Q&A column

Editor: Frederick L. Kiechle, MD, PhD

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Q. If an exfoliative cytology specimen (for example, pleural fluid) is received fresh, how long can it stay refrigerated before it needs to be placed in formalin fixative for cell block preparation? That is, what is the recommended cold ischemic time?

A.June 2021—The CAP does not have an official position on the acceptable cold ischemic time of exfoliative fluid cytology specimens before cell block preparation for immunohistochemistry. The following represents expert opinion based on our collective experience and review of the literature.

The potential for cellular degeneration has led many laboratories to fix effusion samples immediately, commonly with equal volumes of ethanol, rather than submit fresh or refrigerated samples for cytologic examination.

Cold ischemic time refers to the time between specimen collection and placement in tissue fixative. With exfoliative cytology specimens that are collected fresh and submitted to the cytopathology laboratory without added fixative or preservative media, the cold ischemic time needs to be minimized to prevent cellular degeneration and preserve the morphologic, proteomic, and genomic integrity of the specimen. Most studies agree that cytology laboratories should process effusion samples quickly—if not immediately upon receipt—or store the samples in a refrigerator at 4°C until they can be processed.^1,2

If there is an anticipated delay in processing, alcohol-based preservatives may be used for pre-fixation (for example, adding an equal volume of ethanol to the exfoliative cytology specimen or using such media as Cyto­Lyt or CytoRich, which are methanol- and ethanol-based media, respectively). Alcohol pre-fixation precludes sending a sample for flow cytometry, preparing a direct smear, and staining with Diff-Quik. If used, alcohol pre-fixation should be considered when validating IHC and molecular tests on these samples. One may also consider aliquoting out a portion of the specimen for pre-fixation and keeping the remaining sample fresh.

In most situations, preservatives are not needed for these specimens because effusion fluid is rich in nutrients and the cells are floating in their natural milieu.^1,2 A review of the literature identified a handful of studies that evaluated the impact of cold ischemic time on cytologic effusion samples. The studies demonstrated that morphologic detail and immunoreactivity can be preserved for weeks if the specimens are stored in a refrigerator. Manosca, et al., evaluated 10 effusion specimens (four pleural, consisting of three benign and one breast adenocarcinoma, and six peritoneal, consisting of two ovarian cancers, two mesotheliomas, one melanoma, and one atypical mesothelial proliferation) processed after varying periods of refrigerated storage (0, 3, 5, 7, 10, 14 days). They focused on morphology (Diff-Quik and Papanicolaou-stained cytospin and H&E-stained cell block preparations), immunocytochemistry (using leukocyte common antigen, epithelial membrane antigen, AE1/AE3, and calretinin), and DNA viability using PCR amplification.³ They found that while minor changes were seen with morphologic features, prolonged storage did not impact the diagnostic evaluation or DNA amplification of these specimens. Keratin AE1/AE3 and LCA staining remained robust, while there were too few cells stained with EMA or calretinin for the authors to draw a meaningful conclusion. Of note, none of the IHC markers were transcription factors, which, anecdotally, appear to be especially susceptible to preanalytical variables.

Antonangelo, et al., evaluated the cytomorphology of 30 pleural effusion specimens collected in EDTA-coated tubes and stored at room temperature or under refrigeration (4°C) and reported earlier morphological alterations in samples stored at room temperature (day two) than under refrigeration (day four).⁴ Guzman, et al., compared five malignant pleural effusions stored at 4°C with those stored at room temperature. They found that while there was considerably lower cellularity in pleural fluids between day zero and day one, immunocytochemical results using a monoclonal antibody to EpCAM were similar. The authors concluded that effusion specimens can be stored for at least one day before immunocytochemical staining provided that the cells are handled gently during processing.⁵

With a growing clinical need for nucleic acid- and IHC-based predictive marker testing, it is critical to recognize the preanalytical variables that impact the quality of analytes used for downstream testing. An excellent review by Bass, et al., summarizes these preanalytical variables.⁶ Guidelines for breast cancer predictive biomarkers recommend a cold ischemic time of less than one hour for formalin-fixed, paraffin-embedded tissue samples to ensure the accuracy of test results.⁷ Additional studies evaluating such preanalytic variables as the impact of cold ischemia time, storage conditions, and type and duration of fixation on exfoliative fluid cytology samples to assess diagnostic and predictive biomarkers by IHC and DNA- and RNA-based techniques are sorely needed.

1. Engels M, Michael C, Dobra K, Hjerpe A, Fassima A, Firat P. Management of cytological material, pre-analytical procedures and bio-banking in effusion cytopathology. Cytopathology. 2019;30(1):31–38.
2. Michael CW, Davidson B. Pre-analytical issues in effusion cytology. Pleura Peritoneum. 2016;1(1):45–56.
3. Manosca F, Schinstine M, Fetsch PA, et al. Diagnostic effects of prolonged storage on fresh effusion samples. Diagn Cytopathol. 2007;35(1):6–11.
4. Antonangelo L, Vargas FS, Acencio MMP, et al. Effect of temperature and storage time on cellular analysis of fresh pleural fluid samples. Cytopathology. 2012;23(2):103–107.
5. Guzman J, Arbogast S, Bross KJ, Finke R, Costabel U. Effect of storage time of pleural effusions on immunocytochemical cell surface analysis of tumor cells. Anal Quant Cytol Histol. 1992;14(3):203–209.
6. Bass BP, Engel KB, Greytak SR, Moore HM. A review of preanalytical factors affecting molecular, protein, and morphological analysis of formalin-fixed, paraffin-embedded (FFPE) tissue: how well do you know your FFPE specimen? Arch Pathol Lab Med. 2014;138(11):1520–1530.
7. Wolff AC, Hammond MEH, Allison KH, et al. Human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline focused update. Arch Pathol Lab Med. 2018;142(11):1364–1382.

Kaitlin Sundling, MD, PhD
Associate Director, Cytology Section
Wisconsin State Laboratory of Hygiene
Faculty Director, University of Wisconsin Cytotechnology Program
Assistant Professor, University of Wisconsin
School of Medicine and Public Health
Madison, Wis.
Member, CAP Cytopathology Committee

Abiy B. Ambaye, MD
Professor
Medical Director of Histology and Immunohistochemistry
Department of Pathology and Laboratory Medicine
University of Vermont
Larner College of Medicine
Burlington, Vt.
Member, CAP Immunohistochemistry Committee

Sinchita Roy-Chowdhuri, MD, PhD
Associate Professor
Departments of Pathology and Translational Molecular Pathology
University of Texas
MD Anderson Cancer Center
Houston, Tex.
Member, CAP Cytopathology Committee

Q. Are two levels of a control required for a manual reticulocyte count?

A.No, two levels of quality control are not required for a manual reticulocyte count. However, a quality control process is required. The process should include ensuring that the reticulocyte blood smear is uniquely identified, properly stained, free from precipitate, and has good cell distribution. Additionally, there must be a written procedure that includes the method, number of cells counted, and calculations used.

Joan Rose, MT(ASCP)SH
Technical Analyst
CAP Accreditation Programs
College of American Pathologists
Northfield, Ill.

SARS-CoV-2 testing

For many in laboratories, the pandemic has been a time of countless questions about SARS-CoV-2 testing. Here, from the practice of one pathologist, Beverly B. Rogers, MD, are commonly asked questions and their answers—for those to whom they might be of help in their own practices.

Dr. Rogers is chief of pathology, Children’s Healthcare of Atlanta, and adjunct professor of pathology and pediatrics, Emory University School of Medicine.

Q. What are the requirements for obtaining emergency use authorization versus 510(k) clearance?

A.The requirements for an EUA are magnitudes less than those for 510(k) clearance. The “typical” test on the market that detects a viral pathogen has 510(k) clearance from the Food and Drug Administration, but SARS-CoV-2 tests do not. There wasn’t sufficient time for the detailed evaluation required for a 510(k) clearance. An example of the requirements to obtain an EUA for a PCR test for SARS-CoV-2 compared with a 510(k) clearance for a PCR test for another virus is shown (see table). This information is taken from one vendor but generalized to provide estimates that give an idea of the magnitude of difference.