Red blood cell alloimmunization prevalence and hemolytic disease of the fetus and newborn in Israel: A retrospective study.

Hemolytic disease of the fetus and newborn (HDFN) is a severe form of anemia caused by maternal antibodies against fetal red blood cells (RBC) that can cause intrauterine and perinatal morbidity and mortality. The prevalence and specificities of alloantibodies among Israeli pregnant women and clinical outcomes for their fetuses and newborns are unknown. STUDY DESIGN AND METHODS: A retrospective study of women who gave birth between January 1, 2011, and December 31, 2011, was performed. Data were obtained for obstetric admissions from 16 of 27 hospitals, which included results of maternal ABO, D, antibody screens, antibody identification, and requirements for intrauterine or newborn exchange transfusions. RESULTS: Data on 90 948 women representing 70% of all births during 2011 were analyzed. Antibody screen was positive in 5245 (5.8%) women. Alloantibodies, excluding anti-D titer (<16) were identified in 900 (1.0%) women. Of 191 D- women, 75 (39.3%) had anti-D titer of 16 or greater. Other common clinically significant antibodies were anti-E (204, 23%), anti-K (145, 16%), and anti-c (97, 10.8%) alone or in antibody combinations. Multiple alloantibodies were observed in 132 of 900 (15%) of women. Severe HDFN developed in 6.8% (9/132) of these pregnancies. Seventeen fetuses and newborns (0.02% of births) including one set of twins required RBC transfusions. Two fetuses whose mothers had multiple alloantibodies received intrauterine transfusions; one of them was hydropic and died. CONCLUSION: The prevalence of RBC alloantibodies was 1.0% among Israeli pregnant women. Transfusion was required in 0.02% of the fetuses and newborns. Severe HDFN developed in 6.8% of pregnancies with multiple maternal alloantibodies.

Primed polymorphonuclear leukocytes from hemodialysis patients enhance monocyte transendothelial migration.

Increased counts and priming of peripheral polymorphonuclear leukocytes (PMNLs) are associated with future or ongoing atherosclerosis; however, the role of PMNLs in enhancing monocyte transendothelial migration is still unclear. Our aims were to examine endothelial and monocyte activation, transmigration, and posttransmigration activation induced ex vivo by in vivo primed PMNLs and the effect of antioxidants on the activation. A unique ex vivo coculture system of three cell types was developed in this study, enabling interactions among the following: primary human umbilical vein endothelial cells (HUVECs), monocytes (THP-1 cell line), and in vivo primed PMNLs from hemodialysis (HD) patients and healthy control (HC) subjects. The interactions among these cells were examined, and an intervention with superoxide dismutase and catalase was performed. Preexposed HUVECs to HD/HC PMNLs showed a significant monocyte transmigration yield, 120-170% above HCs. Monocyte exposure to HD PMNLs induced pre- and posttransmigration activation. When the three cell types were cocultivated at the same time, monocyte chemoattractant protein-1 protein levels released from HUVECs, and activation markers on HUVECs [CD54 and chemokine (C-X3-C motif) ligand 1] and monocytes [chemokine (C-X3-C) receptor 1 and chemokine (C-C motif) receptor 2] were increased. Monocyte transmigration yield decreased to 70% (compared with HC subjects) due to adherence and accumulation of monocytes to HUVECs. When superoxide dismutase and catalase were used, reduced HUVEC and monocyte activation markers brought the transmigration yields to control levels and abolished accumulation of monocytes, emphasizing the role of superoxide in this process. We conclude that peripheral primed PMNLs play a pivotal role in enhancing monocyte transendotelial migration, the hallmark of the atherosclerotic process. Primed PMNLs can be used as a mediator and a biomarker of atherosclerosis even before plaque formation.NEW & NOTEWORTHY Primed polymorphonuclear leukocytes are key mediators in monocyte transendothelial migration, a new understanding of the initiation of endothelial dysfunction and monocyte activation, transmigration, and accumulation in the subendothelial layer.

Lymphocyte Subset Reference Ranges in Healthy Israeli Adults.

BACKGROUND: Reference ranges for adult peripheral blood lymphocyte subsets have been established in a few countries. To the best of our knowledge no broad lymphocyte subset analysis of the Israeli population has been reported.  Objectives: To establish reference ranges for healthy adults in Israel and to describe age- and gender-specific differences, if present. OBJECTIVES: To establish reference ranges for healthy adults in Israel and to describe age- and gender-specific differences, if present. METHODS: Lymphocyte subsets CD3, CD3/CD4, CD3/CD8, CD3-/CD16+/CD56+, CD3/TCRαß, CD3/TCRγδ, and CD19 were examined by flow cytometry in 326 subjects. Samples were subdivided according to age and gender. RESULTS: Women of all ages had a significantly higher percentage and absolute counts of CD3/CD4 cells than their male counterparts. Higher CD3/CD4 cells were observed also in the older population (> 50 years). CD3/CD8 and CD3-/CD16+/CD56+ were higher in males. Older males had a lower total lymphocyte percentage and CD19 cells compared to younger men. No significant gender-related differences were observed in percent and number of CD19, CD3/TCRαß or CD3/TCRγδ at all ages. CONCLUSIONS: These reference values could be useful in further studies for assessing changes that occur in different populations in human pathology.

Gray platelet syndrome: natural history of a large patient cohort and locus assignment to chromosome 3p.

Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by macrothrombocytopenia and absence of platelet α-granules resulting in typical gray platelets on peripheral smears. GPS is associated with a bleeding tendency, myelofibrosis, and splenomegaly. Reports on GPS are limited to case presentations. The causative gene and underlying pathophysiology are largely unknown. We present the results of molecular genetic analysis of 116 individuals including 25 GPS patients from 14 independent families as well as novel clinical data on the natural history of the disease. The mode of inheritance was autosomal recessive (AR) in 11 and indeterminate in 3 families. Using genome-wide linkage analysis, we mapped the AR-GPS gene to a 9.4-Mb interval on 3p21.1-3p22.1, containing 197 protein-coding genes. Sequencing of 1423 (69%) of the 2075 exons in the interval did not identify the GPS gene. Long-term follow-up data demonstrated the progressive nature of the thrombocytopenia and myelofibrosis of GPS resulting in fatal hemorrhages in some patients. We identified high serum vitamin B(12) as a consistent, novel finding in GPS. Chromosome 3p21.1-3p22.1 has not been previously linked to a platelet disorder; identification of the GPS gene will likely lead to the discovery of novel components of platelet organelle biogenesis. This study is registered at www.clinicaltrials.gov as NCT00069680 and NCT00369421.

Comparison of a modified flow cytometry osmotic fragility test with the classical method for the diagnosis of hereditary spherocytosis.

BACKGROUND: Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia. The flow cytometric test using eosin-5'maleimide (EMA) is a well-established diagnostic method. However, in order to improve HS detection, it is recommended that EMA and an osmotic fragility test (OFT) both be performed. OFT is time consuming and labor intensive. We used a flow cytometric (FOFT) adaptation of the classical OFT reported by Yamamoto. We compare the FOFT to the classical OFT including practical data and propose options for simplifying this method. METHODS: Suspected and known HS patients and controls were tested by the following methods: EMA, OFT, and FOFT including some modifications. RESULTS: The FOFT method is robust and correlates to loss of red blood cells. OFT and FOFT gave similar results in healthy controls and four HS patients. Normal range for FOFT in 70 adults is shown and can be used as a reference value. Neonates should have their own normal range defined. Overnight sample incubation at 37°C did not add information to the FOFT results. CONCLUSION: Our modified Yamomoto FOFT can replace the classic OFT as the addition to EMA for the diagnosis of HS. The use of flow cytometry in both these methods requires small sample volume, is reproducible, simpler, and produces results more rapidly.

The Role of Alpha 2 Macroglobulin in IgG-Aggregation and Chronic Activation of the Complement System in Patients With Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the western world. One of the treatments offered for CLL is immunotherapy. These treatments activate various cellular and biochemical mechanisms, using the complement system. Recently it was shown that the complement system in CLL patients is persistently activated at a low level through the classical pathway (CP). The mechanism of chronic CP activation involves the formation of IgG-hexamers (IgG-aggregates). According to recent studies, formation of ordered IgG-hexamers occurs on cell surfaces via specific interactions between Fc regions of the IgG monomers, which occur after antigen binding. The present study investigated the formation of IgG-hexamers in CLL patients and normal (non-malignant) controls (NC), their ability to activate complement, their incidence as cell-free and cell-bound forms and the identity of the antigen causing their formation. Sera from 30 patients and 12 NC were used for separation of IgG- aggregates. The obtained IgG- aggregates were measured and used for assessment of CP activation. For evaluation of the presence of IgG- aggregates on blood cells, whole blood samples were stained and assessed by flow cytometry. Serum levels of IgG- aggregates were higher in CLL and they activated the complement system to a higher extent than in NC. Alpha 2 macroglobulin (A2M) was identified as the antigen causing the hexamerization/aggregation of IgG, and was found to be part of the hexamer structure by mass spectrometry, Western blot and flow cytometry analysis. The presence of A2M-IgG-hexamers on B-cells suggests that it may be formed on B cells surface and then be detached to become cell-free. Alternatively, it may form in the plasma and then attach to the cell surface. The exact time course of A2M-IgG-hexamers formation in CLL should be further studied. The results in this study may be useful for improvement of current immunotherapy regimens.

Epoetin-alpha: preserving kidney function via attenuation of polymorphonuclear leukocyte priming.

BACKGROUND: Polymorphonuclear leukocyte priming and low grade inflammation are related to severity of kidney disease. Erythropoietin-receptor is present on PMNLs. OBJECTIVESxi: To evaluate the effect of 20 weeks of epoetin-alpha treatment on PMNL characteristics in relation to the rate of kidney function deterioration in patients with chronic kidney disease. METHODS: Forty anemic chronic kidney disease patients, stage 4-5, were assigned to EPO and non-EPO treatment for 20 weeks. A group of 20 healthy controls was also studied. PMNL priming and PMNL-derived low grade inflammation were estimated, in vivo and ex vivo, before and after EPO treatment: The rate of superoxide release, white blood cells and PMNL counts, serum alkaline phosphatase and PMNL viability were measured. EPO-receptor on PMNLs was assayed by flow cytometry. The effect of 20 weeks of EPO treatment on kidney function was related to the estimated glomerular filtration rate. esults: EPO treatment attenuated superoxide release ex vivo and in vivo and promoted PMNL survival ex vivo. Decreased low grade inflammation was reflected by reduced WBC and PMNL counts and ALP activity following treatment. EPO retarded the deterioration in GFR. The percent of PMNLs expressing EPO-R was higher before EPO treatment and correlated positively with the rate of superoxide release. After 20 weeks of EPO treatment the percent of PMNLs expressing EPO-R was down-regulated. CONCLUSIONS: These non-erythropoietic properties of EPO are mediated by EPO-R on PMNLs, not related to the anemia correction. A new renal protection effect of EPO via attenuation of PMNL priming that decreases systemic low grade inflammation and oxidative stress is suggested.

Adult pre B-cell acute lymphoblastic leukemia with unusually large proportion of bone marrow CD45 bright/high SSc blasts.

BACKGROUND: We present a pre B-ALL patient with the rare clinical manifestation of extramedullary disease, and a normal hemogram. This patient's blasts expressed bright CD45 and high side scatter (SSc) placing the cells in the monocyte gate. METHODS: Samples from peripheral blood and bone marrow (BM) aspirate from a 50-year-old female patient were immunophenotyped by multiparametric flow cytometry. RESULTS: Flow cytometry studies of the BM aspirate showed a large monocyte gate with 90-95% of the cells expressing an abnormal B cell phenotype. Peripheral white blood cells count was normal and cytogenetic analysis of the BM revealed a normal karyotype. CONCLUSION: It was not possible, based on CD45/SSc to identify a lymphoblast population in this pre B-ALL patient. Although bright expression of CD45 B-ALL blasts has been associated with poor prognosis to the best of our knowledge, the combination of bright CD45 blasts with high SSc has not been reported. As CD45 expression vs. SSc is routinely measured in the diagnostics of acute leukemias, a possible association between CD45 bright positivity and extramedullary disease or prognosis warrants further exploration. © 2015 International Clinical Cytometry Society.

Exogenous superoxide mediates pro-oxidative, proinflammatory, and procoagulatory changes in primary endothelial cell cultures.

Endothelial dysfunction/activation underlies the development of long-term cardiovascular complications and atherosclerosis. The aim of this study was to examine a direct role for exogenous sublethal flux of superoxide on endothelial cell dysfunction. Human umbilical vein endothelial cells (HUVEC) were exposed to superoxide generated by 0.1 mM xanthine and 4 mU/ml xanthine oxidase for 15 min and essential endothelial functions were examined. Superoxide dismutase and/or catalase was used as scavenger for O(2)(-)/H(2)O(2) to determine the key culprit. HUVEC detachment was determined by neutral red uptake and apoptosis by annexin V binding. Inflammation was estimated by IL-8 mRNA expression and cellular adhesion molecules (CAM). eNOS and iNOS message and eNOS protein served as an indirect measure for NO. Procoagulable state was evaluated by estimating the intracellular tissue factor. Activation of endothelial NADPH oxidase was determined by lucigenin chemiluminescence. Sublethal superoxide dose evoked: (1) proinflammatory state manifested by increased IL-8 mRNA expression and CAM on the endothelial surface, (2) HUVEC apoptosis and activated endothelial NADPH oxidase, (3) increase in intracellular tissue factor, and (4) decrease in eNOS mRNA and protein and up-regulation of iNOS mRNA. We conclude that extracellular low flux of superoxide exhibits pleiotropic characteristics, triggering activation/dysfunction of endothelial cells.

Heparin Interaction with the Primed Polymorphonuclear Leukocyte CD11b Induces Apoptosis and Prevents Cell Activation.

Heparin is known to have anti-inflammatory effects, yet the mechanisms are not completely understood. In this study, we tested the hypothesis that heparin has a direct effect on activated polymorphonuclear leukocytes (PMNLs), changing their activation state, and can explain its anti-inflammatory effect. To test our hypothesis, we designed both in vitro and ex vivo studies to elucidate the mechanism by which heparin modulates PMNL functions and therefore the inflammatory response. We specifically tested the hypothesis that priming of PMNLs renders them more susceptible to heparin. Amplified levels of CD11b and increased rate of superoxide release manifested PMNL priming. Increase in cell priming resulted in a dose-dependent increase in heparin binding to PMNLs followed by augmented apoptosis. Blocking antibodies to CD11b inhibited heparin binding and abolished the apoptotic response. Moreover, heparin caused a significant dose-dependent decrease in the rate of superoxide release from PMNLs, which was blunted by blocking antibodies to CD11b. Altogether, this study shows that the interaction of heparin with the PMNL CD11b results in cell apoptosis and explains heparin's anti-inflammatory effects.

A link between polymorphonuclear leukocyte intracellular calcium, plasma insulin, and essential hypertension.

BACKGROUND: Intracellular ionized calcium ([Ca2+]i) is a key mediator in the activation and oxidant production by peripheral polymorphonuclear leukocytes (PMN). Primed PMN contribute to oxidative stress (OS) and inflammation in essential hypertension (EH). Elevated [Ca2+]i has been described in insulin-resistant states and in various cell types in EH but not in EH PMN. The aim of this study was to evaluate the levels of [Ca2+]i in peripheral EH PMN in relation to plasma insulin levels and blood pressure (BP). METHODS: The PMN were separated from blood of 20 nonsmoking, nonobese untreated EH patients, age range 20 to 60 years and from 20 age- and gender-matched healthy individuals (NC). Plasma glucose and insulin levels 2 h after a 75-g oral glucose load, reflected insulin resistance. PMN [Ca2+]i was measured by flow cytometry in isolated cells stained with Fluo-3. RESULTS: The EH PMNs showed significantly increased [Ca2+]i compared to NC PMN. Eighty percent of EH patients showed significantly higher plasma insulin levels after glucose load. Linear regression analysis showed significant correlation between 1) PMN [Ca2+]i and mean arterial pressure (MAP) (r = 0.5, P < .006); 2) PMN [Ca2+]i and fasting plasma insulin (r = 0.7, P < .005); and 3) fasting plasma insulin and MAP (r = 0.4, P < .04). CONCLUSIONS: This study adds PMN to previously described cells exhibiting elevated [Ca2+]i, contributing to OS and inflammation. The correlation of individual BP with both PMN [Ca2+]i and plasma insulin levels, together with the fact that elevated [Ca2+]i mediates PMN priming, suggest that elevated [Ca2+]i and insulin are involved in the pathogenesis of hypertension-induced vascular injury in EH.

Comparison of two techniques for the evaluation of fetomaternal hemorrhage in RhD-negative women: gel agglutination and haemoglobin F determination by flow cytometry.

BACKGROUND: In the treatment of RhD-negative women, it is clinically important to adjust the RhD immunoglobulin dose to the volume of the fetal-maternal hemorrhage (FMH). The present study compared a standard flow cytometry technique for FMH quantification to a simple alternative, the gel agglutination test. METHODS: Blood samples were collected from 118 RhD negative women after delivery, and were analysed for the amount of FMH by both flow cytometry and the gel agglutination test. Events associated with increased FMH in a previous and current pregnancy, and with neonatal complications, were correlated to the results. RESULTS: A FMH of 0.1 ml or more was detected in all 118 women in the study group by flow cytometry (mean 2.0+/-1.2 ml), but in only 31 women (35.6% of 87 with RhD positive infant) (mean FMH 0.76+/-1.48 ml) by the gel agglutination test (p<0.001). On multivariate regression analysis, only gestational age was a weak significant independent positive predictor for FMH (r (2)=0.037, p=0.047). CONCLUSION: The gel agglutination technique, as used in the range of 0.1-10 ml, is not sensitive enough to detect FMH.

Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury.

Peripheral polymorphonuclear leukocytes (PMNL) in hemodialysis (HD) patients are primed, continually releasing and exposing the vascular endothelium to soluble factors such as reactive oxygen species and inflammatory mediators. To mimic the close proximity between PMNL and the endothelial monolayer and to monitor and characterize the influence of soluble mediators released from PMNL, we developed a novel cocultivation system using primary human umbilical vein endothelial cell (HUVEC) cultures and PMNL, with a sieve separating the two cell types to prevent direct adhesive effects. PMNL (10(6)) from HD patients or from healthy normal controls were cocultivated with HUVEC (10(5)) for 15 min, and endothelial cell injury was assessed by HUVEC morphology, cell detachment, and apoptosis. Proinflammatory changes were estimated by expression of HUVEC adhesion molecule P-selectin and by endothelial IL-8 and endothelial nitric oxide synthase mRNA. The levels of intracellular tissue factor reflected the procoagulant state, whereas NADPH oxidase activity served as an indicator for prooxidative changes in HUVEC. Mediators released from the primed PMNL triggered activation/dysfunction of endothelial cells, causing 1) an increase in endothelial cell detachment and apoptosis, 2) a proinflammatory state manifested by increased IL-8 mRNA expression and P-selectin on the endothelial surface, 3) activation of endothelial NADPH oxidase, 4) an increase in endothelial cell tissue factor that directly correlated with PMNL priming index, and 5) a decrease in endothelial nitric oxide synthase mRNA. Our data support a pathogenic link between PMNL priming and endothelial dysfunction, suggesting that PMNL priming is a potential new nontraditional risk factor for the development of atherosclerosis.

Primed peripheral polymorphonuclear leukocyte: a culprit underlying chronic low-grade inflammation and systemic oxidative stress in chronic kidney disease.

This study characterizes the causal relationship between peripheral polymorphonuclear leukocyte (PMNL) priming, systemic oxidative stress (OS), and inflammation in patients with varying degrees of renal insufficiency (chronic kidney disease [CKD] not on renal replacement therapy [RRT]: continuous ambulatory peritoneal dialysis or hemodialysis [HD]) and healthy control subjects. Rate of superoxide release was measured after stimulation of PMNL with phorbol 12-myristate 13-acetate or zymosan. Priming was estimated by the rate of superoxide release after phorbol 12-myristate 13-acetate stimulation. Systemic OS was related to PMNL priming and intracellular myeloperoxidase activity. Inflammation was linked to peripheral white blood cells and PMNL counts, PMNL apoptosis, and PMNL ex vivo survival in autologous and heterologous sera. PMNL priming and counts were related to the severity of renal failure in CKD not on RRT. Compared with control subjects, PMNL from all CKD patients showed increased priming, highest in HD, with a significant decrease in their response to zymosan. PMNL myeloperoxidase activity and apoptosis were increased in all renal failure patients. Decreased ex vivo cell survival and elevated leukocyte counts were found in all patients, highest in HD. Both PMNL priming and counts correlated negatively with the GFR. A positive significant correlation was shown between PMNL counts and their priming in all groups, suggesting that the increased PMNL count in peripheral blood is an adaptive response to PMNL priming. Hence, PMNL priming is a key mediator of low-grade inflammation and OS associated with renal failure, occurring before the onset of RRT and further augmented in chronic HD.