USAF JBAIDS/M1M USAF JBAIDS/M1M Proficiency Test ProgramProficiency Test Program

Ketan Patel, Ph.D.Ketan Patel, Ph.D.

SAFMLSSAFMLS

Feburary 11, 2008Feburary 11, 2008

Points of Interest for Points of Interest for USAF PT Program FY08USAF PT Program FY08

JBAIDS PT Program DetailsJBAIDS PT Program DetailsSubmitting Results for JBAIDS PTSubmitting Results for JBAIDS PTJBAIDS PT FailuresJBAIDS PT FailuresM1M Proficiency Test M1M Proficiency Test AFIP supportAFIP support

JBAIDS PT: Program DetailsJBAIDS PT: Program Details Approximately 53 Sites Approximately 53 Sites Testing FormatTesting Format

2 Unknowns (multiple simulated matrices)2 Unknowns (multiple simulated matrices) 4 Organisms (B.a (2 targets), F.t (1), Y.p (2), 4 Organisms (B.a (2 targets), F.t (1), Y.p (2),

Orthopox(1))Orthopox(1)) 4 Runs per sample (PTC, NTC, Unk1, Unk2; pos, 4 Runs per sample (PTC, NTC, Unk1, Unk2; pos,

neg, and unks ran in dup)neg, and unks ran in dup) 4 Extraction Procedures4 Extraction Procedures

QFlow QFlow ScoopScoop VibeVibe SwipeSwipe

JBAIDS PT: Program DetailsJBAIDS PT: Program Details

Three PT events per fiscal yearThree PT events per fiscal yearSupplies Supplies

Unknown samples (2) Unknown samples (2) DNA or heat-killed bacteria DNA or heat-killed bacteria

Variety of matricesVariety of matrices Sterility reports kept on file at AFIPSterility reports kept on file at AFIP

Provided by AFIPProvided by AFIP Shipped via FEDEXShipped via FEDEX Notification of receipt of samples required from HLDsNotification of receipt of samples required from HLDs

JBAIDS PT: Program DetailsJBAIDS PT: Program Details

ReagentsReagentsExtraction Kits (Extraction Kits (QQFLOW, Scoop, VIBE, and FLOW, Scoop, VIBE, and

Swipe)Swipe) Provided by individual sites Provided by individual sites

JBAIDS Assay TargetsJBAIDS Assay Targets Lyophilized reagents require reconstitutionLyophilized reagents require reconstitution

Reconstitution water provided in assay kitReconstitution water provided in assay kit Reconstitution buffer provided in assay kitReconstitution buffer provided in assay kit

Provided by individual sites Provided by individual sites

JBAIDS PT: Program DetailsJBAIDS PT: Program Details

RT-PCR Using JBAIDS PlatformRT-PCR Using JBAIDS PlatformUnknown sample QC done by AFIPUnknown sample QC done by AFIP

Pre-shipment QCPre-shipment QCPost-shipment QCPost-shipment QC

Results submitted to PT CoordinatorResults submitted to PT CoordinatorDue 10 business days from the time of receipt Due 10 business days from the time of receipt

of PT samples by HLDof PT samples by HLD

Changes With The JBAIDS PT & Changes With The JBAIDS PT & InstrumentationInstrumentation

Sites provide their own reagents for PTSites provide their own reagents for PTJBAIDS software differs from RAPID JBAIDS software differs from RAPID

User-friendlyUser-friendlySpecific positioning of samplesSpecific positioning of samplesAutomatically makes result callsAutomatically makes result callsNotifies user of failed QCsNotifies user of failed QCsProduces a “combined call” based on QC Produces a “combined call” based on QC

results and individual sample resultsresults and individual sample results

Changes With The JBAIDS PT & Changes With The JBAIDS PT & Instrumentation (cont.)Instrumentation (cont.)

Multiple extraction protocols for different Multiple extraction protocols for different matricesmatricesQFlow (replaced Flow in Fall 07)QFlow (replaced Flow in Fall 07)ScoopScoopSwipeSwipeVIBE VIBE

QFlow Sample ExtractionQFlow Sample Extraction

MatricesMatricesWhole blood (DNA)Whole blood (DNA)Food (DNA)Food (DNA)

WaterWaterTunaTunaGround BeefGround BeefSaladSalad

Gastric Wash (DNA)Gastric Wash (DNA)Water (DNA)Water (DNA)Air into PBS (DNA/RNA)Air into PBS (DNA/RNA)

Scoop & Vibe ExtractionsScoop & Vibe Extractions

Matrices for Scoop ExtractionMatrices for Scoop ExtractionSoil (DNA)Soil (DNA)Stool (DNA)Stool (DNA)

Matrices for Vibe ExtractionMatrices for Vibe ExtractionWhole Blood (DNA)Whole Blood (DNA)Nasal Swab (RNA)Nasal Swab (RNA)Sputum (DNA/RNA)Sputum (DNA/RNA)

Swipe Sample ExtractionSwipe Sample Extraction

MatricesMatricesNasal/Pus SwabNasal/Pus SwabSurface SwabSurface SwabPowder Powder Culture SamplesCulture Samples

Phases of JBAIDS ExtractionsPhases of JBAIDS Extractions

Reconstitution of samplesReconstitution of samples Sample lysisSample lysis

Bead BeatingBead Beating

DNA/RNA Bound to filterDNA/RNA Bound to filter Buffer 1Buffer 1

DNA/RNA Washed of inhibitorsDNA/RNA Washed of inhibitors Buffer 2Buffer 2

DNA/RNA Eluted from the filterDNA/RNA Eluted from the filter Buffer 3Buffer 3

Submitting ResultsSubmitting Results

Fax Result Forms to AFIPFax Result Forms to AFIPFax Conditions & Progress Logs to AFIPFax Conditions & Progress Logs to AFIPFax JBAIDS report to AFIPFax JBAIDS report to AFIPForward JBAIDS run files (.ixo) to AFIPForward JBAIDS run files (.ixo) to AFIPEvaluation of PTEvaluation of PT

Grades will be determined based on:Grades will be determined based on:JBAIDS reportJBAIDS reportResult form responses Result form responses

>>80% constitute passing grade for PT80% constitute passing grade for PT

FailuresFailures

JBAIDS Failure policyJBAIDS Failure policy (Insufficient) Less than 80% in a PT period (Insufficient) Less than 80% in a PT period

requires:requires:Investigation of failureInvestigation of failurePerform any appropriate action to correct problemPerform any appropriate action to correct problemComplete Exception response formComplete Exception response formReturn form to AFIP PT CoordinatorReturn form to AFIP PT Coordinator

(Poor) Less than 80% in two out of three (Poor) Less than 80% in two out of three consecutive PT periods requires:consecutive PT periods requires:The discontinuation of testing until MAJCOM The discontinuation of testing until MAJCOM

approves resumptionapproves resumptionInvestigation of failureInvestigation of failure

Failure (cont.)Failure (cont.)

Perform any appropriate action to correct problemPerform any appropriate action to correct problemComplete Exception response formComplete Exception response formForward completed Exception report including Forward completed Exception report including

investigation results, corrective action proposals, investigation results, corrective action proposals, etc. to MAJCOM for review and signatureetc. to MAJCOM for review and signature

MAJCOM will decide if appropriate action has MAJCOM will decide if appropriate action has been taken and if testing should resumebeen taken and if testing should resume

Forward forms to AFIP PT Coordinator after Forward forms to AFIP PT Coordinator after MAJCOM approvalMAJCOM approval

Within three days of resumption of testing forward Within three days of resumption of testing forward Exception response form to the CCLM officeException response form to the CCLM office

Failures (cont.)Failures (cont.)

(Unsatisfactory) Less than 80% in three (Unsatisfactory) Less than 80% in three out of four consecutive PT periods out of four consecutive PT periods requires:requires:Complete the steps for poor performance Complete the steps for poor performance

listed previously in addition to listed previously in addition to The Director of the CCLM will review investigations The Director of the CCLM will review investigations

and other corrective action documentationand other corrective action documentationThe decision to resume testing will be made by the The decision to resume testing will be made by the

Director of the CCLMDirector of the CCLM

Current Status of JBAIDS PT ‘08Current Status of JBAIDS PT ‘08

PT events prior to ’08 were educational PT events prior to ’08 were educational challangeschallanges

JBAIDS PT ‘08 #1 JBAIDS PT ‘08 #1 Shipped on January 4, 2008Shipped on January 4, 2008Awaiting final reportAwaiting final report

Common errors from Past PTsCommon errors from Past PTs

Common errors Common errors Failure to follow OIFailure to follow OIFailure to detect DNA (technical)Failure to detect DNA (technical)Blanks left on result form (clerical)Blanks left on result form (clerical)

Common Errors for JBAIDS PTCommon Errors for JBAIDS PT

Failure to follow SOPFailure to follow SOPRunning Target 2 with Target 1 Running Target 2 with Target 1 Target 2 should be ran only when Target 1 is Target 2 should be ran only when Target 1 is

positivepositiveUsing wrong extraction kitUsing wrong extraction kitTranscription errorsTranscription errors

Switching answersSwitching answersSkipping answers (leaving results blank)Skipping answers (leaving results blank)

Current Current

Changes for fall of 2007Changes for fall of 2007 Introduction of inhibition controls for each Introduction of inhibition controls for each

assayassay Introduction of extraction controls for each Introduction of extraction controls for each

extractionextractionSites will stock the following assays onlySites will stock the following assays only

Ba target #1 and #2Ba target #1 and #2Yp target #1 and #2Yp target #1 and #2F. tularensisF. tularensisOrthopoxOrthopoxSmallpox Smallpox

M1M Proficiency TestingM1M Proficiency Testing

Approximately 27 participantsApproximately 27 participantsTesting FormatTesting Format

One unknownOne unknownThree target toxinsThree target toxins

M1M Test Menu for HLDM1M Test Menu for HLDRicinRicinBot ABot ASEB (Staphylococcus enterotoxin B)SEB (Staphylococcus enterotoxin B)

M1M Proficiency TestM1M Proficiency Test(Cont.)(Cont.)

SuppliesSupplies UnknownsUnknowns

Non-infectious, lyophilized materialNon-infectious, lyophilized material Provided by AFIPProvided by AFIP

Reagents and Instrument suppliesReagents and Instrument supplies Provided by individual sitesProvided by individual sites

Frequency and Delivery MethodsFrequency and Delivery Methods Three times per fiscal yearThree times per fiscal year M1M PT will be delivered with JBAIDS PTM1M PT will be delivered with JBAIDS PT

M1M PT will be scored independently of JBAIDS PTM1M PT will be scored independently of JBAIDS PT

M1M Proficiency Test M1M Proficiency Test (Cont.)(Cont.)

Submitting resultsSubmitting resultsFax result form to AFIPFax result form to AFIPFax conditions and progress logs to AFIPFax conditions and progress logs to AFIP

GradingGradingPass/Fail ScoringPass/Fail Scoring

Based on number of unknowns and assaysBased on number of unknowns and assaysWith only 3 possible answers, one incorrect result With only 3 possible answers, one incorrect result

causes the percentage to fall below 80%causes the percentage to fall below 80%

Common Errors with M1M PTsCommon Errors with M1M PTs

Problems that occurredProblems that occurredLack of package inserts with controlsLack of package inserts with controlsLack of OILack of OILack of “key” to coded reagentsLack of “key” to coded reagentsError in reconstitution directions for PT Error in reconstitution directions for PT

materialmaterial

AFIP SupportAFIP Support

AFIP PT CoordinatorAFIP PT Coordinator Notification of shipment of PT thru emailNotification of shipment of PT thru email Technical supportTechnical support

Via emailVia email Via phoneVia phone

Tracking information for shipments will be made Tracking information for shipments will be made available upon requestavailable upon request

Distribution of results to individual sites and MAJCOM Distribution of results to individual sites and MAJCOM consultants via emailconsultants via email

Limited number of replacement PT samples will be Limited number of replacement PT samples will be available upon requestavailable upon request

AFIP Proficiency Testing TeamAFIP Proficiency Testing Team

Dr. Ketan PatelMolecular Chief

Ms. Ellen LaMorenaAF PT Coordinator

Dr. S. FrancesconiMicrobiology Chief

Major Patrick KennedyScience Coordinator

Dr. Robert CrawfordDivision Chief

AFIP

AFIP Contact InformationAFIP Contact Information Dr. Robert CrawfordDr. Robert Crawford

(202)782-1825(202)782-1825 crawfordr@afip.osd.milcrawfordr@afip.osd.mil

Dr. Ketan PatelDr. Ketan Patel (202) 782-2847(202) 782-2847 patelk@afip.osd.milpatelk@afip.osd.mil

Major Patrick KennedyMajor Patrick Kennedy (202) 782-1863(202) 782-1863 kennedyp@afip.osd.milkennedyp@afip.osd.mil

Dr. Stephen FrancesconiDr. Stephen Francesconi (202)782-1834(202)782-1834 francesconis@afip.osd.milfrancesconis@afip.osd.mil

Ms. Ellen LaMorenaMs. Ellen LaMorena (202) 782-1861(202) 782-1861 dnicuolam@afip.osd.mildnicuolam@afip.osd.mil

Questions?Questions?

Comments?Comments?