Differences in detecting blasts between ADVIA 2120 and Beckman-Coulter LH750 hematology analyzers.

The complete blood count is often used as a screening tool to detect hematologic abnormalities in the peripheral blood. In patients with suspected or known leukemia, blast cells serve as an indicator of disease pathology. We compared the analytical performance of the Siemens ADVIA 2120 to the Beckman-Coulter LH750 in the detection of blasts. In the study, 390 blood samples were analyzed from a general hospital population, which included oncology patients. The presence of blasts, as indicated by the analyzers' flags, was compared with the results of a manual differential (regarded as the reference method). The ADVIA 2120 demonstrated 100% sensitivity at detecting blasts compared with 62% using the LH750. This improved sensitivity came at the expense of a lower specificity (49% vs. 86%). The effect of the false-positive and false-negative samples on the laboratory's manual review was partially (false-positive) or completely (false-negative) mitigated by triggering of other criteria. Detecting blasts in the peripheral blood depends on the performance characteristics of the hematology analyzer, in conjunction with the stringency of the laboratory's manual review criteria.

Inhibitory autoantibodies against ADAMTS-13 in patients with thrombotic thrombocytopenic purpura bind ADAMTS-13 protease and may accelerate its clearance in vivo.

BACKGROUND: Many patients with acquired thrombotic thrombocytopenic purpura (TTP) harbor autoantibodies that may bind and/or inhibit ADAMTS-13 proteolytic activity and accelerate its clearance in vivo. METHODS: To test this hypothesis, we determined ADAMTS-13 activity and antigen levels in parallel plasma samples from patients clinically diagnosed with TTP. Collagen binding, GST-VWF73 and FRETS-VWF73 assays were used to determine ADAMTS-13 activity and to detect inhibitory autoantibodies. Enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation plus Western blotting (IP/WB) were used to detect total anti-ADAMTS-13 IgG (inhibitory and non-inhibitory). RESULTS: Among 40 patients with TTP (21 idiopathic and 19 non-idiopathic), inhibitory autoantibodies were detected (by FRETS-VWF73) in 52% of idiopathic and 0% of non-idiopathic TTP patients. In contrast, non-inhibitory IgG autoantibodies were detected in 29% of idiopathic and 50% of non-idiopathic TTP patients. The concentration of inhibitory IgG autoantibody in idiopathic TTP patients was significantly higher than that of non-inhibitory IgG in either idiopathic or non-idiopathic TTP patients. Idiopathic TTP patients demonstrated significantly reduced ADAMTS-13 activity compared with non-idiopathic patients, but only slightly lower ADAMTS-13 antigen levels. Interestingly, patients with inhibitory autoantibodies exhibited significantly lower ADAMTS-13 antigen levels than those with only non-inhibitory IgG autoantibodies or no autoantibody. Serial plasma exchanges increased levels of ADAMTS-13 activity and antigen concurrently in patients with inhibitory autoantibodies. CONCLUSION: The identification of severe ADAMTS-13 deficiency and autoantibodies or inhibitors appears to be assay-dependent; the inhibitory IgG autoantibodies, in addition to binding and inhibiting ADAMTS-13 proteolytic activity, may accelerate ADAMTS-13 clearance in vivo.

Decreased corticotropin-releasing factor receptor expression and adrenocorticotropic hormone responsiveness in anterior pituitary cells of Wistar-Kyoto rats.

The Wistar-Kyoto (WKY) rat shows signs of persistent activation of the hypothalamic-pituitary-adrenal axis, but the cause and site of this activation is not yet known. Chronically activated corticotrophs generally show blunted adrenocorticotropic hormone (ACTH) response to corticotropin releasing factor (CRF); therefore, the anterior pituitary responsiveness to ACTH secretagogues, CRF and vasopressin, was compared in male WKY and Wistar rats. Anterior pituitary CRF binding and CRF receptor mRNA expression was significantly decreased in WKY rats. ACTH response to CRF or vasopressin was markedly impaired, and vasopressin failed to potentiate the CRF-stimulated ACTH release in cultured WKY anterior pituitary cells. In contrast, CRF and vasopressin alone and in combination stimulated large, concentration-dependent increases in ACTH release in Wistar anterior pituitary cells. By contrast to the decreased ACTH secretory responses, steady-state anterior pituitary pro-opiomelanocortin mRNA levels were approximately 12-fold greater in WKY rats compared to Wistar rats, and they further increased in response to CRF stimulation. These findings suggest that, although the WKY rat corticotroph is under a chronic state of activation or disinhibition, the in vitro secretory responses to classic ACTH secretagogues are impaired.

Fetal alcohol exposure alters serotonin transporter sites in rat brain.

This study examined the effects of fetal alcohol exposure (FAE) on serotonin transporter (5-HTT) binding sites in the brains of developing male and female rat offspring using the technique of quantitative autoradiography. Time-pregnant dams were fed liquid ethanol diet, isocaloric diet without ethanol or normal rat chow. Male and female offspring were sacrificed at 21, 40 and 60 days of age, brains removed and sectioned for analysis of 5-HTT sites. FAE led to distinct effects on 5-HTT sites depending on the age and gender of the offspring. FAE increased 5-HTT binding sites in cortical layers 5, 6, hippocampal layers CA(2,3), lateral nucleus of the amygdala and in the dorsal raphe nucleus. FAE decreased 5-HTT binding sites in the medial nucleus of amygdala, dorsomedial and ventromedial nuclei of the hypothalamus. FAE decreased 5-HTT binding sites temporarily in the ventromedial nucleus of the hypothalamus in the 21-day-old female; this effect was found to disappear by day 40. In contrast, FAE increased 5-HTT sites in the lateral nucleus of the amygdala in the adult animal, suggesting that ethanol exposure in utero may alter serotonin neurotransmission in discrete brain regions permanently.

Glucocorticoid and mineralocorticoid regulation of angiotensin II type 1 receptor binding and inositol triphosphate formation in WB cells.

Mineralocorticoids, glucocorticoids, and angiotensin II (AngII) act cooperatively to maintain body fluid homeostasis. Mineralocorticoids, such as aldosterone and deoxycorticosterone-acetate (DOCA), function synergistically with AngII in the brain to increase salt appetite and blood pressure. In addition, glucocorticoids increase AngII-induced drinking and pressor responses and may also facilitate the actions of aldosterone on salt appetite. The AngII Type 1 (AT1) receptor mediates many of the physiological and behavioral actions of AngII. This receptor is coupled to the G-protein Gq, which mediates AngII-induced inositol triphosphate (IP3) formation. The WB cell line, a liver epithelial cell line that expresses the AT1 receptor, was used to examine the cellular basis of glucocorticoid and mineralocorticoid regulation of AT1 function. In this study corticosterone and dexamethasone treatments increased the number of AT1 receptors by activating the glucocorticoid receptor (GR). This increase in AT1 binding resulted in enhanced AngII-stimulated IP3 formation. However, only supraphysiological doses of aldosterone or DOCA increased AT1 binding, and this effect also was mediated by GR activation. Furthermore, despite evidence that mineralocorticoids and glucocorticoids function together to increase AngII-stimulated actions in vivo, aldosterone and dexamethasone did not act synergistically to affect AT1 binding, Gq expression, or IP3 formation. These results indicate that GR activation, and the subsequent increases in AT1 binding and in AngII-stimulated IP3 formation, may represent a cellular mechanism underlying the synergy between adrenal steroids and AngII.

Mineralocorticoids and glucocorticoids cooperatively increase salt intake and angiotensin II receptor binding in rat brain.

Mineralocorticoids, such as deoxycorticosterone acetate (DOCA), and angiotensin II (AngII) act synergistically in the brain to elicit salt appetite. Glucocorticoids, such as dexamethasone (DEX), also may enhance the behavioral effects of DOCA and AngII. However, the brain regions involved in these behavioral interactions have not been elucidated. This study tested the hypothesis that DEX potentiates the effects of DOCA on AngII binding, especially at the AT1 receptor. We confirmed that DEX potentiated the effects of DOCA on salt appetite. Concomitantly, steroid-specific and region-specific changes in AT1 binding were noted. Specifically, in the hypothalamic paraventricular nucleus, treatment with DEX or DOCA + DEX increased AT1 binding. In the subfornical organ (SFO) and area postrema, there was an increase in AT1 binding when both steroids were combined, but not when given individually. However, there was no change in AT2 binding in any brain region studied and no change in AT1 or AT2 binding to either receptor subtype in the pituitary. The results indicate that DOCA and DEX may increase the sensitivity of the brain to the behavioral and physiological actions of AngII by upregulating AT1 receptors in the SFO and area postrema.

Adrenal steroid regulation of central angiotensin II receptor subtypes and oxytocin receptors in rat brain.

The neuropeptides angiotensin II (AngII) and oxytocin (OT) play important but opposing roles in the regulation of sodium appetite in the rat, AngII as a stimulatory peptide and OT as an inhibitory peptide. Adrenal steroids increase the density of AngII receptors in brain following in vivo administration, although the neuroanatomical and subtype specificity have not been thoroughly examined. Furthermore, previous studies demonstrate that adrenalectomy (ADX) leads to a reduction in OT receptors, although regions associated with sodium appetite remain to be examined. In the present study, quantitative receptor autoradiography was used to locate regions where perturbations in circulating adrenal steroids affect the density of oxytocin receptors and the angiotensin receptor subtypes AT1 and AT2. The results show that ADX results in a small, but significant decrease in AT1 expression in the paraventricular nucleus of the hypothalamus, subfornical organ, and the area postrema. That this effect is reversed by either aldosterone or low-dose corticosterone replacement suggests that occupancy of the mineralocorticoid receptor is responsible for the steroid effect. No changes were observed in AT2 or OT receptors in nuclei associated with sodium appetite, indicating that perturbations in adrenal steroids did not affect these receptors in brain regions implicated in the control of salt appetite.

Analysis of angiotensin type 2 receptors in vasopressinergic neurons and pituitary in the rat.

Previous functional studies indicated that an angiotensin type 2 (AT2) receptor subtype may participate in the regulation of vasopressin release by angiotensin II (AngII). In the present study, AT2 receptor-directed antiserum immunohistochemically detected AT2 receptors within the hypothalamic paraventricular (PVN) and the supraoptic nuclei (SON) of the rat brain, more specifically, in identified vasopressinergic neurons. Considering the lack of AT2 binding in the PVN and the SON using receptor autoradiography, we tested the hypothesis that these AT2 receptors are transported to the posterior pituitary. Western blot analysis detected AT2 immunoreactivity in the posterior pituitary. However, no AT2 binding was detected in posterior pituitary membranes, and no AT2 binding was detected with quantitative receptor autoradiography in the neurohypophysis. Thus, if AT2 receptors are transported from the magnocellular vasopressin neurons to the posterior pituitary, their role in AngII regulation of vasopressin release at the neurohypophyseal terminals remains to be clarified.

Exposure to dehydroepiandrosterone in utero affects T-cell function in males only.

Maternal stress can affect the growth, behavior and immune function of the offspring. Some of these effects are thought to be mediated by maternal glucocorticoids secreted in response to stress. The purpose of the present study was to establish whether dehydroepiandrosterone (DHEA), a weak androgen that is also secreted in response to stress, contributes to the sequelae of prenatal stress in the offspring. Therefore, pregnant rats were treated with different doses of DHEA in the drinking water from day 8 of gestation until parturition, when DHEA treatment was discontinued. Body weights of prepubertal male and female offspring of DHEA-treated dams were significantly and dose responsively lower than those of controls. In contrast, thymic weights were significantly higher in the DHEA offspring. Prenatal DHEA treatment reduced the splenic lymphocyte proliferative response to the T cell mitogen concanavalin A in prepubertal males only, while decreasing the mRNA levels of interleukin-2 receptor alpha and gamma subunits. The anterior pituitary expression of pro-opiomelanocortin (POMC) mRNA was increased in prepubertal males only. These data suggest that decreased body weight seen after prenatal stress can be mediated by either DHEA or glucocorticoids. The persistent increase in thymus weight is a new finding that may be very specific to prenatal DHEA treatment. In conclusion, the decreased T cell function and increased POMC expression found in this study indicate that prenatal-stress-induced immune suppression and altered hypothalamic-pituitary-adrenal activity can be, at least in part, DHEA mediated.