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ctca breakout session: lab issues of in vitro diagnostic testing

Outline. 1. QuantiFERON

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ctca breakout session: lab issues of in vitro diagnostic testing

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    1. CTCA Breakout Session:Lab Issues of In Vitro Diagnostic Testing Sally Liska, DrPH, Director San Francisco PH Laboratory SLiska@sfdph.org

    3. Principle of the Test 1. Collect whole heparinized blood. 2. Incubate 1ml w/ 4 different antigens. 3. Measure production of interferon-? {IFN-?} using EIA {enzyme immunoassay}. 4. Compare IFN-? levels in the 2 mycobacterial peptide wells to the 2 controls wells.

    4. Principle of the Test 1. Collect whole heparinized blood Use Na or Li heparin (green top) tubes Need 5 mls ? Unsatisfactory: QNS Use 10 ml tube Invert the tube several times to dissolve the heparin and mix with the blood

    5. Principle of the Test 2. Incubate 1ml aliquots w/ 4 different antigens Incubate 16-24 hrs Within 12 hrs of blood collection Four antigens: Nil Control (PBS) Mitogen Control ESAT-6* CFP-10* *proteins which stimulate IFN-? production in those who have been infected w/ M. tb complex. Also present in M. sps kansasii, szulgai & marinum.

    6. Principle of the Test 3. Measure production of interferon-? using EIA methodology. Prepare IFN-? standard dilution series and test in duplicate Aliquot each of the test plasma samples to microtiter plate well Incubation time 2-3 hrs, total time 5-6 hrs

    7. Principle of the Test 4. Compare IFN-? levels in the 2 mycobacterial protein wells to the 2 controls wells.

    8. Inconclusive Results A. Indeterminate ? Can be due to: 1-Technical factors – incomplete washing of EIA plate, mixing Ag with blood 2-Host factors – Peptide Ag results not more than 50% of Nil Mitogen < 0.5, peptide results < 0.35 and Nil is <0.7 IU B. Unsatisfactory ? Can be due to: Lipemic or clotted specimen

    9. Lab Logistics Personnel – Lab Technician, PH Micro Test Kit Ancillary Supplies – 24 well culture plates, pipettes, pipet tips, etc. Equipment Rocker for blood samples Multi-channel pipette Microplate shaker Microplate washer, reader

    10. Reimbursement 1. Bill another program (occupational health, jail health program, etc.) 2. Bill private insurance, submitter 3. Bill MediCal CPT code 86849 Reimbursement amt $20.08 Submit paper bill 4. Bill Medicare CPT code 86480 Reimbursement amt $86.58

    11. Come on in – The water’s fine

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