Biomedical subjects

Thomas Montag

Publications and source records attributed to Thomas Montag.

9 recordsLinked to original sources

PubMed2006

International validation of pyrogen tests based on cryopreserved human primary blood cells.

Pyrogens as fever-inducing agents can be a major health hazard in parenterally applied drugs. For the control of these contaminants, pyrogen testing for batch release is required by pharmacopoeias. This has been done either by the in vivo rabbit pyrogen test (since 1942) or the limulus amoebocyte lysate test (LAL), since 1976. New approaches include cell-based assays employing in vitro culture of human immune cells which respond e.g. by cytokine production (IL-1beta; IL-6) upon contact with pyrogens. Six variants of these assays have been validated in a collaborative international study. The recent successful development of cryopreservation methods promises to make standardized immunoreactive primary human blood cells available for widespread use. Furthermore, the pretesting of donors for infectious agents such as HIV or hepatitis has made it possible to develop a safe and standardised reagent for pyrogen testing. Using a total of 13 drugs, we have validated the pyrogen test based on fresh and cryopreserved human whole blood in four laboratories. The test reached >90% sensitivity and specificity. In contrast to the LAL, the test was capable of detecting non-endotoxin pyrogens derived from Gram-positive bacteria or fungi.

Animal Testing Alternatives↗

PubMed2006

Basics of flow cytometry-based sterility testing of platelet concentrates.

BACKGROUND: Flow cytometry (FACS) is a common technique in blood banking. It is used, for example, for the enumeration of residual white blood cells in plasma and in cellular blood products. It was investigated whether it can also be applied for sterility testing of buffy coat-derived platelet concentrates (PCs). STUDY DESIGN AND METHODS: Plasma-reduced PCs were spiked with bacteria and stored at 20 to 24 or 37 degrees C for various times. The following 10 species were used: Bacillus cereus, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Propionibacterium acnes, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, and Yersinia enterocolitica. Bacterial DNA was stained with thiazole orange. After the platelets were lysed, bacteria were enumerated by FACS. RESULTS: All bacteria species used were detectable by FACS. The lower detection limit was approximately 100 bacteria per microL, that is, 10(5) per mL. In general, the titers measured were 1.2- to 3-fold higher than those determined by colony forming assay. In one case (K. pneumoniae) in which the dot plot of the bacteria cloud overlapped with that of bacteria debris, they were consistently lower. When PC samples were inoculated with approximately 1 colony-forming unit per mL of bacteria and kept at 37 degrees C, most species were detected within 21 hours or less. Exceptions were E. cloacae and P. acnes, which were detected after 24 to 40 and 64 hours, respectively. At 20 to 24 degrees C, the detection times were strongly prolonged. CONCLUSION: Sterility testing of PCs by FACS is a feasible approach. The present data suggest incubating PC samples for 20 to 24 hours at 37 degrees C before testing. For slow-growing bacteria, the incubation period must be prolonged by 1 to 2 days.

Bacteria↗

PubMed2006

Sterility testing of platelet concentrates prepared from deliberately infected blood donations.

BACKGROUND: In general the bacterial count in freshly donated blood is low and even lower in the corresponding platelet concentrates (PCs). By use of flow cytometry (FACS) for sterility testing, the reliability of early versus later sampling times was evaluated. STUDY DESIGN AND METHODS: Blood donations were spiked with various numbers of Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, and Klebsiella pneumoniae. The corresponding PCs were prepared by the buffy-coat method and stored at 22 degrees C. A 20-mL sample was collected from each PC directly after preparation and after 8 hours. Samples were stored at 35 degrees C. Sterility testing of both PCs and samples was by FACS analysis at different time points. RESULTS: All stored PCs were found positive by FACS analysis, with detection times ranging between 8 and 24 hours (K. pneumoniae, B. cereus), 8 and 91 hours (S. aureus), and 144 hours (S. epidermidis). In the samples incubated at 35 degrees C, bacteria were detected after 8 to 19 hours (K. pneumoniae, B. cereus), 8 to 67 hours (S. aureus), and 19 to 43 hours (S. epidermidis). Some of the samples did not contain bacteria. CONCLUSION: Detection times for slow-growing bacteria are significantly shortened when PC samples are incubated at 35 degrees C: the numbers of bacteria in freshly prepared PCs may, however, be so low that the samples drawn for sterility testing do not contain a single bacterium. Our results do not support a shortening of the 24-hour or greater sampling time recommended by the manufacturers of established test systems, because also for consistent detection by FACS, bacteria need to grow in the PCs to sufficient numbers.

Bacteria↗

PubMed2006

A comparison of three rapid bacterial detection methods under simulated real-life conditions.

BACKGROUND: Bacterial screening of all produced platelet concentrates (PCs) is implemented in many countries to reduce the risk of transfusion-transmitted sepsis. This study compares three rapid bacterial detection methods by imitating real-life conditions. STUDY DESIGN AND METHODS: The sensitivity of a solid-phase scanning cytometer (optimized Scansystem, Hemosystem), fluorescence-activated cell sorting (FACS) analysis, and 16S RNA in-house nucleic acid testing (NAT) was evaluated by spiking PCs with four transfusion relevant bacteria (Staphylococcus aureus, Bacillus cereus, Klebsiella pneumoniae, and Escherichia coli ). Two different inocula (10 colony-forming units [CFUs]/mL and 10 CFUs/bag) were used to simulate real-life conditions. Samples were taken at 12, 16, 20, and 24 hours after spiking. RESULTS: With the high inoculum, NAT had a 100 percent rate of positive testing for all four types of bacteria (10/10 replicates) at each time point. With the exception of E. coli, the sensitivity of FACS and optimized Scansystem was comparable for the high inoculum. With the low inoculum, 60 percent of E. coli, 80 percent of B. cereus, 90 percent of K. pneumoniae, and 100 percent of S. aureus were NAT-positive 12 hours after spiking. In contrast, only 20 percent of E. coli, 10 percent of B. cereus, and 70 percent of K. pneumoniae were FACS-positive with the low inoculum 12 hours after spiking. CONCLUSIONS: In summary, the preliminary data revealed a higher sensitivity for NAT in comparison to FACS and optimized Scansystem under the defined study conditions. To imitate real-life conditions, further spiking studies with a low inoculum (10 CFUs/bag) and slower growing organisms should be conducted to examine the sensitivity of available detection systems.

Bacterial Infections↗

PubMed2005

Optimized Scansystem platelet kit for bacterial detection with enhanced sensitivity: detection within 24 h after spiking.

BACKGROUND AND OBJECTIVES: The prevention and detection of bacterial contamination of platelet concentrates remains a major challenge for transfusion medicine. To be suitable for blood-transfusion services, the contamination detection method must be highly sensitive, easy to perform and preferably of low cost. In this spiking study, we evaluated the new optimized Scansystem Platelet Kit detection method for use on apheresis platelets. STUDY DESIGN AND METHODS: Apheresis platelet concentrates (APCs) were individually spiked with 10 colony-forming units (CFU)/ml of one of 10 different strains of bacteria. The spiked APCs were analysed at specific time-points during incubation by using the optimized Scansystem Platelet Kit. Bacterial enumeration was performed by plating onto blood agar. RESULTS: All the bacterial strains tested were detected by using the optimized Scansystem Platelet Kit when sampled 24 h after spiking. Compared to the Scansystem standard kit, sensitivity was increased to < 50 CFU/ml. The identity of the spiked bacteria was confirmed by Gram staining and DNA fingerprinting. CONCLUSION: The optimized Scansystem Platelet Kit was able to reliably detect, within 70 min, 10 transfusion-relevant bacterial species in APCs when a sample volume was taken 24 h after spiking. This is the first study carried out by using the optimized Scansystem bacterial detection that was found to have an enhanced sensitivity compared to the standard kit.

Bacteria↗

PubMed2005

Optimisation of pyrogen testing in parenterals according to different pharmacopoeias by probabilistic modelling.

The rabbit test to detect pyrogenic contamination in parenterals is crucial to ensure patient safety. The pharmacopoeial tests in Europe, the US and Japan are based on the fever reaction of rabbits, but differ in their experimental design and in their algorithms to assess contamination. Employing an international reference endotoxin, fever can be induced in rabbits. Data from 171 rabbits built the base for probabilistic modelling of the fever reaction and for the comparison of the pharmacopoeial tests. The rabbit fever reaction could be modelled as a function of the amount of injected endotoxin (per kg body weight) by linear regression. Combining the pharmacopoeial algorithms of the rabbit pyrogen test with the developed model allowed analysis of differences regarding test results and animal consumption. This showed that the assessment of pyrogenic contamination strongly depends on the respective pyrogen test stipulated by regulations. Additionally, the approach was used to develop a new experimental design. Two specific versions of this design resulted in a reduction of the number of animals used by about 30% while the safety of the test was maintained. A need for harmonisation is evident, allowing optimisation of the experimental design, which promotes animal welfare.

Algorithms↗

PubMed2003

Cloning, sequencing and recombinant expression of the open reading frame encoding a novel member of the Sarcocystis muris (Apicomplexa) microneme lectin family.

Micronemes are characteristic secretory organelles located within the apical cell region of apicomplexan parasites. The protein contents are exocytosed during an early phase of host cell invasion and contribute to parasite motility and the invasion of target cells. We report here on the cloning and heterologous expression of a novel member of the Sarcocystis muris microneme lectin family. The deduced amino acid sequence is in total agreement with that obtained after sequencing the native protein and is characterized by two copies of the apple domain motif. The recombinant polypeptide is expressed in a biologically active conformation as demonstrated by its galactose binding properties.

Amino Acid Sequence↗

PubMed2002

Comparative study of rabbit pyrogen test and human whole blood assay on human serum albumin.

A comparative study of rabbit pyrogen test and human whole blood assay was performed on released preparations of human serum albumin. In addition, the samples were spiked with 5 IU/ml (in whole blood 0.5 IU/ml too) and 10 IU endotoxin/ml. The unspiked samples were negative in both assays. The human whole blood test resulted in the same level of security for the products as the rabbit pyrogen test did. Both, the borderline 5 IU/kg and the 10 IU/kg-Spike partially lead to results of the rabbit test which would cause further testing with additional animals. In contrast, the human whole blood assay resulted in a 100% detection for the 5 IU/ml and 10 IU/ml-Spike. We designed a study protocol for a minimised number of test animals and were able to show the general usefulness of the human whole blood assay.

Animal Testing Alternatives↗

PubMed2001

[Effect of the preservative thiomersal on the release of interleukin-1 beta from human peripheral blood cells].

Several vaccines have been tested in the human whole blood pyrogen test as an alternative to the rabbit pyrogen test. As reported previously by our group, the alternative test system is basically applicable to vaccines. The widely used conservative thiomersal is influencing the test system. Surprisingly, the induction of fever inducing cytokines by endotoxin contaminations and other pyrogens can be suppressed by thiomersal.

Animal Testing Alternatives↗