ISHAGE PROTOCOL dr suman (2).pptx

ISHAGE PROTOCOL SUMAN SMITH ADHIKARI MODERATOR: DR. JASMITA DASS CD34 Transmembrane glycoprotein expressed in hematopoietic stem cells, progenitor cells and endothelial cells Almost all pluripotent and committed stem cells in the CFUs express CD34 The uncommitted progenitor cells ar...

ISHAGE PROTOCOL SUMAN SMITH ADHIKARI MODERATOR: DR. JASMITA DASS CD34 Transmembrane glycoprotein expressed in hematopoietic stem cells, progenitor cells and endothelial cells Almost all pluripotent and committed stem cells in the CFUs express CD34 The uncommitted progenitor cells are CD38- while the committed progenitor cells are CD38+ In normal conditions, CD34 positive cells form 1-2% of the total bone marrow cells Principles of immunophenotyping, Atlas of  SCHEMA FOR IMMUNOPHENOTYPING OF STEM AND PROGENITOR CELLS Short-Term Storage of Mobilized Peripheral Blood Stem Cells in a Closed System Changes the Microenvironment and May Affect the Quantity of CD34+ and CD34+CD38-CD45RA-CD90+ Cells. Transplantation and cellular therapy, Issue 2, p112, E1-112, E9,  NEED FOR ENUMERATION OF CD34 STEM CELLS Adequate number of peripheral blood stem cells are required for successful rescue of hematopoiesis after myeloablation Absolute count correlates with engraftment Approximately 2x10^6 CD34 cells/kg body weight are required for adequate reconstitution of hematopoiesis with a lower limit of 1x10^6 CD34 cells/kg body weight PBSCs are collected by apheresis after mobilization into peripheral blood by growth factor (G-CSF) and plerixafor(novel SDF-1/CXCR4 antagonist) Short-Term Storage of Mobilized Peripheral Blood Stem Cells in a Closed System Changes the Microenvironment and May Affect the Quantity of CD34 + and CD34+CD38-CD45RA-CD90+ Cells. Transplantation and cellular therapy, Issue 2, p112, E1-112, E9, February 2023  Routine clinical assay for hematopoietic progenitor stem cells should meet the following criteria a)Simplicity- to allow widespread applicability b)Sensitivity- since hematopoietic stem cells are rare events c)Accuracy- to provide clinically relevant results d)Reproducibility- to provide clinically reliable results e)Speed- to provide real time and online analysis The ISHAGE protocol is a simple gating strategy which uses light scatter and two-colour immunofluorescence analysis using a pan CD45 and pan CD34 monoclonal antibody combination  TYPES OF EPITOPES 1) CD34 is a heavily glycosylated cell surface molecule that gives rise to different kinds of epitopes 2) Monoclonal antibodies have been divided into three classes based on their epitope recognition and sensitivity to enzymatic cleavage with different glycoproteases 3) The class III epitope is more widely distributed both on endothelial cells and a large proportion of hematopoietic progenitor cells PASTEURELLA CHYMOPAPAIN HEMOLYTICA NEURAMINIDASE DERIVED GLYCOPREOTEASE CLASS I EPITOPE SENSITIVE SENSITIVE SENSITIVE CLASS II EPITOPE RESISTANT SENSITIVE SENSITIVE CLASS III EPITOPE RESISTANT RESISTANT RESISTANT The Role of the Different CD34 Epitopes in Detection and Positive Selection of CD34+ Bone Marrow and Peripheral Blood Stem Cells A. J. CROOCKEWIT, R. A. P. RAYMAKERS, F. W. M. B. PREIJERS, G. VIERWINDEN & T. J. M. DE WITTE Division of Hematology, Department of Medicine, University Hospital Nijmegen, The Netherlands MODIFICATIONS DONE IN 1998 SINGLE PLATFORM CD34 ENUMERATION Single platform for absolute CD34 count Usage of three colours/dyes Pan CD45 monoclonal antibody Pan CD34 monoclonal antibody Viability dye - 7-AAD Flow CountTM Fluorospheres Sequential Boolean gating strategy to identify true CD34+ cells Keeney M et al. Single platform flowcytometric CD34+ cell counts based on ISHAGE guidelines. Cytometry 1998;34:61-7 7-AAD 7- Amino Actinomycin D Fluorescent chemical compound with strong affinity for DNA Strongly binds to GC rich regions of DNA It is an impermeant dye: cells with intact cell membrane(viable cells) don’t allow the dye to pass through Pediatric Hematology and Oncology, Early Online:1–16, 2012 Copyright C Informa Healthcare USA, Inc. ISSN: 0888-0018 print / 1521-0669 online DOI: 10.3109/08880018.2012.717172  SAMPLES G-CSF mobilized peripheral blood Fresh/frozen cord blood samples Leukapheresis products Bone marrow sample Specimens with leukocyte count exceeding 30x103/µL are diluted with PBS Enumeration of viable CD34+ cells by flow cytometry in blood, bone marrow and cord blood: Results of a study of the novel BD™â., ¢ stem cell enumeration kit, November 2010, Cytotherapy 13(4):449-58 PRINICIPLE: Known values of sample is added to the fluorochrome labelled antibodies The fluorochrome labelled antibodies bind to the cell surface antigens 7-AAD dye is added to assess the viability NH4Cl based lysing solution is added to lyse the red blood cells Known concentration of beads of same volume as that of sample is added to get the absolute count of CD34+ cells A minimum of 75000 CD45+ events with 100 CD34+ events are acquired (recommended according to 1996 PROCEDURE: 100µL of sample is added to the BD trucount tubes The sample is incubated for 20 minutes with 20ul of premixed antibody cocktail and 20ul of 7-AAD dye at room temperature The sample is added by reverse pipetting method to improve accuracy After incubation, red blood cells are lysed for 10 minutes by 2ml of ammonium chloride lysing reagent Acquisition is done by flow cytometry immediately Selection of the viable cells The viable cells show lack of expression of 7-AAD Gating CD45+ cells  Gating the region of CD45+ lymphocytes  Gating CD34+ progenitor cells  All CD34+ events showing high expression of CD45 are excluded Using lymphocytes as reference population for size and scatter, debris and events with low FSC values are excluded The CD34 gated parameters superimposed on the gated lymphocytes confirm true CD34+ cells having similar scatter properties as that of lymphocytes and progenitor cells Identification and enumeration of beads Refinement of the beads CALCULATION: Viable CD34 cells/ µL = (Number of viable CD34+cells x number of beads per TRUCOUNT tube x dilution factor) / (total number of beads counted x sample volume)  CONCLUSION The accurate enumeration of CD34+ cells is paramount for successful hematopoietic stem cell transplantation The single platform ISHAGE protocol is a simple and efficient method for enumeration of CD34 cells Cell viability can be efficiently assessed by employing 7- AAD dye in flow cytometry The single platform assay has led to decrease in inter- laboratory variability THANK YOU