A dual microtiter plate (192 sample) luminometer employing computer-aided single-photon imaging applicable to cellular luminescence and luminescence immunoassay.

In spite of the increasing applications of luminescence measurements, no instrumentation is yet commercially available that permits sensitive luminescence measurements to be performed simultaneously on a large number of samples. Based on the principle of single-photon imaging, we describe here the performance of a computer-aided image luminometer with a capacity for two microtiter plates. Applications to the study of chemiluminescence by a B lymphocyte cell line, and to an IgE luminescence immunoassay are used to exemplify the capabilities of the system, which we have termed 'chemiluminescence multiwell analyser'.

Near infrared spectroscopy (NIRS): a new tool to study hemodynamic changes during activation of brain function in human adults.

In healthy human adults, cerebral concentrations of oxygenated hemoglobin ([HbO2]) and deoxygenated hemoglobin ([HbR]) were assessed during brain activation using near infrared spectroscopy (NIRS). Measurements were made either in the frontal cortex (n = 10) during performance of cognitive tasks or in the occipital cortex (n = 6) during visual stimulation (flash-light exposure, picture observation). The typical findings during brain activation were an increase in [HbO2] and a decrease in [HbR]. We demonstrate that these findings are not due to alterations in skin blood flow. NIRS is a simple bedside technique for the assessment of hemodynamic alterations accompanying brain activation.

Evidence of ex vivo and in vitro impaired neutrophil oxidative burst and phagocytic capacity in type 1 diabetes mellitus.

In this study neutrophil (PMN) oxidative burst activity was investigated ex vivo and in vitro in comparison to the PMN-phagocytic functions ingestion and bacterial killing in poorly-controlled type 1 diabetic patients. Luminol enhanced chemiluminescence in response to phorbolesters as a measure of oxidative burst was assessed in a parallel detecting microtiterplate luminometer in 40 poorly-controlled type 1 diabetic subjects. PMN ingestion was measured with [3H]thymidine-labelled Staphylococcus aureus in a kinetic radiometric assay. Microbicidal activity was determined by pure plate counting of surviving bacteria (colony forming units, cfu) after defined pmn challenge. PMNs of type 1 diabetic subjects showed a highly significant reduction of peak CL response in response to PMA compared to nondiabetic controls (P < 0.001) and PMN ingestion (51.8 +/- 4.6%) and bacterial killing (28.6 +/- 3.2%) were reduced as well (78.2 +/- 5.2% (IN) and 18.4 +/- 4.1% (BK), P < 0.01, respectively). The in vitro data displayed impaired PMN oxidative burst activity at glucose concentrations > or = 13.8 mmol/l whereas PMN IN and BK were significantly reduced at glucose levels > or = 27.75 mmol/l. In the control group there was a positive correlation of peak CL response and IN as well as BK (P < 0.05); in type 1 diabetic patients this was also true, but did not reach statistical significance. The data obtained in this study clearly demonstrated impaired PMN oxidative burst activity and markedly reduced ingestion and bacterial killing in type 1 diabetic patients ex vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of radioactivity on microtiterplates in situ using a photon-counting imager.

A position-sensitive single-photon counting imaging system, which can determine intensity and location of a light source, has been used for the detection of 125I-labeled Interleukin-1 and [3H]thymidine in 96 wells of a microtiter plate (MTP) simultaneously. 4 Bq (1 Bq = 1 Bequerel = 1 disintegration/s), 22 and 150 Bq of 125I and 3, 10 and 100 Bq of 3H were visualized and quantified by transforming the radioactivity into light in the visible range by means of Xtalscint, a solid scintillator. After only 1 min of photon accumulation time, the highest radioactivity of both isotopes could be clearly distinguished from background. Photon counts correlate well with radioactivity measured in a beta-counter (for 3H) and a gamma-counter (for 125I). The overall counting efficiency was about 5% for 125I and 3% for 3H.

Noninvasive assessment of cerebral hemodynamics and tissue oxygenation during activation of brain cell function in human adults using near infrared spectroscopy.

Near Infrared Spectroscopy (NIRS) was employed to noninvasively and continuously (temporal resolution 0.5 s) assess changes in cerebral hemodynamics and oxygenation during various functional states of the adult human brain. During cognitive stimulation (performing calculations) a frontal increase in local cerebral blood volume and oxygenated hemoglobin concentration was observed in most (10 of 12) subjects. During visual stimulation (observing a picture) this was demonstrated in the occipital region in all three subjects. Deoxygenated hemoglobin either decreased, remained unchanged or slightly increased during these procedures. Epileptic patients were examined during spontaneously occurring complex-partial seizures. During these seizures extremely large increases in blood volume and oxygenated hemoglobin concentration were measured. In conclusion, this feasibility study indicates that NIRS might become a useful and simple bedside tool to assess brain function.

[Near infrared spectroscopy. Limits and problems in the intensive care unit]. / Nahinfrarot-Spektroskopie. Grenzen und Probleme an der Intensivstation.

Near infrared spectroscopy is a non-invasive method of calculating changes in cerebral oxygenation. We have now evaluated a new near infrared spectroscope, the NIRO-500 (Hamamatsu Photonics, Japan), in an intensive care unit setting. The influence of extracerebral and intracerebral physical-technical, physiological and pathophysiological parameters was investigated. The results showed that the method is very sensitive to changes in oxygenation and perfusion in the cerebral vasculature. However, the influence of other factors can be considerable and not always easy to detect and interpret.

The application of a photon-counting camera in very sensitive, bioluminescence-enhanced detection systems for protein blotting. Ultrasensitive detection systems for protein blotting and DNA hybridization, II.

A relatively simple, very sensitive bioluminescence-enhanced detection system for protein blots was described recently. This method utilizes antibodies conjugated with alkaline phosphatase. Alkaline phosphatase releases D-luciferin (Photinus pyralis) from D-luciferin-O-phosphate. Liberated D-luciferin reacts with luciferase, ATP and oxygen with light emission. The light produced is measured with a very sensitive photon counting camera (Argus 100), permitting visualization and localization of the alkaline phosphatase-conjugated antibodies on nitrocellulose sheets. Under non-optimized conditions the limit of detection is at present 5 to 500 fg of protein (rabbit immunoglobulin G), corresponding to 30 to 3 amol. The method is therefore 10(5) times more sensitive than other used at present.

Monitoring of polymorphonuclear leukocyte functions in diabetes mellitus--a comparative study of conventional radiometric function tests and low-light imaging systems.

In this study neutrophil (PMN) phagocytic capacity was investigated using a conventional radiometric ingestion assay (IN) in comparison with PMN respiratory burst activity assessed by luminol-enhanced chemiluminescence (LCL) in response to phorbolesters and LCL induction during phagocytosis of opsonized Staphylococcus aureus (STLCL) in diabetes mellitus and healthy controls. PMN ingestion was measured with 3H-thymidine-labelled S. aureus in a kinetic radiometric assay. LCL and STLCL were assessed in a parallel detecting microtitre-plate luminometer (MTP-Reader). PMN of diabetic subjects showed a highly significant reduction of peak LCL in response to PMA as well as during phagocytosis of S. aureus (STLCL) compared to non-diabetic controls (p < 0.001 respectively). PMN ingestion in diabetic patients (51.8 +/- 4.6%) was significantly reduced compared to controls (78.3 +/- 6.2%) (p < 0.01). The in vitro data displayed impaired PMN oxidative burst activity at glucose concentrations > or = 13.8 mmol/L, whereas PMN IN was significantly reduced at glucose levels > or = 27.75 mmol/L. The control group showed a positive correlation of peak LCL response and IN (p < 0.05) but not of STCL and IN; in diabetic patients this was also true, but did not reach statistical significance. The data obtained in this study clearly demonstrated impaired PMN respiratory burst activity and markedly reduced phagocytic PMN functions in diabetic patients ex vivo and in vitro as measured by LCL and by ingestion of 3H-thymidine-labelled S. aureus suggesting inhibitory effects of elevated glucose concentrations on various PMN-functions, which might be of clinical importance concerning altered host defence.

[Impaired induction of chemiluminescence and function of polymorphonuclear neutrophilic granulocytes in diabetes mellitus]. / Eingeschränkte Chemilumineszenzinduktion und Funktion polymorphkerniger neutrophiler Granulozyten bei Diabetes mellitus.

The aim of this study was to investigate PMN-chemiluminescence response as a measure of respiratory burst activity and the phagocytic PMN function "ingestion" with regard to metabolic control parameters in diabetes mellitus (d.m.) in comparison to healthy controls. Our findings demonstrated a significant reduction of chemiluminescence response and ingestion in diabetic patients compared to controls (p less than 0.01 resp.); further an inverse relation of metabolic control parameters in d.m. and PMN impairment, suggesting inhibitory effects on PMN function, thus leading or contributing at least in part to altered host defense.

New, sensitive, radioactive-free bioluminescence-enhanced detection system in protein blotting and nucleic acid hybridization.

A relatively simple, very sensitive bioluminescence-enhanced detection system for protein blotting and nucleic acid hybridization is described. The method utilizes antibodies conjugated with alkaline phosphatase or nucleotide probes complexed with alkaline phosphatase. Then the alkaline phosphatase takes part in a reaction by releasing D-luciferin (Photinus pyralis) from D-luciferin-O-phosphate. Liberated D-luciferin reacts with luciferase, ATP and oxygen under light emission. Light is measured using the Argus-100 a photon counting camera system or photographic films. Bound alkaline phosphatase conjugated antibodies or hybridized nucleotide probes can be visualized. The limit of detection is at present 5 to 50 fg of protein (IgG), corresponding, for example to 30 to 300 x 10(-21) mol. This means a much higher sensitivity of the detection system in comparison to systems used at present. Experiments concerning nucleic acid hybridization and visualization of the emitted light by a photon counting camera (Argus-100) are under investigation.

Low light level in vitro monitoring of cellular and antigen-antibody reactions using a photon detection camera system--new perspectives for clinical diagnosis and research.

This article briefly describes the use of a photon counting system (ARGUS-100) in the detection of low levels of light. The ARGUS-100 was used in determining ATP in cell sections from tumor tissues and in measuring a luminescence-enhanced immunoluminometric assay, using ferritin as the analyte, based on the luminol-peroxide-4-iodophenol reaction with peroxidase as the enzyme. The aim is not so much the presentation of data, but rather to show the potentials of the photon counting camera in increasing our knowledge of the cellular and subcellular levels, as well as lowering the detection limits in already sensitive systems, such as immunoassays.

Simultaneous detection of whole blood chemiluminescence in microtitre plates.

The measurement of reactive oxygen species provides a simple method for monitoring the degree of activation of leukocytes in various disorders, and for determining the effects of drugs on this activation. The present report describes the determination of luminol- or lucigenin-amplified chemiluminescence of whole blood in a microtitre plate assay with a 96-well luminometer (HAMAMATSU MTP reader). Using heparinized venous human blood from healthy donors, optimal chemiluminescence intensities were determined at a blood dilution of 1/100 in a total volume of 0.25 ml of Hank's balanced salt solution, containing 0.4 mmol/l luminol as enhancer and either opsonized zymosan (1 milligram) or the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (10(-6) mol/l), as stimuli. The in vitro effects of nordihydroguaiaretic acid, diphenylene iodonium, and diclofenac were tested. After preincubation of the diluted whole blood with these drugs for 15 min, the zymosan-stimulated chemiluminescence was diminished in all cases. The specific NADPH oxidase inhibitor, diphenylene iodonium, was most effective (half maximal inhibition at 1.5 x 10(-8) mol/l), whereas higher concentrations of the antioxidant, nordihydroguaiaretic acid (1.6 x 10(-6) mol/l), or the nonsteroidal antiinflammatory drug, diclofenac (about 10(-5) mol/l), were needed to achieve half maximal inhibition. In addition to its usefulness in the rapid screening of drug effects this assay system seems to be very beneficial for the clinical diagnosis of congenital disorders. Furthermore, it is suited as an effective and simple method for the continuous determination of the phagocyte functional state in patients in pathophysiological situations and during therapy.