A summary review provided by the American Red Cross Blood Services Regions serving the North Atlantic Area July, 2003 Bacterial Contamination in Blood

A summary review provided by the A summary review provided by the

American Red Cross Blood Services Regions American Red Cross Blood Services Regions

serving the North Atlantic Areaserving the North Atlantic Area

July, 2003July, 2003

Bacterial Contamination Bacterial Contamination in Blood Productsin Blood Products

Risks, Prevention and DetectionRisks, Prevention and Detection

Bacterial Contamination Bacterial Contamination in Blood in Blood

ProductsProductsAgendaAgenda

What is the Problem? What are the Risks?

What Organisms are Associated with Bacterial Contamination?

What are the Sources of Contamination? What Corrective Actions are Planned?

Recent Advances in Recent Advances in Testing TechnologyTesting Technology

1990-20031990-2003

Anti-HCV (1990) Multi-antigen anti-HCV (1992)

Anti-HIV1/2, replacing anti-HIV-1 (1992) HIV-1 p24 antigen (1996) HCV/HIV NAT (IND) (1999)

Licensed NAT (2003) West Nile Virus (IND) (2003)

Comparison of Residual RisksComparison of Residual Risks

HIVHIV

HBVHBV

HCVHCV

19961996199419941992199219901990198819881986198619841984

1:1001:100

1:10001:1000

1:101:10 000000

1:100 0001:100 000

1:1 000 0001:1 000 000

19981998 20002000

Transmission risk, Transmission risk, per unitper unit

Updated from: Goodnough LT Updated from: Goodnough LT e t al. NEJMe t al. NEJM 1999;341:126- 1999;341:126-77

20022002

BacterialBacterialContaminationContamination

(platelets)(platelets)

SepticSepticFatalitiesFatalities(platelets)(platelets)

ClinicalSepsis

(platelets)

Bacterial Contamination Bacterial Contamination of Blood Products of Blood Products First recognized infectious risk of blood

transfusion Risk greatly reduced in the 1960s by the

use of closed, sterile systems for the collection and storage of blood

Recent dramatic improvements in safety from viral screening and testing have reduced the risks from Hepatitis and HIV

Bacterial sepsis is now the most common infectious disease event following transfusion

Bacterial Contamination Bacterial Contamination of Blood Productsof Blood Products

Bacterial contamination occurs primarily in room-temperature stored products (platelets) but can occur in red blood cells and plasma also

The blood banking community is taking steps to improve prevention and detection of bacterial contamination

The American Association of Blood Banks, as well as the College of American Pathologists have established compliance criteria for transfusion services

Bacterial Contamination in Bacterial Contamination in Blood ProductsBlood Products

The American Association of Blood Banks has issued two new standards (March, 2003):

“5.1.5.1 The blood bank or transfusion service shall have methods to limit and detect bacterial contamination in all platelet components.”

“5.1.5.1.1 Standard 5.1.5.1 shall be implemented by March 1, 2004”

“5.6.2 The venipuncture site shall be prepared so as to minimize the risk of bacterial contamination. Green soap shall not be used”

Bacterial Contamination Bacterial Contamination in Blood Products in Blood Products

College of American Pathologist’s Accreditation Checklist (December, 2002):

“TRM.44955 Phase 1 Does the laboratory have a system to detect the presence of bacteria in platelet components?”

Bacterial Contamination of Bacterial Contamination of Blood Products Blood Products

What are the Risks?

Risk of clinical sepsisRisk of clinical sepsis Ness et al, Transfusion 2001;41:857-

60. Identified clinical cases of transfusion

associated sepsis over a 12 year period, with conversion from 51.7% random donor platelets to 99.4% SDPs

The #donors/septic event remained constant at 15,000 throughout the 12 year period, despite the conversion to SDPs

Pooled random donor platelets were 5.5-times more likely to cause sepsis than SDPs due to pool size

Pooling issuesPooling issues

Bacterial Contamination of Bacterial Contamination of Blood ProductsBlood Products

What Bacterial Organisms are associated with Blood Product

Contamination?

Bacterial species in platelets Bacterial species in platelets implicated in clinical sepsisimplicated in clinical sepsis

S. epidermidis, 30.2%

S. aureus, 10.5%

E. coli, 9.3%

B. cerus, 9.3%

S. cholerae-suis, 8.1%

E. cloacae, 5.8%

B-hem. Strep, 5.8%

E. aerogenes, 2.3%

10 others, 1.3% each

n = 86n = 86

Compilation of data from Clin Micro Rev Compilation of data from Clin Micro Rev 1994; 7:290-302; Transfusion 2001;41:1493-1994; 7:290-302; Transfusion 2001;41:1493-99; www.shot.demon.co.uk/toc99; www.shot.demon.co.uk/toc

S. epidermidis 9.6%S. aureus 17.3%E. coli 5.7%Bacillus 5.7%Salmonella 7.7%Enterobacter 5.7%Streptococcus 7.7%Klebsiella 17.3%Serratia 15.4%P. mirabilis 2.2%

n = 52

Bacterial species in platelets Bacterial species in platelets implicated in septic fatalities implicated in septic fatalities

reported to the FDA (1976-1998)reported to the FDA (1976-1998)

Differences between the Differences between the species implicated in septic species implicated in septic morbidity and mortality in morbidity and mortality in

platelet componentsplatelet components S. epidermidis is less commonly observed

in septic fatalities and more commonly observed in septic reactions

Klebsiella is commonly observed in septic fatalities

Gram negative organisms are implicated in more fatalities (60%) than gram positive organisms (40%); gram positives cause a majority of septic reactions (56%)

Organisms implicated in Organisms implicated in sepsis from platelets sepsis from platelets

Approximately 30% are associated with normal skin flora

Approximately 56% are gram positive All are aerobic or facultative anaerobes

A rare (single case) exception: Clostridium perfringens fatality from a pooled platelet unit Trans Med 1998;8:19-22


What are the Sources of Bacterial Contamination?

Sources of Bacterial Sources of Bacterial ContaminationContamination

Skin Surface Contamination Phlebotomy Core Donor Bacteremia Containers and Disposables Environment

Avoiding Skin Avoiding Skin ContaminationContamination

Diversion of the initial blood flow

Improvement in pre-phlebotomy skin cleansing

Skin sourceSkin source

Diversion of initial blood Diversion of initial blood flowflow

Diversion of initial blood flow into sampling tubes

Reduces the load of skin-associated bacteria entering blood container

Phlebotomy “core” directed into sampling pouch instead of blood container

Clinical data supporting Clinical data supporting diversion of initial blood diversion of initial blood

flowflow de Korte et al. Vox de Korte et al. Vox Sang 2002;83:13-16Sang 2002;83:13-16 Collected blood Collected blood

normally or diverted normally or diverted the first 10mL of the first 10mL of whole blood into a whole blood into a satellite bagsatellite bag

Performed bacterial Performed bacterial testing by automated testing by automated blood culture blood culture (BacT/Alert) in a (BacT/Alert) in a laminar flow hoodlaminar flow hood

Total bacterial prevalen

ce

S. epidermidisprevalenc

e

StandardStandard

CollectionCollection0.35%(0.27-0.44)

0.14%

Collection Collection with with

diversiondiversion

0.21%(0.12-0.35)

0.03%

P-valueP-value <0.05 <0.02

Skin disinfection methodsSkin disinfection methods

Some agents may reduce the number of surface bacteria more than others

Method of application and applicator may have some impact on the extent of reduction of surface bacteria

Minimum scrub of 30 seconds required to be effective

Impact of Skin Disinfection on surface Impact of Skin Disinfection on surface bacteriabacteria

CFU per plate

PVPI Isopropyl Alcohol + Tincture

of Iodine

Chlor-hexidine

Gluconate

Green soap +

Isopropyl alcohol

0 34-40% 60% 0%

1-10 35-43% 34% 25% 17%

11-100 10-14% 2% 12% 47%

>100 0-13% 1% 3% 36%

Goldman et al, Transfusion 1997;37:309-12Goldman et al, Transfusion 1997;37:309-12

63%

Recurrent contamination from the Recurrent contamination from the dimpled skin of one plateletpheresis dimpled skin of one plateletpheresis

donordonor Anderson et al., Am J Med 1986;405-11.

One donor gave 17 plateletpheresis donations from a scarred dimpled site in the right antecubital fossa

Two units were implicated in septic events traced to this donor

Four units, including the two units linked to the septic event, were culture positive with coagulase negative Staphylococcus

Follow up blood samples obtained from the non-scarred left antecubital fossa were routinely culture negative

Recurrent contamination from an Recurrent contamination from an asymptomatic bacteremic donorasymptomatic bacteremic donor

Rhame et al., Ann Intern Med 1973;78:633-41. One plateletpheresis donor was linked to

7 cases of Salmonella cholerae-suis transfusion associated bacterial sepsis; 2 cases were fatal

Three of the cases were linked to positive culture of the platelet units

The donor had a low-grade bacteremia and unknowingly had Salmonella osteomyelitis of the tibia

Donor bacteremiaDonor bacteremia

Multiple cases of sepsis from Multiple cases of sepsis from contaminated blood contaminated blood

containerscontainers Heltberg et al. Transfusion 1993;33:221-7 Högman et al. Transfusion 1993;33:189-91.

Serratia marcescens was cultured from three septic patients and their implicated units in Denmark

All units were collected using the same lot of blood containers

11 of 1,515 blood products collected using the implicated lot were positive for Serratia marcescens

An organism of the same ribotype was isolated from the manufacturing plant

The same containers were implicated in Sweden

Container sourceContainer source

A fatal case of A fatal case of Clostridium Clostridium perfringensperfringens sepsis from a platelet sepsis from a platelet

poolpool McDonald et al., Transfusion Medicine 1998;8:19-22. C. Perfringens is a spore forming facultative

anaerobe, found in soil and human intestinal tract Organism recovered from platelet pool; septic

recipient was on antibiotics; no organism recovered

Patients death was considered a septic event The same serotype of Clostridium was isolated

from the arm of 1 of the 4 donors; a subsequent culture of the same arm 6 months later yielded fecal flora

The donor was a mother who carried her two toddlers in the crook of her arm

Environmental sourceEnvironmental source

Bacterial Contamination Bacterial Contamination in Blood Products in Blood Products

What Options exist to Prevent and Detect Bacterial Contamination?

Bacterial Contamination of Bacterial Contamination of PlateletsPlatelets

Prevention and Detection OptionsPrevention and Detection Options Donor screening – not feasible except for arm screening. Can’t detect asymptomatic bacteremic donors

Arm Preparation-Limited effectiveness of arm scrub

Pathogen reduction – not yet available. May not inactivate spore forming organisms

Better phlebotomy methods and initial blood diversion

Bacterial detection offers best confirmatory option

Bacterial Detection Options Bacterial Detection Options in Platelet Productsin Platelet Products

Visual examination for discoloration, clumping or abnormal morphology

Microscopy Gram stain Acridine orange

Measuring Biochemical changes Lowered pH Reduced Glucose

Bacterial culture Detection through oxygen consumption Detection through CO2 production


Visual Examination

Inspect product prior to transfusion for discoloration or abnormal clumping

Perform “swirl” procedure to detect morphologic changes in platelets Normal shaped platelets will align with fluid flow

and “shimmer” when swirled Contaminated platelets, among others, lose

discoid shape and do not “shimmer” when swirled– Not a specific marker for contamination

SwirlingSwirling

Low pHLow pHMetabolic disturbanceMetabolic disturbance

No alignment with flowNo alignment with flow

Alignment with flowAlignment with flow

SENSITIVITY: 75%SPECIFICITY: 95%

Leach MF Leach MF et al. Vox Sanget al. Vox Sang 1998;74(suppl 1):1180. 1998;74(suppl 1):1180.


Microscopic Methods

Gram Stain or Acridine Orange preferred methods

Limitations: Must be performed by the Transfusion

Service prior to product issue for transfusion

Lack sensitivity with low bacterial load

Bacterial Detection Options Bacterial Detection Options in Platelet in Platelet

ProductsProducts Measuring Biochemical Changes

Measure changes in glucose consumption against a control. Variances of >2 S.D. may indicate bacterial contamination

“Dipstick” testing Limitations:

Both this method and staining methods are subjective, require high levels of contamination, and must be performed prior to issue by the Transfusion Service

0102030405060708090

100

0 1 2 3 4 5

Control

Bacillus

Klebsiella

Glucose, % Day 0

Storage Time, dafter Burstain JM after Burstain JM et al. Transfusionet al. Transfusion 1997;37:255-8. 1997;37:255-8.

-2 SD-2 SD

Detecting Bacteria in Platelets:Detecting Bacteria in Platelets:Biochemical ChangesBiochemical Changes

Chemical Tests - Chemical Tests - DipsticksDipsticks

Must be performed immediately before issue because of itsrelative insensitivity and the need for high bacterial counts


Blood Culture Methods

Two methodologies presently approved by FDA for Quality Control use bioMeriuex BacT/Alert System Pall Biomedical BDS System

Bacterial Detection Bacterial Detection Options in Platelet Options in Platelet

ProductsProductsbioMeriuex BacT/Alert System

Detects bacterial growth in culture bottles by measuring CO2 production

Automated reader continuously monitors samples Sampling interval of >24 hours post phlebotomy Culturing interval of >24 hours post sampling

(aerobic and anaerobic cultures) Cultures incubate for 5-7 days; may identify

positive cultures post-transfusion FDA-Approved for Q.C. purposes only on

Leukoreduced Apheresis Platelets

Practical Application of Culturing Practical Application of Culturing in a Transfusion Service Laboratoryin a Transfusion Service Laboratory

Aubuchon, DartmouthAubuchon, Dartmouth

Experience in first 3 years:Experience in first 3 years:3,927 apheresis units cultured 3,927 apheresis units cultured (5 mL into aerobic bottle, BacT/Alert automated system)(5 mL into aerobic bottle, BacT/Alert automated system)

23 initial positives 23 initial positives (0.5%) in 28 h (10-69)(0.5%) in 28 h (10-69)

14 not confirmed on repeat culture14 not confirmed on repeat culture5 not able to be recultured5 not able to be recultured4 confirmed positives4 confirmed positives

RATE = 1/1,000 units RATE = 1/1,000 units (95% CI: to 1/600)(95% CI: to 1/600)

Filter: StopsFilter: StopsWBCs+PltsWBCs+Plts

Passes: BacteriaPasses: Bacteria

Gas impermeable bagGas impermeable bag

24 h24 h

24 h at 35C24 h at 35C

Measure %OMeasure %O22

in headspacein headspace

Limit: 19.5%Limit: 19.5%

Detecting Bacteria in Platelets:Detecting Bacteria in Platelets:Detection of Growth by ODetection of Growth by O22 Consumption Consumption

Pall BDS system


Pall Biomedical BDS System

Detects bacterial contamination by measuring O2 consumption

Automated reader measures O2 levels in headspace of culture pouch

Sampling interval of >24-48 hours Culture performed for >24-30 hours FDA-Approved for Q.C. on leukoreduced platelet

concentrates and leukoreduced apheresis platelets


Limitations of Blood Culture Methods

Early sampling/testing may not detect small # bacteria per bag. Approved methods require 24-30 hour wait before sampling

Two FDA-Approved methods require bacteria to grow up after sampling to detectable levels, so culture must be done well before planned transfusion (Blood Center)

The two time intervals (collection to sampling and sampling to release/transfusion) dominate the logistic considerations

Bacterial Detection Options in Bacterial Detection Options in Platelet ProductsPlatelet Products

Limitations of Blood Culture Methods

Both options require leukoreduced platelets

BacT/Alert requires continued culture after product release

Release and recall (BacT/ALERT) or hold to end of culture to release (PALL BDS)

Bacterial Detection Options in Bacterial Detection Options in Platelet ProductsPlatelet Products

Limitations of Blood Culture Methods Need to balance the risk of platelet shortages

versus the risk of platelet contamination The two available devices are FDA-Approved

for Q.C, and not approved as pre-release tests Cost Probable negative impact on outdates Possible extension of platelet storage to seven

days or pooling/storing whole blood derived platelets

Bacterial Contamination in Bacterial Contamination in Transfusable Blood Transfusable Blood

ProductsProductsAABB Guidance

Association Bulletin #03-07 issued May 16, 2003

Provides guidance for methods to limit contamination and to detect contamination

AABB Association Bulletin AABB Association Bulletin #03-07#03-07

May 16, 2003May 16, 2003Methods to Limit Contamination:

Careful phlebotomy – No green soap prep

Iodine based scrub recommended Consider phlebotomy diversion –

“sample first” technologies Consider increased use of apheresis

platelets

AABB Association Bulletin AABB Association Bulletin #03-07#03-07

May 16, 2003May 16, 2003Methods to Detect Contamination:

Culture methods optimal. Two approved products cited. Other culture methods can be validated. No label claims allowed

Due to insensitivity, staining and dipstick methods should be used as close in time to issue as possible

Validation of all methods is required “Swirl” procedure useful for inspection but

does not by itself meet AABB Standard 5.1.5.1


American Red Cross Bacterial Prevention and Detection Strategy

Implement prevention and detection strategies to meet the requirements and timelines of the AABB and CAP

Solicit customer feedback to develop efficient and cost-effective implementation strategies

Keep customers well-informed during the pre-implementation period

Documents

A summary review provided by the American Red Cross Blood Services Regions serving the North Atlantic Area July, 2003 Bacterial Contamination in Blood