Applications of imaging flow cytometry in the diagnostic assessment of red cell membrane disorders.

BACKGROUND: Red cell membranopathies refers to phenotypically and morphologically heterogeneous disorders. High throughput imaging flow cytometry (IFC) combines the speed, sensitivity, and phenotyping abilities of flow cytometry with the detailed imagery and functional insights of microscopy to produce high content image analysis with quantitative analysis. We have evaluated the applications of IFC to examine both the morphology as well as fluorescence signal intensity in red cell membranopathies. METHODS: Fluorescence intensity of eosin-5-maleimide (EMA) labeled red cells was measured for diagnosis of RBC membrane protein defect on Amnis ImageStreamX followed by Image analysis on IDEAS software to study features such as circularity and shape ratio. RESULTS: The hereditary spherocytosis (HS) group showed significantly decreased MFI (52,800 ± 9,100) than normal controls (81,100 ± 4,700) (p < .05) whereas non-HS showed 78,300 ± 9,900. The shape ratio of hereditary elliptocytosis (HE) was significantly higher (43.8%) than normal controls (14.6%). The circularity score is higher in HS (64.15%) than the normal controls (44.3%) whereas the circularity score was very less in HE (10%) due to the presence of elliptocytes. CONCLUSIONS: The advantages of the IFC over standard flow cytometry is its ability to provide high-content image analysis and measurement of parameters such as circularity and shape ratio allow discriminating red cell membranopathies (HS and HE) due to variations in shape and size. It could be a single, effective, and rapid IFC test for detection and differentiation of red cell membrane disorders in hematology laboratories where an IFC is available.

Assessment of erythrocyte damage and in-line pressure changes associated with simulated transfusion of canine blood through microaggregate filters.

OBJECTIVE: To determine whether passage of whole blood through a microaggregate filter by use of a syringe pump would damage canine erythrocytes. SAMPLE: Blood samples obtained from 8 healthy client-owned dogs. PROCEDURES: Whole blood was passed through a standard microaggregate filter by use of a syringe pump at 3 standard administration rates (12.5, 25, and 50 mL/h). Prefilter and postfilter blood samples were collected at the beginning and end of a simulated transfusion. Variables measured at each time point included erythrocyte osmotic fragility, mean corpuscular fragility, RBC count, hemoglobin concentration, RBC distribution width, and RBC morphology. In-line pressure when blood passed through the microaggregate filter was measured continuously throughout the simulated transfusion. After the simulated transfusion was completed, filters were visually analyzed by use of scanning electron microscopy. RESULTS: Regardless of administration rate, there was no significant difference in mean corpuscular fragility, RBC count, hemoglobin concentration, or RBC distribution width between prefilter and postfilter samples. Additionally, there were no differences in in-line pressure during the simulated transfusion among administration rates. Echinocytes were the erythrocyte morphological abnormality most commonly observed at the end of the transfusion at administration rates of 12.5 and 25 mL/h. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that regardless of the administration rate, the microaggregate filter did not alter fragility of canine RBCs, but may have altered the morphology. It appeared that the microaggregate filter would not contribute to substantial RBC damage for transfusions performed with a syringe pump.

Assessment of the red blood cell proteome in a dog with unexplained hemolytic anemia.

A 7-year-old female neutered Jack Russell Terrier was presented to Langford Vets, the University of Bristol, with a history of chronic intermittent lethargy. Investigations and clinical course were compatible with hereditary hemolysis due to a red blood cell membrane defect. Proteomics was used to explore protein alterations in the presence of a hypothesized red blood cell membrane protein deficiency. Proteomic analysis revealed downregulation of the band 3, and alpha- and beta-adducin proteins, and alterations in the red blood cell proteome consistent with previous reports of changes due to the presence of reticulocytosis and ongoing hemolysis. The spectrum of protein alterations identified in the affected dog may be homologous to a band 3 protein deficiency secondary to hereditary spherocytosis, as described in people.

Morphology, membrane nanostructure and stiffness for quality assessment of packed red blood cells.

Transfusion of packed red blood cells (PRBC) to patients in critical states is often accompanied by post-transfusion complications. This may be related with disturbance of properties of PRBC and their membranes during long-term storage in the hemopreservative solution. The purpose of our work is the study of transformation of morphology, membranes stiffness and nanostructure for assessment of PRBC quality, in vitro. By atomic force microscopy we studied the transformation of cell morphology, the appearance of topological nanodefects of membranes and by atomic force spectroscopy studied the change of membrane stiffness during 40 days of storage of PRBC. It was shown that there is a transition period (20-26 days), in which we observed an increase in the Young's modulus of the membranes 1.6-2 times and transition of cells into irreversible forms. This process was preceded by the appearance of topological nanodefects of membranes. These parameters can be used for quality assessment of PRBC and for improvement of transfusion rules.

Time Dependent Assessment of Morphological Changes: Leukodepleted Packed Red Blood Cells Stored in SAGM.

Usually packed red blood cells (pRBCs) require specific conditions in storage procedures to ensure the maximum shelf life of up to 42 days in 2-6°C. However, molecular and biochemical consequences can affect the stored blood cells; these changes are collectively labeled as storage lesions. In this study, the effect of prolonged storage was assessed through investigating morphological changes and evaluating oxidative stress. Samples from leukodepleted pRBC in SAGM stored at 4°C for 42 days were withdrawn aseptically on day 0, day 14, day 28, and day 42. Morphological changes were observed using scanning electron microscopy and correlated with osmotic fragility and hematocrit. Oxidative injury was studied through assessing MDA level as a marker for lipid peroxidation. Osmotic fragility test showed that extended storage time caused increase in the osmotic fragility. The hematocrit increased by 6.6% from day 0 to day 42. The last 2 weeks show alteration in the morphology with the appearance of echinocytes and spherocytes. Storage lesions and morphological alterations appeared to affect RBCs during the storage period. Further studies should be performed to develop strategies that will aid in the improvement of stored pRBC quality and efficacy.

A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six-color immunophenotyping.

Abnormal DNA ploidy is a valuable prognostic factor in many neoplasms, especially in hematological neoplasms like B-cell acute lymphoblastic leukemia (B-ALL) and multiple myeloma (MM). Current methods of flow-cytometric (FC) DNA-ploidy evaluation are either technically difficult or limited to three- to four-color immunophenotyping and hence, challenging to evaluate DNA-ploidy in minute tumor population with background rich of its normal counterpart cells and other hematopoietic cells. We standardized a novel sensitive and easy method of simultaneous evaluation of six- to seven-color immunophenotyping and DNA-ploidy using a dye-FxCycle Violet (FCV). Linearity, resolution, and coefficient of variation (CV) for FCV were studied using chicken erythrocyte nuclei. Ploidy results of FCV were compared with Propidium iodide (PI) in 20 samples and intra-assay variation for FCV was studied. Using this six-color immunophenotyping & FCV-protocol DNA-ploidy was determined in bone-marrow samples from 124 B-ALL & 50 MM patients. Dilution experiment was also conducted to determine the sensitivity in detection of aneuploidy in minute tumor population. FCV revealed high linearity and resolution in 450/50 channel. On comparison with PI, CV of Go/G1-peak with FCV (mean-CV 4.1%) was slightly higher than PI (mean-CV 2.9%) but had complete agreement in ploidy results. Dilution experiment showed that aneuploidy could be accurately detected up to the limit of 0.01% tumor cells. Intra-assay variation was very low with CV of 0.005%. In B-ALL, hypodiploidy was noted in 4%, hyperdiploidy in 24%, near-hyperdiploidy in 13% and remaining 59% were diploid. In MM, hypodiploidy was in 2%, hyperdiploidy in 58%, near-hyperdiploidy in 8% and remaining 30% were diploid. FCV-based DNA-ploidy method is a sensitive and easy method for simultaneous evaluation of six-color immunophenotyping and DNA analysis. It is useful in DNA-ploidy evaluation of minute tumor population in cases like minimal residual disease and MM precursor conditions.

NMR of (133)Cs(+) in stretched hydrogels: One-dimensional, z- and NOESY spectra, and probing the ion's environment in erythrocytes.

(133)Cs nuclear magnetic resonance (NMR) spectroscopy was conducted on (133)Cs(+) in gelatin hydrogels that were either relaxed or stretched. Stretching generated a septet from this spin-7/2 nucleus, and its nuclear magnetic relaxation was studied via z-spectra, and two-dimensional nuclear Overhauser (NOESY) spectroscopy. Various spectral features were well simulated by using Mathematica and the software package SpinDynamica. Spectra of CsCl in suspensions of human erythrocytes embedded in gelatin gel showed separation of the resonances from the cation inside and outside the cells. Upon stretching the sample, the extracellular (133)Cs(+) signal split into a septet, while the intracellular peak was unchanged, revealing different alignment/ordering properties of the environment inside and around the cells. Differential interference contrast light microscopy confirmed that the cells were stretched when the overall sample was elongated. Analysis of the various spectral features of (133)Cs(+) reported here opens up applications of this K(+) congener for studies of cation-handling by metabolically-active cells and tissues in aligned states.

Surfactins modulate the lateral organization of fluorescent membrane polar lipids: a new tool to study drug:membrane interaction and assessment of the role of cholesterol and drug acyl chain length.

The lipopeptide surfactin exhibits promising antimicrobial activities which are hampered by haemolytic toxicity. Rational design of new surfactin molecules, based on a better understanding of membrane:surfactin interaction, is thus crucial. We here performed bioimaging of lateral membrane lipid heterogeneity in adherent living human red blood cells (RBCs), as a new relevant bioassay, and explored its potential to better understand membrane:surfactin interactions. RBCs show (sub)micrometric membrane domains upon insertion of BODIPY analogs of glucosylceramide (GlcCer), sphingomyelin (SM) and phosphatidylcholine (PC). These domains exhibit increasing sensitivity to cholesterol depletion by methyl-ß-cyclodextrin. At concentrations well below critical micellar concentration, natural cyclic surfactin increased the formation of PC and SM, but not GlcCer, domains, suggesting preferential interaction with lipid assemblies with the highest vulnerability to methyl-ß-cyclodextrin. Surfactin not only reversed disappearance of SM domains upon cholesterol depletion but further increased PC domain abundance over control RBCs, indicating that surfactin can substitute cholesterol to promote micrometric domains. Surfactin sensitized excimer formation from PC and SM domains, suggesting increased lipid recruitment and/or diffusion within domains. Comparison of surfactin congeners differing by geometry, charge and acyl chain length indicated a strong dependence on acyl chain length. Thus, bioimaging of micrometric lipid domains is a visual powerful tool, revealing that intrinsic lipid domain organization, cholesterol abundance and drug acyl chain length are key parameters for membrane:surfactin interaction. Implications for surfactin preferential location in domains or at their boundaries are discussed and may be useful for rational design of better surfactin molecules.

Assessment of genotoxicity induced by 7,12-dimethylbenz(a)anthracene or diethylnitrosamine in the Pig-a, micronucleus and Comet assays integrated into 28-day repeat dose studies.

As part of the Stage 3 of the Pig-a international trial, we evaluated 7,12-dimethylbenz(a)anthracene (DMBA) for induction of Pig-a gene mutation using a 28-day repeat dose study design in Sprague-Dawley rats. In the same study, chromosomal damage in peripheral blood and primary DNA damage in liver were also investigated by the micronucleus (MN) assay and the Comet assay, respectively. In agreement with previously published data (Dertinger et al., [2010]: Toxicol Sci 115:401-411), DMBA induced dose-dependent increases of CD59-negative erythrocytes/reticulocytes and micronucleated reticulocytes (MN-RETs). However, there was no significant increase in DNA damage in the liver cells when tested up to 10 mg/kg/day, which appears to be below the maximum tolerated dose. When tested up to 200 mg/kg/day in a follow-up 3 dose study, DMBA was positive in the liver Comet assay. Additionally, we evaluated diethylnitrosamine (DEN), a known mutagen/hepatocarcinogen, for induction of Pig-a mutation, MN and DNA damage in a 28-day study. DEN produced negative results in both the Pig-a mutation assay and the MN assay, but induced dose-dependent increases of DNA damage in the liver and blood Comet assay. In summary, our results demonstrated that the Pig-a mutation assay can be effectively integrated into repeat dose studies and the data are highly reproducible between different laboratories. Also, integration of multiple genotoxicity endpoints into the same study not only provides a comprehensive evaluation of the genotoxic potential of test chemicals, but also reduces the number of animals needed for testing, especially when more than one in vivo genotoxicity tests are required.

Evaluation of oxidative DNA lesions in plasma and nuclear abnormalities in erythrocytes of wild fish (Liza aurata) as an integrated approach to genotoxicity assessment.

Genetic lesions (8-hydroxy-2'-deoxyguanosine (8-OHdG) and erythrocytic nuclear abnormalities (ENA) were seasonally quantified in the blood of Liza aurata caught at Ria de Aveiro (Portugal), a multi-contaminated aquatic system. Thus, five critical sites were assessed and compared with a reference site (Torreira). Oxidative DNA damage was found in Gafanha (harbour-water area), Laranjo (metal-contaminated) and Vagos (contaminated with PAHs) in the spring; Rio Novo do Principe (near a former paper-mill effluent) in the autumn; Rio Novo do Principe and Vagos in the winter. ENA were higher than Torreira at VAG (spring and winter). Torreira did not display seasonal variation neither in terms of 8-OHdG or total ENA. A positive correlation between 8-OHdG and ENA was found, suggesting oxidative stress as a mechanism involved in the formation of ENA. This study clearly demonstrates the presence of DNA-damaging substances in Ria de Aveiro and recommends the use of 8-OHdG and ENA as biomarkers of environmental contamination.

Safety assessment of a novel ingredient for removable chewing gum.

Rev7 is an indigestible gum polymer used for the manufacturing of chewing gum. It allows for the formulation of chewing gum with low adhesion; thus can be readily removed from surfaces such as sidewalks, clothing, carpets and furniture. In a toxicological safety assessment, Rev7 was found to be non-mutagenic in the AMES assay. The highest concentration tested in a mouse lymphoma thymidine kinase locus gene mutation assay induced a slight but biologically relevant increase in mutations under non-metabolic activation conditions after 24h. Because of this finding, a mouse micronucleus assay was performed, and the test article was found to be negative for inducing chromosomal damage. A 28-day repeated oral toxicity study resulted in a NOAEL of 80,000 ppm; the highest concentration tested. Rev7 was found to be free from contaminants such as heavy metals, monomers, and solvents. Lastly, Rev7 did not demonstrate skin-sensitizing properties in the murine local lymph node assay.

[Computer assessment of membrane structure in various erythrocyte forms].

The photometric computer image analysis method is described. It is based on the creation of the gallery of the images of various erythrocyte forms (discocytes, ecchinocytes, target cells and degenerative forms). Using the Bio Vision program, the structure of membranes of each type of erythrocytes was studied. It was found that the morpho-functional changes of erythrocytes of various degrees were accompanied by the alterations in the relative content of condensed membrane protein-lipid complexes.

[The assessment of biocenosis contamination in the region of radioactive waste storage (RWS) placing and influence of this storage on rodents].

The results of radiative and of chemical monitoring show definite contamination of this zone by 90Sr and toxic metals. The essential local contaminations of geosystems (up to 2.3 x 10(4) Bk/kg of soil) require in environmental condition assessment at biocenosis level. Biotesting found the increase of metallothioneines levels in kidney (up to 15.63 microg/g of tissue) and liver (up to 19.22 microg/g of tissue) of rodents inhabited in the region of RWS placing as compared with the control group (3.51 and 4.44 microg/g of tissue accordingly). Besides, the decrease of total quantity of leucocytes (by 14.5% as compared with the control group) and absolute quantity all forms of them in animal blood were noted. It was assumed the increase of protein--MT is the result of complex influence by ionizing radiation and toxic metals.

Ultrastructural assessment of Plasmodium falciparum in age-fractionated thalassaemic erythrocytes.

Culture of Plasmodium falciparum in age-fractionated thalassaemic red blood cells (RBC) has shown evidence of parasite damage on light microscopy in older cells during the third culture cycle (96-144 h). In this report, parasites growing in thalassaemic trait and normal RBC were examined ultrastructurally from 96 to 144 h. All parasite stages in old thalassaemic RBC showed evidence of damage worsening with culture duration. There were cytoplasmic alterations with ribosomal damage, and parasite cytoplasm became increasingly loose and grainy, with multiple fissures. Discontinuity of the nuclear membrane with an abnormal nucleolus was seen at l20 h. Cytosomes remained normal, but damage to the food vacuole and shrunken disintegrating parasites were observed at 144 h. These changes are compatible with cellular degeneration and developmental retardation and would account for the schizont maturation arrest and reduced reinvasion rates previously reported. Increased free radicals associated with thalassaemic erythrocytes would explain these changes, further supporting the role for oxidant stress in the protective mechanism.

Simulations of molecular diffusion in lattices of cells: insights for NMR of red blood cells.

The pulsed field-gradient spin-echo (PGSE) nuclear magnetic resonance (NMR) experiment, conducted on a suspension of red blood cells (RBC) in a strong magnetic field yields a q-space plot consisting of a series of maxima and minima. This is mathematically analogous to a classical optical diffraction pattern. The method provides a noninvasive and novel means of characterizing cell suspensions that is sensitive to changes in cell shape and packing density. The positions of the features in a q-space plot characterize the rate of exchange across the membrane, cell dimensions, and packing density. A diffusion tensor, containing information regarding the diffusion anisotropy of the system, can also be derived from the PGSE NMR data. In this study, we carried out Monte Carlo simulations of diffusion in suspensions of "virtual" cells that had either biconcave disc (as in RBC) or oblate spheroid geometry. The simulations were performed in a PGSE NMR context thus enabling predictions of q-space and diffusion tensor data. The simulated data were compared with those from real PGSE NMR diffusion experiments on RBC suspensions that had a range of hematocrit values. Methods that facilitate the processing of q-space data were also developed.

Orthogonal polarisation spectral imaging as a new tool for the assessment of antivascular tumour treatment in vivo: a validation study.

Tumour angiogenesis plays a key role in tumour growth, formation of metastasis, detection and treatment of malignant tumours. Recent investigations provided increasing evidence that quantitative analysis of tumour angiogenesis is an indispensable prerequisite for developing novel treatment strategies such as anti-angiogenic and antivascular treatment options. Therefore, it was our aim to establish and validate a new and versatile imaging technique, that is orthogonal polarisation spectral imaging, allowing for non-invasive quantitative imaging of tumour angiogenesis in vivo. Experiments were performed in amelanotic melanoma A-MEL 3 implanted in a transparent dorsal skinfold chamber of the hamster. Starting at day 0 after tumour cell implantation, animals were treated daily with the anti-angiogenic compound SU5416 (25 mg kg x bw(-1)) or vehicle (control) only. Functional vessel density, diameter of microvessels and red blood cell velocity were visualised by both orthogonal polarisation spectral imaging and fluorescence microscopy and analysed using a digital image system. The morphological and functional properties of the tumour microvasculature could be clearly identified by orthogonal polarisation spectral imaging. Data for functional vessel density correlated excellently with data obtained by fluorescence microscopy (y=0.99x+0.48, r2=0.97, R(S)=0.98, precision: 8.22 cm(-1) and bias: -0.32 cm(-1)). Correlation parameters for diameter of microvessels and red blood cell velocity were similar (r2=0.97, R(S)=0.99 and r2=0.93, R(S)=0.94 for diameter of microvessels and red blood cell velocity, respectively). Treatment with SU5416 reduced tumour angiogenesis. At day 3 and 6 after tumour cell implantation, respectively, functional vessel density was 4.8+/-2.1 and 87.2+/-10.2 cm(-1) compared to values of control animals of 66.6+/-10.1 and 147.4+/-13.2 cm(-1), respectively. In addition to the inhibition of tumour angiogenesis, tumour growth and the development of metastasis was strongly reduced in SU5416 treated animals. This new approach enables non-invasive, repeated and quantitative assessment of tumour vascular network and the effects of antiangiogenic treatment on tumour vasculature in vivo. Thus, quantification of tumour angiogenesis can be used to more accurately classify and monitor tumour biologic characteristics, and to explore aggressiveness of tumours.

Assessment of the effect of Maytenus ilicifolia (espinheira santa) extract on the labeling of red blood cells and plasma proteins with technetium-99m.

We are trying to develop a model to assess properties of products utilized in popular medicine. Maytenus ilicifolia is used in herbal medicine. Red blood cells (RBC) labeled with technetium-99m (99mTc) are employed in nuclear medicine. This labeling procedure depends on a reducing agent and stannous chloride is used. There is evidence that this labeling may be altered by drugs. We have investigated the possibility of M. ilicifolia extract being capable to alter the labeling of blood elements with 99mTc. Blood was incubated with M. ilicifolia extract. Stannous chloride solution and Tc-99m were added. Blood was centrifuged and plasma (P) and blood cells (C) were isolated. Samples of P or C were also precipitated, centrifuged and insoluble (IF) and soluble (SF) were separated. The percentages of radioactivity (%ATI) in C, IF-P and IF-C was calculated. The %ATI decreased on C from 93.6+/-2.3 to 29.0+/-2.7, on IF-P from 77.6+/-1.2 to 7.5+/-1.0 and on IF-C from 80.0+/-3.4 to 12.6+/-4.8. Once in RBC labeling procedure with 99mTc depends on the presence of stannous (+2) ions, the substances of the M. ilicifolia extract could increase the valence these ions to stannic (+4). This fact would decrease the %ATI on blood elements and indicate the presence of oxidant agents in the M. ilicifolia extract.

[Scanning electron microscopy in the assessment of erythrocyte shape in peripheral blood of patients with anthracotic tuberculosis and pulmonary tuberculosis]. / Rastvovaia élektronnaia mikroskopiia v otsenke konfiguratsii éritrotsitov perifericheskoi krovi bol'nykh koniotuberkulezom i tuberkulezom legkikh.

Morphology of peripheral blood red cells was investigated in the course of enterosorption in patients with anthracotic and lung tuberculosis by means of scanning electron microscopy. Application of enterosorbent noticeably reduced the number of normocytes, this evidencing stimulating effect of detoxication. The proportion of deformed red cells lowered in parallel to inhibiting activity of tuberculous process activity in the lungs as a result of detoxicating effect of the treatment.

An assessment of the in vivo clastogenicity of erythrosine.

In an investigation of the in vivo clastogenic potential of the food colouring erythrosine (ER), male B6C3F1 mice were treated by ip injection at doses of 50, 100 and 200 mg/kg, repeated 24 hr apart. Signs of toxicity were observed at the highest dose of ER administered. The three cytogenetic endpoints analysed were sister chromatid exchanges (SCEs) in peripheral blood lymphocytes (PBLs), micronuclei in bone marrow polychromatic erythrocytes (PCEs), and micronuclei in peripheral blood reticulocytes (PBRs). SCE frequencies in PBLs were 4.13, 4.58, 4.33 and 4.60 SCE/cell at 0, 50, 100 and 200 mg ER/kg, respectively. At the same doses, the frequencies of micronucleated PCEs were 3.5, 3.2, 2.0 and 2.5/1000 PCEs. Micronuclei in PBRs ranged from 1.2 to 3.6 and from 1.4 to 3.0/1000 PBRs in control and treated mice, respectively. These results indicate that ER is inactive as a clastogen in mouse blood and marrow cells. This result supports the hypothesis of a non-genotoxic mechanism for ER carcinogenicity.