Q&A column

Q. We are hoping to validate a procedure for the fixation, decalcification, and staining of bone marrow specimens including bone marrow core biopsy, a service we do not currently provide. Our problem is we will not be able to access fresh marrow specimens for our decalcification validation. Could you recommend an alternative tissue we could use to validate the preservation of tissue morphology and antigenicity after decalcification? We have several other tissue samples we could potentially access, including bone, gastrointestinal, dermatologic, and breast. Could any of these tissues be used? If not, what specimen do you recommend?

A. Consideration of effects of alternative fixation and decalcification protocols on morphology, antigenicity, and even DNA-RNA preservation is important in surgical and hematopathology. A variety of commercial and homebrew chemical decalcification methods are available and tend to differ widely between laboratories. In general, acid-based protocols are more deleterious to immunostaining.^1,2 Testing and validation of such protocols are covered in CAP immunohistochemistry validation guidelines.³ Recommendation No. 8, from Table 3 of the CAP IHC guidelines, states: “If IHC is regularly done on decalcified tissues, laboratories should test a sufficient number of such tissues to ensure that assays consistently achieve expected results. The laboratory medical director is responsible for determining the number of positive and negative tissues and the number of predictive and nonpredictive markers to test.”³ The example of bone marrow biopsies discussed in the CAP IHC guideline encompasses the following: “To assess the influence of their decalcification procedure on IHC test results in bone marrows, laboratories should test a selected set of commonly ordered markers (eg, CD3, CD20, CD138) in a series of cases. The results may be correlated with expected results in routinely processed (control) tissues and with other applicable test results (eg, flow cytometry, IHC testing of lymph node in same patient).”³

Test tissue might include other bone specimens containing marrow elements together with other hematolymphoid tissues. Autopsy might offer another source of bone marrow biopsies, although it is not available to all laboratories. We find that tonsil, thymus, and spleen are available in many laboratories⁴; spleen with extramedullary hematopoiesis might provide an abundant source of non-lymphoid and hematopoietic precursor cells. Construction of multi-tissue block(s) could provide a substrate for testing of multiple relevant antibodies on paired decalcified and nondecalcified tissue with increased efficiency.

In an attempt to validate the direct effects of decalcification on antigens, we have incubated a commonly used immunohistochemistry protocol control tissue (i.e. tonsil tissue) in decalcification agents, followed by image analysis to quantify the effects.⁴ We also compared the effect of decalcification on breast tissue, obtained at reduction mammoplasty⁵; others have used excess breast tumor tissue.^2,6,5 These may provide ancillary validation of the effects of decalcifying agents. Importantly, these studies showed that not all antigens from tonsil or breast tissue were affected in the same way as bone marrow. Further, demonstrating that decalcification does not impair CK7 staining would not translate to a CD61 stain. Thus, testing each antibody to be used in decalcified tissue would be considered best practice. In addition, it is critical that the tissue type to be used for validation, including those proposed here, include the cellular or disease process target for a given marker.

In summary, there is no easy way to provide optimal controls for all antigens used in IHC testing, and the individual laboratory should exercise diligence in characterizing the effects of decalcification on bone tissue.

1. Arber JM, Arber DA, Jenkins KA, Battifora H. Effect of decalcification and fixation in paraffin-section immunohistochemistry. Applied Immunohistochemistry. 1996;4(4):241–248.
2. Schrijver WA, van der Groep P, Hoefnagel LD, et al. Influence of decalcification procedures on immunohistochemistry and molecular pathology in breast cancer. Mod Pathol. 2016;29(12):1460–1470.
3. Fitzgibbons PL, Bradley LA, Fatheree LA, et al. Principles of analytic validation of immunohistochemical assays: guideline from the College of American Pathologists Pathology and Laboratory Quality Center. Arch Pathol Lab Med. 2014;138(11):1432–1443.
4. Shvartsbeyn M, Salama ME, Dewar R. Histologic evaluation of the effects of tissue decalcification on immunohistochemical markers in bone marrow specimen. 105th Annual Meeting of the USCAP; March 12–18, 2016; Seattle. Mod Pathol. 2016;29(Suppl2):​504A.
5. Hathuc VM, East E, Pang J, Jorns JM, Dewar R. A practical quality control procedure for decalcified bone specimens in evaluation of ER/PR/HER-2 immunohistochemistry. 106th Annual Meeting of the USCAP; March 4–10, 2017; San Antonio. Mod Pathol. 2017;30(Suppl2):508A.
6. Yoest J, Clark BZ, Onisko A, Dabbs DJ. Breast carcinoma biomarkers: effects of hydrochloric acid and formic acid decalcification on immunohistochemistry and in situ hybridization. 106th Annual Meeting of the USCAP; March 4–10, 2017; San Antonio. Mod Pathol. 2017;30(Suppl2):522A.

Megan Troxell, MD, PhD
Professor, Department of Pathology, Stanford University School of Medicine, Stanford, Calif.
Chair, CAP Immunohistochemistry Committee

Mohamed El-Sayed Salama, MD
Medical Director, Mayo Medical Laboratories, Mayo Clinic, Rochester, Minn.
Member, CAP Immunohistochemistry, Committee

Rajan Dewar, MD, PhD
Associate Professor, Director, Hematology Laboratory
University of Michigan, Ann Arbor
Member, CAP Surgical Pathology Committee