This 12 week program focuses on laboratory practices and interpretation of flow cytometry and molecular biology tests for diagnosis, prognosis, and evaluation of treatment effectiveness during hematological disorder follow-ups.
Through didactic lecture presentations, case study discussion and hands-on laboratory work, the trainee will understand practice issues involved in flow cytometry and molecular diagnostics for hematological disorders. The diagnostic work-up in each disease type will be discussed with academic staff from the Department of Hematology and the Flow Cytometry and Molecular Biology Laboratory. Trainees may attend the weekly Hematology Department Meeting and follow up with patients diagnosed in the Flow Cytometry and Molecular Biology Lab.
During flow cytometry training candidates will participate in the triage of new cases that arrive to the lab as well as follow up on procedures and protocols to solve the cases and assist faculty as they analyze and report them. During the molecular biology training candidates will learn how to perform basic molecular techniques (DNA/RNA extraction, nucleic acid quantification, PCR, agarose gel electrophoresis) and will become familiar with the molecular testing used in hematopathology. For each molecular test, the trainees will learn about appropriate test utilization, limitations of techniques and final interpretations.
Specific activities for each week
- Week 1: Procedures and protocols used in the flow cytometry lab and the instrument set up
- Daily QC and standardization procedure using CS&T and rainbow beads
- Flow cytometer operation sample acquisition
- Sample triage and antibody panel selection according to clinical data
- Sample preparation: protocols for blood and BM, cerebrovascular fluid, FNA samples, tissues biopsies
- Data display, storage and transfer
- Data analysis. Gating.
- Surface and intracellular staining protocols. Bulk lysis protocols
- Week 2: Acute Myeloid Leukemia
- Recognizing normal in myeloid differentiation in bone marrow
- Diagnostic work-up for AML
- Acute myeloid leukemia immunophenotyping. Diagnosis and minimal residual disease
- Myelodysplastic syndrome. Score systems
- Week 3: Acute Lymphoid Leukemia
- Recognizing normal in lymphoid differentiation in bone marrow and lymph nodes
- ALL: Immunophenotyping for diagnosis and minimal residual disease.
- Diagnostic work-up for ALL
- Week 4-5: B, T and NK chronic lymphoid disorders
- Screening for mature lymphoid disorders. Differential diagnosis
- Minimal residual disease for chronic lymphocytic leukemia
- Paucicellular samples. Cerebrospinal fluid analysis for detection blasts and lymphoma cells
- Week 6: Plasma cell disorders: MGUS, Myeloma and Waldestrom macroglobulinemia
- Screening for PC disorders.
- Minimal residual disease for myeloma. Concept of next generation flow
- Week 7: Research Module
- Immune cell subsets in peripheral blood, intracellular cytokine detection, bead-based immunoassays
- Endothelial progenitor cells and circulating endothelial cells
- Week 8: Orientation of the molecular laboratory:
- Hands –on activity: DNA and RNA extraction. Quantitification
Agarose gel preparation
Overview of PCR and variants
- Protocols and procedures: PCR /RFLP and allele specific PCR
- Thrombophilia testing : FV Leiden and FII 20210A
- Week 9: Protocols and procedures: Mutation analysis using amplicon sizing by capillary electrophoresis and fragment length analysis
- Molecular tests for diagnosis and prognosis assessment in chronic myeloproliferative disorders:
BCR-ABL t(9;22) , JAK2-V617F, JAK2-Exón 12, Calreticuline (CALR), MPL, ABL-T315, c-KIT-D816V
- Molecular testing algorithm for myeloproliferative neoplasms
- Week 10: Protocols and procedures: Sequencing analysis
- Molecular tests for diagnosis and prognosis assessment in acute myeloid and lymphoid leukemia:
FLT3-D835 , FLT3-ITD, FLT3-ITD ratio, NPM1 mutations, CEBPA, RUNX1, ASXL1, P53
- Molecular testing algorithm for acute leukemias
- Week 11: Protocols and procedures: RQ PCR
- Molecular tests for treatment response assessment in chronic myeloid leukemia:
BCR-ABL t(9;22) by Q-RTPCR
- Week 12: Molecular testing in other molecular laboratories: diagnostic tests for infectious diseases and solid tumors.
Requirements for Candidates to this Program:
- The candidate must represent an institution that currently owns and operates a flow cytometer.
- The candidate must present a support letter from his or her home institution signed by the candidate’s supervisor that verifies support of leave for training and confirms that the required equipment is available.
- The candidate must meet all other eligibility requirements listed at the top of the LATP application.