Implementation of a new platelet pooling system for platelet concentrates led to a higher corrected count increment after transfusion: a comparative observational study of platelet concentrates before and after implementation.

OBJECTIVES: To study the effect of extended storage of platelet concentrates (PCs) and the implementation of a new platelet pooling system for PCs on corrected count increment (CCI) after transfusion. BACKGROUND: Due to new developments and changes in processes or procedures, one should remain alert for the effects of these changes. Besides in vitro studies and validation, in vivo studies are also important, as it has been shown that in vitro results do not always predict in vivo outcomes. METHODS/MATERIALS: After introduction of extended storage of PCs for 5-7 days prepared from five buffy coats and plasma, transfusion monitoring for transfusions of PCs in haemato-oncological patients was set up. After 9 months, a new pooling system for PCs was implemented, Composelect instead of Optipure PLT, and transfusion monitoring was continued for another 8 months. The CCI was used as primary outcome. RESULTS: In total, 93 patients were included and transfused with PCs prepared in the Optipure PLT system (262 transfusions) or in the Composelect system (127 transfusions). Extended storage of PCs for 7 days had no significant effect on CCI. Although the implementation of the Composelect system did not influence the CCI1 h (13.8 ± 6.0 vs. 13.0 ± 5.8; n.s.), it seemed to have a positive effect on CCI24 h (7.0 ± 4.9 vs. 4.7 ± 4.5; P < 0.05). CONCLUSION: Although the influence of confounders could not be excluded, it seemed that implementation of the Composelect system for PCs led to an improved CCI24 h and that extended storage of PCs did not influence the CCI.

A positive effect of immune suppression on corrected count increment after platelet transfusion at 1 but not at 24h.

The effects of patient characteristics on corrected count increment (CCI) in hemato-oncology patients were studied. CCI values were 13.6 ± 6.5 (1h, n=79) and 6.3 ± 5.3 (24h, n=69). With concomitant immune suppression CCI was higher at 1h (20.0 ± 7.9 versus 13.2 ± 6.3, p=0.023), but not at 24h (5.9 ± 6.7 versus 6.3 ± 5.2; p=0.88). The observed effect is short lived, potentially benefiting bleeding patients, but may not increase intervals between transfusions. Further, CCI1h was lower if fever was present (9.7 ± 3.6 versus 14.3 ± 6.7; p=0.002), and corresponding CCI24h values were 3.7 ± 6.3 versus 6.7 ± 5.0 (p=0.09). At 24h an effect for previous transfusions was observed, 6.7 ± 5.1 (with) versus 1.6 ± 5.4 (without p=0.02).

Platelet concentrates from fresh or overnight-stored blood, an international study.

BACKGROUND: Whole blood and also buffy coats (BCs) can be held for a few hours or overnight before processing into blood components or platelet concentrates (PCs). Individual studies have reported a range of outcomes regarding in vitro variables for PCs prepared from fresh and stored whole blood. In this multicenter study, effects of storage of whole blood or BCs on the in vitro quality of PCs were studied. STUDY DESIGN AND METHODS: The leukoreduced BC PCs were prepared from fresh BCs (2-8 hr after collection; fresh/fresh), from BCs at 20 to 24 hours after collection (fresh/stored), or from BCs prepared from whole blood stored for 20 to 24 hours (stored/fresh). PCs were stored on a flat-bed shaker at 20 to 24°C for 7 days. PCs were tested on Days 0 (only fresh/fresh), 1, 5, and 7 for in vitro quality. There were six participating centers that tested all three conditions with n = 6 per condition. RESULTS: In comparison to fresh/stored and stored/fresh PCs, fresh/fresh PCs exhibited a lower platelet (PLT) count (Day 1-220 × 10(9) ± 70 × 10(9) vs. 324 × 10(9) ± 50 × 10(9) and 368 × 10(9) ± 56 × 10(9) PLTs/PC), lactate, pCO(2), and hypotonic shock response (HSR; Days 5 and 7; Day 7-50 ± 13% vs. 57 ± 12 and 63 ± 11%) and a higher pH, glucose, pO(2), and CD62P expression (than stored/fresh PCs only; Day 7-33 ± 10% vs. 28 ± 12 and 24 ± 11%; p < 0.05). No differences were observed for volume, swirling effect, white blood cell count, annexin V binding, or aggregation between these conditions. CONCLUSION: Based on PLT count, HSR, and PLT activation, PCs are best prepared after 20 to 24 hours hold of the whole blood or BCs.

White blood cell fragments in platelet concentrates prepared by the platelet-rich plasma or buffy-coat methods.

BACKGROUND AND OBJECTIVES: White blood cell (WBC) fragments in platelet concentrates (PCs) may induce allo-immunization in the recipient. MATERIALS AND METHODS: As the level of WBC fragments can differ between PCs produced using different methods, we compared PCs prepared by using the buffy-coat method (BC-PCs) in plasma or platelet additive solution (Composol) and PCs prepared using the platelet-rich plasma method (PRP-PCs). RESULTS: Post-filtration results revealed identical levels of WBC, but significantly higher CD62p expression and a significantly lower amount of total DNA, cell-free DNA and number of WBC fragments (0.6 vs. 4.2 WBC equivalents/microl) in PRP-PCs than in BC-PCs in either plasma or Composol. CONCLUSIONS: The number of WBC fragments is significantly higher in BC-PCs than in PRP-PCs.

Development of white blood cell fragments, during the preparation and storage of platelet concentrates, as measured by using real-time polymerase chain reaction.

BACKGROUND AND OBJECTIVES: White blood cell (WBC) fragments may cause human leucocyte antigen (HLA) immunization in recipients. We investigated the occurrence and production of WBC fragments in platelet concentrates (PCs) and plasma units, during storage and filtration, by using real-time polymerase chain reaction (PCR) and flow cytometry. MATERIALS AND METHODS: To study the occurrence of WBC fragments, 'male' WBCs were spiked into double-filtered 'female' PCs in a concentration series of 0.03-100 WBCs/microl (n = 4 per level). To study the production of WBC fragments, 'male' WBCs were spiked into 'female' plasma units to 4 x 10(9) WBCs/l and stored at room temperature prior to filtration (n = 4 per storage time; t = 0, 24 or 48 h). DNA was measured by both albumin real-time PCR and Y real-time PCR. Intact WBCs were counted by using flow cytometry. The number of WBC fragments was calculated by subtracting cell-free DNA (real-time PCR on supernatant) and intact WBCs (flow cytometry) from the total DNA amount (real-time PCR). RESULTS: Spiking of 'male' WBCs into 'female' PCs showed that the Y real-time PCR is linear and has a reproducible quantitative range down to 0.03 WBC/microl, but that the albumin-PCR, in unspiked samples, revealed a total of 6-10 WBC equivalents/microl (eq/microl). After centrifugation, half of this was observed as cell-free DNA in the supernatant, suggesting that the remaining DNA is derived from WBC fragments. The number of intact WBCs, amount of cell-free DNA and number of WBC fragments after filtration increased significantly when filtration was delayed for up to 48 h, from 0.1 WBC/microl, 1.3 WBC eq/microl and 0.6 WBC eq/microl at t = 0 h to 25 WBC/microl, 38 WBC eq/microl and 57 WBC eq/microl at t = 48 h, respectively. CONCLUSIONS: WBC fragments occur in WBC-reduced PCs and increase when products are stored, prior to filtration, up to levels that are equivalent to the amounts of intact WBCs that induce HLA immunization (i.e. > 5 x 10(6)/unit).

Correlation between the extent of platelet activation in platelet concentrates and in vitro and in vivo parameters.

BACKGROUND AND OBJECTIVES: Platelet activation, which is necessary to stop bleeding, also occurs in vitro during the storage of platelet concentrates (PCs). However, it is unknown whether in vitro-activated platelets are able to reduce blood loss in the patient. We studied correlations between platelet activation in PCs and in vitro parameters (pH, platelet count, swirling effect, storage time). In addition, we studied the correlation between platelet activation and in vivo parameters [the volume of thorax drain fluid as a measure of blood loss, platelet count, international normalized ratio (INR), and activated partial thrombin time (APTT)] in a clinical pilot study. MATERIALS AND METHODS: White blood cell-reduced PCs prepared from five buffy coats and one plasma unit (n = 55; storage time: median, 5 days; range, 2-12 days) were sampled. Platelet activation (CD62p, CD63, CD42b), as measured by flow cytometry, pH, platelet count, swirling effect and storage time, was determined. For the in vivo pilot study, PCs (n = 21) stored for 2-7 days were also checked for the above parameters and transfused into patients (n = 21) immediately after coronary artery bypass graft surgery. The volume of thorax drain fluid was measured for up to 12 h after surgery, and the platelet count, INR and APTT were measured < 1 h and 16-24 h postsurgery. RESULTS: A good correlation (r2 > 0.5) was observed between CD62p and CD63, between CD62p and CD42b, between CD62p or CD63 and pH and between CD62p or CD63 and swirling effect. Also, a significant increase in platelet activation was observed for PCs stored for > or = 8 days (mean +/- standard deviation: CD62p, 41.6 +/- 30.7; CD63, 23.8 +/- 18.6), compared to PCs stored for 2-7 days (mean +/- standard deviation: CD62p, 12.3 +/- 4.8; CD63, 10.4 +/- 3.6). No correlation (r2 < or = 0.1) was observed between platelet activation and the in vivo parameters. CONCLUSIONS: Although a correlation between platelet activation and in vitro parameters was observed, no correlation was found between platelet activation and in vivo parameters. Possible explanations for this are a too low variance in platelet activation in transfused PCs, and too small a number of patients.

Influence of cell-free DNA in plasma on real-time polymerase chain reaction for determination of residual leucocytes in platelet concentrates.

BACKGROUND AND OBJECTIVES: Real-time quantitative (RQ) polymerase chain reaction (PCR) can be used to determine the number of residual leucocytes in leucocyte-reduced platelet concentrates (LR-PCs), which should contain < 3.3 leucocytes/ micro l. In this study we investigated the extent to which cell-free DNA, known to be present in plasma, might interfere with this determination. In this study, RQ-PCR was employed to determine the following: the influence of filtration of platelet concentrates (PCs) on the amount of cell-free DNA; the variation in concentration of cell-free DNA between the buffy coats (BCs) of different donors; and the amount of cell-free DNA during storage and processing of whole blood. MATERIALS AND METHODS: PCs were sampled before and after filtration (n = 5), BCs were sampled (n = 100) and whole blood units were sampled < 2 h and 16-20 h after collection, and the BCs were also sampled after processing the whole blood (n = 10). Samples were centrifuged to obtain cell-free plasma in which the amount of cell-free DNA was determined using an RQ-PCR for the albumin gene. RESULTS: The amount of cell-free DNA was not influenced by filtration of the PCs [1.7 +/- 0.8 vs. 1.5 +/- 0.8 leucocyte-equivalents (eq)/ micro l]. However, the amount of cell-free DNA in plasma of the BCs varied considerably, from 0.1 to 18.2 leucocyte-eq/ micro l (median = 1.5 leucocyte-eq/ micro l; mean +/- SD: 2.2 +/- 2.4 leucocyte-eq/ micro l). In 18% of the BCs the amount cell-free DNA was > 3.3 leucocyte-eq/ micro l. The amount of cell-free DNA increased during storage, from 0.3 +/- 0.3 leucocyte-eq/ micro l (< 2 h after collection) to 0.9 +/- 0.6 leucocyte-eq/ micro l (16-20 h after collection) and, after processing the whole blood, to 2.0 +/- 2.0 leucocyte-eq/ micro l. CONCLUSIONS: Variable amounts of cell-free DNA in plasma will interfere if RQ-PCR is applied to estimate leucocyte numbers in leucocyte-reduced PCs.

In vivo PLT increments after transfusions of WBC-reduced PLT concentrates stored for up to 7 days.

BACKGROUND: Bacterial screening and improvement of storage conditions of leukoreduced PLT concentrates (LR-PCs) allows extension of their storage period from 5 to 7 days. STUDY DESIGN AND METHODS: For in vitro studies, 40 LR-PCs made from five buffy coats and plasma were studied for 8 days. For in vivo studies, routinely produced LR-PCs stored for 2 to 7 days after blood collection were administered to clinically stable thrombocytopenic patients. CI1 h was calculated after 353 transfusions (67 patients), and CCI1 h, after 195 transfusions (55 patients), with pretransfusion PLT counts of not greater than 20 x 10(9) per mL. RESULTS: Storage experiments showed that the pH of LR-PCs remained greater than 6.8 for 8 days, provided that the PLT concentration was less than 1.3 x 10(9) per mL. Routinely produced LR-PCs had a volume of 282 +/- 15 mL (n = 10,193) and contained 329 x 10(9)+/- 40 x 10(9) PLTs (n = 3467). For 7-day-old LR-PCs, 76 of 78 (97%) of the transfusions resulted in a CI1 h of at least 10 and 37 of 39 (95%) in a CCI1 h of at least 7.5, which indicated levels for successfulness. Mean +/- SE values of CI1 h and CCI1 h of 7-day-old LR-PCs were 28.7 +/- 2.3 (n = 78) and 19.0 +/- 2.0 (n = 39), respectively. No significant differences were observed between 5- and 7-day-old LR-PCs transfused with respect to CI1 h and CCI1 h values. CONCLUSION: In vitro and in vivo studies showed that LR-PCs can be stored for up to 7 days with excellent clinical results, provided that they are routinely screened for bacterial contamination.

Comparison of various dimethylsulphoxide-containing solutions for cryopreservation of leucoreduced platelet concentrates.

BACKGROUND AND OBJECTIVES: Leucoreduced platelet concentrates (LR-PCs) can be stored at 20-24 degrees C for 5-7 days. When LR-PCs are cryopreserved they can be stored for several years. For cryopreservation to become applicable in blood-bank practice, an off-the-shelf cryoprotectant is needed that can be added to the LR-PC in a sterile manner. For this, we varied the composition of the cryopreservation medium and studied various parameters of cryopreserved LR-PCs for up to 24 h after thawing at room temperature. MATERIALS AND METHODS: LR-PCs in plasma or Composol were concentrated and divided into 2 units. To each unit, an equal part of 10% dimethylsulfoxide (DMSO) in plasma, Composol with or without 5% albumin, or GPO (pasteurized plasma-protein solution) was added. Freezing occurred at 1 degrees C/min and LR-PCs were placed in the vapour phase of nitrogen. LR-PCs were thawed at 37 degrees C and stored at room temperature. LR-PCs were tested for morphology, platelet recovery, swirling effect, and activation antigens at various time-points thereafter. RESULTS: LR-PCs in 100%, 65% and 50% plasma supplemented with Composol showed good morphology scores (>250), limited CD62P expression (<35%), low CD63 expression (<20%) and a swirling effect of about 2, at 24 h after thawing. At the same time-point, platelet recovery was >80% under all conditions and CD42b expression varied between 70 and 85%. Results of LR-PCs in 15% plasma and Composol, with or without plasma substitutes, were not acceptable at 24 h after thawing, i.e. the morphology score was <200 and the CD62P expression was >40%. CONCLUSIONS: A minimum of 50% plasma in the cryopreserved LR-PC is necessary to maintain an acceptable in vitro quality of platelets up to 24 h after thawing. Composol is a good candidate for using to prepare an off-the-shelf cryoprotectant.

Crossover study of three methods for low-level white blood cell counting on two types of flow cytometer.

BACKGROUND: Flow cytometric methods were previously shown to be preferable to microscopic and volumetric methods for counting residual white blood cells (WBCs). In this study, three flow cytometric, low-level WBC counting methods were cross compared using two flow cytometers. METHODS: Double-filtered red cell and platelet concentrates were spiked with different amounts of WBC to obtain panels of unspiked and 0.3, 1.0, 3.3, and 10.0 WBC/microl. The methods of BD Biosciences (BDB), Beckman-Coulter (BC), and an in-house method were performed on flow cytometers from BDB and BC. Samples were measured in ninefold. We required that (a) r(2) be at least 0.98 (linearity), (b) at least 80% of observations fell within 20% of expected values (accuracy), and (c) the coefficients of variation be at least 20% (precision) for samples containing at least 3.3 WBC/microl. RESULTS: For the red cell panel, our requirements were met by the BDB method on both flow cytometers and by the BC and in-house methods on the BDB flow cytometer only. For the platelet panel, our requirements were met on all combinations of methods and flow cytometers, except for the in-house method on the BDB flow cytometer. Intra-assay variation was lowest for the BDB method, irrespective of the type of flow cytometer used. CONCLUSION: Based on accuracy and precision, the BDB method on the BDB flow cytometer produced the best results for counting low-level WBCs.

WBC-reduced platelet concentrates from pooled buffy coats in additive solution: an evaluation of in vitro and in vivo measures.

BACKGROUND: The use of a platelet additive solution (PAS-II, Baxter) may have benefits over plasma for storage of platelets. It was the aim of this study to develop a method to produce WBC-reduced platelet concentrates (PCs) in PAS-II with >240 x 10(9) platelets and <1 x 10(6) WBCs per unit, which can be stored for 5 days at pH >6.8 and that will give sufficient platelet increments after transfusion: a 1-hour CCI of >7.5 and a 20-hour CCI of >2.5. STUDY DESIGN AND METHODS: PCs were made from five pooled buffy coats and 250 g of PAS-II. After centrifugation the PCs were WBC-reduced with a filter (Autostop BC, Pall Biomedical) and stored in a 1000-mL polyolefin container. CCIs were assessed in stable hemato-oncologic patients after 5-day old PCs were transfused. RESULTS: Routinely produced PCs contained a median of 310 x 10(9) platelets (n = 5,363) with 3.5 percent containing <240 x 10(9) platelets, in a median volume of 320 mL (n = 11,834). The median number of WBCs was <0.03 x 10(6) (n = 694). The WBC count exceeded 1 x 10(6) in three PCs, but it was always <5 x 10(6), giving 99-percent confidence that more than 99.5 percent of the units will contain <1 x 10(6) WBCs. The pH remained >6.8 on Day 8, provided the concentration was below 1.1 x 10(9) platelets per mL (n = 32). After 28 transfusions in 28 patients, the 1-hour CCI was 12.6 +/- 4.3 (mean +/- SD, with 2/28 CCIs <7.5) and the 20-hour CCI was 8.9 +/- 5.6 (with 4/28 CCIs <2.5). Limitations of this study include the absence of a control group of patients receiving platelets stored in plasma and of in vivo radiolabeled survival studies, but a comparison of these data with previously published data suggested that the in vivo survival of platelets stored in PAS-II is less than that of platelets stored in plasma. CONCLUSION: The WBC-reduced PCs conformed to specifications. These WBC-reduced PCs could be stored at least 5 days with maintenance of pH, and they gave sufficient increments after transfusion to patients.

Comparison of five platforms for enumeration of residual leucocytes in leucoreduced blood components.

The need for quality control of leucoreduction of blood products has led to the development of various methods to count low levels of residual leucocytes. We compared five platforms side-by-side: the Nageotte haemocytometer and four based on fluorescent staining of nuclei: two flowcytometers (Beckman Coulter, BD Biosciences) with methods based on counting beads, a volumetric flow cytometer (Partec) and the microvolumic fluorimeter ImagN2000 (BD Biosciences), all according to their manufacturers' recommended methods. Analysis of double-filtered red cell concentrates (RCCs) and platelet concentrates (PCs), spiked with various numbers of leucocytes, revealed good linearity for all methods over the range of 1.6-32.7 leucocytes/microl, all with r(2) > 0.99. At the rejection level of leucocyte-reduced blood components, i.e. 1 x 10(6) per unit corresponding with approximately 3.3 leucocytes/microl, the Nageotte haemocytometer had low accuracy (0% for RCCs, 56% for PCs), and was relatively imprecise [coefficient of variance (CV) of 34% and 30% respectively]. The Partec flow cytometer gave good results for RCCs (accuracy 67%, CV 22%), but not for PCs (accuracy 0%, CV 25%). The ImagN2000 had an accuracy of 44% for RCCs and 89% for PCs, but the precision was variable (CV 32% for RCCs, 15% for PCs). The best results were obtained with the Beckman Coulter (RCCs: accuracy 86%, CV 13%, PCs: accuracy 67%, CV 16%), and BD Biosciences platforms (RCCs: accuracy 100%, CV 10%; PCs: accuracy 89%, CV 11%). We conclude that, at the rejection level of 1 x 10(6) leucocytes per unit, the widely used Nageotte haemocytometer performs poorly in terms of inaccuracy and imprecision, and that both counting-bead-based, flow cytometric methods performed best.

Characterization of mouse thrombin-activatable fibrinolysis inhibitor.

Based on in vitro studies, thrombin-activatable fibrinolysis inhibitor (TAFI) has been hypothesized as a link between coagulation and fibrinolysis, but the physiological role of TAFI in vivo has not yet been established. To anticipate on the availability of genetically modified mouse models, we studied the endogenous expression of TAFI in mice. Functional TAFI was found in mouse plasma. TAFI mRNA was only detectable in the liver, showing a hepatocyte-specific expression with a pericentral lobular distribution pattern. The murine TAFI cDNA was cloned and sequenced. The deduced amino acid sequence revealed that murine TAFI is highly identical to human TAFI. The murine cDNA was stably expressed and the activated recombinant protein was functionally active; it converted the substrate hippuryl-arginine, and prolonged the clot lysis time of TAFI depleted plasma. We conclude that mice have functional TAFI in plasma, which is highly similar to human TAFI. Therefore, genetically modified mice may provide useful models to study the role of TAFI in vivo.

Evidence against a direct role of the integrin alpha2beta1 in collagen-induced tyrosine phosphorylation in human platelets.

In the present study we have investigated whether the collagen receptor alpha2beta1 (GPIa-IIa; GP, glycoprotein) regulates protein tyrosine phosphorylation in platelets directly through activation of tyrosine kinases or indirectly through modification of the response to GPVI. The interaction of collagen with alpha2beta1 was inhibited in two distinct ways, using the metalloprotease jararhagin, which cleaves the beta1 subunit, or the antibody P1E6 which competes with binding of collagen to the integrin. The two inhibitors caused a shift to the right in the collagen concentration response curves for protein tyrosine phosphorylation and platelet activation consistent with a causal relationship between the two events. There was no change in the overall pattern of tyrosine phosphorylation in response to high concentrations of collagen in the presence of alpha2beta1 blockade demonstrating that the integrin is not required for this event. In contrast, jararhagin and P1E6 had a small, almost negligible inhibitory effect against responses to the GPVI-selective agonist collagen-related peptide (CRP) and the G protein-coupled receptor agonist thrombin. Crosslinking of alpha2beta1 in solution or by adhesion to a monolayer using a variety of antibodies to either subunit of the integrin did not induce detectable protein tyrosine phosphorylation in whole cell lysates. The snake venom toxin trimucytin-stimulated a similar pattern of tyrosine phosphorylation to that induced by crosslinking of GPVI which was maintained in the presence of jararhagin. Trimucytin may therefore induce activation via GPVI rather than alpha2beta1 as previously thought. These observations show that the integrin alpha2beta1 is not required for regulation of tyrosine phosphorylation by collagen.

Characterization of transgenic mice that secrete functional human protein C inhibitor into the circulation.

Protein C inhibitor (PCI) is a heparin binding serine protease inhibitor in plasma, which exerts procoagulant activity by inhibiting thrombomodulin-bound thrombin or activated protein C (APC). Since the role of PCI in vivo is largely unknown we generated genetically modified mice with expression of human PCI mRNA in hepatocytes only. Three transgenic lines have been characterized. Transgenic mice did not show gross developmental abnormalities. Two lines showed a pericentral and one line showed a periportal expression pattern of human PCI mRNA in the liver. Genetically modified mice secreted a functional transgenic protein into the circulation (3-5 microg/ml plasma in heterozygous mice and 10 microg/ml in homozygous mice), which inhibited human APC activity in the presence of heparin. Interestingly, transgenic mice in which human PCI was expressed periportally in the liver had the highest specific activity. Endogenous mouse PCI mRNA could only be detected in the male and female reproductive system, but not in the liver, indicating that endogenous PCI levels in the circulation are low or even absent in mice. These results demonstrate that the human PCI transgenic mice are a suitable model for studying the in vivo role of PCI in blood coagulation.