Optimization of cord blood unit sterility testing: impact of dilution, analysis delay, and inhibitory substances

Abstract

BACKGROUND: Different methods are used by cord blood banks to prepare samples for sterility testing. Suboptimal methods can result in the release of contaminated products. In our organization, samples are prepared by diluting the final product in RPMI-1640 medium. In this work, we have compared our method with different approaches to verify whether optimization should be sought. STUDY DESIGN AND METHODS:Cord blood units (n 5 6 units per bacterial strain) characterized to contain inhibitory substances or not were inoculated (10 colonyforming units/mL) with Streptococcus agalactiae, Staphylococcus epidermidis, Klebsiella pneumoniae, Escherichia coli, or Bacteroides fragilis. After plasma and red blood cell removal, stem cell concentrates were diluted in RPMI-1640, thioglycollate, or the unit's plasma. These products, as well as final product, plasma, and red blood cell fractions, were held from 0 to 72 hours at 20 to 248C before inoculation in culture bottles and detection using the BacT/ALERT 3D system. RESULTS: Dilution of cell concentrates in RPMI-1640allowed bacterial detection in 93.3% of noninhibitory cord blood samples after a 24-hour storage period. Thioglycollate medium better promoted bacterial growth in inhibitory cord blood samples that were held for 72 hours before testing (66.7%) compared with RPMI-1640 (45.0%). Less than 33% of all spiked plasma samples were detected by the BacT/ALERT 3D system. CONCLUSION:Diluting cord blood samples in culture medium containing bacterial growth promoting substances is a suitable option for sterility testing, whereas the use of plasma should be proscribed, because it might lead to false-negative results. Because inhibitory substances affect bacterial growth, inoculation of culture bottles should be done rapidly after sample preparation. U mbilical cord blood (CB) transplantation is indicated for severely immunocompromised patients. These patients have a high risk of contracting infections associated with the transplant. Given the nonsterile collection environment and the numerous procedures carried out before transplantation, microbial contamination of cord blood units (CBUs) is more frequent than for any other blood product.1-3 Consequently, the efficient microbial screening of CB donations remains a primary safety concern for cord blood banks (CBBs) while also being required by international accreditation organizations, such as the American Association of Blood Banks and the Foundation for the Accreditation of Cellular Therapy. 4,5 The development of standardized procedures for microbial screening of CBUs is still being awaited. The lack of consensus standards led to CBBs developing their own method for microbial screening. Generally, a sample of the final volume-reduced CB product is used for sterility testing; however, postprocessing plasma, red blood cell (RBC) fraction, a mixture of these by-products, or a small volume of the final stem cell concentrate (which includes the cryopreservation solution) are also used for microbial ABBREVIATIONS: ATCC 5 American Ty...

Development of a proof of principle for universal neutralization of antibiotics in cord blood by‐products used for sterility testing

“…Our validation of microbial testing of CB units at Canadian Blood Services' Cord Blood Bank revealed that the BacT/ALERT 3D system is able to detect bacterial contamination in a mix of the by‐products plasma and RBCs obtained during CB processing if the units do not contain antibiotics . It is reported that approximately 30% of CB donated in Canada and the United States contain antibiotics . Furthermore, it has been documented that CB units believed to be microbe free could indeed be contaminated, posing an infectious risk to transplant recipients .…”

“…Although serious septic cases in transplant patients are rare because of patient antibiotic treatment, there have been reports of transplantation reactions, ranging from nonserious to fatal, due to contaminated hematopoietic stem cell products . Importantly, isolation of antibiotic‐resistant bacteria from CB units, which would pose a high septic risk to transplantation patients, has been reported …”

“…[16][17][18][19][20][21] Importantly, isolation of antibiotic-resistant bacteria from CB units, which would pose a high septic risk to transplantation patients, has been reported. 2,11 The incidence of contaminated CB varies widely, and the rate of contamination is higher when open collection systems are used in comparison to collections by venipuncture into a closed CB collection bag. 5 Overall, microbial safety of CB units possesses challenges that are only partly addressed with current testing practices.…”

“…In a recent study, Girard and colleagues clearly described the challenges to optimize sterility CB testing methods, which are attributable to inhibitory substances, including antibiotics, present in CB units. 11 Antibiotic neutralization is achieved in most CB banks by the use of resin-containing culture bottles specific for the system used for CB sterility testing at the bank. Two commercial systems offer the option of using culture bottles with antibiotic neutralizing properties, the BACTEC and the BacT/ ALERT systems.…”

“…9 The most commonly used antibiotics include b-lactams such as penicillin and aminoglycosides such as gentamicin. [9][10][11] Neutralization of antimicrobials in CB units is recommended for an optimal sterility testing process, 12 a practice followed by several CB banks, including CB banks at H ema-Qu ebec 9 and Australia, 5 and the St Louis Cord Blood Bank & Cellular Therapy Laboratory. 13 The present study was aimed at developing an in-house method that can be used in any culture testing system for neutralizing antibiotics in the mixture of by-products plasma and red blood cells (RBCs) used for CB sterility testing at Canadian Blood Services' Cord Blood Bank.…”