Transcript Screening of Pooled Platelets

Bacterial Contamination and
Screening of Platelet Concentrates
James X. Gray, BSc(Hons), PhD, MD
Haematology Registrar
The Alfred Hospital
Case from SHOT annual report 2004
A 75 year old female patient with chronic lymphatic leukaemia
developed rigors, vomiting and pyrexia following transfusion
of a 5-day old pooled platelet unit. The transfusion was
terminated and the patient recovered. An identical strain of
S.epidermidis was isolated from the transfused platelet pack
and from the venepuncture site of one of the four contributing
donors. However, the organism was not isolated from the
recipient following the reaction. This is evidence of bacterial
contamination 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.
Differential Centrifugation and Separation
Whole Blood + Preservative
(450 ml +/- 45 ml)
Specific Gravity
RBC
1.08 – 1.09
Platelet 1.03 – 1.04
Plasma 1.023
Hard Spin
Red Cells
Buffy Coat
Plasma
Pooled x 4
Soft Spin
Pelleted Red Cells
Platelets
Blood Product Testing
Sydney Red Cross(TGA accreditation)
Pathogen Screen test
window / d
Risk
HIV 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 330,000
HCV
serology / NAT
07
1 in 3.7 x 106
HBV
serology
45
HTLV I&II serology
51 <1 in 2.4 x 106
CMV
serology
Syphilis
serology
Malaria
serology
vCJD
questionairre
none reported
Bacterial culture of Platelets (20%)
Quality Control Specifications
Platelet Concentrates: pooled x 4, leukodepleted
TGA requirements (Council of Europe Guidelines):
Parameter
Volume
Specification Acceptance
160-240 ml
Sampling
75 %
1%
Platelets
(per unit)
> 240 x 109
75 %
1%
Leucocytes
(per unit)
pH
< 0.8 x 106
90 %
1%
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 of
Platelets With the BacT/ALERT System:
A 30-Month Experience From the
Sanquin 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 of
Platelet Concentrates:
A 4-Year Experience in
Belgian Blood Centers
L Muylle
Red Cross-Flanders Blood Service, Mechelen, and
University of Antwerp, Antwerp, Belgium
Muylle, (Belgium)
Transfusion Medicine Reviews, vol 19 (4)
Arm disinfection
Closed system for platelet preparation
Pooled (5-6 patients)
Apheresis platelets
No diversion
Automated culture - Bact T/alert
100% screening
108,000 platelet concentrates over 4 years
Test Results of the Bacterial Screening of Platelet Units
for 5 days using BacT/alert detection
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%)
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 clinicians
12 transfusion reactions - sepsis x 2
fever
hypotension
rigors
rash
x6
x1
x1
x2
Bacterial Screening of Buffy Coat–Derived
Platelet 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 Coat
Platelet 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% screening
False 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 safety
of platelet transfusions
Morris A. Blajchman , Mindy Goldmanc and Federico Baezad
a
Departments of Pathology and Medicine,
McMaster University, Hamilton, Ontario, Canada
b Canadian Blood Services, Hamilton, Ontario, Canada
c Canadian Blood Services, Ottawa, Canada
d 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 Systems
Detection 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
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 UVA
Demonstrated 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 screening
Donor questionnaire for subclinical bacteremia
Improve venepuncture site disinfection
Antiseptic - quality
- quantity
- mode of application
Diversion Pouch –
13.5 ml to 30 ml
40%, 72%, 90%
Reducing Risk of Platelet Contamination 2
Platelet storage
most TA septic events occur with units > 3d
early use while bacterial load is sub-clinical.
Universal Leukoreduction (also removes bacteria)
Apheresis derived platelets
8 std units from a single procedure
Reducing transfusion triggers
Clinical 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.