Multiplex IHC – Tech Talk

Multiplex IHC

Biocare™ Medical is the proven leader in providing Multiplex antibody cocktails and detection platforms that enable simultaneous IHC staining with multiple antibodies on a single slide. Our Multiplex detection system simplifies procedures by conserving precious tissue, saving valuable technician time, reducing the number of reagents and steps in the protocol, and allowing for simultaneous evaluation of multiple targets on one slide.

Prostate cancer stained with CK HMW + p63 + AMACR (RM)

Antibody Cocktails

Early on, Biocare recognized the market need for evaluating morphologically distinct markers to aid in solving complex clinical problems and simplifying interpretation, all while conserving precious patient tissue.

The most glaring clinical need was determined to be the ability to differentiate between prostatic intraepithelial neoplasia (PIN) and carcinoma of the prostate. There also needed to be a way to clearly identify any microinvasion or micrometastasis into adjacent prostate tissue. Additionally, this would need to be performed in a clinical laboratory, with superior accuracy and specificity. All of this whilst conserving the limited tissue (prostate needle biopsies are thin filaments of tissue) and reducing the time to result.

With these constraints and needs in mind, Biocare developed our flagship Multiplex IHC cocktail: CK HMW + p63 + AMACR (RM) [formerly known as PIN-4]. Studies have shown that combinations of CK HMW [34βE12], p63, and/or AMACR may be useful in the evaluation of normal prostate glands, prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma1,2,3. In prostate, CK HMW [34βE12] has been shown to be a useful marker of basal cells of normal glands and PIN, a precursor lesion to prostatic adenocarcinoma; whereas invasive prostatic adenocarcinoma typically lacks a basal cell layer3-5. p63, a homolog of the tumor suppressor p53, has been detected in nuclei of the basal epithelium in normal prostate glands; however, it was not expressed in malignant tumors of the prostate6. α-Methylacyl coenzyme A racemase (AMACR), also known as P504S, has been shown to be a specific marker of prostatic adenocarcinoma7-10. Additionally, prostate glands involved in PIN have been found to express AMACR, whereas AMACR was nearly undetectable in benign glands10-11.

  • Conserve tissues, time and money = Cost-effective
    • A single Multiplex IHC can replace up to 5 single Ab stains; reducing labor and reagent costs by at least 50%
  • Accurate analysis and easy interpretation of staining results
  • Increase predictive value by combining highly sensitive & highly specific antibodies on one slide
  • More rapidly exclude or diagnose disease states
  • Eliminate multiple slides to evaluate antigen ratios

Multiplex IHC Products

We have also developed Multiplex IHC antibody cocktails for additional tissue applications: Breast, Bladder, Lung, Skin, Colon, Lymphoma, Tumors of Unknown Origin, and Cell Death vs. Proliferation.

Tissue Multiplex Product
Prostate CK HMW + p63 + AMACR (RM)
Breast CK5/14 + p63 + CK7/18
Bladder Uro-2
Lung Desmoglein 3 + p40 (M) + Napsin A (RM)
Skin Pan Melanoma + Ki-67
Colon CDX2 (M) + CDH17 (RM)
Lymphoma Kappa (M) + Lambda (P)
Tumor of Unknown Origin CDX2 + CK7
Death vs. Proliferation Ki-67 + Caspase 3

Clinical References:
1. Shah RB, et al. Am J Clin Pathol. 2004 Oct; 122(4):517-23. 2. Sung MT, et al. Hum Pathol. 2007 Feb; 38(2):332-41. 3. Bostwick DG, Qian J. Mod Pathol. 2004 Mar; 17(3):360-79. 4. Humphrey PA. J Clin Pathol. 2007 Jan; 60(1):35-42. 5. Shah RB, et al. Am J Surg Pathol. 2002 Sep; 26(9):1161-8. 6. Signoretti S, et al. 2000 Dec; 157(6):1769-75. 7. Xu J, et al. Cancer Res. 2000 Mar 15; 60(6):1677-82. 8. Rubin MA, et al. JAMA. 2002 Apr 3; 287(13):1662-70. 9. Luo J, et al. Cancer Res. 2002 Apr 15; 62(8):2220-6. 10. Zhou M, et al. Am J Surg Pathol. 2002 Jul; 26(7):926-31. 11. Wu CL, et al. Hum Pathol. 2004 Aug; 35(8):1008-13.

Biocare Medical PDL1 (CAL10) – Stands Up to the Competition


Several publications have compared Biocare’s PD-L1 (CAL10) antibody for immunohistochemistry to FDA approved PD-L1 clones SP263 and 28-8 in both rare and prevalent cancer types. Positive and significant correlation of expression, intensity and concordance resulted in both studies. Below is a recent abstract published in the Journal of Clinical Oncology using these different PD-L1 clones in malignant Thymoma and Thymic Carcinoma.  Also included is a link to a study published in Human Pathology using the different PD-L1 clones in Breast Cancer.


Immunohistochemical staining for programmed cell-death ligand 1 (PD-L1) in malignant thymoma and thymic carcinoma. Alexei Shimanovsky, Richard Cartun, Mary Fiel-Gan, Daniza Mandich, Jonathan Earle, Andrew L. Salner… Abstract e20003

Background: Recent development of anti-PD-1/L1 antibodies has demonstrated activity in various neoplasms. Thymic malignancies (TMS) are rare and treatment in advanced disease is limited. To evaluate the potential impact of anti-PD-1/L1 therapy in TMS, we examined the expression of PD-L1 in previously resected thymoma (TM) and thymic carcinoma (TC). Methods: We examined resected specimens from patients at Hartford Hospital with TM and TC between 2000 and 2014. Expression of PD-L1 was evaluated on formalin-fixed paraffin-embedded tissue. Immunohistochemical testing was done using four different clones of PD-L1 antibodies on the Leica Bond Max automated platform. The four clones include: E1L3N (Cell Signaling Technology), 28-8 (Epitomics) and SP142 (Spring Bioscience), and CAL10 (BioCare). PD-L1 expression was evaluated based on the percentage of tumor cells positive and their intensity graded as negative, weak (1+), moderate (2+), and strong (+3). The scoring was performed by three pathologists and was blinded for clinicopathologic data and antibody clones. Results: We evaluated a total of 29 patients, including 26 patients with TM and 3 with TC. Among the 29 available specimens, 12 had completed PD-L1 expression assessment at the time of submission. PD-L1 expression is present in 75-100% of the evaluated patients. All had positive PD-L1 staining by SP142 and CAL10. Three patients showed strong intensity by CAL10, and one by SP142. E1L3N and 28-8 had positive PD-L1 expression in 9 and 8 patients respectively with weak/moderate intensity. SP142 and CAL10 demonstrated the strongest concordance (R2 = 0.91) but there was significant variation between antibodies (R2 = 0.31-0.91). No correlation was detected between tumor grade and PD-L1 expression. There were focal areas that lacked expression in all of the evaluated specimens. Conclusions: There is increased expression of PD-L1 in TMS. The level of PD-L1 expression varies between the four PD-L1 antibodies. Increased PD-L1 expression provides evidence for the use of PD-L1 inhibitors in TMS. The variable staining highlights the heterogeneity of TMS and challenges in developing predictive biomarker in this cancer.

Citations: Immunohistochemical staining for programmed cell-death ligand 1 (PD-L1) in malignant thymoma and thymic carcinoma. Alexei Shimanovsky, Richard Cartun, Mary Fiel-Gan, Daniza Mandich, Jonathan Earle, Andrew L. Salner, Katrina Collins, Gregory Alan Otterson, and Benjamin F. Chu. Journal of Clinical Oncology 2017 35:15_suppl, e20003-e20003