Enhancement of Endogenous Biotin Staining 175Letter

175

Address correspondence toDr. Amitabh Srivastava,Department of Pathology,Tufts-New England MedicalCenter, 750 Washington St.,Boston, MA. E-mail:asrivastava@tufts-nemc.org

Endocrine Pathology, vol. 15,no. 2, 175–178, Summer 2004© Copyright 2004 by HumanaPress Inc. All rights of anynature whatsoever reserved.1046–3976/04/15:175–178/$25.00

Endogenous Biotin Stainingas an Artifact of Antigen Retrievalwith Automated Immunostaining

To assess whether the reactivity was aresult of endogenous biotin content ornon-specific binding promoted by theheat-driven reactions on the automatedstainer, we performed primary incubationswith each antibody (CG, SYN, CAL, PSA)in four different ways: on a Ventanaimmunostainer as a routine run; an over-night incubation at 4°C; on the automatedstainer following pretreatment with abiotin-blocking kit (Ventana); and manu-ally using the Envision kit (DAKO). A cellblock from a patient with hepatic metasta-sis from a pulmonary small cell carcinomawas stained similarly for comparison. Nega-tive controls were run with and withoutantigen retrieval and in each instance theprimary antibody was replaced with theappropriate commercially available mouse orrabbit IgG negative control antisera(Ventana).

The results are summarized in Table 1.No difference in staining intensity ordistribution was seen between the 4°Covernight and the routine run on theautomated stainer in any case. Negativecontrols run without antigen retrievalwere completely negative (Fig. 1B). Strik-ingly, however, negative controls run afterantigen retrieval showed staining of thesame intensity and distribution as the spu-rious immunoreactivity on the test cases(Figs. 1A,C). In addition, the false posi-tive reactivity in the three test cases wascompletely abolished when primary incu-bation was done after treatment with the

We wish to highlight certain artifacts ofantigen retrieval that we encounteredrecently while using automated immuno-histochemistry. Immunohistochemicalanalysis for routine diagnostic purposes isincreasingly being performed on auto-mated stainers. The recognition of spuri-ous immunoreactivity in such proceduresis critically dependent on proper negativecontrols run with every batch of staining.Negative controls used most often involveeither the omission of primary antibodyor replacement of the primary antibodywith normal serum at equivalent concen-tration. While most manufacturers provideappropriate negative control antisera in theform of irrelevant mouse and rabbit IgG tobe used in place of the primary antibody,the role of antigen retrieval in negative con-trols is seldom highlighted. The phenom-enon of heat induced endogenous biotinactivity has been described previously [1–3] and can be a pitfall in the interpretationof immunohistochemical results.

We recently studied three cases withimmunoreactivity for chromogranin (CG)and synaptophysin (SYN) that were dis-cordant with the morphological andclinical data and included two hepatocel-lular carcinomas (one biopsy and one cellblock) and one resected follicular carci-noma of the thyroid gland. The thyroidtumor was additionally positive for calci-tonin (CAL) and one of the hepatocellularcarcinomas showed reactivity with pros-tate-specific antigen (PSA).

176 Endocrine Pathology Volume 15, Number 2 Summer 2004

Fig. 1. Follicular carcinoma thyroid with spurious immunoreactivity for calcitonin (A). The negative control run without antigen retrievalshows no staining (B). A similar staining pattern was seen with chromogranin and synaptophysin as well. The same negative control runafter antigen retrieval shows staining similar in intensity as the test case (C).

Enhancement of Endogenous Biotin Staining 177

Fig. 2. The spurious positive result with calcitonin and the other antibodies was not seen after treatment with the biotin-blocking kit (A)or when using the Envision kit (B). The pulmonary small cell carcinoma stained with the biotin block was still strongly positive for synaptophysin (C).

178 Endocrine Pathology Volume 15, Number 2 Summer 2004

Table 1. Results of Immunoreactivity with Neuroendocrine Markers

Test cases Negative control

Routine Without With antigenDiagnosis (Antibody)a (Ventana) 4°C overnight Biotin block Envision kit retrieval retrieval

FTC (CAL, CG, SYN) + + – – – +HCC (CG, SYN) + + – – – +HCC (CG, SYN) + + – – – +SCC (CG, SYN) + + + + – –

aFTC: follicular thyroid carcinoma; HCC: hepatocellular carcinoma; SCC: small cell carcinoma, lung; CAL: calcitonin; CG: chromogranin;SYN: synaptophysin.

biotin-blocking kit (Fig. 2A). No immuno-reactivity for CAL, SYN, and CG was seenin the follicular carcinoma thyroid whenstained with the Envision kit (Fig. 2B).Finally, staining for neuroendocrine mark-ers CG and SYN persisted with the sameintensity even after biotin-block treat-ment in the metastatic small cell carcinomaindicating specific reactivity for neuro-endocrine markers (Fig. 2C).

The cases described above illustrate thatit is imperative that negative controls onautomated immunostainers be run withantigen retrieval to detect spuriousimmunoreactivity due to endogenousbiotin content. Any discordant immuno-histochemical results on test materials canbe verified by pretreatment with biotinblocking kits before the primary incuba-tion or by using detection systems that donot use a biotinylated secondary antibodysuch as the Envision kit.

References

1. Herrmann ME, LiVolsi VA, Pasha TL, Rob-erts SA, Wojcik EM, Baloch ZW. Immuno-histochemical expression of galectin-3 inbenign and malignant thyroid lesions. ArchPathol Lab Med 126:710–713, 2002.

2. Bussolati G, Gugliotta P, Volante M, Pace M,Pappoti M. Retrieved endogenous biotin:a novel marker and a potential pitfall in diag-nostic immunohistochemistry. Histopathol-ogy 31:400–407, 1997.

3. Kashima K, Yokoyama S, Daa T, NakayamaI, Nickerson PA, Noguchi S. Cytoplasmicbiotin like activity interferes with immuno-histochemical analysis of thyroid lesions:a comparison of antigen retrieval methods.Mod Pathol 10:515–519, 1997.

Amitabh Srivastava, MD

Department of Pathology,Tufts-New England Medical Center

Boston, MA

Arthur S. Tischler, MD

Department of Pathology,Tufts-New England Medical Center

Boston, MA

Ronald A. Delellis, MD

Department of Pathology,Lifespan Academic Center

Providence, RI