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ReaPan Micro


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ReaMetrix Rapid bacterial detection presentation

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ReaPan Micro

  1. 1. ReaPanMicro: Presence/Absence Test for Bacteria in Urine
  2. 2. What is ReaPanMicro? A diagnostic test to confirm the absence of bacteria in urine, based on the principle of nucleic acid detection in bacteria on a fluorescence event detector such as a flow cytometer <ul><li>What is the information that the test provides? </li></ul><ul><li>Provides a doctor information that rules </li></ul><ul><li>out bacterial Infection </li></ul><ul><li>Key features of the test </li></ul><ul><li>High sensitivity ( Nearly 100% Negative </li></ul><ul><li>Predictive Value ) </li></ul><ul><li>Time to result: Within 15 min of sample </li></ul><ul><li>reaching a flow cytometer </li></ul><ul><li>Easy to use, simple assay workflow </li></ul>By Culture 119 43 TN 99.2 NPV 67.7% PPV 1 Negative by Flow 90 Positive by Flow TP Overall
  3. 3. Initial Studies on Clinical Urine Samples
  4. 4. Experimental Design: Staining of Clinical Urine Samples Training Set 6 culture negative (<10,000 CFU/mL) clinical urine samples 6 highly positive (>100,000 CFU/mL) clinical urine samples These 12 samples were used to determine the ideal gating strategy Staining Protocol using Reagents Filter 1 mL urine sample through a 11 um Syringe Filter into a 1.5 mL eppendorf tube Add 900 uL of the filtered urine to FACStube filled with 50 uL of nucleic acid stain and 10,000 volume control beads. Add 50 uL of PFA to fix Vortex and incubate for 5 min Run the sample on the Flow Cytometer using pre-set template Filtering is done only to remove any uric acid crystals, and “debris”
  5. 5. Running the Test: Workflow Envisaged ID’ing FACSTube with dried down reagent: 1 min Filtering 900 uL of urine sample into FACStube using enclosed syringe filter 2 min Vortex and incubate sample at RT 5 min Run MilliQ water tube thru FACScan 1 min Acquire data on FACScan with vortexed patient sample 2 min Time to Result: Within 15 min of sample reaching instrument
  6. 6. Calibration Beads Before the gain on the PMT’s are adjusted After the gain on the PMT’s are adjusted Setting up the template : Intensity Control Beads Determine Appropriate Gain Settings on the Instrument Gate where bacterial events are collected Gate where bacterial events are collected
  7. 7. Gating Strategy Optimized using 12 Training Set Samples Counts (Arbitrary Units) Culture Negative (Less than 10,000 CFU/mL) Culture Positive (More than 100,000 CFU/mL) ReaMetrix Confidential 85.24 counts per unit volume 40083 counts per unit volume Counts (Arbitrary Units) Region R6 gate has been applied
  8. 8. Statistical Analysis to Determine Decision Threshold using N = 253 samples Counts Scatter Plots of Bacterial Counts from Flow Cytometry N = 91 N = 162 N = 91 N = 162 Counts Mean Counts N = 91
  9. 9. Statistical Analysis to Determine Decision Threshold using N = 253 samples Zooming in : Exception Analysis and Identifying the Gray Region This definitely could not have been negative by culture. We will look at other clinical investigations that were done on these samples N = 29 (18% of negatives) N = 68 (75% of positives) Counts Gray Region N = 17 (11% of negatives) N = 22 (24% of positives) Could these samples have all been similar in some respect? 0-3000 counts 3000-10000 counts > 10000 counts N = 116 (72% of negatives) N = 1 (1% of positives)
  10. 10. Statistical Analysis of 253 Clinical Samples tested thus far Threshold defined using the training set. Threshold was set to ensure that NPV was 100% TP = True Positive at that level using culture as Gold Standard TN = True Negative at that level using culture as Gold Standard PPV - Positive Predictive Value NPV - Negative Predictive Value By Culture     By Culture By Flow CFU/mL Arb Units Negative < 10000 < 3,000 Positive > 100000 > 3,000 119 43 TN 99.2 NPV 67.7% PPV 1 Negative by Flow 90 Positive by Flow TP Overall
  11. 11. Samples Stained with just TO without formaldehyde and stored at 4C overnight (N=22 samples) Y Axis: Counts measured on samples immediately after staining X Axis: Counts measured on samples stained and kept at 4 C for 24 hrs. No fixative was used Red Square Points: Indicative of False Positive samples as per culture results False Positive Zone: Samples that lie in this rectangle refer to those that show high counts deeming them positive, 24 hrs later, but were negative when counted immediately after staining False Negative Zone: Samples that lie in this rectangle refer to those that show low counts deeming them negative, 24 hrs later, but were positive when counted immediately after staining We find no samples in the False Positive or False Negative Zones. Thus the stained samples can be transported after fixing
  12. 12. Transferring the assay to Diagnostic Lab : Instrument Set Up Protocol using ReaTCount Beads User will open SSC, FL1, FL2, FL3 histograms in a template and set the gains on these channels to get channel numbers within the specifications given below. On 5 different days the maximum variation seen for the same gain is no more than 2 channel numbers on any of the channels We ran experiments to determine , what is the variation that is tolerable (that does not impact counts by more than 10%) We have found that up-to 3 channel numbers variation about either side of the mean does not impact the counts seen in urine samples If the set up is within spec, the threshold is then set on FL1 at channel number 215
  13. 13. Quantifying the Maximum Variability in Counts Seen with Assay Repeats (Assessing the robustness of the finalized assay protocol) 6 culture positive and 4 culture negative samples were filtered and stained in triplicate, to determine the assay variability seen with the finalized protocol. The assay variability is typically less than 15%. Thus the gray region for the threshold of 3000 is nearly +/- 450 counts
  14. 14. ReaTCount Volume Control Beads FL3 – LDS stained cells are gated on R9 R9 gate is applied on FL1 to get counts using M1 Proof of concepts experiments – Dual Staining of Bacteria using LDS and Thiazole Orange NEGATIVE URINE SAMPLES POSITIVE URINE SAMPLES
  15. 15. Conclusion <ul><li>We anticipate ReaPanMicro to enable the doctor to rule out bacterial infection quickly – without the need for results of culture (that typically takes 24-48 hrs) </li></ul><ul><li>The assay has been validated with Anand Diagnostics at Bangalore </li></ul>
  16. 16. Manufactured by ReaMetrix India Pvt. Ltd. 50-B, II Phase, Peenya Industrial Area Peenya, Bangalore 560058, India Ph: +91-80-28378693/5, Fax: +91-80-41172451 E-mail: Further details: