Tissue array technology for testing interlaboratory and interobserver reproducibility of immunohistochemical estrogen receptor analysis in a large multicenter trial.

Author(s): von Wasielewski R, Mengel M, Wiese B, Rüdiger T, Müller-Hermelink HK, Kreipe H

Publication: Am J Clin Pathol, 2002, Vol. 118, Page 675-82

PubMed ID: 12428786 PubMed Review Paper? No

Purpose of Paper

This paper performed a comparative assessment of estrogen receptor (ER) immunohistochemistry among (1) different fixation parameters (fixative and duration, pre-fixation delay), and (2) multiple institutions using tissue microarrays containing negative, low, moderate, and high ER expressing specimens. Confounding sources of variations were also explored.

Conclusion of Paper

Optimal ER immunostaining was observed among specimens fixed in neutral buffered formalin for 24 h, with robust reductions observed in specimens subjected to cryosectioning pre-fixation, a pre-fixation delay at 4 degrees C, or fixation in ethanol. Agreement among institutions concerning ER classification varied in relation to the magnitude of ER expression among tumor specimens, with low and intermediate ER expressing tumor specimens generating the most discordance, 56 and 72% agreement, respectively. The authors hypothesize that differences in antigen retrieval protocols may be a confounding variable among institutions.

Studies

Study Purpose

The purpose of this study was to assess the influence of different fixatives, fixation durations, and a pre-fixation delay on quantifiable ER immunoreactivity in tissue microarrays. Tissue microarray specimens included breast, stomach, lymph nodes, and skin.

Summary of Findings:

Optimal ER immunostaining, defined by the percentage of positive nuclei and staining intensity, was observed after immediate fixation in neutral buffered formalin for 24 h, with significant reductions (45-60% that of optimally fixed specimens) observed among specimens post-fixed after cryosectioning, storage at 4 degrees C overnight prior to fixation, or ethanol fixation; conversely, fixation in buffered or unbuffered formalin for as little as 6 h or as long as 72 h resulted in modest reductions in immunostaining (80-100% that of optimally fixed specimens).

Biospecimens

Preservative Types

Formalin
Ethanol

Diagnoses:

Neoplastic - Carcinoma
Neoplastic - Lymphoma
Neoplastic - Sarcoma
Neoplastic - Melanoma

Platform:

Analyte	Technology Platform
Protein	Tissue microarray
Protein	Immunohistochemistry

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Biospecimen Preservation	Time in fixative	6 h 24 h 72 h 96 h 168 h 384 h
Biospecimen Acquisition	Cold ischemia time	Overnight at 4 degrees C Fixed immediately (unspecified time)
Biospecimen Preservation	Type of fixation/preservation	Ethanol Formalin (buffered) Formalin (unbuffered)
Biospecimen Preservation	Fixative pH	5 7

Study Purpose

The purpose of this study was to assess the extent of inter-institutional agreement among 172 institutions for ER immunohistochemistry using tissue microarrays, and to determine if antigen retrieval may be a potential source of variation. TIssue microarray specimens included breast, stomach, skin, and lymph nodes.

Summary of Findings:

The extent of inter-institutional agreement concerning ER classification was dependent upon the extent of the immunoreactive score (defined by the percentage of positively stained nuclei and staining intensity), with 95%, 72%, and 56% agreement among high, intermediate, and low ER-expressing tumor specimens. The authors note that subjecting tissue microarrays to antigen retrieval (autoclaving for 10 min in citrate buffer) improved the intensity of staining without affecting background.

Biospecimens

Preservative Types

Formalin

Diagnoses:

Neoplastic - Carcinoma
Neoplastic - Lymphoma
Neoplastic - Melanoma
Neoplastic - Sarcoma

Platform:

Analyte	Technology Platform
Protein	Immunohistochemistry
Protein	Tissue microarray

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Analyte Extraction and Purification	Antigen retrieval	Citrate buffer, 10 min of autoclaving No treatment

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