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

2. Center for Disease Detection
Mohammad Al-Ghoul
Center for Disease Detection

3. Agenda New tests implemented by CDD New HIV testing algorithm
4th generation HIV test
HIV-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 used
Tips for optimal sample collection
List of tests available
Q & A

4. 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

5. 4th Generation HIV Assay
FDA-approved in June 2010
The 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
Ability to Detect HIV Infection Earlier

6. 4th Generation HIV Assay Detects Infection in Acute Phase
Acute retroviral symptoms
HIV RNA
HIV Abs
0-11 days,
Infection Undetectable:
No tests to close the gap
4th generation improvement11-22 days
HIV p24 Ag
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 patient’s 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. 10 20 30 40
168 11 16 22
(Days)
Viral Load by HIV RNA or other NAAT
4th – generation (p24 Ag+Ab Combination) EIA
HIV Antibody 3rd gen
50% of new HIV infections acquired from persons with Acute HIV Infection (Day 0 – Day 20)

7. Advantages 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.

8. Western Blot Vs. Multispot
HIV-1 Positive: Preliminary positive for HIV-1 Ab
HIV-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 Ag
HIV-2 Positive: Preliminary positive for HIV-2 antibodies
Indeterminate: Collect another specimen and send for NAAT testing
HIV Positive (Undifferentiated) Additional testing recommended

9. Limitations of Current Algorithm
Antibody tests do not detect infection in ~10% of infected persons at highest risk of transmission
Cannot 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
because infected persons may not learn their test results and become lost to follow up
Detection of Acute HIV Infection in Two Evaluations of a New HIV Diagnostic Testing Algorithm — United States, 2011–2013, CDC, MMWR June 21, 2013 / 62(24);

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

11. New HIV Diagnostic Algorithm
A1: 4th generation HIV-1/2 immunoassay
A1+
A1(-)
Negative for HIV-1 and HIV-2 antibodies and p24 Ag
Repeat in duplicate
A2: HIV-1/HIV-2 differentiation immunoassay (Multispot)
HIV-1 +
HIV-1 antibodies detected Initiate care
HIV-1&2 (-)
HIV-2 +
HIV-2 antibodies detected Initiate care
RNA
RNA Acute HIV-1 infection Initiate care
RNA (-) Negative for HIV-1
Proposed in March 2010
CDC APHL HIV Diagnostic conference

12. Conclusion Earlier detection of infection
Differentiation between HIV-1 and HIV-2
Faster turnaround times for reports

13. Agenda New tests implemented by CDD New HIV testing algorithm
4th generation HIV test
HIV-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 used
Tips for optimal sample collection
Test List
Q & A

14. 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

15. CDC Screening Recommendations CT/NG Specimen Types
Vaginal swabs: preferred specimen type for testing using NAAT
Urine: preferred specimen type for testing males using NAAT
Sexually Transmitted Diseases Treatment Guidelines, 2010; CDC, December 17, 2010

16. Testing for CT/GC by the Following
Vaginal Swab (Clinical Setting)
Collector Stability (RT 365 days)
Urine
Collector Stability (RT 365 day)
PreservCyt (ThinPrep)
Collector Stability (RT 21 days)
Coming Soon:
Endocervical Swab
CT/GC results are reported within 24 hours of specimen receipt

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

18. Urine Rejection Criteria
0.35%
0.175%
0.083%
No Blood
Reject cobas® Urine specimens for CT/NG testing if whole blood concentration is >0.35%.

19. PreservCyt (ThinPrep) Rejection Criteria
1.5%
0.75%
0.037%
No Blood
Compare PreserveCyt® specimens to the images above.
Reject specimens for CT/GC testing if whole blood concentration is >1.5%.

20. Agenda New tests implemented by CDD New HIV testing algorithm
4th generation HIV test
HIV-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 used
Tips for optimal sample collection
Test List
Q & A

21. Reliability and Validity of Urine Toxicology Specimen
More than 70% of lab errors are due to specimen collection issues

22. Urine Drug Testing Limitation
Urine drug testing only indicates prior use
Not useful for determining:
Time since last use
Extent/frequency of use
Additional use since last positive test
Current impairment

23. Testing Methods Screening: Enzyme Immunoassay (EIA)
Confirmation: Gas Chromatography/Mass Spectrometry (GC/MS)
“Gold-Standard” for Drug Testing
NO BIOLOGICAL FALSE POSITIVES

24. Test Menu and Turnaround Times
Job Corps Toxicology Panel
AMP/Meth-AMP Panel (72h)
Cannabinoids (48h)
Cocaine (48h)
Opiates (72h)
PCP (48h)
NEW
Toxicology orders now have their own accession numbers.
Discuss the
Negative screening results are released the same day the specimen is received at CDD

25. Spice/K2
K2 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 cannabinoids

26. John W. Huffman, PhD "People who use it are idiots.”
JWH-018
JWH-133
"People who use it are idiots.”
"You don't know what it's going to do to you."

27. Spice Test (Synthetic Cannabinoids)
Current test detects three metabolites
JWH-018 M5
JWH-018 M6
JWH-073 M6

28. New Spice Test Screening Confirmation for 16 separate metabolites
Detected / Non detected
Confirmation for 16 separate metabolites
AM2201, 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

29. Agenda New tests implemented by CDD New HIV testing algorithm
4th generation HIV test
HIV-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 used
Tips for optimal sample collection
Test List
Q & A

30. Center for Disease Detection
Joe Lyons
Center for Disease Detection

31. Sample Collection Devices
Blood Collectors
Urine Collectors
Swab Collection Device

32. Blood Collection Devices
7.5 mL Tiger Top
No Fill Line Mark
Additive – serum clot activator

33. Blood Collection Devices
10% Above
Fill Mark
10% Below
4 mL SST and Red Top
Additive – serum clot activator

34. Blood Collection Devices
3 mL Purple Top
CBC, H/H
Sample Fill Line
Additive - Ethylenediaminetetraacetic acid (EDTA)
10% Above
Fill Mark
10% Below

35. Urine Collection Devices
Yellow Top
TOX and NY CT/GC
Sample fill line
No Additive
No fill requirement other than amount necessary to run test
Fill Mark

36. Urine Collection Devices
Do Not Exceed this line
Red and Yellow Top
Sample fill lines (stay within lines)
Additive – urine preservative
Do Not fill lower than this line

37. Urine Collection Devices
cobas® PCR Urine collector
Sample fill lines (stay within lines)
Additive – PCR media
A drop DOES NOT make a difference
Do not exceed this line
Do not fill lower than this line
Mention 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.

38. Swab Collection Device
cobas® PCR Female Swab
Patient collected
Sample fill lines NOT used
Additive – PCR media
Discard extra swab
Swab down (bacteria in media)

39. The best result begins with the SPECIMEN
Women – Vaginal specimen
Men – Urine Specimen
Does not require use of the cleaning swab
Discard one swab
Insert one swab approximately 2 inches into the vagina
Gently turn the swab for about 30 seconds while rubbing the swab against the wall of the vagina
Remove swab and place into the cobas® PCR media tube
Break shaft at etch mark and recap tube
Note: Swab must be at bottom of tube
Patient should not urinate 1 hour prior to sampling
Collect 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 specimen
Transfer appropriate volume of urine to the cobas® PCR media tube
We have full collection instructions that are available from your account manager.

40. Phlebotomy Tips
Place arm in a downward position.

41. Phlebotomy Tips
Use an insertion angle of 10 to 20 degrees.

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

43. Phlebotomy Tips Make a fist, do not pump (pumping increases potassium)
Avoid tapping the venipuncture site
Warm the venipuncture site
Wait an extra few seconds when it appears blood flow has stopped
Ensure all additive tubes are gently rocked 8-10 times

44. Phlebotomy Tips To avoid Hemolysis
Do 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

45. Agenda New tests implemented by CDD New HIV testing algorithm
4th generation HIV test
HIV-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 used
Tips for optimal sample collection
Test List
Q & A

46. Center for Disease Detection
Marian Tully
Center for Disease Detection

47. Account Managers
Barbara Castro, Account Manager (888) , Ext. 236
Veronica Rodriguez, Account Manager (888) , Ext. 250
Marian Tully, Sr. Account Manager (888) , Ext. 208

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

49. Helpful Links

50. Thank You