Thermometry of red blood cell concentrate: magnetic resonance decoding warm up process.

PURPOSE: Temperature is a key measure in human red blood cell concentrate (RBC) quality control. A precise description of transient temperature distributions in RBC units removed from steady storage exposed to ambient temperature is at present unknown. Magnetic resonance thermometry was employed to visualize and analyse RBC warm up processes, to describe time courses of RBC mean, surface and core temperatures by an analytical model, and to determine and investigate corresponding model parameters. METHODS: Warm-up processes of 47 RBC units stored at 1-6°C and exposed to 21.25°C ambient temperature were investigated by proton resonance frequency thermometry. Temperature distributions were visualized and analysed with dedicated software allowing derivation of RBC mean, surface and core temperature-time courses during warm up. Time-dependence of mean temperature was assumed to fulfil a lumped capacitive model of heat transfer. Time courses of relative surface and core temperature changes to ambient temperature were similarly assumed to follow shifted exponential decays characterized by a time constant and a relative time shift, respectively. RESULTS: The lumped capacitive model of heat transfer and shifted exponential decays described time-dependence of mean, surface and core temperatures close to perfect (mean R(2) were 0.999±0.001, 0.996±0.004 and 0.998±0.002, respectively). Mean time constants were τmean = 55.3±3.7 min, τsurface = 41.4±2.9 min and τcore = 76.8±7.1 min, mean relative time shifts were Δsurface = 0.07±0.02 and Δcore = 0.04±0.01. None of the constants correlated significantly with temperature differences between ambient and storage temperature. CONCLUSION: Lumped capacitive model of heat transfer and shifted exponential decays represent simple analytical formulas to describe transient mean, surface and core temperatures of RBC during warm up, which might be a helpful tool in RBC temperature monitoring and quality control. Independence of constants on differences between ambient and storage temperature suggests validity of models for arbitrary storage and ambient temperatures.

Four-dimensional temperature distributions in red blood cells withdrawn from storage and exposed to ambient temperature: a magnetic resonance thermometry study.

BACKGROUND: Recommended by current guidelines, red blood cell (RBC) temperature should not exceed 10°C during transport. Since warming is a generically three-dimensional process that is not homogeneous, it is necessary to clarify the term "temperature during warming." The purpose of this study was therefore to investigate laws and relations between surface, mean, and core temperature and the corresponding times when they exceed 10°C during warm-up. STUDY DESIGN AND METHODS: Time-resolved three-dimensional temperature distributions of 53 resuspended RBC units (mean volume, 253 ± 17 mL) were measured noninvasively by magnetic resonance thermometry. Warm-up temperature maps were visualized and analyzed by dedicated software. RESULTS: Mean times when surface, mean, and core temperature exceeded 10°C were 16 ± 4, 24 ± 5, and 36 ± 7 minutes, respectively. Times strongly correlated with each other (r = 0.78-0.95) and their variances mainly depended on RBC storage temperature and RBC pouch width (R(2) = 0.81-0.89). Measured mean temperature time courses were well described by a lumped capacitive model of heat transfer with a sample width-dependent time constant τ(RBC) = 56.3 ± 3.5 minutes (mean R(2) = 0.996). CONCLUSION: Times when RBC surface, mean, and core temperature exceed 10°C can be estimated from each other. Moreover RBC mean temperature can be calculated for arbitrary storage and ambient temperatures. Findings might serve as a helpful tool in RBC temperature monitoring.

Thrombin receptor levels in platelet concentrates during storage and their impact on platelet functionality.

BACKGROUND: Quality control of platelet (PLT) concentrates is challenging, due to PLT lesions, which are difficult to detect with routine methods. The search for reliable PLT lesion biomarkers is focused on the role of PLTs in primary hemostasis. PLT transfusions also have a significant impact on secondary hemostasis. In this phase, responsiveness of PLTs to small amounts of thrombin is crucial. PAR1 and PAR4 are protease-activated receptors and are responsible for thrombin reactivity of human PLTs. This study should elucidate if levels of those two receptors are changing in PLT concentrates during storage and if those changes have an impact on PLT aggregation and support of thrombin generation. STUDY DESIGN AND METHODS: PLT concentrates from buffy coat preparations were stored in SSP+ solution for 9 days at 22±2°C on a horizontal flatbed agitator, and samples were taken daily for analysis. PAR1 and PAR4 levels were evaluated using Western blot analysis. PLT aggregation was measured using Born aggregometry and specific PAR1 or PAR4 agonists. Thrombin generation was measured using calibrated automated thrombography. RESULTS: Levels of both receptors (PAR1 and PAR4) started to decrease after 5 days of storage. PAR1-mediated PLT aggregation remained constant, whereas PAR4-mediated PLT aggregation decreased with storage time. Rate of thrombin generation was accelerated after 5 days of storage. CONCLUSION: Decreasing levels of PARs in PLT concentrates after 5 days of storage influenced PAR4-mediated, but not PAR1-mediated, aggregation. Thrombin generation with senescent PLTs was increased, which may be attributed to other mechanisms promoting increased phosphatidylserine exposure.

Impact of 13.56-MHz radiofrequency identification systems on the quality of stored red blood cells.

BACKGROUND: Radiofrequency identification (RFID) technology is emerging as one of the most pervasive computing technologies due to its broad applicability. Storage of red blood cells (RBCs) is a routine procedure worldwide. Depending on the additive solution, RBCs can be stored at 4 ± 2°C up to 49 days. To support the decision of discarding or further using a blood product, temperature measurement of each unit could be provided by RFID application. The safety evaluation of RFID devices was demonstrated in a regulatory agency required study. It has been concluded in limit tests that high frequency-based RFID technology performed safely for blood products; therefore, a longer exposure of radiofrequency (RF) energy on blood units was performed in this study to detect any biologic effects in RBC samples. STUDY DESIGN AND METHODS: Buffy coat-depleted, in line-filtered RBCs were used as standard products in all tests. Various variables like pH, potassium, glucose, lactate, hemoglobin (Hb), hematocrit, free Hb, and hemolysis rate were measured in a test group with RFID tags placed on their surface and continuously radiated with 13.56-MHz RFID reader radiation for 42 days while stored at 4 ± 2°C and compared to a control group by two-sample t test. RESULTS: In both groups glucose and pH levels decreased while lactate, free Hb, and potassium increased within the expected levels. The hemolysis rate showed increase after the 25th day but remained below the maximum acceptable threshold of 0.8%. CONCLUSION: It is feasible to implement RFID-enabled processes, without detecting any known biologic effects of longer exposure of RF energy on the quality of RBCs.

CD160: a unique activating NK cell receptor.

Here we discuss CD160 an essential NK cell activating receptor that remains poorly understood. CD160 receptor exhibits a number of unique structural and functional characteristics that are not common to other killer immunoglobulin-like receptors that recognize major histocompatibility complex (MHC) class I molecules: (1) In addition to humans and mice, the cd160 gene is conserved in several other mammal species; (2) cd160 is located outside the NK gene complex and the Leukocyte Receptor Complex in humans; (3) CD160 expression is associated to the CD56(dim) CD16+ cytotoxic NK cell phenotype; (4) both human and mouse CD160 recognize MHC class Ia and Ib molecules; (5) unlike the other MHC class I-dependent activating NK receptors, CD160 is a glycosylphosphatidylinositol-anchored molecule with a single immunoglobulin-like domain, and does not bear immunoreceptor tyrosine-based activation motifs. Consequently, CD160 cannot signal by itself, requiring the recruitment of adaptor proteins. CD160 recruits phosphoinositide-3 kinase to trigger cytotoxicity and cytokine secretion; (6) specific engagement of NK CD160 receptor expressed by circulating NK cells produces proinflammatory cytokines IFN-γ, TNF-α, and, most notably, IL-6 and IL-8 as well as MIP1-ß chemokine. The level of CD160-mediated IFN-γ production is always higher than the one observed after engagement of the CD16 receptor.

Soluble HLA-G in IVF/ICSI embryo culture supernatants does not always predict implantation success: a multicentre study.

Several reports have described an association between the presence of soluble human leukocyte antigen G (sHLA-G) in human embryo culture supernatants (ES) and implantation success. However, not all studies agree with these findings. To further document this debate, a multicentre blinded study was performed to investigate, on a large number of IVF ES and ICSI ES, whether sHLA-G is a useful criterion for embryo selection before transfer. A total of 1405 ES from 355 patients were collected from three assisted reproductive technique (ART) centres and evaluated for their sHLA-G content in a single laboratory, using a chemiluminescence enzyme-linked immunosorbent assay. In only one centre was a significant association between sHLA-G-positive ES and successful implantation established (P = 0.0379), whereas no such association was observed in the other centres. It was found that the percentages and concentrations of sHLA-G-positive ES varied between centres, depending on culture media and ART conditions. The percentage of sHLA-G-positive ES was significantly higher in IVF ES than ICSI ES (P < 0.001 and P < 0.01 for two centres). These data demonstrate that substantial variations of sHLA-G content in ES occur between different ART centres, highlighting the influence of several technical parameters that differ from one centre to another.

sHLA-G production by human IVF embryos: can it be measured reliably?

A number of reports have demonstrated that sHLA-G can be detected in the culture medium of human IVF embryos and that levels correlate with the potential of an embryo to implant. This has aroused considerable interest in the IVF field. If sHLA-G can be used as a non-invasive marker of embryo quality, it will facilitate selection of the best embryos to transfer to the mother and thereby increase IVF pregnancy rates. However, there have been concerns about some aspects of these studies, including the sensitivity of the sHLA-G ELISAs used, the IVF culture conditions and the levels of sHLA-G which have been reported. A recent study by Sageshima et al. [J. Reprod. Immunol. 75, 11-22, 2007] attempts to address some of these concerns. However, despite using a sensitive ELISA, they were unable to detect sHLA-G in 111 embryo culture supernatants, or sHLA-G secretion by less than 10,000 sHLA-G transfected cells. They concluded that it is not possible to measure sHLA-G production by human embryos. This study has highlighted technical differences between IVF culture techniques and sHLA-G ELISAs that are currently confounding the system. Further collaboration between the research groups involved is required to establish robust reproducible systems that function identically in all laboratories.

Progesterone-induced blocking factor activates STAT6 via binding to a novel IL-4 receptor.

Progesterone-induced blocking factor (PIBF) induces Th2-dominant cytokine production. Western blotting and EMSA revealed phosphorylation as well as nuclear translocation of STAT6 and inhibition of STAT4 phosphorylation in PIBF-treated cells. The silencing of STAT6 by small interfering RNA reduced the cytokine effects. Because the activation of the STAT6 pathway depends on the ligation of IL-4R, we tested the involvement of IL-4R in PIBF-induced STAT6 activation. Although PIBF does not bind to IL-4R, the blocking of the latter with an Ab abolished PIBF-induced STAT6 activation, whereas the blocking of the IL-13R had no effect. PIBF activated suppressor of cytokine signaling-3 and inhibited IL-12-induced suppressor of cytokine signaling-1 activation. The blocking of IL-4R counteracted all the described effects, suggesting that the PIBF receptor interacts with IL-4R alpha-chain, allowing PIBF to activate the STAT6 pathway. PIBF did not phosphorylate Jak3, suggesting that the gamma-chain is not needed for PIBF signaling. Confocal microscopic analysis revealed a colocalization and at 37 degrees C a cocapping of the FITC PIBF-activated PIBF receptor and PE anti-IL-4R-labeled IL-4R. After the digestion of the cells with phosphatidylinositol-specific phospholipase C, the STAT6-activating effect of PIBF was lost, whereas that of IL-4 remained unaltered. These data suggest the existence of a novel type of IL-4R composed of the IL-4R alpha-chain and the GPI-anchored PIBF receptor.

The progesterone-induced blocking factor modulates the balance of PKC and intracellular Ca.

PROBLEM: Progesterone-induced blocking factor (PIBF) induces Th2 biased cytokine production; therefore, this study investigates the effects of PIBF on the protein kinase C (PKC)/Ca(++) system - which plays a key role in Th1/Th2 differentiation. METHOD OF STUDY: Proteins from PIBF-treated cells were reacted on Western blots with phospho-specific antibodies recognizing different PKC izoforms. Intracellular free calcium was measured by flow cytometry. RESULTS: Both interleukin (IL)-4 and PIBF induced PKC phosphorylation, which was abrogated by anti-IL-4Ralpha or anti-PIBF immunoglobulin G pre-treatment. PIBF treatment did not alter intracellular Ca(++) levels. Inhibition of PKCzeta or PKCtheta phosphorylation, but not that of PKCalpha/beta resulted in the loss of STAT6 and Jak1 phosphorylation by PIBF. CONCLUSIONS: Our data show that PIBF phosphorylates PKC via binding to the IL-4R, without affecting intracellular Ca(++). Phosphorylation of PKCzeta and PKCtheta is required for Jak1 and STAT6 activation, whereas PKCalpha/beta is not involved. These findings explain the mechanism by which PIBF supports a Th2 dominant cytokine pattern.