A comparative analysis of two antigen retrieval techniques: Microwave oven and pressure cooker for immunoexpression of estrogen and progesterone receptors in breast cancer tissue

INTRODUCTION

Immunohistochemistry (IHC) is used to characterize intracellular proteins or various cell surface receptors in tissues. Individual markers, or a panel of markers, can be used to characterize various tumor subtypes, confirm tissue of origin, distinguish metastases from primary tumors, and provide additional information regarding prognosis, predicting response to therapy or evaluating residual tumor post-treatment.^[1] The analysis of estrogen receptor (ER) and progesterone receptor (PR) expression levels by IHC is an important part of the initial evaluation of breast cancer and is critically important in treatment planning.^[2] There has been a gradual development of IHC methodologies, which have allowed the identification of specific and highly selective cellular epitopes, in formalin-fixed paraffin-embedded tissues with an antibody and appropriate labeling system.^[3] Immunohistochemical methods ultimately depend on the good preservation of the specimen and of the target molecule. Long-term formalin tissue fixation results in antigen masking, probably through the aldehydic linkage between proteins and fixative molecules.^[4,5]

Antigen retrieval (AR) refers to any technique in which the masking of an epitope is reversed and epitope antibody binding is restored. Heat-induced epitope retrieval (HIER), also known simply as AR, was pioneered in the early 1990s ^[6,7] after it had become apparent that the use of enzyme digestion alone to improve IHC staining was inadequate. As a general rule, all HIER procedures involve heating slide-mounted specimen material in a buffer solution, followed by a cooling-off period. Heat causes cross-linked protein epitopes to “unfold” (in a manner similar to deoxyribonucleic acid denaturation), while buffer solutions aid in maintaining the conformation of the unfolded protein.^[8] However, HIER is better for unmasking epitopes because it is easier to use and produces better results compared to enzyme treatment.^[9]

Although it is not possible to standardize AR methods by manual IHC methods after the evolution of automated IHC but this type of AR can be used in limited resource settings as it is cost-effective, does not require much space, and is flexible with regard to options of using different buffers and times at different temperatures.

There are limited numbers of centers offering immunohistochemical evaluation, and automated IHC may not be affordable by small-scale laboratories. In the present study, we sought to compare the two widely used heat-induced AR methods-microwave ovens and pressure cookers using household appliances to study the effect on ER and PR immunoexpression in breast cancer.

MATERIALS AND METHODS

This was a prospective study conducted on 30 breast resection specimens.

Exclusion criteria

• Review slides/Blocks with suboptimal processing were excluded from the study

• Post-neoadjuvant chemotherapy (NACT) cases were excluded from the study.

Diagnosed cases of breast carcinoma on FNAC or core biopsies irrespective of their grade, stage, and age were included in the study. Cases that had received NACT were excluded from the study. All specimens were adequately fixed, grossed, and processed as per standard protocol. The stained sections were examined for the presence of tumors. Appropriate tumor sections were then subjected to IHC testing for ER and PR receptors.

4 µm thick sections were cut for ER and PR IHC. Two sections were taken from each case on charged slides along with external positive control. Tissue sections were then dried for 10 min at room temperature and were subjected to overnight incubation at 37° C in the incubator. On the next day, the tissue sections were incubated at 65° for 30 min. Sections were deparaffinized with xylene and hydrated with graded alcohols. Sections were then treated with 3% methanol/hydrogen peroxide solution for 30 min to prevent non-specific background staining. Tumor sections from each case were subjected to two different AR techniques. Slides were placed in a domestic microwave oven [Figure 1a and b] for AR in sodium citrate buffer solution (pH 6.0) in three cycles:

• First cycle (minimum heating): 5 min at 350W and then cooled at room temperature for 5 min

• Second cycle (moderate heating): 5 min at 500W and then cooled at room temperature for 5 min

• Third cycle (maximum heating): 5 min at 700W and then cooled at room temperature for 5 min.

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Figure 1:

(a and b) Microwave oven antigen retrieval method. (c and d) Pressure cooker antigen retrieval method.

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The second set of slides was subjected to a domestic pressure cooker [Figure 1c and d] for AR in sodium citrate buffer (pH 6.0). First, the pressure cooker was preheated for 5 min. Afterward, a plastic rack holding slides and the appropriate amount of buffer solution was kept inside the pressure cooker and AR was done until the pressure cooker is fully pressurized (two whistles).

The slides were then rinsed in Tris wash buffer for 5 min. For immunostaining, sections were treated with 100 µL of primary ER antibody (SP1) and PR antibody (1E2) for 60 min and then rinsed with TRIS wash buffer for 5 min. The slides were then treated with 100 µL of standard horseradish peroxidase-labeled polymer for 30 min. In the end, Diaminobenzidine (DAB) substrate solution was applied on the tissue section for 5 min, rinsed in running tap water for 1–2 min, and counterstained with Harris hematoxylin for 10 s. Slides were then dehydrated in graded alcohols, cleared in xylene, and mounted in Dibutylpathalate Xylene (DPX).

The slides were then assessed on microscopy by the Pathologist and the immunoexpression of ER and PR Receptors was scored based on the “Allred score.”^[10] Expression of ER and PR in at least 1% of tumor cells was taken as positive and the Allred score was calculated in each case as per American society of clinical oncology (ASCO) and the college of American pathologists (CAP) guidelines^[10]. The immunoexpression of the ER and PR receptors on the initial core biopsies/resection specimens of the cases which were subjected to automated IHC on Autoimmunostainer Ventana benchmark XT was taken as the reference. The following parameters were used for comparing the staining in both the AR methods: Uniformity of nuclear staining, nuclear staining quality, internal control staining, background staining, and Allred score. The sensitivity and specificity and positive and negative predictive values for each method were calculated.

RESULTS

The results were divided into technique-based and microscopy-based. Both techniques have their advantages and disadvantages which are outlined in Table 1. On microscopy, the quality and uniformity of nuclear staining and background staining, as well as Allred scores, were comparable in both methods [Table 2 and Figure 2a-d]. Uniformity of staining was found to be better in microwave oven AR as compared to pressure cooker AR. Sensitivity for ER IHC performed manually on microwave oven as well as on pressure cooker was 94% and specificity was 100%. Sensitivity and specificity for PR IHC performed manually on microwave oven were calculated and it was 94% and 100% and on pressure cooker and was 88% and 100%, respectively. The positive and negative predictive values were also calculated in both AR methods [Table 3].

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Figure 2:

(a) ER immunoexpression by microwave oven method. (b) PR Immunoexpression by microwave oven method. (c) ER immunoexpression by pressure cooker method. (d) PR immunoexpression by pressure cooker method. ER: Estrogen receptor, PR: Progesterone receptor.

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There was no false positive case in both the methods, while there was one false negative case in both ER and PR using the microwave oven method. On using the pressure cooker method, one false negative case was found in ER while two false negative cases were found in PR. Comparison of the individual Allred scores of manual and automated IHC revealed similar results in most cases with both ER and PR. In three out of 30 cases, a higher Allred score was observed in manual IHC as compared to the automated IHC.

DISCUSSION

The analysis of ER and PR expression levels by IHC is an important part of the initial evaluation of breast cancer and helps in assisting treatment planning.^[2]

The present study was undertaken on 30 cases of breast carcinoma to compare two methods of heat-induced AR -microwave oven and pressure cooker for manual IHC in the assessment of expression of ER and PR.

On comparing the expression of ER and PR in both methods, we found a similar rate of expression for ER in both methods (57%) while there was only a marginal increase in PR expression by microwave oven (53%) as compared to the pressure cooker (50%).

This contrasts with the findings of Qadir et al.^[11] who demonstrated a higher ER expression with a pressure cooker as compared to microwave heating. They did not study PR expression in their study. Furthermore, the quality and uniformity of nuclear staining were comparable in both methods. While no false positive was found in either of the two methods, one false negative each was observed in both ER and PR using a microwave oven. On using the pressure cooker method, one false negative was found in ER while two false negatives were found in PR^[12] (Neves et al.) also found no false-positive results in their study. They however found more false negatives with the microwave oven and no false negatives with the pressure cooker.

Out of the false negatives in PR expression, one occurred with the microwave oven method while two occurred with the pressure cooker method. The case which was a false negative on the microwave oven also turned out to be a false negative on the pressure cooker. For this case, even the ER expression was reduced with both the methods. The reference Allred score for this case was 7/8 in ER and 6/8 in PR. Manual IHC in two different tumor sections was performed but similar results were seen. One of the reasons for this false negativity for PR expression and reduction in ER expression could be attributed to improper fixation as the internal control was also not stained in this case. Improper fixation is an important cause of false negatives.^[10]

Improper fixation might also be the most probable reason for the single false negative observed in ER expression in both microwave oven and pressure cooker methods which occured in the same case. The other false negative observed in PR expression occurred using the pressure cooker method. The reference Allred score for this case was 7/8. This case had strong ER expression using both microwave oven and pressure cooker. PR expression with microwave oven was also strong and the Allred score was 5/8. In this case, the false negative could be procedure-related.

Sensitivity and specificity were also calculated for ER and PR in both methods using the automated IHC Allred score as a reference. For expression of ER, we observed a sensitivity of 94% for both microwave oven and pressure cooker. The sensitivity for PR expression was also comparable between a microwave oven and pressure cooking (94% vs. 88%). On comparing the individual Allred scores of manual and automated IHC, almost similar Allred scores were observed in most cases in both ER and PR. In a few cases, a higher Allred score was observed in manual IHC as compared to the automated IHC.

CONCLUSION

With the advent of automated IHC platforms, the need for manual retrieval techniques is diminishing. However, in a developing country like India with many small-scale laboratories with limited resources, manual IHC is still cost-effective, does not require much space, and is very flexible with regards to experimenting with different buffers, time, and temperature.

In such circumstances, manual IHC with proper standardization can be utilized. Manual staining offers an advantage pertaining to flexibility in choosing reagents and retrieval methods and the possibility of applying subtle variations in technique when optimizing a staining protocol.^[13] On technical aspects, a pressure cooker offers the advantage of being more convenient due to the possibility of simultaneous handling of more slides and being more cost-effective and less time-consuming.

The retrieval methods need to be decided keeping the needs of the laboratory in mind along with the cost factor. Following standard protocols at all steps would ensure optimum AR and in turn ensure accurate results which ultimately impacts patient management.