Determination of Thiopurine S-Methyltransferase (TPMT) Activity in Human Erythrocytes by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the metabolism of thiopurine medications that are used in the treatment of multiple malignant and nonmalignant immunologic conditions. Polymorphisms in the TPMT gene associated with low enzyme activity can produce pronounced pharmacologic effects during therapy. The determination of TPMT erythrocyte activity is a valuable adjunct test to genotyping for the assignment of TPMT phenotype, especially in the presence of indeterminate genotypes. We present a method for the determination TPMT activity in human erythrocytes whereby we utilize LC-MS/MS to measure the amount of 6-methylmercaptopurine formed in red blood cell (RBC) lysates using 6-mercaptopurine as substrate and S-adenosylmethionine (SAM) as methyl donor. This method has been successfully implemented and proven to be reliable for use in human specimens.

Quantification of Thiopurine Metabolites in Human Erythrocytes by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

The thiopurine drugs, azathioprine, mercaptopurine, and thioguanine, are widely used in the treatment of several malignant and nonmalignant diseases. These inactive prodrugs undergo extensive metabolism to form active cytotoxic metabolites, which act mainly by incorporating into DNA and affecting cell replication. Thiopurine methyltransferase is a highly variable cytosolic enzyme that catalyzes the S-methylation of the thiopurine bases-an inactivating pathway. Patients with low-activity variants of TPMT can be affected by pronounced pharmacologic effects when receiving thiopurine medications. Clinical studies have reported significant interpatient variability in intracellular thiopurine metabolite concentrations in patients receiving thiopurine therapy. In this chapter, we present an LC-MS/MS method to monitor the thiopurine metabolites: 6-thioguanine nucleotides and 6-methylmercaptopurine derivatives in human erythrocytes. This method utilizes acid hydrolysis to release the bases and improves upon previously published procedures by utilizing stable isotope internal standards and a more efficient chromatographic separation.

Oxidative and osmotolerant effects in Salvator merianae (Squamata: Teiidae) red blood cells during hibernation / Efeitos de oxidação e osmotolerância dos eritrócitos de Salvator merianae (Squamata: Teiidae) durante a hibernação

Abstract Hibernation is a natural condition of animals that lives in the temperate zone, although some tropical lizards also experience hibernation annually, such as the lizard native from South America, Salvator merianae, or "tegu" lizard. Even though physiological and metabolic characteristic associated with hibernation have been extensively studied, possible alterations in the red blood cells (RBC) integrity during this period remains unclear. Dehydration and fasting are natural consequences of hibernating for several months and it could be related to some cellular modifications. In this study, we investigated if the osmotic tolerance of RBCs of tegu lizard under hibernation is different from the cells obtained from animals while normal activity. Additionally, we indirectly investigated if the RBCs membrane of hibernating tegus could be associated with oxidation by quantifying oxidized biomolecules and the activity of antioxidant enzymes. Our findings suggest that RBCs are more fragile during the hibernation period, although we did not find evidence of an oxidative stress scenario associated with the accentuated fragility. Even though we did not exclude the possibility of oxidative damage during hibernation, we suggested that an increased RBCs volume as a consequence of hypoosmotic blood during hibernation could also affect RBCs integrity as noted.

Resumo A hibernação é uma condição natural dos animais que vivem na zona temperada, embora alguns lagartos tropicais também experenciem hibernação anualmente, como é o caso do lagarto nativo da América do Sul, Salvator merianae ou "teiú". Embora as características fisiológicas e metabólicas associadas à hibernação tenham sido amplamente estudadas, possíveis alterações na integridade das hemácias durante esse período ainda permanecem obscuras. A desidratação e o jejum são consequências naturais da hibernação por vários meses e podem estar relacionadas a algumas modificações celulares. Neste estudo, investigamos se a tolerância osmótica de hemácias do lagarto teiú sob hibernação são diferentes das células obtidas de animais em atividade normal. Além disso, investigamos indiretamente por meio da quantificação de biomoléculas oxidadas e da atividade de enzimas antioxidantes se a membrana das hemácias dos teiús em hibernação poderia estar associada à oxidação. Nossos resultados sugerem que as hemácias possuem maior fragilidade durante o período de hibernação, embora não tenhamos encontrado evidências de um cenário de estresse oxidativo associado à essa fragilidade acentuada. Embora não tenhamos excluído a possibilidade de dano oxidativo durante a hibernação, sugerimos que um aumento no volume das hemácias como consequência de sangue hipoosmótico durante a hibernação também poderia afetar a integridade de hemácias, tal como foi observado.

Genotoxic and cytotoxic evaluation of venlafaxine in an acute and a subchronic assay in mouse / Avaliação genotóxica e citotóxica da venlafaxina em ensaio agudo e subcrônico em camundongos

Abstract The present research was made to determine the micronuclei and cytotoxic capacity of the antidepressant venlafaxine in an in vivo acute and subchronic assays in mouse. In the first study, we administered once 5, 50, and 250 mg/kg of the drug, and included a negative and a daunorubicin treated group. Observations were daily made during four days. The subchronic assay lasted 5 weeks with daily administration of venlafaxine (1, 5, and 10 mg/kg) plus a negative and an imipramine administered groups. Observations were made each week. In the first assay results showed no micronucleated polychromatic erythrocytes (MNPE) increase, except with the high dose at 72 h. The strongest cytotoxic effect was found with 250 mg/kg at 72 h (a 51% cytotoxic effect in comparison with the mean control level). In the subchronic assay no MNPE increase was found; however, with the highest dose a significant increase of micronucleated normochromatic erythrocytes was observed in the last three weeks (a mean of 51% respect to the mean control value). A cytotoxic effect with the two high doses in the last two weeks was observed (a polychromatic erythrocyte mean decrease of 52% respect to the mean control value). Results suggest caution with venlafaxine.

Resumo A presente pesquisa foi feita para determinar a capacidade micronuclei e citotóxica do antidepressivo venlafaxina em ensaios agudos e subcrônicos in vivo em camundongos. No primeiro estudo, administramos uma vez 5, 50 e 250 mg/kg do medicamento e incluímos um grupo negativo e um grupo tratado com daunorubicina. As observações foram feitas diariamente durante quatro dias. O ensaio subcrônico durou cinco semanas com administração diária de venlafaxina (1, 5, e 10 mg/kg) mais um grupo negativo e um grupo administrado de imipramina. As observações foram feitas a cada semana. No primeiro ensaio, os resultados não mostraram aumento de eritrócitos policromáticos micronucleados (MNPE), exceto com a dose elevada a 72 h. O efeito citotóxico mais forte foi encontrado com 250 mg/kg a 72 h (um efeito citotóxico de 51% em comparação com o nível médio de controle). No ensaio subcrônico não foi encontrado aumento de MNPE; entretanto, com a dose mais alta, um aumento significativo de eritrócitos normocromáticos micronucleados foi observado nas últimas três semanas (média de 51% em relação ao valor médio de controle). Foi observado um efeito citotóxico com as duas altas doses nas últimas duas semanas (uma diminuição média de 52% em relação ao valor médio de controle dos eritrócitos policromáticos). Os resultados sugerem cautela com a venlafaxina.

Diagnosis of diabetes mellitus using high frequency ultrasound and convolutional neural network.

The incidence of diabetes mellitus has been increasing, prompting the search for non-invasive diagnostic methods. Although current methods exist, these have certain limitations, such as low reliability and accuracy, difficulty in individual patient adjustment, and discomfort during use. This paper presents a novel approach for diagnosing diabetes using high-frequency ultrasound (HFU) and a convolutional neural network (CNN). This method is based on the observation that glucose in red blood cells (RBCs) forms glycated hemoglobin (HbA1c) and accumulates on its surface. The study incubated RBCs with different glucose concentrations, collected acoustic reflection signals from them using a custom-designed 90-MHz transducer, and analyzed the signals using a CNN. The CNN was applied to the frequency spectra and spectrograms of the signal to identify correlations between changes in RBC properties owing to glucose concentration and signal features. The results confirmed the efficacy of the CNN-based approach with a classification accuracy of 0.98. This non-invasive diagnostic technology using HFU and CNN holds promise for in vivo diagnosis without the need for blood collection.

New Insights into Clinical Management for Sickle Cell Disease: Uncovering the Significant Pathways Affected by the Involvement of Sickle Cell Disease.

One of the severe monogenic conditions with the highest prevalence in the globe is sickle cell disease. Although the significance of chronic anemia, hemolysis, and vasculopathy has been established, hemoglobin polymerization, which results in erythrocyte stiffness and Vaso-occlusion, is important to the pathophysiology of this disease. Clinical management is elementary, and there is scant reliable data for many treatments. The onset of cerebrovascular illness and cognitive impairment are two of the major issues associated with sickle cell disease in children, and it is only now that researchers are beginning to understand how blood transfusions and hydroxycarbamide can prevent these complications. When Vaso occlusion and inflammation occur repeatedly, the majority of organs are gradually damaged, including the brain, kidneys, lungs, bones, and cardiovascular system. This damage worsens with age. In our study, we focused on the specific pathways which are affected by the involvement of effected genes. Firstly, we retrieved the gene datasets from the publically available data source website DisGNET. Using literature-based genes, we identified 290 highly regulated genes that are directly associated with sickle cell disease. We subsequently performed a gene expression analysis and extracted a gene set using GEO2R analysis, which was then used to prune 290 differentially expressed genes (DEGs). After pruning we got 60 highly expressed genes. After identification of DEGs, we used these genes for pathway analysis. For the pathway analysis, we used Reactome software and we found that these DEGs are directly associated with 7 different pathways, which are alpha beta signaling pathways, 15 antiviral mechanism, Oligoadenylate synthetase (OAS) antiviral response, interleukin 1 signaling pathways, interleukin 4 and 13, interleukin 10 signaling pathway, and aspirin ADME pathway. After pathway analysis, we can exactly relate how sickle cell disease alters the gene expression and how these genes affect the different pathways. Additionally, we performed gene ontology of 60 genes and identified the gene biological process, cellular component, and molecular functions as we mentioned in our results. With the help of our study data, there is a chance for pre-identification of sickle cell disease person. Our gene result was used as a biomarker of sickle cell disease. In this paper, our result is the primary approach for sickle cell disease; with the help of this paper any researcher can get their primary data and use that for further research.

Multielement analysis of single red blood cells by single cell - inductively coupled plasma tandem mass spectrometry.

A method for the analysis of essential metals (Fe, Cu, Mg, and Zn) and non-metals (P, S) in single red blood cells was developed by single cell (SC)-ICP-MS. The use of a triple quadrupole configuration (MS/MS) enabled an effective elimination of polyatomic interferences, which affect the accuracy of ICP-MS analysis using a single quadrupole mass analyzer. Fixation with glutaraldehyde for at least 90 days was developed to improve the quantification of elements in a single red blood cell. The experimental conditions were optimized while special attention was paid to the residence time of analytes in the plasma. Addition of a surfactant (0.05% (v/v) Tween80®) improved quantification of elements in fixed red blood cells. The detection limits obtained by SC-ICP-MS/MS were lower than for ICP-MS, especially for S and P (3 fg and 1.7 fg. cell-1 instead of 163 and 6.3 fg. cell-1, respectively).

Association of hemoglobin-to-red blood cell distribution width ratio and depression in older adults: A cross sectional study.

BACKGROUND: The association between hemoglobin-to-red blood cell distribution width ratio (HRR) and the depression in old adults was not clear. METHODS: We extracted data on depression, general characteristics, lifestyle, medical history, drug use, and blood indicators from the National Health and Nutrition Examination Survey 2005-2018 to investigate the relationship between HRR and depression. RESULTS: A total of 4141 individuals were evaluated, among whom 266 (6.4 %) were identified as having depression. HRR was significantly lower in the low depression group, and Spearman correlation analysis revealed an inverse association between HRR and depression scores (r = -0.148, P < 0.001). Multiple linear regression showed that HRR was associated with depression after adjusted for general characteristics, life style, medical history, drug use and blood indicators (P = 0.010). ROC analysis demonstrated that in participants with depression, the area under the curve (AUC) for HRR was 0.612, surpassing both Hb(0.586) and RDW(0.401). These findings were statistically significant (P < 0.05). LIMITATIONS: Only participants aged 65-79 years are selected for this study and this was a cross-sectional study that can only represent an association between HRR and depression, but not a cause-and-effect relationship. CONCLUSIONS: HRR, being more potent than Hb or RDW, emerges as an independent risk factor for depression. It has the potential to facilitate early depression detection, aiding in the prevention of clinical deterioration or relapses, and could also serve as a viable treatment target.

DIA-based serum proteomics revealed the protective effect of modified siwu decoction against hypobaric hypoxia.

ETHNOPHARMACOLOGICAL RELEVANCE: Siwu decoction (SWD) is a common traditional formula for nourishing blood, and its derived formulas are also widely used in traditional Chinese medicine (TCM) clinic. However, the protective effects of SWD and its derived formulas on blood deficiency and blood stasis caused by rushing to the plateau are rarely reported, and the underlying mechanism has not been fully elucidated. AIM OF THE STUDY: This study explores the pharmacological effects and mechanisms of modified siwu decoction (MSWD) adding Persicae Semans (Prunus persica (L.) Batsch) and Carthami Flos (Carthamus tinctorius L.) against hypobaric hypoxia (HH). The acute toxicity of MSWD was also evaluated to further validate the potential of MSWD as a therapeutic candidate for HH. MATERIALS AND METHODS: Hypoxic models of C57BL/6 J and KM male mice were used to evaluate the pharmacological effect of MSWD. 2 µL serum sample of C57BL/6 J mice was digested into peptide mixtures and analyzed with DIA mode on an Orbitrap Fusion Lumos mass spectrometer after LC separation. The peptide and protein identifications were limited to a 1% FDR. Screening of differential expressed proteins, correlation analysis, hierarchical clustering analysis, principal components analysis and Mfuzz analysis were all performed by R packages. The protein-protein interaction network was analyzed using the STRING website and constructed with Cytoscape software. RESULTS: MSWD showed a protective effect against acute hypoxia exposure through increasing the number of red blood cells and improving hemodynamics indexes in mice. Meanwhile, the biochemical results showed that MSWD could reduce the inflammation and oxidative stress, reduce the content of organ injury biomarkers and significantly improve the high-intensity exercise ability of mice. Subsequently, serum DIA proteomic results revealed significant changes in proteomic characteristics after MSWD intervention. Specifically, proteins related to oxidative stress and ubiquitin-proteasome system, such as Sod1, Gstp1, Vcp and Usp14, were down-regulated after MSWD intervention, suggesting that the protective effect of MSWD involved the reduction of oxidative stress and energy expenditure. MSWD also intervened in energy metabolism and lipid metabolism processes by altering the expression levels of Eno1, Sphk1 and Apoa1 to ameliorate hypoxia-induced disorders. At the same time, MSWD acute toxicity test showed no obvious toxicity. CONCLUSIONS: MSWD has a good protective effect against HH by ameliorating hypoxia-induced disorders of energy and lipid metabolism, supporting MSWD as a safe drug candidate for the prevention and treatment of acute hypoxia fatigue.

Cellular Blood Flow Modeling with HemoCell.

Many of the intriguing properties of blood originate from its cellular nature. Bulk effects, such as viscosity, depend on the local shear rates and on the size of the vessels. While empirical descriptions of bulk rheology are available for decades, their validity is limited to the experimental conditions they were observed under. These are typically artificial scenarios (e.g., perfectly straight glass tube or in pure shear with no gradients). Such conditions make experimental measurements simpler; however, they do not exist in real systems (i.e., in a real human circulatory system). Therefore, as we strive to increase our understanding on the cardiovascular system and improve the accuracy of our computational predictions, we need to incorporate a more comprehensive description of the cellular nature of blood. This, however, presents several computational challenges that can only be addressed by high performance computing. In this chapter, we describe HemoCell ( https://www.hemocell.eu ), an open-source high-performance cellular blood flow simulation, which implements validated mechanical models for red blood cells and is capable of reproducing the emergent transport characteristics of such a complex cellular system. We discuss the accuracy and the range of validity, and demonstrate applications on a series of human diseases.

Association of red blood cell distribution width to albumin ratio with the prognosis of acute severe pulmonary embolism: A cohort study.

Red blood cell distribution width (RDW) to human serum albumin (ALB) ratio (RDW/ALB Ratio, RAR) is a prognostic factor for adverse outcomes in different disease populations. However, the relationship between RAR and pulmonary embolism outcomes remains unclear. Therefore, this study set out to investigate the association between RAR and the risk of all-cause death in acute pulmonary embolism (APE) patients admitted to the intensive care unit (ICU). This is a retrospective study based on the MIMIC-IV database. The primary outcome was all-cause mortality among patients with APE (in-hospital and 1-year mortality). The relationship between RAR and all-cause mortality was assessed using Cox regression analysis. The survival curve was drawn to evaluate the predictive value of RAR for patient mortality. Correlations and threshold effects between RAR and all-cause mortality were analyzed using the generalized additive model (GAM). The study included 773 patients, and fully adjusted Cox regression models showed that RAR was associated with higher all-cause mortality in the hospital and one year later (all P < .05). In the GAM, the relationship between RAR and all-cause mortality was shown to be nonlinear, with a positive association between RAR and all-cause mortality in APE patients when RAR values were at low to moderate levels. This study revealed a significant association between RAR and the risk of all-cause day death in patients with pulmonary embolism. Higher RAR value was associated with increased in-hospital mortality and 1-year mortality.

Marginated aberrant red blood cells induce pathologic vascular stress fluctuations in a computational model of hematologic disorders.

Red blood cell (RBC) disorders such as sickle cell disease affect billions worldwide. While much attention focuses on altered properties of aberrant RBCs and corresponding hemodynamic changes, RBC disorders are also associated with vascular dysfunction, whose origin remains unclear and which provoke severe consequences including stroke. Little research has explored whether biophysical alterations of RBCs affect vascular function. We use a detailed computational model of blood that enables characterization of cell distributions and vascular stresses in blood disorders and compare simulation results with experimental observations. Aberrant RBCs, with their smaller size and higher stiffness, concentrate near vessel walls (marginate) because of contrasts in physical properties relative to normal cells. In a curved channel exemplifying the geometric complexity of the microcirculation, these cells distribute heterogeneously, indicating the importance of geometry. Marginated cells generate large transient stress fluctuations on vessel walls, indicating a mechanism for the observed vascular inflammation.

Comparative spatial proteomics of Plasmodium-infected erythrocytes.

Plasmodium parasites contribute to one of the highest global infectious disease burdens. To achieve this success, the parasite has evolved a range of specialized subcellular compartments to extensively remodel the host cell for its survival. The information to fully understand these compartments is likely hidden in the so far poorly characterized Plasmodium species spatial proteome. To address this question, we determined the steady-state subcellular location of more than 12,000 parasite proteins across five different species by extensive subcellular fractionation of erythrocytes infected by Plasmodium falciparum, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. This comparison of the pan-species spatial proteomes and their expression patterns indicates increasing species-specific proteins associated with the more external compartments, supporting host adaptations and post-transcriptional regulation. The spatial proteome offers comprehensive insight into the different human, simian, and rodent Plasmodium species, establishing a powerful resource for understanding species-specific host adaptation processes in the parasite.

Exploration of a Novel Noninvasive Prenatal Testing Approach for Monogenic Disorders Based on Fetal Nucleated Red Blood Cells.

BACKGROUND: Due to technical issues related to cell-specific capture methods, amplification, and sequencing, noninvasive prenatal testing (NIPT) based on fetal nucleated red blood cells (fNRBCs) has rarely been used for the detection of monogenic disorders. METHODS: Maternal peripheral blood was collected from 11 families with hereditary hearing loss. After density gradient centrifugation and cellular immunostaining for multiple biomarkers, candidate individual fetal cells were harvested by micromanipulation and amplified by whole-genome amplification (WGA). Whole-exome sequencing/whole-genome sequencing (WGS) and Sanger sequencing were performed on the identified fNRBCs to determine the fetal genotype. The impact of single-cell and pooled WGA products on the sequencing quality and results was compared. A combined analysis strategy, encompassing whole-exome sequencing/WGS, haplotype analysis, and Sanger sequencing, was used to enhance the NIPT results. RESULTS: fNRBCs were harvested and identified in 81.8% (9/11) of families. The results of cell-based-NIPT (cb-NIPT) were consistent with those of invasive prenatal diagnosis in 8 families; the coincidence rate was 88.9% (8/9). The combined analysis strategy improved the success of cb-NIPT. The overall performance of pooled WGA products was better than that of individual cells. Due to a lack of alternative fetal cells or sufficient sequencing data, cb-NIPT failed in 3 families. CONCLUSIONS: We developed a novel fNRBC-based NIPT method for monogenic disorders. By combining multiple analysis strategies and multiple fetal cell WGA products, the problem of insufficient genome information in a single cell was remedied. Our method has promising prospects in the field of NIPT for the detection of monogenic disorders.

Different PfEMP1-expressing Plasmodium falciparum variants induce divergent endothelial transcriptional responses during co-culture.

The human malaria parasite Plasmodium falciparum is responsible for the majority of mortality and morbidity caused by malaria infection and differs from other human malaria species in the degree of accumulation of parasite-infected red blood cells in the microvasculature, known as cytoadherence or sequestration. In P. falciparum, cytoadherence is mediated by a protein called PfEMP1 which, due to its exposure to the host immune system, undergoes antigenic variation resulting in the expression of different PfEMP1 variants on the infected erythrocyte membrane. These PfEMP1s contain various combinations of adhesive domains, which allow for the differential engagement of a repertoire of endothelial receptors on the host microvasculature, with specific receptor usage associated with severe disease. We used a co-culture model of cytoadherence incubating human brain microvascular endothelial cells with erythrocytes infected with two parasite lines expressing different PfEMP1s that demonstrate different binding profiles to vascular endothelium. We determined the transcriptional profile of human brain microvascular endothelial cells (HBMEC) following different incubation periods with infected erythrocytes, identifying different transcriptional profiles of pathways previously found to be involved in the pathology of severe malaria, such as inflammation, apoptosis and barrier integrity, induced by the two PfEMP1 variants.

Initial investigation on the feasibility of porcine red blood cells from genetically modified pigs as an alternative to human red blood cells for transfusion.

The decline in blood donation rates and the ongoing shortage of blood products pose significant challenges to medical societies. One potential solution is to use porcine red blood cells (pRBCs) from genetically modified pigs as an alternative to human red blood cells (hRBCs). However, adverse immunological reactions remain a significant obstacle to their use. This study aimed to evaluate the compatibility of diverse genetically modified pRBCs with human serum. We acquired human complement-competent serum, complement 7 (C7)-deficient serum, and hRBCs from all ABO blood types. Additionally, we used leftover clinical samples from health checkups for further evaluation. pRBCs were collected from wild-type (WT) and genetically modified pigs: triple knockout (TKO), quadruple KO (QKO), and TKO/hCD55.hCD39 knockin (hCD55.hCD39KI). The extent of C3 deposition on RBCs was measured using flow cytometry after incubation in C7-deficient serum diluted in Ca++-enriched or Ca++-depleted and Mg++-enriched buffers. The binding of immunoglobulin (Ig) M/IgG antibody to RBCs after incubation in ABO-type human serum was evaluated using flow cytometry. Naïve human serum- or sensitized monkey serum-mediated hemolysis was also evaluated. Phagocytosis was assessed by incubating labeled RBCs with the human monocytic cell line THP-1 and measurement by flow cytometry. All three genetic modifications significantly improved the compatibility of pRBCs with human serum relative to that of WT pRBCs. The extent of IgM/IgG binding to genetically modified pRBCs was lower than that of WT pRBCs and similar to that of O-type hRBCs. Total and alternative pathway complement activation in all three genetically modified pRBCs was significantly weaker than that in WT pRBCs and did not differ from that in O-type hRBCs. The extent of serum-mediated hemolysis and phagocytosis of these genetically modified pRBCs was low and similar to that of O-type hRBCs. Sensitized monkey serum-mediated hemolysis in QKO and TKO/hCD55.hCD39KI pRBCs was higher than in O-type hRBCs but lower than in TKO pRBCs. The elimination of porcine carbohydrate antigens in genetically modified pigs significantly enhanced pRBC compatibility with naïve human sera, which was comparable to that of O-type hRBCs. These findings provide valuable insights into the development of pRBCs as potential alternatives to hRBCs.

Transfusion Practice: Hemolysis Markers After In Vitro Infusion of Packed Red Blood Cells by the Gravitational Method in Peripheral Catheter.

The objective of this study was to compare hemolysis marker levels after in vitro infusion of red blood cells (RBCs) according to storage time, infusion rate, and peripheral intravenous catheter size. This is an experimental study with randomly administered RBCs in quintuplicate, according to storage time shorter than and longer than 14 days, as well as infusion rate (50 mL/h and 100 mL/h) using catheters with calibers of 14-, 18-, and 20-gauge. Aliquots were collected from RBCs (V1), after equipment and catheter (V2) free-flow filling and after controlled infusion through the catheter (V3). The hemolytic markers analyzed were degree of hemolysis (%), hematocrit (Ht) (%), total hemoglobin (THb) (g/dL), free hemoglobin (FHb) (g/dL), potassium (K) (mmol/L), and lactate dehydrogenase (LDH) (U/L), considering a probability of error ≤5%. Sixty experiments were performed with the analysis of 180 aliquots. When RBCs aged <14 days were used, all catheters tended to increase THb, FHb, and K; while >14 days, RBCs presented increased FHb and degree of hemolysis with catheters of 18-gauge and THb levels at 14-gauge. Among the conditions analyzed, only 20-gauge catheters (the smallest) did not influence changes in hemolysis markers, regardless of RBC storage time.

Immunohematological testing and transfusion management of the prenatal patient.

The primary indication for immunohematological testing in the prenatal patient is to detect and identify maternal red cell antibodies. If there are antibodies that are expected to hemolyze the fetus' red cells, their strength of reactivity must be tested, and the fetus' antigen status determined. After delivery, testing is performed to assess the extent of fetomaternal hemorrhage, as a large hemorrhage may require other therapeutic interventions. Another major role for immunohematological testing is to select blood components appropriately when intrauterine transfusion is required for fetal anemia resulting from maternal alloimmunization or some other cause. Supplementation with molecular methods has transformed the practice of immunohematology, particularly as it applies to typing for the D antigen of the Rh blood group system. Notwithstanding the advances in testing, close coordination and communication between the transfusion service and the obstetrics service are the foundation for ensuring the finest care for prenatal patients, and for new mothers and their infants. This review describes testing and transfusion practices for prenatal patients, using case presentations to highlight the management of selected immunohematological findings. It also includes a discussion of key patient management topics that are currently unresolved.

Numerical analysis of oxygen uptake processes by red blood cells in stopped-flow measurements: Effects of cell shape, membrane permeability and unstirred layer.

The transport process of oxygen and other gas species across red blood cell (RBC) membrane is of great importance for better understanding the critical biological functions of RBCs, and the stopped-flow experiments have often been employed for such investigations. In previous stopped-flow analyses, the RBC had usually been represented by a spherical capsule based on the RBC volume, and an assumed unstirred layer (USL) thickness had been used to determine the membrane permeability. In this research, unlike these previous studies, we simulate the oxygen uptake process with different RBC shapes (shperical, ellipsoidal and biconcave) and examine the effects of USL thickness and membrane permeability over broad ranges based on literature values. Our results show that the excess membrane area can greatly improve the oxygen transport efficiency, and a same uptake half-time can be obtained using different combinations of USL thickness and membrane permeability. These findings raise concerns on the reliability and uncertainty for the results and conclusions in previous studies, and also call for more complete numerical models, for example, with the fluid flow and cell deformation considered, and more in-depth investigations on the oxygen transport processes.

NK cell-induced damage to P.falciparum-infected erythrocytes requires ligand-specific recognition and releases parasitophorous vacuoles that are phagocytosed by monocytes in the presence of immune IgG.

Natural killer (NK) cells lyse virus-infected cells and transformed cells through polarized delivery of lytic effector molecules into target cells. We have shown that NK cells lyse Plasmodium falciparum-infected red blood cells (iRBC) via antibody-dependent cellular cytotoxicity (ADCC). A high frequency of adaptive NK cells, with elevated intrinsic ADCC activity, in people chronically exposed to malaria transmission is associated with reduced parasitemia and resistance to disease. How NK cells bind to iRBC and the outcome of iRBC lysis by NK cells has not been investigated. We applied gene ablation in inducible erythrocyte precursors and antibody-blocking experiments with iRBC to demonstrate a central role of CD58 and ICAM-4 as ligands for adhesion by NK cells via CD2 and integrin αMß2, respectively. Adhesion was dependent on opsonization of iRBC by IgG. Live imaging and quantitative flow cytometry of NK-mediated ADCC toward iRBC revealed that damage to the iRBC plasma membrane preceded damage to P. falciparum within parasitophorous vacuoles (PV). PV were identified and tracked with a P.falciparum strain that expresses the PV membrane-associated protein EXP2 tagged with GFP. After NK-mediated ADCC, PV were either found inside iRBC ghosts or released intact and devoid of RBC plasma membrane. Electron microscopy images of ADCC cultures revealed tight NK-iRBC synapses and free vesicles similar in size to GFP+ PV isolated from iRBC lysates by cell sorting. The titer of IgG in plasma of malaria-exposed individuals that bound PV was two orders of magnitude higher than IgG that bound iRBC. This immune IgG stimulated efficient phagocytosis of PV by primary monocytes. The selective NK-mediated damage to iRBC, resulting in release of PV, and subsequent phagocytosis of PV by monocytes may combine for efficient killing and removal of intra-erythrocytic P.falciparum parasite. This mechanism may mitigate the inflammation and malaria symptoms during blood-stage P. falciparum infection.