Anatomic Pathology / MINIMUM FORMALIN FIXATION TIME FOR CONSISTENT IMMUNOHISTOCHEMICAL STAINING

To identify the minimum time necessary for consistent immunohistochemical estrogen receptor (ER) results in our laboratory, we evaluated results in timed fixation blocks and cases with disparate and similar needle core biopsy and partial mastectomy specimens. Tissue sections of 24 ER-positive, invasive breast carcinomas were fixed for 3, 6, 8, and 12 hours and 1, 2, and 7 days. ER values were quantified using the Q score (0-7). In timed fixation blocks, the mean Q score per block was 2.46 for blocks fixed for 3 hours, 5.75 for blocks fixed for 6 hours, and 6.70 for blocks fixed for 8 hours (P < .001). The difference between the case maximum and mean block Q scores was a plateau of almost 0 at 6 to 8 hours of formalin fixation. For needle core biopsy specimen fixation times, the means for specimens with ER-disparate and ER-similar results were 1.2 and 6.3 hours, respectively (P = .01). The minimum formalin fixation time for reliable immunohistochemical ER results is 6 to 8 hours in our laboratory, regardless of the type or size of specimen. Immunohistochemical analysis is the standard detection method for evaluating estrogen receptor (ER) expression levels in invasive breast carcinoma cells. Consistent ER results are important because they are integral in clinical therapeutic decisions.1-3 The application of heat antigen retrieval pretreatment improves ER staining.3-5 A threshold in the amount of heat energy applied during this step can reduce or eliminate staining variations caused by irregular formalin fixation.4-7 Most studies have focused on issues pertaining to antigen retrieval of carcinomas that were fixed for 24 hours or longer.8,9 Prolonged formalin fixation is rarely a problem in our laboratory. Rapid turnaround times requested by clinicians result in minimal fixation. Most specimens were processed the same day they were excised, allowing only several hours of fixation. Several cases of disparate ER results between needle core specimens received late in the afternoon and the subsequent partial mastectomy specimen prompted us to question whether a threshold of insufficient fixation had been crossed. We studied relationships between short formalin fixation times and ER results and identified the minimum formalin fixation time to yield consistent ER results in our laboratory. Materials and Methods The study had 2 parts. Part 1: Timed Fixation Tissue Blocks In part 1 of the study, we evaluated ER levels using timed fixation tissue blocks from 24 large carcinomas that had strongly ER-positive results (stained nuclear area, >90%; Anatomic Pathology / ORIGINAL ARTICLE Am J Clin Pathol 2003;120:86-92 87 87 DOI: 10.1309/QPHDRB00QXGMUQ9N 87 © American Society for Clinical Pathology Q score, 6 or 7) in corresponding needle core biopsy specimens. We retrieved the carcinomas from the operating room immediately when they were removed from the patients. Tissue sections from the periphery of the carcinoma that included adjacent normal breast parenchyma were placed simultaneously in 10% neutral-buffered formalin. None of the patients had undergone neoadjuvant therapy. All carcinomas were index neoplasms and from the initial resection; none were recurrences. Seventeen neoplasms were ductal (not otherwise specified) carcinomas, 6 were lobular carcinomas, and 1 was a tubular carcinoma. The tissue sections were removed after fixing for precisely 3, 6, 8, 10, and 12 hours and 1, 2, and 7 days. The tissue sections were temporally stored in 100% cold ethanol until transfer to the automated tissue processor instruments. The blocks were fixed for an additional 90 minutes in heated (40°C) 20% formalin on the tissue processor before being placed in dehydration solutions. Tissue sections 3 to 4 μm thick were placed on charged slides and stored in a refrigerator until they were stained immunohistochemically. ER staining was assessed semiquantitatively by using the Q score method. This method incorporates intensity and distribution of reactivity.2,9 Intensity was scored as follows: 0, negative (no staining of any nuclei at high magnification); 1, weak (staining visible only at high magnification); 2, moderate (staining readily visible at low magnification); or 3, strong (staining strikingly positive at low magnification). The proportion of stained cells was scored as follows: +0, 0%; +1, 1% to 25%; +2, 26% to 50%; +3, 51% to 75%; or +4, >75%. Intensity and proportion of stained cells were added for the Q score, which had a range of 0 to 7. Part 2: Needle Core Biopsy Specimens The second part of the study evaluated the clinical impact of the shortened fixation time for needle core biopsy specimens on immunohistochemical ER results. Needle core biopsy specimens from 9 patients with disparate results (ERnegative needle core biopsy specimens and ER-positive partial mastectomy specimens) were identified retrospectively from the files of William Beaumont Hospital, Royal Oak, MI, for the period July 1, 1999, through December 30, 2001. The control group was 36 randomly selected, needle core biopsy specimens with ER values that were similar in the needle core biopsy and resection specimens. Twelve of the 36 (33%) control group needle core biopsy specimens had invasive carcinoma with a stained nuclear area of less than 10% (ER-negative), 12 (33%) had invasive carcinoma with a stained nuclear area of 20% to 50% (ER-positive, low), and 12 (33%) had invasive carcinomas with a stained nuclear area of more than 80% (ER-positive, high). All needle core biopsy specimens were unfixed when they arrived in surgical pathology, where they were immediately accessioned and placed into formalin. An approximate fixation time for the needle core biopsy specimens was recorded in each case by using the accession time and the tissue processor start time. A CAS 200 Image Analyzer (Becton Dickinson, Elmhurst, IL) was used to quantify the stained nuclear area of invasive carcinoma cells compared with the total nuclear area. Immunohistochemical Analysis Slides from every tissue block were stained immunohistochemically using 2 procedures that differed only in the length of time for antigen retrieval. The standard procedure used 40 minutes of antigen retrieval with a 0.001-mol/L concentration of EDTA buffer (pH 8) in a 95°C water bath. The second immunohistochemical staining procedure used 25 minutes, instead of 40, for antigen retrieval. The second staining procedure was used to amplify any possible signal alterations induced by the length of formalin fixation. After cooling for 20 minutes on the counter, the slides were removed from the antigen retrieval containers, washed with tap water, and incubated with 3% hydrogen peroxide for 15 minutes. The slides were transferred to an autostainer (DAKO, Carpinteria, CA) in which the primary ER antibody (clone 1D5, 1:50 dilution; DAKO) was incubated over the slides for 1 hour. After washing, the chromogenic components of the DAKO Envision system were used with diaminobenzidine. The slides were counterstained with methyl green, dehydrated, and coverslipped using synthetic mounting media. Statistical analysis was performed using the Systat computer program (version 10, SPSS, Chicago, IL). Mean values were compared by using the paired t test. Analysis of variance was used to compare differences in the mean Q score values of the 2 protocols. Linear regression was used to compare the difference in immunohistochemical ER staining in needle core biopsy specimens and resection specimens with the fixation period for the needle core biopsy specimens.