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G E Hoffmann

Publications and source records attributed to G E Hoffmann.

At least 19 recordsLinked to original sources

Concepts for the third generation of laboratory systems.

This paper briefly describes the history of laboratory systems and discusses some of the recent concepts. The third generation of laboratory systems, which appeared around 1990, encompasses most of the pre-analytical, analytical and post-analytical procedural steps of the laboratory workflow, thus eliminating much of the so-called "3 D tasks" (dull, dirty, dangerous). These automation systems enable humans to focus on work of higher value such as result validation or development of tests in emerging areas. The new development started in Japan in 1981 and reached the Western hemisphere around 1995. Currently there are between 800 and 900 installations world-wide that meet the above criteria. The majority of them automate hematology, whereas systems that automate more complex areas such as clinical chemistry, immunochemistry, coagulation and urinalysis, represent only about one third. More than 60% of the world-wide system base has been installed in Japan. Future growth in the West and high market saturation in Japan are likely to decrease this percentage during the next few years. The two key concepts of third generation systems are "consolidation" and "integration". The following definitions are suggested: * Consolidation: Combining different analytical technologies or strategies on one instrument or on one group of connected instruments. * Integration: Linking analytical instruments or groups of instruments with pre- and post-analytical devices. Examples for the technical realization of both concepts and practical aspects of how to apply them in an individual laboratory are given. Components, which are specifically new in the context of laboratory automation, are conveyor belts, stationary and floor-running robots, and software for process control. The most attractive options to be considered when automating a laboratory are primary tube sorting and the use of secondary samples to increase speed and to avoid sample carryover. Other applications include automatic centrifugation (esp. for hospital laboratories), decapping (esp. for clinical chemistry), automatic loading and unloading of analyzers (esp. for large laboratories), automatic rerun and reflex testing as well as computer-based sample retrieval, result validation, and interpretation. Specific recommendations for automation planning include the need to see and discuss working installations either during site visits or via video documentations, and to conduct computer simulation experiments on the basis of a "virtual laboratory" model.

Automation↗

Phospholipase A activity in Pseudomonas aeruginosa.

Our study describes the production, purification and properties of an enzyme from Pseudomonas aeruginosa displaying the properties of phospholipase A. Maximal amounts of enzyme could be detected in the culture supernatant when the bacterium was grown for 3 to 5 days at 37 degrees C in stirred flask cultures containing brain heart infusion. The enzyme was purified by polyethylenimine precipitation and ammonium sulfate precipitation followed by gel filtration. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme preparation exhibited two bands with molecular weights of 13.5 and 60 kD, respectively. Correspondingly, two peaks of the same molecular weight could be demonstrated by high performance size exclusion chromatography. The activity toward the sn-2 ester binding of phospholipids was characterized and found to be highest towards phosphatidylcholine. Enzymatic activity was not influenced by the addition of calcium or EDTA while magnesium and strontium caused a decrease of activity. The lyophilized enzyme was found to be stable when stored at -70 degrees C and most active at pH 8.0.

Fatty Acids↗

Low-dose hydrocortisone infusion attenuates the systemic inflammatory response syndrome. The Phospholipase A2 Study Group.

There is increasing evidence that the hypercortisolemia in inflammatory diseases suppresses the elaboration of proinflammatory cytokines, thus protecting the host from its own defence reactions. In severe sepsis and septic shock cortisol levels are usually elevated, but some patients may have relative adrenal insufficiency. This may contribute to the overwhelming systemic inflammatory response syndrome. We evaluated the impact of low-dose hydrocortisone infusion (10 mg/h) on the course of the systemic inflammatory response syndrome. This dose corresponds to a maximum secretory rate of cortisol achieved in corticotropin-stimulated healthy humans. In a prospective observational study 57 surgical patients with severe sepsis or septic shock were studied, of which in addition to the conventional treatment 12 patients were infused with low-dose hydrocortisone, and 45 were treated without any corticosteroid. In the longitudinal analysis the systemic inflammatory response--as judged by body temperature, cardiovascular response, and kinetics of inflammatory mediators such as phospholipase A2, C-reactive protein, and neutrophil elastase--started to differ in favor of the hydrocortisone-treated patients after 2 days of treatment (P < 0.05, Mann-Whitney U test). The difference disappeared after withdrawal of exogenous cortisol. Shock reversal was achieved in all patients treated with low-dose hydrocortisone. The data provide evidence that low-dose hydrocortisone infusion attenuates the systemic inflammatory response in human septic shock. From an immunological point of view a relative cortisol deficiency may contribute to the amplified immune response in systemic inflammatory diseases. A randomized clinical trial must clarify the impact of low-dose hydrocortisone infusion on the clinical course and outcome of septic shock patients.

Adrenal Insufficiency↗

Realistic modeling of clinical laboratory operation by computer simulation.

An important objective of laboratory management is to adjust the laboratory's capability to the needs of patients' care as well as economy. The consequences of management may be changes in laboratory organization, equipment, or personnel planning. At present only one's individual experience can be used for making such decisions. We have investigated whether the techniques of operations research could be transferred to a clinical laboratory and whether an adequate simulation model of the laboratory could be realized. First we listed and documented the system design and the process flow for each single laboratory request. These input data were linked by the simulation model (programming language SIMSCRIPT II.5). The output data (turnaround times, utilization rates, and analysis of queue length) were validated by comparison with the current performance data obtained by tracking specimen flow. Congruence of the data was excellent (within +/- 4%). In planning experiments we could study the consequences of changes in order entry, staffing, and equipment on turnaround times, utilization, and queue lengths. We conclude that simulation can be a valuable tool for better management decisions.

Chemistry, Clinical↗

Differentiation of human phospholipase A2 isoenzymes in serum and other body fluids with use of monoclonal antibodies.

Elevated phospholipase A2 activities in serum were measured in patients suffering from acute pancreatitis or various inflammatory diseases. The photometric phospholipase A assay of Hoffmann & Neumann (Klin. Wochenschr. 67 (1989) 106-109) was combined with immunoabsorption by different monoclonal antibodies directed against pancreatic phospholipase A2. Pancreatic phospholipase A2 was purified from human duodenal juice. Monoclonal antibodies were prepared by fusion of spleen cells from immunized mice with P3X63-Ag8-653 myeloma cells. Samples with phospholipase A2 activity were incubated in monoclonal antibody-coated microtitre plates. Phospholipase A2 activities were determined in the monoclonal antibody-treated samples as well as in control samples. The method allows the determination of the fraction of human phospholipase A2 isoenzymes in various biological materials. For pancreatic phospholipase A2 the specific binding capacity was about 60-80%, the unspecific binding was 5-30%. Practically no cross-reactivity was seen with partially purified serum phospholipase A2, with recombinant platelet phospholipase A2, or with the sera of patients with non-pancreatic diseases. In conclusion, the present study confirmed the presence of pancreatic phospholipase A2 in human duodenal juice and in the ascites of necrotizing pancreatitis. However, pancreatic isoenzyme was absent in non-pancreatic inflammatory diseases. Therefore, elevated phospholipase activities in non-pancreatic inflammatory diseases cannot be attributed to the pancreas.

Aged↗

Increased phospholipase A activities in sera of intensive-care patients show sn-2 specificity but no acyl-chain selectivity.

Phospholipase A (PLA) activities were measured by high-performance liquid chromatography in two enzyme preparations purified from human duodenal juice and a serum pool as well as in 52 sera from 31 intensive-care patients with various diseases. On the basis of a position-specific fatty acid analysis of the natural substrate ("soybean lecithin") from a commercial PLA kit, serum activities of PLA1 could be clearly distinguished from those of PLA2, which is not possible in the usual measurements made with single-label radioactive substrates. Independent of the type of disease, all sera with highly increased PLA activities (40-200 U/L) showed nearly pure PLA2 characteristics without any preference among oleic, linoleic, and linolenic acid in the sn-2 position of the glycerophospholipid substrate. Nevertheless, very low PLA1 activities (< or = 5 U/L, most likely due to heparin perfusion therapy) could also be detected by palmitic and stearic acid release from the sn-1 position, leading to small changes in fatty acid release patterns of sera with low PLA activities. Measurements with sera from heparin-treated volunteers demonstrated that heparin therapy may initially contribute as much as 22 U/L to increased PLA1 activities but is not important under prolonged therapy. The absence of selectivity with respect to acyl-chain desaturation supports the concept of serum PLA2 as an acute-phase protein rather than a regulator of the arachidonic acid cascade.

Adult↗

Characterization of cellular and elevated serum phospholipase A2 activities with a comparison of two methods.

The phospholipase A assay of Hoffmann et al based on the enzymatic photometric determination of the fatty acids liberated from soy-bean phospholipids was compared with the fluorometric assay of Thurén et al. where a synthetic pyrene-labelled substrate is used. Sera from patients with suspected pancreatitis or sepsis were studied. High values compared well while the Hoffmann method was not sensitive enough to detect slightly elevated values in sera from patients with suspected pancreatitis. The phospholipase A2 activities from enzymes purified from human duodenal juice, human sera from patients with sepsis and rat liver mitochondria were characterized in regard to activity towards several synthetic pyrene-labelled substrates, activation by Ca2+ and inhibition by Sr2+ and Mg2+. The enzyme from serum was distinctly different from both the pancreatic secretory and the mitochondrial ones, both in its substrate specificity pattern and in being most strongly inhibited by Mg2+.

Acute Disease↗

Characterization of a phospholipase A2 in human serum.

Elevated catalytic activities of serum phospholipase A2 were measured in patients with inflammatory diseases. In contrast to human pancreatic phospholipase A2, the enzyme in serum of patients with non-pancreatic diseases was rather heat labile and showed a broad pH-optimum in the neutral range. Molecular sieving experiments revealed the existence of macro-molecular forms of the enzyme in serum which were cleaved into monomers of M(r) 14,000 in the presence of high salt concentrations. More than 100-fold purification was achieved by gel filtration chromatography on Sephadex G-100 in the presence of 2 mol/l KCl. Whether this serum phospholipase A2 and a secretory liver and platelet isoenzyme are identical remains to be established.

Blood Platelets↗

Overestimation of albumin in heparinized plasma.

Monochromatic measurement of albumin by the bromcresol green method leads to overestimation of albumin in the presence of heparin. The interference seems to be caused by fibrinogen, which, in the presence of heparin, produces an almost constant increase of the measuring signal over the whole range of suitable wavelengths (550 to 700 nm). The albumin overestimation can be eliminated by using a bichromatic modification. An application of this procedure for the automated analyzer RAXT, RA-1000 (Bayer Diagnostik, Technicon) is presented. We recommend using only bichromatic methods for albumin determination with bromcresol green to avoid unexpected analytical artifacts.

Bromcresol Green↗

Hypothalamic glucocorticoid implants prevent fetal ovine adrenocorticotropin secretion in response to stress.

We evaluated the role of the hypothalamic paraventricular nucleus (PVN) in control of ACTH secretion in fetal sheep. Dexamethasone (DEX, 700 micrograms) (n = 6) or cholesterol (CHOL, 700 micrograms) (n = 5) implants were placed bilaterally 2 mm lateral to PVN of fetal sheep at 108 to 111 days of gestation (dga). After 5 days recovery, fetuses were challenged with: 1) hypotension (50% drop of blood pressure), 2) hypoxemia (fall of greater than 5 mm Hg in fetal PaO2), and 3) corticotropin-releasing hormone (CRH) (10 micrograms iv, single injection to fetus). Hypotension and hypoxemia were repeated after 125 dga. Compared with CHOL, DEX fetuses had lower average concentrations of ACTH in plasma after hypotension [23 +/- 0.5 vs. 149 +/- 83.8 and 31 +/- 13.1 vs. 101 +/- 31.3 pg ml-1 at less than 125 and more than 125 dga, respectively (mean +/- SEM, P less than 0.05)] and during hypoxemia [11 +/- 1.6 vs. 292 +/- 152.8 and 33 +/- 9.4 vs. 304 +/- 91.3 pg ml-1 at less than 125 and more than 125 dga, respectively (P less than 0.05)]. DEX and CHOL responses to CRH at 122 to 127 dga (10 micrograms iv) were not different (38 +/- 23.9 vs. 92 +/- 26.7 pg ml-1, respectively). Immunocytochemistry demonstrated that CRH was decreased in PVN and eliminated from median eminence in DEX, but not in CHOL fetuses. Arginine vasopressin (AVP) immunostaining of PVN of DEX and CHOL fetuses was similar; however, unlike CHOL, DEX fetuses showed no AVP immunostaining of the external zone of median eminence. These results show that, in fetal sheep, high concentrations of glucocorticoid near the fetal PVN prevent increases in plasma ACTH secretion seen in controls in response to hypotension and hypoxemia, and exert at least part of their effect at the level of the CRH- and AVP-producing neurons located in the PVN.

Adrenocorticotropic Hormone↗

Determination of human serum phospholipase A2. Comparison of two methods.

A photometric assay for serum phospholipase A is compared with a radiometric assay specific for phospholipase A2. The methods show a high correlation. The increased phospholipase A activity in sera of patients with inflammatory and non-pancreatic necrotizing diseases is attributable to a neutral-active, calcium-sensitive phospholipase A2 but not to pancreatic phospholipase A2. Phospholipase A1 did not seem to contribute significantly to the overall activity of phospholipase A.

Evaluation Studies as Topic↗

Serum phospholipase A and prognosis in intensive care patients.

The clinical value of phospholipase measurements in serum is as yet an open question, particularly with respect to the prognosis quoad vitam. Sensitivities and specificities at different decision levels for unselected intensive care patients are reported and discussed.

Adolescent↗

Serum phospholipase A in hospitalized patients.

Threehundred and thirty sera from hospitalized patients were analyzed for phospholipase A (PLA) activity. About 30% of unselected patients showed values above the normal range (0 to 10 U/l). The ratio of pathological to normal results was even higher in intensive care patients (around 1:1) and in patients with severe infections (2:1). 'Hyperphospholipasemia' was not typical for any defined organ system. From an etiological viewpoint, infectious diseases were most often related to increased PLA, followed by myocardial infarction and insufficiency and by malignant diseases. It is suggested that phospholipase A is a marker enzyme for the phagocytic activity in inflammation and necrosis.

Bacterial Infections↗

Purification of pancreatic phospholipase A2 from human duodenal juice.

Phospholipase A2 (EC 3.1.1.4) was purified from delipidated human duodenal juice by hydrophobic and cation exchange chromatography, followed by molecular sieving on an HPLC column. The resulting enzyme preparation of phospholipase A2 had a molecular weight of 14 kDa, a specific activity of 2000 U/mg protein, and an N-terminal amino acid sequence which was characteristic for human pancreatic phospholipase A2.

Amino Acid Sequence↗

Modified photometric method for the determination of phospholipase A activities.

A modified photometric method for the determination of phospholipase A activities which is based on a previously published reaction principle is described. The modified assay uses a lyophilized substrate emulsion rather than a freshly prepared phospholipid emulsion and a new chromogen (tribromohydroxybenzoic acid and 4-amino-antipyrine) with a high molar absorption coefficient (1,930 m2/mol at 546 nm, 2,900 m2/mol at 512 nm wavelength). The new test is more practicable with respect to pipetting volumes and incubation times. Preliminary results of a method evaluation indicate that the modified assay fulfills the usual criteria for clinical chemical enzyme measurements.

Humans↗

Serum phospholipase--regulatory and pathophysiological aspects.

The development of a photometric procedure to measure phospholipase A activity has extended previous observations that this enzyme activity increases in several pathological states including pancreatic and inflammatory diseases. Serum phospholipase A in pancreatitis was characterized as a mixture of the pancreatic enzyme and a different phospholipase with a pH optimum at 8.0. The latter enzyme was also observed in nonpancreatic diseases like septicemia and acute lung failure which are characterized by an increase in tissue phagocyte activity. The possible pathogenic role of phospholipase(s) A, their intracellular regulation and the proposed mechanisms of release into the blood stream are discussed with respect to the present pathobiochemical knowledge. This includes the mechanism of activation of phagocytosis and the possible role of lipocortins known to be stimulated by glucocorticoid treatment.

Acute Disease↗

Serum activities of phospholipase A in acute posttraumatic pulmonary insufficiency.

The time courses of serum phospholipase A (PLA) and arterioalveolar oxygen differences (AaDO2) were compared in a group of 30 patients with severe multiple injuries. Evaluation of 391 measuring points revealed that high PLA (above 50 U/l) was associated with increased AaDO2 (above 150 mm Hg). Twelve patients who died having the symptoms of acute respiratory distress syndrome (ARDS) exhibited high serum PLA levels with individual peak values between 65 and 363 U/l (normal range 0-10 U/l). Analysis of individual time courses showed some striking parallelism between PLA and AaDO2. In five cases, however, AaDO2 increases preceded those of PLA by 1 to 4 days, while in one patient, impairment of the pulmonary function and subsequent recovery followed the corresponding PLA values with a 5-day delay. Our study supports present theories assuming some association between lung failure and the release of PLA into the circulation. Regarding the obvious time dissociation between both events, the nature of this relationship seems, however, to be complex so that pathophysiological conclusions should be drawn with caution.

Adolescent↗

Serum phospholipase A in hematological diseases.

Inflammatory cells, e.g., neutrophils, monocytes, and macrophages are presumed to be a source of circulating phospholipase A in nonpancreatic diseases. Therefore, we investigated in a preliminary study whether serum phospholipase A activity is related to leukocyte counts in 43 patients with hematological diseases. Serum PLA activity was significantly increased in patients with Hodgkin's disease, acute monocytic leukemia, myelofibrosis with myeloid metaplasia, and polycythemia vera when compared with patients with chronic myelogenous leukemia, chronic lymphocytic leukemia, and acute myelogenous leukemia, but did not correlate with total leukocyte counts.

Adult↗