Verax The Platelet Pan Genera Detection PGD Test

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Verax The Platelet Pan Genera Detection (PGD)
Test
Somchai Amornwacharapong Abbott Diagnostics
March 14, 2008
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BackgroundCausing agent to be transmitted by
transfusion
However, in regular, blood units were tested only
for
Parasites
Bacteria
Viruses
HIV, HCV, HBV, CMV, HTLV

• Malaria,
• T. cruzi,
• T. gondii

T. pallidum, R.rickttessi Etc.
Prion
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Platelets units
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Current Methodology

• Culture
• Pall eBDS (Oxygen consumption)
• BacT/ALERT (CO2 measurement)
• Hemoculture systems (Florescence)

• Swirling
• pH
• Glucose
• Gram Stain

Apheresis (Single Donor Platelets, SDPs)
Random Donor Platelets (RDPs) (Whole Blood
Derived)
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Culture Requires sampling early in platelet life
Sampling early in life limits Clinical Sensitivity
False Negatives Clinical Sensitivity 50
Source

Goodnough LT et al. NEJM 1999,
Klein et al.JAMA, vol 274, issue 1368 1373,
November 1,2005, Goodman et al. Bacterial
Contamination of blood Components Risk, Strategy
and regulation, Hematology 2003
Culture sample _at_ 24 hours
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STORAGE
Platelet products are stored at room temperature,
5-7 days, rather than in the cold.
Source American Society of Hematology (
Bacterial Contamination of Blood Components
Risks , Strategies, and Regulation)
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Bacterial vs. Virus Infectious risk in the
blood supply
Hepatitis B
HBsAg 1971
Anti HBsAg 1987
HIV
P 24 1996
HIV PCR 2000
HIV Ab 1985
Hepatitis C
HCV Ab 1990
HCV PCR 2000
HTLV 1/2
HTLV 1/2 1989
West Nile Virus
WNV NAT 2003
No Practical Screening Tests Currently Available
Bacteria - Platelets
12,000
Bacteria - Red Cells
120,000
Source

Ilert WE, et al. Transfusion Medicine
1995, 557-61
Brecher et al. Clinical Microbiology Reviews,
2005, 195-204
1100,000
11,000,000
Incidence rate
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Typical Platelet Contaminants Skin Colonizers
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Potential Sources of Contaminations

• Donor skin at blood donation
• Unapparent donor bacteremia
• Contamination during collection or processing
• Environment
• Equipment
• Disposable supplies

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The Problems of Bacterial Contamination
Identification of At-Risk Situations
Bacteremia Enteric organisms Prototype Yersinia
MOST LETHAL
Surface contamination Skin commensals Prototype
Staphylococci
MOST COMMON
Unit contamination Environmental
organisms Example Pseudomonas
Source James P. AuBuchon, MD How reliable are
our Bacterial Detection Methods
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Why test at point of issue ?
Wide variation in rate of log Phase Growth
Wide variation in duration of Lag Phase
Source

Goodnough LT et al. NEJM 1999,
Klein et al.JAMA, vol 274, issue 1368 1373,
November 1,2005, Goodman et al. Bacterial
Contamination of blood Components Risk, Strategy
and regulation, Hematology 2003
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Verax Platelet PGD Test
Product Information
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Verax Platelet PGD Test
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Intended Use
The Verax Platelet PGD Test A rapid,
qualitative immunoassay for the detection of
Aerobic and Anaerobic Gram-positive and
Gram-negative bacteria in leukocyte reduced
apheresis platelets (LRAP) and platelets derived
from buffy coat as a quality control test.
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Pan Genera Detection (PGD)
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Verax rapid Platelet PGD Test
Single-use disposable test
1. Rapid 25 minutes (3 min hands on)
2. Positives typically lt 10 minutes
3. Sensitivity 103 CFU/ mL
4. Specificity gt 99.7
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Pan Genera Rapid Test Format
Test Features
Procedural Control
Procedural Control
1. ABS housing holding GP/GN test strips
2. Shared 300 uL sample addition well
3. Separate GP and GN read windows
4. Procedural controls for GP GN
Sample Well
Gram-Negative Read Window
Gram-Positive Read Window
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Verax Platelet PGD Test Procedure
Centrifugation
Re-suspension
Reading
B.
C.
A.
Fill well and Incubate at room temperature
until procedural controls change color ( 20 min)
Add 8 drops of Reagent
Aspirate pellet with disposable pipette and
triterate 4 to 8 times Leave any remaining pellet
in tube
Cap and gently invert 1-2 times until solution
turns GREEN
Add 500 uL platelet sample to microfuge tube and
8 drops of Reagent
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Solution should Be BLUE
Interpret results
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Negative
GP
GN
Positive
C
C
Solution turns YELLOW Vortex until any remaining
pellet completely dissolves
Add 4 drops of Reagent
GP
GN
C
Invalid
Spin for 5 minutes _at_ 13,400 RPM
Decant supernatant
C
3
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Examples of Reactive PGD Test Results
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Examples of Non-reactive PGD Test Results
Example of a non-reactive result with no visible
line in either window
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Growth Study Result

• All types of tested bacteria (10 spp.) were
  detectable by the Platelet PGD test system at 48
  or 72 hours

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Verax Platelet PGD Test
Methodology Comparison
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Detection Strategies for Fast and Slow Growing
Bacteria
Fast growing bacteria
Slow growing bacteria
BACTERIAL LOAD (log CFU/ML)
99.8
Sensitivity of culture
35-50
0 1 2 3
4 5 6
7 STORAGE DAYS
Source

Goodnough LT et al. NEJM 1999,
Klein et al.JAMA, vol 274, issue 1368 1373,
November 1,2005, Goodman et al. Bacterial
Contamination of blood Components Risk, Strategy
and regulation, Hematology 2003
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Methods Comparison
Source Biomerieux and Pall Medical web site.
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TRANSFUSION Vol 48, Feb 2008, p.282-285
N 2,000 Random-donor platelets
Found 0.4 by aerobic culturing
Bacteria Predominantly Gram ve (62.5)
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Isolated microorganisms and time of storage
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Recent Example
Updated 831 a.m. CT July 27, 2006
KANSAS CITY, Mo. - A hospital patient died after
receiving a unit of blood platelets tainted with
E. coli bacteria, the Community Blood Center in
Kansas City said.
???????????????????????????????????????????
????????? platelet ???????????????? E.Coli
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South China Morning Post,Jan 8th 2008Hong Kong
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Man dies after blood transfusion reaction
Tainted blood baffles experts
Found Pseudomonas fluorescens bacterium in the
blood bag
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AABB Letter to the FDA
To US-FDA on approval strategies for
point-of-release testing for bacterial
contamination
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Q A ?
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THANK YOU !
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Back up
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Verax Growth Study Results
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Verax Growth Study Results
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PGD Immunoassay Format two tests run in parallel
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Verax Platelet PGD Test
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Examples of Invalid PGD Test Results
Example GN procedural control failure
Example of failure of GP and GN Test Result
Windows to clear to white or light pink
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Results and Interpretation
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Verax Platelet PGD Test
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Methods Comparison
Source Biomerieux and Pall Medical web site.
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Methods Comparison
Source

Ilert WE, et al. Transfusion Medicine
1995, 557-61
Brecher et al. Clinical Microbiology Reviews,
2005, 195-204
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Buffy Coat Background
Buffy coat, produced from a whole blood donation,
is a combination of platelets and white blood
cells (WBCs), as well as small amounts of red
blood cells (RBCs) and plasma. Whole blood
donations are separated into components via
gravitational force in a centrifuge. The force of
the centrifuge causes the whole blood to separate
into layers based on cell density. The upper
layer captured in the centrifuge is plasma. The
middle layer is the buffy coat. The bottom,
heaviest layer is packed red blood cells. In the
mid-1970's, European blood centers began to
remove the buffy coat from separated RBCs to
prevent transfusion reactions reactions mainly
caused by the presence of WBCs. Researchers then
made a valuable discovery the buffy coat
contained a high concentration of platelets.