CAP Laboratory Improvement Programs
Immunohistochemistry Practices of Cytopathology Laboratories A Survey of Participants in the College of American Pathologists Nongynecologic Cytopathology Education Program Andrew H. Fischer, MD; Mary R. Schwartz, MD; Ann T. Moriarty, MD; David C. Wilbur, MD; Rhona Souers, MS; Lisa Fatheree, BSCT; Christine N. Booth, MD; Amy C. Clayton, MD; Daniel F. I. Kurtyz, MD; Vijayalakshmi Padmanabhan, MD; Barbara A. Crothers, DO
Context.—Immunohistochemistry (IHC) is important for cytology but poses special challenges because preanalytic conditions may differ from the conditions of IHC-positive controls. Objectives.—To broadly survey cytology laboratories to quantify preanalytic platforms for cytology IHC and identify problems with particular platforms or antigens. To discover how validation guidelines for HER2 testing have affected cytology. Design.—A voluntary survey of cytology IHC practices was sent to 1899 cytology laboratories participating in the College of American Pathologists Nongynecologic Cytopathology Education Program in the fall of 2009. Results.—A total of 818 laboratories (43%) responded to the survey by April 2010. Three hundred fourty-five of Accepted for publication November 27, 2013. Published as an Early Online Release May 19, 2014. From the Department of Pathology, University of Massachusetts Memorial Health Care, Worcester (Dr Fischer); the Department of Pathology and Genomic Medicine, The Methodist Hospital, Houston, Texas (Dr Schwartz); the Department of Pathology, AmeriPath Indiana, Indianapolis (Dr Moriarty); the Department of Pathology, Massachusetts General Hospital, Boston (Dr Wilbur); the Departments of Statistics/Biostatistics (Ms Souers) and Cytology Surveys (Ms Fatheree), College of American Pathologists, Northfield, Illinois; the Department of Anatomic Pathology, Cleveland Clinic Foundation, Cleveland, Ohio (Dr Booth); the Department of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota (Dr Clayton); the Department of Cytology, Wisconsin State Laboratory of Hygiene, Madison (Dr Kurtyz); the Department of Pathology, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire (Dr Padmanabhan); and the Department of Pathology and Area Laboratory Services, Walter Reed Army Medical Center, Washington, District of Columbia (Dr Crothers). Dr Fischer is an inventor of the Cellient Automated Cell Block System, licensed to Hologic, Inc, and he receives royalties for sales of this technology. The other authors have no relevant financial interest in the products or companies described in this article. Presented in part as a poster abstract at the 58th Annual Scientific Meeting of the American Society of Cytopathology; November 2010; Boston, Massachusetts. Corresponding author: Andrew H. Fischer, MD, Department of Pathology, University of Massachusetts Memorial Health Care, 1 Innovation Dr, Worcester, MA 01605 (e-mail: Fischa01@ummhc. org). Arch Pathol Lab Med—Vol 138, September 2014
791 respondents (44%) performed IHC on cytology specimens. Seventeen different fixation and processing platforms prior to antibody reaction were reported. A total of 59.2% of laboratories reported differences between the platforms for cytology specimens and positive controls, but most (155 of 184; 84%) did not alter antibody dilutions or antigen retrieval for cytology IHC. When asked to name 2 antibodies for which staining conditions differed between cytology and surgical samples, there were 18 responses listing 14 antibodies. A total of 30.6% of laboratories performing IHC offered HER2 testing before publication of the 2007 College of American Pathologists/American Society of Clinical Oncologists guidelines, compared with 33.6% afterward, with increased performance of testing by reference laboratories. Three laboratories validated a nonformalin HER2 platform. Conclusions.—The platforms for cytology IHC and positive controls differ for most laboratories, yet conditions are uncommonly adjusted for cytology specimens. Except for the unsuitability of air-dried smears for HER2 testing, the survey did not reveal evidence of systematic problems with any antibody or platform. (Arch Pathol Lab Med. 2014;138:1167–1172; doi: 10.5858/arpa.2013-0259-CP)
T
he goal of cytology is to use the smallest possible biopsy for diagnosis, thereby reducing risk and discomfort for patients, facilitating population-based cancer detection programs, allowing faster diagnosis than can be achieved with larger biopsies, and saving money for the health care system. Even with large-sized surgical biopsies, immunohistochemistry (IHC) is often needed for the diagnosis and determination of prognostic markers. The cytology literature documents the suitability of cytology specimens for IHC1 and the importance of IHC in allowing patients to realize the benefits of cytology.2–4 Working with minimally sized specimens poses a number of interesting technical challenges for laboratories, and the solutions to these challenges can result in a variety of potential preanalytic IHC ‘‘platforms.’’ For example, during adequacy assessment, much or all of a diagnostic cytology Immunohistochemistry Practices in Cytology—Fischer et al 1167
Table 1.
Preanalytical Platforms Used for Cytology Immunohistochemistry (IHC)
Cytology Platforms Cell block: material collected in physiologic transport media and then fixed in formalin Cell block: material fixed in ThinPrep media and postfixed in formalina Smear fixed in 95% alcohol Liquid-based: CytoLyte/PreservCyta Cytospin fixed in 95% alcohol Smear, air dried Smears fixed in another manner (formalin, CytoLyte, spray, acetone, ‘‘Davine’s lysing solution,’’ methanol) Cytospin air dried Cell block: material fixed in ThinPrep media without formalin exposure Cell block: material fixed in SurePath media and postfixed in formalinb Liquid based: Cytorich Red/SurePathb Cell block: material fixed in SurePath media without formalin exposure a b
No. of Laboratories Laboratories Listing This Using Platform as Principal Platform, %
Median Annual Volume of IHC Testing per Laboratory for the Primary Platform
187
25.5
240
185 151 144 128 82
19.1 17.0 10.9 6.5 7.6
120 168 120 240 180
57 50
5.3 1.5
216 228
46
2.1
120
38 31
2.1 2.6
300 120
11
0.0
0
ThinPrep media, CytoLyte, and PreservCyt are proprietary fixatives of Hologic Inc (Bedford, Massachusetts). SurePath and Cytorich Red are proprietary fixatives of Becton, Dickinson and Company (Franklin Lakes, New Jersey).
specimen may need to be smeared onto slides, leaving only the smears as the material available for performance of IHC. Blood contamination is another problem in working with minimal samples. Nonformalin fixatives have been developed specifically to overcome the dilution of diagnostic fragments by blood and precipitated serum proteins. There are many reports of successful IHC staining on cytology specimens using a variety of preanalytic ‘‘platforms,’’ including direct smears5; various monolayer preparations, such as ThinPrep (Hologic Inc, Marlborough, Massachusetts)6,7; SurePath (BD Diagnostics, Franklin Lakes, New Jersey)8; cytospin preparations1,9; and cells removed from a stained, cover-slipped smear and transferred to another slide10,11; or paraffin-embedded cell blocks prepared with or without formalin.9,12–15 There are no data on the number of laboratories using these different preanalytic IHC platforms in the United States. Positive and negative controls are required for IHC to assure valid staining results, and failure to document the appropriate use of positive and negative controls is a phase 2 deficiency that can affect laboratory accreditation by the College of American Pathologists (CAP). Regarding positive control material, CAP states: ‘‘Ideally, the positive control tissue would be the same specimen type as the patient test specimen’’ (for example, formalin-fixed, paraffin-embedded [FFPE] tissues). The small size of cytology specimens makes it difficult for specimens to be saved for use as positive controls, and CAP notes accordingly that ‘‘. . .for most laboratories, it is not practical to maintain separate positive control samples to cover every possible combination of fixation, processing, and specimen type. Thus, it is reasonable for a laboratory to maintain a bank of formalin-fixed tissue samples as its positive controls. . .[provided] that the laboratory can show that these patient specimens exhibit equivalent immunoreactivity’’ (italics added for emphasis).16 The purpose of this study was to examine the number of different cytology IHC platforms used by participants in the CAP Nongynecologic Cytopathology Education Program. Because validation of cytology platforms can involve 1168 Arch Pathol Lab Med—Vol 138, September 2014
considerable expense and time for laboratories, a second goal was to survey a large number of laboratories to identify problems with particular antibodies or preanalytic platforms that would be expected to be uncovered when laboratories addressed the CAP checklist question that relates to verifying the comparability of their cytology platform to their positive controls. MATERIALS AND METHODS 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.
RESULTS 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 Immunohistochemistry Practices in Cytology—Fischer et al
Table 2.
Volume of Cytology Immunohistochemistry (IHC) Testing and Number of Antibodies Offered No. of Respondents
5th Percentilea
25th Percentile
50th Percentile
75th Percentile
95th Percentile
307
1
5
15
35
125
298
2
25
50
85
150
Estimated No. of IHC tests performed on cytology specimens per month No. of different IHC antibodies available for testing cytology specimens a
Percentile distribution.
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 Table 3.
(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 ¼ 26) or an alteration of antibody titers (n ¼ 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 (v2 test; P , .001). Four laboratories attempted validation of a non-FFPE platform. Three laboratories
Differences in the Platform Between Cytology Specimens and Positive Controls, and Adjustments of Antigen-Retrieval Conditions or Antibody Titers No. (%) of responses
Are the positive IHC controls used for cytology samples fixed or processed differently than the cytology samples being tested? Yes, for all antibodies Yes, for some antibodies Noa Is the antigen-retrieval process for any of the cytology IHC stains different from that used for the surgical pathology process for the same antigen? Yes No Is the antibody dilution used for any of the cytology IHC stains different from that used for the surgical pathology dilutions used for the same antigen? Yes No
139 (44.7) 45 (14.5) 127 (40.8) 26 (14.1) 158 (85.9) 3 (1.6) 181 (98.4)
Abbreviation: IHC, immunohistochemistry. a Respondents were asked to skip the next 2 questions. Arch Pathol Lab Med—Vol 138, September 2014
Immunohistochemistry Practices in Cytology—Fischer et al 1169
Table 4. Preanalytic Platforms and Antibodies for 11 Laboratories That Reported a Difference in Either Antigen Retrieval or Antibody Dilution for Cytology Specimens Compared to Surgical Specimens Primary Preparation
Antibody 1
Direct smears fixed in 95% alcohol Direct smears fixed in 95% alcohol Direct smears fixed in 95% alcohol Direct smears fixed in 95% alcohol Direct smears fixed in 95% alcohol Direct smears fixed in a way other than by air drying or with 95% alcohol Liquid-based preparation using CytoLyte/PreservCyt Liquid-based preparation using CytoLyte/PreservCyt Cell block sample fixed in ThinPrep media and postfixed in formalin Air dried Not stated
CK7 TTF-1 Pneumocystis jiroveci Cytokeratin AE1/AE3 Pancytokeratin ER ‘‘Cytoplasmic membrane’’ CD3 CK20 LCA Vimentin
Antibody 2 CK20 Calretinin Cytokeratin AE1/AE3 TTF-1 LMWK—low molecular weight ER Chromogranin
Abbreviations: CK, cytokeratin; ER, estrogen receptor; LCA, leucocyte common antigen; LMWK, low molecular weight keratin; TTF, thyroid transcription factor. A participant did not list a second antibody, resulting in the blank spaces.
successfully validated an alcohol-fixed, nonembedded platform for HER2 testing. One laboratory found air-dried smears could not be successfully validated. COMMENT 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
Table 5. HER2 Testing by Laboratories That Perform Immunohistochemistry (IHC) on Cytology Specimens No. (%) of Responses Is HER2 IHC testing currently offered on cytology samples? Yes, within the institution/laboratory Yes, sent to reference laboratory No Did you previously offer HER2 IHC testing on cytology samples prior to publication of the CAP/ASCO guidelines in 2007? Yes, within the institution/laboratory Yes, sent to reference laboratory No Has the laboratory attempted validation of a nonformalin fixative for HER2 testing on cytology samples? Yes No Which nonformalin fixatives has the laboratory tried to validate? 95% alcohol—successful (direct smear [1]; cytocentrifuged prep [1]; not specifically stated [1]) Air dry—not successful
69 (21.1) 41 (12.5) 217 (66.4) 67 (21.1) 30 (9.5) 220 (69.4) 4 (1.2) 319 (98.8) 3 1
Abbreviations: ASCO, American Society of Clinical Oncologists; CAP, College of American Pathologists. 1170 Arch Pathol Lab Med—Vol 138, September 2014
Immunohistochemistry Practices in Cytology—Fischer et al
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 lowvolume 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 Arch Pathol Lab Med—Vol 138, September 2014
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 staining22–25 that led to new IHC testing guidelines17,26 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 nonFFPE 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 Immunohistochemistry Practices in Cytology—Fischer et al 1171
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. References 1. Kirbis IS, Maxwell P, Fleˇzar MS, Miller K, Ibrahim M. External quality control for immunocytochemistry on cytology samples: a review of UK NEQAS ICC (cytology module) results. Cytopathology. 2011;22(4):230–237. 2. Skoog L, Tani E. Immunocytochemistry: an indispensable technique in routine cytology. Cytopathology. 2011;22(4):215–229. 3. Schmitt F, Barroca H. Role of ancillary studies in fine-needle aspiration from selected tumors. Cancer Cytopathol. 2011;120(3):145–160. 4. Fowler LJ, Lachar WA. Application of immunohistochemistry to cytology. Arch Pathol Lab Med. 2008;132(3):373–383.
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Colasacco C, Mount S, Leiman G. Documentation of immunocytochemistry controls in the cytopathologic literature: a meta-analysis of 100 journal articles. Diagn Cytopathol. 2010;39(4):245–250. 20. NordiQC immunohistochemistry quality control for pan keratin. NordiQC We b s i t e . h t t p : / / w w w. n o r d i q c . o r g / R u n - 3 6 - B 1 4 - H 2 / A s s e s s m e n t / assessment-36-CK-PAN.htm. Accessed November 20, 2013. 21. NordiQC immunohistochemistry quality control for PAX8. NordiQC Web site. http://www.nordiqc.org/Run-34/Assessment/assessment-34-PAX8.htm. Accessed November 20, 2013. 22. Paik S, Bryant J, Tan-Chiu E, et al. Real-world performance of HER2 testing– National Surgical Adjuvant Breast and Bowel Project experience. J Natl Cancer Inst. 2002;94(11):852–854. 23. Perez EA, Suman VJ, Davidson NE, et al. HER2 testing by local, central, and reference laboratories in specimens from the North Central Cancer Treatment Group N9831 intergroup adjuvant trial. J Clin Oncol. 2006;24(19):3032–3038. 24. Roche PC, Suman VJ, Jenkins RB, et al. Concordance between local and central laboratory HER2 testing in the breast intergroup trial N9831. J Natl Cancer Inst. 2002;94(11):855–857. 25. Gelber RD, Gelber S. Facilitating consensus by examining patterns of treatment effects. Breast. 2009;18(suppl 3):S2–S8. 26. Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Arch Pathol Lab Med. 2010;134(7):e48–e72. 27. Wolff AC, Hammond MEH, Hicks DG, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update [published online ahead of print October 7, 2012]. Arch Pathol Lab Med. 2014;138(2):241–256. 28. Fischer AH, Savas L, Smith K, et al. Validation of Cellient-type processing of FNA’s without formalin for Her2/Neu testing. Cancer Cytopathol. 2008; 114(suppl 5):351. 29. Hardy LB, Fitzgibbons PL, Goldsmith JD, et al. Immunohistochemistry validation procedures and practices: a College of American Pathologists survey of 727 laboratories. Arch Pathol Lab Med. 2013;137(1):19–25. 30. Kurtycz DF, Logrono R, Leopando M, Slattery A, Inhorn SL. Immunocytochemistry controls using cell culture. Diagn Cytopathol. 1997;17(1):74–79.
Immunohistochemistry Practices in Cytology—Fischer et al
Immunohistochemistry Practices of Cytopathology Laboratories A Survey of Participants in the College of American Pathologists Nongynecologic Cytopathology Education Program Andrew H. Fischer, MD; Mary R. Schwartz, MD; Ann T. Moriarty, MD; David C. Wilbur, MD; Rhona Souers, MS; Lisa Fatheree, BSCT; Christine N. Booth, MD; Amy C. Clayton, MD; Daniel F. I. Kurtyz, MD; Vijayalakshmi Padmanabhan, MD; Barbara A. Crothers, DO
Context.—Immunohistochemistry (IHC) is important for cytology but poses special challenges because preanalytic conditions may differ from the conditions of IHC-positive controls. Objectives.—To broadly survey cytology laboratories to quantify preanalytic platforms for cytology IHC and identify problems with particular platforms or antigens. To discover how validation guidelines for HER2 testing have affected cytology. Design.—A voluntary survey of cytology IHC practices was sent to 1899 cytology laboratories participating in the College of American Pathologists Nongynecologic Cytopathology Education Program in the fall of 2009. Results.—A total of 818 laboratories (43%) responded to the survey by April 2010. Three hundred fourty-five of Accepted for publication November 27, 2013. Published as an Early Online Release May 19, 2014. From the Department of Pathology, University of Massachusetts Memorial Health Care, Worcester (Dr Fischer); the Department of Pathology and Genomic Medicine, The Methodist Hospital, Houston, Texas (Dr Schwartz); the Department of Pathology, AmeriPath Indiana, Indianapolis (Dr Moriarty); the Department of Pathology, Massachusetts General Hospital, Boston (Dr Wilbur); the Departments of Statistics/Biostatistics (Ms Souers) and Cytology Surveys (Ms Fatheree), College of American Pathologists, Northfield, Illinois; the Department of Anatomic Pathology, Cleveland Clinic Foundation, Cleveland, Ohio (Dr Booth); the Department of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota (Dr Clayton); the Department of Cytology, Wisconsin State Laboratory of Hygiene, Madison (Dr Kurtyz); the Department of Pathology, Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire (Dr Padmanabhan); and the Department of Pathology and Area Laboratory Services, Walter Reed Army Medical Center, Washington, District of Columbia (Dr Crothers). Dr Fischer is an inventor of the Cellient Automated Cell Block System, licensed to Hologic, Inc, and he receives royalties for sales of this technology. The other authors have no relevant financial interest in the products or companies described in this article. Presented in part as a poster abstract at the 58th Annual Scientific Meeting of the American Society of Cytopathology; November 2010; Boston, Massachusetts. Corresponding author: Andrew H. Fischer, MD, Department of Pathology, University of Massachusetts Memorial Health Care, 1 Innovation Dr, Worcester, MA 01605 (e-mail: Fischa01@ummhc. org). Arch Pathol Lab Med—Vol 138, September 2014
791 respondents (44%) performed IHC on cytology specimens. Seventeen different fixation and processing platforms prior to antibody reaction were reported. A total of 59.2% of laboratories reported differences between the platforms for cytology specimens and positive controls, but most (155 of 184; 84%) did not alter antibody dilutions or antigen retrieval for cytology IHC. When asked to name 2 antibodies for which staining conditions differed between cytology and surgical samples, there were 18 responses listing 14 antibodies. A total of 30.6% of laboratories performing IHC offered HER2 testing before publication of the 2007 College of American Pathologists/American Society of Clinical Oncologists guidelines, compared with 33.6% afterward, with increased performance of testing by reference laboratories. Three laboratories validated a nonformalin HER2 platform. Conclusions.—The platforms for cytology IHC and positive controls differ for most laboratories, yet conditions are uncommonly adjusted for cytology specimens. Except for the unsuitability of air-dried smears for HER2 testing, the survey did not reveal evidence of systematic problems with any antibody or platform. (Arch Pathol Lab Med. 2014;138:1167–1172; doi: 10.5858/arpa.2013-0259-CP)
T
he goal of cytology is to use the smallest possible biopsy for diagnosis, thereby reducing risk and discomfort for patients, facilitating population-based cancer detection programs, allowing faster diagnosis than can be achieved with larger biopsies, and saving money for the health care system. Even with large-sized surgical biopsies, immunohistochemistry (IHC) is often needed for the diagnosis and determination of prognostic markers. The cytology literature documents the suitability of cytology specimens for IHC1 and the importance of IHC in allowing patients to realize the benefits of cytology.2–4 Working with minimally sized specimens poses a number of interesting technical challenges for laboratories, and the solutions to these challenges can result in a variety of potential preanalytic IHC ‘‘platforms.’’ For example, during adequacy assessment, much or all of a diagnostic cytology Immunohistochemistry Practices in Cytology—Fischer et al 1167
Table 1.
Preanalytical Platforms Used for Cytology Immunohistochemistry (IHC)
Cytology Platforms Cell block: material collected in physiologic transport media and then fixed in formalin Cell block: material fixed in ThinPrep media and postfixed in formalina Smear fixed in 95% alcohol Liquid-based: CytoLyte/PreservCyta Cytospin fixed in 95% alcohol Smear, air dried Smears fixed in another manner (formalin, CytoLyte, spray, acetone, ‘‘Davine’s lysing solution,’’ methanol) Cytospin air dried Cell block: material fixed in ThinPrep media without formalin exposure Cell block: material fixed in SurePath media and postfixed in formalinb Liquid based: Cytorich Red/SurePathb Cell block: material fixed in SurePath media without formalin exposure a b
No. of Laboratories Laboratories Listing This Using Platform as Principal Platform, %
Median Annual Volume of IHC Testing per Laboratory for the Primary Platform
187
25.5
240
185 151 144 128 82
19.1 17.0 10.9 6.5 7.6
120 168 120 240 180
57 50
5.3 1.5
216 228
46
2.1
120
38 31
2.1 2.6
300 120
11
0.0
0
ThinPrep media, CytoLyte, and PreservCyt are proprietary fixatives of Hologic Inc (Bedford, Massachusetts). SurePath and Cytorich Red are proprietary fixatives of Becton, Dickinson and Company (Franklin Lakes, New Jersey).
specimen may need to be smeared onto slides, leaving only the smears as the material available for performance of IHC. Blood contamination is another problem in working with minimal samples. Nonformalin fixatives have been developed specifically to overcome the dilution of diagnostic fragments by blood and precipitated serum proteins. There are many reports of successful IHC staining on cytology specimens using a variety of preanalytic ‘‘platforms,’’ including direct smears5; various monolayer preparations, such as ThinPrep (Hologic Inc, Marlborough, Massachusetts)6,7; SurePath (BD Diagnostics, Franklin Lakes, New Jersey)8; cytospin preparations1,9; and cells removed from a stained, cover-slipped smear and transferred to another slide10,11; or paraffin-embedded cell blocks prepared with or without formalin.9,12–15 There are no data on the number of laboratories using these different preanalytic IHC platforms in the United States. Positive and negative controls are required for IHC to assure valid staining results, and failure to document the appropriate use of positive and negative controls is a phase 2 deficiency that can affect laboratory accreditation by the College of American Pathologists (CAP). Regarding positive control material, CAP states: ‘‘Ideally, the positive control tissue would be the same specimen type as the patient test specimen’’ (for example, formalin-fixed, paraffin-embedded [FFPE] tissues). The small size of cytology specimens makes it difficult for specimens to be saved for use as positive controls, and CAP notes accordingly that ‘‘. . .for most laboratories, it is not practical to maintain separate positive control samples to cover every possible combination of fixation, processing, and specimen type. Thus, it is reasonable for a laboratory to maintain a bank of formalin-fixed tissue samples as its positive controls. . .[provided] that the laboratory can show that these patient specimens exhibit equivalent immunoreactivity’’ (italics added for emphasis).16 The purpose of this study was to examine the number of different cytology IHC platforms used by participants in the CAP Nongynecologic Cytopathology Education Program. Because validation of cytology platforms can involve 1168 Arch Pathol Lab Med—Vol 138, September 2014
considerable expense and time for laboratories, a second goal was to survey a large number of laboratories to identify problems with particular antibodies or preanalytic platforms that would be expected to be uncovered when laboratories addressed the CAP checklist question that relates to verifying the comparability of their cytology platform to their positive controls. MATERIALS AND METHODS 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.
RESULTS 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 Immunohistochemistry Practices in Cytology—Fischer et al
Table 2.
Volume of Cytology Immunohistochemistry (IHC) Testing and Number of Antibodies Offered No. of Respondents
5th Percentilea
25th Percentile
50th Percentile
75th Percentile
95th Percentile
307
1
5
15
35
125
298
2
25
50
85
150
Estimated No. of IHC tests performed on cytology specimens per month No. of different IHC antibodies available for testing cytology specimens a
Percentile distribution.
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 Table 3.
(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 ¼ 26) or an alteration of antibody titers (n ¼ 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 (v2 test; P , .001). Four laboratories attempted validation of a non-FFPE platform. Three laboratories
Differences in the Platform Between Cytology Specimens and Positive Controls, and Adjustments of Antigen-Retrieval Conditions or Antibody Titers No. (%) of responses
Are the positive IHC controls used for cytology samples fixed or processed differently than the cytology samples being tested? Yes, for all antibodies Yes, for some antibodies Noa Is the antigen-retrieval process for any of the cytology IHC stains different from that used for the surgical pathology process for the same antigen? Yes No Is the antibody dilution used for any of the cytology IHC stains different from that used for the surgical pathology dilutions used for the same antigen? Yes No
139 (44.7) 45 (14.5) 127 (40.8) 26 (14.1) 158 (85.9) 3 (1.6) 181 (98.4)
Abbreviation: IHC, immunohistochemistry. a Respondents were asked to skip the next 2 questions. Arch Pathol Lab Med—Vol 138, September 2014
Immunohistochemistry Practices in Cytology—Fischer et al 1169
Table 4. Preanalytic Platforms and Antibodies for 11 Laboratories That Reported a Difference in Either Antigen Retrieval or Antibody Dilution for Cytology Specimens Compared to Surgical Specimens Primary Preparation
Antibody 1
Direct smears fixed in 95% alcohol Direct smears fixed in 95% alcohol Direct smears fixed in 95% alcohol Direct smears fixed in 95% alcohol Direct smears fixed in 95% alcohol Direct smears fixed in a way other than by air drying or with 95% alcohol Liquid-based preparation using CytoLyte/PreservCyt Liquid-based preparation using CytoLyte/PreservCyt Cell block sample fixed in ThinPrep media and postfixed in formalin Air dried Not stated
CK7 TTF-1 Pneumocystis jiroveci Cytokeratin AE1/AE3 Pancytokeratin ER ‘‘Cytoplasmic membrane’’ CD3 CK20 LCA Vimentin
Antibody 2 CK20 Calretinin Cytokeratin AE1/AE3 TTF-1 LMWK—low molecular weight ER Chromogranin
Abbreviations: CK, cytokeratin; ER, estrogen receptor; LCA, leucocyte common antigen; LMWK, low molecular weight keratin; TTF, thyroid transcription factor. A participant did not list a second antibody, resulting in the blank spaces.
successfully validated an alcohol-fixed, nonembedded platform for HER2 testing. One laboratory found air-dried smears could not be successfully validated. COMMENT 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
Table 5. HER2 Testing by Laboratories That Perform Immunohistochemistry (IHC) on Cytology Specimens No. (%) of Responses Is HER2 IHC testing currently offered on cytology samples? Yes, within the institution/laboratory Yes, sent to reference laboratory No Did you previously offer HER2 IHC testing on cytology samples prior to publication of the CAP/ASCO guidelines in 2007? Yes, within the institution/laboratory Yes, sent to reference laboratory No Has the laboratory attempted validation of a nonformalin fixative for HER2 testing on cytology samples? Yes No Which nonformalin fixatives has the laboratory tried to validate? 95% alcohol—successful (direct smear [1]; cytocentrifuged prep [1]; not specifically stated [1]) Air dry—not successful
69 (21.1) 41 (12.5) 217 (66.4) 67 (21.1) 30 (9.5) 220 (69.4) 4 (1.2) 319 (98.8) 3 1
Abbreviations: ASCO, American Society of Clinical Oncologists; CAP, College of American Pathologists. 1170 Arch Pathol Lab Med—Vol 138, September 2014
Immunohistochemistry Practices in Cytology—Fischer et al
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 lowvolume 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 Arch Pathol Lab Med—Vol 138, September 2014
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 staining22–25 that led to new IHC testing guidelines17,26 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 nonFFPE 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 Immunohistochemistry Practices in Cytology—Fischer et al 1171
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. References 1. Kirbis IS, Maxwell P, Fleˇzar MS, Miller K, Ibrahim M. External quality control for immunocytochemistry on cytology samples: a review of UK NEQAS ICC (cytology module) results. Cytopathology. 2011;22(4):230–237. 2. Skoog L, Tani E. Immunocytochemistry: an indispensable technique in routine cytology. Cytopathology. 2011;22(4):215–229. 3. Schmitt F, Barroca H. Role of ancillary studies in fine-needle aspiration from selected tumors. Cancer Cytopathol. 2011;120(3):145–160. 4. Fowler LJ, Lachar WA. Application of immunohistochemistry to cytology. Arch Pathol Lab Med. 2008;132(3):373–383.
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Immunohistochemistry Practices in Cytology—Fischer et al
