Bacterial Contamination and Screening of Platelet Concentrates James X. Gray, BSc(Hons), PhD, MD Haematology Registrar The Alfred Hospital.

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.