Computer vision quantitation of erythrocyte shape abnormalities provides diagnostic, prognostic, and mechanistic insight.

Examination of red blood cell (RBC) morphology in peripheral blood smears can help diagnose hematologic diseases, even in resource-limited settings, but this analysis remains subjective and semiquantitative with low throughput. Prior attempts to develop automated tools have been hampered by their poor reproducibility and limited clinical validation. Here, we present a novel, open-source machine-learning approach (denoted as RBC-diff) to quantify abnormal RBCs in peripheral smear images and generate an RBC morphology differential. RBC-diff cell counts showed high accuracy for single-cell classification (mean AUC, 0.93) and quantitation across smears (mean R2, 0.76 compared with experts, interexperts R2, 0.75). RBC-diff counts were concordant with the clinical morphology grading for 300 000+ images and recovered the expected pathophysiologic signals in diverse clinical cohorts. Criteria using RBC-diff counts distinguished thrombotic thrombocytopenic purpura and hemolytic uremic syndrome from other thrombotic microangiopathies, providing greater specificity than clinical morphology grading (72% vs 41%; P < .001) while maintaining high sensitivity (94% to 100%). Elevated RBC-diff schistocyte counts were associated with increased 6-month all-cause mortality in a cohort of 58 950 inpatients (9.5% mortality for schist. >1%, vs 4.7% for schist; <0.5%; P < .001) after controlling for comorbidities, demographics, clinical morphology grading, and blood count indices. RBC-diff also enabled the estimation of single-cell volume-morphology distributions, providing insight into the influence of morphology on routine blood count measures. Our codebase and expert-annotated images are included here to spur further advancement. These results illustrate that computer vision can enable rapid and accurate quantitation of RBC morphology, which may provide value in both clinical and research contexts.

Evaluation of the CellaVision Advanced RBC Application for Detecting Red Blood Cell Morphological Abnormalities.

BACKGROUND: The Advanced RBC Application of the CellaVision DM9600 system (CellaVision AB, Lund, Sweden) automatically characterizes and classifies red blood cells (RBCs) into 21 morphological categories based on their size, color, shape, and inclusions. We evaluated the diagnostic performance of the CellaVision Advanced RBC Application with respect to the classification and grading of RBC morphological abnormalities in accordance with the 2015 International Council for Standardization in Haematology (ICSH) guidelines. METHODS: A total of 223 samples, including 123 with RBC morphological abnormalities and 100 from healthy controls, were included. Seven RBC morphological abnormalities and their grading obtained with CellaVision DM9600 pre- and post-classification were compared with the results obtained using manual microscopic examination. The grading cut-off percentages were determined in accordance with the 2015 ICSH guidelines. The sensitivity and specificity of the CellaVision DM9600 system were evaluated using the manual microscopic examination results as a true positive. RESULTS: In pre-classification, >90% sensitivity was observed for target cells, tear drop cells, and schistocytes, while >90% specificity was observed for acanthocytes, spherocytes, target cells, and tear drop cells. In post-classification, the detection sensitivity and specificity of most RBC morphological abnormalities increased, except for schistocytes (sensitivity) and acanthocytes (specificity). The grade agreement rates ranged from 35.9% (echinocytes) to 89.7% (spherocytes) in pre-classification and from 46.2% (echinocytes) to 90.1% (spherocytes) in post-classification. The agreement rate of samples with within-one grade difference exceeded 90% in most categories, except for schistocytes and echinocytes. CONCLUSIONS: The Advanced RBC Application of CellaVision DM9600 is a valuable screening tool for detecting RBC morphological abnormalities.

Spicule movement on RBCs during echinocyte formation and possible segregation in the RBC membrane.

We use phase contrast microscopy of red blood cells to observe the transition between the initial discocyte shape and a spiculated echinocyte form. During the early stages of this change, spicules can move across the surface of the cell; individual spicules can also split apart into pairs. One possible explanation of this behaviour is that the membrane forms large scale domains in association with the spicules. The spicules are formed initially at the rim of the cell and then move at speeds of up to 3 µm/min towards the centre of the disc. Spicule formation that was reversed and then allowed to proceed a second time resulted in spicules at reproducible places, a shape memory effect that implies that the cytoskeleton contributes towards stopping the spicule movement. The splitting of the spicules produces a well-defined shape change with an increase in membrane curvature associated with formation of the daughter pair of spicules; the total boundary length around the spicules also increases. Following the model in which the spicules are associated with lipid domains, these observations suggest an experimental procedure that could potentially be applied to the calculation of the line tension of lipid domains in living cells.

Neonates with suspected microangiopathic disorders: performance of standard manual schistocyte enumeration vs. the automated fragmented red cell count.

OBJECTIVES: To enhance the diagnosis of schistocyte-producing conditions, we compared routine manual schistocyte enumeration with automated fragmented red cell counts (FRCs). STUDY DESIGN: In neonates "suspected" of having sepsis, NEC, or DIC we compared manual schistocyte estimates vs. automated FRC counts. When the two disagreed, we used a "gold standard" from a  ≥ 1000 RBC differential. We also assessed the diagnostic accuracy of the FRC count in diagnosing sepsis, NEC, or DIC. RESULTS: We collected 270 CBCs from 90 neonates. The methods agreed in 63% (95% CI 55%-70%) of the CBCs. Among the 37% where they disagreed, the FRC count was more accurate in 100% (95% CI 88-100%). An elevated FRC count was specific for sepsis, and was sensitive and specific for necrotizing enterocolitis and DIC. CONCLUSIONS: Automated FRC counts have advantages over routine manual evaluation, larger sample size, lower expense, and superior accuracy in diagnosing schistocyte-producing conditions.

Mercury leads to abnormal red blood cell adhesion to laminin mediated by membrane sulfatides.

Exposure to mercury is associated with numerous health problems, affecting different parts of the human body, including the nervous and cardiovascular systems in adults and children; however, the underlying mechanisms are yet to be fully elucidated. We investigated the role of membrane sulfatide on mercuric ion (Hg2+) mediated red blood cell (RBC) adhesion to a sub-endothelial matrix protein, laminin, using a microfluidic system that mimics microphysiological flow conditions. We exposed whole blood to mercury (HgCl2), at a range of concentrations to mimic acute (high dose) and chronic (low dose) exposure, and examined RBC adhesion to immobilized laminin in microchannels at physiological flow conditions. Exposure of RBCs to both acute and chronic levels of Hg2+ resulted in elevated adhesive interactions between RBCs and laminin depending on the concentration of HgCl2 and exposure duration. BCAM-Lu chimer significantly inhibited the adhesion of RBCs that had been treated with 50 µM of HgCl2 solution for 1 h at 37 °C, while it did not prevent the adhesion of 3 h and 24 h Hg2+-treated RBCs. Sulfatide significantly inhibited the adhesion of RBC that had been treated with 50 µM of HgCl2 solution for 1 h at 37 °C and 0.5 µM of HgCl2 solution for 24 h at room temperature (RT). We demonstrated that RBC BCAM-Lu and RBC sulfatides bind to immobilized laminin, following exposure of RBCs to mercuric ions. The results of this study are significant considering the potential associations between sulfatides, red blood cells, mercury exposure, and cardiovascular diseases.

Quantifying Shear-Induced Deformation and Detachment of Individual Adherent Sickle Red Blood Cells.

Vaso-occlusive crisis, a common painful complication of sickle cell disease, is a complex process triggered by intercellular adhesive interactions among blood cells and the endothelium in all human organs (e.g., the oxygen-rich lung as well as hypoxic systems such as liver and kidneys). We present a combined experimental-computational study to quantify the adhesive characteristics of sickle mature erythrocytes (SMEs) and irreversibly sickled cells (ISCs) under flow conditions mimicking those in postcapillary venules. We employed an in vitro microfluidic cell adherence assay, which is coated uniformly with fibronectin. We investigated the adhesion dynamics of SMEs and ISCs in pulsatile flow under well-controlled hypoxic conditions, inferring the cell adhesion strength by increasing the flow rate (or wall shear stress (WSS)) until the onset of cell detachment. In parallel, we performed simulations of individual SMEs and ISCs under shear. We introduced two metrics to quantify the adhesion process, the cell aspect ratio (AR) as a function of WSS and its rate of change (the dynamic deformability index). We found that the AR of SMEs decreases significantly with the increase of WSS, consistent between the experiments and simulations. In contrast, the AR of ISCs remains constant in time and independent of the flow rate. The critical WSS value for detaching a single SME in oxygenated state is in the range of 3.9-5.5 Pa depending on the number of adhesion sites; the critical WSS value for ISCs is lower than that of SMEs. Our simulations show that the critical WSS value for SMEs in deoxygenated state is above 6.2 Pa (multiple adhesion sites), which is greater than their oxygenated counterparts. We investigated the effect of cell shear modulus on the detachment process; we found that for the same cell adhesion spring constant, the higher shear modulus leads to an earlier cell detachment from the functionalized surface. These findings may aid in the understanding of individual roles of sickle cell types in sickle cell disease vaso-occlusion.

Automated Quantification of Fragmented Red Blood Cells: Neonatal Reference Intervals and Clinical Disorders of Neonatal Intensive Care Unit Patients with High Values.

BACKGROUND: Schistocytes are circulating erythrocyte fragments. They can be identified microscopically from a blood smear; but automated systems evaluate more cells and avoid inconsistencies in microscopy. Studies using adult subjects indicate that automated quantification of schistocytes can be clinically useful. However, reference intervals for automated schistocyte counts of neonates have not been published, and the relevance of a high automated schistocyte count from neonates has not been reported. OBJECTIVES: Using retrospective automated neonatal complete blood count (CBC) data, we created reference intervals for fragmented red cells (FRCs) and sought to discover the clinical conditions of neonates with high FRCs (above the upper reference interval). RESULTS: We created reference intervals based on 39,949 CBCs from 15,655 neonates 0-90 days old. The lower reference interval was 0 FRC/µL and the upper interval was 100,000/µL. The highest FRCs (96 CBCs from 44 neonates) were > 250,000/µL. These neonates clustered into the following groups: 37% had sepsis, 29% had disseminated intravascular coagulation (DIC), 17% had a genetic syndrome, 14% necrotizing enterocolitis (NEC), and 7% had iron deficiency (some had more than one diagnosis). Based on the reference intervals, we divided the 39,949 FRC values into 3 groups: (1) < 100,000/µL ("normal"), (2) 100,000-200,000/µL ("moderately elevated"), and (3) > 200,000/µL ("extremely elevated"). The odds that a microangiopathic condition (DIC, sepsis, NEC) or a microcytic disorder (iron deficiency) were present were significantly higher in the moderately elevated, and more so in the extremely elevated group. CONCLUSIONS: Our study suggests that a high FRC could prompt investigation into, or inform follow-up of, a neonatal microangiopathic or extremely microcytic disorder.

Importance of methodological standardization for the ektacytometric measures of red blood cell deformability in sickle cell anemia.

Red blood cell (RBC) deformability is severely decreased in patients with sickle cell anemia (SCA), which plays a role in the pathophysiology of the disease. However, investigation of RBC deformability from SCA patients demands careful methodological considerations. We assessed RBC deformability by ektacytometry (LORRCA MaxSis, Mechatronics, The Netherlands) in 6 healthy individuals and 49 SCA patients and tested the effects of different heights of the RBC diffraction patterns, obtained by altering the camera gain of the LORRCA, on the result of RBC deformability measurements, expressed as Elongation Index (EI). Results indicate that the pattern of RBCs from control subjects adopts an elliptical shape under shear stress, whereas the pattern of RBCs from individuals with SCA adopts a diamond shape arising from the superposition of elliptical and circular patterns. The latter represent rigid RBCs. While the EI measures did not change with the variations of the RBC diffraction pattern heights in the control subjects, we observed a decrease of EI when the RBC diffraction pattern height is increased in the SCA group. The differences in SCA EI values measured at 5 Pa between the different diffraction pattern heights correlated with the percent of hemoglobin S and the percent of sickled RBC observed by microscopy. Our study confirms that the camera gain or aperture of the ektacytometer should be used to standardize the size of the RBC diffraction pattern height when measuring RBC deformability in sickle cell patients and underscores the potential clinical utility of this technique.

Control of metazoan heme homeostasis by a conserved multidrug resistance protein.

Several lines of evidence predict that specific pathways must exist in metazoans for the escorted movement of heme, an essential but cytotoxic iron-containing organic ring, within and between cells and tissues, but these pathways remain obscure. In Caenorhabditis elegans, embryonic development is inextricably dependent on both maternally derived heme and environmentally acquired heme. Here, we show that the multidrug resistance protein MRP-5/ABCC5 likely acts as a heme exporter, and targeted depletion of mrp-5 in the intestine causes embryonic lethality. Transient knockdown of mrp5 in zebrafish leads to morphological defects and failure to hemoglobinize red blood cells. MRP5 resides on the plasma membrane and endosomal compartments and regulates export of cytosolic heme. Together, our genetic studies in worms, yeast, zebrafish, and mammalian cells identify a conserved, physiological role for a multidrug resistance protein in regulating systemic heme homeostasis. We envision other MRP family members may play similar unanticipated physiological roles in animal development.

Developmental plasticity of red blood cell homeostasis.

Most human physiologic set points like body temperature are tightly regulated and show little variation between healthy individuals. Red blood cell (RBC) characteristics such as hematocrit and mean cell volume are stable within individuals but can vary by 20% from one healthy person to the next. The mechanisms for the majority of this inter-individual variation are unknown and do not appear to involve common genetic variation. Here, we show that environmental conditions present during development, namely in utero iron availability, can exert long-term influence on a set point related to the RBC life cycle. In a controlled study of rhesus monkeys and a retrospective study of humans, we use a mathematical model of in vivo RBC population dynamics to show that in utero iron deficiency is associated with a lowered threshold for RBC clearance and turnover. This in utero effect is plastic, persisting at least 2 years after birth and after the cessation of iron deficiency. Our study reports a rare instance of developmental plasticity in the human hematologic system and also shows how mathematical modeling can be used to identify cellular mechanisms involved in the adaptive control of homeostatic set points.

Quantitative detection of target cells using unghosted cells (UGCs) of DxH 800 (Beckman Coulter).

BACKGROUND: In the Retic channel of DxH 800 (Beckman Coulter), the red blood cells (RBCs) resistant to hemoglobin clearing are counted as unghosted cells (UGCs). The aim of this study was to evaluate that the UGC is a surrogate marker for both the detection and counting of target cells. METHODS: In total, 1181 samples including 22 from iron deficiency anemia (IDA) patients, 95 from jaundice, 2 from sickle cell anemia, 3 from thalassemia, 1 cord blood, and 269 from normal controls were analyzed. Slides were prepared from all samples except normal controls and target cells were counted for correlation analysis of target cell counts to UGCs. RESULTS: The normal control samples showed 0.01% (0%-0.01%) UGCs, and the reference range was set at ≤0.02%. The IDA samples showed 0.015% (0.01%-0.03%) UGC count and 0.05% (0%-0.2%) target cell count. The jaundice samples showed 0.98% (0.1%-5.36%) UGC count, and 1.4% (0.1%-7.0%) target cell count. The two sickle cell anemia samples showed 0.41% and 3.74% UGC counts and 0.4% and 11.5% target cell counts. A cord blood sample showed 0.01% UGCs and 0% target cells. The three thalassemia samples showed 0.01%, 1.99%, and 7.82% UGC counts and 0%, 1.4%, and 15.5% target cell counts. The samples showing poikilocytosis other than target cells showed normal UGC count (≤0.02%). The positive predictive value of UGCs was 58.2% (124/213) and the negative predictive value was 96.8% (674/696). The UGC counts were well correlated to the manual target cell counts (r=0.944, p=0.000). CONCLUSIONS: This study demonstrates for the first time in the literature that a hematological parameter obtained automatically every time a reticulocyte counting is performed can be used to both screen for the presence of target cells and reliably quantify them.

The passage of a hemodialysis filter affects hemorheology, red cell shape, and platelet aggregation.

We investigated the influence of the passage of a hemodialysis filter on red blood cells (RBCs), platelets, and hemorheological parameters. After one hour of hemodialysis, blood was drawn from 15 patients immediately ahead and behind the dialysis filter. RBCs were fixed for morphological analysis. Blood viscosity was measured with a Couette viscometer (LS-30, Contraves), RBC aggregation with a Myrenne aggregometer, platelet aggregation in flowing whole blood and in platelet rich plasma. The passage of the hemodialysis filter increased the hematocrit from 34.0 ± 3.8 to 44.6 ± 8.7% (p < 0.01). Discocytes decreased from 73 ± 9 to 60 ± 15%, while echinocytes/knizocytes were more abundant 24 ± 9% and 38 ± 15%, respectively, p < 0.01). Blood viscosity increased from 3.77 ± 0.52 to 6.75 ± 2.21 mPa.s (p < 0.01). The RBC aggregation index decreased from 25.8 ± 5.0 to 20.9 ± 5.6 (p < 0.05). These changes were less pronounced when the blood flow rate was reduced from 350 to 100 ml/min. Platelet aggregation was slightly increased in flowing whole blood, but decreased in platelet rich plasma. At the end of hemodialysis, a small increase in abnormally shaped RBCs, hematocrit, and whole blood viscosity persisted; platelet aggregation in flowing whole blood was reduced in all patients. We conclude that the passage of a hemodialysis filter induced RBC shape changes, increased the hematocrit, whole blood and plasma viscosity, decreased RBC aggregation, and affected platelet aggregation.

Erythrocyte dysplasia in peripheral blood smears from 5 thrombocytopenic dogs treated with vincristine sulfate.

Secondary dyserythropoiesis has been associated with vincristine administration in dogs. Evaluation of bone marrow aspirates for the presence of morphologic abnormalities in the erythroid lineage aids in the diagnosis. However, morphologic features of circulating erythroid precursors in these cases have not been described previously. The purpose of this report was to describe the cytologic features of dyserythropoiesis in peripheral blood and also bone marrow smears in a case series of dogs with immune-mediated thrombocytopenia (IMT) treated with vincristine sulfate. Nineteen dogs receiving vincristine for treatment of IMT were identified by retrospectively searching a computerized medical record system. There were 5 dogs that had dysplastic erythroid precursors in peripheral blood smears within 7 days of vincristine treatment. Two of those 5 dogs also had evidence for erythrodysplasia in modified Wright's-stained bone marrow smears obtained postvincristine administration. Morphologic changes included bizarre or inappropriate mitotic figures, abnormal nuclear configurations (fragmentation, elongation, indentation, and binucleation), atypical nuclear remnants (Howell-Jolly bodies), or nuclear and cytoplasmic asynchrony within the erythroid precursors. A brief review of the literature with discussion of the etiologies for dyserythropoiesis is provided. The dyserythropoiesis was clinically insignificant in all 5 cases and resolved. However, pathologists and clinicians should be aware of these potential findings to prevent misdiagnosis of other conditions.

Fragmented red blood cells automated measurement is a useful parameter to exclude schistocytes on the blood film.

INTRODUCTION:   The diagnosis of thrombotic microangiopathies (TMA) or disorders that may mimic their features remains difficult. Mechanical hemolytic anemia with the detection of shistocytes on the blood smear is a cornerstone finding to assess the diagnosis, but microscopic evaluation of shistocytes is still problematic with wide interobserver variations. Some of the latest generation automated blood cell counters (ABCC) propose an original quantitative approach of fragmented red cells (FRC), aiming to be equivalent to the microscopic count. This parameter has been poorly evaluated. METHODS:   To assess the predictive value (PV) of this test, we conducted studies comparing automated and microscopic counts of FRC/schistocytes, based on the analysis of thousands samples in four university hospitals and using the 2 ABCC currently available (Siemens ADVIA series, Sysmex XE-2100). RESULTS: Reference range for FRC was <0.3% for the ADVIA and <0.5% for the XE-2100. The presence of FRC below a threshold determined at 1% (ADVIA and XE-2100) had a negative PV close to 100% to exclude the presence of schistocyte on the blood smear, but in relationship with a poor PV value. CONCLUSIONS: Our study validated the utility of the immediately available FRC parameter on ABCC to exclude schistocytes and the diagnosis of TMA.

Reconstruction of erythrocyte shape during modified morphological response.

Changes in erythrocyte shape during morphological response modified by benzalkonium chloride (BzA) were studied in sucrose solutions. Fixation of the cells with glutaraldehyde- and formaldehyde-containing fixatives at some time points is usually inadequate to maintain the current cell shape. Considering the reconstruction of erythrocyte shape, which takes into account the mode of fixative action, we showed that the echinocyte-forming activity of BzA depends on the concentration of this surfactant. It can induce a direct spherostomatocyte-spheroechinocyte transition without altering the near-spherical shape of the cells. On the other hand, the reverse spheroechinocyte-spherostomatocyte transition was always accompanied by some flattening of the cells, although in some instances discoidal shape was not achieved. The data point to asymmetric shape transitions of erythrocytes in sucrose solution, which contradicts the continuum and bilayer-couple models of shape regulation. It seems that the nonuniform structure of native erythrocyte membrane plays a more important role in morphological transitions of these cells than suggested earlier.

Genotoxic effect of epirubicin in mouse bone marrow in vivo.

The genotoxic effect of epirubicin, a semisynthetic anthracycline antibiotic which has been used as an anticancer drug, was investigated in vivo on bone marrow cells of Swiss albino mice using the micronucleus test. To determine the incidence of micronuclei, mice were injected intraperitoneally with the drug at single doses of 4, 6, 8, and 10 mg/kg body weight. Then, bone marrow was sampled 18, 24, 36, and 48 h after the treatment. Polychromatic and normochromatic erythrocytes were examined for the presence of micronuclei. Epirubicin significantly increased the frequency of micronucleated polychromatic erythrocytes (MNPCEs) for all treatment periods compared with the negative control (P < 0.001). The frequency of MNPCEs increased with the dose, but at the highest dose used (which is considered to be quite toxic), the frequency of MNPCEs was rather lower. Epirubicin also decreased the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) for all sampling intervals, which is indicative of bone marrow cytotoxicity. It can be concluded from the present study that the anticancer drug epirubicin has genotoxic effects on mouse bone marrow cells.

Effect of interleukin-8 and RANTES on the Gardos channel activity in sickle human red blood cells: role of the Duffy antigen receptor for chemokines.

We investigated the effects of the chemokines IL-8 and RANTES on the activity of the Gardos channel (GC) of sickle red blood cells (SSRBCs). SSRBCs expressing the Duffy antigen receptor for chemokines (DARC) incubated under oxygenated conditions exhibit GC activation. The deoxygenation-stimulated K(+) loss via the GC is activated by the chemokines in the Duffy-positive SSRBCs. The percentage of cells with high density is 17 times higher in the Duffy-positive group. These findings are consistent with a greater susceptibility of Duffy-positive SSRBCs to inflammatory chemokines leading to GC activation and cellular dehydration and suggest a coupling, promoted by the sickling process, between DARC and the GC.

Statistical dynamics of flowing red blood cells by morphological image processing.

Blood is a dense suspension of soft non-Brownian cells of unique importance. Physiological blood flow involves complex interactions of blood cells with each other and with the environment due to the combined effects of varying cell concentration, cell morphology, cell rheology, and confinement. We analyze these interactions using computational morphological image analysis and machine learning algorithms to quantify the non-equilibrium fluctuations of cellular velocities in a minimal, quasi-two-dimensional microfluidic setting that enables high-resolution spatio-temporal measurements of blood cell flow. In particular, we measure the effective hydrodynamic diffusivity of blood cells and analyze its relationship to macroscopic properties such as bulk flow velocity and density. We also use the effective suspension temperature to distinguish the flow of normal red blood cells and pathological sickled red blood cells and suggest that this temperature may help to characterize the propensity for stasis in Virchow's Triad of blood clotting and thrombosis.

Sickle red cells induce adhesion of lymphocytes and monocytes to endothelium.

Infusion of epinephrine-activated human sickle erythrocytes (SS RBCs) into nude mice promotes both SS RBC and murine leukocyte adhesion to vascular endothelium in vivo. We hypothesized that interaction of epinephrine-stimulated SS RBCs with leukocytes leads to activation of leukocytes, which then adhere to endothelial cells (ECs). In exploring the underlying molecular mechanisms, we have found that coincubation in vitro of epinephrine-treated SS RBCs with human peripheral blood mononuclear cells (PBMCs) results in robust adhesion of PBMCs to ECs. Sham-treated SS RBCs had a similar but less pronounced effect, whereas neither sham- nor epinephrine-treated normal RBCs activated PBMC adhesion. PBMC activation was induced via at least 2 RBC adhesion receptors, LW and CD44. In response to SS RBCs, leukocyte CD44 and beta2 integrins mediated PBMC adhesion to ECs, a process that involved endothelial E-selectin and fibronectin. SS RBCs activated adhesion of both PBMC populations, lymphocytes and monocytes. Thus, our findings reveal a novel mechanism that may contribute to the pathogenesis of vaso-occlusion in sickle cell disease, in which SS RBCs act via LW and CD44 to stimulate leukocyte adhesion to endothelium, and suggest that RBC LW and CD44 may serve as potential targets for antiadhesive therapy designed to prevent vaso-occlusion.

Effects of triploidy on the Caspian salmon Salmo trutta caspius haematology.

The aim of this study was a comparison of key haematological features of diploid (2n) and triploid (3n) Caspian salmon (Salmo trutta caspius). Morphometric indices of erythrocytes were determined on blood smears by light microscopy. Triploidy significantly (P < 0.001) increased all morphometric indices measured in the erythrocytes including cell size, cell surface area, and cell volume. The increase in cell size was larger for the major (27%) axis than for the minor (22%) axis, thus making erythrocytes of 3n Caspian salmon more ellipsoidal. The estimated increase in erythrocyte nuclear volume (87%) was bigger than the theoretical expected 50% increase. Haematological indices were measured manually by hemocytometry. Triploids had lower numbers of red blood cells (RBC: 1,120,000 cells/mL in 2n vs. 700,000 cells/mL in 3n; P < 0.001) but they were larger in size (mean erythrocytic volume [MEV]: 363.1 nm3 in 2n vs. 483.3 nm3 in 3n; P < 0.001). The decrease in RBC number was not compensated by the increase in MEV and, thus, triploidy affected the haematocrit (Hct: 38.8% in 2n vs. 33.06% in 3n; P < 0.05). Total blood hemoglobin concentration was lower in triploid fish (Hb: 9.9 g/dL in 2n vs. 8.9 g/dL in 3n; P < 0.05). In contrast, mean erythrocytic hemoglobin (MEH: 95 mug in 2n vs. 133.2 mug in 3n; P < 0.001) was higher for 3n Caspian salmon as a result of their larger erythrocytes, although MEH concentration (MEHC: 0.26 g/dL in 2n vs. 0.27 g/dL in 3n) did not significantly differ (P > 0.05). White blood cell (WBC) counts (lymphocytes and neutrophiles) were measured and WBC/RBC ratios were calculated. There were no significant differences in WBC (15,710 cells/mL in 2n vs. 12,683 cells/mL in 3n; P > 0.05), lymphocytes, and neutrophils as %WBC as well as WBC/RBC ratios between two ploidy levels (P > 0.05). Triploid Caspian salmon showed higher erythrocyte abnormalities such as 'twisted', 'tailed', and 'anucleated' cells as well as high portions of immature RBC in blood smears in comparison with diploids (P < 0.001).