BD-Plts

1. Bacterial detection of PlateletsBacterial detection of Platelets
Dr. RAFIQ AHMADDr. RAFIQ AHMAD
Regional Laboratory & Blood BankRegional Laboratory & Blood Bank
Dammam, K.S.A.Dammam, K.S.A.

2. 2
Bacterial Contamination ofBacterial Contamination of
Blood ComponentsBlood Components
 Known to occur since 1900 when using ventedKnown to occur since 1900 when using vented
glass bottles for whole blood collectionglass bottles for whole blood collection
 Continued with advent of plastic bloodContinued with advent of plastic blood
containers in 1950scontainers in 1950s
 Platelets stored refrigerated had less of aPlatelets stored refrigerated had less of a
contamination problem, but were notcontamination problem, but were not
efficacious on transfusion*!efficacious on transfusion*!

3. 3
Whole Blood Collection Set: 1900Whole Blood Collection Set: 1900 Whole Blood Glass Bottle: 1940Whole Blood Glass Bottle: 1940
Today!Today!

4. 4
Bacterial Contamination ofBacterial Contamination of
Blood ComponentsBlood Components
 Bacterial contaminated blood components causeBacterial contaminated blood components cause
of >10% (77/694) of recipient fatalities reportedof >10% (77/694) of recipient fatalities reported
to FDA from 1985-1999*to FDA from 1985-1999*
 11stst
multicenter study of bacterial contaminationmulticenter study of bacterial contamination
of blood components published by Americanof blood components published by American
Red Cross**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/workshop-
min.htm.
**Kuehnert MJ, et al. Transfusion-transmitted bacterial infection in the United States,
1998 through 2000. Transfusion 2001;41:1493-99

5. 5
Bacterial Contamination ofBacterial Contamination of
PlateletsPlatelets
 US Food & Drug Administration (FDA) viaUS Food & Drug Administration (FDA) via
blood regulatory department Center forblood regulatory department Center for
Biologics Evaluation & Research (CBER) heldBiologics Evaluation & Research (CBER) held
industry workshops in 1995 & 1999 to discussindustry workshops in 1995 & 1999 to discuss
platelet bacterial contaminationplatelet bacterial contamination
 Literature continues to report increasing plateletLiterature continues to report increasing platelet
bacterial contamination (BaCon study ofbacterial contamination (BaCon study of
American Red Cross Blood Services)American Red Cross Blood Services)

6. 6
Bacterial Contamination ofBacterial Contamination of
Blood ComponentsBlood Components
 BaCon studyBaCon study
 23,711,169 RBCs23,711,169 RBCs
 1,804,725 single donor1,804,725 single donor
platelets (SDP)platelets (SDP)
 1,033,671 Pooled platelets1,033,671 Pooled platelets
(WBDP)(WBDP)

7. 7
Bacterial Contamination ofBacterial Contamination of
PlateletsPlatelets
 Additional studies on platelet storage medium, storageAdditional studies on platelet storage medium, storage
temperature, pH, type of agitation, volume of plateletstemperature, pH, type of agitation, volume of platelets
in container and size of container, plastic used forin container and size of container, plastic used for
platelet bag from 1960s-1980splatelet bag from 1960s-1980s
 Changes included:Changes included:
 Gentle horizontal agitationGentle horizontal agitation
 Room temperature (RT) 20-22 degree CRoom temperature (RT) 20-22 degree C
 Storage- 3 days to 5 days (1981); 5 days to 7 days (1984)Storage- 3 days to 5 days (1981); 5 days to 7 days (1984)
Reduction from 7 day to 5 day RT storage (1986)*Reduction from 7 day to 5 day RT storage (1986)*

8. 8
Bacterial Contamination ofBacterial Contamination of
PlateletsPlatelets
 Transfusion-transmitted bacteremia rates:Transfusion-transmitted bacteremia rates:
 SDP= 1:100,000SDP= 1:100,000
 WBDP= 1:100,000WBDP= 1:100,000
 RBC= 1:8,000,000RBC= 1:8,000,000

9. 9
Bacterial Contamination ofBacterial Contamination of
PlateletsPlatelets
Sources of Bacterial ContaminationSources of Bacterial Contamination
Skin Surface Contamination
Phlebotomy Core
Donor Bacteremia
Containers and Disposables
Environment

10. Bacterial Contamination ofBacterial Contamination of
PlateletsPlatelets
10
Organisms involvedOrganisms involved
ExogenousExogenous Normal skinNormal skin
Staphylococcus epidermidisStaphylococcus epidermidis
Staphylococcus aureusStaphylococcus aureus
Diphteroids sppDiphteroids spp
Micrococcus sppPseudomonas sppMicrococcus sppPseudomonas spp
Bacillus cereusBacillus cereus
Propionibacterium acnesPropionibacterium acnes
Flavobacterium sppFlavobacterium spp

11. 11
Bacterial Contamination ofBacterial Contamination of
PlateletsPlatelets
Prevention and Detection OptionsPrevention and Detection Options
 Donor screening – not feasible except for arm screening.Donor screening – not feasible except for arm screening.
Can’t detect asymptomatic bacteremic donorsCan’t detect asymptomatic bacteremic donors
 Arm Preparation-Limited effectiveness of arm scrubArm Preparation-Limited effectiveness of arm scrub
 Better phlebotomy methods and initial blood diversionBetter phlebotomy methods and initial blood diversion
 Bacterial contamination testing offers best confirmatoryBacterial contamination testing offers best confirmatory
optionoption
• Pathogen Inactivation!!!Pathogen Inactivation!!!

12. 12
CBAHI RegulationsCBAHI Regulations
 To develop policy & procedures to limit bacterialTo develop policy & procedures to limit bacterial
contamination of platelets and investigate any positivecontamination of platelets and investigate any positive
casecase
 Relates to current good manufacturing practice (cGMP)Relates to current good manufacturing practice (cGMP)
of blood components - safety, quality, purity, potency,of blood components - safety, quality, purity, potency,
identificationidentification
 CBAHI- 07465: plateletsCBAHI- 07465: platelets
 Subsections itemize various manufacturing regulations, -Subsections itemize various manufacturing regulations, -
source, donor suitability, collections, QCsource, donor suitability, collections, QC
 QC elements- platelet count of donor, donor weight,QC elements- platelet count of donor, donor weight, pH at outdatepH at outdate
(≥6.2),(≥6.2), sterility testingsterility testing, etc, etc
 Manufacturer’s requirements for automated plateletpheresisManufacturer’s requirements for automated plateletpheresis
collectioncollection

13. 13
CAP Accreditation ChecklistCAP Accreditation Checklist
 11stst
accreditation group to require bacterial testingaccreditation group to require bacterial testing
of platelets!of platelets!
 CAP Checklist - 2016CAP Checklist - 2016
 Phase I deficiencyPhase I deficiency
 Revised 08/17/2016 toRevised 08/17/2016 to Phase IIPhase II deficiencydeficiency

14. 14
CAP Accreditation Checklist 2008CAP Accreditation Checklist 2008
 TRM.44955 Phase IIPhase 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.

15. 15
CAP Accreditation Checklist 2016CAP Accreditation Checklist 2016
 In US two commercial systems have been cleared by the FDA for
in-process 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.

16. 16
AABB AccreditationAABB Accreditation
 AABB Standards for Blood Banks &AABB Standards for Blood Banks &
Transfusion Services, 23Transfusion Services, 23rdrd
ed, Effective May 1,ed, Effective May 1,
2004 reviewed in AABB public conference on2004 reviewed in AABB public conference on
23/08/201223/08/2012
 Implement standard 5.1.5.1:Implement standard 5.1.5.1:
The blood bank or transfusion service shall have methodsThe blood bank or transfusion service shall have methods
to limit and detect bacterial contamination in all plateletto limit and detect bacterial contamination in all platelet
components. Standard 5.6.2 appliescomponents. Standard 5.6.2 applies
 Standard 5.6.2- Protection Against Contamination:Standard 5.6.2- Protection Against Contamination:
The venipuncture site shall be prepared so as to minimizeThe venipuncture site shall be prepared so as to minimize
risk of bacterial contamination. Green soap shall not berisk of bacterial contamination. Green soap shall not be
used.used.

17. 17
AABB Accreditation con’tAABB Accreditation con’t
 Standard 5.1.5.1.1 (Standard 5.1.5.2, 25Standard 5.1.5.1.1 (Standard 5.1.5.2, 25thth
ed, 2008)ed, 2008)
 When a true-positive result is obtained and an appropriateWhen a true-positive result is obtained and an appropriate
specimen is available, additional testing to identify thespecimen is available, additional testing to identify the
organism shall be performed. Additional testing and follow-organism shall be performed. Additional testing and follow-
up shall be defined. Standards 5.2.2 and 7.1 to 7.1.4 apply.up shall be defined. Standards 5.2.2 and 7.1 to 7.1.4 apply.
 Standard 5.2.2: Donor Notification of AbnormalStandard 5.2.2: Donor Notification of Abnormal
Findings and Test Results (Standard 5.2.3, 25Findings and Test Results (Standard 5.2.3, 25thth
ed, 2008)ed, 2008)
 Standards 7.1-7.1.4: Non-conformancesStandards 7.1-7.1.4: Non-conformances

18. 18
AABB Accreditation con’tAABB Accreditation con’t
 Standard 5.6.6.1 (35Standard 5.6.6.1 (35thth
ed, 2017)ed, 2017)
Blood collection containers with draw line (inlet)Blood collection containers with draw line (inlet)
diversion pouches shall be used for any collection ofdiversion pouches shall be used for any collection of
platelets, including whole blood from which plateletsplatelets, including whole blood from which platelets
are made.are made.

19. 19
Bacterial Contamination ofBacterial Contamination of
PlateletsPlatelets
 Manual tests- validation required for pH glucoseManual tests- validation required for pH glucose
& Swirling& Swirling
 pHpH
 GlucoseGlucose
 SwirlingSwirling

20. 20

21. 21
Bacterial Contamination ofBacterial Contamination of
Blood ComponentsBlood Components
Bacterial detection tests
 3 devices are cleared for quality control monitoring of
platelet collection process of leukoreduced platelets
BioMeriuex BacT/ALERT®BioMeriuex BacT/ALERT®
Pall eBDSPall eBDS
HemoSystems Scansystem™HemoSystems Scansystem™
 Other non approved and non validated methods are
also being used to meet the AABB standard for
bacterial detection

22. 22
BioMeriuex BacT/ALERT®BioMeriuex BacT/ALERT®
 ColorimetricColorimetric
technology/Sensortechnology/Sensor
Culture bottlesCulture bottles
 COCO22 release causes sensorrelease causes sensor
bottle to turn yellowbottle to turn yellow
 Instrument measures &Instrument measures &
detects color change,detects color change,
analyzes data to determineanalyzes data to determine
positivity, alerts whenpositivity, alerts when
positive culturepositive culture

23. 23
Devices approved for QC detection of plateletDevices approved for QC detection of platelet
bacterial contamination-bacterial contamination- Pall BDSPall BDS

24. 24
Pall eBDSPall eBDS
 Sample set/Oxygen AnalyzerSample set/Oxygen Analyzer
 Sterile weld platelet component to setSterile weld platelet component to set
 Fill pouch with ~3 mL of productFill pouch with ~3 mL of product
 Disconnect sample pouch from set and incubateDisconnect sample pouch from set and incubate
at 35°C for 24-30 hrsat 35°C for 24-30 hrs
 Measure the OMeasure the O22 content in the air above thecontent in the air above the
plasma sample with insertion of analyzer probeplasma sample with insertion of analyzer probe
into pouchinto pouch
 LED display will readLED display will read PASSPASS oror FAILFAIL

25. 25
HemoSystems Scansystem™HemoSystems Scansystem™
 Scansystem PlateletScansystem Platelet
Kit/Scansystem AnalyzerKit/Scansystem Analyzer
 After sample processing inAfter sample processing in
platelet kit, bacteria are stainedplatelet kit, bacteria are stained
with a green fluorescence andwith a green fluorescence and
retained on the surface of aretained on the surface of a
dedicated membrane (plateletsdedicated membrane (platelets
are aggregated and lysed)are aggregated and lysed)
 Membrane is inserted inMembrane is inserted in
analyzer and scanned by ananalyzer and scanned by an
Argon laserArgon laser
 Each fluorescent spot on theEach fluorescent spot on the
membrane will be detected andmembrane will be detected and
analyzedanalyzed
 Analyze 3 platelet componentsAnalyze 3 platelet components
(SDP, WBDP)(SDP, WBDP)

26. 26
Preparing Platelets for CulturePreparing Platelets for Culture

27. 27
Aliquoting Platelet SampleAliquoting Platelet Sample
for Bacterial Detection Testingfor Bacterial Detection Testing

28. 28
Sterile Connection PortSterile Connection Port

29. 29
FDA Approved Device for TransfusionFDA Approved Device for Transfusion
ServicesServices
 The Abbott Verax PlateletThe Abbott Verax Platelet
PGDPGD®® TestTest
 A rapid, qualitativeA rapid, qualitative
immunoassay for theimmunoassay for the
detection of Aerobic anddetection of Aerobic and
Anaerobic; Gram-positiveAnaerobic; Gram-positive
and Gram-negative bacteriaand Gram-negative bacteria
in leukocyte reducedin leukocyte reduced
apheresis platelets (SDP)apheresis platelets (SDP)
 ~30 minute TAT~30 minute TAT
 SDP must have undergoneSDP must have undergone
QC culture by bloodQC culture by blood
supplier!!supplier!!

30. 30
New Device for Transfusion ServicesNew Device for Transfusion Services

31. 31
Device for Transfusion ServicesDevice for Transfusion Services
1. Rapid ~ 25 minutes (3 min hands on)
3. Sensitivity ~ 103
CFU/ mL
Single-use disposable testSingle-use disposable test
Verax rapid Platelet PGD®
Test
2. Positives typically < 10 minutes
4. Specificity > 99.7%

32. 32
Methods ComparisonMethods Comparison
Verax PGDVerax PGD BacT/ALERTBacT/ALERT Pall eBDSPall eBDS
TechnologyTechnology
Conserved Bacterial AgConserved Bacterial Ag
ImmunoassayImmunoassay
CultureCulture
CO2 MeasureCO2 Measure
Aerobic CultureAerobic Culture
O2 measureO2 measure
Sample VolumeSample Volume 500 uL500 uL 4-20 mLs4-20 mLs 3-5 mLs.3-5 mLs.
Time to ResultTime to Result
10 – 30 min (positives10 – 30 min (positives
typically within 10typically within 10
minutes)minutes)
24 – 96 hours24 – 96 hours 24 – 30 hours24 – 30 hours
Detect Aerobic andDetect Aerobic and
Anaerobic bacteria?Anaerobic bacteria?
YesYes Yes, but time variesYes, but time varies Misses AnaerobesMisses Anaerobes
Clinical SpecificityClinical Specificity 99.7%99.7% 99.2-99.8%99.2-99.8% ~ 99%~ 99%
Source: Abbott, Biomerieux and Pall Medical web site.

33. 33
ConclusionConclusion
 pH & glucose tests are analytically insensitive (Yomatovian R,pH & glucose tests are analytically insensitive (Yomatovian R,
Brecher ME. Transfusion 2005;45:647-8)Brecher ME. Transfusion 2005;45:647-8)
 Validation requiredValidation required
 Variable plastic bagsVariable plastic bags
 Variable anticoagulantsVariable anticoagulants
 Variable handlingVariable handling
 Gram stain insensitive unless 10Gram stain insensitive unless 1066
CFU/mLCFU/mL
 Facilities may not have FDA approved equipmentFacilities may not have FDA approved equipment
 Validate for QC useValidate for QC use
 SensitivitySensitivity
 Types of organismsTypes of organisms
 Growth timeGrowth time

34. 34
ConclusionConclusion
 Bacterial contamination of blood componentsBacterial contamination of blood components
continues to pose a threat to transfusioncontinues to pose a threat to transfusion
recipientsrecipients
 Progress to prevent adverse reaction to bloodProgress to prevent adverse reaction to blood
transfusions due to bacterial contaminationtransfusions due to bacterial contamination
continues to be seencontinues to be seen

35. 35
Thank youThank you
The End!!!The End!!!