 steps has not been established. Although preanalytic steps can alter the relative intensity of IHC results,⁴  there is clear evidence from the literature that appropriate staining can nevertheless be achieved using a wide variety of platforms.^1,7,10,15  For example, in a large, predominantly European study, participants sent their stained positive controls used for cytology specimens for an independent assessment.¹  Of 1482 positive control slides stained for a variety of antigens, 66% were FFPE tissue, 6% were cell blocks, 18% were cytospins, 5% were other liquid-based, nonembedded specimens, and 5% were direct smears. Interestingly, the quality of the immunostains for these different platforms was judged to be similar. Acetone was judged to be inferior as a fixative, not for the immunostaining per se, but because the underlying cytomorphology was not well preserved.¹ 

One goal of this survey was to capture the collective expertise of many individual laboratories. As described in the CAP laboratory accreditation checklists, IHC validations or verifications are conducted individually by each laboratory,¹⁶  and the literature on IHC validity on cytology specimens is dominated by the relatively small experiences of individual laboratories. Our study cohort performed an estimated 146 000 IHC stains per year as reported by 307 laboratories, providing a broad, unbiased screen for problems with certain platforms or antibodies. Problems could be expected to be uncovered if laboratories fulfilled the CAP checklist requirement by having assessed at least a subset of antibodies for the platforms that did not match the platform for their control slides. Laboratories also presumably would have identified problems with cytology IHC through the CAP requirement to perform correlation with available histologic material. Although it is possible that the surveyed laboratories simply were not able to gauge the quality and validity of the cytology IHC,¹  there are few indications of problems with particular platforms or antibodies. The fact that so many different platforms were used at relatively similar frequencies suggests that there has not been a convergence toward an optimal platform. The least commonly used platform—paraffin-embedded specimens fixed in SurePath media—was still used at least sometimes by 11 CAP-accredited laboratories. Thus, for each of the various platforms used, there were multiple independent opportunities to identify needed adjustments of antibody titers or antigen-retrieval conditions. The survey shows that relatively few adjustments were made for cytology specimens. Only 26 of 184 laboratories (14%) reported that they used different antigen-retrieval methods for at least some cytology specimens compared with their surgical pathology specimens. Only 3 of 184 laboratories (1.6%) altered the titer of an antibody for cytology specimens compared with surgical specimens. Laboratories that adjusted conditions should be expected to have been vigilant regarding problems with cytology IHC. When asked to list 2 antibodies that required an alteration of either antigen retrieval or titer, 11 laboratories responded by listing 14 antibodies (Table 4). Five different platforms were used by the laboratories that responded. Except for possible problems with pankeratin or cytokeratin AE1/AE3 (listed by 3 laboratories, all of which used direct smears fixed with 95% alcohol), no other antibodies were uniformly identified by laboratories as causing problems when used with cytology specimens.

These data suggest that the optimal conditions for FFPE may generally be close to the optimal conditions for any non-FFPE cytology platform, a finding supported by the results of the external quality control initiative in Europe.¹  Many reports specified that the same (or “virtually” the same) staining protocol was used for FFPE specimens as for non-FFPE cytology specimens.^{5–7,9,10,12,14}  In their survey of 28 laboratories (primarily European laboratories), Schmitt et al¹⁸  found that IHC conditions were identical for more than 50% of laboratories for cytology specimens compared with surgical specimens, even though cell blocks were used by only 20% of laboratories.¹⁸ 

Cytology IHC shares with surgical pathology the same potential sources of error in the analytic and postanalytic aspects of IHC, and there is clear evidence that major variability in IHC results can be attributed to analytic or postanalytic factors. For example, the organization Nordic immunohistochemical Quality Control (NordiQC, Aalborg, Denmark) has surveyed 202 laboratories regarding their cytokeratin AE1/AE3 antibody staining on uniformly prepared FFPE tissue microarrays, and only 65% of laboratories produced “optimal” or “good” results; 10% of laboratories produced “borderline” results; and 25% produced “poor staining” results.²⁰  Likewise, a NordiQC survey found that PAX8 staining of FFPE tissue gave “borderline” or “poor” results in 37% of 35 laboratories.²¹  One can also point out that the problems with inaccurate estrogen receptor/progesterone receptor and HER2 staining^22–25  that led to new IHC testing guidelines^17,26  were uncovered when other laboratories restained the same blocks of tissue. These results prove that clinically serious variation in IHC is not related to the preanalytical platform. Nevertheless, guidelines that emerged from discovering these analytic errors focused their strongest attention on preanalytic conditions for HER2 and estrogen receptor/progesterone receptor IHC.

Our survey offers insights into the effects of validation guidelines on practice patterns. The 2007 consensus guidelines¹⁷  (recently updated²⁷) for HER2 testing require extensive validation if FFPE is not used as the test platform. Only a minority of laboratories offered HER2 testing on cytology specimens (34% of responding laboratories). Surprisingly, HER2 testing on cytology specimens was offered by slightly more laboratories after the publication of these guidelines. This small increase is likely the result of laboratories sending specimens to a reference laboratory for HER2 testing, the proportion of which increased from 9.5% to 12.5% in our survey. A presumption is that reference laboratories have performed validations of the various non-FFPE cytology platforms. It is possible that laboratories sent out HER2 testing to avoid the cost of validating the test in their own facilities. The cost for 1 laboratory to perform validations of 2 non-FFPE fixatives for fluorescent in situ hybridization and IHC detection of HER2 status was estimated to be $26 000, based on Medicare reimbursement rates for only the Common Procedural Terminology code technical components.²⁸  Only 4 laboratories reported an attempt at formal validation of HER2 testing on non-FFPE material, and 3 of the 4 successfully validated non-FFPE platforms. Air-dried smears were found not to be a suitable platform for HER2 IHC.

Laboratories independently face the problem of adapting IHC protocols to cytology platforms that do not match their positive controls. Given that valid IHC results can be obtained from a variety of cytology platforms,¹  and that the analytic and postanalytic components of IHC remain problematic for FFPE specimens as well as cytology specimens, what are the most effective steps that can be taken to help ensure valid IHC results throughout anatomic pathology? A review of IHC validation procedures and practices²⁹  suggested that uniformity in the approach that individual laboratories take for IHC validation should lead to more consistently accurate IHC results. The authors of the review noted that the scarcity of appropriate control materials is a serious issue, particularly for cytology. In vitro cell lines³⁰  could be an optimal control because their antigen content can be precisely quantified, and they can be grown in sufficiently large quantities to establish national, or even international, IHC standards. Such cell lines could be provided unfixed and frozen, ready to be prepared with any preanalytic platform, including FFPE cell blocks. Use of standardized cell lines could be of major benefit to labs that use FFPE tissues as the platform for IHC, because different laboratories use different positive control tissues that can have different amounts of antigen. For the approximately 25% of responding laboratories that appear to create their own cytology controls, there could also be intra-institution variation between the cytology and surgical pathology controls. Standardizing the control materials between laboratories, or between cytology and surgical pathology laboratories, would provide needed solid ground for validation efforts.

Another way of efficiently ensuring accurate cytology IHC results could be to use a consortium approach, whereby laboratories share their protocols for the analytic aspects of IHC and share the burden of validating their cytology IHC platforms. A consortium approach to help laboratories use proper protocols has been successfully developed for formalin-fixed surgical specimens by NordiQC, a European-based nonprofit organization (http://www.nordiqc.org/; last accessed October 10, 2013), and by the European organization NEQAS (National External Quality Assessment Service).¹ 

The relative importance of preanalytical, analytical, and post-analytical steps in achieving clinically valid IHC results is uncertain. The finding of a wide variety of preanalytical platforms used by laboratories in our survey, without evidence of particular problems, suggests that the analytical and post-analytical steps deserve relatively more attention, for both surgical pathology and cytopathology.