77
and subjected to Southern blotting.3,4 Monoclonal integration of HTLV-1 proviral DNA was detected. The rearrangement of the TCR P and y genes and a deletion ofgene were consistent with the phenotypic studies. The ATL cells described here expressed neither CD4 nor CD8 antigens, but expressed both CD3 and the TCR fxp heterodimer.
This patient had double-negative ATL. Double-negative T cells bearing the TCR a(3 heterodimer were initially detected as a distinct subset of thymocytes in mice, and were found to express Vp8 preferentially. Cells bearing similar phenotypes were later found in normal adult human skin and in peripheral blood.6,7 Though the functional role of these cells has not been clarified, it is unlikely that they are the precursors of CD3 +, CD4 + or CD3 +, CD8 + cells because these clones did not alter their phenotypes over a 12-month period in culture" and because of their appearance late in the development of murine thymocytes .5 Clinicopathologically, this case could be diagnosed as a leukaemic conversion of malignant lymphoma of mucosaassociated lymphoid tissue, though most of such cells have been considered to be B-cell origin. However, eight cases of enteropathy-associated T-cell lymphoma have been reported; all lacked CD4 and CD8 and expressed CD7, though CD3 antigen expression by neoplastic cells was variable.8 The leukaemic cells described here suggest a novel T-cell subset (CD4-, CD8-, TCRex&bgr;+) in the gastrointestinal tract of adult man, where it plays an undefined immunological role. Infections of this subset with HTLV-1 may alter or suppress host immunity. The frequent infestation by Strongyloides stercoralis-a parasite affecting colon, lungs, and other organs that is found in HTLV-1 carriers-could be one manifestation.9 REFERENCES T, Uchiyama T, Toibana T, Takatsuki K, Uchino H. Surface phenotype of Japanese adult T-cell leukemia characterized by
1. Hattori
monoclonal antibodies. Blood 1981; 58: 645-47. 2. Shirono K, Hattori T, Hata H, Nishimura K, Takatsuki K. Profiles of expression of activated cell antigens on peripheal blood and lymph node cells from different clinical states of adult T-cell leukemia. Blood 1989; 73: 1664-71. 3. Matsuoka M, Hagiya M, Hattori T, et al. Gene rearrangements of T cell receptor &bgr; and y chains in HTLV-I infected primary neoplastic T cells. Leukaemia 1988; 2: 84-90. 4. Asou N, Hattori T, Matsukoka M, Kawano F, Takatsuki K. Rearrangement of T-cell antigen receptor &dgr; chain gene in hematologic neoplasms. Blood 1989; 74: 2707-12. 5. Fowlkes BJ, Kruisbeek AM, Ton-That H, et al. A novel population of T-cell receptor &agr;&bgr;-bearing thymocytes which predominantly express a single V&bgr; gene family. Nature 1987; 329: 251-54. 6. Groh V, Fabbi M, Hochstenbach F, Maziarz RT, Strominger JL. Double-negative (CD4- CD8-) lymphocytes bearing T-cell receptor &agr; and &bgr; chains in normal human skin. Proc Natl Acad Sci (USA) 1989; 86: 5059-63. 7. Londei M, Verhoef A, DeBeradinis P, et al. Definition of a population of CD4-8- T cells that express the &agr;&bgr; T-cell receptor and respond to interleukins 2,3, and 4. Proc Natl Acad Sci (USA) 1989; 86: 8502-06. 8. Spencer J, Cerf-Bensussan N, Jarry A, et al. Enteropathy-associated T cell lymphoma (malignant histiocytosis of the intestine) is recognized by a monoclonal antibody (HML-1) that defines a membrane molecule on human mucosal lymphocytes. Am J Pathol 1988; 132: 1-5. 9. Nakada K, Yamaguchi K, Furugen S, et al. Monoclonal integration of HTIV-I proviral DNA in patients with strongyloidiases. Int J Cancer 1987; 40: 145-48.
ADDRESSES Second Department of Internal Medicine, Kumamoto University Medical School, Kumamoto 860, Japan (T. Hattori, MD, N Asou, MD, H Suzushima, MD, Prof K. Takatsuki, MD); and First Department of Internal Medicine, Kurume University School of Medicine (K. Tanaka, MD, K. Naito, MD, H. Natori, MD, Prof K. Oizumi, MD). Correspondence to Dr Toshio Hattori
Identification of pathological mineral deposits by Raman
microscopy
Raman
microscopy—the analysis of scattered
photons after excitation—is well established in non-biological sciences for the identification of crystals. It shows promise in biological (clinical) specimens also, as demonstrated here in studies of synovial fluid, synovium, and gouty tophus, selected for their known content of sodium urate and calcium
pyrophosphate crystals. In most
crystal-related diseases, such as gout and kidney gallbladder stones, little is known about what controls crystal formation or about the contribution mineral deposits make to the associated tissue damage.12 The accurate identification of tissue mineral deposits is difficult because the crystals deposited are usually small and more than one or
type of mineral may be involved.3 Current methods include polarised light microscopy, analytical electron microscopy, infra-red spectroscopy, and X-ray diffraction, but all have disadvantages: for example, large amounts of the mineral are required for spectroscopy and X-ray diffraction and microscopic methods may lack specificity and involve complex processing of tissues or fluids. Raman spectroscopy provides a method for obtaining the characteristic spectrum of vibrational frequencies of a molecule. The Raman effect requires illumination of the sample by high intensity (10), monochromic radiation and analysis of the energy elastically scattered Rayleigh photons of intensity around 10-510 and of inelastically scattered Raman photons of intensity around 101. Raman spectroscopy is widely used in non-biological sciences,4and is beginning to be applied to the investigation of biomineralisation. We have applied a variation, Raman microscopy, to the identification of crystals in connective tissue and joint fluid. Raman scattered photons due to promotion of a vibrational transition in the sample have a lower energy than the incident photons (Stokes Raman scatter); those due to abstraction of a vibrational quantum have a higher energy (anti-Stokes Raman scatter) but these photons are less probable. It is usual to analyse Stokes Raman scatter. In Raman microscopy" microscope optics are used to focus a laser beam to a spot about 1 /ffi1 in diameter on the surface of the sample and the Raman scattered light is collected and analysed. White light epi-illumination and a videocamera allow precise positioning of the laser spot onto the region of interest and permit the use of polarised light microscopy to locate crystal samples. The deposits most frequently identified in connective tissue are monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD), and they can be identified by polarised light microscopy. Samples containing these crystals were used to evaluate the potential of Raman microscopy. Currently the instrumentation for Raman microscopy is confined to research groups and industrial laboratories, in part because it costs over 100 000. However, developments such as low noise, high sensitivity detectors and small, cheap, air-cooled lasers’ may increase the availability. Reference minerals-MSUM crystals grown from aqueous solutions and CPPD grown in polyacrylamide gels8-were
78
Spex ’Datamate’ data acquisition system.6 Excitation was with the 514-5 nm line from a Spectra Physics 142 argon ion laser supplying about 10 mW at the sample. The spectral resolution was 6 cm-1 and acquisition times varied from 1 to 30 min. Crystals were located in transmitted light by crossed-polarising filters. A x 100 objective was used to obtain spectra from the smaller crystals; for larger ones x 40 was used.
a
Fig 1-Raman microscopy of MSUM samples. Raman spectra in region 610-650 cm-1 recorded from synthetic MSUM (a), small cluster of MSUM crystals in synovial fluid smear (b), and sample of gouty tophus (c). examined by polarised light microscopy, infra-red spectrometry, and X-ray powder diffraction. Synovial fluid was obtained from the knees of a 57-year-old man with acute gout and a 78-year-old woman with acute pseudogout, and smears were prepared on clean glass slides and air dried; a gouty tophus was taken from a 79-year-old patient with gout; and synovial tissue from a 72-yearold woman with pyrophosphate arthropathy having a total knee replacement. A BGSC RM III Raman microscope coupled to a Spex 1403 double monochromator with photon counting from a mixed alkali Hamamatsu R928 photomultiplier tube, was used with
Raman spectra from the reference samples of MSUM and CPPD accorded with the published data. The intense bands at 631 cm-1 for MSUM and at 1050 cm’for CPPD were used to identify the crystals in the biological samples, narrowing the spectrum to be scanned and reducing the analysis time. In particular instances the full spectrum was checked to confirm identification. All spectra illustrated are from single crystals 2-10 ltm long and confirm the identity of MSUM (fig 1) and CPPD (fig 2) in the biological samples. The frequencies (crri 1) of the more intense bands, which were uniquely characteristic of each mineral, were: 490, 589, 631, 788, 873, 1010, 1061, 1208, 1420, and 1446, for MSUM; and 351, 494, 515, 535, 560, 756, 1050, 1079, 1118, and 1184 for CPPD. Raman spectroscopy can thus be used to identify single crystals in pathological samples from patients with crystal deposition disease. The samples were chosen to see if Raman spectroscopy detects crystals in circumstances in which there was no doubt about the presence or nature of the deposits. To avoid contamination with material likely to give strong Raman spectra dried smears of unprocessed synovial fluid and tophaceous material were used and synovial tissue was washed free of paraffin and not stained. The spectra in the biological samples were of lower intensity and had poorer signal/noise ratios than of pure reference samples, and experiments are underway to reduce the background scattering from organic material. The potential, however, is considerable. Samples can be examined under ordinary or polarised light microscopy without the complex processing that might alter the deposit. Individual particles seen under the microscope can then be analysed in situ and mixed deposits can be accurately analysed. With further development Raman microscopy could overcome the problems of other analytical techniques and provide a powerful new tool for the investigation of disorders related to crystal formation and of physiological mineralisation. REFERENCES pathologic calcification. Clin Rheum Dis N
1. Anderson HC. Mechanisms of
Am 1988; 14: 303-19. Dieppe P, Watt I. Crystal deposition in osteoarthritis: an opportunistic event? Clin Rheum Dis 1985; 11: 367-92. 3. Dieppe P, Campion GG, Doherty. Mixed crystal deposition. Clin Rheum
2.
Dis Am 1988; 14: 415-26. 4. Gardiner DJ, Graves PR, eds. Practical Raman spectroscopy. Berlin: Springer, 1989. 5. Etz ES, Tomazic BB, Brown WE. Micro-Raman characterisation of atherosclerotic and bioprosthetic calcification. Microbeam Anal 1986; 21: 39-46. 6. Gardiner DJ, Bowden M, Graves PR. Novel applications of Raman microscopy. Phil Trans Roy Soc 1986; A320: 295-306. 7. Bowden M, Birnie J, Donaldson P, Gardiner DJ, Southall J. Raman imaging of a diamond film using microline focus spectrometry. Procedings of XIIth International Conference on Raman Spectroscopy (Columbia, South Carolina). New York: Wiley, 1990: 844-45. 8. Harries JE, Dieppe PA, Heap P, Gilgead J, Mather M, Shar JS. In vitro growth of calcium pyrophosphate crystals in polyacrylamide gels. Ann Rheum Dis 1983; 42 (suppl): 100-01.
Fig 2-Raman microscopy of CPDD samples. Raman spectra in region 1030-1070 cm-’ recorded from: synthetic CPPD (a), synovial fluid smear (b), cartilage section (c), and synovium (d)
ADDRESSES Rheumatology Unit, Bristol Royal Infirmary (N McGill, FRACP, Prof P A Dieppe, FRCP); and Department of Chemical and Life Sciences, Newcastle upon Tyne Polytechnic, Newcastle upon Tyne NE1 8ST, UK (M Bowden, PhD, Prof D J Gardiner, PhD, M Hall, BSc). Correspondence to Prof D J Gardiner
and subjected to Southern blotting.3,4 Monoclonal integration of HTLV-1 proviral DNA was detected. The rearrangement of the TCR P and y genes and a deletion ofgene were consistent with the phenotypic studies. The ATL cells described here expressed neither CD4 nor CD8 antigens, but expressed both CD3 and the TCR fxp heterodimer.
This patient had double-negative ATL. Double-negative T cells bearing the TCR a(3 heterodimer were initially detected as a distinct subset of thymocytes in mice, and were found to express Vp8 preferentially. Cells bearing similar phenotypes were later found in normal adult human skin and in peripheral blood.6,7 Though the functional role of these cells has not been clarified, it is unlikely that they are the precursors of CD3 +, CD4 + or CD3 +, CD8 + cells because these clones did not alter their phenotypes over a 12-month period in culture" and because of their appearance late in the development of murine thymocytes .5 Clinicopathologically, this case could be diagnosed as a leukaemic conversion of malignant lymphoma of mucosaassociated lymphoid tissue, though most of such cells have been considered to be B-cell origin. However, eight cases of enteropathy-associated T-cell lymphoma have been reported; all lacked CD4 and CD8 and expressed CD7, though CD3 antigen expression by neoplastic cells was variable.8 The leukaemic cells described here suggest a novel T-cell subset (CD4-, CD8-, TCRex&bgr;+) in the gastrointestinal tract of adult man, where it plays an undefined immunological role. Infections of this subset with HTLV-1 may alter or suppress host immunity. The frequent infestation by Strongyloides stercoralis-a parasite affecting colon, lungs, and other organs that is found in HTLV-1 carriers-could be one manifestation.9 REFERENCES T, Uchiyama T, Toibana T, Takatsuki K, Uchino H. Surface phenotype of Japanese adult T-cell leukemia characterized by
1. Hattori
monoclonal antibodies. Blood 1981; 58: 645-47. 2. Shirono K, Hattori T, Hata H, Nishimura K, Takatsuki K. Profiles of expression of activated cell antigens on peripheal blood and lymph node cells from different clinical states of adult T-cell leukemia. Blood 1989; 73: 1664-71. 3. Matsuoka M, Hagiya M, Hattori T, et al. Gene rearrangements of T cell receptor &bgr; and y chains in HTLV-I infected primary neoplastic T cells. Leukaemia 1988; 2: 84-90. 4. Asou N, Hattori T, Matsukoka M, Kawano F, Takatsuki K. Rearrangement of T-cell antigen receptor &dgr; chain gene in hematologic neoplasms. Blood 1989; 74: 2707-12. 5. Fowlkes BJ, Kruisbeek AM, Ton-That H, et al. A novel population of T-cell receptor &agr;&bgr;-bearing thymocytes which predominantly express a single V&bgr; gene family. Nature 1987; 329: 251-54. 6. Groh V, Fabbi M, Hochstenbach F, Maziarz RT, Strominger JL. Double-negative (CD4- CD8-) lymphocytes bearing T-cell receptor &agr; and &bgr; chains in normal human skin. Proc Natl Acad Sci (USA) 1989; 86: 5059-63. 7. Londei M, Verhoef A, DeBeradinis P, et al. Definition of a population of CD4-8- T cells that express the &agr;&bgr; T-cell receptor and respond to interleukins 2,3, and 4. Proc Natl Acad Sci (USA) 1989; 86: 8502-06. 8. Spencer J, Cerf-Bensussan N, Jarry A, et al. Enteropathy-associated T cell lymphoma (malignant histiocytosis of the intestine) is recognized by a monoclonal antibody (HML-1) that defines a membrane molecule on human mucosal lymphocytes. Am J Pathol 1988; 132: 1-5. 9. Nakada K, Yamaguchi K, Furugen S, et al. Monoclonal integration of HTIV-I proviral DNA in patients with strongyloidiases. Int J Cancer 1987; 40: 145-48.
ADDRESSES Second Department of Internal Medicine, Kumamoto University Medical School, Kumamoto 860, Japan (T. Hattori, MD, N Asou, MD, H Suzushima, MD, Prof K. Takatsuki, MD); and First Department of Internal Medicine, Kurume University School of Medicine (K. Tanaka, MD, K. Naito, MD, H. Natori, MD, Prof K. Oizumi, MD). Correspondence to Dr Toshio Hattori
Identification of pathological mineral deposits by Raman
microscopy
Raman
microscopy—the analysis of scattered
photons after excitation—is well established in non-biological sciences for the identification of crystals. It shows promise in biological (clinical) specimens also, as demonstrated here in studies of synovial fluid, synovium, and gouty tophus, selected for their known content of sodium urate and calcium
pyrophosphate crystals. In most
crystal-related diseases, such as gout and kidney gallbladder stones, little is known about what controls crystal formation or about the contribution mineral deposits make to the associated tissue damage.12 The accurate identification of tissue mineral deposits is difficult because the crystals deposited are usually small and more than one or
type of mineral may be involved.3 Current methods include polarised light microscopy, analytical electron microscopy, infra-red spectroscopy, and X-ray diffraction, but all have disadvantages: for example, large amounts of the mineral are required for spectroscopy and X-ray diffraction and microscopic methods may lack specificity and involve complex processing of tissues or fluids. Raman spectroscopy provides a method for obtaining the characteristic spectrum of vibrational frequencies of a molecule. The Raman effect requires illumination of the sample by high intensity (10), monochromic radiation and analysis of the energy elastically scattered Rayleigh photons of intensity around 10-510 and of inelastically scattered Raman photons of intensity around 101. Raman spectroscopy is widely used in non-biological sciences,4and is beginning to be applied to the investigation of biomineralisation. We have applied a variation, Raman microscopy, to the identification of crystals in connective tissue and joint fluid. Raman scattered photons due to promotion of a vibrational transition in the sample have a lower energy than the incident photons (Stokes Raman scatter); those due to abstraction of a vibrational quantum have a higher energy (anti-Stokes Raman scatter) but these photons are less probable. It is usual to analyse Stokes Raman scatter. In Raman microscopy" microscope optics are used to focus a laser beam to a spot about 1 /ffi1 in diameter on the surface of the sample and the Raman scattered light is collected and analysed. White light epi-illumination and a videocamera allow precise positioning of the laser spot onto the region of interest and permit the use of polarised light microscopy to locate crystal samples. The deposits most frequently identified in connective tissue are monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD), and they can be identified by polarised light microscopy. Samples containing these crystals were used to evaluate the potential of Raman microscopy. Currently the instrumentation for Raman microscopy is confined to research groups and industrial laboratories, in part because it costs over 100 000. However, developments such as low noise, high sensitivity detectors and small, cheap, air-cooled lasers’ may increase the availability. Reference minerals-MSUM crystals grown from aqueous solutions and CPPD grown in polyacrylamide gels8-were
78
Spex ’Datamate’ data acquisition system.6 Excitation was with the 514-5 nm line from a Spectra Physics 142 argon ion laser supplying about 10 mW at the sample. The spectral resolution was 6 cm-1 and acquisition times varied from 1 to 30 min. Crystals were located in transmitted light by crossed-polarising filters. A x 100 objective was used to obtain spectra from the smaller crystals; for larger ones x 40 was used.
a
Fig 1-Raman microscopy of MSUM samples. Raman spectra in region 610-650 cm-1 recorded from synthetic MSUM (a), small cluster of MSUM crystals in synovial fluid smear (b), and sample of gouty tophus (c). examined by polarised light microscopy, infra-red spectrometry, and X-ray powder diffraction. Synovial fluid was obtained from the knees of a 57-year-old man with acute gout and a 78-year-old woman with acute pseudogout, and smears were prepared on clean glass slides and air dried; a gouty tophus was taken from a 79-year-old patient with gout; and synovial tissue from a 72-yearold woman with pyrophosphate arthropathy having a total knee replacement. A BGSC RM III Raman microscope coupled to a Spex 1403 double monochromator with photon counting from a mixed alkali Hamamatsu R928 photomultiplier tube, was used with
Raman spectra from the reference samples of MSUM and CPPD accorded with the published data. The intense bands at 631 cm-1 for MSUM and at 1050 cm’for CPPD were used to identify the crystals in the biological samples, narrowing the spectrum to be scanned and reducing the analysis time. In particular instances the full spectrum was checked to confirm identification. All spectra illustrated are from single crystals 2-10 ltm long and confirm the identity of MSUM (fig 1) and CPPD (fig 2) in the biological samples. The frequencies (crri 1) of the more intense bands, which were uniquely characteristic of each mineral, were: 490, 589, 631, 788, 873, 1010, 1061, 1208, 1420, and 1446, for MSUM; and 351, 494, 515, 535, 560, 756, 1050, 1079, 1118, and 1184 for CPPD. Raman spectroscopy can thus be used to identify single crystals in pathological samples from patients with crystal deposition disease. The samples were chosen to see if Raman spectroscopy detects crystals in circumstances in which there was no doubt about the presence or nature of the deposits. To avoid contamination with material likely to give strong Raman spectra dried smears of unprocessed synovial fluid and tophaceous material were used and synovial tissue was washed free of paraffin and not stained. The spectra in the biological samples were of lower intensity and had poorer signal/noise ratios than of pure reference samples, and experiments are underway to reduce the background scattering from organic material. The potential, however, is considerable. Samples can be examined under ordinary or polarised light microscopy without the complex processing that might alter the deposit. Individual particles seen under the microscope can then be analysed in situ and mixed deposits can be accurately analysed. With further development Raman microscopy could overcome the problems of other analytical techniques and provide a powerful new tool for the investigation of disorders related to crystal formation and of physiological mineralisation. REFERENCES pathologic calcification. Clin Rheum Dis N
1. Anderson HC. Mechanisms of
Am 1988; 14: 303-19. Dieppe P, Watt I. Crystal deposition in osteoarthritis: an opportunistic event? Clin Rheum Dis 1985; 11: 367-92. 3. Dieppe P, Campion GG, Doherty. Mixed crystal deposition. Clin Rheum
2.
Dis Am 1988; 14: 415-26. 4. Gardiner DJ, Graves PR, eds. Practical Raman spectroscopy. Berlin: Springer, 1989. 5. Etz ES, Tomazic BB, Brown WE. Micro-Raman characterisation of atherosclerotic and bioprosthetic calcification. Microbeam Anal 1986; 21: 39-46. 6. Gardiner DJ, Bowden M, Graves PR. Novel applications of Raman microscopy. Phil Trans Roy Soc 1986; A320: 295-306. 7. Bowden M, Birnie J, Donaldson P, Gardiner DJ, Southall J. Raman imaging of a diamond film using microline focus spectrometry. Procedings of XIIth International Conference on Raman Spectroscopy (Columbia, South Carolina). New York: Wiley, 1990: 844-45. 8. Harries JE, Dieppe PA, Heap P, Gilgead J, Mather M, Shar JS. In vitro growth of calcium pyrophosphate crystals in polyacrylamide gels. Ann Rheum Dis 1983; 42 (suppl): 100-01.
Fig 2-Raman microscopy of CPDD samples. Raman spectra in region 1030-1070 cm-’ recorded from: synthetic CPPD (a), synovial fluid smear (b), cartilage section (c), and synovium (d)
ADDRESSES Rheumatology Unit, Bristol Royal Infirmary (N McGill, FRACP, Prof P A Dieppe, FRCP); and Department of Chemical and Life Sciences, Newcastle upon Tyne Polytechnic, Newcastle upon Tyne NE1 8ST, UK (M Bowden, PhD, Prof D J Gardiner, PhD, M Hall, BSc). Correspondence to Prof D J Gardiner
