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CARCINOMA-IN-SITU GERM-CELLS EXFOLIATED FROM SEMINIFEROUS EPITHELIUM INTO SEMINAL FLUID
530 reduction
(p<0-001, xz) in the rate compared with previous years (table).
of
pneumothorax in 1987
We believe that this system, which allows infants to breathe at "their own rate" and removes the need for paralysing or sedative agents, represents an important advance in ventilation of the
premature infant. Special Care Baby Unit, Northwick Park Hospital, and Clinical Research Centre, Harrow, Middlesex HA1 3UJ 1 Mehta
A, Wright BM, Callan K, newborn. Lancet 1986; ii 17—19
R. D. CLIFFORD G. WHINCUP R. THOMAS Stacey
TE
Patient-triggered ventilation
in
the
CARCINOMA-IN-SITU GERM-CELLS EXFOLIATED FROM SEMINIFEROUS EPITHELIUM INTO SEMINAL FLUID
SIR,-Invasive testicular cancer can be prevented if the malignancy is diagnosed at the stage of carcinoma-in-situ (CIS).I.2 However, screening for CIS of the testis has been hampered by lack of a convenient non-invasive diagnostic method.2 Normal germcells from the seminiferous epithelium have been found in semen of fertile and infertile men.3 We postulated that CIS germ-cells, which are also components of the seminiferous epithelium, may be likewise exfoliated.4 But, using conventional light-microscopy, we have not detected malignant germ-cells in semen of patients with documented CIS of the testis (unpublished data). -However, morphologically abnormal germ-cells have been found in semen of patients with manifest testicular cancer.4 The use of specific immunological markers may facilitate detection of ejaculated germ-cells which are often poorly preserved after their epididymal passage. Recently a novel marker for CIS germ-cells, monoclonal antibody M2A, has been characterised (ref 5 and our data to be published). We have examined semen of men with CIS of the testis for M2A positive cells. We studied four ejaculates from a 38-year-old man with isolated CIS of the testis and one ejaculate each from 3 men (aged 34, 37, and 43) who had CIS associated with invasive cancer of the testis (two seminomas and one embryonal carcinoma and seminoma). Single semen samples from 10 consecutive men attending an infertility clinic served as controls. After liquefaction and within 2 h of ejaculation, the semen sample was diluted with an equal part of 0.9% NaCl (pH 6).4 After centrifugation the pellet was resuspended at a sperm concentration of 05-LOxlO/ml in phosphate-buffered saline (PBS). Smears were prepared in a Shandon cytocentrifuge, air dried overnight, and stored at - 20°C. After fixation in Stieve’s fluid for 30 min at 4°C, the streptavidinbiotin technique was used for immunoperoxidase staining. Inhibition of endogenous peroxidase activity in 5 % H202/methanol was followed by incubation for 1 h at room temperature with M2A diluted 1/800 in PBS. Biotinylated rabbit anti-mouse antibody was the secondary reagent followed by the streptavidin-peroxidase conjugate. The colour reaction was developed by incubation with
diaminobenzidine-H202 and the smears were counterstained with iron-haematoxylin. In every smear 100 visual fields were examined under a light-microscope. M2A positive cells were identified by dark brown staining in all four smears from the patient with isolated CIS of the testis and in every smear from the 3 men having CIS associated with invasive cancer (figure). The positively stained cells had these characteristics of CIS germ cells: the nucleus was large (diameter at least 10 J..lffi) and irregularly shaped with coarse chromatin clumps and cytoplasm was abundant. No similar M2A positive cells were identified in ejaculates from the 10 controls. However, in two control smears one or two small cells with sparse cytoplasm and a round, regularly shaped nucleus (diameter about 5 pm) were stained non-specifically. The median number of M2A positive cells found in the 7 smears from men with testicular neoplasia was 5 (range 2-9). Based on sample volume and dilution factor the estimated median number of M2A positive cells per ejaculate was 104 (34-600). The slides were coded and another investigator judged the semen smears "blind". He was informed that 7 of the smears were from patients with CIS and 10 were from controls. Correct identification
Immunocytochemical staining of ejaculate from patient with CIS of testis.
Dark brown-stained CIS germ-cell with and abundant cytoplasm ( x 1200).
large, irregularly shaped nucleus
made in 15 of the 17 cases (6 patients and 9 controls; Fisher’s text, p=0007). We assumed that the M2A positive cells in semen of the 4 patients with CIS of the testis were CIS cells because we have shown that M2A is a marker for these cells. Furthermore, the M2A positive cells had morphological characteristics of CIS germ-cells.’ Since M2A also reacts with seminomas,6 we cannot exclude the possibility that the M2A positive cells in the semen of the 3 patients with seminomas or mixed-type tumours were, in fact, invasive tumour cells, and not CIS germ-cells. This, however, seems unlikely because invasive tumour cells are located outside the seminiferous tubules whereas CIS germ-cells are located inside the tubules, and therefore may be exfoliated into the semen. Careful microscopy was necessary for detecting M2A positive cells because their number per smear was low. However, we did find that CIS germ-cells are exfoliated into semen and can be identified on the basis of combined and morphological immunocytochemical criteria with immunoperoxidase staining by M2A. The technique may provide a convenient screen for detection of both pre-invasive and invasive testicular neoplasia. was
exact
This study was supported by the Danish Cancer Society (grants 84-007, 86-017, 86-044, 86-065, 87-002, 87-030, 87-041, 87-055, and 87-111), the Daell Fund, and the PSI Foundation of Ontario.
Laboratory of Reproductive Biology, Rigshospitalet, Copenhagen
ALEKSANDER GIWERCMAN
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada
ALEXANDER MARKS
Laboratory of Reproductive Biology, Rigshospitalet, Copenhagen, and University Department of Paediatrics, Hvidovre Hospital, 2650 Hvidovre, Denmark
NIELS E. SKAKKEBAEK
1. Skakkebaek NE. Possible carcinoma-in-situ of the testis. Lancet 1972, i: 516-17. 2. Editorial. Carcinoma-in-situ of the testis. Lancet 1987, ii: 545-46 3. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction Cambridge: Cambridge University Press, 1987 4. Czaplicki M, Rojewska J, Pykalo R, Szymanska K Detection of testicular neoplasms by cytological examination of seminal fluid.J Urol 1987; 138: 787-88 5. Bailey D, Baumal R, Law J, et al Production of monoclonal antibody specific for seminomas and dysgermmomas. Proc Natl Acad Sci USA 1986; 83: 5291-95.
(p<0-001, xz) in the rate compared with previous years (table).
of
pneumothorax in 1987
We believe that this system, which allows infants to breathe at "their own rate" and removes the need for paralysing or sedative agents, represents an important advance in ventilation of the
premature infant. Special Care Baby Unit, Northwick Park Hospital, and Clinical Research Centre, Harrow, Middlesex HA1 3UJ 1 Mehta
A, Wright BM, Callan K, newborn. Lancet 1986; ii 17—19
R. D. CLIFFORD G. WHINCUP R. THOMAS Stacey
TE
Patient-triggered ventilation
in
the
CARCINOMA-IN-SITU GERM-CELLS EXFOLIATED FROM SEMINIFEROUS EPITHELIUM INTO SEMINAL FLUID
SIR,-Invasive testicular cancer can be prevented if the malignancy is diagnosed at the stage of carcinoma-in-situ (CIS).I.2 However, screening for CIS of the testis has been hampered by lack of a convenient non-invasive diagnostic method.2 Normal germcells from the seminiferous epithelium have been found in semen of fertile and infertile men.3 We postulated that CIS germ-cells, which are also components of the seminiferous epithelium, may be likewise exfoliated.4 But, using conventional light-microscopy, we have not detected malignant germ-cells in semen of patients with documented CIS of the testis (unpublished data). -However, morphologically abnormal germ-cells have been found in semen of patients with manifest testicular cancer.4 The use of specific immunological markers may facilitate detection of ejaculated germ-cells which are often poorly preserved after their epididymal passage. Recently a novel marker for CIS germ-cells, monoclonal antibody M2A, has been characterised (ref 5 and our data to be published). We have examined semen of men with CIS of the testis for M2A positive cells. We studied four ejaculates from a 38-year-old man with isolated CIS of the testis and one ejaculate each from 3 men (aged 34, 37, and 43) who had CIS associated with invasive cancer of the testis (two seminomas and one embryonal carcinoma and seminoma). Single semen samples from 10 consecutive men attending an infertility clinic served as controls. After liquefaction and within 2 h of ejaculation, the semen sample was diluted with an equal part of 0.9% NaCl (pH 6).4 After centrifugation the pellet was resuspended at a sperm concentration of 05-LOxlO/ml in phosphate-buffered saline (PBS). Smears were prepared in a Shandon cytocentrifuge, air dried overnight, and stored at - 20°C. After fixation in Stieve’s fluid for 30 min at 4°C, the streptavidinbiotin technique was used for immunoperoxidase staining. Inhibition of endogenous peroxidase activity in 5 % H202/methanol was followed by incubation for 1 h at room temperature with M2A diluted 1/800 in PBS. Biotinylated rabbit anti-mouse antibody was the secondary reagent followed by the streptavidin-peroxidase conjugate. The colour reaction was developed by incubation with
diaminobenzidine-H202 and the smears were counterstained with iron-haematoxylin. In every smear 100 visual fields were examined under a light-microscope. M2A positive cells were identified by dark brown staining in all four smears from the patient with isolated CIS of the testis and in every smear from the 3 men having CIS associated with invasive cancer (figure). The positively stained cells had these characteristics of CIS germ cells: the nucleus was large (diameter at least 10 J..lffi) and irregularly shaped with coarse chromatin clumps and cytoplasm was abundant. No similar M2A positive cells were identified in ejaculates from the 10 controls. However, in two control smears one or two small cells with sparse cytoplasm and a round, regularly shaped nucleus (diameter about 5 pm) were stained non-specifically. The median number of M2A positive cells found in the 7 smears from men with testicular neoplasia was 5 (range 2-9). Based on sample volume and dilution factor the estimated median number of M2A positive cells per ejaculate was 104 (34-600). The slides were coded and another investigator judged the semen smears "blind". He was informed that 7 of the smears were from patients with CIS and 10 were from controls. Correct identification
Immunocytochemical staining of ejaculate from patient with CIS of testis.
Dark brown-stained CIS germ-cell with and abundant cytoplasm ( x 1200).
large, irregularly shaped nucleus
made in 15 of the 17 cases (6 patients and 9 controls; Fisher’s text, p=0007). We assumed that the M2A positive cells in semen of the 4 patients with CIS of the testis were CIS cells because we have shown that M2A is a marker for these cells. Furthermore, the M2A positive cells had morphological characteristics of CIS germ-cells.’ Since M2A also reacts with seminomas,6 we cannot exclude the possibility that the M2A positive cells in the semen of the 3 patients with seminomas or mixed-type tumours were, in fact, invasive tumour cells, and not CIS germ-cells. This, however, seems unlikely because invasive tumour cells are located outside the seminiferous tubules whereas CIS germ-cells are located inside the tubules, and therefore may be exfoliated into the semen. Careful microscopy was necessary for detecting M2A positive cells because their number per smear was low. However, we did find that CIS germ-cells are exfoliated into semen and can be identified on the basis of combined and morphological immunocytochemical criteria with immunoperoxidase staining by M2A. The technique may provide a convenient screen for detection of both pre-invasive and invasive testicular neoplasia. was
exact
This study was supported by the Danish Cancer Society (grants 84-007, 86-017, 86-044, 86-065, 87-002, 87-030, 87-041, 87-055, and 87-111), the Daell Fund, and the PSI Foundation of Ontario.
Laboratory of Reproductive Biology, Rigshospitalet, Copenhagen
ALEKSANDER GIWERCMAN
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada
ALEXANDER MARKS
Laboratory of Reproductive Biology, Rigshospitalet, Copenhagen, and University Department of Paediatrics, Hvidovre Hospital, 2650 Hvidovre, Denmark
NIELS E. SKAKKEBAEK
1. Skakkebaek NE. Possible carcinoma-in-situ of the testis. Lancet 1972, i: 516-17. 2. Editorial. Carcinoma-in-situ of the testis. Lancet 1987, ii: 545-46 3. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction Cambridge: Cambridge University Press, 1987 4. Czaplicki M, Rojewska J, Pykalo R, Szymanska K Detection of testicular neoplasms by cytological examination of seminal fluid.J Urol 1987; 138: 787-88 5. Bailey D, Baumal R, Law J, et al Production of monoclonal antibody specific for seminomas and dysgermmomas. Proc Natl Acad Sci USA 1986; 83: 5291-95.