| Abstract|| |
BACKGROUND: Posttransfusion bacterial sepsis is mostly attributed to platelets and rarely red blood cell concentrates (RBCC). However, bacterial screening of both of these blood components can mitigate the risk of transfusion-associated hazards.
AIMS: The study was aimed at evaluating the prevalence of bacterial contamination in whole blood-derived platelets and RBCC in a regional blood bank in northern Pakistan.
SETTINGS AND DESIGN: A cross-sectional study was carried out at a regional transfusion center in Rawalpindi from January 2019 to 2020.
MATERIALS AND METHODS: A total of 400 cellular blood components were tested aerobically and anaerobically for bacterial contamination using BD BACTEC™ FX 40 automated culture system. Over the period of incubation, culture vials that showed positive signals were subcultured onto a set of solid media followed by bacterial identification.
STATISTICAL ANALYSIS USED: Data analysis was performed using SPSS version 21.0 (Chicago, Illinois, USA). The overall prevalence of bacterial contamination was reported as a percentage of positive whole blood-derived platelets and RBCC.
RESULTS AND CONCLUSION: Of 400 cultured components, 1% whole blood-derived platelets were contaminated with Gram-positive bacteria including coagulase-negative Staphylococcus species and Staphylococcus aureus, whereas none of the RBCC were found to be contaminated with any bacteria. A significant percentage of platelets had bacterial contamination, whereas no unit of red blood cells was found to be bacterially contaminated. Therefore, strict adherence to standard operating procedures is required to avoid the risk of contamination of blood products.
Keywords: Blood platelet transfusion, blood platelets, red blood cell transfusion
|How to cite this URL:|
Rathore MA, Naeem MA, Javed A, Raja MI. Bacterial contamination of platelets and red blood cell concentrates: A regional transfusion center study in Pakistan. Asian J Transfus Sci [Epub ahead of print] [cited 2022 Dec 4]. Available from: https://www.ajts.org/preprintarticle.asp?id=345978
| Introduction|| |
Transfusion-transmissible infections (TTIs) are a public health problem, especially in countries with a high prevalence of these diseases in the general population. Although TTIs caused by viruses including hepatitis B virus, hepatitis C virus, and human immunodeficiency virus are on the decline because of employment of very sensitive detection assays over the years, the bacterial infections post transfusion still pose a significant threat to safe blood supply. Platelet concentrate (PCs) transfusion is a vital lifesaving therapy in different pathological conditions such as thrombocytopenia, bleeding, or malignancies and is associated with greater risk of bacterial sepsis and death owing primarily to conducive storage conditions (22°C) as compared to red blood cell concentrates (RBCC) which are stored a 4°C. The initial low bacterial inoculum in blood products at the time of collection can reproduce exponentially within a short period of time. Therefore, 1 in 5000 platelet units carries a risk of bacterial contamination.,, Transfusion of bacterially contaminated blood units may have variable clinical sequelae depending on the type of contaminant, recipient's condition, and timing of transfusion. The predominant bacterial contaminants implicated in platelet contamination are resident and transient skin flora comprising Gram-positive bacteria such as Staphylococcus species, Propionibacterium, and Bacillus species; however, enteric contaminants are also detected in rare but fatal cases. Among these, biofilm-forming species can adhere to biological and nonbiological surfaces and remain undetected by automated culture screening systems. However, RBCC screening is usually not performed routinely although septic reactions following contaminated RBCC transfusion have been reported. Spectrum of red blood cell contaminants varies as most bacteria do not survive at low temperature. Psychrophilic Gram-negative bacterial species are the major implicated contaminants that replicate slowly under refrigerated conditions and require extended incubation periods. Several preventive strategies have been adopted to mitigate the risk of bacterial contamination encompassing donor screening, initial aliquot diversion, effective skin disinfection, pathogen reduction techniques, and bacterial testing of blood components by automated detection systems, but still, it is difficult to achieve zero risk transfusion. A study from the National Institute of Blood Disease and Bone Marrow Transplantation Karachi, Pakistan, demonstrated that among acute posttransfusion reactions, 12.5% were reported due to bacterial contamination of platelet products, while red blood cells were found free of any contamination. The pretransfusion bacterial testing of blood components in Pakistan is limited due to financial constraints associated with expensive automated culture methods and relatively longer periods required for incubation. Hence, this study was intended to determine the prevalence of bacterial contamination in cellular blood products by an automated blood culture system.
| Materials and Methods|| |
A single-center cross-sectional study was conducted at a regional transfusion center in Rawalpindi after approval was granted from the ethics review board of the institute. The study period was spanned from January 2019 to January 2020. The sample size was calculated using Kish Leslie's formula with 95% confidence interval, margin of error d = 0.01, and anticipated prevalence P = 1%. The sample size determined was 396 and a total of 400 blood products, being derived from Whole bood (WB) by centrifugation method, were randomly selected for this study. Collection bags (Triple Blood Bags without Diversion pouch, Terumo, Vietnam) containing 63 mL CPDA-1 anticoagulant were used. A two-step disinfection process was performed during phlebotomy. Antecubital fossa site was first swabbed with 7% povidone-iodine for 30 s followed by 70% isopropyl alcohol for another 30 s according to the World Health Organization protocol. Subsequently, whole blood was collected and processed into components. Sampling was performed after a storage period of 2 days for PCs and 28 days for RBCC that were negative on routine screening of TTIs including HIV-I/HIV-II (anti-HIV, p24), HBV (HBsAg), HCV (anti-HCV), and syphilis (anti-treponema pallidum antibodies). Blood products were mixed well by inversion and the site of sample collection was disinfected using 70% isopropyl alcohol. A 10 mL sample from stored units of PC and RBCC was collected using sterile syringes (STAR, CHINA) and inoculated aseptically into BD BACTEC™ plus aerobic and anaerobic blood culture vials. These vials were incubated in the automated BACTEC™ FX 40 (Becton, Dickinson and Company, USA) broth culture system at 35°C until positive and up to 7 days for negative vials. Negative controls were run at the same time by inoculating 10 mL of sterile distilled water into BD BACTEC™ FX 40 aerobic vials. Culture vials that showed positive signals were seeded onto blood agar, MacConkey agar, and chocolate agar. Incubation conditions for culture plates were as follows: blood and MacConkey agar plates were aerobically incubated at 37°C for 24–48 h, while chocolate agar plates were placed in candle jars at 37°C for 48 h. Bacterial identification was carried out using standard microbiological tests. The blood products were discarded after sampling according to the institutional standard operating procedures (SOPs).
| Statistical analysis|| |
Data analysis was performed using SPSS version 21.0 (Chicago, Illinois, USA). The overall prevalence of bacterial contamination was reported as a percentage of positive whole blood derived platelets and RBCC.
| Results|| |
A total of 400 stored units of blood components (PCs n = 200, RBCC n = 200) were randomly collected and cultured using an automated BD BACTEC™ FX 40 culture system. The overall prevalence of bacterial contamination in cellular blood components was observed as 0.5% (n = 2/400). This was statistically significant with P = 0.0001 (P < 0.05) calculated by statistix version 9.0 using Chi-square test. Of these, 1% of platelets which correspond to two platelet bags were contaminated, whereas no bacterial contamination was detected in any unit of red blood cells. The instrument detected positivity only in aerobic culture vials inoculated with platelet sample against which graph is shown [Figure 1]. The subcultured platelet specimen revealed the growth of Gram-positive bacterial contaminants which were subsequently identified as coagulase-negative Staphylococcus species (CoNS) and Staphylococcus aureus.
|Figure 1: The graph of a positively flagged culture vial showing distinct phases of bacterial growth such as lag, exponential, and stationary phase. The Y-axis represents fluorescence units (x, s) and positivity readings (small squares), whereas X-axis represents time-to-detection which is shown as days; hours (dd; hh). The level of fluorescence units corresponded to the amount of carbon dioxide (CO2) generated by the metabolic activities of cultured microorganism. The positivity line shifted from low to high across the fluorescence readings when positivity was declared|
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| Discussion|| |
Safe blood supply is still a major challenge. RBCC screening is not a routine practice; therefore, detection of bacterial contamination in only PCs is the main objective of several studies. This is due to platelet storage at room temperature (22°C ± 2°C) under continuous agitation which effectively promotes the bacterial growth as compared to red blood cell concentrates which are stored at 4°C. The predefined objective of our study was to detect bacterial contamination in WB derived PCs and RBCC. In the present cross-sectional study, the rate of contamination in PCs was detected as 1% (n = 2/200) which is comparable with the findings of a study conducted by Noorulamin et al. in the same country. However, a study from Zimbabwe illustrated the highest susceptibility of platelets toward bacterial contamination than any other blood component. The rate of contamination of tested platelets was reported as 0.2% in a study from Iran followed by the identification of diverse microbial species implicated in contamination. By contrast, various studies from developed countries demonstrated relatively low prevalence such as 0.04% in New Zealand, 0.18% in Australia, and 0.22% in China.,, In disagreement with the current study, a study from North America (9%) and Nigeria (8.8%) demonstrated the highest prevalence of bacterial contamination in platelets., This comparison illustrates that the prevalence of bacterial contamination varies considerably, being very low in developed countries and high in developing countries. Nevertheless, variability in sample size, culture procedures, true positivity criteria, and type of PC may also account for differences in the contamination rate.
The PCs can be either obtained from whole blood collections or apheresis procedure. The bacterial contamination rate can vary by the type of PCs. Pooling of whole blood-derived PCs from 4 or 5 donors could expectedly increase chances of bacterial contamination as compared to apheresis PCs which are from single donor. Referring to recent meta-analysis, apheresis PCs showed lower contamination rate than WBDPCs. However, study from Canada showed no difference in contamination rate between WBDPCs and apheresis platelets.
Transfusion of contaminated platelets in immunocompromised individuals poses an additional problem and can lead to severe adverse reactions. Bacterial contamination might occur during any stage of whole blood collection, storage, processing, transport, or can originate from contaminated collection equipment and asymptomatic bacteremia in donor. Type of contaminants isolated in this study included Gram-positive bacteria such as CoNS and S. aureus. Implicated CoNS suggest that the most likely source of contamination was inadequate disinfection of the donor's arm during phlebotomy and skin plug removal by the collection needle. In addition, S. aureus also colonizes forearm skin and is commonly isolated from donors with skin diseases which emphasizes the need for deferral of donors with skin conditions.
Red blood cell units are less susceptible to contamination by these bacteria due to cold storage conditions (2°C–6°C) which suppress their growth by reducing their growth kinetics. The detection strategy was implemented depending on the growth characteristics of different bacterial species contaminating the blood supply. Bacterial concentrations are initially very low immediately after blood collection within 16–22 h, which is the reason for postdonation hold period for platelets to carry out effective sampling and inoculation. However, psychrophilic Gram-negative bacterial species which exhibit long lag phase have been known to cause significant contamination at low temperatures (4°C). The long lag phase influences the reliability of screening test, thus RBCC was sampled after 28 days for better detection. There is a paucity of recently published literature on the detection of RBCC contamination and prevalence is either based on posttransfusion septic reactions or data retrieved during sterility testing. Although rare, fatalities due to transfusion of contaminated RBCC have been documented by the Food and Drug Administration. This demonstrates that pretransfusion testing of RBCC should be considered as a mandatory measure to improve blood safety. The most recent study from northern Ethiopia showed a 4.6% rate of contamination in fresh red cell concentrate units among other blood components which are in sharp contrast to the current study.
These findings underline the need for various approaches including diversion of an initial aliquot of whole blood, stringent donor selection to avoid the risk of bacteremia, visual inspection of blood bags prior to release, and deferring donors with skin conditions or scarred phlebotomy site due to which sterile venipuncture cannot be achieved. Especially, the use of diversion pouches during phlebotomy has become a standard practice to mitigate the risk of bacterial contamination. Diversion of an initial aliquot of whole blood, approximately 20–30 mL, into diversion pouches attached to the blood collection system may help to reduce the transmission of skin bacterial flora into the primary collection bag and blood components., In a study from Japan, the contamination rate was reduced from 0.17% to 0.05% for PCs. This serves as an effective strategy to reduce the rate of bacterial contamination.
Apart from these procedures, pathogen reduction technologies should be implemented after carrying out cost-effective analysis. Therefore, it is necessary to monitor and guide the phlebotomy services and to motivate the staff for adhering to SOPs, especially during phlebotomy. This is imperative because septic transfusion reactions are mostly underreported and unrecognized due to the lack of hemovigilance programs. The prevalence data related to posttransfusion septic episodes are usually not established and improvement in transfusion safety can be achieved by improving the quality of blood products rather than assessing clinical consequences.
Limitation of the study
We conducted a single-center study; therefore, multicenter study with a large sample size is needed to achieve sufficient evidence to confirm these findings.
| Conclusion|| |
The results of this study showed that a significant percentage of platelets had bacterial contamination, whereas no RBCC had bacterial contamination. The prevalence of bacterial contamination in platelets stipulates active surveillance with strict adherence to standard protocols to avoid contamination during whole blood collection and processing procedures. We recommend detection of bacterial contamination of blood components using an automated culture system in combination with the above-mentioned sampling strategy. Furthermore, a single-culture test would be a cost-effective strategy owing to resource constraints in our country.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Muhammad Ali Rathore,
Armed Forces Institute of Transfusion, Sher Khan Road, Rawalpindi
Source of Support: None, Conflict of Interest: None