ASC statements EFCS survey

Summary Recommendations on Specimen Collection, Transport, and Fixation conditions

Participant name*

Email contact*

Country*

Department*

Academic/university
Regional/community
Private laboratory

Do you perform molecular pathology*

At your laboratory
At your institution
Outsourced
Not performed

Collection and Transport

1. When collecting cytology and small biopsy specimens, cold ischemic time should be*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

1a. Specimens collected fresh should be transported to the laboratory immediately after collection.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

1b. Specimens that cannot be immediately transferred, including those from distant referral sites, should be refrigerated at 4°C and transported to the Pathology laboratory under temperature-controlled conditions, minimizing delay to the extent possible.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

1c. Small biopsy specimens should be collected directly into 10% neutral buffered formalin (NBF) to minimize any delay in fixation (see section on Fixation below).*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

1d. Laboratories should try to document the cold ischemic time (i.e. time between collection and fixation of the specimen), when possible.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

1e. Unknown cold ischemic time should be documented in a disclaimer but may not necessarily exclude a specimen from testing.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

1f. Laboratory information systems should support convenient documentation of cold ischemic time.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

1g. Direct smear preparation slides should be placed in fixative immediately (if wet- fixed) or air dried and then fixed prior to further processing and use for ancillary studies.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

2. When collecting cytology specimens, a variety of collection/transport media may be utilized depending on anticipated downstream processing, including tissue culture media (e.g. RPMI), balanced salt solutions (e.g. saline, Hanks), liquid based cytology (LBC) commercial solutions (e.g. CytoLyt®, CytoRich®, PreservCyt®), nucleic acid preserving media (e.g. RNAlater™), and 10% NBF. However, for specimens collected in media that do not contain a preservative/pre-fixative/fixative reagent, the delay to fixation should be minimized to the extent possible.*

A. AGREE
B. AGREE with COMMENT
C. DISAGREE
D. DISAGREE with COMMENT

Comment*

3. When collecting cytology specimens for molecular testing in specific transport media, special considerations may be required, for example, storage in a container to minimize exposure to light and/or at room temperature.*

A. AGREE
B. AGREE with COMMENT
C. DISAGREE
D. DISAGREE with COMMENT

Comment*

4. When fresh or viable cells are needed for downstream ancillary testing (e.g. flow cytometry), or when the need for ancillary testing is unknown, additional collection/transport media options may be incorporated.*

A. AGREE
B. AGREE with COMMENT
C. DISAGREE
D. DISAGREE with COMMENT

Comment*

5. When collecting serous cavity fluids, such as pleural effusions or ascites, in which cells are typically present in a nutrient-rich milieu, the need for immediate addition of transport media may not be necessary; however, the specimen should be refrigerated at 4°C if there is a delay in transport to the laboratory.*

A. AGREE
B. AGREE with COMMENT
C. DISAGREE
D. DISAGREE with COMMENT

Comment*

Preservative/pre-fixative/fixative agents

6. When collecting small biopsy specimens, the recommended standard fixative is 10% NBF as it provides a standardized workflow for performing downstream ancillary studies.*

A. AGREE
B. AGREE with COMMENT
C. DISAGREE
D. DISAGREE with COMMENT

Comment*

7. When collecting fine needle aspiration (FNA) specimens for possible ancillary studies, laboratories may consider collecting part of the aspirate directly into formalin for cell block preparation, providing standardized workflow to minimize cold ischemic time and harmonize validation of subsequent ancillary studies on formalin-fixed paraffin- embedded (FFPE) tissue. However, for ancillary studies that require non-formalin fixed workflows, alternate handling may need to be considered.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

8. When collecting cytology specimens for LBC that use alcohol-based media, including methanol-based media (PreservCyt®, Cytolyt®) or combination ethanol and formaldehyde media (CytoRich Red®), the need for alternate immediate fixation may not be necessary.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

9. When collecting FNA specimens for possible molecular and in-situ hybridization (ISH) studies, high concentration alcohol fixatives (95% ethanol or Carnoy’s solution) may be superior in preserving DNA and RNA compared to formalin fixed specimens or those fixed in methanol or low concentration alcohol (40% EtOH). Dedicated RNA preservatives may be used for collecting cytology specimens when RNA-based testing (e.g. mRNA, miRNA-based expression assays) are being considered.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

10. If alcohol fixation is considered for molecular and ISH studies, some caution may be needed when using IHC staining protocols that are validated on FFPE tissue. Specimens pre-fixed in alcohol or fully alcohol-based cell block preparations (e.g. Cellient™) may have altered antigenicity for some immunohistochemical (IHC) stains and may require optimization of staining protocols that are validated on FFPE tissue.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

Additives/Decalcification agents

11. The use of reagents such as B5 fixative, Bouin’s solution, Zenker's fixative, B plus fixative, Acetic Acid-Zinc-Formalin (AZF) or dyes such as erythrosine are not recommended for specimen processing as they interfere with molecular and/or ISH studies.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

12. The use of strong inorganic acids such as nitric or hydrochloric acid are not recommended for decalcification of bone specimens. Alternatives such as EDTA may be considered instead for small biopsy bone specimens.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

13. When collecting serous cavity fluids, adding anticoagulants are not necessary, containers lined with EDTA may be used, if needed.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

Fixative volume and Time in fixative

14. An adequate volume of fixative should be used for cytology and small biopsy specimens. The preferred volume of fixative to tissue ratio (approximately 15-20 parts formalin to 1 part tissue/specimen volume) should be included in the laboratory collection/handling and submission procedures.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

15. For all specimens fixed using 10% NBF, a minimum of 6 hours and a maximum of 72 hours is recommended, which is considered optimal for IHC staining. While no data was available for specific recommendations for alcohol-fixed specimens, standardized fixation times for all specimens should be established and documented within individual laboratory standard operating procedure manuals.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Note: Specimens with unknown or sub-optimal fixation time should be documented in a disclaimer but may not necessarily exclude a specimen from testing.

Specify objection*

Summary Recommendations on Cell Block and Tissue Block Preparation

16. For specimens processed as cell block preparation, a variety of methods may be used, including plasma thrombin, HistoGel, sedimentation, automated (Cellient™), collodion bag, etc. However, caution should be exercised when using cell block preparations that have known limitations (for example, potential genomic DNA contamination in plasma thrombin blocks, or alcohol-based fixative in Cellient blocks with altered antigenicity for some IHC assays). See detailed discussion on the different cell block preparation methods in prior recommendations from the American Society of Cytopathology Clinical Practice Committee.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

Paraffin type

17. For specimens that are paraffin-embedded for block preparation, a high-quality paraffin with a low melting point (<60°C) is recommended.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

Summary Recommendations on Slide and Block Related Considerations Stains and Destaining

18. When using stained cytology smear and/or cytospin preparations for molecular studies, both Romanowsky and Papanicolaou stained slides may be used. However, some studies have indicated higher DNA yield and quality from Romanowsky stained smears, while others have noted better next-generation sequencing (NGS) quality control metrics when using Papanicolaou stained smears. Stained H&E slides are not recommended for ancillary studies as they tend to have a lower nucleic acid yield.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

19. When using cytology smears and/or cytospin for molecular testing such as NGS, destaining is typically not needed. However, some DNA-based testing may require destaining for optimal performance. Specifically, some cartridge based real-time PCR assays have reported high background signal with stained smears. Soaking the smears in xylene, followed by destaining in HCl/ethanol may be performed to reduce background.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

20. When using stained cytology smear and/or cytospin preparations for IHC, optimization and validation should be performed as false positive results can occur with some antibodies.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

21. When using stained cytology smear and/or cytospin preparations for ISH, optimization and validation should be performed as stained smear preparation can sometimes generate autofluorescence that may limit interpretation of the fluorescence in-situ hybridization (FISH) probes.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

22. When using cytology smears and/or cytospin preparations for DNA-based molecular testing, positively charged, non-frosted, and frosted tip slides are preferred over fully frosted slides. Insufficient data was available to recommend the type of slides for RNA- based testing or IHC-based testing on smears and/or cytospin preparations.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

23. When using cytology smears and/or cytospin preparations for DNA-based molecular testing, non-xylene based mounting media may be used for coverslipping to enhance yield. However, xylene-based mounting media are commonly used for FISH slides due to their ability to preserve samples and maintain fluorescence.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

24. When using cytology smears and/or cytospin preparations for molecular testing, soaking in xylene up to 48 hours at room temperature may be used for decoverslipping. Alternative methods include freezing the slide at -20°C for 1-2 minutes or using a hot plate at 120°C for ≤30 seconds (for fresh slides) or 170°C for 2 minutes (for older slides) to remove the coverslip without exposing the tissue to additional xylene.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

Slide/Block Processing and Storage

25. Glass slides and blocks should be properly stored in a temperature and humidity controlled, pest-free, organized manner that ensures preservation and accessibility.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

26. For specimens that are paraffin-embedded for block preparation, unstained sections for IHC and ISH studies are recommended to be cut at 4-5 microns. Note: Some tissues may require thinner/thicker sections, and the thickness of the paraffin section to be used for ancillary studies is at the discretion of the laboratory director.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

27. For specimens that are paraffin-embedded, unstained sections are recommended to be dried at 58-60°C for at least 15-30 minutes. Note: Special techniques, such as IHC or ISH may require longer drying times.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

28. When using cell block sections for molecular testing, unstained slides should ideally be used within 3-6 months if stored at room temperature, or up to 1 year if refrigerated. For predictive IHC biomarkers, unstained slides are not recommended to be stored at room temperature for longer than 3 months. For ISH testing, unstained sections are not recommended to be stored more than 6 weeks prior to testing.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

29. When using cytology smears and/or cytospin preparations for DNA based testing, coverslipped glass slides may be stored at room temperature up to 10 to 16 years. Insufficient data was available for RNA-based testing. Note: Digital archiving of stained cytology smear preparations is recommended when utilized for molecular studies, if available.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

30. When using cytology smears and/or cytospin preparations for IHC based testing, glass slides stored over one year are not recommended.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

Summary for Organ-Specific Special Considerations Urine specimens

31. Voided urine specimens should be collected fresh and refrigerated or processed within 12 hours. If refrigeration cannot occur within four hours, the specimen should be collected in a preservative solution and then processed within 72 hours. For samples collected for FISH, processing should be performed within 72 hours if collected in a preservative solution or within 3 hours if prepared from a fresh specimen.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

32. When collecting specimens for NGS based molecular testing, the minimum volume recommended should be at least 15mL fresh urine or at least 25mL if diluted in preservative solution. For samples that are not immediately processed and requiring storage beyond 24 hours, the sample should be centrifuged and stored as a frozen pellet at -80°C.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

33. When collecting urine specimens for ancillary studies, the sample may be split with at least 25mL undiluted sample reserved for microscopic analysis. For specimens processed using the SurePath system, residual specimens can be used for ancillary studies and the splitting of specimen prior to processing may be unnecessary.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

34. When collecting urinary tract washings and barbotage specimens, sterile water or normal saline may be used as irrigants and standard collection and laboratory storage protocols should be followed.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

Cerebrospinal fluid specimens

35. When collecting cerebrospinal fluid for ancillary studies, specimens should be collected in sterile, RNase/DNase-free tubes and transported to the laboratory immediately for processing. If delay in processing is anticipated, samples may be stored at 4°C short- term, but for longer storage, freezing at -80°C is recommended.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

36. When collecting cerebrospinal fluid for molecular profiling, cell-free DNA is freezing at -80°C is recommended avoiding multiple freeze-thaw cycles.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

37. When collecting samples for circulating tumor cell analysis, specimens should not be frozen. Samples should be refrigerated until processing; however, collecting in CellSave™ Preservative Tubes will allow for specimen storage at room temperature for up to 96 hours.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

Hematologic specimens

38. When collecting cytology specimens that require flow cytometric analysis, fresh fluid should be transported to the laboratory immediately after collection for testing.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

39. For specimens undergoing flow cytometry, specimen can be transported at room temperature if processed the same day or shipped on ice if processed later. All specimens should be processed within 72 hours to avoid specimen degradation. For specimens that cannot be processed immediately, an aliquot in at least 1mL of RPMI or EDTA (but not to exceed 10mL) can be prepared and stored at 4°C for up to 5 days for flow cytometry testing.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

Pancreaticobiliary specimens

40. When collecting pancreatic cyst fluid from cystic pancreatic lesions for ancillary studies, fresh undiluted specimens may be collected if transported to the laboratory immediately.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

41. When collecting pancreatic cyst fluid for molecular testing, liquid based commercial media (e.g. PancreaSeq media, CytoLyt,) or nucleic acid preserving media (e.g. RNAlater) may be used as recommended by the testing laboratory. Specimens collected fresh without a preservative/pre-fixative/fixative reagent should be processed same day for optimal test results.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

42. For samples collected for commercial molecular analysis (e.g. PancreaSeq, PancraGen/Interpace Diagnostics, etc.), the appropriate commercial collection/transport media should be used.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

43. When collecting bile duct brushing specimens for molecular testing and/or ISH studies, each individual brush should ideally be placed into a CytoLyt vial or other appropriate non-formalin preservative/media containing 15-20mL of solution and transferred to the cytology lab to be processed within 24 hours. If sending out for commercial molecular analysis, the appropriate commercial collection/transport media should be used.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

Thyroid specimens

44. When collecting thyroid FNA samples for commercial molecular analysis, the appropriate vendor guidelines should be used.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

Exfoliative respiratory specimens

45. When collecting sputum specimens for ancillary testing, sputum preservation reagents maybe used to prevent sample degradation.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

46. When collecting bronchoalveolar fluid, containers that limit cellular adherence to its surface such as silicone coated glass, polypropylene, or other plastics for suspension tissue cultures are recommended.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*

47. When collecting bronchial brushings, specimens should be placed into alcohol-based fixative immediately; however, if specimens are collected in other media (e. g., saline, RPMI) they should be transported to the laboratory immediately or refrigerated at 4°C and transported under temperature-controlled conditions.*

A. FEASIBLE, APPLICABLE
B. FEASIBLE, APPLICABLE with OBJECTION
C. NOT FEASIBLE, NOT APPLICABLE

Specify objection*