What’s Cooking? Detection of Important Biomarkers in HOPE-Fixed, Paraffin-Embedded Tissues Eliminates the Need for Antigen Retrieval

To the Editor-in-Chief:

Recently, the immunohistochemical assessment of biological parameters has become an integral part of both investigative as well as diagnostic pathology. Cell proliferation is often measured in tumors and the determination of hormone receptors is mandatory in the reporting of breast cancer. 1–3 Finally, there are FDA-approved immunohistochemical assays which are decisive for adjuvant therapy, eg, the HercepTest. To our knowledge, however, there are no assay systems available applicable to routinely formalin-fixed and paraffin-embedded material without antigen retrieval, which in most cases means, in effect, cooking of the specimens. The mechanism underlying this is poorly understood. Moreoever, the procedure often leads to a loss in the morphological quality and the repeated heating and cooling are very difficult to standardize. 4 Recently it has been described that the HOPE (Hepes-glutamic acid buffer mediated Organic solvent Protection Effect, German patent DE 10021390 C2) fixation and subsequent direct paraffin-embedding results in a morphological preservation of human soft tissues which is well comparable with routinely formalin-fixed and paraffin-embedded specimens. 5 As part of an investigation aimed at elucidating the possibilities and limitations of HOPE-fixation, we analyzed the immunohistochemical assessment of cell proliferation using the common MIB-1 antibody. 2 From the group of important and relevant hormone receptors we studied the detectability of estrogen- and progesterone-receptors 6 and of FDA-approved assays we applied the HercepTest. 7 The assessment of all these important biomarkers in paraffin sections requires cooking when formalin fixation is used.

HOPE-solution was obtained from Dr. J. Olert (contact: olert@kinderpatho.klik.uni-mainz.de). Formalin-fixed and correspondingly HOPE-fixed, paraffin-embedded tissues of mammary carcinoma specimens were cut into 4-μm sections. Sections were deparaffinized and the primary antibodies were applied in moist chambers. 5 Endogenous peroxidase was inactivated by 3% H₂0₂ for 10 minutes at ambient temperature. Negative controls were included from every specimen. Formalin-fixed samples from the same tumors as the HOPE-fixed were included, conventionally deparaffinized and treated or not treated with the appropriate antigen retrieval techniques. Specimens were blocked for non-specific binding by incubation in normal, heat-inactivated pig serum (1:30 in Tris-buffered saline) for 10 minutes at ambient temperature. The applied dilutions and incubation times of the primary antibodies were: (a) 1 hour at ambient temperature: MIB-1 (1 μg/ml for formalin-fixed sections, 333 ng/ml for HOPE-fixed sections); and (b) overnight at 4°C: estrogen-receptor (Dako, 1D5, 1:75 dil. for formalin-fixed sections; 1:225 dil for HOPE-fixed sections), progesterone-receptor (Dako, PgR 636, 1:100 dil. for formalin-fixed sections: 1:300 dil. for HOPE-fixed sections)

Detection of HOPE-treated material was achieved by LSAB2 (Dako) diluted 1:3 for MIB-1, estrogen- and progesterone-receptor, or EnVision (Dako) diluted 1:3 for the HercepTest. In the case of formalin-fixed specimens, detection was achieved by LSAB2 or Envision according to the manufacturer’s recommendations. Sections treated with LSAB were developed using 3-amino9-ethylcarbazole/H₂0₂, the HercepTest was developed with diaminobenzidine/H₂0₂ according to the protocol of the manufacturer. Subsequently all specimens were counterstained with hemalum and mounted.

The formalin-fixed specimen without antigen retrieval remained negative staining (not shown). As demonstrated in Figure 1 ▶ we achieved very good immunostaining for all important biomarkers investigated in HOPE-fixed paraffin sections without antigen retrieval. It is important to note that in terms of quantity and intensity of positively stained cells there was almost no difference between the established protocols for formalin-fixed materials using antigen retrieval and the HOPE-protocol without the latter, with the HOPE-fixed specimens requiring only one-third of the primary antibodies as well as one-third of the respective detection system.

Figure 2.

Immunohistochemistry applying HOPE-technique without antigen retrieval on breast cancer sections. A: Ki-67 (MIB-1, ×200). B: Estrogen-receptor (1D5, ×400). C: Progesterone-receptor (PgR 636, ×400). D: c-erb B2/neu (HercepTest, ×200).

Thus HOPE-fixation, due to the proved excellent preservation of antigens, the highly standardized tissue-sampling, and the well preserved morphology meets the new and increasing demands in the expanding field of molecular diagnostics, and we propose that it should be considered in this respect as an improved alternative fixation method in routine immunohistology. 8–10 Appropriate large scale studies are underway.