by Dr J. Filipe, Dr D. V. Pereira and Dr S. André
Granular cell tumours of the breast are rare, mostly benign neoplasms derived from Schwann cells. Clinical and radiological features may be worrisome, raising the possibility of malignancy. The only method for diagnosis is histopathological evaluation of the lesion, mandatory for the appropriate management of patients.

Background

Granular cell tumours (GCTs) were first described in the tongue in 1926 by the Russian pathologist Abrikossoff, who suggested a myofibroblastic origin [1]. He also reported the first case of breast GCT in 1932 [2]. The 5th edition of the World Health Organization classification of breast tumors considers GCTs as mostly benign neoplasms with neuroectodermal origin, derived from Schwann cells [3]. Rare cases of granular cell malignant tumours are reported in the literature, associated with poor prognosis with lymph node and lung metastases [4].
Breast tumours represent 8% of all GCTs, which are more common in the head and neck, proximal extremities, gastrointestinal and respiratory tracts [3]. Usually, breast GCTs are single, but as many as 18% can be multicentric [3]. They arise more often in young adult African-American women, with earlier presentation in this population (mean age: 41|years as compared with 54|years in white-Americans) [3].
The majority of cases occur in the upper inner quadrant and it is suggested that GCTs grow through intracanalicular branches of the supraclavicular nerve, around Schwann cells [5].
Most cases are sporadic but breast GCTs have been reported in association with other conditions in Bannayan–Riley–Ruvalcaba syndrome, neurofibromatosis type|1 and Noonan syndrome (also known as LEOPARD syndrome) [3]. There are also reports of breast GCT associated with mastectomy scars and simultaneous GCT and breast carcinoma [3].
Loss-of-function mutations in ATP6AP1 and ATP6AP2 genes, involved in endosomal pH regulation, are frequently found in GCTs, leading to the accumulation of intracytoplasmic granules [3, 6]. These mutations are found irrespective of anatomic localization and are present in both benign and malignant tumours. As loss-of-function mutations in ATP6AP1 and ATP6AP2 genes were not yet described in other tumours, as far as it is known, they seem to be pathognomonic of GCTs [6].

Diagnosis

According to recommendations by EUSOMA (the European Society of Breast Cancer Specialists), in the preoperative phase, histopathology evaluation of core biopsy is the gold standard for GCT diagnosis [5], as clinical and radiological features of breast GCTs mimic breast carcinoma [3, 7].
In fact, GCTs present as irregular firm masses, typically superficial and mobile, but may rarely be adherent to the pectoralis fascia. They may cause skin thickening, retraction and nipple inversion [3]. On mammography, ultrasound and magnetic resonance imaging they are poorly defined masses with spiked margins but no microcalcifications [3, 7]. Despite these features usually associated with carcinomas only 1–2% of breast GCT cases show histological malignant change [3]. It is uncertain if malignant GCTs are malignant transformations from benign lesions or occur ‘de novo’ [4].

Pathologic findings

1. Macroscopic appearance

Grossly, GCTs are white or tan, firm and homogeneous, with regular or spiked margins, that can reach up to 5|cm (Fig.|1) [3].

2. Histopathologic features

Different sampling methods allow histological evaluation of GCTs: (1) needle-core biopsy (Fig.|2a); (2) vacuum-assisted breast biopsy (Fig.|2b); (3) surgical specimens, such as lumpectomies (Fig.|2c).
Histologically, breast GCTs are poorly defined tumours with infiltrative borders (Fig.|2c), composed of sheets, clusters or trabeculae of large, round and polygonal cells, separated by collagenous bands (Fig.|3a). The cells have indistinct borders and may have a syncytial pattern (Fig.|3b).
The hallmark feature is the presence of abundant eosinophilic cytoplasm with granular appearance (Fig.|3b). The nucleus is centrally located and it is usually round, small, hyperchromatic, rarely vesicular and with prominent nucleoli. Mitoses are usually absent. Perineural and perivascular invasion is frequently present. When GCTS are localized in the dermis, they may be associated with pseudoepitheliomatous hyperplasia. In small and superficial cutaneous biopsies, this sometimes can be confused with squamous cell carcinoma [3, 6].
The finely granular cytoplasm is derived from lysosome accumulation. Larger intracytoplasmic granules with clear haloes, named pustulo-ovoid bodies of Milian, are usually periodic acid–Schiff stain (PAS) positive and diastase resistant [3, 6].
The rare malignant GCTs morphologically vary from having a sarcomatous appearance to relatively bland features [6]. The histological criteria suggestive of malignancy are: increased cellularity, spindling, high nuclear to cytoplasm ratio, marked pleomorphism, vesicular nuclei with prominent nucleoli, more than two mitoses per 2|mm2 and geographical necrosis [4]. Larger tumours (>5|cm) and local recurrence are also features favouring malignancy [6].
Differential diagnosis
The histological differential diagnosis of breast GCTs includes reactive histiocytic lesions, dermatofibroma, epithelial tumours such as apocrine neoplasms and invasive carcinomas, melanocytic lesions (nevi and melanoma), hibernoma and alveolar soft part sarcoma [3, 6].
Morphological and immunohistochemical characterization are crucial in the differential diagnosis.
GCTs are strong and diffusely immunoreactive for S100 protein (Fig.|4a), CD68, CD63 antigen and neuron-specific enolase (NSE). The cells are also positive for transcription factor SOX-10 (SOX-10) (Fig.|4b), calretinin and inhibin A, and show diffuse nuclear expression of transcription factor E3 (TFE3) and microphthalmiaassociated transcription factor (MTIF). The cytoplasmatic granules are PAS positive and diastase resistant. Usually, there is no expression of cytokeratins (Fig.|4c), glial fibrillary acidic protein (GFAP), melanoma antigen recognized by T-cells 1 (Melan-A), HMB45- reactive antigen (HMB45), estrogen and progesterone receptors and receptor tyrosine-protein kinase erbB-2 (ERBB2) [3, 6].
S100 protein and SOX-10 are markers of Schwann cells but they may also be expressed in melanocytic lesions and primary breast carcinomas. The absence of cytokeratin expression excludes an epithelial lesion and the absence of Melan-A and HMB45 expression renders less likely a melanocytic lesion.
NSE is also a marker of neuroectodermal cells and is not specific for GCTs.
Inflammatory cells such as histiocytes can express CD68, as well as CD63 antigen; the last may also be present in neural derived tissue.
Dermatofibroma is also a benign infiltrative lesion commonly located at the dermis or subcutis with an identifiable grenz zone. It has spindle cells with scant cytoplasm and elongated nuclei dispersed among collagen bundles. Unlike GCTs, the cells are negative for S100 protein, SOX-10 and NSE.
Hibernoma is a benign, richly-vascularized adipose neoplasm composed by large brown fat cells with eosinophilic or pale multivacuolated cytoplasm that is granular with central nucleus, admixed with white adipose tissue. The cells are also positive for S100 protein, but staining is more variable. Unlike GCT, hibernoma is platelet endothelial cell adhesion molecule (CD31) positive.
Alveolar soft part sarcoma is a rare tumour of the soft tissue. It has an alveolar architecture and it is composed of large epithelioid polygonal cells with granular eosinophilic cytoplasm and prominent nucleoli. It has strong nuclear expression of TFE3, and may be focally positive for S100 protein; however, in contrast to GCTs, the cytoplasm has crystalline material and the cells are positive for actin and desmin. Furthermore, it is characterized by an ASPSCR1-TFE3 fusion gene.

3. Cytology

Fine-needle aspiration (FNA) is often inconclusive. Smears are hypercellular, with large polygonal cells with fragile membranes and abundant eosinophilic granular cytoplasm and small regular nuclei; no stromal or myoepithelial components are present (Fig.|5) [3, 8].
The cytoplasm features can frequently be interpreted as apocrine benign lesions, histiocytic lesions or even apocrine, lobular and secretory invasive carcinomas [3, 8]. In contrast to apocrine cells, the cells of GCTs are larger, with more granular cytoplasm and poorly defined borders. Some cases may also have prominent nuclear atypia. The use of cellblock and immunohistochemistry can be crucial.
Moreover, owing to the rarity of breast GCTs, pathologists lack experience in its cytological evaluation [8]. This method is no longer used for diagnosis of breast GCTs [5, 8].

Treatment

Treatment of GCTs relies on local excision [3, 5]. The best approach is lumpectomy (Fig.|2c) or even excision by vacuum-assisted breast biopsy in small lesions, a relatively safe and minimally invasive procedure (Fig.|2b) [3, 9]. Sentinel lymph node biopsy is not recommended but may be considered in the rare cases of malignant GCTs [5]. Metastasis have been described in 50% of malignant GCTs [6].
The recurrence rate is very low, even when excised with positive margins.
There is no need for adjuvant therapy in the benign cases, but long-term follow-up is recommended [5].

Summary

GCTs of the breast are rare neoplasms with neuroectodermal origin. Despite the fact that they are mostly benign tumours, they can mimic malignancies owing to their clinical and radiological features. Histopathology evaluation is the gold standard for diagnosis. Morphological and immunophenotypical features are characteristic, however differential diagnoses must be kept in mind.
Multidisciplinary approach is essential in breast tumours, and close contact between clinicians, radiologists and pathologists is vital for the correct management of patients.

The authors

Juliana Filipe* MD, Daniela Vinha Pereira MD, Saudade
André MD Serviço de Anatomia Patológica, Instituto Português
de Oncologia de Lisboa Francisco Gentil, Lisboa, Portugal

*Corresponding author
E-mail: jffilipe91@gmail.com

by Dr Jody M.¦W. van den Ouweland and Dr Rob Janssen
Desmosine is a promising biomarker for estimating elastin degradation activity in chronic obstructive pulmonary disease patients and provides a means to test the beneficial effects of therapeutic interventions. LC-MS/MS has emerged as a goldstandard method for accurate and sensitive measurement of desmosine in various body fluids, including plasma, urine, bronchoalveolar lavage fluid and sputum.

Introduction

Chronic obstructive pulmonary disease (COPD) is one of the major health problems in the world, and currently the third leading cause of death by disease in the USA. COPD is a progressive lung disease defined by persistent airflow limitation predisposing the patient to exacerbations and serious illness. Distinct COPDphenotypes can be identified such as chronic bronchitis and emphysema. The disease is characterized by a low-grade inflammation and involves the release of enzymes that have the capacity to degrade the pulmonary elastic fibre network. Diagnosis is based on a combination of clinical symptoms and abnormalities in lung function tests. Chest radiology and arterial blood gas analysis are often used to establish disease severity. Validated lab tests that can be used in the management of COPD, however, are lacking. The current standard for determining COPD progression is through assessment of the decline of forced expiratory volume in one second (FEV1). As the rate of elastic fibre degradation is accelerated in COPD, matrix elastin degradation products may be effective biomarkers for estimating disease activity and to study the effect of therapeutic interventions [1]. Elastin degradation is not unique for COPD and is also accelerated in several other chronic pulmonary conditions, including COPD, cystic fibrosis and tobacco use.

Principle of elastin synthesis and degradation

Elastin is a unique protein providing elasticity and resilience to dynamic organs, such as lungs and arteries and is thereby a basic requirement for both respiration and circulation. Elastin is synthesized in various cells which secrete the soluble precursor, monomer tropoelastin, into the extracellular matrix, which is then cross-linked mainly through formation of two amino acids, desmosine and isodesmosine (Dl), which are derived from the condensation of four lysine residues of elastin molecules by lysyl-oxidase (Fig. 1). The DI pyridinium ring has three allysyl side chains and one unaltered lysyl side chain (Fig. 2). Cross-linking transforms the soluble tropoelastin to the insoluble cross-linked mature elastin fibre. DI, as a cross-linker of elastin, gives elasticity to the tissue (Fig. 1). DI occurs only in mature elastin and its presence in body fluids is an indicator of degradation of mature elastic fibres [1].

Desmosine as a biomarker of elastin degradation

DI is one of the oldest discovered biomarkers and was developed in the 1960s, but the first time it was correlated to lung elastin content was in the 1980s. As the concentrations of DI in body fluids are extremely low, their precise and specific measurements have been a challenge. Initially, DI measurements in biological samples, particularly urine, relied on immunological techniques such as radioimmunoassay or ELISA as well as on spectrophotometric methods, all of them with limited selectivity and sensitivity, and inconsistencies in measured concentrations. Progressively, these methods have been replaced by more selective and sensitive methods such as capillary electrophoresis laser-induced fluorescence or liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowing measurement of DI in body fluids, including urine, plasma, bronchoalveolar lavage fluid and sputum [2]. Moreover, LC-MS/MS has shown much better inter-method agreement than other assays.

LC-MS/MS measurement of desmosine in body fluids

It has shown to be possible with LC-MS/MS to accurately measure DI in body fluids, including urine, plasma, bronchoalveolar lavage fluid and sputum. The assay procedure for measuring total DI is rather laborious comprising three major steps including acid hydrolysis, solid phase extraction (SPE) with drying/resuspension, and LC-MS/MS. In brief, it starts with adding an equal volume of concentrated hydrochloric acid to plasma, urine or other body fluid including isotopically-labelled desmosine-d4 as internal standard, followed by a 24-hour incubation at 110|°C to liberate DI covalently bound to DI-containing peptides. Next, cellulose SPE is performed to extract total DI from plasma or urine after which the extract is dried and resuspended. Chromatographic separation of both isomers is achieved on a C18 column by addition of an ion-pairing reagent to the mobile phase, followed by selected reaction monitoring by mass spectrometry.
What was not anticipated were the many hurdles in the developmental process, taking years before the assay was ready to be used for clinical research in our hospital. First, the harsh acidic conditions used in sample preparation resulted in corrosion of stainless steel needles in the SPE manifold and in the dry-down heating block with consequent loss of peak signals. Second, discontinuation of critical SPE material by the manufacturer led to a long-lasting search for suitable alternatives. Finally, a twofold difference in measured concentrations of DI was observed when compared to data obtained from literature that could be traced back to an error in designation of the DI standard concen-tration by the supplier. Since then, our LC-MS/MS assay appears robust with performance of over 3000 analyses in various specimens and clinical application areas. The assay has a broad measuring range of 0.14–210|μg/L for DI enabling measurement in various body fluids.

Clinical application areas

We started our quest for an intervention to decelerate elastic fibre degradation. We studied the effect of vitamin|D administration on DI levels in COPD patients but did not find a favourable effect. From vitamin|D, we became interested in vitamin|K and were the first to demonstrate an inverse correlation between vitamin|K status and plasma DI levels [3]. We found this association in patients with COPD and idiopathic pulmonary fibrosis (IPF) as well as in subjects using vitamin|K antagonists as anticoagulant medication. We are currently planning intervention trials in COPD to evaluate whether vitamin|K supplementation reduces DI levels. Elastic degradation accelerates during ageing and is particularly pronounced in COPD and IPF. Reference values for DI increase during ageing and have been established for non-smokers and smokers without lung diseases as well as for patients with COPD and IPF. DI levels appeared to be equally increased in IPF as in COPD [4]. In cystic fibrosis patients, plasma DI correlated with lung function, exacerbation frequency and disease progression, suggesting that measuring DI levels in body fluids by LC-MS/MS may be an effective strategy of monitoring disease progression in cystic fibrosis patients [5].
A large study in 1177 COPD patients investigated the association between plasma DI and emphysema severity/progression, coronary artery calcium score and mortality [6]. It was found that in COPD, excess elastin degradation relates to cardiovascular comorbidities, atherosclerosis, arterial stiffness, systemic inflammation and mortality, but not to emphysema or emphysema progression. The latter may be due to the heterogenicity of the study population including distinct COPD-phenotypes from chronic bronchitis to emphysema. Indeed, elastin is not only present in alveolar walls but also in airways and plasma DI does not therefore specifically reflect emphysema formation. This can well explain why plasma DI was not correlated with emphysema progression in this heterogenous COPD population. Accelerated elastin degradation could potentially contribute to both the pulmonary and extrapulmonary disease manifestations of COPD and may represent a mechanistic link between COPD and the increased risk of cardiovascular disease.
Plasma DI levels correlate with emphysema severity on CT scan in patients with the genetic disorder alpha-1 antitrypsin deficiency (AATD). These patients have insufficient or absent AAT to protect elastic fibres from degradation by proteases, in particular neutrophil elastase. Weekly administration of alpha-1 antitrypsin reduced plasma DI levels [7]. Given that loss of lung parenchyma is irreversible, early initiation in subjects with AATD and elevated plasma DI levels may be an attractive strategy to prevent permanent lung function decline. A plausible reason why plasma DI was correlated with emphysema in AATD patients and not in a heterogenous group of COPD patients, is that AATD patients are a rather homogeneous group with a common predominant form of panlobular emphysema in the basal lung fields.
Finally, in a recent study in SARS-CoV-2 patients, we found impaired vitamin|K-dependent matrix-Gla-protein activation, as a measure of extrahepatic vitamin|K status, linked to accelerated elastic fibre degradation and premorbid vascular calcifications as measured by DI in plasma [8]. We are currently planning intervention trials in COVID-19 patients to evaluate whether vitamin|K supplementation improves outcome of SARS-CoV-2 infections.
In conclusion, the detection and measurement of DI as a means to study elastin degradation has been used for almost 30|years; however, recent methodological advances by our group and others have aided DI detection, as the concentrations present in body fluids are extremely low.

Conflict of interest statement

JO and RJ are owners of Desmosine.com. RJ discloses application of a patent for vitamin|K status as a prognostic and therapeutic biomarker in COVID-19.

The authors

Jody M.W. van den Ouweland*1 PhD and Rob Janssen2 MD
1Department of laboratory Medicine, Canisius-Wilhelmina Hospital, 6532, SZ, Nijmegen, The Netherlands
2Department of Pulmonary Medicine, Canisius-Wilhelmina Hospital, 6532, SZ, Nijmegen, The Netherlands

*Corresponding author
E-mail: j.v.d.ouweland@cwz.nl

micromotor-based immunoassay for on-the-move determination of sepsis biomarkers in blood samples from very low birth weight infants

by Prof. A. Escarpa, Prof. M. A. López, Dr A. Molinero-Fernández, Dr M. Moreno-Guzmán and Dr L. Arruza
Sepsis is a condition that can develop and become life threatening very quickly. The key to obtaining the best possible outcome is early diagnosis and quick treatment, which can be challenging when treating very low birth weight infants and limited volume blood samples. This article describes how micromotors, tiny particles that can propel themselves autonomously, can be adapted to achieve immunoassays for sepsis diagnosis in a very short time and with a very tiny sample volume.

Introduction

Sepsis in general and particularly neonatal sepsis in the highly vulnerable population of very low birth weight infants (VLBWIs), is still a major cause of mortality and morbidity. Despite the significant advances in neonatal care and the increased understanding of the pathogenesis of the disease in recent years, the ability to intervene and modify the path of the disease has been only partially successful.
An early sepsis diagnosis and timely initiation of the therapy improves patient outcomes significantly. However, the diagnosis of sepsis is still one of the fundamental challenges in healthcare worldwide [1]. Early diagnosis is especially challenging in neonates owing to the low specificity and high variability of the symptoms, the lack of ideal sepsis biomarkers and the absence of optimal diagnostic tests [2]. Blood culture continues to be the gold standard method for diagnosing sepsis. However, its low sensitivity, the delay in obtaining results and the relatively large sample volume needed from VLBWIs, make it unsuitable for an early sepsis diagnostic. Likewise, the limitations in the available volume of blood in newborns can negatively affect the performance of the blood culture, because of the low rate of bacteremia in most cases.
As a result of the aspects cited above, new diagnostic tools are much desired by clinicians. These analytical methods should provide specificity, sensitivity, multiplexed analysis and fast results at the bedside. Moreover, they should also account for the limitations in blood volume availability in VLBWIs.

Micromotor-based immunoassays: a new clinical analysis tool

To help in solving these challenges, the use of catalytic self-propelled micromotors, adequately functionalized with relevant specific antibodies, is proposed as a new paradigm in the clinical assay scenario for the analysis of validated sepsis biomarkers, such as procalcitonin (PCT) and C-reactive protein (CRP). The pairing of the well-known features of immunoassays and the great potential of micromotors provides a synergistic combination to develop highly interesting point-of-care-testing (POCT) devices for sepsis diagnosis. A brief introduction to the nature of micromotors is described before further elaborating on this specific application.
Micromotors are microscopic-sized nanotechnological particles that have the ability to move autonomously, offering a plethora of new possibilities in clinical analysis and other fields [3]. Nature has provided many examples of its own machinery, such as kinesins, dynein, myosin or motor proteins to drive flagella and cilia in bacteria, sperm cells, etc. However, until the arrival of nanotechnology, the miniaturization of macroscopic objects was nothing more than a science fiction fantasy.
One of the main challenges of such micromachinery involves the power supply required for their propulsion. Although this energy can be provided by an external field or stimuli (ultrasound, magnetic and electrical field, light source, pH, temperature), the majority of micromotors developed so far harvest energy from the surrounding environment and are known as fuel-driven micromotors [4]. These devices are constructed with an inner layer that is composed of a catalytic material which triggers a chemical reaction upon interaction with another chemical substance present in the liquid environment (fuel). Among others, the most widely explored mechanism for creating movement is bubble propulsion. In this case, a favourable geometry is selected for the design of the micromotor, such as a tube with one closed end and one open end. Then, decomposition of the fuel micromotor-based immunoassay for on-the-move determination of sepsis biomarkers in blood samples from very low birth weight infants Sepsis Diagnosis by Prof. A. Escarpa, Prof. M. A. López, Dr A. Molinero-Fernández, Dr M. Moreno-Guzmán and Dr L. Arruza A potential new clinical analysis tool for sepsis diagnosis: September 2020 21 | (i.e. hydrogen peroxide) on the inner catalytic surface of the micromotor tube causes the formation of gaseous product (oxygen) that creates a stream of bubbles. The exiting of the stream of bubbles from the open end of the tube creates a strong thrust, propelling the micromotor in the opposite direction. Bubble-propelled micromotors can move with high speed of up to several millimetres per second [5].
However, micromotor construction can be customized to perform the desired application. In this case, a layer-by-layer architecture was easily electro-synthetized via a template-assisted method to provide the required functionalities [6]. Constructed in three layers, (Fig. 1) the external layer must contain carboxy moieties that can be further functionalized with the desired antibody to selectively recognize and capture the target protein and be able to successfully carry out all the immunoassay steps. The middle magnetic layer allows the guidance and stoppedflow operations of the self-propelled micromotors, providing easy manipulation/collection of the micromotor. The inner layer consists of the catalyst, which allows the bubble formation creating propulsion of the micromotor. The nanomaterials used for their construction can be chosen/selected accordingly to the desired properties [7].

Application of micromotors to sepsis diagnosis

With the knowledge of the techniques described above, our research group decided to apply this amazing technology to help solve the problem of sepsis diagnosis. Micromotor-based immunoassays have been used for the determination of both CRP and PCT levels, which are biomarkers relevant to sepsis diagnosis. Different nanomaterials have been used to construct the micromotors and different detection techniques have been tried, which demonstrates the versatility and potential of this technology.

Micromotor immunoassay detection of CRP

In a first approach, electrochemical detection of CRP using carbon-based micromotors was designed [8]. Self-propelled catalytic micromotors functionalized with anti-CRP specific antibodies were designed for capture and electrochemical detection of CRP using a sandwich format, and horseradish peroxidase (HRP)-labelled tracer. Micromotors with different carbon-based outer layer compositions were evaluated as active supports for antibody immobilization while maintaining propulsion efficiency. Among them, reduced graphene oxide (rGOx) resulted in the highest affinity and the best immunoassay performance. Platinum nanoparticles forming the inner layer catalysed the oxygen bubble generation by the decomposition of hydrogen peroxide on its surface.
Once the successful micromotors have been synthetized, the immunoassay can be developed. The number of rGOx micromotors, the immunoassay performance (antibody concentrations, non-specific adsorption incubation times), and propulsion conditions have to be studied in depth. In contrast with conventional immunosensors, where the analyte interacts with the usually immobilized specific antibody by diffusion or external stirring of the solution, self-propelled micromotors actively move around the sample to bind the analyte. Besides the expected efficient movement of the micromotors, the generated microbubble tails can enhance the fluid mixing, and consequently improve the efficiency of the biorecognition event. In this sense, the analysis time can be decreased significantly (<10|min) and extremely low sample volumes (7|μL) can be used, in which other stirring mechanisms are not effective enough.
These features are especially relevant when small volume samples are available, such as those from preterm babies with suspected sepsis.
Briefly, the CRP-micromotor immunoassay (MIm) procedure consists of the addition of 10|μL of micromotor suspension (around 2000 micromotors) modified with the capture antibody into a test tube. Then, the sample and detection antibody were added (10|μL total volume, of which 7|μL was the sample) to perform the sandwich immunocomplex in one step. In order to generate movement of the micromotors, the fuel was also added. This fuel mix contained surfactant to lower the interfacial tension as well as H2O2 as fuel, which allows the catalytic reaction responsible for the bubble formation in the inner layer of the micromotor. Under these conditions, the modified micromotors swim around the sample to find and capture the specific analyte for 5|min, boosting the biological interaction thanks to their autonomous movement in such a small volume. As a result of their magnetic characteristics (intermediate magnetic layer of nickel), the micromotors can be stopped and retained while the supernatant was removed. Once the immunoaffinity interactions were complete, electrochemical detection can be easily accomplished, re-suspending the micromotor–immunocomplexes in just 45|μL of hydroquinone solution (electrochemical mediator) and magnetically captured onto the surface of a portable and disposable screen-printed carbon electrode. Amperometric measurements were performed after the addition of hydrogen peroxide solution as enzymatic substrate.
Under these circumstances, the obtained analytical characteristics are highly competitive with other CRP immunoassays reported in the literature [8]. A linear working range from 2 to 100|μg/mL, (r|=|0.992), limit of detection (LOD) and limit of quantification (LOQ) of 0.8|μg/mL and 2.0|μg/mL, respectively, were obtained. Inter-assay precision for two different concentration values was 8%, whereas inter-assay precision was lower than 15% (n|=|5 days) for micromotors produced in different batches.
In order to evaluate the applicability in neonate clinical samples, the propulsion of the micromotors was tested in serum and plasma media, to compare with their behaviour in buffer. Although a diminished speed was observed in serum and plasma, it did not affect their function and indeed propulsion was improved in plasma by adjusting the fuel concentration. Hence, serum certified reference material (SCRM) and unique plasma samples from neonates with suspected sepsis were analysed.
The achieved results revealed an excellent accuracy of our MIm (Er|=|1%). Moreover, the results obtained by our MIm in comparison with those reported by the Hospital laboratory using standard analytical methods (BRAHMS CRPus KRYPTOR) for preterm neonate plasma samples, did not show significant differences (P<0.05), allowing the analysis of clinically relevant samples.
Seeking to fulfil all the requirements for performing on-site/bedside assays, the micromotor-based immunoassay for CRP described above was fully integrated into a microfluidic platform [9]. In this new approach, both main immunoassays steps, immunocomplex formation and detection steps, were performed in the same microfluidic system, in a fully automated way, and so dispensing with the need for human handling. This on-chip MIm approach blends the advantages of the micromotors with those of microfluidic technology, giving place to a new alternative for sepsis POCT development. Among other advantages, this CRP assay is fast (<10|min), easy-to-use, automated, requires low sample volume (7|μL) and presents an improvement of an analytical characteristic (LOD|=|0.54|μg/mL). Furthermore, the good results obtained after SCRM (Er<10%) and the analysis of neonate samples demonstrated its applicability in a real clinical environment.

MIm detection of PCT

As previously stated, the flexibility of the technology allowed changing the target analyte by simply modifying the specific antibody bound to the outer layer of the micromotors, and the detection technique using an adequate label. In this sense, the second approach deals with a micromotor-based fluorescence immunoassay for PCT determination in samples from neonates [10]. This time, different polymeric outer layers were tested, with COOH-polypyrrol being the polymer producing a higher degree of antibody functionalization and improving the sensitivity of the assay. The operating mode is similar to the previously developed CRP-MIm. However, in this case, detection is carried out on a fluorescence microscope by directly positioning 1|μL of the previously formed micromotor–immunocomplexes onto a microscope slide to perform the fluorescence measurements at excitation and emission wavelengths of 490|nm and 520|nm, respectively. These fluorescence signals were recorded and analysed by the high-resolution camera and its software associated with the microscope. A wide working linear range between 0.50 and 150|ng/mL, and LOD and LOQ of 0.07 and 0.50|ng/mL were obtained, respectively. Precision, even for intra- or inter-assay was lower than 9%. The applicability to real clinical samples was evaluated by analysing plasma samples from very low birth weight neonates. The results obtained by our MIm did not show significant differences with the PCT levels declared by the Hospital (BRAHMS PCT) (P<0.05).

Summary

In summary, these new micromotor-based immunoassay approaches exhibited key advantages such as simplicity, rapidity, miniaturization, automatization potential and reliability of analysis, using extremely low sample volumes and covering the entire range of concentrations involved in the clinical sepsis diagnosis (Fig. 2). Therefore, MIms are presented as future tools for early diagnostics, which is essential for timely treatment and the adequate guidance of antibiotic therapy as well as in the development of POCT devices.

The authors

Alberto Escarpa*1,2 , Miguel Ángel López 1,2 , Águeda Molinero-Fernández 1
PhD, María Moreno-Guzmán 3 and Luis Arruza 4
1Department of Analytical Chemistry, Physical Chemistry and Chemical
Engineering, University of Alcalá, Alcalá de Henares, E-28871 Madrid, Spain
2Chemical Research Institute Andres M. del Rio, University of Alcalá,
E-28871 Madrid, Spain
3Department of Chemistry in Pharmaceutical Sciences, Analytical Chemistry,
Faculty of Pharmacy, Universidad Complutense de Madrid, E-28040 Madrid,
Spain
4Division of Neonatology, Instituto del Niño y del Adolescente, Hospital
Clinico San Carlos – IdISSC, E-28040 Madrid, Spain

*Corresponding author
E-mail: alberto.escarpa@uah.es