Multiplex IHC for Increased Efficiency, Diagnostic Accuracy

Vol. 20 • Issue 6 • Page 33


Advanced multiplex testing has become more common due to the significant efficiencies and critical diagnostic information that these techniques present to the laboratory. Multiplex techniques, which enable simultaneous testing for two or more analytes, enable laboratories to multiply instrument throughput, reduce sample preparation and minimize overall labor time per result.

More recently, multiplex paradigms and efficiencies are being applied to routine immunohistochemistry (IHC) testing. Antibodies must be carefully chosen so that they are morphologically distinct; that is, they bind to markers located in different cellular compartments or cell types or that indicate different disease states. This ensures that the clinical picture is clear and easy to interpret, giving additional information to the pathologist and increasing confidence in the result.

Technological Advances

Development of a true multiplex IHC detection technology that detects two to five antibodies simultaneously and in about the same time as it takes to do a single antibody IHC procedure has been critical to its success in the laboratory. Advanced technologies, such as Biocare Medical’s MACH 2 Double Stain kits, allow for a single application of each reagent, eliminating the need to apply primary and secondary antibodies as well as tertiary (polymer) detection reagents twice or more. (Many non-multiplex IHC methods have offered staining for two antibodies essentially by performing the IHC process two or even three times. In a clinical laboratory setting, this approach is impractical since it extends the time-to-result by several hours, reducing instrument throughput.)

Lean, Financial Benefits

Lean and financial benefits of simultaneous multiplex IHC include use of approximately half as many detection reagents since the reagents consist of a combination of two or more primary antibodies, secondary antibodies or tertiary detection. This decreases steps, consistent with Lean principles, while reducing staining time and waste disposal costs. Importantly, throughput of instrumentation can be increased by two- to three-fold since there are several reimbursable tests per slide, so a 50-slide staining system does the work of a 100- or 150-slide staining platform.

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Because only one slide, not two or three, needs to be cut, dried and deparaffinized, and antigen retrieved and put through the 2.5- to 4-hour IHC process, both reagent and labor costs are greatly reduced.

Finally, use of a single tissue section helps preserve precious tissue in core needle core biopsies.

Slide Interpretation for Breast Cancer

Multiplex IHC technology is more effective when supported by a library of clinically validated antibody cocktails directed at solving the most challenging clinical problems. One example in the breast cancer area is the complex differentiation of atypical ductal hyperplasia (ADH; borderline) or ductal carcinoma in situ (DCIS; pre-invasive) from usual ductal hyperplasia (UDH; benign). ADH or DCIS diagnosis carries a 4-10% likelihood of progression to breast cancer, so treatment usually involves surgical excision and radiation therapy for DCIS patients.1 UDH, however, carries minimal risk of progression to breast cancer, so no further treatment is necessary. Thus, this differentiation is critical to optimize patient care, reduce side effects and complications of surgery and radiation therapy, and reduce costs to patient and the healthcare system.

Antibody Cocktail

A multiplex IHC test that is specifically designed to aid in the critical differentiation of UDH from ADH and DCIS is ADH-5 (Biocare Medical). The ADH-5 multiplex IHC antibody cocktail contains antibodies for CK5/14 (high molecular weight cytokeratin), p63 and CK7/18 (low molecular weight cytokeratin). UDH is associated with a polyclonal proliferation of cells, including differentiated luminal epithelial cells expressing CK7/18 and more primitive epithelial cells expressing CK5/14 and p63. Presence of a monoclonal proliferation of CK7/18 expressing cells is indicative of ADH or DCIS.

Sunil Badve, MD, FRCPath, professor, Departments of Pathology and Internal Medicine at Indiana University, and his team conducted a study comparing diagnostic agreement with hematoxylin and eosin (H&E) stain alone versus H&E plus the ADH-5 multiplex IHC cocktail. This study found that inter- and intra-pathologist variation is typically quite high for differentiation of UDH versus ADH and DCIS. Seven or more of the nine pathologists in the study agreed on a diagnosis of UDH, ADH or DCIS only 47% of the time using H&E stains alone, whereas the level of agreement was increased to 63% of cases when the ADH-5 stain was added.1,2

Dr. Badve says, “We have shown that diagnostic agreement for noninvasive epithelial breast proliferations such as ADH, UDH and DCIS based on morphology alone is only mediocre. However, this agreement can be significantly improved through use of a multiplex IHC cocktail such as ADH-5 that combines high and low molecular weight cytokeratins. More importantly, it gives the pathologists confidence in diagnosing lesions with minor degrees of architectural or cytological atypia and helps to reduce overdiagnosis of atypical ductal hyperplasia lesions.”

Solving a Dilemma

Sensitivity and specificity for differentiation of adenocarcinoma from squamous cell carcinoma in NSCLC patients have typically been in the range of 80%. This is a critical diagnostic problem since use of VEGF-inhibiting drugs such as Avastin® is associated with fatal lung hemorrhage in 30% of patients with non-squamous, non-small cell lung cancer (NSCLC).

This intriguing challenge in lung cancer diagnosis has been addressed with multiplex IHC cocktails such as PulmoPanel (Biocare Medical). This new multiplex IHC test consists of Desmoglein 3 + Napsin A, TTF-1 + CK5 and p63 + TRIM29.

Incorporation of multiple highly specific new markers for differentiation of adeno- and squamous cell carcinoma such as Desmoglein 3 (100% specific) and Napsin A (94% specific) add power to PulmoPanel-above that achieved using more traditional markers alone (TTF-1; CK5). This results in overall sensitivity of 92.9% and 100% specificity for this important differentiation.3

Mark Cross is senior director of Sales and Marketing at Biocare Medical.


1. Jain et al. Atypical ductal hyperplasia: interobserver and intraobserver variability. Mod Pathol advance online publication, April 29, 2011;doi:10.1038/modpathol.2011.66 (also appearing in July 2011 Mod Pathol).

2. Jain et al. Atypical Hyperplasia at 25 years-Interobserver and intraobserver variability. Modern Pathol February 2010;23:1;53A.

3. Tacha et al. An Evaluation of Sensitivity and Specificity and Correlation of Tumor Grade for Lung Squamous Cell Carcinoma vs. Lung Adenocarcinoma. Mod Path 2011;24;1;425A.

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