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Background: Bacterial sepsis is still a complication in patients transfused with stored platelets (PLTs). We have recently demonstrated that selected antimicrobial peptides (AMPs) have bactericidal activity in bacteria-spiked PLTs. In a subsequent preclinical study, we have also shown that these AMPs do not elicit antibody response in rabbits and treatment of PLTs before transfusion does not affect their in vivo recovery and survival in severe combined immunodeficient mice. Here we have selected two such AMPs, Arg-Trp (RW) repeats of tri- and tetra-peptides (RW3 and RW4) in combination (i.e., cocktail), and evaluated their effect on the in vitro properties of PLTs., Study Design and Methods: Leukoreduced ABO- and D-identical whole blood-derived PLT concentrates were pooled and divided into two 60-mL aliquots in CLX storage bags. On Day 0, one bag received a peptide cocktail of RW3 plus RW4 at 0.01 mmol/L final concentration (test) and the other bag received only phosphate-buffered saline (PBS), the AMP solvent (control). The treated PLTs were stored for 7 days at 20 to 24°C. Samples were collected on Days 1, 5, and 7 to evaluate the in vitro properties of PLTs with standard assays., Results: In vitro properties of the RW3 plus RW4 cocktail-treated PLTs were similar to those incubated with PBS only. There were no significant differences between the control and test PLTs during the 7-day storage., Conclusion: Leukoreduced whole blood-derived PLTs treated with a mixture of RW3 and RW4 peptides maintain their in vitro properties during 7 days of storage., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Nitric oxide (NO), a potent signaling molecule, is known to inhibit platelet (PLT) function in vivo. We investigated how the levels of NO and its metabolites change during routine PLT storage. We also tested whether the material of PLT storage containers affects nitrite content since many plastic materials are known to contain and release nitrite., Study Design and Methods: For nitrite and nitrate measurement, leukoreduced apheresis PLTs and concurrent plasma (CP) were collected from healthy donors using a cell separator. Sixty-milliliter aliquots of PLT or CP were stored in CLX or PL120 Teflon containers at 20 to 24°C with agitation and daily samples were processed to yield PLT pellet and supernatant. In a separate experiment, PLTs were stored in PL120 Teflon to measure NO generation using electron paramagnetic resonance (EPR)., Results: Nitrite level increased markedly in both PLT supernatant and CP stored in CLX containers at a rate of 58 and 31 nmol/L/day, respectively. However, there was a decrease in nitrite level in PLTs stored in PL120 Teflon containers. Nitrite was found to leach from CLX containers and this appears to compensate for nitrite consumption in these preparations. Nitrate level did not significantly change during storage., Conclusion: PLTs stored at 20 to 24°C maintain measurable levels of nitrite and nitrate. The nitrite decline in nonleachable Teflon containers in contrast to increases in CLX containers that leach nitrite suggests that it is consumed by PLTs, residual white blood cells, or red blood cells. These results suggest NO-related metabolic changes occur in PLT units during storage., (© 2012 American Association of Blood Banks.)

Background: A previous study demonstrated that several in vitro storage properties of apheresis platelets (PLTs) that are isolated by sedimentation against the collection container and subsequently resuspended can benefit from a rest period before continuous agitation. This study examines whether the in vitro storage properties of apheresis PLTs isolated by collection from the buffy coat layer benefit from a rest period before agitation., Study Design and Methods: Freshly collected apheresis PLTs (Trima, GambroBCT) were divided into five 60-mL aliquots. One aliquot was immediately placed on a flat-bed agitator; the other aliquots were held on a laboratory bench for 1, 2, 4, and 6 hours before continuous agitation. Samples were obtained on Days 1, 5, and 7 for standard in vitro PLT assays. The experiment was repeated 12 times., Results: For each sampling day, no significant differences were observed in aliquots held with or without a rest period for any of the following PLT properties: PLT content, mean PLT volume, pH, pCO2, bicarbonate, glucose, lactate, hypotonic shock response, extent of shape change, aggregation, morphology, CD62P, CD63, and CD42b. Although regression analysis identified several in vitro properties whose mean levels appeared to improve with increasing length of the rest period, maximum differences in mean levels were small (<6%)., Conclusion: The in vitro storage properties of Trima apheresis PLTs isolated from the buffy coat layer do not benefit from a rest period., (© 2012 American Association of Blood Banks.)

Background: A comparative study evaluated the retention of apheresis platelet (A-PLT) in vitro properties prepared with PLT additive solution (PAS)-III or 100% plasma and stored with continuous agitation (CA) and without continuous agitation (WCA)., Study Design and Methods: PLTs collected with the Amicus cell separator (Fenwal, Inc.) were utilized to prepare two matched components, each with approximately 4 × 10(11) PLTs. In the primary study, one component contained 65% PAS-III/35% plasma and the other 100% plasma. Four storage scenarios were used, one with CA and three with periods without agitation under simulated shipping conditions. In vitro assays were used early and after 5 days of storage., Results: pH levels after 5 days with CA were less with PAS-III components than 100% plasma components, with levels always above 6.6 in any component. With CA, a number of other variables were reduced even early during storage with PAS-III including morphology, extent of shape change, hypotonic stress response, adhesion, and aggregation. Storage WCA resulted in only a limited increase in the magnitude of the assay differences between PAS-III and 100% plasma components. Periods WCA did not reduce the pH below 6.6. The thromboelastograph variable associated with the strengthening of clots by PLTs was essentially comparable with PAS-III and plasma components throughout storage with CA or WCA., Conclusion: The data indicate that a 100% plasma medium provides for better retention of specific in vitro PLT properties, with CA and WCA, although the clinical significance of these in vitro decrements due to PAS-III is unknown., (© 2011 American Association of Blood Banks.)

Background: Amicus apheresis platelets (PLTs) can be collected using either a single- (SN) or a double-needle (DN) procedure. To investigate whether the method of PLT collection using the same instrument influences PLT quality, the in vitro storage properties of Amicus PLTs were evaluated in the same donors collected by SN and DN procedures., Study Design and Methods: Single apheresis PLT collections with concurrent plasma were performed on donors using the Amicus with a target yield of 4 × 10(11). A PLT unit was collected from a donor assigned to either a SN or a DN procedure; a subsequent donation from the same individual was collected by the other procedure (n = 10). Units were stored at 20 to 24°C with continuous agitation, assayed for 19 PLT storage variables, and analyzed by paired t test, with differences between values obtained with SN and DN collections considered significant with p values of less than 0.001., Results: PLT units collected by SN procedure had contents and concentrations similar to those collected by DN procedures (4.1 × 10(11) ± 0.3 × 10(11) vs. 4.0 × 10(11) ± 0.3 × 10(11) and 1396 × 10(9) ± 131 × 10(9) vs. 1367 × 10(9) ± 110 × 10(9) PLTs/L). On Day 7, SN and DN PLTs had comparable pH values (7.07 ± 0.09 vs. 6.99 ± 0.17), morphology (52.4 ± 18.7% vs. 56.0 ± 13.3% discoid), aggregation (87.1 ± 11.5% vs. 91.3 ± 5.4%), and activation (45. ± 11.9% vs. 48.2 ± 8.7% CD62P), as well as all other variables (p > 0.05; Day 7 CO(2) , p = 0.0304)., Conclusion: The in vitro storage properties of apheresis PLTs collected from the same donors using a SN and DN procedure with the Amicus instrument were maintained through 7 days of storage and yielded comparable results., (© 2011 American Association of Blood Banks.)

Background: The platelets (PLTs) in PLT concentrates are counted with hematology analyzers, but varying results among different hematology analyzers are observed, making comparisons very difficult. Due to the absence of red blood cells in PLT concentrates, the International Council for Standardization in Hematology (ICSH) reference method was modified to be used for PLT concentrates and validated in an international comparative study., Study Design and Methods: Five PLT samples were shipped to eight participating centers of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative and counted on the same day. PLTs were stained with fluorescein isothiocyanate-labeled anti-CD41a in tubes (TruCount, BD Biosciences), measured on a flow cytometer, and analyzed with a uniform template. These samples were also counted on 15 hematology analyzers., Results: The ICSH method and newly developed BEST method yielded PLT counting results with less than 1% difference (not significant). The intercenter coefficient of variation (CV) of the BEST method was on average 6.3% versus 7.6% on average for hematology analyzers. The CV of individual hematology analyzers was on average 0.9%, which was considerably lower than for the flow cytometers with a mean of 3.7%., Conclusion: The BEST flow cytometric method has a smaller intercenter CV and a smaller center-to-center deviation from the group mean compared to hematology analyzers. Conversely, individual hematology analyzers are more precise than the flow cytometric method. Thus, the flow cytometric method provides a calibration tool to allow comparisons between centers, but there is no need to replace routine counting with hematology analyzers., (© 2011 American Association of Blood Banks.)

Background: pH measurements of platelet (PLT) components remain a key parameter when assessing how storage and shipping conditions influence the retention of PLT properties. Studies were conducted to characterize variations in pH measured with two pH meters and a blood gas analyzer., Study Design and Methods: Samples were obtained from apheresis PLT units that were stored with or without continuous agitation to measure a range of pH values. pH values were determined with pH meters at room temperature (20-24°C) upon placing of samples in 5-mL sterile polypropylene tubes and with the blood gas analyzer at 37°C upon injection of identical samples, with conversion to 22°C., Results: The calculated coefficient of variation (%CV) of pH measurements using pH meters (n = 10) was 0.43% or less. The %CV values were comparable with different samples having pH values ranging from 6.0 to 7.4. The %CV levels with the blood gas analyzer were comparable to those observed with the pH meters. The difference in the mean pH values for the two pH meters was no greater than 0.10 units, with 9 of 10 samples having differences in values of 0.05 or less; however, greater differences of values (0.1 to 0.2) were observed between pH measured using the blood gas analyzer and pH meters., Conclusion: Our data show good precision and comparability of pH measurements with two pH meters. Differences in pH values were greater on comparison of the blood gas analyzer with the pH meters., (© 2011 American Association of Blood Banks.)

Background: Apheresis platelet (PLT) units are not routinely agitated during transit. Our study compared the in vitro properties of apheresis PLT units that were stored with continuous agitation (CA) and without continuous agitation (WCA) during two separate periods, immediately after collection and between Day 2 and Day 3 of storage., Study Design and Methods: Two identical apheresis PLTs units were prepared from collections with Amicus (n = 11, Fenwal, Inc.) and Trima (n = 10, CaridianBCT) cell separators. One apheresis PLT unit was continuously agitated, starting routinely within 30 minutes of collection, and an identical apheresis PLT unit was held without agitation initially for 7 to 8 hours and subsequently for 24 hours between Day 2 and Day 3 of storage. The apheresis PLT units were maintained WCA at 20 to 24 °C in a shipping box. In vitro PLT properties were evaluated on Day 1 (day after collection), after 5 and 7 days of storage., Results: With both Amicus and Trima apheresis PLT units, the mean PLT content and concentration of CA and WCA were comparable and essentially constant throughout storage. Mean pH levels (± 1 SD) after 5 days for Amicus apheresis PLT units were 6.97 ± 0.20 (WCA) and 7.13 ± 0.16 (p < 0.001, CA) and for Trima apheresis PLT units 6.97 ± 0.21 (WCA) and 7.22 ± 0.17 (p < 0.001, CA). In vitro variables, including percentage of disc PLTs, extent of shape change, and hypotonic stress levels, after 5 days of storage, showed mean differences between WCA and CA that were less than 15%., Conclusion: The in vitro results show that apheresis PLT units can be stored without agitation for 7 to 8 hours immediately after collection and also subsequently during storage for 24 hours with minimal influence on in vitro PLT properties compared to continuously agitated PLTs., (© 2010 American Association of Blood Banks.)

Background: The Advia 120 (Siemens Diagnostics) hematology analyzer is different from other hematology analyzers in that it requires platelets (PLTs) to be "effectively spherical" to be counted. Our study evaluated how PLT counts with this hematology analyzer and two other models were influenced by the holding of PLT product samples., Study Design and Methods: Samples were prepared from apheresis PLT products (APs) collected in ACD-A and from whole blood-derived PLT concentrates (PCs) in CP2D or ACD-A. Samples were stored in K(2) and K(3) ethylenediaminetetraacetate (EDTA) tubes at room temperature (RT) and in the cold. PLT counts were determined immediately, after 1 and 4 hours, and after an overnight hold, using Advia 120, XE-2100D, and Cell-Dyn 3700 hematology analyzers., Results: A time-dependent increase in PLT counts was observed with AP samples held at RT using the Advia 120, but not with the other two hematology analyzers. AP samples held in the cold did not show a substantial time-dependent increase with any of the hematology analyzers. With the Advia 120, the PLT counts in the immediate samples were approximately 14% lower compared to those in cold or overnight-held RT samples. PC samples with all holding conditions and hematology analyzers did not show any substantial time-dependent increase in counts., Conclusions: With the Advia 120 hematology analyzer, the time-dependent increase in PLT counts with RT-held samples may be related to the need to have effectively sphered PLTs unlike that with the other two hematology analyzers. The absence of a holding effect with PC samples may indicate that only AP samples have population(s) that are slow to convert to spherical PLTs., (© 2010 American Association of Blood Banks.)

Background: The activation marker CD40 ligand (CD40L) has recently been demonstrated to be released from the cytoplasm of platelets (PLTs) during storage. CD40L may be associated with some adverse transfusion reactions including febrile responses and transfusion-related acute lung injury. CD62P has been traditionally measured to assess PLT activation. This study compares the surface levels of CD40L and CD62P and accumulation of the soluble forms of these activation markers in the plasma of stored PLTs, prepared by PLT-rich plasma (PRP) or buffy coat (BC) methods and with two apheresis instruments., Study Design and Methods: Individual PLT concentrates (PCs) were prepared in 100 percent plasma from a pool of two ABO-identical whole-blood units. Apheresis PLTs (APs) were prepared in 100 percent plasma using one of two commercially available cell separators (Amicus, Baxter Healthcare; and Trima, Gambro BCT). Surface expression of CD40L and CD62P was measured by flow cytometry, and secretion of soluble CD40L (sCD40L) and soluble CD62 (sCD62) was measured by enzyme-linked immunosorbent assay during 7 days of PLT storage., Results: Secretion of sCD40L was greater in Amicus APs than in Trima APs during the first 3 days of storage. It was also greater in the PRP-PC preparations than in BC-PC preparations through the first day of storage. Surface expression of CD40L was low in all PLT preparations. Secretion of sCD62P was greater in Amicus APs than in Trima APs during the entire storage period and greater in PRP-PC than in BC during the first 5 days of storage. The percentage of CD62P-positive PLTs was greater in Amicus units than Trima units and greater in PRP-PC than BC-PC preparations during the first 5 and 3 days of storage, respectively., Conclusion: The kinetics of the secretion of CD40L are influenced by the method used to prepare PLTs for storage. The patterns for CD40L membrane association and secretion are different than those observed for CD62P during storage.

Background: The testing of cord blood (CB) progenitor and stem cell units for transplantation suitability involves enumeration of total nucleated cells before freezing. CD34+ cell counts may also be a means of determining suitability. Studies have been conducted to evaluate how specific storage conditions influence cell counts., Study Design and Methods: CB units were processed by hydroxyethyl starch volume reduction. Cryopreserved-thawed samples were diluted 1:3 without washing. CD34+ cells were measured with three commercially available assay methods. In specific studies, apoptosis-indicating reagents were included. CB units were analyzed for nucleated cells, aldehyde dehydrogenase-containing cells, and progenitor colonies., Results: CD34+ cell levels and nucleated cells were retained during storage in test tubes at 1 to 6 degrees C for 3 days. Cryopreserved-thawed samples showed a reduction in CD34+ cells relative to prefreeze levels with the largest decrease with the Stem-Kit (Beckman Coulter) restricted gating procedure. Prefreeze samples contained minimal numbers of presumed apoptotic cells detected with 7-aminoactinomycin D or SYTO16, but after cryopreservation-thawing there was an increase. Nucleated cell levels determined with a hematology analyzer or flow cytometry were reduced after thawing. Cryopreservation-thawing reduced the percentage of CD34+ cells positive for the presence of aldehyde dehydrogenase and the number of progenitor colonies. These differences were significant., Conclusion: These studies indicate that CD34+ cell counts were maintained when CB samples were stored at 1 to 6 degrees C in test tubes for 3 days. Cryopreservation-thawing resulted in changes in a number of parameters including the percentage of CD34+ cells that were aldehyde dehydrogenase(+) and the number of 7-aminoactinomycin D(+) cells and SYTO16(low) cells.

Background: Understanding the variability in results obtained by multiple laboratories is important because cord blood units are distributed worldwide for transplantation., Study Design and Methods: Four exercises were conducted by multiple laboratories to assess assay variability on nucleated cell (NC), mononuclear cell (MNC) by hematology analyzers [HAs], and CD34+ cell (flow cytometry) measurements. Exercise 1 was an intralaboratory exercise in which the reproducibility of cell measurements was determined. Exercises 2 and 3 involved the shipment of identical processed cord blood samples. In Exercise 2, laboratory-specific methods were utilized. In Exercise 3, two commercial CD34+ cell methods (Stem-Kit and TruCOUNT) were used. In Exercise 4, CD34+ cell levels were determined on repetitive regating of identical list-mode files., Results: Intralaboratory reproducibility was highest for NC measurements and lowest for CD34+ cell measurements. In Exercise 2, all laboratories except one utilized HA with an impedance technology and determined comparable results for NC and MNC levels, whereas the other laboratory utilized a HA with an optical counting method. Substantial variation was observed on measuring CD34+ cells with ranges of 32 to 141, 32 to 66, and 25 to 116 CD34+ cells per microL for the three identical samples. In Exercise 3, on the use of one specific commercial assay, the ranges of CD34+ levels were 214 to 411 and 62 to 178 cells per microL for the two identical samples. Nearly all participating laboratories determined comparable CD34+ levels on the use of identical list-mode files., Conclusion: These studies indicate that substantial variability in CD34+ cell levels were determined with flow cytometry. The variability in NC and MNC levels was minimal with HA methodology.