The incidence of donor white blood cell survival (transfusion-associated microchimerism) in Australian pediatric patients.

INTRODUCTION: Donor leucocyte survival following red blood cell (RBC) transfusion, known as transfusion-associated microchimerism (TAM), can occur in some patients. In Australia, despite the introduction of leucocyte filtration (leucodepletion) during RBC manufacture, TAM has been detected in adult trauma patients. However, the incidence of TAM in Australian pediatric patients has not been analyzed. METHODS: Patients aged 0-16 years were recruited across two cohorts. Retrospective participants had RBC transfusion between January 1, 2002 and November 15, 2017 and prospective participants received RBC transfusion between December 1, 2016 and November 25, 2020. Twelve bi-allelic insertion/deletion (InDel) polymorphisms were used to detect microchimerism amplification patterns using real-time PCR (RT-PCR) and droplet digital PCR (ddPCR). RESULTS: Of the retrospective cohort (n = 40), six patients showed amplification of InDel sequences indicating potential microchimerism. For three patients, minor InDel sequences were detected using RT-PCR only, two patients had minor InDel amplification using ddPCR only, and one patient had minor InDel amplification that was confirmed using both techniques. Amplification of minor sequences occurred in three patients who had received a bone marrow transplant in addition to RBC transfusion. In the prospective cohort (n = 25), no InDel amplification indicating potential microchimerism was detected using RT-PCR. DISCUSSION: Cell-based therapies had been administered in three patients where microchimerism amplification patterns were detected. Three patients have microchimerism that may be attributed to RBC transfusion. In prospective patients, who received leucodepleted and gamma-irradiated RBC units, no potential microchimerism amplification were detected. ddPCR may be a suitable technique for TAM analysis but requires further evaluation.

5WBF: a low-cost and straightforward whole blood filtration method suitable for whole-genome sequencing of Plasmodium falciparum clinical isolates.

BACKGROUND: Whole-genome sequencing (WGS) is becoming increasingly helpful to assist malaria control programmes. A major drawback of this approach is the large amount of human DNA compared to parasite DNA extracted from unprocessed whole blood. As red blood cells (RBCs) have a diameter of about 7-8 µm and exhibit some deformability, it was hypothesized that cheap and commercially available 5 µm filters might retain leukocytes but much less of Plasmodium falciparum-infected RBCs. This study aimed to test the hypothesis that such a filtration method, named 5WBF (for 5 µm Whole Blood Filtration), may provide highly enriched parasite material suitable for P. falciparum WGS. METHODS: Whole blood was collected from five patients experiencing a P. falciparum malaria episode (ring-stage parasitaemia range: 0.04-5.5%) and from mock samples obtained by mixing synchronized, ring-stage cultured P. falciparum 3D7 parasites with uninfected human whole blood (final parasitaemia range: 0.02-1.1%). These whole blood samples (50 to 400 µL) were diluted in RPMI 1640 medium or PBS 1× buffer and filtered with a syringe connected to a 5 µm commercial filter. DNA was extracted from 5WBF-treated and unfiltered counterpart blood samples using a commercial kit. The 5WBF method was evaluated on the ratios of parasite:human DNA assessed by qPCR and by sequencing depth and percentages of coverage from WGS data (Illumina NextSeq 500). As a comparison, the popular selective whole-genome amplification (sWGA) method, which does not rely on blood filtration, was applied to the unfiltered counterpart blood samples. RESULTS: After applying 5WBF, qPCR indicated an average of twofold loss in the amount of parasite template DNA (Pf ARN18S gene) and from 4096- to 65,536-fold loss of human template DNA (human ß actin gene). WGS analyses revealed that > 95% of the  parasite nuclear and organellar genomes were all covered at ≥ 10× depth for all samples tested. In sWGA counterparts, the organellar genomes were poorly covered and from 47.7 to 82.1% of the nuclear genome was covered at ≥ 10× depth depending on parasitaemia. Sequence reads were homogeneously distributed across gene sequences for 5WBF-treated samples (n = 5460 genes; mean coverage: 91×; median coverage: 93×; 5th percentile: 70×; 95th percentile: 103×), allowing the identification of gene copy number variations such as for gch1. This later analysis was not possible for sWGA-treated samples, as a much more heterogeneous distribution of reads across gene sequences was observed (mean coverage: 80×; median coverage: 51×; 5th percentile: 7×; 95th percentile: 245×). CONCLUSIONS: The novel 5WBF leucodepletion method is simple to implement and based on commercially available, standardized 5 µm filters which cost from 1.0 to 1.7€ per unit depending on suppliers. 5WBF permits extensive genome-wide analysis of P. falciparum ring-stage isolates from minute amounts of whole blood even with parasitaemias as low as 0.02%.

The expression of CD47 and its association with 2,3-DPG levels in stored leuco-reduced blood units.

BACKGROUND: Red blood cell (RBC) aging in transfusion medicine is characterized by alteration of many biochemical and morphological integrity of the cell referred to as red cell storage lesion (RCSL), CD47 is a protective marker expressed on RBCs that salvage the cell from phagocytosis. 2,3-diphosphoglycerate (2,3-DPG) tends to have a greater affinity towards deoxygenated hemoglobin. Any oxygen unloading at tissue capillaries are facilitated by 2,3-DPG, and any alterations in its levels can significantly interfere with oxygen release. Alteration of both CD47 expression and 2,3-DPG levels during red cell storage may serve as markers in the development of RCSL. The aim of this study was to validate the impact of storage time and leuco-depletion on CD47 expression on the RBCs, which could be a prospective marker for detection of RBCs viability and to clarify if the changes in CD47 expression and 2,3-DPG levels are correlated during storage of Packed RBCs. SUBJECTS AND METHODS: One hundred samples from Packed RBCs units were divided into two groups [Group 1 comprised unfiltered packed red cell units (n=50), whereas Group 2 included filtered "leuco-reduced" red cell units (n=50)]. Collection of samples was executed on days 0, 1 and 21. Each sample was measured for 2,3-DPG and alteration of CD47 expression on RBC using flow cytometry. RESULTS: Decreased CD47 expression along the storage period was statistically significant in both groups (P<0.05). Interestingly, the expression of CD47 was significantly higher in group 2 than group 1 on day zero, 1st and 21st days (P<0.05). Additionally, a statistically significant decrease in 2,3-DPG level was detected at day 21 of storage in group 1 compared to group 2 with a P-value of <0.001. There was a significant positive correlation (r=0.570, P<0.001) between CD47 MFI on RBC during storage and the level of 2,3-DPG at day 21 from packed RBCs storage. CONCLUSION: Older unfiltered RBC possesses lower expression of CD47 and low levels of 2,3-DPG, however filtration (leucoreduction) of RBCs units may help to retain considerable levels of 2,3-DPG and CD47 and hence sustains preservation of RBCs through reduction of phagocytosis.

Comparison of Flow Cytometric Methods for the Enumeration of Residual Leucocytes in Leucoreduced Blood Products: A Multicenter Study.

The BD FACSVia™ System features novel designs in hardware, software, and instrument QC. We compared the performance of the BD FACSVia System using the BD Leucocount™ kit with the BD FACSCalibur™ flow cytometer. Leucoreduced platelet (PLT, n = 252) and red blood cell (RBC, n = 278) specimens were enrolled at four sites. Each specimen was stained in four tubes using the BD Leucocount kit reagents and acquired on the two systems. BD Leucocount Control cells (high and low) were used to evaluate the inter-site reproducibility on the BD FACSVia System at three sites over 20 days. Deming regression and Bland-Altman analysis were performed to determine the WBC absolute counts on the BD FACSVia System vs. the BD FACSCalibur system. Assay accuracy for the range of 0-350 WBCs/µl was adequate. For samples with <25 WBCs/µl, the bias with 95% limits of agreement was 0.136 (-1.897 to 2.169) WBC/µl for PLTs (n = 184) and 0.170 (-2.025 to 2.365) WBC/µl for RBCs (n = 193). For inter-site reproducibility, the CV% was 6.46% (upper 95% CI 7.16%) for the PLT high control and 9.49% (10.52%) for the PLT low control. The CV% was 7.51% (8.32%) for the RBC high control and 10.76% (11.92%) for the RBC low control. The BD FACSVia System reported equivalent results of WBC absolute counts for leucoreduced PLT and RBC samples compared to the BD FACSCalibur system. The inter-laboratory reproducibility of the BD FACSVia System met study specifications. © 2018 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.

Changes in circulating inflammatory markers following febrile non-haemolytic transfusion reactions to leucoreduced red cells.

It would be desirable to be able to distinguish fever as a result of febrile non-haemolytic transfusion reactions (FNHTR) from other febrile conditions. To further characterize the inflammatory feature of FNHTR, we measured a large panel of inflammatory markers in pre- and posttransfusion plasma samples from patients with and without FNHTR following the transfusion of leucoreduced red blood cells. As FNHTR patients only displayed a significant increase in IL-6, we conclude that changes in plasma cytokine levels during FNHTR are unlikely to be used diagnostically. An incidental finding of a distinct cytokine pattern in pretransfusion samples from FNHTR patients warrants further investigations, as it might be used to characterize the nature of FNHTR and to predict the risk of these adverse events.

The residual risk of transfusion-transmitted cytomegalovirus infection associated with leucodepleted blood components.

BACKGROUND AND OBJECTIVES: Cytomegalovirus poses a risk to transfusion safety as its transmission to an immunocompromised recipient may lead to significant clinical sequelae. Once infection is established, it is lifelong and generally asymptomatic. Strategies to reduce the risk of transfusion-transmitted CMV (TT-CMV) include donor serological testing and blood component leucodepletion to deplete the transmissible reservoir. We estimate the residual risk for non-CMV antibody screened, leucodepleted (LD-only) fresh blood components. MATERIALS AND METHODS: We established an approach to estimate the risk of TT-CMV under various scenarios. We estimated the probability of an infectious component, for both red cells and platelets, as a function of the observed WBC filter failure rate and the probability that such a unit was also contaminated with infectious virus. RESULTS: Using this model, the estimated combined residual risk of LD-only red cell and platelet units was very low, 1 in 13 575 000 (95%CI:1 in 1 344 167 000-1 in 1 730 000) as was the individual residual risk estimate for LD-only red cells, 1 in 7 790 000 (95%CI: 1 in 771 307 000-1 in 993 000) and LD-only platelets, where a zero risk was estimated (95%CI: 0-1 in 1 074 000). CONCLUSION: We describe a novel approach to assess the residual risk of LD-only components. This can be applied generally using local data. Our risk estimate for LD-only blood components in Australia is below the threshold of 1 in 1 million, generally considered negligible. This provides a useful indicator of the relative safety of LD-only components to assist clinical decisions when serologically screened inventory is unavailable.

Comparison of a new microscopic system for the measurement of residual leucocytes in apheresis platelets with flow cytometry and manual counting.

BACKGROUND AND OBJECTIVES: Since 2001, all blood components in Germany must be leucocyte depleted. Recently, a new method for quality control of depletion was introduced. Our study aimed at the validation of the method for routine use in apheresis platelet concentrates. MATERIALS AND METHODS: We compared the new ADAM-rWBC device with manual counting in the Nageotte chamber and flow cytometry, two standard methods, by measuring residual leucocytes in 40 units of apheresis platelet concentrates and in six geometrical dilution series. RESULTS: Cell counts of residual leucocytes in the 40 units were below 10(6) cells per component with all methods, although mean cell counts were approximately 5 and 6 times higher in flow cytometry and ADAM-rWBC, respectively, compared to the Nageotte chamber. No unit with <10(6) leucocytes was regarded as contaminated. The dilution series showed acceptable accuracy, especially in the range around the cut-off (approximately 4·5 cells/µl in components with a volume of 220 ml) for regarding a concentrate as contaminated with leucocytes. No sample spiked with more than 4·5 cells/µl was counted as having less. CONCLUSION: In comparison with manual counting and flow cytometry, the ADAM-rWBC device performed equally. The method is suitable for routine screening of leucocyte contamination of apheresis platelets.

Comparative Study of Predeposit and Bedside Leucodepletion Filters.

BACKGROUND: Removal of leucocytes from cellular blood components is associated with reduction of several transfusion associated adverse reactions. METHODS: A total of 400 units of packed red blood cells (RBCs) were subjected to leucodepletion at room temperature and 4°C using different commercially available prestorage and bedside filters (Terumo Penpol Immugard III and Pall Medical BPF-4). Pre-filtration and post-filtration parameters were compared to assess the efficacy of prestorage leucodepletion vis-à-vis bedside leucodepletion and the requirement of universal leucodepletion. RESULT: Mean post-filtration red cell recovery ranged from 88.49-93.49% with all bags showing more than 85% red cell recovery. Mean post-filtration residual leucocyte count ranged from 0.205 × 10(6)-0.338 × 10(6)/bag with all bags showing more than log 3 leucoreduction. Prestorage leucoreduction achieved by the polyurethane filter was better than that achieved by the polyester filter. Red cell recovery with the bedside filters at room temperature was significantly less than that with prestorage filters at either temperature. CONCLUSION: This study suggests that prestorage leucoreduction is preferable over bedside leucoreduction and that polyurethane filters are better than polyester filters since leucodepletion achieved with the former is higher. We recommend selective log 3 leucodepletion using polyurethane prestorage filters for patients with specific indications.

Seroprevalence of Cytomegalovirus among Voluntary Blood Donors.

BACKGROUND: Primary cytomegalovirus (CMV) infection in immunocompetent host is self limiting infection, leading to latency of virus. However congenital CMV and CMV infections in immunocompromised patients are associated with high morbidity and mortality. Transfusion transmitted-cytomegalovirus (TT-CMV) infection in low birth weight neonate and immunocompromised transfusion recipients is being increasingly reported. Studies recommended transfusion of CMV free or CMV safe blood in prevention of TT-CMV. In this background, the study was undertaken to assess the CMV seroprevalence in blood donor. METHODS: A prospective study was conducted in which 431 voluntary blood donors were screened for CMV IgG and IgM by EIA (Enzyme Immuno Assay). RESULT: A total of 379 (87.9 %) voluntary blood donors were seropositive for CMV IgG. There was no statistical difference of CMV seropositivity and age. Further, seven (1.6%) subjects were both CMV IgM and IgG seropositive. CONCLUSION: High seroprevalence of CMV in our donor population is a threat to the blood safety. Strategies in reducing the risk of TT- CMV are discussed. Use of prestorage leucodepleted 'CMV safe' blood components along with judicious use of blood is recommended in prevention of TT-CMV in high risk recipients.

Leucodepletion and Blood Products.

BACKGROUND: Leucoreduction of blood products is increasingly being employed to produce blood products with residual WBCs < 5 × 10(6) per unit (99.9 percent or a log 3 leucoreduction). Clinical data suggests that non-haemolytic febrile transfusion reactions can be prevented by leucodepletion. The procedure also prevents alloimmunisation to HLA antigens in patients who will repeatedly require transfusion of blood/blood products. METHOD: Of the methods available to reduce the number of WBC in blood products washing of red cells, freezing and deglycerolisation are effective and yield a product with only a 24 hour shelf life. Other methods such as leucodepletion filters are relatively inexpensive, simple and the final product has a normal shelf life. Modern generation of leucoreduction filters and apheresis machines can provide greater than 4 log reduction of WBC. RESULTS: After the introduction of leucodepletion of blood for Thalassemics at our center in 2003, the incidence of non haemolytic febrile transfusion reactions (NHFTR) fell from 4% in 2002 to 1% in 2003. CONCLUSION: In patients undergoing long-term blood transfusion therapy e.g. Thalassemics, alloimmunisation against the HLA antigens on donor white cells is prevented by leucodepletion and prevents NHFTRs.

Transfusion of leukocyte-depleted blood in pediatric open heart surgery / 中国输血杂志

Objective To evaluate the efffects of leukocyte-depleted blood transfusion the protection of cardio pulmonary tissue and the reduction of postoperative bacterial infection.Methods Three hundred and forty-four pediatric patients were allocated to one of 3 groups based on transfusing different components of blood during open heart surgery:control group(n=109),routing blood transfusion group(RBT,n=113),leukocyte depleted blood transfusion group (LDBT,n=122). Blood samples were taken before operation,at 6h,and 24h after operation fir determination of creatininee kinase MB( CK MB),aspartate transaminase(AST),lactate dehydrogenase(LDH)and MDA. At the same time,we monitored changes of TPR and RI,and recorded changes of body temperature and days of utilizing antibiotics after operation.Results After operation serum concentrations of CK MB,AST, LDH and MDA significantly increased in all groups as compared to the values before operation,but the values of those in group LDBT were significantly loert than those in group RBT(P0.05).Conclusions Leukocyte depleted blood transfusion can effectively prevent the patients from cardion pulmonary ischemia-reperfusion injury and reduce postoperative bacterial infection in pediatric open heart surgery.

EVALUATION OF PRESTORAGE LEUCODEPLETION IN MASSIVE BLOOD TRANSFUSION / 解放军医学杂志

The patients received massive blood transfusion in General Hospital of the Chinese Pepole's Liberation Army between 1999to 2001were reviewed. The following items of leucodepleted blood transfusion (LDBT)group and routing blood transfusion(RBT) group at pretransfusion, the first day, third day, and seventh day after blood transfusion were recorded and compared: glutamic pyruvic transaminase(GPT), glutamic oxaloacetic transaminase(GOT), lactate dehydrogenase(LDH), total bilirubin(TB), direct bilirubin(DB), alkalescence phosphorylase(ALP), total protein(TP), albumin(ALB), glucose (GLU), Ca 2+ , total CO 2, pH and febrile nonhemolytic transfusion reactions(FNHTR). The results showed that compared with RBT group, GPT in LDBT group on the third day after blood transfusion was significantly lower ( P 0 05). When antiallergic medicine was given, FNHTR rate of massive blood transfusion in RBT and LDBT groups was 27% and 1 9%( P