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DIAGNÓSTICO Y CLASIFICACIÓN DE LEUCEMIAS AGUDAS CON LOS PANELES EUROFLOW

DIAGNOSIS OF CLONAL HAEMATOLOGICAL DISORDERS

Clinical symptoms and signs

Laboratory findings

Morphology + cytochemistry

CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN)

Cytogenetics Immunophenotyping

Curso Avanzado de Actualización en Oncohematología por Citometria de Flujo,Molecular biology/FISH

Buenos Aires, 31 de mayo de 2011

DIAGNOSTICS IN HEMATO-ONCOLOGY
1. Making the diagnosis Normal ↔ reactive/regenerating ↔ malignant
Annually > 300,000 new patients with a hematological malignancy in developed countries

REQUIRED DEVELOPMENTS IN FLOW CYTOMETRY
(status in 2005) Immunobeads – introduce combined cellular/immunobead assays – special immunobead forleukemias Novel antibodies – test new (academic) antibodies for application in intracellular stainings – development of new antibodies against oncoproteins and aberrant signalling pathways Multicolor flow cytometry: 8 color comprehensive panels – inclusion of solid state violet laser – selection of appropriate fluorochromes – compare conjugated antibodies (multiple companies) Development of novelsoftware for complex pattern recognition – combining multiple tubes: calculate data & multivariate analyses – mapping of diagnosis and follow-up leukemia samples against templates of reference “normal/control” samples

2. Classification of hematopoietic malignancies - relation with prognosis - relevance of risk-group definition in treatment protocols
Based on differentiation characteristicsand particularly on chromosome aberrations, resulting in fusion gene transcripts or aberrantly (over) expressed genes

3. Evaluation of treatment effectiveness Detection of minimal residual disease (MRD):
MRD-based risk-group stratification (treatment reduction or treatment intensification) Annually > 400,000 follow-up samples in leukemia patients (ALL, AML, CML)
Prepared by JJM van DongenTHE EUROFLOW APPROACH TO LEUKEMIA/LYMPHOMA IMMUNOPHENOTYPING
Clinical question Knowledge Experience Evaluation

SET UP OF A FCM LABORATORY FOR LEUKEMIA /LYMPHOMA TYPING: CONVENTIONAL PANEL DESIGN
Clinical request/need New indications

Reference profiles

Diagnostic screening tube

Purchase a flow cytometer

Majority of diseases? Majority of cases? New disease entities? Training Designof MAb panels
(Disease category vs cell lineage oriented)

“Diagnostic classification” panel
14 Major groups

MRD monitoring

154 Nosologic entities

Immunophenotypic diagnostic activity started

≥

Experience

Panel optimization

Experience

1

STANDARDIZATION EFFORTS FOR IMMUNOPHENOTYPIC STUDIES
- CLSI (Clinical Laboratory Standards Institute):
- Stetler-Stevenson etal.: Clinical flow cytometric analysis of neoplastic hematolymphoid cells; Approved guideline. CLSI document H43-A2. CLSI, 2007

LEUKEMIA /LYMPHOMA IMMUNOPHENOTYPING: EVALUATION OF ANTIBODY PANELS
Single center panel Multicenter panel

Single center experience/evaluation

- CCS (Clinical Cytometry Society):
- Davis et al: 2006 Bethesda International Consensus recommendations on the flowcytometric immunophenotypic analysis of hematolymphoid neoplasias. Clin Cytometry, 72B, 2007. Experience-based
(subjective)

Multicenter evaluation possible

- ESCCA (European Society for Clinical Cell Analysis:
www.escca.eu)

Long time required
Limited by new: instruments techniques markers

Consensus recommendations
Shared experience objectivity

- European Leukemia Net(www.leukemia-net.org) - Consenso Latinoamericano (Clin Cytometry, 1998 y 2006)

CONSTRUCTION OF EUROFLOW LEUKEMIA/ LYMPHOMA IMMUNOPHENOTYPING ANTIBODY PANEL Clinical request/need
Medical indication Proposed strategy

CONSTRUCTION OF EUROFLOW PANELS: MEDICAL INDICATION ORIENTATION/SCREENING & CLASSIFICATION PANELS
Acute leukemia Cytopenia Lymphocytosis LN involvement Monoclonal component Eosinophilia...