Laboratory testing for lupus anticoagulants

The diagnosis of LA requires special attention to specimen processing and careful determination of a reference interval for the screening procedures. A sensitive APTT reagent should be used as the routine screen with appropriate secondary screening procedures available for the difficult patient (e.g., KCT and dRVVT). Appropriate attention to the ratio of patient to normal plasma in mixing studies is imperative. The ratio of four parts patient to one part normal is recommended. In some instances, confirmatory tests utilizing either the PNP or other test systems may be ambiguous. In these cases it is necessary to perform factor assays for a definitive diagnosis. There is a need for reference plasmas and a common means of expressing LA titers.     

Laboratory testing of a lethal ovitrap for Aedes aegypti

Laboratory tests were conducted to determine the feasibility of making the mosquito ovitrap lethal to Aedes aegypti (L.) when they attempt to oviposit in the trap. Heavy-weight velour paper strips (2.54 x 11 cm) were used as an alternative to the wooden paddle normally provided as a substrate for mosquito oviposition. The paper strips were pretreated with insecticide solutions and allowed to dry before being used in oviposition cups of 473 ml capacity, filled with water initially to within 2.5 cm of the brim. Insecticides chosen for their quick knock-down efficacy were bendiocarb 76% WP (1.06 mg a.i./strip) and four pyrethroids: permethrin 25% WP (0.16 mg a.i./strip), deltamethin 4.75% SC (0.87 mg a.i./strip), cypermethrin 40% WP (2.81 mg a.i./strip), and cyfluthrin 20% WP (0.57 mg a.i./ strip). For experimental evaluation, two oviposition cups (one with an insecticide-treated strip and one with an untreated strip) were placed in cages (cubic 30 cm) with gravid female Ae. aegypti mosquitoes (aged 6-8 days) from a susceptible laboratory strain. Mortality-rates of female mosquitoes were 45% for bendiocarb, 47% for permethrin, 98% for deltamethrin, 100% for cypermethrin, and 100% for cyfluthrin. Young instar larvae added to the treated cups died within 2h. After water evaporation from the cups for 38 days, fresh mosquito females had access to previously submerged portions of the velour paper paddle, and mortality rates of 59% or more occurred. Cups that had water (360 ml) dripped into them, to simulate rain, produced female mosquito mortality rates of > 50% and all larvae died within 3 h of being added. These tests demonstrate that the ovitrap can be made lethal to both adults and larvae by insecticidal treatment of the ovistrip. Field efficacy trials are underway in Brazil to access the impact of this simple, low-cost, environmentally benign approach on populations of the dengue vector Ae. aegypti.     

Laboratory testing of clinically approved drugs against Balamuthia mandrillaris

Balamuthia mandrillaris is a free-living protist pathogen that can cause life-threatening granulomatous amoebic encephalitis. Given the lack of effective available drugs against B. mandrillaris encephalitis with a mortality rate of more than 90 %, here we screened drugs, targeting vital cellular receptors and biochemical pathways, that are already in approved clinical use for their potential clinical usefulness. Amoebicidal assays were performed by incubating B. mandrillaris with drugs (3 × 10⁵ cells/0.5 mL/well) in phosphate buffered saline for 24 h and viability was determined using Trypan blue exclusion staining. For controls, amoebae were incubated with the solvent alone. To determine whether effects are reversible, B. mandrillaris were pre-exposed to drugs for 24 h, washed twice, and incubated with human brain microvascular endothelial cells, which constitute the blood–brain barrier as food source, for up to 48 h. Of the ten drugs tested, amlodipine, apomorphine, demethoxycurcumin, haloperidol, loperamide, prochlorperazine, procyclidine, and resveratrol showed potent amoebicidal effects, while amiodarone and digoxin exhibited minimal effectiveness. When pre-treated with these drugs, no viable trophozoites re-emerged, suggesting that drugs destroyed parasite irreversibly. Based on the in vitro assay, amlodipine, apomorphine, demethoxycurcumin, haloperidol, loperamide, prochlorperazine, procyclidine, and resveratrol are potential antimicrobials for further testing against B. mandrillaris encephalitis. These findings may provide novel strategies for therapy but further research is needed to determine clinical usefulness of aforementioned drugs against granulomatous amoebic encephalitis caused by B. mandrillaris, and other free-living amoebae, such as Acanthamoeba spp., and Naegleria fowleri.     

Laboratory performance in a food microbiology proficiency testing scheme

Results from two shipments in a proficiency testing scheme in which almost 200 food microbiology laboratories participated are summarized. Freeze-dried mixtures of bacteria were used as simulated food samples. Four and six samples, respectively, were examined. The statistical procedures used to evaluate the performance of participating laboratories are described. It is shown that laboratories which had been in the scheme for a long time perform, on average, better than those that had been in the scheme for a short time. The former laboratories produced fewer false and outlying results, and were more accurate and precise in their determinations.     

Laboratory performance in a food microbiology proficiency testing scheme.

Results from two shipments in a proficiency testing scheme in which almost 200 food microbiology laboratories participated are summarized. Freeze-dried mixtures of bacteria were used as simulated food samples. Four and six samples, respectively, were examined. The statistical procedures used to evaluate the performance of participating laboratories are described. It is shown that laboratories which had been in the scheme for a long time perform, on average, better than those that had been in the scheme for a short time. The former laboratories produced fewer false and outlying results, and were more accurate and precise in their determinations.     

The Computerized Laboratory Notebook concept for genetic toxicology experimentation and testing

We describe a microcomputer system utilizing the Computerized Laboratory Notebook (CLN) concept developed in our laboratory for the purpose of automating the Battery of Leukocyte Tests (BLT). The BLT was designed to evaluate blood specimens for toxic, immunotoxic, and genotoxic effects after in vivo exposure to putative mutagens. A system was developed with the advantages of low cost, limited spatial requirements, ease of use for personnel inexperienced with computers, and applicability to specific testing yet flexibility for experimentation. This system eliminates cumbersome record keeping and repetitive analysis inherent in genetic toxicology bioassays. Statistical analysis of the vast quantity of data produced by the BLT would not be feasible without a central database. Our central database is maintained by an integrated package which we have adapted to develop the CLN. The clonal assay of lymphocyte mutagenesis (CALM) section of the CLN is demonstrated. PC-Slaves expand the microcomputer to multiple workstations so that our computerized notebook can be used next to a hood while other work is done in an office and instrument room simultaneously. Communication with peripheral instruments is an indispensable part of many laboratory operations, and we present a representative program, written to acquire and analyze CALM data, for communicating with both a liquid scintillation counter and an ELISA plate reader. In conclusion we discuss how our computer system could easily be adapted to the needs of other laboratories.     

Laboratory testing and computer simulation of blue stain growth on and in wood coatings

Wood coatings are susceptible to fungal growth which might cause a reduction of the performance and ensuing service life apart from aesthetical depreciation. Therefore, manufacturers strive for highly resistant systems ensuring sufficient quality protection. Experimental tests complemented with growth modelling help to achieve insight in pattern development. The purpose of this work is twofold. First, the influence of different wood species and weathering of coatings are examined and secondly the fungal growth on coated pine sapwood is simulated in silico. Therefore several coating systems were applied on different wood species and samples were subjected to the EN 152 reverse blue stain method. Scots pine sapwood samples were analysed in-depth with a 3-D reconstruction technique. Moreover, a new mycological test, accelerating growth of fungi, was also implemented. Second, for computer modelling cellular automata were used. The reverse method appears to be a more realistic approach of exterior conditions and offers the possibility of resistance calculation. The new mycological test gave promising results and data were comparable with those of the reverse method.     

Laboratory method for testing side-effects of pesticides on the rove beetleAleochara bilineata — Adults

Tests were conducted in glass cells containing moistened sand sprayed with the concentration recommended for the pesticide or with demineralised water (controls). Adult females (1–2 weeks old) ofA. bilineata were placed on the fresh residue in the cells and provided daily with fresh fly eggs as food. The duration of the test was 5 days, mortality being recorded every day. The side effects of the pesticide were expressed as reduction in egg production ofA. bilineata during the whole test period. Results permitted classification of the pesticides according to the 4 categories of harmfulness used by the IOBC working group. 23 pesticides were tested, and the reproducibility of the test appeared to be satisfactory. Generally, insecticides/acaricides had a pronounced effect on the beetles whereas fungicides and herbicides were less detrimental. Exceptions were the insecticides Tedion V 18 and Kilval, classified as harmless, and the fungicides Morestan, Pomarsol forte and Afugan rated as moderately harmful. Among growth regulators Prosevor 85 was highly detrimental while Cycocel Extra and Rhodofix had no effects. Excepting Morestan, Pomarsol forte and Ustinex PA the pesticides did not affect the hatching of eggs. The work was financed by the Danish Environmental Protection Agency.     

Laboratory culture of Chironomus tentans for use in toxicity testing: optimum initial egg-stocking densities

The midge Chironomus tentans Fabricius is a commonly used freshwater invertebrate in sediment toxicity tests. Rigorous laboratory culturing techniques are needed to provide organisms of uniform quality and known age for use in testing and for the continuation of the culture itself. This study was conducted to determine the effect of initial culture stocking density on: (1) post-hatch (larval) dry weight, body length and head-capsule width at 10 and 20 days; (2) time to emergence; (3) number and sex of emergent adults; (4) number of larvae and pupae at test termination (day 42 post hatch); and (5) adult dry weight. Three egg stocking densities were used 690 (1.1 eggs cm^–2), 1043 (1.7 eggs cm^–2) and 1463 (2.4 eggs cm^–2). Mean weight of larvae at 10 days in high density tanks (0.13 mg/organism) was significantly higher (P=0.003) than both the medium and low density tanks (0.10 and 0.09 mg/organism, respectively). No significant differences between the three stocking densities were observed for the body length or head-capsule width at either 10 or 20 days post-hatch. Although not statistically significant, larval dry weight decreased with increased stocking density at day 20. A significantly (P=0.02) greater number of females (173±28) emerged from the low stocking density compared to both the medium and high stocking densities (123±45 and 118±54, respectively). Peak adult emergence for the low and medium stocking densities occurred between days 22 and 25 post-hatch, whereas peak adult emergence occurred between days 30 and 33 for the high stocking density. Survival relative to the initial number of eggs stocked was significantly greater (P=0.007) in the low density treatment compared to that in either the medium or the high density treatments. Mean adult weight exhibited an inverse relationship with initial stocking densities. At test end, there was not a significant difference in the mean number of organisms surviving and emerging in the three density levels. The central tendency for number of organisms surviving for all three treatments was 504 organisms per tank (0.82 organisms cm^–2). The results of this experiment suggest that an optimal egg stocking density of 1.0 egg cm^–2 (600 eggs/tank) be used with the feeding rate identified. This would ensure uniform larvae at the appropriate developmental stage (2nd–3rd instar) needed for toxicological research/testing (e.g. 10 days post-hatch), as well as producing sufficient emergence of males and females for future culture establishment.     

Laboratory host systems for extraneous agent testing in avian live virus vaccines: Problems encountered

The regulation for batch testing of avian live viral vaccines for extraneous agents was modified recently in order to reduce animal use, and routine batch testing had to be adapted to this new regulation. As a result, however, some tests have become more complicated and time-consuming. Due to systematic technical problems, the new methods required could only be applied to a small proportion of the batches produced. As a consequence, the majority of batches are still tested in animals.     

Laboratory policies and practices for the genetic testing of children: a survey of the Helix network.

In order to discover whether laboratories have policies regarding the testing of unaffected children, we surveyed all laboratories registered with Helix, a national net-work of DNA diagnostic laboratories. Of 186 laboratories asked to respond anonymously to a four-page questionnaire, 156 (84%) replied. A screening question removed 51 laboratories that provided no clinical services. Of the remaining 105, 92% said that their requisition forms asked the person's age. Substantial minorities had policies for the testing of minors for late-onset disorders (46%), for carrier status for recessive disorders (33%), or for disorders for which the test offers no medical benefit within 3 years (33%). Most laboratories are responsive to parental requests. For 12 of 13 late-onset disorders, the majority of laboratories that offered testing had had requests to test children. The majority had tested healthy children, <12 years of age, for eight disorders. Approximately 22% had tested children, <12 years of age, for Huntington disease. Majorities had received requests to test healthy children for carrier status for 10 of 15 recessive or X-linked disorders and had tested children, <12 years of age, for 6 of these disorders, including cystic fibrosis, hemophilia A, fragile X syndrome, and Duchenne muscular dystrophy. Approximately 45% of the laboratories occasionally had provided tests directly to consumers. In view of the possibility that the harms of presymptomatic diagnoses of children sometimes may outweigh the benefits, our results suggest a need for consistent laboratory policies designed for the best interests of the child and the family.