Cytokine levels as performance indicators for white blood cell reduction of platelet concentrates.

BACKGROUND AND OBJECTIVES: With the implementation of universal white blood cell (WBC) reduction in the UK, in-process WBC-reduction filters for pooled buffy coat (BC)-derived platelet concentrates (PCs) and apheresis methods are used routinely for the production of WBC-reduced PCs. While these strategies meet the specification for WBC reduction (< 5 x 10(6) WBCs/unit), the products from these processes may differ depending on the process employed and its performance. The aim of this study was therefore to investigate whether PCs prepared using various WBC-reduction processes are sufficiently depleted of WBCs to limit cytokine accumulation during storage and to assess if cytokine levels detected in platelet products can serve as indicators of acceptable platelet activation as a result of the WBC-reduction process. MATERIALS AND METHODS: We measured the levels of cytokines predominantly derived from WBCs [e.g. interleukin-8 (IL-8)] and platelets [e.g. regulated on activation, normal, T-cell expressed, and secreted (RANTES) and transforming growth factor-beta(1) (TGF-beta(1))] under the present experimental conditions in different WBC-reduced PCs, i.e. PCs prepared from three different WBC-reduction filters and control non-filtered PCs using pooled BCs from the same donors and three apheresis types. Supernatant plasma was collected at the beginning (day 1) and end (day 5) of the shelf life of each PC, and the cytokine content was determined using appropriate enzyme-linked immunosorbent assays (ELISAs). Process efficiency was assessed by platelet yield and residual WBC count. RESULTS: We found that products from the apheresis process involving a filtration step (Haemonetics MCS+) showed a lower cytokine content on both day 1 and day 5 in comparison with the fluidized bed (COBE Spectra) or elutriation (Amicus) processes. WBC reduction of BC-PCs of the same origin using three different filters showed comparable levels of cytokines on day 1 in all units. After storage for 5 days, the levels of IL-8 remained essentially unchanged in filtered BC-PCs but increased by more than threefold in control non-filtered BC-PCs, suggesting IL-8 release by residual WBCs present in the control PCs. The concentration of platelet-derived cytokines such as RANTES and TGF-beta(1), however, increased significantly in all filtered and control non-filtered PCs during the storage period. CONCLUSION: These results show that markers of cytokine release from both WBCs and platelets are useful indicators of the performance and efficacy of the WBC-reduction process and of platelet quality.

The quality of MCS+ version C2 double dose platelet concentrate with leucodepletion through a continuous filtration process.

182 full double donations were collected, using 7-9 cycles with an in-house guideline based on donor HCT, and platelet count. 95.6% of donations were within specifications for platelet yield and 100% were leucodepleted. To overcome the potential for red cell spill-over when the platelet peak is too small, automated continuous monitoring of machine performance has been introduced reducing the potential for spill-over. To understand the impact of the continuous exposure of platelets to the filter, the quality of the first and second part of the collection was monitored using a new set of markers for the platelet storage lesion (activation, microvesiculation, cellular injury, complement and response to ADP). The results were equivalent and compared well with routine practice. Our new protocol allows a wide range of donors to be selected and the highest percentage of products to meet the UK specification with consistency. It is recommended that it can be used for double dose platelets with routine SPM.

Cytokines as quality indicators of leucoreduced red cell concentrates.

Different types of filters are currently used for leucodepletion of red cell concentrates. These filters meet the specification for leucoreduction (<5 x 10(6) leucocytes/ATD) but the quality of the final product may differ depending on the performance of the filters for effective removal of both leucocytes, platelets and possibly cytokines which are associated with transfusion reactions. We measured the levels of three representative cytokines: IL-8, RANTES and TGF-beta1 in red cell concentrates prior to and subsequent to the filtration procedure on day 1 and after a storage period of 35 days. Low levels of IL-8 (10-24 pg/ml) in the control unfiltered concentrates on day 1 which increased by approximately twofold on storage. Filtration reduced the levels of IL-8 on day 1 and day 35, in filtered concentrates in comparison with their control unfiltered counterparts. Leucoreduced concentrates produced by three different filters showed similar IL-8 levels on day 1 and day 35. However, concentrates prepared using another type of process showed a twofold increase in IL-8 levels on storage in comparison with day 1. None of the concentrates tested contained any detectable RANTES and TGF-beta1 suggesting a minimal platelet content. These results indicate that a combination of IL-8, RANTES and TGF-beta1 are useful quality indicators for validation of leucoreduced red cell preparations.

Studies on the characterisation of the cause of leucoreduction failures, with particular reference to extra gatal events.

The causes of leucodepletion failure are multifactoral and can be related to haematological variability in blood donors or donation, defective filters, poor specimen handling or ageing, and/or the presence of non-adhering leucocyte/platelets. Since refiltering removes all types of leucocytes, including the populations appearing as extra gated events, we have developed a practical method for refiltering the failed leucodepleted components on standard filters and back-flushing the second filter to assess the nature of the WBC sub-population. In practice, recovered leucocytes from red cell filters and whole blood mainly consist of neutrophils. Those from platelet and plasma filters were mainly lymphocyte with considerable differences depending on the type of leucodepletion process. Atypical leucocytes are often seen in some pre-/post-cellular leucofiltered components. These appear characteristically as small WBC with a lower affinity for filter matrix, or as cell fragment, pinched leucocyte or apoptotic cells. Different reagents in use show variable sensitivity in identifying these extra gatal events. Storage of leucodepleted samples also induces different types of abnormality in leucocyte dot plot. A useful practical approach for characterisation of the nature of leucocyte sub-populations causing failure in leucodepleted components is provided.

The role of maternal serum C-reactive protein and white blood cell count in the prediction of chorioamnionitis in women with premature rupture of membranes.

This study was performed to evaluate the diagnostic performance of maternal serum C-reactive protein, maternal white blood cell (WBC), and neutrophil counts in the detection of histologic chorioamnionitis. One hundred and twenty six pregnant women after at least 28 weeks of gestation with premature rupture of membranes (PROM) were studied. Blood samples for C-reactive protein, WBC and neutrophil counts were taken at delivery. Placental histology was evaluated for histologic chorioamnionitis. Maternal and neonatal complications were observed. Among women with and without histologic chorioamnionitis, the maternal WBC and neutrophil counts were different (P<0.05) but the maternal serum C-reactive protein was not. Cutoff values for C-reactive protein, WBC, and neutrophil counts were 0.5 mg/dL, 15,000 cell/mm3, and 80 per cent, respectively. Sensitivity and specificity were 56 per cent and 58 per cent for C-reactive protein, 60 per cent and 63 per cent for WBC count, and 62 per cent and 54 per cent for neutrophil count, respectively. In conclusion, the maternal serum C-reactive protein, WBC, and neutrophil counts have poor diagnostic performance for histologic chorioamnionitis.

Leukoreduction of sickle cell trait blood: an unresolved issue.

With the increasing demand for leukoreduction of blood components and the implementation of universal leukoreduction in several countries, the problems associated with leukocyte filtration of sickle cell trait blood have been revisited. Currently, there is no unified standard practice for sickle cell trait donors. Different blood centers adopt different policies. While some defer these donors from red cell component donation, some do not. Some screen all ethnic African donors for hemoglobin S (Hb S), others do not. Furthermore, there are differences in views of whether sickle cell trait red cells should be considered as equivalent to non-sickle cell trait red cells. Some blood centers do not give red cells from a sickle cell trait donor to the newborn or patients undergoing general anesthesia as a preventative measure. In this presentation, we discuss the epidemiology of the sickle gene, the sickling process, problems associated with leukoreduction of sickle cell trait whole blood and red cells, and some unresolved issues concerning donor referral and the usage of sickle cell trait blood.

Platelet storage lesion and apoptosis: are they related?

The relationship between the platelet storage lesion (PSL) and programmed cell death (apoptosis) is poorly understood. Nevertheless, there is some experimental evidence that platelets contain most of the components of the apoptosis machinery and both the apoptotic process and the PSL lead to platelet activation and microvesiculation with expression of phosphatidyl serine (PS) on the outer layer of cell membrane, a hallmark of all nucleated cells. The PS exposure is believed to contribute to the development of inflammatory or immunomodulation process, to the regulation of haemostatic balance and the ultimate clearance of dead or fragmented cells from the circulation. While there is no doubt that apoptosis, as a form of genetically encoded programmed cell death in nucleated cells, is triggered by several signalling stimuli at the nuclear level, there is some doubt as to whether platelets, as enucleated cells have retained the memory of the "parental" megakaryocytes for apoptosis or whether platelet mitochondrial DNA has a major role in both the apoptotic process and the PSL. The storage lesion occurs during processing and storage subsequent to mechanical trauma, hypoxic conditions or exposure to cold. In this brief report some observational evidence is provided in support of the notion that the PSL and apoptosis may be related to each other, despite the fact that, in contrast to the 'parental' megakarocyte, the platelets appear to survive upon stimulation with a high concentration of protein kinase inhibitors such as staurosporine (STS), in the presence of cycloheximide (CHX) which inhibit protein synthesis. This is a model which is often used to regulate the level of survival signals. The possible relevance of platelet microvesiculation to transfusion practice is briefly discussed.

Studies on the improvement of leucodepletion performance of the Haemonetics MCS+ for production of leucodepleted platelet concentrate.

With the implementation of universal leucodepletion, an in-line, negatively charged LRF6H leucodepleting filter became an essential part of the Haemonetics MCS+ plateletpheresis system. A larger-scale (968) study using the standard protocol revealed a 2.79% leucodepletion failure rate (standard < 5 x 10(6) leucocytes per adult therapeutic dose). Factors influencing the efficacy of the filter were investigated. The pH of the filtrate was 7.0, the temperature 28 degrees C and filtration rate 80 ml/min. Reduction of the filtration rate to 30 ml/min (784 doses) reduced leucodepletion failure to 0.38%. Measurement of the leucocyte count, pre- and post-filtration of the platelet products, revealed that donations from 1% of donors contained substantially larger numbers of leucocytes in pre-filter samples (300-1500/microl) than in control samples (35-70/microl). This number tends to increase progressively with subsequent donations in these individuals, leading to leucodepletion failure, whilst peripheral leucocyte counts remain normal. The new continuous filtration protocol (version C) using a less impact filter LRF-XL and a lower (7 ml/min) head pressure was also effective but failure still occurred twice on one of the donors who persistently showed high pre-filter count. We conclude that leucodepletion failures in the Haemonetics system are related to both donor leucocyte (i.e., being light and non-adherent) and operational/filter performance.

A case of leucodepletion failure in Cobe spectra plateletpheresis: further studies on the identification of the potential cause.

A 54-year-old double dose donor failed leucodepletion by Cobe LRS for five sequential donations, without triggering the "machine failure alarm". All collections were uneventful but final products appeared lipaemic. The last two donations were studied: (i) by collecting the first half of each donation into one bag and the other half into a second bag. In both cases, the product in bag one was leucodepleted and that in bag two "failed" leucodepletion. (ii) By examining the filterability of the final products using an LRF6 filter. Leucodepletion was satisfactory but showed a slightly higher level of leucocytes as compared to control or lipaemic donations, possibly due to the presence of an interfering substance in the platelet product. (iii) By examining the cellular content of the LRS cone and comparing it with those obtained from lipaemic donations from other donors. Only the test cone contained a large mass of waxy white material. It is postulated that this material was gradually filling the cone and increasingly interfering with its leucodepleting function as the donation progressed. (iv) To monitor pre/post whole blood as well as the products for the presence of abnormal leucocyte subsets. The only difference was the presence of some extra abnormal dot plot. The intensity of these extra events which was reduced both upon donation and filtration. We conclude that, in this case, failure of leucodepletion was clearly donor rather than machine or process related. The implementation of this investigative study may help in the characterisation of the potential cause of leucodepletion failures.

The effect of leucocyte depletion on the quality of fresh-frozen plasma.

The aim of this study was to evaluate the quality of leucodepleted (LD) fresh-frozen plasma (FFP) produced using one of five whole blood filters (Baxter RS2000 & RZ2000, NPBI T2926, Macopharma LST1 and Terumo WBSP) or two plasma filters (Pall LPS1 and Baxter FGR7014). Whole blood or plasma was filtered within 8 h of collection at an ambient temperature. Samples were taken pre- and post filtration for analysis of coagulation factors and complement activation (n = 7--12 for each type of filter). All filtered units (209--286 ml) contained < 5 x 10(6) residual leucocytes and < 30 x 10(9)/l platelets. Statistically significant losses of factors V, VIII, IX, XI and XII and increases in markers of coagulation activation were observed (0--21%), which were dependent on filter type. None of the filters had a significant effect on von Willebrand factor (VWF) multimeric distribution or the activity of VWF and factors II, VII or X. The effect on levels of C3a appeared to be related to the filter surface charge: positively charged filters resulted in C3a generation, whereas negatively charged resulted in C3a removal. None of the observed changes are likely to be clinically significant unless subsequent processing of plasma (such as pathogen inactivation) results in further losses of coagulation factors.

Platelet storage lesion of WBC-reduced, pooled, buffy coat-derived platelet concentrates prepared in three in-process filter/storage bag combinations.

BACKGROUND: With the implementation of universal WBC reduction in the United Kingdom, in-process WBC-reduction filters for pooled buffy coat (BC)-derived platelet concentrates (PCs) are used in routine production. The effects of three filter/storage bag combinations on platelet activation and microvesiculation and on the activation of coagulation were investigated. STUDY DESIGN AND METHODS: Using pooled BCs from the same donors, three filter/storage bag combinations (Autostop BC/CLX, Pall Biomedical; Sepacell PLX5/PL2410, Asahi Medical; and Imugard III-PL 4P/Teruflex, Terumo) were compared with unfiltered controls for their effects on microvesiculation and other storage-induced changes in platelets. Process efficiency was measured by platelet yield and residual WBC count. The storage changes were assessed: pH, activation of platelets measured by CD62P on the platelet surface and in supernatant plasma, quantitation of platelet-derived and RBC-derived microvesicles, cellular injury measured by annexin V in the supernatant plasma, and activation of the coagulation system measured by kallikrein-like and thrombin-like activities, prothrombin fragment 1+2, and thrombin-antithrombin complex. RESULTS: All three filters were comparable in terms of platelet recovery and WBC removal, and none induced immediate platelet activation or microvesiculation. With storage, platelet activation or microvesiculation increased in platelets prepared by all three filters and in unfiltered controls, but these effects were significantly less in the Imugard PCs than in controls. These findings were consistent with those for annexin V in the supernatant plasma, which were lower in Imugard PCs than in other products. Sepacell and Imugard filters reduced RBC-derived microvesicles to 50 percent of control levels, but the Autostop filter had no effect. On storage, levels of RBC-derived microvesicles in filtered products remained static, but levels in the unfiltered control doubled. Kallikrein- and thrombin-like activities were generated only by the Autostop filter without any further increment on storage. CONCLUSION: WBC-reduced pooled BC-PCs prepared by various filter/bag combinations were equivalent on Day 1 but differed during storage in terms of platelet activation or microvesiculation.

Counting of residual WBCs in WBC-reduced blood components: a multicenter evaluation of a microvolume fluorimeter by the United Kingdom National Blood Service.

BACKGROUND: Implementation of universal WBC reduction of blood components means that automated analytical methods may be the only satisfactory way for production laboratories to meet increased testing requirements. STUDY DESIGN AND METHODS: A multicenter study on the performance of a microvolume fluorimeter (IMAGN 2000, Becton Dickinson) was undertaken on 519 RBC, 353 platelet, and 27 fresh plasma units. RESULTS: WBC counts for the RBC samples ranged from 0.02 to 6.94 x 10(6) per unit (mean, 0.57) as determined by FC and from 0.02 to 5.53 x 10(6) per unit (mean, 0.40) as determined by MVF with a mean FC bias of +0.15 x 10(6) WBCs per unit, and discrepancies outside the 95% limits of agreement were mainly associated with higher FC counts. The series of platelet samples showed means of 0.90 (range, 0.06-19.45) and 0.66 (range, 0.01-18.95) x 10(6) WBCs per unit for FC and MVF methods, respectively. FC and MVF results were in good agreement at low counts, although significant discrepancies were noted at higher counts. Overall, for the platelet units, there was a mean FC bias of +0.34 x 10(6) WBCs per unit. The intermethod agreement exceeded 99 percent for both types of blood component when the single (both UK and United States) decision point of 5.0 x 10(6) WBCs per unit was applied. The mean WBC counts for the 27 analyzed fresh plasma units were 61.8, 56.0, and 46.0 per microL by Nageotte hemocytometry, FC, and MVF, respectively. CONCLUSIONS: This evaluation found that the level of intersite consistency for FC was relatively poor compared to that for MVF. The results nevertheless validated the broad equivalence of FC and MVF results for the current Council of Europe and UK/US decision points of <1.0 and <5.0 x 10(6) WBCs per unit.

Statistical process monitoring of WBC-reduced blood components assessed by two types of software.

BACKGROUND: Statistical process control is required for monitoring of the WBC-reduction process. This study focused on some factors that may influence the outcomes of statistical process monitoring, such as WBC-reduction technologies, the anticoagulant used, and WBC-counting technologies, by using two types of software. STUDY DESIGN AND METHODS: Data were collected from January to September 1999, before the implementation of universal WBC reduction. The effects of three major factors were investigated: methods of preparation, the addition of EDTA to the sample, and the WBC-counting technologies used (microvolume fluorimetry, flow cytometry, and Nageotte chamber). The WBC-reduction process capability was assessed by two types of software, EZQC (Gambro BCT) and NWA (Northwest Analytical). In addition, the differences between various sets of results were compared by the t test or ANOVA. RESULTS: There was no statistical difference (at the 0.05 level of significance) in WBC content when the three types of platelets in citrate samples were compared with EDTA samples. In general, the Nageotte chamber appeared to count the lowest, and microvolume fluorimetry appeared to count lower than flow cytometry. There were minor but significant methodologic differences between the software packages. However, these differences had negligible effects on the percentage of conforming components at both <1 x 10(6) and <5 x 10(6) WBCs per unit. CONCLUSION: Only the counting technologies were sufficiently different to warrant consideration. This difference may make unacceptable the interchange of results obtained from various counting methods.

Residual red cell and platelet content in WBC-reduced plasma measured by a novel flow cytometric method.

BACKGROUND: The levels of residual red blood cells (RBC) and platelets (PLT) in WBC-reduced plasma are often below the lower detection limit of automated blood cell counters. This study established a novel flow cytometric method for the enumeration of residual RBC and PLT in plasma. Furthermore, their levels in WBC-reduced plasma prepared by using various filters were investigated. MATERIALS AND METHODS: WBC-reduced plasma was prepared from two sources: (i) filtration of buffy-coat reduced plasma using dock-on Baxter, Pall and Maco Pharma plasma filters; (ii) filtered whole blood using integral Asahi RZ2000, Maco Pharma LST1, NPBI, and Pall WBF2 whole blood filters. Residual RBC and PLT counts were assessed by using a TruCount tube (Becton Dickinson) containing a known number of lyophilized fluorescent beads. RBC and PLT were labelled with dual monoclonal antibodies, anti-CD41-R-phycoerythrin and anti-glycophorin A-fluorescein isothiocyanide, and analyzed by flow cytometer. RESULTS: The flow cytometric method used in this method can detect residual RBC, PLT as well as RBC-MV simultaneously. The sensitivity of the assay was 50 x 10(6) cells/l with the coefficient of variations < or = 10%. Baxter and Maco Pharma plasma filters consistently reduced both RBC, RBC-MV and PLT to below 50 x 10(6/)l. Plasma derived from day 1 RZ2000 filtered whole blood contained PLT below 50 x 10(6) cells/l, whereas day 0 NPBI filtered whole blood showed the highest level of residual PLT. CONCLUSION: A sensitive and accurate method for the detection of low levels RBC, RBC-MV, and PLT was established to measure their levels in WBC-reduced plasma. The procedure is simple and practical for routine quality monitoring of plasma, as well as for setting a new specification for WBC-reduced plasma.

What's happening? The quality of methylene blue treated FFP and cryo.

It is currently unclear to what degree methylene blue in combination with removal, of cells from plasma, by filter, can directly influence the loss of active components of plasma and whether the co-precipitation of FVIII/vWf with fibrinogen/fibronectin is affected by combined methylene blue and light treatment (MBLT). These questions are investigated using the Fenwal system. Our results indicate that up to 15% of the FVIII and IX are lost due to exposure of plasma to filters and methylene blue (MB). The illumination leads to a further 10-15% loss of all other major clotting factors. Factor XI appears to be highly sensitive to the MBLT-process, while inhibitors of the coagulation system are less affected. MBLT did not grossly influence the distribution of fVIII/vWf:Ag between cryoprecipitate and cryosupernatant using a paired control/test protocol, although the fVIII/vWf recovery is reduced in MBLT samples. The three commercially available MBLT processes differ in terms of operational aspects. These may have some impact on overall quality/safety and bioequivalency.

Coagulation factor content of cryoprecipitate prepared from methylene blue plus light virus-inactivated plasma.

Levels of factor VIII (FVIII) and fibrinogen were assessed in control cryoprecipitate and cryoprecipitate prepared in two centres from plasma subjected to methylene blue (MB) photochemical virus inactivation. The level of coagulation FVIII activity was reduced in plasma by approximately 30% after MB photoinactivation, with only 44% (centre A) and 31% (centre B) of units meeting the current UK specification of 0.7 iu/ml. A revised specification of 0.5 iu/ml is suggested. Losses of less than 11% were seen for von Willebrand factor (VWF)-related activities. Cryoprecipitate prepared from group O or group A MB-treated plasma contained 27-40% less FVIII than control units. This reflected the lower levels in MB-treated plasma. The concentrating power of the cryoprecipitation process was not reduced for FVIII or fibrinogen in MB-treated units. MB cryoprecipitate from centre A still met the UK guideline specification for FVIII and fibrinogen content, whereas at centre B only 62.5% of the group O cryoprecipitates contained > 70 iu FVIII/unit. This may reflect the lower product volume and lower FVIII content of group O plasma used at centre B and suggests that maintenance of total coagulation factor recovery in MB-treated cryoprecipitate will require the higher product volume.

The role of in process qualification in quality improvement of the haemonetics MCS plus leucodepleted platelet concentrate.

With the implementation of universal leucodepletion in UK all leucodepletion processes have gone through a standard process qualification and quality improvement. The Haemonetics MCS system is a well established automated platelet collection system for the production of double dose leucoreduced platelet concentrate (WBC approximately 70x10(6)/dose). Recently an automated post collection filtration harness system has been introduced (MCS plus LDP) in which platelets are filtered, using an in-line PALL polyester filter (LRFH6 PALL) to reduce the WBC level to below 5x10(6) WBC/dose. This system passed our Phase I evaluation process based on 20-40 runs. However, some changes in the final volume of the products were needed to conform to national guidelines. Large scale trials using the new volume adjusted protocol revealed occasional failure in the leucocyte content. Therefore, 100% testing had to be implemented on all products. A national evaluation was carried out to determine whether changing the filter to a more efficacious one, the LRFXL (PALL) or slowing the filtration flow rate can influence the overall outcome. To reduce donor variability, known donor population were used with identical apheresis conditions. A more consistent and systematic drop in leucocyte content was observed by reducing the flow rate whereas a similar failure (i.e. 1-3%) rate was found both in controls and LRFXL when using the standard head pressure, which is recommended by the manufacturer. A similar failure rate was found using three different low leucocyte counting technologies (Nageotte, flow cytometry and Imagn 2000). It is recommended that a process qualification/validation program should be implemented when even a small modification in the collection system is introduced.