Time from apheresis platelet donation to cold storage: Evaluation of platelet quality and bacterial growth.

BACKGROUND: Cold storage reduces posttransfusion survival of platelets; however, it can improve platelet activation, lower risk of bacterial contamination, and extend shelf-life compared to room temperature (RT) storage. To facilitate large-scale availability, manufacturing process optimization is needed, including understanding the impact of variables on platelet potency and safety. Short time requirements from collection to storage is challenging for large blood centers to complete resuspension and qualify platelets for production. This study evaluated the impact of time from platelet component collection to cold storage on in vitro properties and bacterial growth. STUDY DESIGN AND METHODS: Double-apheresis platelet components were collected from healthy donors, suspended in 65% PAS-III/35% plasma, and split into 2 equal units. One unit was placed into cold storage within 2 h and the other unit after 8 h. Eight matched pairs were evaluated for 12 in vitro parameters. Twenty-four matched pairs were evaluated with 8 bacterial strains tested in triplicate. Samples were tested throughout 21 days of storage. RESULTS: In vitro properties were not different between 2 and 8 h units, and trends throughout storage were similar between arms. Time to cold storage did not significantly impact bacterial growth, with <1 log10 difference at all timepoints between units. DISCUSSION: Our studies showed that extending time to cold storage from 2 to 8 h from collection did not significantly increase the bacterial growth, and the platelet component quality and function is maintained. The ability to extend the time required from collection to storage will improve blood center logistics to feasibly produce CSPs.

Flow-based analysis of cell division identifies highly active populations within plasma products during mixed lymphocyte cultures.

BACKGROUND: Leukoreduction to eliminate mononuclear cells within blood products is necessary to prevent graft-versus-host disease after transfusion. Published reports document low concentrations of mononuclear cells leftover in fresh-frozen plasma products, however the phenotype and the proliferative potential of these cells has not been tested. MATERIALS AND METHODS: We investigated residual cellular components contained within fresh and fresh-frozen plasma products and characterised their proliferative potential in co-cultures with unrelated allogeneic cells. We designed a flow-based assay to phenotype cells and quantify cell division by measuring the dilution of fluorescently labeled protein as cells divide. Leukocytes from consenting donors were purified from fresh liquid or fresh-frozen plasma units and cultured for three to seven days with unrelated irradiated allogeneic targets. RESULTS: We discovered a median of 1.6×107 viable lymphocytes were detectable in fresh plasma units after collection (n=8), comprised of a mixture of CD3+ CD8+ and CD3+ CD4+ cells. Furthermore, we identified a median of 8.4% of live CD3+ plasma lymphocytes divided as early as Day 4 when co-cultured with unrelated allogeneic cells, expanding to a median 88.8% by Day 7 (n=3). Although freezing the plasma product reduced the total number of viable leukocyte cells down to 2.3×105 (n=10), residual naive CD3+ cells were viable and demonstrated division through Day 7 of co-culture. DISCUSSION: The evidence of viable proliferative lymphocytes in fresh and fresh-frozen plasma products derived from centrifugation suggests that additional leukoreduction measures should be investigated to fully eradicate reactive lymphocytes from centrifuged plasma products.

Fatal sepsis associated with a storage container leak permitting platelet contamination with environmental bacteria after pathogen reduction.

BACKGROUND: Pathogen reduction technology and enhanced bacterial culture screening promise to significantly reduce the risk of transfusion-associated septic reactions due to contaminated platelets. Recent reports suggest that these interventions lack efficacy for post-collection and processing contamination with environmental organisms if the storage bag integrity is compromised. CASE REPORT: We report a fatal septic transfusion reaction in a 63-year-old patient with chronic kidney and liver disease who received a pathogen reduced platelet transfusion in anticipation of surgery. METHODS: The residual platelet concentrate was cultured, with the detected microorganisms undergoing 16S genotype sequencing. Separate pathogen reduction studies were performed on the recovered bacteria, including assessment for amotosalen photoproducts. The storage container was subjected to pressure testing and microscopic examination. Environmental culture screening was performed at the hospital. RESULTS: Gram negative rods were detected in the platelet unit and cultures of both platelet component and the patient's blood grew Acinetobacter baumannii complex, Leclercia adecarboxylata and Staphylococcus saprophyticus. These strains were effectively inactivated with >7.2, 7.7, and >7.1 log10 kill, respectively. The platelet storage container revealed a leak visible only on pressure testing. Hospital environmental cultures were negative and the contamination source is unknown. A. baumannii complex and S. saprophyticus 16S genotyping sequences were identical to those implicated in a previously reported septic reaction. CONCLUSION: Findings are compatible with post-processing environmental contamination of a pathogen reduced platelet concentrate via a non-visible, acquired storage container leak. Efforts are warranted to actively prevent damage to, and detect defects in, platelet storage containers, and to store and transport components in clean environments.

Establishment of transfusion-relevant bacteria reference strains for red blood cells.

BACKGROUND AND OBJECTIVES: Red blood cell concentrates (RBCC) are susceptible to bacterial contamination despite cold storage. A reliable evaluation of strategies to minimize the risk of RBCC-associated bacterial transmission requires the use of suitable reference bacteria. Already existing Transfusion-Relevant Bacteria Reference Strains (TRBRS) for platelet concentrates fail to grow in RBCC. Consequently, the ISBT TTID, Working Party, Bacterial Subgroup, conducted an international study on TRBRS for RBCC. MATERIALS AND METHODS: Six bacterial strains (Listeria monocytogenes PEI-A-199, Serratia liquefaciens PEI-A-184, Serratia marcescens PEI-B-P-56, Pseudomonas fluorescens PEI-B-P-77, Yersinia enterocolitica PEI-A-105, Yersinia enterocolitica PEI-A-176) were distributed to 15 laboratories worldwide for enumeration, identification, and determination of growth kinetics in RBCC at days 7, 14, 21, 28, 35 and 42 of storage after low-count spiking (10-25 CFU/RBCC). RESULTS: Bacterial proliferation in RBCC was obtained for most strains, except for S. marcescens, which grew only at 4 of 15 laboratories. S. liquefaciens, S. marcescens, P. fluorescens and the two Y. enterocolitica strains reached the stationary phase between days 14 and 21 of RBCC storage with a bacterial concentration of approximately 109  CFU/ml. L. monocytogenes displayed slower growth kinetics reaching 106 -107  CFU/ml after 42 days. CONCLUSION: The results illustrate the importance of conducting comprehensive studies to establish well-characterized reference strains, which can be a tool to assess strategies and methods used to ameliorate blood safety. The WHO Expert Committee on Biological Standardization adopted the five successful strains as official RBCC reference strains. Our study also highlights the relevance of visual inspection to interdict contaminated RBC units.

Validation of a SCID mouse model for transfusion by concurrent comparison of circulation kinetics of human platelets, stored under various temperature conditions, between human volunteers and mice.

BACKGROUND: Initial evaluation of new platelet (PLT) products for transfusion includes a clinical study to determine in vivo recovery and survival of autologous radiolabeled PLTs in healthy volunteers. These studies are expensive and do not always produce the desired results. A validated animal model of human PLTs in vivo survival and recovery used pre-clinically could reduce the risk of failing to advance product development. STUDY DESIGN AND METHODS: An immunodeficient (SCID) mouse model to evaluate recovery of human PLTs was compared to a radiolabeling study in human volunteers. Autologous apheresis PLTs stored for 7 days at room temperature (RT), thermo-cycled (TC), and cold temperature (CT) were radiolabeled and infused into healthy humans (n = 16). The same PLTs, non-radiolabeled, were also infused into mice (n = 160) on the same day. Blood samples from humans and mice were collected to generate clearance curves of PLTs in circulation. Flow cytometry was used to detect human PLTs in mouse blood. RESULTS: Human and mouse PLTs were cleared with one phase exponential clearance. Relative differences for initial recovery and AUC, expressed as ratio of test and control PLTs, were similar in humans and mice. The initial recovery ratio of TC/RT was 0.73 ± 0.07 in humans and 0.67 ± 0.14 in mice. The ratio for CT/TC was 0.53 ± 0.06 in humans and 0.75 ± 0.18 in mice. CONCLUSION: The SCID mouse model can provide information on relative differences of initial in vivo recovery and AUC between control and alternatively stored/processed human PLTs that is predictive of performance in healthy human volunteers.

Preliminary characterization of the properties of cold-stored apheresis platelets suspended in PAS-III with and without an 8-hour room temperature hold.

BACKGROUND: Use of extended cold storage of platelets promises to increase PLT availability and the bacterial safety of bleeding patients. No information is currently available on the preservation of apheresis PLT in vitro quality parameters when PLTs are held at room temperature early in the storage period prior to transfer to cold storage. STUDY DESIGN AND METHODS: Double units of platelets suspended in 35% plasma/65% PAS-III were collected from normal consenting research donors and rested at room temperature for 1-2 hours. One of the units was then stored at 1-6°C while the other unit was placed on an agitator at 20-24°C. Eight hours after collection, the unit stored at room temperature was transferred to 1-6°C storage without agitation. Units were sampled for an array of PLT in vitro parameters on Days 1, 7, 14, and 21. RESULTS: As expected, PLTs held for 8 hours at 20-24°C prior to 1-6°C storage had greater lactate levels and reduced glucose levels and pH compared to PLTs subjected to a 1-2-hour room temperature hold prior to cold storage (P < .05). Unexpectedly, platelets held for 8 hours at room temperature had less aggregation response to collagen, ADP, and TRAP compared to PLTs held 1-2 hours at room temperature prior to cold storage (P < .05, n = 8). CONCLUSION: Decline of aggregation response should be considered when evaluating longer than necessary room temperature holds prior to cold storage of platelets.

Sepsis from an apheresis platelet contaminated with Acinetobacter calcoaceticus/baumannii complex bacteria and Staphylococcus saprophyticus after pathogen reduction.

BACKGROUND: Strategies to reduce platelet (PLT) bacterial contamination include donor screening, skin disinfection, sample diversion, bacterial culture, pathogen reduction (PR), and day-of-transfusion tests. We report bacterial sepsis following a pathogen-reduced PLT transfusion. CASE REPORT: An adult male with relapsed acute lymphoblastic leukemia was successfully treated for central catheter-associated Staphylococcus aureus bacteremia. A peripherally inserted central catheter (PICC) was placed. Chills, rigors, and flushing developed immediately after PICC-infused pathogen-reduced PLTs, progressing to septic shock requiring intensive care management. METHODS: PICC and peripheral blood (PB), transfused bag saline flushes (TBFs), environmental samples, and the pathogen-reduced untransfused co-component (CC) were cultured. Plasma metagenomic and bacterial isolate whole-genome sequencing; PLT mitochondrial DNA (mtDNA) testing of untransfused CC and TBF; CC testing for amotosalen (S-59)/S-59 photoproducts; isolate PR studies (INTERCEPT); and TBF polymerase chain reaction for recipient Y-chromosome DNA were performed. RESULTS: PB and PICC cultures grew Acinetobacter calcoaceticus/baumannii complex (ACBC). TBF was gram-positive; mass spectrometry identified ACBC and Staphylococcus saprophyticus (SS). CC Gram stain and cultures were negative. Environmental cultures, some done after decontamination, were ACBC/SS negative. Posttransfusion patient plasma and TBF ACBC sequences were genetically identical. No Y-chromosome signal was detected in TBF. S-59 photoproducts and evidence of mtDNA amplification inhibition were found in the CC. Spiking PR studies showed >5.9-log inactivation for both isolates. Donor skin cultures for Acinetobacter were negative. CONCLUSION: CC sterility, PR studies, residual S-59 photoproducts, and mtDNA amplification inhibition suggest successful PR. Unidentified environmental sources and inherent or acquired bag defects may have contributed to postmanufacturing pathogen-reduced PLT contamination.

Existing and Emerging Blood-Borne Pathogens: Impact on the Safety of Blood Transfusion for the Hematology/Oncology Patient.

Despite measures to mitigate risk of transfusion-transmitted infections, emerging agents contribute to morbidity and mortality. We outline the epidemiology, risk mitigation strategies, and impact on patients for Zika virus, bacteria, Babesia, and cytomegalovirus. Nucleic acid testing of blood has reduced risk of Zika infection and reduced transfusion-transmitted risk of Babesia. Other collection and testing measures have reduced but not eliminated the risk of sepsis from bacterially contaminated blood components. Cytomegalovirus has almost been eliminated by high-efficiency leukoreduction, but residual transmissions are difficult to distinguish from community-acquired infections and additional antibody testing of blood may confer further safety of susceptible recipients.

Sepsis Attributed to Bacterial Contamination of Platelets Associated with a Potential Common Source - Multiple States, 2018.

During May-October 2018, four patients from three states experienced sepsis after transfusion of apheresis platelets contaminated with Acinetobacter calcoaceticus-baumannii complex (ACBC) and Staphylococcus saprophyticus; one patient died. ACBC isolates from patients' blood, transfused platelet residuals, and two environmental samples were closely related by whole genome sequencing. S. saprophyticus isolates from two patients' blood, three transfused platelet residuals, and one hospital environmental sample formed two whole genome sequencing clusters. This whole genome sequencing analysis indicated a potential common source of bacterial contamination; investigation into the contamination source continues. All platelet donations were collected using apheresis cell separator machines and collection sets from the same manufacturer; two of three collection sets were from the same lot. One implicated platelet unit had been treated with pathogen-inactivation technology, and two had tested negative with a rapid bacterial detection device after negative primary culture. Because platelets are usually stored at room temperature, bacteria in contaminated platelet units can proliferate to clinically relevant levels by the time of transfusion. Clinicians should monitor for sepsis after platelet transfusions even after implementation of bacterial contamination mitigation strategies. Recognizing adverse transfusion reactions and reporting to the platelet supplier and hemovigilance systems is crucial for public health practitioners to detect and prevent sepsis associated with contaminated platelets.

Sodium citrate contributes to the platelet storage lesion.

BACKGROUND: Sodium citrate has become the preferred anticoagulant used for apheresis collection and has been included in commercial platelet additive solutions (PASs) since PAS-II. It was suggested that citrate be included in PASs to prevent spontaneous aggregation. Reports in cell lines and cord blood have demonstrated that concentrations of citrate present in PAS formulations (10 mM) cause apoptosis. We evaluated whether the removal of citrate from PAS-III could improve platelet storage. STUDY DESIGN AND METHODS: Study 1 evaluated the effects of a citrate dose response on the storage of platelets in 65% PAS containing sodium chloride, sodium acetate, and phosphate. Study 2 compared the cell quality and function of platelets stored in 65% citrate-free PAS-III or PAS-III containing 10 mM of citrate. Measurements included cell count, blood gases, flow cytometry analysis of surface activation markers, and aggregation. RESULTS: Study 1 identified that inclusion of citrate in PAS resulted in a dose-dependent increase in glucose utilization, lactate formation, P-selectin expression, phosphatidylserine (PS) exposure, and reactive oxygen species (ROS) formation. Study 2 showed similar results in which platelets stored in citrate-free PAS-III benefited through better maintenance of glucose utilization with less lactate production, P-selectin expression, PS exposure, and ROS formation compared to citrate-containing PAS-III. Platelets stored in citrate-free PAS-III had aggregation responses that were at least 10% greater than those platelets stored in PAS-III. CONCLUSION: Storage of apheresis platelets in citrate-free PAS-III improved multiple storage parameters including glucose utilization, lactate production, P-selection expression, PS exposure, and ROS formation and resulted in a modest increase in aggregation.

Influence of apheresis collection device and container on the storage properties of platelets in 90% PAS-5/10% plasma.

BACKGROUND: The storage properties of apheresis platelets suspended in the experimental additive solution PAS-5 and 10% plasma may be affected by the collection instrument or storage container. METHODS AND EXPERIMENTAL DESIGN: The same consenting 12 donors provided A or T platelets with concurrent plasma on four occasions in 100% plasma. Following collection and resting, the platelets were centrifuged, and plasma was expressed and resuspended in PAS-5 to yield units with 10% plasma. Platelets were either maintained in the original storage container or transferred to another of the manufacturer's storage containers. On days 1, 5 and 7, units were assayed for an array of in vitro tests. RESULTS: Average unit volume, yield and percent plasma was 291±11 mL, 3.7±0.4×1011, and 10.3±0.7%, respectively, and were comparable between collections with either of the apheresis instruments and stored with either of the manufacturer's containers. Day 1 platelet activation (CD62P+) was 40±22% and was similar in either of the collection instruments or containers. Except for pH (days 1, 5), CO2 (days 1, 5, 7), and extent of shape change (day 5), every other in vitro parameter was similar between apheresis platforms or the manufacturer's container. pH values of all units on all days of storage were ≥6.8, except one unit that was collected on T and stored in an A container, which had pH values of 6.8 and 5.7 on days 5 and 7, respectively. DISCUSSION: Storage of platelets suspended in PAS-5 with 10% plasma is feasible in the original manufacturer's container for seven days. Based on CO2 levels, T containers have greater gas exchange than A containers.

Temperature cycling during platelet cold storage improves in vivo recovery and survival in healthy volunteers.

BACKGROUND: Room temperature (RT) storage of platelets (PLTs) can support bacterial proliferation in contaminated units, which can lead to transfusion-transmitted septic reactions. Cold temperature storage of PLTs could reduce bacterial proliferation but cold exposure produces activation-like changes in PLTs and leads to their rapid clearance from circulation. Cold-induced changes are reversible by warming and periodic rewarming during cold storage (temperature cycling [TC]) has been proposed to alleviate cold-induced reduction in PLT circulation. STUDY DESIGN AND METHODS: A clinical trial in healthy human volunteers was designed to compare in vivo recovery, survival, and area under the curve (AUC) of radiolabeled autologous apheresis PLTs stored for 7 days at RT or under TC or cold conditions. Paired comparisons of RT versus TC and TC versus cold PLTs were conducted. RESULTS: Room temperature PLTs had in vivo recovery of 55.7 ± 13.9%, survival of 161.3 ± 28.8 hours, and AUC of 5031.2 ± 1643.3. TC PLTs had recovery of 42.6 ± 16.4%, survival of 48.1 ± 14.4% hours, and AUC of 1331.3 ± 910.2 (n = 12, p < 0.05). In a separate paired comparison, cold PLTs had recovery of 23.1 ± 8.8%, survival of 33.7 ± 14.7 hours, and AUC of 540.2 ± 229.6 while TC PLTs had recovery of 36.5 ± 12.9%, survival of 49.0 ± 17.3 hours, and AUC of 1164.3 ± 622.2 (n = 4, AUC had p < 0.05). CONCLUSION: TC storage for 7 days produced PLTs with better in vivo circulation kinetics than cold storage but is not equivalent to RT storage.

Increase of plasma concentration to 10% improves a number of in vitro storage parameters of apheresis platelets suspended in a bicarbonate-containing additive solution and stored with a 24-hour interruption of agitation.

BACKGROUND: A previous study demonstrated several pH failures during 7-day storage of platelets suspended in 5% plasma/95% PAS-5 following a 24-hour interruption of agitation. The aim of this study was to investigate whether pH control improves in platelets stored in PAS-5 with 10% plasma following interruption of agitation. MATERIALS AND METHODS: Four aliquots were prepared from a single unit of apheresis platelets: two each with 5% and 10% plasma. After resting for 1 hour, the aliquots were placed on an agitator. On day 2, agitation of one aliquot with 5% plasma and another with 10% plasma was interrupted for 24 hours before the aliquots were returned to agitator. The two control aliquots remained on the agitator. An array of platelet parameters was measured on days 2, 5 and 7. RESULTS: On day 7, aliquots containing 10% plasma and subjected to interruption of agitation had a significantly higher mean pH compared to those of similarly treated aliquots containing 5% plasma (6.80±0.54 vs 6.41±0.57, p≤0.05). Platelets containing 10% plasma/95% PAS-5 subjected to interruption of agitation had a greater hypotonic stress response, greater shape change, higher mitochondrial membrane potential, decreased glucose utilisation and lower CD62P levels compared to those of similarly treated platelets suspended in 5% plasma. DISCUSSION: Increasing plasma concentration to 10% improves pH control and some in vitro platelet properties during 7 days of storage of platelets suspended in PAS-5 after a 24-hour interruption of agitation compared to those of similarly treated platelets suspended in 5% plasma/95% PAS-5.