Band 36, Heft 4

Gene therapy has enormous potential and could in the near future involve the clinical biochemist in monitoring its efficacy. The involvement of clinical biochemists in this field could be not only in evaluating the impact of a gene-based strategy on disease progression, but also in measuring the expression of the products of therapeutic genes in treated individuals. Indeed, gene therapy presents new possibilities for the treatment of many diseases and, in particular, merits consideration in the treatment of a fatal disease like AIDS. The aim of this paper is to review the biochemical basis and clinical relevance of the gene therapy approaches directed towards the inhibition of human immunodeficiency virus type-1. We discuss the goals which have been achieved, the problems which have occurred and the efforts that are being made to solve them. In this regard, particular attention is paid to new strategies targeting ‘therapeutic’ enzymes to human immunodeficiency virus type-1 nucleic acids.

Reticulated platelets are a fraction of newly released circulating elements characterized by a residual amount of RNA. It has been suggested that the reticulated platelet count, providing an estimate of thrombopoiesis in the same way as erythrocyte reticulocyte count is a measure of erythropoiesis, may be useful in the study of thrombocytopenic disorders. Reticulated red cells and platelets can be analyzed by flow cytometry using specific stains for nucleic acids such as Thiazole Orange and Auramine-O. The aim of our work was to perform the simultaneous evaluation of reticulated elements in whole blood using a standard flow cytometer and to correlate the results obtained with a dedicated cytometer. A group of 14 patients with abnormal absolute reticulocyte counts (range 1.1–11%) and a group of 41 patients showing a platelet discrimination error when analyzed with a dedicated flow cytometer (Sysmex R1000) were enrolled. Linear amplification of both scatter and fluorescence was used to perform reticulocyte count. A gate was set on platelet dimensions, and logarithmic amplification of scatter and fluorescence was used to count reticulated platelets. A good correlation was obtained both for results of reticulocyte count (r 2 = 0.9825) and for reticulated platelets (r 2 = 0.8717) between our method and those using dedicated instruments. These data show that reticulated platelet count may be easily introduced in clinical laboratories that routinely perform reticulocyte count by flow cytometry.

Salivary cortisol was measured as an alternative to serum cortisol as a marker for adrenocortical function following insulin tolerance test, corticotropin-releasing-hormone stimulation and adreno-corticotrophic hormone stimulation. During insulin tolerance test and corticotropin-releasing-hormone stimulation adreno-corticotrophic hormone was also measured. The tests were performed on healthy control subjects as well as on patients under investigation for various disturbances in the hypothalamic-pituitary-adrenocortical axis (insulin tolerance test: 3 controls on two occasions and 14 patients; corticotropin-releasing-hormone stimulation: 4 controls and 18 patients; adreno-corticotrophic hormone stimulation: 6 controls and 10 patients). Five patients underwent both insulin tolerance test and corticotropin-releasing-hormone stimulation. Using criteria for adequate cortisol response in serum, the patients were classified as good or poor responders. In 42 of the 45 tests performed the same conclusion as to cortisol status was drawn when based on serum and salivary cortisol responses. In healthy subjects and good responders the mean cortisol relative increase was greater in saliva than in serum in all three tests (p < 0.05). Characteristic of the results for the insulin tolerance test was a significant initial mean decrease (p < 0.05), not found in serum, and the highest observed salivary cortisol value was delayed for at least 30 minutes compared to that in serum. Plasma adreno-corticotrophic hormone correlated significantly with the cortisol concentrations determined 15 minutes later in serum (r = 0.54–0.64) and in saliva (r = 0.76–0.85). The more pronounced cortisol response in saliva than in serum and its closer correlation with adreno-corticotrophic hormone offer advantages over serum cortisol, suggesting salivary cortisol measurement may be used as an alternative parameter in dynamic endocrine tets.

Monitoring of testosterone replacement therapy requires a reliable method for testosterone measurement. Determination of salivary testosterone, which reflects the hormone's biologically active plasma fraction, is a superior technique for this purpose. The aim of the present study was to establish a new sensitive time-resolved fluorescence immunoassay for the accurate measurement of testosterone levels in saliva and to validate it by monitoring testosterone replacement therapy in eight hypogonadal men. A clinical phase I-study with the new ester testosterone buciclate was performed to search for new testosterone preparations to produce constant serum levels in the therapy of male hypogonadism. After two control examinations eight male patients with primary hypogonadism were randomly assigned to two treatment groups (n= 2×4) and given single doses of either 200 mg (group I) or 600 mg (group II) testosterone buciclate intramuscularly. Saliva and blood samples were obtained 1, 2, 3, 5 and 7 days post injection and then weekly for three months. The time-resolved fluorescence immunoassay for salivary testosterone shows a detection limit of 16 pmol/l, an intra-assay CV of 8.9 % (at a testosterone concentration of 302 pmol/l), an inter-assay CV of 8.7 % (at a testosterone concentration of 305 pmol/l) and a good correlation with an established radioimmunsassay of r=0.89. The sample volume required by this method is only 180 μl for extraction and duplicate determination. The assay procedure requires no more than three hours. In group I (200 mg) testosterone did not increase to normal levels either in saliva or in serum. However, in group II, androgen levels increased significantly and were maintained in the normal range for up to 12 weeks with maximal salivary testosterone levels of 303 ±18 pmol/l (mean ±SE) and maximal testosterone levels of 13.1 ±0.9 nmol/l (mean±SE) in serum in study week 6 and 7. The time-resolved fluorescence immunoassay for salivary testosterone provides a useful tool for monitoring androgen status in men and women and is well suited for the follow-up of testosterone replacement therapy on an outpatient basis. The long-acting ester testosterone buciclate is a promising agent for substitution therapy of male hypogonadism and in combination with testosterone monitoring in saliva offers an interesting new perspective for male contraception.

The Reference Institute of Bioanalysis of the German Society of Clinical Chemistry has performed the first external assessment of molecular genetics methods used in medical diagnosis. The following procedures were tested: (I) DNA preparation from whole blood, (II) PCR amplification using “standard” primers, and (III) submarine agarose gel electrophoresis. Out of 50 participants, 45 returned samples for evaluation.

Conventional laboratory investigations of haemostasis like prothrombin time and activated partial thromboplastin time are not useful in predicting and managing intra-operative bleeding complications. In order to establish a possible “perioperative reference range” for thrombin generation prothrombin fragment F1+2 (F1+2) and fibrin degradation (D-dimer) markers, we measured F1+2 and D-dimer concentrations before surgery (but after induction of anaesthesia), 30 minutes into surgery, 10 minutes after the event expected to induce the maximal activation of the haemostatic systems, 90 minutes after surgery and on postoperative days 1 and 2 in 226 consecutive patients. Samples were collected from arterial lines. Twenty patients developed a clinically defined, intraoperative disorder of haemostasis, 206 did not. Patients with an intraoperative disorder of haemostasis had significantly higher preoperative F1+2 and D-dimer concentrations. Preoperative values for F1+2 and D-dimer concentrations above the 75th percentile of patients without an intraoperative disorder of haemostasis indicated a 2.70 to 2.88 fold risk of developing an intraoperative disorder of haemostasis (odds ratios were 3.04, 3.12 and 3.29 for D-dimer, ELISA, F1+2, and D-dimer latex tests, respectively with 95% confidence intervals from 1.20 to 8.46) with negative predictive values of 94%, but positive predictive values of only 16% to 26%. These data suggest that preoperative determination of molecular markers might be helpful in identifying a group of patients at high risk for intraoperative disorder of haemostasis by exclusion of low risk patients. Validation of such an approach requires a prospective trial.

The treatment of tuberculosis usually includes the antibiotic rifampicin, especially in patients with concomitant human immunodeficiency virus infection. Some of these patients are in withdrawal therapy for drug abuse. When opiate screening is carried out in patients receiving rifampicin, false positive results are detected with the kinetic interaction of microparticles in solution method. We evaluated this interference in a Cobas-Integra analyzer and found a 12% cross-reactivity of rifampicin for antibiotic concentrations ranging from 0.19 to 6.08 μmol/l (156 to 5000 μg/l). This effect is not explained by the colour of the rifampicin solutions. Calculations assuming first order kinetics of elimination show that more than 18 hours after a single oral dose of 600 mg of rifampicin, a false positive result for opiates could be obtained. This indicates that the risk of a false positive result must always be considered when urine samples from these patients are analyzed.

With foetal sonography prenatal detection of tumours has become more frequent. To evaluate and treat these infants it is necessary to identify the tumour postnatally. Elevated neuron-specific enolase is a biochemical marker of neuroblastoma. Since conditions during birth may influence neuron-specific enolase concentration in foetal serum, specific reference values in cord blood are required. Cord blood samples were taken from 192 healthy term newborns and concentration of neuron-specific enolase was measured by enzyme immunoassay (EIA). Median neuron-specific enolase concentration in the reference group was 8.0 μg/l and the 5th–95th percentiles were 4.8–19.4 μg/l. No differences between male and female newborns were detected (p = 0.13). Measurement of neuron-specific enoloase in cord blood, in comparison with our reference values, offers an early postnatal possibility of confirming the diagnosis of neuroblastoma.