Effect of decalcification on immunohistochemical staining of trichorhinophalangeal syndrome type 1

To evaluate the effect of different decalcification solutions on the immunohistochemical staining of trichorhinophalangeal syndrome type 1 (TRPS1), and to provide a reliable basis for the accurate diagnosis of bone metastasis of breast cancer. Due to the limited biopsy samples of bone metastatic cancer, 20 cases of invasive breast cancer were selected to simulate bone metastatic biopsy, dividing into four groups: undecalcified group, 30% formic acid group, 10% hydrochloric acid group and 10% nitric acid group. Immunohistochemical staining was performed after treatment for 2, 6, 18 and 24 h. There was no change in the proportion and intensity of TRPS1 cells in the formic acid decalcification group within 18 h, but decreased significantly after 24 h. The staining intensity of TRPS1 and the proportion of stained cells were significantly decreased in the hydrochloric acid decalcification group since 2 h treatment. The percentage and intensity of positive cells in the nitric acid decalcification group changed little or no change within 6 h, and then gradually decreased with the extension of time. Another three invasive breast cancer samples were used to compare the effects of different decalcification solutions on the same case. In brief, we conclude that the effect of 30% formic acid decalcification on TRPS1 staining is small when the time is less than 18 h. 10% nitric acid decalcification should be controlled within 6 h, which has little effect on TRPS1 staining, and 10% hydrochloric acid decalcification will lead to significantly less positive TRPS1 staining, so it is not recommended for breast bone metastasis tissue decalcification.

Immunohistochemical staining was performed after treatment for 2, 6, 18 and 24 h.
There was no change in the proportion and intensity of TRPS1 cells in the formic acid decalcification group within 18 h, but decreased significantly after 24 h.The staining intensity of TRPS1 and the proportion of stained cells were significantly decreased in the hydrochloric acid decalcification group since 2 h treatment.The percentage and intensity of positive cells in the nitric acid decalcification group changed little or no change within 6 h, and then gradually decreased with the extension of time.Another three invasive breast cancer samples were used to compare the effects of different decalcification solutions on the same case.In brief, we conclude that the effect of 30% formic acid decalcification on TRPS1 staining is small when the time is less than 18 h.10% nitric acid decalcification should be controlled within 6 h, which has little effect on TRPS1 staining, and 10% hydrochloric acid decalcification will lead to significantly less positive TRPS1 staining, so it is not recommended for breast bone metastasis tissue decalcification.

| INTRODUCTION
Trichorhinophalangeal syndrome type 1 (TRPS1) is a highly sensitive and specific marker of breast cancer, which is more beneficial than other markers in the diagnosis of breast cancer metastases. 1,2Bone is the most common site of distant metastasis in breast cancer, accounting for about 65%-75% of all metastatic breast cancer patients, and once metastasis occurs, the 5-year survival rate will drop to 10%. 3,4id decalcification is commonly used in clinical practice to decalcify bone metastases. 5While the effect of decalcification on the detection performance of breast cancer marker TRPS1 is largely unknown, this study evaluated the effect of several of the most common acid decalcification solutions on TRPS1 staining.

| Materials
Breast cancer tissues of 23 cases were collected, washed with normal saline and fixed with 10% neutral formalin solution for 24 h.The collected breast cancer tissues are samples that have been diagnosed as invasive breast cancer by freezing and rapid pathology.Generally, the maximum diameter of the mass should be at least 1 cm before collecting the samples, so as not to affect the issuance of clinicopathological examination reports.A piece of cancer tissue with the size of 10 Â 10 Â 3 mm 3 was taken from each sample, and each tissue was evenly divided into 4 parts, the size of which was about 2.5 Â 10 Â 3 mm 3 .
The 23 breast cancer patient's age ranged from 30 to 84 years.
All the specimens were invasive ductal carcinoma.The maximum diameter of the tumors ranged from 1.3 to 5.2 cm, and lymph node metastasis occurred in 13 cases.The expression of ER/PR/HER2 can be seen in the following Table 1.

| Methods
Breast cancer tissues were randomly divided into four groups: undecalcified group, 30% formic acid group, 10% hydrochloric acid group and 10% nitric acid group, with 5 cases in each group.The three groups of breast cancer tissues were placed in the corresponding decalcified solution at room temperature for 2, 6, 18 and 24 h, respectively, and washed with running water for 3 h, then routine paraffin embedding section staining was performed.Immunohistochemistry was performed to detect TRPS1, and the influence of different decalcification solutions on TRPS1 staining was evaluated.
The standard methodology for metastatic bones in hospital are as follows: The most commonly used 10% nitric acid treatment of bone metastases about 16-18 h, with pin puncture without obstruction as the standard.

| Immunohistochemical staining
Immunohistochemical EnVision two-step method was used for staining, paraffin specimens were sliced for 3 μm and baked at 65 C for 30 min, then dewaxed to water.Citric acid antigen repair solution (pH 6.0) high pressure hot repair for 12 min.After it was cooled to room temperature, it was blocked for 10 min to eliminate endogenous peroxidase activity, and then TRPS1 primary antibody was added (Abcam, UK, 1:8000).Incubate in a refrigerator at C overnight for about 16 h, incubate in room temperature for 30 min with secondary antibody, and wash with phosphate buffer (PBS) for three times after each step.Finally, DAB color development, redyeing, differentiation, gradient ethanol dehydration, xylene transparent, neutral gum seal.

| Immunohistochemical results
The TRPS1 protein is localized in the nucleus.No positive staining was 0 points, 1 point for 1%-10%, 2 points for 11%-30%, and 3 points for 51%-100%.Negative staining intensity of positive cells was 0 points, weak positive was 1 points, medium positive was 2 points, strong positive was 3 points, and the final score was the proportion of positive cells multiplied by positive cell staining intensity value, 0-1 was negative, 2 was low expression, 3-4 was medium expression, and 6-9 was high expression.3 | RESULTS

| Effect of formic acid decalcification on TRPS1 immunohistochemical staining
30% formic acid decalcification treatment had the least effect on the staining strength of TRPS1 within 18 h, and when decalcification was extended to 24 h, the staining strength of TRPS1 significantly decreased, as shown in Figure 1.

| Effect of decalcification of hydrochloric acid on immunohistochemical staining of TRPS1
Decalcification with hydrochloric acid had the greatest effect on TRPS1, which showed a significant decrease in TRPS1 staining intensity and proportion of stained cells after 2 h treatment.It is not recommended to use 10% hydrochloric acid as decalcification fluid for bone metastases in clinic, so as not to affect subsequent tests, as shown in Figure 2.   can accelerate the speed of bone decalcification, and 8%-15% hydrochloric acid is the most widely used rapid decalcification agent.Shawn

C. Maclary et al. reported an abnormal decrease in antigen expression
of prognostic markers of breast cancer after 2 h of treatment with HCL based decalcification solution. 6This is also consistent with the conclusion of our current study, which once again confirmed that strong acid decalcification can destroy the antigen immune activity of tumor tissue.
Studies have found that exposing soft tissue to decalcifiers, as well as soft tissue with additional bone, there was no significant difference between the two samples. 6However, tumor cells surrounded by bone calcified matrix cannot be fully simulated, which is a major limitation of this study.TRPS1 is a nuclear positive protein marker, and to a certain extent, it also weakens the influence of tumor surrounding matrix on its detection.Therefore, we think that using tumor tissue to simulate decalcification samples can replace bone metastasis samples to a certain extent, and evaluate the influence of decalcification on TRPS1 expression in tumor cells.
In summary, we conclude that the intensity and time of decalcification directly affect the staining of TRPS1.Although short periods of decalcification result in little to no change, decalcification of even weak formic acid for more than 18 h can cause significant adverse effects.
These two factors should be considered when deciding to decalcify samples that may require immunohistochemistry, particularly TRPS1.

GraphPad Prism 6
software (GraphPad Software, San Diego, CA, USA) was used to conduct all the statistical analyses.Alteration of expression of the proteins and the behavioral responses were tested with one-way analysis of variance (ANOVA).Results are expressed as mean ± SEM. p values <.05 were considered significant.F I G U R E 1 Effects of 30% formic acid decalcification on the immunohistochemistry of TRPS1.Samples were decalcified with 30% formic acid for 2, 6, 12 and 24 h.Immunohistochemical images (A) and scores (B) of TRPS1 in breast cancer tissue.The scores were the proportion of positive cells multiplied by positive cell staining intensity value.Total magnification: 40Â and 100Â.Significant differences were revealed following oneway ANOVA (*p < .05,**p < .01,***p < .001and ****p < .0001;Bonferroni post hoc tests).
Effects of 10% hydrochloric acid decalcification on the immunohistochemistry of TRPS1.Immunohistochemical images (A) and scores (B) of TRPS1 in breast cancer tissue.The scores were the proportion of positive cells multiplied by positive cell staining intensity value.Total magnification: 40Â and 100Â.Significant differences were revealed following one-way ANOVA (*p < .05,**p < .01,***p < .001and ****p < .0001;Bonferroni post hoc tests).

3. 4 |
Effects of different decalcification solutions on immunohistochemical staining of TRPS1 in the same sample We further used the same breast tissue to verify the effects of different decalcification solutions on immunohistochemical staining of TRPS1.As shown in Figure 4, after decalcification with formic acid (18 h), hydrochloric acid (2 h) or nitric acid (6 h), the expression of F I G U R E 3 Effects of 10% nitric acid decalcification on the immunohistochemistry of TRPS1.Immunohistochemical images (A) and scores (B) of TRPS1 in breast cancer tissue.The scores were the proportion of positive cells multiplied by positive cell staining intensity value.Total magnification: 40Â and 100Â.Significant differences were revealed following one-way ANOVA (*p < .05,**p < .01,***p < .001and ****p < .0001;Bonferroni post hoc tests).TRPS1 in formic acid group and nitric acid group did not change significantly due to decalcification treatment, but the decalcification of hydrochloric acid had a greater effect on TRPS1, and the expression of TRPS1 was significantly reduced.4| DISCUSSIONTRPS1 has been found to be a highly specific and sensitive marker for all types of breast cancer, especially triple negative breast cancer (TNBC), and can help to confirm whether metastasis originates in the breast with less immunohistochemical tests on limited biopsies.Its nuclear positive staining is also easier to read than the cytoplasmic staining of GCDFP-15 and Mammaglobin.In the process of decalcification of bone metastases, acid treatment may change the antigenicity of tumor cells, thus affecting the immunomarker status of tumor at the site of metastasis.And the effect of decalcification on TRPS1 staining, as far as we know, no studies have been published on this topic.Our results suggest that different decalcification methods have different effects on the detection of TRPS1.Firstly, 30% formic acid decalcification could better preserve the antigenic immune activity of TRPS1, while hydrochloric acid decalcification treatment had the greatest effect on TRPS1, and significantly damaged the antigenic immune activity of TRPS1 after only 2 h of treatment.However, 10% nitric acid, the most commonly used decalcification solution in clinicopathological tests, showed little or no change in the percentage and intensity of TRPS1 positive within 6 h, and then gradually decreased with the extension of decalcification time.The acid decalcification solution used in this experiment is mainly formic acid, hydrochloric acid and nitric acid.Formic acid is the organic acid closest to the critical point of inorganic acid and organic acid, when the concentration reaches 30%, its decalcification effect is the best。Hydrochloric acid and nitric acid are both strong acids that F I G U R E 4 Effect of nitric acid decalcification on immunohistochemical staining of TRPS1.Immunohistochemical images (A) and scores (B) of TRPS1 in breast cancer tissue.The scores were the proportion of positive cells multiplied by positive cell staining intensity value.Total magnification: 40Â and 100Â.Significant differences were revealed following one-way ANOVA (*p < .05,**p < .01,***p < .001and ****p < .0001;Bonferroni post hoc tests).
Characteristics of the BC cases.