Rapid diagnosis of hereditary haemolytic anaemias using automated rheoscopy and supervised machine learning

Haemolytic anaemias arise when red blood cell (RBC) integrity is compromised, eventually resulting in premature clearance or lysis and leading to anaemia when these effects cannot be sufficiently compensated by the capacity of the bone marrow to produce new cells. Hereditary anaemia occurs as a consequence of genetic mutation (e.g. affecting membrane complex or cytoskeletal proteins, haemoglobin or metabolic enzymes), and diagnosing affected patients is a complex process since, given the wide variety of possible genetic causes, multiple examinations must be performed and an unambiguous result is usually reached only after DNA sequencing. Furthermore, phenotypic severity can vary widely not just among individuals with different mutations but also among individuals suffering from the same mutation, thereby complicating diagnosis

The biodistribution of triamcinolone acetonide injections in severe keloids:an exploratory three-dimensional fluorescent cryomicrotome study

Intralesional corticosteroid injections are a first-line treatment for keloids; yet clinical treatment results are highly variable and often suboptimal. Variation in triamcinolone acetonide (TAC) biodistribution may be an important reason for the variable effects of TAC treatment in keloids. In this exploratory study we investigated the biodistribution of TAC in keloids and normal skin using different drug delivery techniques. Fluorescent-labeled TAC suspension was administered into keloids and normal skin with a hypodermic needle and an electronic pneumatic jet injector. TAC biodistribution was represented by the fluorescent TAC volume and 3D biodistribution shape of TAC, using a 3D-Fluorescence-Imaging Cryomicrotome System. Twenty-one keloid and nine normal skin samples were analyzed. With needle injections, the mean fluorescent TAC volumes were 990 µl ± 479 in keloids and 872 µl ± 227 in normal skin. With the jet injector, the mean fluorescent TAC volumes were 401 µl ± 252 in keloids and 249 µl ± 67 in normal skin. 3D biodistribution shapes of TAC were highly variable in keloids and normal skin. In conclusion, TAC biodistribution in keloids is highly variable for both needle and jet injection. This may partly explain the variable treatment effects of intralesional TAC in keloids. Future research is needed to confirm this preliminary finding and to optimize drug delivery in keloids.</p

PIEZO1 gain-of-function mutations delay reticulocyte maturation in hereditary xerocytosis

Dehydrated hereditary stomatocytosis 1 or hereditary xerocytosis (HX, OMIM 194380) is a rare hereditary autosomal dominant disorder characterized by hemolytic anemia and red blood cell (RBC) dehydration. The occurrence of HX is linked with gain-of-function mutations in PIEZO1, the gene encoding for the mechanosensitive non-specific cation channel PIEZO121 which is activated by shear-stress and in concert with other ion channels (particularly the Gardos potassium calcium-activated channel, KCNN4) regulates cell volume homeostasis and metabolic activity in the RBC.3 Intriguingly, PIEZO1 gain-of-function mutations have recently been reported to occur at a much higher frequency within the population than had been previously described and have also been implicated in malaria resistance,4 suggesting that the mechanisms underpinning HX may merit further investigation. Since reticulocytosis is one of the hallmarks of HX,5 we sought to determine whether altered reticulocyte maturation could be a causative agent of this phenotype. We characterize reticulocytes and erythrocytes from 10 HX patients in comparison to healthy controls, revealing alterations in deformability and vesicle content that implicate a maturational defect in HX. We further demonstrate that HX patients suffer from impaired reticulocyte maturation as assayed through differences in the extent and rate of loss of CD71 and RNA content over time and that this effect can be recapitulated in healthy reticulocytes upon chemically-induced PIEZO1 overactivation, providing a functional link to the reticulocytosis phenotype present in HX

Reticulocyte and red blood cell deformation triggers specific phosphorylation events

The capacity to undergo substantial deformation is a defining characteristic of the red blood cell (RBC), facilitating transit through the splenic interendothelial slits and microvasculature. Establishment of this remarkable property occurs during a process of reticulocyte maturation that begins with egress through micron-wide pores in the bone marrow and is completed within the circulation. The requirement to undertake repeated cycles of deformation necessitates that both reticulocytes and erythrocytes regulate membrane-cytoskeletal protein interactions in order to maintain cellular stability. In the absence of transcriptional activity, modulation of these interactions in RBCs is likely to be achieved primarily through specific protein posttranslational modifications, which at present remain undefined. In this study, we use high-throughput methods to define the processes that underlie the response to deformation and shear stress in both reticulocytes and erythrocytes. Through combination of a bead-basedmicrosphiltration assay with phosphoproteomics we describe posttranslational modification of RBC proteins associated with deformation. Using microsphiltration and microfluidic biochip-based assays, we explore the effect of inhibiting kinases identified using this dataset. We demonstrate roles for GSK3 and Lyn in capillary transit and maintenance of membrane stability following deformation and show that combined inhibition of these kinases significantly decreases reticulocyte capacity to undergo repeated deformation. Finally, we derive a comprehensive and integrative phosphoproteomic dataset that provides a valuable resource for further mechanistic dissection of the molecular pathways that underlie the RBC's response to mechanical stimuli and for the study of reticulocyte maturation

Non-muscle myosin ii drives vesicle loss during human reticulocyte maturation

The process of maturation of reticulocytes into fully mature erythrocytes that occurs in the circulation is known to be characterized by a complex interplay between loss of cell surface area and volume, removal of remnant cell organelles and redundant proteins, and highly selective membrane and cytoskeletal remodeling. However, the mechanisms that underlie and drive these maturational processes in vivo are currently poorly understood and, at present, reticulocytes derived through in vitro culture fail to undergo the final transition to erythrocytes. Here, we used high-throughput proteomic methods to highlight differences between erythrocytes, cultured reticulocytes and endogenous reticulocytes. We identify a cytoskeletal protein, non-muscle myosin IIA (NMIIA) whose abundance and phosphorylation status differs between reticulocytes and erythrocytes and localized it in the proximity of autophagosomal vesicles. An ex vivo circulation system was developed to simulate the mechanical shear component of circulation and demonstrated that mechanical stimulus is necessary, but insufficient for reticulocyte maturation. Using this system in concurrence with non-muscle myosin II inhibition, we demonstrate the involvement of non-muscle myosin IIA in reticulocyte remodeling and propose a previously undescribed mechanism of shear stress-responsive vesicle clearance that is crucial for reticulocyte maturation

Rapid diagnosis of hereditary haemolytic anaemias using automated rheoscopy and supervised machine learning

Haemolytic anaemias arise when red blood cell (RBC) integrity is compromised, eventually resulting in premature clearance or lysis and leading to anaemia when these effects cannot be sufficiently compensated by the capacity of the bone marrow to produce new cells. Hereditary anaemia occurs as a consequence of genetic mutation (e.g. affecting membrane complex or cytoskeletal proteins, haemoglobin or metabolic enzymes), and diagnosing affected patients is a complex process since, given the wide variety of possible genetic causes, multiple examinations must be performed and an unambiguous result is usually reached only after DNA sequencing. Furthermore, phenotypic severity can vary widely not just among individuals with different mutations but also among individuals suffering from the same mutation, thereby complicating diagnosis

Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing

Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassaemia or sickle-cell disease. With repeated transfusion, alloimmunisation often occurs due to incompatibility at the level of minor blood group antigens. We use CRISPR-mediated genome editing of an immortalised human erythroblast cell line (BEL-A) to generate multiple enucleation competent cell lines deficient in individual blood groups. Edits are combined to generate a single cell line deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (Bombay phenotype), Rh (Rh null ), Kell (K 0 ), Duffy (Fy null ), GPB (S−s−U−). These cells can be differentiated to generate deformable reticulocytes, illustrating the capacity for coexistence of multiple rare blood group antigen null phenotypes. This study provides the first proof-of-principle demonstration of combinatorial CRISPR-mediated blood group gene editing to generate customisable or multi-compatible RBCs for diagnostic reagents or recipients with complicated matching requirements

Translation of 3D Anatomy to 2D Radiographic Angle Measurements in the Ankle Joint: Validity and Reliability

Background: The objective consisted of 2 elements, primarily to define 2 bone geometry variations of the ankle that may be of prognostic value on ankle instability and secondly to translate these bone variations from a 3D model to a simple 2D radiographic measurement for clinical use. Methods: The 3D tibial and talar shape differences derived from earlier studies were translated to two 2D radiographic parameters: the medial malleolar height angle (MMHA) and talar convexity angle (TCA) respectively to ensure clinical use. To assess validity, the MMHA and TCA were measured on 3D polygons derived from lower leg computed tomographic (CT) scans and 2D digitally reconstructed radiographs (DRRs) of these polygons. To assess reliability, the MMHA and TCA were measured on standard radiographs by 2 observers calculating the intraclass correlation coefficient (ICC). Results: The 3D angle measurements on the polygons showed substantial to excellent agreement with the 2D measurements on DRR for both the MMHA (ICC 0.84-0.93) and TCA (ICC 0.88-0.96). The interobserver reliability was moderate with an ICC of 0.58 and an ICC of 0.64 for both the MMHA and TCA, respectively. The intraobserver reliability was excellent with an ICC of 0.96 and 0.97 for the MMHA and the TCA, respectively. Conclusion: Two newly defined radiographic parameters (MMHA and TCA) are valid and can be assessed with excellent intraobserver reliability on standard radiographs. The interobserver reliability was moderate and indicates training is required to ensure uniformity in measurement technique. The current method may be used to translate more variations in bone shape prior to implementation in clinical practice. Level of Evidence: Level III, cohort study

The accuracy of 3D virtual bone models of the pelvis for morphological sex estimation

It is currently unknown whether morphological sex estimation traits are accurately portrayed on virtual bone models, and this hampers the use of virtual bone models as an alternative source of contemporary skeletal reference data. This study determines whether commonly used morphological sex estimation traits can be accurately scored on virtual 3D pelvic bone elements. Twenty-seven intact cadavers from the body donation program of the Amsterdam UMC, University of Amsterdam, were CT scanned; this data was used to produce virtual bone models. Thereafter, the dry bones were obtained. Three traits by Klales (2012) and five traits from the Workshop of European Anthropologists (WEA) (1980) were scored on the virtual bone models and their dry skeletal counterparts. Intra- and inter-observer agreement and the agreement between the scores for each virtual bone model-dry bone pair were calculated using weighted Cohen’s kappa (K). For all Klales (2012) traits, intra- and inter-observer agreement was substantial to almost perfect for the virtual- and dry bones (K = 0.62–0.90). The agreement in scores in the virtual-dry bone pairs ranged from moderate to almost perfect (K = 0.58–0.82). For the WEA (1980) traits, intra-observer agreement was substantial to almost perfect (K = 0.64–0.91), but results were less unambiguous for inter-observer agreement (K = 0.24–0.88). Comparison of the scores between the virtual bone models and the dry bones yielded kappa values of 0.42–0.87. On one hand, clinical CT data is a promising source for contemporary forensic anthropological reference data, but the interchangeability of forensic anthropological methods between virtual bone models and dry skeletal elements needs to be tested further.http://link.springer.com/journal/414hj2020Anatom