A 73-year-old Greek woman presented with symptomatic anaemia requiring red cell transfusion in the setting of progressive chronic lymphocytic leukaemia (CLL). Based on a negative antibody screen, two units of red blood cells (RBCs) were provided for transfusion. During the transfusion, the patient developed an acute haemolytic transfusion reaction (HTR), but recovered with supportive measures. Subsequent antibody investigation confirmed that the patient had an anti-Wb antibody and that the implicated RBC unit was Wb-positive. This is the first report of an anti-Wb causing a clinically significant acute HTR in the literature.
]]>There are manual and automated methods to separate whole blood (WB) available. The Atreus whole blood processing system is an automated method, which combines centrifugation and expression of components into a single device. A major difference to conventional methods is that centrifugation temperature is not controlled at 22°C. The aim of this study was to examine the influence of increased centrifugation temperatures on the quality of red-blood-cell concentrates (RCC) after active cooling of WB prior to processing.
A total of 28 WB were processed: 16 at centrifugation temperatures of up to 28°C (1st protocol) and 12 at 34°C (2nd protocol). RCC quality parameters were tested weekly for 42 days.
Red-blood-cell concentrates (RCC) quality complied with the European and German guidelines. Haemolysis was not significantly different throughout storage. Significant statistical differences were detected between both protocols in potassium concentration at the end of storage and in ATP levels at the day of processing.
Centrifugation temperatures of up to 34°C are well tolerated by the red blood cells with minimal interference with the RCC quality parameters.
Red blood cell (RBC) transfusion is a life-saving intervention for critically ill patients; however, it has been linked to increased morbidity and mortality. We hypothesize that a number of important proteins accumulate during routine storage of RBCs, which may explain some of the adverse effects seen in transfused patients.
Five RBC units were drawn and divided (half prestorage leucoreduced (LR-RBC) and half left as an unmodified control (RBC). The supernatant was separated on days 1 and 42 of storage and proteomic analyses completed with in-gel tryptic digestion and nano-liquid chromatography tandem mass spectrometry.
In RBC supernatants, 401 proteins were identified: 203 increased with storage, 114 decreased, and 84 were unchanged. In LR-RBC supernatant, 231 proteins were identified: 84 increased with storage, 30 decreased, and 117 were unchanged. Prestorage leucoreduction removed many platelet- and leucocyte-derived structural proteins; however, a number of intracellular proteins accumulated including peroxiredoxins (Prdx) 6 and latexin. The increases were confirmed by immunoblotting, including the T-phosphorylation of Prdx-6, indicating that it may be functioning as an active phospholipase. Active matrix metalloproteinase-9 also increased with a coinciding decrease in the metalloproteinase inhibitor 1 and cystatin C.
We conclude that a number of proteins increase with RBC storage, which is partially ameliorated with leucoreduction, and transfusion of stored RBCs may introduce mediators that result in adverse events in the transfused host.
Since 2000, Quality Assurance (QA) exercises for the detection and identification of granulocyte antibodies and DNA typing for human neutrophil antigens (HNA) have been distributed within the International Granulocyte Immunobiology Workshops, which are linked to International Society of Blood Transfusion. The exercises were standardised at the outset to enable laboratory performance to be monitored. Between 2000 and 2012, nine exercises were distributed to 20 laboratories. Overall, 45 examples of 42 unique samples containing defined granulocyte reactive antibodies were distributed for serological analysis together with 20 samples for HNA genotyping. The level of satisfactory serological performance was initially set at 50% and later increased to 70%, while the ‘cut-off’ for HNA genotyping was set at 100% after 2008. Failure to achieve the minimum score in the QA exercises in consecutive years resulted in temporary exclusion. In 2000, the 15 participating laboratories had a mean score of 56.1% for serological analysis and 13 laboratories attempted HNA-1a and -1b genotyping, while 11 attempted HNA-1c typing. Steady improvements in proficiency for serological testing and HNA typing occurred in subsequent exercises. In 2012, the mean score for serology was 88.5% and 12/13 laboratories scored 100% for HNA-1a, -1b, -1c, -3a, -3b, -4a, -4bw, -5a and -5bw genotyping. These QA exercises have provided an invaluable tool to monitor and improve the standard of granulocyte immunology investigations for participating laboratories, thereby enhancing performance for both clinical investigations and donor screening programmes to reduce the incidence of TRALI.
Idiopathic thrombotic thrombocytopenic purpura (TTP) is a rare, clinically diagnosed disorder characterized by widespread intravascular platelet thrombosis. The pathophysiology involves acquired deficiency of ADAMTS13 (A disintegrin and metalloprotease with thrombospondin type 1 repeats), the enzyme responsible for cleavage of high molecular weight vonWillebrand factor multimers. Disease mortality is high, although prompt treatment with plasma exchange is generally effective. A readily available and highly reliable method of identifying ADAMTS13-deficient patients for appropriate plasma exchange is therefore of interest.
Our initial study involved the assessment of multiple clinical and laboratory variables in patients with clinically suspected TTP for whom ADAMTS13 assay was performed. Five variables were found to be of significant predictive power. This enabled the development of a point-based scoring system to efficiently determine the likelihood of TTP and response to plasma exchange in a given patient. This current study involved a separate validation cohort of patients with clinically suspected TTP who underwent ADAMTS13 testing within two large healthcare systems in Utah between 2009 and 2011. The previously derived score was applied to this cohort and its performance was analysed. Additionally, the original and validation cohorts were combined to revisit the predictive power of individual variables and the five-variable prediction score.
A total of 84 (11 paediatric cases excluded) patients comprised the validation population. The percentage of TTP diagnoses in this group (10%) was identical to that in the initial cohort. Using an ADAMTS13 activity of <10% of normal, our original score correctly predicted or excluded severe ADAMTS13 deficiency in all patients in the second cohort when data for all variables was available. Individual variables retained predictive power and the performance of a three-variable parsimonious model, as well as the ultimate diagnoses for patients in the second cohort are described.
This work confirms the predictive power of a simple point-based score to exclude TTP as evidenced by severe ADAMTS13 deficiency in appropriately selected patients. It may enable clinicians to rapidly begin plasma exchange or to pursue an alternative cause of thrombotic microangiopathy.
Haemochromatosis (HC) is a disorder of iron metabolism, requiring frequent phlebotomy to normalize high serum iron levels. There is currently no consensus relating to the eligibility of these patients to donate blood for transfusion. To gain a better understanding of the policies worldwide, a survey amongst blood services was performed.
A web-based questionnaire was developed and distributed among 44 blood services in 41 countries to identify the different policies relating to patients with HC and blood donation.
Respondents from 35 blood services (80%) of 33 countries completed the questionnaire. In 24 blood services among them (69%), individuals with genetic susceptibility for HC and/or patients with HC are accepted as blood donors. In approximately one-third of these blood centres (33%), genetic carriers/patients are allowed to donate blood more frequently than regular donors. Prescription from/approval by the patient's treating physician and/or a donor physician is required in the majority of the blood services (87%). Similar policies were identified in a few countries; however, in general, the policies regarding blood donation from patients with HC remain widely variable.
The results of our survey demonstrate large differences in the blood donation policies regarding carriers/patients with HC illustrating the need for uniform evidence-based and cost-effective policies which could benefit both HC patients and the blood supply around the world.
HPA-1a antibodies account for 70–80% of cases of fetal–neonatal alloimmune thrombocytopenia (FNAIT) in Caucasians. However, numerous workshops have demonstrated variability in their detection. We recently showed that exposure of αIIbβ3 to ethylene diamine tetraacetic acid (EDTA) affected binding of many anti-αIIbβ3 monoclonal, and HPA-1a allo-, antibodies; this adversely affected sensitivity of the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay and indirect platelet immunofluorescence test (PIFT). This study presents results from an international workshop studying the impact of cation chelation on HPA-1a antibody detection in routine diagnostic laboratories.
Serum and EDTA-anticoagulated plasma samples containing anti-HPA-1a were distributed to 39 laboratories. Participants were asked to detect and identify any HPA antibodies present.
2/39 (5·1%) participants were able to detect and identify anti-HPA-1a in the serum, but not in the plasma sample. EDTA plasma reduced MAIPA assay sensitivity by ≥20% in 17/24 (70·8%) laboratories and by ≥50% in 9/24 (37·5%) when using HPA-1a1a platelets (mean: 27·7%, range 0–85·1%); when using HPA-1a1b platelets 3/4 (75%), participants reported ≥50% loss of sensitivity (mean 65·6%, range 0–96·6%). A small but significant increase in optical densities was observed in antigen capture ELISA assays when using plasma (mean difference: 0·081, P < 0·01). Insufficient PIFT data were returned to draw firm conclusions.
Use of EDTA plasma significantly affects the sensitivity of the MAIPA assay and can affect detection of even potent, FNAIT-causing examples of anti-HPA-1a. These data highlight the importance of use of αIIbβ3 in an appropriate conformation for the sensitive detection of anti-HPA-1a.
Despite growing demand for transfusion, the number of voluntary young blood donors has steadily decreased over recent years in Japan. This study aimed to develop an easy-to-use survey tool to assess barriers and motivators to blood donation among Japanese university students.
We conducted cross-sectional studies at two universities in Fukushima Prefecture, Japan, in December 2011 (Stage 1) and February 2012 (Stage 2) using self-administered questionnaires. A short list of motivators and barriers to blood donation was developed from the open-ended questions asked of 50 students in Stage 1. In the Stage 2, we asked 105 students how important these items were when they decided whether or not to donate blood. Items showing a significant difference between donors and non-donors were kept in the final list.
Overall, 56% of the 100 participants analysed in Stage 2 were men, and ages ranged from 19 to 24 with a median of 20 years. Comparison of motivators and barriers between donors and non-donors revealed that only barrier item 8 (‘Frightened by blood donation’) showed a significant difference (P = 0·0006) in an expected direction and with a consistency between two universities.
This study identified fear as being the most significant barrier to blood donation among Japanese university students, which could be used as a single convenient indicator to assess their readiness to donate. More academic and clinical efforts are needed to understand and address students' fear towards blood donation in order to increase the donor pool in Japan.
Clinical use of plasma rich in growth factors requires biochemical product control. We aimed to measure and modulate concentrations of growth factors in solutions deriving from platelet apheresis or whole blood.
Growth factor concentrations were measured 5’, 10’, 20’, 30’, 60’ after CaCl2 was added at 40°C to platelet-apheresis products (n = 39) or after 60’ in platelet concentrates from whole blood (n = 13). Growth factor release was also obtained in platelet apheresis a) by incubation at 22°C or 40°C for 10’ or 30’ (n = 4); b) by repeated freeze–thaw (n = 9).
Fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) isoforms AA and AB and transforming growth factor beta (TGF-β) concentrations (pg/109 plt) were 25–60% higher in growth factors solutions from whole blood compared to platelet apheresis. Vascular endothelial growth factor (VEGF), TGF-β and PDGF isoforms were released early (5–10’) during incubation: TGF-β concentration increased also at 30’. FGF and epidermal growth factor (EGF) were released only after 30’. Incubation at 40°C/10’ increased VEGF (+70%) and decreased EGF (−30%) and PDGF-BB (−50%) versus 22°C/30’. Shock significantly increased TGF-β (1·6-fold), EGF (1·5-fold), FGF (4·5-fold) and lowered PDGF isoforms (0·2- to 0·5-fold) versus prolonged incubation at 40°C.
Platelets from platelet apheresis and whole-blood release all investigated growth factors. The release can be regulated controlling incubation time and/or temperature and performing cell lysis.
The haemoglobin level of prospective blood donors is usually performed on blood obtained by from the finger pulp by fingerstick with a lancet and filling a capillary tube with a sample. New noninvasive methods are now available for rapid, noninvasive predonation haemoglobin screening.
Prospective blood donors at our blood centre were tested, in two different trials, as follows: by the NBM 200 (OrSense) test (n = 445 donors) and by the Pronto-7 (Masimo) test (n = 463 donors). The haemoglobin values of each trial and the haemoglobin of finger pulp blood obtained by fingerstick with a lancet (HemoCue) were compared with the haemoglobin values obtained from a venous sample on a Cell Counter (Beckman Coulter).
Comparison of Beckman Coulter Cell Counter and OrSense and results showed a bias of 0·29 g/dl, the standard deviation of the differences (SDD) of 0·98 and 95% limits of agreement from −1·64 to 2·21, using Bland and Altman statistical methodology. Comparison of Masimo and Beckman Coulter Cell Counter results showed a bias of −0·53 g/dl, SDD of 1·04 and 95% limits of agreement from −2·57 to 1·51. Cumulative analysis of all 908 donors, as tested by the usual fingerstick test showed a bias of 0·83 g/dl, SDD of 0·70 and 95% limits of agreement from −0·54 to 2·20 compared with the Coulter Cell Counter. Compared with the Coulter Counter, the specificity of the methods was 99·5% for fingerstick, 97% for OrSense and 83% for Massimo, and the sensitivity was 99, 98 and 93%, respectively.
Analysis of finger pulp blood by either direct sampling by fingerstick and Hemocue, or by noninvasive haemoglobin tests does not replicate the results of cell counter analysis of venous samples. Compared with fingerstick, noninvasive haemoglobin tests eliminate pain and reduce stress, but have a lower level of specificity and sensitivity.
Errors in administration of blood products can lead to poor patient outcomes including fatal ABO incompatible transfusions. This pilot study sought to establish whether the use of two-dimensional (2D) barcode technology combined with patient identification software designed to assist in blood administration improves the bedside administration of transfusions in an Australian tertiary hospital.
The study was conducted in a Haematology/Oncology Day Clinic of a major metropolitan hospital, to evaluate the use of 2D barcode technology and patient safety-software and hand-held PDAs to assist nursing staff in patient identification and blood administration. Comparative audits were conducted before and after the technology's implementation.
The preimplementation transfusion practice audits demonstrated a poor understanding of the blood checking process, with focus on the product rather than patient identification. Following the implementation of 2D barcode technology and patient safety-software, there was significant improvement in administration practice. Positive, verbal patient identification improved from 57% (51/90) to 94% (75/80). Similarly, the cross-referencing of the patient's identification with the patient's wristband improved from 36% (32/90) to 94% (75/80), and the cross-referencing of patient ID on the compatibility tag to wristbands improved from 48% (43/90) to 99% (79/80). Importantly, the 2D barcode technology and patient safety-software saw 100% (80/80) of checks being conducted at the patient bedside, compared with 76% (68/90) in the preimplementation audits.
This pilot study demonstrates that 2D barcode technology and patient safety-software significantly improves the bedside check of patient and blood product identification in an Australian setting.
To evaluate the clinical effect of platelet concentrate (PC) transfusions after PC storage time reduction to 4 days.
This was a single-centre cohort study comparing two 3-month periods of time, before and after the reduction of PC storage time from 5 to 4 days. Seventy-seven consecutive patients with PC transfusions were enrolled after blood stem cell transplantation. Corrected platelet count increment (CCI) on the morning after transfusion, time to next platelet transfusion, need for red blood cell (RBC) transfusion and clinical bleeding symptoms were compared.
Platelet concentrate storage time was reduced between period 1 (storage for up to 5 days, median storage time 78 h, range 11–136 h) and period 2 (storage for up to 4 days, median storage time 53 h, range 11–112 h). Patients were comparable for age, weight, body surface area, underlying disorder, type of transplantation and transfused platelet dose. The CCI increased from a median of 4 (range 0–20) to 8 (0–68) × 109/l per 1011 platelets/m2 (P < 0·0001). Time to next PC transfusion increased from 1·1 to 2·0 days (P < 0·0001). Any bleeding symptom was noted in 20 of 36 patients (56%) vs. 9/41 patients (22%, P < 0·01). Nose bleeds, haematuria and bleeding at more than one site were significantly reduced. Frequency of RBC transfusion within 5 days after PC transfusion was reduced from 74 to 58% (P < 0·0001).
Platelet concentrate storage time shortening was associated with highly significant CCI increase, reduced RC needs and lower patient numbers with bleeding events.
Haemoglobin A1c (HbA1c) represents a key biomarker in diabetes diagnosis and management, as it is indicative of recent blood glucose concentrations. Glycation of haemoglobin is a non-enzymatic irreversible process that is promoted by the prolonged exposure of erythrocytes to high glucose concentrations, a condition that is known to occur under blood banking conditions. However, controversial data indicate no clear hint as to whether and to which extent HbA1c accumulates during red blood cell storage. Hereby, we propose the application of a validated MALDI-TOF mass-spectrometry-based method to this issue and report the observation about HbA1c levels apparently increasing over storage progression.
]]>Bleeding is increasingly considered an important end point in clinical platelet transfusion studies. Accurate recording and adjudication into well-defined bleeding grades, however, remains a major challenge.
We developed a computer algorithm for automatic adjudication. The algorithm's results were compared to those of three independent adjudicators.
For one of 1186 bleeding days, the clinical report form (CRF) was filled out incorrectly, and the algorithm therefore missed one grade-1 skin bleed. For two bleeding days, the adjudicators incorrectly classified a grade-2 skin bleed as grade-1 while the algorithm correctly classified these days. The algorithm saved approximately six person-hours of adjudication time for the adjudication of 1186 days from 60 patients.
The algorithm can be an invaluable tool for adjudicating large amounts of bleeding data.
Although risk factors for HIV infection are known, it is important for blood centres to understand local epidemiology and disease transmission patterns. Current risk factors for HIV infection in blood donors in Brazil were assessed.
A case–control study was conducted at large public blood centres located in four major cities between April 2009 and March 2011. Cases were persons whose donations were confirmed positive by enzyme immunoassays followed by Western blot confirmation. Audio computer-assisted structured interviews (ACASI) were completed by all cases and controls. Multivariable logistic regression was used to estimate adjusted odds ratios (AORs) and associated 95% confidence intervals (CIs).
There were 341 cases, including 47 with recently acquired infection, and 791 controls. Disclosed risk factors for both females and males were sex with an HIV-positive person AOR 11·3, 95% CI (4·1, 31·7) and being an IVDU or sexual partner of an IVDU [AOR 4·65 (1·8, 11·7)]. For female blood donors, additional risk factors were having male sex partners who also are MSM [AOR 13·5 (3·1, 59·8)] and having unprotected sex with multiple sexual partners [AOR 5·19 (2·1, 12·9)]. The primary risk factor for male blood donors was MSM activity [AOR 21·6 (8·8, 52·9)]. Behaviours associated with recently acquired HIV were being a MSM or sex partner of MSM [13·82, (4·7, 40·3)] and IVDU [11·47, (3·0, 43·2)].
Risk factors in blood donors parallel those in the general population in Brazil. Identified risk factors suggest that donor compliance with selection procedures at the participating blood centres is inadequate.
LDL apheresis is used to treat patients with familial hypercholesterolaemia, and low-volume plasmapheresis for plasma donation may similarly lower cholesterol levels in some donors. This study was designed to assess the effect of plasmapheresis on total, LDL and HDL cholesterol levels in a plasma donor population.
This was a prospective, unblinded longitudinal cohort study in which a blood sample was obtained for analysis before each donation. Data from 663 donors were analysed using a multivariable repeated measures regression model with a general estimating equations approach with changes in cholesterol as the primary outcome measure.
The model predicted a significant decrease in total and LDL cholesterol for both genders and all baseline cholesterol levels (P < 0·01). The greatest total cholesterol decreases (women, −46·8 mg/dL; men, −32·2 mg/dL) were associated with high baseline levels and 2–4 days between donations. Small but statistically significant increases (P ≤ 0·01) in HDL cholesterol were predicted for donors with low baseline levels.
These results suggest that, in donors with elevated baseline cholesterol levels, total and LDL cholesterol levels may decrease during routine voluntary plasmapheresis.
Transfusion of blood products in particular older products is associated with patient morbidity. Previously, we demonstrated a higher incidence of acute lung injury in lipopolysaccharide-treated sheep transfused with stored blood products. As transfusion following haemorrhage is more common, we aimed to determine whether a ‘first hit’ of isolated haemorrhage would precipitate similar detrimental effects following transfusion and also disrupt haemostasis.
Anaesthetized sheep had 33% of their total blood volume collected into Leukotrap bags (Pall Medical), which were processed into packed red blood cells and cross-matched for transfusion into other sheep. After 30 mins, the sheep were resuscitated with either: fresh (<5 days old) or stored (35–42 days old) ovine blood followed by 4% albumin to replacement volume, albumin alone or normal saline alone and monitored for 4 h.
The first hit of haemorrhage precipitated substantial decreases in mean arterial pressure however haemostasis was preserved. Transfusion of stored ovine blood induced (1) transient pulmonary arterial hypertension but no oedema and (2) reduced fibrinogen levels more than fresh blood, but neither induced coagulopathy. Thus, transfusion of stored blood affected pulmonary function even in the absence of overt organ injury.
The fact that stored blood transfusions: (1) did not induce acute lung injury in contrast to previous lipopolysaccharide-primed animal models identifies the ‘first hit’ as an important determinant of the severity of transfusion-mediated injury; (2) impaired pulmonary dynamics verifies the sensitivity and vulnerability of the pulmonary system to injury.
Although orthotopic liver transplantation (OLT) is nowadays considered standard practice at experienced centres, it can still be affected by a significant risk of massive bleeding and its related complications. Solvent/detergent plasma (S/D Plasma) has been proposed as an alternative to fresh frozen plasma (FFP) to curtail such complications. This study aimed at evaluating the efficacy of S/D Plasma in OLT patients by comparing it to FFP.
Sixty-three OLT patients were randomized into two groups depending on whether they were transfused with FFP or S/D plasma. A thromboelastography-based protocol aimed at achieving and maintaining predetermined coagulation goals was used to guide plasma transfusions. At the beginning and the end of surgery, standard laboratory coagulation tests were performed together with the assessment of the VII, VIII, V, XII factors and S protein blood levels.
The two study groups equally achieved the thromboelastography goals but with a reduced amount of transfusions in the S/D plasma group (P < 0·0001). At the end of surgery, factors V and XII and S protein blood levels were lower in the S/D plasma patients who also showed lower INR, aPTT and antithrombin III levels.
In cirrhotic patients undergoing OLT, the use of S\D plasma associated with thromboelastography allows the same clinical results but with a significant reduction in the amount of plasma transfusions.
An adequate donor population is of key importance for the entire blood transfusion chain. For good donor management, a detailed overview of the donor database is therefore imperative. This study offers a new description of the donor cycle related to the donor management process. It also presents the outcomes of a European Project, Donor Management IN Europe (DOMAINE), regarding the segmentation of the donor population into donor types.
Blood establishments (BEs) from 18 European countries, the Thalassaemia International Federation and a representative from the South-Eastern Europe Health Network joined forces in DOMAINE. A questionnaire assessed blood donor management practices and the composition of the donor population using the newly proposed DOMAINE donor segmentation. 48 BEs in 34 European countries were invited to participate.
The response rate was high (88%). However, only 14 BEs could deliver data on the composition of their donor population. The data showed large variations and major imbalances in the donor population. In 79% of the countries, inactive donors formed the dominant donor type. Only in 21%, regular donors were the largest subgroup, and in 29%, the proportion of first-time donors was higher than the proportion of regular donors.
Good donor management depends on a thorough insight into the flow of donors through their donor career. Segmentation of the donor database is an essential tool to understand the influx and efflux of donors. The DOMAINE donor segmentation helps BEs in understanding their donor database and to adapt their donor recruitment and retention practices accordingly. Ways to use this new tool are proposed.
The risk of transfusion-transmitted HIV infection under (i) permanent exclusion and (ii) 12-month deferral of MSM in England and Wales during 2005–2007 was estimated. Assuming equal compliance with both scenarios, estimated risk under a 12-month deferral (0.228/million donations [range 0·168–0·306/million donations]) was only marginally greater (0·5%) than that under lifetime exclusion (0·227/million donations [range 0·157–0.318/million donations]), with one extra-HIV infectious donation every 455 years. Poorer compliance of MSM with a 12-month deferral would be expected to increase the estimated risk, whereas improved compliance could decrease risk by up to 29·1%.
]]>Due to an increasing number of reported thromboembolic events (TEE) after the administration of one intravenous immunoglobulin (IVIG) and one subcutaneous immunoglobulin (SCIG), pharmacovigilance and laboratory data were collected to analyse the root cause and assess the reporting frequency of TEEs for various IG products.
Paul-Ehrlich-Institut retrospectively analysed 228 reports of TEEs associated with six different IG products and estimated annual TEE-reporting rates based on worldwide sale figures over a period of 6 years (2006–2011). In addition, non-activated partial thromboplastin time (NAPTT) testing was performed to capture pro-coagulant potential of six IG products (four IVIG and two SCIG).
For three IVIGs, the drug-related TEE-reporting rates remained stable from 2006 to 2011 (0–0·83 cases per 1000 kg IVIG distributed). In contrast, the TEE rate of one IVIG increased significantly from 0·33 cases in 2006 to nearly nine cases in 2010 (P < 0·001).
The NAPTT testing of IG products with a low TEE rate revealed a NAPTT time >200 s and a NAPTT ratio >0·8, whereas TEE-associated batches of IG products with an increased TEE rate had a NAPTT ratio <0·8. After modifications of manufacturing processes, a normalization of NAPTT results and a decrease in TEE rates could be demonstrated.
The ‘30-min rule’ requires discarding red blood cells (RBCs) exposed to uncontrolled temperatures for >30 min to ensure safe RBC transfusion. This study was aimed at determining whether multiple room temperature (RT) exposures promote bacterial growth.
Pooled and split RBC units were inoculated with ~1 CFU/ml of Serratia marcescens, Yersinia enterocolitica, Escherichia coli or Staphylococcus epidermidis. Control units remained in storage, while test units were exposed to RT for six 30-min or three 60-min intervals. Bacterial concentrations and endotoxin levels were determined after each exposure and at 42 days of storage. RBC core temperature and RT were monitored in mock units with Escort iLog temperature loggers. A mixed model was used for statistical analyses.
Red blood cell core temperature reached 10·7 ± 0·4°C and 14·2 ± 0·2°C during 30- and 60-min exposures, respectively. Staphylococcus epidermidis and E. coli did not grow in either control or exposed RBCs. Yersinia enterocolitica concentration and endotoxin levels were similar in both control and test units. Serratia marcescens concentration and endotoxin levels were higher in exposed units; however, differences between units exposed for 30 min or 60 min were not observed.
There is no added risk to RBC safety by increasing RT exposures to 60 min with each removal from storage for up to a total of 3 h during RBC shelf life. Therefore, extending the 30-min limitation in RBCs exposed to uncontrolled temperatures to 60 min should be considered by regulatory agencies.
There have been great advances over the last decades in haematopoietic stem cell (HSC) transplantation, using either bone marrow, peripheral blood or cord blood-derived stem cells. The coming into force of the European legislation on tissues and cells and the consequent transposition of Directives into national laws have required the health authorities in the Member States (MS) and the scientific societies to review the transplantation activities to ensure the circulation of safe HSC products. Here, the regulatory inspection process performed by the Competent Authorities and the professional voluntary accreditation process of the Transplant Programmes active in Italy is compared.
]]>A number of DNA-based methods to genotype the alleles coding for HNA have been described, but all require the separate amplification and analysis of each allele. The aim was to develop a DNA-based method for simultaneous detection of HNA-1, HNA-3, HNA-4 and HNA-5 alleles.
An allele-specific primer extension method was used in combination with magnetic beads from Luminex technology. PCR–sequence-specific primers (SSP) was used to resolve the presence of the HNA-1b allele in samples assigned by the Luminex bead assay as HNA-1a/-1b/-1c or HNA-1b/-1c. HNA allele frequencies were determined in a panel of 140 randomly selected English Caucasoid blood donors.
HNA allelic types were compared with historical results, and 100% concordance was found. Only eight of the 97 samples used in the validation required additional testing by PCR–SSP. Allele frequencies were determined in the blood donor population as follows: 0·318 for HNA-1a, 0·668 for HNA-1b, 0·014 for HNA-1c, 0·768 for HNA-3a, 0·232 for HNA-3b, 0·882 for HNA-4a, 0·118 for HNA-4b, 0·736 for HNA-5a and 0·264 for HNA-5b.
A multiplex Luminex bead assay for the simultaneous detection of HNA-1, HNA-3, HNA-4 and HNA-5 alleles is described that enables rapid typing of donors to support HNA alloimmunized patients who require HNA-compatible blood products.
Despite long being a mainstay in describing platelet activation via degranulation, interlaboratory variation remains an issue in measurement of membrane CD62P by flow cytometry. Our objective was to identify actions that may minimize this variation.
Sixteen laboratories participated in an international comparative study. Two sets of platelet samples were prepared in one laboratory. Set 1 was stained and fixed; set 2 was fixed and required staining at participating laboratories. A single-staining method was used, and platelet populations were selected based on forward scatter/side scatter characteristics. Calibration beads were used to standardize measurement across different instruments.
There was a large discrepancy in reported CD62P values among study sites [interlaboratory coefficient of variance (CV): 36–78%]. When electronic data were re-analysed by a single analyst using a consistent gating strategy and a stable reference point, variation decreased markedly (CV < 12%), indicating a problem with isotype control samples, possibly related to sample fixation or shipment.
Consensus regarding gating strategies and use of a reliable reference point would greatly improve agreement in interlaboratory CD62P measurement.
Therapeutic leucodepletion plays an established role in the initial treatment of patients with acute myeloid leukaemia (AML) and possibly other leukaemias presenting with leucostasis. Recently, a new leucodepletion technology, Spectra Optia IDL, has become available that differs from its predecessor, COBE Spectra MNC, by a variety of electronic supports, including by electronic adjustment of buffy coat positioning at the collection port. Given the paucity of patients in need of leucodepletions and marked differences in clinical presentation as well as blast properties (e.g. size, density), formal clinical trials comparing leucodepletion technologies have never been executed.
Here, we present aggregate data from eight leucodepletions performed in AML patients with clinical signs of leucostasis between 11/2011 and 07/2012 with the new device and compare the apheresis outcomes with those from fifteen leucodepletions performed with the old technology between 06/2010 and 10/2011.
Patients did not differ with respect to epidemiological data. Pre-apheresis leucocyte count (WBC) was significantly higher in Spectra Optia IDL patients. Tolerability was excellent with both devices. Basic apheresis denominators such as duration, processed volume, inlet pump rate, ACD-A consumption and product volume were very similar. A negative correlation between pre-apheresis WBC and collection efficiency was noted. Mean collection efficiency for leucocytes with Spectra Optia IDL (47·3%) was similar to that with COBE Spectra MNC (50·5%). Platelet attrition was similar with both devices, approximately 30%.
The novel, electronically guided leukapheresis system is suitable for leucodepletion.
Peripheral blood stem cell cryopreservation is associated with cell damage and decreased viability. We evaluated the impact of up to 10 years of cryopreservation (5% DMSO) on viability of CD34+ cells utilizing graft samples of consecutive patients (2002–2012) with different malignancies who underwent stem cell collection and transplantation. Viability of CD34+ cells from oncohaematological patients measured after 5 weeks (97·2 ± 0·6%) or after 9–10 years of cryopreservation (95·9 ± 0·5%) was unaffected. Haemoglobin, granulocyte and platelet recovery after transplantation of long-term cryopreserved grafts occurred within 8–13 days. CD34+ stem cells can be safely stored up to 9–10 years, without affecting cell viability and clinical effectiveness.
]]>In fetal/neonatal thrombocytopenia, maternal alloimmunization is diagnosed by the identification of the maternal alloantibody and the offending paternal antigen inherited by the foetus/neonate. Today, for practical reasons, most laboratories perform platelet genotyping instead of phenotyping. Here, we report the case of a human platelet antigen (HPA)-5 genotype/phenotype discrepancy observed in a mother who delivered a mildly thrombocytopenic newborn.
Platelet antibody detection and platelet phenotyping were performed using the MAIPA assay; platelet genotypes were determined using BeadChip technology (BioArray), PCR-SSP, PCR-RFLP and sequencing.
Serological investigations revealed the presence of maternal anti-GPIIbIIIa autoantibodies. No alloantibodies were detected. No feto-maternal platelet incompatibility was observed for HPA-1 to -21. The mother and newborn were genotyped as HPA-5aa using BeadChips, but as HPA-5a (weak b) with PCR-SSP and HPA-5ab with PCR-RFLP. Mother's platelets were phenotyped as HPA-5b(+). GPIa exon 13 sequencing confirmed the HPA-5ab genotype of the mother and newborn, and revealed an NM_002203.3:c.1594A>C mutation near the HPA-5 polymorphism (5′ side), leading to an I503L amino acid change.
Feto-maternal alloimmunization was ruled out: the neonatal thrombocytopenia probably resulted from maternal anti-GPIIbIIIa autoantibodies. This case highlights that platelet typing should be performed using two different methods to avoid false diagnosis.
Interventions to change physician transfusion behavior are often evaluated by examining the amount of red blood cell (RBC) units transfused or the proportion of patients transfused before and after the intervention. The pre-transfusion haemoglobin concentration is a sensitive measure of transfusion practice, but has not been used to evaluate behavioral interventions. We examined the effect of a Danish National Board of Health December 2007 transfusion guideline on the behavior of clinicians treating acute myeloid leukaemia (AML). We compared the effect of the guideline on pre-transfusion haemoglobin concentrations with other measures of transfusion behavior, including use of RBC units and proportion of patients transfused. No change in transfusion behavior could be demonstrated by examining amount of RBC units transfused and proportion of patients transfused. Conversely, the pre-transfusion haemoglobin concentration fell significantly. Pre-transfusion haemoglobin determination is a sensitive measure of the effect of an intervention to change physician transfusion behaviour.
During storage, red blood cells (RBCs) lose their membrane stability, leading to haemolysis and microparticle (MP) formation. The use of RBCs stored for more than 28 days has been associated with an increased incidence of deep vein thrombosis. However, the exact mechanism by which coagulation activation is enhanced in stored RBCs is still unknown.
To investigate the relevant potential procoagulant activities of MPs and study the relative procoagulant factors for initiating the coagulation on MPs in stored RBCs.
MPs were isolated from the plasma of RBC units stored in citrate–phosphate–dextrose–adenine. At seven storage time-points (d0, d7, d14, d21, d28, d35 and d42), MPs were morphologically observed, quantified and analysed for tissue factor, factor XI (FXI) and their thrombin-generating potential.
MPs were observed using electron microscopy. The size of the MPs ranged from 0·272 μm to 0·973 μm in diameter. During the storage of RBCs in plastic bags, the MP concentration increased from 3389 ± 218/μl at day 0 to 61 586 ± 2237/μl at d42. Thrombin generation was dependent on the total number of MPs (r = 0·987). Anti-human FXI antibody inhibited thrombin concentrations by 50·3% compared with control plasma, whereas antitissue factor and antitissue factor pathway inhibitor failed to reduce thrombin concentrations.
Our study provides evidence that MP formation due to RBC storage might propagate coagulation not only by exposing phosphatidylserine, but also by initiating thrombin generation independently of tissue factor in a FXI -dependent manner.
Glucose and acetate have been proposed to be required elements in platelet storage media. This study investigated the role of these compounds on the varied elements that comprise the platelet storage lesion.
For each replicate, four pooled and split ABO group–specific buffy coat–derived platelet concentrates were suspended in an in-house additive solution with minimal plasma and varying final concentrations of acetate or glucose. Units were sampled on days 2, 3, 6, 8 and 10 and tested for markers of platelet morphology, activation, function, metabolism and indicators of cell death.
The absence of glucose was associated with a decrease in ATP, falling to a mean of 1·1 ± 0·1 μmol/1011 plts in units with no added glucose compared with 4·2 ± 0·6 μmol/1011 plts (P < 0·001) in units with 30 mm glucose. As glucose became depleted, the decrease in ATP to levels below 3 μmol/1011 plts was associated with an increase in both annexin V binding and intracellular free calcium. In units lacking exogenous acetate, ATP levels on day 10 were 5·2 ± 1·5 μmol/1011 plts compared with 2·7 ± 0·9 μmol/1011 plts in units with 56 mm acetate (P = 0·006). Higher concentrations of exogenous acetate were associated with a lower hypotonic shock response and higher surface expression of CD62P suggestive of a dose dependency.
Under current physical storage conditions, glucose appears necessary for the maintenance of platelets stored as concentrates in minimal volumes of plasma. The addition of acetate was associated with increased platelet activation and reduced ATP levels.
Background and Objectives ABO blood group accounts for up to 40% of the variability in plasma von Willebrand factor (VWF) levels, which vary in the rank order AB > B > A > O > Bombay. This may be due in part to the influence of ABO-associated oligosaccharides on the proteolysis of VWF by the metalloprotease ADAMTS13, which is markedly deficient in thrombotic thrombocytopenic purpura (TTP). Using ABO blood group as a surrogate for baseline VWF levels as well as susceptibility to proteolysis by ADAMST13, we set out to determine whether ABO blood group influences the clinical course of TTP.
Methods We conducted a retrospective analysis of the clinical course of 76 patients with primary, sporadic TTP treated at two institutions over the past 10 years.
Results We found no significant differences between group O and non-O patients with respect to presenting platelet count and lactate dehydrogenase concentration, maximum serum creatinine concentration, and total number of therapeutic plasma exchanges per episode.
Conclusions Substrate-related contributors to the highly variable phenotype and clinical course of TTP warrant further investigation.
‘Safe blood’ is and has always been the major concern in transfusion medicine. Plasma can undergo virus inactivation treatments based on physicochemical, photochemical or thermal methodologies for pathogen inactivation. The validation of these treatments is essentially based on clottability assays and clotting factors’ titration; however, their impact on plasma proteins at the molecular level has not yet been evaluated. Proteomics appears as particularly adapted to identify, to localize and, consequently, to correlate these modifications to the biological activity change. At the crossroads of biology and analytical sciences, proteomics is the large-scale study of proteins in tissues, physiological fluids or cells at a given moment and in a precise environment. The proteomic strategy is based on a set of methodologies involving separative techniques like mono- and bidimensional gel electrophoresis and chromatography, analytical techniques, especially mass spectrometry, and bioinformatics. Even if plasma has been extensively studied since the very beginning of proteomics, its application to transfusion medicine has just begun. In the first part of this review, we present the principles of proteomics analysis. Then, we propose a state of the art of proteomics applied to plasma analysis. Finally, the use of proteomics for the evaluation of the impact of storage conditions and pathogen inactivation treatments applied to transfusion plasma and for the evaluation of therapeutic protein fractionated is discussed.
]]>Background and Objectives In Australia, the risk of transfusion-transmitted malaria is managed through the identification of ‘at-risk’ donors, antibody screening enzyme-linked immunoassay (EIA) and, if reactive, exclusion from fresh blood component manufacture. Donor management depends on the duration of exposure in malarious regions (>6 months: ‘Resident’, <6 months: ‘Visitor’) or a history of malaria diagnosis. We analysed antibody testing and demographic data to investigate antibody persistence dynamics. To assess the yield from retesting 3 years after an initial EIA reactive result, we estimated the proportion of donors who would become non-reactive over this period.
Materials and Methods Test results and demographic data from donors who were malaria EIA reactive were analysed. Time since possible exposure was estimated and antibody survival modelled.
Results Among seroreverters, the time since last possible exposure was significantly shorter in ‘Visitors’ than in ‘Residents’. The antibody survival modelling predicted 20% of previously EIA reactive ‘Visitors’, but only 2% of ‘Residents’ would become non-reactive within 3 years of their first reactive EIA.
Conclusion Antibody persistence in donors correlates with exposure category, with semi-immune ‘Residents’ maintaining detectable antibodies significantly longer than non-immune ‘Visitors’.
Background and Objectives The outbreak of vCJD in the UK leads to concern regarding the potential for human-to-human transmission of this agent. Plasma-derived products such as albumin, immunoglobulin and coagulation factors were manufactured by BPL from UK plasma up until 1999 when a switch to US plasma was made. In the current study, the capacity of various manufacturing processes that were in use both prior to and after this time to remove the TSE agent was tested.
Materials and Methods Small-scale models of the various product manufacturing steps were developed. Intermediates were spiked with scrapie brain extract and then further processed. Samples were assayed for the abnormal form of prion protein (PrPSC) by Western blotting, and the reduction in the amount of scrapie agent determined.
Results Many of the manufacturing process steps produced significant reduction in the scrapie agent. Particularly effective were steps such as ethanol fractionation, depth filtration, ion-exchange and copper chelate affinity chromatography. Virus retentive filters, of nominal pore size 15 or 20 nm, removed >3 log. The total cumulative reduction capacity for individual products was estimated to range from 7 to 14 log. In the case of factor VIII (8Y), the total removal was limited to 3 log.
Conclusion All the processes showed a substantial capacity to remove the TSE agent. However, this was more limited for the intermediate purity factor VIII 8Y which included fewer manufacturing steps.
Background and Objectives In many jurisdictions, blood donors who have an atypical pulse rate are temporarily deferred. This practice is not supported by evidence. We evaluated whether accepting donors with an atypical pulse rate increases their risk of cardiac ischaemic events.
Methods We measured the cumulative incidence of hospitalizations and deaths for coronary heart disease within 1 year of follow-up among donors who, between 2002 and 2006, were temporarily deferred because of an atypical pulse (<50 beats/min, >100 beats/min, or irregular). We compared this incidence to that observed among donors who also had an atypical pulse but who were allowed to donate, following a change in our deferral policy in 2007. The occurrence of cardiac events was determined through hospital discharge and death registries.
Results Among 6076 donors who were temporarily deferred for an atypical pulse, the 1-year rate of hospitalization or death for cardiac ischaemic events was 3·5/1000, compared to 2·4 in donors who had an atypical pulse but who were allowed to donate (n = 10 671), for an adjusted odds ratio of 1·7 (95% CI, 0·9–3·0, P = 0·08).
Conclusion Regardless of the clinical significance of an atypical pulse rate, our data show that accepting donors with this condition does not increase the occurrence of serious cardiac ischaemic events. We conclude that pulse rate measurement in prospective donors is not warranted.
To determine the accuracy of fingerstick haemoglobin assessment in blood donors, the performance of a portable haemoglobinometer (HemoCue Hb 201+) was prospectively compared with that of an automated haematology analyzer (Cell-Dyn 4000). Haemoglobin values obtained by the latter were used as the ‘true’ result.
Capillary fingerstick samples were assayed by HemoCue in 150 donors. Fingerstick samples from two sites, one on each hand, were obtained from a subset of 50 subjects. Concurrent venous samples were tested using both HemoCue and Cell-Dyn devices.
Capillary haemoglobin values (HemoCue) were significantly greater than venous haemoglobin values (HemoCue), which in turn were significantly greater than venous haemoglobin values by Cell-Dyn (mean ± SD: 14·05 ± 1·51, 13·89 ± 1·31, 13·62 ± 1·23, respectively; P < 0·01 for all comparisons among groups). Nine donors (6%) passed haemoglobin screening criteria (≥12·5 g/dl) by capillary HemoCue, but were deferred by Cell-Dyn values (false-pass). Five donors (3%) were deferred by capillary sampling, but passed by Cell-Dyn (false-fail). Substantial variability in repeated fingerstick HemoCue results was seen (mean haemoglobin 13·72 vs. 13·70 g/dl, absolute mean difference between paired samples 0·76 g/dl). Hand dominance was not a factor.
Capillary samples assessed via a portable device yielded higher haemoglobin values than venous samples assessed on an automated analyzer. False-pass and false-fail rates were low and acceptable in the donor screening setting, with ‘true’ values not differing by a clinically significant degree from threshold values used to assess acceptability for blood donation.
The purpose of our studies was to determine the effects of extended platelet storage on poststorage platelet viability.
Normal subjects were recruited to donate platelets using two different apheresis systems: either the COBE Spectra (n = 58) or the Haemonetics MCS+ (n = 84). Platelet recovery and survival data from the two systems were compared with each other and with in vitro measurements of the stored platelets.
There were no significant differences in either platelet recoveries or survivals between the two machines between 1 and 8 days of storage. Combining the data from both machines, platelet recoveries decreased by 2·6% and survivals by 0·3 days/storage day. In vitro assays did not predict either platelet recoveries or survivals during storage for 5–8 days. After 9 days of storage, pHs were unacceptable (≤ 6·1), suggesting that 8 days will be the longest possible storage time.
These data suggest that, if stored platelet bacterial contamination issues are resolved, significant extension of platelet storage times is possible.
Previous data that showed maintenance of reticulocyte percentage in whole blood stored in CPDA-1 have led to the assumption that reticulocyte maturation becomes arrested during refrigerated storage. However, reticulocyte behaviour in red-blood-cell units stored in additive solutions has not yet been studied. This study was thus aimed at determining reticulocyte count and reticulocyte subtypes in red-blood-cells units stored in AS-1.
Reticulocyte percentage and subtypes were determined by flow cytometry with thiazole orange in six red-blood-cells units stored in AS-1.
Reticulocyte count was 26·8 ± 4·6 × 109/l at week 0·5 and 8·2 ±2·9 × 109/l at week 6. Total haemolysis during storage was 0·19 ± 0·08%. High-fluorescence reticulocytes were 2·0 ± 3·2 × 109/l at week 0·5 and decreased by weeks 2, 4 and 6. Low-fluorescence reticulocytes were 22·1 ± 3·1 × 109/l at week 0·5 and decreased by weeks 4 and 6.
A significant decrease in reticulocytes occurred during red-blood-cells units’ storage in AS-1. Even if it were assumed that all of haemolysed cells during storage were reticulocytes, there are a number of them whose disappearance cannot be explained by this mechanism. Changes observed in reticulocyte subtypes suggest that they mature during storage.
Our objective was to compare the frequency of adverse events (AEs) due to any of the 4 types of fresh-frozen plasma (FFP) prepared and delivered by the French Blood Establishment (EFS) over a 10-year period. Surveillance of AEs and vigilance was performed according to a homogeneous policy. The four types of FFP comprised of one type (methylene blue [MB) that was stopped since then and of another type [amotosalen (AI)] that was recently introduced, along with two conventional products [quarantine (Q) and solvent–detergent (SD)].
This is a retrospective study based on the national AE reporting database and on the regional database system for deliveries. AEs recorded after the delivery of 1 of the 4 types of FFP were pairwise compared, with appropriate statistical corrections.
105 964 FFP units were delivered (38·4% Q, 17·9% SD, 9·7% MB and 34% AI).
Statistical comparisons of AEs identified only a difference in AE rates between quarantine and solvent–detergent plasma.
FFP was confirmed to be extremely safe in general, especially if one considers ‘severe’ AEs. All types of FFP were associated with extremely low occurrences of AEs. Q, SD, MB and AI led, respectively, to 7·14, 4·86, 1·05 and 4·16 AEs per 10 000 deliveries.
Plasma transfusions are commonly used in adult and paediatric intensive care units. Recent data suggest an association between plasma transfusions and worse clinical outcome in adult trauma patients. To date, no prospective paediatric study has addressed this issue. Our objective was to prospectively analyse the association between plasma transfusions and clinical outcome of critically ill children.
Prospective, observational and single centre study that includes all consecutive admissions to a tertiary level multidisciplinary paediatric critical care unit over a 1-year period. The primary outcome measure was the incidence after transfusion of new or progressive multiple organ dysfunction syndrome. Secondary outcome measures included nosocomial infections, intensive care unit length of stay and 28-day mortality. Odds ratios were adjusted for weight, severity of illness, coagulopathy, plasma transfusions prior to admission, need for extracorporeal life support and transfusion of other labile blood products.
A total of 831 patients were enrolled, among which 94 (11%) received at least one plasma transfusion. In the latter group of patients, the adjusted odds ratio for an increased incidence of new or progressive multiple organ dysfunction syndrome was 3·2 (P = 0·002). There was also a significant difference in the occurrence of nosocomial infections and intensive care unit length of stay, but no significant difference in the 28-day mortality.
In critically ill children, plasma transfusions seem to be independently associated with an increased occurrence of new or progressive multiple organ dysfunction syndrome, nosocomial infections and prolonged length of stay.