Cliff Threlkeld, D.O. Central Florida Research
A New Diagnostic Test for Lyme Disease <ul><li>Introduction – using flow cytometry to detect  Borrelia burgdorferi (Bb) </...
A New Diagnostic Test for Lyme Disease <ul><li>Basics of flow cytometry </li></ul><ul><ul><li>Typical flow cytometer  - po...
Typical Flow Cytometer Computer System Detector & Mechanical Power
Cytometer Power PC & Printer Specimen Tube
Injector  Tip Fluorescence signals Focused laser beam <ul><li>Sheath </li></ul><ul><li>fluid </li></ul>
Flow Cytometry PMT  FITC PMT  PE PMT  ECD PMT  PC5 Dichroic Filters Laser  1 2 3 4 Flow cell Bandpass Filters
FS SS How do Abs work? 3 19 ZAP 70 5
A New Diagnostic Test for Lyme Disease <ul><li>Application of flow cytometry to Lyme disease testing </li></ul><ul><ul><li...
 
 
A New Diagnostic Test for Lyme Disease <ul><li>Comparison of FC to WB and PCR </li></ul><ul><ul><li>Phase 1 study compared...
A New Diagnostic Test for Lyme Disease <ul><li>Comparison of FC to PCR </li></ul><ul><ul><li>Phase 2 compared FC to PCR in...
 
A New Diagnostic Test for Lyme Disease <ul><li>Sensitivity and specificity of FC and WB compared </li></ul><ul><ul><li>Sen...
A New Diagnostic Test for Lyme Disease <ul><li>Sensitivity and specificity of FC and PCR compared </li></ul><ul><ul><li>Se...
 
A New Diagnostic Test for Lyme Disease <ul><li>Availability of flow cytotometry LD testing </li></ul><ul><ul><li>Central F...
A New Diagnostic Test for Lyme Disease <ul><li>Future applications </li></ul><ul><ul><li>Testing for multiple tick-borne d...
 
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Lyme disease-antigen-test-flow-cytometry

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Lyme Disease antigen test performed using flow cytometry used to detect Lyme disease and Borrelia burgdorferi

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  • We specifically measure ZAP-70 in CLL cells defined by CD19 and CD5 coexpression. We also measure ZAP-70 in T cells defined by CD3 and CD5 coexpression. Including FS and SS, each cell is defined by 5 parameters.
  • Lyme disease-antigen-test-flow-cytometry

    1. 1. Cliff Threlkeld, D.O. Central Florida Research
    2. 2. A New Diagnostic Test for Lyme Disease <ul><li>Introduction – using flow cytometry to detect Borrelia burgdorferi (Bb) </li></ul><ul><ul><li>Basic principles of flow cytometry </li></ul></ul><ul><ul><li>Application to Lyme disease testing </li></ul></ul><ul><ul><li>Comparison of flow cytometry to other methods of Lyme disease testing </li></ul></ul><ul><ul><li>Availability of flow cytometry Lyme disease testing </li></ul></ul>
    3. 3. A New Diagnostic Test for Lyme Disease <ul><li>Basics of flow cytometry </li></ul><ul><ul><li>Typical flow cytometer - power, mechanical and detection, and computer system </li></ul></ul><ul><ul><li>Typical setup </li></ul></ul><ul><ul><li>Flow cell </li></ul></ul><ul><ul><li>How are events detected? </li></ul></ul><ul><ul><li>How antibodies work </li></ul></ul>
    4. 4. Typical Flow Cytometer Computer System Detector & Mechanical Power
    5. 5. Cytometer Power PC & Printer Specimen Tube
    6. 6. Injector Tip Fluorescence signals Focused laser beam <ul><li>Sheath </li></ul><ul><li>fluid </li></ul>
    7. 7. Flow Cytometry PMT FITC PMT PE PMT ECD PMT PC5 Dichroic Filters Laser 1 2 3 4 Flow cell Bandpass Filters
    8. 8. FS SS How do Abs work? 3 19 ZAP 70 5
    9. 9. A New Diagnostic Test for Lyme Disease <ul><li>Application of flow cytometry to Lyme disease testing </li></ul><ul><ul><li>Traditional two-test protocol antibody testing - ELISA and Western Blot (WB) </li></ul></ul><ul><ul><li>Bacterial detection methods – Culture and polymerase chain reaction (PCR) – Flow cytometry underutilized </li></ul></ul><ul><ul><li>Using flow cytometry to detect Borrelia burgdorferi in patient blood </li></ul></ul><ul><ul><ul><li>Antibody with fluorescein label (goat, KPL) added to aliquot of patient blood (EDTA) </li></ul></ul></ul><ul><ul><ul><li>Incubation for 1 hour at 37 degrees C </li></ul></ul></ul><ul><ul><ul><li>Analyze by FC – Bb are bright fluorescent events </li></ul></ul></ul>
    10. 12. A New Diagnostic Test for Lyme Disease <ul><li>Comparison of FC to WB and PCR </li></ul><ul><ul><li>Phase 1 study compared FC with WB </li></ul></ul><ul><ul><li>66 patients with clinical diagnosis of LD </li></ul></ul><ul><ul><ul><li>5 positive by WB, 4 of those positive by FC </li></ul></ul></ul><ul><ul><ul><li>An additional 28 patients positive by FC </li></ul></ul></ul>
    11. 13. A New Diagnostic Test for Lyme Disease <ul><li>Comparison of FC to PCR </li></ul><ul><ul><li>Phase 2 compared FC to PCR in 42 patients with a clinical diagnosis of LD </li></ul></ul><ul><ul><li>16 patients positive by FC and 18 positive by PCR </li></ul></ul><ul><ul><ul><li>8 patients positive by PCR and FC </li></ul></ul></ul><ul><ul><ul><li>Gene sequencing performed on 14 PCR positive patients (5 FC positive) </li></ul></ul></ul><ul><ul><ul><li>8 Bb sensu stricto (4 FC) </li></ul></ul></ul><ul><ul><ul><li>7 Bb sensu lato (new subgroup, 2 by FC) </li></ul></ul></ul>
    12. 15. A New Diagnostic Test for Lyme Disease <ul><li>Sensitivity and specificity of FC and WB compared </li></ul><ul><ul><li>Sensitivity FC = 66% Specificity = 92% </li></ul></ul><ul><ul><li>Sensitivity WB = 52% Specificity = 100% </li></ul></ul>
    13. 16. A New Diagnostic Test for Lyme Disease <ul><li>Sensitivity and specificity of FC and PCR compared </li></ul><ul><ul><li>Sensitivity FC = 62% Specificity = 100% </li></ul></ul><ul><ul><li>Sensitivity PCR = 64% Specificity = 100% </li></ul></ul>
    14. 18. A New Diagnostic Test for Lyme Disease <ul><li>Availability of flow cytotometry LD testing </li></ul><ul><ul><li>Central Florida Research, Winter Haven, Florida </li></ul></ul><ul><ul><ul><li>Web site – centralfloridaresearch.com </li></ul></ul></ul>
    15. 19. A New Diagnostic Test for Lyme Disease <ul><li>Future applications </li></ul><ul><ul><li>Testing for multiple tick-borne disease pathogens simultaneously </li></ul></ul><ul><ul><li>Add antibodies to European Borrelia species </li></ul></ul><ul><ul><li>Look for Bb antigens within monocytes </li></ul></ul>
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