ARAM: an automated image analysis software to determine rosetting parameters and parasitaemia in Plasmodium samples.

BACKGROUND: Rosetting is associated with severe malaria and a primary cause of death in Plasmodium falciparum infections. Detailed understanding of this adhesive phenomenon may enable the development of new therapies interfering with rosette formation. For this, it is crucial to determine parameters such as rosetting and parasitaemia of laboratory strains or patient isolates, a bottleneck in malaria research due to the time consuming and error prone manual analysis of specimens. Here, the automated, free, stand-alone analysis software automated rosetting analyzer for micrographs (ARAM) to determine rosetting rate, rosette size distribution as well as parasitaemia with a convenient graphical user interface is presented. METHODS: Automated rosetting analyzer for micrographs is an executable with two operation modes for automated identification of objects on images. The default mode detects red blood cells and fluorescently labelled parasitized red blood cells by combining an intensity-gradient with a threshold filter. The second mode determines object location and size distribution from a single contrast method. The obtained results are compared with standardized manual analysis. Automated rosetting analyzer for micrographs calculates statistical confidence probabilities for rosetting rate and parasitaemia. RESULTS: Automated rosetting analyzer for micrographs analyses 25 cell objects per second reliably delivering identical results compared to manual analysis. For the first time rosette size distribution is determined in a precise and quantitative manner employing ARAM in combination with established inhibition tests. Additionally ARAM measures the essential observables parasitaemia, rosetting rate and size as well as location of all detected objects and provides confidence intervals for the determined observables. No other existing software solution offers this range of function. The second, non-malaria specific, analysis mode of ARAM offers the functionality to detect arbitrary objects. CONCLUSIONS: Automated rosetting analyzer for micrographs has the capability to push malaria research to a more quantitative and statistically significant level with increased reliability due to operator independence. As an installation file for Windows © 7, 8.1 and 10 is available for free, ARAM offers a novel open and easy-to-use platform for the malaria community to elucidate resetting.

The use of modified Cunningham chambers for the enumeration of H rosettes, E rosettes, and lymphocyte-tumour cell conjugates.

In this paper, we have described the use of a slide-coverslip Cunningham chamber which is ideal for counting delicate rosettes and lymphocyte-tumour cell conjugates. The ease of construction, facility of use, and economy of cost in comparison with hemocytometers make the use of these chambers the method of choice for rosette counts in general. In contrast to standard double-slide Cunningham chambers, the design of these rosette chambers is such that they can be used with ordinary light microscope objectives (including oil immersion), they can be more easily loaded without trapping air bubbles, and they can be sealed without tilting the slide. As an example of one potential application of these chambers to tumour immunology, data have been presented showing the high proportion of lymphocyte-tumour cell conjugates which can be formed by human peripheral blood cells. The ability of any particular tumour cell to form conjugates did not necessarily reflect the susceptibility of that cell type to natural killer (NK) cell-mediated lysis, nor was there any evidence that E rosette-forming cells preferentially formed conjugate in comparison to non E rosette-forming cells.

The enhancing effect of multiple sclerosis brain homogenates on the active E-rosette forming lymphocytes in neurological disorders.

Peripheral blood lymphocytes from 23 out of 27 (85%) patients with multiple sclerosis responded to MS brain homogenates by increased formation of active E-rosettes. Lymphocytes from only six out of 78 (8%) patients with other neurological diseases responded to MS brain homogenates. The possible value of the test in the diagnosis of MS is discussed.

Bioassay for thymic extracts: guinea pig spleen lymphocytes-rabbit red blood cells rosette method.

An assay is described for assessing the potency of thymic extracts by the capacity of guinea pig spleen lymphocytes to form rosettes with rabbit red blood cells. Its sensitivity and reproducibility have been evaluated by statistical analysis of the data obtained testing different batches of the calf thymus extract TP-1.