Abstract
To avoid the radiobiological hazards linked to the use of {sup 3}H-thymidine, several workers have performed in vitro incubations of biopsy material in the presence of this labelled DNA -precursor. Attempts were made to define the proliferative pattern of the tissue from the distribution and intensity of labelling in autoradiographs. However, for true estimation of the cell cycle length, one has to know the S-phase (DNA-synthesis phase) duration, since the labelling index reflects only the part taken by this phase in the whole cell cycle. We chose the ''double-labelling technique'' with two dose levels of {sup 3}H-thymidine, which was developed by Maurer's group. By administering two pulses of the tritiated precursor (a weak dose and a heavy dose) at a given time interval and submitting the treated tissue to the autoradiographic process, one can calculate the S-phase duration(s) after the formula N{sub h}/N{sub w} = S/t. N{sub h} is the number of heavily labelled nuclei (i.e. those nuclei that were engaged in S-phase during the second pulse labelling); N{sub w} is the number of weakly labelled nuclei, corresponding to the cells which left S-phase during the time interval (t) between the two pulse labellings. From the percentage of heavily labelled
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Galand, P.
[1]
- Faculte de Medecine de l'Universite Libre de Bruxelles, Bruxelles (Belgium)
Citation Formats
Galand, P.
Use of Biopsy Tissue Samples, Incubated In Vitro for Autoradiographic Measurement of Cell Proliferation in Human Tissues; Utilisation de Biopsies Incubees In Vitro pour la Mesure Autoradiographique de la Proliferation Cellulaire de Tissus Humains.
IAEA: N. p.,
1970.
Web.
Galand, P.
Use of Biopsy Tissue Samples, Incubated In Vitro for Autoradiographic Measurement of Cell Proliferation in Human Tissues; Utilisation de Biopsies Incubees In Vitro pour la Mesure Autoradiographique de la Proliferation Cellulaire de Tissus Humains.
IAEA.
Galand, P.
1970.
"Use of Biopsy Tissue Samples, Incubated In Vitro for Autoradiographic Measurement of Cell Proliferation in Human Tissues; Utilisation de Biopsies Incubees In Vitro pour la Mesure Autoradiographique de la Proliferation Cellulaire de Tissus Humains."
IAEA.
@misc{etde_22205098,
title = {Use of Biopsy Tissue Samples, Incubated In Vitro for Autoradiographic Measurement of Cell Proliferation in Human Tissues; Utilisation de Biopsies Incubees In Vitro pour la Mesure Autoradiographique de la Proliferation Cellulaire de Tissus Humains}
author = {Galand, P.}
abstractNote = {To avoid the radiobiological hazards linked to the use of {sup 3}H-thymidine, several workers have performed in vitro incubations of biopsy material in the presence of this labelled DNA -precursor. Attempts were made to define the proliferative pattern of the tissue from the distribution and intensity of labelling in autoradiographs. However, for true estimation of the cell cycle length, one has to know the S-phase (DNA-synthesis phase) duration, since the labelling index reflects only the part taken by this phase in the whole cell cycle. We chose the ''double-labelling technique'' with two dose levels of {sup 3}H-thymidine, which was developed by Maurer's group. By administering two pulses of the tritiated precursor (a weak dose and a heavy dose) at a given time interval and submitting the treated tissue to the autoradiographic process, one can calculate the S-phase duration(s) after the formula N{sub h}/N{sub w} = S/t. N{sub h} is the number of heavily labelled nuclei (i.e. those nuclei that were engaged in S-phase during the second pulse labelling); N{sub w} is the number of weakly labelled nuclei, corresponding to the cells which left S-phase during the time interval (t) between the two pulse labellings. From the percentage of heavily labelled nuclei in the whole population (L.I.), the generation time (G.T.) is then given by the formula G.T. = S/L.I. x 100. Human rectal and gastric biopsies were submitted to this procedure which, because of its brevity, requires simple survival conditions. Normal and pathologic cases were studied. Endometrial tissue is also currently under study. In parallel, experiments on animal tissues were also performed in order to make a comparison for the same individual between in vitro and in vivo measurements. This latter was realized by the classic method of the wave of labelled mitoses. The following aspects were examined: (1) Proper choice of the dose range, for valid recognition of ''weakly'' and ''heavily'' labelled cells; (2) choice of the region of the sample where homogeneous labelling occurs after the diffusion of the labelled thymidine, and (3) tests on the stability of the biopsy during incubation. This was done by using varied time interval between {sup 3}H-thymidine administrations. The good correlation between the results obtained in this way also gives supports to the validity of our distinction between the two kinds of labelling intensity. In conclusion, this method could be applied to several types of tissues, provided they can be obtained by biopsy, and with proper safeguards, namely in the choice of the doses range and in the definition of the proliferating population. (author) [French] Afin d'eviter les risques d'effets radiobiologiques lies a l'utilisation de thymidine tritiee, plusieurs chercheurs ont procede a des incubations de biopsies in vitro en presence de ce precurseur radiomarque de l'ADN. Ils s' efforcaient de definir le taux de proliferation cellulaire d'apres la distribution et l' intensite du marquage dans les autoradiographies. Toutefois, pour evaluer exactement la duree du cycle mitotique ou temps de generation, il est necessaire de connaitrez duree de la phase S (phase de synthese de l'ADN), car l'index de marquage ne depend que de l'importance de cette phase par rapport a la duree totale du cycle mitotique. L'auteur a choisi la methode du 'double marquage', utilisant deux differentes doses de thymidine tritiee, qui a ete mise au point par Maurer et ses collaborateurs. En administrant deux doses du precurseur tritie (une dose faible et une dose forte) a un intervalle de temps donne et en soumettant le tissu traite au processus autobiographique, on peut calculer la duree (s) de la phase S d'apres la formule N{sub F}/N{sub f} = S/t. Np est le nombre de noyaux fortement marques (c'est-a-dire les noyaux qui etaient en phase S pendant le deuxieme marquage); Nf est le nombre de noyaux faiblement marques, correspondant aux cellules qui ont quitte la phase S pendant l'intervalle de temps (t) entre les deux marquages. D'apres le pourcentage de cellules fortement marquees par rapport a la population totale (LI), on peut obtenir le temps de generation (TG) d'apres la formule: TG = S/LI x 100. Des biopsies rectales et gastriques de patients ont ete etudiees a l'aide de cette methode qui, etant donne la brievete du temps d'experience, exige des conditions de simple survie. Des cas normaux et des cas pathologiques ont ete etudies. Des tissus d'endometre font aussi actuellement l'objet d'etudes. Parallelement, des experiences ont ete executees sur des tissus animaux afin de comparer, pour le meme sujet, les mesures in vitro et in vivo. Les mesures in vivo ont ete effectuees au moyen de la methode classique de la 'courbe des mitoses marquees'. Les questions suivantes ont ete etudiees: 1) Choix des doses de thymidine tritiee permettant de distinguer les cellules 'faiblement' et 'fortement' marquees; 2) Determination de la zone de l'echantillon ou le marquage des noyaux est homogene apres diffusion de la thymidine tritiee; 3) Etude de la stabilite de la biopsie pendant l'incubation. A cette fin, l'auteur a fait varier l'intervalle de temps entre les deux administrations de thymidine tritiee. La concordance des resultats obtenus de cette maniere constitue egalement un argument en faveur de la validite de la methode du double marquage. L'auteur conclut que cette methode pourrait etre appliquee a plusieurs types de tissus, a condition que ceux-ci puissent etre preleves par biopsie et que les precautions necessaires soient prises, en ce qui concerne l'intervalle des doses et la definition de la population de cellules qui prolifere. (author)}
place = {IAEA}
year = {1970}
month = {Feb}
}
title = {Use of Biopsy Tissue Samples, Incubated In Vitro for Autoradiographic Measurement of Cell Proliferation in Human Tissues; Utilisation de Biopsies Incubees In Vitro pour la Mesure Autoradiographique de la Proliferation Cellulaire de Tissus Humains}
author = {Galand, P.}
abstractNote = {To avoid the radiobiological hazards linked to the use of {sup 3}H-thymidine, several workers have performed in vitro incubations of biopsy material in the presence of this labelled DNA -precursor. Attempts were made to define the proliferative pattern of the tissue from the distribution and intensity of labelling in autoradiographs. However, for true estimation of the cell cycle length, one has to know the S-phase (DNA-synthesis phase) duration, since the labelling index reflects only the part taken by this phase in the whole cell cycle. We chose the ''double-labelling technique'' with two dose levels of {sup 3}H-thymidine, which was developed by Maurer's group. By administering two pulses of the tritiated precursor (a weak dose and a heavy dose) at a given time interval and submitting the treated tissue to the autoradiographic process, one can calculate the S-phase duration(s) after the formula N{sub h}/N{sub w} = S/t. N{sub h} is the number of heavily labelled nuclei (i.e. those nuclei that were engaged in S-phase during the second pulse labelling); N{sub w} is the number of weakly labelled nuclei, corresponding to the cells which left S-phase during the time interval (t) between the two pulse labellings. From the percentage of heavily labelled nuclei in the whole population (L.I.), the generation time (G.T.) is then given by the formula G.T. = S/L.I. x 100. Human rectal and gastric biopsies were submitted to this procedure which, because of its brevity, requires simple survival conditions. Normal and pathologic cases were studied. Endometrial tissue is also currently under study. In parallel, experiments on animal tissues were also performed in order to make a comparison for the same individual between in vitro and in vivo measurements. This latter was realized by the classic method of the wave of labelled mitoses. The following aspects were examined: (1) Proper choice of the dose range, for valid recognition of ''weakly'' and ''heavily'' labelled cells; (2) choice of the region of the sample where homogeneous labelling occurs after the diffusion of the labelled thymidine, and (3) tests on the stability of the biopsy during incubation. This was done by using varied time interval between {sup 3}H-thymidine administrations. The good correlation between the results obtained in this way also gives supports to the validity of our distinction between the two kinds of labelling intensity. In conclusion, this method could be applied to several types of tissues, provided they can be obtained by biopsy, and with proper safeguards, namely in the choice of the doses range and in the definition of the proliferating population. (author) [French] Afin d'eviter les risques d'effets radiobiologiques lies a l'utilisation de thymidine tritiee, plusieurs chercheurs ont procede a des incubations de biopsies in vitro en presence de ce precurseur radiomarque de l'ADN. Ils s' efforcaient de definir le taux de proliferation cellulaire d'apres la distribution et l' intensite du marquage dans les autoradiographies. Toutefois, pour evaluer exactement la duree du cycle mitotique ou temps de generation, il est necessaire de connaitrez duree de la phase S (phase de synthese de l'ADN), car l'index de marquage ne depend que de l'importance de cette phase par rapport a la duree totale du cycle mitotique. L'auteur a choisi la methode du 'double marquage', utilisant deux differentes doses de thymidine tritiee, qui a ete mise au point par Maurer et ses collaborateurs. En administrant deux doses du precurseur tritie (une dose faible et une dose forte) a un intervalle de temps donne et en soumettant le tissu traite au processus autobiographique, on peut calculer la duree (s) de la phase S d'apres la formule N{sub F}/N{sub f} = S/t. Np est le nombre de noyaux fortement marques (c'est-a-dire les noyaux qui etaient en phase S pendant le deuxieme marquage); Nf est le nombre de noyaux faiblement marques, correspondant aux cellules qui ont quitte la phase S pendant l'intervalle de temps (t) entre les deux marquages. D'apres le pourcentage de cellules fortement marquees par rapport a la population totale (LI), on peut obtenir le temps de generation (TG) d'apres la formule: TG = S/LI x 100. Des biopsies rectales et gastriques de patients ont ete etudiees a l'aide de cette methode qui, etant donne la brievete du temps d'experience, exige des conditions de simple survie. Des cas normaux et des cas pathologiques ont ete etudies. Des tissus d'endometre font aussi actuellement l'objet d'etudes. Parallelement, des experiences ont ete executees sur des tissus animaux afin de comparer, pour le meme sujet, les mesures in vitro et in vivo. Les mesures in vivo ont ete effectuees au moyen de la methode classique de la 'courbe des mitoses marquees'. Les questions suivantes ont ete etudiees: 1) Choix des doses de thymidine tritiee permettant de distinguer les cellules 'faiblement' et 'fortement' marquees; 2) Determination de la zone de l'echantillon ou le marquage des noyaux est homogene apres diffusion de la thymidine tritiee; 3) Etude de la stabilite de la biopsie pendant l'incubation. A cette fin, l'auteur a fait varier l'intervalle de temps entre les deux administrations de thymidine tritiee. La concordance des resultats obtenus de cette maniere constitue egalement un argument en faveur de la validite de la methode du double marquage. L'auteur conclut que cette methode pourrait etre appliquee a plusieurs types de tissus, a condition que ceux-ci puissent etre preleves par biopsie et que les precautions necessaires soient prises, en ce qui concerne l'intervalle des doses et la definition de la population de cellules qui prolifere. (author)}
place = {IAEA}
year = {1970}
month = {Feb}
}