ARTICLE |
Correspondence to: Adrien Negoescu, Groupe de Recherche sur le Cancer du Poumon, Institut Albert Bonniot, Faculté de Médecine, Domaine de la Merci, 38706 Grenoble cedex, France.
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Summary |
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TUNEL, i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, has become a widely used staining method to assist in detection of apoptotic cells in tissue sections. However, despite its apparent simplicity, this technique has led to considerable disappointment because of its serious limitations in sensitivity and, even more, in specificity. We reviewed the limitations and artifacts of TUNEL and designed a comprehensive protocol to reassess the various procedures in use for five crosslinking and/or precipitating fixatives. By introducing microwave heating in extreme pH-value solutions (pH 3 for formalin and pH 10.6 for Bouin's fixative) coupled with proteolysis, we obtained an intense staining of 7080% of apoptotic cells and bodies on archival tissue blocks, with little or no background. Owing to the enhanced sensitivity, early stages of apoptosis could be visualized and may enlarge our vision of the apoptotic cell beyond the mere image of shrinkage necrosis. We conclude that TUNEL remains a technique as useful as it is delicate, requiring critical interpretation of the staining. This study points out that, on archival tissues, despite the technical improvements we propose no protocol can be the final answer to all problems. Technique must be readjusted for any variation in tissue processing. However, step-by-step progress has rendered this method not only applicable but also performable within the constraints of archival surgical pathology specimens. (J Histochem Cytochem 46:327334, 1998)
Key Words: TUNEL, sensitivity, specificity, microwaves, pH, preapoptosis
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Introduction |
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In the absence of a universal specific molecular tracer of apoptosis, structural DNA alterations provide the basis for labeling systems: double-strand fragmentation for TUNEL (
By introducing microwaves to TUNEL staining of cultured cells, we obtained considerable gains in sensitivity without impairment of specificity (
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Materials and Methods |
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We proceeded in two steps: After having adjusted TUNEL using various fixatives, pretreatments, and labeling protocols on the same Graves' thyroid specifically prepared for this purpose, the optimized protocol was put to the test of a retrospective investigation of 35 archival cases of Graves' thyroid. The results, markedly less satisfactory, stressed the extreme sensitivity of TUNEL to tissue processing. Technical investigation was taken up again, now targeted at the two fixatives used for most archival tissues, formalin and Bouin's liquid.
Fixation
Prospective Study.
Fragments of thyroid parenchyma measuring 58 mm on each side and 3 mm thick (less than 0.5 cm3) were fixed by immersion. Five fixatives were used: 4% (w/v) paraformaldehyde (PFA) in 0.01 M PBS, pH 7.4; 4% (v/v) formaldehyde (formalin) in PBS (pH 7.4); B5 [8% (w/v) mercury chloride in 4% formaldehyde] (pH 6.5); Bouin's liquid [2.5% (w/v) copper acetate, 4% (w/v) picric acid in 3.5% (v/v) formaldehyde, 1.5% (v/v) acetic acid in distilled water] (pH 3.8); and Methacarn [10% (v/v) acetic acid, 30% (v/v) chloroform in methanol] (pH 5.0). Fixation lasted about 20 hr at room temperature (RT) for all fixatives except Methacarn (2 hr).
Retrospective Study. Archival paraffin-embedded specimens measured 15 cm2 and were 1.53 mm thick. Each had been fixed at RT by immersion, dispatched between formalin and Bouins' liquid, 20 hr to 4 days, with no specification given as to duration.
Pretreatments
Prospective study.
TUNEL was performed with or without pretreatment. For detergent, Triton X-100 (Sigma; Saint Quentin Fallavier, France) was used at 0.1% (v/v) in 0.1% (w/v) sodium citrate (Merck; Darmstadt, Germany) in water for 2 min on ice. Proteinase K (Boehringer; Mannheim, Germany) was used at 20 µg/ml in PBS for 15 min at RT. Other concentrations (2, 10, 20, and 40 µg/ml) and durations (5, 10, and 15 min) were tested.
For microwave irradiation, an AEG Micromat 120 oven (2.45 GHz) with nine power settings (0850 W) was used. Best results were obtained with Setting 4 (376 W), 5 min in 200 ml of 0.01 M citrate buffer, pH 6 (
As a combination pretreatment (
No inhibition of endogenous peroxidase was performed because H2O2 weakens TdT activity (
Retrospective Study. Detergent treatment was performed as in the previous step but was always combined with other pretreatments. The proteolytic enzyme was used at 15, 20, 25, or 40 µg/ml for 15 min at RT. For microwave irradiation, the slides (8 to 15) were immersed in 200 ml of the following solutions: 0.01 M citrate buffer (pH 3 and 6), 0.01 M sodium hydrogen carbonate (Merck; pH 9), 0.05 M tris- [hydroxymethyl]aminomethane (Tris; Sigma, pH 10.6) or a 1/1 (v/v) mixture of citrate buffer (pH 6) and Tris solution (pH 10.6) to a final pH of 9. Citrate buffer and the citrate/Tris mixture were heated for one or two cycles of 5 min at Setting 8 (752 W). To avoid section detachement in carbonate and Tris solutions, slides were first irradiated for 3 min at Setting 8 and then maintained at Setting 4 for 222 min. Boiling temperature was 99.6C for all solutions, reached in 3 min at Setting 8.
Pretreatment combinations included the following: (a) Triton followed by proteinase K (15, 20, and 25 µg/ml); (b) microwaving followed by Triton; (c) microwaving followed by proteinase K (15, 20, and 25 µg/ml); and (d) microwaving followed by Triton and then by proteinase K (15, 20, and 25 µg/ml).
TUNEL
Prospective Study.
Three modalities of TUNEL were tested: a laboratory protocol and kits from Boehringer Mannheim (In Situ Cell Death Detection Kit, Peroxidase) and from Oncor (ApopTag Plus; Gaithersburg, MD). The laboratory protocol was adapted from
Retrospective Study. The Boehringer Mannheim kit alone was used.
Cell Counting and Statistical Analysis
Prospective Study.
Once established, each optimal method was reproduced in triplicate and quantification was performed on a slide representative of each working condition. Thyreocytes in the vesicle lumina were assessed for morphology and staining, according to
Retrospective Study. The tissue sections were about 10 times larger than those used in the first step and we noted uneven labeling from one area to another. Therefore, a semiquantitative evaluation was performed, with four parameters (Table 1): fraction of apoptotic cells labeled; labeling intensity; non-apoptotic cell coloration; and connective tissue background.
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Results |
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Prospective Study (see Figure 1)
TUNEL Protocols.
The three modalities of TUNEL we tested (laboratory protocol, ApopTag kit, and Boehringer Mannheim kit) yielded no significantly different results, with the exception of proteinase K-pretreated tissue sections fixed with either Bouin or Methacarn, for which the Boehringer Mannheim kit proved more sensitive than the two others, having a good signal-to-background ratio and being the easiest to use; therefore, it was used for the retrospective study.
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Fixatives, Cells, and Labeling Morphology. The highest resolution of nuclear morphology was provided by Bouin's fluid (Figure 2D). Nevertheless, no difficulty was encountered in identifying the criteria of Kerr and Wyllie for apoptotic cells, no matter what the fixative. Some of the apoptotic bodies in the vesicle lumina were in secondary necrosis with loss of nuclear and cytoplasmic outlines. These were not included in the counts.
Effect of Pretreatments. TUNEL performed without any pretreatment yielded unsatisfactory results, with 851% of apoptotic-appearing cells being stained. The best pretreatment proved to be proteinase K (20 µg/ml, 15 min at RT) (p<0.05), with about 70% labeled apoptotic cells for aldehyde fixatives and Methacarn and about 60% for Bouin's fluid, without increasing the proportion of morphologically normal stained cells. Triton and microwaving led to a smaller gain in specific staining (p<0.05) while increasing the percentage of labeled cells with a non-apoptotic appearance. Combined microwaveproteinase K pretreatment (according to Sträter's protocol) did not prove to be more efficient than proteinase K alone (p>0.05). No important differences were noted between classically and rapidly cooled slides. Therefore, classic cooling (15 min) was preferred for its convenience.
Controls and Background Treatment (data not shown). Lack of TdT in the TUNEL mix abolished labeling. Positive controls exhibited many stained, condensed nuclei and micronuclei, either isolated or within the cytoplasm of phagocytic cells. The connective stroma of the thyroid was colored by TUNEL, sometimes intensely. This collagen-bound background was abolished in TdT-lacking controls. Therefore, to investigate the possibility of calf TdTcollagen interaction, we introduced a 20% normal bovine serum incubation before TUNEL, which significantly decreased background.
Retrospective Study (Table 1; Figure 2 and Figure 3)
The protocol retained as best adapted (proteinase K 20 µg/ml, 15 min at RT, followed by TUNEL performed with the Boehringer Mannheim kit) to light fixation of small tissue blocks, when applied to an archival series of 35 formalin- and Bouin-fixed Graves' thyroids, yielded disappointing results. Overall, three situations were encountered, either alone or combined on the same section: no staining, general staining of all nuclei (apoptotic or not), or "inverse staining," i.e., lack of or low staining of apoptotic cells surrounded by intense staining of virtually all normal nuclei (Figure 2A and Figure 2B). At best, the sensitivity was about 40% in formalin-fixed tissues and was less than 10% in Bouin-fixed samples (Figurea 2C and 2D). TUNEL was not improved by varying proteinase K concentration or by adding a detergent step to the enzymatic digestion. For microwaving, of the various pH values investigated with different irradiation times, the following did not improve TUNEL when used either alone or combined with Triton, proteinase K, or both: the carbonate solution (pH 9) altered the histological structures and frequently detached sections; the citrate/Tris mixture (same pH of 9 but less aggressive for the tissues); and citrate buffer (pH 6; Figure 2E and Figure 2F).
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Significant improvement was achieved when extreme pH values were explored (Figure 2G and Figure 2H). For formalin-fixed samples, citrate buffer (pH 3), twice for 5 min at 752 W, abolished background. Sensitivity remained stable compared to proteinase K digestion, and therefore the readability of the labeling was improved.
An increment in sensitivity to about 80% of apoptotic cells was achieved by addition of a second pretreatment with proteinase K (20 µg/ml). Labeling morphology was influenced by pretreatments. In the absence of proteolysis, nuclear labeling was faint and the cytoplasm of apoptotic cells was sometimes darker than the nuclei. Triton favored staining of apoptotic cell cytoplasm, together with an extremely clean general background. Proteinase K enabled the labeling to concentrate in the nuclei, the cytoplasm being either stained or unstained. Therefore, we adopted acidic pH and the three-pretreatment combination as our best protocol for formalin-fixed tissues. For Bouin-fixed sections, a similar improvement resulted from the use of the basic Tris solution (pH 10.6) with a comparable energy input (3 min at 752 W plus 7 min at 376 W), followed by Triton and proteinase K (20 µg/ml). The analysis of apoptosis in the 35-case series treated according to these protocols will be reported in the future (LabatMoleur et al., manuscript in preparation). The DAB color reaction was optimal when it was short (10 sec to 1 min) and was carefully monitored, under the microscope, slide by slide, to avoid background.
Thyreocytes with large nuclei (about 1.5 times as large as those of surrounding cells) and with a thin rim of marginated and condensed chromatin were observed in the vesicular epithelium in the vicinity of highly apoptotic foci. These dilated nuclei were quite numerous and their chromatin rim was as intensely TUNEL-positive as that of condensed apoptotic bodies. These cells may represent an early stage of apoptosis (Figure 2I).
In summary, among the fixation variations of our archival blocks, the best results were obtained with combined pretreatment: (a) microwave irradiation (around 10 min) at acidic pH (pH 3) for aldehyde-fixed tissues and basic pH (pH 10.6) for Bouin's-fixed tissues; and (b) proteinase K, 15 min at 20 µg/ml, the conditions used by most authors. Indeed, our tests revealed no gain when this parameter was varied. Triton proved to be optional. The optimal color reaction was brief, 10 sec to 1 min.
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Discussion |
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We attempted in this investigation to overcome the main drawbacks of TUNEL detection of apoptotic cells in tissue sections. Several authors (
We suggest separating artifactual staining of necrosis and mitosis from the body of criticism. Several authors (
The major problem with TUNEL, as recurrently emerging in the critical evaluations devoted to the technique and confirmed by our results, can be summarized as follows (Figure 2 and Figure 3). (a) Without pretreatments, TUNEL sensitivity is far too low. (b) Pretreatments with acknowledged efficiency, i.e., proteinase K and microwaves, easily induce general labeling of morphologically normal nuclei (
This points up the fact that TUNEL is an ambitious approach whose target, apoptotic DNA breaks, is less accessible than breaks that occur in non-apoptotic DNA. In fact, many known causes of DNA breaks can be listed: DNA recombination, replication, repair or compactionrelaxation during mitosis, tissue electrocoagulation, autolysis, fixation, paraffin embedding, cutting, and pretreatments with H2O2, detergent, proteinase K, and microwaves (
Even when optimal, pretreatments do not guarantee lack of background. Therefore, we agree with the trend to carefully control the detection system (
When fixation is controlled and can be kept homogeneous and light, as in prospective studies, proteinase K alone may be sufficient for all the crosslinking aldehyde fixatives we tested (paraformaldehyde, formalin, B5), with more than 70% apoptotic cells labeled (Figure 1). When tissues were fixed with precipitating solutions (Methacarn, Bouin) only about half of the apoptotic cells were stained. A comparison of proteinase K efficiency between the prospective and retrospective steps illustrates the dramatic drop in sensitivity (75% to 40%, 57% to 5% for Bouin) linked to the variation of block size and fixation duration (frequently drastic overfixation). In this situation, the combined use of microwaves plus proteinase pretreatment suggested by
As a consequence of enhanced sensitivity and specificity of TUNEL staining, the constant and intense labeling of dilated nuclei within a peculiar environment of vesicular remodeling adjacent to apoptosis-rich areas (manuscript in preparation) can be accepted as specific (Figure 2I). This image probably corresponds to the "swelling cells," stage 3 of the model proposed by
In conclusion, despite its hardly accessible target, step-by-step TUNEL improvements enable accurate identification of apoptosis, even within the constraints of archival surgical pathology specimens. Nevertheless, TUNEL is so sensitive to tissue processing that no unique protocol can be proposed. Whenever possible, fixation must be standardized. For archival samples, adaptative tests must be performed for each particular case series. Standardization of TUNEL is a critical issue that requires further studies based on control tissues or cell lines with known quantitative information concerning apoptosis. Such control tissues should be used to monitor the TUNEL procedure so as to achieve more accurate delineation of apoptosis.
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Acknowledgments |
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A. Negoescu acknowledges financial support from the COMARES (Comité des Maladies Respiratoires de l'Isère).
We thank Dr Ruth GriffinShea, who kindly revised the English version of this manuscript. We are indebted to Catherine Pépin and Jean-Michel Lasserre for the iconography.
Received for publication June 2, 1997; accepted September 30, 1997.
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