Preparation Of Red Blood Cells Research Articles

The selection and preparation of red cell components for intrauterine transfusion: A national survey.

The practice regarding the selection and preparation of red blood cells (RBCs) for intrauterine transfusion (IUT) is variable reflecting historical practice and expert opinion rather than evidence-based recommendations. The aim of this survey was to assess Canadian hospital blood bank practice with respect to red cell IUT. A survey was sent to nine hospital laboratories known to perform red cell IUT. Questions regarding component selection, processing, foetal pre-transfusion testing, transfusion administration, documentation and traceability were assessed. The median annual number of IUTs performed in Canada was 109 (interquartile range, 103-118). RBC selection criteria included allogeneic, Cytomegalovirus seronegative, irradiated, fresh units with most sites preferentially providing HbS negative, group O, RhD negative, Kell negative and units lacking the corresponding maternal antibody without extended matching to the maternal phenotype. Red cell processing varied with respect to target haematocrit, use of saline reconstitution (n = 4), use of an automated procedure for red cell concentration (n = 1) and incorporation of a wash step (n = 2). Foetal pre-transfusion testing uniformly included haemoglobin measurement, but additional serologic testing varied. A variety of strategies were used to link the IUT event to the neonate post-delivery, including the creation of a unique foetal blood bank identifier at three sites. This survey reviews current practice and highlights the need for standardized national guidelines regarding the selection and preparation of RBCs for IUT. This study has prompted a re-examination of priorities for RBC selection for IUT and highlighted strategies for transfusion traceability in this unique setting.

Noninvasive hemoglobin monitoring for maintaining hemoglobin concentration within the target range during major noncardiac surgery: A randomized controlled trial

Study objectiveThe effect of noninvasive CO-oximetry hemoglobin (SpHb) monitoring on the clinical outcomes of patients undergoing surgery remains unclear. This trial aimed to evaluate whether SpHb monitoring helps maintain hemoglobin levels within a predefined target range during major noncardiac surgeries with a potential risk of intraoperative hemorrhage. DesignA single-center, prospective, randomized controlled trial. SettingUniversity hospital. PatientsOne hundred and thirty patients undergoing elective noncardiac surgery with a potential risk of hemorrhage. InterventionsPatients were randomly allocated to undergo either SpHb-guided management (SpHb group) or usual care (control group). MeasurementsThe primary outcome was the rate of deviation of the total hemoglobin concentration (determined from laboratory testing) from a pre-specified target range (8–14 g/dL). This was defined as the number of laboratory tests revealing such deviations divided by the total number of laboratory tests performed during the surgery. Main resultsThe primary outcome occurred significantly less frequently in the SpHb group as compared to that in the control group (15/555 [2.7%]) vs. 68/598 [11.4%]; relative risk, 0.24; 95% confidence interval, 0.13–0.41; P < 0.001). Fewer point-of-care blood tests were performed in the SpHb group than in the control group (median [interquartile range], 2 [1–4] vs. 4 [2–5]; P < 0.001). There were no significant intergroup differences in the number of patients who received red blood cell transfusions during surgery (SpHb vs. control, 33.8% vs. 46.2%; P = 0.201). The incidence of unnecessary red blood cell preparation (>2 units) was lower in the SpHb group than in the control group (3.1% vs. 16.9%; P = 0.024). ConclusionsCompared with routine care, SpHb-guided management resulted in significantly lower rates of hemoglobin deviation outside the target range intraoperatively in patients undergoing major noncardiac surgeries with a potential risk of hemorrhage. Clinical trial registrationClinicalTrials.gov (identifier: NCT03816514).

Stepwise options for preparing therapeutic plasma proteins from domestic plasma in low- and middle-income countries.

Industrial plasma fractionation, a complex and highly regulated technology, remains largely inaccessible to many low- and middle-income countries (LMICs). This, combined with the limited availability and high cost of plasma-derived medicinal products (PDMPs), creates deficiency of access to adequate treatment for patients in resource-limited countries, and leads to their suffering. Meanwhile, an increasing number of LMICs produce surplus plasma, as a by-product of red blood cell preparation from whole blood, that is discarded because of the lack of suitability for fractionation. This article reviews pragmatic technological options for processing plasma collected from LMICs into therapies and supports a realistic stepwise approach aligned with recent World Health Organization guidance and initiatives launched by the Working Party for Global Blood Safety of the International Society of Blood Transfusion. When industrial options based on contract or toll plasma fractionation programme and, even more, domestic fractionation facilities require larger volumes of quality plasma than is produced, alternative methods should be considered. In-bag minipool or small-scale production procedures implementable in blood establishments or national service centres are the only realistic options available to gradually reduce plasma wastage, provide safer treatments for patients currently treated with non-pathogen-reduced blood products and concurrently improve Good Manufacturing Practice (GMP) levels with minimum capital investment. As a next step, when the available volume of quality-assured plasma reaches the necessary thresholds, LMICs could consider engaging with an established fractionator in a fractionation agreement or a contract in support of a domestic fractionation facility to improve the domestic PDMP supply and patients' treatment.

Associations of Perioperative Red Blood Cell Transfusion With Outcomes of Kidney Transplantation in Korea Over a 16-Year Period.

This study investigated the associations between transfusion of different types of red blood cell (RBC) preparations and kidney allograft outcomes after kidney transplantation (KT) over a 16-year period in Korea using a nationwide population-based cohort. We investigated the reported use of RBCs during hospitalization for KT surgery, rejection, and graft failure status using nationwide data from the National Health Information Database (2002-2017). The associations between the type of perioperative RBC product and transplant outcomes were evaluated among four predefined groups: no RBC transfusion, filtered RBCs, washed RBCs, and packed RBCs (pRBCs). A total of 17,754 KT patients was included, among which 8,530 (48.0%) received some type of RBC transfusion. Of the patients who received RBC transfusion, 74.9%, 19.7%, and 5.4% received filtered RBCs, pRBCs, or washed RBCs, respectively. Regardless of the type of RBC products, the proportions of acute rejection and graft failure was significantly greater in patients receiving transfusion (P < 0.001). Cox proportional hazards regression analyses showed that the filtered RBC and pRBC groups were significantly associated with both rejection and graft failure. The washed RBC group also had hazard ratios greater than 1.0 for rejection and graft failure, but the association was not significant. Rejection-free survival of the pRBC group was significantly lower than that of the other groups (P < 0.001, log-rank test), and graft survival for the no RBC transfusion group was significantly greater than in the other groups (P < 0.001, log-rank test). Perioperative RBC transfusion was associated with poor graft outcomes. Notably, transfusion of pRBCs significantly increased transplant rejection. Therefore, careful consideration of indications for RBC transfusion and selection of the appropriate type of RBCs is necessary, especially for patients at high risk of rejection or graft failure.

Abnormal uterine bleeding in adolescence - three cases of heavy menstrual bleeding (HMB)

Heavy menstrual bleeding (HMB) is profuse, acyclic bleeding from the genital tract, with or without clots, usually lasting more than 10 days. They are the most common cause of abnormal uterine bleeding in adolescence. The following are used in the treatment: iron preparations, antifibrinolytic drugs, NSAIDs, estrogen-progestagen therapy or progestagen therapy. In cases where the Hb concentration is &lt;7-8 g/dl, red blood cells preparation should be transfused. In the article we present a description of three cases of patients with heavy menstrual bleeding (HMB) who were treated in the Pediatric Department of the Independent Public Provincial Specialist Hospital in Chełm.

Feasibility of a mean platelet volume standard: an international council for standardization in hematology (ICSH) inter-laboratory study

We have evaluated a commercial-fixed porcine platelet preparation (with and without added fixed human red blood cells (RBC)) for the potential standardization of mean platelet volume (MPV) measurements. The standards (Biotechne) were distributed internationally to 19 laboratories including all major hematology instrument manufacturers and academic/pathology laboratories. Overall, the standards demonstrated excellent stability up to 1 month within both MPV values and platelet counts when stored at 4°C. The presence of RBC significantly increased the platelet count and MPV values compared to platelets alone. However, as expected, there were differences in MPV values between different instruments and manufacturers. MPV values were also significantly higher in the whole blood standard compared to the platelet standard in the majority of instruments except with some instruments, where MPV values were significantly higher in the platelet only preparation. To further investigate this phenomenon, two different Platelet MPV preparations (with low and high MPV) in combination with 3 different RBC MCV preparations (with low, normal or high MCVs) were tested to try and further elucidate how RBC populations may impact upon platelet analysis (count, MPV, and PDW) using a single impedance analyzer. Both MPV and MCV values showed good stability over the course of the study for up to 50 days. As expected, the RBC preparation with the lowest MCV had the greatest impact on the MPV. However, this was not observed with an increase in MCV of the RBC or by a larger MPV of the platelet population. To further understand how different gating strategies may also influence results, we investigated the effect of either fixed or floating gate strategies upon MPV raw data from patient samples in a single impedance analyzer. Overall, it was clear that floating and fixed gate strategies also significantly impact upon MPV values. In conclusion, we have demonstrated the potential of an MPV standard with good stability characteristics for calibrating and comparing full blood counters that use different analysis principles, gating and MPV calculations. This may facilitate future instrument calibration and harmonization of results between different technologies.

Human and rodent red blood cells do not demonstrate xanthine oxidase activity or XO-catalyzed nitrite reduction to NO

A number of molybdopterin enzymes, including xanthine oxidoreductase (XOR), aldehyde oxidase (AO), sulfite oxidase (SO), and mitochondrial amidoxime reducing component (mARC), have been identified as nitrate and nitrite reductases. Of these enzymes, XOR has been the most extensively studied and reported to be a substantive source of nitric oxide (NO) under inflammatory/hypoxic conditions that limit the catalytic activity of the canonical NOS pathway. It has also been postulated that XOR nitrite reductase activity extends to red blood cell (RBCs) membranes where it has been immunohistochemically identified. These findings, when combined with countervailing reports of XOR activity in RBCs, incentivized our current study to critically evaluate XOR protein abundance/enzymatic activity in/on RBCs from human, mouse, and rat sources. Using various protein concentrations of RBC homogenates for both human and rodent samples, neither XOR protein nor enzymatic activity (xanthine → uric acid) was detectable. In addition, potential loading of RBC-associated glycosaminoglycans (GAGs) by exposing RBC preparations to purified XO before washing did not solicit detectable enzymatic activity (xanthine → uric acid) or alter NO generation profiles. To ensure these observations extended to absence of XOR-mediated contributions to overall RBC-associated nitrite reduction, we examined the nitrite reductase activity of washed and lysed RBC preparations via enhanced chemiluminescence in the presence or absence of the XOR-specific inhibitor febuxostat (Uloric®). Neither addition of inhibitor nor the presence of the XOR substrate xanthine significantly altered the rates of nitrite reduction to NO by RBC preparations from either human or rodent sources confirming the absence of XO enzymatic activity. Furthermore, examination of the influence of the age (young cells vs. old cells) of human RBCs on XO activity also failed to demonstrate detectable XO protein. Combined, these data suggest: 1) that XO does not contribute to nitrite reduction in/on human and rodent erythrocytes, 2) care should be taken to validate immuno-detectable XO by demonstrating enzymatic activity, and 3) XO does not associate with human erythrocytic glycosaminoglycans or participate in nonspecific binding.

Liposome-encapsulated methemoglobin as an antidote against cyanide poisoning

Cyanide induces acute lethal poisoning resulting from inhibition of cytochrome c oxidase located in the complex IV (Complex IV) of mitochondria. However, current therapies for cyanide poisoning using hydroxocobalamin and nitrous acid compounds remain a clinical issue. Here, we show that liposome-encapsulated methemoglobin (metHb@Lipo), nanosized biomimetic red blood cells, replicate the antidotal mechanism of nitrous acid compounds against cyanide poisoning, achieving superior efficacy and fast action with no adverse effects. The structure of metHb@Lipo, which consists of concentrated methemoglobin in its aqueous core and a lipid membrane resembling the red blood cell membrane, provides favorable characteristics as a cyanide antidote, such as binding properties and membrane permeability. Upon cyanide exposure, metHb@Lipo maintained the mitochondrial function in PC12 cells, resulting in a cell viability comparable to treatment with nitrous acid compounds. In a mouse model of cyanide poisoning, metHb@Lipo treatment dramatically improved mortality with a rapid recovery from the symptoms of cyanide poisoning compared to treatment with nitrous acid compounds. Furthermore, metHb@Lipo also possesses satisfactory pharmacokinetic properties without long-term bioaccumulation and toxicity. Our findings showed a novel concept to develop drugs for cyanide poisoning and provide a promising possibility for biomimetic red blood cell preparations for pharmaceutical applications.

S-nitrosothiols, and other products of nitrate metabolism, are increased in multiple human blood compartments following ingestion of beetroot juice

Ingested inorganic nitrate (NO3⁻) has multiple effects in the human body including vasodilation, inhibition of platelet aggregation, and improved skeletal muscle function. The functional effects of oral NO3⁻ involve the in vivo reduction of NO3⁻ to nitrite (NO2⁻) and thence to nitric oxide (NO). However, the potential involvement of S-nitrosothiol (RSNO) formation is unclear. We hypothesised that the RSNO concentration ([RSNO]) in red blood cells (RBCs) and plasma is increased by NO3⁻-rich beetroot juice ingestion. In healthy human volunteers, we tested the effect of dietary supplementation with NO3⁻-rich beetroot juice (BR) or NO3⁻-depleted beetroot juice (placebo; PL) on [RSNO], [NO3⁻] and [NO2⁻] in RBCs, whole blood and plasma, as measured by ozone-based chemiluminescence. The median basal [RSNO] in plasma samples (n = 22) was 10 (5–13) nM (interquartile range in brackets). In comparison, the median values for basal [RSNO] in the corresponding RBC preparations (n = 19) and whole blood samples (n = 19) were higher (p < 0.001) than in plasma, being 40 (30–60) nM and 35 (25–80) nM, respectively. The median RBC [RSNO] in a separate cohort of healthy subjects (n = 5) was increased to 110 (93–125) nM after ingesting BR (12.8 mmol NO3⁻) compared to a corresponding baseline value of 25 (21–31) nM (Mann-Whitney test, p < 0.01). The median plasma [RSNO] in another cohort of healthy subjects (n = 14) was increased almost ten-fold to 104 (58–151) nM after BR supplementation (7 × 6.4 mmol of NO3⁻ over two days, p < 0.01) compared to PL. In conclusion, RBC and plasma [RSNO] are increased by BR ingestion. In addition to NO2⁻, RSNO may be involved in dietary NO3⁻ metabolism/actions.

Process mapping of the urgent red cell exchange procedure for patients with severe complications of sickle cell disease at a centralized hemapheresis service.

Sickle cell disease (SCD) patients require urgent red cell exchange (RCE) procedures for acute chest syndrome (ACS), demanding a coordinated effort of multiple clinical services. Execution of RCE is a multistep process from the time the procedure is requested to the time the procedure is initiated. A retrospective review of patients with SCD requiring urgent RCE for ACS and stroke from 2012 to 2017 was performed at a centralized hemapheresis service covering a multihospital healthcare system. A total of 30 urgent RCE procedures performed on 28 patients were evaluated. The time required for red blood cell (RBC) preparation was the longest step in the process (median 3.8 hours). Furthermore, RBC preparation time was longer for sickle cell patients with RBC alloimmunization compared with nonalloimmunized patients (8.6 vs 3.8 hours, P = .03). One mortality event occurred in Ab- group. This study identified potentially modifiable factors, which impact the time to implementation of RCE in one service area. It highlights the importance of a structured and coordinated approach for the efficient and timely delivery of this vital treatment modality.

Preparation of Glutaraldehyde-Treated Red Blood Cells Expressing Weak ABO and RhD Antigens for Use in Tube Methods

Preparation of Glutaraldehyde-Treated Red Blood Cells Expressing Weak ABO and RhD Antigens for Use in Tube Methods

Evaluation of cytochrome P450-based drug metabolism in hemorrhagic shock rats that were transfused with native and an artificial red blood cell preparation, Hemoglobin-vesicles

Hemoglobin-vesicles (Hb-V) are being developed as red blood cell (RBC) substitutes. In this study, we report on quantitative and qualitative alterations of hepatic cytochrome P450 (CYPs) and the pharmacokinetics of CYP-metabolizing drugs, with a focus on four CYP isoforms (CYP1A2, CYP2C11, CYP2E1 and CYP3A2), after Hb-V resuscitation from a massive hemorrhage. The results of proteome analysis and western blot data indicate that resuscitation with both Hb-V and packed RBC (PRBC) resulted in a decrease in the protein levels of CYPs. Along with a decrease in the protein expression of CYPs, pharmacokinetic studies showed that the elimination of CYP-metabolizing drugs was prolonged in the Hb-V and PRBC resuscitation groups. It is also noteworthy that the CYP-metabolizing drugs in the Hb-V resuscitation group was retained for a longer period compared to the PRBC resuscitation group, and this is attributed to the CYP isoforms having a lower metabolic activity in the Hb-V resuscitation group than that for the PRBC resuscitation group. These findings suggest that resuscitation with Hb-V after a massive hemorrhage has a slight but not clinically significant effect on drug metabolism via CYPs in the liver due to decreased protein levels and the metabolic activity with respect to the CYPs.

Assessing cytochrome P450-based drug-drug interactions with hemoglobin-vesicles, an artificial red blood cell preparation, in healthy rats

Hemoglobin-vesicles (Hb-V), hemoglobin encapsulated within a liposome, were developed as an artificial red blood cell (RBC). When Hb-V becomes clinically available in the future, patients would presumably be co-administered with one or more drugs. Since drug-drug interactions can cause serious adverse effects and impede overall curative effects, evidence regarding the risk associated with drug-drug interactions between Hb-V and such simultaneously administered drugs is needed. Therefore, we report on cytochrome P450 (CYP)-based drug interactions with Hb-V in healthy rats. At 1 day after the saline, Hb-V or packed RBC (PRBC) administration, the blood retention of CYP-metabolizing drugs (caffeine, chlorzoxazone, tolbutamide and midazolam) were moderately prolonged in the case of the Hb-V group, but not the PRBC group, compared to saline group. The results of a proteome analysis revealed that the Hb-V administration had only negligible effects on the protein expression of CYPs in the liver. Hb-V administration, however, clearly suppressed the CYP metabolic activity of the four target CYP isoforms compared with the saline and PRBC group. However, these alterations were nearly recovered at 7 day after the Hb-V administration. Taken together, these results suggest that the administration of Hb-V slightly and transiently affects the CYP-based metabolism of the above drugs.

Blood Group Antigen Shielding Facilitated by Selective Cell Surface Engineering.

Production of red blood cells (RBCs) without immunogenicity of blood group antigens is of special interest in blood transfusion therapy in clinical chemistry. In this study, a selective cell surface engineering method was developed for the preparation of antigen-shielded RBCs based on molecular imprinting. Using an epitope imprinting method, biocompatible molecularly imprinted nanogels (MIgels) were prepared with a high affinity to the blood group antigens of RBCs. The antigen-shielded RBCs could avoid the agglutination caused by blood group mismatch, resulting in the antigen-shielded RBCs in efficiently substituting RBCs in case of a shortage of blood supply. Moreover, the antigen-shielded RBCs could maintain the normal physiological structure and functions of the original RBCs. We believe that the selective cell surface engineering presented in this work may offer significant benefits in specific cell protection for biomedical application.

Intrauterine transfusion: an international review of practice and policy

BackgroundIntrauterine transfusion (IUT) is a treatment for fetal anemia. This technique involves ultrasound guided needle placement followed by transfusion of a specially prepared blood component. The most common indications include hemolytic disease of the fetus and newborn (HDFN) and prenatal infection.MethodsThe literature was reviewed, and blood component characteristics and preparatory techniques identified. An international survey of IUT was conducted.ResultsComponent preparation for IUT differs from preparation for other transfusions. Differences relate to unique characteristics of the fetus including a small blood volume, invasive access for transfusion and immune deficiency. The techniques for preparation of red blood cells (RBC) are variable with limited evidence. Most use fresh concentrated RBC. Irradiation is uniformly recommended. RBC that are antigen negative for the maternal alloantibody are provided, and the product is selected or modified to minimize risk for cytomegalovirus transmission. Selection of ABO group and the degree of additional prophylactic phenotype matching are variable. While maternal RBC transfusion may be used, compatible allogeneic RBC are more common. For maternal antibodies to high prevalence antigens rare phenotype deglycerolized RBC may be used. There are challenges in providing RBC for IUT. Pre and post transfusion blood samples must be correctly managed in the medical record. Hemovigilance requires that both the fetus and the mother be documented as recipients. Preparation may be urgent, and sterile technique is required. Records of IUT must be conveyed to neonatal care providers as the transfused blood may mask the true blood group for weeks to months post transfusion, and component modifications may be required for any postnatal transfusion in neonates who were treated with IUT.

Preparation of packed red blood cell units in the blood bank: Alteration in red blood cell deformability

BackgroundDonated blood is stored in the blood bank as packed red blood cell units. In the process of packed cells preparation, the red blood cells (RBCs) are subjectedto high level of shear stress, which can induce alterations in their properties.In the present study, we examined the effect of packed RBCs preparation (which included leuko-filtration) on red cell deformability. MethodsBlood samples were collected from 25 healthy donors and from corresponding units of packed RBCs. The portion of undeformable cells (%UDFC) was determined for each sample. ResultsThe median value of %UDFC was equal to 6.75 ± 0.70 %, for freshly-donated RBCs, and to 6.36 ± 0.51 %, for packed cells. Wherein, %UDFC may increase or decrease following packed cells preparation, depending upon the initial portion of undeformable cells. ConclusionLikely, exposure of RBCs to high shear stress, during packed cells preparation, induces opposing effects: (a) removal/destruction of rigid (undeformable) cells, thereby reducing their total amount (i.e., decreasing the %UDFC) on the one hand, and (b) mechanical damage to the cell membrane and subsequent reduction of the cell deformability (thereby increasing the %UDFC) on the other. As a consequence, the final impact of packed cells preparation is primarily determined by the initial state of erythrocytes in the blood of the donor.

Neonatal red blood cell transfusion practices in Switzerland: national survey and review of international recommendations.

The aim of the study was to analyse Swiss practices in blood transfusion services and neonatal units for the preparation and administration of red blood cells in newborns. Two questionnaires were developed and their results analysed. A first questionnaire was developed for the Swiss blood transfusion services and local hospital haematology laboratories, and a second for the neonatal units in Switzerland. 18/25 (72%) of laboratories and 26/29 (90%) of neonatal units performing red blood cell transfusions participated. Responses revealed a lack of consensus for the majority of questions. Differences were found in all steps of the process, from preparation (testing, irradiation, splitting, shelf life and storage of blood bags) to indication and administration (volume, speed, vascular access, patient monitoring) of red blood cells. Forty-six percent of neonatal units stated that they apply internal guidelines. Nevertheless, all but two would welcome the establishment of national recommendations. This study confirmed the large variety in neonatal red blood cell transfusion practices in Switzerland. In the absence of clear evidence, national guidelines &ndash; as applied in other countries &ndash; would foster a common policy among Swiss neonatologists and facilitate the implementation of a national database for future research and comparison with international literature.

Electron paramagnetic resonance oximetry as a novel approach to monitor the effectiveness and quality of red blood cell transfusions.

The goal of red blood cell transfusion is to improve tissue oxygenation. Assessment of red blood cell quality and individualised therapeutic needs can be optimised using direct oxygen (O2) measurements to guide treatment. Electron paramagnetic resonance oximetry is capable of accurate, repeatable and minimally invasive measurements of tissue pO2. Here we present preclinical proof-of-concept of the utility of electron paramagnetic resonance oximetry in an experimental setting of acute blood loss, transfusion, and post-transfusion monitoring. Donor rat blood was collected, leucocyte-reduced, and stored at 4 °C in AS-3 for 1, 7 and 14 days. Red blood cell morphology, O2 equilibrium, p50 and Hill numbers from O2 binding and dissociation curves were evaluated in vitro. Recipient rats were bled and maintained at a mean arterial pressure of 30-40 mmHg and hind limb muscle (biceps femoris) pO2 at 25-50% of baseline. Muscle pO2 was monitored continuously over the course of experiments to assess the effectiveness of red blood cell preparations at different stages of blood loss and restoration. Red blood cell morphology, O2 equilibrium and p50 values of intra-erythrocyte haemoglobin were significantly altered by refrigerated storage for both 7 and 14 days. Transfusion of red blood cells stored for 7 or 14 days demonstrated an equivalently impaired ability to restore hind limb muscle pO2, consistent with in vitro observations and transfusion with albumin. Red blood cells refrigerated for 1 day demonstrated normal morphology, in vitro oxygenation and in vivo restoration of tissue pO2. Electron paramagnetic resonance oximetry represents a useful approach to assessing the quality of red blood cells and subsequent transfusion effectiveness.

Relationship of Iron Deposition to Calcium Deposition in Human Aortic Valve Leaflets

BackgroundIntraleaflet hematomas are associated with advanced stages of aortic valve calcification and suspected to be involved in disease progression. However, the mechanism by which the entry of blood cells into the valves affects the biology of aortic valvular interstitial cells (VICs) remains to be elucidated. ObjectivesThis study sought to evaluate the putative link between intraleaflet hematoma and aortic valve calcification and to assess its pathophysiological implications. MethodsThe spatial relationship between calcium deposits and intraleaflet hematomas was analyzed by whole-mount staining of calcified and noncalcified human aortic valves, obtained in the context of heart transplantation and from patients who underwent surgical valve replacement. Endothelial microfissuring was evaluated by en face immunofluorescence and scanning electron microscopic analyses of the fibrosa surface. Red blood cell (RBC) preparations were used in vitro to assess, by immunofluorescence microscopy and Alizarin red staining, the potential impact of intraleaflet hematomas on phenotypic changes in VICs. ResultsIntraleaflet hematomas, revealed by iron deposits and RBCs into the fibrosa, secondary to endothelial microfissuring, were consistently found in noncalcified valves. The contact of primary VICs derived from these valves with RBCs resulted in a global inflammatory and osteoblastic phenotype, reflected by the up-regulation of interleukin-6, interleukin-1β, bone sialoprotein, osteoprotegerin, receptor activator of nuclear factor kappa B, bone morphogenic protein 2, and muscle segment homeobox 2, the production of osteocalcin, and the formation of calcium deposits. ConclusionsThe acquisition of an osteoblastic phenotype in VICs that come into contact with the senescent RBCs of intraleaflet hematomas may play a critical role in the initiation of calcium deposition into the fibrosa of human aortic valves.

Blood penetration into human aortic valves consecutive to endothelial micro-fissuring of the fibrosa determines the site of calcium deposition: Physiopathological implications

Intraleaflet hematoma has been associated with advanced stages of aortic valve stenosis. Its presence is suspected to be involved in faster disease progression. However, the mechanism whereby the entry and the presence of blood elements into the valves would have an impact on the biology of aortic valvular interstitial cells (VICs) remains to be deciphered. To evaluate the presence of intraleaflet hematoma, in respect to calcium deposition, at all stages of aortic valve calcification (AC) and to assess its putative pathophysiologic implication. The spatial relationship between calcium deposits and intraleaflet hematomas was analyzed by whole-mount staining of calcified and non-calcified human aortic valves, obtained in the context of heart transplantation and from patients who underwent surgical valve replacement. Endothelial micro-fissuring was evaluated by en face immunofluorescence and scanning electron microscope analyses of the fibrosa surface. Red blood cell preparations (RBCs) were used in vitro in order to assess, by immunofluorescence microscopy and Alizarin red staining, the potential impact of intraleaflet hematomas on the phenotype change of human VICs. Intraleaflet hematomas revealed by ferric iron deposits and extensive penetration of RBCs into the fibrosa, secondary to endothelial micro-fissuring, were found both in calcified and non-calcified valves. Primary VICs derived from non-calcified valves and cultured in the presence of senescent RBCs acquired an osteoblastic phenotype, as demonstrated by the production of osteocalcin and the formation of calcium deposits. Intraleaflet hematomas occurring through endothelial micro-fissuring may play a critical role in the initiation of calcium deposition into human aortic valves by driving the acquisition of an osteoblastic phenotype by the VICs that enter in contact with the infiltrated RBCs.