Q&A column

Editor: Frederick L. Kiechle, MD, PhD

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Q. For many years, breast cancers were tested routinely for HER2 and estrogen and progesterone receptors in an attempt to find actionable treatments. Then it was discovered that over- and underfixation with formalin could give erroneous results. Have standards been developed to address this issue with the latest actionable IHC markers, such as PD-L1? Shouldn’t this be addressed for all actionable IHC markers?

A.March 2021—The short answer is yes. Time to fixation (i.e. cold ischemia time) and duration of fixation should be monitored and probably recorded for all specimens, especially tumor specimens. American Society of Clinical Oncology and College of American Pathologists evidence-based guidelines address this for HER2¹ and ER/PR.² There is little data on the impact of these preanalytical variables on PD-L1 immunohistochemistry, though the International Association for the Study of Lung Cancer pathology committee released specimen-handling recommendations in a 2020 PD-L1 testing update.³ The IASLC recommends that biopsies or cell blocks be fixed for six to 48 hours and resection specimens for 24 to 48 hours, though fixation for up to 72 hours is acceptable for the latter. This recommendation is based on data from a study on lung cancer that found delayed fixation had negative effects across a range of diagnostic and predictive markers.⁴ For PD-L1, decreased staining was observed in some samples after a cold ischemia time of only one hour. In this same study, prolonged fixation of up to one week had little to no deleterious effect.

The impact of delayed fixation, underfixation, and overfixation is best studied in breast biomarkers and Ki-67.^5-7 Delayed fixation could lead to autolysis and resulting loss of antigenicity. Given the pressure for a fast turnaround time, it may be tempting to rush tissues to the processor. Underfixed tissues undergo variable combinations of formalin fixation, coagulation fixation due to alcohol on the processor penetrating the tissue, and autolysis (i.e. raw, unfixed tissue), with unpredictable consequences. The negative impact of prolonged fixation has largely been overcome by modern antigen-retrieval techniques.

Taking its cue from the ASCO/CAP breast biomarker guidelines, the joint College of American Pathologists/National Society for Histotechnology “Practical guide to specimen handling in surgical pathology,” updated in April 2020, states, “As a general recommendation, when using 10% NBF [neutral buffered formalin] ALL clinical tissue specimens should be fixed for a minimum of 6 hours and a maximum of 72 hours.”⁸

1. Wolff AC, Hammond ME, Hicks DG, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol. 2013;31(31):3997–4013.
2. Allison KH, Hammond MEH, Dowsett M, et al. Estrogen and progesterone receptor testing in breast cancer: ASCO/CAP guideline update. J Clin Oncol. 2020;38(12):1346–1366.
3. Lantuejoul S, Sound-Tsao M, Cooper WA, et al. PD-L1 testing for lung cancer in 2019: perspective from the IASLC pathology committee. J Thorac Oncol. 2020;15(4):499–519.
4. van Seijen M, Brcic L, Gonzales AN, et al. Impact of delayed and prolonged fixation on the evaluation of immunohistochemical staining on lung carcinoma resection specimen. Virchows Arch. 2019;475(2):191–199.
5. Yildiz-Aktas IZ, Dabbs DJ, Bhargava R. The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma. Mod Pathol. 2012;25(8):1098–1105.
6. Neumeister VM, Anagnostou V, Siddiqui S, et al. Quantitative assessment of effect of preanalytic cold ischemic time on protein expression in breast cancer tissues. J Natl Cancer Inst. 2012;104(23):1815–1824.
7. De Marzo AM, Fedor HH, Gage WR, Rubin MA. Inadequate formalin fixation decreases reliability of p27 immunohistochemical staining: probing optimal fixation time using high-density tissue microarrays. Hum Pathol. 2002;33(7):756–760.
8. Lott R, Tunnicliffe J, Sheppard E, et al. Practical guide to specimen handling in surgical pathology. College of American Pathologists. Revised April 2020. https://cap.objects.frb.io/documents/practical-guide-specimen-handling.pdf

David L. Rimm, MD, PhD
Director of Yale Pathology Tissue Services
Yale University School of Medicine
New Haven, Conn.
Member, CAP Immunohistochemistry Committee

Andrew Michael Bellizzi, MD
Clinical Professor
Department of Pathology
University of Iowa Hospitals and Clinics
Iowa City, Iowa
Chair, CAP Immunohistochemistry Committee

Q. In our hospital, respiratory therapy runs most of the blood gas tests on instruments in centralized locations. Staff are able to enter into the blood gas instrument, which is connected to the LIS, to whom they gave critical results. However, staff do not have a way to document that a result was read back. The majority of these critical results happen in the neonatal intensive care and intensive care units, where respiratory therapy is a part of the care team, so results are given in person. Is documenting a read-back necessary when critical results are communicated verbally? How does the CAP checklist COM.30100 relate to point-of-care testing?

A.The CAP checklist requirements COM.30000 Critical Result Notification and COM.30100 Critical Result Read-Back apply to all laboratory testing sites, including point of care. COM.30000 states, “In the point-of-care setting, the identity of the testing individual and person notified need not be recorded when the individual performing the test is the same person who treats the patient. In this circumstance, however, there must be a record of the critical result, date, and time in the test report or elsewhere in the medical record.” Documenting a critical result notification and read-back is required in all other circumstances. If the LIS does not have the capability to document read-back or other required elements, the person conducting the testing must document them using another mechanism, such as by manually recording the information on a log sheet.

Susan Jacobs, MHA, BSMT(ASCP)
Inspection Specialist
College of American Pathologists
Northfield, Ill.

Q. What is the correct nomenclature for HbA1c in printed test reports? Different laboratories use different names and abbreviations for the same test—hemoglobin A1c, Hgb A1c, glycated hemoglobin, glycated Hb, etc.

A.Different names for the same laboratory test may impact the timely delivery of proper medical care as providers may not select the right test. The LOINC (Logical Observation Identifiers Names and Codes) system was developed to standardize test names—but it is used by computers, not people.

Part of the confusion regarding hemoglobin A1c stems from the fact that carbohydrate may become fixed to protein in two major ways. The formation of glycoproteins involves glycosylation, the enzymatic addition of carbohydrate to amino acid residues in the protein during post-translational processing in the cell. Glycated proteins are formed when carbohydrate reacts chemically (nonenzymatically) with amino acid residues long after the protein has been released from the cell that made it. Glycosylation is a much more familiar process, so it seems natural that people may refer to glycated hemoglobin as glycosylated. But it is the slow nonenzymatic addition of glucose to hemoglobin as red blood cells endure rising and falling levels of blood glucose that makes glycated hemoglobin an important marker for diagnosing and monitoring patients with diabetes mellitus.

There are tests for other glycated blood proteins, such as glycated albumin, but their role is unclear and may be limited. After early landmark studies showed the benefit of better glycemic control using glycated hemoglobin, this marker became the standard.