CDD Updates on Specimen Collection & Processing

Mohammad Al-Ghoul Joe Lyons Marian Tully CDD Updates on Specimen Collection & Processing

Center for Disease Detection

Mohammad Al-Ghoul

Agenda New tests implemented by CDD New HIV testing algorithm4th generation HIV testHIV-1/HIV-2 Discriminatory assay (Multispot test) CT/GC testing using cobas 4800 system Toxicology testing process and test changes Recent changes with CDD processes and tubes usedTips for optimal sample collectionList of tests availableQ & A

HIV New Testing Algorithm Advantages of using the fourth generation HIV assay

Advantages of using the HIV-1/HIV-2 Discriminatory assay (Multispot test)

Explain the new testing algorithm

Ability to Detect HIV Infection Earlier

4th Generation HIV Assay FDA-approved in June 2010The first diagnostic assay that:Detects HIV antibodies and HIV-1 p24 antigen Can be used on children 2 years A new testing algorithm that replaces WB test with Multispot

5Viral Load by HIV RNA or other NAAT4th generation (p24 Ag+Ab Combination) EIAHIV Antibody 3rd gen1101020304016816220-11 days, Infection Undetectable:No tests to close the gapHIV AbsHIV RNAHIV p24 AgAcute retroviral symptoms4th generation improvement11-22 days50% of new HIV infections acquired from persons with Acute HIV Infection (Day 0 Day 20)(Days)4th Generation HIV Assay Detects Infection in Acute Phase To consider efficacy of current HIV detection methods, we must understand that acute infection or the diagnostic window is defined as the interval from the initial infection to the time that the antibody to HIV is detectable. Viral Load testing by HIV RNA or other NAAT testing method is the most sensitive test method. At the time of infection until about 11 days post-infection, HIV RNA would not be detectable in the patients plasma. This window cannot be closed with current technology. On average, about 5 days later (or Day 16), p 24 Ag would be detected in patient serum. After about three weeks, Ab would be detected in patient serum.

In summary, while the WB method was more sensitive at the time of implementation of the CDC/APHL testing algorithm in 1989, WB is actually less sensitive than the third and fourth generation HIV assays (with WB blot detection occurring at about 4 weeks). The 4th generation assay improves the diagnostic window and is capable of detecting infection within 5 days after Viral Load by HIV RNA or other NAAT testing. 6Advantages of 4th Gen. Assay Detects HIV-1/-2 antibodies and HIV-1 p24 antigen simultaneously

Identifies HIV infection 2-20 days earlier than HIV antibody tests alone

Detects acute, as well as latent infections using a time and cost-saving strategy.

The p24 antigen is produced during the first few weeks and is detectable 7-9 days earlier than the appearance of HIV antibodies. As a result, the p24 antigen is an ideal marker to aid in early HIV diagnosis.7Western Blot Vs. Multispot Western BlotMultispotHIV-1 Positive: Preliminary positive for HIV-1 AbHIV-1 Negative: No Ab detected for HIV-1 Indeterminate: Repeat testing or collect another specimen and send for NAAT testing

HIV-1 Positive: Preliminary Positive for HIV-1 Antibodies and HIV-1 P24 AgHIV-2 Positive: Preliminary positive for HIV-2 antibodiesIndeterminate: Collect another specimen and send for NAAT testingHIV Positive (Undifferentiated) Additional testing recommended

Limitations of Current AlgorithmAntibody tests do not detect infection in ~10% of infected persons at highest risk of transmissionCannot detect acute infections and misclassifies approximately 60% of HIV-2 infections as HIV-1, based on HIV-1 WB results. Three day turnaround time for confirmation

Detection of Acute HIV Infection in Two Evaluations of a New HIV Diagnostic Testing Algorithm United States, 20112013, CDC, MMWR June 21, 2013 / 62(24);489-494because infected persons may not learn their test results and become lost to follow up9

Characteristics of an Ideal HIV Diagnostic AlgorithmDetects HIV acute and latent infections Differentiates HIV-1 from HIV-2Eliminates indeterminate results due to cross reactivity of HIV-2 Ab with HIV-1 WB Reduces the diagnostic window Faster turnaround times

A1: 4th generation HIV-1/2 immunoassay

A2: HIV-1/HIV-2 differentiation immunoassay (Multispot) A1(-)Negative for HIV-1 and HIV-2 antibodies and p24 AgA1+HIV-1 +HIV-1 antibodies detected Initiate care HIV-2 + HIV-2 antibodies detected Initiate careHIV-1&2 (-)RNARNA+ Acute HIV-1 infection Initiate careRNA (-) Negative for HIV-1Proposed in March 2010 CDC APHL HIV Diagnostic conference Repeat in duplicate

New HIV Diagnostic Algorithm

11Conclusion Earlier detection of infectionDifferentiation between HIV-1 and HIV-2Faster turnaround times for reports

12Agenda New tests implemented by CDD New HIV testing algorithm4th generation HIV testHIV-1/HIV-2 Discriminatory assay (Multispot test) CT/GC testing using cobas 4800 system Toxicology testing process and test changes Recent changes with CDD processes and tubes usedTips for optimal sample collectionTest List Q & A

CT/GC Testing: New Methodology The cobas 4800 system recently approved by the FDA to test for HPV and CT/GC Increased Sensitivity/Specificity Provides inhibitory control

Sexually Transmitted Diseases Treatment Guidelines, 2010; CDC, December 17, 2010http://www.aphl.org/aphlprograms/infectious/std/documents/ctgclabguidelinesmeetingreport.pdfVaginal swabs: preferred specimen type for testing using NAAT

Urine: preferred specimen type for testing males using NAATCDC Screening RecommendationsCT/NG Specimen TypesTesting for CT/GC by the FollowingVaginal Swab (Clinical Setting)Collector Stability (RT 365 days)UrineCollector Stability (RT 365 day)PreservCyt (ThinPrep) Collector Stability (RT 21 days)

CT/GC results are reported within 24 hours of specimen receipt Coming Soon:Endocervical Swab

16Vaginal Swab Rejection Criteria

10%5%2.5%No BloodReject cobas vaginal swab specimens for CT/NG testing if the whole blood concentration is >10.0% (dark red or brown coloration).Urine Rejection Criteria

0.35%0.175%0.083%No BloodReject cobas Urine specimens for CT/NG testing if whole blood concentration is >0.35%.PreservCyt (ThinPrep) Rejection Criteria

1.5%0.75%0.037%No BloodCompare PreserveCyt specimens to the images above. Reject specimens for CT/GC testing if whole blood concentration is >1.5%.Agenda New tests implemented by CDD New HIV testing algorithm4th generation HIV testHIV-1/HIV-2 Discriminatory assay (Multispot test) CT/GC testing using cobas 4800 system Toxicology testing process and test changes Recent changes with CDD processes and tubes usedTips for optimal sample collectionTest List Q & A

More than 70% of lab errors are due to specimen collection issues

Reliability and Validity of Urine Toxicology Specimen

Urine Drug Testing LimitationUrine drug testing only indicates prior use

Not useful for determining:Time since last useExtent/frequency of useAdditional use since last positive testCurrent impairment

Testing MethodsScreening: Enzyme Immunoassay (EIA)

Confirmation: Gas Chromatography/Mass Spectrometry (GC/MS)Gold-Standard for Drug TestingNO BIOLOGICAL FALSE POSITIVES

Negative screening results are released the same day the specimen is received at CDD

NEWToxicology orders now have their own accession numbers.Test Menu and Turnaround Times Job Corps Toxicology Panel AMP/Meth-AMP Panel (72h) Cannabinoids (48h) Cocaine (48h)Opiates (72h) PCP (48h)

Discuss the 24Spice/K2K2 or Spice is a mixture of herbs, spices or shredded plant material that is typically sprayed with a synthetic compound chemically similar to THC

At least 41 states and Puerto Rico have legislatively banned synthetic cannabinoidshttp://www.ncsl.org/research/civil-and-criminal-justice/synthetic-cannabinoids-enactments.aspx

JWH-133

JWH-018"People who use it are idiots."You don't know what it's going to do to you."

John W. Huffman, PhD

Spice Test (Synthetic Cannabinoids)Current test detects three metabolitesJWH-018 M5JWH-018 M6JWH-073 M6

27New Spice Test Screening Detected / Non detected

Confirmation for 16 separate metabolitesAM2201, AM694, JWH 018, JWH 019, JWH 073, JWH 081, JWH 122, JWH 203, JWH210, JWH 250, JWH 398, MAM 2201, RCS-4, 2XUR-144, XLR11

Agenda New tests implemented by CDD New HIV testing algorithm4th generation HIV testHIV-1/HIV-2 Discriminatory assay (Multispot test) CT/GC testing using cobas 4800 system Toxicology testing process and test changes Recent changes with CDD processes and tubes usedTips for optimal sample collectionTest List Q & A

Center for Disease Detection

Joe Lyons

Sample Collection DevicesBlood CollectorsUrine CollectorsSwab Collection Device

7.5 mL Tiger TopNo Fill Line MarkAdditive serum clot activator

Blood Collection Devices

Fill Mark10% Above10% Below

4 mL SST and Red TopAdditive serum clot activator

Blood Collection Devices

Fill Mark10% Above10% Below3 mL Purple TopCBC, H/HSample Fill LineAdditive - Ethylenediaminetetraacetic acid (EDTA)

Blood Collection DevicesYellow TopTOX and NY CT/GCSample fill lineNo AdditiveNo fill requirement other than amount necessary to run test

Fill Mark

Urine Collection DevicesRed and Yellow TopSample fill lines (stay within lines)Additive urine preservative

Do Not Exceed this lineDo Not fill lower than this line

Urine Collection Devicescobas PCR Urine collectorSample fill lines (stay within lines)Additive PCR mediaA drop DOES NOT make a difference

Do not exceed this lineDo not fill lower than this line

Urine Collection DevicesMention that we have seen people using the temperature gauge as a fill line and filling to the small area between the arrow and fill line.37

cobas PCR Female SwabPatient collectedSample fill lines NOT usedAdditive PCR mediaDiscard extra swabSwab down (bacteria in media)

Swab Collection Device

The best result begins with the SPECIMENWomen Vaginal specimenMen Urine SpecimenDoes not require use of the cleaning swabDiscard one swabInsert one swab approximately 2 inches into the vaginaGently turn the swab for about 30 seconds while rubbing the swab against the wall of the vaginaRemove swab and place into the cobas PCR media tubeBreak shaft at etch mark and recap tubeNote: Swab must be at bottom of tube

Patient should not urinate 1 hour prior to samplingCollect urine at the beginning of the urine stream. This is not a mid-stream collection.Collect first 10 to 50 mL of urine, additional volume may begin to dilute the specimenTransfer appropriate volume of urine to the cobas PCR media tube

We have full collection instructions that are available from your account manager.39Place arm in a downward position.

Phlebotomy TipsUse an insertion angle of 10 to 20 degrees.

Phlebotomy TipsA frequent cause of no blood flow is that the tip is no longer in the vein lumen.

Phlebotomy Tips

Phlebotomy TipsMake a fist, do not pump (pumping increases potassium)Avoid tapping the venipuncture siteWarm the venipuncture siteWait an extra few seconds when it appears blood flow has stopped Ensure all additive tubes are gently rocked 8-10 times

Phlebotomy TipsTo avoid HemolysisDo not apply tourniquet too tightly or for too long.Do not shake the sample instead gently rock it 8-10 times.Do not delay separation of cells from serum by centrifugation(>3 hours)Too long or too high centrifugation

Agenda New tests implemented by CDD New HIV testing algorithm4th generation HIV testHIV-1/HIV-2 Discriminatory assay (Multispot test) CT/GC testing using cobas 4800 system Toxicology testing process and test changes Recent changes with CDD processes and tubes usedTips for optimal sample collectionTest List Q & A

Center for Disease Detection Marian Tully

Account ManagersBarbara Castro, Account Manager(888) 858-8663, Ext. 236barbara.castro@cddmedical.comVeronica Rodriguez, Account Manager(888) 858-8663, Ext. 250veronica.rodriguez@cddmedical.comMarian Tully, Sr. Account Manager(888) 858-8663, Ext. 208marian.tully@cddmedical.com

Note: The Department of Labor is only contracted for Chlamydia, HIV and Toxicology.List of Tests48

Helpful Links49Thank You