Establishment of transfusion-relevant bacteria reference strains for red blood cells.

BACKGROUND AND OBJECTIVES: Red blood cell concentrates (RBCC) are susceptible to bacterial contamination despite cold storage. A reliable evaluation of strategies to minimize the risk of RBCC-associated bacterial transmission requires the use of suitable reference bacteria. Already existing Transfusion-Relevant Bacteria Reference Strains (TRBRS) for platelet concentrates fail to grow in RBCC. Consequently, the ISBT TTID, Working Party, Bacterial Subgroup, conducted an international study on TRBRS for RBCC. MATERIALS AND METHODS: Six bacterial strains (Listeria monocytogenes PEI-A-199, Serratia liquefaciens PEI-A-184, Serratia marcescens PEI-B-P-56, Pseudomonas fluorescens PEI-B-P-77, Yersinia enterocolitica PEI-A-105, Yersinia enterocolitica PEI-A-176) were distributed to 15 laboratories worldwide for enumeration, identification, and determination of growth kinetics in RBCC at days 7, 14, 21, 28, 35 and 42 of storage after low-count spiking (10-25 CFU/RBCC). RESULTS: Bacterial proliferation in RBCC was obtained for most strains, except for S. marcescens, which grew only at 4 of 15 laboratories. S. liquefaciens, S. marcescens, P. fluorescens and the two Y. enterocolitica strains reached the stationary phase between days 14 and 21 of RBCC storage with a bacterial concentration of approximately 109  CFU/ml. L. monocytogenes displayed slower growth kinetics reaching 106 -107  CFU/ml after 42 days. CONCLUSION: The results illustrate the importance of conducting comprehensive studies to establish well-characterized reference strains, which can be a tool to assess strategies and methods used to ameliorate blood safety. The WHO Expert Committee on Biological Standardization adopted the five successful strains as official RBCC reference strains. Our study also highlights the relevance of visual inspection to interdict contaminated RBC units.

Familial acquired thrombotic thrombocytopenic purpura in siblings - no immunogenetic link with associated human leucocyte antigens.

Acquired immunoglobulin G (IgG)-mediated thrombotic thrombocytopenic purpura (TTP) has not yet been described in non-twin siblings. We report two cases of acquired TTP in Caucasian sisters with inactive ADAMTS13 metalloprotease due to ADAMTS13 autoantibodies suggesting a role of genetic determinants in this life-threatening disease. However, human leucocyte antigen (HLA) class II types presumably associated with acquired TTP were not identified in the patients, indicating that HLA class II typing may not be useful in acquired TTP risk assessment of family members.

Comparison of three noninvasive methods for hemoglobin screening of blood donors.

BACKGROUND: To prevent phlebotomy of anemic individuals and to ensure hemoglobin (Hb) content of the blood units, Hb screening of blood donors before donation is essential. Hb values are mostly evaluated by measurement of capillary blood obtained from fingerstick. Rapid noninvasive methods have recently become available and may be preferred by donors and staff. The aim of this study was to evaluate for the first time all different noninvasive methods for Hb screening. STUDY DESIGN AND METHODS: Blood donors were screened for Hb levels in three different trials using three different noninvasive methods (Haemospect [MBR Optical Systems GmbH & Co. KG], NBM 200 [LMB Technology GmbH], Pronto-7 [Masimo Europe Ltd]) in comparison to the established fingerstick method (CompoLab Hb [Fresenius Kabi GmbH]) and to levels obtained from venous samples on a cell counter (Sysmex [Sysmex Europe GmbH]) as reference. The usability of the noninvasive methods was assessed with an especially developed survey. RESULTS: Technical failures occurred by using the Pronto-7 due to nail polish, skin color, or ambient light. The NBM 200 also showed a high sensitivity to ambient light and noticeably lower Hb levels for women than obtained from the Sysmex. The statistical analysis showed the following bias and standard deviation of differences of all methods in comparison to the venous results: Haemospect, -0.22 ± 1.24; NBM, 200 -0.12 ± 1.14; Pronto-7, -0.50 ± 0.99; and CompoLab Hb, -0.53 ± 0.81. CONCLUSION: Noninvasive Hb tests represent an attractive alternative by eliminating pain and reducing risks of blood contamination. The main problem for generating reliable results seems to be preanalytical variability in sampling. Despite the sensitivity to environmental stress, all methods are suitable for Hb measurement.

Impact of the blood group on postoperative CRP and leukocyte levels after primary total hip and knee arthroplasty.

BACKGROUND: C-reactive protein (CRP)- and leukocyte levels are common parameters to evaluate the inflammatory response after orthopaedic surgery and rule out infectious complications. Nevertheless, both parameters are vulnerable to disturbing biases and therefore leave room for interpretation. OBJECTIVE: Since blood groups are repeatedly discussed to influence inflammatory response, our aim was to observe their impact on CRP and leukocyte levels after total hip and knee arthroplasty (THA/TKA). METHODS: Short term postoperative CRP and leukocyte levels of 987 patients, who received either primary TKH (n= 479) or THA (n= 508), were retrospectively correlated with their blood group. ABO, Rhesus and a combination of both blood groups were differentiated. RESULTS: CRP levels after TKA were significantly higher in blood type AB than in type A and O on day 2-4 and also than in type A on day 6-8. Leukocyte levels after THA were significantly higher in blood group type O than in type A on day 6-8 while still remaining in an apathological range. We observed no significant differences between Rhesus types and Rhesus types and CRP or leukocyte levels. CONCLUSION: We observed significantly increased CRP levels after TKA in patients with blood group AB. Since the elevated CRP levels do not account for early periprosthetic infection, surgeons should include this variation in their postoperative evaluation.

Implication of IL-2/IL-21 region in systemic sclerosis genetic susceptibility.

OBJECTIVE: The interleukin 2 (IL-2) and interleukin 21 (IL-21) locus at chromosome 4q27 has been associated with several autoimmune diseases, and both genes are related to immune system functions. The aim of this study was to evaluate the role of the IL-2/IL-21 locus in systemic sclerosis (SSc). PATIENTS AND METHODS: The case control study included 4493 SSc Caucasian patients and 5856 healthy controls from eight Caucasian populations (Spain, Germany, The Netherlands, USA, Italy, Sweden, UK and Norway). Four single nucleotide polymorphisms (rs2069762, rs6822844, rs6835457 and rs907715) were genotyped using TaqMan allelic discrimination assays. RESULTS: We observed evidence of association of the rs6822844 and rs907715 variants with global SSc (pc=6.6E-4 and pc=7.2E-3, respectively). Similar statistically significant associations were observed for the limited cutaneous form of the disease. The conditional regression analysis suggested that the most likely genetic variation responsible for the association was the rs6822844 polymorphism. Consistently, the rs2069762A-rs6822844T-rs6835457G-rs907715T allelic combination showed evidence of association with SSc and limited cutaneous SSc subtype (pc=1.7E-03 and pc=8E-4, respectively). CONCLUSIONS: These results suggested that the IL-2/IL-21 locus influences the genetic susceptibility to SSc. Moreover, this study provided further support for the IL-2/IL-21 locus as a common genetic factor in autoimmune diseases.

Cell quality of apheresis-derived platelets treated with riboflavin-ultraviolet light after resuspension in platelet additive solution.

BACKGROUND: Previously, we evaluated the Mirasol pathogen reduction technology (PRT) system on platelet (PLT) function before resuspension. We now evaluated this system in the presence of PLT additive solution (PAS). STUDY DESIGN AND METHODS: Double-dose PLTs (n = 15) were generated using a commercially available apheresis system (Trima, Version 5.2, CaridianBCT) allowing for the resuspension in SSP+ (MacoPharma) immediately after collection. Paired units (n = 30) were PRT treated (M) or remained untreated (C) and analyzed for metabolism (pH, pO(2) , glucose, lactate, adenosine triphosphate [ATP]), swirl, hypotonic shock response (HSR), turbidometric aggregation, CD62P expression, annexin A5 and lactate dehydrogenase (LDH) release, mitochondrial enzymatic reduction activity (MTS), transmembrane mitochondrial potential (Δψ), and surface coverage (SC) during shear-induced adhesion throughout 8 days of storage. RESULTS: As seen previously, PRT treatment of PLT units, containing a mean of 3.9 × 10(11) ± 0.3 × 10(11) PLTs in 397 ± 10 mL with a 32% to 34% plasma carryover, was associated with significantly (p < 0.001) increased cell activation, acidity, and glycolytic flux. PRT treatment appeared to up regulate both oxidative pathway and adhesional properties as evidenced by significantly higher MTS reduction, oxygen consumption, and shear-induced SC on Day 1 (p ≤ 0.016). While no significant differences were found for LDH release and ATP content (except for Day 8), M units were significantly inferior (p ≤ 0.021) for aggregation (TRAP-6); for Δψ and annexin A5 release (by Day 5); and for swirl, HSR, and MTS reduction (by Day 7). CONCLUSION: PRT treatment in the presence of PAS was comparable to PRT treatment before resuspension preserving ATP content and mitochondrial function.

Platelet derived cytokine accumulation in platelet concentrates treated for pathogen reduction.

BACKGROUND: Pathogen reduction technologies (PRTs) prevent replication and proliferation of pathogens in platelet (PLT) concentrates (PCs) by modifying nucleic acids. Due to increased cell activation, PRT may also lead to increased cytokine release from α granules and promote adverse transfusion reactions in the recipient. DESIGN: Fifteen double-dose leukoreduced apheresis PCs were collected on the Trima Accel platform (vs. 5.2.) allowing for the resuspension in PLT additive solution (PAS) immediately after collection. After a 2-h resting period (1st hour without, 2nd hour with agitation), splitting was performed: one unit remained untreated to serve as control (C), while the other was riboflavin-UVB treated using the Mirasol-PRT system according to the manufacturer's instructions (M). During 8 days of storage, PCs were analyzed for contaminating white and red blood cells, bacterial growth, PLT activation, LDH and cytokine release (MIP-1 α, RANTES, PF4, and TGF-ß-1). Results obtained were opposed to a former study, where triple-dose PCs underwent Mirasol-PRT prior to resuspension or the INTERCEPT BLOOD SYSTEM (psoralen-UVA) or remained untreated. RESULTS: Despite similar LDH release, PRT treatment was associated with significantly higher (p<0.05) cell activation but only slightly higher cytokine accumulation during storage. Differences became significant only for PF4 and RANTES at day 8 of storage. On the other hand, in the investigation on triple-dose PCs (yielding higher cytokine levels), TGF beta-1 and RANTES remained significantly (p<0.05) lower after PRT treatment compared to untreated units. CONCLUSION: Factors, such as collection modality, onset of resuspension and additional amounts of magnesium/potassium in the PAS used may be of equal or even greater impact for cytokine accumulation in stored PCs than PRT treatment.

Plasma exchange with COVID-19 convalescent plasma in a patient with severe ANCA-associated vasculitis and COVID-19 pneumonia after rituximab therapy.

The combination of coronavirus disease 2019 (COVID-19) pneumonia and pulmonary-renal syndrome due to ANCA-associated vasculitis (AAV) poses diagnostic uncertainty and a therapeutic dilemma. According to current limited knowledge of COVID-19, the application of commonly used drugs in AAV, cyclophosphamide (CYC) and rituximab (RTX), must be weighed carefully in active COVID-19 infection. We report a case of a 52-year-old male patient with concurrent severe COVID-19 pneumonia and acute relapse of pulmonary-renal syndrome due to AAV after recent RTX maintenance dose. The patient presented with severe hypoxaemia, complete B-cell depletion and severe acute respiratory syndrome coronavirus 2 viraemia. He was successfully treated with therapeutic plasma exchange employing COVID-19 convalescent plasma.

Annexin V Release and Transmembrane Mitochondrial Potential during Storage of Apheresis-Derived Platelets Treated for Pathogen Reduction.

BACKGROUND: In vitro function of stored platelet (PLT) con-centrates was analyzed after applying two different techniques of pathogen reduction technology (PRT) treatment, which could increase cellular injury during processing and storage. METHODS: Nine triple-dose PLT apheresis donations were split into 27 single units designated to riboflavin-UVB (M) or psoralen-UVA (I) treatment or remained untreated (C). Throughout 8 days of storage, samples were analyzed for annexin V release, the mitochondrial transmembrane potential (Deltapsi) and some classical markers of PLT quality (pH, LDH release, hypotonic shock response (HSR)). RESULTS: PLT count and LDH release of all units maintained initial ranges. All units exhibited a decrease in pH and HSR and an increase in annexin V release and Deltapsi disruption. Notably, throughout the entire storage period, annexin V release re-mained lowest in M units. Throughout 7 days of storage, M units remained comparable to C units (p > 0.05), whereas inferior values were observed with I units. Here, differences to C units reached significance by day 1 (pH: p < 0.0001), day 5 (annexin V release: p < 0.014), and day 7 (HSR, Deltapsi: p

Platelet function assessed by shear-induced deposition of split triple-dose apheresis concentrates treated with pathogen reduction technologies.

BACKGROUND: Pathogen inactivation/reduction technology (PRT) may alter quality of stored platelet (PLT) concentrates (PCs). Therefore, PLT adhesion and aggregation should be studied before transfusion of PRT-treated PLTs. STUDY DESIGN AND METHODS: A three-arm in vitro study on triple-dose apheresis PCs (n = 9) was conducted. Split single units were designated to PRT treatment with either a riboflavin (M)- or a psoralen (I)-based technique and compared to untreated controls (C). Samples were taken on Days 0, 1, 5, 7, and 8 to assess PLT function via a cone and plate(let) analyzer, flow cytometric P-selectin expression, and turbidometric aggregation response to thrombin receptor-activating peptide 6 (TRAP-6). RESULTS: P-selectin expression increased and TRAP-6-inducible expression decreased steadily in all units until reaching a plateau on Day 5 of storage. PRT-treated units demonstrated significant (p < or = 0.008) differences to C units due to a more pronounced upregulation in P-selectin expression after PRT treatment. The same was true for TRAP-6 after Day 5 of storage. C units were significantly superior over PRT-treated units (p < or = 0.002), among which M yielded higher values than I (p < or = 0.008). Although M demonstrated increased shear-induced PLT deposition that remained stable during storage (p = 0.082), surface coverage significantly declined in C (p = 0.047) and especially in I (p = 0.003), but differences between M, C, and I did not reach significance. All units exhibited a slight increase in aggregate size that remained comparable throughout storage (p > or = 0.141). CONCLUSIONS: Irrespective of storage-related changes in PLT activation and turbidometric aggregation response, riboflavin-based PRT seemed to benefit shear-induced PLT adhesion. The impact of this finding for PLT function and thrombogenesis in vivo must await clinical evaluation.

Cell viability during platelet storage in correlation to cellular metabolism after different pathogen reduction technologies.

BACKGROUND: The objective of this study was to evaluate if pathogen reduction technologies (PRTs) affect platelet (PLT) viability by alteration of PLT metabolism during storage. STUDY DESIGN AND METHODS: Twenty-seven split triple-dose apheresis PLTs were PRT treated using ultraviolet light with either riboflavin-UVB (M) or psoralen-UVA (I) or remained untreated (C). Samples were taken on Days 0, 1, 5, 7, and 8 and analyzed for annexin V release (enzyme-linked immunosorbent assay), mitochondrial enzymatic activity (MTS assay), transmembrane mitochondrial potential (Deltapsi; JC-1 assay), and metabolism based on pH, pO(2), glucose, lactate, and ATP content. RESULTS: During storage, Deltapsi and MTS reduction activity decreased, while annexin V release and acidity increased in all units, more pronounced, however, after PRT treatment, which led to higher lactate accumulation due to acceleration in glycolytic flux. No significant differences were found between C and M, whereas I was significantly different by Day 1 (pH value), Day 5 (annexin V release), and Day 7 (Deltapsi) of storage. Intracellular ATP content remained similar between C and M but was significantly lower in end-stored I units. Cell viability markers of I units were highly correlated with the oxidative pathway, which appeared impaired in I but up regulated in M units. CONCLUSION: PRT treatment using M increased both anoxidative glycolytic flux and oxidative phosphorylation. The I-based technique was associated with an impaired mitochondria-based respiration. During terminal storage, this resulted in significantly lower maintenance of ATP and cell viability. The impact of these findings for storage prolongation or clinical use must await further evaluation.

Cell integrity and mitochondrial function after Mirasol-PRT treatment for pathogen reduction of apheresis-derived platelets: Results of a three-arm in vitro study.

BACKGROUND: Mirasol pathogen reduction technology (PRT) treatment uses riboflavin (vitamin B(2)) in combination with ultraviolet light (UV) to inactivate pathogens in platelet concentrates (PCs). This treatment has been reported to increase glycolytic flux, which could result from damage to mitochondria and/or increased ATP demand. DESIGN: Triple-dose PCs were collected by the Trima Accel device. Immediately after splitting, single units were designated to Mirasol-PRT treatment (M), gamma irradiation (X) or remained untreated (C). Platelet (PLT) mitochondrial transmembrane potential (Deltapsi) was evaluated (JC-1 assay) as well as mitochondrial enzymatic activity (MTS assay). LDH release, p selectin expression, glucose/oxygen consumption and lactate production rates were quantified and compared among study groups during 7days of storage. RESULTS: Immediately after PRT treatment, no significant changes were found in JC-1 signal, MTS activity, and LDH release indicating that PRT treatment did not alter functional/structural cell or mitochondrial integrity as evidenced by LDH release comparable to untreated study groups. In parallel to significantly higher p selectin expression, treated PLTs exhibited significantly accelerated oxygen and glucose consumption rates associated with increased acidity due to higher lactate production rates throughout storage. Despite larger cell populations with depolarized Deltapsi particularly at days 5 and 7, mitochondrial reduction activity of M units as measured by the MTS assay was maintained and appeared to be up-regulated relative to untreated and irradiated controls. CONCLUSION: Mirasol-PRT treated PLTs increased both glycolytic flux as well as respiratory/enzymatic mitochondrial activity. An increased demand for ATP due to increased alpha granule degranulation may be the driving force for these observations.

Evaluation of White Blood Cell- and Platelet-Derived Cytokine Accumulation in MIRASOL-PRT-Treated Platelets.

BACKGROUND: Soluble mediators in platelet concentrates (PCs) released from contaminating white blood cells (WBCs) and platelets (PLTs) themselves are supposed to promote allergic and non-hemolytic febrile transfusion reactions in the recipient. Pathogen reduction technologies (PRTs) prevent replication and proliferation of pathogens as well as of WBCs, and may reduce cytokine accumulation in PCs during storage and prevent adverse events after PLT transfusion. On the other hand, such treatments may also lead to increased cytokine production by stimulation of WBCs or PLTs due to the photochemical or photodynamical process itself. MATERIAL AND METHODS: 12 triple-dose PLT apheresis collections were leukoreduced by the process-controlled leukoreduction system of the Trima Accel machine and split into 3 units undergoing Mirasol-PRT treatment (M) or gamma irradiation (X) or remaining untreated (C). During storage for up to 7 days, PLT activation, WBC-derived Th-1/2, and inflammatory as well as PLT-derived cytokines were measured by cytometric bead array and enzymelinked immunosorbent assay, respectively. RESULTS: Independent of treatment, all PLT products exhibited low levels of WBC-associated cytokines near or below assay detection limits. WBC-associated cytokines were not elevated by Mirasol-PRT treatment. PLT-derived cytokines were detected at higher levels and increased significantly during storage in all units. Most likely due to higher PLT activation, M units showed significantly higher levels of PLT-derived cytokines compared to untreated and gamma-irradiated units on day 5 of storage. CONCLUSION: In all PCs, PLTs themselves were the main source of cytokine release. Mirasol-PRT treatment was associated with a significantly increased PLT activation and accumulation of PLT-derived cytokines during storage, without affecting WBC-derived cytokines relative to controls.

Evaluation of Four Bedside Test Systems for Card Performance, Handling and Safety.

SUMMARY: OBJECTIVE: Pretransfusion ABO compatibility testing is a simple and required precaution against ABO-incompatible transfusion, which is one of the greatest threats in transfusion medicine. While distinct agglutination is most important for correct test interpretation, protection against infectious diseases and ease of handling are crucial for accurate test performance. Therefore, the aim of this study was to evaluate differences in test card design, handling, and user safety. DESIGN: Four different bedside test cards with pre-applied antibodies were evaluated by 100 medical students using packed red blood cells of different ABO blood groups. Criteria of evaluation were: agglutination, labelling, handling, and safety regarding possible user injuries. Criteria were rated subjectively according to German school notes ranging from 1 = very good to 6 = very bad/insufficient. RESULTS: Overall, all cards received very good/good marks. The ABO blood group was identified correctly in all cases. Three cards (no. 1, no. 3, no. 4) received statistically significant (p < 0.008) prominence (mean values shown) concerning clearness of agglutination (1.7-1.9 vs. 2.4 for no. 2). Systems with dried antibodies (no. 2, no. 4) outmatched the other systems with respect to overall test system performance (2.0 vs. 2.8-2.9), labelling (1.5 vs. 2.2-2.4), handling (1.9-2.0 vs. 2.5), and user safety (2.5 vs. 3.4). Analysis of card self-explanation revealed no remarkable differences. CONCLUSION: Despite good performance of all card systems tested, the best results when including all criteria evaluated were obtained with card no. 4 (particularly concerning clear agglutination), followed by cards no. 2, no. 1, and no. 3.

Probability of success in the search for a related bone marrow donor in Cologne, Germany using HLA-A, -B and -DRB1 haplotype frequencies.

Between 2004 and 2013, 603 patients and their relatives (n = 1297) were typed as part of the search for a suitable HLA-matched donor in their nuclear and extended families at the central service provider for transfusion medicine at the University Hospital of Cologne. The high success rate in finding donors over the years at our center (38.1%) led us to examine our database retrospectively in order to evaluate the donor search and haplotype frequencies (HFs) in the sample. Our goal was to identify the factors contributing to this high success rate and also to compare the HFs we observed with other reported haplotype frequency estimations (HFE) for the Cologne area. Probability estimations for a successful donor search were constructed based on the HFEs for the sample.

Prospective evaluation of double RBC collection using three different apheresis systems.

BACKGROUND: Automated component collection systems offer the possibility of increasing blood supply and improving transfusion safety. DESIGN: 30 blood donors were randomly assigned to double RBC collection with either the Baxter Alyx (AX), the Haemonetics MCS Plus (MCS+), or the Gambro Trima Accel (TA). Procedures were prospectively evaluated focussing on yield, time, efficiency, citrate donor load, and in vitro quality. RESULTS: All units showed sufficient in vitro quality throughout 42 days of storage and complied with international requirements. Donor reactions were limited to mild citrate reactions. AX was the fastest and most efficient system* * (* *p approximately 0.001) attaining the highest yield* * from similar amounts of whole blood. The drawbacks were a higher RBC loss* (*p < 0.05) and accelerated citrate infusion* *. Due to lower collection rates* * * (* * *p < 0.001), MCS+ was slower than TA* * * but compensated with lower citrate load * * *. CONCLUSION: Double RBC apheresis was performed safely and efficiently with all three instruments. AX had advantages for most parameters evaluated.

Biochemical and molecular study of mentally retarded patient with partial deficiency of hypoxanthine-guanine phosphoribosyltransferase.

Nucleotide metabolism was studied in erythrocytes of a mentally retarded child and family members. Partial hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency was found in the propositus and an asymptomatic maternal uncle. Studies in crude lysates demonstrated decreased apparent V(max) and slightly decreased apparent K(m) for hypoxanthine in both HPRT-deficient subjects. Genomic DNA analysis revealed a single nucleotide change with leucine-147 to phenylalanine substitution in both subjects; mother and grandmother were heterozygous carriers of the same defect. This new variant has been termed HPRT(Potenza). Increased erythrocyte concentration of NAD and rate of synthesis by intact erythrocytes were found in the patient; increased activities of nicotinic acid phosphoribosyltransferase (NAPRT) and NAD synthetase (NADs) were demonstrated in erythrocyte lysates, with normal apparent K(m) for their substrates and increased V(max). These alterations were not found in any member of the family, including the HPRT-deficient uncle. These findings show multiple derangement of nucleotide metabolism associated with partial HPRT deficiency. The enzyme alteration was presumably not the cause of neurological impairment since no neurological symptoms were found in the HPRT-deficient uncle, whereas they were present in the propositus' elder brother who had normal HPRT activity.