Review Article
Biospecimen Repositories and Cytopathology Savitri Krishnamurthy, MD
Biospecimen repositories are important for the advancement of biomedical research. Literature on the potential for biobanking of fine-needle aspiration, gynecologic, and nongynecologic cytology specimens is very limited. The potential for biobanking of these specimens as valuable additional resources to surgically excised tissues appears to be excellent. The cervicovaginal specimens that can be used for biobanking include Papanicolaou-stained monolayer preparations and residual material from liquidbased cytology preparations. Different types of specimen preparations of fine-needle aspiration and nongynecologic specimens, including Papanicolaou-stained and Diff-Quik–stained smears, cell blocks. and dedicated passes/residual material from fineneedle aspiration stored frozen in a variety of solutions, can be used for biobanking. Because of several gaps in knowledge regarding the standard of operative procedures for the procurement, storage, and quality assessment of cytology specimens, further studies as well as national conferences and workshops are needed not only to create awareness but also to facilitate the C 2014 American Cancer Society. use of cytopathology specimens for biobanking. Cancer (Cancer Cytopathol) 2015;123:152-61. V
KEY WORDS: biobanking; cytopathology; fine-needle aspiration; gynecologic specimens.
INTRODUCTION Human biospecimens form the foundation of basic and translational biomedical research. Biobanks are biorepositories that collect, process, store, and distribute biospecimens and associated relevant data for conducting research. Innovations and discoveries in biomedical research cannot be facilitated or advanced without the aid of well established biospecimen repositories to provide the needed tissues obtained from individuals for conducting the research. Today, tissue banking is essential to all aspects of biomedical research and is crucial to the development of personalized medicine. The establishment of tissue banks has emerged as a global priority in view of the rapid advances and availability of new technological platforms that have increased the demand for tissues for research in genomics and proteomics. In recent years, the field of biorepository and biospecimen science has emerged for advancing our knowledge regarding all issues related to biobanking. The journal Cancer Epidemiology, Biomarkers and Prevention is based on this emerging field. Regulatory requirements, including adherence to the Health Insurance Portability and Accountability Act, institutional review board approval, and patient consent documentation, have added to the complexity in the functioning of biobanks. The establishment of biospecimen repositories entails a concerted effort by a multidisciplinary team of experts from several specialties, including pathology, surgery, medical oncology, and internal medicine experts and ethical, legal, and technology professionals. To start with, great effort must be taken to document that the patients consent to the use of their tissues for future research. Tissues must be collected and processed according to standards that safeguard the quality of the specimens, and they should be annotated with the appropriate patient-related and biospecimen-specific information. Realizing that biospecimens form the cornerstone of all biomedical research endeavors, the National Cancer Institute (NCI) leadership has implemented a strategy to Corresponding author: Savitri Krishnamurthy, MD, Department of Pathology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030; Fax: (713) 792-3738; [email protected] Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas Received: September 17, 2014; Revised: November 24, 2014; Accepted: November 24, 2014 Published online December 18, 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cncy.21505, wileyonlinelibrary.com
152
Cancer Cytopathology
March 2015
Cytology Biobanking/Krishnamurthy
create sustainable, standardized biospecimen resources.1–5 In 2005, the NCI established the Office of Biorepositories and Biospecimen Research (OBBR), which functions as the focal point for addressing all issues related to biobanking. Since its inception, the OBBR has convened several national workshops on best practices for biorepositories that support cancer research, covering biospecimen ethical, legal, and policy issues; biospecimen custodianship and ownership issues; biorepository economics; and other biospecimen-related topics. In 2007, the OBBR initiated the Biospecimen Research Network, which funds research in biospecimen science with the goal of gathering an evidence base for biobanking practices.1–3 Through these initiatives and the collective and intensive efforts of experts, including input from the public, a comprehensive document titled Best Practices for Biospecimen Resources was published by the NCI.6 Guidelines for biospecimen reporting for improved study quality are available, and adherence to these guidelines can be useful for improving the quality of research using human specimens.7 The practices outlined in the document Best Practices for Biospecimen Resources, which are followed by current biobanking establishments, are focused on the collection, storage, and disbursal of tissues, including blood, plasma, serum, and tissues collected from surgically resected specimens. There is very limited literature regarding the potential for biobanking of cytology specimens, such as fineneedle aspiration (FNA) specimens and both gynecologic and nongynecologic cytology specimens obtained from procedures like brushing, washing, lavage, and effusions. Cytology specimens form a significant component of the archival hospital pathology specimens. However, the potential of these specimens as an important resource in addition to surgically excised tissues has been under recognized to date. Only recently, with the increased understanding of the role of cytology specimens in molecular testing, has there been some awareness regarding the possible incorporation of cytology specimens as a biobanking resource. This review outlines the potential utility and current status of gynecologic, nongynecologic, and FNA cytology specimens for biobanking. Biobanking of Cervicovaginal Cytology Specimens
Most existing reports on the biobanking of cytology specimens relate to the establishment of cervical cytology biobanks (CCBs) in Europe. The CCB established in Europe Cancer Cytopathology
March 2015
is considered an extension of the clinical standard of care cytopathology laboratory practice and includes the systematic storage of cervicovaginal specimens obtained from women who participate in cervical cancer screening programs.8 The primary objectives of this CCB are to facilitate future scientific research and to improve the quality of preventive services targeted against cervical cancer. The CCB is linked to the cervical cancer registry and to a cervical cancer screening and human papillomavirus (HPV) vaccination registry that offers a unique resource for conducting cytologic studies and investigating the natural history and molecular pathways of cervical carcinogenesis. The CCB is clearly a prodigious resource for fundamental and applied biologic research for the investigation of the natural history of HPV infection, HPVinduced lesions and cancers, HPV screening effectiveness, and exploration of new biomarkers. The European CCB stores Papanicolaou (Pap)stained cervicovaginal smears and residual samples of liquid-based cytology (LBC) preparations of gynecologic specimens.8 Archived Pap-stained smears are used for conducting molecular studies, such as amplification of human or viral genomic sequences and for HPV genotyping, using the DNA that can be extracted from scraping the cells in the smear.9,10 It is reported that the methods of specimen fixation and staining are more important than the length of storage for achieving optimal recovery of DNA from the scraped cells. The inclusion of acetic acid in the fixative solution reportedly reduces the length of amplifiable DNA fragments. The optimal storage conditions for collecting residual LBC samples in the European CCB are yet to be established. The CCB centrifuges the LBC specimens, retains the cell pellets after removing the supernatant, and subsequently freezes the specimens at 280 C in the biobank. Because several alcohol components in the fixative fluid may interfere with subsequent biomolecular testing, the supernatant is discarded, and the cell pellet alone is retained.11 Storage of LBC specimens at ambient temperature decreases the stability of nucleic acids compared with frozen specimens.11 The recovery of DNA and RNA from specimens collected using ThinPrep (Hologic, Boxborough, Mass) reportedly is good.12,13 Protocols for the efficient recovery of nucleic acids from specimens processed using SurePath (Becton, Dickinson and Company, Franklin Lakes, NJ) also have been reported.14,15 Long-term storage of 153
Review Article
DNA and RNA extracted from the LBC specimens at 280 C also is used for biobanking of cervicovaginal cytology specimens in this CCB. Because of all the confounding factors that can affect the quality of this extracted DNA and RNA, checking the length of the DNA fragments or the amount of ribosomal RNA to evaluate the quality of DNA and RNA in these stored samples is recommended before using these specimens from the biobank. There are also reports of a separate CCB established in Sweden. The Swedish CCB is a national initiative for the establishment of a prospective repository of LBC cervical samples from women who participate in organized cervical cancer screening programs.16,17 The Swedish CCB is embedded in a comprehensive cytology diagnostic registry that is linked to the national cancer registry in Sweden. The development and implementation of a nationally standardized method for the handling and long-term storage of LBC cervical samples has been the primary goal for this Swedish hub of the biobanking and molecular resource infrastructure. The sample handling protocol that the Swedish CCB follows was developed through a review of biobanking processes reported in the literature, wide consultation within the academic community, and various verification assays in collaboration with clinical cytology laboratories in Sweden. To store the LBC samples, the Swedish CCB follows a dual sedimentation procedure that was established after initial verification assays for each of its steps, and the procedure was capable of retrieving 80% of the cells in the original ThinPrep vial. The first step consists of aspirating 4 mL of cell solution from the ThinPrep vial and then transferring the solution to a conical tube and allowing it to sediment for 30 minutes; then, 300 lL of the sediment are transferred to the storage vial. The storage vials are displayed in a 96well format and are stored at 225 C, a temperature that was chosen for storing the vials because preliminary studies demonstrated that the cells, including their DNA and RNA, remained intact at this temperature and were well preserved for cytologic, immunohistochemical, and molecular studies. Specifically, 98% of the biobanked samples in the Swedish CCB were identified as satisfactory for conventional cytopathologic examination. Similarly, polymerase chain reaction (PCR) amplification of b-globin fragments indicated that 98% of the biobanked samples in the CCB contained adequate amounts of bglobin, indicating well preserved DNA in the samples. 154
The biobanked samples in the Swedish CCB are assessed twice each year for cellular morphology and DNA preservation. Biobanking of FNA Cytology Specimens
The feasibility and application of FNA specimens for different kinds of molecular testing are evolving at a rapid pace. The contribution of the results from molecular testing performed on FNA specimens and other types of cytology specimens that guide the clinical management team in developing personalized cancer therapy for the patient is now very well recognized.18–29 FNA specimens are particularly valuable for settings in which they are the only type of specimens available for testing. With increasing realization of the utility of FNA and other cytology specimens for molecular testing, there is currently a lot of interest in understanding the advantages and limitations of different types of specimen preparations for molecular testing. There are few investigators who have contributed toward our understanding of the potential of FNA specimens for biobanking by providing data from the results of molecular testing that were obtained by using archival specimens. Those studies and several recent reports highlighting the utility of FNA specimens for molecular testing form the basis for considering FNA specimens as yet another valuable biobanking resource. The FNA specimen preparations that have been studied and have exhibited potential utility for biobanking include smears stained with the Pap and Diff-Quik methods and material collected directly into a variety of solutions. Killian and colleagues reported the remarkable preservation of high-molecular-weight DNA in archival FNA smears that had been stored for >10 years.30 The DNA extracted from those archival smears could be used successfully for high-resolution comparative genomic hybridization arrays, DNA methylation, and single-nucleotide polymorphism genotyping. In that study, significant differences in DNA preservation and DNA integrity were observed between Pap-stained and Diff-Quik–stained archival FNA smears. The authors hypothesized that these differences were related to the DNA-damaging effect of hematoxylin in the Pap stain. On the basis of their successful molecular testing results and the retrieval of highquality DNA from smears, Killian et al reported the excellent potential of FNA smears, particularly the air-dried, Diff-Quik–stained smears, for biobanking purposes. Subsequently, other investigators also observed the excellent Cancer Cytopathology
March 2015
Cytology Biobanking/Krishnamurthy
preservation of high-molecular-weight DNA in both Papstained and Diff-Quik–stained archival smears.31–38 The study by Dejmek et al is perhaps the first systematic study to compare DNA yield and the quality of different cytologic preparations using human lung carcinoma cell lines for epidermal growth factor receptor (EGFR) genotyping.39 Those authors demonstrated that, although reliable EGFR genotyping could be achieved with any of the cytologic preparations, there were differences resulting from the use of different fixatives, staining, and mounting media. They observed that spray or ethanol-fixed Pap slides provided superior results in terms of DNA yield and fragment length compared with Romanowsky-stained, air-dried smears. Cells fixed in CytoLyt solution (Cytyc Corporation, Marlborough, Mass) yielded 5-fold higher DNA than cells fixed in CytoRich Red fluid (Fisher Scientific UK Ltd., Loughborough, UK), most likely because of the formaldehyde content in the latter fixative solution. Significantly higher DNA yield was obtained using slides mounted with Ecomount (Biocare Medical LLC, Concord, Calif), an organic, polymer-based mounting medium, compared with the xylene-based mounting medium Pertex (Leica Biosystems, Buffalo Grove, Ill). The collection of FNA specimens directly into buffer with and without RNA stabilizing agents also has been suggested for biobanking purposes. Ladd and colleagues demonstrated that both cellular morphology and RNA integrity could be maintained for up to 27 weeks after cryopreservation of FNA samples collected in RNAlater (Ambion RNA isolation kit; Applied Biosystems, Dallas, Tex) or in cryopreservation media (80% fetal bovine serum plus 20% RPMI 1640; Invitrogen, Carlsbad, Calif) with added cryoprotectants, such as dimethyl sulfoxide.40 Those authors clearly demonstrated that the cell morphology was sufficient to evaluate tumor cellularity when cells were prepared from a portion of the thawed specimen using their protocol, and RNA integrity was preserved for the extraction of high-quality RNA suitable for RNA-based testing. They observed that keeping the devitalization time to
Biospecimen Repositories and Cytopathology Savitri Krishnamurthy, MD
Biospecimen repositories are important for the advancement of biomedical research. Literature on the potential for biobanking of fine-needle aspiration, gynecologic, and nongynecologic cytology specimens is very limited. The potential for biobanking of these specimens as valuable additional resources to surgically excised tissues appears to be excellent. The cervicovaginal specimens that can be used for biobanking include Papanicolaou-stained monolayer preparations and residual material from liquidbased cytology preparations. Different types of specimen preparations of fine-needle aspiration and nongynecologic specimens, including Papanicolaou-stained and Diff-Quik–stained smears, cell blocks. and dedicated passes/residual material from fineneedle aspiration stored frozen in a variety of solutions, can be used for biobanking. Because of several gaps in knowledge regarding the standard of operative procedures for the procurement, storage, and quality assessment of cytology specimens, further studies as well as national conferences and workshops are needed not only to create awareness but also to facilitate the C 2014 American Cancer Society. use of cytopathology specimens for biobanking. Cancer (Cancer Cytopathol) 2015;123:152-61. V
KEY WORDS: biobanking; cytopathology; fine-needle aspiration; gynecologic specimens.
INTRODUCTION Human biospecimens form the foundation of basic and translational biomedical research. Biobanks are biorepositories that collect, process, store, and distribute biospecimens and associated relevant data for conducting research. Innovations and discoveries in biomedical research cannot be facilitated or advanced without the aid of well established biospecimen repositories to provide the needed tissues obtained from individuals for conducting the research. Today, tissue banking is essential to all aspects of biomedical research and is crucial to the development of personalized medicine. The establishment of tissue banks has emerged as a global priority in view of the rapid advances and availability of new technological platforms that have increased the demand for tissues for research in genomics and proteomics. In recent years, the field of biorepository and biospecimen science has emerged for advancing our knowledge regarding all issues related to biobanking. The journal Cancer Epidemiology, Biomarkers and Prevention is based on this emerging field. Regulatory requirements, including adherence to the Health Insurance Portability and Accountability Act, institutional review board approval, and patient consent documentation, have added to the complexity in the functioning of biobanks. The establishment of biospecimen repositories entails a concerted effort by a multidisciplinary team of experts from several specialties, including pathology, surgery, medical oncology, and internal medicine experts and ethical, legal, and technology professionals. To start with, great effort must be taken to document that the patients consent to the use of their tissues for future research. Tissues must be collected and processed according to standards that safeguard the quality of the specimens, and they should be annotated with the appropriate patient-related and biospecimen-specific information. Realizing that biospecimens form the cornerstone of all biomedical research endeavors, the National Cancer Institute (NCI) leadership has implemented a strategy to Corresponding author: Savitri Krishnamurthy, MD, Department of Pathology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030; Fax: (713) 792-3738; [email protected] Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas Received: September 17, 2014; Revised: November 24, 2014; Accepted: November 24, 2014 Published online December 18, 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cncy.21505, wileyonlinelibrary.com
152
Cancer Cytopathology
March 2015
Cytology Biobanking/Krishnamurthy
create sustainable, standardized biospecimen resources.1–5 In 2005, the NCI established the Office of Biorepositories and Biospecimen Research (OBBR), which functions as the focal point for addressing all issues related to biobanking. Since its inception, the OBBR has convened several national workshops on best practices for biorepositories that support cancer research, covering biospecimen ethical, legal, and policy issues; biospecimen custodianship and ownership issues; biorepository economics; and other biospecimen-related topics. In 2007, the OBBR initiated the Biospecimen Research Network, which funds research in biospecimen science with the goal of gathering an evidence base for biobanking practices.1–3 Through these initiatives and the collective and intensive efforts of experts, including input from the public, a comprehensive document titled Best Practices for Biospecimen Resources was published by the NCI.6 Guidelines for biospecimen reporting for improved study quality are available, and adherence to these guidelines can be useful for improving the quality of research using human specimens.7 The practices outlined in the document Best Practices for Biospecimen Resources, which are followed by current biobanking establishments, are focused on the collection, storage, and disbursal of tissues, including blood, plasma, serum, and tissues collected from surgically resected specimens. There is very limited literature regarding the potential for biobanking of cytology specimens, such as fineneedle aspiration (FNA) specimens and both gynecologic and nongynecologic cytology specimens obtained from procedures like brushing, washing, lavage, and effusions. Cytology specimens form a significant component of the archival hospital pathology specimens. However, the potential of these specimens as an important resource in addition to surgically excised tissues has been under recognized to date. Only recently, with the increased understanding of the role of cytology specimens in molecular testing, has there been some awareness regarding the possible incorporation of cytology specimens as a biobanking resource. This review outlines the potential utility and current status of gynecologic, nongynecologic, and FNA cytology specimens for biobanking. Biobanking of Cervicovaginal Cytology Specimens
Most existing reports on the biobanking of cytology specimens relate to the establishment of cervical cytology biobanks (CCBs) in Europe. The CCB established in Europe Cancer Cytopathology
March 2015
is considered an extension of the clinical standard of care cytopathology laboratory practice and includes the systematic storage of cervicovaginal specimens obtained from women who participate in cervical cancer screening programs.8 The primary objectives of this CCB are to facilitate future scientific research and to improve the quality of preventive services targeted against cervical cancer. The CCB is linked to the cervical cancer registry and to a cervical cancer screening and human papillomavirus (HPV) vaccination registry that offers a unique resource for conducting cytologic studies and investigating the natural history and molecular pathways of cervical carcinogenesis. The CCB is clearly a prodigious resource for fundamental and applied biologic research for the investigation of the natural history of HPV infection, HPVinduced lesions and cancers, HPV screening effectiveness, and exploration of new biomarkers. The European CCB stores Papanicolaou (Pap)stained cervicovaginal smears and residual samples of liquid-based cytology (LBC) preparations of gynecologic specimens.8 Archived Pap-stained smears are used for conducting molecular studies, such as amplification of human or viral genomic sequences and for HPV genotyping, using the DNA that can be extracted from scraping the cells in the smear.9,10 It is reported that the methods of specimen fixation and staining are more important than the length of storage for achieving optimal recovery of DNA from the scraped cells. The inclusion of acetic acid in the fixative solution reportedly reduces the length of amplifiable DNA fragments. The optimal storage conditions for collecting residual LBC samples in the European CCB are yet to be established. The CCB centrifuges the LBC specimens, retains the cell pellets after removing the supernatant, and subsequently freezes the specimens at 280 C in the biobank. Because several alcohol components in the fixative fluid may interfere with subsequent biomolecular testing, the supernatant is discarded, and the cell pellet alone is retained.11 Storage of LBC specimens at ambient temperature decreases the stability of nucleic acids compared with frozen specimens.11 The recovery of DNA and RNA from specimens collected using ThinPrep (Hologic, Boxborough, Mass) reportedly is good.12,13 Protocols for the efficient recovery of nucleic acids from specimens processed using SurePath (Becton, Dickinson and Company, Franklin Lakes, NJ) also have been reported.14,15 Long-term storage of 153
Review Article
DNA and RNA extracted from the LBC specimens at 280 C also is used for biobanking of cervicovaginal cytology specimens in this CCB. Because of all the confounding factors that can affect the quality of this extracted DNA and RNA, checking the length of the DNA fragments or the amount of ribosomal RNA to evaluate the quality of DNA and RNA in these stored samples is recommended before using these specimens from the biobank. There are also reports of a separate CCB established in Sweden. The Swedish CCB is a national initiative for the establishment of a prospective repository of LBC cervical samples from women who participate in organized cervical cancer screening programs.16,17 The Swedish CCB is embedded in a comprehensive cytology diagnostic registry that is linked to the national cancer registry in Sweden. The development and implementation of a nationally standardized method for the handling and long-term storage of LBC cervical samples has been the primary goal for this Swedish hub of the biobanking and molecular resource infrastructure. The sample handling protocol that the Swedish CCB follows was developed through a review of biobanking processes reported in the literature, wide consultation within the academic community, and various verification assays in collaboration with clinical cytology laboratories in Sweden. To store the LBC samples, the Swedish CCB follows a dual sedimentation procedure that was established after initial verification assays for each of its steps, and the procedure was capable of retrieving 80% of the cells in the original ThinPrep vial. The first step consists of aspirating 4 mL of cell solution from the ThinPrep vial and then transferring the solution to a conical tube and allowing it to sediment for 30 minutes; then, 300 lL of the sediment are transferred to the storage vial. The storage vials are displayed in a 96well format and are stored at 225 C, a temperature that was chosen for storing the vials because preliminary studies demonstrated that the cells, including their DNA and RNA, remained intact at this temperature and were well preserved for cytologic, immunohistochemical, and molecular studies. Specifically, 98% of the biobanked samples in the Swedish CCB were identified as satisfactory for conventional cytopathologic examination. Similarly, polymerase chain reaction (PCR) amplification of b-globin fragments indicated that 98% of the biobanked samples in the CCB contained adequate amounts of bglobin, indicating well preserved DNA in the samples. 154
The biobanked samples in the Swedish CCB are assessed twice each year for cellular morphology and DNA preservation. Biobanking of FNA Cytology Specimens
The feasibility and application of FNA specimens for different kinds of molecular testing are evolving at a rapid pace. The contribution of the results from molecular testing performed on FNA specimens and other types of cytology specimens that guide the clinical management team in developing personalized cancer therapy for the patient is now very well recognized.18–29 FNA specimens are particularly valuable for settings in which they are the only type of specimens available for testing. With increasing realization of the utility of FNA and other cytology specimens for molecular testing, there is currently a lot of interest in understanding the advantages and limitations of different types of specimen preparations for molecular testing. There are few investigators who have contributed toward our understanding of the potential of FNA specimens for biobanking by providing data from the results of molecular testing that were obtained by using archival specimens. Those studies and several recent reports highlighting the utility of FNA specimens for molecular testing form the basis for considering FNA specimens as yet another valuable biobanking resource. The FNA specimen preparations that have been studied and have exhibited potential utility for biobanking include smears stained with the Pap and Diff-Quik methods and material collected directly into a variety of solutions. Killian and colleagues reported the remarkable preservation of high-molecular-weight DNA in archival FNA smears that had been stored for >10 years.30 The DNA extracted from those archival smears could be used successfully for high-resolution comparative genomic hybridization arrays, DNA methylation, and single-nucleotide polymorphism genotyping. In that study, significant differences in DNA preservation and DNA integrity were observed between Pap-stained and Diff-Quik–stained archival FNA smears. The authors hypothesized that these differences were related to the DNA-damaging effect of hematoxylin in the Pap stain. On the basis of their successful molecular testing results and the retrieval of highquality DNA from smears, Killian et al reported the excellent potential of FNA smears, particularly the air-dried, Diff-Quik–stained smears, for biobanking purposes. Subsequently, other investigators also observed the excellent Cancer Cytopathology
March 2015
Cytology Biobanking/Krishnamurthy
preservation of high-molecular-weight DNA in both Papstained and Diff-Quik–stained archival smears.31–38 The study by Dejmek et al is perhaps the first systematic study to compare DNA yield and the quality of different cytologic preparations using human lung carcinoma cell lines for epidermal growth factor receptor (EGFR) genotyping.39 Those authors demonstrated that, although reliable EGFR genotyping could be achieved with any of the cytologic preparations, there were differences resulting from the use of different fixatives, staining, and mounting media. They observed that spray or ethanol-fixed Pap slides provided superior results in terms of DNA yield and fragment length compared with Romanowsky-stained, air-dried smears. Cells fixed in CytoLyt solution (Cytyc Corporation, Marlborough, Mass) yielded 5-fold higher DNA than cells fixed in CytoRich Red fluid (Fisher Scientific UK Ltd., Loughborough, UK), most likely because of the formaldehyde content in the latter fixative solution. Significantly higher DNA yield was obtained using slides mounted with Ecomount (Biocare Medical LLC, Concord, Calif), an organic, polymer-based mounting medium, compared with the xylene-based mounting medium Pertex (Leica Biosystems, Buffalo Grove, Ill). The collection of FNA specimens directly into buffer with and without RNA stabilizing agents also has been suggested for biobanking purposes. Ladd and colleagues demonstrated that both cellular morphology and RNA integrity could be maintained for up to 27 weeks after cryopreservation of FNA samples collected in RNAlater (Ambion RNA isolation kit; Applied Biosystems, Dallas, Tex) or in cryopreservation media (80% fetal bovine serum plus 20% RPMI 1640; Invitrogen, Carlsbad, Calif) with added cryoprotectants, such as dimethyl sulfoxide.40 Those authors clearly demonstrated that the cell morphology was sufficient to evaluate tumor cellularity when cells were prepared from a portion of the thawed specimen using their protocol, and RNA integrity was preserved for the extraction of high-quality RNA suitable for RNA-based testing. They observed that keeping the devitalization time to
