J Clin Pathol 65:437-440 doi:10.1136/jclinpath-2011-200533
  • Original article

The value of triple antibody (34βE12 + p63 + AMACR) cocktail stain in radical prostatectomy specimens with crushed surgical margins

  1. Theodorus H van der Kwast2
  1. 1Department of Pathology and Microbiology, Jordan University of Science and Technology, Irbid, Jordan
  2. 2Department of Pathology, University Health Network, Toronto, Canada
  1. Correspondence to Dr N Al Daoud, Department of Pathology and Microbiology, Jordan University of Science and Technology, BO Box 3030, Irbid 22110, Jordan; naglaadaoud{at}
  1. Contributors All authors have contributed to the paper. NAD is the guarantor of the study, the study design was by THvdK and NAD, collection of samples by NAD, GL and AJE. The draft was written by NAD, edited by TvdK and AE. The interpretation of the staining was by NA, TvdK and AJE. The content of the manuscript was approved by all authors.

  • Accepted 5 January 2012
  • Published Online First 31 January 2012


Background Triple antibody cocktail immunohistochemical staining is routinely used as an ancillary method to establish a diagnosis of prostate cancer in biopsies with small foci of atypical glands. Crush artefact can distort surgical margins in radical prostatectomy specimens, occasionally making it difficult to diagnose a positive margin.

Aim To investigate the ability of a cocktail stain to distinguish carcinoma from benign prostatic glands at the edge of crushed margins in prostatectomy specimens.

Methods 10 radical prostatectomy specimens with crushed benign glands at the surgical margins, and 20 with crushed margins positive for carcinoma were retrieved from the pathology archives. The latter included 16 (80%) with positive apical margins, 2 (10%) incised intraprostatic margins, and 1 (5%) soft tissue margin. Two-colour triple antibody stain using a cocktail of antibodies against α-methylacyl coenzyme A racemase (AMACR), high molecular weight keratin and p63 was performed on all the selected cases.

Results In 10/10 specimens with crushed benign glands, basal cell staining continued to be detectable, while AMACR staining was negative in all cases (0/10). In the positive margin cases, none of the crushed glands expressed basal cell marker staining (0/20), whereas 14/20 (70%) of the cases showed variable levels of AMACR positivity at the inked margin.

Conclusion Two-colour triple antibody cocktail stain is useful in the assessment of most, but not all, surgical margins with crushed artefact in prostatectomy specimens by helping to establish whether glands are malignant or benign.


A positive surgical margin after prostatectomy treatment for clinically localised prostate cancer is an independent predictor of disease recurrence, in addition to Gleason score and pathological stage.1–4 Further, among patients with advanced prostate cancer, those with a positive margin after prostatectomy benefit most from adjuvant radiotherapy.5 As a consequence, the demonstration of a positive surgical margin often prompts adjuvant radiotherapy, in particular in patients with intermediate and high risk prostate cancer. Several factors contribute to the presence of a positive margin in prostatectomy specimens, including those related to the surgeon and the pathologist. Accurate identification of positive surgical margins (PSM) by pathologists is of great clinical relevance.6

Previous work from our group and others has established that there is interobserver variation between pathologists concerning the interpretation of surgical margins in prostatectomies,7 8 and in certain situations it may sometimes be impossible for pathologists to give a robust opinion on the surgical margin status.9 The occurrence of crush artefact along an inked margin, produced by the surgical removal of the prostate, may cause considerable confusion. The associated morphological distortion of acinar structures adjacent to or on the actual margin may prevent the pathologist from distinguishing carcinoma from benign glands or even benign stromal structures. When a small acinar lesion is present in a prostate biopsy, immunostaining with a cocktail of antibodies specific for high molecular weight cytokeratin (HMWK) and p63 as basal cell marker and for α-methylacyl coenzyme A racemase (AMACR) for neoplastic cells is commonly used as an ancillary tool in suspect small lesions to differentiate between carcinoma and benign acini.10 11 We considered that the use of such a triple antibody cocktail immunostaining might also be helpful in terms of clarifying the surgical margin status in problematic cases.

Materials and methods

Twenty cases with positive crushed margins and 10 cases with crushed benign glands at the surgical margins of radical prostatectomy specimens were retrieved from the pathology archives of Toronto General Hospital. Fourteen specimens with crushed positive margins were identified from men with pT2 disease, with only an apical positive margin. The remaining six cases with crushed PSM and the 10 cases with crushed benign glands at the surgical margin were selected through a database search for radical prostatectomy specimens. The slides of both sets were reviewed by two pathologists (TvdK, NAD), and cases with a moderate to severe crush or traumatic artefact were selected, characterised by severe distortion of glands to such an extent that it was a challenge to unequivocally identify their nature. In the presence of crush artefact on H&E sections, a positive margin was defined when clearly identifiable carcinoma, with no obviously admixed benign glands, tracked into a crushed area and appeared to extend out to the inked edge. Sixteen (80%) of the PSM were apical margins, 2 (10%) were incised intraprostatic margins, and 1 (5%) was a soft tissue margin. Two-colour triple antibody stain using a cocktail of antibodies against AMACR, HMWK and p63 was performed on all the selected cases. The procedure was performed using an automated immunostainer (NexES protocol Editor-BenchMark XT IHC/ISH Staining Module). HMWK (34βE12) is a mouse monoclonal predilute antibody from Ventana: (Tucson, AZ, USA), and p63 (Vector, Burlingame, CA, USA) is a mouse monoclonal antibody at a dilution of 1:50. Both were mixed and then applied to the sections for 40 min, followed by an ultra-wash, and then antibody denaturation by heating at 90° for 4 min. AMACR (13H4, a rabbit monoclonal antibody from Zeta Corporation) at a dilution of 1:100 was then applied for 36 min. The chromogen kit for HMWK/p63 is based on an anti-mouse horseradish peroxidase labelling (diamino-benzidine), and for AMACR on an anti-rabbit alkaline phosphatase labelling (Fast Red), allowing the visualisation of the basal cell markers in brown and AMACR in red. For the calculation of 90% CI of the sensitivity of the markers, the SAS V.9.2 Mayo Clinic %bnmlci was used, based on the binomial distribution.


All 10 samples with crushed benign glands at the surgical margin retained the basal cell marker staining (sensitivity 100%; 90% CI 74% to 100%), while the AMACR staining was negative (figure 1). Among the 20 cases with crushed glandular structures at the site of the positive surgical margin, none expressed a basal cell marker, whereas 14/20 (sensitivity 70%, 90% CI 49% to 86%) cases showed variable levels of AMACR positivity (figure 2), and 6/20 (30%) were AMACR negative (figure 3). The intensity of AMACR positivity was weaker at the margin than the underlying tumour, but, particularly at higher magnification, traces of the red AMACR visualisation product remained detectable. In the cases negative for AMACR staining, the underlying adenocarcinoma was entirely negative in one case, and weak and focal in the remaining five cases.

Figure 1

(A) Surgical margin with crushed benign glands. (B) Crushed benign acini are positive for basal cell markers and negative for α-methylacyl coenzyme A racemase, with inset showing a higher magnification of the crushed glands.

Figure 2

(A) Photomicrograph of an apical positive surgical margin with crushed acini. (B) Crushed acini negative for basal cell markers and positive for α-methylacyl coenzyme A racemase, with inset showing a higher magnification of the crushed acini at the margin.

Figure 3

(A) Photomicrograph of an apical positive margin with crushed acini at the margin. (B) Crushed acini are negative for both basal cell markers and α-methylacyl coenzyme A racemase.


A positive surgical margin is defined as the presence of tumour cells touching the inked surface of the surgically removed prostate.12 13 The reported incidence of PSM varies tremendously in the literature from 4% to 45%,14 due to variation in surgical technique and probably also due to variation in interpretation by the pathologist during histological examination of the specimen.7–9 Although the apex is the most common site of a positive surgical margin in some studies,15 in other studies the posterolateral margin was most commonly involved by cancer.4 A previous study by our group demonstrated that crush artefact created during the surgical removal of the prostate created significant interobserver variability among expert urological pathologists with respect to calling margins positive or negative for carcinoma.9 In our series, we noted that a crushed margin was most often seen in the apex, likely resulting from shear forces exerted on this site during surgery.

We demonstrate here that the use of a triple immunohistochemical cocktail stain can be helpful in distinguishing benign from malignant glands at an inked distorted/crushed margin. The combination of the negativity for the basal cell markers together with the positivity for AMACR gives an additional diagnostic value by highlighting the presence of malignant acini. In this study, we have shown that the performance of both AMACR staining and the basal cell markers p63 and 34βE12 is largely unaffected by crush artefact. Of the three markers, it was our experience that the performance of the basal cell stains was more robust than that of AMACR when applied to crushed margins. In 30% of the specimens with crushed positive margins, the malignant acini showed no AMACR positivity, but they failed to express basal cell markers, rendering a diagnosis of malignant acini most likely, as all of the crushed benign glands in our study consistently expressed basal cell markers (table 1). Another issue that may hamper the unequivocal demonstration of a surgical positive surgical margin may be that the cauterised glands may resemble stromal cells, including capillaries. We believe that the application of the triple stain may also help solve this issue in most, but not all instances.

Table 1

Triple antibody cocktail immunostaining results for surgical margins with crushed benign and malignant prostate glands

An additional benefit of using the cocktail stain in crushed PSM cases is that by highlighting the malignant acini, it may be easier for pathologists to confidently measure the linear extent of positive margins. According to the recommendations of the 2009 ISUP Consensus Conference on prostatectomy reporting,13 this parameter should be documented in the pathology report as it is a predictor of biochemical recurrence.4

The use of ancillary immunohistochemical staining to assist with the evaluation of crushed margins has several real or theoretical limitations. First, AMACR staining may be negative in up to 30% of the specimens with a positive crushed surgical margin based on H&E morphology. It is known that AMACR can be negative in up to 20% of prostate cancers in biopsies that are free of crush artefact.11 It is also acknowledged that AMACR positivity is less sensitive than negative staining with basal cell markers for the diagnosis of prostate cancer.10 16 Second, the use of the triple cocktail stain usually requires cutting deeper levels and it is possible that the area of interest at the crushed margin may partially or completely disappear on those levels. Third, the possibility of false positive staining due to the so-called ‘edge effect’ should be considered. This ‘edge effect’ may be the consequence of the lifting of tissue edges from the glass slide, allowing exposure to the immunoperoxidase reaction to both sides of the tissue,17 or due to inadequate dehydration of the tissue when alcohol solutions have been used for too long a time.18 The current more frequent application of automated tissue processing equipment has virtually abolished the latter cause for the ‘edge effect’. Finally, HMWK/34 βE12 may be falsely negative in benign glands, depending on the length of time the specimen spent in formalin prior to histological processing.19 This should not be an issue, however, if the more consistent p63 is also included in the cocktail.20 Despite these limitations, our results suggest that the triple cocktail will add confidence to the interpretation of crushed margins in a prostatectomy specimen being positive or negative for carcinoma in the majority of cases.

Take-home message

Immunostaining with a cocktail of antibodies specific for basal cell markers and α-methylacyl coenzyme A racemase can help to establish a diagnosis of positive or negative surgical margin in a prostatectomy specimen with severe crush artefacts.

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  • Competing interests None.

  • Provenance and peer review Not commissioned; externally peer reviewed.