Counting platelets in platelet concentrates on hematology analyzers: a multicenter comparative study

BACKGROUND: Hematology analyzers are designed to count whole blood samples, but are also used by blood centers to perform quality control on blood components. In platelet (PLT) concentrates, the number of PLTs is approximately fivefold higher and red blood cells are absent, causing variable PLT counting results. It was our aim to compare currently used hematology analyzers for counting PLTs in PLT concentrates using fixed human PLTs.
STUDY DESIGN AND METHODS: PLT samples were fixed, diluted into seven concentration levels (plus one blank), aliquoted, and shipped to 68 centers. Evaluable data were obtained for 89 hematology analyzers. All samples were counted six times, and results were reported to the coordinating center. The overall group mean was calculated, and the percentage deviation from this mean was calculated for each analyzer.
RESULTS: At PLT levels relevant for blood centers, 750 × 10⁹ to 2000 × 10⁹ per L, analyzers gave results that were between 35 percent lower and 16 percent higher than the overall group mean. Within a group of analyzers, results were comparable with coefficient of variations usually below 10 percent, indicating that the observed differences were caused by instrument characteristics. A smaller study with fresh, unfixed PLT samples showed that analyzers behaved similarly for fixed and fresh PLTs.
CONCLUSION: With a wide array of currently used hematology analyzers, a marked difference was determined for the PLT counts of fixed human-based identical samples provided to 68 laboratories by a centralized facility. A gold standard method is needed to allow for more valid interlaboratory comparisons between hematology analyzers.     

In vitro pH effects on in vivo recovery and survival of platelets: an analysis by the BEST Collaborative

BACKGROUND: The pH environment of stored platelet (PLT) products is recognized as an important factor and is generally used as a key surrogate measure of PLT viability. It is the only in vitro measurement that has been translated into industry standards and regulatory rules or specifications for storage of PLT products. The objective of this study was to evaluate the effect of in vitro pH on the in vivo recovery and survival of autologous PLT products.
STUDY DESIGN AND METHODS: Data from individual autologous radiolabeled PLT kinetic studies were solicited from independent laboratories. PLTs stored for at least 5 days in 100 percent autologous plasma with a pH_22°C of at least 6.2 were analyzed. Data were fit to a mixed-effects regression model with fixed effects of pH_22°C, time of storage, and preparation method-storage bag combination.
RESULTS: Eight research laboratories reported 476 individual recovery and survival results with associated pH before labeling from a variety of autologous, radiolabeled PLT kinetic studies from September 1999 to March 2005. These results are from 254 individual subjects who donated a total of 386 PLT units, with up to nine collections per subject reported. The effect of pH on either PLT recovery (p = 0.86) or survival (p = 0.55) was not significant. Time of storage and the method-bag combination both had significant effects on these outcomes (p < 0.0001).
CONCLUSION: These data suggest that there is no relationship between in vitro pH at a pH_22°C of at least 6.2 and in vivo PLT viability as measured by radiolabeled recovery and survival of autologous PLTs.     

Bacterial contamination of platelet concentrates: results of a prospective multicenter study comparing pooled whole blood-derived platelets and apheresis platelets

BACKGROUND: The GERMS Group initiated a prospective multicenter study to assess prevalence and nature of bacterial contamination of pooled buffy-coat platelet concentrates (PPCs) and apheresis platelet concentrates (APCs) by routine screening with a bacterial culture system. STUDY DESIGN AND METHODS: In nine centers overall, 52,243 platelet (PLT) concentrates (15,198 APCs, 37,045 PPCs) were analyzed by aerobic and anaerobic cultures (BacT/ALERT, bioMérieux). RESULTS: In 135 PLT concentrates (PCs; 0.26%), bacteria could be identified in the first culture (0.4% for APCs vs. 0.2% for PPCs; p < 0.001). In 37 (0.07%) of these PC units, the same bacteria strain could be identified in a second culture from the sample bag and/or the PC unit. The rate of confirmed-positive units did not differ significantly between APC (0.09%; 1/1169) and PPC units (0.06%; 1/1544). Bacteria from skin flora (Propionibacterium acnes, Staphylococcus epidermidis) were the most prevalent contaminants. Median times to first positive culture from start of incubation were 0.7 and 3.7 days in aerobic and anaerobic cultures for confirmed-positive units. With a "negative-to-date" issue strategy, most PC units (55%) had already been issued by time of the first positive culture. CONCLUSION: The rate of confirmed bacterial contamination of PC units was low. Nevertheless, clinicians must be aware of this risk. The risk of bacterial contamination does not warrant universal preference of APCs. It must be questioned whether routine bacterial screening by a culture method can sufficiently prevent contaminated products from being transfused due to the delay until a positive signal in the culture system and due to false-negative results.     

The effect of temperature and mode of agitation on the resuspension of platelets during preparation of platelet concentrates

A problem arose in a blood bank in which about 10 percent of platelet concentrates (PC) observed at day 1 had a large number of macroscopic aggregates. The aggregates were clumps of platelets that did not go into suspension during preparation of PC. These units were set aside for observation. A maximum of 25 percent were stored on a circular rotator; the remainder were stored on either an elliptical rotator or horizontal shaker. By the end of storage, 6 percent of the units still had a large number of clumps. This percentage was reduced to 2.4 percent, when all clumped units were stored on a circular rotator. The number of clumped units observed at day 1 and at the end of storage were reduced dramatically when the temperature of the laboratory (18-19 degrees C), centrifuge (20 degrees C) and storage environmental chamber (22 degrees C) were increased to 24 degrees C. The units with clumps at day 5 were reduced from 225 (out of 8316) to 25 (out of 7137).     

The effect of interruption of agitation on in vitro measures of platelet concentrates in additive solution

BACKGROUND: Interruption of agitation results in lower in vitro quality of platelet concentrates (PCs). The rates at which the deleterious effects occur, however, are unknown. Therefore, in vitro parameters of PCs in additive solution (AS) during various periods without agitation have been investigated. STUDY DESIGN AND METHODS: PCs from five buffy coats in AS (Composol, Fresenius HemoCare) were white cell (WBC)-reduced by filtration. Four PCs were pooled and divided to obtain paired samples. Beginning immediately after processing, three PCs were stored without agitation and placed on an agitator after 16, 20, and 24 hours. The fourth PC was agitated throughout storage and served as reference (n = 10 paired experiments). RESULTS: pH(37 degrees C) on Day 7 was greater than 6.8 in reference PCs, and in PCs that were not agitated for 16 hours, longer interruption resulted in lower pH values. During interruption of agitation, metabolic rates were significantly higher in the study groups: glucose consumption was 12.5 +/- 1.6 micromol per 10(11) platelets (PLTs) per hour in PCs during the first 24 hours without interruption versus 2.0 +/- 0.4 micromol per 10(11) PLTs per hour in the reference group (p < 0.01). Lactate formation was 24.7 +/- 4.2 versus 3.9 +/- 0.4 micromol per 10(11) PLTs per hour in the above-mentioned groups, respectively (p < 0.01). Once replaced on the agitator, the metabolic rates lowered, but remained significantly elevated during consecutive storage days compared to the reference. CONCLUSION: WBC-reduced PCs in Composol AS may experience 16 hours without agitation with no permanent effects on in vitro measures compared to reference units. During interruption of agitation, glucose and lactate metabolism is elevated, resulting in lower pH values in the subsequent storage period.     

Isolation of leukocyte-free platelets from standard platelet concentrates by centrifugation

We evaluated centrifugation techniques to isolate leukocyte-free platelets from standard platelet concentrates. Among various settings of centrifugation force and time, we found the setting at 300 X g for 10 minutes or 370 X g for 5 minutes to be efficient and reproducible for recovering sufficient quantities of platelets free of leukocytes.     

Platelet factor 4 release from the platelets stored in platelet concentrates

Many consider that granular components of platelets should remain intact during storage of platelet concentrates (PCs) in order to participate in hemostasis when infused. The present investigations were done to determine factors affecting the release of an alpha-granular component, platelet factor 4 (PF-4), from platelets stored as PCs. Sedimentation of the pelleted platelet button and stronger flat-bed agitation induced a significant PF-4 release. After 1 day of storage at 22 degrees C, the percent PF-4 release correlated roughly with a pH fall in PCs (r = 0.520, y = 0.014 + 7.204). Rates of pH fall were roughly proportional to the platelet count. In addition, there was a positive relationship between PF-4 release and the platelet count per unit volume (r = 0.615, y = 0.045 + 1.327). The results indicated that one of the critical factors which determines the amount of PF-4 release during storage is concentrated pH. Lowering the platelet count per unit volume in PCs is better for the maintenance of granular components in platelets during storage because of the intact platelet metabolism.     

Practices associated with ABO‐incompatible platelet transfusions: a BEST Collaborative international survey

BACKGROUND: There is a lack of evidence for guiding the best strategy for ABO selection of platelet (PLT) transfusions. As a baseline for future studies, the BEST Collaborative performed an international survey of current practices in this area. STUDY DESIGN AND METHODS: An international survey was sent via BEST members to transfusion services and hospitals requesting the demographics of the transfused patient population, ABO matching policies, anti‐A and anti‐B measurements in PLT concentrates (PCs), and practices regarding ABO‐incompatible PC transfusions to adult and pediatric patients. RESULTS: We received 126 responses from 14 countries, 59% from Europe. Most of them were from local/community (42%) and university hospitals (39%) serving between 500 and 1500 beds; 50.4% transfused fewer than 1000 PCs per year. One‐fifth of respondents (19.4%, mainly local/community hospitals) did not have a written policy for selecting ABO‐incompatible PCs. Significant practice variation was reported when ABO‐mismatched PLTs were given to adults: for PCs suspended in 100% plasma, 29% to 43% of respondents selected any ABO group available; 52% to 61% selected units with compatible supernatant; and, in the case of minor ABO incompatibility, 43% to 54% did not take any specific action. In contrast if ABO‐identical PCs were not available for a pediatric recipient, for PCs resuspended in 100% plasma, 71% to 82% selected PCs so the supernatant plasma would be compatible with patient's red blood cells. CONCLUSION: Considerable practice variation exists when transfusing ABO‐incompatible PCs, suggesting an opportunity for research to inform evidence‐based practices.     

Effect of microaggregate blood filtration on platelet concentrates in vitro

In order to determine if platelet concentrate could be transfused through microaggregate filters, platelets collected in CPD and CPDA-1 preservatives were passed through the currently available microaggregate blood filters. Both clean filters and filters preloaded with two units of outdated whole blood were studied. Prefiltration and postfiltration testing included determination of platelet count, per cent maximal ADP aggregation, release of B-thromboglobulin, and morphologic examination using electron microscopy. Compared to the prefiltration values, those for postfiltration showed no significant differences (p > 0.05) for any of the microaggregate filters tested. Filtration itself did not appear to affect platelet morphology. Unlike the platelets in stored concentrates, however, a significant (p < 0.05) number of the platelets contained in fresh whole blood (27 to 45%) were removed by depth-type microaggregate filters. Platelets remaining in the filter residual volume could be readily eluted by rinsing with crystalloid or colloid solutions. Due to development of an in vitro storage lesion, platelet concentrates can be transfused through new or used microaggregate filters without loss of platelet number or changes in platelet function as measured in vitro.     

The effects of pH and agitation on platelet preservation

Platelet concentrates made with an initial pH of 7.85 or 6.85 by addition of alkali or acid were stored at 22 degrees C on tumbler or horizontal agitators. The combination of a high pH and use of a tumbler rotator was associated with a 40 percent reduction in platelet count, fragmentation of platelets, release of lactate dehydrogenase, and a marked loss in response to adenosine diphosphate. Similar, but less striking changes occurred in acidified platelet concentrates stored on the tumbler rotator. Preservation was good for both alkaline and acidified concentrates stored on the horizontal agitator. These in vitro studies showed that, even at a pH of up to 7.7, gently agitated platelets were relatively undamaged for up to 60 hours, while vigorous agitation destroyed platelets at the same pH.     

False-positive alarms for bacterial screening of platelet concentrates with BacT/ALERT new-generation plastic bottles: a multicenter pilot study

BACKGROUND: The microbial detection system BacT/ALERT (bioMérieux) is widely used to monitor bacterial contamination of platelet concentrates (PCs). Recently, the manufacturer introduced polycarbonate culture bottles and a modified pH-sensitive liquid emulsion sensor as microbial growth indicator. This reconfigured assay was investigated in a routine setting. STUDY DESIGN AND METHODS: In each of eight transfusion centers, samples from 500 consecutive PCs were monitored for 1 week. For all PCs with a positive BacT/ALERT signal, retained samples and, if available, original PC containers and concomitant red blood cell concentrates were analyzed independently. Initially BacT/ALERT-positive PCs without bacterial identification in any sample were defined as false-positive. BacT/ALERT-positive PCs with bacteria in the first sample only were called potentially positive. PCs with bacteria in the first sample and the same strain in at least one additional sample were accepted as positive. RESULTS: Five PCs (0.13%) were positive, 9 PCs (0.23%) were potentially positive, and 35 PCs (0.9%) were false-positive. The rate of false-positive BacT/ALERT results varied substantially between centers (<0.2%-3.2%). Tracings from false-positive cultures lacked an exponential increase of the signal during incubation. Most of these false-positives were due to malfunctioning cells in various BacT/ALERT incubation units. CONCLUSION: Careful assessment of individual tracings of samples with positive signals helps to identify malfunctioning incubation units. Their early shutdown or replacement minimizes the high rate of unrectifiable product rejects attributed to false-positive alarms and avoids unnecessary concern of doctors and patients after conversion to a reconfigured BacT/ALERT assay.     

Effects of high platelet concentration in collecting and freezing dry platelets concentrates

OBJECTIVE: To evaluate, in vitro, the effects of collecting and cryopreserving fresh dry platelet concentrates (PCs). MATERIAL AND METHODS: Standard and dry PCs were collected in the same apheresis procedure. PCs were evaluated by mean platelet volume (MPV), pH, glucose and LDH levels. Activation was examined by flow cytometry using anti-CD41, anti-CD42 and anti-CD62p monoclonal antibodies and annexin binding assay. Platelet function was assessed by aggregation using ADP, collagen and arachidonic acid as agonists. Dry PCs were compared to standard PCs and to cryopreserved dry PCs. We also compared the use of ThromboSol to 5% DMSO as cryoprotectives. RESULTS: Dry PCs presented a significantly reduced pH and glucose (p<0.001), increased LDH levels and CD62p expression (p<0.001) and diminished aggregation response to ADP (p<0.001). Platelet cryopreservation was associated with platelet lysis, activation and loss of function. Dry PCs cryopreserved with TS were associated with statistically higher LDH levels (p<0.001) and a higher percentage of annexin binding (p=0.005), in addition to a lower number of CD42 positive platelets (p=0.01). CONCLUSION: Dry PCs should be rapidly frozen after collection to avoid a fall in pH and platelet activation. 5% DMSO performed better than TS to cryopreserve dry PCs.     

Periodic alternating interface positioning to lower WBC contamination of apheresis platelet concentrates: a multicenter evaluation

BACKGROUND: A new software version of a cell separator (AS TEC 204, Fresenius) providing WBC-reduced single-donor plateletpheresis concentrates was tested. STUDY DESIGN AND METHODS: Dual-needle apheresis procedures (n = 621) were performed in three centers, using either fixed interface positioning (FIP) or periodic alternating interface positioning (PAIP). The other separation parameters (e.g., anticoagulant:whole-blood ratio, and blood flow) were set individually. All platelet concentrates were evaluated for platelet yields and contaminating WBCs. RESULTS: The introduction of the PAIP resulted in a significant (p<0.001) reduction in contaminating WBCs (median, 30,000) from the numbers seen with FIP (median, 2,300,000) while maintaining the separation efficacy (47%) and separation time. Ninety-eight percent of all concentrates contained less than 5 x 10(6) WBCs per concentrate and 92 percent contained less than 1 x 10(6). CONCLUSION: Plateletpheresis using the AS TEC 204 cell separator with PAIP is a valid alternative to WBC reduction by filtration. It may provide WBC-reduced platelet concentrates without the additional cost of filters. However, the reliability of the WBC reduction is not yet advanced enough that PAIP can be employed without any monitoring of the end product.     

Pooled platelet concentrates provide a small benefit over single-donor platelets for patients with platelet refractoriness of any etiology

BackgroundAt our institution, patients with platelet refractoriness (of any etiology) are sometimes switched from apheresis platelets to pooled platelets before human leukocyte antigen (HLA)-matched units become available.Study design and methodsSeven patients were analyzed. Platelet counts were available from 57 single-unit transfusions (26 pooled, 31 apheresis). A mixed linear effects model was used and significance was determined using a likelihood ratio test.ResultsWhen analyzed as the only fixed effect in the model, the use of pooled versus single-donor units and time from transfusion to post-transfusion blood sampling each showed a significant effect on platelet count increments. A mixed linear effect model including both factors showed that transfusing a pooled unit correlated with a 4500±2000/µL greater platelet count increment compared with a single-donor unit, and an increase in time from transfusion to post-transfusion blood sampling lowered the platelet count increment by 300±100/µL per hour.ConclusionA small but potentially clinically relevant benefit was observed in transfusing pooled random-donor platelets compared with single-donor units for patients with platelet refractoriness (of any etiology).     

Storage characteristics of cord blood progenitor cells: report of a multicenter study by the cellular therapies team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative

BACKGROUND: Most hematopoietic progenitor cell (HPC) products are infused or processed shortly after collection, but in some cases this may be delayed for up to 48 hours. A number of variables such as temperature and cell concentration are of critical importance for the integrity of HPCs during this time. STUDY DESIGN AND METHODS: We evaluated critical variables using cord blood HPC units that were divided equally and stored at 4°C versus room temperature (RT) for up to 96 hours. Total nucleated cell (TNC) and mononuclear cell (MNC) counts, viable CD34+ cell counts, and CD45+ cell viability as well as colony‐forming unit–granulocyte‐macrophage (CFU‐GM) present over time at each temperature were determined. RESULTS: Overall, the data indicate that with the exception of viable CD34+ cells, there was a significant decrease in each variable measured for 72 to 96 hours and, with the exception of viable CD34+ cells and CFU‐GM, the reductions were significantly greater in RT units than 4°C units. There was an increase in viable CD34+ count for units where TNC count was greater than 8.5 × 10⁹/L, compared with units where TNC count was less than 8.5 × 10⁹/L, that was different for each storage temperature. CONCLUSIONS: Cord blood HPC collections maintained at 4°C retained higher TNC counts, MNC counts, and CD45+ cell viability over a 72‐ to 96‐hour storage period.     

保存期内单采血小板体外特性的初步研究

目的探讨保存期间不同含量和纯度的单采血小板体外特性的变化。方法根据血小板的含量和纯度,将单采血小板分为Ⅰ组、Ⅱ组和Ⅲ组,(22±2)℃震荡条件下,100%血浆保存7 d,分别于d 0、d 1、d 5、d 7取样检测血小板数(Plt)、血小板形态、pH、葡萄糖、乳酸含量、CD62p、血小板聚集功能(PAgT)和细菌培养等指标。结果血小板保存到d 7时,各组乳酸生成量、MPV、CD62p阳性表达率显著增加,pH、PAgT显著降低(P<0.05),但Ⅱ组各项指标的变化程度比Ⅰ、Ⅲ组低,且pH仍>6.7;与d 5相比,Ⅱ组血小板的新陈代谢指标、形态学、活化与体外功能指标并无统计学差异(P>0.05),亦未见细菌生长。结论在血小板保存过程中,确实存在保存损伤,不同含量和纯度的血小板制品损伤程度不一,只要将每袋血小板计数控制在(1.0—5.0)×1011,白细胞污染率控制在1.0×106以下,可以将保存时间由目前的d 5延长到d 7。     

Comparative in vitro evaluation of apheresis platelets stored with 100% plasma or 65% platelet additive solution III/35% plasma and including periods without agitation under simulated shipping conditions

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 × 10¹¹ 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.     

A multicenter evaluation of reproducibility of swirling in platelet concentrates. Biomedical Excellence for Safer Transfusion (BEST) Working Party of the International Society of Blood Transfusion

BACKGROUND: Eleven laboratories participated in a study investigating whether swirling can be used in monitoring platelet discoid morphology in platelet concentrates (PCs). STUDY DESIGN AND METHODS: In each laboratory, two readers observed the swirling pattern in 1- to 5-day- old PCs, in PCs containing spherical platelets obtained by 4 degrees C storage (spherical PCs), and in PCs containing known proportions of spherical and discoid platelets. Swirling was reported as positive, intermediate, or negative. Moreover, platelet morphology was evaluated by oil-phase microscopy. RESULTS: Swirling of spherical PCs was found to be positive and negative in 5 and 68 percent of cases, respectively. Conversely, swirling of fresh PCs was positive and negative in 83 and 2 percent of cases, respectively. Results in PCs containing known amounts of added spherical platelets were intermediate between those in fresh and spherical PCs. The observed agreement between the two readers (number of concordant readings/total readings) was 67 percent. Most disagreements concerned positive versus intermediate or intermediate versus negative evaluations, whereas a positive versus negative disagreement was observed in only 1 percent of cases. The percentages of discs found by microscopy showed greater variation among the different laboratories, indicating that this measurement should be better standardized. CONCLUSION: Evaluation of swirling seems promising for large-scale quality control of PCs. Further studies are needed to confirm this hypothesis.     

White cell reduction in platelet concentrates and packed red cells by filtration: a multicenter clinical trial. The Trap Study Group

BACKGROUND: Most previous studies on white cell (WBC) reduction by filtration have been small-scale studies conducted under tightly controlled laboratory conditions. Their results would be the ideal, rather than what might be expected during routine operation. STUDY DESIGN AND METHODS: To obtain information on routine filtration of blood components, data have been collected from a large-scale, ongoing, multicenter clinical trial designed to determine the effectiveness of WBC reduction in or ultraviolet B radiation of platelet concentrates before transfusion in preventing platelet alloimmunization and platelet transfusion refractoriness. The WBC content of blood components both before and after filtration was determined by automated cell counters and a manual propidium iodide-staining method, respectively. Platelet and red cell losses during filtration were measured. RESULTS: The average platelet losses after filtration were 24 +/? 15 percent and 20 +/? 9 percent for apheresis platelets and pooled platelets, respectively. The frequencies at which filtered platelet concentrates contained high levels of residual WBCs (> 5 × 10(6)) were 7 percent and 5 percent for apheresis platelets and pooled platelets, respectively. Further analysis of the platelet filtration data showed that greater numbers of total initial WBCs in the pooled platelets were associated with increased percentages of filtration failure (> 5 × 10(6) residual WBCs). For packed red cells, the average losses during filtration were 23 +/? 4 percent and 15 +/? 3 percent for CPDA-1 units and Adsol units, respectively. The frequencies at which filtered red cells contained > 5 × 10(6) residual WBCs were 2.7 percent for one type of filter and 0.3 percent for another type of filter. CONCLUSION: There were significant losses of platelets during filtration, which could add to the costs of WBC reduction and lead to possible increases in donor exposures. Filtration failures still occurred, despite careful observation of the standard filtration procedures. The number of total WBCs in pooled platelets before filtration has been identified as an important factor in determining the success of WBC reduction.