Lyme disease-antigen-test-flow-cytometry
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Transcript of Lyme disease-antigen-test-flow-cytometry
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Cliff Threlkeld, D.O.Central Florida Research
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A New Diagnostic Test for Lyme Disease
Introduction – using flow cytometry to detect Borrelia burgdorferi (Bb)Basic principles of flow cytometryApplication to Lyme disease testingComparison of flow cytometry to other methods
of Lyme disease testingAvailability of flow cytometry Lyme disease
testing
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A New Diagnostic Test for Lyme Disease
Basics of flow cytometryTypical flow cytometer - power, mechanical
and detection, and computer systemTypical setupFlow cellHow are events detected?How antibodies work
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Typical Flow CytometerTypical Flow Cytometer
Computer SystemDetector &Mechanical
Power
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Cytometer
Power
PC & Printer
Specimen Tube
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Injector Tip
FluorescenceFluorescencesignalssignals
Focused laserFocused laserbeambeam
•Sheath• fluid
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Flow CytometryFlow Cytometry
PMT FITC
PMT PE
PMT ECD
PMT PC5
DichroicFilters
Laser
1
2
3
4
Flow cell
Bandpass Filters
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319
ZAP70
5
FS
SS
How do Abs work?
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A New Diagnostic Test for Lyme DiseaseApplication of flow cytometry to Lyme disease
testingTraditional two-test protocol antibody testing - ELISA
and Western Blot (WB)Bacterial detection methods – Culture and polymerase
chain reaction (PCR) – Flow cytometry underutilizedUsing flow cytometry to detect Borrelia burgdorferi in
patient blood Antibody with fluorescein label (goat, KPL) added to aliquot
of patient blood (EDTA) Incubation for 1 hour at 37 degrees C Analyze by FC – Bb are bright fluorescent events
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A New Diagnostic Test for Lyme Disease
Comparison of FC to WB and PCRPhase 1 study compared FC with WB66 patients with clinical diagnosis of LD
5 positive by WB, 4 of those positive by FC An additional 28 patients positive by FC
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A New Diagnostic Test for Lyme Disease
Comparison of FC to PCRPhase 2 compared FC to PCR in 42 patients
with a clinical diagnosis of LD16 patients positive by FC and 18 positive by
PCR 8 patients positive by PCR and FC Gene sequencing performed on 14 PCR positive
patients (5 FC positive) 8 Bb sensu stricto (4 FC) 7 Bb sensu lato (new subgroup, 2 by FC)
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A New Diagnostic Test for Lyme Disease
Sensitivity and specificity of FC and WB comparedSensitivity FC = 66% Specificity = 92%Sensitivity WB = 52% Specificity = 100%
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A New Diagnostic Test for Lyme Disease
Sensitivity and specificity of FC and PCR comparedSensitivity FC = 62% Specificity = 100%Sensitivity PCR = 64% Specificity = 100%
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A New Diagnostic Test for Lyme Disease
Availability of flow cytotometry LD testingCentral Florida Research, Winter Haven,
Florida Web site – centralfloridaresearch.com
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A New Diagnostic Test for Lyme Disease
Future applicationsTesting for multiple tick-borne disease
pathogens simultaneouslyAdd antibodies to European Borrelia speciesLook for Bb antigens within monocytes
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