Beta thalassemia IVS-I-5(G-->C) heterozygosity masked by the presence of HbJ-Meerut in a Dutch-Indian patient.

We describe the genotype/phenotype correlation in a 35 year old anemic female referred to our laboratory because a fast eluting minor fraction on HPLC, mild hemolysis and hematological parameters suggesting a Thalassemia trait, eventually in combination with iron depletion. Direct sequencing of the alpha globin genes revealed heterozygosity for HbJ-Meerut, a Glu-->Ala substitution at residue 120 not justifying the hematological parameters. No other point mutations were found on the alpha genes and Gap-PCR excluded the 6 common deletion defects. Direct sequencing of the beta-globin genes revealed the IVS-I-5 (G-->C) transversion in absence of the elevated HbA2 levels usually measured in carriers of this beta-Thalassemia mutation. The HbA2 tetramer in the presence of HbJ-Meerut divides in two parts. One alphaN2/delta2 migrating on the right spot on HPLC. The other alphaJ2/delta2 migrating under the HbA fraction. Classic alkaline electrophoresis and the modern capillary electrophoresis CE showed these two tetramers and the reduction of the elevated HbA2 level of the beta-Thalassemia trait by at least 20% due to HbA2 Meerut.

C-reactive protein and D-dimer with clinical probability score in the exclusion of pulmonary embolism.

This study evaluated the diagnostic value of C-reactive protein (CRP) combined with a clinical decision rule in the exclusion of pulmonary embolism (PE) and compared this with D-dimer. In 363 consecutive outpatients CRP and D-dimer test were performed and clinical probability of PE was assessed. Patients with D-dimer levels<500 microg/l and clinical probability indicating 'PE unlikely' were followed for 3 months. Ventilation-perfusion scan or spiral computerized tomography was performed in patients with D-dimer levels>or=500 microg/l or clinical probability indicating 'PE likely'. The CRP had a sensitivity of 95.7% [95% confidence interval (CI): 90-100] and negative predictive value (NPV) of 98.4% (96-100). CRP<5 mg/l with clinical probability score indicating 'PE unlikely' (n=108, 30%), had a sensitivity of 96.7% (90-100), a specificity of 43.0% (37-49) and NPV of 99.1% (97-100). D-dimer<500 microg/l with clinical probability score indicating 'PE unlikely' (n=170, 51%), had a sensitivity of 96.7% (90-100), a specificity of 67.9% (62-74) and NPV of 99.4% (98-100). Based on retrospective data it was concluded that a standard CRP test can potentially be used to safely exclude PE, either as a sole test or combined with clinical probability assessment. Prospective studies are needed to confirm these findings.

The science of systematic reviewing studies of diagnostic tests.

BACKGROUND: Systematic reviews have gradually replaced single studies as the highest level of documented effectiveness of health care interventions. Systematic reviewing is a new scientific method, concerned with the development and application of methods for identifying relevant literature, analysing the material while increasing validity and precision, and presenting and discussing the results in a way that does justice to the research question and to the available evidence. The objective of this study was to review the systematic reviews in laboratory medicine, to evaluate the methods applied in these reviews and the applicability of guidelines of the Cochrane Methods Working Group on Screening and Diagnostic Tests, and identify areas for future research. METHODS: All the systematic reviews in the field of clinical chemistry and laboratory haematology that could be identified in Medline, EMBASE and other literature databases up to December 1998, were evaluated. RESULTS: We studied 23 reviews of diagnostic trials. Although all reviews share the same basic methodology, there was a wide variation in the methods applied. There was no consensus on the quality criteria for inclusion of primary studies. The results of the primary studies were heterogeneous in most cases. This was partly due to design flaws in the primary studies, but was also inherent in the diverse study designs in diagnostic trials. We observed differences in the analysis of the factors that cause heterogeneity of the results, and in the summary statistics used to pool the data from the primary studies. The additional diagnostic value of a test, after other test results are taken into consideration, was only addressed in one study. CONCLUSION: This overview of 23 reviews of diagnostic trials identifies areas in the methods of systematic reviewing where consensus is lacking, such as quality rating of primary studies, analysis of heterogeneity between primary studies and pooling of data. Guidelines need to be improved on these points.

Diagnostic value of the mean corpuscular volume in the detection of vitamin B12 deficiency.

In clinical practice, the finding of an elevated mean corpuscular volume (MCV), macrocytic anaemia or specific neurological symptoms is often the reason to test for vitamin B12 (B12) deficiency. Use of the MCV as a test for the detection or exclusion of B12 deficiency is only justified if the diagnostic accuracy is sufficiently high. However, the sensitivity and specificity are not well known. We performed a systematic review of the diagnostic value of an elevated MCV for B12 deficiency in both anaemic and non-anaemic patients. Of approximately 3500 titles and/or abstracts that were screened, 37 original papers contained usable data. The population under study proved to be the characteristic of major influence on the study outcome. Pooling of data from different studies was performed in subsets of the data corresponding to the different populations studied. The cut-off levels of both MCV and serum B12 had a significant influence on the study outcomes. The data, however, were pooled without taking these cut-off levels into account. The pooled estimates should be interpreted with this limitation in mind. The reference standards were (1) a low serum B12 concentration and (2) a B12 deficiency confirmed by low serum B12 combined with additional diagnostic investigations. In the population that was randomly screened for low serum B12, the sensitivity of the MCV for B12 deficiency was 17%, whereas the sensitivity was 30% for B12 deficiency in patients with anaemia. When measurement of serum B12 was ordered to exclude B12 deficiency as part of the patients' treatment, the sensitivity was 30% for low serum B12 concentration, 58% for B12 deficiency and 75% for B12 deficiency in patients with anaemia. In the population with pernicious anaemia, the sensitivity was far from perfect (77%). In the five studies that reported data on the positive predictive value of the MCV for B12 deficiency, this ranged from 0% (0/6) to 55% (11/20). This systematic review shows that a considerable number of B12-deficient patients will remain unnoticed when the MCV is used to rule in patients for further evaluation. Depending on the population studied, up to 84% of cases will than be missed. The MCV can be used to make the diagnosis of B12 deficiency more--or less--probable. An elevated MCV justifies the measurement of serum B12. The MCV should not be used as the only parameter ruling out the diagnosis of B12 deficiency.

Antithrombin acts as a negative acute phase protein as established with studies on HepG2 cells and in baboons.

Patients with sepsis or after major surgery have decreased plasma levels of the anticoagulant protein antithrombin. In such patients elevated levels of interleukin-6 (IL-6) are present and this interleukin is known to induce positive and negative acute phase responses. To investigate the possibility that antithrombin acts as a negative acute phase response-protein we performed studies on the human hepatoma cell line HepG2 in vitro and baboons in vivo. HepG2 cells were treated with recombinant human IL-6, IL-1beta, or combinations of the latter two, and tested for production of antithrombin, fibrinogen and prealbumin (transthyretin). This treatment resulted in a dose dependent increase in fibrinogen concentration (with a maximum effect of 2.8-2.9-fold) and a dose dependent decrease in prealbumin (with a maximum effect of 0.6-0.7-fold) and antithrombin concentrations (with a maximum effect of 0.6-0.8-fold). Simultaneous treatment of the HepG2 cells with IL-6 (1,000 pg/ml or 2,500 pg/ml) and IL-1beta (25 pg/ml), provided more extensively decreased prealbumin (0.8 and 0.6-fold, respectively) and antithrombin concentration (0.7 and 0.6-fold, respectively) compared to the single interleukin treatment at these concentrations. Baboons treated with 2 microg IL-6 x kg body-weight(-1) x day(-1) showed increased plasma CRP levels (59-fold, p <0.05) and decreased prealbumin (0.9-fold, p <0.05) and antithrombin (0.8-fold, p <0.05) plasma levels, without evidence for coagulation activation. Our results indicate that antithrombin acts as a negative acute phase protein, which may contribute to the decreased antithrombin plasma levels observed after major surgery or in sepsis.

Ligand-dependent enhancement of human antithrombin gene expression by retinoid X receptor alpha and thyroid hormone receptor beta.

We studied potential modulators of antithrombin gene expression. A putative hormone response element (HRE) was identified by sequence similarity analysis of the antithrombin promoter, situated between nucleotides -92 and -54 relative to the transcription start site. This HRE contains three hexa-nucleotide motifs with an AGGTCA consensus, which are potential targets of members of the steroid/thyroid superfamily of nuclear receptors. Stimulation of the hepatoma cell line HepG2 with the receptor ligands L-3,5,3'-tri-iodothyronine, all-trans retinoic acid, or their combination, increased production of antithrombin into the culture medium by 1.3-, 1.6-, and 2.0-fold, respectively. In contrast, the receptor ligand 1,25-dihydroxy-cholecalciferol[1,25-(OH)2VitD3] did not influence antithrombin production. Analysis of promoter chloramphenicol acetyl-transferase (CAT) constructs, showed that the first 86 bp of the antithrombin promoter region are sufficient for basal transcription. The DNA length polymorphism of 32 bp or 108 bp, located upstream of position -276, did not influence anti-thrombin promoter activity. The antithrombin promoter activity dropped to background values when deleting the region -97/-49 of promoter fragment -453/+57. Transactivation of the antithrombin promoter by retinoid X receptor alpha (RXR alpha) (5-7-fold) or thyroid hormone receptor beta (TR beta) (4-5-fold) was only observed when at least -167/+57 bp of the promoter region is present in CAT constructs, and when the appropriate ligand of the nuclear receptor was added. This transactivation was not observed upon deletion of the antithrombin promoter region -97/-49. With three copies of the antithrombin promoter fragment -109/-42 in front of the thymidine kinase minimal promoter, transactivation was only obtained with RXR alpha, and not with TR beta. In conclusion, these results indicate that the ligand-dependent enhancement of antithrombin gene expression is regulated by RXR alpha as well as by TR beta. Transactivation of antithrombin gene expression by RXR alpha and TR beta appears to be dependent upon the presence of promoter region up to nucleotide -167. The HRE segment (-109/-42) only confers RXR alpha responsiveness to a heterologous promoter. Further study is needed to unravel the exact nature of this HRE and its 5'-flanking sequences.

Fetal and neonatal development of antithrombin III plasma activity and liver messenger RNA levels in sheep.

In healthy term human newborns a unique hemostatic balance exists with reduced plasma concentrations of several coagulant and anticoagulant proteins, including antithrombin III (AT III). In preterm newborns even lower AT III concentrations are observed, with an associated thromboembolic risk. As part of our study program on the gene regulation of AT III, we investigated whether the increase in plasma AT III activity during fetal and neonatal development is particularly controlled at the transcriptional level. Plasma AT III activity and liver AT III mRNA content between the 8th wk of gestation and the 4th wk after birth were determined in sheep. AT III activity gradually increased from 34% of the mean adult level at 8-10 wk of gestation to 86% (2.5-fold) at term (21 wk), and remained in the adult range after birth. The mean body weight, and thus plasma volume, increased 57-fold. Therefore, the total plasma AT III activity increased 140-fold. The total liver AT III mRNA content increased only 14-fold between these fetal stages, mainly due to increased liver weight. Therefore, the total plasma AT III activity increased 10-fold more than the liver AT III mRNA content. In the neonatal period between d 1-3 and 28, the total plasma AT III activity increased only 2-fold more than the liver AT III mRNA content. We conclude that the increase in plasma AT III activity during the fetal period, and similarly the neonatal period, is not regulated at the transcriptional level. Furthermore, a unique fetal isoform of AT III was detected in sheep. This isoform had a 2500-D higher molecular mass compared with the other fetal, neonatal, and adult AT III isoform, and disappeared from the circulation between d 2 and 7 after birth. These AT III isoforms differ in their carbohydrate moiety.

A point mutation in an invariant splice acceptor site results in a decreased mRNA level in a patient with severe coagulation factor XIII subunit A deficiency.

Amplification and sequencing of exons I-XV of the gene encoding subunit A of coagulation factor XIII (FXIII) in a patient with severe subunit A deficiency revealed a single G-->A base substitution at the last position of intron E, mutating the invariant AG dinucleotide splice acceptor site to AA. Northern blot analysis of FXIII subunit A mRNA levels in peripheral mononuclear leukocytes showed that this mutation leads to an undetectable FXIII subunit A mRNA level, suggesting that the mutant transcript is either highly unstable or only spliced at low efficiency. Despite this low mRNA level we were able to amplify cDNA fragments containing the exonV-exonVI junction. Sequence analysis showed that the AA dinucleotide is not recognized by the splicing machinery. Instead, an AG dinucleotide located seven bases downstream of the mutated splice acceptor site is used as alternative acceptor. The resulting, alternatively spliced, FXIII subunit A transcript contains a deletion of the first seven bases of exon VI, while translation continues out of frame and leads to a premature stop codon 27 bases thereafter.

The influence of insulin, beta-estradiol, dexamethasone and thyroid hormone on the secretion of coagulant and anticoagulant proteins by HepG2 cells.

As a basis for regulatory studies on the influence of hormones on (anti)coagulant protein production by hepatocytes, we examined the amounts of the plasma proteins antithrombin III (AT III), protein C, protein S, factor II, factor X, fibrinogen, and prealbumin produced by the hepatoma cell line HepG2, into the culture medium, in the absence and presence of insulin, beta-estradiol, dexamethasone and thyroid hormone. Without hormones these cells produced large amounts of fibrinogen (2,452 +/- 501 ng/mg cell protein), AT III (447 +/- 16 ng/mg cell protein) and factor II (464 +/- 31 ng/mg cell protein) and only small amounts of protein C (50 +/- 7 ng/mg cell protein) and factor X (55 +/- 5 ng/mg cell protein). Thyroid hormone had a slight but significant effect on the enrichment in the culture medium of the anticoagulant protein AT III (1.34-fold) but not on protein C (0.96-fold) and protein S (0.91-fold). This hormone also significantly increased the amounts of the coagulant proteins factor II (1.28-fold), factor X (1.45-fold) and fibrinogen (2.17-fold). Insulin had an overall stimulating effect on the amounts of all the proteins that were investigated. Neither dexamethasone nor beta-estradiol administration did substantially change the amounts of these proteins. We conclude that the HepG2 cell is a useful tool to study the hormonal regulation of the production of (anti)coagulant proteins. We studied the overall process of protein production, i.e., the amounts of proteins produced into the culture medium. Detailed studies have to be performed to establish the specific hormonal effects on the underlying processes, e.g., transcription, translation, cellular processing and transport, and secretion.

Sequence characterization of a sheep cDNA for antithrombin III.

We determined the cDNA sequence of the mRNA for antithrombin III (AT III) from sheep liver. It encodes a protein of 465 amino acids, including a signal peptide of 32 amino acids. The amino acid sequence of the mature protein shows a sequence identity of 89.1%, 95.6% and 85.0% to the human, bovine and rabbit equivalents, respectively. Cysteine residues involved in disulfide bonds as well as potential glycosylation sites are conserved between the four species. In contrast, the amino acid sequence of the signal peptide shows a smaller identity, i.e., 68.7% and 56.3% compared to the human and rabbit preprotein, respectively.

DNA methylation as a regulatory mechanism in rat gamma-crystallin gene expression.

We have investigated the methylation state of the rat gamma-crystallin genes in DNA from lens cells at different developmental stages as well as from kidney and heart cells. A clear correlation between the extent of demethylation of the promoter and 5' gene regions and the expression of these genes was observed. No change in the methylation state of the far upstream or 3' regions of the genes was seen. The demethylation of the promoter region was shown to occur during the differentiation from the lens epithelial to the lens fiber cell. The effect of cytosine methylation on gamma-crystallin promoter activity was tested by measuring gamma-crystallin promoter/chloramphenicol acetyltransferase fusion gene expression after in vitro primed repair synthesis of the promoter region in the presence of either dCTP or 5mdCTP. The hemimethylated promoter was no longer capable of promoting high CAT activity after introduction into lens-like cells. Taken together, our data suggest that DNA demethylation may be the determining step in the developmental stage-specific expression of the rat gamma-crystallin genes.