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Immunocytochemistry Performed on the Cell-Transferred Direct Smears of the Fine-Needle Aspirates
A Comparison Study With the Corresponding Formalin-Fixed Paraffin-Embedded Tissue

Howard H. Wu MD, Kelly J. Jones SCT (ASCP), Harvey M. Cramer MD
DOI: http://dx.doi.org/10.1309/AJCP8O7VIGSIXIVS 754-758 First published online: 1 June 2013

Abstract

Immunocytochemistry (ICC) performed on the cell-transferred cytologic smears (CTCS) of fine-needle aspiration (FNA) is useful when the cell blocks lack adequate material. The comparison of the ICC results from the CTCS of FNA with the corresponding formalin-fixed paraffin-embedded tissue (FFPE) has not been reported previously. We applied 12 commonly used ICC antibodies on 160 pieces of ethanol-fixed, cell-transferred Papanicolaou-stained smears obtained from 42 FNA specimens and compared the staining results with the corresponding FFPE on which the same panel of immunostains was performed. Of the 160 pieces of transferred materials, only 3 (1.9%) were lost during specimen processing. In total, 153 of 157 (97.5%) showed staining results that agreed with the corresponding FFPE, including 78 of 81 positive staining and 75 of 76 negative staining cases. ICC performed on the cell-transferred FNA smears is reliable and shows staining results highly comparable with the corresponding FFPE tissue.

Key Words:
  • Immunocytochemistry
  • Fine-needle aspiration
  • Cytology
  • Cell-transfer

Immunocytochemistry (ICC) performed on the cell-transferred direct smears prepared from fine-needle aspiration (FNA) smears is an extremely helpful technique if conventional cell blocks lack adequate cellularity.13 It is not uncommon for cell blocks prepared from FNAs to be insufficiently cellular, thereby making confirmatory conventional immunostaining impossible, unless other alternative methods are employed. Gong et al1 have validated the cell-transfer technique through comparison with those obtained previously from nontransferred cytologic materials of the same specimens. Similarly, Elsheikh and Corbin2 validated the technique by using touch imprint materials derived from fresh tumors for comparison. However, a comparison of immunostaining results performed on the cell-transferred FNA cytologic smears with the accepted gold standard of formalin-fixed paraffin-embedded (FFPE) tissue has not been evaluated previously. In this study, we applied the cell-transfer technique to a panel of ICC on both cell-transferred cytologic smears (CTCS) and the corresponding sections cut from the FFPE tissue from the patients’ original tumors.

Materials and Methods

This study was approved by the Indiana University Institutional Review Board (IRB study no. EX1012-23). A computerized search of our laboratory informatics system was performed for a 3-year period (January 1, 2007, through December 31, 2009) to identify FNA cases having both adequate direct smears and adequate FFPE tissue blocks of the patients’ original carcinomas. A total of 42 FNA specimens were identified, including 6 cases of adenocarcinoma, 12 cases of squamous cell carcinoma, 6 cases of small cell carcinoma, 13 cases of metastatic urothelial carcinoma, and 5 cases of metastatic ductal carcinoma of the breast. The corresponding FFPE tissue was obtained from 11 biopsy specimens and 31 resection specimens, of which 21 were from the same biopsy site as the FNA, whereas 21 were from the original tumors removed from different anatomic sites. We used a panel of ICC including cytokeratin (CK) 7, CK20, TTF1, CDX2, p63, and AE1/3 on cases diagnosed as adenocarcinoma or squamous cell carcinoma; a panel including TTF1, chromogranin, synaptophysin, and CK-cocktail AE1/CAM5.2 on the small cell carcinoma cases; a panel including CK7, CK20, and p63 on metastatic urothelial carcinomas; and a panel including estrogen receptor (ER), progesterone receptor (PR), and HER2-neu on metastatic ductal carcinomas of the breast. In total, 12 commonly used ICC antibodies were applied to 160 pieces of ethanol-fixed, cell-transferred Papanicolaou-stained smears and the corresponding FFPE tissue sections.

Cell-transfer Technique

One representative ethanol-fixed Papanicolaou-stained direct smear containing adequate tumor cells was selected for ICC. The cell-transfer technique was performed as follows: (1) The coverslip was removed using histologic grade xylene (Thermo Fisher Scientific, Pittsburgh, PA). (2) A thin layer of Mount Quick media (Daido Sangyo, Tokyo, Japan) was spread uniformly over the top of the cellular material. (3) The slide was then placed in a 60°C heated oven for approximately 2 to 3 hours (or until hardened to the touch). (4) A Sharpie marker was used on the surface of the dried media to divide the slide into multiple areas of interest. (5) The slide was then placed into a Coplin jar of deionized water and submerged into a warm water bath at 45 ± 3°C for 30 minutes to 2 hours, or until the media were soft enough to easily peel away from the slide. (6) The media were cut along the marked areas, and each section to be tested was carefully arranged on separate, positively charged glass slides (Leica InJet Plus, Leica Microsystems, Buffalo Grove, IL). (7) Gentle pressure was then applied to each section using a moistened gauze pad. (8) The slides with the newly transferred material were left to dry in a warm oven (37–60°C) for at least 2 hours, or until the media were dry to the touch. (9) The dried slides were soaked in 4 exchanges of xylene (15 minutes each) to remove the mounting media. (10) The slides were then rehydrated using 2 exchanges of absolute alcohol, 2 exchanges of 95% alcohol, and 2 exchanges of deionized water. (11) ICC staining was performed.

ICC Procedure

The ICC procedure was identical to that used for regular immunohistochemistry for FFPE tissue. No destaining was applied in our process. The ICC was stained using the DAKO Autostainer Link 48 (DAKO, Carpinteria, CA) with antigen retrieval, with the exception of polyclonal chromogranin, for which no retrieval was used. The same panel of immunostains was also performed on sections cut from a representative paraffin block of the corresponding original tumor. Twelve antibodies were used for immunostains: CK7, CK20, CDX2, TTF1, AE1/3, p63, chromogranin, synaptophysin, CK-cocktail AE1/CAM5.2, ER, PR, and HER2-neu. The sources and the dilutions of the antibodies are listed in Table 1.

Interpretation of Immunostaining Results

The immunostaining results were recorded using a numeric scale: 0 (no staining), 1+ (1%–33% staining), 2+ (34%–66% staining), and 3+ (>66% staining). The staining results from CTCS of the FNAs were compared with the FFPE from the original tumors. Positive was defined as at least 1+ staining. Negative was defined as absence of staining. Results were considered discrepant when the difference between CTCS and FFPE was positive vs negative.

View this table:
Table 1

Results

A total of 160 pieces of ethanol-fixed, cell-transferred materials were immunostained. Only 3 (1.9%) of the 160 pieces were lost during the cell-transfer and staining process. A total of 153 of 157 pieces (97.5%) showed staining results that agreed with the corresponding FFPE, including 78 of 81 positive staining cases and 75 of 76 negative staining cases Table 2. In 1 case of metastatic squamous cell carcinoma to the soft tissue near the iliac bone, the CTCS showed 3+ positive staining with CK7 and 1+ staining with CK20, with the corresponding core biopsy specimen demonstrating negative staining for both markers. Two metastatic urothelial carcinoma cases were noted to have discrepant results on the CK20 immunostain. In 1 case, the CTCS from metastatic urothelial carcinoma demonstrated 1+ staining for CK20, and the FFPE tissue from the original urothelial carcinoma was negative for CK20. In another case, the FFPE tissue from the original urothelial carcinoma expressed 2+ staining, and the CTCS from the metastatic urothelial carcinoma was negative for CK20.

View this table:
Table 2

Discussion

ICC performed on cell-transferred direct smears prepared from FNA is very useful if conventionally prepared cell blocks lack adequate cellularity. Technically, the processing steps are not complex, and either a histology or cytology technologist can easily be trained to perform the procedure. Cell-transfer can be performed in any laboratory, and no special equipment is necessary. Furthermore, the additional cost is relatively low, and multiple immunostains can be performed from a single cellular smear using the cell-transfer technique.13 Gong et al1 had successfully performed ICC on a variety of different tumor samples using the cell-transfer technique and obtained adequate results in 97% of cases. The cell-transfer technique was also validated by Elsheikh and Corbin,2 who used touch imprints from fresh tumors and demonstrated a 100% concordance rate of immunostaining results between Papanicolaou-stained cell-transferred pieces treated with antigen retrieval and the corresponding formalin-fixed tissue. In the current study, we have performed immunostaining for 12 markers—CK7, CK20, CDX2, TTF1, AE1/3, p63, chromogranin, synaptophysin, CK-cocktail AE1/CAM5.2, ER, PR, and HER2-neu—using the cell-transfer technique on selected, adequately cellular, alcohol-fixed, archived Papanicolaou-stained FNA direct smears. The ICC on the cell-transferred direct smears was easy to interpret, and the qualities of the immunostaining patterns were comparable to that of the corresponding formalin-fixed tissue Image 1 and Image 2.

In 50% of specimens (21/42), we performed the same immunostaining panel on CTCS of metastatic tumors and on FFPE tissue of the original tumors. Only 2 cases of urothelial carcinoma showed discrepant results on CK20 staining. Antibodies to CK7 and CK20 are commonly used in the evaluation of urothelial carcinomas. Greater than 90% of cases of urothelial carcinoma are positive for CK7, while a variable percentage (30%–90%) also coexpresses CK20.4,5 The discrepancy of CK20 immunostaining is probably due to the smaller sample size on the FNA, or the expression of CK20 by the urothelial carcinoma might have been changed in the metastatic process. Overall, there was a 97.5% concordance rate between the cell-transferred FNA smears and the sections cut from the original FFPE tissue. This high concordance rate confirms that most of the metastatic carcinomas have maintained the same immunoprofile as the original primary tumors.6

One limitation to the technique is that only alcohol-fixed direct smears can be used. Air-dried slides previously have been found to show a high rate of false negativity (approximately 30%) as well as high background nonspecific staining.1 Another limitation is that this is a manual procedure, requiring 5 to 7 hours to complete the cell-transfer, which may add 1 more day to the turnaround time for the case.

Since validation of the cell-transfer technique, it has been used in approximately 1% of the FNA cases in our laboratory and is performed whenever immunostaining is required but the cell blocks are unavailable, hypocellular, or acellular. Even archived direct smears or liquid-based preparations that are months or years old can be used as sources of cellular material for the cell-transfer technique. By using this technique, we are able to further subtype non–small cell carcinomas of the lung into either adenocarcinoma or squamous cell carcinoma; identify the origin of a metastatic tumor through the application of ICC on the cell-transferred smears; and evaluate for ER, PR, and HER2-neu status for patients with breast carcinoma.7 The cell-transfer technique can also be applied on histologic sections of the small biopsy or core biopsy specimens for immunostaining, especially when the tissue is too scant and recut sections have exhausted the areas of interest. For example, in the examination of the prostate core biopsy specimen, we were able to perform p63 and 34βE12 immunohistochemical stains on the transferred histologic sections and upgrade our diagnosis from atypical acinar cell proliferation to an unequivocal diagnosis of prostatic adenocarcinoma.

Image 1

A, Fine-needle aspiration cytology of a metastatic urothelial carcinoma (Papanicolaou stain, ×400). B, Tumor cells on the cell-transferred smear demonstrating strong cytokeratin 7 cytoplasmic staining (3+) (immunocytochemistry, ×400).

Image 2

A, Section from a formalin-fixed paraffin-embedded block of primary urothelial carcinoma (H&E stain, ×400). B, Tumor cells on the formalin-fixed section demonstrating strong cytokeratin 7 cytoplasmic staining (3+) (immunohistochemistry, ×400).

In summary, ICC performed on cell-transferred FNA smears is very accurate and comparable to the results obtained on corresponding FFPE tissue, and it provides a very useful option when the FNA cell blocks lack adequate cellular material.

CME/SAM

Upon completion of this activity you will be able to:

  • apply the cell-transfer technique to daily practice of fine-needle aspiration cytopathology.

  • be able to manually perform the cell-transfer technique and submit targeted cellular material for ancillary immunocytochemical testing.

  • predict the rate of immunoreactivity that can be expected from samples prepared by the method of cell transfer from direct smears.

The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module.

The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Questions appear on p 827. Exam is located at www.ascp.org/ajcpcme.

Footnotes

  • This study was supported by the Department of Pathology of Laboratory Medicine, Indiana University School of Medicine, Indianapolis.

References

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