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Updated: Dec 10, 2025
Routine Screening Method for Microparticles in Platelet Transfusions
Published on: January 31, 2018
Optimized broad-range real-time PCR-based method for bacterial screening of platelet concentrates
F Alexandrino1, J S Malgarin1, M A Krieger1
1Instituto de Biologia Molecular do Paraná - IBMP, Rua Prof. Algacyr Munhoz Mader, 3775, CEP 81350-010, Curitiba, PR, Brasil.
Abstract:
Bacterial contamination of blood components remains a major challenge in transfusion medicine, particularly, platelet concentrates (PCs) due to the storage conditions that support bacterial proliferation. In this study, we develop a rapid, sensitive and specific real-time PCR protocol for bacterial screening of PCs. An internally controlled real-time PCR-based method was optimized and validated with our proprietary 16S Universal PCR Master Mix (IBMP/Fiocruz), which targets a conserved region of the bacterial 16S rRNA gene. Nonspecific background DNA was completely eliminated by treating the PCR Master Mix with ethidium monoazide (EMA). A lower limit of detection was observed for 10 genome equivalents with an observed Ct value of 34±1.07 in calibration curve generated with 10-fold serial dilutions of E. coli DNA. The turnaround time for processing, including microbial DNA purification, was approximately 4 hours. The developed method showed a high sensitivity with no non-specific amplification and a lower time-to-detection than traditional microbiological methods, demonstrating it to be an efficient means of screening pre-transfusion PCs.
Insights
A new real-time PCR method rapidly screens platelet concentrates (PCs) for bacterial contamination. This sensitive assay provides results in approximately 4 hours, enhancing transfusion safety.
Area of Science:
- Transfusion Medicine
- Microbiology
- Molecular Diagnostics
Background:
- Bacterial contamination of blood components, especially platelet concentrates (PCs), is a significant risk in transfusion medicine.
- Storage conditions for PCs can promote bacterial growth, increasing the risk of transfusion-transmitted infections.
Purpose of the Study:
- To develop and validate a rapid, sensitive, and specific real-time PCR protocol for screening bacterial contamination in platelet concentrates.
- To improve the safety of transfused blood products by enabling timely detection of bacterial presence.
Main Methods:
- Development of an internally controlled real-time PCR assay targeting the bacterial 16S rRNA gene.
- Optimization using a proprietary 16S Universal PCR Master Mix and ethidium monoazide (EMA) to eliminate background DNA.
- Validation of the assay's sensitivity, specificity, and turnaround time.
Main Results:
- The real-time PCR protocol demonstrated high sensitivity, with a lower limit of detection at 10 genome equivalents (Ct value of 34±1.07).
- The method exhibited no non-specific amplification, ensuring specificity.
- The total processing and detection time, including DNA purification, was approximately 4 hours.
Conclusions:
- The developed real-time PCR method is a rapid, sensitive, and specific tool for screening bacterial contamination in platelet concentrates.
- This assay offers a significant reduction in detection time compared to traditional microbiological methods.
- Implementation of this method can enhance the safety of pre-transfusion platelet products.
