Advanced Flow Workshop Shumway Murphey

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Workshop 2: Advanced Flow Workshop- Wayne Shumway, CHT, CHS and Cathi Murphey, MT(ASCP), CHS, BLM Electronics Amplifier Log / Linear Linear Scaling (“normal numbers”: : movement along scale according to amount of increase) Log “Mode”: 1024 data points in log relation (Log Amp) Channels 256 512 Background 2x 4x 200 End of 1 st decade 400 End of 2 nd decade 1 2 3 4 5 6 7 8 910 Fluorescence Intensity 20 30 40 50 60 70 100 200 400 600 800 1000 Fluorescence Intensity Integers that directly represent quantities of fluorescence CHANNELS USED FOR “BINNING” DATA Variations in light intensity identifiable by PMT ~infinite Represented as a finite, 4 log range for practicality Entire range = 1024 increments (Channels) Log Mode: 1 X 1.009 X1.009 X1.009 X1.009 Electronics Amplifier Log / Linear Background 2x 4x 8x 16x Log Scaling (movement along scale according to proportion of increase) 32x 128x 64x 256x 512x 1024x 2048x Moving from the origin (10 0 ) to the end of the scale (10 4 ), each subsequent channel serves as a “bin” for a higher range of values. 200 400 600 800 1000 Fluorescence Intensity CHANNEL SHIFTS AND THE MULTIPLES OF THE “STARTING VALUE” (BACKGROUND FLUORESCENCE) THAT THEY REPRESENT MULTIPLE 2 3 4 5 6 7 8 9 10 1024 SCALE 77 122 154 179 199 216 231 244 256 256 SCALE 19 31 39 45 50 54 58 61 64 ** Bottom Line – channel shifts represent a PROPORTIONAL relationship! ** ** Bottom Line – channel shifts represent a PROPORTIONAL relationship! ** THEREFORE: “Channel Shift” and “MFI Ratio” are EXACTLY the SAME method of analysis! MFI Ratio 2 3 4 5 6 7 8 9 10 MCF Shift (1024) 77 122 154 179 199 216 231 244 256 (256) 19 31 39 45 50 54 58 61 64 4
Workshop 2: Advanced Flow Workshop- Wayne Shumway, CHT, CHS and Cathi Murphey, MT(ASCP), CHS, BLM XM: DONOR + RECIPIENT SELECTION Best donor choice = least fluorescence from secondary antibody 3 detected. FCXM #1 3 The quantity of signal XM: above background is of - Bkgnd: 36 200% of background significance (because it Aby: 3 (77 ch. . Shift) directly reflects the Y number of patient FCXM #2 1 antibodies bound), the XM: 1 proportion of background - Bkgnd: 12 200% of background is IRRELEVANT*! Aby: 1 (77 ch. . Shift) *with regard to the number of antibodies present Y Y Y Y Y Y Y Channel Shifts and the Illusion of Increased Sensitivity When Using Pronase Y Y The same number of Untreated B Cells antibody molecules XM: binding against a lower - Bkgnd: background produce a 36 greater channel shift – Aby: 3 but remain the same 77 ch. . Shift number of antibody molecules! Pronase Treated B Cells Linear data reflects the XM: true quantity above - Bkgnd: 14 background – a value Aby: 3 that is independent of 154 ch. . Shift the background. Y Y Y Y Y 3 Y Y 1 Y Y Y 3 background 1 background Relative Linear Values (RLV) are frequently referred to as MFI (Median or Mean Fluorescence Intensity) values. It is these RLV / MFI values that are “numbers” in the conventional sense (NOT the channel values!), they have simply been plotted on a log scale. Events with an MFI value of 600 are twice as bright as those with an MFI of 300 (the expected, conventional, “linear” relationship). HOWEVER, if an event reads at channel 600, an event half as bright will read at channel 523. So how do we convert the channels to the “numbers”? CHANNEL VALUE Calculate “R” = the channel value divided by channels per decade 512 512/256 = 2 End of 1 st decade End of 2 nd decade End of 3 rd decade 256 512 300 768 1024 200 400 600 600 800 End of 4 th decade 1000 10 R = RELATIVE LINEAR VALUE (RLV – aka MFI) 10 2 = 100 1 RLV 10 100 1000 Fluorescence Intensity 10,000 W H E N I N L O G “M M O D E” E : CHANNEL VALUES BD = “Log Data” Coulter = “Linear Data” RELATIVE LINEAR VALUES BD = “Linear Data” Coulter = “Log Data” End of 1 st decade End of 2 nd decade End of 3 rd decade End of 4 th decade 256 512 768 1024 RELATIVE LINEAR VALUE is a much better term than Median Fluorescence Intensity 1 RLV 200 400 600 800 1000 10 100 1000 10,000 5
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Advanced Flow Workshop Shumway Murphey

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