Hemoglobin Binding to the Red Blood Cell (RBC) Membrane Is Associated with Decreased Cell Deformability.

The deformability of red blood cells (RBCs), expressing their ability to change their shape as a function of flow-induced shear stress, allows them to optimize oxygen delivery to the tissues and minimize their resistance to flow, especially in microcirculation. During physiological aging and blood storage, or under external stimulations, RBCs undergo metabolic and structural alterations, one of which is hemoglobin (Hb) redistribution between the cytosol and the membrane. Consequently, part of the Hb may attach to the cell membrane, and although this process is reversible, the increase in membrane-bound Hb (MBHb) can affect the cell's mechanical properties and deformability in particular. In the present study, we examined the correlation between the MBHb levels, determined by mass spectroscopy, and the cell deformability, determined by image analysis. Six hemoglobin subunits were found attached to the RBC membranes. The cell deformability was negatively correlated with the level of four subunits, with a highly significant inter-correlation between them. These data suggest that the decrease in RBC deformability results from Hb redistribution between the cytosol and the cell membrane and the respective Hb interaction with the cell membrane.

Red Blood Cell Deformability Is Expressed by a Set of Interrelated Membrane Proteins.

Red blood cell (RBC) deformability, expressing their ability to change their shape, allows them to minimize their resistance to flow and optimize oxygen delivery to the tissues. RBC with reduced deformability may lead to increased vascular resistance, capillary occlusion, and impaired perfusion and oxygen delivery. A reduction in deformability, as occurs during RBC physiological aging and under blood storage, is implicated in the pathophysiology of diverse conditions with circulatory disorders and anemias. The change in RBC deformability is associated with metabolic and structural alterations, mostly uncharacterized. To bridge this gap, we analyzed the membrane protein levels, using mass spectroscopy, of RBC with varying deformability determined by image analysis. In total, 752 membrane proteins were identified. However, deformability was positively correlated with the level of only fourteen proteins, with a highly significant inter-correlation between them. These proteins are involved in membrane rafting and/or the membrane-cytoskeleton linkage. These findings suggest that the reduction of deformability is a programmed (not arbitrary) process of remodeling and shedding of membrane fragments, possibly mirroring the formation of extracellular vesicles. The highly significant inter-correlation between the deformability-expressing proteins infers that the cell deformability can be assessed by determining the level of a few, possibly one, of them.

Post-operative anterior diaphragmatic hernias in children with Trisomy 21 after cardiac surgery.

Morgagni hernias account for less than 5% of congenital diaphragmatic hernias. They are characteristically retrosternal and bilateral, with right-sided predominance. An association between Trisomy 21 and diaphragmatic hernias resembling Morgagni hernia has been reported, but the effect of cardiac surgery on its formation has not been investigated. The purpose of this study was to determine whether there is a higher incidence of anterior diaphragmatic hernias in children with Trisomy 21 after cardiac surgery. We compared the prevalence of anterior diaphragmatic hernias in 92 patients with Trisomy 21 who underwent cardiac surgery with its prevalence in 100 children without Trisomy 21 who underwent cardiac surgery. All available CXRs of all children underwent revision for the presence of an anterior diaphragmatic hernia by a pediatric radiologist. Within the study group, four cases of an anterior diaphragmatic hernia were detected, all upon presentation to the emergency room due to breathing difficulties. No cases of an anterior diaphragmatic hernia were found in the control group (P = 0.0094). CONCLUSIONS: A high index of suspicion for an anterior diaphragmatic hernia should be maintained in children with Trisomy 21 who have undergone cardiac surgery and present with breathing difficulty. If CXR findings are uncertain, UGI series and\or CT should be performed. In light of our findings, the surgical technique has been modified in patients with DS in our medical center. WHAT IS KNOWN: • Several studies reported an association between Trisomy 21 and diaphragmatic hernia resembling Morgagni hernia, but the effect of cardiac surgery on its formation has not been investigated. WHAT IS NEW: • There is a higher incidence of anterior diaphragmatic hernia resembling a Morgagni hernia in children with Trisomy 21 after cardiac surgery. • A high index of suspicion for an anterior diaphragmatic hernia should be maintained in children with Trisomy 21 who have undergone cardiac surgery and present with breathing difficulty. If CXR findings are uncertain, UGI series and\or CT should be performed.

The inhibition of glucose uptake to erythrocytes: microwave dielectric response.

Dielectric spectroscopy has been used in the study and development of non-invasive glucose monitoring (NIGM) sensors, including the range of microwave frequencies. Dielectric relaxation of red blood cell (RBC) cytosolic water in the microwave frequency band has been shown to be sensitive to variations in the glucose concentration of RBC suspensions. It has been hypothesized that this sensitivity stems from the utilization of D-glucose by RBCs. To verify this proposition, RBCs were pretreated with inhibitors of D-glucose uptake (cytochalasin B and forskolin). Then their suspensions were exposed to different D-glucose concentrations as measured by microwave dielectric spectroscopy (MDS) in the 500 MHz-40 GHz frequency band. After incubation of RBCs with either inhibitor, the dielectric response of water in the cytoplasm, and specifically its relaxation time, demonstrated minimal sensitivity to the change of D-glucose concentration in the medium. This result allows us to conclude that the sensitivity of MDS to glucose uptake is associated with variations in the balance of bulk and bound RBC cytosolic water due to intracellular D-glucose metabolism, verifying the correctness of the initial hypothesis. These findings represent a further argument to establish the dielectric response of water as a marker of glucose variation in RBCs.

Bowel Resection Through a Single Umbilical Incision: A Case Series.

Background: The advent of laparoscopy has revolutionized surgery. The surgeon strives to minimize incisions and their adverse consequences. Although laparoscopy has gained widespread popularity, several advantages in open surgery are thereby lost. Tactile sensation of the tissue, hand-sewn anastomosis, and nonthermic vascular control are most prominent. To combine both approaches, single incision laparoscopic-assisted surgery (SILS) was advanced, trying to combine the best in both worlds. This approach is widely used in appendectomies. After having gained experience in this approach, we expanded the indications and hereby present our experience with bowel resections utilizing SILS. Patients and Methods: Data were collected retrospectively from operations performed during the past 3 years. We found 11 cases of SILS bowel resections: 3 sigmoidectomies, 3 small bowel atresia repairs, 1 subtotal colectomy, 1 Meckel's diverticulectomy, and 3 resections of bowel duplications. The age of the patients ranged from 2 days to 17 years. In all cases, a working 10 mm scope was inserted through the umbilicus, the bowel was extracted outside the abdomen through the umbilicus, dissection and resection with anastomosis were performed outside the abdomen in the classic open approach, and the bowel was returned to the abdomen. Results and Conclusions: All patients recovered promptly with no need for further intervention. There were no cases of wound infection, leak, or intra-abdominal abscess formation. Cosmesis was excellent with a small umbilical scar. We conclude that this approach is feasible and safe in a select population.

Deformability of cord blood vs. newborns' red blood cells: implication for blood transfusion.

AIM: About 50% of premature neonates (PN) are treated with transfusion of packed red blood cells (PRBC) collected from adult donors, which has been suggested to potentially provoke PN pathologies, characterized as blood circulation disorders. RBC have properties that are key determinants of blood circulation, primarily the cell deformability. In previous studies we have shown that transfusion of RBC with reduced deformability impaired the transfusion outcome. Although RBC of PN (PN-RBC) are larger, and their microvessels are narrower than those of adults, their blood circulation is very efficient, pointing to the possibility that the deformability of adults' PRBC is inferior to that of PN-RBC, and that treating PN with PRBC transfusion might, therefore, introduce a risk to the recipients. This would infer that PN should be given RBC with high deformability. However, since using PN-RBC is not feasible, the use of cord blood RBC (CB-RBC) is a sound alternative, assuming that the deformability of CB-RBC is comparable to that of PN-RBC.The present study is aimed at testing this hypothesis. METHODS: We compared the deformability of (1) RBC of PN vs. the PRBC they received, and (2) PN-RBC vs. their autologous CB-RBC. RESULTS: 1. The deformability of the transfused PRBC is indeed inferior to that of PN-RBC. 2. The deformability of CB-RBC is equivalent to that of PN-RBC. CONCLUSION: This study supports the notion that treating PN with transfusion of adults' PRBC has the potential to introduce a circulatory risk to the recipients, while CB-RBC, with their superior deformability, provides a safer and more effective PN-specific transfusion therapy.

Determination of red blood cell adhesion to vascular endothelial cells: A critical role for blood plasma.

Red blood cell (RBC) adhesion to vascular endothelial cells (EC) is considered a potent effector of circulatory disorders, and its enhancement is implicated in the pathophysiology of numerous conditions, mainly hemoglobinopathies. The actual RBC/EC interaction is determined by both cellular and plasmatic factors, and the differentiation between them is essential for understanding its physiological implications. Yet, RBC/EC adhesion has been studied predominantly in protein-free media. To explore the plasma contribution to RBC/EC adhesion, we examined the adhesion of human RBC to human vascular endothelial cells in the presence of fresh frozen plasma (FFP) and compared it to that in a protein-free phosphate-buffered saline (PBS). RBC from blood samples freshly-collected from five healthy donors and from fifteen units of packed RBC units were used. The same FFP sample was used in all measurements. In FFP, the RBC form strongly adherent aggregates, which are dispersed as the shear stress (τ) increases to 3.0 Pa, and even at 5.0 Pa a large portion of the RBC are still adherent. In PBS, the RBC are singly dispersed and their adhesion becomes insignificant already at τ = 0.5 Pa. No cross-correlation was found between the adhesion in PBS vs. that in FFP at the same τ. However, in both media, under conditions that form singly dispersed adherent RBC, an inverse correlation between RBC/EC adhesion in PBS vs. that in FFP was observed. This study clearly implies that for understanding the physiological relevance of RBC/EC adhesion it should be determined in plasma.

Do We Store Packed Red Blood Cells under "Quasi-Diabetic" Conditions?

Red blood cell (RBC) transfusion is one of the most common therapeutic procedures in modern medicine. Although frequently lifesaving, it often has deleterious side effects. RBC quality is one of the critical factors for transfusion efficacy and safety. The role of various factors in the cells' ability to maintain their functionality during storage is widely discussed in professional literature. Thus, the extra- and intracellular factors inducing an accelerated RBC aging need to be identified and therapeutically modified. Despite the extensively studied in vivo effect of chronic hyperglycemia on RBC hemodynamic and metabolic properties, as well as on their lifespan, only limited attention has been directed at the high sugar concentration in RBCs storage media, a possible cause of damage to red blood cells. This mini-review aims to compare the biophysical and biochemical changes observed in the red blood cells during cold storage and in patients with non-insulin-dependent diabetes mellitus (NIDDM). Given the well-described corresponding RBC alterations in NIDDM and during cold storage, we may regard the stored (especially long-stored) RBCs as "quasi-diabetic". Keeping in mind that these RBC modifications may be crucial for the initial steps of microvascular pathogenesis, suitable preventive care for the transfused patients should be considered. We hope that our hypothesis will stimulate targeted experimental research to establish a relationship between a high sugar concentration in a storage medium and a deterioration in cells' functional properties during storage.

Inter-donor variability in deformability of red blood cells in blood units.

OBJECTIVE: This study aimed to examine the donor-to-donor variability in the deformability of red blood cells (RBCs) from freshly collected blood donations (F-RBC) and packed RBCs. BACKGROUND: Packed RBCs are supplied for transfusion by the first-in-first-out (FIFO) criterion, assuming that their quality is the same for packed RBCs with equal storage duration. To challenge this notion, we determined the deformability of F-RBC and packed RBCs stored for different durations. METHODS: Three RBC groups were employed: A. 79 samples of F-RBC; B. 76 samples of packed RBC units, randomly used for transfusion at different storage durations; and C. 65 samples of outdated packed RBCs stored for 35 to 37 days. All packed RBC units were non-leukofiltrated and stored in Citrate-phosphate-dextrose solution with adenine (CPDA-1). RBC deformability was determined using a computerised cell-flow properties analyser, which monitors the shape change of cells directly visualised in a narrow-gap flow chamber and provides the cells' deformability distribution in a large RBC population. RESULTS: The F-RBC deformability exhibited a wide-range inter-donor variability. The cold storage of packed RBCs exerted a mild reduction of deformability, which became significant, compared to the initial inter-donor variability, only after 3 weeks of storage. CONCLUSION: Packed RBCs are generally supplied for transfusion by the FIFO criterion based on the assumption that the storage duration is a key factor of RBC quality. This study demonstrates that the deformability of red blood cells is significantly different in donors, and substantial variability persists throughout the entire process of their storage. Therefore, the FIFO criterion is not sufficient for assessing the RBC deformability, which should, therefore, be specifically characterised for each unit.

The dielectric spectroscopy of human red blood cells during 37-day storage: ß-dispersion parameterization.

This study exploits dielectric spectroscopy to monitor the kinetics of red blood cells (RBC) storage lesions, focusing on those processes linked to cellular membrane interface known as ß-dispersion. The dielectric response of RBC suspensions, exposed to blood-bank cold storage for 37 days, was studied using time-domain dielectric spectroscopy in the frequency range 500 kHz to 200 MHz. The measured dielectric processes are characterized by their dielectric strength (Δε) and their relaxation times (τ). Changes in the dielectric properties of the RBC suspensions, due to storage-related biophysical changes, were evaluated. For a quantitative characterization of RBC vitality, we characterized the shape of fresh and stored RBC and measured their deformability as expressed by their average elongation ratio, which was achieved under a shear stress of 3.0 Pa. During the second week of storage, an increment in the evolution of the relaxation times and in the dielectric permittivity strength of about 25% was observed. We propose that the characteristic increment of ATP, during the second and third weeks of storage, is responsible for the raise of the specific capacitance of cell membrane, which in turn explains the changes observed in the dielectric response when combined with the influence of the shape changes.

Unit-to-unit variability in the deformability of red blood cells.

BACKGROUND: In blood banking practice, the storage duration is used as the primary criterion for inventory management, and usually, the packed red blood cells (PRBC) units are supplied primarily according to first-in-first-out (FIFO) principle. However, the actual functionality of individual PRBC units is mostly ignored. One of the main features of the RBCs not accounted for under this approach is the deformability of the red cells, i.e., their ability to affect the recipients' blood flow. The objective of the study was to analyze unit-to-unit variability in the deformability of PRBCs during their cold storage. METHODS: RBC samples were obtained from twenty leukoreduced PRBC units, stored in SAGM. The deformability of cells was monitored from the day of donation throughout 42 days. RBC deformability was determined using the computerized cell flow-properties analyzer (CFA) based on cell elongation under a shear stress of 3.0 Pa, expressed by the elongation-ratio (ER). The image analysis determines the ER for each cell and provides the ER distribution in the population of 3000-6000 cells. RESULTS: The deformability of freshly-collected RBCs exhibited marked variability already on the day of donation. We also found that the aging curve of PRBC deformability varies significantly among donors. SIGNIFICANCE: The present study has demonstrated that storage duration is only one of the factors, and seemingly not even the major one, affecting the PRBCs functionality. Therefore, the FIFO approach is not sufficient for assessing the potential transfusion outcome, and the PRBC functionality should be determined explicitly for each unit.

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

BACKGROUND: Donated 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. METHODS: Blood 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. RESULTS: The 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. CONCLUSION: Likely, 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.

Is It Possible to Reverse the Storage-Induced Lesion of Red Blood Cells?

Cold-storage of packed red blood cells (PRBCs) in the blood bank is reportedly associated with alteration in a wide range of RBC features, which change cell storage each on its own timescale. Thus, some of the changes take place at an early stage of storage (during the first 7 days), while others occur later. We still do not have a clear understanding what happens to the damaged PRBC following their transfusion. We know that some portion (from a few to 10%) of transfused cells with a high degree of damage are removed from the bloodstream immediately or in the first hour(s) after the transfusion. The remaining cells partially restore their functionality and remain in the recipient's blood for a longer time. Thus, the ability of transfused cells to recover is a significant factor in PRBC transfusion effectiveness. In the present review, we discuss publications that examined RBC lesions induced by the cold storage, aiming to offer a better understanding of the time frame in which these lesions occur, with particular emphasis on the question of their reversibility. We argue that transfused RBCs are capable (in a matter of a few hours) of restoring their pre-storage levels of ATP and 2,3-DPG, with subsequent restoration of cell functionality, especially of those properties having a more pronounced ATP-dependence. The extent of reversal is inversely proportional to the extent of damage, and some of the changes cannot be reversed.

[ADVANCES IN FETAL THERAPY IN COMPLICATED MONOCHORIONIC PREGNANCIES AND IN SEVERE CONGENITAL DIAPHRAGMATIC HERNIA: FIVE YEARS EXPERIENCE].

INTRODUCTION: : Minimally invasive fetal therapeutic procedures reduce the morbidity and mortality in monochorionic (MC) twins and in fetuses with congenital diaphragmatic hernia (CDH). MC pregnancies share their blood systems due to communicating vessels over their single placenta and may develop specific complications: Twin-to-Twin transfusion syndrome (TTTS), Selective intrauterine growth restriction (sIUGR), Twin Anemia-Polycythemia Sequence (TAPS), Twin Reverse Arterial Perfusion Syndrome (TRAP) or anomalies in one. Half of complicated MC require intrauterine interventions. Severe CDH is linked to a high rate of neonatal death due to pulmonary hypoplasia. Fetoscopic tracheal occlusion (FETO) with a balloon improves postnatal outcome. AIMS: A fetal therapy center was established in the Hadassah Medical Centers, Jerusalem in 2011 for intrauterine interventions. We report our 5 years' experience. METHODS: This prospective cohort follows the outcome of MC pregnancies and cases of severe CDH which underwent therapeutic fetal procedures in Hadassah between the years 2011-16. RESULTS: Out of 114 procedures, 95 were in MC: 84 monochorionic diamniotic twins, 7 monochorionic monoamniotic twins, 2 dichorionic triamniotic triplets and 2 monochorionic triplets. We treated 65 TTTS cases with fetoscopy and laser ablation of communicating vessels. The survival rate of both twins was 58.5% and at least one survived in 81.5% of the cases. In 2nd/3rd trimesters selective termination of 15 cases the survival rate of the remaining twin was 87%. In 11 cases of TRAP sequence treated with laser ablation of the feeding vessel the survival of the remaining twin was 91%. In 19 fetoscopies in severe CDH, 12 were for balloon insertion and 7 for removal. Endotracheal balloon was successfully placed in 11 of 12 cases (10 left-sided, 1 right-sided CDH). Balloon removal was prenatally performed by elective fetoscopy (n=7) or by intrapartum urgent puncture. There were no intrauterine fetal deaths. In isolated left-sided CDH the survival was 57%, whereas none survived in non-isolated left-sided CDH and right sided CDH. CONCLUSIONS: In-utero procedures are safe for the mother and increase newborn survival in MC pregnancies, thus specialized clinics are life-saving. FETO is a therapeutic option for severe CDH. Our results meet similar achievements reported by other leading world centers.

Hemodynamic Functionality of Transfused Red Blood Cells in the Microcirculation of Blood Recipients.

The primary goal of red blood cell (RBC) transfusion is to supply oxygen to tissues and organs. However, due to a growing number of studies that have reported negative transfusion outcomes, including reduced blood perfusion, there is rising concern about the risks in blood transfusion. RBC are characterized by unique flow-affecting properties, specifically adherence to blood vessel wall endothelium, cell deformability, and self-aggregability, which define their hemodynamic functionality (HF), namely their potential to affect blood circulation. The role of the HF of RBC in blood circulation, particularly the microcirculation, has been documented in numerous studies with animal models. These studies indicate that the HF of transfused RBC (TRBC) plays an important role in the transfusion outcome. However, studies with animal models must be interpreted with reservations, as animal physiology may not reflect human physiology. To test this concept in humans, we have directly examined the effect of the HF of TRBC, as expressed by their deformability and adherence to vascular endothelium, on the transfusion-induced effect on the skin blood flow and hemoglobin increment in ß-thalassemia major patients. The results demonstrated, for the first time in humans, that the TRBC HF is a potent effector of the transfusion outcome, expressed by the transfusion-induced increase in the recipients' hemoglobin level, and the change in the skin blood flow, indicating a link between the microcirculation and the survival of TRBC in the recipients' vascular system. The implication of these findings for blood transfusion practice and to vascular function in blood recipients is discussed.

Intestinal tight junctions are severely altered in NEC preterm neonates.

BACKGROUND & AIMS: Necrotizing Enterocolitis (NEC) is a severe inflammatory disorder of the intestine endangering the health and survival of preterm infants. It is well established that the gut barrier is severely damaged in NEC patients, nonetheless an in depth investigation of modifications at the transcriptional and translational levels of tight junction genes and proteins during NEC are still missing. The aim of this study was to investigate changes in the expression of tight junctions and other associated proteins during NEC and determine their correlation to the disease severity. METHODS: We examined intestinal specimens from six NEC patients and compared them with six control specimens from patients that underwent surgeries for reasons other than NEC. The expression of genes was analyzed by real time PCR and protein expression by immunohistochemistry. RESULTS: The tight junction genes ZO-1, occludin, cingulin and claudin-4 were significantly down regulated in NEC. Furthermore TLR4, BAX and SIRT1 genes were found to be significantly down regulated while HIF-1A showed a trend of up regulation in NEC patients. These changes were found to correlate with the severity of the disease. Additionally we demonstrated in an ex-vivo model that hypoxic conditions initiated a destructive process of the epithelial barrier. We also showed that the expression of the tight junction proteins ZO-1 and occludin were significantly down regulated in NEC specimens. CONCLUSIONS: The expression of tight junction proteins and their encoding genes are significantly altered in NEC. We surmise that SIRT1 and HIF-1A may play a role in controlling these effects.