Immunohistochemical studies, immunofluorescence and in situ hybridization. New methodologies and application in diagnosis

Immunohistochemical and in situ hybridization studies make it possible to identify specific phenotypic characteristics of different cell types or molecular alterations, both at the level of protein expression and at the level of DNA or RNA. The incorporation of immunohistochemical techniques into the clinical routine of pathological anatomy services occurred more than 30 years ago, and the entry of semi-quantitative tests into the diagnostic routine also represented a great advance in this technique. In the same way, in situ hybridization techniques were incorporated more than 15 years ago, useful for the diagnosis of some types of sarcomas and currently, they are also essential for the detection of predictive biomarkers.

In recent years, the relevance that these techniques have acquired, as part of the diagnostic method, have made it necessary to have an exhaustive control of pre-analytical and analytical factors, and to incorporate quality assurance programs.

At present these techniques are essential in any pathological anatomy service, for the correct classification of different tumors based on their phenotype and the detection of new prognostic and predictive markers of targeted therapy.

This course aims to carry out a comprehensive approach to these three essential techniques in our Pathological Anatomy services, from its theoretical base knowing its foundation and the new existing methodologies, passing through needs to know the standardization processes and the performance of controls of quality, to finally know its application in the diagnosis and in the determination of prognostic and predictive biomarkers.


Who is it addressed to:

Pathological Anatomy Technicians, Biologists and Pathological Anatomy Resident Physicians.

* Continuing education credits are not valid for resident physicians.


School periods available for enrollment:

2nd edition: from September 1, 2021 to November 28, 2021

3rd edition: from December 1, 2021 to February 27, 2022

4th edition: from March 1, 2022 to May 29, 2022

Topic I: Immunohistochemical Techniques

1. Introduction to immunohistochemical techniques: types of IHC, technical procedures, need for internal and external controls. Detection and correction of errors, importance of preanalytical factors ( Rubén López Aguado)

2. Evolution and automation of immunohistochemical techniques ( Rubén López Aguado)

3. Validation and fine-tuning of IHC techniques ( Sandra Pérez Buira)

4. Multiple immunohistochemistry: IHC and special stains; Double and triple IHC in clinical practice (Sandra Pérez Buira)


Topic II. Immunofluorescence techniques

1. Fundamentals of immunofluorescence: Introduction, types of IF, technical procedures and internal and external controls. (Elena Gonzalvo)

2. IF in samples for paraffin, fine-tuning of the technique. (Elena Gonzalvo)

3. Detection and correction of errors in the technique. Importance of pre-analytical factors. (Elena Gonzalvo)

4. IF display systems. Multiple IF. (Laura Comerma )


Topic III. In situ hybridization study

1. Introduction to in situ hybridization techniques: Objective: introduction of the technique, methodology and rationale, explaining the molecular alterations that allow identifying in situ hybridization (Carlos Casterá)

2. In situ hybridization in bright field. Technique and types (chromogenic and silver) (RNA / DNA, RNAscope) (Carlos Casterá)

3. Fluorescent in situ hybridization. Technique, type of samples (blood / FFPE) and type of probes (Marta Salido / María Rodríguez)

4. Detection and correction of errors in the technique. Importance of pre-analytical factors ( María Rodríguez)

5. Application of in situ hybridization in hemato-lymphoid pathology (Marta Salido)


Topic IV. Quality in immunohistochemical, immunofluorescence and in situ hybridization techniques (Teresa Hernández)

1. Internal-external quality controls

2. Certification and accreditation processes


Topic V. Application of immunohistochemical, immunofluorescence and in situ hybridization techniques in diagnosis: developing IHC / IF or ISH that are used in the diagnosis of the different pathologies, it must be explained which ones, with diagnostic, prognostic and / or predictive markers that are requested in the routine, why / for what they are requested, and basic notions of assessment that the technique is correct

1. Pulmonary pathology (Fernando López Ríos)

2. Digestive pathology (Mar Iglesias- Laura Comerva)

3. Breast pathology (Octavio Burgués)

4. Renal pathology (Pablo Cannata)

5. Gynecological pathology (Franklin Idrovo)

6. Urological pathology (Nuria Juanpere)

7. Hemato-Lymphoid (Juan Fernando García)

8. Dermatopathology (Maite Fernández Figueras)

9. Sarcomas (Isidro Machado)

10. CNS tumors (Cristina Carrato)

11. Immunohistochemistry in carcinomas / Tumors of unknown origin (Eva Musulen)


Topic VI. Application of immunohistochemical, immunofluorescence and in situ hybridization techniques in personalized medicine: prognostic and predictive biomarkers (Sandra Zazo)

Teaching Hours
75 €

Solicitada la acreditación a la Comisión de Formación Continuada de las Profesiones Sanitarias de la Comunidad de Madrid.

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Dra. Carolina Martínez Ciarpaglini

Dra. Carolina Martínez Ciarpaglini

Médica Patóloga Adjunta al Servicio de Anatomía Patológica
HU de Valencia
Nº Colegiado: 464622652
Colegio de Médicos de Valencia


Dra. Sandra Zazo

Dra. Sandra Zazo

Doctora en biología molecular. Bióloga Adjunta al servicio de anatomía patológica.
Hospital Universitario Fundación Jiménez Díaz, Madrid
Nº de colegiado: 20013-M
Colegio oficial de biólogos de la Comunidad de Madrid