**Role of Biological Markers for Cerebral Bleeding Risk STRATification in Patients with Atrial Fibrillation on Oral Anticoagulants for Primary or Secondary Prevention of Ischemic Stroke (Strat-AF Study): Study Design and Methodology**

**Anna Poggesi 1,2,3,\* , Carmen Barbato 2 , Francesco Galmozzi 2 , Eleonora Camilleri 4, Francesca Cesari 5, Stefano Chiti 6, Stefano Diciotti 7, Silvia Galora 4, Betti Giusti 4, Anna Maria Gori 4, Chiara Marzi 7 , Anna Melone 2, Damiano Mistri 2, Francesca Pescini 1, Giovanni Pracucci 2, Valentina Rinnoci 1,2,3, Cristina Sarti 1,2, Enrico Fainardi 8, Rossella Marcucci 4 and Emilia Salvadori 1,2,3**

Received: 19 July 2019; Accepted: 19 September 2019; Published: 23 September 2019

**Abstract:** *Background and Objectives:* In anticoagulated atrial fibrillation (AF) patients, the validity of models recommended for the stratification of the risk ratio between benefits and hemorrhage risk is limited. Cerebral small vessel disease (SVD) represents the pathologic substrate for primary intracerebral hemorrhage and ischemic stroke. We hypothesize that biological markers—both circulating and imaging-based—and their possible interaction, might improve the prediction of bleeding risk in AF patients under treatment with any type of oral anticoagulant. *Materials and Methods*: The Strat-AF study is an observational, prospective, single-center hospital-based study enrolling patients with AF, aged 65 years or older, and with no contraindications to magnetic resonance imaging (MRI), referring to Center of Thrombosis outpatient clinic of our University Hospital for the management of oral anticoagulation therapy. Recruited patients are evaluated by means of a comprehensive protocol, with clinical, cerebral MRI, and circulating biomarkers assessment at baseline and after 18 months. The main outcome is SVD progression—particularly microbleeds—as a selective surrogate marker of hemorrhagic complication. Stroke occurrence (ischemic or hemorrhagic) and the progression of functional, cognitive, and motor status will be evaluated as secondary outcomes. Circulating biomarkers may further improve predictive potentials. *Results*: Starting from September 2017, 194 patients (mean age 78.1 ± 6.7, range 65–97; 61% males) were enrolled. The type of AF was paroxysmal in 93 patients (48%), and persistent or permanent in the remaining patients. Concerning

the type of oral anticoagulant, 57 patients (29%) were on vitamin K antagonists, and 137 (71%) were on direct oral anticoagulants. Follow-up clinical evaluation and brain MRI are ongoing. *Conclusions*: The Strat-AF study may be an essential step towards the exploration of the role of a combined clinical biomarker or multiple biomarker models in predicting stroke risk in AF, and might sustain the incorporation of such new markers in the existing stroke prediction schemes by the demonstration of a greater incremental value in predicting stroke risk and improvement in clinical outcomes in a cost-effective fashion.

 Introduction**

Thromboprophylaxis with oral anticoagulation effectively reduces stroke risk in patients with atrial fibrillation (AF). Benefits must be balanced against the risk of bleeding, with intracranial hemorrhage being the most feared. Stroke and bleeding risk stratification schemes are aimed at identifying patients who may benefit most from different types of oral anticoagulation (vitamin K antagonists vs. direct oral anticoagulants). Currently, such schemes (e.g., CHADS2VASC2 and HASBLED scores) rely only on clinical information, the validity of which remains controversial and needs to be improved. In AF patients, advanced imaging technology such as magnetic resonance imaging (MRI), has led to the increased detection of asymptomatic brain changes, mainly those related to small vessel disease (SVD), which is the pathologic substrate for primary intracerebral hemorrhage (ICH) [1–6]. These changes have also been proven to strongly predict stroke risk [2,5]. Such imaging findings, obtained both at baseline and in terms of lesion progression over time, could be used as additional markers for risk stratification [3,7]. Furthermore, markers of coagulation activation, including prothrombin fragment 1+2, thrombin–antithrombin complex, D-dimer, time in therapeutic range for warfarin, and drug dosage for new anticoagulants, may be also studied as cofactors. Preliminary data suggest that circulating biomarkers of endothelial dysfunction, hypercoagulable state, and inflammation may further enhance risk prediction [4].

The validity of the currently recommended models for the stratification of risk ratio between benefits and hemorrhagic risk in anticoagulated AF patients is limited. These models do not specifically take intracranial hemorrhage into account, which is indeed the most severe hemorrhagic complication [8]. Our hypothesis is that biological markers—both circulating and imaging-based, and their possible interaction—might improve the prediction of bleeding risk in AF patients treated with any type of oral anticoagulant. Neuroimaging biomarkers of SVD—particularly microbleeds—may serve as a selective surrogate marker of hemorrhagic complications. Circulating biomarkers assessed together with imaging may further improve predictive potentials.

In this scenario, we set up the prospective observational Strat-AF study, primarily aimed at investigating circulating biomarkers and MRI markers (baseline and progression) of SVD as surrogate markers for the prediction of cerebral bleeding in a cohort of patients with AF on oral anticoagulants. Secondary outcomes included stroke occurrence (either ischemic or hemorrhagic) and the progression of functional, cognitive, and motor status.

#### **2. Materials and Methods**

Stratification of Cerebral Bleeding Risk in AF, Strat-AF, Strat-AF is an observational, prospective, single-center hospital-based study enrolling patients with AF, referring to the Center of Thrombosis outpatient clinic of our University Hospital for the management of oral anticoagulation therapy.

#### *2.1. Inclusion Criteria*

#### *2.2. Exclusion Criteria*

All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Careggi University Hospital (Project identification code 16RFAP, approved on March 2017).

Sample size was estimated based on a feasibility criterion (i.e., the real flow of patients to the Thrombosis outpatient clinic of Careggi Hospital). Yearly, approximately 600 patients with AF on oral anticoagulation refer to the outpatient clinic. Considering a foreseeable rate of ineligible patients and refusals, we originally estimated to contact approximately 300 patients in one year fulfilling the inclusion criteria. Starting from September 2017, consecutive eligible patients were invited to participate in the study. The initial foreseen period for enrollment was 12 months, but in order to augment the number of included patients, we extended the enrollment period to 18 months.

#### *2.3. Clinical Assessment*

All enrolled patients were assessed at baseline by means of a standard clinical/functional protocol collecting information on vascular risk factors (particularly hypertension and diabetes), dietary habits, previous cerebrovascular events, general neurological and functional status, cognitive performances, mood and gait disorders, and general neurological examination.

In detail, clinical data were collected about social and medical history; a standard cardiovascular and neurological examination, including office blood pressure measurement, were performed. Functional status was assessed using the Activities of Daily Living scale and the Instrumental Activities of Daily Living scale [9,10]; mood assessment using the Geriatric Depression Scale [11]; motor performance using the Short Physical Performance Battery [12]; daytime sleepiness using the Epworth Sleepiness Scale [13]; and quality of life using the EuroQol Visual Analog Scale [14]. Dietary habits were assessed by means of the questionnaire on the adherence to the Mediterranean diet [15].

A comprehensive multi-domain cognitive assessment, including global functioning, orientation, memory, attention, executive functions, language, speed, and motor control was administered to each included patient.

The cognitive domains assessed by means of the extensive battery of tests were as follows:

mean number of elements correctly remembered on the immediate and delayed recalls (score range 0–28).

	- i. Semantic verbal fluency test: a test allowing the semantic evaluation of lexical access [23]. Three categories were tested: car brands, fruits, and animals. The total score was given by the sum of the number of words produced for each category in one minute.
	- ii. Sentence construction test: a test used to evaluate the verbal ability to construct a meaningful sentence starting from a set of two or three words (score range 0–25) [19].

All raw test scores were demographically corrected according to Italian population normative data, and adjusted scores were then recoded as normal, borderline, or abnormal according to equivalent scores (ES) methodology [24]. ES methodology is a non-parametric norming method based on percentiles and is independent from the distribution form. ES is an ordinal 5-point scale (ranging from 0 to 4), and the main point of ES methodology is to fix the outer tolerance limit of the left queue of the adjusted scores so that it is possible to assess, with a known risk of error (<5%), the cut-off splitting the bottom 5% of the population and representing pathological performance (ES = 0). At the other end of the scale, ES = 4 indicates an optimal performance (equal to or better than the median). ES = 1 indicates a borderline performance (an adjusted score between the outer and inner confidence limits for the fifth centile of the normal population), while the remaining ES scores of 2 and 3 represent normal performances.

The diagnosis of MCI requires at least one altered score (ES = 0) plus one borderline score (ES = 1) in any cognitive test included in the neuropsychological battery.

#### *2.4. Cerebral Magnetic Resonance Imaging Assessment*

Cerebral MRI scans were performed at baseline, and again 18 months after enrollment. The brain imaging protocol was planned and set up by imaging personnel with different expertise and skills, as suggested by current guidelines [25].

Baseline and follow-up MRI examinations are performed on an Ingenia 1.5-Tesla MRI unit (Philips Healthcare, Eindhoven, The Netherlands). Our standardized MRI protocol consisted of the following sequences: sagittal T1-weighted spin-echo (repetition time (TR) = 547 ms; echo time (TE) = 12 ms; slice thickness = 5 mm; interslice spacing = 0.5 mm; matrix size = 320 × 250; field of view (FOV) = 23 cm × 23 cm; number of signals averaged (NSA) = 1), coronal T2-weighted fast spin-echo (TR = 3347 ms; TE = 110 ms; slice thickness = 5 mm; interslice spacing = 0.5 mm; matrix size = 512 × 322; FOV = 22 cm × 22 cm; NSA = 2); axial fluid-attenuated inversion recovery (FLAIR) (TR = 11,000 ms; TE = 125 ms; inversion time (TI) = 2800 ms; slice thickness = 5 mm; interslice spacing = 0.5 mm; matrix size = 384 × 204; FOV = 23 cm × 23 cm; NSA = 2); axial gradient-echo T2\* (GRE) (TR = 534 ms; TE = 23 ms; flip angle (FA) = 18; slice thickness = 5 mm; interslice spacing = 0.5 mm; matrix size = 256 × 185; FOV = 23 cm × 23 cm; NSA = 1); axial diffusion-weighted imaging (DWI) (TR = 3891 ms; TE = 75 ms; slice thickness = 5 mm; interslice spacing = 0.5 mm; matrix size = 164 × 162; FOV = 23 cm × 23 cm; NSA = 2); gradient-echo 3D T1-weighted (TR = 7.5 ms; TE = 3.4 ms; TI = 950, slice thickness = 1 mm; matrix size = 256 × 241; FOV = 25.6 cm × 25.6 cm; NSA = 1) followed by multiplanar reconstruction (MPR) in axial, coronal, and sagittal planes. DWI images were obtained by single-shot echo-planar spin-echo sequences according to the Stejskal–Tanner method. The diffusion

gradients were applied in three orthogonal directions (*x*, *y*, *z*) with two b-values (0 and 1000 s/mm2) to form the isotropic DWI images at b 1000 s/mm2. Apparent diffusion coefficient (ADC) maps were generated automatically by the software provided by the manufacturer from isotropic DWI images and concurrent images with a b value of 0 s/mm2 by using the following equation: ADC = -ln [S(b)/S(0)]/b, where b indicates the b value and S(b) and S(0) are the signal intensities of images with b values equal to 1000 and 0, respectively.

Each MRI scan was evaluated by an expert neuroradiologist, and a written medical report was provided to patients. Neurologists involved in the project revised the report before returning it to patients, so that any incidental findings could be handled according to individual needs.

Qualitative and quantitative analyses of SVD-related features on MRI are under way.

The following features related to SVD will be assessed: i) lacunar infarcts, either silent or not; ii). white matter hyperintensities; iii) microbleeds; iv) dilated perivascular spaces; v) cortical and subcortical atrophy; vi) superficial siderosis [5].

Non-lacunar infarcts will be visually assessed in terms of number and location of lesions.

#### *2.5. Circulating Biomarker Assessment*

Venous blood samples were collected after enrollment and placed at -80 ◦C for long-term storage. Besides routine parameters (complete blood count (CBC), prothrombin time (PT), activated partial thromboplastin time (APTT), D-dimer, fibrinogen, low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides, glucose, creatinine, glomerular filtration rate (GFR), alanine aminotransferase, C-reactive protein (CRP), N Terminal pro B-type natriuretic peptide, Lp(a), troponin), several circulating biomarkers will be evaluated:

The list of biomarkers under investigation is detailed in Table 1. The biological material (serum, plasma, DNA) is properly conserved to allow the evaluation of further biomarkers by specific or global assessment strategies (e.g., metabolomics profiling, targeted or whole-exome sequencing).

Proteomic and genetic biomarkers will be evaluated with high-multiplex immunoassays or high-throughput technologies such as Bio-Plex Multiplex Immunoassay System (BioRad), Proximity Extension Assay (Olink) technology, H-nuclear magnetic resonance spectroscopy (Bruker BioSpin), real-time PCR (Life Technologies), droplet digital PCR (BioRad), and high-throughput sequencing (Illumina).

Biosamples were processed using standard and harmonized operating procedures.

Clinical data are registered electronically in the web-based registry (http://www.strat-af.it/). Quality controls are done on a weekly basis. Imaging data are instantly checked for protocol conformity.

The same clinical/functional assessment and brain MRI will be repeated 18 months after enrollment. An interim telephone follow-up interview will be scheduled approximately three months before the final clinical assessment.

**Table 1.** Circulating biomarkers under investigation in the Strat-AF study.

#### *2.6. Primary Endpoint*

The primary study endpoint will be SVD progression, evaluated by means of the control MRI performed 18 months after enrollment. The progression of lacunar infarcts and microbleeds will be evaluated as the appearance of at least one new lesion, respectively. White matter hyperintensities progression will be rated by means of the visual Rotterdam Progression Scale (score range from 0 to 9) [26]. Absence or presence of progression (0 or 1, respectively) will be rated in three periventricular regions (frontal caps, occipital caps, bands), four subcortical white matter regions (frontal, parietal, occipital, temporal), basal ganglia, and the infratentorial region. Post-processing and ratings will be centralized and performed by expert and reliable observers, blinded to clinical data.

#### *2.7. Secondary Endpoints*

Secondary endpoints will be:

be further categorized in subtypes according to the TOAST classification system [27]. Hemorrhagic strokes will be classified according to lesion type and location.

#### *2.8. Statistical Analyses*

Multivariate regression analyses will be performed in order to identify independent predictors of SVD and its progression. Among the variables of interest, apart from the clinical ones and those assessed by means of conventional MRI, special attention will be paid to the relationship with advanced neuroimaging features and circulating biological markers.

Cerebral SVD and its progression—particularly microbleeds, which are considered the main expression of a bleeding-prone state—will be studied as outcome variables in multivariate regression models, considering the independent effect of all relevant markers, including major vascular risk factors and anticoagulant treatment with different types of anticoagulants (vitamin K antagonists vs. direct thrombin antagonists or factor Xa antagonists), and circulating biomarkers.

Interactions of circulating biomarkers with imaging markers, and types of oral anticoagulants will also be analyzed. Bonferroni correction will be applied in multivariate models to counteract the problem of multiple comparisons.

The multivariable prediction of the risk of SVD progression in this cohort of AF patients on oral anticoagulants will be studied as the methodological background for clinical research in the setting of stroke prevention in patients with AF (i.e., for designing and sample sizing studies potentially adopting the above indicated biomarkers as surrogate markers of the clinical outcomes).

#### **3. Results**

Starting from September 2017 until March 2019, 617 patients referring to the outpatient clinic of the Center of Thrombosis for oral anticoagulants therapy control were screened for inclusion in the study. As shown in Figure 1, 423 patients (68%) were excluded because of MRI contraindications (*n* = 227) or refusal (*n* = 196). The remaining 194 patients were enrolled. Demographic and clinical characteristics of the baseline sample are shown in Table 2: mean age was 78.1 ± 6.7 (range 65–97) years, 118 (61%) were males, and mean education was 9.1 ± 4.3 (range 2–19) years. The type of AF was paroxysmal in 93 patients (48%), and persistent or permanent in the remaining patients. Concerning the type of oral anticoagulant, 57 patients (29%) were on vitamin K antagonists, and 137 (71%) were on direct oral anticoagulants.

**Figure 1.** Strat-AF flow diagram.

#### **4. Discussion**

The Strat-AF study is an ongoing, single-center, longitudinal observation study evaluating elderly patients with AF on oral anticoagulation for primary or secondary prevention of stroke. The project foresees, in consecutive elderly patients with AF attending the Center for Thrombosis outpatient clinic of Careggi University Hospital for management of oral anticoagulation therapy, the implementation of a comprehensive neurological, neuropsychological, and functional evaluation together with blood sample collection for the determination of circulating biological markers (in relation to the hemorrhagic risk profile) and brain MRI for the determination of brain parenchyma lesions as surrogate markers of a bleeding-prone state. The Strat-AF cohort will allow the evaluation of the possible role of biological markers, including clinical, circulating, and neuroimaging-based, and their interaction, on the prediction of bleeding risk in AF patients under treatment with any type of oral anticoagulant. Neuroimaging biomarkers of SVD, particularly microbleeds, will be tested as a selective surrogate marker of such complication. Circulating biomarkers assessed together with imaging might further improve the predictive potentials. Active enrollment started from September 2017 and ended in March 2019. The baseline study cohort included 194 elderly patients. Follow-up assessments are now ongoing.

One first major comment arises from the fact that in order to reach the number of enrolled patients, more than 600 patients had to be screened. Of these, more than half were excluded. Main reasons for exclusion were refusal (46%) and MRI contraindications (54%). Thus, the method under evaluation (i.e., brain MRI) will not be feasible for all AF patients, but just for those without contraindication. According to our experience, about one-quarter of patients would not be included in such a new stratification risk schema.

Overall, the study protocol seems feasible, and nearly all included patients completed the evaluation.

Our results will provide a unique opportunity to achieve preliminary data about SVD progression as a surrogate marker of the effect of antithrombotic treatments used for stroke prevention in patients with AF. The longitudinal design of the study may also provide clues about the possible association of SVD and its progression with clinical endpoints. Thrombo-embolic and bleeding risk will be assessed using the clinical risk scores CHADS2VASC2 and HAS-BLED respectively. Such predictive models will be completed studying the effect of neuroimaging features, particularly those related to SVD, and circulating biological markers, in order to provide preliminary knowledge about the incremental value of such markers.

Conclusive evidence about the predictive value of these markers in single patients can only come from adequately powered large prospective follow-up studies, and few efforts are already under way. Prospective multicenter studies such as the CROMIS 2 (clinical relevance of microbleeds in stroke; ClinicalTrials.gov Identifier NCT02513316), ICH because of oral anticoagulants: prediction of the risk by MRI (HERO, Hirulog Early Reperfusion/Occlusion Trial; ClinicalTrials.gov Identifier NCT02238470), and CMB-NOW (CMBs during NOACs or warfarin therapy in non-valvular AF patients with acute ischemic stroke; ClinicalTrials.gov Identifier NCT02356432) are ongoing. Main results from CROMIS-2 have been recently published: among the 1490 patients with AF enrolled after their ischemic stroke, the presence of CMBs significantly increased the risk of symptomatic ICH (adjusted hazard ratio 3.67, 95% CI 1.27–10.60), confirming that CMBs are independently associated with symptomatic ICH risk, and could be used to inform anti-coagulation decisions. In this study, after 24 months of follow-up, 14 patients had an intracerebral hemorrhage. The very low number of events did not allow the establishment of whether a CMBs threshold exists. Moreover, the study does not foresee a control MRI, so no information is available concerning the possible role of CMBs progression. A recent meta-analysis assessed the association between CMBs and future ICH risk in ischemic stroke patients with AF taking oral anticoagulants. The authors concluded that the presence of CMBs on MRI and the dichotomized cutoff of ≥5 CMBs might identify subgroups of patients with high ICH risk [28], but these data need to be confirmed before they can be used in clinical practice.

#### **5. Conclusions**

Long-term oral anticoagulation is the mainstay therapy for ischemic stroke prevention in patients with AF. Available stroke and bleeding risk stratification schemes are aimed at identifying patients who may benefit most from oral anticoagulation [29]. Such schemes (e.g., CHADS2VASC2, HAS-BLED scores) currently rely only on clinical information, the validity of which remains controversial and needs to be improved. Attempts have been made to refine the risk stratification scores by the addition of various biomarkers (blood, urine, cardiac, and cerebral imaging), but data are still inconclusive as to whether the costs are justified [30].

The Strat-AF study may be an essential step towards the exploration of the role of a combined clinical biomarker or multiple biomarker models in predicting stroke risk in AF, and might sustain the incorporation of such new markers in the existing stroke prediction schemes by the demonstration of a greater incremental value in predicting stroke risk and improvement in clinical outcomes in a cost-effective fashion.

**Author Contributions:** Conceptualization, A.P., F.C., S.D., B.G., A.M.G., R.M., E.S.; methodology, A.P., F.C., S.C., S.D., B.G., A.M.G., G.P., E.F., R.M., E.S.; formal analysis, A.P., G.P., E.S.; investigation, A.P., C.B., F.G., E.C., S.G., C.M., A.M., D.M., V.R., E.F., R.M., E.S.; data curation, A.P., G.P., E.S.; writing—original draft preparation, A.P., C.B., F.G., E.S.; writing—review and editing, A.P., C.B., F.G., E.C., F.C., S.C., S.D., S.G., B.G., A.M.G., A.M., D.M., F.P., G.P., V.R., C.S., E.F., R.M., E.S.; supervision, A.P.; project administration, A.P., F.C., S.D., F.P, R.M., E.S.; funding acquisition, A.P., F.C., S.D., F.P, E.S.

**Funding:** This research was funded by Tuscany region and Italian Ministry of Health under Grant Aimed Research Call "Bando Ricerca Finalizzata 2013" GR-2013-02355523. Title of the project "Role of biological markers for cerebral risk stratification in patients with atrial fibrillation on oral anticoagulants for primary or secondary prevention of ischemic stroke".

**Acknowledgments:** The authors would like to thank Graziella Terranova and the staff of Careggi office for the administrative support.

**Conflicts of Interest:** The authors declare no conflicts of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**6. Some selected medicinal plants with antiulcerogenic properties in Nigeria**

In Nigeria, SU remains a significant public health challenge affecting people of all ages. While its management through orthodox medicine has recorded substantial successes over the years, a considerable proportion of the populace still rely exclusively on complementary and alternative medicine (CAM) in seeking aid to treat and manage SU. This may be due to the ease of accessibility, affordability and minimal side effect associated with the use of medicinal plants [22]. A compilation of selected antiulcerogenic medicinal plants in Nigeria revealed that the most widely used plants in the management of SU are *Occimum basillicum*, *M. paradisiaca*, *Aloe vera*, *Azadiracter indica*, *Brassica oleracae* and *Carica papaya* [78]. Others include but not limited to the following: *Diodia sarmentosa*, *Cassia nigricans*, *Ficus exasperate*, *Synclisia*  *scabrida*, *Artocarpus heterophyllus* Lam., *Blighia sapida* Konig., *Dialium guineense* Willd., *Emblica officinalis* Gaertn., *Gongronema latifolium*, *Ageratum conyzoides*, *Aloe vera*, *Artocarpusaltilis*, *Aspilia africana*, *Bryophyllum pinnatum*, *Fluerya aestuans*, *Musa paradiasiaca*, *Musa sapientum*, *Persea Americana*, *Talinum triangulare*, *Fluerya aetuans*, *Brassica oleracae, Acacia nilotica* L.*, Alchornea cordifolia Schum & thonn, Anacardium occidentale* L., *Balanites aegyptiaca* L., *Bridelia ferruginea* Benth, *Carica papaya* Linn, *Ficus thonningii* Blume, *Guiera senegalensis* J. F. Gmel, *Hibiscus sabdariffa* L., *Mangifera indica* L., *Momordica charantia* L., *Ocimum gratissium* L., *Piliostigma reticulatum* (DC) Hochst, *Pisidium guajava* L., *Scoparia dulcis*, *Vernonia kotschyana* Sch. Bip., *Zingiber officinale* Rosc [79].

## **7. The role of medicinal plants in oxidative gastropathy**

Following the experimental demonstration that many medicinal plants are endowed with good antiulcerogenic activity with relatively lesser adverse effect compared with the conventional drugs, further steps have been taken in presenting a good number of them for clinical trials. Despite this giant stride, not many of the medicinal plants have passed market entry stage. To the best of our knowledge, of the many presented for developmental evaluations in 2004, only *Azadirachta indica* (Family: Meliaceae) received remarkable attention at its advance stage of clinical trial. It exhibited significant therapeutic potency by reducing gastric hyperse-

Several phytonutrients have proven health benefits and have been reported to elicit significant antiulcerogenic potential in both humans and experimental animal models [73]. While steroid glycosides, tannins, terpenoids and flavonoids have been shown to preserve gastric mucosal against oxidative insults of reactive metabolites and oxidative stress [22, 59], the tendency of phenolic compounds and alkaloids to regulate gastric acid secretion and protect the gastric mucosal epithelia against erosion and other aggressive factors in different ulcer models have been demonstrated [77]. While **Table 1** also presents some of these phytonutrients as being responsible for the elicited antiulcerogenic properties of the plants, several others have also been identified and isolated from diverse plants. Some of these include; saponins, phobaphenes, glucose, luvangetin, tartarate, potash, nimbidin, quercetin, apigenin, papain, chymopapain, pectin, carposide, carotenoids, antheraxanthin, carpaine, resin, euphorbon, caoutchouc, rutin, anthocyanins, cyanindin, kaempferol, sterols, mucilage, terpenoids, kaepferom, ash, starch, fats, proteins, glycosides, ellagic acid, beta sitosterol, gallic acid, limonene, pinene, albuminous matter, cellulose, chlorophyll, mineral salts, myricitin, triterpenes, and sorbitol [37]. These compounds have either been elucidated to decrease acid/pepsin secretion or confer cytoprotection via effective modulation on mucosal

**6. Some selected medicinal plants with antiulcerogenic properties in**

In Nigeria, SU remains a significant public health challenge affecting people of all ages. While its management through orthodox medicine has recorded substantial successes over the years, a considerable proportion of the populace still rely exclusively on complementary and alternative medicine (CAM) in seeking aid to treat and manage SU. This may be due to the ease of accessibility, affordability and minimal side effect associated with the use of medicinal plants [22]. A compilation of selected antiulcerogenic medicinal plants in Nigeria revealed that the most widely used plants in the management of SU are *Occimum basillicum*, *M. paradisiaca*, *Aloe vera*, *Azadiracter indica*, *Brassica oleracae* and *Carica papaya* [78]. Others include but not limited to the following: *Diodia sarmentosa*, *Cassia nigricans*, *Ficus exasperate*, *Synclisia*

cretion, gastroesophageal and gastroduodenal ulcers [76].

defensive factors.

**Nigeria**

32 Stomach Disorders

**5. Phytonutrients associated with antiulcerogenic activity**

Reactive oxygen species (ROS) are a by-product of normal metabolism and have roles in cell signaling and homeostasis [80]. Mechanisms exist that regulate cellular levels of ROS, as their reactive nature may otherwise cause damage to key cellular components including DNA, protein, and lipids [81]. A good number of NSAIDS have been implicated in cellular toxicity leading to oxidative gastropathy [82]. Despite the use of NSAIDS as antipyretic and antiinflammation agents, and in the treatment of rheumatic, musculoskeletal, and cardiovascular diseases [83], gastrointestinal toxicity through ROS formation has limited their application [84, 85]. It has been proposed that NSAID-mediated gastrointestinal lesions involve the uncoupling of oxidative phosphorylation and inhibition of electron transport chain causing incomplete reduction of oxygen [82]. This they do by tenaciously binding to a site near complex I and ubiquinone, thus facilitating events leading to ROS generation [86, 87]. Subsequently, when the gastric antioxidant capacity is overwhelmed, the epithelia mitochondrial aconitase is inhibited, resulting in the release of iron that reacts with H2 O2 , producing hydroxyl radical. These cascades of event amplify gastric oxidative stress whose consequential effect is manifested as gastropathy [88]. Oxidative stress-induced functional loss is well correlated with numerous disease states including cardiovascular, neurological, cancer, aging processes and gastropathy [83] and is also implicated in a variety of drug-induced toxicities such as SU [2]. Antioxidative and free radical scavenging mechanisms play an important role in the protection against ROS mediated toxicities [89]. Over the past decades, interests in medicinal plants, especially the antioxidative ones, have increased appreciably and they have been elucidated to significantly either protect against or ameliorate ROS-mediated oxidative gastropathy [22, 90]. Such annihilation of ROS in SU diseases have been achieved through induction of enzymic antioxidants (superoxide dismutase, catalase, glutathione reductase and peroxidase) and optimization of reduced glutathione (GSH) contents [2, 22, 59]. Harmonizing the foregoing, a probable mechanism of antioxidative and gastroprotective activities of medicinal plants may be idealized as illustrated in **Figure 1**. This ultimately involves induction and optimization of preventive (catalase, glutathione peroxidase) and chain-breaking (superoxide dismutase, glutathione reductase) antioxidants that subsequently improve gastric GSH level, annihilate liberated reactive metabolite and effectively scavenge ROS (O2 − , OH− ) (**Figure 1**). This may also be opined to regulate mucosal fluidity and strengthens defensive mechanisms against oxidative gastric damage.

**Figure 1.** Probable mechanism of antioxidative and gastroprotective capabilities of medicinal plants. The dotted arrows represent sites of induction and optimization by the plants that consequently promote scavenging of O2 − and OH− . This will normalize and increase gastric reduced glutathione (GSH) content and promotes its mobilization toward detoxification of the liberated reactive metabolites. NSAIDS, nonsteroidal anti-inflammatory drugs; MP, medicinal plants; GPx, glutathione peroxidase; GRx, glutathione reductase; GSSG, oxidized glutathione.

## **8. Conclusion**

Globally, SU is a devastating disease posing serious threat to the quality of life of humans. It affects significant proportion of the populace in both developed and developing countries. Although, conventional drugs have been used to manage and treat SU sufferers, affordability and inherent side effects have limited their application. Consequently, alternatives are being sought in medicinal plants, which provide a potential source of antiulcerogenic drugs and are widely used in traditional systems of medicine. Several medicinal plants have been investigated for their proven health benefits in SU management with their phytonutrients playing significant roles. Of the phytonutrients, tannins seem to top the list and has suggested probable focus on their characterization for antiulcer therapy. In spite of the impressive experimental evaluation of medicinal plants for the treatment of SU, very few have reached clinical trials and not very many have been marketed. This indicates that the intended benefits of CAM research are not yet having far-reaching effect. Nevertheless, the continuous search for antiulcerogenic agents of plant origin (available as gifts of nature) is imperative. This will ultimately and eventually present effective and globally competitive exciting opportunities for the development of new lead therapeutics for SU and other related disorders.


**4.6 Treatment of duodenal obstruction**

Superior mesenteric artery syndrome is a rare condition that results from intermittent functional obstruction of the third part of the duodenum. The diagnostic criteria are clinical, radiological and endoscopic. The classic approach has been open surgery [123]. There are case reports of robotic Roux-en-Y duodenojejunostomy as a surgical option for the treatment of this condition [106, 124].

Robotic repair of congenital duodenal atresia may help overcome the obstacles presented by the use of traditional rigid laparoscopic instruments, due to the difficulty in constructing a precise duodenal anastomosis, with robotic surgery the procedure is relatively straightforward [107]. About gastroduodenal obstruction due to trichobezoar in children and laparoscopy, we found several reports. We operated with RAS on a 12-year-old girl weighing 23 kg with pica and psychological disorder, with success and without postoperative morbidity [14].


*Review* **Attenuation Factors in Molecular Electronics: Some Theoretical Concepts**

### **Yannick J. Dappe**

SPEC, CEA, CNRS, Université Paris-Saclay, CEA Saclay, 91191 Gif-sur-Yvette, CEDEX, France<EMAIL_ADDRESS>
Received: 30 July 2020; Accepted: 1 September 2020; Published: 4 September 2020

**Abstract:** Understanding the electronic transport mechanisms in molecular junctions is of paramount importance to design molecular devices and circuits. In particular, the role of the different junction components contributing to the current decay—namely the attenuation factor—is yet to be clarified. In this short review, we discuss the main theoretical approaches to tackle this question in the non-resonant tunneling regime. We illustrate our purpose through standard symmetric junctions and through recent studies on hybrid molecular junctions using graphene electrodes. In each case, we highlight the contribution from the anchoring groups, the molecular backbone and the electrodes, respectively. In this respect, we consider different anchoring groups and asymmetric junctions. In light of these results, we discuss some perspectives to describe accurately the attenuation factors in molecular electronics.

**Keywords:** molecular junctions; attenuation factor; density functional theory; graphene

### **1. Introduction**

One of the main goals of molecular electronics is to mimic standard electronic circuits using molecules instead of p-n junctions like components based on silicon [1]. To do so, the first property to achieve in a molecular junction is to favor and understand at the fundamental level the circulation of the electronic current through the molecule. The problem of the electronic conduction mechanism in a molecule connected to metallic electrodes is very complex, and the possibility of many different regimes has been well presented theoretically by Reed et al. [2,3]. In particular, according to the molecular length, different regimes are observed, which exhibit different dependences in voltage and temperature. Hence, in the frame of elastic transport (considering that inelastic interactions only occur in the electrodes), for big molecular chains (longer than 5 nm), the electronic transport lies in an activated regime, called hopping regime, which is thermally activated. Indeed, in this regime, the conductance evolution of the molecular junction can be written as G ~ exp(−EA/kBT), where EA represents the hopping activation energy, around 0.5 eV, kB is the Boltzmann constant and T the temperature [4]. With respect to the molecular length, the conductance decreases linearly, which is easily understandable since the electrons have to jump (hop) from one molecular site to the nearest neighbor one [5,6]. This behavior is characteristic of Ohm's law, which we can observe at the macroscopic scale.

For smaller molecular chains, (i.e., below 5 nm) [7], the regime is not activated anymore and corresponds to the direct tunneling of the electrons through the molecular junction, provided that the applied voltage is lower than the characteristic electronic barrier of the system. In this case, there is no temperature dependence, and it is a reasonable approximation to say that the current varies linearly with the voltage at low bias. Indeed, the current–voltage relation is often non-linear for a significant range of voltages, before the molecular level is brought into alignment with the Fermi level of the electrodes. The transition between both regimes has been observed around 4 nm in conjugated polymers [8]. Note that, for larger voltages, this regime is generalized to the Fowler–Nordheim tunneling, where the current varies like the inverse of the voltage [9].

A fundamental problem in electronic transport in molecules lies in the length dependence of the conductance in the molecular junction. This dependence is reflected in the so-called attenuation factor, which is representative of the electronic current propagation in the molecular junction. Since a molecular junction is generally not metallic, the electronic conductance decreases as a function of the molecular length. Obviously, bearing in mind that the main application of molecular electronics is to design new devices for future electronics, one is interested in the lowest possible attenuation factor in order to increase the current in the molecular circuit. In the direct tunneling regime that we will particularly consider here, the conductance decays exponentially with respect to the molecular length, as we will detail in the next section. The main goal is therefore to reduce this exponential decrease as much as possible in order to optimize the electronic flow in the molecular circuit.

Hence, many experimental studies have been devoted to the determination of attenuation factors in different types of molecular junctions. In the meantime, theoretical methods have been developed to determine the electronic and transport properties of molecular junctions. The aim of this short review is not to describe extensively what has been done in the field, which would lead to an unreasonable amount of references, but more to discuss, in light of some representative systems, the progress and perspectives in theoretical methods to characterize the electronic transport in molecular junctions and to determine the attenuation factors.

In this respect, this review is organized as follows: in the first section, I will present a short state of the art of experimental determinations of attenuation factors in molecular junctions, and I will stress the most important results. Then, I will discuss the commonly used theoretical approaches, pointing out the corresponding strengths and weaknesses. In the fourth section, I will illustrate this discussion with the standard case of alkane-based molecular junctions, considering first the role of the anchoring groups in a symmetric junction with metallic electrodes and then by breaking this symmetry by using either different electrodes or different anchoring groups at each molecule sides. Obviously, since the anchoring groups are present in all the systems considered here, their influence will be analyzed in coordination with the different electrodes, molecular backbones and symmetry breakings. The underlying physical mechanisms will be addressed in light of these non-symmetric junctions. To complete this section, I will also discuss the role of the molecular backbone and its potential influence on the attenuation factor. Finally, I will summarize and draw some conclusions in the last section.

### **2. Some Attenuation Factors of Standard Molecular Junctions**

In this review, we will consider specifically the non-resonant tunneling regime, which deals with small molecular lengths in the junction (typically below 5 nm [7]) and also low applied bias. In this case, the evolution of the conductance with respect to the molecular length can be written in the following form:

$$\mathbf{G} = \mathbf{A} \exp\left(-\beta \mathbf{L}\right),\tag{1}$$

where L is the molecular length, A is the pre-exponential factor representative of the contact resistance at the molecule–metal interface, and β is the attenuation factor [10]. This attenuation factor is representative of the current attenuation within the molecular junction, with respect to the molecular length. In order to redefine the context, the attenuation factor is zero for a pure metallic junction, as no attenuation occurs, whereas it is of the order of 25 nm−1 for the vacuum where the current is fully attenuated.

A standard example of attenuation factor measurement is alkane-based molecular junctions. Many studies have been devoted to this system, and the attenuation factor currently lies around 7–9 nm−1 [10–12]. It has to be noted that this value is strongly dependent on the chemical nature of the molecular wire, since substituting carbon by silicon to constitute what is called oligosilane chains reduces the attenuation factor to 3.9 nm−1 [13]. This effect is even more enhanced considering germanium-based molecular wire, to form a germane chain, leading to β ~ 3.6 nm−1 [14]. In this respect, we can speculate that going down the lines of the periodic table, i.e., increasing the atomic number of

the chain constituents, would lead to an important reduction in the attenuation factor—in other words, a higher conductance, potentially related to the increasing number of electrons per atom. On the other hand, still considering non-conjugated molecular chains, the opposite behavior is found in siloxane chains, where the repetition unit is based on a Si-O dimer, with a very important attenuation factor of β ~ 12.3 nm−1 [15]—in other words, almost no conductance.

Beyond the chemical nature of the molecular chain elements, the nature of the molecular bonding plays an important role. For example, coming back to carbon elements, aromatic chains present reduced attenuation with respect to alkane chains, with β ~ 2.5 nm−1 [16]. Similarly, carotenoid polyenes present β of 2.2 nm−1 [17] and oligothiophenes have β between 2 and 3 nm−1 [18]. Consequently, one can deduce that the aromaticity of the molecular chain helps in reducing the attenuation factor; however, it is more or less always in the same range.

Besides the above, some more complex molecular systems also exhibit even lower attenuation factors, around 10 times lower than the polyenes discussed previously. For example, polymethine dyes, which are π-conjugated compounds with an odd number of carbons, have attenuation factor β ~ 0.4 nm−1 [19]. This is mainly attributed to greater electronic delocalization, in comparison to standard polyenes, enhanced by a smaller degree of bond order alternation. However, this behavior seems to be limited by the molecular length, leading to a more resistive junction for long molecular chains. In addition, the more exotic case of porphyrin polymers is very interesting. Indeed, when considering oligo-porphyrin molecular junctions, based on Zn porphyrins with pyridine ligands, one can observe a similar reduced attenuation factor β ~ 0.4 nm−1 [20]. Actually, it seems that the molecular conductance has a strong temperature dependence and a weak length dependence, even though it does not correspond to the hopping mechanism described in the introduction. It is rather consistent with phase-coherent tunneling through the whole molecular junction. Even more surprising is the result obtained by Leary et al., who have studied molecular chains of fused porphyrins [21]. Namely, in the considered porphyrin oligomers, the monomers connect directly with their nearest neighbor through the porphyrin cycle. In this case, contrary to the previous cases discussed here, the conductance increases with the distance, by more than a factor of 10, when a small bias (~0.7 V) is applied to the junction. This exceptional behavior is due to the evolution of the HOMO-LUMO gap that rapidly decreases with the molecular length, which compensates for the increased tunneling distance. Finally, the last example to be cited with respect to low attenuation factors is the work conducted by Brooke et al., who show a structural control on the electronic transport resonance in HS(CH2)n[1,4 −C6H4](CH2)nSH (*n* = 1, 3, 4, 6) metal–molecule–metal junctions, leading to very small attenuation factors [22]. This work offers very promising perspectives in gating the transport resonance in order to modulate the molecular junction behavior.

In this discussion, we have mainly considered the case of single molecule junctions. An interesting question that arises is this: what happens if we consider not only a single molecule junction but also a large molecular area? In this case, several experiments have found that the attenuation factor β mainly remains the same, and only the prefactor of the conductance is modified, as for molecular junctions based on biphenyl, nitrophenyl, ethynyl benzene, anthraquinone, etc., where one can observe attenuation factors around 2.7 nm−1 [23] (see Figure 1).

**Figure 1.** Overlay of attenuation plots for eight different molecules constructed from J–V curves with different thicknesses of each structure (the length of the error bars is two standard deviations). The lines are least squares regression lines for aromatic (2.7 nm−1) molecules. The different abbreviations stand for the aromatic molecular names: NP = nitrophenyl, AQ = anthraquinone, NAB = nitroazobenzene, BrB = bromophenyl, EB = ethynylbenzene, AB = azobenzene and BTB = bisthienylbenzene. Adapted figure from [23] and with permission. Copyright (2010) American Chemical Society.

As an important consequence, these results show that the study of single molecule junctions is not only important at the fundamental level. The understanding of the transport mechanism at the single molecule level and, in particular, the determination of the attenuation factor may be extrapolated to large molecular areas, which are currently used in molecular electronics devices. Another important aspect that can be stressed is that, below 5 nm, it seems that there is no significant difference in the attenuation factor with respect to the molecular backbone of the aromatic molecules considered in the junction. This might be attributed to the relative positions of the molecular electronic levels, which remain constantly pinned near the Fermi level at around 1.3 eV [23,24]. We will see in the next sections that this position of the molecular levels is an important parameter to determine the attenuation factor. However, it has to be noted also that most of these experimental results have been obtained using molecular junctions in symmetric configurations, namely with the same electrode at each side of the junction (most of the time, metallic electrodes made of noble metals) and similar anchoring groups. Very few studies have considered non-symmetric junctions—for example, with different anchoring groups at each molecular end—which might have an influence on the position of the molecular levels and yield other kinds of information about the transport mechanism [25,26]. In addition, as we will detail later, some recent experiments have started to use different electrodes at each end as well as

non-metallic electrodes. Obviously, the question that arises is this: what is the fundamental element in a molecular junction that determines the attenuation factor? Is it the chemical nature of the molecular backbone? Is it the anchoring groups? Is it the electrode or the coupling to the electrode? Alternatively, is it a mix of all these aspects? To answer these questions, theoretical modeling of the molecular junction and of the electronic transport through the junction is of paramount importance. In particular, it should help to discriminate these different contributions. In the next section, we will consider the most common theoretical approaches to characterize the electronic transport in a molecular junction and to determine the attenuation factor.

### **3. Theoretical Approaches**

In this section, we will discuss the most common theoretical approaches used to determine the electronic transport and in particular the attenuation factor in a molecular junction. An extensive review of electronic transport calculations using ab initio and density functional theory (DFT) based methods and Green functions can be found in [27]. Unfortunately, a direct relationship with the attenuation factor and overall a deep interpretation is not necessarily straightforward to deduce from these calculations. The objective here is to present the main contribution from the different parts of the molecular junctions that are characterized theoretically in each approach.

Hence, the most common description of the electronic current in a molecular junction has been shown by Simmons [1,28], considering the tunnel effect between metallic electrodes and a thin insulating film. In this respect, the current and the electronic conductance follow an exponential decay, as proposed in Equation (1), where the attenuation factor β can be expressed as β ∼ - (2mϕ/-, with m the mass of the electron, the reduced Planck constant and where ϕ represents the electronic potential barrier of the system. In a molecular junction, this barrier is nothing other than the energy difference between the closest (non-resonant) molecular level to the Fermi level of the electrode and the Fermi level [1,10]. Namely, if we consider electron transport, β will depend on the ELUMO–EFermi difference, whereas, if we consider hole transport, β will depend on the EFermi–EHOMO (ELUMO, EHOMO and EFermi being, respectively, the energy position of the LUMO, HOMO and Fermi level of the system). The immediate consequence of this interface property is that, in principle, beyond the nature of the molecular backbone, one could modulate the attenuation factor by changing the specific anchoring groups forming the bond between the molecule and the electrode. Indeed, the position of the molecular levels with respect to the Fermi level is mainly influenced by the anchoring groups used to connect the molecule to the electrodes. This aspect will be detailed later when considering properly the influence of the anchoring groups in well-established examples. However, this heuristic model is inspired by scanning tunneling microscope (STM) experiments, or the standard evolution of a wave function through a potential barrier in quantum mechanics, and represents the simplest expression of the Simmons model [28], currently used in molecular electronics to describe conductance attenuation in molecular systems. It works rather well for molecular junctions where the levels are close to the Fermi level but requires more ingredients when this is not the case.

Other more sophisticated models highlight the role of the molecular backbones without taking into account the interfaces between the molecule and the electrode. For example, from an atomistic point of view, the conductance of the molecular wire can be understood using a simple tight-binding model, with N sites of energy ε1, ε2 ... εN coupled through hoppings t1,2, t2,3, ... . tN-1,N and bridging the two electrodes [29] (see Figure 2).

**Figure 2.** Schematic representation of the bridge model with N sites of energy ε1, ε2 ... εN coupled through hoppings t1,2, t2,3, ... . tN-1,N and bridging the left and right electrodes. Reprinted figure from [1] and with permission. Copyright (2010) World Scientific.

Notice that these energy sites are independent of the coupling at the interfaces, which automatically eliminates the role of the anchoring groups. Moreover, we can consider that all these energies are identical, namely to ε0, and the same for the hoppings, which are all equal to t. In this approach, the attenuation factor can be expressed as:

$$
\beta(\mathbf{E}) = (2/a) \ln |(\mathbf{E} - \varepsilon\_0)/\mathbf{t}| \tag{2}
$$

where *a* measures the segment size, for a total molecular length of N*a* [1]. Considering the energy E as the Fermi level, and average values of |(E − ε0)/t| = 10 and a = 5 Å, this expression gives typical values of β of around 9 nm−1, independently of the anchoring groups or the electrodes. Note that a similar result can be deduced from the dispersion relation of an infinite linear chain:

$$\text{tr}\, 2\text{t}\, \cosh(\kappa a) = \text{E}\_{\text{Fermi}} - \varepsilon\_{0\prime} \tag{3}$$

with κ = β/2, (EFermi − ε0)/t >> 1, and where the hyperbolic cosine stands for the exponential behavior at each side of the molecular chain where the electronic wavefunction tunnels from or to the electrode. In other words, this approach is also a generalization of the Simmons model. However, here, the energy difference EFermi−ε0 corresponds to the potential barrier of an infinite molecular chain without anchoring groups or electrodes, namely ε0 being an orbital of the infinite molecular backbone and not, for example, the HOMO level of the junction.

Finally, the last important model lies in the determination of the attenuation factor through a complex bandstructure calculation [30,31]. Similarly to what happens in solid state physics, an infinite molecular chain (analog to an infinite crystal) is considered and its bandstructure is calculated. Real wavevectors correspond to the usual molecular spectrum of the junction.

The attenuation factor is determined from the calculation of evanescent states of the electronic wavefunction inside the molecule, namely the electronic states corresponding to complex wavevectors. These evanescent states, which correspond to the Schockley surface states in solid crystals [32], correspond to the different potential conduction channels of the molecular junction, originating from the different orbital hybridizations. Obviously, among all these different channels, the current will follow the channel with the lowest attenuation factor, similarly to the macroscopic behavior. Therefore, the value of the attenuation factor can be read on the complex bandstructure for the evanescent state with the smallest extension in the gap, as represented in Figure 3. This approach also leads to attenuation factors around 8 nm−1 for alkane chains. Notice that an interesting mathematical derivation establishes a link between this result and a square root variation for the attenuation factor similar to

the one of the Simmons model. However, this expression is valid again only for the infinite molecule and not for the one whose HOMO and LUMO levels are determined by the coupling at the interface.

**Figure 3.** Calculated complex bandstructure for an infinite alkane molecular chain. The left and right part of the graph correspond to the imaginary and real wavevectors, respectively. The attenuation factor can be read on the horizontal axis, in the imaginary wavevector part, considering the localized state with the smallest extension (semielliptical-like curve). Reprinted figure from [30] and with permission. Copyright (2002) by the American Physical Society.

To summarize the different theoretical approaches presented in this section, we can observe that each approach stresses a specific contribution. Hence, the model considers either the anchoring groups/electrodes (namely the coupling of the molecule at the interface) through the relative position of the molecular levels with respect to the Fermi level or the molecular backbone (namely the chemical nature of the molecular chain), in a rather exclusive manner. Consequently, it remains difficult to determine accurately and for all kinds of molecular junctions the corresponding attenuation factor. As such, a universal model does not seem to exist yet. Therefore, the debate remains open regarding whether the attenuation factor is determined by interface effects or only by the molecular backbone or by a combination of both. A large experimental consensus seems however to be established in favor of a major influence of the molecular backbone. As a remark, we can stress nevertheless that all of these theoretical studies (as already discussed in the previous section for most of the experimental studies) have only considered symmetric cases, where electrodes and anchoring groups at each side were similar. The study of a non-symmetric case through these models would be of high interest to test their range of applicability.

In the next section, we will illustrate these different models with standard test cases, namely alkane molecules sandwiched between metallic electrodes, and we will investigate the respective roles of the electrodes, the anchoring groups and the molecular backbone, by investigating asymmetries in the junctions.

### **4. A Test Case: Alkane Chains**

### *4.1. Role of the Anchoring Groups: Symmetric Junctions*

Alkane polymers probably constitute the most common and the simplest molecular chains that have been studied in molecular electronics. Indeed, from their simple chemical nature, their easy availability and ability to connect to different chemical groups or metals and despite their very large gap, which would in principle reduce the conductance, it represents a toy model for electrical conduction in molecular systems. Hence, it is no surprise that this system has been measured in numerous works using different metallic electrodes (mainly noble metals Au, Ag, Cu) and different anchoring groups. Here, we briefly present the well-documented results in the literature on symmetric alkane-based molecular junctions, before exploring further the underlying physical mechanisms through the study of hybrid junctions. For example, anchoring groups like thiol (-SH) [33], amine (-NH2) [34], carboxylic acid (-COOH) [35], isocyanide (-NC) [36], methyl sulfide (-SMe) [37], etc., have been studied extensively. What is particularly interesting is the comparison of the measured attenuation factors for several junctions with different anchoring groups. For example, Chen et al. [38] performed such a comparison between thiol, amine and carboxylic acid anchoring groups. The first important difference in the respective electronic properties of these junctions lies in the contact resistance, which is inversely related to the prefactor A in Equation (1). Indeed, the contact resistance is smaller for thiol, bigger for amine and even bigger for carboxylic acid, which yields overall conductance higher for thiol than for amine and then higher for amine than for carboxylic acid. This is due obviously to the different kinds of electronic coupling at the interface, namely of covalent nature for the thiol, much weaker for the amine and related to a deprotonation process of the carboxylic acid to contact the electrode. In this respect, geometries at the interfaces are also affected and the involved symmetries are different, as we will see in more detail later. In addition, the electronic properties seem to be rather different, since the HOMO level is located at around 2.0 eV from the Fermi level in the thiol case, 5.5 eV for the amine case and 1.1 eV in the carboxylic acid case. These differences in HOMO level positions are attributed to different symmetry couplings at the interfaces, as will be discussed in the next section. However, despite these important differences, the attenuation factors seem to remain rather similar for the three junctions, around 0.8–0.9 per C atom (or –CH2 unit). Considering such similarities in the attenuation factors for different anchoring groups, one can wonder what the real impact of the anchoring groups on the attenuation factor is. From these first results, the only incidence that we can deduce is a variation in the contact resistance and consequently a variation in the overall conductance. In the next section, we will consider the role of the electrode, using different anchoring groups, in the electronic transport in molecular junctions. The main idea is to compare the influence of the anchoring groups with the same or different electrodes.

### *4.2. Role of the Electrodes: Hybrid Junctions Using a Graphene Electrode*

Most of the conductance measurements in molecular electronics have used metallic electrodes and, in particular, noble metals, either using mechanically controlled break junctions (MCBJ) [39], scanning tunneling microscopy (STM) [40], conductive probe atomic force microscopy (CP-AFM) [41] or also the STM-based I(s) method developed by Nichols et al. [42]. Nevertheless, an important number of recent works have been devoted to the use of carbon electrodes or even graphene substrates [43,44]. In this respect, we will discuss here the attenuation factors measured experimentally and determined theoretically on hybrid molecular junctions with a gold electrode at one molecular end and a graphene electrode at the other. We will also consider the role of different anchoring groups using this graphene electrode.

First, we start by considering alkanedithiol molecules of different lengths, probed experimentally using the I(s) method and modeled using DFT and electronic transport calculations to interpret the obtained results [45]. An atomic model of the system is represented in the left part of Figure 4 for a butanedithiol sandwiched between a gold and a graphene electrode, forming a hybrid

metal/molecule/graphene junction. The conductance has been measured for different molecular lengths between 2 and 12 –CH2 groups, allowing us to deduce the length dependence of the conductance. In this respect, the use of a graphene electrode does not affect the exponential conductance decay. In parallel, electronic transmission spectra and conductance have been calculated after DFT structural optimization of the junctions and density of states calculations. In addition, the contact resistance has been determined to be significantly larger than what is obtained for a standard symmetric junction with two gold electrodes. This is due to the weak coupling (of van der Waals nature) at the graphene–molecule interface.

**Figure 4.** (**Left**) Schematic representation of the hybrid metal–molecule junction formed in the experiment for conductance measurements. (**Right**) Evolution of the conductance as a function of the molecular length: experimental measurements on asymmetric metal–graphene junction (black), experimental measurements on symmetric metal–metal junction (blue) and conductance calculation on asymmetric metal–graphene junction (red). Reprinted figure from [40] and with permission. Copyright (2016) American Chemical Society.

The length evolution of the experimental and theoretical conductance is represented on the right part of Figure 4.

As a result, the attenuation factor for this hybrid junction is approximately half of the one obtained for its symmetric metal–molecule–metal counterpart. Namely, the attenuation factor now lies at around 0.4 per carbon atom. Hence, even though the contact resistance is quite high for the hybrid junction due to the weakly coupled interface with graphene, for molecules longer than ~1 nm, the metal–molecule–graphene junction turns to be more conductive than the standard metal–molecule–metal one. Moreover, for similar molecular lengths, the hybrid junction is also longer than the standard one, due to the van der Waals contact which requires a 3 Å distance between the molecule and graphene. Therefore, the question arises: how can such unusual behavior occur? We will try to understand it in the frame of the Simmons model, the most common theoretical approach to determine an attenuation factor that we have discussed in Section 3. Hence, the electronic behavior can be explained as follows by the different couplings at the electrode–molecule interfaces and the different molecular level alignments. When a thiol-terminated alkane is adsorbed on a gold surface, due to the strong interface electrostatic dipole, there is an important charge transfer, which partially depopulates the molecule and moves the HOMO level toward the Fermi level of the surface. If the same molecule is contacted between two gold electrodes, there is an interface dipole at each molecular extremity, in opposite directions, leading to a cancellation of the two dipoles. Then, the HOMO level

remains far from the Fermi level, at around 2 eV, as discussed previously, yielding an important electronic barrier that is reflected in a high attenuation factor (β ~ 8.6 nm-1 or 0.8 per C atom [1,11]). Note that Brooke et al. have also studied the case of symmetric junctions, where it was argued that dipoles in symmetric junctions can result in the HOMO level being dragged down in energy [22]. If the second electrode is now a graphene plane, the interface dipole at the graphene side ruled by van der Waals interaction is much weaker than the one at the gold side, which is not cancelled in this situation. This results in an important charge transfer at the gold–molecule interface and a shift of the HOMO level toward the Fermi level. Consequently, the energy difference EFermi–EHOMO is reduced to 0.4 eV, which reduces considerably the attenuation factor, to 0.4 per C atom, as observed experimentally. In this case, as we can see, the simple Simmons model is perfectly able to describe the electronic behavior of the junction. Moreover, we have observed that the use of a graphene electrode at one extremity breaks the electrostatic symmetry of the system, leading to a reduced attenuation factor for thiol groups, as compared to the same junction and two gold electrodes.

Now, in order to extend the comparison to symmetric junctions, and to probe the theoretical model used for the thiol case, we will consider another hybrid junction with different anchoring groups, namely amine groups [46]. The same procedure has been applied here with I(s) measurements and DFT based electronic transport calculations. The corresponding results are presented in Figure 5. First, we consider the evolution of the conductance as a function of the molecular length, in a logarithmic scale. Similarly to the case of the thiol Au–graphene junction, an attenuation factor of 0.4 per C atom is also found in the amine case. This behavior is consistent with what happens with Au–Au junctions, as we have seen in the previous subsection. Indeed, for all the anchoring groups, the attenuation factor remains practically the same. Here, the introduction of a graphene electrode seems to result in a similar effect, but it is reduced to around half its value with respect to Au–Au junctions. What is more surprising here is the direct comparison of the conductance with the Au–Au junction and the same amine groups. Indeed, while, for the thiol case, we observed greater conductance above a certain molecular length due to the reduced attenuation factor, we can observe for the amine group a more important conductance independently of the molecular length is, as shown in Figure 5. Even more surprisingly, while, for the Au–Au junction, the thiol case presents higher conductance than the amine one (due to better contact and lower contact resistance), in the Au–graphene junction, the amine case presents higher conductance than the thiol one.

**Figure 5.** (**Left**) Evolution of the conductance as a function of the molecular length for molecular junctions with different anchoring groups and electrodes: conductance calculation on Au–graphene junctions with amine group (green), conductance measurements on Au–graphene junctions with amine group (black), conductance measurements on Au–graphene junctions with thiol group (red) and conductance measurements on Au–Au junctions with amine group (blue). (**Right**) Density functional

theory (DFT) calculated density of states (DOS) for Au–graphene and Au–Au molecular junctions with amine anchoring groups. Reprinted figure from [46] and with permission. Copyright (2017) American Chemical Society.

In order to understand this particular behavior, we have calculated the density of states (DOS) for the alkane-based junction with amine anchoring groups, using Au–Au or Au–graphene electrodes. The result is represented in the right part of Figure 5.

The calculated DOS allows us to determine the position of the HOMO level (which is the closest level to the Fermi level and therefore the conductive one in the junction, as for the thiol junction), so that we can apply the Simmons model used previously to determine the attenuation factor and the overall conductance. However, the calculated DOS indicates here a HOMO level located not around 0.4 eV below the Fermi level but 3.5 eV below the Fermi level for the amine anchoring group. As a consequence, the use of such a big potential barrier in the Simmons model would necessarily lead to a huge attenuation factor, which is not in agreement with the experimental observations. Therefore, this simple example highlights some limitations of the models presented in Section 3. Indeed, other models based on complex bandstructure calculations would not help either in determining correctly the attenuation factor, as electrode asymmetry cannot be taken into account. Note that the determination of the attenuation factor for the symmetric Au–Au junction is complicated as well, since, in that case, the energy barrier is about 5.5 eV, which does not fit into the Simmons model either. From this system, we can deduce that there is no universal model to calculate accurately the attenuation factors in molecular junctions. Hence, the Simmons model can be used only for specific ranges of electronic potential barriers, and the other models seem to be valid only for symmetric junctions, without properly considering the influence of the anchoring groups or the electrodes.

In [46], this problem has been solved by considering that, between the Au–Au and the Au–graphene amine junctions, the HOMO level has been relocated from the same amount as in the Au–Au and the Au–graphene thiol junctions, namely around 2 eV (as indicated by the green arrow on Figure 5). Since the attenuation factors are the same for both anchoring groups in Au–Au junctions, it can be deduced that they should be the same for the Au–graphene junctions. The underlying reason is the fact that the coupling to the electrode and the molecular conduction are different for thiol and amine anchoring groups. The thiol groups connect very well to the electrodes, as of π symmetry, which explains the small electronic barrier, whereas the amine groups connect poorly to the electrodes, as of σ symmetry. Conversely, the thiol groups connect poorly to the alkane molecular backbone, yielding low intramolecular electronic propagation, whereas the amine groups connect much better, which increases the intramolecular propagation. One effect compensating the other, both anchoring groups lead to the same attenuation factor, mainly depending on the electrodes. This balance between coupling to the molecular backbone, leading to a good intramolecular conductance, and coupling to the electrode, which reduces the interface potential energy barrier, also explains the similar attenuation factors for different anchoring groups in alkane-based symmetric junctions between gold electrodes.

One last point has to be clarified regarding the overall conductance of the amine junction, and the explanation is found again in the DOS calculation. Indeed, the HOMO level is different in the case of Au–graphene electrodes, due to a level splitting with respect to the Au–Au junction. Indeed, the introduction of the graphene electrode breaks the symmetry of the molecular junction (this effect was also observed for the thiol junction, where the symmetry breaking was seen in the non-compensation of the electric dipoles) and splits the original HOMO level, leading to a reduced molecular gap (by about 2 eV). Consequently, since the overall conductance depends on the self-energies that couple the molecule to the electrodes, and these self-energies vary as the inverse of the molecular gap, this important reduction of the gap leads to a much higher molecular conductance. This is why the Au–graphene alkanediamine junction presents more important conductance than its Au–Au counterpart but also than the alkanedithiol junction, where the introduction of the graphene electrode shifts the molecular levels without any gap reduction.

### *4.3. The Case of Platinum–Graphene Hybrid Junctions*

In the previous subsection, we have considered hybrid Au–graphene electrodes for two different anchoring groups in alkane-based molecular junctions and we have observed an important reduction in the attenuation factor. Here, we will observe the effect of a change in the metallic electrode by substituting the gold electrode with a platinum one. As a matter of comparison, we consider again the alkanedithiol molecular junction, following the same experimental and theoretical procedure [47]. Similarly to the Au–graphene alkanedithiol molecular junction, the conductance presents an exponential decay, still within the framework of the non-resonant tunneling. The corresponding attenuation factor is slightly lower, around 0.3 per C atom, which is attributed mainly to the difference in work function between gold and platinum. It has to be noticed that this system is again very well modeled through the Simmons approach, because of the strong coupling to the electrode and the reduced electronic potential barrier.

Moreover, we can deduce from this result, combined with the results obtained on Au–graphene junctions, that the reduction of the attenuation factor is caused by the symmetry breaking of the graphene introduction in the system. This is not related to the nature of metallic electrode, but rather to the difference of interaction strength at each electrode interface. Indeed, the metallic electrode is coupled covalently to the molecule through the anchoring group, while the graphene electrode is coupled in a much weaker manner through van der Waals interaction. This is particularly true in the case of the thiol anchoring group, which deprotonates at the gold interface, leading to a –S radical (thiolate) that is very reactive with the gold electrode, whereas it remains in the thiol form –SH at the graphene interface, leading to van der Waals contact. Consequently, this is the interaction symmetry breaking at each molecular end, covalent/van der Waals, which leads to the important decrease in the attenuation factor and the increase in the molecular conductance. In this respect, one can anticipate that a molecule weakly coupled to two graphene electrodes through van der Waals interaction would probably present a similar attenuation factor as the one obtained for symmetric Au–Au junctions. As a remark, a similar junction has been studied using a graphitic tip for the I(s) measurements and the usual graphene electrode. The resulting attenuation factor was found to be very similar to the one of the Au–graphene junction, around 0.4 per C atom. This was due again to a coupling difference, since the graphitic tip was very reactive in this situation and coupled covalently to the molecule, whereas the graphene counter electrode coupled weakly to the molecule [48].

### *4.4. Role of the Anchoring Groups: Asymmetric Junctions*

As we know now, symmetry effects are very important in electronic transport in molecular junctions. In particular, we have seen that it has a strong influence on the attenuation factor. For this reason, it is interesting also to see what would be the influence of considering different anchoring groups at each molecular side to connect the electrodes to the molecules. In this respect, we highlight here the interesting case of hybrid Au–S–alkane–COOH–graphene molecular junctions, where thiol and carboxylic acid groups have been used at each extremity of the junction with gold and graphene electrodes [49]. An atomic representation of the molecular junctions studied here is represented in Figure 6.

Obviously, from the theoretical considerations discussed above, there was no valid approach until now that was able to describe accurately such an asymmetric junction. Moreover, this type of junction has also been studied previously using Au–Au electrodes. As a reminder, the attenuation factor of junctions using only thiol or only carboxylic acid as anchoring groups are very similar, around 0.8–0.9 per C atom. Then, the corresponding attenuation factor for an asymmetric junction using different anchoring groups was found to be of the same magnitude, so without important change with respect to the symmetric junctions [25]. When considering junctions with Au–graphene electrodes, the situation is slightly different. Indeed, for a symmetric junction with carboxylic acid at both ends, the attenuation factor is around 0.7 per carbon atom, probably due to the low coupling of the carboxylic acid to the electrodes, which does not make a lot of difference in the situation of two gold electrodes. However, the situation is slightly different in the Au–graphene case, where the attenuation factor is around 0.4 per carbon atom, namely the same value as that found previously for thiol and amine groups in Au–graphene junctions. Interestingly, the electronic behavior is rather different. In thiol overall, but also in amine molecular junctions, the electronic transport is achieved through the HOMO level which is the closest level to the Fermi level. Here, as shown in Figure 7, the Fermi level is rather located near the middle of the HOMO and LUMO gap of the molecule, similarly to what is observed in the same junction with carboxylic acid at each molecular end. In terms of electronic transport, it means that there is no dominant molecular level (HOMO or LUMO) to the molecular conductance.

**Figure 6.** Atomic representation of the different hybrid Au–graphene alkane-based molecular junctions using thiolate and carboxylic acid anchoring groups at each extremity. Reprinted figure from [49] and with permission. Copyright (2019) Wiley-VCH Verlag GmbH & Co.

**Figure 7.** DFT calculated electronic transmissions for Au–graphene molecular junctions with carboxylic acid anchoring groups at each extremity (pink) and a thiol group at one extremity and a carboxylic acid at the other one (blue). Reprinted figure from [49] and with permission. Copyright (2019) Wiley-VCH Verlag GmbH & Co.

From this result, we can deduce that molecular junctions with asymmetries in the anchoring groups see their electronic transport driven by the anchoring group that couples the most, even though the electronic structure is also strongly affected by the less coupled anchoring group. In other words, the current flows along the most conductive channel, similarly to what happens at the macroscopic

scale, namely the channel which presents the best coupling at the molecule–electrode interface or the lowest contact resistance. Certainly, this constitutes only one particular study of asymmetric junctions in terms of anchoring groups and many other examples should be probed to extract trends that are more general. Nevertheless, it appears rather intuitive that the most conductive anchoring group will favor the most conductive channel and therefore the lowest attenuation factor.

### **5. Importance of the Molecular Backbone: Conjugated Molecular Wires**

In the previous section, we have focused the discussion on alkane molecular chains and we have considered the different parameters which can influence the electronic transport in the junction and consequently the attenuation factor. In particular, we have considered the anchoring groups, in symmetric or asymmetric junctions, and the electrodes, with different metals and with a graphitic tip. Without being exhaustive, it is important also to consider what happens when using another molecular backbone, like conjugated molecular wires. To this end, we chose to have a short look at polyphenylene chains, which are also very common and popular for molecular electronics. These polymers are very interesting, since, as presented in Figure 1 and [15], their attenuation factors are around three times smaller than for the alkane chains, making these molecular wires much more conductive. Hence, with what we have seen by using graphene electrodes, we can expect an even higher conductance for these polymers used in hybrid molecular junctions.

Therefore, hybrid Au–graphene and standard Au–Au molecular junctions have been studied through I(s) measurements and DFT calculations, with polyphenylenes, using thiol and amine anchoring groups, as a direct comparison with alkane-based molecular junctions [50].

Surprisingly, the electronic behavior is very different from what was expected. Indeed, either experimentally or theoretically, almost no difference has been found for the attenuation factors determined for Au–Au and Au–graphene junctions. Moreover, the different anchoring groups, thiol and amine, did not bring any significant difference. Again, this can be better understood from the DOS calculations represented in Figure 8. The projected DOS on the molecular part (including the anchoring group) reveals that there is almost no difference in the electronic structure of each molecular junction. This means that, in this case, the use of a graphene electrode does not break the symmetry, as was the case for the alkane chains. This is further illustrated in Figure 8 with the representation of the spatial extension of the HOMO level for both Au–Au and Au–graphene junctions. In both cases, we can observe a molecular orbital of π symmetry which propagates well along the molecular chain. However, we can also observe that the coupling to the electrodes remains the same in both junctions, despite the introduction of the graphene electrode. In other words, for this system, there is no symmetry breaking induced by the introduction of the graphene electrode. Consequently, the coupling to the electrode or the anchoring group used will have no effect on the electronic structure of the system and therefore on the electronic transport. Hence, the polyphenylene molecular chain solely drives the attenuation factor. From a theoretical point of view, in this case, the Simmons model would not help in discriminating the effect of the anchoring group, whereas a complex bandstructure calculation would probably give the correct attenuation factor, independently of the electrodes or the anchoring groups.

**Figure 8.** (**Left**) DFT calculated projected density of states for Au–graphene and Au–Au triphenyl molecular junctions using thiol groups. (**Right**) Representation of the corresponding HOMO wavefunction for both junctions. Reprinted figure from [50] and with permission. Copyright (2019) American Chemical Society.

### **6. Summary**

In this review, we have introduced the main electronic transport mechanisms in molecular junctions, to discuss mainly the non-resonant tunneling regime. In particular, we have considered as a characteristic of this regime the attenuation factor in the current exponential decay, observed experimentally and determined theoretically. In this respect, we have discussed the main theoretical approaches proposed to date to describe the attenuation factors. As we have seen, there is unfortunately no universal model to characterize the electronic transport in a molecular junction. Beyond performant DFT calculations, which allow us to calculate the electronic transmission and to deduce the attenuation factor, a full understanding of the electronic transport in a molecular junction remains complicated. Indeed, each present theoretical approach describes either the role of the anchoring group, by considering an accurate evaluation of the molecular levels position with respect to the Fermi level, or the role of the molecular backbone, without considering properly the coupling to the electrode or the interface states related to the anchoring groups. In addition, most of the studies consider symmetric junctions with the same electrodes and same anchoring groups at each molecular side.

Then, we have illustrated these aspects by shortly reviewing experimental and theoretical studies on alkane-based molecular junctions. Starting with standard junctions with gold electrodes at each end, we have also considered a hybrid junction where one gold electrode is substituted by a graphene electrode. In this case, and for different anchoring groups, we have obtained a much reduced attenuation factor. In addition, for some specific anchoring groups, it is even possible to increase the overall conductance of the molecular junction. By considering platinum instead of gold, yielding the same effect, we have deduced that the attenuation factor reduction can be attributed to symmetry breaking induced by the graphene electrode in the molecular junction. Indeed, at one side, the molecule is coupled covalently to the electrode, whereas it is weakly coupled (through van der Waals interactions) at the other side. Another interesting case to consider is a full asymmetric junction, with different anchoring groups and different electrodes at each side. As a result, the most conductive anchoring group, similarly to what happens at the macroscopic scale, mainly drives the conductance. Finally, hybrid junctions with conjugated molecular wires have been studied as well. A much lower attenuation factor could have been expected since these molecules are very conductive, unfortunately, due to bad coupling to the electrodes, the electronic transport properties remain unchanged, independently of the electrodes or the anchoring groups.

To conclude, despite the recent advances in the measurement techniques as well as in the theoretical and computational approaches, a full understanding of the electronic transport mechanism in molecular junctions has not been reached yet. In particular, the theoretical determination of the attenuation factor of the current remains a difficult task due to the lack of universal model to treat this problem. As we have seen, the Simmons model applies in very specific cases where the HOMO level is close to the Fermi level, as in the case of alkanedithiol in hybrid gold–graphene junctions. For other anchoring groups, the difference in symmetry couplings strongly modifies the energetic position of the molecular states, and the Simmons model does not apply anymore. Other theoretical approaches do not apply either since they consider infinite molecular chains and no effect of anchoring groups or electrodes. Therefore, the attenuation factors for these systems are theoretically deduced from this first case in comparison with the standard symmetric junctions, which are well documented in the literature. The attenuation factors of these hybrid junctions are all very similar, due to compensation effects between coupling and conduction in the molecular backbone and coupling to the electrodes. Moreover, we have also observed that the complex bandstructure approach may apply to aromatic molecular chains since the attenuation factors do not differ from the one determined in symmetric junctions. Consequently, a challenging task and an ideal perspective for theoretical approaches in determining attenuation factors in molecular junctions would be to consider, in the meantime, the intrinsic properties of the molecular backbone and the coupling to the electrodes through the anchoring groups that determines the positions of the molecular levels with respect to the Fermi level.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

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**7. HLA-matching in kidney allocation from deceased donors**

Most renal transplant programs preferentially allocate kidneys from deceased donors to transplant candidates with favourable HLA compatibility. The current allocation of deceaseddonor kidneys in most countries, including Australia and the Eurotransplant group (Germany, The Netherlands, Belgium, Luxembourg, Slovenia, and Austria), is weighted largely on the degree of mismatched antigens at the HLA-A, -B and -DR loci, with less emphasis on other factors such as time on dialysis, prior sensitization and even ischaemic time. When a potential deceased-donor kidney is available in Australia, transplant candidates on the wait-list are ranked according to an allocation score calculated from a combination of factors including the number of HLA-mismatches, age of recipient, degree of sensitization and time on wait-list [91]. Approximately 20% of deceased donor kidneys are allocated on a national level to highly sensitized transplant candidates (around 20% of kidneys allocated) but the remaining 80% of deceased donor kidneys are allocated through individual state allocation algorithms.

**Study Cohort Rejection Graft survival**

Vascular: 19% T-B- vs 32% T-B+ (p=0.01); DSA was a significant predictor for vascular or glomerular rejection

The presence of SAB HLA-DSA on the peak and current serum has a PPV for AMR of 35% and 32% respectively. Prevalence of AMR 1% in patients with MFI <465, 19% MFI between 466 and 3000, 36% MFI between 3001 and 6000, and 51% MFI "/>6000. Peak HLA-DSA Luminex MFI predicted AMR better than current HLA-DSA MFI.

The incidence of AMR among patients with preformed DSA was 35%, 9-fold higher than in patients without DSA (3%) (p <

Overall incidence of clinical/ subclinical rejection (i.e., AMR and acute T-cell mediated rejection) at day 200 posttransplant was significantly higher in patients with HLADSA (48/67; 71%) than in patients without HLA-DSA (94/267; 35%).

HLA – human leukocyte antigen, DD – deceased donor, LD – live-donor, CDC-XM – complement dependent cytotoxicity cross-match, DSA – donor-specific antibodies, SAB – single antigen bead, AMR – antibody mediated rejection, DCGS –

Most renal transplant programs preferentially allocate kidneys from deceased donors to transplant candidates with favourable HLA compatibility. The current allocation of deceased-

death-censored graft survival, MFI – mean fluorescent intensity, PPV – positive predictive value.

**7. HLA-matching in kidney allocation from deceased donors**

**Table 1.** Association between donor-specific antibodies and graft outcomes.

0.001).

Graft loss: T-B+ - 44%; T-B- 27%

5 and 8-year DCGS were 89% and 84% in non-sensitized patients, 92% and 92% in sensitized patients with no peak HLA-DSA, and 71% and 61% in patients with peak HLA-DSA. Relative risk (RR) for graft loss for patients who had an episode of AMR was 4.1 (95% CI 2.2 to 7.7) as compared with patients without

Overall graft survival at 8 years was 68% in patients with DSA and 77% in those with no DSA. Graft survival of patients with DSA and AMR was significantly worse than in DSA patients without AMR and in non-

DCGS at 5 years was 89% in those without DSA, 87% with DSA but no AMR and 68% with DSA and AMR.

(especially class I DSA)

AMR.

DSA patients.

83 T-B+ XM vs 386 T-B-XM; IgG HLA DSA in 33% of T-B+ XM patients

358 Current Issues and Future Direction in Kidney Transplantation

83 (21%) positive DSA by Luminex by peak sera vs 76 (19%) by current sera

All negative T and B-cell CDC-XM. 27% class I or II anti-HLA antibody with

332 negative T and B cell CDC-XM, 67 DSA vs 267 no DSA by Luminex

52% DSA.

*Eng H et al* (n=471 DD renal transplant recipients) [*79*]

*Lefaucheur C et al* (n=402 DD renal transplant recipients)

*Lefaucheur C et al* (n=237 LD and DD renal transplant recipients) [*81*]

*Amico P et al (n=334 LD and DD renal transplant recipients)*

*[105]*

[*80*]

Despite efforts to achieve equity of access to transplantation in many countries, the inclusion of HLA matching in the allocation of deceased donor kidneys is believed to disadvantage transplant candidates with uncommon HLA phenotypes [92]. Consequently, indigenous populations and ethnic minorities often have a much longer transplant wait-list time and are less likely to receive well-matched kidneys [97-100]. The elimination of the allocation priority for HLA-B mismatches has been shown to improve the transplant potential of ethnic minorities but this approach has not been widely adopted by other countries [61].

In Australia, unacceptable class I HLA-mismatches are defined using the Luminex platform and the presence of class I DSA against HLA-A and -B antigens with >2000 mean fluorescent intensity (MFI) excludes transplant candidates from receiving these donor kidneys, independ‐ ent of the CDC-cross match results. At present, class II DSA is not explicitly considered in the allocation of kidneys from deceased donors in Australia but many centres have already adopted the policy of avoiding transplantation of kidneys into transplant candidates with high levels of class II DSA.