Técnicas de Imunologia

Apresentação em tema: "Técnicas de Imunologia"— Transcrição da apresentação:

1 Técnicas de Imunologia
Citometria de Fluxo: Aplicações Prof.Doutor José Cabeda

2 Identificação Celular por Dispersão FS e SS
Monócitos Granulócitos Linfócitos Prof. Doutor José Cabeda

3 Prof. Doutor José Cabeda
Imunofenotipagem Prof. Doutor José Cabeda

4 IOTest 3 Combinations List
24 Relevant Tubes 2 Controls One tube is for intra-cellular testing Kappa & Lambda polyclonals from Dako FITC- and PE-conjugations performed at IOT Prof. Doutor José Cabeda

5 IOTest 3 Combinations List + Target Diseases
Prof. Doutor José Cabeda

6 Prof. Doutor José Cabeda
IOTest 3 Combinations List + Target Diseases Prof. Doutor José Cabeda

7 Quantificação antigénica
Prof. Doutor José Cabeda

8 Prof.Doutor José Cabeda
Quantificação de antigénios em Citometria de Fluxo Prof.Doutor José Cabeda

9 BioCytex MARQUIS TECHNIQUE
Beads contain calibrated amounts of anti-mouse IgG molecules. Size mimics size of target molecule/receptor. Amount of molecules on beads reflects expression level of target molecule/receptor. Prof. Doutor José Cabeda

10 CELLQUANT CD38/CD8-PE Kit
-Flow Analysis Procedure Calibration Beads Prof. Doutor José Cabeda

11 CELLQUANT CD38/CD8-PE Kit
-Flow Analysis Procedure Negative Control Prof. Doutor José Cabeda

12 CELLQUANT CD38/CD8-PE Kit
-Flow Analysis Procedure CD38 Positive Sample Prof. Doutor José Cabeda

13 CELLQUANT CD38/CD8-PE Kit
-Data Analysis Procedure Prof. Doutor José Cabeda

14 Pesquisa de populações raras
Prof. Doutor José Cabeda

15 Prof. Doutor José Cabeda
Estudos Cinéticos Time (Seconds) 36 72 108 144 180 Stimulation Calcium Flux - Indo-1 Prof. Doutor José Cabeda

16 Análise de Ciclo Celular
Count 200 400 600 800 1000 G0 /G1 S G2 /M DNA content 2N 4N G2 M G0 G1 S Prof. Doutor José Cabeda

17 Prof. Doutor José Cabeda
Apoptose Prof. Doutor José Cabeda

18 Detectores de Fluorescências Tubos para Recolha do Produto Separado
Separação Celular Sensor Fs Detectores de Fluorescências 488 nm laser Formação de gotículas Tempo Coincidência - Pureza e Eficiência + Placas Carregadas Tubos para Recolha do Produto Separado - Prof. Doutor José Cabeda

19 QC Workflow Patient Analysis Basic Elements of Daily QC Run Patient
Alignment Check Fix Problem Obtain Results Call Service Fluorescent Standard Generate Report TroubleshootMaintenance Color Compensation Process/ Method Control Test Reimbursement FAIL PASS Prof. Doutor José Cabeda

20 Prof. Doutor José Cabeda
AUTOMATIZAÇÃO Q-Prep ™ TQ-Prep ™ PrepPlus Prof. Doutor José Cabeda

21 TQ-Prep / ImmunoPrep Reagent
Sistema automático de lisis de eritrocitos No requiere lavado Preserva la morfología Utiliza carruseles compatibles con XL-MCL, FC500, PrepPlus y CellPrep Prof. Doutor José Cabeda

22 Prof. Doutor José Cabeda
PrepPlus – TQ-Prep Sistema automático de carga de muestra y reactivos para citometría de flujo Utiliza tubos primarios de cualquier marca Rack de hasta 24 tubos primarios con agitación para homogeneizar la muestra Controlado por el TQ-Prep Prof. Doutor José Cabeda

23 Prof. Doutor José Cabeda
PrepPlus – TQ-Prep Posibilidad de carga desde tubo abierto 3 soportes diferentes para reactivos Puede cargar anticuepos monoclonales, FlowCount y reactivos de control de calidad Prof. Doutor José Cabeda

24 Prof. Doutor José Cabeda
CellPrep Sistema de lavado celular por fibra hueca Sustituye a la centrifugación Mantiene la viabilidad celular Mantiene la morfología y preserva células lábiles No activa ni destruye plaquetas Utiliza sangre entera, sangre lisada y cualquier suspensión celular Prof. Doutor José Cabeda

25 Prof. Doutor José Cabeda
CellPrep Fibras huecas con poros de 0.65 µm, que permiten la salida de moléculas y líquido de la suspensión original Permite la concentración final de la muestra Diferentes programas pregrabados, así como posibilidad de crear programas propios Prof. Doutor José Cabeda

26 Citometria de Imagem: Uma nova Tecnologia no apoio à Anatomia Patológica Prof. Doutor José Cabeda

27 Tipos de amostras possiveis de analisar por Citometria de Imagem:
Etanol Glutaraldeido Tecido fixado Tecido a “fresco” Tecido “criopreservado” Tecido sólido Tecido líquido Sem preservação Com preservação Tecido a “fresco” congelado Paraformaldeido Formaldeido e incluído em parafina Amostra Prof. Doutor José Cabeda

28 Parâmetros possíveis de analisar por Citometria de Imagem:
Total de partículas Forma Tamanho Área Diâmetro Perímetro “Perfeição” Irregularidade Prof. Doutor José Cabeda

29 Requisitos Básicos num Citómetro de Imagem:
Prof. Doutor José Cabeda

30 Captação dos Parâmetros de Análise:
Núcleo Marcado Branco Preto Prof. Doutor José Cabeda

31 Prof. Doutor José Cabeda
Citometria de Imagem: Prof. Doutor José Cabeda

32 Protocolo para Citometria de Imagem:
Partindo de um fragmento de tecido sólido, fixado em formol, processado e incluído em parafina. Corte a 8m, extensão em lâminas de gelatina Desparafinar Pós fixar com fixador de Bohm Hidratar Efectuar uma Hidrólise Ácida Marcar as células com reagente de Shiff Lavar com SO2 Desidratar Montar Quantificar o conteúdo em ADN e o ciclo celular no Citómetro de Imagem Prof. Doutor José Cabeda

33 Prof. Doutor José Cabeda
MetaCyte - concept microscope slide is scanned field by field at lowest possible magnification without gaps objects of interest are automatically identified; gallery images, object features and positions are recorded The fundamental concept of slide scanning is shown on this slide: A microscope slide is scanned in a systematic pattern at relatively low optical magnification. The magnification is a compromise between number of fields to scan and thus speed on one hand and the resolution on the other hand. It is important that the captured fields do slightly overlap in order to avoid gaps which could lead to missing objects. Depending on the specific application and the objects of interest appropriate criteria or classifiers have to be used to detect the objects of interest. To detect metaphases, the software has to analyze the morphology and to find object clusters with a certain number and shape of individual objects. The coordinates of the found objects, measured object features as well as gallery images are recorded during the scan. Prof. Doutor José Cabeda

34 Prof. Doutor José Cabeda
MetaCyte - concept on screen review of analyzed cells false positives can be rejected cells suitable for analysis are preselected with a mouse click preselected objects can be automatically relocated at 100x to capture high resolution images for analysis Once the scan has been completed, the image gallery is used to reject false positives and to mark objects that seem suitable for analysis (shown here for metaphases captured in transmitted light). With a single mouse click any object can be relocated under the microscope if necessary. For analysis, automatic relocation of the preselected objects is usually done at high optical magnification to capture images of maximum resolution. Prof. Doutor José Cabeda

35 Prof. Doutor José Cabeda
MetaCyte - concept histogram or scatter plot display of measured features gating for selection of subpopulations relocation of cells detected in a previous scan after re-staining Once the scan has been completed, the image gallery is used to reject false positives and to mark objects that seem suitable for analysis (shown here for metaphases captured in transmitted light). With a single mouse click any object can be relocated under the microscope if necessary. For analysis, automatic relocation of the preselected objects is usually done at high optical magnification to capture images of maximum resolution. Prof. Doutor José Cabeda

36 Prof. Doutor José Cabeda
MetaCyte - concept CCD Camera MetaCyte PC Win 9x/NT image analysis microscope control Motorized stage (x,y) What are the main components of the scanning system? A motorized microscope, the Axioplan2 mot is required that needs to be equipped with a motorized scanning stage. The standard stage used has a capacity of 8 slides. The microscope is controlled by a computer that does also digitize and analyze the images taken by the CCD camera. For fluorescence scanning the CCD camera needs long time integration capability and the microscope needs to be equipped with a motorized filter turret or excitation filter wheel. Motorized microscope (z) Prof. Doutor José Cabeda