Transcript Bacterial Testing of Platelet Components: 2008 Update

Bacterial Testing of Platelet
Components: 2008 Update
William B. Lockwood, PhD, MD
Clinical Professor
Department of Pathology & Laboratory Medicine
University of Louisville
Director, Transfusion Services & Tissue/Bone Bank
University of Louisville Hospital
Director, Transfusion Services, Tissue/Bone Bank & Coagulation Lab
Norton Hospital & Kosair Children’s Hospital
Louisville, Kentucky
[email protected]
502-852-5857
Objectives
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Describe the current FDA regulations
and accreditation agency standards for
testing platelet collections for bacterial
contamination
Compare & contrast 3 methods of
bacterial detection in platelet collections
Describe a validation plan for use of
non-FDA approved bacterial detection
methods
PACE Program Number: 362-001-09
SCACM is approved as a provider of
continuing education programs in the
clinical laboratory sciences by the
ASCLS P.A.C.E. Program.
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Whole Blood Collection Set
ca. 1900
Whole Blood Glass Bottle
ca. 1940
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TODAY!
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Bacterial Contamination of
Blood Components
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Known to occur since 1900 when using
vented glass bottles for whole blood
collection
Continued with advent of plastic blood
containers in 1950s
Platelets stored refrigerated had less of a
contamination problem, but were not
efficacious on transfusion*!
*Murphy S, Gardner FH. NEJM 1969;380:1094-98
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Bacterial Contamination of
Blood Components
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Additional studies on platelet storage medium,
storage temperature, pH, type of agitation, volume
of platelets in container and size of container,
plastic used for platelet bag from 1960s-1980s
Changes included:
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Gentle horizontal agitation
Room temperature (RT) storage- 3 days to 5 days (1981); 5
days to 7 days (1984)
Reduction from 7 day to 5 day RT storage (1986)*
*Shiffer CA et al. Blood 1986;67:1591-94
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Bacterial Contamination of
Blood Components
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US Food & Drug Administration (FDA) via
blood regulatory department Center for
Biologics Evaluation & Research (CBER)
held industry workshops in 1995 & 1999 to
discuss platelet bacterial contamination
Literature continues to report increasing
platelet bacterial contamination (BaCon
study of American Red Cross Blood
Services)
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Bacterial Contamination of
Blood Components
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Bacterial contaminated blood components
cause of >10% (77/694) of recipient
fatalities reported to FDA from 1985-1999*
1st multicenter study of bacterial
contamination of blood components
published by American Red Cross**
*Workshop on bacterial contamination of platelets.Bethesda: FDA, Center for Biologics Evaluation
and Research,September 24, 1999. Available from http://www.fda.gov/cber/minutes/workshopmin.htm.
**Kuehnert MJ, et al. Transfusion-transmitted bacterial infection in the United States,
1998 through 2000. Transfusion 2001;41:1493-99
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Bacterial Contamination of
Blood Components
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BaCon study
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23,711,169 RBCs
1,804,725 single donor
platelets (SDP)
1,033,671 Pooled platelets
(WBDP)
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Bacterial Contamination of
Blood Components
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Transfusion-transmitted bacteremia rates:
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SDP= 1:100,000
WBDP= 1:100,000
RBC= 1:8,000,000
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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 Ab
1985
HIV PCR
2000
Hepatitis C
HCV Ab
1990
HCV PCR
2000
HTLV 1/2
HTLV 1/2
1989
West Nile Virus
WNV NAT
2003
Bacteria - Platelets
Bacteria - Red Cells
Source:
Ilert WE, et al. Transfusion Medicine 1995, 5:57-61
Brecher et al. Clinical Microbiology Reviews, 2005,
195-204
No Practical
Screening Tests
Currently
Available
1:2,000
1:20,000
1:100,000
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1:1,000,000
Incidence rate
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Bacterial Contamination of
Blood Components
Sources of Bacterial Contamination
Skin Surface
Contamination
Phlebotomy Core
Donor Bacteremia
Containers and
Disposables
Environment
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Bacterial Contamination of
Blood Components
ORGANISMS INVOLVED
Exogenous
Normal skin
Staphylococcus epidermidis
Staphylococcus aureus
Diphteroids spp
Micrococcus spp
Pseudomonas spp
Bacillus cereus
Propionibacterium acnes
Flavobacterium spp
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Bacterial Contamination of
Blood Components
ORGANISMS INVOLVED
Endogenous
Teeth
Osteomyelitis
Staphylococcus
S. cholera suis
Staphylococcus spp
Streptococcus viridans
Serratia liquefaciens
Yersinia enterocolitica
Intestines Salmonella spp
Campylobacter spp
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Bacterial Contamination of
Blood Components
Prevention and Detection Options
• Donor screening – not feasible except for arm
screening. Can’t detect asymptomatic bacteremic
donors
• Arm Preparation-Limited effectiveness of arm scrub
• Better phlebotomy methods and initial blood
diversion
• Bacterial contamination testing offers best
confirmatory option
• Pathogen Inactivation--INTERCEPT
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FDA Regulations
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FDA is silent about Whole-blood Derived Platelets
(WBDP) bacterial testing guidelines [FDA
guidelines are recommendations but are not
legally binding until published as a Code of
Federal Regulations (CFR)]
HOWEVER- establishments requesting a
Biological License Application (BLA) or
Supplement (BLS) must follow the Title 21 CFR
Parts 200 and 600
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FDA Regulations
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Part 200 relates to Good Manufacturing Practice,
current (cGMP) of biologics
Part 600 relates to blood components - safety,
quality, purity, potency, identification
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Part 640- platelets
Subsections itemize various manufacturing regulations, source, donor suitability, collections, QC
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QC elements- platelet count of donor, donor weight, pH at outdate
(≥6.2), sterility testing, etc
Manufacturer’s requirements for automated plateletpheresis collection
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FDA Regulations
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Revised regulation effective in 2008 (1st was in 1988,
draft in 2005!!)
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“Revisions to the Requirements Applicable to Blood, Blood
Components and Source Plasma”*
Pertains to those establishments that want to license their
blood component
THEREFORE, FDA regulates platelet
“manufacturing” through the BLA/BLS!!
Also, approval of manufacturer’s devices through
Pre-Market Approval 510(k) application regulates
equipment used in all phases of blood component
production
*Federal Register: August 16, 2007 (Volume 72, Number 158)
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CAP Accreditation Checklist
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1st accreditation group to require bacterial
testing of platelets!
CAP Checklist - 2002
Phase I deficiency
Revised 12/29/2004 to Phase II deficiency
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CAP Accreditation Checklist 2008
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TRM.44955
Phase II
N/A YES NO
Does the laboratory have a validated system to
detect the presence of bacteria in platelet
components?
NOTE: For random donor platelets, any of the following testing
methods satisfy this checklist question: detection of decreased pH
or glucose by analytic instrument or dipstick; gram stain; acridine
orange stain. Though of low sensitivity, these methods may detect
units that are heavily contaminated by bacteria. Culture or
FDA-approved commercial detection systems have greater
sensitivity. The swirling technique is not recommended because of
its very low sensitivity.
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CAP Accreditation Checklist 2008
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Two commercial systems have been cleared by the FDA for inprocess quality control culturing of platelet units; one detects the
growth of bacteria by their generation of CO2, and the other
detects growth by their consumption of O2. Another system has
been cleared for bacterial detection by fluorescent staining. If this
testing is performed by the supplier of platelet components, the
transfusion service can satisfy this checklist requirement by having
an agreement with the supplier to notify the transfusion service if
any units suspected of containing bacteria have been transferred
to the transfusion service.
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AABB Accreditation
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AABB Standards for Blood Banks &
Transfusion Services, 23rd ed, Effective May
1, 2004
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Implement standard 5.1.5.1:
The blood bank or transfusion service shall have methods
to limit and detect bacterial contamination in all platelet
components. Standard 5.6.2 applies
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Standard 5.6.2- Protection Against Contamination:
The venipuncture site shall be prepared so as to minimize
risk of bacterial contamination. Green soap shall not be
used.
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AABB Accreditation con’t
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Standard 5.1.5.1.1 (Standard 5.1.5.2, 25th ed, 2008)
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When a true-positive result is obtained and an appropriate
specimen is available, additional testing to identify the
organism shall be performed. Additional testing and followup shall be defined. Standards 5.2.2 and 7.1 to 7.1.4 apply.
Standard 5.2.2: Donor Notification of Abnormal
Findings and Test Results (Standard 5.2.3, 25th ed,
2008)
Standards 7.1-7.1.4: Nonconformances
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AABB Accreditation con’t
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Standard 5.6.6.1 (25th ed, 2008)
Blood collection containers with draw line (inlet)
diversion pouches shall be used for any collection of
platelets, including whole blood from which platelets
are made.
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Bacterial Contamination of
Blood Components
Bacterial detection tests
•3 devices are cleared for quality control monitoring of
platelet collection process of leukoreduced platelets
BioMeriuex BacT/ALERT®
Pall eBDS
hemoSystems Scansystem™
•Other non approved and non validated methods are
also being used to meet the AABB standard for bacterial
detection
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Bacterial Contamination of
Blood Components
FDA concerns with bacterial detection as
currently applied (Vostal JG, Jan. 28, 2005 CBER workshop)
• Test performance characteristics unknown
• Use of non-validated tests (glucose and pH by dipstick,
swirling)
• Non-standardized methodology even with culture-based
devices
• Potential for excessive false positives or negatives
• Less reliable methods are used on whole blood derived
platelets creating a two tiered safety system for apheresis and
whole blood derived platelets
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Bacterial Contamination of
Blood Components
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Manual tests- validation required for pH &
glucose
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pH
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Glucose
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Swirling
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Devices approved for QC detection of platelet
bacterial contamination- Pall BDS
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Pall eBDS
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Sample set/Oxygen
Analyzer
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Sterile weld platelet component
to set
Fill pouch with ~3 mL of
product
Disconnect sample pouch
from set and incubate at 35°C
for 24-30 hrs
Measure the O2 content in the
air above the plasma sample
with insertion of analyzer
probe into pouch
LED display will read PASS or
FAIL
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Pall eBDS
Pall Medical eBDS Brochure, 2004
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BioMeriuex BacT/ALERT®
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Colorimetric
technology/Sensor
Culture bottles
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CO2 release causes sensor
bottle to turn yellow
Instrument measures &
detects color change,
analyzes data to
determine positivity, alerts
when positive culture
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hemoSystems Scansystem™
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Scansystem Platelet
Kit/Scansystem Analyzer
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After sample processing in
platelet kit, bacteria are stained
with a green fluorescence and
retained on the surface of a
dedicated membrane (platelets
are aggregated and lysed)
Membrane is inserted in
analyzer and scanned by an
Argon laser
Each fluorescent spot on the
membrane will be detected and
analyzed
Analyze 3 platelet components
(SDP, WBDP)
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hemoSystems Scansystem™
Scansystem™ Brochure accessed at www.hemosystem.com
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Preparing Platelets for Culture
Sterile Connection Device
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Connecting Platelets to Syringe Set
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Tubing from syringe set
Tubing from platelet bag
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Aliquoting Platelet Sample
from Transfusion Bag
(Suspected Transfusion Reaction)
Syringe set
Transfusion set
SDP
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Aliquoting Platelet Sample
for Bacterial Detection Testing
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Aliquoting Platelet Sample
for Bacterial Detection Testing
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Inoculating Culture Bottle
(pediatric size)
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Sterile Connection Port
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Bacterial Contamination of
Blood Components-2008
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ARC Study of bacterial
contamination before &
after implementation of
sample diversion pouch
(Pall Acrodose with eBDS)
Prestorage pooling of
WBDP with culture testing
Jan 2003-December 2006
Benjamin RJ, et al. Transfusion 2008;48:2348-55
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Bacterial Contamination of
Blood Components-2008
Benjamin RJ, et al. Transfusion 2008;48:2348-55
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Bacterial Contamination of
Blood Components-2008
Benjamin RJ, et al. Transfusion 2008;48:2348-55
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Bacterial Contamination of
Blood Components-2008
PSP=prestorage pooled WBDP
Benjamin RJ, et al. Transfusion 2008;48:2348-55
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Blood Transfusion SafetyFDA Blood Transfusion Fatality Report
http://www.fda.gov/Cber/
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Why test at point of issue ?
Wide variation in duration
of Lag Phase
Wide variation in rate
of log Phase Growth
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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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New FDA Approved Device for
Transfusion Services
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The Abbott 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
(SDP)
~30 minute TAT
SDP must have undergone
QC culture by blood
supplier!!
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New Device for Transfusion Services
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New Device for Transfusion Services
Verax rapid Platelet PGD® Test
Single-use disposable test
1. Rapid ~ 25 minutes (3 min hands on)
2. Positives typically < 10 minutes
3. Sensitivity ~ 103 CFU/ mL
4. Specificity > 99.7%
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Pan Genera Rapid Test Format
Procedural
Control
Procedural
Control
Test
Features
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-Positive
Read Window
Gram-Negative
Read Window
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Methods Comparison
Verax PGD
BacT/ALERT
Pall eBDS
Technology
Conserved Bacterial Ag
Immunoassay
Culture
CO2 Measure
Aerobic Culture
O2 measure
Sample Volume
500 uL
4-20 mLs
3-5 mLs.
Time to Result
10 – 30 min (positives
typically within 10
minutes)
24 – 96 hours
24 – 30 hours
Detect Aerobic and
Anaerobic bacteria?
Yes
Yes, but time varies
Misses Anaerobes
Clinical Specificity
99.7%
99.2-99.8%
~ 99%
Source: Abbott, Biomerieux and Pall Medical web site.
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Validation of
Bacterial Detection Methods
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pH & glucose tests are analytically insensitive (Yomatovian
R, Brecher ME. Transfusion 2005;45:647-8)
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Validation required
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Variable plastic bags
Variable anticoagulants
Variable handling
Gram stain insensitive unless 106 CFU/mL
Facilities may not have FDA approved equipment
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Validate for QC use
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Sensitivity
Types of organisms
Growth time
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Snyder JW, et al. BACTEC detection of bacteria in platelet pools. ASM 05-GM-A-4146; 2005
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SUMMARY
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Bacterial contamination of
blood components
continues to pose a threat
to transfusion recipients
Progress to prevent
adverse reaction to blood
transfusions due to
bacterial contamination
continues to be seen
Microbiologists now play a
major role in this
progress!!
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UNIVERSITY OF LOUISVILLE MEDICAL CENTER, LOUISVILLE, KENTUCKY
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