Apheresis Platelet Units Research Articles

Evaluation of the Mirasol platelet reduction technology system against Babesia microti in apheresis platelets and plasma

Babesia microti is an intraerythrocytic parasite, transmitted naturally to humans by infected ixodid ticks, that causes babesiosis. In recent years, B. microti has been identified as a growing public health concern that has also emerged as a critical blood safety issue in the absence of appropriate interventions to reduce transmission by blood transfusion. Thus, we evaluated the ability of the Mirasol pathogen reduction technology (PRT; CaridianBCT), which uses riboflavin (RB) and ultraviolet (UV) light, to diminish the presence of B. microti in apheresis plasma and platelets (PLTs). Apheresis plasma and PLT units were spiked with B. microti-infected hamster blood and subsequently treated using the Mirasol PRT system. Control and experimental samples were collected at different stages during the treatment process and injected into hamsters to detect the presence of viable parasites. Four weeks postinoculation, hamster blood was tested for B. microti infection by blood smear and real-time polymerase chain reaction analysis. None of the blood smears from animals injected with samples from PRT-treated plasma or PLT units were positive by microscopy, while all the non-PRT-treated plasma and PLT units were demonstrably parasitemic. Parasite load reduction in hamsters ranged between 4 and 5 log in all PRT-treated units compared to untreated controls. The data indicate that the use of RB and UV light efficiently reduces the presence of viable B. microti in apheresis plasma and PLT products, thereby reducing the risk of transfusion-transmitted Babesia potentially associated with these products. Based on this observed "proof of principle," future studies will determine the efficacy of the Mirasol PRT in whole blood.

Platelet transfusions: trigger, dose, benefits, and risks

Over the last half century, platelet transfusion has been an effective therapy for the prevention and treatment of bleeding, particularly in patients with hematologic malignancies. Recent randomized trials have demonstrated that current practices may be suboptimal in a number of ways. The rationale for parsimony in the use of this powerful therapy includes previously described severe and fatal adverse outcomes (including refractoriness, hemolysis from ABO-mismatched transfusions, acute lung injury, and bacterial sepsis), newly described serious potential risks (including thrombosis and earlier leukemic recurrence), difficulty in maintaining adequate supplies of platelets, the need to place volunteer donors on cell separators to provide the product, and cost. Recent findings demonstrate that the platelet count threshold for prophylactic transfusion can be as low as 10,000/µL, and a therapeutic rather than a prophylactic strategy of transfusion for bleeding manifestations only may be equally safe for most patients. Another recently completed study suggests that very low doses of platelet transfusions (the equivalent of half a unit of apheresis platelets or two to three units of whole blood-derived platelets) are as effective at preventing bleeding as much higher doses. One question for which there are no randomized trial data is at what threshold prophylactic platelet transfusion should be given before invasive procedures or major surgery. The typically recommended threshold of 50,000/µL is based only on expert opinion, and substantial observational data indicate that this threshold leads to many transfusions that are likely unnecessary and therefore represent risk with little or no additional benefit.

Collection and Storage of Apheresis Platelets Results in a Significant Decline in the Percentage of Coated-Platelets.

Abstract 23 Introduction:Platelets initiate hemostasis by adhering to the site of vascular injury and providing a negatively charged surface for thrombin generation. A subset of platelets, referred to as coated-platelets, has an enhanced ability to generate thrombin. Coated-platelets, observed in vitro upon dual agonist stimulation with collagen and thrombin, retain several serotonin-derivatized proteins on their surface and express phosphotidylserine, creating a highly active prothrombinase complex. Experimental studies have concluded that dogs deficient in coated-platelets manifest with excessive nasal, intraocular and post-operative bleeding, suggesting a key role for coated-platelets in hemostasis. The fraction of coated-platelets made by normal individuals is approximately 30% with a range of 15% – 55%. Platelet concentrates are stored at room temperature to preserve their viability and must be transfused within five days of collection secondary to a high risk of contamination. We conducted a pilot study in healthy platelet donors to evaluate how the process of apheresis and in vitro storage affect the ability to generate coated-platelets. Secondarily, we aimed to evaluate the coated-platelet potential of apheresis platelet units transfused in clinical practice. Methods: Healthy voluntary platelet donors presenting at the Oklahoma Blood Institute were enrolled and demographic data such as age, sex, smoking status, donation history, past medical history and medications were noted. A Pre-donation blood sample was obtained. Thereafter, donors underwent apheresis using the Caridian BCT Trima system via continuous centrifugation in a closed system per guidelines outlined by the American Association of Blood Banks (AABB). A Post-donation sample was drawn from the platelet concentrate bag 30 minutes after apheresis. Units were packaged, processed and stored per routine procedures. A third platelet sample was obtained when the unit was released for transfusion. Platelet rich plasma was prepared and coated-platelets were assayed as previously described (J. Thromb. Haemostasis 6:609, 2008). Statistical analysis was performed using paired t-test to compare the change in percentage of coated-platelets between the three samples. Results: Nine donors were enrolled. The mean percentage of coated-platelets in the Pre-donation samples was 40.4% (20.7%–59.7%). Thirty minutes after the apheresis collection procedure, the mean percentage of coated-platelets in the concentrate was 34.3% (17.3%–49.0%) suggesting a 15% decline in percentage of coated-platelets with the process of apheresis (p<0.01). Eight units were transfused within one to four days of collection. The mean percentage of coated-platelets in the transfused product was 17.1 % (10.1%–28.4%); a 52% decline in the percentage of coated-platelets during the process of storage was noted (p<0.001). [Figure A and B]. [Display omitted] Conclusion:Coated-platelets are a recently recognized component of hemostasis, and the ability of stored platelets to retain coated-platelet potential is unknown. Our preliminary results indicate that the process of platelet apheresis and storage significantly decreases the ability to generate coated-platelets. Current quality control guidelines for platelet storage measure platelet viability with factors such as pH, pCO2, pO2, lactate and platelet morphology; however, there is no evaluation of the hemostatic function of stored platelets relative to coated-platelets. Our results reveal that during storage following current transfusion guidelines, the percentage of coated-platelets decreases. Part of this decline appears to be associated with the apheresis process itself and further progresses with storage. Our study provides the first evidence that retention of coated platelet levels during preparation and storage is problematic and may be an important factor in evaluating the hemostatic potential of infused platelets. Disclosures:No relevant conflicts of interest to declare.

Comparison between the BACTEC 9240 and the Pall eBDS system for detection of bacterial platelet concentrate contamination

Only two commercially available automated systems have been cleared by the FDA for screening of bacterial contamination in platelet (PLT) products. These are the Pall eBDS (Pall Corp.), based on measurement of oxygen consumption by contaminant organisms, and the BacT/ALERT (bioMérieux), revealing increasing carbon dioxide concentration due to bacterial growth. The authors compared the performance of the Pall eBDS with the BACTEC 9240 (bioMérieux) in detecting PLT contamination. Serial dilutions of 10 bacterial species frequently associated with PLT contamination were prepared in an apheresis PLT unit per organism. Units were from single donors. After 30 minutes from seeding PLT units, a volume of suspension achieving a final bacterial concentration of 1 to 10 colony-forming units/mL for each unit was inoculated in two Pall bags and a BACTEC bottle, and the same was done after 24 hours from seeding. Measurements were performed at 24 and 30 hours. Significant differences between the two instruments were only found when screening PLT units after 24 hours from seeding. The Pall system showed a higher sensitivity than BACTEC 9240, because it revealed 97 and 98% of positive samples at 24 and 30 hours of incubation, respectively, whereas the second detected 86 and 90% of contaminated products. Significance was lost after 35-hour incubation with the BACTEC 9240. By comparing the two instruments, their performances were found to be comparable; the Pall system appeared as a more suitable method when using 24 to 30 hours as times for readings, but the significant difference was lost after 35-hour incubation.

Lack of Impact on Platelet Increments by Number of Apheresis Platelet Units Transfused

Introduction: Prophylactic platelet transfusions are often given when a predetermined platelet count is reached and the amount given is that necessary to achieve a desired post count. This method of transfusion ignores the question of whether the patient receives increased benefit from transfusion when more than one unit is given. To determine what effect this practice has had on transfusion outcome and platelet utilization, we have reviewed 5 years of platelet transfusion data to look specifically at transfusion events when more than one single donor platelet was given during a 24 hour period.Methods: Transfusion data was obtained from that used for utilization review 2002 –2006. The data was sorted to identify records indicating that 2 or more units of apheresis platelets were received in a 24 hour period. This information was further selected to include only those with hematologic diseases. The platelet counts identified for inclusion were the counts prior to the release of the first unit given during the day and the first count after the release of the last unit. The change in the platelet count for the number of units was recorded and averaged for each unit increment for each record. Statistical analysis was performed and was not statistically significant.Results For the 5 year period, 86% of all platelet transfusions at our institution were given to patients with hematologic diseases and 27% of these were given as 2, 3, or 4 units within a 24 hour period. 716 records had 2377 days with multiple transfusions; 1802, 428, and 147 episodes of 2, 3, and 4 units respectively. The increase in platelet count for a single unit transfusion at our institution averages 32.3 ×109/L. The platelet increments for 2, 3, and 4 units showed no statistical increase from this number or each other. There is no trend for the final platelet count to be greater when more units are given. These results do not take patient condition or the use of specialized products into consideration.Discussion: Our experience suggests that transfusions of multiple platelet units may not have a greater impact on platelet increments than that when fewer units are transfused. To fully understand this data and its potential impact on patient care, further subgroup analysis within the platelet increments and correlation with patient condition and the prescribed transfusion parameters is needed. [Display omitted]

Concentration changes to counteract the effects of bacteriological sampling on PLT yields

Bacterial culturing of apheresis platelet (PLT) units appeared to increase the incidence of low yield products of <3.0 x 10(11) PLT (LYP) and decrease the incidence of multiple PLT products. To determine the effects of sampling and subsequent modifications in collections on PLT yield and adverse reactions, a retrospective analysis was performed. Four time periods were examined: baseline, after sampling implementation, after concentration change (Conc. Delta) implementation, and after concentration and yield target change (Target + Conc. Delta) implementation. Collections were performed on the Gambro Trima Accel. PLT concentration settings were changed from 1,550 to 1,200 (single and double PLT products) or 1,610 to 1,500 (triple PLT products). A 3.2 x 10(11) target was eliminated and a 9.4 x 10(11) target added. Donors in all groups were comparable. Average incidence of LYP per week was significantly higher for Sampling (8.1%) than Baseline (2.0%) and declined significantly in the Conc. Delta (3.4%) and Target + Conc. Delta (2.0%) groups. Average PLT products/donor per week for Sampling (1.7) was significantly lower than Baseline (1.8), equivalent to Conc. Delta (1.7), and significantly lower than Target + Conc. Delta (1.9). Average weekly incidence of triple PLT products was significantly higher for Target + Conc. Delta (17%) compared with Baseline (12.4%), Sampling (10.7%), and Conc. Delta (10.8%). Donor reaction incidence was not significantly different among the groups. There was no change in recipient reactions. The use of a yield target change and PLT concentration reduction compensated for changes associated with bacterial culturing without increasing donor or recipient reactions.

Evaluation of platelet transfusion triggers in a tertiary‐care hospital

Our 1100-bed referral hospital uses approximately 12,000 units of random-donor platelets (PLTs) and 1,900 units of single-donor apheresis PLTs per year with a mean of 23 percent outdating. An analysis of patterns of utilization has been undertaken to evaluate practice. Over a 9-month period, data were collected on a total of 1682 transfusion episodes in 464 patients. When the pretransfusion count was greater than 10 x 10(9) per L an attempt was made to identify the specific indications for PLT transfusions such as bleeding. The majority (78%) of PLTs were transfused when the counts were above 10 x 10(9) per L. The mean pretransfusion counts for different services were: bone marrow transplant (BMT) 17.4 x 10(9) per L, hematology-oncology 14.6 x 10(9) per L, the Heart Institute 3 x 10(9) per L, and other services 36 x 10(9) per L. The percentage of transfusions given to patients with a count greater than 10 x 10(9) per L varied by service with 79 percent in BMT, 60 percent in hematology and oncology, 98 percent at the Heart Institute, and 81 percent in other services. Routine monitoring of counts shows a mean increment of 10.2 x 10(9) per L per transfusion. One hour posttransfusion counts, 24-hour posttransfustion counts, and documentation of clinical justification for transfusions was often not available. The data show that most patients who receive PLTs have pretransfusion counts of more than 10 x 10(9) per L and more than one-third have pretransfusion counts of greater than 20 x 10(9) per L. The medical literature supports prophylactic PLT transfusion based solely on the count when the PLT number is 10 x 10(9) per L or less. Above this level additional justification is needed although there are different points of view concerning the appropriate triggers. Our data suggest that there is a need for clear hospital transfusion guidelines and ongoing monitoring of PLT use.

Development of proficiency testing for detection of bacterial contamination of platelet products

Accrediting agencies now require quality control for minimizing platelet (PLT) bacterial contamination (PBC). A proficiency testing (PT) strategy for PBC testing was developed. During Phase 1, validation of the pH meter and an enhanced bacteria detection system (Pall eBDS)--methods used in our blood bank--was accomplished with two aliquots of an apheresis PLT unit inoculated with Escherichia coli (ATCC 25922, 70 CFU/mL) or Staphylococcus aureus (ATCC 27217, 46 CFU/mL) with an uninoculated aliquot serving as a negative control. Quantitative plate culture was the reference method. PLTs were stored on a rotator at 22 degrees C. Units were sampled in duplicate at 0, 24, and 48 hours. pH testing was considered positive when the pH value was less than 6.6. eBDS was positive when the oxygen concentration was less than 9.4 percent. During Phase 2, synchronized PT of pH and eBDS was performed at four independent sites. PLT samples were inoculated and incubated as above, and aliquots were removed at Time 0 for eBDS testing and at 48 hours for pH testing. In Phase 1, on inoculated bags eBDS was positive at all time periods but pH was positive only at 48 hours. In Phase 2, synchronized results showed positive eBDS at Time 0 and positive pH at 48 hours on inoculated bags with agreement between paired sites. This strategy may serve as a useful model for developing PT materials for PBC detection. eBDS was able to identify low levels of PBC, and pH testing, only much higher levels. It is important to carefully coordinate and standardize handling of PT materials and reporting of results.

Use of Blood Bank Services in a Burn Unit

Burn patients may need transfusions, especially during surgery. The purpose of the study was to determine blood bank services utilization at an urban burn center. This was a retrospective review (March 1999 to May 2004) of burn patient data on blood utilization. There were 1615 admissions; 800 (49.5%) of the patients had a type and screen (T and S); 180 of 1615 (11%) were transfused with 1691 red blood cell (RBC) units; 616 units of fresh- frozen plasma (FFP), 399 units of pooled platelets, and 38 units of apheresis platelets. Of the 180 transfused patients, 140 (79%) received RBCs, 11 (6%) received RBCs plus FFP, 23 (13%) received RBCs plus FFP plus platelets, and 3 (2%) each received RBCs plus platelets and FFP. Overall, 3393 red cell units were cross-matched, and 1691 units were transfused for a cross-match/transfused unit (C/T) ratio of 2.01. Seventy-three (5.7%) of 1282 of patients with 0% to 10% TBSA required transfusions; 42 (21%) of 193 with 11% to 20% TBSA; 24 (39%) of 61 with 21% to 30% TBSA; and 62% of patients with 31% or greater TBSA required transfusion (P = .001). As %TBSA increased, more of the cross-matched units were transfused: 37% in the 0% to 10% TBSA to 59% in the 30% or greater TBSA burn. The C/T ratio in the 0% to 10% TBSA was 2.69; only 18% of patients with a T&S received blood. Although most patients who underwent transfusion were in the less than 30% TBSA group, more of the red cell units were used in patients with a 30% or greater TBSA burn. Elimination of admission T&S and cross matches in burn patients with less than 20% TBSA may improve the C/T ratio in this cohort.

Evaluation of the enhanced bacterial detection system for screening of contaminated platelets

The Pall third-generation enhanced bacterial detection system (eBDS) was recently approved for detection of bacterial contamination in leukoreduced platelets (PLTs). The method is based on the measurement of the oxygen content as a marker for bacteria. eBDS incorporates major modifications including removal of the sample-set filter, modification of the culture medium, and incubation with agitation of the sample pouch. Ten whole blood-derived random-donor PLT units collected on Day 1 after donation and 10 single-donor apheresis PLT units were spiked with low levels of bacteria in three different blood transfusion centers. Inoculation was performed at a final concentration of 5 to 50 colony-forming units per mL with reference strains of five organisms involved in severe transfusion-associated infections. PLT units were stored at 22 degrees C for 24 hours before sampling. Six sample sets were then sterile-connected to each unit and placed on a horizontal agitator at 35 degrees C for 18 or 24 hours of incubation. No false-positive results were obtained, indicating a 100 percent specificity of the assay. Of 126 spiked sample pouches tested, 61 of 63 (96.82%) and 63 of 63 (100%) were detected positive after 18 or 24 hours of incubation, respectively. In the two missed cases that failed to detect Bacillus cereus, the measured oxygen was slightly above the detection threshold but was markedly different from the negative samples. The eBDS method allows definitive testing of PLTs as soon as 42 hours after collection and offers an alternative culture method to the BacT/ALERT system.

Evaluation of Immunophenotypes and Activities of Cell Membrane Microparticles in Apheresis Platelets: Impact of Seven Day Storage.

Seven day storage of platelets has become a reality in transfusion management. Cell membrane microparticles (MP), 0.2–1.0 μm in size are released from stimulated platelets, blood- and endothelial cells. The pathogenic potential of MP has been documented. Since a portion of platelet transfusion adverse effects is likely not antibody related, MP are good candidates to study. We have previously demonstrated the presence of MP derived from platelets, blood-, and endothelial cells in apheresis platelet units (APU) on day 6 of storage. Here we investigate the pathophysiological activities of MP in APU and the impact of extended storage on MP immunophenotype and activity profiles. We analyzed MP in non-frozen APU supernatants (n=28) on day 6 and 8 of storage. A three-color flow cytometry assay of MP (Simak et al, British J Haematol, 2004) was used, and several functional assays were performed. Plasma from healthy volunteers (HVP) (n=38) was used for comparison. On day 6, the median platelet count in APU was 1514x103/μL (range: 1144x103–2248x103) and pH 7.28 (6.67–7.51). The pH decreased significantly to 6.97 (6.19–7.40) on day 8. All APU bacterial cultures were negative. When comparing APU on day 6 and 8 of storage, platelet MP (CD41+), endothelial MP (CD144+), red blood cell MP (CD235a+), and proinflammatory CD154+MP did not change significantly. In contrast, an increase of leukocyte MP, including leukocyte MP exposing tissue factor (TF, CD142) (CD142+CD45+MP; p=0.001) was found. Since both platelet CD41a+CD142+ and endothelial CD144+CD142+ MP tend to decrease during storage, the total CD142+MP (detected by V1C7 and HFT-1 Mab) showed only a slight increase. All studied MP phenotypes were significantly higher in APU on day 6 and 8 when compared to HVP. TF activity (using FVIIa, FX and FXa substrate) of washed MP was 619 nM TF (35–393) in APU on day 6 vs. 629 nM TF (38–2443) on day 8. MP associated TF activity correlated with counts of CD142+MP (r=0.76; p=0.02) and was in some individual APU over 10x the median value of HVP MP, 190 nM TF (20–440). The effect of APU MP on intracellular free Ca2+ concentration [Ca2+]i was also studied, using a ratio fluorometry in GT1-7 cells loaded with a Ca2+-sensitive probe FURA-2AM. Washed APU MP induced a rapid MP concentration dependent peak of [Ca2+]i, followed by a sustained slow increase of [Ca2+]i. The activity was significantly higher in day 6 vs. day 8 APU MP (p=0.001) and could be inhibited by EGTA, suggesting that extracellular Ca2+ predominate in APU MP stimulated calcium flux. No significant [Ca2+]i increase was achieved even with 3x concentrated MP from HVP. To test apoptotic activity (TUNEL), washed APU MP were incubated overnight with endothelial cells (HUVEC). About 25% of APU MP induced significant apoptotic activity which correlated with CD154+CD45−CD41a+MP (r=0.81; p=0.002) and CD45+MP (r=0.75; p=0.009) and was higher in day 6 vs. day 8 APU MP vs. HVP MP (p=0.03). In addition, cell cycle analysis showed that APU MP induced G1 arrest. In conclusion, APU MP exhibited various pathophysiological activities in vitro. Some individual units showed a high MP content, and correlations were found between certain MP phenotypes and APU MP activities. The 7-day storage of effectively leukoreduced APU should not have a dramatic impact on a potentially pathogenic MP content. Studies investigating an association of high MP counts in some APU with clinical adverse effects of APU transfusions are warranted. The views of the authors should not be construed as FDA policy.

In‐house validation of the BACTEC 9240 blood culture system for detection of bacterial contamination in platelet concentrates

At present, only two commercially available automated culture systems are cleared by the FDA for the purpose of quality control (QC) testing for bacterial contamination of platelet (PLT) concentrates: the BacT/ALERT blood culture system (bioMérieux) and the Pall eBDS (Pall Corporation), both of which allow testing of leukoreduced apheresis as well as whole blood-derived PLTs. After the decision of the AABB to institute universal QC testing of PLT concentrates for evidence of bacterial contamination, in-house validation of the performance of our current blood culture system, the BACTEC 9240, was carried out for this purpose. Serial dilutions of nine species of bacteria commonly associated with PLT contamination were prepared in one single-donor apheresis PLT unit per organism. Four mL of dilutions containing less than 1 to greater than 10(3) colony-forming units (CFUs) per mL was inoculated into blood culture bottles (Standard 10 Aerobic/F, Becton-Dickinson Diagnostic Systems) and incubated in a BACTEC 9240 continuously monitored blood culture system. Positive bottles were removed from the system and subcultured to insure the identity of bacterial growth. With the exception of Streptococcus mitis, the BACTEC system provided a detection sensitivity of less than 10 CFUs per mL for Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Bacillus cereus, Enterobacter cloacae, and Pseudomonas aeruginosa. The limit of detection for the S. mitis test strain was 61 CFUs per mL. Detection of positive bottles ranged from 6.5 to 17.6 hours depending on the species tested and the cell density of the inoculum. Ongoing use of this system for bacterial detection yielded two true-positive samples from 3879 apheresis PLT products collected at our hospital-based donor center over 9 months. This study validates the use of the BACTEC 9240 continuously monitored blood culture system for the detection of low-level bacterial contamination in single donor apheresis PLTs in less than 24 hours.

The Influence of Various Hematology Analyzers on Component Platelet Counts

Hematology analyzers designed to count platelets in samples of whole blood are used to enumerate the total number of platelets in components prepared for transfusion. This report addresses the issue of variability in platelet counts obtained with different models of hematology analyzers. The influence of a common calibration procedure, involving one level of porcine platelets, on the extent of variability was also evaluated. Identical sets of samples of simulated and apheresis-derived human platelets were counted by multiple laboratories in 3 separate studies. In the first 2 exercises, 7 samples of both porcine platelets and modified goat erythrocytes with targeted platelets counts from 0.2 to 4.0 x 10(12)/L were counted without prior dilution. In both exercises, the samples were counted multiple times after routine calibration using instructions provided by the manufacturers of the various hematology analyzers used. In the second exercise, the samples were recounted after the hematology analyzers were recalibrated with a common calibrant consisting of porcine platelets at a targeted concentration of 0.5 x 10(12)/L. In the first and second exercises, 20 and 18 hematology analyzers were used, respectively. In the third exercise, 6 samples prepared from a single unit of apheresis platelets with targeted counts from 0.2 to 1.64 x 10(12)/L were shipped by an overnight courier and counted in triplicate on the day of arrival. Eleven hematology analyzers were used. The influence of recalibration was evaluated statistically by using the 95% prediction interval for the mean of a future set of observations. The platelet counts measured with a specific type of hematology analyzer provided the data to calculate the 95% prediction interval. With routine calibration, a wide variability in platelet counts was observed with all levels of both simulated and apheresis-derived human platelets. For example, with porcine platelets at a targeted level of 0.4 x 10 (12)/L, the platelet counts ranged from 0.31 to 0.47 x 10(12)/L. Recalibration reduced the extent of variability observed with all levels of simulated and apheresis-derived human platelets by increasing the observed platelet counts determined with a subset of hematology analyzers that produced platelet counts in the lower portion of the range. With recalibration, the mean platelet counts obtained with most hematology analyzers, especially with samples having targeted platelet levels no greater than 1.0 x 10(12)/L, were within or near the 95% prediction interval determined with the instruments that provided the highest platelet counts with routine calibration. With recalibration, the reproducibility of the platelet counts was considered to be good for all hematology analyzers with all levels of simulated and apheresis-derived human platelets for most of the instruments. The coefficient of variance did not exceed 6%, with most of the values ranging from 1% to 3%. This study therefore found that the platelet counts of platelet concentrates can be markedly influenced by the type of hematology analyzer used. A common calibration procedure designed specifically for the range of platelet counts in platelet products may be beneficial considering that many different hematology analyzers are being used to count platelets.

Evaluation of the Scansystem method for detection of bacterially contaminated platelets

Platelet (PLT) bacterial contamination occurs in approximately 1 in 2000 PLT units. The College of American Pathologists recommends and AABB requires procedures to detect PLT bacterial contamination. Although two methods, BacT/ALERT (bioMerieux) and Pall BDS (Pall Corporation), have FDA approval for quality control testing, additional methods are in development. One such method was evaluated, the Scansystem (Hemosystem), which has been developed for use on leukoreduced PLT components between 30 and 72 hours after collection. Leukoreduced, single-donor apheresis PLT units (LR-SDPs) were inoculated with 10 bacterial species (low and high inocula) associated with PLT contamination. Bacterial detection was compared with the Scansystem and BacT/ALERT. Testing was initiated (10 replicates performed) when LR-SDPs were experimentally inoculated with bacteria. The Scansystem was evaluated 30 hours later, the shortest manufacturer recommended time after PLT collection. All replicates were positive with the Scansystem at 30 hours and with the BacT/ALERT, at 9.3 to 24.0 hours after inoculation. The Scansystem detected bacteria in 83 of 200 replicates (42%) at the time of inoculation indicating a potential for earlier application. The Scansystem, used to test LR-SDPs 30 hours after bacterial inoculation, detected all 20 replicates with a sensitivity equal to the BacT/ALERT system. Based on use of Scansystem with LR-SDPs 30 hours after collection and the BacT/ALERT being inoculated 24 hours after collection and incubated for an additional 24 hours before being determined to be negative, the Scansystem will potentially provide results at an earlier time point (32 hr) than provided by the BacT/ALERT system (48 hr).

Blood components should be labeled for content

Blood components should be labeled for content

High Counts of Potentially Pathogenic Cell Membrane Microparticles in Apheresis Platelets.

Moderate to severe reactions to platelet transfusion have been documented in more than 20% of recipients, and a significant portion of the adverse effects is likely not antibody mediated. Cell membrane microparticles (MP), 0.2–1.0 μm in size, are released into the circulation from stimulated platelets, blood or endothelial cells, and the elevated MP in blood are associated with various vascular pathologies. Moreover, MP potential for different pathogenic activities has been documented. We analyzed MP in apheresis platelet units (APU) (n=26) sent to our laboratory from blood centers nationwide. The analysis was performed on day 6 of the platelet storage period. The median platelet count and pH in APU were 1386x103/μL (range: 697x103–1612x103), and 7.38 (6.82–7.51), respectively. A three-color flow cytometry assay (Simak et al, British J Haematol 125, 804–813, 2004) was used for analyzing MP in the non-frozen platelet supernatants. Plasma from healthy volunteers (HVP) (n=12) was used for comparison. To identify MP of platelet, white blood cell (WBC), red blood cell (RBC), and endothelial cell (EC) origin, the antibodies to CD41, CD45, CD235a, and CD105 or CD144, respectively, were used. We also analyzed the expression of CD54 (ICAM 1), a likely marker of proinflammatory status. To study procoagulant phenotypes of MP, phosphatidylserine-positive MP were assayed using the annexin V binding (AVB), and the tissue factor (CD142) was detected with the VIC7 Mab clone. As expected, we found a nine-fold higher platelet CD41+MP in APU (median: 16830/μL; range: 5860–65210) vs. HVP (1910/μL; 1020–2200; p<0.001). Surprisingly, the RBC CD235a+MP were two-fold increased in APU (14650/μL; 10390–21410) vs. HVP (7350/μL; 6160–8960; p<0.001), whereas WBC CD45+MP were about five times higher in APU (4310/μL; 2620–10960) vs. HVP (880/μL; 670–1330; p=0.018). In addition, EC MP (CD105+CD45-MP) were about three times higher in APU (2790/μL; 840–9870) vs. HVP (930/μL; 710–1930; p=0.002). Moreover, the tissue factor specific Mab VIC7 detected in APU about a three times higher count of CD142+MP (4670/μL; 1830–11150) than in HVP (1580/μL; 1070–2510; p<0.001). Counts of CD142+AVB+MP were 2080/μL (730–8040) in APU, and 1330/μL (720–1700; p=0.042) in HVP. Unexpectedly, only 20% of the CD142+MP were CD142+CD45+MP in both, APU and HVP samples, while over 50% were CD142+CD41+MP, and about 20% were CD142+CD144+MP. Although counts of CD54+MP in the APU group (1760/μL; 700–4760) were not significantly different from the HVP group (1350/μL; 970–1560), several APU samples exceeded nearly four times the HVP median. In a functional assay, the overnight incubation of washed APU MP with cultured EC (HUVEC) induced a significant increase in EC apoptosis (TUNEL), and an increase in the cell expression of CD54 and CD142, as compared to control. In conclusion, we found in APU significantly elevated counts of MP derived from platelets, WBC, RBC, and EC, and also MP of procoagulant phenotypes, when compared to HVP. Moreover, APU MP exhibited potentially pathogenic activities on cultured EC. Our results warrant further studies to investigate if the outlying high counts of MP populations with procoagulant or proinflammatory phenotypes, observed in some APU samples, could be related to the clinical adverse effects of APU transfusions. In addition, the potential impact of collection, processing, and storage steps on MP generation in APU should be investigated. The views of the authors represent a scientific opinion and should not be construed as FDA policy.

Unappreciated risk factors for transplant patients: HLA antibodies in blood components

One of the more aggressive approaches in renal transplantation is the use of plasmapheresis (PP) and intravenous immunoglobulin to eliminate donor-directed human leukocyte antigen (HLA) alloantibodies. A potential complication of a PP protocol is iatrogenic hypocoagulability resulting from the removal of coagulation factors. To prevent bleeding, hypocoagulable patients may require transfusions with fresh frozen plasma (FFP) and/or cryoprecipitate (Cryo). Although HLA alloantibodies in these components have been linked to complications, such as transfusion-related acute lung injury (TRALI), whether they cause complications following transfusion into allograft recipients is unknown. The incidence of complications would be dependent, in part, upon the frequency of HLA alloantibodies in the various blood components. In this study, segments from 77 units of FFP, 66 units of Cryo, 106 units of packed red blood cells (RBCs), and 59 units of apheresis platelets (Plts) were tested for antibodies to HLA class I and class II antigens using FlowPRA, an HLA antigen-specific flow cytometric assay. On average, 22% of blood components tested contained HLA alloantibodies, tenfold greater than previously reported. This unappreciated frequency of HLA alloantibodies in blood components may pose a risk to transplant patients requiring transfusions by promoting allograft dysfunction or loss.

Preliminary validation of a new standard of efficacy for stored platelets.

Platelet preparation and storage systems, unlike those for RBC, lack an objective, absolute performance criterion to determine acceptability. Recently, a criterion based on paired comparison with the radiolabeled recovery and survival of "fresh" platelets has been proposed, namely, recovery = two-thirds and survival = half of "fresh" platelets. Eleven normal subjects donated a unit of leukoreduced apheresis platelets using a standard, approved system. They received an aliquot radiolabeled with 111In or 51Cr (random selection) 4 to 20 hours after donation and, using the other radioisotope, on Day 5 of storage. The recovery was calculated based on the injectate radioactivity. The survival was determined using the multiple-hit model. The area under the platelet survival curve was calculated using the COST program. Reinfusion of platelets less than 20 hours after collection resulted in a recovery of 74.7 +/- 12.3 percent and a survival time of 7.5 +/- 1.1 days. Reinfusion on Day 5 resulted in a recovery of 58.2 +/- 12.0 percent and a survival time of 6.9 +/- 1.4 days, values that were 77.9 +/- 9.5 percent and 91.8 +/- 16.1 percent of the observation using "fresh" platelets, respectively. The area under the curve using Day 5 platelets was 67.8 +/- 11.5 percent of that using "fresh" platelets. The proposed criterion for objective evaluation of platelet preparation and storage systems appears applicable to a commonly accepted approach, leukoreduced apheresis platelets stored in plasma for 5 days, and merits evaluation using other collection, treatment, and storage systems.

Storage of platelets in additive solutions: a multicentre study of the in vitro effects of potassium and magnesium.

In a preliminary study, the presence of potassium and magnesium in a modified synthetic medium (PAS-III) was found to have a significant influence on platelet metabolism (using apheresis-derived, as well as buffy-coat-derived platelets) when compared with standard PAS-III. The differences included reduced glycolysis, as evidenced by lower consumption of glucose and lower production of lactate, but also better preservation of pH and hypotonic shock response reactivity. The results suggested that storage in modified PAS-III containing 20% plasma was comparable to storage in standard PAS-III containing 30% plasma. To confirm the preliminary results and to evaluate the effects of different preparation protocols, an international multicentre study, which included 11 different sites, was conducted. Platelets from 30 pools of approximately 20 buffy coat (BC) units each and 24 pooled apheresis platelet units were aliquoted for storage in plasma (reference) or synthetic medium using either a specific additive solution (PAS-III) containing 30% plasma or a modification of PAS-III containing 5.0 mm potassium and 1.5 mm magnesium (PAS-IIIM) and either 30% or 20% plasma. Units were stored at room temperature with agitation for 7 days during which in vitro testing was carried out for biochemical, haematological and functional parameters. Storage of platelets in PAS-IIIM resulted in a reduction in the rate of glycolysis and better retention of pH and hypotonic shock response reactivity. Storage in PAS-IIIM containing 20% plasma appeared to result in the retention of in vitro properties, similar to those observed during storage in standard PAS-III containing 30% plasma. The results of this study confirm the preliminary results. Similar results were seen with platelets prepared by BC and apheresis methods, despite differences in equipment, the preparation technique and in the final platelet contents achieved in the platelet units. Storage of platelets in PAS-IIIM should be considered to improve platelet function and allow plasma reduction to 20%.

Evaluation of the 3D BacT/ALERT automated culture system for the detection of microbial contamination of platelet concentrates.

Bacterial transmission remains the major component of morbidity and mortality associated with transfusion-transmitted infections. Platelet concentrates are the most common cause of bacterial transmission. The BacT/ALERT 3D automated blood culture system has the potential to screen platelet concentrates for the presence of bacteria. Evaluation of this system was performed by spiking day 2 apheresis platelet units with individual bacterial isolates at final concentrations of 10 and 100 colony-forming units (cfu) mL-1. Fifteen organisms were used which had been cited in platelet transmission and monitoring studies. BacT/ALERT times to detection were compared with thioglycollate broth cultures, and the performance of five types of BacT/ALERT culture bottles was evaluated. Sampling was performed immediately after the inoculation of the units, and 10 replicates were performed per organism concentration for each of the five types of BacT/ALERT bottles. The mean times for the detection of these 15 organisms by BacT/ALERT, with the exception of Propionibacterium acnes, ranged from 9.1 to 48.1 h (all 10 replicates were positive). In comparison, the time range found using thioglycollate was 12.0-32.3 h (all 10 replicates were positive). P. acnes' BacT/ALERT mean detection times ranged from 89.0 to 177.6 h compared with 75.6-86.4 h for the thioglycollate broth. BacT/ALERT, with the exception of P. acnes, which has dubious clinical significance, gave equivalent or shorter detection times when compared with the thioglycollate broth system. The BacT/ALERT system detected a range of organisms at levels of 10 and 100 cfu mL-1. This study validates the BacT/ALERT microbial detection system for screening platelets. Currently, the system is the only practically viable option available for routinely screening platelet concentrates to prevent bacterial transmission.