Viable Bacteria Associated with Red Blood Cells and Plasma in Freshly Drawn Blood Donation

Christian Damgaard, Karin Magnussen, Christian Enevold, Martin Nilsson, Tim Tolker-Nielson, Palle Holmstrup, Claus Henrik Nielsen.

Copenhagen University Hospital , Denmark.

​

SN NORLIANI ABDUL KADIR

10 APRIL 2018

​

​

Objective

• Infection remains a leading cause of post-transfusion mortality and morbidity
• The aim study was to identify viable bacteria in standard blood-pack units, with particular focus on bacteria from the oral cavity.
• Determine the distribution of bacteria revealed in plasma and in the red blood cell(RBC)-fraction

Design

• Cross-sectional study
• Blood were separated into plasma and RBC-suspensions, which were incubated anaerobically or aerobically for 7 days on trypticase soy blood agar (TSA) or blue lactose plates.
• For identification colony PCD was performed using primers targeting 16SrDNA

Setting

• Blood donors attending Capital Region Blood Bank,Copenhagen University Hospital, Rigshospitalet, Hvidovre, Denmark, October 29^th to December 10^th 2013

Participants

• 60 donor (≥ 50 years old)
• Self-reported medically healthy

Result

• Bacterial growth was observed on plates inoculated with plasma or RBCs from 62% of the blood donations.
• Growth was evident in 21 (35%) of 60 RBC-fractions and in 32 (53%) of 60 plasma-fraction versus 8 of 60 negative controls (p=0.005 and p=2.6 x 10⁻⁶, respectively.
• Propionibacterium acnes was found in 23% of the donations, staphylococcus epidermidis 38%
• The majority of bacteria identified in the present study were either facultative anaerobic(59.5%),or anaerobic (27.8%) species .
• Which are not likely to be detected during current routine screening.

Conclusions

• Viable bacteria are present in blood from donors self-reported as medically healthy
• Indicating that conventional test systems employed by blood banks insufficiently detect bacteria in plasma
• Further investigation is needed to determine whether routine testing for anaerobic bacteria and testing of RBC-fraction for adherent bacteria should be recommended.

Introduction

​

• Hypothesized that the high frequency of post-transfusional infections is due to unrevealed contamination of donor blood, including RBC fraction that is routinely subjected to screening.
• The aim of study was therefore
• To identify viable bacteria in standard blood-pack units with particular focus on bacteria from the oral cavity
• To determine the distribution of bacteria revealed in plasma and in the RBC-fraction

Methods

1.Sample size

• The present study is cross-sectional
• Sample size was estimated using a two sided power analysis with μ(0) = 40, μ(1) = 10, S = 50, α = 0.005
• The total sample size required for the study was 56.
• The final sample size was , however, adjusted to a total of 60 participants, allowing a rate of 7.5% of eligible subjects, who could withdraw their consent.

2.Ethics

• The study and consent procedure were approved by The Ethics Committee for The capital Region of Denmark
• All donors attended the Capital Region Blood Bank, Copenhagen university Hospital, Rigshospitalet,Hvidovre, Denmark, october 29^th to december 10^th2013
• All donor gave informed written consent prior to blood donation

3.Blood specimen collection

• Blood was drawn from 60 donor self-reported as medically healthy (age 50 years or older)
• From the antecubital vein after topical disinfection with a combination of 20% chlorhexidine gluconate and 70% isopropyl alcohol for 30 second, followed by 30 second drying time, in accordance with who guidelines.
• 30 ml blood drawn was collected into a pre sample bag to minimize the risk of contamination from insertion of the needle.
• The following 450 ml of blood was drawn into triple blood-pack units containing citratephosphate dextrose solution
• The tube connecting the needle with pre sample bag and the 450 ml triple blood pack units was welded off
• The triple blood pack units were then stored at room temperature until fractionation and culturing (with 9 hours)
• The pre sample bag was discarded.

4.Blood fractionation

• The bottom hose on the blood pack unit was disinfected twice with 85% alcohol and afterwards cut with a sterile scissor.
• First 30 ml blood was discarded to minimize risk of contamination from cutting of the hose
• Following were poured directly into two sterile 15 ml tubes, which were then fractioned under sterile conditions into plasma and blood cells by centrifugation.
• The RBC-fraction was washed twice in sterile phosphate buffered saline

5.Isolation of viable bacteria from blood

• 0.5 ml of plasma and 0.5 ml washed RBC-suspension were plated out separately under sterile condition on trypticase soy blood agar (TSA) plates, containing 5mg/L hemin and 50 /L vitamin k, and intubated at 37C under anaerobic conditions in the presence of 10% CO2, 10%H2 and 80% N2, Aaerobically in the presence of 5% CO2
• 0.5ml of each fraction was handles similarly and incubated on blue lactose plates under aerobic condition were incubated at Department of Clinical Microbiology, Rigshospitalet,Copenhagen denmark for 7 days at 37 0c

​

6.Detection of colony forming units

• All plates were examined for colonies after 7 days of incubation
• If positive , the number of colonies was counted and the plate was photographed
• Colonies were then individually transferred to fresh plated to obtain monocultures for identification of species
• The re-plated colonies were incubated for 4 days

7.Colony PCR and 16S rDNA sequence analysis

• For identification of bacteria, colony PCR was performed using primers targeting the bacteria 16S rDNA isolate sequences were compared with taxon sequences in the Human Oral Microbiome database (HOMD), the NCBI database and the Ribosomal Database Project

​

​

8.Statistical methods

• Using fishers exact test, bacterial growth observed on plates with plasma, RBCs or negative control CPD, was compared.
• McNemar test (paired) was used to evaluate differences in number of plates with bacterial growth.
• Mann-Whitney test was used to examine differences between the number of colony forming units on plates with RBC- and plasma fraction, versus growth-positive controls

Result

​

• The species isolated included
• Propionibacterium acnes 23%
• Staphylococcus epidermidis 38%
• Staphylococcus caprae 8%
• Micrococcus Luteus 5%
• Acinetobacter Iwoffii 3%

Discussion

• Several previous studies have focussed on identification of bacteria in patient blood or transfused blood components, once patients developed clinical symptoms TTIs is fever, chills, drop in blood pressure, shock, isolated dyspnea, malaise, anxiety and digestive distress
• In direct blood agar culture 77% were positive for bacteria and bacteria found in blood components were Gram-negative rods in 46 cases, Gram positive cocci in 28% and Gram positive rods in 21%

• Studies based on recognized TTIs likely to underestimate the frequency of bacteremia for a number of reason
• Studies are often based on standard methods for bacterial screening only
• If none of the transfused blood components are available, patient are excluded from the studies
• Patient may be receiving antimicrobials at the time of blood culture.
• Participation may be non-uniform and often voluntary
• Fever and other symptoms may not be interpreted as caused by bacteria
• Bacteremia is presumably often asymptomatic in immunocompetent individuals

​

• At lease 3 factors may contribute to the high frequency of contaminated blood product found in study. Donor has 50 year and older , criterion increase the risk of unreported infections such as periodontitis, which might explain the high prevalence of bacterial growth detected.
• The majority of bacteria identified in the present study were either facultative anaerobic 59.5% or anaerobic 27.8% species which are not likely to be detected using current screening procedures.
• The RBC fraction is not routinely tested for contamination.

​

​

​

​

• Larger studies are required to confirm our findings, and since symptomatic TTIs are rare, screening of RBC preparation and cultivation under anaerobic conditions are probably not recommendable in general

​

CASES IN PDN

• Have 6 case in 2017
• 2 positive case.
• 1 case with Diptheroids
• 1 case with corynebacterium spp (ubiguitous in nature and commonly colonies human skin and mucous membrane)

Bacterial contamination case in pdn

• Possible source of contamination
• Blood donor
• Unwell during donation/ asymptomatic bacteremia
• Phlebotomist
• Improper technique of infection control measure
• Equipment storage
• Contamination of blood bag or glove prior to blood collection
• Processing
• Improper blood preparation causing contamination
• Blood storage
• Contamination during storage of blood in production and inventory division
• Quality laboratory
• Improper sampling of blood component during culture

​

Recommendation

• Blood collection unit
• To provide adequate training to all staffs regarding infection control on regular basis by infection control team.
• Audit of infection control to be check on adherence of staffs in infection control SOP during blood collection including
• aseptic technique hand rub before and after donation,
• Use glove
• skin disinfection with alcohol swab until clean before blood collection
• Use new gloves were used in contact with each donor
• Proper storage of glove and avoid contamination

​