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Tissue microarray technology and findings for diagnostic immunohistochemistry
Pathology (January 2013) 45(1), pp. 71–79
TECHNICAL REPORT
Tissue microarray technology and findings for diagnostic immunohistochemistry ANTHONY
VAN
ZWIETEN
Department of Anatomical Pathology, Pathology Queensland – The Prince Charles Hospital, Chermside, Queensland, Australia
Summary Aims: This study was undertaken to determine the validity and viability of tissue microarray (TMA) technology in assessing diagnostic immunohistochemistry (IHC) at a single laboratory site. Methods: IHC using 57 primary antibodies was performed on a TMA paraffin block containing 89 cores of duplicate previously identified 1 mm diameter tissue specimens. IHC was interpreted by a histology scientist with IHC experience, with pathologist assistance if required. Review of the literature was performed to investigate cases of unexpected immunoreactivity. Results: 55 of 57 antibodies had expected positive staining against the TMA tissue panel that correlated with the original paraffin blocks. Immunostaining of duplicate 1 mm cores correlated with the originally sourced paraffin block in 42 of 43 (98%) tissue types. Some antibodies had unexpected positive immunoreactivity. Discussion: TMA technology can be utilised effectively in the diagnostic IHC laboratory as a universal positive multiple tissue control block for routine IHC, and can provide valuable information for the benefit of histology scientists and pathologists with respect to interpretation of IHC staining. Key words: Immunohistochemistry, immunoreactivity, napsin A, quality control, tissue microarray. Received 1 April, revised 19 August, accepted 22 August 2012
INTRODUCTION Tissue microarrays (TMAs) consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in an array fashion to allow multiplex histological analysis. The key benefit of utilising TMA technology is that hundreds of tissue samples can be analysed on one microscope slide. This allows cost savings to research teams by reducing reagent use and the manual labour required to section large numbers of paraffin blocks.1 This technology has been widely used in the research area for a number of years, whilst its use in the diagnostic laboratory is slowly increasing with an example being its use in the Royal College of Pathologists of Australasia (RCPA) External Quality Assurance Program (QAP) immunohistochemistry (IHC) survey testing program. The use of an extensive TMA block for quality control (QC) in the diagnostic histopathology laboratory has been limited, as a small core of tissue is considered not to be as representative as a whole paraffin block. Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0b013e32835b7b99
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In the TMA technique, a hollow needle is used to remove tissue cores as small as 0.6 mm in diameter from regions of interest in paraffin embedded tissues such as clinical biopsies or tumour samples. These tissue cores are then inserted in a recipient paraffin block in a precisely spaced-array pattern. Sections from this block are cut using a microtome, mounted on a glass slide and analysed by any standard histological method. Each TMA block potentially can be cut into hundreds of sections, which can be subjected to independent tests including IHC2 and fluorescence in situ hybridisation (FISH).3 Manufacturer kit inserts for purchased in vitro diagnostic antibodies supplied by several companies include an assessment of cellular and tissue immunoreactivity, but this often does not encompass all of the tissue types encountered in our diagnostic laboratory. There is an onerous requirement for the anatomical pathologist to keep up-to-date with the myriad of immunoreactivity patterns which can occur when primary antibodies are applied to normal and abnormal tissue types. Validating currently used in vitro diagnostic antibodies against a large number of different tissue types can assist the pathologist with interpretation of IHC staining, and provides access slides to examples of unexpected, rarely observed immunoreactivity. The histology scientist trained in IHC methods is responsible for maintaining appropriate QC measures. A skilled, knowledgeable scientist who is able to confidently and accurately assess a large volume of IHC stained slides which are known to contain pathologist-identified areas of interest, is an asset to the laboratory.1 This study explores the hypotheses that TMA technology gained as firsthand experience can be used for (a) retention of antigenicity in 1 mm tissue cores from known tissue paraffin blocks, (b) establishing QC resource information for the diagnostic IHC laboratory, and (c) screening of new clones of primary antibodies clones and comparing with the currently used clones, to continually improve IHC staining. TMA technology could also be used to design a single tissue positive control paraffin block for IHC at the testing laboratory. By using small amounts of control material, there would be a reduced reliance on sourcing a large variety of normal and abnormal tissue, which can be rare, from storage archives. There is also an increasing demand for archived tumour tissue to be sampled for molecular testing requests, for example epidermal growth factor receptor (EGFR) and Kristen rat sarcoma (KRAS) with the aim of patient benefit through new targeted therapy regimens. The histopathology laboratory should not be reliant on sourcing these tumour tissue types for diagnostic IHC control material.
2012 Royal College of Pathologists of Australasia
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
Fallopian tube Stomach Fallopian tube Stomach
ADC, adenocarcinoma; BCL, B-cell lymphoma; Ca, carcinoma; CRC, colorectal carcinoma; EWS, Ewing’s sarcoma; GIST, gastrointestinal stromal tumour; MCL, mantle cell lymphoma; RCC, renal clear cell carcinoma; SCC, squamous cell carcinoma; SCLC, small cell lung cancer; TCL, T-cell lymphoma.
Oesophagus Liver Skin Thymoma Lung normal Skin Thymoma Lung normal Small intestine EWS Vas deferens Small intestine EWS Vas deferens Cervix Teratoma
Ovarian serous Ca Appendix Lymph node Ovarian serous Ca Appendix Lymph node
Cervix Teratoma
Uterus Breast Ca MCL Uterus Breast Ca MCL Heart Lung ADC TCL Heart Lung ADC TCL Placenta Tonsil BCL
Spleen CRC
Breast Mesothelioma (biphasic) Kidney Lung SCC SCLC minimal tissue Small intestine Schwannoma Breast Mesothelioma (biphasic) Kidney Lung SCC SCLC minimal tissue Small intestine Schwannoma Colon Mesothelioma (epithelial) Thyroid GIST Pancreas
Map of paraffin TMA block Table 1
Fig. 1 Example of tissue microarray slides and paraffin TMA block used in this study.
Spleen CRC
Prostate Melanoma
Prostate Melanoma
Testis Ovary
Testis Ovary
Lung inflamed RCC
Tissue control material was obtained from Anatomical Pathology – The Prince Charles Hospital (TPCH) tissue archives. Forty-three samples were sourced from 2007 to 2011 as shown in Supplementary Table 1 (http://links.lww.com/ PAT/A8). All tissue used in this study was formalin fixed, paraffin embedded (FFPE) according to standard protocols employed by our laboratory. Intermediate sample blocks were prepared from 43 tissue types. Each intermediate block contained eight 4 mm cores with duplicate cores from four tissue types. Each intermediate block was sectioned at 4 mm and stained with haematoxylin and eosin (H&E) to identify and mark the area of interest. The tissue composition of the intermediate sample blocks is described further in Supplementary Table 2 (http://links.lww.com/PAT/A8). The intermediate tissue blocks and corresponding marked H&E slides were forwarded to University of Queensland’s Centre for Clinical Research (UQCCR) to prepare the final TMA blocks as follows: two 1 mm cores from each 4 mm tissue sample were taken (exceptions were small intestine sampled into 4 1 mm cores and skin sampled into 3 1 mm cores), and duplicate TMA blocks of 89 1 mm cores were constructed using a manual tissue arrayer (MTA) machine (Beecher instruments, USA). Each paraffin TMA block (Fig. 1) contained adjacent duplicate 1 mm tissue cores from the same intermediate 4 mm tissue core. A detailed TMA map is shown in Table 1. A total of 114 slides containing 3–4 mm sections of a TMA block using the same semi-automated microtome (Leica, Germany) were placed onto positively charged coated slides (SuperFrost Plus; Menzel-Glaser, Germany) containing positive and negative tissue controls. The slides were air-dried and heat-fixed at 608C for 30 min. A pre-soaking method in 2% aqueous Tween-20 (BDH) was used on the TMA paraffin block where microtomy was difficult due to hard or brittle tissue cores. Fifty-seven primary antibodies within the recommended manufacturer’s expiry date were applied against duplicate TMA slides using automated IHC. The dilutions of required primary antibody (0.15 mL on each slide) and heatinduced epitope retrieval methods employed are described in Table 2. Slides were de-waxed and stained using the Bond Max Automated immunostainer (Leica Microsystems) with the Refine Detection Kit DS9800 (Leica; Lot #15283, expiry date 26/02/2013) for in vitro diagnostic use as per the manufacturer’s instructions, with minor modifications. In brief, the 114 slide sections of FFPE tissue were deparaffinised with Bond Dewax Solution (Leica Microsystems). Epitope retrieval was performed using either Enzyme Solution (Leica Microsystems), Bond Epitope Retrieval 1 (pH 6) Solution (Leica Microsystems), or Bond Epitope Retrieval 2 (pH 9) Solution (Leica Microsystems) for varying time periods and temperatures (Table 2). The sections were incubated for 20 min at room temperature with all antibodies, using a biotin-free, polymeric horseradish peroxidase-linker antibody conjugate system on the BondMax Immunostainer. Nuclei were counterstained with haematoxylin. Following staining, slides were dehydrated through graded alcohols, cleared in xylene and mounted in Pertex medium (CellPath, UK) (Fig. 1). Sections were cut and stained on the same day (maximum 30 per day) in batches, over a period of 4 consecutive days. This was done to prevent loss of antigenicity through excessive storage of unstained sectioned slides.
Placenta Tonsil BCL
Lung inflamed RCC
MATERIALS AND METHODS
Oesophagus Liver Skin
Pathology (2013), 45(1), January
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Colon Mesothelioma (epithelial) Thyroid GIST Pancreas
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TISSUE MICROARRAY FOR DIAGNOSTIC IMMUNOHISTOCHEMISTRY
Table 2
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Antibody data summary
Antibody
Clone, manufacturer and product code
Dilution
Retrieval
Control
Staining pattern
Actin (SMA) AE1/3 aFP Bcl-2 Calretinin Cam 5.2 C4D CD 3 CD 5 CD 10 CD 15 CD 20 CD 23 CD 30 CD 31 CD 34 CD 43 CD 45 LCA CD 45 RO CD 56 CD 68 CD 79a CD 99 CD 117 (C-kit) CDX2 CEA Chromogranin CK 5/6 CK 7 CK14 CK18 CK 20 CK 34bE12 CK 34bE12(PIN 4) CMV Cyclin D1 Desmin EBV E-cad EMA ER hCG HMB45 Ki-67 Melan A Napsin A p63 P504S (Racemase) PLAP PR PSA S100 Synaptophysin Thrombomodulin TTF-1 Vimentin WT-1 (6F-H2) WT-1 (WT49)
1A4; Dako; M0851 AE1/AE3; Dako; M3515 Poly; Dako; IS500 Ready To Use 124; Dako; M0887 DAK-Calret 1; Dako; M7245 Cam 5.2; Becton Dickinson; 349205 Poly; Cell Marque; 404A-15 Poly; Dako; A0452 4C7; Leica; NCL-CD5-4C7 C656; Leica; NCL-L-CD10-270 Carb-3; Dako; M3631 L26; Dako; M0755 1B12; Leica; NCL-L-CD23-1B12 Ber-H2; Dako; M0751 JC70A; Dako; M0823 QBEnd/10; Leica; NCL-END DF-T1; Dako; M0786 2B11 þ PD7/26; Dako; M0701 UCHL1; Dako; M0742 123C3; Invitrogen; 180152 PG-M1; Dako; M0876 JCB117; Dako; M7050 12E7; Dako; M3601 Poly; Dako; A4502 DAK-CDX2; Dako; M3636 II-7; Dako; M7072 5H7; Leica; NCL-CHROM-430 D5/16 B4; Dako; M7237 OV-TL 12/30; Dako; M7018 LL002; Leica; NCL-L-LL002 DC10; Dako; M7010 K220.8; Dako; M7019 34bE12; Dako; M0630 34bE12; Dako; M0630 CCH2 þ DDG9; Dako; M0854 SP4; Thermo; RM-9104-S D33; Dako; IS606 Ready To Use CS 1-4; Dako; IS753 Ready To Use NCH-38; Dako; M3612 E29; Dako; M0613 6F11; Leica; NCL-L-ER-6F11 Poly; Dako; IS508 Ready To Use HMB45; Dako; M0634 MIB-1; Dako; M7240 A103; Dako; M7196 Poly; Cell Marque; 352A-75 4A4; Dako; M7247 13H4; Dako; M3616 8A9; Dako; M7191 PgR 636; Dako; M3569 Poly; Dako; A0562 Poly; Dako; Z0311 SY38; Dako; M0776 1009; Dako; M0617 8G7/G3/1; Dako; M3575 V9; Dako; M0725 6F-H2; Dako; M3561 WT49; Leica; PA0562
1/2000 1/100 1/2 1/400 1/200 1/2 1/400 1/150 1/200 1/50 1/100 1/400 1/100 1/50 1/700 1/100 1/200 1/500 1/1000 1/300 1/100 1/500 1/100 1/800 1/50 1/100 1/200 1/50 1/400 1/75 1/200 1/100 1/250 1/300 1/200 1/25 1/4 1/8 1/50 1/1500 1/50 1/4 1/200 1/200 1/200 1/200 1/500 1/200 1/200 1/400 1/20,000 1/2000 1/200 1/75 1/400 1/700 1/400 RTU
ER 1 20 min ER 1 20 min ER 2 20 min ER 2 20 min ER 2 20 min ENZ 1 10 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 1 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 1 20 min ER 1 20 min ER 1 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 30 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 30 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 5 min ER 2 20 min ER 2 5 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min
Heart; uterus Skin; colon; lung Foetal liver Tonsil Testis Colon; lung; liver Allograft rejected kidney Tonsil; appendix Tonsil; appendix Tonsil; breast Hodgkin’s lymphoma Tonsil; appendix Tonsil; appendix Hodgkin’s lymphoma Heart; uterus; skin Heart; uterus; skin Tonsil; appendix Tonsil; appendix Tonsil; appendix Pancreas Lung, liver Tonsil; appendix Ewing’s sarcoma; thymus; liver GIST; skin Colon Colon Pancreas Skin; prostate Lung; liver Skin Liver; colon Colon Normal prostate; skin Normal prostate Infected lung Tonsil; mantle cell lymphoma Heart; uterus Infected tissue Breast carcinoma; cervix Breast Breast; uterus; cervix Placenta Skin; melanoma Lymph node Skin; melanoma Lung Skin; breast; prostate Prostate ADC Placenta Breast; uterus; cervix Prostate Skin; melanoma Pancreas Heart; uterus Thyroid; lung Kidney Fallopian tube; ovary Fallopian tube; ovary
Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic/nuclear Cytoplasmic Vascular membranous Membranous/cytoplasmic Membranous Membranous Membranous/cytoplasmic Membranous Membranous Membranous/cytoplasmic Membranous/cytoplasmic Membranous/cytoplasmic Membranous Membranous Cytoplasmic Cytoplasmic Membranous/cytoplasmic Membranous/cytoplasmic Membranous Membranous/cytoplasmic Nuclear Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic/nuclear Nuclear Cytoplasmic Membranous/cytoplasmic Membranous/cytoplasmic Membranous/cytoplasmic Nuclear Cytoplasmic Cytoplasmic Nuclear Cytoplasmic Cytoplasmic Nuclear Cytoplasmic Cytoplasmic Nuclear Cytoplasmic Cytoplasmic Cytoplasmic Membranous/cytoplasmic Nuclear Cytoplasmic Nuclear / cytoplasmic Nuclear
GIST, gastrointestinal stromal tumour; RTU, ready to use.
The slides were assessed using a light microscope (Olympus BX45) at an initial magnification of 100, with further assessment at 200 if required. Unexpected staining results and/or difficult interpretation were referred to pathologists at TPCH for assistance. Results were recorded as positive (50% of cells), negative, focally positive (<50% of cells) or non-assessable. True positive staining was assessed as having the required cellular staining pattern, i.e., nuclear, cytoplasmic, membranous or a combination of these for the relevant antibody (described in Table 2). With regards to Wilms’ Tumour 1 protein (WT-1), positive IHC staining was assessed as cytoplasmic, nuclear or a combination of both as activated expression occurs
in the nucleus, with cytoplasmic expression present following phosphorylation and deactivation.4 On conclusion of microscopic assessment, a review of the literature including journals, textbooks, antibody marker specification data sheets, IHC quality assurance resources and the PathIQ Immunoquery website (http://www.immuno query.com/) was performed to identify reasons for initially unexpected results. This was defined as staining not described in the manufacturer’s antibody specification data summary provided with purchased antibody concentrate vials. Interesting staining results and patterns were photographed using a microscope digital camera (Olympus DP21; Olympus, Japan).
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Pathology (2013), 45(1), January
RESULTS The sectioning of a TMA paraffin block containing 89 1 mm cores resulted in 2387 of 2451 (43 tissue types 57 antibodies) or 97.4% of tissue IHC staining results being assessable as positive, focally positive or negative. The remaining 64 (2.6%) were non-assessable due to excessive folding or missing sections. This was more prevalent in tissue types with minimal tissue, e.g., small cell lung cancer (SCLC) and T-cell lymphoma. Sections of highly cellular tissues that were less than 50% folded were still able to be accurately assessed (Fig. 2). The entire list of antibodies reacted as expected with known positive and negative tissue controls (Supplementary Table 3, http://links.lww.com/PAT/A8). There were antibodies that reacted with tissue types that were not specified in their respective manufacturer’s product specification data sheet supplied. There were two antibodies that failed to react with any TMA tissue panel section, namely alpha-fetoprotein (AFP) and cytomegalovirus (CMV). Initially, unexpected results were observed with some antibodies. An example was p63, with positive cytoplasmic staining observed within myocardial cells (Fig. 3). Another example was strong staining of mucin within gastrointestinal epithelium with progesterone receptor (PR), including normal colon (Fig. 4), normal small intestine (Fig. 4, inset) and colonic adenocarcinoma (Fig. 5A). Positive cytoplasmic immunoreactivity was observed for PR in lung adenocarcinoma (Fig. 5B). Positive nuclear immunoreactivity was observed for oestrogen receptor (ER) in melanoma (Fig. 6). Another instance of unexpected immunoreactivity was positivity for napsin A in primary colonic adenocarcinoma (Fig. 7A,B). The same case was subsequently stained for thyroid transcription factor (TTF-1) and an alternative napsin A clone IP64 (Leica Microsystems) (Fig. 8) using the same heat-induced epitope retrieval method and dilution as the initial
Fig. 2 Focal AE1/3 immunoreactivity within heart myocardial cells; note folded section.
Fig. 3 Cytoplasmic p63 staining of heart tissue.
polyclonal antibody (Cell Marque, USA). The aim of this was to confirm or remove a possible diagnosis of metastatic pulmonary adenocarcinoma. These subsequent staining results were negative. The only tested tissue sample that strongly reacted with the Epstein–Barr virus latent membrane protein (EBV LMP1) antibody was melanoma (Fig. 9). Our BCL-2 antibody showed immunoreactivity with all of the tissue types. There were multiple tissue types that had a positive cytoplasmic reaction with WT-1 clone 6FH2 (Dako). This cytoplasmic immunoreactivity was not observed when using WT-1 clone WT49 (Leica Microsystems), where nuclear staining was interpreted as positive. The most immunoreactive tissue type with strong positivity encountered in this study was colon (30/57), followed by appendix (24/57) and tonsil (22/57). Forty-two of 43 (98%) of the duplicate tissue core samples achieved expected IHC staining results compared with the original paraffin tissue control block. The exception was appendix, where one of the tissue cores contained epithelial and lymphoid cells (Fig. 10A), whereas the other contained mostly muscle and connective tissue (Fig. 10B).
Fig. 4 Progesterone receptor (PR) staining of mucin in normal colon epithelial mucosa and small intestine (inset).
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TISSUE MICROARRAY FOR DIAGNOSTIC IMMUNOHISTOCHEMISTRY
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Fig. 7 Napsin A (Cell Marque polyclonal) antibody staining colon adenocarcinoma. (A) TMA core. (B) Whole block section.
DISCUSSION Fig. 5 Progesterone receptor (PR) staining. (A) Mucin in colonic adenocarcinoma. (B) Pulmonary adenocarcinoma tumour cells.
The overall cost to complete this study with two TMA blocks and 114 slides was approximately 25 h of manual labour (2 h to source blocks and prepare intermediate blocks; 8 h to construct 2 TMA paraffin blocks; 2 h to section slides; 11 h to assess slides microscopically) and AU$1300 for a Leica Bond Refine Detection Kit (DS9800) capable of staining up to 200 IHC slides. Consumables costs (slides, 0.3 mL of diluted primary antibody) were covered by our laboratory’s cost centre.
Fig. 6 Oestrogen receptor staining of melanoma.
This investigation has shown that comprehensive IHC crossreactivity data of an antibody can be obtained from a single slide, and was achieved with significant savings of cost, labour and time. By choosing TMA technology over sectioning and staining multiple whole block sections, scientists and pathologists within a diagnostic laboratory are able to improve their knowledge and understanding of IHC staining without impacting on routine workload. Future studies with the aim of further validating this data, and continually improving IHC QC in the diagnostic laboratory, would be to include an extended sample number of specific tissue types as a constructed TMA paraffin block. This process would be of particular benefit for optimising IHC staining for
Fig. 8 Napsin A (IP64) colon adenocarcinoma whole block section.
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Fig. 9 EBV LMP1 staining melanoma.
tumours with a broad range of heterogeneity and associated varying levels of antigenic expression, for example ER and PR staining in breast carcinoma5 and napsin A and TTF-1 staining in pulmonary adenocarcinoma.6 Regarding tumour heterogeneity within a single paraffin block, it has been demonstrated that two 0.6 mm cores adequately represent the staining of an entire histological section of
Fig. 10 Appendix duplicate core sampling discrepancy. (A) Epithelium (stained with AE1/AE3) and lymphoid component (stained with haematoxylin) present in TMA core. (B) Connective tissue elements only in adjacent TMA core.
Pathology (2013), 45(1), January
tumour.2,5 However this study focused on one tissue sample as an example of expected antigen-antibody reaction to assess IHC staining of a broad range of different tissue and cellular types on only one stained microscope slide. Future studies would include assessment of additional examples of the tissue types used in this study, with the aim to reproduce cases of identified unexpected immunoreactivity. Tumour heterogeneity and its impact on TMA sampling could be investigated further, with breast carcinoma and pulmonary adenocarcinoma possible tumours to be studied. TMA block section quality is dependent on a higher level of technical skill than of a whole tissue section.1 As described, there were slides that contained some folded (Fig. 1) and/or missing cores. The reasons for this include minimal or no tissue remaining in the paraffin block, or varying amounts of dense connective tissue in the different tissue types which renders microtomy technically difficult. The same scientist used the same semi-automated microtome to cut sections for this study, which guaranteed a consistent section thickness. Fortunately, most of the folded core sections were less than 50% affected, and in these instances a staining result was able to be interpreted confidently. In the routine histology laboratory, it is commonplace to presoak hard or brittle tissue within a paraffin block prior to sectioning. This method did little to improve the section quality in this study. A possible alternative to improve section quality of TMA blocks containing such a large number of cores is to adopt a tape transfer technique.7 This would also eliminate the need for a water bath adjacent to the microtome for floatation of sections. The only tissue sample that strongly reacted with the EBV LMP1 antibody was the melanoma. This was a surprising initial observation, but a review of the literature revealed that up to 90% of melanomas react with this antibody. This immunoreactivity is not related to viral infection, but is due to unidentified protein cross-reactivity.8 In 2001, it was argued that positive staining of melanoma by EBV LMP1 was due to the particular avidin biotin complex (ABC) IHC staining method performed.9 However, this study used a polymeric-based IHC detection system which also demonstrates positive staining, indicating that the ABC IHC staining technique is not responsible for the observed positive immunostaining. For the pathologist to avoid this staining phenomenon it is recommended that appropriate tissue controls and routine antibody panel selection for diagnostic IHC be followed. Napsin A is an aspartic proteinase involved in the maturation of surfactant protein B. It is detected in the cytoplasm of type 2 pneumocytes and alveolar macrophages and is a putative marker for pulmonary adenocarcinomas.10 Many studies have shown that napsin A does not stain non-pulmonary adenocarcinomas11,12 and the PathIQ Immunoquery website (http://www.immunoquery.com/) reports that zero of 28 (0%) colorectal carcinomas were positive for napsin A. The only other known positive IHC staining identified in the literature is expression in renal tubular epithelial cells and most papillary renal carcinomas.11,13 When our laboratory began to optimise Napsin A staining, a section of metastatic colorectal carcinoma was included as a negative control, which was validated in-house. This marker has been in use since 2010 at this laboratory site. Therefore, it was surprising to identify positive staining of primary colonic adenocarcinoma in the TMA with the Cell Marque polyclonal Napsin A. Recently has there been another report of positive staining of two of 95 (2%) cases
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TISSUE MICROARRAY FOR DIAGNOSTIC IMMUNOHISTOCHEMISTRY
of colonic adenocarcinoma using a different clone of napsin A (TMU-Ad02).14 However, the authors dismissed this staining as false positive due to their negative control showing stronger staining than the tumour cells. It is interesting that they used an antibody dilution of 1:100, while the dilution used in the current study was 1:200. The positive cytoplasmic staining pattern (Fig. 7A,B) is reproducible and not artefactual. Recently we have seen a liver core biopsy case with metastatic colorectal adenocarcinoma that has stained with a similar pattern. These staining results and the limited studies to date, indicate that more investigation into napsin A IHC and colonic adenocarcinoma is required to provide increased reliability of antibody staining performance. Our laboratory recently purchased a newly available alternative napsin A clone IP64 (Leica Microsystems). Initial titrating and optimising of this antibody clone mirrored that of the Cell Marque polyclonal antibody, with a dilution of 1/200 with heatinduced epitope retrieval of ER2 20 min at 1008C. The case of primary colon adenocarcinoma that was used in the TMA staining experiments was stained as a whole block section in parallel with both the new napsin A IP64 clone and the polyclonal Cell Marque antibody. The section stained with the Cell Marque polyclonal antibody stained as per the TMA result, while the section did not stain with the IP64 monoclonal antibody. This result brings to question the validity of antibody clones that are distributed by manufacturers and marketed as highly sensitive, specific markers for IHC. The manufacturer of the polyclonal napsin A has been contacted for comment regarding the unexpected observed staining patterns. The above finding indicates that unless an adequate antibody marker selection panel is used for IHC, the possibility of a false positive diagnosis of pulmonary adenocarcinoma is increased, especially on small biopsy and cell block material. As always recommended, IHC is a valuable tool for pathologists and correlation with clinicopathological findings is most important to achieve the correct diagnosis. It is noted that a large percentage of the unexpected immunoreactivity observed in this study are unlikely to be encountered by the clinical pathologist during routine IHC testing. As an example, EBV LMP1 IHC would not usually be requested for a case suspected to be malignant melanoma. The heart tissue used in this study was sent to our histology laboratory for examination for pathology that may contraindicate donation and was assessed as being within normal limits. This tissue demonstrated the presence of a lipofuscin-type pigment within the myocardial cells on H&E staining, but this was the not the cause of the positive cytoplasmic staining with p63 (Fig. 3). This antibody is used in our laboratory as a nuclear staining marker for proliferative epithelial cells in squamous cell carcinomas and aids in the diagnosis of prostate adenocarcinoma. The literature review initially performed into p63 could not locate any reference to heart cytoplasmic p63 staining, but it was found that heart-derived mesenchymal cells can support epidermal regeneration in keratinocytes, and if these cells are vimentin positive, also express some cytokeratins (CK) antibody markers and p63.15 Only recently has there been a report of cytoplasmic p63 staining in normal myocardium as well as rhabdomyosarcomas and rhabdomyomas.16 The results in our study support these recent findings and that p63 is involved in cardiogenesis17 and is a sensitive marker for skeletal muscle differentiation. Cells containing mucin are also reported to have stained positively for PR.18 This marker is known to bind to an epitope
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on the O-acetylated sialomucin molecule, and is specific to normal colonic epithelial mucosa.19 It is important to note that this staining pattern is not cellular, and should be regarded as an artefact of IHC staining and not as true positive PR expression. Positive nuclear IHC staining with ER in melanoma and cytoplasmic PR staining in pulmonary adenocarcinoma is not a novel finding.20,21 However, these particular results serve as a reminder to scientists and pathologists about another interpretive pitfall of IHC. With WT-1, nuclear staining in the absence of cytoplasmic staining was observed using the clone WT49 (Leica Microsystems). This clone has been the most commonly requested WT-1 marker in this laboratory by pathologists, due in part to instances of the 6F-H2 clone non-specifically staining connective tissue cytoplasm in patient tumour biopsy specimens. The range of immunoreactivity observed with our BCL-2 antibody and the tissue cores in the TMA block was expected.22 This marker is used primarily in our laboratory as a lymphoma marker in establishing a preliminary diagnosis, so it was initially surprising to our scientists and pathologists to observe cross-immunoreactivity on such a large scale. C4d IHC staining is performed by our laboratory when assessing patient heart biopsies post-transplant with a negative histological diagnosis of cellular rejection. This antibody positively stains in cardiothoracic tissue venules and arterioles as a result of perioperative activation of the complement system.23 Positive capillary staining is regarded as an indication of cardiac antibody-mediated rejection (AMR), with positive venule and arteriole staining considered as an excellent internal control for assessing the quality and intensity of C4d IHC staining.24 In our laboratory, lung endothelium would be an appropriate control for C4d IHC staining. This staining pattern was also observed in other tissue vascular endothelium (colon, prostate and appendix). The author hypothesises that perioperative complement activation is the reason for this staining reaction, and its presence is made aware to the scientist and pathologist performing IHC quality control. Further investigation into the observed unexpected immunoreactivity results would be to adjust heat-induced epitope retrieval methods and antibody dilutions. It was decided not to proceed with this, as these results are indicative of staining results that could be encountered by our laboratory when performing diagnostic IHC. Selecting initial 4 mm tissue cores from whole paraffin blocks ensured that sampling of smaller 1 mm cores contained sufficient representative tissue for IHC staining. This is the first described instance of performing intermediate sampling prior to construction of a TMA block. In this study, there was only one instance of sampling discrepancy (appendix) where the duplicate tissue cores in the same block did not have a similar representative architecture (Fig. 10A,B). Future investigations From the results of this exercise, it is proposed that a universal control block be used at our laboratory for daily IHC QC. This block would contain nine tissue types consisting of placenta, tonsil, appendix, colon, melanoma, lung, testis, cervix and prostate. These would be included in a control block arranged as 3 mm cores in three rows of three (Fig. 11) and could be constructed manually. By adopting a tissue control block with these tissue types, 55 of 57 antibodies used in our laboratory will stain positively, with most antibodies having at least two
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Fig. 11 Proposed ‘universal’ TMA block for routine IHC quality control containing 9 3 mm diameter tissue cores.
positive and two negative tissue control sections (Supplementary Tables 4 and 5, http://links.lww.com/PAT/A8). Using 3 mm cores will improve the quality of microtomy and also reliability of sampling tissue with multiple cellular elements required for consistent IHC staining performance. For example, appendix and tonsil contains both epithelium and lymphoid cells. Pre-cut 3–4 mm sections of the universal control block could be stored in slide boxes at room temperature. It is our laboratory’s routine that freshly cut control sections are used when staining six of our antibodies (Ki-67, ER, PR, WT-1, cyclin-D1, and TTF-1). This is done to prevent loss of immunoreactivity due to variables such as oxidation and ultraviolet light exposure that has been documented,25 and should continue as a standard method at this laboratory. IHC staining in our laboratory routinely contains a positive and negative tissue control on the same slide as the tested section. The universal control block proposed in this study will not change this routine procedure, despite containing nine tissue cores. By having a control section on the same slide as the test section, reproducibility and repeatability of staining performance is maintained. In rare instances where the test section and control section cannot be placed on the same slide, then they should be stained on separate adjacent slides and stained in parallel. Melanoma would be the only tumour tissue included in the universal control. This means that for our laboratory to adopt the universal tissue block as its control for IHC, only one tumour type is to be continually sourced. This provides the laboratory benefit in not relying on sourcing rare tumour tissue as IHC control material, as there is an increasing number of molecular profiling requests (e.g., EGFR and KRAS) on archived tumour tissue being performed to potentially improve patient management through targeted therapies. The use of melanoma as the only tumour tissue control for IHC avoids this sourcing issue to a degree. Our laboratory is situated within the population with the highest worldwide incidence of this disease26 and minimal pathologist permitted sampling of archived
Pathology (2013), 45(1), January
melanoma tissue would not compromise any future molecular profiling requests. This is the first report to recommend melanoma tissue as a control for EBV LMP1 IHC staining. However, appropriate quality control measures are required to validate this method. It is suggested that any primary and/or metastatic melanoma tumour tissue to be incorporated in the proposed universal control block is first immunostained with EBV LMP1, as 10% of cases are documented to negatively stain.8 The use of a TMA slide containing known EBV infected tissue as a validation tool would be useful in instances where melanoma control tissue negatively stains with this antibody. In this instance, EBV infected tissue would be used as a separate control for EBV LMP1 IHC staining. Eight of the nine tissue types to be included in the universal tissue control block are normal. This is ideal as normal tissue contains consistent antigenic expression levels compared to heterogeneous tumour tissue. In the past, tumour tissue has been used as IHC control tissue in our laboratory, but heterogeneity of tumour subtypes removes the consistency of antigenic expression. Having this consistent antigenicity using normal tissue will allow for reliability of IHC staining performance through standardised antibody optimisation. For instances where antibody performance requires troubleshooting due to poor performance or possible antibody crosscontamination and for optimising new and existing antibodies when new batches are received, it is proposed that a TMA block similar to that used in this study be utilised for antibody titration for diagnostic IHC validation. Other tissue types that are encountered more commonly by the laboratory could also be added into the TMA block to produce further IHC immunoreactivity data. When recording and comparing IHC staining results for standardisation between multiple observers, there is an argument for utilising a whole slide digital scanner. These instruments are an ideal alternative to manual IHC scoring as they are able to provide a clear image of a freshly stained section that will not fade, which can be observed when staining results of individual slides are performed by multiple observers over a period of time. An example of this is when slides are exposed to UV light, the intensity of DAB staining may be reduced. The other advantage of adopting this instrument is that the same scanned image can be scored by multiple observers simultaneously, saving time. This study was based on the observations of primarily one scientist, and the use of a whole slide digital scanner would have enabled multiple observers to record the staining results of this project. At present it is not an economically viable option to purchase such an instrument for many pathology laboratories (including ours), but in the future this would be a valuable tool for IHC staining standardisation and validation between laboratory sites. The proposed universal TMA block is based on the interpretation of one observer, a histology scientist with extensive experience in diagnostic IHC. On-site pathologist assistance was sought when the observer encountered unexpected staining patterns. The results were not audited externally, which justifiably raises some concern about certain recommendations; for example, the suggested controls for C4d and EBV LMP1 staining. This study recommends that the proposed universal control block be adopted by our laboratory site initially, with future studies involving the collaboration of external laboratories and the RCPA QAP to follow before other laboratories utilise this diagnostic IHC tool.
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TISSUE MICROARRAY FOR DIAGNOSTIC IMMUNOHISTOCHEMISTRY
Financial considerations The overall cost to complete this study with two TMA blocks and 114 slides was approximately 25 h of manual labour and AU$1300 for a Leica Bond Refine Detection Kit (DS9800) capable of staining up to 200 IHC slides. Consumables costs (slides, 0.3 mL of diluted primary antibody) were covered by our laboratory’s cost centre. The alternative method would be to section and stain 43 whole paraffin block sections in duplicate against 57 antibodies. This would mean that 4902 slides would require increased manual labour and 24 Bond Refine Detection Kits at a cost of AU$31 200. The initial outlay to purchase a manual tissue microarrayer machine capable of constructing a block containing hundreds of tissue cores (AU$17 000) would be saved through reducing labour and time for the IHC scientist and other laboratory staff to source tissue control material and maintaining a large number of catalogued control blocks and storage of pre-cut sections. Alternatively, there are disposable, partly constructed TMA blocks (approximately AU$285 per paraffin block) available for purchase. These still require manual insertion of tissue cores before laboratory use, and would not be more cost-effective long-term as a manual tissue microarrayer machine.
CONCLUSION This study has established preliminary data of immunoreactivity between a large range of tissue types and antibody markers used at this laboratory, whilst reminding pathologists and histology scientists that IHC is a tool that can be used to assist diagnosis. TMA technology has proven to be a cost-effective, comprehensive and efficient screening tool in the diagnostic IHC laboratory and is now used by external QAP organisations for inter-laboratory assessments of IHC and ISH techniques Managers of pathology laboratories focus on minimising expenditure to remain viable in a competitive, highly regulated industry while retaining skilled scientific and technical staff through providing quality educational resources. TMA technology in the diagnostic IHC laboratory fulfils both of these criteria. Acknowledgements: The author would like to thank the pathologists, scientists and all staff of the Department of Anatomical Pathology, Pathology Queensland – The Prince Charles Hospital for their assistance, especially Dr Belinda Clarke and Dr Kayla Tran for peer review. Thanks also to Ms Annette Lane from UQCCR for construction of the TMA blocks. Conflicts of interest and sources of funding: Financial support was provided by the Pathology Queensland Study, Education and Research Trust Fund. The author states that there are no conflicts of interest to disclose. Address for correspondence: Mr A. van Zwieten, Department of Anatomical Pathology, Pathology Queensland - The Prince Charles Hospital, Rode Road, Chermside, Qld 4032, Australia. E-mail: [email protected]. gov.au
References 1. Camp RL, Neumeister V, Rimm DL. A decade of tissue microarrays: Progress in the discovery and validation of cancer biomarkers. J Clin Oncol 2008; 26: 5630–7.
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2. Torhorst J, Bucher C, Kononen J, et al. Tissue microarrays for rapid linking of molecular changes to clinical endpoints. Am J Pathol 2001; 159: 2249– 56. 3. Diaz LK, Gupta R, Kidwai N, Sneige N, Wiley EL. The use of TMA for interlaboratory validation of FISH testing for detection of HER2 gene amplification in breast cancer. J Histochem Cytochem 2004; 52: 501–7. 4. Kumar-Singh S, Segers K, Rodeck U, et al. WT1 mutation in malignant mesothelioma and WT1 immunoreactivity in relation to p53 and growth factor receptor expression, cell-type transition, and prognosis. J Pathol 1997; 181: 67–74. 5. Camp RL, Charette LA, Rimm DL. Validation of tissue microarray technology in breast carcinoma. Lab Invest 2000; 80: 1943–9. 6. Stoll LM, Johnson MW, Gabrielson E, Askin F, Clark DP, Li QK. The utility of napsin-A in the identification of primary and metastatic lung adenocarcinoma among cytologically poorly differentiated carcinomas. Cancer Cytopathol 2010; 118: 441–9. 7. Rimm DL, Camp RL, Charette LA, Olsen DA. Provost E Amplification of tissue by construction of tissue microarrays. Exp Mol Pathol 2001; 70: 255–64. 8. Bertalot G, Biasi MO, Gramegna M, et al. Immunoreactivity for latent membrane protein 1 of Epstein-Barr virus in nevi and melanomas is not related to the viral infection. Virchows Arch 2000; 436: 553–9. 9. Van den Oord JJ, Delabie J. Binding of rabbit immunoglobulins to melanoma cells: a pitfall in the immunohistochemical study of malignant melanoma. Virchows Arch 2001; 438: 421–3. 10. Chuman Y, Bergman A, Ueno T, et al. Napsin A, a member of the aspartic protease family, is abundantly expressed in normal lung and kidney tissue and is expressed in lung adenocarcinomas. FEBS Lett 1999; 462: 129–34. 11. Schauer-Vukasinovic V, Bur D, Kling et al. Human napsin A. Expression, immunochemical detection, and tissue localization. FEBS Lett 1999; 462:135–139. 12. Bishop JA, Sharma R, Illei PB. Napsin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Hum Pathol 2010; 41: 20–5. 13. Kim JH, Kim YS, Choi YD, et al. Utility of napsin A and thyroid transcription factor 1 in differentiating metastatic pulmonary from non-pulmonary adenocarcinoma in pleural effusion. Acta Cytol 2011; 55: 266–70. 14. Turner BM, Cagle PT, Sainz IM, Fukuoka J, Shen SS, Jagirdar J. Napsin a, a new marker for lung adenocarcinoma, is complementary and more sensitive and specific than thyroid transcription factor 1 in the differential diagnosis of primary pulmonary carcinoma: evaluation of 1674 cases by tissue microarray. Arch Pathol Lab Med 2012; 136: 163–71. 15. Aoki S, Takezawa T, Uchihashi K, Sugihara H, Toda S. Non-skin mesenchymal cell types support epidermal regeneration in a mesenchymal stem cell or myofibroblast phenotype-independent manner. Pathol Int 2009; 59: 368–75. 16. Martin SE, Temm CJ, Goheen MP, Ulbright TM, Hattab EM. Cytoplasmic p63 immunohistochemistry is a useful marker for muscle differentiation: an immunohistochemical and immunoelectron microscopic study. Mod Pathol 2011; 24: 1320–6. 17. Rouleau M, Medawar A, Hamon L, et al. TAp63 is important for cardiac differentiation of embryonic stem cells and heart development. Stem Cells 2011; 29: 1672–83. 18. Shepherd NA, Healey CJ, Warren BF, Richman PI, Thomson WH, Wilkinson SP. Distribution of mucosal pathology and an assessment of colonic phenotypic change in the pelvic ileal reservoir. Gut 1993; 34: 101–5. 19. Richman PI, Bodmer WF. Monoclonal antibodies to human colorectal epithelium: markers for differentiation and tumour characterization. Int J Cancer 1987; 39: 317–28. 20. de Giorgi V, Mavilia C, Massi D, et al. Estrogen receptor expression in cutaneous melanoma: a real-time reverse transcriptase-polymerase chain reaction and immunohistochemical study. Arch Dermatol 2009; 145: 30–6. 21. Ollayos CW, Riordan GP, Rushin JM. Estrogen receptor detection in paraffin sections of adenocarcinoma of the colon, pancreas, and lung. Arch Pathol Lab Med 1994; 118: 630–2. 22. Hockenbemy DM, Zutter M, Hickey W, Nahm M, Korsemeyer SJ. bcl-2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci USA 1991; 88: 699l. 23. Kirklin JK, Westaby S, Blackstone EH, Kirklin JW, Chenoweth DE, Pacifico AD. Complement and the damaging effects of cardiopulmonary bypass. J Thorac Cardiovasc Surg 1983; 86: 845–57. 24. Berry GJ, Angelini A, Burke MM, et al. The ISHLT working formulation for pathologic diagnosis of antibody-mediated rejection in heart transplantation: evolution and current status (2005–2011). J Heart Lung Transplant 2011; 30: 601–11. 25. Blind C, Koepenik A, Pacyna-Gengelbach M, et al. Antigenicity testing by immunohistochemistry after tissue oxidation. J Clin Pathol 2008; 61: 79–83. 26. Baxter AJ, Hughes MC, Kvaskoff M, et al. The Queensland study of melanoma: environmental and genetic associations (Q-MEGA); study design, baseline characteristics, and repeatability of phenotype and sun exposure measures. Twin Res Hum Genet 2008; 11: 183–96.
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TECHNICAL REPORT
Tissue microarray technology and findings for diagnostic immunohistochemistry ANTHONY
VAN
ZWIETEN
Department of Anatomical Pathology, Pathology Queensland – The Prince Charles Hospital, Chermside, Queensland, Australia
Summary Aims: This study was undertaken to determine the validity and viability of tissue microarray (TMA) technology in assessing diagnostic immunohistochemistry (IHC) at a single laboratory site. Methods: IHC using 57 primary antibodies was performed on a TMA paraffin block containing 89 cores of duplicate previously identified 1 mm diameter tissue specimens. IHC was interpreted by a histology scientist with IHC experience, with pathologist assistance if required. Review of the literature was performed to investigate cases of unexpected immunoreactivity. Results: 55 of 57 antibodies had expected positive staining against the TMA tissue panel that correlated with the original paraffin blocks. Immunostaining of duplicate 1 mm cores correlated with the originally sourced paraffin block in 42 of 43 (98%) tissue types. Some antibodies had unexpected positive immunoreactivity. Discussion: TMA technology can be utilised effectively in the diagnostic IHC laboratory as a universal positive multiple tissue control block for routine IHC, and can provide valuable information for the benefit of histology scientists and pathologists with respect to interpretation of IHC staining. Key words: Immunohistochemistry, immunoreactivity, napsin A, quality control, tissue microarray. Received 1 April, revised 19 August, accepted 22 August 2012
INTRODUCTION Tissue microarrays (TMAs) consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in an array fashion to allow multiplex histological analysis. The key benefit of utilising TMA technology is that hundreds of tissue samples can be analysed on one microscope slide. This allows cost savings to research teams by reducing reagent use and the manual labour required to section large numbers of paraffin blocks.1 This technology has been widely used in the research area for a number of years, whilst its use in the diagnostic laboratory is slowly increasing with an example being its use in the Royal College of Pathologists of Australasia (RCPA) External Quality Assurance Program (QAP) immunohistochemistry (IHC) survey testing program. The use of an extensive TMA block for quality control (QC) in the diagnostic histopathology laboratory has been limited, as a small core of tissue is considered not to be as representative as a whole paraffin block. Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0b013e32835b7b99
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In the TMA technique, a hollow needle is used to remove tissue cores as small as 0.6 mm in diameter from regions of interest in paraffin embedded tissues such as clinical biopsies or tumour samples. These tissue cores are then inserted in a recipient paraffin block in a precisely spaced-array pattern. Sections from this block are cut using a microtome, mounted on a glass slide and analysed by any standard histological method. Each TMA block potentially can be cut into hundreds of sections, which can be subjected to independent tests including IHC2 and fluorescence in situ hybridisation (FISH).3 Manufacturer kit inserts for purchased in vitro diagnostic antibodies supplied by several companies include an assessment of cellular and tissue immunoreactivity, but this often does not encompass all of the tissue types encountered in our diagnostic laboratory. There is an onerous requirement for the anatomical pathologist to keep up-to-date with the myriad of immunoreactivity patterns which can occur when primary antibodies are applied to normal and abnormal tissue types. Validating currently used in vitro diagnostic antibodies against a large number of different tissue types can assist the pathologist with interpretation of IHC staining, and provides access slides to examples of unexpected, rarely observed immunoreactivity. The histology scientist trained in IHC methods is responsible for maintaining appropriate QC measures. A skilled, knowledgeable scientist who is able to confidently and accurately assess a large volume of IHC stained slides which are known to contain pathologist-identified areas of interest, is an asset to the laboratory.1 This study explores the hypotheses that TMA technology gained as firsthand experience can be used for (a) retention of antigenicity in 1 mm tissue cores from known tissue paraffin blocks, (b) establishing QC resource information for the diagnostic IHC laboratory, and (c) screening of new clones of primary antibodies clones and comparing with the currently used clones, to continually improve IHC staining. TMA technology could also be used to design a single tissue positive control paraffin block for IHC at the testing laboratory. By using small amounts of control material, there would be a reduced reliance on sourcing a large variety of normal and abnormal tissue, which can be rare, from storage archives. There is also an increasing demand for archived tumour tissue to be sampled for molecular testing requests, for example epidermal growth factor receptor (EGFR) and Kristen rat sarcoma (KRAS) with the aim of patient benefit through new targeted therapy regimens. The histopathology laboratory should not be reliant on sourcing these tumour tissue types for diagnostic IHC control material.
2012 Royal College of Pathologists of Australasia
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Fallopian tube Stomach Fallopian tube Stomach
ADC, adenocarcinoma; BCL, B-cell lymphoma; Ca, carcinoma; CRC, colorectal carcinoma; EWS, Ewing’s sarcoma; GIST, gastrointestinal stromal tumour; MCL, mantle cell lymphoma; RCC, renal clear cell carcinoma; SCC, squamous cell carcinoma; SCLC, small cell lung cancer; TCL, T-cell lymphoma.
Oesophagus Liver Skin Thymoma Lung normal Skin Thymoma Lung normal Small intestine EWS Vas deferens Small intestine EWS Vas deferens Cervix Teratoma
Ovarian serous Ca Appendix Lymph node Ovarian serous Ca Appendix Lymph node
Cervix Teratoma
Uterus Breast Ca MCL Uterus Breast Ca MCL Heart Lung ADC TCL Heart Lung ADC TCL Placenta Tonsil BCL
Spleen CRC
Breast Mesothelioma (biphasic) Kidney Lung SCC SCLC minimal tissue Small intestine Schwannoma Breast Mesothelioma (biphasic) Kidney Lung SCC SCLC minimal tissue Small intestine Schwannoma Colon Mesothelioma (epithelial) Thyroid GIST Pancreas
Map of paraffin TMA block Table 1
Fig. 1 Example of tissue microarray slides and paraffin TMA block used in this study.
Spleen CRC
Prostate Melanoma
Prostate Melanoma
Testis Ovary
Testis Ovary
Lung inflamed RCC
Tissue control material was obtained from Anatomical Pathology – The Prince Charles Hospital (TPCH) tissue archives. Forty-three samples were sourced from 2007 to 2011 as shown in Supplementary Table 1 (http://links.lww.com/ PAT/A8). All tissue used in this study was formalin fixed, paraffin embedded (FFPE) according to standard protocols employed by our laboratory. Intermediate sample blocks were prepared from 43 tissue types. Each intermediate block contained eight 4 mm cores with duplicate cores from four tissue types. Each intermediate block was sectioned at 4 mm and stained with haematoxylin and eosin (H&E) to identify and mark the area of interest. The tissue composition of the intermediate sample blocks is described further in Supplementary Table 2 (http://links.lww.com/PAT/A8). The intermediate tissue blocks and corresponding marked H&E slides were forwarded to University of Queensland’s Centre for Clinical Research (UQCCR) to prepare the final TMA blocks as follows: two 1 mm cores from each 4 mm tissue sample were taken (exceptions were small intestine sampled into 4 1 mm cores and skin sampled into 3 1 mm cores), and duplicate TMA blocks of 89 1 mm cores were constructed using a manual tissue arrayer (MTA) machine (Beecher instruments, USA). Each paraffin TMA block (Fig. 1) contained adjacent duplicate 1 mm tissue cores from the same intermediate 4 mm tissue core. A detailed TMA map is shown in Table 1. A total of 114 slides containing 3–4 mm sections of a TMA block using the same semi-automated microtome (Leica, Germany) were placed onto positively charged coated slides (SuperFrost Plus; Menzel-Glaser, Germany) containing positive and negative tissue controls. The slides were air-dried and heat-fixed at 608C for 30 min. A pre-soaking method in 2% aqueous Tween-20 (BDH) was used on the TMA paraffin block where microtomy was difficult due to hard or brittle tissue cores. Fifty-seven primary antibodies within the recommended manufacturer’s expiry date were applied against duplicate TMA slides using automated IHC. The dilutions of required primary antibody (0.15 mL on each slide) and heatinduced epitope retrieval methods employed are described in Table 2. Slides were de-waxed and stained using the Bond Max Automated immunostainer (Leica Microsystems) with the Refine Detection Kit DS9800 (Leica; Lot #15283, expiry date 26/02/2013) for in vitro diagnostic use as per the manufacturer’s instructions, with minor modifications. In brief, the 114 slide sections of FFPE tissue were deparaffinised with Bond Dewax Solution (Leica Microsystems). Epitope retrieval was performed using either Enzyme Solution (Leica Microsystems), Bond Epitope Retrieval 1 (pH 6) Solution (Leica Microsystems), or Bond Epitope Retrieval 2 (pH 9) Solution (Leica Microsystems) for varying time periods and temperatures (Table 2). The sections were incubated for 20 min at room temperature with all antibodies, using a biotin-free, polymeric horseradish peroxidase-linker antibody conjugate system on the BondMax Immunostainer. Nuclei were counterstained with haematoxylin. Following staining, slides were dehydrated through graded alcohols, cleared in xylene and mounted in Pertex medium (CellPath, UK) (Fig. 1). Sections were cut and stained on the same day (maximum 30 per day) in batches, over a period of 4 consecutive days. This was done to prevent loss of antigenicity through excessive storage of unstained sectioned slides.
Placenta Tonsil BCL
Lung inflamed RCC
MATERIALS AND METHODS
Oesophagus Liver Skin
Pathology (2013), 45(1), January
VAN ZWIETEN
Colon Mesothelioma (epithelial) Thyroid GIST Pancreas
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TISSUE MICROARRAY FOR DIAGNOSTIC IMMUNOHISTOCHEMISTRY
Table 2
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Antibody data summary
Antibody
Clone, manufacturer and product code
Dilution
Retrieval
Control
Staining pattern
Actin (SMA) AE1/3 aFP Bcl-2 Calretinin Cam 5.2 C4D CD 3 CD 5 CD 10 CD 15 CD 20 CD 23 CD 30 CD 31 CD 34 CD 43 CD 45 LCA CD 45 RO CD 56 CD 68 CD 79a CD 99 CD 117 (C-kit) CDX2 CEA Chromogranin CK 5/6 CK 7 CK14 CK18 CK 20 CK 34bE12 CK 34bE12(PIN 4) CMV Cyclin D1 Desmin EBV E-cad EMA ER hCG HMB45 Ki-67 Melan A Napsin A p63 P504S (Racemase) PLAP PR PSA S100 Synaptophysin Thrombomodulin TTF-1 Vimentin WT-1 (6F-H2) WT-1 (WT49)
1A4; Dako; M0851 AE1/AE3; Dako; M3515 Poly; Dako; IS500 Ready To Use 124; Dako; M0887 DAK-Calret 1; Dako; M7245 Cam 5.2; Becton Dickinson; 349205 Poly; Cell Marque; 404A-15 Poly; Dako; A0452 4C7; Leica; NCL-CD5-4C7 C656; Leica; NCL-L-CD10-270 Carb-3; Dako; M3631 L26; Dako; M0755 1B12; Leica; NCL-L-CD23-1B12 Ber-H2; Dako; M0751 JC70A; Dako; M0823 QBEnd/10; Leica; NCL-END DF-T1; Dako; M0786 2B11 þ PD7/26; Dako; M0701 UCHL1; Dako; M0742 123C3; Invitrogen; 180152 PG-M1; Dako; M0876 JCB117; Dako; M7050 12E7; Dako; M3601 Poly; Dako; A4502 DAK-CDX2; Dako; M3636 II-7; Dako; M7072 5H7; Leica; NCL-CHROM-430 D5/16 B4; Dako; M7237 OV-TL 12/30; Dako; M7018 LL002; Leica; NCL-L-LL002 DC10; Dako; M7010 K220.8; Dako; M7019 34bE12; Dako; M0630 34bE12; Dako; M0630 CCH2 þ DDG9; Dako; M0854 SP4; Thermo; RM-9104-S D33; Dako; IS606 Ready To Use CS 1-4; Dako; IS753 Ready To Use NCH-38; Dako; M3612 E29; Dako; M0613 6F11; Leica; NCL-L-ER-6F11 Poly; Dako; IS508 Ready To Use HMB45; Dako; M0634 MIB-1; Dako; M7240 A103; Dako; M7196 Poly; Cell Marque; 352A-75 4A4; Dako; M7247 13H4; Dako; M3616 8A9; Dako; M7191 PgR 636; Dako; M3569 Poly; Dako; A0562 Poly; Dako; Z0311 SY38; Dako; M0776 1009; Dako; M0617 8G7/G3/1; Dako; M3575 V9; Dako; M0725 6F-H2; Dako; M3561 WT49; Leica; PA0562
1/2000 1/100 1/2 1/400 1/200 1/2 1/400 1/150 1/200 1/50 1/100 1/400 1/100 1/50 1/700 1/100 1/200 1/500 1/1000 1/300 1/100 1/500 1/100 1/800 1/50 1/100 1/200 1/50 1/400 1/75 1/200 1/100 1/250 1/300 1/200 1/25 1/4 1/8 1/50 1/1500 1/50 1/4 1/200 1/200 1/200 1/200 1/500 1/200 1/200 1/400 1/20,000 1/2000 1/200 1/75 1/400 1/700 1/400 RTU
ER 1 20 min ER 1 20 min ER 2 20 min ER 2 20 min ER 2 20 min ENZ 1 10 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 1 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 1 20 min ER 1 20 min ER 1 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 30 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 30 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 5 min ER 2 20 min ER 2 5 min ER 2 20 min ER 2 20 min ER 2 20 min ER 2 20 min
Heart; uterus Skin; colon; lung Foetal liver Tonsil Testis Colon; lung; liver Allograft rejected kidney Tonsil; appendix Tonsil; appendix Tonsil; breast Hodgkin’s lymphoma Tonsil; appendix Tonsil; appendix Hodgkin’s lymphoma Heart; uterus; skin Heart; uterus; skin Tonsil; appendix Tonsil; appendix Tonsil; appendix Pancreas Lung, liver Tonsil; appendix Ewing’s sarcoma; thymus; liver GIST; skin Colon Colon Pancreas Skin; prostate Lung; liver Skin Liver; colon Colon Normal prostate; skin Normal prostate Infected lung Tonsil; mantle cell lymphoma Heart; uterus Infected tissue Breast carcinoma; cervix Breast Breast; uterus; cervix Placenta Skin; melanoma Lymph node Skin; melanoma Lung Skin; breast; prostate Prostate ADC Placenta Breast; uterus; cervix Prostate Skin; melanoma Pancreas Heart; uterus Thyroid; lung Kidney Fallopian tube; ovary Fallopian tube; ovary
Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic/nuclear Cytoplasmic Vascular membranous Membranous/cytoplasmic Membranous Membranous Membranous/cytoplasmic Membranous Membranous Membranous/cytoplasmic Membranous/cytoplasmic Membranous/cytoplasmic Membranous Membranous Cytoplasmic Cytoplasmic Membranous/cytoplasmic Membranous/cytoplasmic Membranous Membranous/cytoplasmic Nuclear Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic/nuclear Nuclear Cytoplasmic Membranous/cytoplasmic Membranous/cytoplasmic Membranous/cytoplasmic Nuclear Cytoplasmic Cytoplasmic Nuclear Cytoplasmic Cytoplasmic Nuclear Cytoplasmic Cytoplasmic Nuclear Cytoplasmic Cytoplasmic Cytoplasmic Membranous/cytoplasmic Nuclear Cytoplasmic Nuclear / cytoplasmic Nuclear
GIST, gastrointestinal stromal tumour; RTU, ready to use.
The slides were assessed using a light microscope (Olympus BX45) at an initial magnification of 100, with further assessment at 200 if required. Unexpected staining results and/or difficult interpretation were referred to pathologists at TPCH for assistance. Results were recorded as positive (50% of cells), negative, focally positive (<50% of cells) or non-assessable. True positive staining was assessed as having the required cellular staining pattern, i.e., nuclear, cytoplasmic, membranous or a combination of these for the relevant antibody (described in Table 2). With regards to Wilms’ Tumour 1 protein (WT-1), positive IHC staining was assessed as cytoplasmic, nuclear or a combination of both as activated expression occurs
in the nucleus, with cytoplasmic expression present following phosphorylation and deactivation.4 On conclusion of microscopic assessment, a review of the literature including journals, textbooks, antibody marker specification data sheets, IHC quality assurance resources and the PathIQ Immunoquery website (http://www.immuno query.com/) was performed to identify reasons for initially unexpected results. This was defined as staining not described in the manufacturer’s antibody specification data summary provided with purchased antibody concentrate vials. Interesting staining results and patterns were photographed using a microscope digital camera (Olympus DP21; Olympus, Japan).
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VAN ZWIETEN
Pathology (2013), 45(1), January
RESULTS The sectioning of a TMA paraffin block containing 89 1 mm cores resulted in 2387 of 2451 (43 tissue types 57 antibodies) or 97.4% of tissue IHC staining results being assessable as positive, focally positive or negative. The remaining 64 (2.6%) were non-assessable due to excessive folding or missing sections. This was more prevalent in tissue types with minimal tissue, e.g., small cell lung cancer (SCLC) and T-cell lymphoma. Sections of highly cellular tissues that were less than 50% folded were still able to be accurately assessed (Fig. 2). The entire list of antibodies reacted as expected with known positive and negative tissue controls (Supplementary Table 3, http://links.lww.com/PAT/A8). There were antibodies that reacted with tissue types that were not specified in their respective manufacturer’s product specification data sheet supplied. There were two antibodies that failed to react with any TMA tissue panel section, namely alpha-fetoprotein (AFP) and cytomegalovirus (CMV). Initially, unexpected results were observed with some antibodies. An example was p63, with positive cytoplasmic staining observed within myocardial cells (Fig. 3). Another example was strong staining of mucin within gastrointestinal epithelium with progesterone receptor (PR), including normal colon (Fig. 4), normal small intestine (Fig. 4, inset) and colonic adenocarcinoma (Fig. 5A). Positive cytoplasmic immunoreactivity was observed for PR in lung adenocarcinoma (Fig. 5B). Positive nuclear immunoreactivity was observed for oestrogen receptor (ER) in melanoma (Fig. 6). Another instance of unexpected immunoreactivity was positivity for napsin A in primary colonic adenocarcinoma (Fig. 7A,B). The same case was subsequently stained for thyroid transcription factor (TTF-1) and an alternative napsin A clone IP64 (Leica Microsystems) (Fig. 8) using the same heat-induced epitope retrieval method and dilution as the initial
Fig. 2 Focal AE1/3 immunoreactivity within heart myocardial cells; note folded section.
Fig. 3 Cytoplasmic p63 staining of heart tissue.
polyclonal antibody (Cell Marque, USA). The aim of this was to confirm or remove a possible diagnosis of metastatic pulmonary adenocarcinoma. These subsequent staining results were negative. The only tested tissue sample that strongly reacted with the Epstein–Barr virus latent membrane protein (EBV LMP1) antibody was melanoma (Fig. 9). Our BCL-2 antibody showed immunoreactivity with all of the tissue types. There were multiple tissue types that had a positive cytoplasmic reaction with WT-1 clone 6FH2 (Dako). This cytoplasmic immunoreactivity was not observed when using WT-1 clone WT49 (Leica Microsystems), where nuclear staining was interpreted as positive. The most immunoreactive tissue type with strong positivity encountered in this study was colon (30/57), followed by appendix (24/57) and tonsil (22/57). Forty-two of 43 (98%) of the duplicate tissue core samples achieved expected IHC staining results compared with the original paraffin tissue control block. The exception was appendix, where one of the tissue cores contained epithelial and lymphoid cells (Fig. 10A), whereas the other contained mostly muscle and connective tissue (Fig. 10B).
Fig. 4 Progesterone receptor (PR) staining of mucin in normal colon epithelial mucosa and small intestine (inset).
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TISSUE MICROARRAY FOR DIAGNOSTIC IMMUNOHISTOCHEMISTRY
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Fig. 7 Napsin A (Cell Marque polyclonal) antibody staining colon adenocarcinoma. (A) TMA core. (B) Whole block section.
DISCUSSION Fig. 5 Progesterone receptor (PR) staining. (A) Mucin in colonic adenocarcinoma. (B) Pulmonary adenocarcinoma tumour cells.
The overall cost to complete this study with two TMA blocks and 114 slides was approximately 25 h of manual labour (2 h to source blocks and prepare intermediate blocks; 8 h to construct 2 TMA paraffin blocks; 2 h to section slides; 11 h to assess slides microscopically) and AU$1300 for a Leica Bond Refine Detection Kit (DS9800) capable of staining up to 200 IHC slides. Consumables costs (slides, 0.3 mL of diluted primary antibody) were covered by our laboratory’s cost centre.
Fig. 6 Oestrogen receptor staining of melanoma.
This investigation has shown that comprehensive IHC crossreactivity data of an antibody can be obtained from a single slide, and was achieved with significant savings of cost, labour and time. By choosing TMA technology over sectioning and staining multiple whole block sections, scientists and pathologists within a diagnostic laboratory are able to improve their knowledge and understanding of IHC staining without impacting on routine workload. Future studies with the aim of further validating this data, and continually improving IHC QC in the diagnostic laboratory, would be to include an extended sample number of specific tissue types as a constructed TMA paraffin block. This process would be of particular benefit for optimising IHC staining for
Fig. 8 Napsin A (IP64) colon adenocarcinoma whole block section.
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Fig. 9 EBV LMP1 staining melanoma.
tumours with a broad range of heterogeneity and associated varying levels of antigenic expression, for example ER and PR staining in breast carcinoma5 and napsin A and TTF-1 staining in pulmonary adenocarcinoma.6 Regarding tumour heterogeneity within a single paraffin block, it has been demonstrated that two 0.6 mm cores adequately represent the staining of an entire histological section of
Fig. 10 Appendix duplicate core sampling discrepancy. (A) Epithelium (stained with AE1/AE3) and lymphoid component (stained with haematoxylin) present in TMA core. (B) Connective tissue elements only in adjacent TMA core.
Pathology (2013), 45(1), January
tumour.2,5 However this study focused on one tissue sample as an example of expected antigen-antibody reaction to assess IHC staining of a broad range of different tissue and cellular types on only one stained microscope slide. Future studies would include assessment of additional examples of the tissue types used in this study, with the aim to reproduce cases of identified unexpected immunoreactivity. Tumour heterogeneity and its impact on TMA sampling could be investigated further, with breast carcinoma and pulmonary adenocarcinoma possible tumours to be studied. TMA block section quality is dependent on a higher level of technical skill than of a whole tissue section.1 As described, there were slides that contained some folded (Fig. 1) and/or missing cores. The reasons for this include minimal or no tissue remaining in the paraffin block, or varying amounts of dense connective tissue in the different tissue types which renders microtomy technically difficult. The same scientist used the same semi-automated microtome to cut sections for this study, which guaranteed a consistent section thickness. Fortunately, most of the folded core sections were less than 50% affected, and in these instances a staining result was able to be interpreted confidently. In the routine histology laboratory, it is commonplace to presoak hard or brittle tissue within a paraffin block prior to sectioning. This method did little to improve the section quality in this study. A possible alternative to improve section quality of TMA blocks containing such a large number of cores is to adopt a tape transfer technique.7 This would also eliminate the need for a water bath adjacent to the microtome for floatation of sections. The only tissue sample that strongly reacted with the EBV LMP1 antibody was the melanoma. This was a surprising initial observation, but a review of the literature revealed that up to 90% of melanomas react with this antibody. This immunoreactivity is not related to viral infection, but is due to unidentified protein cross-reactivity.8 In 2001, it was argued that positive staining of melanoma by EBV LMP1 was due to the particular avidin biotin complex (ABC) IHC staining method performed.9 However, this study used a polymeric-based IHC detection system which also demonstrates positive staining, indicating that the ABC IHC staining technique is not responsible for the observed positive immunostaining. For the pathologist to avoid this staining phenomenon it is recommended that appropriate tissue controls and routine antibody panel selection for diagnostic IHC be followed. Napsin A is an aspartic proteinase involved in the maturation of surfactant protein B. It is detected in the cytoplasm of type 2 pneumocytes and alveolar macrophages and is a putative marker for pulmonary adenocarcinomas.10 Many studies have shown that napsin A does not stain non-pulmonary adenocarcinomas11,12 and the PathIQ Immunoquery website (http://www.immunoquery.com/) reports that zero of 28 (0%) colorectal carcinomas were positive for napsin A. The only other known positive IHC staining identified in the literature is expression in renal tubular epithelial cells and most papillary renal carcinomas.11,13 When our laboratory began to optimise Napsin A staining, a section of metastatic colorectal carcinoma was included as a negative control, which was validated in-house. This marker has been in use since 2010 at this laboratory site. Therefore, it was surprising to identify positive staining of primary colonic adenocarcinoma in the TMA with the Cell Marque polyclonal Napsin A. Recently has there been another report of positive staining of two of 95 (2%) cases
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of colonic adenocarcinoma using a different clone of napsin A (TMU-Ad02).14 However, the authors dismissed this staining as false positive due to their negative control showing stronger staining than the tumour cells. It is interesting that they used an antibody dilution of 1:100, while the dilution used in the current study was 1:200. The positive cytoplasmic staining pattern (Fig. 7A,B) is reproducible and not artefactual. Recently we have seen a liver core biopsy case with metastatic colorectal adenocarcinoma that has stained with a similar pattern. These staining results and the limited studies to date, indicate that more investigation into napsin A IHC and colonic adenocarcinoma is required to provide increased reliability of antibody staining performance. Our laboratory recently purchased a newly available alternative napsin A clone IP64 (Leica Microsystems). Initial titrating and optimising of this antibody clone mirrored that of the Cell Marque polyclonal antibody, with a dilution of 1/200 with heatinduced epitope retrieval of ER2 20 min at 1008C. The case of primary colon adenocarcinoma that was used in the TMA staining experiments was stained as a whole block section in parallel with both the new napsin A IP64 clone and the polyclonal Cell Marque antibody. The section stained with the Cell Marque polyclonal antibody stained as per the TMA result, while the section did not stain with the IP64 monoclonal antibody. This result brings to question the validity of antibody clones that are distributed by manufacturers and marketed as highly sensitive, specific markers for IHC. The manufacturer of the polyclonal napsin A has been contacted for comment regarding the unexpected observed staining patterns. The above finding indicates that unless an adequate antibody marker selection panel is used for IHC, the possibility of a false positive diagnosis of pulmonary adenocarcinoma is increased, especially on small biopsy and cell block material. As always recommended, IHC is a valuable tool for pathologists and correlation with clinicopathological findings is most important to achieve the correct diagnosis. It is noted that a large percentage of the unexpected immunoreactivity observed in this study are unlikely to be encountered by the clinical pathologist during routine IHC testing. As an example, EBV LMP1 IHC would not usually be requested for a case suspected to be malignant melanoma. The heart tissue used in this study was sent to our histology laboratory for examination for pathology that may contraindicate donation and was assessed as being within normal limits. This tissue demonstrated the presence of a lipofuscin-type pigment within the myocardial cells on H&E staining, but this was the not the cause of the positive cytoplasmic staining with p63 (Fig. 3). This antibody is used in our laboratory as a nuclear staining marker for proliferative epithelial cells in squamous cell carcinomas and aids in the diagnosis of prostate adenocarcinoma. The literature review initially performed into p63 could not locate any reference to heart cytoplasmic p63 staining, but it was found that heart-derived mesenchymal cells can support epidermal regeneration in keratinocytes, and if these cells are vimentin positive, also express some cytokeratins (CK) antibody markers and p63.15 Only recently has there been a report of cytoplasmic p63 staining in normal myocardium as well as rhabdomyosarcomas and rhabdomyomas.16 The results in our study support these recent findings and that p63 is involved in cardiogenesis17 and is a sensitive marker for skeletal muscle differentiation. Cells containing mucin are also reported to have stained positively for PR.18 This marker is known to bind to an epitope
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on the O-acetylated sialomucin molecule, and is specific to normal colonic epithelial mucosa.19 It is important to note that this staining pattern is not cellular, and should be regarded as an artefact of IHC staining and not as true positive PR expression. Positive nuclear IHC staining with ER in melanoma and cytoplasmic PR staining in pulmonary adenocarcinoma is not a novel finding.20,21 However, these particular results serve as a reminder to scientists and pathologists about another interpretive pitfall of IHC. With WT-1, nuclear staining in the absence of cytoplasmic staining was observed using the clone WT49 (Leica Microsystems). This clone has been the most commonly requested WT-1 marker in this laboratory by pathologists, due in part to instances of the 6F-H2 clone non-specifically staining connective tissue cytoplasm in patient tumour biopsy specimens. The range of immunoreactivity observed with our BCL-2 antibody and the tissue cores in the TMA block was expected.22 This marker is used primarily in our laboratory as a lymphoma marker in establishing a preliminary diagnosis, so it was initially surprising to our scientists and pathologists to observe cross-immunoreactivity on such a large scale. C4d IHC staining is performed by our laboratory when assessing patient heart biopsies post-transplant with a negative histological diagnosis of cellular rejection. This antibody positively stains in cardiothoracic tissue venules and arterioles as a result of perioperative activation of the complement system.23 Positive capillary staining is regarded as an indication of cardiac antibody-mediated rejection (AMR), with positive venule and arteriole staining considered as an excellent internal control for assessing the quality and intensity of C4d IHC staining.24 In our laboratory, lung endothelium would be an appropriate control for C4d IHC staining. This staining pattern was also observed in other tissue vascular endothelium (colon, prostate and appendix). The author hypothesises that perioperative complement activation is the reason for this staining reaction, and its presence is made aware to the scientist and pathologist performing IHC quality control. Further investigation into the observed unexpected immunoreactivity results would be to adjust heat-induced epitope retrieval methods and antibody dilutions. It was decided not to proceed with this, as these results are indicative of staining results that could be encountered by our laboratory when performing diagnostic IHC. Selecting initial 4 mm tissue cores from whole paraffin blocks ensured that sampling of smaller 1 mm cores contained sufficient representative tissue for IHC staining. This is the first described instance of performing intermediate sampling prior to construction of a TMA block. In this study, there was only one instance of sampling discrepancy (appendix) where the duplicate tissue cores in the same block did not have a similar representative architecture (Fig. 10A,B). Future investigations From the results of this exercise, it is proposed that a universal control block be used at our laboratory for daily IHC QC. This block would contain nine tissue types consisting of placenta, tonsil, appendix, colon, melanoma, lung, testis, cervix and prostate. These would be included in a control block arranged as 3 mm cores in three rows of three (Fig. 11) and could be constructed manually. By adopting a tissue control block with these tissue types, 55 of 57 antibodies used in our laboratory will stain positively, with most antibodies having at least two
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Fig. 11 Proposed ‘universal’ TMA block for routine IHC quality control containing 9 3 mm diameter tissue cores.
positive and two negative tissue control sections (Supplementary Tables 4 and 5, http://links.lww.com/PAT/A8). Using 3 mm cores will improve the quality of microtomy and also reliability of sampling tissue with multiple cellular elements required for consistent IHC staining performance. For example, appendix and tonsil contains both epithelium and lymphoid cells. Pre-cut 3–4 mm sections of the universal control block could be stored in slide boxes at room temperature. It is our laboratory’s routine that freshly cut control sections are used when staining six of our antibodies (Ki-67, ER, PR, WT-1, cyclin-D1, and TTF-1). This is done to prevent loss of immunoreactivity due to variables such as oxidation and ultraviolet light exposure that has been documented,25 and should continue as a standard method at this laboratory. IHC staining in our laboratory routinely contains a positive and negative tissue control on the same slide as the tested section. The universal control block proposed in this study will not change this routine procedure, despite containing nine tissue cores. By having a control section on the same slide as the test section, reproducibility and repeatability of staining performance is maintained. In rare instances where the test section and control section cannot be placed on the same slide, then they should be stained on separate adjacent slides and stained in parallel. Melanoma would be the only tumour tissue included in the universal control. This means that for our laboratory to adopt the universal tissue block as its control for IHC, only one tumour type is to be continually sourced. This provides the laboratory benefit in not relying on sourcing rare tumour tissue as IHC control material, as there is an increasing number of molecular profiling requests (e.g., EGFR and KRAS) on archived tumour tissue being performed to potentially improve patient management through targeted therapies. The use of melanoma as the only tumour tissue control for IHC avoids this sourcing issue to a degree. Our laboratory is situated within the population with the highest worldwide incidence of this disease26 and minimal pathologist permitted sampling of archived
Pathology (2013), 45(1), January
melanoma tissue would not compromise any future molecular profiling requests. This is the first report to recommend melanoma tissue as a control for EBV LMP1 IHC staining. However, appropriate quality control measures are required to validate this method. It is suggested that any primary and/or metastatic melanoma tumour tissue to be incorporated in the proposed universal control block is first immunostained with EBV LMP1, as 10% of cases are documented to negatively stain.8 The use of a TMA slide containing known EBV infected tissue as a validation tool would be useful in instances where melanoma control tissue negatively stains with this antibody. In this instance, EBV infected tissue would be used as a separate control for EBV LMP1 IHC staining. Eight of the nine tissue types to be included in the universal tissue control block are normal. This is ideal as normal tissue contains consistent antigenic expression levels compared to heterogeneous tumour tissue. In the past, tumour tissue has been used as IHC control tissue in our laboratory, but heterogeneity of tumour subtypes removes the consistency of antigenic expression. Having this consistent antigenicity using normal tissue will allow for reliability of IHC staining performance through standardised antibody optimisation. For instances where antibody performance requires troubleshooting due to poor performance or possible antibody crosscontamination and for optimising new and existing antibodies when new batches are received, it is proposed that a TMA block similar to that used in this study be utilised for antibody titration for diagnostic IHC validation. Other tissue types that are encountered more commonly by the laboratory could also be added into the TMA block to produce further IHC immunoreactivity data. When recording and comparing IHC staining results for standardisation between multiple observers, there is an argument for utilising a whole slide digital scanner. These instruments are an ideal alternative to manual IHC scoring as they are able to provide a clear image of a freshly stained section that will not fade, which can be observed when staining results of individual slides are performed by multiple observers over a period of time. An example of this is when slides are exposed to UV light, the intensity of DAB staining may be reduced. The other advantage of adopting this instrument is that the same scanned image can be scored by multiple observers simultaneously, saving time. This study was based on the observations of primarily one scientist, and the use of a whole slide digital scanner would have enabled multiple observers to record the staining results of this project. At present it is not an economically viable option to purchase such an instrument for many pathology laboratories (including ours), but in the future this would be a valuable tool for IHC staining standardisation and validation between laboratory sites. The proposed universal TMA block is based on the interpretation of one observer, a histology scientist with extensive experience in diagnostic IHC. On-site pathologist assistance was sought when the observer encountered unexpected staining patterns. The results were not audited externally, which justifiably raises some concern about certain recommendations; for example, the suggested controls for C4d and EBV LMP1 staining. This study recommends that the proposed universal control block be adopted by our laboratory site initially, with future studies involving the collaboration of external laboratories and the RCPA QAP to follow before other laboratories utilise this diagnostic IHC tool.
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Financial considerations The overall cost to complete this study with two TMA blocks and 114 slides was approximately 25 h of manual labour and AU$1300 for a Leica Bond Refine Detection Kit (DS9800) capable of staining up to 200 IHC slides. Consumables costs (slides, 0.3 mL of diluted primary antibody) were covered by our laboratory’s cost centre. The alternative method would be to section and stain 43 whole paraffin block sections in duplicate against 57 antibodies. This would mean that 4902 slides would require increased manual labour and 24 Bond Refine Detection Kits at a cost of AU$31 200. The initial outlay to purchase a manual tissue microarrayer machine capable of constructing a block containing hundreds of tissue cores (AU$17 000) would be saved through reducing labour and time for the IHC scientist and other laboratory staff to source tissue control material and maintaining a large number of catalogued control blocks and storage of pre-cut sections. Alternatively, there are disposable, partly constructed TMA blocks (approximately AU$285 per paraffin block) available for purchase. These still require manual insertion of tissue cores before laboratory use, and would not be more cost-effective long-term as a manual tissue microarrayer machine.
CONCLUSION This study has established preliminary data of immunoreactivity between a large range of tissue types and antibody markers used at this laboratory, whilst reminding pathologists and histology scientists that IHC is a tool that can be used to assist diagnosis. TMA technology has proven to be a cost-effective, comprehensive and efficient screening tool in the diagnostic IHC laboratory and is now used by external QAP organisations for inter-laboratory assessments of IHC and ISH techniques Managers of pathology laboratories focus on minimising expenditure to remain viable in a competitive, highly regulated industry while retaining skilled scientific and technical staff through providing quality educational resources. TMA technology in the diagnostic IHC laboratory fulfils both of these criteria. Acknowledgements: The author would like to thank the pathologists, scientists and all staff of the Department of Anatomical Pathology, Pathology Queensland – The Prince Charles Hospital for their assistance, especially Dr Belinda Clarke and Dr Kayla Tran for peer review. Thanks also to Ms Annette Lane from UQCCR for construction of the TMA blocks. Conflicts of interest and sources of funding: Financial support was provided by the Pathology Queensland Study, Education and Research Trust Fund. The author states that there are no conflicts of interest to disclose. Address for correspondence: Mr A. van Zwieten, Department of Anatomical Pathology, Pathology Queensland - The Prince Charles Hospital, Rode Road, Chermside, Qld 4032, Australia. E-mail: [email protected]. gov.au
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