ARAM: an automated image analysis software to determine rosetting parameters and parasitaemia in Plasmodium samples
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.
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.
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.
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 rosetting.
KeywordsMalaria Plasmodium falciparum Parasitaemia Rosetting Automatic analysis Software Image analysis Cell detection
To date the standard procedure to determine rosette rate and size is manual counting of a stained culture in the microscope. This is time consuming and susceptible for operator dependent systematic errors. Especially when working with anti-rosetting drugs requiring large number of samples the manually performed analysis is a bottleneck. For the significance of the findings in all studies the objectivity of the operator and a sufficiently high number of analysed RBC is crucial. To reduce analysis time and improve the uniformity as well as accuracy of the results automation of the whole process is highly desirable.
A very efficient way to determine the parasitaemia of a specimen is to use fluorescence activated cell sorting (FACS). The determination of parasitemia only with the help of FACS is automated, fast and possibly to prefer if access to a FACS is granted, but the set-up is very costly and also expensive to maintain.
Another way is the fully automated analysis of microscope images. There are several publications describing software and automated algorithms to determine at least one of the before named measures at a time. One of the first approaches of automatic image analysis of stained blood smears is the MATLAB ® -based script MalariaCount, developed by Sio et al. [17, 18]. The analysis depends on command line input as the tool has no graphical user interface (GUI). Furthermore, the script needs an installation of MATLAB ®. MalariaCount only gives parasitaemia as result. The source code is available on the journal website. With a three-step algorithm Dìaz et al.  determine parasitaemia and the parasites infection stage. This algorithm needs image preprocessing, color images for segmentation and a set of classifiers to identify the erythrocytes infection stage. Unfortunately, it is not mentioned how this software is implemented or where to get its source code. Frean uses the free software ImageJ  in combination with a script to determine parasitaemia in thick Giemsa-stained blood smear microscope images . Therefore, he combined the particle counting algorithm of ImageJ and tunes its parameters in a semi-automatic way. His script is available in the publication. Tek et al.  gives an overview about literature concerning automated blood film analysis for malaria and similar infectious diseases.
Despite these publications a great number of malaria research laboratories still estimates parasitaemia and rosetting rate by manual counting of pRBC and RBC. This is due to a lack of a capable, convenient, free software that is easy to operate.
In this paper a free, easy-to-use, stand-alone software that allows to detect parasitaemia, rosetting rate and rosette size distribution via a self-explaining, graphical user interface is presented. The automated rosetting analyzer for micrographs (ARAM) analysis itself is performed automatically, significantly faster than by hand and without additional operator input or preprocessing of the images. Additionally, in contrast to FACS, ARAM offers the possibility to check the detection results on each analysed frame.
Plasmodium falciparum laboratory strains are cultivated according to standard protocols  under shaking conditions during cultivation. By enrichment in a Ficoll-gradient solution or through enrichment with monoclonal antibodies (mAbs)  the rosetting phenotype is maintained.
Image acquisition and analysis
For the manual analysis the parasite culture is stained with 10 µg/ml of acridine orange for 5 min at room temperature (RT). For analysis with ARAM the same preparation process is used but the parasite culture gets diluted to a haematocrit of 0.2 % to prevent cell overlapping. In addition, the slides are left at RT for a total of 10 min to allow the cells to settle down prior to microscopic investigation.
In rosette disruption assays 45 µl of cultures are mixed with 5 µl of heparin (10× final concentration) and are then incubated at room temperature for 60 min. For the mixing wide pipettes tips are used to not mechanically interfere with rosettes.
Image acquisition routine
In the manual analysis 500 RBC are counted for parasitaemia and 100 pRBC for rosetting rate determination like described in . Therefore, a Nikon Optishot-2 microscope with 40× objective is used. Neighboring fields are selected for counting all parameters by moving from the upper left corner of the slide to the lower right and from the upper right corner to the lower left corner to ensure compensation for potential uneven distribution of pRBC and rosettes on the slide. For the ARAM analysis a Nikon Eclipse80i microscope with 40× objective is utilized. The images are captured by a Hamamatsu ORCA ER camera with NIS element software. The same neighbouring fields selection method as described above is used and 50 or 100 images are taken with a FITC filter with simultaneously applied bright light (exposure time 1 s). For further analysis, the images are exported in jpg-file format.
With the goal of creating a free, stand-alone application MATLAB ® is chosen as the underlying software framework. The collection of scripts that make up ARAM is then compiled and accessible as executable file. This allows ARAM to run without a MATLAB ® installation. The basic concept in detecting objects on the images is to separate them from the background. Therefore, an algorithm transforms the image into a binary map of the original, where pixels that are part of the cell object are white, and background pixels are black. There are different methods to identify objects. In default mode ARAM works with a derivation-based algorithm to detect parasitaemia, rosetting rate and rosette size distribution from micrographs with fluorescent pRBC. A second operation mode is included which can use an additional threshold based algorithm to detect size distribution and location of arbitrary objects, such as nano-particles or vesicles. During development of the software a third background based algorithm for object identification was created but is not implemented in ARAM: as this algorithm utilizes the most apparent procedure it is described in the following paragraph to explain why it does not meet the requirements of an automated detection method.
To separate cell objects from the background this algorithm creates a background-only version of the analysed image by line wise fitting of intensity values, that is subtracted from the original image. To account for non-conformity of illumination in typical micrographs the algorithm applies two threshold operations near the mean intensity of the image to isolate the background. Areas that do not meet the threshold range are filled with the mean intensity value. The resulting matrix is line wise fitted with a polynomial to create a smooth background-only image that includes non-uniform illumination. The algorithm subtracts this new background-version from the original image leaving a black background and lighter cells (as shown in Additional file 1: Figure S1). For highly non-uniform backgrounds the algorithm is slowed down due to the great number of necessary polynomial fits.
For very uniformly lit backgrounds and high contrast between objects and background a threshold-based algorithm delivers the best object detection results. Therefore, every pixel of the grey value image is compared to a threshold value: pixels with higher intensity are displayed white, pixels with lower intensity are displayed black. The analysis part of the algorithm then searches the resulting binary image for the detected objects. An example for this algorithm is shown in the Additional file 2: Figure S2. ARAM optionally uses this algorithm in the object counting mode but not in the default mode as the background is lighter than the cells but darker than the pRBC, which would need two threshold operations simultaneously.
Gradient based algorithm
The advantages of this algorithm are the ability to handle a wide range of object shapes, sizes and structure without the need for an image dependent pre-processing. For images with pronounced noise a blur- or smooth-preprocessing step helps to avoid erroneous edge-detections. Since ARAM is intended to be fast, applicable to all kinds of images without significant preprocessing and without the operator input the gradient-based algorithm is used for detecting cell objects on micrographs as default detection algorithm in both above named operation modes.
Detecting parasitized RBC and rosettes
To assure the quality of the results ARAM performs an error analysis. Uncertainties in the analysis are small compared to the influence of statistical deviations in e.g. cell size. Since a few microscope images with a small amount of cell objects do not represent the entire population of cells and cell size distribution in the sample a statistical approach is necessary. This way it is possible to quantify the impact of non-uniform cell and rosette distribution throughout the specimen. Additionally, the software does not have algorithm induced errors and detects 100 % of objects and observables in ideal images. A measurement or algorithm error would depend on the analysed image series and is hard to quantify. Therefore, ARAM calculates error margins ε for a given confidence probability.
Results and discussion
Validation of ARAM
Application of ARAM: rosette inhibition study
As an example for the application of ARAM, a study of the inhibitory effect of heparin on rosetting rate and size is performed. Three different heparin concentrations are chosen. Each sample is analysed by manual counting in the microscope, manual counting from the images (50 images each) and ARAM analysis (same 50 images each) to compare the results. Heparin interacts with the surface expressed P. falciparum erythrocyte membrane protein 1 (PfEMP1) competing out the receptor on the RBC involved in the formation of rosettes. This leads to the disruption of already formed rosettes or inhibiting rosette formation [25, 26]. The detected parasitaemia is independent on heparin concentration within the confidence intervals, as can be seen in Fig. 5. The values are 6.21 ± 0.68, 7.84 ± 1.04 and 6.74 ± 0.88 %. The rosette rate is decreasing with increasing inhibitor concentration as analysed with any of the three methods, see Fig. 5 top right part. The differences between automatic and manual analysis of the images are much smaller than between manual image analysis and manual analysis directly performed with the microscope, probably due to the inhomogeneous distribution of RBC, pRBC and rosettes throughout the slide. By performing the analysis on a greater amount of samples this deviation is reduced, but without automation this is a time consuming task. The mean rosette size is greater for the specimen without inhibitor and so is the interquartile range of the rosette size distribution. The heparin treated specimens show smaller rosettes with a more narrow size distribution. The rosette size distribution (projection area) is depicted in the lower right part of Fig. 5. The trend of each measurement can be described by a rapidly decreasing function towards greater rosette size (positive x-axis). Additionally, a higher inhibitor concentration reduces the size of rosettes.
To the authors’ knowledge, currently there are no reports on rosette size distribution within in vivo or in vitro studies. Hence, the basic form of such a distribution is estimated with a simple model (see Additional file 4: Supplementary information). As argued, it is plausible to assume a Gaussian distribution of rosette size in terms of volume as indicated in Fig. 3 bottom bar chart. It is important to note that with the definition in Eq. 6 this Gaussian distribution is cut at the left side when there are non-zero values for rosette sizes smaller than the threshold. Since there are no studies about rosette size distribution, ARAM for the first time offers an easy way to analyse those and determine the amount of rosette objects necessary to get reliable and quantitative results of this highly important measure.
One major convenience of ARAM is the simplicity of performing an analysis: in a standard analysis the algorithm does not need additional input. Nevertheless, to ensure this simple operation the images have to meet some specifications. There obviously is a limitation for high cell density and low contrast. For the edge detection via Prewitt-filtering there needs to be a sufficient difference between the grey value of background and cell wall pixels and edges should not be blurred. For the detection of the pRBC, the fluorescence needs to be bright enough and clearly visible compared to the image background. To identify a cell cluster from the first analysis step (object detection with Prewitt-filter) as a rosette there has to be a detected pRBC and at least two RBC within the cluster. The size cut-off value m (see Eq. 6) can be adjusted in the configuration file. This might be necessary when rosettes appear too small and are therefore not detected.
The possibility to save the analysed images with rosettes and detected RBC marked, allows easy verification of the results by the operator and readjustment of the analysis with help of the configuration-file. The text export transferring and saving of the calculated values makes data collection easy and time-saving. Additional scripts could read those text files and create automated plots if desired.
Above-mentioned published software [17, 19, 21, 22] calculate parasitaemia from images, while  claim to additionally determine the parasites stage. ARAM not only calculates parasitaemia but also gives the statistical error to estimate the quality of the results. Beyond that ARAM is the only available software solution to analyse rosetting rate and rosette size distributions. The easy usage of ARAM is supported by its independence from other software, its graphical user interface and its results-text-export functionality, features the other solutions do not include. With these abilities ARAM is the only software suitable for laboratory work-flows to automatically analyse and characterize parasitaemia and rosetting in malaria infected specimens.
Summing up ARAM produces accurate and reproducible results for parasitaemia, rosetting rate and rosette size distribution. This analysis is fast and performed automatically without the operators in depth knowledge of the software. Overall ARAM is a quick and simple way to reduce manual cell counting and produce reliable results for cell population monitoring and the study of drug influence on P. falciparum and its rosetting behaviour.
To improve the quality of measuring results and reduce time spent by the operator, the experiment can be further optimized, standardized and made operator independent. For now, the most time consuming part of this analysis is image acquisition. Consequently, a promising approach is the use of a motorized sample stage allowing to automatically vary the sample position within the slide, focus and take the images. Furthermore, real time analysis could use the existing functionality to determine the necessary sample size.
Concluding, ARAM has the evident potential to serve as tool in daily laboratory work as well as a platform for studies of so far inaccessible measures like precise rosette size distribution. All interested operators can download this new powerful tool for free from the following website: http://nanoquakes.de/ARAM/. ARAM is tested for 64-bit Windows © 7, 8.1 and 10.
PK developed the software, analysed the data and drafted the manuscript. KM carried out the experiments, took the micrographs, analysed the data manually and helped to draft the manuscript. MW helped to conceive the study, participated in its design and coordination and to draft the manuscript. AW participated in the design of the study and helped to draft the manuscript. CW participated in design and coordination of the study, analysed data and helped to develop the software and to draft the manuscript. All authors read and approved the final manuscript.
CW likes to acknowledge funding by Nanosystems Initiative Munich (NIM) via their Seed Funding Program. PK and CW thank Alexander Hupfer and Andreas Spörhase for technical support and Thomas Geislinger and Dominik Breyer for fruitful discussions. MW acknowledges funding from the Swedish Strategic Foundation and the Swedish Research Council (VR 2012-2015/521-2011-3377).
The authors declare that they have no competing interests.
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