immunostaining for thyroid transcription factor-1 on fine-needle aspiration specimens of lung tumors...

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Immunostaining for Thyroid Transcription Factor-1 on Fine-Needle Aspiration Specimens of Lung Tumors A Comparison of Direct Smears and Cell Block Preparations Jing Liu, M.D., Ph.D. Anwar Farhood, M.D. Department of Pathology and Laboratory Medicine, The University of Texas Health Science Center at Houston Medical School, Houston, Texas. Presented in poster form at the 92nd Annual Meet- ing of the United States and Canadian Academy of Pathology, Washington, DC, March 22–28, 2003. The authors thank Bonnie Price Whitaker, H.T. (ASCP), I.H.C., for her expert technical assistance in immunocytochemistry and immunohistochemistry. Address for reprints: Jing Liu, M.D., Ph.D., Depart- ment of Pathology and Laboratory Medicine, The University of Texas Health Science Center at Hous- ton Medical School, 6431 Fannin Street, MSB 2.260A, Houston, TX 77030; Fax: (713) 500-0732; E-mail: [email protected] Received July 7, 2003; revision received Novem- ber 1, 2003; accepted January 5, 2004. BACKGROUND. Fine-needle aspiration (FNA) is used commonly for the diagnosis of pulmonary neoplasms. It has been reported that thyroid transcription factor-1 (TTF-1) is a sensitive and specific marker for certain primary lung tumors. To the authors’ knowledge, the use of TTF-1 immunostaining on FNA smears has not been documented in the literature. This study was designed to examine the utility of TTF-1 immunostaining on FNA specimens from various types of lung tumors by comparing the expression rates on Papanicolaou (Pap)-stained and Diff-Quik (DQ)-stained smears with the expression rates on cell block (CB) sections. METHODS. Forty-three FNA specimens of lung tumors were studied, including 34 primary tumors (14 adenocarcinomas, 12 squamous carcinomas, and 8 small cell carcinomas) and 9 metastatic tumors. One Pap-stained slide and one DQ-stained slide were selected from each tumor. The cytologic material from the slides was transferred to positively charged slides. Unstained recuts were obtained from the CB sections. All slides were stained with TTF-1 monoclonal antibody using heat- induced epitope retrieval and a labeled polymer detection system. RESULTS. Twelve of 14 pulmonary adenocarcinomas were positive for TTF-1 (11 specimens on both CB sections and Pap-stained smears and 1 specimen on all 3 preparations, including the DQ-stained smear). Two of 14 adenocarcinomas were negative for TTF-1. Of 12 pulmonary squamous carcinomas, only 1 was positive for TTF-1 (on the CB section and the Pap-stained smear); the others were negative in all 3 preparations. Of eight small cell lung carcinomas, six specimens showed positive staining for TTF-1 on both CB and Pap-stained preparations; one of those also was positive on the DQ-stained smear. The remaining two small cell lung carcinomas were negative for TTF-1 in all three preparations. All metastatic tumors were negative for TTF-1. CONCLUSIONS. TTF-1 immunostaining of pulmonary neoplasms was applicable to FNA smears that were stained previously with the Pap technique, and the rate of positive staining in each tumor type was identical to the rate of positive staining in CB sections and was comparable to that reported in previous publications. Smears previously stained with the DQ method were unreliable for TTF-1 immunostaining. Cancer (Cancer Cytopathol) 2004;102:109 –14. © 2004 American Cancer Society. KEYWORDS: cytology, Diff-Quik method, fine-needle aspiration, immunoperoxidase technique, lung neoplasms, Papanicolaou technique, thyroid transcription factor-1. T hyroid transcription factor-1 (TTF-1) is a nuclear protein that is expressed almost exclusively in the normal thyroid and lung and in most carcinomas derived from those organs. TTF-1 is a 38-kilodal- ton (kD) member of the NKx2 family of homeodomain transcription factors. In the normal lung, TTF-1 regulates the transcription of lung-specific genes for surfactant and Clara cell secretory proteins. 1 109 CANCER CYTOPATHOLOGY © 2004 American Cancer Society DOI 10.1002/cncr.20110

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Page 1: Immunostaining for thyroid transcription factor-1 on fine-needle aspiration specimens of lung tumors : A comparison of direct smears and cell block preparations

Immunostaining for Thyroid Transcription Factor-1 onFine-Needle Aspiration Specimens of Lung TumorsA Comparison of Direct Smears and Cell Block Preparations

Jing Liu, M.D., Ph.D.

Anwar Farhood, M.D.

Department of Pathology and Laboratory Medicine,The University of Texas Health Science Center atHouston Medical School, Houston, Texas.

Presented in poster form at the 92nd Annual Meet-ing of the United States and Canadian Academy ofPathology, Washington, DC, March 22–28, 2003.

The authors thank Bonnie Price Whitaker, H.T.(ASCP), I.H.C., for her expert technical assistance inimmunocytochemistry and immunohistochemistry.

Address for reprints: Jing Liu, M.D., Ph.D., Depart-ment of Pathology and Laboratory Medicine, TheUniversity of Texas Health Science Center at Hous-ton Medical School, 6431 Fannin Street, MSB2.260A, Houston, TX 77030; Fax: (713) 500-0732;E-mail: [email protected]

Received July 7, 2003; revision received Novem-ber 1, 2003; accepted January 5, 2004.

BACKGROUND. Fine-needle aspiration (FNA) is used commonly for the diagnosis of

pulmonary neoplasms. It has been reported that thyroid transcription factor-1

(TTF-1) is a sensitive and specific marker for certain primary lung tumors. To the

authors’ knowledge, the use of TTF-1 immunostaining on FNA smears has not

been documented in the literature. This study was designed to examine the utility

of TTF-1 immunostaining on FNA specimens from various types of lung tumors by

comparing the expression rates on Papanicolaou (Pap)-stained and Diff-Quik

(DQ)-stained smears with the expression rates on cell block (CB) sections.

METHODS. Forty-three FNA specimens of lung tumors were studied, including 34

primary tumors (14 adenocarcinomas, 12 squamous carcinomas, and 8 small cell

carcinomas) and 9 metastatic tumors. One Pap-stained slide and one DQ-stained

slide were selected from each tumor. The cytologic material from the slides was

transferred to positively charged slides. Unstained recuts were obtained from the

CB sections. All slides were stained with TTF-1 monoclonal antibody using heat-

induced epitope retrieval and a labeled polymer detection system.

RESULTS. Twelve of 14 pulmonary adenocarcinomas were positive for TTF-1 (11

specimens on both CB sections and Pap-stained smears and 1 specimen on all 3

preparations, including the DQ-stained smear). Two of 14 adenocarcinomas were

negative for TTF-1. Of 12 pulmonary squamous carcinomas, only 1 was positive for

TTF-1 (on the CB section and the Pap-stained smear); the others were negative in

all 3 preparations. Of eight small cell lung carcinomas, six specimens showed

positive staining for TTF-1 on both CB and Pap-stained preparations; one of those

also was positive on the DQ-stained smear. The remaining two small cell lung

carcinomas were negative for TTF-1 in all three preparations. All metastatic tumors

were negative for TTF-1.

CONCLUSIONS. TTF-1 immunostaining of pulmonary neoplasms was applicable to

FNA smears that were stained previously with the Pap technique, and the rate of

positive staining in each tumor type was identical to the rate of positive staining in

CB sections and was comparable to that reported in previous publications. Smears

previously stained with the DQ method were unreliable for TTF-1 immunostaining.

Cancer (Cancer Cytopathol) 2004;102:109 –14. © 2004 American Cancer Society.

KEYWORDS: cytology, Diff-Quik method, fine-needle aspiration, immunoperoxidasetechnique, lung neoplasms, Papanicolaou technique, thyroid transcription factor-1.

Thyroid transcription factor-1 (TTF-1) is a nuclear protein that isexpressed almost exclusively in the normal thyroid and lung and

in most carcinomas derived from those organs. TTF-1 is a 38-kilodal-ton (kD) member of the NKx2 family of homeodomain transcriptionfactors. In the normal lung, TTF-1 regulates the transcription oflung-specific genes for surfactant and Clara cell secretory proteins.1

109CANCERCYTOPATHOLOGY

© 2004 American Cancer SocietyDOI 10.1002/cncr.20110

Page 2: Immunostaining for thyroid transcription factor-1 on fine-needle aspiration specimens of lung tumors : A comparison of direct smears and cell block preparations

The value of TTF-1 immunostaining has been investi-gated in histologic sections of surgical specimens andcell block (CB) sections of cytologic material. The re-sults have demonstrated that TTF-1 is both a sensitiveand specific marker for certain types of primary lungtumors.2–7

Fine-needle aspiration (FNA) is used frequently inthe diagnosis of lung lesions. It is preferred especiallywhen surgery may not be the primary treatment op-tion (e.g., in the diagnosis of small cell lung carcinoma[SCLC] and metastatic lung tumors). Although 95%ethanol-fixed, Papanicolaou (Pap)-stained smears andair-dried, Diff-Quik (DQ)-stained smears are used forlight microscopic diagnosis in routine cytology prac-tice, pathologists prefer using CB preparation for im-munostains because the procedure is similar to that ofroutinely fixed and processed histology sections. How-ever, the amount of material obtained during FNA ofthe lung frequently is limited because of the risk ofpneumothorax. There is often insufficient material forCB preparation.

To our knowledge, the reliability of TTF-1 immu-nostaining using routinely fixed and stained FNAsmears from lung tumors has not been investigatedfully. The current study was designed to compareTTF-1 immunostaining on Pap-stained and DQ-stained FNA smears with TTF-1 immunostaining onCB sections and to evaluate the diagnostic applicationof TTF-1 in various types of lung tumors.

MATERIALS AND METHODSStudy MaterialWe reviewed our computer files for a 5-year periodand found 43 FNA specimens (all from different pa-tients) of lung neoplasms with both Pap-stained andDQ-stained smears and CB sections available. Of the43 specimens, 34 specimens were primary pulmonarycarcinomas and 9 were metastatic tumors. Of 34 pri-mary lung tumors, 14 were adenocarcinomas, 12 weresquamous carcinomas, and 8 were SCLC. The ninemetastatic tumors included adenocarcinomas frombreast (three tumors), colon (two tumors), endocervix(one tumor), and stomach (one tumor) as well as oneadenoid cystic carcinoma of the tongue and one mel-anoma. All FNA procedures were performed withcomputed tomography guidance by radiologists.Smears either were fixed in modified Carnoy solution(7 parts 95% ethanol in 0.5 parts glacial acetic acid)and stained with the Pap technique or were air driedand stained with the DQ method. Material for CBpreparations was fixed in 10% neutral buffered forma-lin, subjected to routine histology processing, andthen embedded in paraffin blocks. For this study, weselected one Pap-stained smear and one DQ-stained

smear from each specimen. Unstained sections wereobtained from the CB sections.

ImmunochemistryCytologic material from the original smears was trans-ferred to positively charged slides using the followingprocedure. The area containing diagnostic materialwas outlined on the coverslip with a marking pen andthe corresponding lines were drawn on the undersideof the slide with a diamond pen. The slides weresoaked in xylene for coverslip removal, with additionalimmersion in xylene to ensure complete removal ofmounting media after removing the coverslips. Theslides then were completely covered with liquid cov-erglass medium (Mount Quick; Newcomer Supply,Middleton, WI) and placed horizontally in a 60 °C ovenfor 2 hours to allow the coverglass medium to dry andharden. The resulting slides were immersed in a 45 °Cwater bath for at least 1 hour or as long as necessary toeasily pry off the medium from the edges of the slidesusing a scalpel blade. The sliver on each slide then wasdivided into segments corresponding to the scoring ofthe underside of the slide prior to final removal. Aftermoistening with water, each segment was placed on aseparately moistened, positively charged slide and wasmounted on the same side as the original slide. Theslides then were dried in a horizontal position in a 60°C oven for 1 hour.

Prior to immunostaining, the coverglass mediumwas removed by immersing the slides in 3 changes ofxylene for 4 minutes each time. The slides then werehydrated by sequential immersion in absolute ethanol(twice), 95% ethanol, 70% ethanol, and water. Un-stained sections from the CB sections were subjectedto the same steps for removal of paraffin and rehydra-tion.

The immunohistochemical procedures for thesmears and the CB sections were identical. The pro-cess was initiated with application of heat-inducedepitope retrieval using a vegetable steamer (HandySteamer Plus; Black & Decker, Shelton, CT). The slideswere placed in 1 mM ethylenediamine tetraacetic acidbuffer (pH 8), steamed for 40 minutes, and then al-lowed to cool to room temperature in the buffer for 20minutes. The Dako Autostainer (Dako Corporation,Carpinteria, CA) was used for the remaining steps. Theslides were incubated in 0.9% hydrogen peroxide inmethanol for 20 minutes to quench any endogenousperoxidase activity and were then incubated with a1:50 dilution of TTF-1 antibody (clone 8G7G3/1; DakoCorporation) for 32 minutes at room temperature. TheEnVision� (Dako Corporation) detection system wasused with diaminobenzidine as the chromogen. Tu-

110 CANCER (CANCER CYTOPATHOLOGY) April 25, 2004 / Volume 102 / Number 2

Page 3: Immunostaining for thyroid transcription factor-1 on fine-needle aspiration specimens of lung tumors : A comparison of direct smears and cell block preparations

mors were considered positive for TTF-1 if � 10% ofthe tumor cells demonstrated nuclear staining.

ControlsIdentically prepared slides from nonneoplastic lungand thyroid FNA smears and histologic sections frompulmonary adenocarcinoma and nonneoplastic lungtissue were used as positive controls. Negative con-trols consisted of FNA smears and CB sections inwhich Tris buffer was substituted for the primary an-tibody.

RESULTSThe mean patient age was 68 years (range, 44 –77years). Of 43 patients, 19 patients were women, and 24patients were men. The diagnoses of primary lungcarcinoma and metastatic tumor were made based onclinical, radiologic, and pathologic findings.

ControlsThe alcohol fixed, Pap-stained FNA smears of nonneo-plastic lung and thyroid and the histologic sectionsfrom nonneoplastic lung and pulmonary adenocarci-noma showed moderate to strong nuclear immunore-activity for TTF-1. Air-dried, DQ-stained smears fromlung specimens were nonreactive, and similarlystained smears from thyroid specimens showed weaknuclear immunoreactivity. Figure 1 illustrates thethree types of control slides.

Immunoreactivity for TTF-1 in Cytologic MaterialThe results of TTF-1 immunostaining in 34 specimensof primary lung carcinomas are shown in Tables 1–3and Figure 2. TTF-1 positivity was observed in 12 of 14adenocarcinomas, 6 of 8 SCLCs, and 1 of 12 squamouscarcinomas and was detectable on both CB sectionsand Pap-stained smears in all the positive specimens.Material from air-dried, DQ-stained preparations waslargely nonreactive for TTF-1, with the exception ofone lung adenocarcinoma and one SCLC. All tumorsthat were positive for TTF-1 showed moderate tostrong nuclear immunoreactivity. The poorly pre-served areas of necrotic/degenerating SCLC cells orareas with crush artifact were negative or only weaklypositive for TTF-1 (Fig. 3). TTF-1 immunoreactivitywas not detected in any of the preparations from allnine metastatic lung neoplasms. No cytoplasmicstaining was observed in any of the specimens.

DISCUSSIONA variety of neoplasms arise from the lung, and thelung also frequently is the site for metastatic neo-plasms. Primary and metastatic lung tumors mayshare similar microscopic features. TTF-1 has recently

FIGURE 1. Controls for thyroid transcription factor-1 immunostaining on (A)

a thyroid fine-needle aspiration (FNA) smear previously stained using the

Papanicolaou (Pap) method showing positive staining; (B) a thyroid FNA smear

previously Diff-Quik (DQ) stained showing weakly positive staining; (C) a lung

FNA smear previously Pap stained showing positive staining; (D) a lung FNA

smear previously DQ stained showing no immunoreactivity; and (E) a formalin

fixed, paraffin embedded lung adenocarcinoma tissue section showing positive

staining. Original magnification � 400.

TTF-1 Immunostaining on Lung FNA Smears/Liu and Farhood 111

Page 4: Immunostaining for thyroid transcription factor-1 on fine-needle aspiration specimens of lung tumors : A comparison of direct smears and cell block preparations

been recognized for its utility in the diagnosis of cer-tain types of primary lung carcinomas, most notablyadenocarcinoma and SCLC. Based on the literature,the TTF-1 expression rate in histologic specimensranges from 81–100% (mean, 89%) in SCLC and from75–100% (mean, 82%) in adenocarcinoma.2–12 TTF-1immunoreactivity on CB sections appears less, with areported range of 33–93% (mean, 62%) in SCLC and19 – 89% (mean, 62%) in adenocarcinoma.13–16

Although FNA is used frequently to obtain lesionalmaterial for the diagnosis of lung nodules, it is notalways possible to obtain sufficient cytologic materialfor preparation of CB sections or to anticipate which

FNA smear(s) will contain adequate cellularity prior tostaining. Thus, immunostaining, if necessary, mayhave to be performed on cytologic smears that arefixed previously and stained with conventional cyto-logic methods, such as alcohol fixed, Pap-stainedsmears and air-dried, DQ-stained smears.

In the current study, nuclear immunoreactivity forTTF-1 was observed in 12 of 14 specimens (86%) ofprimary lung adenocarcinoma, 6 of 8 specimens (75%)of SCLC, 1 of 12 specimens (8%) of primary lungsquamous carcinoma, and 0 of 9 specimens (0%) ofmetastatic neoplasms to the lung. None of the meta-static tumors were of thyroid origin. These results aresimilar to those reported in the literature. The TTF-1positivity on CB sections always was accompanied bypositivity on the smears that were stained previouslywith the Pap technique. In contrast, most smears thatpreviously had been air-dried and stained with DQ didnot show TTF-1 immunoreactivity.

TTF-1 immunoreactivity in primary lung adeno-carcinoma is both sensitive and specific, with multipleinvestigations consistently showing that, except forthyroid carcinomas, extrapulmonary adenocarcino-mas are nonimmunoreactive for this marker.2,8 –10,13,14

Distinguishing primary lung adenocarcinoma frommetastatic adenocarcinoma is a frequent challengebecause of the clinical, radiographic, and pathologicoverlap in presentation. For therapeutic purposes,surgical resection usually is the treatment of choice forprimary lung adenocarcinoma when the tumor is re-sectable; however, chemotherapy and/or other non-invasive procedure(s) may be indicated for metastatic

TABLE 1Thyroid Transcription Factor-1 Expression in Different Preparationsof Fine-Needle Aspiration Specimens of Primary LungAdenocarcinoma

Case

TTF-1 expression

CB section Pap-stained smear DQ-stained smear

1 � � �2 � � �3 � � �4 � � �5 � � �6 � � �7 � � �8 � � �9 � � �10 � � �11 � � �12 � � �13 � � �14 � � �

TTF-1: thyroid transcription factor-1; CB: cell block; Pap: Papanicolaou; DQ: Diff-Quik; �: positive; �:

negative.

TABLE 2Thyroid Transcription Factor-1 Expression in Different Preparationsof Fine-Needle Aspiration Specimens of Small Cell Lung Carcinoma

Case

TTF-1 expression

CB section Pap-stained smear DQ-stained smear

1 � � �2 � � �3 � � �4 � � �5 � � �6 � � �7 � � �8 � � �

TTF-1: thyroid transcription factor-1; CB: cell block; Pap: Papanicolaou; DQ: Diff-Quik; �: negative; �:

positive.

TABLE 3Thyroid Transcription Factor-1 Expression in Different Preparationsof Fine-Needle Aspiration Specimens of Primary Lung SquamousCarcinoma

Case

TTF-1 expression

CB section Pap-stained smear DQ-stained smear

1 � � �2 � � �3 � � �4 � � �5 � � �6 � � �7 � � �8 � � �9 � � �10 � � �11 � � �12 � � �

TTF-1: thyroid transcription factor-1; CB: cell block; Pap: Papanicolaou; DQ: Diff-Quik; �: negative; �:

positive.

112 CANCER (CANCER CYTOPATHOLOGY) April 25, 2004 / Volume 102 / Number 2

Page 5: Immunostaining for thyroid transcription factor-1 on fine-needle aspiration specimens of lung tumors : A comparison of direct smears and cell block preparations

disease. TTF-1 and other markers (e.g., cytokeratin 7,cytokeratin 20, thyroglobulin, and estrogen and pro-gesterone receptors) now greatly facilitate this distinc-tion.

SCLC reportedly has a consistently high expres-sion rate of TTF-1 immunostaining on histologic sec-tions. In contrast, 4 studies of TTF-1 immunostainingon CB sections resulted in a wide range of immuno-reactivity (33–93%).13–16 In the current study, TTF-1was detected in 6 of 8 specimens (75%) of SCLC, whichis within the reported range. Both the quantity and thequality of FNA samples may contribute to the slightlylower expression rate of TTF-1 in cytologic specimenscompared with surgical specimens.13 In general, theamount of cytologic material present, even in CBpreparations, is usually less compared with theamount of material from surgical specimens. The cur-rent study also showed that poorly preserved tumorcells, due to necrosis or the crush artifact commonly

seen in SCLC, more frequently were negative for TTF-1immunostaining. In addition, patchy or focal expres-sion of antigen by tumor cells as well as the inability toselect the most optimal tissue section for immunocy-tochemistry, which is possible in surgical specimens,may contribute to a lower sensitivity for TTF-1 detec-tion in cytologic specimens.11 It is important to notethat the TTF-1 immunoreactivity has been observed insome of extrapulmonary small cell carcinomas, with arange of 11– 80%.6,11,12,17 Thus, unlike adenocarcino-mas, TTF-1 cannot be used to distinguish SCLC fromextrapulmonary small cell carcinoma. Nevertheless,TTF-1 may be helpful when it is used in conjunctionwith clinical and radiologic findings.

In the current study, primary lung squamous car-cinomas had a TTF-1 expression rate of 8%. This rateis consistent with that found in previous studies usinghistologic or CB sections (mean, 9%; range,0 –38%).4,9,10,13–16

TTF-1 is a nuclear marker. It is noteworthy thatcytoplasmic staining with TTF-1 has been reported in6.3% of carcinomas from various sites.18 In one study,71% of hepatocellular carcinomas showed TTF-1 cy-toplasmic staining.19 In addition, normal liver cellsfrequently show this cytoplasmic staining. The basisfor this pattern of staining is not known. None of thespecimens in the current study showed cytoplasmicstaining.

Although the immunostaining technique can beapplied directly to Pap-stained FNA smears, it is ad-vantageous to transfer the cytologic material to posi-tively charged slides to minimize cell loss if theoriginal slides were not coated with adhesive. Further-more, the material from a slide that contains high

FIGURE 3. Crushed small cell lung carcinoma cells are negative or weakly

positive for thyroid transcription factor-1 (TTF-1), whereas the intact tumor

cells are positive for TTF-1 on a cell block section. Original magnification �

400.

FIGURE 2. Thyroid transcription factor-1 immunostaining in fine-needle

aspiration (FNA) material from primary lung adenocarcinoma showing (A)

positive staining on a previously Papanicolaou-stained FNA smear, (B) no

immunoreactivity on a previously Diff-Quik-stained FNA smear, and (C) positive.

staining on a cell block section. Original magnification � 400.

TTF-1 Immunostaining on Lung FNA Smears/Liu and Farhood 113

Page 6: Immunostaining for thyroid transcription factor-1 on fine-needle aspiration specimens of lung tumors : A comparison of direct smears and cell block preparations

cellularity can be divided selectively into several por-tions and transferred to multiple slides so that moreimmunochemical markers may be investigated ifneeded. A destaining step is not necessary.

Utilizing air-dried, DQ-stained smears resulted infalse negativity for TTF-1 in most of our specimens oflung adenocarcinoma and SCLC, and only weak im-munoreactivity was noted in control specimens of thy-roid cytologic material. A previous study demon-strated that air drying decreased immunoreactivity forAE1/AE3 and that the type of fixative affected immu-noreactivity to some degree.20 Air-dried cells may ex-hibit an overall lower antigen density. It is unclearwhether methanol, which is the fixative used for air-dried smears, affects TTF-1 immunoreactivity. Thy-roid tissue may have a higher TTF-1 antigen densitycompared with lung tissue. Further studies are neededto assess whether this false-negative or weak immu-noreactivity for TTF-1 on air-dried, DQ-stainedsmears can be corrected by extending the antibodyand/or chromogen incubation time, by using a differ-ent epitope-retrieval technique, or by using reagentsthat are more sensitive.

In the current study, the immunoreactivity ob-served in the FNA material that was stained previouslywith the Pap technique was identical to the immuno-reactivity observed in the CB preparations. The detec-tion rates of TTF-1 in various lung neoplasms werewithin the ranges reported previously in the literature.FNA smears that have been air-dried and DQ stainedshould not be used for TTF-1 immunostaining. Inaddition, in certain circumstances, the smear transferis necessary for facilitating the immunostaining onpreviously routinely fixed and stained cytologicsmears.

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