Performance of the CellaVision DC-1 digital cell imaging analyser for differential counting and morphological classification of blood cells.

AIMS: Recently, a new automated digital cell imaging analyser (Sysmex CellaVision DC-1), intended for use in low-volume and small satellite laboratories, has become available. The purpose of this study was to compare the performance of the DC-1 with the Sysmex DI-60 system and the gold standard, manual microscopy. METHODS: White blood cell (WBC) differential counts in 100 normal and 100 abnormal peripheral blood smears were compared between the DC-1, the DI-60 and manual microscopy to establish accuracy, within-run imprecision, clinical sensitivity and specificity. Moreover, the agreement between precharacterisation and postcharacterisation of red blood cell (RBC) morphological abnormalities was determined for the DC-1. RESULTS: WBC preclassification and postclassification results of the DC-1 showed good correlation compared with DI-60 results and manual microscopy. In addition, the within-run SD of the DC-1 was below 1 for all five major WBC classes, indicating good reproducibility. Clinical sensitivity and specificity were, respectively, 96.7%/95.9% compared with the DI-60% and 96.6%/95.3% compared with manual microscopy. The overall agreement on RBC morphology between the precharacterisation and postcharacterisation results ranged from 49% (poikilocytosis) to 100% (hypochromasia, microcytosis and macrocytosis). CONCLUSIONS: The DC-1 has proven to be an accurate digital cell imaging system for differential counting and morphological classification of WBCs and RBCs in peripheral blood smears. It is a compact and easily operated instrument that can offer low-volume and small satellite laboratories the possibilities of readily available blood cell analysis that can be stored and retrieved for consultation with remote locations.

Haemolytic anaemia after nitrofurantoin treatment in a pregnant woman with G6PD deficiency.

We present a normotensive, pregnant woman with severe haemolytic anaemia in the third trimester of pregnancy. Owing to normal platelet count diagnoses other than HELLP syndrome were considered and investigated. The patient was treated with nitrofurantoin 3 weeks before presentation and she turned out to have a deficiency of glucose-6-phosphate dehydrogenase. After treatment with blood transfusion, vitamin B12 and folic acid the patient recovered completely. Caesarean delivery was performed because of maternal hypertension and fetal distress at 33 weeks' gestation.

Clinical performance evaluation of the CellaVision Image Capture System in the white blood cell differential on peripheral blood smears.

INTRODUCTION: Differential counting and morphological analysis of peripheral blood smears is of great diagnostic importance to the clinician. For economic and time-saving reasons, automated imaging processes have been successfully introduced over the years. The aim of this study was to investigate whether a new morphology system, the CellaVision Image Capture System (CICS), can be used to perform a white blood cell (WBC) differential on peripheral blood smears. METHODS: WBCs in 200 peripheral blood smears were analysed with the CICS method and compared with the DM96 method to establish accuracy, short-term imprecision and clinical sensitivity and specificity for morphology. For establishing long-term imprecision, two blood smears were analysed for 20 days with the CICS method. RESULTS: Evaluation of accuracy in 199 samples demonstrated a good correlation for the CICS when compared with the postclassification on the DM96. Regression coefficients ranged from 0.97 for monocyte counts to 0.99 for neutrophil counts. All regression lines showed slopes with 1 and intercepts with 0 within the 95% CI. Long-term imprecision was less than 5% for neutrophils, eosinophils, basophils, lymphocytes and monocytes. Comparison of the short-term imprecision demonstrated that the SD did not differ by more than 1.1% between the DM96 method and the CICS method. Clinical sensitivity of the CICS was 93.5% and specificity was 97.8%. Specificity and sensitivity for blasts was 100%. CONCLUSIONS: The CICS has proven to be a reliable and accurate tool in the differential WBC count on peripheral blood smears and provides small laboratories with a 24 h available real-time digital differential WBC count on peripheral blood smears and consultation for patients in remote locations.

Molecular marker differences relate to developmental position and subsets of mesodiencephalic dopaminergic neurons.

The development of mesodiencephalic dopaminergic (mdDA) neurons located in the substantia nigra compacta (SNc) and ventral tegmental area (VTA) follow a number of stages marked by distinct events. After preparation of the region by signals that provide induction and patterning, several transcription factors have been identified, which are involved in specifying the neuronal fate of these cells. The specific vulnerability of SNc neurons is thought to root in these specific developmental programs. The present study examines the positions of young postmitotic mdDA neurons to relate developmental position to mdDA subset specific markers. MdDA neurons were mapped relative to the neuromeric domains (prosomeres 1-3 (P1-3), midbrain, and hindbrain) as well as the longitudinal subdivisions (floor plate, basal plate, alar plate), as proposed by the prosomeric model. We found that postmitotic mdDA neurons are located mainly in the floorplate domain and very few in slightly more lateral domains. Moreover, mdDA neurons are present along a large proportion of the anterior/posterior axis extending from the midbrain to P3 in the diencephalon. The specific positions relate to some extent to the presence of specific subset markers as Ahd2. In the adult stage more of such subsets specific expressed genes are present and may represent a molecular map defining molecularly distinct groups of mdDA neurons.

Alterations in serotonin signalling are involved in the hyperactivity of Pitx3-deficient mice.

Pitx3 deficiency in mice causes a dramatic loss of dopaminergic neurones located in the substantia nigra pars compacta during development. This early disruption of the nigrostriatal pathway in Pitx3-deficient mice is characterized by increased spontaneous home-cage activity levels during the habitual sleep phase of these animals. These findings are reminiscent of the spontaneous hyperactivity in mice neonatally lesioned with 6-hydroxydopamine, which is caused by an extensive serotonergic hyperinnervation of the striatum. The present study investigated whether an imbalance between dopamine (DA) and serotonin (5-HT) signalling is involved in the behavioural phenotype of Pitx3-deficient mice. Serotonergic hyperinnervation was demonstrated by increased [3H]-citalopram autoradiographic binding specifically in the dorsal striatum of adult Pitx3-deficient mice, indicating alterations in 5-HT transporter levels that correlated to DA dysfunction in Pitx3 deficiency. In addition, stimulus-induced release of DA and 5-HT indicated an altered balance between these neurotransmitters in the dorsal striatum of Pitx3-/- mice. To determine whether the increased 5-HT signalling was involved in the spontaneous hyperactivity during the light phase observed in Pitx3 deficiency, we treated Pitx3-deficient and control mice with the selective irreversible tryptophan hydroxylase inhibitor p-chlorophenylalanine to decrease 5-HT levels. Reduction of 5-HT levels in Pitx3-deficient mice decreased their locomotor activity to normal levels, whereas the same treatment increased the locomotor activity levels of control mice. Taken together, our results indicate alterations in 5-HT signalling in Pitx3-deficient mice that underlie their spontaneous hyperactivity.

Retinoic acid counteracts developmental defects in the substantia nigra caused by Pitx3 deficiency.

Selective neuronal loss in the substantia nigra (SNc), as described for Parkinson's disease (PD) in humans and for Pitx3 deficiency in mice, highlights the existence of neuronal subpopulations. As yet unknown subset-specific gene cascades might underlie the observed differences in neuronal vulnerability. We identified a developmental cascade in mice in which Ahd2 (Aldh1a1) is under the transcriptional control of Pitx3. Interestingly, Ahd2 distribution is restricted to a subpopulation of the meso-diencephalic dopaminergic (mdDA) neurons that is affected by Pitx3 deficiency. Ahd2 is involved in the synthesis of retinoic acid (RA), which has a crucial role in neuronal patterning, differentiation and survival in the brain. Most intriguingly, restoring RA signaling in the embryonic mdDA area counteracts the developmental defects caused by Pitx3 deficiency. The number of tyrosine hydroxylase-positive (TH+) neurons was significantly increased after RA treatment in the rostral mdDA region of Pitx3-/- embryos. This effect was specific for the rostral part of the developing mdDA area, and was observed exclusively in Pitx3-/- embryos. The effect of RA treatment during the critical phase was preserved until later in development, and our data suggest that RA is required for the establishment of proper mdDA neuronal identity. This positions Pitx3 centrally in a mdDA developmental cascade linked to RA signaling. Here, we propose a novel mechanism in which RA is involved in mdDA neuronal development and maintenance, providing new insights into subset-specific vulnerability in PD.

Developmental origin and fate of meso-diencephalic dopamine neurons.

Specific vulnerability of substantia nigra compacta neurons as compared to ventral tegmental area neurons, as emphasized in Parkinson's disease, has been studied for many years and is still not well understood. The molecular codes and mechanisms that drive development of these structures have recently been studied through the use of elegant genetic ablation experiments. The data suggested that specific genes at specific anatomical positions in the ventricular zone are crucial to drive development of young neurons into the direction of the dopaminergic phenotype. In addition, it has become clear the these dopaminergic neurons are present in the diencephalon and in the mesencephalon and that they may contain a specific molecular signature that defines specific subsets in terms of position and function. The data indicate that these specific subsets may explain the specific response of these neurons to toxins and genetic ablation.

Molecular and cellular alterations in the Pitx3-deficient midbrain dopaminergic system.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by loss of midbrain dopaminergic (mDA) neurons in the substantia nigra compacta (SNc). In order to provide insights into adaptive mechanisms of the mDA system in pathology, specific molecular and cellular parameters of the mDA system were studied in Pitx3-deficient Aphakia (ak) mice, which suffer from severe developmental failure of SNc mDA neurons. Here, we demonstrate differential changes in striatal gene expression, reflecting the specific neuronal loss in these mice. In addition, the neuronal activity of remaining mDA neurons in the ventral tegmental area (VTA) was significantly increased in ak mice. In conclusion, ak mice display specific molecular and cellular alterations in the mDA system that provide new insights in compensatory mechanisms present in mDA-associated disorders such as PD.

The neuregulin receptor, ErbB4, is not required for normal development and adult maintenance of the substantia nigra pars compacta.

Degeneration of dopaminergic neurons in the substantia nigra is associated with one of the most prominent human neurological disorders, Parkinson's disease. It is therefore of high interest to identify molecules with trophic effects on this neuronal population. We show here that the neuregulin receptor ErbB4 is differentially expressed in mesencephalic dopaminergic neurons, found in the substantia nigra and in a subregion of the ventral tegmentum but not in the retrorubral field. Early developmental onset and continued expression of ErbB4 into the adult and the presence of two high affinity ligands, neuregulin-1 and betacellulin, in the basal ganglia, suggested that these molecules might participate in the differentiation and/or maintenance of the nigrostriatal system. In order to address this hypothesis, we used a loxP flanked ErbB4 allele in combination with a nestin-Cre transgene and generated brain-specific ErbB4 null mice. These mutant animals survived into adulthood. The distribution of dopaminergic cell bodies in the midbrain, the expression of numerous genes specific to mesencephalic dopaminergic neurons, and the axonal projection to the basal ganglia all appeared normal. Finally, an assessment of their motor function revealed no behavioral deficits. The apparent lack of any mutant phenotype suggests the presence of a strong compensatory mechanism.

Homeobox gene Pitx3 and its role in the development of dopamine neurons of the substantia nigra.

The homeobox gene Pitx3 plays an important part in the development and function of vertebrate midbrain dopaminergic neurons. Re-localization of the genetic defect in the mouse mutant aphakia to the Pitx3 locus, together with the subsequent identification of two deletions causing the gene to be silent, has been the hallmark of several studies into the role of Pitx3. In this review, we summarize the data and reflect on the role of Pitx3 in the development of dopamine neurons in the midbrain. The data indicate that Pitx3 is essential for the survival of dopamine neurons located in the substantia nigra compacta during development. Molecular analysis of the underlying mechanisms might provide new insights for understanding the selective degeneration observed in Parkinson patients.

Species differences in brain pre-pro-neurotensin/neuromedin N mRNA distribution: the expression pattern in mice resembles more closely that of primates than rats.

We have used in situ hybridization to determine the distribution of pre-pro-neurotensin/neuromedin N (NT/N) mRNA in the brain of mice and rats. In rats and mice, expression was observed in the lateral septal nucleus, nucleus accumbens, medial preoptic area, bed nucleus of the stria terminalis, lateral hypothalamus, central amygdaloid nucleus and the subicilum. However, several differences in the NT/N mRNA distribution were observed between rats and mice in other brain areas. In mice, NT/N expression was detected in the subthalamic nucleus and geniculate nucleus, whereas expression was not observed in these brain areas in rats. Surprisingly, expression was not observed in mouse mesencephalic dopaminergic (mesDA) neurons and the CA1 area of the hippocampus, areas known to contain NT/N mRNA in the rat brain. Taken together, these results show that although the brain NT/N mRNA distribution largely overlaps in mice and rats, species differences exist in specific brain areas in rodents. Moreover, these data indicate that the distribution in mice resembles most that of primates than rats.

Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3.

The mesencephalic dopamine (mesDA) system is involved in the control of movement and behavior. The expression of Pitx3 in the brain is restricted to the mesDA system and the gene is induced relatively late, at E11.5, a time when tyrosine hydroxylase (Th) gene expression is initiated. We show here that, in the Pitx3-deficient aphakia (ak) mouse mutant, the mesDA system is malformed. Owing to the developmental failure of mesDA neurons in the lateral field of the midbrain, mesDA neurons are not found in the SNc and the projections to the caudate putamen are selectively lost. However, Pitx3 is expressed in all mesDA neurons in control animals. Therefore, mesDA neurons react specifically to the loss of Pitx3. Defects of motor control where not seen in the ak mice, suggesting that other neuronal systems compensate for the absence of the nigrostriatal pathway. However, an overall lower activity was observed. The results suggest that Pitx3 is specifically required for the formation of the SNc subfield at the onset of dopaminergic neuron differentiation.

Molecular mechanisms underlying midbrain dopamine neuron development and function.

The mesencephalic dopaminergic system is involved in the control of multiple brain functions including movement control and emotion and is of clinical importance because it is implicated in several psychiatric disorders, of which many are considered to have a neurodevelopmental origin. Studies into the developmental pathways of these neurons have led to the identification of the transcription factors En1, Pitx3, Nurr1 and Lmx1b, all shown to be important for the development of the mesencephalic dopaminergic system. In this paper, we discuss the consequences of genetic ablation of essential developmental genes. Furthermore, we discuss the consequences of changes in dopamine homeostasis for the function of the mesencephalic dopaminergic system. Finally, we analyse the potential of the mesencephalic dopaminergic system to adapt to gene dysfunction.

Involvement of Nurr1 in specifying the neurotransmitter identity of ventral midbrain dopaminergic neurons.

The mesencephalic dopaminergic (mesDA) system is involved in many brain functions including motor control and motivated behaviour, and is of clinical importance because of its implication in psychiatric disorders and Parkinson's disease. Nurr1, a member of the nuclear hormone receptor superfamily of transcription factors, is essential for establishing the dopaminergic phenotype, because expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, requires Nurr1. In addition, Nurr1 plays an important role in the maintenance of mesDA neurons. Neonatal Nurr1 knockout mice lack expression of the dopamine transporter (DAT), the vesicular monoamine transporter 2 (VMAT2) and l-aromatic amino acid decarboxylase (AADC) in addition to TH specifically in mesDA neurons. It is unclear whether the lack of expression of these dopaminergic markers is caused by a maintenance defect or whether the induction of these markers depends on Nurr1 expression. To address this problem, the expression of DAT, VMAT2 and AADC was analysed at embryonic day 12.5 and 14.5. Here we demonstrate that induction of VMAT2 and DAT specifically in mesDA neurons requires Nurr1 expression, whereas AADC expression in mesDA neurons is induced independently of Nurr1 function.