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Quality Control in Immunocytochemistry: Experiences with the Oestrogen Receptor Assay J Clin Pathol: First Published As 10.1136/Jcp.45.2.120 on 1 February 1992

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Quality Control in Immunocytochemistry: Experiences with the Oestrogen Receptor Assay J Clin Pathol: First Published As 10.1136/Jcp.45.2.120 on 1 February 1992 120 J Clin Pathol 1992;45:120-124 Quality control in immunocytochemistry: Experiences with the oestrogen receptor assay J Clin Pathol: first published as 10.1136/jcp.45.2.120 on 1 February 1992. Downloaded from F T Bosman, A F P M de Goeij, M Rousch Abstract ted specificity to be produced, and the develop- Aims: To evaluate the feasibility of an ment of very sensitive second step reagents. As interlaboratory quality control pro- a consequence, almost every surgical pathology gramme in immunohistochemistry. laboratory routinely uses immunocyto- chemistry on a daily basis. As recently Methods: Several pathology laboratories ' were asked to carry out immunohisto- indicated by Elias et al, however, an important chemical oestrogen receptor staining on a source of variability in the performance of set of freeze dried cryostat sections of immunocytochemistry techniques is the vari- breast cancer tissue. The sections and ation among laboratories in tissue processing protocols for staining and semi- and histochemical procedures. This could be quantitative scoring were mailed to the improved by standardisation, monitored by participating laboratories in two trials. quality control programmes. The oestrogen receptor content of the A typical example of an immunocyto- breast cancer samples was determined by chemical test which requires such an approach radioligand binding assay on the tumour is the evaluation of the oestrogen receptor cytosol. content in breast cancer. About 60% of Results: In the first trial 11 laboratories patients with an oestrogen receptor positive (response rate 60%) participated. Eight tumour will respond to some form ofendocrine (73%) of the participants scored within a treatment; less than 10% of oestrogen receptor 95% confidence interval and all but one negative neoplasms respond.23 Expression of correctlyclassified thetumour as receptor oestrogen receptor protein may also be regar- positive. In the second trial all 20 partici- ded as a hallmark of a differentiated state,4 and pating laboratories (response rate 55%) many studies have shown that the prognosis in correctly scored one tumour sample as terms of disease free interval and survival in negative and 18 of them (90% of respon- oestrogen receptor positive breast cancer is dents) correctly classified the two other more favorable than that of oestrogen receptor http://jcp.bmj.com/ tumour samples as receptor positive. In a negative breast cancer.5 quantitative evaluation a histochemical In order for it to be clinically useful a reliable score within 95% confidence interval technique for oestrogen receptor determina- limits was provided by eight (40%) and 12 tion is required. For two decades now, the (60%) of the participants. standard steroid receptor assay has been the of dextran coated charcoal (DCC) assay, used on Conclusions: Semiquantitative scoring on September 28, 2021 by guest. Protected copyright. immunocytochemical staining is valu- cytosols of tumour tissue. Although reliable in able for performing correlative inter- principle, this ligand binding assay has been laboratory studies, although this scoring shown to lead to high inter- and intra-assay protocol may not be required for diag- variability. Only the introduction of quality nosis or prognosis. Significant inter- control programmes in the clinical chemistry laboratory variability exists, leading to laboratories in Europe, under the auspicies of qualitatively correct receptor classifica- the EORTC has led to a steroid receptor assay tion in 100% ofreceptor negative and 80% with reasonable widescale reproducibility.67 of receptor positive cases, and quanti- Replacement of this technique with an tative agreement in only about half of the immunocytochemical assay, which would be cases. The perceived variability is not preferable in view of the lesser quantity of caused by systematic differences in the tissue required, avoidance of the use of choice of the immunocytochemical tech- radiolabelled ligands, the possibility oflocating of freeze dried the receptor protein specifically in tumour cells, nique, or the mailing in Department of sections. Quality control programmes and the ability to take tumour heterogeneity Pathology, should be included in the standard pro- receptor content into account have all been University of immunohisto- emphasised repeatedly."'0 The development of Limburg, cedures of each diagnostic allowed steroid Medical Faculty, chemistry laboratory. monoclonal antibodies has PO Box 616, receptor assays based on antigen-antibody 6200 MD Maastricht, interactions to be introduced." It is clear, The Netherlands that immunocytochemical receptor F T Bosman Over the past decade immunocytochemistry however, A F P M de Goeij has become essential in surgical pathology. The determination can only be justified if: (a) the M Rousch methodological requirements of repro- results of the immunocytochemical assay have Correspondence to: ducibility and specificity have been met with the been validated against those of the ligand Dr A F P M de Goeij technique, which allows large binding assay; and (b) similar quality control Accepted for publication hybridoma validate the intra- and inter- 3 February 1991 quantities ofmonoclonal antibodies with selec- programmes Quality control in oestrogen receptor immunocytochemistry 121 laboratory reproducibility of the technique. The sections were weakly counterstained in The first requirement has repeatedly been diluted haematoxylin and mounted in Entellan. met.41213 This study was designed to test the After the sections had been returned 11 of J Clin Pathol: first published as 10.1136/jcp.45.2.120 on 1 February 1992. Downloaded from feasibility of a quality control programme to the 20 laboratories indicated that they had used meet the second requirement. the ERICA staining protocol, as outlined in the instructions supplied with the commercial kit (ERICA, Abbott Laboratories). One partici- Methods pant indicated that the protocol used was a local TISSUES AND MAILING OF THE SECTIONS modification ofthe ERICA protocol. In the first Samples of breast cancer tissue were snap- pilot study two participants used the ERICA frozen in isopentane, cooled with dry ice. (Abbott) protocol. Tumours with a high, intermediate, or zero receptor content, as determined by radio- SEMIQUANTITATIVE ANALYSIS chemical (DCC) assay on cytosol, were selec- The stained sections were scored semiquanti- ted. Frozen sections (6.um) were cut on a tatively, according to a modification of the cryostat microtome and mounted on gelatin scoring by McCarty et al,15 as described chromealum coated slides. The slides were earlier.'6 The resulting histochemical score was freeze dried and subsequently stored in a obtained as follows: plastic airtight container over silica gel at room i=4 Histochemical score = I P(i) x i temperature. Each participating laboratory i=O received four sections of each tumour and one where i = staining intensity, which may vary section of a rabbit uterus which served as a between 0 (no staining) and 4 (strongest stain- receptor positive standard tissue. This was ing), and Pi = percentage of stained tumour found to be an acceptable approach after it was cell nuclei in category i (0-100). The maximum shown that when kept absolutely dry, the attainable score is 400 by definition. For reliable sections could be kept at ambient temperatures visual scoring at least three cohorts of 100 for up to several weeks without deterioration of tumour cells had to be scored in different high the immunoreactivity of oestrogen receptor power fields (objective x 40). protein. A receptor score ofless than 35 was regarded In the first trial 11 pathology laboratories as negative, based on earlier correlative participated, 20 in the second trial. This studies'6 between immunohistochemical recep- represented a response rate of 60% and 55%, tor assays and radioligand binding assays. In respectively. these studies a cut-offvalue of 10 fmol oestrogen receptor/mg cytosol protein was used. IMMUNOHISTOCHEMISTRY Together with the mailing of the sections a OESTROGEN RECEPTOR ANALYSIS IN CYTOSOL staining protocol was provided that differed TISSUE from the protocol included in the ERICA kit Oestrogen receptors were quantitated in http://jcp.bmj.com/ with respect to the working dilution ofthe anti- tumour cytosols, prepared from a tumour sam- oestrogen receptor serum (1 in 4 instead of ple directly adjacent to the sample used for undiluted) and to the conditions of incubation with this antibody (overnight at 4°C instead of one hour at room temperature). Before immunostaining, the sections were fixed (10 minutes at 4°C) in picric acid-para- 400- on September 28, 2021 by guest. Protected copyright. formaldehyde'4 or in phosphate buffered 4% paraformaldehyde (pH 7 4). Subsequently the sections were rinsed (three times for five I minutes at room temperature) in phosphate 300 buffered saline (PBS). Before incubation the sections were exposed to 20% normal goat I serum minutes at room .o0 (15 temperature). After i- T U blotting the section, the primary antibody 0 7nSW-I U_ _ Uw T (ERICA kit, kindly provided by Abbott 0 -w-B---- U) I1 Laboratories, Diagnostic Division, The I Netherlands) was applied (diluted 1 in 4 in 1% U bovine serum albumin in PBS) and the sections 100- were incubated overnight in a humid chamber at 4°C. Subsequently the sections were washed in PBS (three times for five minutes each) and 0 S incubated (30 minutes at room temperature) I I I I with goat-anti rat IgG (included in the ERICA 0 5 10
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