taller de discusión de figuras, interpretación de

TALLER DE DISCUSIÓN DE FIGURAS, INTERPRETACIÓN DE
DATOS Y RESULTADOS ENTRE GRUPOS DE INVESTIGACIÓN EN
MICRONÚCLEOS.
Luz Stella Hoyos Giraldo.
lshoyos@gmail.com
Departamento de Biología
Facultad de Ciencias Naturales, Exactas y de la Educación.
Universidad del Cauca.
MICRONÚCLEOS EN CÉLULAS EXFOLIADAS
VALIDACIÓN DEL
BIOMARCADOR DE MN EN
CÉLULAS BUCALES
AUMENTO DE
PUBLICACIONES EN LOS
ÚLTIMOS AÑOS.
2
USO DEL ENSAYO CITÓMICO EN DIVERSOS ESTUDIOS
BÚSQUEDA EN ISI WEB OF KNOWLEDGE
BÚSQUEDA EN ISI WEB OF KNOWLEDGE
BÚSQUEDA EN ISI WEB OF KNOWLEDGE
NATURE PROTOCOLS
2009
SEQUENTIAL ORIGINS AND SPATIO-TEMPORAL SEQUENCE OF THE VARIOUS CELL
TYPES IN THE BMCyt ASSAY
Thomas et al,. 2009
DIAGRAMMATIC REPRESENTATION OF THE VARIOUS CELL TYPES SCORED IN
THE BMCyt ASSAY
De acuerdo con
Tolbert et al,. 1992
Thomas et al,. 2009
CRITERIA FOR CLASSIFICATION OF BMCyt CELL CYTOME ASSAY CELL TYPES
BASAL CELLS
• Large nucleus: cytoplasm ratio relative to differentiated cell
• Smaller and more oval than differentiated cells
• Uniformly stained nucleus
• Darker green cytoplasm relative to differentiated cell when
viewed under transmitted light
DIFFERENTIATED CELLS
• Smaller nuclear: cytoplasmic ratio
• More angular and flatter than basal cells
• Uniformly stained round nucleus
MICRONUCLEATED CELLS
• Contains both main nucleus and micronucleus
• Micronuclei are round or oval with similar stain intensity as
main nucleus
• Micronuclei usually have 1/3–1/16 diameter of main nucleus
• Micronuclei must be located in cellular cytoplasm
• Scored in basal and differentiated cells only
Thomas et al,. 2009
CRITERIA FOR CLASSIFICATION OF BMCyt CELL CYTOME ASSAY CELL TYPES
NUCLEAR BUD CELLS
• Main nucleus has a sharp constriction forming a bud
• Bud is attached to main nucleus
• Bud has similar staining intensity as main nucleus
• Bud diameter can be quarter to half nuclear diameter
BINUCLEATED CELLS
• Cells contain two main nuclei
• Nuclei are of similar size and staining intensity
CONDENSED CHROMATIN CELLS
• Nucleus shows areas of aggregated chromatin
• Distinct areas of nucleus are more intensely stained
• Nucleus exhibits striated pattern
KARYORRHECTIC CELLS
• Nucleus has extensive aggregated chromatin
• Nuclear fragmentation may be evident
Thomas et al,. 2009
CRITERIA FOR CLASSIFICATION OF BMCyt CELL CYTOME ASSAY CELL TYPES
PYKNOTIC CELLS
• Cell has small shrunken nucleus
• Nucleus is uniformly and intensely stained
• Nucleus diameter is 1/3–2/3 diameter of normal nucleus
KARYOLITIC CELLS
• Nucleus is depleted of DNA
• Nucleus is not stained by Feulgen
Thomas et al,. 2009
IMAGES OF THE DIFFERENT CELL TYPES STAINED USING FEULGEN AND LIGHT
GREEN SCORED IN THE BMCyt ASSAY VIEWED BY TRANSMITTED LIGHT OR
UNDER FLUORESCENCE WITH A FAR RED FILTER
(a) Basal cell; (b) differentiated cell; (c) early differentiated cell with micronucleus (arrow);
(d) late differentiated cell with micronucleus (arrow); (e) differentiated cell with nuclear bud
(arrow); (f) binucleated cell; (g) condensed chromatin cell; (h) karyorrhectic cell; (i) pyknotic
cell; (j) Karyolytic cell. Upper panels light microscopy, lower panels fluorescence microscopy.
All images were taken at 1000x magnification.
Thomas et al,. 2009
2013
BUCCAL MICRONUCLEUS CYTOME MODEL: DIAGRAMMATIC REPRESENTATION
OF THE VARIOUS BUCCAL CELL TYPES AND MECHANISMS OF THEIR ORIGIN
Bolognesi et al,. 2013
CRITERIOS PARA LA IDENTIFICACIÓN Y REGISTRO DE DIFERENTES
TIPOS DE CÉLULAS
CÉLULAS BASALES:
• Su frecuencia refleja la actividad proliferativa de la mucosa bucal y puede
depender del método de toma de muestras y su intensidad.
CÉLULAS DIFERENCIADAS:
• Células diferenciadas transicionales: para personas no experimentadas no
son fáciles de clasificar por eso se recomienda agruparlas con las
terminales.
• Células diferenciadas terminales.
Bolognesi et al., 2013
BASAL CELLS
Photomicrographs
of
typical
basal
cells
stained
with
feulgen and light green viewed at 1000x magnification under
transmitted light (a–c) and fluorescence with a far red filter (d–f).
Bolognesi et al., 2013
Criteria:
1. They are the smallest cells
amongst the exfoliated buccal cell
types being approximately 1/4th to
1/3rd
the
size
of
fully
differentiated cells.
2. They have the largest nucleus:
cytoplasm area ratio relative to
transitional and differentiated
cells.
3. Their
cytoplasm
shape
is
sometimes spherical and less
angular than differentiated cells.
4. The nucleus is typically uniformly
stained and has a round or oval
shape.
5. The cytoplasm is stained darker
green relative to differentiated
cells
when
viewed
under
transmitted light.
6. These cells may contain MNi
and/or NBUDs.
TRANSITIONAL DIFFERENTIATED CELLS
Photomicrographs of typical transitional cells stained with Feulgen and
Light Green viewed at 1000 magnification under transmitted light (a–c)
and fluorescence with a far red filter (d–f).
Bolognesi et al., 2013
Criteria:
1. They are larger than basal cells and
smaller than fully differentiated
cells being greater than 1/3rd but
less than 2/ 3rd the size of a
terminally differentiated cell.
2. They may be more angular in
shape than basal cells.
3. The nucleus:cytoplasm area ratio is
less than that of basal cells due to
their larger cytoplasm but greater
than that of fully differentiated
cells.
4. The nucleus is typically uniformly
stained and has a round to oval
shape.
5. They have a lighter green
cytoplasm relative to basal cells
when viewed under transmitted
light.
6. These cells may contain MNi and
NBUDs.
TERMINALLY DIFFERENTIATED CELLS
Criteria:
1. The cytoplasm is typically angular
and flat in shape
2. The cytoplasm is sometimes
folded at the edges.
3. They have a smaller nucleus:
cytoplasmic area ratio relative to
basal cells and to transitional
cells.
4. The nucleus is typically uniformly
stained and round or oval in
shape.
5. The cells have a lighter green
cytoplasm relative to basal cells.
6. These cells may contain MNi
and/or NBUDs.
Photomicrographs of typical differentiated cells stained with Feulgen
and Light Green viewed at 1000 magnification under transmitted light
(a–c) and fluorescence with a far red filter (d–f).
Bolognesi et al., 2013
CRITERIOS PARA LA IDENTIFICACIÓN Y REGISTRO DE TIPOS DE
CÉLULAS CON ANOMALÍAS ASOCIADAS A LA MUERTE CELULAR
CÉLULAS CON CROMATINA CONDENSADA:
•La estructura de la cromatina condensada tiene poca o ninguna actividad
transcripcional y se ha asociado con el proceso de apoptosis.
CÉLULAS CARIORÉTICAS:
• Su patrón nuclear moteado densamente indica desintegración nuclear típica de etapas
tardías de apoptosis.
CÉLULAS PICNÓTICAS:
• Picnosis representa una condensación irreversible de la cromatina en el núcleo de las
células sometidas a apoptosis y se cree que precede a cariorrexis.
• El significado biológico de las células picnóticas y el mecanismo que conduce a su
formación en células bucales no se entienden completamente.
CÉLULAS CARIOLÍTICAS:
• Cariolisis es la etapa en que la desintegración del núcleo es completa y se produce en
las etapas posteriores a la necrosis y la apoptosis.
Bolognesi et al., 2013
CONDENSED CHROMATIN CELLS
Criteria:
1. They are typically angular and
flat in shape usually with the
size
of
a
terminally
differentiated cell.
2. The nuclei show a striated
pattern of parallel tracts of
condensed chromatin.
3. The distinct areas of condensed
chromatin in the nucleus are
more intensely stained than
the rest of the nucleus.
Photomicrographs of typical condensed chromatin cells stained with
Feulgen and Light Green viewed at 1000 magnification under
transmitted light (a–c) and fluorescence with a far red filter (d–f).
Bolognesi et al., 2013
CONDENSED CHROMATIN CELLS
Photomicrographs of typical examples of nuclei of condensed chromatin cells stained with Feulgen
and Light Green viewed at 1000 magnification under transmitted light (a–c) and fluorescence with
a far red filter (d–f).
Bolognesi et al., 2013
KARYORRHEXIS CELLS
Criteria:
1. They are typically angular and
flat in shape usually with a size
of a terminally differentiated
cell.
2. The nucleus contains more
densely aggregated chromatin
than that in condensed
chromatin cells.
3. The nucleus may also exhibit
extensive
fragmentation
indicative of advanced nuclear
fragmentation.
Photomicrographs of karyorrhectic cells stained with Feulgen and Light
Green viewed at 1000 magnification under transmitted light (a–c) and
fluorescence with a far red filter (d–f).
Bolognesi et al., 2013
KARYORRHEXIS CELLS
Photomicrographs of typical examples of nuclei of karyorrhectic cells stained with Feulgen and
Light Green viewed at 1000 magnification under transmitted light (a– c) and fluorescence with
a far red filter (d–f).
Bolognesi et al., 2013
PYKNOTIC CELLS
Criteria:
1. They are angular and flat in
shape with a cytoplasmic area
the size of a terminally
differentiated cell.
2. The nucleus is small and
shrunken with a diameter that
is 1/3rd to 2/3rd of that in a
fully differentiated viable cell.
3. The nucleus is uniformly and
intensely stained.
Photomicrographs of pyknotic cells stained with Feulgen and Light Green
viewed at 1000 magnification under transmitted light (a–c) and
fluorescence with a far red filter (d–f).
Bolognesi et al., 2013
KARYOLYTIC CELLS
Criteria:
1. They are angular and flat in
shape
usually
with
a
cytoplasmic area that is the size
of a terminally differentiated
cell.
2. A ‘‘ghost’’ image of the nucleus
is often apparent in the
cytoplasm
suggesting
the
remnant presence of the
nuclear scaffold proteins.
3. They do not have a DNAcontaining nucleus or other
structures that stain with
Feulgen.
Photomicrographs of karyolytic cells stained with Feulgen and Light Green
viewed at 1000
magnification under transmitted light (a–c) and
fluorescence with a far red filter (d–f).
Bolognesi et al., 2013
CRITERIOS PARA LA IDENTIFICACIÓN Y REGISTRO
DE CÉLULAS BINUCLEADAS
CÉLULAS BINUCLEADAS:
• El mecanismo más probable para su formación es el fracaso de la citocinesis, ya sea
debido a defectos en la formación del anillo de microfilamentos o la detención del ciclo
celular debido a la mala segregación de los cromosomas o disfunción del telómero.
• La proporción de células binucleadas: mononucleadas es un biomarcador útil que
indica insuficiencia en la citocinesis y susceptibilidad a aneuploidía .
Bolognesi et al., 2013
BINUCLEATED CELLS
Criteria:
1. Two main nuclei within a single
cell
2. The nuclei are of similar size
and staining intensity.
3. The nuclei may be either
separated or touching each
other.
Photomicrographs of binucleated cells stained with Feulgen and Light
Green viewed at 1000 magnification under transmitted light (a–c) and
fluorescence with a far red filter (d–f).
Bolognesi et al., 2013
CRITERIA FOR IDENTIFYING AND SCORING CELL TYPES WITH NUCLEAR
ABNORMALITIES INDICATIVE OF CHROMOSOMAL INSTABILITY OR DNA DAMAGE
CÉLULAS CON MICRONÚCLEOS (MN):
• MN son observados y registrados generalmente en células diferenciadas
transitorias o diferenciadas terminales, pero también pueden producirse a veces
en células basales.
• En células con cromatina condensada, células carioréticas y células cariolíticas
no se registra la presencia de MN para evitar confusión con cuerpos apoptóticos
de núcleos en desintegración.
• La frecuencia basal de células bucales con micronúcleos en sujetos sanos no
expuestos a agentes genotóxicos está por lo general dentro del rango de 0.30 a
1.70 por 1000 células diferenciadas.
Bolognesi et al., 2013
CRITERIA FOR IDENTIFYING AND SCORING CELL TYPES WITH NUCLEAR
ABNORMALITIES INDICATIVE OF CHROMOSOMAL INSTABILITY OR DNA DAMAGE
CÉLULAS CON BROTES NUCLEARES (NBUDs):
• Su estructura sugiere un proceso de gemación implicado en la eliminación de
exceso de materiales nucleares tales como complejos de reparación del ADN no
finalizados o ADN amplificado a partir de su segregación a la periferia del
núcleo.
• Existen brotes nucleares que casi alcanzan el tamaño del núcleo principal
(broken egg, Tolbert et. al, 1992).
Bolognesi et al., 2013
CELLS WITH MICRONUCLEI (MNi)
Criteria:
1. They contain both a main
nucleus and one or more MNi.
2. MNi are round or oval.
3. MNi
are
Feulgen-positive
bodies.
4. MNi have the same texture and
staining intensity as the main
nucleus.
5. MNi are 1/3–1/16 diameter of
the main nucleus.
6. MNi are not connected to the
main nucleus.
7. The nuclear boundary of the
micronucleus should be clearly
distinguishable from that of the
main nucleus.
Photomicrographs of mononucleated cells with one or more micronuclei
stained with Feulgen and Light Green viewed at 1000 magnification under
transmitted light (a–c) and fluorescence with a far red filter (d–f).
Bolognesi et al., 2013
CELLS WITH MICRONUCLEI (MNi)
Photomicrographs of binucleated cells with micronuclei stained with Feulgen and Light Green viewed at
1000 magnification under transmitted light (a–d) and fluorescence with a far red filter (d–f).
Bolognesi et al., 2013
CELLS WITH NUCLEAR BUDS (NBUDs)
Photomicrographs of cells with broken eggs stained with Feulgen and Light
Green viewed at 1000 magnification under transmitted light (a–c) and
fluorescence with a far red filter (d–f).
Bolognesi et al., 2013
Criteria:
1. The main nucleus has a sharp
constriction forming a bud of
nuclear material.
2. NBUDs are attached to the
main nucleus by a narrow or
wide nucleoplasmic bridge.
3. NBUDs and their associated
nucleoplasmic bridges have
similar staining intensity as the
main nucleus
4. NBUDs usually have a diameter
that is 1/3rd-1/16th that of the
main nucleus but in some rarer
case (broken eggs) could be
even greater and almost up to
the same size as the main
nucleus.
CELLS WITH NUCLEAR BUDS (NBUDs)
Photomicrographs of typical nuclear buds (a, b and d, e) and broken eggs (c and f) stained
with Feulgen and Light Green viewed at 1000 magnification under transmitted light and
fluorescence with a far red filter respectively.
Bolognesi et al., 2013
ESTUDIOS EPIDEMIOLÓGICOS MOLECULARES
Biomarcadores de Exposición
ICH
Ensayo Cometa
Biomarcadores de Efecto
MNs en
Linfocitos
ACs
MNs en células
de Vejiga
MNs en
células nasales
MNs en células
bucales
Biomarcadores de Susceptibilidad
Genes del Metabolismo
CYP2E1
GSTM1,GSTT1
Genes de Reparación
XRCC1194/399
XRCC1280,
XRCC3241
MNs en Peces
Ensayo Citómico en
Celulas Bucales
Epigenética
Perfiles de Metilación de GST
ENSAYO DE MICRONÚCLEOS EN CÉLULAS DEL EPITELIO BUCAL
CYTOME ASSAY
Tabla 1: Principales variables de los
estudios incluidos en la base de datos del
HUMNXL (N= 5424).
Tabla 2: Frecuencia basal de micronúcleos
(MN/1000) en células exfoliadas del epitelio
bucal.
Total
Número de
Individuos
Frecuencia basal
encontrada
Rango de la
Frecuencia basal
Promedio de Edad
35.55
5424
0.74
0.3 – 1.7
Fumadores N (%)
1381 (25.5)
Consumidores de
alcochol N (%)
1222 (22.5)
Variables
Tinción N (%)
Específica para ADN
No específica para
ADN
Número de células
registradas
≤ 1000
≤ 2000
≥ 2001
Tabla 3: Frecuencia basal de biomarcadores del
Cytome Assay en células exfoliadas del epitelio
bucal evaluados con la base de datos del HUMNXL
Biomarcador
Número de
Individuos
Frecuencia
basal
Brotes Nucleares
789
1.36
Células Binucleadas
852
3.04
Células Cariorréticas
408
2.23
Células Picnóticas
210
4.38
3375 (62.2)
2049 (37.8)
777
3021
1505
(Bonassi et al., 2011)
ENSAYO DE MICRONÚCLEOS EN CÉLULAS UROTELIALES EXFOLIADAS DE LA VEJIGA
Tabla 4: Frecuencia basal de micronúcleos (MN/1000) en células uroteliales
exfoliadas de la vejiga reportada en diferentes estudios de 1995 a 2004
Referencia
Pais
Número
de
Individuos
Tinción
Frecuencia Basal
(Basu et al.,
2004)
India
154
Giemsa
1.41
(Basu et al.,
2002)
India
21
Giemsa
0.56
(Tian et al.,
2001)
China
13
Feulgen-Fast
Green
0.53
(Murray et al.,
1999)
Australia
18
Diff-Quik
6.90
(Hofseth et
al., 1996)
Columbia
Británica
26
Feulgen-Fast
Green
0.098*
(Burgaz et al.,
1995)
Turquía
20
Feulgen-Fast
Green
0.66*
* Frecuencia de micronúcleos reportada 100 células
ANÁLISIS ESTADISTICO
SPSS 13.0
Unidad de Muestreo: El Individuo (Albertini et al 2000)
Distribución Normal
Homogeneidad de Varianzas
Independencia de Datos
Kolmogorov Smirnof
Levene
Rachas
PRUEBAS NO PARAMÉTRICA
DESCRIPTIVO
Primer Objetivo
Frecuencias Genotípicas y Alélicas
Desarrollo Binomial (p+q)2 = p2 + 2pq+q2
Verificar Equilibrio de Hardy-Weimberg
Bondad de Ajuste de
X²
= ∑ (O-E)²/ E
Kruskal-Wallis: comparar 3 o mas grupos
Frecuencias de ACs/ 100 metafases Vs
Edad de los Individuos
Segundo Objetivo
Frecuencias de ACs estructurales y Gaps
/ 100 metafases
Expuestos – Referentes
U de Mann Whitney: Para comparar 2 muestras
Independientes
Frecuencias de ACs / 100 metafases
“Altas” y “Bajas”
A
Expuestos – Referentes
E
Tablas
de Contingencia
R
X²: Prueba
de Asociación2x2
Frecuencias de ACs / 100 metafases Vs
Rangos de Tiempo de exposición (años)
B
Análisis de Correlación Spearman
(Datos Ordenados)
Tercer Objetivo
INFERENCIAL
Asociación gen-Ambiente → ACs
Alta y Baja Frecuencia de ACs / 100 metafases Vs
Genotipos de c/u de los genes
Expuestos – Referentes
RR IC95% y p<0.05: Significativo si IC (95%) No incluye al 1
LINFOCITOS HUMANOS
CBMN
2006
• The ultimate objective of the HUMN project studies was to test
the hypothesis that an elevated MN frequency in human tissues
is predictive of cancer risk
• This critical validation step is essential to justify the use of such
techniques in human biomonitoring studies in populations
LINFOCITOS HUMANOS
CBMN
Fuente: (Bonassi et al., 2006)
LINFOCITOS HUMANOS
CBMN
Fuente: (Bonassi et al., 2006)
LINFOCITOS HUMANOS
CBMN
Fuente: (Bonassi et al., 2006)
LINFOCITOS HUMANOS
CBMN
Fuente: (Bonassi et al., 2006)
LINFOCITOS HUMANOS
CBMN
2010
LINFOCITOS HUMANOS
CBMN
• Frecuencia basal de MN en linfocitos
• La frecuencia media de MN global en sujetos no expuestos
(normal) fue de 6,5MN/1000celulas.
• Rango intercuartil 3-12MN/1000 células
Incremento de MN
con la edad en ambos
sexos,
incremento se da en
individuos > 30 años
Fuente: (Bonassi et al., 2001)
LINFOCITOS HUMANOS
CBMN
The HUMAN Project manifiesto
The information was used to:
(i) Determine the extent of variation of ‘normal’ values for
different laboratories and the influence of other factors
potentially affecting baseline MN frequency, e.g. age,
gender and lifestyle.
(ii) Provide information on the effect of experimental
protocol variations on MN frequency measurements.
(iii) Design and test optimal protocols for the different cell
types
(iv) Determine the extent to which MN frequency is a valid
biomarker of ageing and risk for diseases such as cancer.
Fuente: (Fenesh et al., 2010)
CÉLULAS BUCALES
“Citome Assay”
The HUMNxL project on MN frequencies in human
buccal cells
The aims of the workshop were to
(i) Discuss current state of knowledge on the buccal MN
assay
(ii) Identify important gaps of knowledge regarding
theory, biology and methods
(iii) Decide on a plan of action to resolve the key
methodological and knowledge gap issues and
(iv) Explore the possibility of the pooling of databases to
determine the most important variables affecting the
assay.
Fuente: (Fenesh et al., 2010)
CÉLULAS BUCALES
“Citome Assay”
The HUMNxL project on MN frequencies in human buccal cells
(i)
(ii)
(iii)
(iv)
It was agreed at the workshop that four activities should
be initiated as soon as possible, namely
A method for collection of databases from different
laboratories
Writing of a protocol based on the most commonly used
and best-validated methods
Development of slide scoring procedures and
An Inter-laboratory slide-scoring exercise, in this order. A
follow-up workshop was held at the 10th International
Conference on Environmental Mutagens in Florence in
2009 in which progress on these activities was reported.
The following has been achieved so far.
Fuente: (Fenesh et al., 2010)
CÉLULAS BUCALES
“Citome Assay”
El HUMN xL proyecto en frecuencias MN en células
bucales humanas
Different confounding factors influencing the MN
frequency in peripheral lymphocytes, such as gender,
age and lifestyle habits have been considered for the
buccal cell MN assay.
However, the majority of studies failed to demonstrate
any influence of age or sex, although only a few studies
have investigated a broad age range.
Fuente: (Fenesh et al., 2010)
FUTURE CHALLENGES
(a) MN assays in other tissues such as erythrocytes, nasal epithelium and hair root
cells
(b) The impact of diet and lifestyle factors and the studies reported to date are
sparse.
(c) Polymorphisms
(d) In MN assays is the adoption of the cytome approach that not only scores MN
but also captures other nuclear abnormalities
(e) Automated scoring of MN
(f) Larger and/or longer studies are required to verify the results of previous
studies concerning the association of MN frequency with pregnancy
complications, cancer and cardiovascular disease
(g) Explore the relationship of MN expression with changes in DNA methylation
and the associated transcriptome, metabolome and proteome profiles to
unravel the underlying molecular mechanisms that correlate with this DNA
damage biomarker.
(h) The ultimate goal is to see the validated MN assays becoming a routine
diagnostic in the new disease prevention paradigms and strategies required for
this new millennium based on personalised prevention of DNA damage.
Fuente: (Fenesh et al., 2010)
CÉLULAS BUCALES
“Citome Assay”
Criterios para el registro
• Coloración de Feulgen y Fast –Green es
recomendada
• Observar en microscopio de luz y
confirmar en fluorescencia.
Preparaciones se conservan por años
CÉLULAS BUCALES
“Citome Assay”
Criterios para el registro
1. Paso: Registrar primero frecuencia de varios
tipos de células (normales y anormales) en al
menos 1000 células.
2. Paso: Registrar biomarcadores de daño en ADN
en un mínimo de 2000 células normales
diferenciadas (incluye células diferenciadas
transicionales y terminalmente diferenciadas).
CÉLULAS BUCALES
“Citome Assay”
(Holland et al., 2008)
• Revisión del estado del conocimiento. Identificó:
1. Vacios en el conocimiento sobre el mecanismo biológico
asociado a la expresión de MN. Explicación biológica
básica de los diferentes tipos de células.
2. Necesidad de estandarizar en las preparaciones
citogenéticas, protocolos de coloración y criterios de
registro.
El significado biológico de los diferentes tipos celulares y
otras anomalías nucleares no están completamente
entendidas.
Aunque hay evidencia sugestiva sobre la asociación de los
tipos de células y anomalías nucleares con:
• Envejecimiento
• Enfermedades neurodegenerativas (Thomas et al, 2008;
Ferreira, 2009; Thomas et al, 2007).
CÉLULAS BUCALES
“Citome Assay”
2011
CÉLULAS BUCALES
“Citome Assay”
Linear correlation of FR's in 19 studies from the literature
that simultaneously evaluated MN in peripheral blood
lymphocytes (PBL) and exfoliated buccal cells in the same
individuals.
Fuente: Ceppi, M., Biasotti, B., Fenech, M., & Bonassi, S. (2010). Human population studies with the
exfoliated buccal micronucleus assay: statistical and epidemiological issues. Mutation research, 705(1), 11–9.
CÉLULAS BUCALES
“Citome Assay”
Fuente: Bonassi, S., Coskun, E., Ceppi, M., Lando, C., Bolognesi, C., Burgaz, S., … Fenech, M. (2011). The HUman MicroNucleus project on
eXfoLiated buccal cells (HUMN(XL)): the role of life-style, host factors, occupational exposures, health status, and assay protocol. Mutation
research, 728(3), 88–97.
CÉLULAS BUCALES
“Citome Assay”
Fuente: Bonassi, S., Coskun, E., Ceppi, M., Lando, C., Bolognesi, C., Burgaz, S., … Fenech, M. (2011). The HUman
MicroNucleus project on eXfoLiated buccal cells (HUMN(XL)): the role of life-style, host factors, occupational
exposures, health status, and assay protocol. Mutation research, 728(3), 88–97.
CÉLULAS BUCALES
“Citome Assay”
Fuente: Bonassi, S., Coskun, E., Ceppi, M., Lando, C., Bolognesi, C., Burgaz, S., … Fenech, M. (2011). The HUman
MicroNucleus project on eXfoLiated buccal cells (HUMN(XL)): the role of life-style, host factors, occupational
exposures, health status, and assay protocol. Mutation research, 728(3), 88–97.
CÉLULAS BUCALES
“Citome Assay”
Fuente: Bonassi, S., Coskun, E., Ceppi, M., Lando, C., Bolognesi, C., Burgaz, S., … Fenech, M. (2011). The HUman
MicroNucleus project on eXfoLiated buccal cells (HUMN(XL)): the role of life-style, host factors, occupational
exposures, health status, and assay protocol. Mutation research, 728(3), 88–97.
CELULAS
UROTELIALES
COLORACION
DE FEULGEN
FAST-GREEN
Prueba de MN
COLORACION DE FEULGEN FAST-GREEN
Coloración para la identificación altamente específica de ácidos nucleicos del ADN
a partir del reconocimiento de los grupos aldehídos de la pentosa (azúcar). Para
esto se realizan 2 pasos fundamentales:
1) Hidrólisis ácida:
Se utiliza para romper los puentes de hidrogeno que unen a la doble hélice,
mediante la desnaturalización de la cadena. Para esto se utiliza ácido clorhídrico
(HCl), que es el componente reactivo mas critico del método, controlando la
concentración del ácido, temperatura y tiempo, que depende del tipo de tejido
(compactación de cromatina) y fijador empleado.
Durante la hidrólisis ácida suave la mayor parte del RNA se divide en sustancias
solubles y se pierde del tejido, sin ser removidos los grupos ribosil debido a la
presencia de un grupo hidroxilo en la posición 2' de la ribosa, por lo que, este
material no reacción posteriormente con el reactivo de Schiff. Mientras tanto el
DNA es parcialmente hidrolizado siendo removidas y liberadas las bases púricas
(A y G) de los residuos de deoxiribosa (depurinización) formando ácido apurínico y
generando grupos aldehído (CHO) en el C1 de la pentosa, donde se encontraba
unida la base nitrogenada.
COLORACION DE FEULGEN FAST-GREEN
CELULAS
UROTELIALES
COLORACION
DE FEULGEN
FAST-GREEN
Prueba de MN
CELULAS
UROTELIALES
COLORACION
DE FEULGEN
FAST-GREEN
Prueba de MN
CELULAS
UROTELIALES
COLORACION
DE FEULGEN
FAST-GREEN
Prueba de MN
CELULAS UROTELIALES
Prueba de MN
Fotografía de Células uroteliales coloreadas con tinción FeulgenFast-Gree
(a-b) Fotografías de células transicionales de vejiga normales.
(c-d) Fotografías de células transicionales de vejiga con
Micronúcleos(MN)
CELULAS UROTELIALES
Prueba de MN