Cold-stored leukoreduced CPDA-1 whole blood: in vitro quality and hemostatic properties.

BACKGROUND: Some jurisdictions require leukoreduction of cellular blood components. The only whole blood collection set with a platelet-saving filter uses citrate-phosphate-dextrose (CPD) as storage solution. Substituting CPD with citrate-phosphate-dextrose-adenine (CPDA-1) increases shelf life from 21 to 35 days. This would simplify prehospital and rural resupply and reduce wastage. We investigated in vitro quality and hemostatic properties of CPDA-1 whole blood leukoreduced with a platelet-saving filter. STUDY DESIGN AND METHODS: CPDA-1 whole blood was leukoreduced using a platelet-saving filter and stored 35 days. EDQM requirements, hematology, metabolic parameters, thromboelastography, light transmission aggregometry, fibrinogen, factor VIII, and interleukin-6 were measured on Days 0, 1, 14, 21, and 35 and compared to non-leukoreduced blood. RESULTS: All units met EDQM requirements. Leukoreduction yielded residual white blood cell count <1 × 106 and 87% platelet recovery on Day 1. It caused reduction in thromboelastography parameters, but not aggregometry response. No hemolysis >0.8% was observed. Factor VIII was higher on Day 35 in the leukoreduced group, 37.9 (95% CI: 26.0, 49.8) versus 13.8 (9.4, 18.2) IU/dL. In both groups, aggregation was significantly reduced by Day 14. Thromboelastography showed remaining platelet activity on Day 35, MA 46.9 (42.1, 51.7) in the leukoreduced and 44.3 (39.6, 49.0) mm in the non-leukoreduced group. Fibrinogen was within reference ranges at Day 35 (>2 g/dL). Interleukin-6 was not detectable. CONCLUSION: Leukoreducing CPDA-1 whole blood with a platelet-saving filter did not compromise hemostatic properties. We encourage development of a single bag CPDA-1 whole blood collection set with in-line platelet-saving filter.

Methods for counting residual leukocytes in leukocyte-depleted plasma-a comparison between a routine hematology instrument, the Nageotte chamber, flow cytometry, and a fluorescent microscopy analyzer.

BACKGROUND: Counting very low levels of leukocytes is technically challenging but mandatory for quality control of leukocyte-depleted plasma. Established assays, such as flow cytometry and counting in the Nageotte chamber, are laborious and expensive. The aim of this study was to test two alternative assays, the cerebrospinal fluid program in the routine hematology analyzer ADVIA 2120 and a fluorescence microscopy analyzer, the ADAM-rWBC. STUDY DESIGN AND METHODS: Linearity, accuracy, and precision were established for the ADVIA 2120, the ADAM-rWBC analyzer and the Nageotte chamber with flow cytometry as the reference method. Two hundred consecutive leukocyte-depleted donor plasma samples were also tested. RESULTS: The ADAM-rWBC analyzer and the Nageotte chamber fulfilled all quality requirements. Flow cytometry fulfilled the requirements for linearity and precision. The ADVIA 2120 analyzer did not fully reach the quality criteria, and flow cytometry did not reach quality criteria on accuracy. No false-positive results on donor plasma samples were recorded. CONCLUSION: The ADAM-rWBC is suitable for the purpose of quality control of residual leukocytes in leukocyte-depleted plasma. For the ADVIA 2120, further improvements and studies are needed to reach the quality requirements stated in this study.

AABB Committee Report: reducing transfusion-transmitted cytomegalovirus infections.

Transfusion-transmitted cytomegalovirus (TT-CMV) is often asymptomatic, but certain patient populations, such as very low birth weight neonates, fetuses requiring intrauterine transfusion, pregnant women, patients with primary immunodeficiencies, transplant recipients, and patients receiving chemotherapy or transplantation for malignant disease, may be at risk of life-threatening CMV infection. It is unclear whether leukoreduction of cellular blood components is sufficient to reduce TT-CMV or whether CMV serological testing adds additional benefit to leukoreduction. The AABB CMV Prevention Work Group commissioned a systematic review to address these issues and subsequently develop clinical practice guidelines. However, the data were of poor quality, and no studies of significant size have been performed for over a decade. Rather than creating guidelines of questionable utility, the Work Group (with approval of the AABB Board of Directors) voted to prepare this Committee Report. There is wide variation in practices of using leukoreduced components alone or combining CMV-serology and leukoreduction to prevent TT-CMV for at-risk patients. Other approaches may also be feasible to prevent TT-CMV, including plasma nucleic acid testing, pathogen inactivation, and patient blood management programs to reduce the frequency of inappropriate transfusions. It is unlikely that future large-scale clinical trials will be performed to determine whether leukoreduction, CMV-serology, or a combination of both is superior. Consequently, alternative strategies including pragmatic randomized controlled trials, registries, and collaborations for electronic data merging, nontraditional approaches to inform evidence, or development of a systematic approach to inform expert opinion may help to address the issue of CMV-safe blood components.

Segments from red blood cell units should not be used for quality testing.

BACKGROUND: Nondestructive testing of blood components could permit in-process quality control and reduce discards. Tubing segments, generated during red blood cell (RBC) component production, were tested to determine their suitability as a sample source for quality testing. STUDY DESIGN AND METHODS: Leukoreduced RBC components were produced from whole blood (WB) by two different methods: WB filtration and buffy coat (BC). Components and their corresponding segments were tested on Days 5 and 42 of hypothermic storage (HS) for spun hematocrit (Hct), hemoglobin (Hb) content, percentage hemolysis, hematologic indices, and adenosine triphosphate concentration to determine whether segment quality represents unit quality. RESULTS: Segment samples overestimated hemolysis on Days 5 and 42 of HS in both BC- and WB filtration-produced RBCs (p < 0.001 for all). Hct and Hb levels in the segments were also significantly different from the units at both time points for both production methods (p < 0.001 for all). Indeed, for all variables tested different results were obtained from segment and unit samples, and these differences were not consistent across production methods. CONCLUSION: The quality of samples from tubing segments is not representative of the quality of the corresponding RBC unit. Segments are not suitable surrogates with which to assess RBC quality.

Real-time amplification of glyceraldehyde-3-phosphate dehydrogenase gene for quality control of leukopoor platelets.

BACKGROUND: Leukoreduction of blood products is crucial to prevent white blood cell (WBC)-associated complications during transfusion. Of the widely accepted methods for quantifying WBCs in blood components, Nageotte hemocytometry is time-consuming and laborious whereas a specialized instrument is required for flow cytometry. A reliable and affordable method to assess WBC count in blood products is of particular interest. STUDY DESIGN AND METHODS: Real-time polymerase chain reaction (PCR) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was developed for quantifying WBCs in leukopoor platelets (LPPs). After normalization by the cell-free prefiltrated and postfiltrated plasma DNA, the relative copy number of GAPDH gene in the platelet (PLT) concentrate and its corresponding LPPs was calculated according to the equation of 2(-ΔΔCt) of which Ct is defined as the threshold cycle. The percentage and the number of WBCs that remained in LPPs were consequently determined. This method was compared to Nageotte hemocytometry and was validated by using serially diluted PLT concentrate and 10 pairs of PLT concentrate-LPP samples. RESULTS: Consistent with the removal of WBCs after filtration, the Ct values for the LPP samples were increased when compared to their corresponding PLT concentrate. As revealed by real-time PCR of GAPDH gene, there is a correlation between the calculated and theoretical WBC count in the serially diluted PLT concentrate (correlation coefficient, 0.9532). The WBC counts for the 10 LPP samples were comparable between Nageotte and real-time PCR method and were all below 3.3 × 10(6) WBCs/L. CONCLUSION: The real-time PCR method we report in this study is applicable for routine quality assurance during leukoreduction process.

Quality control of leucocyte-depleted platelet concentrates obtained by buffy-coat method.

OBJECTIVES: Quality control results on leukoreduced buffy-coat platelet pools during the 7-year period (2005-2011) are presented with the aim to assess their overall quality and trends recorded during the study period. METHODS: Data of the Quality Assurance Department, Croatian Institute of Transfusion Medicine were used in the study. Measurement results of all study parameters are presented for the entire study period, while the rate of controlled platelet pools consistent with the specified quality requirements is additionally presented for each study year. RESULTS: The mean component volume was 50 ± 9 mL per 6 × 10(10) platelets (91.5% of conformable results), mean platelet count 7.81 ± 1.26 × 10(10) per single unit equivalent (97.5% of conformable results), mean leucocyte count 0.01 ± 0.08 × 10(6) per single unit equivalent (99.6% of conformable results), and mean pH 7.45 ± 0.16 (99.4% of conformable results). Bacteriologic testing showed negative result in 99.8% of tested components. CONCLUSION: Study results indicated the results of platelet pool quality control to be highly conformable with the specified quality requirements. It is illustrated how simple interventions in the preparation process can have significant impact on product quality improvement and thus on redefining quality requirements.

Eurobloodpack: a common European design for blood bag systems with integral leucodepletion filters.

Three EBA specified blood bag configurations ('Eurobloodpack') are described which are capable of meeting >80% of its member's requirements. These include a 'top-and-top' and two 'bottom-and-top' packs enabling aseptic, pre-donation collection of up to 40 ml of samples, 427.5-522.5 ml of whole blood and the preparation of an extensive range of blood components. Features currently beyond the scope of ISO standardisation have been controlled including: anticoagulant and additive volumes; collection needle and sampling system; transfer tubing; cross-match line; base label; leucodepletion filter performance; compatibility of access ports and transfusion sets. Eurobloodpack has significant advantages for blood services and blood bag manufacturers.

National French observatory of the quality of blood components for transfusion.

PURPOSE: Since 1998, prestorage leucoreduction of cellular blood components (BC) is mandatory in France. The French Blood Service needs to follow the data on the quality of the BC prepared by blood centers. This article gives an overview of the quality control (QC) data from 2001 to 2006. MATERIAL AND METHODS: QC data are submitted to a central data bank by each centre. The data are stratified according to preparation process for analysis of key performance criteria - residual leukocytes and haemoglobin or platelet content. BC preparation processes, methods for measuring haemoglobin and platelet content, and for counting residual leukocytes are those routinely employed by centers. RESULTS: The preparation process of red cell concentrates (RCC) influences the haemoglobin content: 57.6+/-6.8 g per unit versus 50.9+/-5.4 g per unit for whole blood or RCC filtration, respectively. Apheresis RCC exhibits a reduced variability (51.2+/-3.4 g per unit). For apheresis platelet concentrates, the median residual leukocyte count remains low for all separators (0.019-0.044 x 10(6)leukocytes per unit, in 2006). However, the percentage of units exceeding 1 x 10(6)leukocytes per unit is significantly higher with one separator (1.8% versus 0.8%, in 2006). For pooled buffy-coat derived platelets, we observed a significant increase in platelet recovery throughout the years (0.66-0.77 x 10(11)platelets per buffy-coat in 2001 and 2006, respectively). CONCLUSION: Our QC data show an overall compliance with the requirements for cellular BC. Our data bank is useful to inform on the performance of leucoreduced BC preparation processes carried out with market available devices.

Quality of leucoreduced red blood cell concentrates: 5 years of follow-up in France.

BACKGROUND: Since 1998, prestorage leucoreduction of all cellular blood components has been made mandatory in France. The French blood service needed to follow the data on the quality of the blood components prepared by blood centres. MATERIAL AND METHODS: Quality control (QC) data were submitted to a central data bank by each blood centre. The data were stratified by preparation method for analysis of key performance criteria - residual white blood cell (WBC) and total haemoglobin content. The red blood cell (RBC) preparation processes and the methods for measuring haemoglobin content and residual WBC count were those routinely employed by blood centres. Each year, more than 15,500 RBCs were tested. RESULTS: Red blood cells had a mean haemoglobin content between 53.6 and 54.9 g/unit depending on the year (2001 to 2005). The requirement of 40 g/unit was reached for about 99% of units. The haemoglobin content was influenced by the preparation process: 56.8 +/- 6.9 vs. 50.6 +/- 5.6 g/unit in average for whole blood filtration or RBC filtration, respectively. Apheresis RBCs exhibited a reduced variability (51.8 +/- 3.1 g/unit). The median residual WBC count remained low (0.046 to 0.057 x 10(6) WBCs/unit), and the percentage of RBC units exceeding the 1 x 10(6) WBCs/unit cut-off ranged from 1.5 to 0.6% depending on the year. A seasonal pattern was observed, with a significant increase (P < 0.001) of the median residual WBC count and of the percent of non-conforming units during the summer months. CONCLUSION: Our QC data suggest an overall compliance with the standard. Our data bank is useful to inform on the performance of leucoreduced RBC preparation processes carried out with market available devices.

Why implement universal leukoreduction?

The improvement of transfusion medicine technology is an ongoing process primarily directed at increasing the safety of allogeneic blood component transfusions for recipients. Over the years, relatively little attention had been paid to the leukocytes present in the various blood components. The availability of leukocyte removal (leukoreduction) techniques for blood components is associated with a considerable improvement in various clinical outcomes. These include a reduction in the frequency and severity of febrile transfusion reactions, reduced cytomegalovirus transfusion-transmission risk, the reduced incidence of alloimmune platelet refractoriness, a possible reduction in the risk of transfusion-associated variant Creutzfeldt-Jakob disease transmission, as well as reducing the overall risk of both recipient mortality and organ dysfunction, particularly in cardiac surgery patients and possibly in other categories of patients. Internationally, 19 countries have implemented universal leukocyte reduction (ULR) as part of their blood safety policy. The main reason for not implementing ULR in those countries that have not appears to be primarily concerns over costs. Nonetheless, the available international experience supports the concept that ULR is a process that results in improved safety of allogeneic blood components.

Evaluation of a new apheresis system for the collection of leukoreduced single-donor platelets.

BACKGROUND: The Fresenius COM.TEC cell separator is a new device for producing white cell concentrates (WBCs) and leukoreduced single-donor platelet concentrates (SDPs) and performing therapeutic cytapheresis and plasmapheresis that might replace the Fresenius systems AS104 and AS.TEC 204. This novel system's performance was evaluated for producing leukoreduced SDPs. STUDY DESIGN AND METHODS: In an investigational phase, each of 200 donors underwent plateletpheresis with the AS.TEC 204 and the COM.TEC systems. The collection efficiency (CE) and WBC contamination of the different techniques were compared. After some hard- and software modifications, the system was evaluated in an additional 800 procedures in the confirmatory phase. RESULTS: In the investigational phase, the CE of the COM.TEC device was increased significantly in comparison to the AS.TEC 204 device's CE (by 45 +/- 32% when collecting 1 unit of platelets [PLTs] and 1 unit of fresh-frozen plasma and by 43 +/- 42% when collecting only 1 unit of PLTs). Although all AS.TEC products proved to be leukoreduced, 2 percent of the COM.TEC procedures led to PLT concentrates containing more than 1 x 10(6) WBCs. In the confirmatory phase, all 1300 products from 800 COM.TEC procedures proved to be leukoreduced. Furthermore, the CE increased significantly from 53.5 +/- 4.6 percent in the investigational phase to 55.5 +/- 4.9 percent (p < 0.001) in the confirmatory phase. CONCLUSIONS: These data suggest that the new COM.TEC system offers a significantly and importantly improved CE in plateletpheresis procedures in comparison to the AS.TEC system. In the final version, the PLT products collected with this system fulfill the most stringent criteria for leukoreduced PLTs. This aim was achieved without additional filtration steps and thus without filtration-related PLT loss.

Calidad de hemocomponentes leucorreducidos: la validación del procedimiento de leucorreducción / Quality of WBC-reduced blood components: validation of leukoreduction process

Las Guías Internacionales y Nacionales regulan la práctica de leucorreducción para garantizar la calidad de los hemocomponentes leucorreducidos. La normativa local establece que el procedimiento deberá estar validado y que el nivel máximo de leucocitos residuales en productos leucorreducidos es 5 x 10 elevado a la 6. Para alcanzar dicho objetivo se analizan los factores críticos que influyen sobre el proceso de leucorreducción y se presentan métodos de recuento de leucocitos residuales, planes de muestreo y análisis estadístico. (AU)

Calidad de hemocomponentes leucorreducidos: la validación del procedimiento de leucorreducción / Quality of WBC-reduced blood components: validation of leukoreduction process

Las Guías Internacionales y Nacionales regulan la práctica de leucorreducción para garantizar la calidad de los hemocomponentes leucorreducidos. La normativa local establece que el procedimiento deberá estar validado y que el nivel máximo de leucocitos residuales en productos leucorreducidos es 5 x 10 elevado a la 6. Para alcanzar dicho objetivo se analizan los factores críticos que influyen sobre el proceso de leucorreducción y se presentan métodos de recuento de leucocitos residuales, planes de muestreo y análisis estadístico.

In vitro and in vivo evaluation of LEUKOSEP HRC-600-C leukoreduction filtration system for red cells.

BACKGROUND: Documentation of the benefits of leukoreduction has led to the increased use of this technique and the need for development of efficient and effective techniques for its accomplishment. This study investigated the in vitro properties and in vivo autologous radiolabeled recovery of leukoreduced red cells (RBCs) produced through a leukoreduction filtration system for RBCs (LEUKOSEP HRC-600-C, Hemerus Medical). STUDY DESIGN AND METHODS: Normal subjects donated 36 units of RBCs that were leukoreduced on Days 0, 3, or 5 through a "hands-off" technique. Biochemical studies were performed before and after filtration and at the end of 42 days of storage. Units leukoreduced on Days 0 or 5 were held until Day 42 and used for autologous radiolabeled return to determine recovery with 51Cr single-label radiolabeling techniques. RESULTS: Leukoreduction filtration was accomplished in 16.3 +/- 2 minutes on Day 0 at room temperature or 27 to 30 minutes on Days 3 or 5 after refrigeration. Leukoreduction efficiency was 4.6 +/- 0.6 log with a median residual white blood cell (WBC) content of fewer than 3.3 x 10(4) WBCs per unit. RBC recovery was 90 +/- 2 percent. Hemolysis was 0.34 +/- 0.16 percent at the end of 42 days of storage. The in vivo recovery of radiolabeled RBCs 24 hours after autologous return was 80.6 +/- 4.5 percent for RBC units leukoreduced on Days 0 and 5 combined. CONCLUSION: The LEUKOSEP HRC-600-C WBC reduction filtration system produced leukoreduced RBCs efficiently and effectively with acceptable poststorage biochemical measures and posttransfusion recovery after 42 days of storage.

[Residual leukocyte counting in plasma by a real-time genomic amplification assay]. / Méthode de dénombrement des leucocytes résiduels dans les plasmas par amplification génomique en temps réel.

BACKGROUND: Systematic plasma leukoreduction, which was introduced in France in April 1st 2001, has given rise to more sensitive methods for residual leukocytes counting. The technologies in application at this moment (Nageotte hemocytometers and flow cytometry methods) have been modified by a thirty fold sample concentration prior to analysis, inducing frequent downgrading of the plasma unit. So, in order to improve the detection threshold, we developed a more sensitive assay using "Real-Time Polymerase Chain Reaction" technology. MATERIALS AND METHODS: Real-time polymerase chain reaction was performed on a highly conserved HLADQalpha1 gene sequence. In order to determine the analytical performances of the method (accuracy, sensitivity, linearity and specificity) serial dilutions series ranging from 10(4) to 1 cells/ml were performed. A total of 18 series were prepared from three leukocyte stock solutions, by 1 in 10 serial dilutions in three plasmas completely devoided of white cells (called negative plasmas). To examine the specificity of the assay two negative controls were analyzed in each run. RESULTS: a sensitivity of 10 cells/ml (10(4) leukocytes/l) was achieved and the assay was linear between 10 and 10(4) cells/ml. The slope (-3.72) of the average standard curve calculated from all series, showed an amplification yield of 92.85%. CONCLUSION: we developed a quantitative assay for residual leukocytes in leukodepleted plasma, that agreed with the quality control requirement specifications.

Transfusion of leukoreduced cellular blood components from cytomegalovirus-unscreened donors in allogeneic hematopoietic transplant recipients: analysis of 72 recipients.

Leukoreduction of blood components has been considered a safe alternative to screening donors for CMV. The objective of this study is to analyze the effectiveness of bedside leukoreduction in preventing CMV transmission. We retrospectively studied 72 transplant recipients and donors who were CMV-seronegative pairs. All patients were transfused with CMV-unscreened cellular blood products leukoreduced at the bedside using leukoreduction filters. Quality control measures performed monthly in our leukoreduced blood components consistently demonstrated that at least 95% of the units sampled meet the leukoreduction criterion established by the American Association of Blood Banks standards. The CMV status of the recipients and donors was determined before transplantation by the latex agglutination assay. Recipients were observed for at least 100 days after transplantation. CMV cultures of urine, buffy coat, bone marrow, and bronchial washings were done weekly when indicated. CMV antigenemia testing was performed twice weekly: 11 transplant recipients seroconverted after transplantation. One patient was positive for CMV antigenemia 4 months after transplantation, but did not have CMV infection. Two of 61 patients who were not seroconverted were CMV antigenemia positive and did not have CMV infection: leukoreduction of cellular blood products is an efficient method of preventing CMV infection.

The impact of two whole blood inline filters on markers of coagulation, complement and cell activation.

BACKGROUND AND OBJECTIVES: There exists a current lack of information about the impact of different inline filters, used for the leucoreduction of whole blood (WB), on the levels of clotting factors and markers of coagulation, complement and cell activation in plasma. Only a few small comparisons of different types of WB inline filters have been published to date. MATERIALS AND METHODS: This study compared two plasma types of 200 units each. Both study groups were derived from WB, inline-filtered and held for 2 h at 20 degrees between donation and filtration. Then, 200 units (Group A) were filtered using a positively charged polyester filter (Baxter RZ2000) and the other 200 units (Group B) were filtered using an uncharged polyester filter (Fresenius). After filtration, both groups were analysed for fibrinogen, factors V and VIII:C (FV and FVIII:C, respectively), immunoglobulin G (IgG), residual leucocytes and platelets, and markers of coagulation, complement and cell activation. Predonation plasma samples from CPDA1-anticoagulated blood were obtained from 100 different individuals and served as controls. RESULTS: WB inline filtration did not influence fibrinogen, FV, FVIII:C or IgG levels. Neither filter induced thrombin or fibrin formation. The charged filter caused substantial complement activation and neutrophil elastase and platelet factor 4 release. In contrast, the plasma filtered through the uncharged filter showed markedly lower levels of C3a-desArg, C5a, neutrophil elastase and platelet factor 4, and moderately reduced levels of prothrombin fragments 1+2 and D-dimer, compared with controls. CONCLUSIONS: Filter type has a significant impact on the quality of plasma derived from WB filtered through inline filtration systems. Some filters produce substantial coagulation and complement activation and cell release, while others appear to reduce the plasma levels of activation markers. The clinical significance of these findings remains to be determined.