Bacterial Testing of Platelet Components: 2008 Update William B. Lockwood, PhD, MD Clinical...

Bacterial Testing of Bacterial Testing of Platelet Components: Platelet Components:

2008 Update2008 Update

William B. Lockwood, PhD, MDWilliam B. Lockwood, PhD, MDClinical ProfessorClinical Professor

Department of Pathology & Laboratory MedicineDepartment of Pathology & Laboratory MedicineUniversity of LouisvilleUniversity of Louisville

Director, Transfusion Services & Tissue/Bone BankDirector, Transfusion Services & Tissue/Bone BankUniversity of Louisville HospitalUniversity of Louisville Hospital

Director, Transfusion Services, Tissue/Bone Bank & Director, Transfusion Services, Tissue/Bone Bank & Coagulation LabCoagulation Lab

Norton Hospital & Kosair Children’s HospitalNorton Hospital & Kosair Children’s HospitalLouisville, KentuckyLouisville, Kentucky

[email protected]@gwise.louisville.edu502-852-5857502-852-5857

2SCACM Conference Jan. 20, 2009

ObjectivesObjectives Describe the current FDA Describe the current FDA

regulations and accreditation regulations and accreditation agency standards for testing agency standards for testing platelet collections for bacterial platelet collections for bacterial contaminationcontamination

Compare & contrast 3 methods Compare & contrast 3 methods of bacterial detection in platelet of bacterial detection in platelet collectionscollections

Describe a validation plan for Describe a validation plan for use of non-FDA approved use of non-FDA approved bacterial detection methodsbacterial detection methods

PACE Program Number: PACE Program Number: 362-001-362-001-

0909

SCACMSCACM is approved as a provider is approved as a provider of continuing education of continuing education programs in the clinical programs in the clinical laboratory sciences by the laboratory sciences by the ASCLS P.A.C.E. Program.ASCLS P.A.C.E. Program.


Whole Blood Collection SetWhole Blood Collection Set ca. 1900ca. 1900

Whole Blood Glass BottleWhole Blood Glass Bottle ca. 1940ca. 1940

TODAY!TODAY!


Bacterial Contamination of Bacterial Contamination of Blood ComponentsBlood Components

Known to occur since 1900 when Known to occur since 1900 when using vented glass bottles for using vented glass bottles for whole blood collectionwhole blood collection

Continued with advent of plastic Continued with advent of plastic blood containers in 1950sblood containers in 1950s

Platelets stored refrigerated had Platelets stored refrigerated had less of a contamination problem, less of a contamination problem, but were not efficacious on but were not efficacious on transfusion*!transfusion*!*Murphy S, Gardner FH. NEJM 1969;380:1094-98



Additional studies on platelet storage Additional studies on platelet storage medium, storage temperature, pH, type medium, storage temperature, pH, type of agitation, volume of platelets in of agitation, volume of platelets in container and size of container, plastic container and size of container, plastic used for platelet bag from 1960s-1980sused for platelet bag from 1960s-1980s

Changes included:Changes included: Gentle horizontal agitation Gentle horizontal agitation Room temperature (RT) storage- 3 days to 5 Room temperature (RT) storage- 3 days to 5

days (1981); 5 days to 7 days (1984)days (1981); 5 days to 7 days (1984) Reduction from 7 day to 5 day RT storage Reduction from 7 day to 5 day RT storage

(1986)*(1986)**Shiffer CA et al. Blood 1986;67:1591-94



US Food & Drug Administration US Food & Drug Administration (FDA) via blood regulatory (FDA) via blood regulatory department Center for Biologics department Center for Biologics Evaluation & Research (CBER) held Evaluation & Research (CBER) held industry workshops in 1995 & 1999 industry workshops in 1995 & 1999 to discuss platelet bacterial to discuss platelet bacterial contaminationcontamination

Literature continues to report Literature continues to report increasing platelet bacterial increasing platelet bacterial contamination (BaCon study of contamination (BaCon study of American Red Cross Blood Services)American Red Cross Blood Services)



Bacterial contaminated blood Bacterial contaminated blood components cause of >10% components cause of >10% (77/694) of recipient fatalities (77/694) of recipient fatalities reported to FDA from 1985-1999*reported to FDA from 1985-1999*

11stst multicenter study of bacterial multicenter study of bacterial contamination of blood contamination of blood components published by components published by American Red Cross** 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/workshop-min.htm.

**Kuehnert MJ, et al. Transfusion-transmitted bacterial infection in the United States, 1998 through 2000. Transfusion 2001;41:1493-99



BaCon studyBaCon study 23,711,169 RBCs23,711,169 RBCs 1,804,725 single 1,804,725 single

donor platelets donor platelets (SDP)(SDP)

1,033,671 Pooled 1,033,671 Pooled platelets (WBDP)platelets (WBDP)



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


1:100,000 1:1,000,000 Incidence rate

Bacterial vs. Virus: Infectious risk in the blood supply

HIV

Hepatitis C

HTLV 1/2

Bacteria - Platelets

Bacteria - Red Cells

West Nile Virus

Hepatitis B

HIV Ab1985

P 241996

HIV PCR2000

HCV Ab1990

HCV PCR2000

HTLV 1/21989

WNV NAT2003

HBsAg1971

Anti HBsAg 1987

1:2,000

1:20,000

No PracticalScreening Tests

CurrentlyAvailable

No PracticalScreening Tests

CurrentlyAvailable

Source: Ilert WE, et al. Transfusion Medicine 1995, 5:57-61 Brecher et al. Clinical Microbiology Reviews, 2005, 195-204



Sources of Bacterial ContaminationSources of Bacterial Contamination

Skin Surface ContaminationPhlebotomy CoreDonor BacteremiaContainers and DisposablesEnvironment


Bacterial Contamination of Bacterial Contamination of Blood ComponentsBlood ComponentsORGANISMS INVOLVEDORGANISMS INVOLVED

ExogenousExogenous

Staphylococcus Staphylococcus epidermidisepidermidisStaphylococcus aureusStaphylococcus aureusDiphteroids sppDiphteroids sppMicrococcus sppMicrococcus sppPseudomonas sppPseudomonas sppBacillus cereusBacillus cereusPropionibacterium Propionibacterium acnesacnesFlavobacterium sppFlavobacterium spp

Normal skinNormal skin


Bacterial Contamination of Bacterial Contamination of Blood ComponentsBlood ComponentsORGANISMS INVOLVEDORGANISMS INVOLVED

EndogenousEndogenous

OsteomyelitisOsteomyelitisStaphylococcusStaphylococcusS. cholera suisS. cholera suis

Staphylococcus Staphylococcus sppspp TeethTeeth Streptococcus Streptococcus viridansviridans

SerratiaSerratialiquefaciensliquefaciens

YersiniaYersiniaenterocoliticaenterocolitica IntestinesIntestinesSalmonella sppSalmonella spp

Campylobacter Campylobacter sppspp



Prevention and Detection OptionsPrevention and Detection Options• Donor screening – not feasible except Donor screening – not feasible except for arm screening. Can’t detect for arm screening. Can’t detect asymptomatic bacteremic donorsasymptomatic bacteremic donors• Arm Preparation-Limited effectiveness Arm Preparation-Limited effectiveness of arm scrubof arm scrub• Better phlebotomy methods and initial Better phlebotomy methods and initial blood diversionblood diversion• Bacterial contamination testing offers Bacterial contamination testing offers best confirmatory optionbest confirmatory option

• Pathogen Inactivation--INTERCEPTPathogen Inactivation--INTERCEPT


FDA RegulationsFDA Regulations

FDA is silent about Whole-blood FDA is silent about Whole-blood Derived Platelets (WBDP) bacterial Derived Platelets (WBDP) bacterial testing guidelines [FDA guidelines are testing guidelines [FDA guidelines are recommendations but are not legally recommendations but are not legally binding until published as a Code of binding until published as a Code of Federal Regulations (CFR)]Federal Regulations (CFR)]

HOWEVER- establishments requesting HOWEVER- establishments requesting a Biological License Application (BLA) a Biological License Application (BLA) or Supplement (BLS) must follow the or Supplement (BLS) must follow the Title 21 CFR Parts 200 and 600Title 21 CFR Parts 200 and 600


FDA RegulationsFDA Regulations

Part 200 relates to Good Manufacturing Part 200 relates to Good Manufacturing Practice, current (cGMP) of biologicsPractice, current (cGMP) of biologics

Part 600 relates to blood components - Part 600 relates to blood components - safety, quality, purity, potency, safety, quality, purity, potency, identificationidentification Part 640- plateletsPart 640- platelets Subsections itemize various manufacturing Subsections itemize various manufacturing

regulations, - source, donor suitability, collections, regulations, - source, donor suitability, collections, QCQC

QC elements- platelet count of donor, donor weight, QC elements- platelet count of donor, donor weight, pH at pH at outdate (≥6.2),outdate (≥6.2), sterility testingsterility testing, etc, etc

Manufacturer’s requirements for automated plateletpheresis Manufacturer’s requirements for automated plateletpheresis collectioncollection


FDA RegulationsFDA Regulations Revised regulation effective in 2008 (1Revised regulation effective in 2008 (1stst

was in 1988, draft in 2005!!)was in 1988, draft in 2005!!) “ “Revisions to the Requirements Applicable to Revisions to the Requirements Applicable to

Blood, Blood Components and Source Plasma”*Blood, Blood Components and Source Plasma”* Pertains to those establishments that want to Pertains to those establishments that want to

license their blood componentlicense their blood component THEREFORE, FDA regulates platelet THEREFORE, FDA regulates platelet

“manufacturing” through the BLA/BLS!!“manufacturing” through the BLA/BLS!! Also, approval of manufacturer’s devices Also, approval of manufacturer’s devices

through Pre-Market Approval through Pre-Market Approval 510(k)510(k) application regulates equipment used in application regulates equipment used in all phases of blood component all phases of blood component productionproduction

*Federal Register: August 16, 2007 (Volume 72, Number 158)


CAP Accreditation CAP Accreditation ChecklistChecklist

11stst accreditation group to require accreditation group to require bacterial testing of platelets!bacterial testing of platelets!

CAP Checklist - 2002CAP Checklist - 2002 Phase I deficiencyPhase I deficiency Revised 12/29/2004 to Revised 12/29/2004 to Phase IIPhase II

deficiencydeficiency


CAP Accreditation CAP Accreditation Checklist 2008Checklist 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.


CAP Accreditation CAP Accreditation Checklist 2008Checklist 2008

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.


AABB AccreditationAABB Accreditation AABB Standards for Blood Banks & AABB Standards for Blood Banks &

Transfusion Services, 23Transfusion Services, 23rdrd ed, ed, Effective May 1, 2004Effective May 1, 2004 Implement standard 5.1.5.1:Implement standard 5.1.5.1:

The blood bank or transfusion service shall have The blood bank or transfusion service shall have methods to limit and detect bacterial methods to limit and detect bacterial contamination in all platelet components. contamination in all platelet components. Standard 5.6.2 appliesStandard 5.6.2 applies

Standard 5.6.2- Protection Against Standard 5.6.2- Protection Against Contamination:Contamination:The venipuncture site shall be prepared so as to The venipuncture site shall be prepared so as to

minimize risk of bacterial contamination. Green minimize risk of bacterial contamination. Green soap shall not be used.soap shall not be used.


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 When a true-positive result is obtained and an

appropriate specimen is available, additional appropriate specimen is available, additional testing to identify the organism shall be performed. testing to identify the organism shall be performed. Additional testing and follow-up shall be defined. Additional testing and follow-up shall be defined. Standards 5.2.2 and 7.1 to 7.1.4 apply.Standards 5.2.2 and 7.1 to 7.1.4 apply.

Standard 5.2.2: Donor Notification of Standard 5.2.2: Donor Notification of Abnormal Findings and Test Results Abnormal Findings and Test Results (Standard 5.2.3, 25(Standard 5.2.3, 25thth ed, 2008) ed, 2008)

Standards 7.1-7.1.4: NonconformancesStandards 7.1-7.1.4: Nonconformances


AABB Accreditation con’tAABB Accreditation con’t

Standard 5.6.6.1 (25Standard 5.6.6.1 (25thth ed, 2008) ed, 2008)Blood collection containers with draw Blood collection containers with draw

line (inlet) diversion pouches shall be line (inlet) diversion pouches shall be used for any collection of platelets, used for any collection of platelets, including whole blood from which including whole blood from which platelets are made.platelets are made.



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 eBDShemoSystems Scansystem™hemoSystems Scansystem™

•Other non approved and non validated methods are also being used to meet the AABB standard for bacterial detection



FDA concerns with bacterial FDA concerns with bacterial detection as currently applied detection as currently applied ((Vostal JG, Jan. 28, 2005 CBER workshop))• Test performance characteristics unknown• Use of non-validated tests (glucose and pH by Use of non-validated tests (glucose and pH by dipstick, swirling)dipstick, swirling)• Non-standardized methodology even with Non-standardized methodology even with culture-based devicesculture-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



Manual tests- validation required Manual tests- validation required for pH & glucosefor pH & glucose pHpH

GlucoseGlucose

SwirlingSwirling


Devices approved for QC detection Devices approved for QC detection of platelet bacterial of platelet bacterial contaminationcontamination- Pall BDSPall BDS


Pall eBDSPall eBDS Sample set/Oxygen Sample set/Oxygen

AnalyzerAnalyzer Sterile weld platelet Sterile weld platelet

component to setcomponent to set Fill pouch with ~3 mL Fill pouch with ~3 mL

of productof product Disconnect sample Disconnect sample

pouch from set and pouch from set and incubate at 35°C for incubate at 35°C for 24-30 hrs24-30 hrs

Measure the OMeasure the O22 content content in the air above the in the air above the plasma sample with plasma sample with insertion of analyzer insertion of analyzer probe into pouchprobe into pouch

LED display will read LED display will read PASS or FAILPASS or FAIL


Pall eBDSPall eBDS

Pall Medical eBDS Brochure, 2004


BioMeriuex BioMeriuex BacT/ALERT®BacT/ALERT®

Colorimetric Colorimetric technology/Sensor technology/Sensor Culture bottlesCulture bottles COCO22 release causes release causes

sensor bottle to turn sensor bottle to turn yellowyellow

Instrument measures Instrument measures & detects color & detects color change, analyzes change, analyzes data to determine data to determine positivity, alerts positivity, alerts when positive culturewhen positive culture


hemoSystems hemoSystems Scansystem™Scansystem™

Scansystem Platelet Scansystem Platelet Kit/Scansystem Kit/Scansystem AnalyzerAnalyzer After sample processing in After sample processing in

platelet kit, bacteria are platelet kit, bacteria are stained with a green stained with a green fluorescence and retained fluorescence and retained on the surface of a on the surface of a dedicated membrane dedicated membrane (platelets are aggregated (platelets are aggregated and lysed)and lysed)

Membrane is inserted in Membrane is inserted in analyzer and scanned by analyzer and scanned by an Argon laseran Argon laser

Each fluorescent spot on Each fluorescent spot on the membrane will be the membrane will be detected and analyzeddetected and analyzed

Analyze 3 platelet Analyze 3 platelet components (SDP, WBDP)components (SDP, WBDP)


hemoSystems hemoSystems Scansystem™Scansystem™

Scansystem™ Brochure accessed at www.hemosystem.com


Preparing Platelets for Preparing Platelets for CultureCulture

Sterile Connection DeviceSterile Connection Device


Connecting Platelets to Connecting Platelets to Syringe SetSyringe Set


Tubing from syringe setTubing from syringe set

Tubing from platelet bagTubing from platelet bag


Aliquoting Platelet SampleAliquoting Platelet Samplefrom Transfusion Bagfrom Transfusion Bag

(Suspected Transfusion Reaction(Suspected Transfusion Reaction))

Syringe setSyringe set

SDPSDP

Transfusion setTransfusion set


Aliquoting Platelet SampleAliquoting Platelet Samplefor Bacterial Detection for Bacterial Detection

TestingTesting


Inoculating Culture Bottle Inoculating Culture Bottle (pediatric size)(pediatric size)


Sterile Connection PortSterile Connection Port


Bacterial Contamination of Bacterial Contamination of Blood Components-2008Blood Components-2008

ARC Study of ARC Study of bacterial bacterial contamination before contamination before & after & after implementation of implementation of sample diversion sample diversion pouch (Pall Acrodose pouch (Pall Acrodose with eBDS) with eBDS)

Prestorage pooling of Prestorage pooling of WBDP with culture WBDP with culture testingtesting

Jan 2003-December Jan 2003-December 20062006

Benjamin RJ, et al. Transfusion 2008;48:2348-55




PSP=prestorage pooled WBDP


Blood Transfusion Safety-Blood Transfusion Safety-FDA Blood Transfusion Fatality FDA Blood Transfusion Fatality

ReportReport

http://www.fda.gov/Cber/


Why test at point of issue ?

Wide variation in durationof Lag Phase

Wide variation in rateof log Phase Growth

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


New FDA Approved Device New FDA Approved Device for Transfusion Servicesfor Transfusion Services

The Abbott Verax The Abbott Verax Platelet PGDPlatelet PGD®® Test Test

A rapid, qualitative A rapid, qualitative immunoassay for the immunoassay for the detection of Aerobic detection of Aerobic and Anaerobic; Gram-and Anaerobic; Gram-positive and Gram-positive and Gram-negative bacteria in negative bacteria in leukocyte reduced leukocyte reduced apheresis platelets apheresis platelets (SDP) (SDP)

~30 minute TAT~30 minute TAT SDP must have SDP must have

undergone QC culture undergone QC culture by blood supplier!!by blood supplier!!


New Device for Transfusion New Device for Transfusion ServicesServices


New Device for Transfusion New Device for Transfusion ServicesServices

1. Rapid ~ 25 minutes (3 min hands on)

3. Sensitivity ~ 103 CFU/ mL

Single-use disposable testSingle-use disposable testVerax rapid Platelet PGD® Test

2. Positives typically < 10 minutes

4. Specificity > 99.7%


Pan Genera Rapid Test Pan Genera Rapid Test FormatFormat

TestFeatures

2. Shared 300 uL sample addition well3. Separate GP and GN read windows

4. Procedural controls for GP & GN

ProceduralControl

ProceduralControl

1. ABS housing holding GP/GN test strips

SampleWell

Gram-NegativeRead Window

Gram-PositiveRead Window


Methods ComparisonMethods ComparisonVerax PGDVerax PGD BacT/ALERTBacT/ALERT Pall eBDSPall eBDS

TechnologyTechnologyConserved Conserved

Bacterial Ag Bacterial Ag ImmunoassayImmunoassay

CultureCulture

CO2 MeasureCO2 MeasureAerobic 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 10 – 30 min (positives (positives

typically within typically within 10 minutes)10 minutes)

24 – 96 hours24 – 96 hours 24 – 30 hours24 – 30 hours

Detect Aerobic Detect Aerobic and Anaerobic and Anaerobic

bacteria?bacteria?YesYes Yes, but time Yes, but time

variesvaries Misses AnaerobesMisses Anaerobes

Clinical Clinical SpecificitySpecificity 99.7%99.7% 99.2-99.8%99.2-99.8% ~ 99%~ 99%

Source: Abbott, Biomerieux and Pall Medical web site.


Validation of Validation of Bacterial Detection Bacterial Detection

MethodsMethods pH & glucose tests are analytically pH & glucose tests are analytically

insensitive (Yomatovian R, Brecher ME. insensitive (Yomatovian R, Brecher ME. Transfusion 2005;45:647-8)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/mL CFU/mL Facilities may not have FDA approved Facilities may not have FDA approved

equipmentequipment Validate for QC useValidate for QC use

SensitivitySensitivity Types of organismsTypes of organisms Growth timeGrowth time


Snyder JW, et al. BACTEC detection of bacteria in platelet pools. ASM 05-GM-A-4146; 2005


SUMMARYSUMMARY Bacterial Bacterial

contamination of contamination of blood components blood components continues to pose a continues to pose a threat to transfusion threat to transfusion recipientsrecipients

Progress to prevent Progress to prevent adverse reaction to adverse reaction to blood transfusions blood transfusions due to bacterial due to bacterial contamination contamination continues to be seencontinues to be seen

Microbiologists now Microbiologists now play a major role in play a major role in this progress!!this progress!!


UNIVERSITY OF LOUISVILLE MEDICAL CENTER, LOUISVILLE, KENTUCKYUNIVERSITY OF LOUISVILLE MEDICAL CENTER, LOUISVILLE, KENTUCKY

THANK YOU!!!!

THANK YOU!!!!

Bacterial Testing of Platelet Components: 2008 Update William B. Lockwood, PhD, MD Clinical...

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