TINCIN NUCLEAR

METODO PREPARACIN DEL COLORANTE

HEMATOXILINA DE HARRIS
RICHARD BEDON
TECNLOGO MDICO
SERVICIO DE ANATOMA PATOLGICA
HOSPITAL VRG - Hz

OBJETIVOS

El alumno deber identificar los reactivos principales para la

preparacin del colorante Hematoxilina de Harris.

Entender el fundamento de la coloracin nuclear.

Al final de la prctica el alumno deber presentar un mapa

conceptual de lo aprendido

TINCIN NUCLEAR
La

Hematoxilina de Harris esta compuesta por una

mezcla de Hematoxilina (5 g), Alcohol 100% (50 mL),
Alumbre de Potasio (100 g), Agua destilada (1000 mL)
y Oxido de rojo de Mercurio (2,5 g).

TINCIN NUCLEAR

PREPARACIN DE SOLUCIONES:

FUNDAMENTO QUE ES UNA SOLUCIN?

Son mezclas homogneas de dos o mas sustancias, una solucin es la mezcla

de un soluto en un disolvente, entendindose por soluto la sustancia que esta
en menor proporcin y disolvente la sustancia que esta en mayor proporcin

TIPOS DE SOLUCIONES:
Slidas, Lquidas y gaseosas

TINCIN NUCLEAR

Cada uno de los componentes de la Hematoxilina cumple una funcin.

Agua destilada es solvente del mordiente

Alcohol 96% es solvente de la hematoxilina

Alumbre de potasio es mordiente

Oxido de Mercurio que oxida la Hematoxilina a Hemateina que es su ingrediente

activo

Finalmente la Hematoxilina que es el colorante

TINCIN NUCLEAR
La

Hematoxilina necesita de un mordiente

(intermediario) entre el tejido y el colorante,
formando
un
complejo
Tejido-MordienteHematoxilina, compuesto por un complejo de
coordinacin formado entre un ion polivalente de
un metal y el colorante, que no es fcil disociarlo.

TINCIN NUCLEAR
La

Hematoxilina colorante bsico reacciona con

grupos aninicos (cargados negativamente) de
componentes de los tejidos, como grupos fosfato
ionizados (PO3-) de heterocromatina, nuclolos y
cidos nucleicos (ADN y RNA) del ncleo, grupos
sulfato ionizados (SH-) en el citoplasma y matriz del
cartlago, y grupos carbonilo (COOH-) de las
protenas, por lo que son basfilos

TINCIN NUCLEAR
Existen
Se

dos mtodos para teir el ncleo

tie el ncleo con la intensidad de color deseada:

METODO PROGRESIVO

Se

sobre-tie con hematoxilina no acidificada, luego se

remueve el exceso de tincin con cido clorhdrico
diluido:
METODO REGRESIVO

TINCIN NUCLEAR
Grupos

auxocromos de las molculas de hematoxilina

Tonalidad

rojiza pH < 3:

Tonalidad

azulada pH > 3:

En

pH 8 el ncleo adopta un color fuertemente azulado

Reactivo

Hematoxilina

Sulfato de Potasio y
aluminio

cido actico glacial

Funcin del colorante

Funcin en la clula

Componente activo:
Hemateina (carga
negativa) se obtiene de la
maduracin (oxidacin)

Cuando madura toma un

color rojo vinoso, tonalidad
que toman los ncleos

Mordiente le da el color a Suministra cargas positivas

la hematoxilina
que acta como puentes
para unirse a las cargas
negativas de la Hemateina
y del cido fosfrico del
DNA nuclear
Diferenciador, estabiliza el
colorante, previene
oxidacin

Agua destilada

Disuelve el sulfato de
potasio y aluminio por
calentamiento

xido de mercurio (rojo o

amarillo)

Activa la hematoxilina
para obtener por
oxidacin: Hemateina

Incrementa la precisin de
la tincin nuclear

La Hemateina le da la
tonalidad a los ncleos

PROBLEMAS CON LA TINCIN

COLORACIN NUCLEAR
DEMASIADO PLIDA

Deficiente remocin del fijador,

tiempo insuficiente de coloracin,
hematoxilina diluida con agua,
HCl muy concentrado o
demasiadas inmersiones, lavado
con agua corriente con mucho
cloro.

COLORACIN NUCLEAR MUY

OSCURO

Frotis con reas gruesas y finas,

frotis hemorrgicos cambia el pH y
hay mayor afinidad por
hematoxilina, mal enjuague,
pocas inmersiones en HCl o su
concentracin es muy baja.

Definition of cytopathology

Cytopathology is the study of normal and abnormal exfoliated cells in

tissue fluid.

The individual cells reflect the normal and abnormal morphology of the
tissue from which they are derived.

Types of exfoliated cyto-pathology

Natural spontaneous exfoliation

Natural covering epithelium: skin, urinary tract, vagina, and cervix.

Glandular epithelial secretion: Breast (Nipple secretion).

Sputum

Urine

Exudates and transudate:

Pleural fluid
Pericardial fluid

Peritoneal fluid

Artificial enhanced exfoliation:

Scrapings from cervix, vagina, oral cavity, and skin

Brushing and lavage: bronchi, GIT, and urinary tract

Fine needle aspiration (FNA) for:

Body

cavity fluid: pleural, pericardial & peritoneal fluids

Cysts:
Solid

neck, breast & ovary

tissue: body organs, tumors

Role of cytopathology

Early detection of unsuspected diseases (malignant

or pre-malignant lesions).

Confirmation of suspected diseases without surgical

trauma.

Diagnosis of hormonal imbalance.

Useful in flow up the course of disease or

monitoring therapy.

Advantage of Cytopathology

Rapid diagnosis

It is better in evaluating the infectious diseases.

Supplement or replace frozen section or biopsy

No injury to tissue allowing repeated sampling

It is better for hormonal assay

Cytopathological smear cover a wider surface than that

- Inexpensive

involved in surgical biopsy.

- Simple

Disadvantage of Cytopathology

Interpretation of the morphological cellular changes is based only on

individual cell observation.

Not always finally diagnosis, so it is confirmed by histopathology in some

cases.

Not determine the size and type of lesion of some cases.

Factors that determine the appearance of cells

Type of the technique used.

Level of cell maturation at the time of cell collection.

Nature of the parents tissue: soft tissue, cyst, or solid organ.

Medium of the exfoliated cells.

Interval between the stain of the exfoliated cells and collection of samples.

Type of fixative, stain, and processing of the technique used.

PAP smear: named after

Dr. George Papanicolaou (1883-1962)
Vaginal

smears from guinea pigs (1917)

Women

(1920)

Hormonal

cycles

Pathological conditions (1928)

Normal Cervix

Taking the Sample

Liquid Based Cytology lab processing

The Pap Smear

Pap smears are not perfect

For a high grade lesion, the sensitivity of a single pap smear is only
60-80%

Estimated false negative rate is 30-50%

Requires adequate specimen collection

Requires adequate cytological review

Requires adequate patient and physician follow-up

10%

of women with cervical cancer had inappropriate follow-up.

Requires access to care

50%

of women with cervical cancer were never screened and 10% had

not been screened within 5 years of diagnosis.

Who to screen

Any woman with a cervix who has ever had sexual activity.

Screening frequency

Yearly until three consecutive normal pap smears, then may decrease
frequency to every three years

Annual screening for high-risk women is highly recommend.

When to stop routine screening

Age 65 and adequate recent screening

Three

consecutive normal pap smears

No

abnormal pap smears in last 10 years

No

history of cervical or uterine cancer

Hysterectomy for benign disease

Hysterectomy for invasive cervical cancer

Cervical histology

Original Squamous Epithelium

Vagina and outer ectocervix

4 cell layers

Well-glycogenated (pink) unless atrophic

Columnar Epithelium
Upper

and middle endo-cervical canal

Single

layer of columnar cells arranged in folds

Mucin

producing (not true glands)

Squamous Metaplasia

Central ectocervix and proximal endocervical canal

Replacement of columnar cells by squamous epithelium

Progressive and stimulated by

Acidic environment with

onset of puberty

Estrogen causing eversion of

endocervix

Transformation Zone

Zone between original squamo-columnar junction and the new squamocolumnar junction

Nabothian cysts visually identify the transformation zone if present

SquamousEpithelium

Parabasal Cells

Intermediate Cells

Superficial Cells

Endocervix

Endocervical Cells

Endometrial Cells

Non-Epithelial Cells

Lymphocytes

Polymorphs

sperms

Normal smear

Metaplastic Cells