Retention Of Red Blood Cells Research Articles

Reduced red blood cell deformability in vivax malaria.

Reduced deformability of both infected and uninfected red blood cells (RBC) contributes to pathogenesis in falciparum malaria. Whole blood RBC-deformability is not well-characterised in vivax malaria. We used a laser-assisted optical rotational cell analyzer to measure the RBC deformability in fresh whole blood from Malaysian patients with vivax malaria (n=25). Deformability of whole blood RBC, the vast majority of which were uninfected, was reduced in vivax malaria compared to controls (n=15), though not to the same degree as in falciparum malaria (n=90). Reduced RBC-deformability may contribute to the pathogenesis of vivax malaria, including splenic retention of uninfected RBC.

Red blood cell passage through deformable interendothelial slits in the spleen: Insights into splenic filtration and hemodynamics

The spleen constantly clears altered red blood cells (RBCs) from the circulation, tuning the balance between RBC formation (erythropoiesis) and removal. The retention and elimination of RBCs occur predominantly in the open circulation of the spleen, where RBCs must cross submicron-wide inter-endothelial slits (IES). Several experimental and computational studies have illustrated the role of IES in filtrating the biomechanically and morphologically altered RBCs based on a rigid wall assumption. However, these studies also reported that when the size of IES is close to the lower end of clinically observed sizes (less than 0.5 μm), an unphysiologically large pressure difference across the IES is required to drive the passage of normal RBCs, sparking debates on the feasibility of the rigid wall assumption. In this work, We propose two deformable IES models, namely the passive model and the active model, aiming to explore the impact of the deformability of IES on the filtration function of the spleen. In the passive model, we implement the worm-like string model to depict the IES’s deformation as it interacts with blood plasma and allows RBC to traverse. In contrast, the active model involved regulating the IES deformation based on the local pressure surrounding the slit. To demonstrate the validity of the deformable model, we simulate the filtration of RBCs with varied size and stiffness by IES under three scenarios: (1) a single RBC traversing a single slit; (2) a suspension of RBCs traversing an array of slits, mimicking in vitro spleen-on-a-chip experiments; (3) RBC suspension passing through the 3D spleen filtration unit known as’the splenon’. Our simulation results of RBC passing through a single slit show that the deformable IES model offers more accurate predictions of the critical cell surface area to volume ratio that dictate the removal of aged RBCs from circulation compared to prior rigid-wall models. Our biophysical models of the spleen-on-a-chip indicate a hierarchy of filtration function stringency: rigid model > passive model > active model, providing a possible explanation of the filtration function of IES. We also illustrate that the biophysical model of ‘the splenon’ enables us to replicate the ex vivo experiments involving spleen filtration of malaria-infected RBCs. Taken together, our simulation findings indicate that the deformable IES model could serve as a mesoscopic representation of spleen filtration function closer to physiological reality, addressing questions beyond the scope of current experimental and computational models and enhancing our understanding of the fundamental flow dynamics and mechanical clearance processes within in the human spleen.

Clearance of pathogenic erythrocytes is maintained despite spleen dysfunction in children with sickle cell disease.

In children with sickle cell disease (SCD), splenectomy is immediately beneficial for acute sequestration crises and hypersplenism (ASSC/HyS) but portends a long-term risk of asplenia-related complications. We retrieved peripheral and splenic red blood cells (RBCs) from 17 SCD children/teenagers undergoing partial splenectomy for ASSC/HyS, 12 adult subjects without RBC-related disease undergoing splenectomy (controls), five human spleens perfused ex vivo with HbSS- and HbAA-RBC, and quantified abnormal RBC by microscopy, spleen-mimetic RBC filtration, and adhesion assays. Spleens were analyzed by immunohistochemistry and transmission electron microscopy (TEM). In circulating blood of SCD and control subjects, dysmorphic (elongated/spherocytic) RBCs were <2%, while proportions of pocked-RBC were 4.3-fold higher in SCD children than in controls. Compared to controls, splenic RBCs were more frequently dysmorphic (29.3% vs. 0.4%), stiffer (42.2% vs. 12.4%), and adherent (206 vs. 22 adherent RBC/area) in SCD subjects. By TEM, both polymer-containing and homogenous RBC contributed to spleen congestion, resulting in 3.8-fold higher RBC population density in SCD spleens than in control spleens, predominantly in the cords. Perfused spleens with normal function displayed similar congestion and retention of dysmorphic RBC as SCD spleens. The population density of active macrophages was similar in SCD and control spleens, with a relative deficit in phagocytosis of polymer-containing RBC. Despite the existence of hyposplenism, splenectomy in SCD children removes an organ that still efficiently filters out potentially pathogenic altered RBC. Innovative treatments allowing fine-tuned reduction of RBC retention would alleviate spleen congestion, the major pathogenic process in ASSC/HyS, while preserving spleen protective functions for the future.

A combined computational and experimental investigation of the filtration function of splenic macrophages in sickle cell disease.

Being the largest lymphatic organ in the body, the spleen also constantly controls the quality of red blood cells (RBCs) in circulation through its two major filtration components, namely interendothelial slits (IES) and red pulp macrophages. In contrast to the extensive studies in understanding the filtration function of IES, fewer works investigate how the splenic macrophages retain the aged and diseased RBCs, i.e., RBCs in sickle cell disease (SCD). Herein, we perform a computational study informed by companion experiments to quantify the dynamics of RBCs captured and retained by the macrophages. We first calibrate the parameters in the computational model based on microfluidic experimental measurements for sickle RBCs under normoxia and hypoxia, as those parameters are not available in the literature. Next, we quantify the impact of key factors expected to dictate the RBC retention by the macrophages in the spleen, namely, blood flow conditions, RBC aggregation, hematocrit, RBC morphology, and oxygen levels. Our simulation results show that hypoxic conditions could enhance the adhesion between the sickle RBCs and macrophages. This, in turn, increases the retention of RBCs by as much as four-fold, which could be a possible cause of RBC congestion in the spleen of patients with SCD. Our study on the impact of RBC aggregation illustrates a 'clustering effect', where multiple RBCs in one aggregate can make contact and adhere to the macrophages, leading to a higher retention rate than that resulting from RBC-macrophage pair interactions. Our simulations of sickle RBCs flowing past macrophages for a range of blood flow velocities indicate that the increased blood velocity could quickly attenuate the function of the red pulp macrophages on detaining aged or diseased RBCs, thereby providing a possible rationale for the slow blood flow in the open circulation of the spleen. Furthermore, we quantify the impact of RBC morphology on their tendency to be retained by the macrophages. We find that the sickle and granular-shaped RBCs are more likely to be filtered by macrophages in the spleen. This finding is consistent with the observation of low percentages of these two forms of sickle RBCs in the blood smear of SCD patients. Taken together, our experimental and simulation results aid in our quantitative understanding of the function of splenic macrophages in retaining the diseased RBCs and provide an opportunity to combine such knowledge with the current knowledge of the interaction between IES and traversing RBCs to apprehend the complete filtration function of the spleen in SCD.

Evaluation of the Impact of a Naturally Occurring Beta Hemoglobin Variant, Hb G-Makassar, on Mature Red Blood Cell Function and Pathology in a Sickle Cell Disease Mouse Model

Background: Gene based therapy may provide a cure for individuals with sickle cell disease (SCD); base editing is a novel strategy with the ability to directly edit the mutation responsible for disease. The sickle hemoglobin (HbS) point mutation cannot be converted to wild-type HBB by an adenine base editor, however it can be converted to a naturally occurring hemoglobin variant, HbG-Makassar (HbG). HbG is a non-pathogenic, non-sickling hemoglobin variant. In vitro crystal structure analysis of HbG shows that it is highly similar to that of wildtype hemoglobin (HbA). However, few HbGG individuals (with no overt red cell disease) and no HbSG individuals have been identified; thus, the red blood cells (RBCs) of HbGG and HbSG individuals have not been evaluated functionally. The Makassar base editing strategy produces greater than 80% homozygous Hb-G product and has been evaluated in vitro, however erythroid culture of edited hematopoietic stem cells do not produce fully mature, enucleated red cells. Therefore, mouse models of HbGG and HbSG could be used to evaluate the function of mature, enucleated HbGG or HbSG RBCs. Methods: A humanized beta globin locus mouse (Townes) with the Makassar allele was created at by knocking-in the Makassar point mutation to generated WT/Makassar heterozygotes (HbAG) which were backcrossed to generated HbGG homozygotes. HbSG genotypes were subsequently generated by crossing to HbSS Townes mice. We analyzed the red cell function of HbAA, HbAS, HbSS, HbGG, and HbSG mice. Whole blood was collected through terminal enucleation. Complete blood counts were measured on a hematology analyzer (Heska and, ADVIA). Red cell deformability (elongation index, EI) was measured using oxygen gradient ektacytometry (LoRRCA) under normoxic (EImax) and hypoxic (EImin) conditions. Viscosity measurements were obtained on a Beckman cone and plate viscometer. Percent sickle cells were quantified through chemically induced sickling with sodium metabisulfite and counted by an individual blinded to genotype using light microscopy. Kidney, liver, and spleen were fixed, preserved in paraffin, stained using hematoxylin and eosin stains and imaged using a Keyence microscope. Flow cytometry was performed on bone marrow and whole blood to determine the erythroid maturation and mitochondrial retention of RBCs. Analysis was performed with Stata 18.0 (College Station) across genotypes using Dunn Pairwise test with statistically significant values being selected at adjusted p &amp;lt; 0.05. Results Homozygous HbG mice (HbGG) had comparable white blood cell count (WBC) and absolute reticulocyte count (ARC) to that of mice with homozygous HbA (HbAA). HbGG mice had lower hemoglobin and mean corpuscular volume (MCV) in comparison to mice with HbAA. HbGG mice had a higher percentage of dense RBCs than HbAA mice. HbSG mice had comparable values to HbAS mice for MCV but had significantly lower hemoglobin and ARC. HbSG mice had significantly higher values for WBC, and % dense RBCs against HbAS mice. HbSS and HbSG mice had comparable values for hemoglobin, and ARC. Hematocrit to viscosity ratio (HVR) shows comparable oxygen transport effectiveness of HbGG, HbSG, HbAS and HbSS RBC. Further red cell function testing is shown in Figure 1 and Figure 2. Mitochondrial retention was highest in HbSS mice, followed by HbSG, while HbAS, HbAA, and HbGG had similar values. Microscopy analysis of renal sections of HbGG and HbSG mice sacrificed at 24-28 weeks did not show the characteristic morphologic changes seen in HbSS mice at 24-28 weeks. Conclusions- HbGG appears functionally similar to HbAA, with minimal sickling under hypoxia, no anemia, normal spleen size, and no organ damage at 24 weeks of age. HbGG does exhibit signs of dehydration, as evidenced by elevated %DRBC and reduced RBC deformability. RBC function, sickling under hypoxia, Hb, WBC, ARC, and mitochondrial retention measures places HbSG as intermediate in severity between HbAS and HbSS. However, organ pathology, spleen and liver weights for HbSG were more comparable to HbAS than HbSS. Base editing strategies to install Makassar therefore can potentially lead to &amp;gt;90% of RBCs that have therapeutic benefit due to rheological properties comparable to HbAA and can be seen as a promising treatment strategy for patients with sickle cell disease.

Mechanisms of Red Blood Cell Splenic Filtration Revealed By Multiscale Modeling and in-Vitro Microfluidic Experiments

Interendothelial slits in the spleen fulfill the major physiological function of continuously filtering red blood cells (RBCs) from the bloodstream to remove abnormal and aged cells. To date, the process of passage of 8 µm RBCs through 0.3-µm wide slits remains enigmatic. Should the slits increase their caliber during RBC passage as sometimes proposed in the literature? Here, we elucidated the mechanisms that govern the passage dynamics or retention of RBCs in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with slits of defined physiological dimensions, including submicron width. We observed that healthy RBCs pass through 0.28-µm wide rigid slits at body temperature. To achieve this tour de force, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, they must be able to locally unfold their spectrin cytoskeleton inside the slits. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through slits scales with in-slit pressure drop and slit width to the -1 and -3 power, respectively. This transit dynamics is similar to that of a Newtonian fluid in a 2D Poiseuille flow, thus showing that it is controlled by the RBC cytoplasmic viscosity. Altogether, our results clearly show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in-vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery.

Probing interactions of red blood cells and contracting fibrin platelet clots

Contraction of blood clots plays an important role in blood clotting, a natural process that restores hemostasis and regulates thrombosis in the body. Upon injury, a chain of events culminate in the formation of a soft plug of cells and fibrin fibers attaching to wound edges. Platelets become activated and apply contractile forces to shrink the overall clot size, modify clot structure, and mechanically stabilize the clot. Impaired blood clot contraction results in unhealthy volumetric, mechanical, and structural properties of blood clots associated with a range of severe medical conditions for patients with bleeding and thrombotic disorders. Due to the inherent mechanical complexity of blood clots and a confluence of multiple interdependent factors governing clot contraction, the mechanics and dynamics of clot contraction and the interactions with red blood cells (RBCs) remain elusive. Using an experimentally informed, physics-based mesoscale computational model, we probe the dynamic interactions among platelets, fibrin polymers, and RBCs, and examine the properties of contracted blood clots. Our simulations confirm that RBCs strongly affect clot contraction. We find that RBC retention and compaction in thrombi can be solely a result of mechanistic contraction of fibrin mesh due to platelet activity. Retention of RBCs hinders clot contraction and reduces clot contractility. Expulsion of RBCs located closer to clot outer surface results in the development of a dense fibrin shell in thrombus clots commonly observed in experiments. Our simulations identify the essential parameters and interactions that control blood clot contraction process, highlighting its dependence on platelet concentration and the initial clot size. Furthermore, our computational model can serve as a useful tool in clinically relevant studies of hemostasis and thrombosis disorders, and post thrombotic clot lysis, deformation, and breaking.

Computational investigation of flow dynamics and mechanical retention of age-associated red blood cells in the spleen

The shape and deformability of red blood cells (RBCs) change with age, increasing their likelihood of becoming trapped in the splenic interendothelial slits (IESs) and being removed from the circulation. Existing evidence suggests that cell size and viscoelasticity vary with age, which impedes understanding of the mechanical retention of aged RBCs in the spleen. We present a computational study of the flow dynamics, shape changes, and mechanical retention of age-associated RBCs in the splenic IES. The results show that increasing RBC membrane viscosity slows the flow process early in aging, while increased sphericity due to cell size reduction eventually leads to RBC retention.

Utility of White Blood Cell Diluting Fluid (Turk’s Fluid) in Evaluation of Haemorrhagic Thyroid Cytology Smears in a Tertiary Care Centre- A Novel Study

Introduction: Fine Needle Aspiration Cytology (FNAC) has been one of the safest and accurate diagnostic technique in diagnosing thyroid lesions. But due to the vascularity of thyroid tissue, diagnostic cells get entrapped in blood clot unless rapid smearing is done.This results in repeat FNAC which is inconvenient to the patient as well as time consuming to pathologist. Therefore, an alternative is required to avoid repeat FNAC procedure. Aim: To evaluate the role of Turk's fluid in haemorrhagic thyroid FNAC smears in comparison to conventional haemorrhagic smears. Materials and Methods: The present study was a prospective observational study, which was conducted for the period of six months (March-August 2021). A total of 30 cases were included in the study. FNA sampling was done on patients with thyroid swelling. After the needling was done on the swelling, four smears. One was stained using (H &amp; E). Rest of all the smears were stained by Conventional Giemsa (CG) method. One of these CG stained smears was immediately treated with Turk's fluid for 10-20 seconds. The CG stained smears were compared with the Turk's fluid Treated Giemsa (TTG) smears and evaluated for retention of Red Blood Cells (RBCs), visibility of nuclear and cytoplasmic details of thyroid follicular cells. Results: A total of 30 cases were included in the study. The retention of RBC was lesser in TTG compared to CG which was statistically significant (p-value&lt;0.05). The visibility of cytoplasmic and nuclear morphology on TTG was better than that of CG which was statistically significant (p-value&lt;0.05). Conclusion: TTG smears improves visibility of cytomorphological features by reducing background RBCs and hence avoids repeat FNAC.

Rapid clearance of storage-induced microerythrocytes alters transfusion recovery

AbstractPermanent availability of red blood cells (RBCs) for transfusion depends on refrigerated storage, during which morphologically altered RBCs accumulate. Among these, a subpopulation of small RBCs, comprising type III echinocytes, spheroechinocytes, and spherocytes and defined as storage-induced microerythrocytes (SMEs), could be rapidly cleared from circulation posttransfusion. We quantified the proportion of SMEs in RBC concentrates from healthy human volunteers and assessed correlation with transfusion recovery, investigated the fate of SMEs upon perfusion through human spleen ex vivo, and explored where and how SMEs are cleared in a mouse model of blood storage and transfusion. In healthy human volunteers, high proportion of SMEs in long-stored RBC concentrates correlated with poor transfusion recovery. When perfused through human spleen, 15% and 61% of long-stored RBCs and SMEs were cleared in 70 minutes, respectively. High initial proportion of SMEs also correlated with high retention of RBCs by perfused human spleen. In the mouse model, SMEs accumulated during storage. Transfusion of long-stored RBCs resulted in reduced posttransfusion recovery, mostly due to SME clearance. After transfusion in mice, long-stored RBCs accumulated predominantly in spleen and were ingested mainly by splenic and hepatic macrophages. In macrophage-depleted mice, splenic accumulation and SME clearance were delayed, and transfusion recovery was improved. In healthy hosts, SMEs were cleared predominantly by macrophages in spleen and liver. When this well-demarcated subpopulation of altered RBCs was abundant in RBC concentrates, transfusion recovery was diminished. SME quantification has the potential to improve blood product quality assessment. This trial was registered at www.clinicaltrials.gov as #NCT02889133.

Thyroid Hormone Receptor α Mutations Cause Heart Defects in Zebrafish

Background: Mutations of thyroid hormone receptor α1 (TRα1) cause resistance to thyroid hormone (RTHα). Patients exhibit growth retardation, delayed bone development, anemia, and bradycardia. By using mouse models of RTHα, much has been learned about the molecular actions of TRα1 mutants that underlie these abnormalities in adults. Using zebrafish models of RTHα that we have recently created, we aimed to understand how TRα1 mutants affect the heart function during this period. Methods: In contrast to human and mice, the thra gene is duplicated, thraa and thrab, in zebrafish. Using CRISPR/Cas9-mediated targeted mutagenesis, we created C-terminal mutations in each of two duplicated thra genes in zebrafish (thraa 8-bp insertion or thrab 1-bp insertion mutations). We recently showed that these mutant fish faithfully recapitulated growth retardation as found in patients and thra mutant mice. In the present study, we used histological analysis, gene expression profiles, confocal fluorescence, and transmission electron microscopy (TEM) to comprehensively analyze the phenotypic characteristics of mutant fish heart during development. Results: We found both a dilated atrium and an abnormally shaped ventricle in adult mutant fish. The retention of red blood cells in the two abnormal heart chambers, and the decreased circulating blood speed and reduced expression of contractile genes indicated weakened contractility in the heart of mutant fish. These abnormalities were detected in mutant fish as early as 35 days postfertilization (juveniles). Furthermore, the expression of genes associated with the sarcomere assembly was suppressed in the heart of mutant fish, resulting in abnormalities of sarcomere organization as revealed by TEM, suggesting that the abnormal sarcomere organization could underlie the bradycardia exhibited in mutant fish. Conclusions: Using a zebrafish model of RTHα, the present study demonstrated for the first time that TRα1 mutants could act to cause abnormal heart structure, weaken contractility, and disrupt sarcomere organization that affect heart functions. These findings provide new insights into the bradycardia found in RTHα patients.

Fibrinogen and factor XIII: newly recognized roles in venous thrombus formation and composition.

In spite of significant morbidity and mortality associated with venous thromboembolism, the underlying pathogenesis remains poorly understood. Clues to operant pathogenic mechanisms are found in the unique morphology and composition of these thrombi, which have substantial red blood cell and fibrin content. Recent studies have revealed biochemical and biophysical mechanisms that dictate fibrin structure in venous thrombi and promote retention of red blood cells within the contracted clots. These mechanisms include newly recognized contributions of fibrin network structure and factor XIII(a)-mediated fibrin crosslinking to venous thrombus composition, size, and stability. Continued work to elucidate mechanisms by which fibrin(ogen), factor XIII, and red blood cells contribute to venous thrombus formation, structure, and stability may expose novel molecular targets and strategies for reducing thrombosis and thrombotic complications in certain at-risk patients.

Red Blood Cell Deformability, Age, Ethnicity and Susceptibility to Malaria in Africa

Introduction: malaria is one of the most frequent hematological diseases worldwide. Because the malaria parasite Plasmodium falciparum develops mainly in red blood cells (RBC), splenic retention of infected and uninfected RBC is likely a key player in the variable susceptibility of humans to malaria. Age and ethnicity are important determinants of the manifestations of malaria in Africa (Reyburn JAMA 2005, Dolo Am J Trop Med Hyg 2005; Greenwood Ann Trop Med Parasitol 1987; Torcia PNAS 2008), Asia (Price Am J Trop Med Hyg 2001), and in travelers (Seringe Emerg Infect Dis 2011). We had speculated that variations in the splenic sensing of RBC contribute to the innate protection/susceptibility of infants against distinct forms of severe malaria and to the pathogenesis of chronic malaria (Buffet Curr Opin Hematol 2009; Buffet Blood 2011). Here, we explore the deformability and morphology of circulating RBC in populations living in a malaria-endemic area.Materials and methods: experiments were embedded in an integrated study driven by Institut de Recherche pour le Développement, which aims at the identification of genetic, epidemiologic and anthropologic determinants of susceptibility to malaria. IRB approval was obtained from Institut des Sciences Biomédicales Appliquées, Benin. Clinical and biological data were collected at the beginning of the rainy season from 627 individuals, belonging to 4 different ethnic groups living in sympatry in Atakora, North Benin and included age, gender, ethnicity, body temperature, presence and grade of splenomegaly, rapid diagnostic test for malaria (RDT), thick film and rapid hemoglobin determination with HemoCue©. Venous blood was collected for determination of RBC morphology and deformability. Using microsphiltration, a RBC filtering method that uses microsphere layers to mimic the mechanical retention of RBC in the splenic red pulp (Deplaine Blood 2011) we quantified the ability of a mix of labeled and non-labeled RBCs to squeeze between calibrated slits, results being expressed as retention or enrichment rates (RER) of subject’s RBC compared to normal RBC (from a single French O-positive donor) stored in blood bank conditions. Microsphiltration has been adapted to high-throughput experimentation using microplates (Duez AAC 2015). Experiments were performed in a field laboratory established on site; microplates were prepared in Paris and brought to North Benin in luggage with constant care to avoid shocks during transportation. All RBC samples were filtered in triplicate less than 8 hours after blood collection. Up- and downstream samples were brought back to France at 4°C in sealed micro-well plates and analyzed for individual RER calculation in the next 2 weeks by flow cytometry.Results: over 10 days, 262 adults and 249 children were included, 31% Bariba, 17% Gando (genetically related to Bariba), 24% Otamari, 27% Peulhs. Prevalences of splenomegaly, positive RDT, and fever were 13%, 27%, and 2%, respectively. Of 629 blood samples collected, 511 could be analyzed. RER of controls remained stable with time and across 17 microfiltering plates, with a median (IQR) retention rate of 12% (5% - 21%). Ethnicity and age were the only two factors associated with statistically significant differences in RER (figures 1 and 2). Infants (less than 2 year-old) had a more important enrichment than older children and adults (median in 2 years old or less 287%; 3 to 5 years 103%; 6 to 10 years: 64%; more than ten years: 91%; p=0.0161). Peulhs and Otamari also had higher median enrichment rates than Bariba and Gando (RER: 122% and 118%, 75%, 64%, respectively; p=0.0246). Conversely, splenomegaly, gender, positivity of RDT or anemia at the time of sampling were not associated with RER.Discussion: higher averaged enrichment rates in specific ethnic subgroups, namely Peulhs and Otamari, likely result from a more stringent splenic retention, leaving more deformable RBC in circulation. An innate spleen-RBC interaction process was also observed in infants, which is consistent with the higher incidence of severe malarial anemia and splenomegaly observed in this population (Reyburn JAMA 2005; Price Am J Trop Med Hyg 2001). Our results show that innate factors (e.g. ethnicity and age) tend to influence the deformability of RBC, and therefore the phenotypic expression of malaria in Africa. Ongoing experiments aim at deciphering the mechanisms responsible for these differences. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Alteration of the Structure and Dynamics of Venous Clot Formation in Human and Murine Sickle Cell Disease

Sickle cell disease (SCD) is associated with chronic activation of coagulation and an increased risk of venous thromboembolism. Traditionally, it is believed that during venous thrombosis, red blood cells (RBC) are simply trapped within fibrin-rich thrombi and do not actively affect thrombosis. However, a study from our group showed that factor XIII (FXIII) activity is critical for the retention of RBC within clots and directly affects thrombus size. Others reported that during clot contraction polyhedral shaped RBC formed a densely packed core and that SCD alters the formation of polyhedrocytes which may affect clot stability (Strauss et al, 2015, ASH abstract).We further investigated if SCD affects the structure and the dynamics of clot formation. Ex vivo clot retraction was performed using blood from sickle cell patients and Townes mice (a model of SCD). Citrated blood was added to siliconized wells of 96-well plates containing CaCl2 (10mM) and tissue factor (1pM) and incubated for 2 hours at 37°C. The number of RBC extruded from the clot was counted in serum by Hemavet™ and expressed as a percentage of initial RBC number in the anticoagulated blood. Morphology of the clots was evaluated using scanning and transmission electron microscopy (EM). Thrombosis in Townes SS (sickle) and AA (control) mice was studied using femoral vein thrombosis induced by electrolytic injury and inferior vena cava stenosis models.The number of mouse SS RBC in the serum extruded during clot contraction was dramatically reduced compared to the number of AA RBC (0.8±0.8% vs. 19.4±0.8%, n=3, p<0.0001, Fig. A). A similar result was observed for human RBC. Since SS mice and sickle patients have a lower hematocrit (HCT), we investigated if the number of RBC affects the extrusion of these cells during clot retraction. Indeed, lowering HCT in AA mouse blood reduced RBC extrusion from the clots. However, increasing HCT in SS mouse blood to that of AA blood did not increase the number of SS RBC extruded from the clot. Furthermore, inhibition of FXIIIa activity with T101 (10µM) increased the release of AA (by 64%, n=6, p<0.05) but not SS RBC from mouse clots. These data indicate that the entrapment of SS RBCs within the clot is not simply caused by lower RBC number, and is FXIII-independent. Mixing the platelet poor plasma (PPP) and cellular fraction of AA and SS mouse blood revealed that the entrapment of SS RBC is not mediated by PPP (AA blood recombined = 6.9±3.6%; SS blood recombined = 0.4±0.4%; AA cells/SS PPP = 6.7±6.4%; SS cells/AA PPP = 0.1±0.2%, n=3 per group).Clots formed ex vivo from AA blood had a gel-like, soft structure, whereas SS clots were more firm and stiff. EM demonstrated that RBC within AA clots had polyhedral shapes and were tightly packed in the central part of the clot. In contrast, most of the mouse SS RBC did not have polyhedral shapes, underwent sickling and were not compacted within the clot. They also formed long "spicule-like" processes that intertwined with fibrin fibers (Fig. C). Similar results were observed in blood of sickle patients; however, the sickled RBC phenotype was less prominent. Importantly, sickling of RBC was observed in clots formed in the inferior vena cava of SS mice, 2 hours after vessel stenosis (Fig. D). We also performed a tPA challenge assay on clots formed ex vivo from human blood and showed that SS clots challenged with low tPA concentration (0.6nM) were more resistant to fibrinolysis compared to AA clots (clot lysis time, 714±6 vs. 388.3±120.7 minutes, n=6, p=0.08).The electrolytic injury model of venous thrombosis was used to investigate the dynamics of clot formation in SS mice in vivo. Mice were infused with fluorescently labeled antibodies for fibrin (green) and platelets (red). Electrolytic injury was applied to the femoral vein; a relative intensity of fibrin and platelet accumulation was assessed by fluorescence microscopy for one hour at 10-minute intervals. SS mice have increased platelet and fibrin accumulation compared to AA mice (~ 2 fold, n=5-7, p<0.05 for 40, 50 and 60 minute time points). Interestingly, in the AA clots, platelets were mostly localized on the surface, in contrast to their widespread distribution throughout the clot in SS mice (Fig. E, yellow color).Our data demonstrated that SCD alters the structure and dynamics of venous clot formation. Experiments investigating the consequence of these observations in mouse models of stroke and pulmonary embolism are currently ongoing. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Factor XIIIa-dependent retention of red blood cells in clots is mediated by fibrin α-chain crosslinking

AbstractFactor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks γ-chain crosslinking sites, but not in clots that lack α-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block α-, but not γ-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin α-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size.

A biomimetic microfluidic chip to study the circulation and mechanical retention of red blood cells in the spleen.

Red blood cells (RBCs) are deformable and flow through vessels narrower than their own size. Their deformability is most stringently challenged when they cross micrometer-wide slits in the spleen. In several inherited or acquired RBC disorders, blockade of small vessels by stiff RBCs can trigger organ damage, but a functional spleen is expected to clear these abnormal RBCs from the circulation before they induce such complications. We analyzed flow behavior of RBCs in a microfluidic chip that replicates the mechanical constraints imposed on RBCs as they cross the human spleen. Polymer microchannels obtained by soft lithography with a hydraulic diameter of 25 μm drove flow into mechanical filtering units where RBCs flew either slowly through 5- to 2-μm-wide slits or rapidly along 10-μm-wide channels, these parallel paths mimicking the splenic microcirculation. Stiff heated RBCs accumulated in narrow slits seven times more frequently than normal RBCs infused simultaneously. Stage-dependent retention of Plasmodium falciparum-infected RBCs was also observed in these slits. We also analyzed RBCs from patients with hereditary spherocytosis and observed retention for those having the most altered mechanical properties as determined by ektacytometry. Thus, in keeping with previous observations in vivo and ex vivo, the chip successfully discriminated poorly deformable RBCs based on their distinct mechanical properties and on the intensity of the cell alteration. Applications to the exploration of the pathogenesis of malaria, hereditary spherocytosis, sickle cell disease and other RBC disorders are envisioned.

Biochip technology applied to an automated ABO compatibility test at the patient bedside

In the field of blood transfusion, there is a need to improve the bedside pre-transfusion ABO compatibility test. In France, this test is mandatory for each red cell concentrates transfusion. It is performed manually and serious transfusion accidents still occur, principally due to human errors. Therefore, an automated ABO compatibility test is required. Works concerning objective interpretation of ABO compatibility test have been reported but the proposed techniques cannot be easily translated to the patient's bedside. We propose a prototype device which demonstrates the easy use of biochip technology to perform this test: it contains a fluidic system, biochips (two to test the patient and two to test the red cell concentrates) and an optical absorbance detection module. When blood is applied to the biochips, red blood cells are trapped onto the surface if antigens and antibodies are complementary (positive chips). If they are not complementary, very little red blood cells are adsorbed (negative chips). Percentages of surface covered with red blood cells in negative biochips are 2%±2 (red cell concentrates) and 1%±1 (whole blood). This proves that the fluidic configuration leads to an optimum control of fluids flows with little retention of red blood cells in the circuitry. These percentages increase to 96%±3 and 82%±8 for red cell concentrates and whole blood respectively. This demonstrates a strong and specific immunocapture of red blood cells on positive chips. Furthermore, optical detection proves to be efficient at critical red blood cells concentrations (108C/mL) and absorbance strongly correlates to the percentage of red blood cells captured by antibodies.

Fibrin Crosslinking Is Required For Retention Of Red Blood Cells In Venous Thrombi,

Venous thrombi contain high levels of red blood cells (RBC) and fibrin, but little is known about the mechanisms regulating venous thrombus formation and composition. Fibrin acts as a scaffold for leukocytes and platelets that mediate thrombus formation, and cross-linked fibrin networks promote clot stability via their extraordinary extensibility and elasticity. Previous studies have shown that a region of the fibrinogen γ-chain (residues γ390-396) is critical for engagement of the leukocyte integrin receptor αMβ2. Mice expressing a mutant form of fibrinogen with residues 390-396 mutated to a series of alanines (termed Fibγ390-396A) exhibit normal fibrin polymerization and normal hemostasis. However, Fibγ390-396A mice exhibit blunted inflammatory responses and protection from the development of numerous inflammatory diseases (e.g., arthritis, neuroinflammatory disease, colitis). In this study, we analyzed the role of this region of fibrinogen in a stasis-induced model of venous thrombosis. Surprisingly, following inferior vena cava ligation – a model that produces thrombi independent of leukocyte tissue factor activity – Fibγ390-396A mice had 50% smaller thrombi than wild type (WT) mice (9.7±1.3 vs 19.5±1.7 mg, P&lt;0.0003). Reduced thrombus weight was not due to reduced thrombin generation (i.e., TAT levels), and total neutrophil, platelet, and fibrin content within thrombi were similar between groups. Strikingly, Fibγ390-396A thrombi had significantly fewer RBCs than WT thrombi (16.0±4.1 vs 52.9±5.8 arbitrary units [AU], P&lt;0.0001), and thrombus RBC content correlated positively (R=0.90) and significantly (P&lt;0.001) with overall thrombus weight. To determine the mechanism of decreased RBC presence in thrombi from Fibγ390-396A mice, we developed an ex vivo whole blood clot retraction assay. Interestingly, although retraction of platelet-rich plasma clots was indistinguishable for Fibγ390-396A and WT mice (91±1 vs 92±1 %), retraction of whole blood clots resulted in dramatically reduced RBC retention (37.0±8.4 vs 79.0±8.0 % of initial RBCs, P&lt;0.03) and smaller clots (11.6±1.6 vs 53.8±3.6 mg, P&lt;0.003) for Fibγ390-396A mice compared to WT. Reconstitution experiments showed the mechanism of decreased RBC retention was not due to abnormal RBC function. Microfluidic-based adhesion analyses indicated RBCs adhered similarly to both Fibγ390-396A and WT purified fibrinogen (65.7±6.4 vs 76.7±14.8 % cell adhesion, respectively, P=0.55), indicating that RBC extrusion did not result from decreased RBC binding to Fibγ390-396A clots. To test the hypothesis that the Fibγ390-396A mutation disrupts a specific interaction with the fibrin-stabilizing transglutaminase, factor XIII (FXIII), we analyzed levels of FXIII that co-precipitated with WT and Fibγ390-396A fibrinogen. Interestingly, despite normal circulating levels of FXIII in Fibγ390-396A mice, FXIII co-precipitated with WT fibrinogen, but not with Fibγ390-396A fibrinogen. Compared to WT, plasma clots from Fibγ390-396A mice exhibited slower FXIII activation (58.3±19.2 vs 12.2±2.5 AU/min [x10-3], P&lt;0.05) and consequently, slower fibrin crosslinking (γ-γ dimers: 114±14.2 vs 19.5±2.5 AU/min [x10-3], P&lt;0.0001; α polymers: 278±52 vs 46.1±11.4 AU/min [x10-3], P&lt;0.002) and reduced elastic modulus (14.1±0.2 vs 7.8±0.6 G’ [Kd/sec], P&lt;0.0005). Provocatively, whole blood from FXIII-deficient mice and humans phenocopied Fibγ390-396A clots, with reduced RBC retention following clot retraction. Taken together, these studies suggest a critical, yet previously un-described, role for FXIII in mediating RBC retention within clots. Further, these data identify critical residues in fibrinogen that mediate FXIII activation and fibrin crosslinking, and reveal that FXIII-mediated fibrin crosslinking is required for the retention of RBCs in venous thrombi. Disclosures: No relevant conflicts of interest to declare.

A case of chronic kidney disease with thrombotic microangiopathy in a hematopoietic stem cell transplant recipient

A 23-year-old Japanese man who had undergone hematopoietic stem cell transplantation for acute lymphocytic leukemia from an HLA-identical sibling 6 years earlier developed proteinuria and impaired kidney function. Kidney biopsy revealed thrombotic microangiopathy with a moderate increase in mesangial matrices and glomerular microaneurysm featuring retention of red blood cells. The patient's kidney function gradually deteriorated, requiring the institution of treatment with angiotensin II receptor blockers and angiotensin-converting enzyme inhibitors, and progressing to continuous ambulatory peritoneal dialysis 4 years after the initial kidney biopsy. Eventually, kidney transplantation was performed with his mother as the donor. His kidney function is stable on immunosuppressive drugs at 2 years after transplantation. This report reflects the growing number of patients with chronic kidney disease with thrombotic microangiopathy all over the world.

A Rat Model for Sickle Cell-Mediated Vaso-occlusion in Retina

Occlusions of the retinal vasculature are the initiating event in sickle cell retinopathy. In order to understand the mechanism(s) of sickle cell-mediated occlusion, a rat model was developed. Red blood cells (RBCs) from patients homozygous for hemoglobin (Hb) S (SS) or double heterozygous for Hb S and Hb C (SC) were separated on Percoll–Larex continuous density gradients, labeled with fluorescein isothiocyanate (FITC), and delivered via the left ventricle to anesthetized, ventilated rats. Blood gas levels were altered by changing inspired gas and monitored via a femoral arterial catheter. After the RBCs circulated for 5 min, animals were perfused with heparinized saline, the eyes enucleated, and the retinas removed and processed by our ADPase flatmount technique. The retinal vasculature was visualized under dark-field illumination and the FITC-RBCs visualized by fluorescence microscopy. Greater numbers of high-density SS cells (SS4, which consist of dense, dehydrated discocytes and irreversible sickled cells) were retained in the normal rat retinal vasculature than normal-density SS cells (SS2, which have the same density as normal AA cells, but consist of reticulocytes and young cells). Retention of SS4 cells was inversely dependent on the arterial oxygen tension. Most SS4s were retained in capillaries, but a few were observed within precapillary arterioles. The retained RBCs occupied the full lumenal diameter of vessels in most cases. In contrast, very few RBCs from SC donors (normal or high density) were retained in the normal retinal vasculature and retention did not increase significantly with hypoxia. This model demonstrates that high-density SS cells, which include irreversibly sickled cells, are retained in normal rat retinal vessels and that the number retained is oxygen dependent. Furthermore, it appears that trapping, not adhesion, is responsible for retention of RBCs in the normal retinal vasculature because there was preferential retention of SS4 cells, which are known to have lower adherence propensity, and the retained RBCs blocked the full diameter of the vessel. These results also demonstrate that the mechanism of vascular obstruction by SS and SC RBCs is different because low retention of SC cells was observed. The well-known propensity of SC patients to have retinal abnormalities must involve extraerythrocytic factors like increased hematocrit, induction of adhesive molecules and integrins, etc.