Geometry effect in multi-step crossflow microfluidic devices for red blood cells separation and deformability assessment.

The efficient separation of blood components using microfluidic systems can help to improve the detection and diagnosis of several diseases, such as malaria and diabetes. Therefore, a novel multi-step microfluidic device, based on passive crossflow filters was developed. Three different designs were proposed, fabricated and tested in order to evaluate the most suitable geometry to perform, simultaneously, blood cells separation and cell deformability measurements. All the proposed geometries include a main channel and three sequential separation steps, all comprised of symmetrical crossflow filters, with multiple rows of pillars, to reduce the amount of red blood cells (RBCs) flowing to the outlets of the microfluidic device (MD). Sets of hyperbolic constrictions located at the outlets allow the assessment of cells deformability. Based on the proposed geometries, the three correspondent MD were evaluated and compared, by measuring the RBCs velocities, the cell-free layer (CFL) effect through the microchannels and by quantifying the amount of RBCs at the outlets. The results suggest that the proposed MD 3 configuration was the most effective one for the desired application, due to the formation of a wider CFL. As a result, a minor amount of RBCs flow through the hyperbolic contraction at the third separation level of the device. Nevertheless, for all the proposed geometries, the existence of three separation levels shows that it is possible to achieve a highly efficient cell separation. If needed, such microdevices have the potential for further improvements by increasing the number of separation levels, aiming the total separation of blood cells from plasma.

Hemoglobin-based oxygen carriers camouflaged with membranes extracted from red blood cells: Optimization and assessment of functionality.

Despite being an indispensable clinical procedure, the transfusion of donor blood has important limitations including a short shelf-life, limited availability and specific storage requirements. Therefore, a lot of effort has been devoted to developing hemoglobin (Hb)-based oxygen carriers (HBOCs) that are able to replace or complement standard blood transfusions, especially in extreme life-threatening situations. Herein, we employed a Hb-loaded poly(lactide-co-glycolide) core which was subsequently coated with nanozymes to protect the encapsulated Hb from oxidation by reactive oxygen species. To render HBOCs with long circulation in the vasculature, which is a crucial requirement to achieve the high oxygen demands of our organism, the carrier was coated with a red blood cell-derived membrane. Three coating methods were explored and evaluated by their ability to repel the deposition of proteins and minimize their uptake by an endothelial cell line. Preservation of the oxygen carrying capacity of the membrane-coated carrier was demonstrated by an oxygen-binding and releasing assay and, the functionality resulting from the entrapped nanozymes, was shown by means of superoxide radical anion and hydrogen peroxide depletion assays. All in all, we have demonstrated the potential of the membrane-coated nanocarriers as novel oxygen carrying systems with both antioxidant and stealth properties.

HEMATOLOGIC REFERENCE INTEVALS FOR SPIDER MONKEYS (ATELES GEOFFROYI) IN MANAGED CARE WITH RESPECT TO SEX AND AGE.

The determination of health status in spider monkeys (Ateles geoffroyi) that are held in managed care requires periodic evaluation. The present study obtained baseline data on hematologic values, body weight, and length in A. geoffroyi. A total of 118 individuals from three housing centers were included in the study and grouped into two categories by age (92 adults, 26 juveniles) and sex (46 males, 72 females). Body weight, red blood cell (RBC) counts, and hemoglobin counts were significantly higher in adult males than in adult females. No differences in length were found between sex and age groups. The present findings indicate that hematologic values by age and sex in A. geoffroyi are similar to those reported in other spider monkey populations in both managed care and the wild. These results will be useful as a reference for young individuals and adults in future studies of the health of this species.

Potential of cell tracking velocimetry as an economical and portable hematology analyzer.

Anemia and iron deficiency continue to be the most prevalent nutritional disorders in the world, affecting billions of people in both developed and developing countries. The initial diagnosis of anemia is typically based on several markers, including red blood cell (RBC) count, hematocrit and total hemoglobin. Using modern hematology analyzers, erythrocyte parameters such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), etc. are also being used. However, most of these commercially available analyzers pose several disadvantages: they are expensive instruments that require significant bench space and are heavy enough to limit their use to a specific lab and lead to a delay in results, making them less practical as a point-of-care instrument that can be used for swift clinical evaluation. Thus, there is a need for a portable and economical hematology analyzer that can be used at the point of need. In this work, we evaluated the performance of a system referred to as the cell tracking velocimetry (CTV) to measure several hematological parameters from fresh human blood obtained from healthy donors and from sickle cell disease subjects. Our system, based on the paramagnetic behavior that deoxyhemoglobin or methemoglobin containing RBCs experience when suspended in water after applying a magnetic field, uses a combination of magnets and microfluidics and has the ability to track the movement of thousands of red cells in a short period of time. This allows us to measure not only traditional RBC indices but also novel parameters that are only available for analyzers that assess erythrocytes on a cell by cell basis. As such, we report, for the first time, the use of our CTV as a hematology analyzer that is able to measure MCV, MCH, mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), the percentage of hypochromic cells (which is an indicator of insufficient marrow iron supply that reflects recent iron reduction), and the correlation coefficients between these metrics. Our initial results indicate that most of the parameters measured with CTV are within the normal range for healthy adults. Only the parameters related to the red cell volume (primarily MCV and RDW) were outside the normal range. We observed significant discrepancies between the MCV measured by our technology (and also by an automated cell counter) and the manual method that calculates MCV through the hematocrit obtained by packed cell volume, which are attributed to the artifacts of plasma trapping and cell shrinkage. While there may be limitations for measuring MCV, this device offers a novel point of care instrument to provide rapid RBC parameters such as iron stores that are otherwise not rapidly available to the clinician. Thus, our CTV is a promising technology with the potential to be employed as an accurate, economical, portable and fast hematology analyzer after applying instrument-specific reference ranges or correction factors.

Cost of transport, but not gluteus medius and red blood cells monocarboxylate-transporters density differentiated Brazilian Sport Horses at two performance levels.

Cost of transport (COT) and monocarboxylate transporters (MCTs) could affect the ability to perform fast actions during a jumping discipline. This study aimed to compare the COT and evaluate the MCT1, MCT4, and their auxiliary protein CD147 content in the gluteus medius and RBCs of Brazilian sport horses (BH), a breed developed for jumping competitions, with low-level (LL) or intermediate-level (IL) jumping capacities. The physiological difference between the horses was assessed by an incremental jump test (IJT), in which the cost of lactate (COTLAC) and heart rate (COTHR) of running were determined for each animal by the ratio between each variable and the running speed. Western blotting was performed on muscle and RBC membranes to quantify MCT1, MCT4, and CD147. IL showed lower COTLAC and COTHR than LL at all jumping heights. The amount of MCT1, MCT4, and CD147 found in muscle and RBCs were not dependent on performance level. Muscle MCT4 and MCT1 were correlated positively with CD147. We conclude that the relatively small differences between performances did not relevantly influence MCT expression in BH. While MCT analyses are inaccessible for most trainers and veterinarians, the cost of transport measurements is a feasible and sensitive tool to distinguish intermediate and low-level jumping horses.

Growth performance, hematological responses and economic indexes of Colossoma macropomum (Cuvier, 1818) fed graded levels of glycerol.

The aim of this study was to evaluate the performance, hematological responses and economic indicators of juvenile tambaqui Colossoma macropomum fed different levels of partial replacement of corn by glycerol (0%, 25%, 50%, 75%, and 100%). The experiment was conducted for 90 days in the production of aquatic organisms lab at the Nilton Lins University, Manaus, Brazil, in a completely randomized design, and consisted of four treatments, four repetitions and two sampling times. In total, 240 juveniles were used with initial average weight and standard length of 15.32 ± 1,61 g and 8.03 ± 0.22 cm, respectively. The fish were maintained in twenty 310 L water tanks that had a closed system with no reuse, continuous aeration, siphoning and replacement of water every 48 h. Feeding was twice a day with the experimental diets, which contained 28% crude protein. The following parameters were considered: zootechnical checks (weight gain + survival %), welfare (health) (hepatosomatic index + condition factor + viscerosomatic index), economic (Economic efficiency rate + economic profitability index) and hematology (hematology + metabolites + ions). The results of the study show that diets for juvenile tambaqui can contain up to 50% replacement of corn by glycerol without compromising the fishes' development under the conditions studied.

Microfluidic electrical impedance assessment of red blood cell-mediated microvascular occlusion.

Alterations in the deformability of red blood cells (RBCs), occurring in hemolytic blood disorders such as sickle cell disease (SCD), contribute to vaso-occlusion and disease pathophysiology. There are few functional in vitro assays for standardized assessment of RBC-mediated microvascular occlusion. Here, we present the design, fabrication, and clinical testing of the Microfluidic Impedance Red Cell Assay (MIRCA) with embedded capillary network-based micropillar arrays and integrated electrical impedance measurement electrodes to address this need. The micropillar arrays consist of microcapillaries ranging from 12 µm to 3 µm, with each array paired with two sputtered gold electrodes to measure the impedance change of the array before and after sample perfusion through the microfluidic device. We define RBC occlusion index (ROI) and RBC electrical impedance index (REI), which represent the cumulative percentage occlusion and cumulative percentage impedance change, respectively. We demonstrate the promise of MIRCA in two common red cell disorders, SCD and hereditary spherocytosis. We show that the electrical impedance measurement reflects the microvascular occlusion, where REI significantly correlates with ROI that is obtained via high-resolution microscopy imaging of the microcapillary arrays. Further, we show that RBC-mediated microvascular occlusion, represented by ROI and REI, associates with clinical treatment outcomes and correlates with in vivo hemolytic biomarkers, lactate dehydrogenase (LDH) level and absolute reticulocyte count (ARC) in SCD. Impedance measurement obviates the need for high-resolution imaging, enabling future translation of this technology for widespread access, portable and point-of-care use. Our findings suggest that the presented microfluidic design and the integrated electrical impedance measurement provide a reproducible functional test for standardized assessment of RBC-mediated microvascular occlusion. MIRCA and the newly defined REI may serve as an in vitro therapeutic efficacy benchmark for assessing the clinical outcome of emerging RBC-modifying targeted and curative therapies.

Thromboelastometry assessment of mycophenolate mofetil effect on thrombotic risk in dogs with primary and secondary immune-mediated hemolytic anemia and by Erhlichia canis infection / [Avaliação por tromboelastometria do efeito do micofenolato de mofetila no risco trombótico em cães com anemia hemolítica imunomediada primária e secundária à infecção por Erhlichia canis]

This study aimed to identify, by means of thromboelastometry assessment, altered thrombotic risk in dogs with primary and secondary IMHA by E. canis infection after initiating the immunosuppressive therapy with mycophenolate mofetil. The animals' screening was based on complete blood count (CBC), biochemical and urine tests. Dogs with moderate to severe anemia (hematocrit ≤ 25%) which showed symptoms of immune-mediated hemolysis, such as spherocytosis, positive saline agglutination, bilirubinuria and/or hemoglobinuria, were included. Blood and urine samples were collected at two different moments. The first sample (M1) was collected at the time of diagnosis, when hematocrit was lower or equal to 25% before treatment with mycophenolate mofetil (Accord ®); the second sample (M2) was collected after treatment with mycophenolate mofetil, when hematocrit was greater or equal to 30%. Five out of the twelve animals selected died before the end of the study. No reduction in thrombotic risk was observed in the animals treated with mycophenolate mofetil. The animals that presented hypocoagulation at the time of diagnosis showed the worst prognosis, and their reticulocyte count displayed a better prognostic value than their erythrocytes count at the time of diagnosis.(AU)

O objetivo deste estudo foi esclarecer se há alteração do risco trombótico em cães com anemia hemolítica imunomediada primária e secundária a E.canis, avaliado por meio da tromboelastometria, após início de tratamento com micofenolato de mofetila. A seleção dos animais foi baseada na avaliação de hemograma, exame bioquímico e urinálise. Cães com anemia moderada a severa (hematócrito ≤ 25%), com sinais de hemólise imunomediada, como esferocitose, aglutinação em salina positivo, bilirrubinúria e/ ou hemoglobinúria, foram incluídos. As amostras de sangue e urina foram coletadas em dois momentos diferentes. A primeira amostra (M1) foi coletada no momento do diagnóstico, quando o hematócrito era igual ou inferior a 25%, sem fazer uso do micofenolato de mofetila (Accord®), e o segundo momento (M2), após tratamento com micofenolato de mofetila, quando o hematócrito era igual ou maior que 30%. Doze animais foram selecionados, cinco morreram antes do término do estudo. Não houve diminuição do risco trombótico entre os animais tratados com micofenolato de mofetila; os animais que apresentaram menor coagulabilidade apresentaram pior prognóstico, e a contagem de reticulócitos apresentou melhor valor prognóstico do que a contagem de hemácias no momento do diagnóstico.(AU)

Features of modern clinical research blood analysis in the assessment of erythropoiesis in deep-premature newborns.

One of the urgent problems of nursing premature babies is the timely prediction, diagnosis and treatment of anemia, the frequency of which reaches 90%. Of particular importance is the study of reticulocytic blood parameters in deep-premature newborns, since the correct assessment of hemograms is crucial in the management of this category of children. To determine the characteristics of red blood cells and reticulocyte parameters of venous blood hemogram in premature newborns 24-32 weeks of gestation in the dynamics of the neonatal period. 111 newborns were examined at 24-32 weeks of gestation in the early neonatal period (on day 3-7) and at the age of 1 month of life. Along with standard diagnostic procedures, in accordance with current clinical recommendations and standards, 28 parameters of erythrocyte and reticulocyte hemogram parameters were determined for children. Venous blood was examined using an automatic hematological analyzer ADVIA 2120i, Siemens, USA. In deep-premature newborns in the early neonatal period, there is a high activity of erythropoiesis, respectively, the severity of respiratory and metabolic disorders with rejuvenation of reticulocytic subpopulations. Negative values of Delta hemoglobin were found against the background of a decrease in the average amount of hemoglobin in reticulocytes in children 24-27 weeks of gestation, which characterizes the lowest values of iron availability for erythropoiesis in this category of newborns. It is shown that a decrease in reticulocyte counts and normochromia in all examined newborns by the age of one month are accompanied by high levels of immature reticulocyte fraction, while a third of children still have limited iron availability for erythropoiesis.

Quality assessment of red blood cell suspensions derived from pathogen-reduced whole blood.

BACKGROUND: We used laboratory indicators to evaluate the quality of pathogen-reduced red blood cell suspension (RBCS) compared with gamma-irradiated RBCS. MATERIALS AND METHODS: To determine biochemical and metabolic parameters of RBCS, we obtained 50 whole blood units from healthy volunteers and randomized them into 2 groups: 25 were pathogen-reduced, and then, RBCS prepared from them. RBCS from the other 25 was gamma-irradiated. Sampling was carried out on day zero before and after treatment and at 7, 14, 21 and 28 days. To determine lymphocyte inactivation, we collected another 35 whole blood units. Each was sampled to form 3 study groups: untreated, gamma-irradiated and pathogen-reduced. Daily sampling was carried out during 3 days of storage. RESULTS: The quality of RBCS from both groups was largely the same, except for haemolysis and red blood cell fragility, which were more pronounced in the pathogen-reduced group. This finding limited the shelf life of pathogen-reduced RBCS to 14 days. Lymphocyte viability was significantly reduced after both treatments. Proliferation of lymphocytes after pathogen reduction was reduced to the detection limit, while low-level proliferation was observed in gamma-irradiated samples. CONCLUSION: Pathogen-reduced red blood cells have acceptable quality and can be used for transfusion within 14 days. Results of inactivation of lymphocytes demonstrate that pathogen reduction technology, applied on WB, can serve as an alternative to irradiation.

Vibration motor-integrated low-cost, miniaturized system for rapid quantification of red blood cell aggregation.

Human red blood cells (RBCs) aggregate under low shear conditions, which significantly modulates flow resistance and tissue perfusion. A higher aggregation tendency in blood thus serves as an important clinical indicator for the screening of cardiovascular disorders. Conventional ways of measuring RBC aggregation still require large sample volumes, cumbersome manual procedures, and expensive benchtop systems. These inconvenient and high-cost measurement methods hamper their clinical applicability. Here, we propose a low-cost, miniaturized system to overcome the limitations of these methods. Our system utilizes a coin vibration motor (CVM) to generate a localized vortex for disaggregating RBCs in a disposable fluidic chip. The design of the chip was optimized with fluid dynamics simulations to ensure sufficient shear flow in the localized vortex for RBC disaggregation. The time-dependent increase in light transmittance from an LED light source through the plasma gap while the RBCs re-aggregate is captured with a CMOS camera under stasis conditions to quantify the level of RBC aggregation. Our CVM-based aggregometer was validated against a commercial benchtop system for human blood samples under physiological and pathological conditions, and showed an excellent performance with a high intraclass correlation coefficient of 0.995. In addition, we were able to achieve a rapid measurement (<4 min) with the CVM-based aggregometer, requiring only a 6 µl blood sample. These illustrate the potential of our CVM-based aggregometer for low-cost point-of-care diagnostics without compromising the measurement sensitivity.

Assessment of hematological parameters of petrol filling workers at petrol stations in Gondar town, Northwest Ethiopia: a comparative cross-sectional study.

BACKGROUND: Petrol is the non-specific term for petroleum which is used for inside combustion of engines. Petrol filling workers are highly vulnerable to occupational exposure to these harmful substances which lead to hemato-toxicity and blood disorders such as leukemia, aplastic anemia, and dysplastic bone marrow. Thus, this study was aimed to assess hematological parameters of petrol filling workers in Gondar town, Northwest Ethiopia. METHODS: A comparative cross-sectional study was conducted from January to March 2019 in Gondar town, Northwest Ethiopia. A total of 110 study participants comprising 55 study groups and 55 controls group were recruited by a convenient sampling technique. Socio-demographic data were collected using a structured questionnaire, and 3 ml of venous blood was collected for the determination of hematological parameters. The data were entered into Epi info 7.2.0.1 and analyzed by SPSS version of 20. Mean, standard deviation, median, and interquartile ranges were used to present the data. Independent t test and Mann-Whitney U test were used to compare the mean or median difference between parametric and non-parametric hematological parameters, respectively. Moreover, Pearson product-moment and Spearman's rank-order bivariable correlations analyses were used to describe the correlation between hematological parameters and duration of exposure to petrol. A P value of ≤ 0.05 was considered statistically significant. RESULTS: The study revealed that mean red blood cell count and hemoglobin level as well as the median hematocrit, mean cell hemoglobin concentration, platelet count, absolute lymphocytes count, and red cell distribution width values of petrol filling workers showed a significant increment compared with the control group. On the other hand, the mean cell hemoglobin value of petrol filling workers showed a significant decrement compared with healthy controls. Moreover, the duration of exposure to petrol showed a significant positive correlation with red blood cell count and mean cell hemoglobin concentration; however, a significant negative correlation was observed with mean cell volume. CONCLUSION: This study showed that the majority of hematological parameters of petrol filling workers showed an increment compared with healthy controls which might be associated with exposure to petrol chemicals. However, further longitudinal study with a larger sample size should be conducted to explore the impact of petrol exposure on hematopoiesis.

The results of external quality assessment programme on urine leukocyte and erythrocyte counting in Poland.

INTRODUCTION: Urine particle analysis is an important diagnostic tool. The aim of this study was to evaluate the quality of urine leukocyte (WBC) and erythrocyte (RBC) counting results obtained with manual and automated methods in Polish laboratories participating in the international external quality assessment (EQA) programme. MATERIALS AND METHODS: 1400 WBC and RBC counting results were obtained from 183 laboratories in EQA surveys organised by Labquality (Helsinki, Finland) from 2017 to 2019. The between-laboratory coefficient of variation (CV), the percentage difference between the laboratories' results and target values (Q-score (%)), as well as modified Youden plots were analysed. RESULTS: For automated method groups, the medians of inter-laboratory CVs varied from 14% to 33% for WBC counting and from 10% to 39% for RBC counting. For manual method groups, the medians of CV varied from 53% to 71% (WBC) and from 55% to 70% (RBC), and they were significantly higher, in comparison to CVs for most automated method groups (P < 0.001). The highest percentage of results outside the target limits (36%) and the highest range of Q-score (%) (from - 93% to 706%) were observed for laboratories which participated in the surveys for the first or second time. The percentage of deviating results and the ranges of Q-score decreased with an increased frequency of laboratories' participation in the surveys. CONCLUSIONS: The quality of manual methods of urine WBC and RBC counting is unsatisfactory. There is an urgent need to take actions to improve laboratories' performance and to increase harmonisation of the results.

Cost-effectiveness evaluation of the PROPPR trial transfusion protocols.

BACKGROUND: There have been no prior investigations of the cost effectiveness of transfusion strategies for trauma resuscitation. The Pragmatic, Randomized, Optimal Platelet and Plasma Ratios (PROPPR) study was a Phase III multisite, randomized trial in 680 subjects comparing the efficacy of 1:1:1 transfusion ratios of plasma and platelets to red blood cells with the 1:1:2 ratio. We hypothesized that 1:1:1 transfusion results in an acceptable incremental cost-effectiveness ratio, when estimated using patients' age-specific life expectancy and cost of care during the 30-day PROPPR trial period. STUDY DESIGN AND METHODS: International Classification of Diseases, Ninth Revision codes were prospectively collected, and subjects were matched 1:2 to subjects in the Healthcare Utilization Program State Inpatient Data to estimate cost weights. We used a decision tree analysis, combined with standard costs and estimated years of expected survival to determine the cost effectiveness of the two treatments. RESULTS: The 1:1:1 group had higher overall costs for the blood products but were more likely to achieve hemostasis and decreased hemorrhagic death by 24 hours (p = 0.006). For every 100 patients treated in the 1:1:1 group, eight more achieved hemostasis than in the 1:1:2 group. At 30 days, the total hospital cost per 100 patients was $5.6 million in the 1:1:1 group compared with $5.0 million in the 1:1:2 group. For each 100 patients, the 1:1:1 group had 218.5 more years of life expectancy. This was at a cost of $2994 per year gained. CONCLUSION: The 1:1:1 transfusion ratio in severely injured hemorrhaging trauma patients is a very cost-effective strategy for increasing hemostasis and decreasing trauma deaths.

Assessment of oxygenation with polarized light spectroscopy enables new means for detecting vascular events in the skin.

INTRODUCTION: Impaired oxygenation in the skin may occur in disease states and after reconstructive surgery. We used tissue viability imaging (TiVi) to measure changes in oxygenation and deoxygenation of haemoglobin in an in vitro model and in the dermal microcirculation of healthy individuals. MATERIALS AND METHODS: Oxygenation was measured in human whole blood with different levels of oxygenation. In healthy subjects, changes in red blood cell concentration (CRBC,TiVi), oxygenation (ΔCOH,TiVi) and deoxygenation (ΔCDOH,TiVi) of haemoglobin were measured during and after arterial and venous occlusion using TiVi and were compared with measurements from the enhanced perfusion and oxygen saturation system (EPOS). RESULTS: During arterial occlusion, CRBC,TiVi remained unchanged while ΔCOH,TiVi decreased to -44.2 (10.4) AU (p = 0.04), as compared to baseline. After release, CRBC,TiVi increased to 39.2 (18.8) AU (p < 0.001), ΔCOH,TiVi increased to 38.5. During venous occlusion, CRBC,TiVi increased to 28.9 (11.2) AU (p < 0.001), ΔCOH,TiVi decreased to -52.2 (46.1) AU (p < 0.001) compared to baseline after 5 min of venous occlusion. There was a significant correlation between the TiVi Oxygen Mapper and EPOS, for arterial (r = 0.92, p < 0.001) and venous occlusion (r = 0.87, p < 0.001), respectively. CONCLUSION: This study shows that TiVi can measure trends in oxygenation and deoxygenation of haemoglobin during arterial and venous stasis in healthy individuals.

A low-cost, accurate method for detecting reticulocytes at different maturation stages based on changes in the mitochondrial membrane potential.

In the clinical setting, reticulocytes are used as an index for the hematopoietic function of the bone marrow. Different maturation stages of reticulocytes are early markers for bone marrow hematopoietic stem cell transplantation and bone marrow regeneration after chemotherapy. Therefore, we aimed to establish a method for detecting the different reticulocyte maturation stages. Based on the decreases in mitochondrial membrane potential during reticulocyte maturation, we used MitoTracker Green (MTG)/tetramethylrhodamine, ethylester (TMRE) to identify the different reticulocyte maturation stages and used Hoechst33342 to exclude nucleated cells. The results show that this method was universal and could be applied to detect the proportions of reticulocytes in different samples. Their proportion in normal peripheral blood, a blood deficiency model, bone marrow, and spleen were (6 ± 2)%, (38 ± 4)%, (14 ± 4)%, and (3 ± 1)%, respectively. The results obtained using this method were similar to those obtained using the manual counting method (methylene blue); the correlation was good (R = 0.817; p < .01) and the coefficient of variation was lower for the method established. Moreover, reticulocytes in peripheral blood could be further divided into three distinct maturation stages: R1 (MTGneg/TMREhigh), R2 (MTGhigh/TMREhigh), and R3 (MTGhigh/TMREneg). Reticulocytes in the bone marrow and spleen could be further divided into four distinct maturation stages: R1 (MTGneg/TMREhigh), R2-1 (MTGhigh/TMREhigh/FSbig), R2-2 (MTGhigh/TMREhigh/FSsmall), and R3 (MTGhigh/TMREneg). Based on changes in mitochondrial membrane potential, MTG/TMRE/Hoechst33342 staining could be used to detect reticulocytes in different samples and at different maturation stages with low cost and high accuracy.

Utility of HbA1c assessment in people with diabetes awaiting liver transplantation.

AIMS: To investigate the relationship between HbA1c and glucose in people with co-existing liver disease and diabetes awaiting transplant, and in those with diabetes but no liver disease. METHODS: HbA1c and random plasma glucose data were collected for 125 people with diabetes without liver disease and for 29 people awaiting liver transplant with diabetes and cirrhosis. Cirrhosis was caused by non-alcoholic fatty liver disease, hepatitis C, alcoholic liver disease, hereditary haemochromatosis, polycystic liver/kidneys, cryptogenic/non-cirrhotic portal hypertension and α-1-antitrypsin-related disease. RESULTS: The median (interquartile range) age of the diabetes with cirrhosis group was 55 (49-63) years compared to 60 (50-71) years (P=0.13) in the group without cirrhosis. In the diabetes with cirrhosis group there were 21 men (72%) compared with 86 men (69%) in the group with diabetes and no cirrhosis (P=0.82). Of the group with diabetes and cirrhosis, 27 people (93%) were of white European ethnicity, two (7%) were South Asian and none was of Afro-Caribbean/other ethnicity compared with 94 (75%), 16 (13%), 10 (8%)/5 (4%), respectively, in the group with diabetes and no cirrhosis (P=0.20). Median (interquartile range) HbA1c was 41 (32-56) mmol/mol [5.9 (5.1-7.3)%] vs 61 (52-70) mmol/mol [7.7 (6.9-8.6)%] (P<0.001), respectively, in the diabetes with cirrhosis group vs the diabetes without cirrhosis group. The glucose concentrations were 8.4 (7.0-11.2) mmol/l vs 7.3 (5.2-11.5) mmol/l (P=0.17). HbA1c was depressed by 20 mmol/mol (1.8%; P<0.001) in 28 participants with cirrhosis but elevated by 28 mmol/mol (2.6%) in the participant with α-1-antitrypsin disorder. Those with cirrhosis and depressed HbA1c had fewer larger erythrocytes, and higher red cell distribution width and reticulocyte count. This was reflected in the positive association of glucose with mean cell volume (r=0.39) and haemoglobin level (r=0.49) and the negative association for HbA1c (r=-0.28 and r=-0.26, respectively) in the diabetes group with cirrhosis. CONCLUSION: HbA1c is not an appropriate test for blood glucose in people with cirrhosis and diabetes awaiting transplant as it reflects altered erythrocyte presentation.

High-throughput assessment of mechanical properties of stem cell derived red blood cells, toward cellular downstream processing.

Stem cell products, including manufactured red blood cells, require efficient sorting and purification methods to remove components potentially harmful for clinical application. However, standard approaches for cellular downstream processing rely on the use of specific and expensive labels (e.g. FACS or MACS). Techniques relying on inherent mechanical and physical properties of cells offer high-throughput scalable alternatives but knowledge of the mechanical phenotype is required. Here, we characterized for the first time deformability and size changes in CD34+ cells, and expelled nuclei, during their differentiation process into red blood cells at days 11, 14, 18 and 21, using Real-Time Deformability Cytometry (RT-DC) and Atomic Force Microscopy (AFM). We found significant differences (p < 0.0001; standardised mixed model) between the deformability of nucleated and enucleated cells, while they remain within the same size range. Expelled nuclei are smaller thus could be removed by size-based separation. An average Young's elastic modulus was measured for nucleated cells, enucleated cells and nuclei (day 14) of 1.04 ± 0.47 kPa, 0.53 ± 0.12 kPa and 7.06 ± 4.07 kPa respectively. Our identification and quantification of significant differences (p < 0.0001; ANOVA) in CD34+ cells mechanical properties throughout the differentiation process could enable development of new routes for purification of manufactured red blood cells.

Quantitative fetomaternal hemorrhage assessment with the use of five laboratory tests.

INTRODUCTION: In various countries, standard doses of anti-D IgG used for postpartum immunoprophylaxis of hemolytic disease of fetus and newborn (HDFN) vary from 100 µg to 300 µg. There are also different regulations concerning FMH assessment, and opinions about applicable tests are inconclusive. METHODS: Three flow cytometry tests (FCTs) with anti-D, anti-HbF, anti-HbF+CA antibodies, and two modifications of microscopic Kleihauer-Betke test (KBT) were used. RESULTS: In all artificial mixtures with known concentrations, FCTs and KBT with counting 10 000 RBCs had similar satisfying sensitivity and specificity. KBT with counting 2000 RBCs had to be disqualified because of significant discrepancies between expected and measured values of FMH. The test procedure with anti-D was easier and shorter than the remaining tests, but it can be only used for FMH assessment in RhD-negative mothers with RhD-positive newborns. In one clinical sample, it was impossible to distinguish fetal RBCs from maternal F cells in KBT and FC with anti-HbF but other tests were useful. CONCLUSION: In the four tests, correlation between expected and obtained results was appropriate (CCC Ì´1). Each test had some advantage and limitation in any clinical situation. Therefore, it is best to have opportunity to perform two or three assays in the laboratory.