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. 2000 Sep;53(9):666–672. doi: 10.1136/jcp.53.9.666

Laser capture microdissection in pathology

F Fend 1, M Raffeld 1
PMCID: PMC1731250  PMID: 11041055

Abstract

The molecular examination of pathologically altered cells and tissues at the DNA, RNA, and protein level has revolutionised research and diagnostics in pathology. However, the inherent heterogeneity of primary tissues with an admixture of various reactive cell populations can affect the outcome and interpretation of molecular studies. Recently, microdissection of tissue sections and cytological preparations has been used increasingly for the isolation of homogeneous, morphologically identified cell populations, thus overcoming the obstacle of tissue complexity. In conjunction with sensitive analytical techniques, such as the polymerase chain reaction, microdissection allows precise in vivo examination of cell populations, such as carcinoma in situ or the malignant cells of Hodgkin's disease, which are otherwise inaccessible for conventional molecular studies. However, most microdissection techniques are very time consuming and require a high degree of manual dexterity, which limits their practical use. Laser capture microdissection (LCM), a novel technique developed at the National Cancer Institute, is an important advance in terms of speed, ease of use, and versatility of microdissection. LCM is based on the adherence of visually selected cells to a thermoplastic membrane, which overlies the dehydrated tissue section and is focally melted by triggering of a low energy infrared laser pulse. The melted membrane forms a composite with the selected tissue area, which can be removed by simple lifting of the membrane. LCM can be applied to a wide range of cell and tissue preparations including paraffin wax embedded material. The use of immunohistochemical stains allows the selection of cells according to phenotypic and functional characteristics. Depending on the starting material, DNA, good quality mRNA, and proteins can be extracted successfully from captured tissue fragments, down to the single cell level. In combination with techniques like expression library construction, cDNA array hybridisation and differential display, LCM will allow the establishment of "genetic fingerprints"of specific pathological lesions, especially malignant neoplasms. In addition to the identification of new diagnostic and prognostic markers, this approach could help in establishing individualised treatments tailored to the molecular profile of a tumour. This review provides an overview of the technique of LCM, summarises current applications and new methodical approaches, and tries to give a perspective on future developments. In addition, LCM is compared with other recently developed laser microdissection techniques.

Key Words: laser capture microdissection • RNA analysis • DNA analysis • gene expression • profiling • immunohistochemistry

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Figure 1 Schematic representation of laser capture microdissection. (A) Activation of the laser leads to focal melting of the polymer membrane. (B) Lifting of the cap selectively detaches the cells adherent to the activated membrane.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aubele M., Zitzelsberger H., Schenck U., Walch A., Höfler H., Werner M. Distinct cytogenetic alterations in squamous intraepithelial lesions of the cervix revealed by laser-assisted microdissection and comparative genomic hybridization. Cancer. 1998 Dec 25;84(6):375–379. doi: 10.1002/(sici)1097-0142(19981225)84:6<375::aid-cncr10>3.0.co;2-1. [DOI] [PubMed] [Google Scholar]
  2. Banks R. E., Dunn M. J., Forbes M. A., Stanley A., Pappin D., Naven T., Gough M., Harnden P., Selby P. J. The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis--preliminary findings. Electrophoresis. 1999 Apr-May;20(4-5):689–700. doi: 10.1002/(SICI)1522-2683(19990101)20:4/5<689::AID-ELPS689>3.0.CO;2-J. [DOI] [PubMed] [Google Scholar]
  3. Becker I., Becker K. F., Röhrl M. H., Minkus G., Schütze K., Höfler H. Single-cell mutation analysis of tumors from stained histologic slides. Lab Invest. 1996 Dec;75(6):801–807. [PubMed] [Google Scholar]
  4. Bernsen M. R., Dijkman H. B., de Vries E., Figdor C. G., Ruiter D. J., Adema G. J., van Muijen G. N. Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection. Lab Invest. 1998 Oct;78(10):1267–1273. [PubMed] [Google Scholar]
  5. Bianchi A. B., Navone N. M., Conti C. J. Detection of loss of heterozygosity in formalin-fixed paraffin-embedded tumor specimens by the polymerase chain reaction. Am J Pathol. 1991 Feb;138(2):279–284. [PMC free article] [PubMed] [Google Scholar]
  6. Bonner R. F., Emmert-Buck M., Cole K., Pohida T., Chuaqui R., Goldstein S., Liotta L. A. Laser capture microdissection: molecular analysis of tissue. Science. 1997 Nov 21;278(5342):1481–1481,1483. doi: 10.1126/science.278.5342.1481. [DOI] [PubMed] [Google Scholar]
  7. Brown M. R., Chuaqui R., Vocke C. D., Berchuck A., Middleton L. P., Emmert-Buck M. R., Kohn E. C. Allelic loss on chromosome arm 8p: analysis of sporadic epithelial ovarian tumors. Gynecol Oncol. 1999 Jul;74(1):98–102. doi: 10.1006/gyno.1999.5439. [DOI] [PubMed] [Google Scholar]
  8. Böhm M., Wieland I., Schütze K., Rübben H. Microbeam MOMeNT: non-contact laser microdissection of membrane-mounted native tissue. Am J Pathol. 1997 Jul;151(1):63–67. [PMC free article] [PubMed] [Google Scholar]
  9. Chandrasekharappa S. C., Guru S. C., Manickam P., Olufemi S. E., Collins F. S., Emmert-Buck M. R., Debelenko L. V., Zhuang Z., Lubensky I. A., Liotta L. A. Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science. 1997 Apr 18;276(5311):404–407. doi: 10.1126/science.276.5311.404. [DOI] [PubMed] [Google Scholar]
  10. Cossman J., Annunziata C. M., Barash S., Staudt L., Dillon P., He W. W., Ricciardi-Castagnoli P., Rosen C. A., Carter K. C. Reed-Sternberg cell genome expression supports a B-cell lineage. Blood. 1999 Jul 15;94(2):411–416. [PubMed] [Google Scholar]
  11. Darling T. N., Yee C., Bauer J. W., Hintner H., Yancey K. B. Revertant mosaicism: partial correction of a germ-line mutation in COL17A1 by a frame-restoring mutation. J Clin Invest. 1999 May 15;103(10):1371–1377. doi: 10.1172/JCI4338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. DeRisi J., Penland L., Brown P. O., Bittner M. L., Meltzer P. S., Ray M., Chen Y., Su Y. A., Trent J. M. Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat Genet. 1996 Dec;14(4):457–460. doi: 10.1038/ng1296-457. [DOI] [PubMed] [Google Scholar]
  13. Dean-Clower E., Vortmeyer A. O., Bonner R. F., Emmert-Buck M., Zhuang Z., Liotta L. A. Microdissection-based genetic discovery and analysis applied to cancer progression. Cancer J Sci Am. 1997 Sep-Oct;3(5):259–265. [PubMed] [Google Scholar]
  14. Deng G., Lu Y., Zlotnikov G., Thor A. D., Smith H. S. Loss of heterozygosity in normal tissue adjacent to breast carcinomas. Science. 1996 Dec 20;274(5295):2057–2059. doi: 10.1126/science.274.5295.2057. [DOI] [PubMed] [Google Scholar]
  15. Dietmaier W., Hartmann A., Wallinger S., Heinmöller E., Kerner T., Endl E., Jauch K. W., Hofstädter F., Rüschoff J. Multiple mutation analyses in single tumor cells with improved whole genome amplification. Am J Pathol. 1999 Jan;154(1):83–95. doi: 10.1016/S0002-9440(10)65254-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Emmert-Buck M. R., Bonner R. F., Smith P. D., Chuaqui R. F., Zhuang Z., Goldstein S. R., Weiss R. A., Liotta L. A. Laser capture microdissection. Science. 1996 Nov 8;274(5289):998–1001. doi: 10.1126/science.274.5289.998. [DOI] [PubMed] [Google Scholar]
  17. Emmert-Buck M. R., Gillespie J. W., Paweletz C. P., Ornstein D. K., Basrur V., Appella E., Wang Q. H., Huang J., Hu N., Taylor P. An approach to proteomic analysis of human tumors. Mol Carcinog. 2000 Mar;27(3):158–165. [PubMed] [Google Scholar]
  18. Essand M., Vasmatzis G., Brinkmann U., Duray P., Lee B., Pastan I. High expression of a specific T-cell receptor gamma transcript in epithelial cells of the prostate. Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9287–9292. doi: 10.1073/pnas.96.16.9287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fend F., Emmert-Buck M. R., Chuaqui R., Cole K., Lee J., Liotta L. A., Raffeld M. Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis. Am J Pathol. 1999 Jan;154(1):61–66. doi: 10.1016/S0002-9440(10)65251-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fend F., Quintanilla-Martinez L., Kumar S., Beaty M. W., Blum L., Sorbara L., Jaffe E. S., Raffeld M. Composite low grade B-cell lymphomas with two immunophenotypically distinct cell populations are true biclonal lymphomas. A molecular analysis using laser capture microdissection. Am J Pathol. 1999 Jun;154(6):1857–1866. doi: 10.1016/S0002-9440(10)65443-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fink L., Seeger W., Ermert L., Hänze J., Stahl U., Grimminger F., Kummer W., Bohle R. M. Real-time quantitative RT-PCR after laser-assisted cell picking. Nat Med. 1998 Nov;4(11):1329–1333. doi: 10.1038/3327. [DOI] [PubMed] [Google Scholar]
  22. Glasow A., Haidan A., Gillespie J., Kelly P. A., Chrousos G. P., Bornstein S. R. Differential expression of prolactin receptor (PRLR) in normal and tumorous adrenal tissues: separation of cellular endocrine compartments by laser capture microdissection (LCM). Endocr Res. 1998 Aug-Nov;24(3-4):857–862. doi: 10.3109/07435809809032697. [DOI] [PubMed] [Google Scholar]
  23. Glasow A., Haidan A., Hilbers U., Breidert M., Gillespie J., Scherbaum W. A., Chrousos G. P., Bornstein S. R. Expression of Ob receptor in normal human adrenals: differential regulation of adrenocortical and adrenomedullary function by leptin. J Clin Endocrinol Metab. 1998 Dec;83(12):4459–4466. doi: 10.1210/jcem.83.12.5337. [DOI] [PubMed] [Google Scholar]
  24. Going J. J., Lamb R. F. Practical histological microdissection for PCR analysis. J Pathol. 1996 May;179(1):121–124. doi: 10.1002/(SICI)1096-9896(199605)179:1<121::AID-PATH536>3.0.CO;2-D. [DOI] [PubMed] [Google Scholar]
  25. Goldsworthy S. M., Stockton P. S., Trempus C. S., Foley J. F., Maronpot R. R. Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue. Mol Carcinog. 1999 Jun;25(2):86–91. [PubMed] [Google Scholar]
  26. Hiller T., Snell L., Watson P. H. Microdissection RT-PCR analysis of gene expression in pathologically defined frozen tissue sections. Biotechniques. 1996 Jul;21(1):38-40, 42, 44. doi: 10.2144/96211bm07. [DOI] [PubMed] [Google Scholar]
  27. Isenberg G., Bielser W., Meier-Ruge W., Remy E. Cell surgery by laser micro-dissection: a preparative method. J Microsc. 1976 May;107(1):19–24. doi: 10.1111/j.1365-2818.1976.tb02419.x. [DOI] [PubMed] [Google Scholar]
  28. Jin L., Thompson C. A., Qian X., Kuecker S. J., Kulig E., Lloyd R. V. Analysis of anterior pituitary hormone mRNA expression in immunophenotypically characterized single cells after laser capture microdissection. Lab Invest. 1999 Apr;79(4):511–512. [PubMed] [Google Scholar]
  29. Kanzler H., Küppers R., Hansmann M. L., Rajewsky K. Hodgkin and Reed-Sternberg cells in Hodgkin's disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells. J Exp Med. 1996 Oct 1;184(4):1495–1505. doi: 10.1084/jem.184.4.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kohda Y., Murakami H., Moe O. W., Star R. A. Analysis of segmental renal gene expression by laser capture microdissection. Kidney Int. 2000 Jan;57(1):321–331. doi: 10.1046/j.1523-1755.2000.00824.x. [DOI] [PubMed] [Google Scholar]
  31. Kuukasjärvi T., Tanner M., Pennanen S., Karhu R., Kallioniemi O. P., Isola J. Genetic changes in intraductal breast cancer detected by comparative genomic hybridization. Am J Pathol. 1997 Apr;150(4):1465–1471. [PMC free article] [PubMed] [Google Scholar]
  32. Küppers R., Rajewsky K., Zhao M., Simons G., Laumann R., Fischer R., Hansmann M. L. Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):10962–10966. doi: 10.1073/pnas.91.23.10962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Küppers R., Zhao M., Hansmann M. L., Rajewsky K. Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections. EMBO J. 1993 Dec 15;12(13):4955–4967. doi: 10.1002/j.1460-2075.1993.tb06189.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Liang P., Pardee A. B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992 Aug 14;257(5072):967–971. doi: 10.1126/science.1354393. [DOI] [PubMed] [Google Scholar]
  35. Lieberman R., Crowell J. A., Hawk E. T., Boone C. W., Sigman C. C., Kelloff G. J. Development of new cancer chemoprevention agents: role of pharmacokinetic/pharmacodynamic and intermediate endpoint biomarker monitoring. Clin Chem. 1998 Feb;44(2):420–427. [PubMed] [Google Scholar]
  36. Luo L., Salunga R. C., Guo H., Bittner A., Joy K. C., Galindo J. E., Xiao H., Rogers K. E., Wan J. S., Jackson M. R. Gene expression profiles of laser-captured adjacent neuronal subtypes. Nat Med. 1999 Jan;5(1):117–122. doi: 10.1038/4806. [DOI] [PubMed] [Google Scholar]
  37. Luqmani Y. A., Lymboura M. Subtraction hybridization cloning of RNA amplified from different cell populations microdissected from cryostat tissue sections. Anal Biochem. 1994 Oct;222(1):102–109. doi: 10.1006/abio.1994.1460. [DOI] [PubMed] [Google Scholar]
  38. Maitra A., Wistuba I. I., Virmani A. K., Sakaguchi M., Park I., Stucky A., Milchgrub S., Gibbons D., Minna J. D., Gazdar A. F. Enrichment of epithelial cells for molecular studies. Nat Med. 1999 Apr;5(4):459–463. doi: 10.1038/7458. [DOI] [PubMed] [Google Scholar]
  39. Meier-Ruge W., Bielser W., Remy E., Hillenkamp F., Nitsche R., Unsöld R. The laser in the Lowry technique for microdissection of freeze-dried tissue slices. Histochem J. 1976 Jul;8(4):387–401. doi: 10.1007/BF01003828. [DOI] [PubMed] [Google Scholar]
  40. Moskaluk C. A., Kern S. E. Microdissection and polymerase chain reaction amplification of genomic DNA from histological tissue sections. Am J Pathol. 1997 May;150(5):1547–1552. [PMC free article] [PubMed] [Google Scholar]
  41. Natkunam Y., Rouse R. V., Zhu S., Fisher C., van De Rijn M. Immunoblot analysis of CD34 expression in histologically diverse neoplasms. Am J Pathol. 2000 Jan;156(1):21–27. doi: 10.1016/S0002-9440(10)64701-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ornstein D. K., Englert C., Gillespie J. W., Paweletz C. P., Linehan W. M., Emmert-Buck M. R., Petricoin E. F., 3rd Characterization of intracellular prostate-specific antigen from laser capture microdissected benign and malignant prostatic epithelium. Clin Cancer Res. 2000 Feb;6(2):353–356. [PubMed] [Google Scholar]
  43. Peterson L. A., Brown M. R., Carlisle A. J., Kohn E. C., Liotta L. A., Emmert-Buck M. R., Krizman D. B. An improved method for construction of directionally cloned cDNA libraries from microdissected cells. Cancer Res. 1998 Dec 1;58(23):5326–5328. [PubMed] [Google Scholar]
  44. Quintanilla-Martinez L., Fend F., Moguel L. R., Spilove L., Beaty M. W., Kingma D. W., Raffeld M., Jaffe E. S. Peripheral T-cell lymphoma with Reed-Sternberg-like cells of B-cell phenotype and genotype associated with Epstein-Barr virus infection. Am J Surg Pathol. 1999 Oct;23(10):1233–1240. doi: 10.1097/00000478-199910000-00008. [DOI] [PubMed] [Google Scholar]
  45. Radford D. M., Fair K., Thompson A. M., Ritter J. H., Holt M., Steinbrueck T., Wallace M., Wells S. A., Jr, Donis-Keller H. R. Allelic loss on a chromosome 17 in ductal carcinoma in situ of the breast. Cancer Res. 1993 Jul 1;53(13):2947–2949. [PubMed] [Google Scholar]
  46. Schütze K., Lahr G. Identification of expressed genes by laser-mediated manipulation of single cells. Nat Biotechnol. 1998 Aug;16(8):737–742. doi: 10.1038/nbt0898-737. [DOI] [PubMed] [Google Scholar]
  47. Sgroi D. C., Teng S., Robinson G., LeVangie R., Hudson J. R., Jr, Elkahloun A. G. In vivo gene expression profile analysis of human breast cancer progression. Cancer Res. 1999 Nov 15;59(22):5656–5661. [PubMed] [Google Scholar]
  48. Shibutani M., Uneyama C., Miyazaki K., Toyoda K., Hirose M. Methacarn fixation: a novel tool for analysis of gene expressions in paraffin-embedded tissue specimens. Lab Invest. 2000 Feb;80(2):199–208. doi: 10.1038/labinvest.3780023. [DOI] [PubMed] [Google Scholar]
  49. Simone N. L., Bonner R. F., Gillespie J. W., Emmert-Buck M. R., Liotta L. A. Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Trends Genet. 1998 Jul;14(7):272–276. doi: 10.1016/s0168-9525(98)01489-9. [DOI] [PubMed] [Google Scholar]
  50. Simone N. L., Remaley A. T., Charboneau L., Petricoin E. F., 3rd, Glickman J. W., Emmert-Buck M. R., Fleisher T. A., Liotta L. A. Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection. Am J Pathol. 2000 Feb;156(2):445–452. doi: 10.1016/S0002-9440(10)64749-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Suarez-Quian C. A., Goldstein S. R., Pohida T., Smith P. D., Peterson J. I., Wellner E., Ghany M., Bonner R. F. Laser capture microdissection of single cells from complex tissues. Biotechniques. 1999 Feb;26(2):328–335. doi: 10.2144/99262rr03. [DOI] [PubMed] [Google Scholar]
  52. Tam A. S., Foley J. F., Devereux T. R., Maronpot R. R., Massey T. E. High frequency and heterogeneous distribution of p53 mutations in aflatoxin B1-induced mouse lung tumors. Cancer Res. 1999 Aug 1;59(15):3634–3640. [PubMed] [Google Scholar]
  53. To M. D., Done S. J., Redston M., Andrulis I. L. Analysis of mRNA from microdissected frozen tissue sections without RNA isolation. Am J Pathol. 1998 Jul;153(1):47–51. doi: 10.1016/S0002-9440(10)65544-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Vasmatzis G., Essand M., Brinkmann U., Lee B., Pastan I. Discovery of three genes specifically expressed in human prostate by expressed sequence tag database analysis. Proc Natl Acad Sci U S A. 1998 Jan 6;95(1):300–304. doi: 10.1073/pnas.95.1.300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Velculescu V. E., Zhang L., Vogelstein B., Kinzler K. W. Serial analysis of gene expression. Science. 1995 Oct 20;270(5235):484–487. doi: 10.1126/science.270.5235.484. [DOI] [PubMed] [Google Scholar]
  56. Vocke C. D., Pozzatti R. O., Bostwick D. G., Florence C. D., Jennings S. B., Strup S. E., Duray P. H., Liotta L. A., Emmert-Buck M. R., Linehan W. M. Analysis of 99 microdissected prostate carcinomas reveals a high frequency of allelic loss on chromosome 8p12-21. Cancer Res. 1996 May 15;56(10):2411–2416. [PubMed] [Google Scholar]
  57. Weber R. G., Scheer M., Born I. A., Joos S., Cobbers J. M., Hofele C., Reifenberger G., Zöller J. E., Lichter P. Recurrent chromosomal imbalances detected in biopsy material from oral premalignant and malignant lesions by combined tissue microdissection, universal DNA amplification, and comparative genomic hybridization. Am J Pathol. 1998 Jul;153(1):295–303. doi: 10.1016/S0002-9440(10)65571-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Whetsell L., Maw G., Nadon N., Ringer D. P., Schaefer F. V. Polymerase chain reaction microanalysis of tumors from stained histological slides. Oncogene. 1992 Nov;7(11):2355–2361. [PubMed] [Google Scholar]
  59. Zhang L., Cui X., Schmitt K., Hubert R., Navidi W., Arnheim N. Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5847–5851. doi: 10.1073/pnas.89.13.5847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zhuang Z., Bertheau P., Emmert-Buck M. R., Liotta L. A., Gnarra J., Linehan W. M., Lubensky I. A. A microdissection technique for archival DNA analysis of specific cell populations in lesions < 1 mm in size. Am J Pathol. 1995 Mar;146(3):620–625. [PMC free article] [PubMed] [Google Scholar]
  61. Zhuang Z., Merino M. J., Chuaqui R., Liotta L. A., Emmert-Buck M. R. Identical allelic loss on chromosome 11q13 in microdissected in situ and invasive human breast cancer. Cancer Res. 1995 Feb 1;55(3):467–471. [PubMed] [Google Scholar]
  62. Zhuang Z., Vortmeyer A. O., Mark E. J., Odze R., Emmert-Buck M. R., Merino M. J., Moon H., Liotta L. A., Duray P. H. Barrett's esophagus: metaplastic cells with loss of heterozygosity at the APC gene locus are clonal precursors to invasive adenocarcinoma. Cancer Res. 1996 May 1;56(9):1961–1964. [PubMed] [Google Scholar]
  63. d'Amore F., Stribley J. A., Ohno T., Wu G., Wickert R. S., Delabie J., Hinrichs S. H., Chan W. C. Molecular studies on single cells harvested by micromanipulation from archival tissue sections previously stained by immunohistochemistry or nonisotopic in situ hybridization. Lab Invest. 1997 Feb;76(2):219–224. [PubMed] [Google Scholar]

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