• Document: Designing and Implementing a High-Level Multicolor Flow Cytometry Assay. Brent Wood MD PhD Department of Laboratory Medicine University of Washington
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Designing and Implementing a High-Level Multicolor Flow Cytometry Assay Brent Wood MD PhD Department of Laboratory Medicine University of Washington Define Purpose of Assay • Most important question – What information is required? – What information is most important? • Prioritize • Compromises are inevitable – Simplest assay is best Outline • Instrument optimization • Compensation • Reagent selection and optimization • Panel validation • Data analysis Detector Optimization Detector Optimization PMT Voltage Green = 400 V, Blue = 450 V, Red = 500 V, Violet = 550 V Detector Voltage 475 volts 575 volts 675 volts 775 volts 875 volts • Too low reduces sensitivity • Too high pushes bright signals off scale – Dilute with unlabeled antibody • May need to compromise Optimization • Daily optimization not practical • Once optimized, do NOT change instrument settings unless change in performance – New optical filters – New PMT detector • Monitor performance daily – Use stable bright beads – Assess MFI and CV for each detector – Levy-Jennings plots – Define acceptable ranges of performance Fluorochrome Intensity CD4 FITC = Green Pacific Blue = Red PE = Dark Blue A594 = Yellow green PE-TR = Light blue APC = Light green PerCP-Cy5.5 = Magenta A700 = Blue PE-Cy7 = Orange APC-Cy7 = Purple Tandem Fluorochromes Phycoerythrin (PE) PE-Texas Red Tandem Breakdown Whole blood lysis NH4Cl prelyse and wash 1 hour prior Cytometry Part A (2009) 75A:882-890 Fluorochrome Options • PE-Cy5 vs. PerCP-Cy5.5 vs. PE-Cy5.5 vs. PerCP – PerCP dim – PerCP-Cy5.5 brighter but dims with increased laser power – PE-Cy5 brighter but significant comp with APC – PE-Cy5.5 bright, less comp with APC, more comp with PE-Cy7 and APC-A700 • A700 vs. APC-A700 – APC-A700 significantly brighter but more comp with APC • APC-Cy7 vs. APC-H7 vs. APC-A750 – APC-Cy7 undergoes photodegradation but is brightest – APC-H7 dimmer but more stable, low comp with APC – APC-A750 intensity in between but higher comp with APC Compensation • Spectral overlap between fluorochromes • Critical to success of method – For 10 color experiment • Need to determine 90 values for Comp Matrix – Software compensation required • Maximum flexibility • Non-destructive FITC = Green Excitation = Dotted PE = Orange Emission = Solid Compensation - Method • Single stained controls used – One for each individual fluorochrome – One for each individual tandem – As bright as brightest reagent to be used • Samples run without compensation • Compensation calculated in software – Applied either at acquisition or analysis Compensation Correct Undercompensated Overcompensated Compensation Compensation Compensation • Don’t worry unduly about PMT voltage 245% 103% 47.5% 23.0% 12.2% 1.9% 4.7% 10.3% 21.0% 41.0% PE = 400 volts 450 volts 500 volts 550 volts 600 volts PE-TR = 550 volts Compensation values should reflect relative spectral overlap, i.e. detector gains should be equal Compensation Validation Fluorescence minus one “FMO” controls Compensation Validation Fluorescence minus one “FMO” controls Compensation Quality Control • Purist – Set at the time of each experiment • Important when: – Reagents and specimen preparation are inconsistent – Accurate MFI to be measured • Practical – Quality control instrument and reagents using consistent specimen preparation • Compensation is dependent on relative fluorescence intensity • Slight manual adjustment may be required 0.9% 0.9% 1.0% June 2008 Nov 2008 Sept 2009 1.0% 1.0% 1.1% May 2008 Dec 2008 May 2009 6.1% 4.5% 6.1% May 2008 Dec 2008 May 2009 Stem cell evaluation • Antigens to be evaluated: – Early progenitors - CD34, CD117, CD38 – Erythroid - CD71 – B cell - CD19 – pDCs, basophils - CD123, HLA-DR – Myelomonocytic - CD15, HLA-DR – Progenitor gating - CD45 – Test reagents - CD45RA, CD133, CD7, etc. Surface Antigen Titration • Titer for signal to 22 ul ul + + 55 ul ul + 10 ul noise

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