Examination of peripheral blood films using automated microscopy; evaluation of Diffmaster Octavia and Cellavision DM96.

BACKGROUND: Differential counting of peripheral blood cells is an important diagnostic tool. Yet, this technique requires highly trained staff, is labour intensive and has limited statistical reliability. A recent development in this field was the introduction of automated peripheral blood differential counting systems. These computerised systems provide an automated morphological analysis of peripheral blood films, including a preclassification of both red and white cells (RBCs and WBCs, respectively). AIMS: To investigate the ability of two automated microscopy systems to examine peripheral blood smears. METHODS: Two automated microscopy systems, the Cellavision Diffmaster Octavia (Octavia) and Cellavision DM96 (DM96), were evaluated. RESULTS: The overall preclassification accuracy values for the Octavia and the DM96 systems were 87% and 92%, respectively. Evaluation of accuracy (WBC analysis) showed good correlation for both automated systems when compared with manual differentiation. Total analysis time (including post classification) was 5.4 min/slide for the Octavia and 3.2 min/slide for the DM96 (100 WBC/slide) system. The DM96 required even less time than manual differentiation by an experienced biomedical scientist. CONCLUSIONS: The Octavia and the DM96 are automated cell analysis systems capable of morphological classification of RBCs and WBCs in peripheral blood smears. Classification accuracy depends on the type of pathological changes in the blood sample. Both systems operate most effectively in the analysis of non-pathological blood samples.

G20210A is a functional mutation in the prothrombin gene; effect on protein levels and 3'-end formation.

BACKGROUND: The prothrombin G20210A mutation is associated with increased plasma prothrombin levels and risk of thrombosis. The mechanism by which this mutation leads to increased prothrombin expression is as yet unclear and still the subject of debate. OBJECTIVES: The aim of this study was to investigate the effect of the G20210A mutation on mRNA and protein expression. METHODS: We made a set of constructs containing the prothrombin 5'-regulatory region, the firefly luciferase reporter gene and the prothrombin 3'-UTR+ downstream region. The latter element contained either the 20210G or A allele and was inserted either as a single unit (constructs G1 and A1) or in tandem (A1A2, G1G2, A1G2, G1A2). Constructs were transiently expressed in HepG2 cells. Expression was evaluated by luciferase assays and reverse transcriptase-polymerase chain reaction (RT-PCR), followed by quantification of the products and determination of the ratio of poly(A)site usage. RT-PCR sequencing was used for determination of the actual site of polyadenylation in mRNAs from constructs G1 and A1 and from endogenous prothrombin mRNAs from HepG2 cells and human liver tissue. RESULTS: The A1 constructs expressed 1.2-fold more protein than the G1 constructs. The double constructs expressed 1.4-fold more protein (A1A2 vs. G1G2). Similar results were found in a set of constructs in which an SV40 promoter replaced the prothrombin 5'-regulatory region. Ratios of poly(A) site usage (expressed as ratio poly(A) site 1 and 2) for the tandem constructs were similar for constructs with two Gs or As at both poly(A)sites; 2.92 (95% confidence interval 2.39-3.45) and 2.75 (2.55-2.95). pA1/pA2 ratios were 1.46 (1.11-1.81) for G1A2 and 6.29 (5.48-7.10) for A1G2 constructs with different poly(A) sites, indicating that the poly(A)site with the 20210A variant is favored over the normal site. In 20210G mRNAs, the G at 20210 was the last non-A nucleotide in the majority of mRNAs, whereas in most 20210A mRNAs, the last non-A nucleotide was the C at 20209. Over 70% of the prothrombin 20210G mRNAs from HepG2 cells and human liver tissue is polyadenylated at position 20210. CONCLUSIONS: The 20210A variant has a more effective poly(A) site, leading to increased mRNA and protein expression, irrespective of the promoter and gene. It does not affect the position of poly(A) attachment.

Functional characterization of transcription factor binding sites for HNF1-alpha, HNF3-beta (FOXA2), HNF4-alpha, Sp1 and Sp3 in the human prothrombin gene enhancer.

BACKGROUND: Prothrombin is a key component in blood coagulation. Overexpression of prothrombin leads to an increased risk of venous thrombosis. Therefore, the study of the transcriptional regulation of the prothrombin gene may help to identify mechanisms of overexpression. OBJECTIVES: The aim of our study was to localize the regions within the prothrombin enhancer responsible for its activity, to identify the proteins binding to these regions, and to establish their functional importance. METHODS: We constructed a set of prothrombin promoter 5' deletion constructs containing the firefly luciferase reporter gene, which were transiently transfected in HepG2, HuH7 and HeLa cells. Putative transcription factor (TF) binding sites were evaluated by electrophoretic mobility shift assays. The functional importance of each TF binding site was evaluated by site directed mutagenesis and transient transfection of the mutant constructs. RESULTS: We confirmed the major contribution of the enhancer region to the transcriptional activity of the prothrombin promoter. Analysis of this region revealed putative binding sites for hepatocyte nuclear factor HNF4, HNF3-beta and specificity protein(Sp)1. We identified six different TFs binding to three evolutionary conserved sites in the enhancer: HNF4-alpha (site 1), HNF1-alpha, HNF3-beta and an as yet unidentified TF (site 2) and the ubiquitously expressed TFs Sp1 and Sp3 (site 3). Mutagenesis studies showed that loss of binding of HNF3-beta resulted in a considerable decrease of enhancer activity, whereas loss of HNF4-alpha or Sp1/Sp3 resulted in milder reductions. CONCLUSIONS: The prothrombin enhancer plays a major role in regulation of prothrombin expression. Six different TFs are able to bind to this region. At least three of these TFs, HNF4-alpha, HNF3-beta and Sp1/Sp3, are important in regulation of prothrombin expression.

Polymorphisms in the prothrombin gene and their association with plasma prothrombin levels.

To find genetic causes of high plasma prothrombin levels, an established prothrombotic risk factor. we searched for sequence variations in the prothrombin gene. We selected subjects with the 20210-GG genotype (since the 20210-A allele is already known to be associated with high levels) and elevated prothrombin levels (> or = 130 U/dl) from the Leiden Thrombophilia Study (LETS). No mutations were found in the 1 kb promoter region of the prothrombin gene in seven individuals with an isolated high prothrombin level. Comparison of the allelic frequencies of four different polymorphisms in the prothrombin gene in healthy volunteers and in the control subjects among the selected LETS individuals indicated a higher frequency of the 19911-G allele in the latter group (allele frequency 52 vs. 78%, respectively). Homozygous carriers of the 19911-G allele had 8 U/dl higher plasma prothrombin levels than 19911-AA carriers. This difference in prothrombin levels did not affect the thrombotic risk in 20210-GG carriers. In heterozygous 20210-A carriers the odds ratio increased from 1.6 (95% CI: 0.6-4.3) in subjects with 19911-A to 4.7 (1.6-14.0) in subjects with 19911-G on the other prothrombin allele.

Sarcoglycanopathies in Dutch patients with autosomal recessive limb girdle muscular dystrophy.

Within a group of 76 sporadic/autosomal recessive limb girdle muscular dystrophy (LGMD) patients we tried to identify those with LGMD type 2C-E. Muscle biopsy specimens of 40 index patients, who had 22 affected sibs, were analyzed immuno-histochemically for the presence of three subunits: alpha-, beta-, and gamma-sarcoglycans. Abnormal sarcoglycan expression was established in eight patients, with six affected sibs. In one patient gamma-sarcoglycan was absent, and both alpha- and beta-sarcoglycans were reduced. In the remaining seven patients gamma-sarcoglycan was (slightly) reduced, and alpha- and beta-sarcoglycans were absent or reduced. By DNA sequencing mutations were detected in one of the three sarcoglycan genes in all eight cases. Three patients had mutations in the alpha-, three in the beta-, and two in the gamma-sarcoglycan gene. The patients with sarcoglycanopathy comprised the more severely affected cases (P=0.04). In conclusion, sarcoglycanopathy was identified in 23 % (14/62) of the autosomal recessive LGMD patients.