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Identifying and Quantifying Apoptosis: Navigating Technical Pitfalls Megan M Identifying and Quantifying Apoptosis: Navigating Technical Pitfalls Megan M. Garrity, B.S., Lawrence J. Burgart, M.D., Darren L. Riehle, B.A., C.T., Eunice M. Hill, B.S., C.T., Thomas J. Sebo, M.D., Ph.D., Thomas Witzig, M.D. Department of Laboratory Medicine and Pathology (MMG, LJB, DLR, EMH, TJS) and Department of Internal Medicine (TW), Mayo Clinic, Rochester, Minnesota KEY WORDS: Apoptosis, Digital image analysis, Im- Apoptosis or programmed cell death is often age morphometry, Necrosis, TUNEL assay. altered in malignancies and is frequently deter- Mod Pathol 2003;16(4):389–394 mined by the terminal transferase–mediated nick end labeling technique (TUNEL). However, Apoptosis is a physiologic form of cell death that commercially available protocols can produce serves as one aspect of tissue growth and size reg- high background and false-positive staining, ulation (1). The inhibition of apoptosis is believed which renders the distinction between apopto- to play a role in carcinogenesis in one of two ways: sis and necrosis difficult. In an attempt to de- (1) it may allow for the unchecked accumulation of velop a rapid and reproducible method for de- genetic alterations (2, 3) and (2) it may lead to tecting and quantifying apoptosis, we coupled unbalanced proliferation of tumor (4). As such, it optimization of the Apoptag Plus Peroxidase In has become an area of intense interest in oncologic Situ Apoptosis Detection kit with quantitative research. histomorphometric computer imaging software The morphologic criteria for identifying cells un- using the Bacus Laboratories Incorporated Slide dergoing apoptosis are well established and include Scanner (BLISS). Multiple (200–350) unique cytoplasmic condensation, loss of cell–cell contact, and cell shrinkage. This is separate and distinct -40؋ images were scanned using the BLISS sys from necrosis, an alternate form of cell death that tem and downloaded into the WebSlide Browser involves cell swelling and rupture with associated program, creating a permanent, scanned record surrounding tissue damage (5–7). Although cells of the area assessed. The stored images were undergoing apoptosis can be identified using these counted, with the final analysis simultaneously morphologic criteria on a standard hematoxylin taking into account cells that were immunohis- and eosin (H&E) slide, studies have shown that tochemically positive and the histology of the using this method alone may underestimate the surrounding cells to reduce the possibility of rate of apoptosis by 2-fold to 3-fold (8). As a result, false positive and negative staining. In addition, there is increased interest in finding a reliable, re- cells with equivocal staining can be simulta- producible method for identifying and quantifying neously reviewed by other technologists with apoptosis in whole tissue sections. networked WebSlide Browser access to the same Apoptosis is most commonly identified in tissue images. Our data show that the advantages of- using terminal deoxynucleotidyl transferase–medi- fered by the BLISS imaging software greatly re- ated dUTP nick end labeling (TUNEL). TUNEL duce the potential drawbacks of using the TUNEL works on the principle that DNA strand breaks oc- method as a sole means of quantification. cur during apoptosis. Terminal deoxynucleotidyl transferase (Tdt) catalyzes the labeling of these breaks with deoxyuridine triphosphates (dUTPs), which are then detected by immunoperoxidase Copyright © 2003 by The United States and Canadian Academy of techniques (8–10). Several kits are commercially Pathology, Inc. VOL. 16, NO. 4, P. 389, 2003 Printed in the U.S.A. available for TUNEL detection. However, reports Date of acceptance: January 28, 2003. Supported in part by a grant from the National Cancer Institute have indicated incidences of high background and (CA78899). false-positive staining, making interpretation diffi- Address reprint requests to: Megan Garrity, B.S., Mayo Clinic, Department of Laboratory Medicine and Pathology, 200 First Street SW, Rochester, MN cult (6, 8, 9, 11). 55905; e-mail: [email protected]. This high rate of false-positive staining can po- DOI: 10.1097/01.MP.0000062657.30170.92 tentially arise from several factors. First, initial fix- 389 ation of tissue sections that is too extensive, incom- Multiple sections were stained using the Apoptag plete, or delayed can lead to nonspecific staining. Plus Peroxidase In Situ Apoptosis Detection Kit (In- Second, the need for unmasking after formalin fix- tergen Company, Purchase, NY) or the In Situ Cell ation and paraffin embedding, i.e., through pro- Death Detection Kit, AP (Roche Diagnostics Corp, tease addition, can produce artificial strand breaks Indianapolis, IN). Studies were done to evaluate the that are unable to be immunohistochemically dif- effects of proteinase K, Tdt concentration, second- ferentiated from true apoptosis. Finally, necrosis ary substrate (anti-digoxigenin-peroxidase or anti- also results in DNA strand breaks and as such rep- fluorescein antibody AP conjugated) incubation resents a target for Tdt–mediated dUTP labeling (1, and counterstaining on overall reproducibility, sen- 8, 11, 12). Although compensating for deficits in sitivity, and interpretation of stain. For each kit, initial tissue fixation in retrospective studies is proteinase K concentration was varied from 5–30 problematic, addressing the issue of overall optimi- ␮g/mL (5, 10, 15, 20, 25, and 30 ␮g/mL) in phos- zation remains crucial if the role of apoptosis in phate buffered saline (PBS) and incubated from carcinogenesis is to be delineated. In addition, 5–20 minutes (5, 10, 15, and 20 minutes) at each many investigators have suggested that proper in- concentration. After optimizing the time and con- terpretation of TUNEL results requires a second centration for proteinase K (25 ␮g/mL for 20 min- confirmatory methodology (12–14). utes), samples were incubated at both room tem- Initially we compared two commercially avail- perature and 37° C to determine any other able TUNEL kits to determine the ease of optimi- substantive differences. Tdt concentration was var- zation and the reproducibility across serial tissue ied from the recommended dilution to half of that. sections. We then investigated two digital image Anti-digoxigenin-peroxidase or anti-fluorescein an- morphometry systems to see whether either could tibody AP secondary substrate (Apoptag Plus Per- yield reproducible, reliable values for the rate of oxidase In Situ Apoptosis Detection Kit and In Situ apoptosis while also incorporating multiple meth- Cell Death Detection Kit, AP respectively) was in- odologies and technical checks for quantifying ap- cubated at both room temperature and 37° C. Fi- optosis. The two methodologies evaluated were (1) nally, because digital imaging systems delineate morphometric area analysis (MAA), in which the positive and negative cells using the difference in apoptotic index is reported as a percentage of area wavelength between chromogen and counterstain, stained positive versus total area stained using the we tested the effects of diluted hematoxylin, fast Cell Analysis System (CAS 200, Bacus Laboratories, red, and methyl green counterstaining on overall Inc., Lombard, IL) and (2) morphometric cell count readability with the CAS 200 and BLISS. (MCC) analysis, in which the apoptotic index is To test for reproducibility and sensitivity under reported as the total number of apoptotic cells ver- optimal staining conditions, 10 serial sections of sus the total number of cancer cells using the Bacus tonsil tissue were cut from each of two blocks. Five Laboratories Incorporated Slide Scanner (BLISS, of each were then stained using both the Apoptag Bacus Laboratories, Inc.). We evaluated both of Plus Peroxidase In Situ Apoptosis Detection kit (Ap- these methods for quantifying apoptosis as com- optag) or the In Situ Cell Death Detection kit, AP pared with semiquantitative visual scoring of 1, 2, (ISCDD-AP). Similar studies were done on colorec- or 3ϩ to determine how precisely each technique tal adenocarcinoma sections to confirm reproduc- can discriminate between rates of apoptosis. ibility and sensitivity before development of quan- tification techniques. MATERIALS AND METHODS Tissue Samples TUNEL Assay for Development of Quantification Assay Formalin-fixed, paraffin-embedded tissue blocks were obtained and serial 4-␮m sections were pro- Thirty-five colorectal adenocarcinomas were cessed for use in the TUNEL assays. Both tonsil and stained in duplicate using the Apoptag kit previ- colorectal adenocarcinoma tissues were used for ously optimized for formalin-fixed, paraffin- initial TUNEL optimization. Colorectal adenocarci- embedded whole tissue sections. Formalin-fixed, noma tissue was used for subsequent development paraffin-embedded tissue slides were rehydrated of the quantification assays. The use of all samples using xylene to alcohol washings, followed by a hydrogen peroxide–methanol quench. The samples was approved by the institutional review board of ␮ the Mayo Clinic. were treated with 25 g/mL of proteinase K at 37° C for 20 minutes. After washing and incubation with equilibration buffer for 5 minutes, Tdt was diluted TUNEL Optimization 1:3.9 (14 ␮L of Tdt enzyme in 40 ␮L of reaction Serial sections of tonsil tissue were cut from sev- buffer) and incubated on the slides for 1 hour at 37° eral formalin-fixed, paraffin-embedded blocks. C. After applying stop solution for 15 minutes and 390 Modern Pathology washing, the samples were incubated
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