Curriculum and Core Faculty


Cytometry Workshops

Wet Lab Modules with suggested faculty for 2016

LM-1. Solid Tissue Desegregation and Subset Identification. K. Asosingh:  Preparation of single cell suspension is a critical step in flow cytometric analysis of solid tissues such as tumors or organs. In this wet lab, we will cover theoretical principles and demonstrate methods for solid tumor and tissue dissociation to obtain a suspension of single cells with low amounts of debris, cell clumps and high viability.  Cell suspensions will be analyzed for expression of markers associated with cancer stem cells and endothelial, epithelial and inflammatory cells. 


LM-2. Basics of Flow Cytometry.  H. Krishnamurthy, W. Telford: In this lecture and wet lab, we will familiarize those new to flow cytometry with basic hardware components of flow cytometer and their correct usage.  The key components including fluidics, lasers, optics, electronic detectors, analog to digital converters and pulse processors will be described.  In the”Build your own Cytometer” module, Dr. Telford will assemble a  functional flow cytometer from individual components.

LM-3. Cell Cycle, Proliferation and Apoptosis.  A. Krishan, W. Telford, K. Price:  We will demonstrate methods for DNA content and cell cycle analysis and for the detection of proliferating and apoptotic cells. Tissue culture cell lines, tumors, formalin fixed paraffin embedded materials and plant tissues such as leaves and roots may be used for demonstration.

LM-4. Fluorochromes, Spectral Overlap and Compensation.  A. Saluk: The wide range of fluorochromes available has expanded the scope of polychromatic flow cytometry.  However, this has also increased complexicity of using fluorochrome combination due to spectral overlap and need for compensation.   In this lab we will focus on fluorochromes, dyes and proteins used in analysis of marker expression.  An understanding of the theories behind approaches to compensate for spectral spill over will be presented.


LM-5. Panel Development for Multicolor Flow Cytometry.   P. Jain:   Derived from physical facts e.g. fluorochrome brightness and spectra as well as through consideration of biological features such as antigen expression characteristics and patterns, a set of 6 easily applicable stepwise rules will be  developed, discussed and verified through detection of most delicate populations in real samples.

LM-6. Polychromatic Flow Cytometry.  P. Jain, Z. Maciorowski:  In this  lab, we will demonstrate and discuss sample processing and staining protocols used for phenotype analysis of haematological malignancies. We will  review principles of antibody titration, panel design and discuss the relevance of different experimental controls (e.g. isotype, compensation, FMO) in multicolor phenotype analysis.

LM-7. MDS  and Leukemic Cell Phenotype.  B. Wood, S. Gujral, K. Sehgal:  Diagnosis and Sub typing of acute leukemias and chronic lymphoproliferative disorders (CLPD) is important, as treatment and prognosis of different types is different. Although morphology with cytochemistry (myeloperoxidase and non-specific esterase) can diagnose most cases of acute leukemia and CLPD, however, in  a significant minority of cases require the use of immunophenotyping by flow cytometry. Flow cytometry has become a standard method for  evaluation of CLPD, lymphomas and leukemia in most diagnostic laboratories and in detecting minimal residual disease.

LM-8.  Hematopoietic and Mesenchymal Stem Cells.  V. Tanavde: Flow cytometric detection and quantitation of CD34 positive cells is a useful technique for enumeration of hematopoietic stem cells in bone marrow, cord blood or in mobilized peripheral blood used for bone marrow transplantation.  We will discuss and demonstrate the ISHAGE protocol used for enumeration of viable CD34+ cells using single platform flow cytometry. We will also discuss markers used for the identification of mesenchymal stem cells.

LM-9. Stem Cell Assays.  W. Telford, A.Krishan: In this lab, we will review methods available for flow cytometric detection and analysis of stem cells.  We will discuss methods for   cell surface marker expression in tumor stem cell markers and physiological characteristics such as ABC transporter activity using pump-specific efflux probes and aldehyde dehydrogenase activity.

LM-10. Leukemia Lymphoma Phenotype and Minimal Residual Disease: B. Wood,  M. Borowitz: Cases of hematolymphoid neoplasm will be demonstrated and issues /troubleshoots related to multicolor immunophenotyping discussed. This module will describe the principles underpinning flow cytometric evaluation of minimal residual disease with detailed explanation and illustrations of the standardised analysis method.


LM-11.  Paroxysmal nocturnal hemoglobinuria (PNH).  M. Borowitz: PNH is a rare hematopoietic stem cell disorder characterized by a somatic mutation in the PIGA gene, leading to a deficiency of proteins linked to the cell membrane via glycophosphatidylinositol (GPI) anchors.  In this module, we will discuss the background pathophysiology of the disease and  a history of testing by flow cytometry.  Problems occurring with the use of red cells only or with the use of inferior combinations of markers will be reviewed as an introduction to the development of standardized guidelines for performing both routine and high sensitivity testing. 

LM-12.  Infectious and Parasitic Disease Monitoring. M. Chatterjee: In this module, we will use  flow cytometry for monitoring of cell viability of parasites,  measurement of redox status of parasites and/or macrophages in terms of generation of reactive oxygen (CMH2DCFDA)and nitrogen species (DAF2-DA) along with levels of thiols (CMF-DA) in the presence and absence of anti-leishmanial drugs. We will demonstrate the use of JC1 for measurement of mitochondrial membrane potential.


LM-13. Cell Signalling and Phosphoproteins. V. Shankey: Flow cytometric analysis of intracellular phospho-epitopes requires the development of cell fixation and permeabilization techniques that preserve these epitopes, plus cellular light scatter properties and cell surface CD molecules.  We will discuss specific fixation and permeabilization techniques we have developed to study signalling pathways, including MAPK, STAT, and PI3K pathways, and phosphorylation of the ribosomal S6 protein in bone marrows from normal and AML patients.  These approaches provide a unique insight into the biologic heterogeneity of signalling pathways at the single cell level, and offer the potential to monitor individual patients’ responses to targeted agents.

LM-14.  Electrostatic Cell Sorting.  J. Osborne, Z. Maciorowski, J.Trotter:  In this wet lab, we will discuss the ins and outs of using electrostatic flow sorting for collection of cells of interest.  Emphasis will be put on quality control, sterility and validation of sorting parameters. 

LM-15.   Quality Control in Flow Cytometry.  Z. Maciorowski, A. Saluk:  There are four essential variables  that can impact flow cytometric analysis namely, instrument, reagents, sample processing  and data interpretation. In this lecture, we will discuss various components of “instrument related variables” in a flow cytometer and discuss conventional and emerging concepts in characterising instrument performance and tracking it over a period of time to ensure data accuracy and reproducibility.

LM-16.  Chromosome Sorting.  L. S. Cram: The technology associated with sorting of chromosomes will be discussed from the standpoint of fluorescent probes, measurable characteristics, data interpretation, data display, and the techniques of actual sorting for different applications.  These techniques and associated challenges (chromatid peaks, background debris, and sort purity), will be discussed.  The construction of specific FISH probes and the sorting of chromosomes from other species will be discussed.

 LM-17.  Minimal Residual Disease Monitoring (MRD) in Chidhood ALL.  M. Borowitz: We will review the history of important prognostic factors in childhood ALL and discuss the methodology of both flow cytometric and molecular MRD detection.  We will discuss the application and prognostic significance of MRD in 3 generations of Children’s Oncology Group studies showing how use of MRD for risk assignment has evolved. 

LM-18.   NK cells and NK cell cytotoxicity. G. Deniz, S. Devadas: Natural killer (NK) cells are lymphocytes of the innate immune system that can recognize and kill various types of malignant cells and virus infected cells. Flow cytometry is a rapid method for monitoring of NK cells, NK cell subsets and NK cytotoxic activity.  In this module, we will discuss flow cytometric methods for phenotypic detection and functional analysis of NK cells.

LM-19:   Antigen expression monitoring for Organ Transplantation: P. Dorwal: In this module, we will discuss the various flow cytometric assays (such as Flow Cytometric Cross Match,FCXM), screening for Panel Reactive Antibodies by Flow Cytometry, FlowPRA along with other methds  such as the use of  LuminexTM-based assays. The focus will be on study of interesting actual case studies where the synergy of all the modalities will be demonstrated. The various international guidelines on transplant work up and monitoring would also be discussed.

LM-20:  Data Analysis and Presentation: H. Agrawal. During the last few years, the number of parameters (colors) simultaneously used in flow cytometry experiments has increased.  This is enabled by the availability of high-end instruments and powerful data analysis tools.  In this module, we will cover analysis of single or multicolor flow cytometry data and guidelines for the correct data analysis and presentation.                                     

Dr. Mike Borowitz, Baltimore, USA

Leukemia/Lymphoma Immunopathology and PNH

Dr. L. Scott Cram, Los Alamos, USA

Chromosome Analysis and Sorting

Dr. Madhu Dikshit, Lucknow, India

Free Radical Monitoring and Pharma Research

Dr. Sumeet Gujral,  Mumbai, India

Leukemia/Lymphoma Phenotype Analysis

Mr. Ronald Hamelik, Miami, USA

Drug Efflux, Diagnostic Cytology

Dr. Paresh Jain, Gurgoan, India

Phenotype Analysis, Quality Control

Dr. Awtar Krishan, Miami, USA

Cell Cycle, Proliferation, Tumor Markers.

Dr. H. Krishnamurthy, Bangalore, India

Basics of Flow Cytometry, Germ Cell Analysis

Dr. Manisha Madkaikar, Mumbai, India

Immunodeficiency and PNH

Dr. Geoff Osborne, Bellbowrie, Australia

Cell Sorting

Dr. K Pattanpanyasat, Bangkok, Thailand

Parasitic and Viral disease Monitoring

Mr. Alan Saluk, San Diego, USA

Polychromatic Flow Cytometry and Compensation.

Dr. Vincent Shankey, Miami, USA

Signal Transduction and Phosphoproteins

Dr. Vivek Tanavde, Ahmadabad, India

Hematopoietic and Mesenchymal Stem Cells

Dr. William G. Telford, Bethesda, USA

Stem Cell Assays and Lasers

Dr. Brent Wood, Seattle, USA

Leukemia and MRD

Mr. Ronald Hamelik, Miami, 

Cell Cycle, Apoptosis and Proliferation