Post on 17-Dec-2015
Bacterial Contamination and
Screening of Platelet Concentrates
James X. Gray, BSc(Hons), PhD, MD
Haematology RegistrarThe Alfred Hospital
A 75 year old female patient with chronic lymphatic leukaemia developed rigors, vomiting and pyrexia following transfusionof a 5-day old pooled platelet unit. The transfusion was terminated and the patient recovered. An identical strain ofS.epidermidis was isolated from the transfused platelet pack and from the venepuncture site of one of the four contributingdonors. However, the organism was not isolated from the recipient following the reaction. This is evidence of bacterialcontamination of a platelet pool from a donor's arm and suggests arm cleansing was inadequate. Although transmission to the recipient was not confirmed it would seem likely.
Case from SHOT annual report 2004
Differential Centrifugation and Separation
Whole Blood + Preservative(450 ml +/- 45 ml)
Red Cells Buffy Coat Plasma
Pelleted Red Cells Platelets
Soft Spin
Hard Spin
Specific GravityRBC 1.08 – 1.09Platelet 1.03 – 1.04Plasma 1.023
Pooled x 4
Blood Product TestingSydney Red Cross(TGA accreditation)
Pathogen Screen test window / d RiskHIV 1+2 serology only 22 1 in 2.4 x 106
HIV serology + NAT 09 1 in 7.3 x 106
HCV serology only 66 1 in 330,000HCV serology / NAT 07 1 in 3.7 x 106
HBV serology 45HTLV I&II serology 51 <1 in 2.4 x 106
CMV serologySyphilisserologyMalaria serologyvCJD questionairre none reportedBacterial culture of Platelets (20%)
Quality Control SpecificationsPlatelet Concentrates: pooled x 4, leukodepletedTGA requirements (Council of Europe Guidelines):
Parameter Specification Acceptance Sampling
Volume 160-240 ml 75 % 1 %
Platelets
(per unit)
> 240 x 109 75 % 1 %
Leucocytes
(per unit)
< 0.8 x 106 90 % 1 %
pH 6.8 – 7.4 75% 1 %
Microbial
contamination
Negative 5 %
Bacterial Detection of Platelets:Current Problems and Possible Resolutions
Morris A. Blajchman, Erik A.M. Beckers, Ebbe Dickmeiss,Lilly Lin, Gillian Moore and Ludo Muylle
McMaster University and Canadian Blood Services, Hamilton, Ontario, Canada
Sanquin Blood Bank South West Region, Rotterdam, The Netherlands
Copenhagen Blood Transfusion Service Center, Rigshospitalet, Copenhagen, Denmark
Cerus Corporation, Concord, CA
Queen Elizabeth Hospital, King's Lynn, UK
Red Cross-Flanders Blood Service, Mechelen, Belgium
University of Antwerp, Antwerp, Belgium
Transfusion Medicine Reviews Volume 19, Issue 4
October 2005, Pages 259-272
Limitation Of Routine Bacterial Screening ofPlatelets With the BacT/ALERT System:
A 30-Month Experience From theSanquin Blood Bank
Southwest Region of the Netherlands
EAM Beckers
Sanquin Blood Bank Southwest Region, Rotterdam, Netherlands
Beckers, (Holland)Transfusion Medicine Reviews, vol 19 (4)
Donor questionnaire
Disinfect venesection site
Diversion pouch of 30 ml
Pooled buffy coat units (5 donors)
Leukoreduction filtration
Automated culture Bact T/alert
100% screening
Beckers, (Holland)Transfusion Medicine Reviews, vol 19 (4)
Positive cultures 0.76%
172 units (58%) already issued
155 units already transfused
Reported transfusion reactions – Nil
Serious septicemia reported in 2 patients, who received culture negative platelets
Bacterial Screening ofPlatelet Concentrates:A 4-Year Experience inBelgian Blood Centers
L Muylle
Red Cross-Flanders Blood Service, Mechelen, andUniversity of Antwerp, Antwerp, Belgium
Muylle, (Belgium)Transfusion Medicine Reviews, vol 19 (4)
Arm disinfectionClosed system for platelet preparationPooled (5-6 patients)Apheresis plateletsNo diversionAutomated culture - Bact T/alert100% screening108,000 platelet concentrates over 4 years
Number Tested
Screening Positives
False Positives
Confirmed Positives
Pooled (n = 5 or 6) buffy coat platelets
75 829 793 (1.05%) 140 (0.18%) 622 (0.82%)
Single-donor apheresis platelets
31 998 237 (0.74%) 41 (0.13%) 181 (0.57%)
Total 107 827 1030 (0.96%) 181 (0.17%) 803 (0.74%)
Test Results of the Bacterial Screening of Platelet Units for 5 days using BacT/alert detection
Muylle, (Belgium)Transfusion Medicine Reviews, vol 19 (4)
803 units were positive
446 units already transfused
314 units confirmed positive on repeat culture
203 follow-up reports from clinicians12 transfusion reactions - sepsis x 2
fever x 6
hypotension x 1
rigors x 1
rash x 2
Bacterial Screening of Buffy Coat–DerivedPlatelet Concentrates
Findings From a Danish Blood Bank
E Dickmeiss
Copenhagen Blood Transfusion Service Center,Rigshospitalet, Copenhagen, Denmark
Results of the Initial Routine Screening of Pooled Buffy CoatPlatelet Concentrates in the Danish (Copenhagen) Study
No. of buffy coat pooled (n = 4) platelet concentrates monitored
22 165
No. of positive on BacT/ALERT tests 50
No. of positive BacT/ALERT tests with sterile platelet concentrates
16
No. of positive BacT/ALERT tests with bacterial culture–positive platelet concentrates
34 (0.15%)
Time elapsed between seeding of the BacT/ALERT flask and the appearance of a positive signal
22 h (median range, 10-60 h)
No. with a positive culture in 1 of the corresponding RBC units
10 (0.05%)
No. with all corresponding RBC units sterile 24 (0.11%)
Bacteria found in the 34 confirmed positive routine cultures, 6 already transfused
Coagulase-positive Staphylococcus species 28 cases
Corynebacteria 4 cases
B cereus 2 cases
Results from the Copenhagen Study Day 3 Screening(All Units had Tested Negative Upon Initial Screening)
No. of day 3 platelet concentrates screened 2472
No. of confirmed contaminated platelet concentrates 6 (0.24%)
Time elapsed between seeding of the BacT/ALERT flask and the appearance of a positive signal
16 h (median range, 10-25 h)
No. with positive culture in 1 of the corresponding RBC units
1 (0.04%)
No. with all corresponding RBC units sterile 5 (0.20%)
Bacteria found in the confirmed positive cultures
Coagulase-negative Staphylococcus species 5 cases
Corynebacteria species 1 case
Discussion Points 1
Late appearance of positive signal, often after unit is issued and transfused, suggests that the degree of contamination or bacterial load is probably low and often w/o clinical consequence. Six culture-positive units had already been transfused w/o complication.
BacT/Alert culture system relies upon detection of CO2. Sensitivity is highly dependent upon: i) Timing of platelet sampling (day 1 or day 3)ii) Sample volume.
Discussion Points 2
Utility of 100% screeningFalse positives result in unnecessary wastage,False negative results are generally unacceptable
Blocking of platelet units with high bacterial loads
Pathogen inactivation is proactive, can improve blood safety further and potentially permit a longer shelf-life
Improving the bacteriological safetyof platelet transfusions
Morris A. Blajchman , Mindy Goldmanc and Federico Baezad
a Departments of Pathology and Medicine, McMaster University, Hamilton, Ontario, Canadab Canadian Blood Services, Hamilton, Ontario, Canadac Canadian Blood Services, Ottawa, Canadad Baxter Transfusion Therapies Europe, Madrid, Spain
Transfusion Medicine Reviews Volume 18, Issue 1
January 2004, Pages 11-24
Pre-transfusion Bacterial Detection Methods
Transfusion Medicine Reviews January 2004
Visual inspection – swirling and colour
Automated bacterial culture - detection of CO2
Pall BDS (O2 consumption)
DNA/RNA
Fluorescent antibody labeling
Specific peptidoglycan components of cell wall
Dielctrophoresis
Endotoxin detection
Microscopic detection
Automated Bacterial Cultural SystemsDetection of CO2 produced by the growing bacteria; eg.
BacT/Alert.
Culture medium is inoculated with a blood sample, incubated, continuous readings for CO2 production
Smaller inoculums require longer incubation periods and are more likely to give false negative results.
Larger inoculums waste precious resource.
Threshold of detection: 10 CFU/ml
Slow growing bacteria (eg. S. epidermidis) may need 6 to 7 days, Propionibacterium acnes even longer.
Given the need for some incubation periods to be longer than the shelf life of the unit and the likelihood that the unit will already be transfused, underscores the need for a robust communication pathway and audit practice.
Pall Corporation BDS(Bacterial Detection System)
Measures the consumption of O2 by contaminating bacteria, in the absence of platelets or leukocytes.
A 2 – 3 ml sample of platelet concentrate.
In-line filtration to remove leukocytes and platelets, not the bacteria.
Contained within a non-gas-permeable pouch and incubated.
Positive reading when O2 concentration falls.
Detection sensitivity 97% after 24 hrs, when bacterial inoculum 100-500 CFU/ml.
Only a single reading per sample.
Nucleic Acid based detection systems
Detection of bacterial DNA or rRNA
Highly conserved bacterial rRNA sequences, although several probes are required to cover all desired organisms.
PCR based technology
In reported literature ~104 CFU/ml (4-5 days of storage)
Threshold of bacterial load desired 102 CFU/ml
Detection systems based on changes in metabolic parameters during storage.
Abnormal glucose concentrations
Low pH
Significant variations of normal
High levels of bacterial contamination are needed for reliable reproducibility.
Fluorescent Antibiotic Labeling
Antibiotics labeled with fluorescent markers
Binding specificities for bacterial molecular components. Several may be required.
Detection by Flow Cytometry
Threshold ~105 CFU/ml
Detection of specific peptidoglycan components of bacterial cell walls
Bacterial load required ~103 – 104 CFU/ml
Dielectrophoretic Method
Bacteria move in an electric field and platelets don’t
Bacterial load required ~105 CFU/ml
Microscopic examination of stained samples
Bacterial load required ~106 CFU/ml
Endotoxin detection
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Pathogen Inactivation 1
The only approach to achieve absolute bacteriological safety.
L-Carnitine in platelet storage medium, results in a modest inhibition of coag. Neg. Staph.
Gamma Irradiation
GVHD is prevented at doses of 25 – 30 Gy
Bacteria are inactivated at 100 – 150 Gy
Platelet function is compromised at doses above 75 Gy.
Riboflavin plus UVADemonstrated utility in in vitro assays against
strains of Staph. epidermidis, S. aureus, Pseudomonas, Klebsiella, E. coli.
No clinical trials to date.
Amotosalen HCL plus UVA light
Synthetic psoralen intercalates with DNA helices and covalent bonds form in presence of UVA
Demonstrated utility, in RCCT, with bacteria, viruses and malarial parasites.
INTERCEPT Blood System, Cerus Corp, USA
Reducing Risk of Platelet Contamination 1
Improve donor screeningDonor questionnaire for subclinical bacteremia
Improve venepuncture site disinfectionAntiseptic - quality
- quantity
- mode of application
Diversion Pouch – 13.5 ml to 30 ml
40%, 72%, 90%
Reducing Risk of Platelet Contamination 2
Platelet storagemost TA septic events occur with units > 3dearly use while bacterial load is sub-clinical.
Universal Leukoreduction (also removes bacteria)
Apheresis derived platelets8 std units from a single procedure
Reducing transfusion triggersClinical Practice Guidelines
Optimizing Transfusion Indications Audits of blood component usage
Audits by Transfusion Committees
Audits of blood component use have indicated that blood products, both cellular and plasma, are often inappropriately used.