Abnormal Red Blood Cells Research Articles

Hypoxia Impact on Red Blood Cell-Mediated Microvascular Occlusion and Adhesion in Sickle Cell Disease and Sickle Cell Trait

Introduction: Sickle cell disease (SCD) is a recessively inherited anemia caused by a single gene mutation leading to sickle hemoglobin production. Sickle cell trait (SCT) is the carrier state. Abnormal hemoglobin polymerization and resultant red blood cell (RBC) sickling, decreased deformability and increased adhesion, are well-known features of homozygous SCD. However, the overall pathophysiological impact of SCT on the RBC remains incompletely characterized. Here we use microfluidic techniques designed by us, the OcclusionChip and SCD Biochip (previously published), and commercially available ektacytometry to investigate hypoxia impact on RBC biophysical properties in SCT.Methods: Venous blood samples were collected in EDTA from subjects with homozygous HbSS, SCT (HbAS), and non-anemic controls (HbAA) under an IRB-approved protocol. OcclusionChip devices were fabricated using standard soft lithography protocols [1]. RBCs were isolated from whole blood, re-suspended in PBS at 20% hematocrit, and passed through the OcclusionChip device with a constant inlet pressure. Following a wash step, the OcclusionChip microchannel was imaged, and Occlusion Index (OI), a standardized generalizable parameter we developed, representing the overall microcapillary network occlusion, was quantified. SCD Biochip microchannels were fabricated by lamination and were functionalized with human laminin (LN-511) [2]. Undiluted whole blood was injected into the microchannel at 1 dyne/cm 2, a shear stress value typically observed in the post-capillary venules. Following a wash step, the SCD Biochip microchannel was imaged, and the number of adherent RBCs in a 32-mm 2 window was quantified. For hypoxia experiments, a hypoxic setup was fabricated for blood deoxygenation (pO 2 ~45 mmHg) [3, 4]. Ektacytometry measurements were performed according to the manufacturers' specifications (Lorrca Maxsis). Data are reported as mean ± standard deviation (SD).Results: We initially analyzed RBC-mediated microvascular occlusion under normoxia or hypoxia using the OcclusionChip (Figure 1A). Under normoxia, HbSS-containing RBCs had relatively greater OI values compared to HbAA- and HbAS-containing RBCs (Figure 1B, P = 0.057 for HbSS vs HbAA and P = 0.060 for HbSS vs HbAS). However, exposure to hypoxia led to significantly elevated OI values in the HbAS- and HbSS-containing RBCs (Figure 1B, 0.05 ± 0.02% vs 33.62 ± 18.31%, P = 0.015 for HbAS, and 0.27 ± 0.24% vs 49.37 ± 24.47%, P = 0.001 for HbSS, normoxia vs hypoxia). Negligible occlusion was observed in HbAA-containing RBCs (Figure 1B). We then analyzed RBC adhesion to LN under normoxia or hypoxia using the SCD Biochip (Figure 1C). Hypoxia led to greater number of adherent RBCs on LN in the HbSS-containing RBCs (Figure 1D, 141 ± 91 vs 497 ± 392, P = 0.089, normoxia vs hypoxia), but this effect was not present in HbAA- or HbAS-containing RBCs (Figure 1B, 2 ± 1 vs 3 ± 1, P > 0.05 for HbAA, and 10 ± 7 vs 12 ± 3, P > 0.05 for HbAS, normoxia vs hypoxia). Further, under normoxia, we found that the HbAS-containing RBCs had slightly greater number of adherent RBCs on LN compared to the HbAA-containing RBCs (Figure 1D, P = 0.057 for HbAA vs HbAS). As previously reported, HbSS-containing RBCs showed greatest adhesion to LN under normoxia compared to the HbAA- and HbAS-containing RBCs (Figure 1D, P = 0.027 for HbSS vs HbAA and P = 0.033 for HbSS vs HbAS)., Finally, we preformed Lorrca oxyscan and found that ektacytometry is less sensitive to RBC deformability change under hypoxia in SCT (Figure 1E).Conclusions: Findings in this study suggest that although RBCs from subjects with SCT are deformable under normoxia and are able to clear narrow capillaries similar to normal RBCs, hypoxia changes deformability, presumably due to hypoxic polymer formation, and could contribute to microvascular occlusion in SCT. The OcclusionChip is a single cell-based technology, and may be more sensitive to single RBC deformability. Future studies will prospectively focus on analyzing RBC adhesion on activated microvascular endothelial cells in physiologic flow to further interrogate the impact of hypoxia on pathophysiology in SCT.

Whole Blood Adhesion to VCAM-1 and P-Selectin and RBC Mechanical Fragility Can be Compromised in Long COVID-19 Patients with Sickle Cell Disease

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause severe vascular complications associated with endothelial dysfunction and systemic inflammation. COVID19-specific IgG are detectable within a week of infection. Long COVID-19 has been described in patients continuing to exhibit symptoms after the virus is no longer detectable in the respiratory secretions, including fatigue, dyspnea, headache, and brain fog. The recent FAIR Health study reviewed a total of 1,959,982 COVID-19 patients for the prevalence of long COVID symptoms and reported that 23.2% had at least one post-COVID symptom [1]. The underlying biologic mechanisms of long COVID remain unclear, thus treatments are limited to symptomatic relief and supportive care. Many long COVID symptoms are consistent with systemic inflammation and impaired oxygen delivery observed in individuals with sickle cell disease (SCD), in turn associated with elevated blood cell adhesion and decreased red blood cell (RBC) stability. The aim of this study was to determine if deleterious changes in in blood cell properties related to adhesion and membrane stability under stress can be associated with the symptoms of long COVID-19. In this work we evaluated 7 SCD patients that were diagnosed with SARS-Cov-2 and tracked their recovery using semiquantitative IgG and blood cell function assays.Methods: Blood samples were collected by the Foundation for Sickle Cell Disease (SCD) Research from SCD (homozygous SS, n=6) patients coming for regular or urgent clinic visit with SARS-CoV-2 serological and blood cell functions tests performed per the standard of care. Semiquantitative IgG assay was performed using DXi-80 (Beckman Coulter). Flow adhesion of whole blood to VCAM-1 (FA-WB-VCAM)and P-Selectin (FA-WB-Psel) substrates were determined by counting the cells that remain adherent in a microfluidics channel after perfusion with whole blood 1:1 diluted with HBSS buffer and washed by reversed flow at 1 dyne/cm 2. Red blood cell mechanical fragility (RBC MF) was measured as hemolysis induced by an oscillating cylindrical magnet with periodic non-invasive probing of cell-free hemoglobin fraction. Six individuals with SCD recovering from SARS-Cov-2 with biomarker data available both before and for more than 3 months after the infection (179±62 days) were included in the study.Results: IgG levels varied from less than 0.1 to 37, with positive values being defined as IgG > 1. The median estimated half-life of IgG decline was 53 days ranging from 25 to 90 days (the last, for the hospitalized patient). Averaged for IgG positive (IgG+) and IgG negative (IgG-) conditions, combining pre- and post-infection IgG- conditions, values of patient hemoglobin (Hb), FA-WB-VCAM, FA-WB-Psel, and RBC MF cell properties lacked statistical significance (under both a paired t-test and population statistics). Hb levels remained essentially unchanged regardless of the time from infection or IgG status. However, FA-WB-VCAM, FA-WB-Psel, and RBC MF were all significantly elevated after SARS-Cov-2 seroconversion and remained elevated despite declining IgG levels (e.g., Fig. 1). These increases in biomarker values were statistically significant for both FA-WB-VCAM and RBC MF, and were approaching significance for FA-WB-Psel (p<0065). These increases were highly patient-specific with potential return to pe-infection values observed in some cases at about 5-6 months after the infection. A qualitative review of the medical records indicated a new subjective report of fatigue in 5 of 6 patients. Longer observations are required to determine if abnormal blood cell adhesive properties and RBC membrane instability are mechanisms of long-COVID-19 pathophysiology.Conclusions: Whole blood adhesion to both p-selectin and VCAM-1 as well as RBC membrane stability can be significantly impaired in convalescent SARS-Cov-2 patients suggesting an association with long COVID-19. New and emerging treatments that modify whole blood adhesive properties and RBC membrane stability should be investigated for their potential to accelerated recovery from long COVID-19.Health F. A Detailed Study of Patients with Long-Haul COVID: An Analysis of Private Healthcare Claims; White Paper. June 15, 2021 DisclosuresTarasev: Functional Fluidics: Current holder of stock options in a privately-held company. Ferranti: Functional Fluidics: Current holder of stock options in a privately-held company. Allen: Functional Fluidics: Current Employment. Gao: Functional Fluidics: Current Employment. Topping: Functional Fluidics: Current Employment. Ferranti: Functional Fluidics: Current Employment. Makinde-Odesola: Functional Fluidics: Other: conduct research for academic program. Hines: Functional Fluidics: Current holder of stock options in a privately-held company.

Assessment of the Effect of Crizanlizumab on Red Blood Cell Adhesion to Endothelial Cells Using a Standardized Endothelium-on-a-Chip Microfluidic Platform

Background: Chronic upregulation of P-selectin (P-sel) on blood cells and the endothelium leads to abnormal red blood cell (RBC) adhesion to endothelial cells, significantly contributing to vaso-occlusive crises (VOCs), which are a major cause of morbidity and mortality in patients with sickle cell disease (SCD). Crizanlizumab (criz, a.k.a. SEG101) is a humanized anti-P-sel monoclonal antibody and has recently been approved by the Food and Drug Administration to reduce the frequency of VOCs in SCD patients. Here, we report in vitro assessment of the effect of criz on patient-specific RBC adhesion to heme-activated human endothelial cells using a standardized endothelialized microfluidic platform, the Endothelium-on-a-chip.Methods: Whole blood samples were collected from 13 subjects with SCD (13 HbSS and 1 HbSC) in EDTA vacutainers. RBCs were isolated via centrifugation from whole blood and then resuspended in basal cell culture medium (EBM, Lonza, Morristown, USA) at a hematocrit of 20% buffered with 10 mM of HEPES. Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and cultured within the microfluidic channels at 15 dyne/cm 2 for at least 48 hours prior to experiments. For long-term activation, HUVECs were treated with 40 µM heme for 4 hours +/- 100 µg/ml criz for 1 hour followed by injection of blood samples through the microfluidic channels. For short-term activation, blood samples were supplemented with 40 µM heme +/- 100 µg/ml criz and injected through the microfluidic channels for 15 minutes. Thereafter, non-adherent RBCs were rinsed via either only heme-containing EGM or heme- and criz-containing EGM, and the remaining RBCs were quantified based on published methods [1]. Student's t-test was used to calculate statistical significance.Results: We found that 4-hour heme activation of HUVECs resulted in significantly elevated RBC adhesion compared to baseline although adhesion levels were heterogenous among the patient population (Fig. 1A, 1671±522 vs 17±4, p<0.05). Treatment of 4-hour heme-activated HUVECs with criz did not significantly decrease RBC adhesion (Fig. 1A, 1170±413 vs 1671±522, p>0.05), while we observed lower RBC adhesion to criz treated HUVECs for certain subjects (Fig. 1B). By contrast, criz treatment significantly reduced the number of adherent RBCs to 15-min heme-activated HUVECs (Fig. 1C, 135±40 vs 1513±617, p<0.05). Next, we assessed whether criz would disrupt already established adhesive interactions between RBCs and 15-min heme-activated HUVECs. To do so, we first allowed RBCs to adhere to heme-activated HUVECs (for 15-min) and then rinsed the microchannels (at 10 μl/min) via either a heme- or both heme- and criz-containing solution (for 15 min). We then quantified the number of adherent RBCs at min=0 and min=15. While only 10% of the adherent RBCs remained in the microchannels following a 15-minute wash with criz, this ratio was 60% without criz (Fig. 1E).Discussion: Our results show that the magnitude of inhibition of RBC adhesion to HUVECs with criz correlated with the duration of heme-activation (4 hours vs 15 minutes). This is likely due to variable levels of different adhesion molecules on acute or chronically activated HUVECs. For instance, it has been shown that P-selectin is rapidly translocated to the cell surface following heme activation [1], but its concentration on cell surface significantly decays with time. Previous experiments have shown that sickle RBCs can adhere to cell adhesion molecules such as ICAM-1 [2], which mechanistically may play a role in the case of a chronically activated endothelium. We are currently exploring whether criz would also reduce RBC adhesion to acutely activated endothelial cells that are under chronic stress. These preliminary results suggest that the Endothelium-on-a-chip, as partner in novel therapeutic studies, could help monitoring dynamics of targeted therapies in SCD patients during drug development and in clinical trials.Acknowledgements: This work was funded by Novartis. The authors would like to thank the Ohio Third Frontier Technology Validation and Start-up Fund (TVSF) and National Science Foundation Phase-I Small Business Technology Transfer (STTR) award, which supported this work in part. Crizanlizumab was donated by Novartis.

Red Blood Cell Adhesion Index in a Patient with Sickle Cell Disease Prior and Post Hematopoietic Stem Cell Transplant (HSCT)

Introduction: Sickle cell disease (SCD) is one of the most common inherited blood disorders and affects over 100,000 individuals in US only. SCD is caused by a single mutation in the β-globin gene that leads to the production of sickle hemoglobin (HbS). Red blood cells (RBCs) from patients with SCD are sticky, rigid, and prone to hemolysis, resulting in a wide range of acute and chronic complications, such as vaso-occlusive crises, acute chest syndrome, cerebrovascular disease, and multi-organ damage. Hematopoietic stem cell transplant (HSCT) is a curative therapy for SCD, that results in stabilization of organ function and gradual amelioration of cerebrovascular and pulmonary complications, as well vaso-occlusive crises. We report a clinically applicable microfluidic device (SCD Biochip) that enables quantitative evaluation of RBC adhesion to endothelium-associated protein-immobilized microchannels before and after HSCT.Methods: Venous blood samples were collected in EDTA tubes from a 40-year-old male patient with SCD at University Hospitals, Cleveland Medical Center. The sample was tested for abnormal RBCs adhesion with the SCD Biochip at multiple time points from 2016 to 2021. The transplant took place at University Hospitals and we collected the post-transplant blood at two, three and five months. The therapy regimen for the patient before the transplant consisted of transfusions and Hydroxyurea. Fabrication of the microfluidic channels consisted of a glass surface functionalized with Intercellular Adhesion Molecule 1 (ICAM-1) and Laminin (LN), a poly(methyl methacrylate) plastic top (encompassing inlets and outlets), and a sandwiched 50-μm-thick double-sided adhesive tape that defined the height and shape of the microchannels. The samples were injected into the ICAM-1 and LN-immobilized microchannels. An inverted microscope and microscopy camera were used to obtain high-resolution images of whole channel for processing (Adobe Photoshop, San Jose, CA) and quantification of adhered RBCs per unit area (32 mm 2). Adhesion index for ICAM-1 and LN was calculated as the square root of the number of adherent cells counted on the selected areas.Results: RBC adhesion to immobilized ICAM-1 and LN was analyzed under physiologic flow using a standardized microfluidic platform. Three data points were collected both before and after the HCST. The ICAM-1 RBCs adhesion index before and after HSCT is shown in Figure 1a. The LN RBCs adhesion index before and after HSCT is shown in Figure 1b, the green rectangle represents the normal range value for the HbAA controls as established in our previous studies (1,2). The patient had high adhesion indexes in both ICAM-1 and LN before HSCT, a decrease in adhesion indexes to the normal range was observed after HSCT.Conclusion: It has been observed that the adhesion index for ICAM-1 and LN were abnormal in the patient with SCD but they decreased to normal levels post-transplant. These results suggest that the SCD Biochip microfluidic devices could be an effective platform to estimate the RBC adhesion level and to validate the efficacy of HSCT in SCD population.

Influence of red blood cell indices on HbA1c performance in detecting dysglycaemia in a Singapore preconception cohort study

Abnormalities of red blood cell (RBC) indices may affect glycated haemoglobin (HbA1c) levels. We assessed the influence of haemoglobin (Hb) and mean corpuscular volume (MCV) on the performance of HbA1c in detecting dysglycaemia among reproductive aged women planning to conceive. Women aged 18–45 years (n = 985) were classified as normal (12 ≤ Hb ≤ 16 g/dL and 80 ≤ MCV ≤ 100 fL) and abnormal (Hb < 12 g/dL and/or MCV < 80 fL). The Area Under the Receiver Operating Characteristic (AUROC) curve was used to determine the performance of HbA1c in detecting dysglycaemic status (prediabetes and diabetes). There were 771 (78.3%) women with normal RBC indices. The AUROCs for the normal and abnormal groups were 0.75 (95% confidence interval 0.69, 0.81) and 0.80 (0.70, 0.90), respectively, and were not statistically different from one another [difference 0.04 (− 0.16, 0.08)]. Further stratification by ethnicity showed no difference between the two groups among Chinese and Indian women. However, Malay women with normal RBC indices displayed lower AUROC compared to those with abnormal RBC indices (0.71 (0.55, 0.87) vs. 0.98 (0.93, 1.00), p = 0.002). The results suggest that the performance of HbA1c in detecting dysglycaemia was not influenced by abnormal RBC indices based on low Hb and/or low MCV. However, there may be ethnic variations among them.

Valorization of Erythrocyte Indices in the Neonatal Period - A Pilot Study

Abnormal red blood cell indices are frequently missed among newborns that perform blood tests for any clinical reason.

Effects of lead exposure on blood electrical impedance spectroscopy of mice

BackgroundLead is a nonessential heavy metal, which can inhibit heme synthesis and has significant cytotoxic effects. Nevertheless, its effect on the electrical properties of red blood cells (RBCs) remains unclear. Consequently, this study aimed to investigate the electrical properties and the electrophysiological mechanism of lead exposure in mouse blood using Electrical Impedance Spectroscopy (EIS) in 0.01–100 MHz frequency range. Data characteristic of the impedance spectrum, Bodes plot, Nyquist plot and Nichols plot, and Constant Phase Element (CPE) equivalent circuit model were used to explicitly analyze the differences in amplitude–frequency, phase–frequency, and the frequency characteristics of blood in electrical impedance properties.ResultsCompared with the healthy blood in control mice, the changes in blood exposed to lead were as follows: (i) the hematocrit decreased; (ii) the amplitude–frequency and phase–frequency characteristics of electrical impedance decreased; (iii) the characteristic frequencies (f0) were significantly increased; (iv) the electrical impedance of plasma, erythrocyte membrane, and hemoglobin decreased, while the conductivity increased. (v) The pseudo-capacitance of cell membrane (CPE_Tm) and the intracellular pseudo-capacitance (CPE-Ti) were decreased.ConclusionsTherefore, EIS can be used as an effective method to monitor blood and RBC abnormalities caused by lead exposure. The electrical properties of the cells can be applied as an important observation in the evaluation of the toxic effects of heavy metals.

Microplastics-Induced Eryptosis and Poikilocytosis in Early-Juvenile Nile Tilapia (Oreochromis niloticus).

This study aims to assess the impact of microplastics (MPs) on erythrocytes using eryptosis (apoptosis) and an erythron profile (poikilocytosis and nuclear abnormalities), considered to be novel biomarkers in Nile tilapia (Oreochromis niloticus). In this study, four groups of fish were used: The first was the control group. In the second group, 1 mg/L of MPs was introduced to the samples. The third group was exposed to 10 mg/L of MPs. Finally, the fourth group was exposed to 100 mg/L of MPs for 15 days, following 15 days of recovery. The fish treated with MPs experienced an immense rise in the eryptosis percentage, poikilocytosis, and nuclear abnormalities of red blood cells (RBCs) compared with the control group in a concentration-dependent manner. Poikilocytosis of MP-exposed groups included sickle cell shape, schistocyte, elliptocyte, acanthocyte, and other shapes. Nuclear abnormalities of the MPs-exposed groups included micronuclei, binucleated erythrocytes, notched, lobed, blebbed, and hemolyzed nuclei. After the recovery period, a greater percentage of eryptosis, poikilocytotic cells, and nuclear abnormalities in RBCs were still evident in the groups exposed to MPs when crosschecked with the control group. The results show concerning facts regarding the toxicity of MPs in tilapia.

Human Red Blood Cell Abnormalities in Fluoride Endemic Area of Warora Tehsil, Chandrapur District, Maharashtra, India

Human Red Blood Cell Abnormalities in Fluoride Endemic Area of Warora Tehsil, Chandrapur District, Maharashtra, India

Severe Presentation of Congenital Hemolytic Anemias in the Neonatal Age: Diagnostic and Therapeutic Issues.

Congenital hemolytic anemias (CHAs) are a group of diseases characterized by premature destruction of erythrocytes as a consequence of intrinsic red blood cells abnormalities. Suggestive features of CHAs are anemia and hemolysis, with high reticulocyte count, unconjugated hyperbilirubinemia, increased lactate dehydrogenase (LDH), and reduced haptoglobin. The peripheral blood smear can help the differential diagnosis. In this review, we discuss the clinical management of severe CHAs presenting early on in the neonatal period. Appropriate knowledge and a high index of suspicion are crucial for a timely differential diagnosis and management. Here, we provide an overview of the most common conditions, such as glucose-6-phosphate dehydrogenase deficiency, pyruvate kinase deficiency, and hereditary spherocytosis. Although rare, congenital dyserythropoietic anemias are included as they may be suspected in early life, while hemoglobinopathies will not be discussed, as they usually manifest at a later age, when fetal hemoglobin (HbF) is replaced by the adult form (HbA).

Applications of deep learning to the assessment of red blood cell deformability.

Measurement of abnormal Red Blood Cell (RBC) deformability is a main indicator of Sickle Cell Anemia (SCA) and requires standardized quantification methods. Ektacytometry is commonly used to estimate the fraction of Sickled Cells (SCs) by measuring the deformability of RBCs from laser diffraction patterns under varying shear stress. In addition to estimations from model comparisons, use of maximum Elongation Index differences (ΔEImax) at different laser intensity levels was recently proposed for the estimation of SC fractions. Implement a convolutional neural network to accurately estimate rigid-cell fraction and RBC concentration from laser diffraction patterns without using a theoretical model and eliminating the ektacytometer dependency for deformability measurements. RBCs were collected from control patients. Rigid-cell fraction experiments were performed using varying concentrations of glutaraldehyde. Serial dilutions were used for varying the concentration of RBC. A convolutional neural network was constructed using Python and TensorFlow. Measurements and model predictions show that a linear relationship between ΔEImax and rigid-cell fraction exists only for rigid-cell fractions less than 0.2. The proposed neural network architecture can be used successfully for both RBC concentration and rigid-cell fraction estimations without a need for a theoretical model.

Intraoperative Transesophageal Echocardiographic Detection of Large Mobile Aortic Thrombus in a Patient with Iron Deficiency Anemia Presenting as Acute Limb Ischemia

Quantitative and qualitative abnormalities in red blood cells are associated with arterial thrombosis. We report a case of a middle-aged female with iron deficiency anemia (IDA) presenting as bilateral acute limb ischemia (ALI) and on evaluation with transesophageal echocardiography (TEE) a large mobile thrombus was detected in the descending thoracic aorta. Her limbs were salvaged with successful bilateral surgical embolectomy and fasciotomies. The aortic thrombus was resolved with anticoagulation and correction of iron deficiency. We conclude that TEE evaluation of aorta is warranted in middle age females with IDA presenting as ALI.

Severe neonatal haemolytic anaemia caused by compound heterozygous KLF1 mutations: report of four families and literature review

Mutations in the KLF1 gene, which encodes a transcription factor playing a role in erythropoiesis, have recently been demonstrated to be a rare cause of hereditary haemolytic anaemia. We described the genotypic and phenotypic spectra of four unrelated families with compound heterozygous class 2/class 3 KLF1 mutations. All patients had p.G176RfsX179 on one allele and either p.A298P, p.R301H or p.G335R on the other allele. All presented on the first day of life with severe haemolytic anaemia with abnormal red blood cell morphology, markedly increased nucleated red blood cells and hyperbilirubinaemia. Three patients later became transfusion-dependent. All parents with heterozygous KLF1 mutation without co-inherited thalassaemia had normal to borderline mean corpuscular volume (MCV) and normal to slightly elevated Hb F. Fifteen previously reported cases of biallelic KLF1 mutations were identified from a literature review. All except one presented with severe haemolytic anaemia in the neonatal period. Our finding substantiates that compound heterozygous KLF1 mutations are associated with severe neonatal haemolytic anaemia and expands the haematologic phenotypic spectrum. In carriers, the previously suggested findings of low MCV, high Hb A2 and high Hb F are inconsistent; thus this necessitates molecular studies for the identification of carriers.

A study of red blood cell abnormalities in chronic liver disease

In foetal life the liver and spleen are the most important sites of RBC production. Though majority of this function will be transferred to marrow, the nutrients needed for the formation will be dealt in liver itself. The metabolism of heme also occurs in liver up to some extent. So, chronic liver disease is always associated with some red blood disorder. As there are limited number of studies that have been reported in this field this study is one such noble attempt.

Evaluation of Clinical and Morphological Practices in the Diagnosis of Transplant-Associated Microangiopathy: A Study on Behalf of the Complications and Quality of Life Working Party of the EBMT

Introduction: Transplant-associated microangiopathy (TAM) is a severe and frequently life-threatening complication of allogeneic stem cell transplantation (SCT). Thus correct laboratory confirmation and differential diagnosis are important, especially in the context of emerging new therapies, like complement inhibitors. However, among the parameters taken into account when establishing diagnosis, schistocyte count seems to be a subjective and variable one (Zini G. et al., 2012). Therefore, a practice of TAM clinical and morphological diagnosis was investigated in 17 EBMT transplant centers.Methods: The study included two questionnaires, one for transplant physician and one for morphologist, and also a set of 20 blood slide photographs of 20 cases for quantification of proportion of schistocytes. 10 of these cases represented patients with TAM, with diagnosis established based on International Working Group (IWG) criteria at the Pavlov First Saint Petersburg State Medical University and Helsinki university. The remaining 10 were control cases, including diagnosis of myelodysplastic syndrome, autoimmune hemolysis, thalassemia, myelofibrosis and poikilocytosis due to B12 deficiency. 17 EBMT centers participated in the study.Results: Based on physician's questionnaires, 41% of centers use IWG criteria, 41% - “overall TAM” criteria (Cho et al. Transplantation 2010) and 18% physician's decision for the diagnosis of TAM. 88% of centers stated that the differential diagnosis of TAM is complicated and 65% of centers consider schistocyte count a criterion most commonly not fulfilled in suspected cases. For 65% of physicians the percentage of schistocytes required for the diagnosis of TAM is above 4%, for 18% above 1%, for 12% above 2%, and one center considered the level of schistocytes not important for the diagnosis. Based on morphologists's form, the morphological types of abnormal red blood cells that were counted as schistocytes were triangular cells (in 93% of centers), helmet cells (93% of centers), keratocytes (67%), microspherocytes (53%), degmacytes (47%), stomatocytes (13%), acanthocytes (7%) and codocytes (7%). 27% of centers reported that it is impossible to distinguish TAM morphology form autoimmune hemolysis, 13% - from myelodysplastic syndrome, 20% - from poikilocytosis, 7% - from hereditary anemias. 57% of centers considered automated hematology analyzers not applicable for quantification of schistocytes in patients after allogeneic SCT, 36% stated that manual confirmation of results is required, and only one center reported that automated analyzers are accurate for TAM diagnosis. The analysis of blood slides by morphologists revealed significant variability in schistocyte quantification for all the cases. The median number of schistocytes reported was 3% for TAM cases (range 0-19.6%, coefficient of variation 0.83), and 0.6% for control cases (range 0-8.3%, coefficient of variation 1.06). 36% of centers consistently (in at least 18/20 cases) reported number of schistocytes higher than median (5.1±4.1% for all cases), 36% consistently lower than median (0.9±1.3% for all cases), and 28% of centers reported results around median (2.3±2.5% for all cases). The difference might be in part due to inclusion of microspherocytes in the overall count. Centers that classified microspherocytes as schistocytes reported significantly higher values (mean for all cases 6,1±2,6% vs 2,8±2,4%, p=0.035). No significant differences were observed for the other morphological forms. In 31% of the evaluations on TAM cases the reported proportion of schistocytes was below 2%, while in 21% of control cases the proportion was reported to be higher than 2%.Conclusion: The survey identified the need for the standardization of TAM morphological diagnosis and clear criteria to distinguish TAM from other post-transplant complications. Several different methodologies for morphological diagnosis were observed. The existing variability in practices of schistocyte assessment among centers might be an obstacle to reliable multicenter interventional studies in TAM. DisclosuresGreinix:Novartis: Consultancy, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Mallinckrodt: Consultancy, Speakers Bureau. Stoelzel:medac: Other: Travel support.

Evaluation of Abnormal Red Blood Cells in Anemic Patient

Evaluation of Abnormal Red Blood Cells in Anemic Patient

Biostimulation Effects and Temperature Variation in Stimulated Dielectric Substance (Diabetic Blood Comparable to Non-Diabetic Blood) Based on the Specific Absorption Rate (SAR) in Laser Therapy

Human blood exposed to irradiation absorbs electromagnetic energy which consequently effect temperature variation. The evaluation of Specific Absorption Rate (SAR) of human blood helps to ascertain the values for optimum laser power, time, and temperature variation for fair therapy to avoid blood-irradiation pollution but to enhance its rheological properties when using lasers. Prior knowledge of blood SAR evaluating its dielectric properties is significant, but this is under investigation. We investigate the appropriate SAR threshold value as affected by temperature variation using fundamental blood dielectric parameters to optimize the effect of low-level laser therapy based on physiological and morphological changes of the stimulated diabetic blood. Studies were carried out with Agilent 4294A impedance analyser at frequencies (40Hz – 30 MHz) and designed cells (cuvettes) comprises of electrodes were used in the pre- and post-irradiations measurements. At different laser power outputs, blood samples were subjected to various irradiation durations using portable laser diode-pumped solid state of wavelength 532 nm. Results showed laser at low energy is capable of moderating morphologically the proportion of abnormal diabetic red blood cells. Hence, there is a significant effect using a laser at low energy, as non-medicinal therapy in controlling diabetic health conditions. The positive biostimulation effects on the irradiated diabetic blood occurred within absorbance threshold SAR values range of 0.140?0.695 W/kg and average temperatures range of 24.2?28.0 0C before blood saturation absorbance peak. There is morphological stimulation at a laser power of 50 mW for an exposure time of 10–15 minutes and 60 mW for 5–10 minutes of laser therapy that demonstrates better blood rejuvenated conditions. This occurred within the threshold SAR of 0.140?0.695 W/kg and average temperatures range of 24.2?28.0 0C. Therefore, the diabetic blood irradiated using laser output powers of 70 and 80 mW during exposure durations of 5,10, 15 and 20 minutes rather bio-inhibits positive blood stimulation which has resulted to crenation due to excessive irradiation.

Irregular Case of Neuroacanthocytosis Presenting with Late Onset Attention Deficit Hyperactivity Disorder: A Case Report

Neuroacanthocytosis (NA) syndromes are characterized by various neurological abnormalities accompanied by abnormal red blood cells called acanthocytes. Neuroacanthocytosis syndromes may be divided into 4 main subgroups. These subgroups are named as; Core NA syndromes, Degenerative disorders where acanthocytosis is occasionally seen, Paroxysmal dyskinetic disorders and Disorders with reduced blood lipoproteins and acanthocytosis. This report focuses on a patient with Chorea-acanthocytosis who presented with motor signs in the 3rd decade of life. In the later course of disease, the patient developed attention deficit hyperactivity disorder and various tic disorders. We wanted to emphasize the importance of multidisciplinary care in the management of chorea-acanthocytosis.

Concurrent Assessment of Deformability and Adhesiveness of Sickle Red Blood Cells by Measuring Perfusion of an Adhesive Artificial Microvascular Network.

Biomarker development is a key clinical research need in sickle cell disease (SCD). Hemorheological parameters are excellent candidates as abnormal red blood cell (RBC) rheology plays a critical role in SCD pathophysiology. Here we describe a microfluidic device capable of evaluating RBC deformability and adhesiveness concurrently, by measuring their effect on perfusion of an artificial microvascular network (AMVN) that combines microchannels small enough to require RBC deformation, and laminin (LN) coating on channel walls to model intravascular adhesion. Each AMVN device consists of three identical capillary networks, which can be coated with LN (adhesive) or left uncoated (non-adhesive) independently. The perfusion rate for sickle RBCs in the LN-coated networks (0.18 ± 0.02 nL/s) was significantly slower than in non-adhesive networks (0.20 ± 0.02 nL/s), and both were significantly slower than the perfusion rate for normal RBCs in the LN-coated networks (0.22 ± 0.01 nL/s). Importantly, there was no overlap between the ranges of perfusion rates obtained for sickle and normal RBC samples in the LN-coated networks. Interestingly, treatment with poloxamer 188 decreased the perfusion rate for sickle RBCs in LN-coated networks in a dose-dependent manner, contrary to previous studies with conventional assays, but in agreement with the latest clinical trial which showed no clinical benefit. Overall, these findings suggest the potential utility of the adhesive AMVN device for evaluating the effect of novel curative and palliative therapies on the hemorheological status of SCD patients during clinical trials and in post-market clinical practice.

Petechiae and Purpura

Key Points The differential diagnosis for petechiae and/or purpura is broad, but in an ill-appearing child with sepsis must be at the top of the list.In a well-appearing child, petechiae and/or purpura should raise the possibility of a platelet disorder or another hemostatic defect.Facial petechiae after vaginal delivery in a well-appearing newborn likely result from birth trauma, but more diffuse petechiae warrant further investigation.If thrombocytopenia is accompanied by an abnormality of red and/or white blood cells, a bone marrow process should be considered.