Skin toxicity is one of paclitaxel’s adverse effects. However, its real impact on the skin could be underestimated as these alterations can also appear asymptomatic. We have observed that paclitaxel modifies gene and protein expression of skin markers in a 3D epidermis model, and impairs physical, physiological, and biomechanical properties of the skin in gynecologic cancer patients. These subclinical alterations might be avoided by using prophylactic measures during treatment to prevent possible future adverse reactions.Background: Paclitaxel is a microtubule-stabilizing chemotherapeutic agent. Despite its widespread use, it damages healthy tissues such as skin. The goal of this study was to prove that the real impact of paclitaxel-induced skin toxicity could be underestimated because the adverse events might appear asymptomatic. Methods: Gynecological cancer patients were recruited. Skin parameters measurements were taken after three and six paclitaxel cycles. Measurements were conducted using specific probes which measure hydration, transepidermal water loss (TEWL), sebum, elasticity and firmness, erythema, roughness, smoothness, skin thickness, and desquamation levels. Further, a 3D epidermis model was incubated with paclitaxel to analyze gene and protein expression of aquaporin 3, collagen type 1, elastin, and fibronectin. Results: Paclitaxel induced alterations in the skin parameters with no visible clinical manifestations. Gynecological cancer patients under paclitaxel treatment had a decrease in hydration, TEWL, sebum, elasticity, and thickness of the skin, while erythema, roughness, and desquamation were increased. The molecular markers, related to hydration and the support of the skin layers, and analyzed in the 3D epidermis model, were decreased. Conclusions: Results suggest that paclitaxel modifies gene and protein expression of skin-related molecular markers, and impairs different physical, physiological, and biomechanical properties of the skin of cancer patients at a subclinical level. Taxanes are chemotherapeutic agents that produce antitumor activity by causing stabilization of microtubules, thereby inhibiting cell cycle progression . PaclitaIn 1992, paclitaxel was approved by the US Food and Drug Administration (FDA) for the treatment of ovarian cancer. In 1996, a study of the Gynecologic Oncology Group (GOG) showed that paclitaxel-cisplatin was superior in terms of survival to the cyclophosphamide-cisplatin regimen as upfront therapy in stage III–IV ovarian cancer patients . These rAlthough taxanes are tolerable and manageable, their toxic profile includes a wide number of adverse events. Hematological, cardiologic, and neurologic toxicities are very common in taxane-containing regimens . NeutropThere are scarce data regarding the mechanisms that lead to these toxic effects and most remain not understood. Moreover, the real impact of the taxane-induced skin toxicity could be underestimated as the skin adverse events are usually under-reported or paucisymptomatic . The avaOf note, studies on undifferentiated keratinocyte monolayer cultures can lack some of the physiological functions of the stratified keratinocyte epithelium and could misinterpret the results obtained in preclinical studies. Thereby, various three-dimensional (3D) skin equivalents reproducing in vivo conditions have been developed for pharmacologic and toxicologic in vitro testing as an alternative to animal models ,23. One In this study, we aimed to overcome the lack of investigation regarding subclinical alterations induced by PTX. Therefore, we analyzed PTX-induced subclinical skin alterations by measuring different biomechanical properties of the skin in oncologic patients. Secondly, we reconstructed a 3D epidermis cell model to mimic a healthy epidermis and evaluate the effects of paclitaxel in some of the molecular markers associated with skin homeostasis. The results obtained will help understand asymptomatic skin alterations to prevent possible future skin adverse effects.This project was approved by the Research Ethics Committee of Valencia University Clinical Hospital and further authorized by the Valencian Regional Ministry of Health. Informed consent was obtained from each participant before starting the study. Twenty cancer patients and 20 healthy volunteers were recruited from the oncology service at Valencia University Clinical Hospital. The patients’ clinical features can be found in Skin parameter measurements were performed in three visits: before treatment (T1), during treatment after 3 chemotherapy cycles (T2), and at the end of treatment after 6 chemotherapy cycles (T3). The measurements were taken using specific probes following the measurement guidelines ,29,30. A®  was used to obtain cheekbone and forearm hydration values. This probe measures the electrical capacity of the stratum corneum, based on the linear dependency of the electrical property of the epidermis to its hydration. The results are displayed in arbitrary units [Corneometer CM 825ry units .®  was used to provide cheekbone and forearm values of transepidermal water loss (TEWL). The probe measures the vapor pressure and calculates TEWL from the difference between two measurement points using Fick’s law of diffusion. It displays the results in grams per hour per square meter (g/hm2) [Tewameter TM 300 (g/hm2) .®  was used to obtain the forehead sebum value. The probe determines the translucency of a special tape, which becomes transparent after contact with sebum on the skin surface and displays the sebum values in µg per square centimeter (µg/cm2) [Sebumeter SM 815(µg/cm2) .®  was used to determine the forearm erythema based on tissue’s narrow wavelength light absorption. Results are displayed as erythema index in arbitrary units [Mexameter MX 18ry units .® MPA 580. This probe suctions skin and gives a series of R values. The ones that provide more information about elasticity and firmness are R0, R2, R5, and R7. R0 expresses the maximum width of skin and is given in mm. R2 represents the ratio between the maximum width of the skin and its ability to return to its original state after suction (Ua/Uf). R5 represents the ratio between the elasticity of the suction phase and the elasticity of the relaxation phase (Ur/Ue). R7 represents the elastic recovery ratio (Ur/Uf). The closer R2, R5, and R7 are to 1, the greater the elasticity.Skin elasticity and firmness were assessed in the cheekbone with Cutometer® . The device takes grayscale photographs to study the epidermis surface and analyzes the obtained images, where white and bright gray are associated with a bad condition of the skin. The software uses the images to determine the skin topography parameters SELS  [Skin smoothness and roughness were assayed in the cheekbone by Visioscanng skin) . The givng skin) .® was also combined with the Corneofix®  technique to obtain the skin desquamation index on the forehead. Corneofix® is a tape that adheres to the skin and collects corneocytes. Then, the tape is placed on the Visioscan® probe which takes an image and processes it with a determined color grading, depending on the desquamation level. The color scale ranges from cool to warm colors. The software also analyzes the number, size, and thickness of the attached corneocytes to the Corneofix® tape and gives the desquamation percentage.Visioscan® UC22 was used to analyze the thickness of the different layers of the skin on the forearm. The probe takes an ultrasound image of the skin.Finally, UltrascanAll measurements were taken under controlled conditions. Temperature was maintained at 22 ± 2 °C and relative humidity between 40% and 60%. Measurements were taken after patients remained in a 30 min acclimatization period in the same atmospheric conditions.6 BALB/3T3 fibroblasts  were seeded on collagen-coated Millicell inserts  and placed into 6-well plates . Fibroblasts were cultured for 2 days in 1 mL Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal calf serum  and added to the apical and dorsal side of the insert. When fibroblasts reached 60–70% confluence, the monolayer was irradiated with UV light at 0.048 mW for 1 h with UVACUBE 400  to establish the feeder layer. Then, primary adult epidermal keratinocytes  were seeded at a density of 0.5 × 106 cells/cm2. Cultures were grown at 37 °C and 95% air/5% CO2 until approximately 60% confluency and then were switched to Keratinocyte Growth Medium  supplemented with KGM-Gold SingleQuot Kit (Lonza) until confluent. Confluent cultures were raised to the air–liquid interface and cultured for 21 days until epidermal stratification was achieved. To validate the stratification, histological analysis was performed after 21 days. The reconstructed epidermis tissues were fixed with 10% formalin solution, dehydrated, and embedded in paraffin. Six-micrometer-thick sections were cut and stained with hematoxylin–eosin. Random photographs were taken of each sample with a Leica DM6000B microscope .Three-dimensional epidermis cell models were reconstructed using the BALB/3T3 feeder-layer technique adapted from Mak et al.  and Arne® Reagent  following the manufacturer’s instructions. Reverse transcription was performed in 500 ng of total RNA with a TaqMan reverse transcription reagents kit . cDNA was amplified with specific primers and probes predesigned by Applied Biosystems for aquaporin 3 (AQP3) (Hs00185020_m1), collagen type 1 (COL1) (Hs00164004_m1), elastin (ELN) (Hs00355783_m1), and fibronectin (FN1) (Hs01549976_m1) in a QuantStudio™ 5 Real-Time PCR System, using universal master mix . Expression of the target gene was expressed as the fold increase or decrease relative to the expression of β-actin (Hs01060665_g1) as an endogenous control. The mean value of the replicates for each sample was calculated and expressed as the cycle threshold (Ct). Gene expression level was calculated as the difference (ΔCt) between the Ct value of the target gene and the Ct value of β-actin. The fold changes in the target gene mRNA levels were designated 2−ΔCt.The 3D epidermal cell models were incubated for 24 h with PTX within the clinically achievable plasma concentrations of 0.3, 3, and 30 µM ,37,38. A® polyacrylamide gels TGX™  by application of 100 V during 1 h. Proteins were transferred from the gel to a nitrocellulose membrane Trans-Blot® Turbo™ Transfer Pack, using the Trans-Blot® Turbo™ Transfer System . Then, membranes were incubated with 5% bovine serum albumin (BSA) for 2 h and labeled overnight at 4 °C, with various primary antibodies. The secondary antibody was incubated for 1 h at room temperature. The primary antibodies used were the following: AQP3 ; COL1A ; FN1 ; and ELN . To normalize results, the β-actin antibody  was used as housekeeping control. Signal visualization of proteins was carried out by incubating the membranes with chemiluminescence reagents . Densitometry of films was performed using Image J 1.42q software. Results of target protein expression are expressed as the percentage of the densitometry of the endogenous controls β-actin.The 3D epidermal cell models were incubated for 24 h with different PTX concentrations . After incubation, protein extraction was performed incubating samples with lysis buffer  supplemented with a protease inhibitory cocktail complete™ and phenyl-methyl-sulfonyl fluoride (PMSF) . Total protein concentration was quantified using the BCA Protein Assay Kit . Protein electrophoresis was performed to separate proteins according to their molecular weight. Twelve micrograms of denatured proteins along with Rainbow ™ molecular weight marker  were loaded into Mini-PROTEANn experiments; p < 0.05 was considered statistically significant. Normal distribution for each data set was confirmed by the Kolmogorov–Smirnov test. Statistical analysis was carried out by multiple comparisons with analysis of variance (ANOVA) followed by Bonferroni post hoc test. Results from the human in vivo experiments were expressed as the mean ± standard error (SE) of n experiments; p < 0.05 was considered statistically significant. When the comparisons concerned only 2 groups , statistical analysis was carried out by unpaired t-test. Multiple comparisons were analyzed by ANOVA followed by the Bonferroni post hoc test.Results from cellular in vitro experiments were expressed as the mean ± standard error (SE) of The stratification of the 3D epidermis cell model was confirmed by the hematoxylin–eosin staining. As shown in ® on the cheekbone and forearm of the oncologic patients and to the control group (® (The effects of PTX treatment in the hydration molecular marker aquaporin (AQP3) in the 3D skin model were examined. Incubation with PTX for 24 h induced a dose-dependent decrease in gene expression that was statistically significant B. Furtheol group D. No stagroup  and roughness (Ser) parameters were also evaluated on the skin of oncologic patients with the Visioscan the Ser A and Ses the Ser B paramet the Ser A,B, whic® tape sheets with the Visioscan® equipment. Visioscan® software displays a color-graded image of the Corneofix® tape with the attached corneocytes. As shown in the representative images in The effect of PTX on skin desquamation was evaluated by combining the Corneofix® UC22 was used to analyze the thickness of the different layers of the skin in oncologic patients. Epidermis, dermis, and the total thickness of the skin were similar in the healthy group and patients before treatment. After three and six PTX cycles, all layers of skin showed a decrease in thickness, which can be noted in the representative images in The ultrasound technique of UltrascanPaclitaxel is an antineoplastic drug widely used in cancer treatment that has been shown to produce a multitude of skin adverse effects . HoweverIn this study, oncologic patients did not report visible cutaneous symptoms. The values of the skin properties in oncologic patients before treatment (T1) were compared to the values of a healthy control group. In all cases, the parameters were similar and no statistically significant differences were encountered between both groups. This shows that before treatment with PTX, all patients had skin parameters within the normal biological values, representative of the general population. Additionally, data obtained from healthy volunteers and cancer patients in their first visit were within the reference values, as described by the literature ,30,40,41Firstly, we analyzed the hydration levels and TEWL. In the epidermis cell model, treatment with PTX reduced the gene and protein expression of the marker Aquaporin (AQP3). AQP3 is the most abundant skin aquaglyceroporin and is responsible for transporting water and glycerol in the epidermis. Therefore, AQP3 is a key protein in the maintenance of epidermal hydration and differentiation of keratinocytes ,43,44. OThe epidermis cell model was also incubated with PTX to analyze collagen 1 (COL1), elastin (ELN), and fibronectin (FN1) expression. These proteins play a key role in maintaining the elasticity, firmness, and support of the skin layers ,51,52. G® showed that the erythema index had a slight increase after six PTX cycles. It is widely described that treatment with taxanes induces erythema at different areas of skin and in different grades of severity [Erythema is a common adverse effect seen on patients under PTX treatment ,59. In tseverity ,60,61. HAs it was evident that PTX induced changes in the biomechanical properties of skin, roughness and softness were also examined. PTX induced an increase in skin roughness and a decrease in smoothness. Connected to these events, patients showed an increased desquamation percentage. This implies a decrease in the barrier function capacity of the skin after PTX treatment. Similar to the other parameters analyzed, the research data available comes from case reports of patients suffering from PTX-induced toxicities with clinical manifestations. In this case, desquamation had been previously described in patients under PTX treatment, in the palms and soles, necessitating treatment interruption  and assoThere are a few study limitations to be mentioned. Firstly, the heterogeneity of the sample—there are patients with localized disease and patients in different situations of advanced disease. Therefore, the chemotherapy administration schemes differ between patients. However, the heterogeneity of the sample should not have a significant impact since it includes only gynecological tumors with similar treatments. Secondly, considering the treatments used, the drug with the greatest impact at the cutaneous level was PTX. We acknowledge that carboplatin causes skin adverse effects but they are usually associated with hypersensitivity reactions . As the ®) had a protective role against skin reactions [The results of this study show that, although PTX did not cause severe skin adverse reactions, it impaired its physical, physiological, and biomechanical properties with no clinical manifestations. Treatment with PTX induced skin dehydration, a decrease in elasticity, thickness, and sebum levels, and an increase in skin desquamation and erythema. These changes were related to the modulation of gene and protein expression induced by PTX in an epidermis cell model that mimics a healthy epidermis. These results indicate that PTX can alter the skin structure, and impair its barrier function, inducing cutaneous changes that do not become symptomatic. Previous studies have described PTX-induced epithelial damage in zebrafish models ,20 and seactions . HoweverIn conclusion, the results provided by this work suggest the need for prophylactic measures that improve the patient’s quality of life as well to ensure adherence to treatment. Although more studies addressing this matter are needed, early introduction of effective countermeasures including daily skincare  would help in the prevention of future PTX-induced skin toxicities.This work has shown that paclitaxel impairs different physical, physiological, and biomechanical properties of the skin. To our knowledge, this is the first study that has concluded that gynecological cancer patients under paclitaxel treatment show subclinical skin alterations. These subclinical alterations include the decrease of hydration, TEWL, sebum, elasticity, and thickness of the skin, together with an increase in erythema, roughness, and desquamation. Further, this study showed the PTX-induced modulation of molecular markers related to hydration and the support of the skin layers in a 3D epidermis model. These altogether highlight the lack of management measures to prevent skin alterations in patients under taxane treatment. The use of prophylactic measures at the early stages of treatment could be useful to avoid these subclinical alterations and prevent future severe reactions.


Patients with obstructive sleep apnea (OSA) are at a higher risk for atrial fibrillation (AF). Consumer wearable heart rate (HR) sensors may be a means for passive HR monitoring in patients with AF.The aim of this study was to assess the Apple Watch’s agreement with telemetry in measuring HR in patients with OSA in AF.Patients with OSA in AF were prospectively recruited prior to cardioversion/ablation procedures. HR was sampled every 10 seconds for 60 seconds using telemetry and an Apple Watch concomitantly. Agreement of Apple Watch with telemetry, which is the current gold-standard device for measuring HR, was assessed using mixed effects limits agreement and Lin’s concordance correlation coefficient.A total of 20 patients  participated in this study, yielding 134 HR observations per device. Modified Bland–Altman plot revealed that the variability of the paired difference of the Apple Watch compared with telemetry increased as the magnitude of HR measurements increased. The Apple Watch produced regression-based 95% limits of agreement of 27.8 – 0.3 × average HR – 15.0 to 27.8 – 0.3 × average HR + 15.0 beats per minute (bpm) with a mean bias of 27.8 – 0.33 × average HR bpm. Lin’s concordance correlation coefficient was 0.88 (95% CI 0.85-0.91), suggesting acceptable agreement between the Apple Watch and telemetry.In patients with OSA in AF, the Apple Watch provided acceptable agreement with HR measurements by telemetry. Further studies with larger sample populations and wider range of HR are needed to confirm these findings. Atrial fibrillation (AF) is the most common clinically significant cardiac arrhythmia, with a lifetime risk of 1 in 4 among individuals over the age of 40 and about 1 in 3 among individuals over the age of 55, thereby posing substantial public health and economic burden . AF is aOf particularly high risk for developing AF are individuals with sleep breathing disorders, including obstructive sleep apnea (OSA). A strong association between OSA and AF has been consistently observed in both epidemiological and clinical cohorts, with patients with OSA being 2 to 4 times more likely to develop AF compared to those without OSA -8. Gami Recently, the growing prevalence and adoption of digital health tools, including mobile devices with physiologic sensors , have caught the attention of industry giants in the technology sector and clinicians who see opportunities for synergy in subclinical AF detection. This is evidenced by the rapid development and release of wearables for AF detection, including the Apple Watch Series 4 (Apple Inc.), KardiaBand and KardiaMobile (AliveCor), Hexoskin (Carré Technologies Inc.), and QardioCore (Qardio Inc.) . Of thesMany wearables monitor heart rate (HR) through an optic technology known as photoplethysmography (PPG), in which sensors detect and measure pulsatile light absorption in the vasculature beneath the skin as a proxy for the cardiac cycle . While tIn this pilot study, we assessed the Apple Watch’s agreement with telemetry as the gold standard in measuring HR in patients with OSA in AF. We chose to recruit patients with OSA given their higher likelihood of having a co-diagnosis of AF  and becaThis study was approved by the Johns Hopkins Medicine Institutional Review Board. Apple Inc. was not involved in the design, implementation, data analysis, or manuscript preparation of the study.In this prospective pilot study, patients aged 18 and older with OSA in AF episodes confirmed on ECG were identified via electronic health record screening and prospectively recruited prior to cardioversion and AF ablation procedures at Johns Hopkins Hospital between November 2018 and May 2019. Diagnosis of OSA was determined by chart review, and patients with objective clinical documentation of (1) current continuous positive airway pressure (CPAP) device use, (2) polysomnogram results showing OSA, or (3) both were considered eligible. Patients were excluded if they had implantable pacemakers, defibrillators, loop recorders, heart block, or tachycardia not attributable to AF. In addition, patients who were hemodynamically unstable or under contact precautions for infection control were excluded.Eligible patients were approached prior to their procedures and provided informed written consent. AF was confirmed by a 12-lead ECG performed minutes prior to HR data collection. Participants wore a first-generation Apple Watch (model A1554), which was provided by the study team for the duration of data collection. The same device was used for all participants and was cleaned between use with a hospital-grade disinfectant. The Apple Watch face and telemetry monitor  were observed concomitantly under video recording in the presence of a study co-investigator (RS) for 90 seconds. After excluding the first 30 seconds of data to allow time for the watch’s HR monitor to equilibrate, HR measurements were sampled every 10 seconds for 60 seconds, yielding a total of 7 observations per participant per device (Apple Watch and telemetry). In addition, we documented the following relevant clinical data: cardiac history, cardiovascular medications, OSA treatment, nature of AF diagnosis, and demographic characteristics using the electronic health record. Full study flow can be found in Descriptive statistics were performed for the baseline characteristics, using frequencies (percentages) to describe categorical variables and mean (SD) or median (interquartile range) to describe continuous variables. Using the telemetry-determined HR as the gold standard, the Apple Watch was assessed for accuracy by calculating the paired difference between the measures. We first checked the mean constant bias assumption by visualizing the modified Bland–Altman plot accounting for repeated measures per patient . The meaijkDiff represents the jth paired difference in HR between devices in patient i given k value of the true (average) measurement; α is the constant intercept; ir is the random effect of the ith patient; kβ is the fixed effect of average of 2 measurements: and ije is the error for paired difference j on patient i.17 The regression of ijkDiff on the fixed effect of average of measurements gave the following:where P<.05) and further confirmed the average difference was related to the magnitude. We thus calculated the regression-based 95% limits of agreement as 27.7922 – 0.3332A – 1.96 × SD  and 27.7922 – 0.3332A + 1.96 × SD . An estimate of SD (7.6407) was calculated by the square root of total variance for all observations including the estimated between-patient variance and within-patient variance. Data were analyzed using the nlme package of R software version 3.6.1 (R Foundation).The coefficient of –0.3332 was statistically significant . The first participant had 4 out of 14 recordings because the protocol was subsequently changed to capture a greater number of time points over 60 seconds of monitoring. A subsequent participant had 12 out of 14 recordings due to a failure to capture the entire 60 seconds of continuous monitoring on video. HR recordings ranged from 49 to 146 bpm from telemetry and 55 to 127 bpm from the Apple Watch.This study presents a pilot effort to assess the level of agreement in HR measurements between PPG technology using the Apple Watch (1st generation) and telemetry during episodes of AF. We demonstrate that with a Lin’s concordance correlation coefficient of 0.88, the Apple Watch provided acceptable agreement with HR measurements by telemetry even during these episodes. The mean bias between the Apple Watch and telemetry measurements was 0.26 bpm, with 95% of Apple Watch HR measurements falling within 19 bpm of the telemetry measurements.While the Lin’s concordance correlation coefficient is deemed accepted by the literature , we noteOur study is not without limitations. Despite screening 201 patients over a span of 6 months, only 35 patients were eligible, due to the criteria of having objective documentation of OSA. Furthermore, as this was a pilot study and to maximize yield of HR measurements while in AF, we aimed to enroll only 20 patients, yielding 134 HR measurements for each device (268 between the Apple Watch and telemetry) for analysis. Moreover, our small sample population was skewed toward white/Caucasian males. Because enrollment occurred in the preprocedure setting among patients who have established care with an electrophysiologist, the majority of participants demonstrated good rate control, and only 15% (n=3) were in RVR. This makes it difficult to assess the accuracy of PPG technology in measuring elevated HR and detecting periods of RVR, although our data support prior work suggesting that smart watches underestimate HR in these higher ranges . AdditioBecause of its clinically silent nature, AF is difficult to detect, and guideline-directed management involves anticoagulation, rate control, and rhythm control . A user-Although several studies have evaluated the validity of smart watch algorithms to detect AF in healthy adults without cardiovascular disease ,6,23, whMoreover, by providing a larger cohort of data collected over a period in an ambulatory environment rather than within the restrictions of a clinic or hospital setting, smart watches have the potential to empower patients in their conversations with their health care providers regarding the efficacy of their AF therapies, including antiarrhythmic and rate control medications. This has been demonstrated in our clinical practice, where we have had patients with OSA self-identify an AF episode with RVR by a fast HR on their Apple Watch . Our stuThese patient–clinician conversations, informed by patient-generated data, could in turn promote adherence to guideline-directed management . CurrentIn this study, we demonstrated that during AF episodes, HR readings from a commercially available smart watch (first-generation Apple Watch) are in acceptable agreement with HR measurements by telemetry, using patients with OSA as a proxy for a high-risk population. Further studies with larger sample populations and a wider range of HR are needed to confirm these findings. As ownership of smart devices and wearables continues to grow, our work demonstrates that these devices hold promise as tools to monitor efficacy of rate control therapies for patients with AF.


To compare the dynamic corneal response (DCR) and tomographic parameters of thin normal cornea (TNC) with thinnest corneal thickness (TCT) (≤ 500 µm), forme fruste keratoconus (FFKC) and mild keratoconus (MKC) had their central corneal thickness (CCT) matched by Scheimpflug imaging (Pentacam) and corneal visualization Scheimpflug technology (Corvis ST).CCT were matched in 50 eyes with FFKC, 50 eyes with MKC, and 53 TNC eyes with TCT ≤ 500 µm. The differences in DCR and tomographic parameters among the three groups were compared. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic significance of these parameters. Back propagation (BP) neural network was used to establish the keratoconus diagnosis model.P > 0.05). The index of height asymmetry (IHA) and height decentration (IHD) differed significantly among the three groups . IHD also had sufficient strength (area under the ROC curves (AUC) > 0.80) to differentiate FFKC and MKC from TNC eyes. Partial DCR parameters showed significant differences between the MKC and TNC groups, and the deflection amplitude of the first applanation (A1DA) showed a good potential to differentiate (AUC > 0.70) FFKC and MKC from TNC eyes. Diagnosis model by BP neural network showed an accurate diagnostic efficiency of about 91%.Fifty CCT-matched FFKC eyes, 50 MKC eyes and 50 TNC eyes were included. The age and biomechanically corrected intraocular pressure (bIOP) did not differ significantly among the three groups (all The majority of the tomographic and DCR parameters differed among the three groups. The IHD and partial DCR parameters assessed by Corvis ST distinguished FFKC and MKC from TNC when controlled for CCT. Keratoconus (KC) is a corneal ectatic disease, which results in progressive thinning and protrusion of the cornea into a conical shape . The strCorneal visualization Scheimpflug technology (Corvis ST) applies constant airflow to the corneal central area using a high-speed Scheimpflug camera to obtain corneal biomechanical response. Corvis ST is widely used in the diagnosis of KC  and glauIn this study, we aimed to explore the changes in the dynamic corneal response (DCR) and corneal tomographic parameters in a population of eyes with CCT-matched thin normal cornea (TNC) controls, forme fruste keratoconus (FFKC), and mild keratoconus (MKC) with TCT ≤ 500 µm. This will provide a basis for further understanding of the test results produced by Corvis ST, which would aid in distinguishing KC from TNC when the cornea is thin.This retrospective study included 153 eyes of 153 individuals  range: 440–500 µm ), who weAll patients diagnosed with KC in the cornea clinic of the Beijing Tongren Hospital, from January 2013 to December 2019, were eligible for inclusion in this study. Clinical KC was diagnosed if the eye met the following criteria , 17: (1)The institutional review board of the Beijing Tongren Hospital, Beijing, China approved this study, and all participants signed an informed consent form in accordance with the tenets of the Declaration of Helsinki.A comprehensive ocular examination was performed on the eyes of all subjects, including a detailed assessment of uncorrected distance visual acuity, slit-lamp microscopy, fundus examination, tomographic measurements using Pentacam , and biomechanical examination using the Corvis ST . All measurements were taken between 09:00 and 17:00 on the same day and by the same trained ophthalmologists. The Pentacam (software version 1.20r134) reconstructed a three-dimensional image of the entire anterior segment of the eye from the anterior surface of the cornea to the posterior surface of the lens utilizing the high-speed rotating Scheimpflug system. The Corvis ST (software version 1.5r1902) evaluated the dynamic corneal deformation in response to an air-puff pulse. The details and principles of the Pentacam and Corvis ST are described elsewhere , 24. OnlThe Corvis ST output parameters mainly included—First applanation: time from starting until the first applanation A1T), velocity of the corneal apex during the first applanation (A1V), corneal deflection amplitude during the first applanation (A1DA), length at the first applanation (A1L) , MedCalc software  and R . The CCT was matched between the three groups using propensity score matching. Data were evaluated for normality using Shapiro-Wilk test, and when the data was normally distributed, mean ± SD was used to describe data; whilst the data had a non-normal distribution, the median (range of variation) was used to describe the data. The differences between data were evaluated using one-way ANOVA (multiple comparisons between groups were performed using Bonferroni test) or Kruskal-Wallis test (multiple comparisons between groups were performed using Mann-Whitney U test) was used for non-normal data, and gender differences among the three groups were analyzed using the chi-square test. Power of the tests was calculated using the A1DA data among the three groups. A receiver operating characteristic (ROC) curve analysis was constructed to identify the overall predictive accuracy of parameters and analyze the sensitivity and specificity of these parameters. A Based on results of comparison among the three groups, we combined DCR and corneal tomographic parameters to establish the keratoconus diagnosis model with back propagation (BP) neural network . Randomly selected data (70%) was used as the training set, and the rest of the data was used as the verification set. After testing, the three-layer neural network was selected, the number of neurons in each layer was 5, 3 and 1, trainlm was selected as the activation function. Trainlm is a network training function which is suitable for medium-sized networks and have the fastest convergence speed, the learning rate was set to 0.01, target error was set to 0.005, and the maximum number of iterations was set to 1000 times.P > 0.05); and chi-square test showed that there were no statistically significant differences detected for gender, among the three groups (P > 0.05). Furthermore, power of the tests calculated by using the data of A1DA was about 0.99.Table P < 0.001). Except for keratometry from the anterior corneal surface (KmF) and central astigmatism from the anterior corneal surface (Astig F), other tomographic parameters differed significantly between the TNC and MKC groups, FFKC and MKC groups; IHA and IHD were the smallest in TNC groups and the largest in MKC groups , and TNC and MKC groups , respectively. Also, a significant difference was detected for A1DA, A1L, HCDA, HCR, ARTh, and CBI in the FFKC and MKC groups .The DCR parameters obtained by Corvis ST are shown in Table Table The AUC values of some parameters  in the FFKC and TNC groups were between 0.634 and 0.766. The AUC values of parameters with statistical differences between MKC and TNC groups were 0.653 to 0.880, and the AUC of A1DA was > 0.80. Kmax, ISV (index of surface variance), IVA , KI (keratoconus index), IHA, and IHD had sufficient strength (AUC range: 0.83 to 0.981) to differentiate between the MKC and FFKC eyes  were used to establish the keratoconus diagnosis model. Figure Progressive thinning of the cornea is critical for KC development. Intriguingly, thin corneas are prone to corneal ectasia, and the thickness of the cornea is considered while planning the refractive surgery and during the diagnosis of KC. The change in corneal biomechanical properties is considered to be the influencing factor of KC , which mIn our study, nine parameters from Pentacam were compared among the three groups. IHD had sufficient (AUC > 0.80) strength and sensitivity to differentiate FFKC and MKC from the TNC, and MKC from FFKC eyes. Schlegel et al.  also demThe results of this study showed that the AUCs of A1DA and A2DA were higher between FFKC and TNC groups with A1DA, A1L, and HCDA being able to distinguish between MKC from TNC. This phenomenon designated a good distinguishing potential of A1DA to differentiate FFKC and MKC from TNC eyes. Research has found that the topographic parameters of keratoconus patients are related to DCR parameters . CatalánA previous study showed that the resistance of keratoconic corneas to deformation was lower than that of the normal cornea . ResearcP < 0.05) albeit with a low sensitivity (44.0%) for differentiating MKC from TNC. It was found that when CCT and IOP were controlled, the DA of KC was higher than that of TNC, but, there was no ideal cut-off value [P > 0.05). Therefore, the difference of DCR parameters between FFKC and TNC, MKC and TNC may be attributed to the corneal biomechanical properties caused by the change in the corneal microstructure but needs further support from the pathology of FFKC and MKC.The current results show that the DA of the MKC group is greater than that of the TNC group (ff value . In thisKeratoconus diagnosis model combining DCR and corneal tomographic parameters by BP neural network showed a more accurate diagnostic efficiency was about 91.1%. To improve the accuracy and efficiency of early or mild KC diagnosis, some researchers used machine learning to diagnose keratoconus , 40. ZouNevertheless, the present study has some limitations. The maintenance of the cornea depends on the corneal biomechanical properties. Herein, we compared the corneal biomechanical and topography parameters in FFKC and MKC groups, however, the relationship between DCR parameters and topographic map parameters was not explored. Thus, we can further explore the correlation between these parameters to provide guidance for the detection of KC. In this study, the number of patients included was limited, and hence, should be expanded to substantiate these observations.When CCT among TNC, FFKC, and MKC are matched, the majority of the tomographic and DCR parameters were different among the three groups. IHD and partial DCR parameters from Corvis ST showed that FFKC and MKC could be distinguished from TNC.


The cytogenetic and molecular assessment of deletions, amplifications and rearrangements are key aspects in the diagnosis and therapy of cancer. Not only the initial evaluation and classification of the disease, but also the follow-up of the tumor rely on these laboratory approaches. The therapeutic choice can be guided by the results of the laboratory testing. Genetic deletions and/or amplifications directly affect the susceptibility or the resistance to specific therapies. In an era of personalized medicine, the correct and reliable molecular characterization of the disease, also during the therapeutic path, acquires a pivotal role. Molecular assays like multiplex ligation-dependent probe amplification and droplet digital PCR represent exceptional tools for a sensitive and reliable detection of genetic alterations and deserve a role in molecular oncology. In this manuscript we provide a technical comparison of these two approaches with the golden standard represented by fluorescence in situ hybridization. We also describe some relevant targets currently evaluated with these techniques in solid and hematologic tumors. Fluorescence in situ hybridization (FISH) represents the reference method for gene deletion and amplification assessment. Today, this approach and its variants are still the gold standard in many genetics laboratories dealing with deletions and amplifications or rearrangements ,2.TP53), human epidermal growth factor receptor 2 (HER2), anaplastic lymphoma kinase (ALK), baculoviral IAP repeat containing 3 (BIRC3), rearranged during transfection (RET) and many other targets during the years [It is an informative and versatile technique that has been applied to the study of tumor protein 53  and has been shown to be limited in specificity for common targets such as ALK . The tecIn recent years, some molecular techniques have been developed and tested successfully with the purpose of improving the copy number variation (CNV) detection and quantitation. Multiplex ligation-dependent probe amplification (MLPA), droplet digital PCR (ddPCR) and next-generation sequencing (NGS) encompass these techniques.www.bio.rad.com (accessed on 1 April 2021)).MLPA is an accurate and time-efficient approach to detect genomics deletions and insertions, which are frequently causes of cancers. MLPA can successfully detect the CNV of all exons within a gene simultaneously and with high sensitivity. Besides the detection of CNV and rearrangement assessments in cancers and other diseases, another application of MLPA is the analysis of the ploidy in human cells . ddPCR iALK and lysine methyltransferase 2A (KMT2A) in lung carcinoma, anaplastic large cell carcinoma and acute leukemias [Targeted next-generation sequencing approaches (NGS) have proven to be affective in the detection of chromosomal aberrations like translocation of eukemias . Sensitieukemias . NGS haseukemias .However, some limits can be represented by variability in tumor cellularity, input DNA levels and sequencing coverage. Last but not least, NGS requires dedicated bioinformatics platforms and skills, which not all the routine laboratories can afford. Thus, next generation sequencing approaches deserve a dedicated review ,11.EMAP like 4-anaplastic lymphoma kinase-ALK (EML4-ALK) gene rearrangements in the molecular oncology field is commented.In the present manuscript, MLPA and ddPCR are reviewed. Relevant examples of solid and hematologic tumors like breast, lung and colon cancers in addition to chronic lymphocytic leukemia and gliomas are considered. The translational potential of ddPCR for the detection of copy number variations (CNVs) in several examples is reported and CNVs are notable sources of genetic variation in human DNA and result involved in a large number of diseases. For diagnostic purposes the main technique used in routine clinical laboratory is FISH ,13,14.FISH is an approach that uses fluorescently labeled DNA probes to detect chromosomal and cytogenetic alterations like aneusomy, duplication, amplification, deletion and translocation. There are four types of FISH probes: chromosome enumeration probes (CEPs), locus-specific indicator (LSI) probes, repetitive-sequence probes (RSPs) (centromeric and telomeric) and whole chromosome painting (WCP) .FISH probes were first developed in the late 1970s ,17,18. CMLPA is a multiplex amplifiable probe hybridization (MAPH)-related method developed by Mrc-Holland and presented for the first time in 2002. MLPA was developed to bypass the immobilization of the nucleic acid to the membrane, like Southern blot, that is difficult to introduce in routine diagnostics .MLPA are based on a simple quantitative PCR reaction and can be performed using standard laboratory equipment. MLPA probes are comprised of a pair of single-stranded DNA half-probes that need to be joined by ligation before PCR amplification can occur. MLPA have been designed to determine, in one assay, the quantity and the copy number, of a set of specific sequences in a sample of interest. This technique allows the detection of deletions and duplications in genomic DNA in relation to specific genetic diseases. To achieve this, the method works with genomically unique sequences that hybridize to one target sequence only. After ligation, bound probes are coamplified and quantified. To discriminate the individual probes the original protocols encompass size separation by capillary electrophoresis. A MLPA probe set is designed so that the length of each of its amplification products is unique. In a diploid genome, a deletion will result in a 50% signal reduction of one or more probes while a duplication results in a 50% gain of signal ,32,33,34-It is a cost effective way to check for rearrangements, duplications and deletions;-It can be applied to a large number of targets (high throughput);-It can be performed on a large number of samples simultaneously;-It is reproducible, easy to perform, and it is capable of detecting a low quantity of the target;-It requires only 50 ng of human DNA, can distinguish sequences differing by a single nucleotide and can detect small copy number differences.The main advantages of MLPA can be summarized as follows:While being a very robust technique there are some limitations linked to MLPA. These limits are represented by inability to detect anomalies at the single cell level, inability to detect unknown point mutations, sensitivity to contaminants and to novel benign polymorphisms or to polymorphisms located near to a probe ligation site. The procedure is represented in Droplet digital PCR is a robust technique that enables accurate absolute quantification of target molecules at a high degree of sensitivity. It essentially combines the simplicity of traditional end-point PCR and the quantification features of the real-time quantitative PCR (qPCR) methodologies. Unlike qPCR, the quantitation is absolute and does not utilize standards for calibration, leading to a faster, more precise and reproducible process ,36.RAS mutations by partitioning the sample. Digital PCR represents an evolution of the previously developed “limiting dilution PCR”. Digital PCR uses fluorescence instead of gel electrophoresis for the endpoint detection and, eventually, became widespread in the biomedical field due to technological improvements [The term “digital-PCR” was first used in 1999 to describe the application of the technique to the detection of ovements ,38.The basic principle of ddPCR involves an emulsion chemistry, which consists of creating emulsions of aqueous droplets in an oil medium where each droplet functions as a PCR unit ,40. A DNMinimum information for publication of quantitative digital PCR experiments guidelines (MIQE) were eventually published, with the aim of standardizing the experimental protocols. Specific MIQE for ddPCR (dMIQE) have been described and updated in recent papers in order to match the technological improvements ,42.-Reproducibility of the data, given by the absolute quantitation of the target genes;-Sensitivity of the quantitation, obtained through the amplification into an emulsion matrix;-Very low amount of starting material requested for any of the possible applications, due to the high sensitivity of the technique.-The few disadvantages known up to now are:-ddPCR requires special and dedicated instrumentation;-ddPCR detects only known mutations.-The procedure is represented in The most appreciated features of this system are:Starting material is either formalin-fixed, paraffin-embedded tissue, needle aspirates or cell slides;Incubate with 200 µL RNase for 1 h at 37 °CWash slides in 2× saline-sodium citate buffer (SSC) for 5 min, repeat.Rinse slides in 10 mM HCl.Incubate with 200 µL pepsin for 10 min at 37 °C.2O.Rinse slides in deionized HWash slides in 2× SSC for 5 min, repeat.Stabilize slides in paraformaldehyde for 10 min.Wash slides in 2× SSC for 5 min, repeat.Dehydrate slides in an ethanol series: 70%, 80% and 95%; 2 min each.Air dry.Prepare 30 µL hybridization solution per slide containing the specific probes for a target of interest. Heat to 70 °C for 10 min and place on ice.Place 30 µL of hybridization solution on each slide and cover with a plastic cover slip.Denature slide at 65–70 °C for 5 min on heat block.Gradually decrease temperature to 37 °C.Hybridize at 37 °C overnight in humidity chamber.Wash slides in 2× SSC to remove coverslip.Wash slides in wash buffer at 40 °C for 5 min, repeat.Wash slides in 0.1× SSC at 40 °C for 5–15 min.Wash slides in 2× SSC at 40 °C for 5–15 min.Cool slides to room temperature.Equilibrate slides in detection buffer for 5 min.Block in blocking buffer for 20–30 min.Incubate with 50 µL antibody or detection compound for 30–60 min .Wash slides in 2× SSC for 5 min, repeat twice.Counterstain with DAPI solution for 10 min.Rinse briefly and mount in antifade mounting medium.Analyze with a fluorescence microscope.-Incubate 100 ng DNA sample in 5 μL of Tris-EDTA (pH 8) for 5 min at 98 °C.DNA denaturation:-Cool down the samples to room temperature;-Add 3 μL hybridization master mix;-In a thermal cycler incubate 1 min at 95 °C followed by 16 h at 60 °C.Probes hybridization to sample DNA:-Lower thermocycler temperature to 54 °C;-Add 32 μL Ligase-65 master mix, incubate 15 min at 54 °C;-Heat inactivates the ligase enzyme: 5 min at 98 °C.Ligation of hybridized probes:-Cool down the samples to room temperature;-Add 10 μL polymerase master mix at room temperature;Start PCR amplification.PCR amplification of ligated probes:Fragment separation by capillary electrophoresis and data analysis.Prepare the samples by diluting genomic DNA at the desired concentration before setting up the reaction mix ;Prepare master mixes containing the specific reaction buffer for probes, each of the sampling and reference probes, 25 ng DNA/sample and water to a final volume of 20 μL/sample. The master mixes shall be sufficient for three-four technical replicates each target deletion;Mix thoroughly and allow reaction tubes to equilibrate at room temperature for about 3 min;Once the reaction mixtures are ready, load 20 μL of each reaction mix into a sample well of a cartridge, followed by 70 μL of droplet generation oil for probes into the oil wells;Put the cartridge in the automated droplet generator;After droplet generation, carefully transfer droplets into a clean 96-well PCR plate. Seal the plate with an aluminum foil in the PCR plate sealer.Proceed to thermal cycling and PCR amplification, followed by the acquisition of droplets in the QX100 or QX200 droplet reader.Design the experimental template through QuantaSoft™ Software (plate layout). After that the droplets acquisition is complete, data are analyzed with the proper setup for any specific application .These three techniques, as shown, feature significant differences in terms of the number of steps, reagents and time required for the whole procedure. Therefore, they involve different resources in order to complete medical reports. A direct and schematic comparison is shown in A relevant and somehow overlooked issue is represented by the time required to produce a medical report. Regardless of whether the disease is acute or chronic, time is crucial and bears economics implications. The shorter the time required to produce a medical report, the less is the cost related to specialized personnel dedicated to the procedures.In this regard, ddPCR is advantageous over FISH and MLPA when dealing with deletion- or duplication-detection. ddPCR is also very advantageous for the detection of gene rearrangements, useful during the differential diagnosis of different tumor types. In the recent paper by Frazzi R. and coworkers , the estThe analytical sensitivity and specificity of these three techniques is very high, according to the literature. FISH shows analytical specificity and sensitivity above 95%. MLPA is reported to have 95–99% of sensitivity and 93–97% of specificity whereas ddPCR above 98% for both ,44,45,46Cancer represents a major field of application for these three techniques, both for diagnostic and research purposes. To the present day, FISH and MLPA are mainly used for diagnostic purposes while ddPCR for research purposes.Some of the most significant targets investigated by means of these three techniques in cancer are mentioned hereinafter .TP53 gene disruption is a major prognostic and predictive factor in chronic lymphocytic leukemia  are deleted in one or more exons [ia . It hasre exons . FISH anTP53 can be also analyzed by MLPA. MLPA is an improvement of the polymerase chain reaction and can simultaneously detect several alterations like copy number changes, DNA methylation and point mutations of up to 50 genomic DNA sequences in a single experiment [Genomic aberrations of periment .Previous studies have demonstrated that MLPA is a suitable technique to detect genomic aberrations in CLL and exhibits good correlation with FISH results ,50. AddiATM) gene and often also BIRC3, located on the 11q22.2 band, in cis with ATM [Deletion 11q (del11q-) is another relevant karyotypic aberration in CLL associated to unfavorable prognosis . Del11q with ATM ,53.ATM is involved in DNA damage repair whereas BIRC3 is a negative regulator of non-canonical NF-kB signaling [BIRC3 deletion occurs in 83% of del11q cases and always coexists with ATM deletion, as demonstrated by the CLL4 study [ignaling ,54,55. BL4 study .BIRC3 in the 81.8% of cases. In the validation cohort both ATM and BIRC3 were investigated by ddPCR, confirming the ATM deletion and showing the BIRC3 deletion in 75.9% of the cases. The patients with a biallelic lesion of BIRC3 (del and mut) showed a significant shorter time to first treatment when compared to BIRC3-del/wt or wild type patients [In the Italian study mentioned before, del11q- was investigated by FISH in the discovery cohort, where the deleted region included ATM likewise TP53 point mutations were investigated also by MLPA vs. FISH. These studies demonstrated that, although MLPA detected most recurrent copy number genomic aberrations (90.9%), false negative results were found in cases with small-size abnormal clones. False positive MLPA results may be obtained, instead, from samples with point mutations (TP53) or due to an apparent lack of probe specificity. Thus, MLPA may be a useful complementary but not alternative approach for FISH testing, in these cases [se cases .BIRC3 deletions and mutations are rare albeit unfavorable events for CLL patients that can be also associated to fludarabine-chemoresistance and to adverse prognosis in some chemotherapy-treated CLL patients [BIRC3 lesions (del and mut) are an independent marker of inferior progression free survival (PFS) and overall survival (OS) [TP53-, ATM- and BIRC3-deletions would improve the predictive and prognostic opportunities in CLL patients. The introduction of multiplex diagnostic ddPCR panels would reduce considerably the time required to produce a complete report.patients ,57. Furtval (OS) . Thus, tGliomas are the most common tumor of the brain in humans with an incidence rate of 6.03 per 100,000 individuals every year .Low-grade gliomas (LGGs) have an indolent course but may evolve to aggressive, high-grade gliomas  . GlioblaEGFR) amplification, TP53 and cyclin dependent kinase Inhibitor 2A (CDKN2A) deletion as associated to poor prognosis [Gliomas can be classified into subtypes based on distinct molecular signatures. These molecular subtypes are characterized by different prognoses and treatment responses ,65,66. Trognosis ,68,69,70IDH1 and IDH2 gene mutations were correlated with a favorable prognosis [Meanwhile, other markers such as rognosis .EGFR gene, given its correlation to a poor prognosis, is an important analytical marker in gliomas. EGFR is a transmembrane receptor tyrosine kinase that can initiate several intracellular signaling pathways contributing to cancer cell proliferation, inhibition of apoptosis, invasion, metastasis and stimulation of neovascularization [EGFR alterations are at genetic level ) or at protein level (overexpression). EGFR alterations promote proliferation, angiogenesis and invasion and in glioblastoma also resistance to radiotherapy and chemotherapy [The rization ,117. Theotherapy ,73,74.EGFR gene amplification preferentially by FISH [EGFR/CEP7 ratio. A probe hybridizes with chromosomal 7p12 region containing EGFR locus while a second probe hybridizes to the centromeric region 7p11.1. This latter one is used to count the number of copies of chromosome 7 [EGFR specific probe is directly related to the number of copies of this gene. The number of signals corresponding to the CEP7 is directly related to the number of copies of chromosome 7. Normally, one probe is red and one is green. Yellow signals correspond to the overlap between red and green labeling. EGFR amplification is recorded when more than 10% of analyzed cells show an EGFR to CEP7 ratio ≥2 [EGFR protein overexpression is mainly studied by IHC whereas  by FISH . The FISmosome 7 ,77. The ratio ≥2 ,78.EGFR abnormalities in brain tumor tissue can be also assessed by MLPA and ddPCR [EGFR mutations.nd ddPCR ,80. FISHEGFR is a prognostic marker that has to be combined with other molecular markers in order to identify different glioma subtypes. As an example, the codeletion of chromosomes 1p and 19q is strongly associated to oligodendroglioma [KIAA1549:BRAF is a specific molecular marker of the group of pilocytic astrocytoma [As mentioned before, roglioma ,80,81,82rocytoma ,84.Breast cancer, lung cancer and colorectal cancer are three examples of solid and often aggressive tumors. The investigation of specific gene alterations is useful for diagnostic and prognostic purposes.HER2 are associated with poor prognosis in breast cancer and suitable for anti-HER2 therapy. Thus, a correct identification of patients with HER2 amplification is crucial. There are several methods to determine HER2 status, including FISH, MLPA and ddPCR. FISH analysis usually uses a probe to the HER2 gene and another for CEP17, as reference control. Patients with increased HER2 along with increased CEP17 copy numbers might be misclassified as non-amplified HER2 as during polysomy of chromosome 17. Although several studies have reported that true polysomy of chromosome 17 is rare specific reporting guidelines have recently been developed to overcome this issue [HER2 and c-Myc gene amplification also were detected by MLPA and FISH in tissue samples of invasive breast cancer [Amplification and overexpression of is issue ,97,98,99t cancer ,102.HER2 copy number can be determined by ddPCR without the need for calibration. So, ddPCR technique may be useful as an alternative to FISH and MLPA in HER2 diagnosis [Moerland E. and coworkers evidenced a good correlation between MLPA, FISH and chromogenic in situ hybridization (CISH). However, the use of these different methods raises the issue of intra- and inter-laboratory reproducibility. Fixation time in FISH, together with other preanalytic variables, is another parameter affecting this approach . Absolutiagnosis .Colorectal cancer (CRC) is one of the most deadly cancer in the world together with lung, liver and stomach cancer . AdvanceEGFR. The pivotal role of EGFR in cancer progression is used in the treatment of RAS wild-type metastatic CRC by EGFR-targeting monoclonal antibodies cetuximab and panitumumab [EGFR is routinely analyzed by FISH. Likewise, HER2 and EGFR copy number is evaluated by EGFR/CEP7 ratio and can be affected by intra- and inter-laboratories variability [EGFR with other techniques such as MLPA and ddPCR are few and concern gene mutations instead of copy number variations.In CRC the main gene with copy number variation related to the disease is itumumab ,122,123.iability ,125,126.RAS) gene are described more frequently than EGFR in CRC. The literature reports several studies on RAS using the three techniques that we are comparing in this review.The mutations in the rat sarcoma  and neuroblastoma RAS viral oncogene homolog (NRAS). It has also been suggested that NRAS mutations play a role in prognosis since patients harboring NRAS mutations have a significantly shorter survival compared to those bearing wild type NRAS. This is due to the fact that when RAS family members are mutated, the cells have an increased potential of invasion and metastasis [tastasis ,129,130.As mentioned above, another leading cause of cancer-related death in the world is lung cancer. The clinical importance of the molecular phenotype of lung cancer bears therapeutic implications since different subtypes may respond to different targeted treatments.ALK rearrangement represents a molecular target in a subset of non-small cell lung cancers (NSCLCs) [ALK fusion gene, thus defining a tumor group that may be responsive to targeted therapy [ALK with the upstream partner EML4 was found in NSCLC in 2007. This fusion is due to a chromosomal inversion on chromosome 2p, resulting in the formation of EML4-ALK fusion oncogene. Notably, ALK rearrangements occur almost exclusively in adenocarcinomas [ALK-rearranged lung cancer is a unique molecular subgroup with a high sensitivity to ALK inhibitors and mutually exclusive with other well-known oncogenic mutations (EGFR or KRAS) [(NSCLCs) . Approxi therapy . Fusion rcinomas ,145. ALKor KRAS) ,147.ALK-rearranged lung cancer can be identified by immunohistochemistry (IHC), FISH or qRT-PCR with each method having advantages and disadvantages for screening. In 2011 ALK FISH was approved as a diagnostic kit for NSCLC [ALK rearrangement in NSCLC, the ALK FISH test was already being challenged by IHC. However, discrepancies in results obtained by FISH and IHC, such as ALK FISH+/ALK IHC− or ALK FISH−/ALK IHC+, have been reported in some cases [or NSCLC ,149,150.me cases ,154,155.ALK gene fusion, and automation of IHC procedures, a new generation ALK IHC has been developed and is considered as sensitive as ALK FISH for detecting this rearrangement [In the past decade, due to new and better anti-ALK antibodies that detect angement ,158,159.angement .EML4-ALK fusion at the RNA level [ALK transcripts, because the breakpoint in ALK consistently occurs at exon 20 and EML4 (or other fusion partners). The resulting strong expression of ALK kinase domain leads to an unbalanced expression in 5′ and 3′ portions of ALK transcripts [ALK fusion partners and variants and also could be a suitable method for detecting ALK translocations using cytology samples from patients with primary lung cancer, especially when tissue samples are not available [ALK IHC or ALK FISH are approximately 95%-100% [Quantitative RT-PCR (qRT-PCR) has also been applied successfully to the detection of NA level ,161. qRTnscripts ,162. qRT95%-100% ,162.ALK rearrangements. The utility of these NGS-based methods emerged in the identification of ALK rearrangement-positive cases with an atypical ALK FISH signal pattern, even in cases that resulted in being negative by ALK FISH [NGS-based methods are also becoming widely used in the detection of ALK FISH ,164,165.ALK rearrangements. A significant advantage of ddPCR is the ability to detect and quantitate three variants of the EML4-ALK rearrangements that FISH cannot distinguish. Further advantages of ddPCR over FISH are represented by a lower limit of detection (LOD) associated to a lower cost of the test [Finally, also ddPCR based assays have proven to be appropriate for the detection of the test ,167,168.Molecular and cytogenetic techniques are the key to the study and diagnosis of cancer. For this purpose, some of their main applications are copy number variation analysis (deletion and duplication) and genetic rearrangements. Over the years, many targets have been successfully detected through FISH, MLPA and ddPCR (please, see In this article, we compared these three techniques evidencing the potential and characteristics of each one for cancer diagnostics.Although FISH is often the golden standard for many targets, MLPA and ddPCR, according to the published data, can be much more sensitive and accurate. MLPA and ddPCR, however, differ in timing so that ddPCR bears a great potential for future applications.


Blood-based clinical diagnostics require challenging limit-of-detection for low abundance, circulating molecules in plasma. Micro-scale blood plasma separation (BPS) has achieved remarkable results in terms of plasma yield or purity, but rarely achieving both at the same time. Here, we proposed the first use of electrospun polylactic-acid (PLA) membranes as filters to remove residual cell population from continuous hydrodynamic-BPS devices. The membranes hydrophilicity was improved by adopting a wet chemistry approach via surface aminolysis as demonstrated through Fourier Transform Infrared Spectroscopy and Water Contact Angle analysis. The usability of PLA-membranes was assessed through degradation measurements at extreme pH values. Plasma purity and hemolysis were evaluated on plasma samples with residual red blood cell content  corresponding to output from existing hydrodynamic BPS systems. Commercially available membranes for BPS were used as benchmark. Results highlighted that the electrospun membranes are suitable for downstream residual cell removal from blood, permitting the collection of up to 2 mL of pure and low-hemolyzed plasma. Fluorometric DNA quantification revealed that electrospun membranes did not significantly affect the concentration of circulating DNA. PLA-based electrospun membranes can be combined with hydrodynamic BPS in order to achieve high volume plasma separation at over 99% plasma purity. Blood plasma separation is usually required for several blood-based clinical diagnostics . In thisMicrofluidic-based methods for blood plasma separation usually rely on active and passive techniques . Active The correct micro-scale BPS strategy depends on several factors, such as the volume of plasma required for subsequent clinical tests as well as the number and type of tests to be performed on the same sample. For instance, blood gas, electrolytes, and metabolites (BGEM) tests can be carried out on plasma from a drop of blood, typically in the order of 10–200 µL. In this volume range, paper-based technologies or filtration are the most common approaches ,13,14,15If larger blood volumes are needed (milliliter range), such in the case of amplification of rare circulating DNA from plasma , hydrodyThe combination of hydrodynamic and membrane separation in a BPS system is proposed to achieve high yield and high purity simultaneously when processing large volumes of blood by allowing large amount of plasma to be removed with suboptimal purity, followed by removal of residual erythrocytes, of red blood cells (RBCs) by filtration without loss of plasma volume. Membrane filtration at microscale is often used as a sieving process to retain large molecules or particulate matter ,22,23.However, in order to accomplish high performance separation, membranes should meet challenging requirements such as low pressure drop and high plasma purity. The development of engineered membranes could allow the control of chemical, physical, and mechanical properties of biopolymeric highly porous structures susceptible to meet these requirements. Among the several approaches developed for fabricating biopolymeric porous structures, thermally or diffusion induced phase separation (TIPS or DIPS) as well as electrospinning are gaining more attention ,25.TIPS and DIPS techniques are based on thermodynamic de-mixing of a homogeneous polymer-solvent solution into a polymer-rich and a polymer-lean phase. De-mixing can be achieved by changing temperature (TIPS) or changing the solution’s exposure to another immiscible solvent (DIPS) ,25. BothOn the other hand, electrospinning produces ultrafine fibers ranging from micro- to nano-meter in diameters from polymeric solutions or melts . ElectroOne of the most common biopolymers used for these applications is polylactic acid (PLA). Due to its commercial availability at an affordable cost, PLA has been extensively studied and used in many biomedical purposes, including wound healing ,32 and t−1. In addition, we have compared the performance of pristine with surface modified membranes, using commercially available BPS membranes as a benchmark. Finally, non-specific binding of circulating DNA was evaluated for each of the membranes via fluorometric quantification with QubitTM analysis in order to assess their suitability in use in clinical applications.In an effort to develop a rapid and effective residual cell trapping, the efficacy of PLA-based membranes prepared by TIPS, DIPS, and electrospinning for blood plasma separation was assessed. To the best of our knowledge, it is the first time that PLA-based porous membranes have been tested as a microfiltration unit (MFU) for microfluidic blood plasma separation purposes. Initially, a comparative study on PLA membranes degradation at extreme pH values was carried out in order to ascertain their usability in microfluidic protocols requiring the use of non-neutral buffers or after acid or basic treatments. Secondly, plasma purity, free hemoglobin concentration and, pressure drop across the DIPS, TIPS, and ES membranes have been evaluated employing plasma-diluted blood corresponding to output-plasma tested from already existing HBPS systems  i.e., wi® L 209 S  for TIPS and DIPS membranes and Natureworks® 2002D (PLA) for ES membranes. PMMA sheets at different thicknesses were produced by Clarex, Nitto Jushi Kogyo Co. Ltd., Tokyo, Japan and supplied by Weatherall Ltd., Wendover, UK. Hexamethylenediamine (HMDA), methanol (MeOH), 1,4 dioxane, chloroforms (TCM), sodium hydroxide (NaOH), and acetone (Ac) were purchased from Sigma Aldrich, Munich, Germany. 37% Hydrochloric Acid (HCl) solution was purchased from Carlo Erba Reagents. Blood samples were obtained under local Ethics approval from The Scottish National Blood Transfusion Service, Jack Copland Centre, Edinburgh, UK. TIPS, DIPS, and ES PLA membranes were compared with commercially available membranes VividTM GF (Vivid A) and VividTM GX (Vivid B) from Pall. All the reactants were ACS grade (purity > 99%). Two different grades of polylactic acid were chosen for this work: Resomerw/w dioxane-water mixture at 120 °C. The solution, kept above 60 °C to ensure homogeneity, was hot poured in a cylindrical high-density polyethylene (HDPE) mold. The mold filled with the polymer solution was first immersed for 10 min in a 0 °C bath and then moved to a –20 °C bath for 20 min in order to allow the complete freezing of the as-obtained structure. Successively, the foams were extracted from the mold and subjected to a washing step in distilled water. Finally, the foams were dried under vacuum for 24 h to ensure the complete removal of any solvent trace. The subsequently obtained membranes were then cut to obtain slices 1 mm thick.TIPS membranes were prepared by dissolving PLLA (4 wt%) in an 87/13 −1 (dip coating) to ensure a proper final average membrane thickness of 120 µm, based on previous work [DIPS membranes were produced by dissolving PLLA (8 wt%) in neat dioxane at 120°C. A stainless steel slide was immersed into the solution, cooled at 30 °C and then pulled out at a constant speed of 3 cm minous work . The coa−1; distance between the needle tip and the collector, 13 cm; supplied high voltage, 15 kV; temperature, 25 °C and relative humidity, 40%. The nanofibers obtained were collected on a grounded rotary drum  wrapped in an aluminum foil for 180 min in order to obtain membranes approximately 100 μm thick. The collected PLA ES membranes were subsequently dried for at least 2 days under fume hood in order to remove any residual solvents.The polymeric solution for electrospinning was obtained by adding PLA (10 wt%) to 20 mL of TCM:Ac (2:1 v:v), and completely dissolved by stirring overnight at room temperature to obtain homogeneous PLA/TCM:Ac solution . A semi-All the membranes were surface modified through a solution of methanol:water (3:7 v:v) and HMDA (1% w:v). The solution was directly pipetted onto each side of the PLA membranes. A 12 h drying period at room temperature in a fume cupboard followed.The morphology of the nanofiber mats was evaluated by scanning electron microscopy, . The samples were attached on an aluminum stub using adhesive carbon tape.Fiber diameter distribution of ES membranes and pore size distribution of TIPS and DIPS membranes were determined using ImageJ as an image processing software. In particular, the plugin DiameterJ is able to analyze an image obtained by SEM and to find the diameter of nanofibers at every pixel along a fiber axis. The software produces a histogram of these diameters and summarizes statistics such as mean fiber diameter . The hydrolysis of the ES, TIPS and DIPS membranes was performed in 10 mL of solutions  at 25 °C up to 4 h. At fixed times, i.e., 0.5, 1, 2, and 4 h, the membranes were washed thoroughly with distilled water at room temperature, followed by drying under chemical hood for 1 day. −1.The chemical surface properties of the samples were assessed by spectroscopic analysis. FTIR-ATR analysis was carried out by using a PerkinElmer FTIR/NIR Spectrum 400 spectrophotometer; the spectra were recorded in the range 4000–400 cmStatic contact angles were measured on all the samples by using distilled water as fluids with an FTA 1000  instrument. More in detail, 4 μL of distilled water were dropped on the membranes. Images of the water on the surface were taken at a time of 10 s. At least seven spots of each composite nanofiber mat were tested and the average value was taken. 2 laser cutter  equipped with a 2 in lens (10.6 μm beam spot). Residual dust was removed with clean-room tissue wetted with ethanol. Layers were aligned in specific custom-made plates endowed with metallic pins; therefore, individual layers were featured with corresponding alignment holes. The membranes were positioned in the proper layer and then ethanol was spread between PMMA layers with a pipette. The bonding was carried out at 70 °C and 100 bar for 5 min in a Carver laboratory press [The 4 PMMA layers were 7.5 cm × 3.2 cm with different thicknesses selected on their function. The upper (L1) and lower (L4) cover were 2 mm while the inlet channel layer (L2) was 1 mm. The layer containing the membrane holder (L3) was 0.5 mm thick with an engraving of 0.1 mm for DIPS membrane and ES 1L membrane. The same layer was 2 mm thick and 1 mm engraved for TIPS membrane and for ES 10L membranes. The entire membrane holder diameter was 7 mm divided as follows: 5.5 mm cut through and 1.5 mm engraved to ensure the correct adhesion of the membrane and avoid flow leaking to the outlet side due to membrane bypass. The inlet and outlet channels in the second and fourth layers were engraved, while a circle (4.5 mm diameter) at the end of the inlet channel was cut through as input for the membrane. Membranes and PMMA layers with different thicknesses were cut using a COry press .A WPI Al300-220 syringe pump loaded with a 10 mL syringe was used to impose a constant flow through the membranes. Plasma-diluted human blood with initial hematocrit (Hct) was measured through an hemoanalyzer . Separated plasma was collected and analyzed in aliquots at different volume points (inHct) equal to 1%, 3%, and 5% were used for this test. All kinds of functionalized and unfunctionalized membranes were tested. The blood flow rates were 1 mL h−1, 2 mL h−1, and 3 mL h−1, which corresponds to tested output flow rates from existing HBPS system [Hct . Separated plasma was collected and analyzed in aliquots at different volume points :A560, A576.5 and A593 are the absorbance values at 560 nm, 576.5 nm and 593 nm, respectively. Before UV-Vis spectroscopy, each plasma sample was centrifuged for 10 min at 1600 g and only the supernatant absorbance was measured in order to ensure to count only free hemoglobin present in the sample. Absorbance was measured using Jenway 7315 Spectrophotometer in 500–630 nm wavelength range at 1 nm interval.The extent of hemolysis in plasma samples was determined through UV-Vis spectroscopic measurement according to Cripps method. In particular, Cripps number was calculated according to the Equation (3) :(3)H=A57correctedH), the H value of the centrifuged feed (controlH) was subtracted to H value of the extracted volume (outH) according to Equation (4): In order to report hemolysis values only due to the cell damage resulting from the microfluidic membranes  were used to obtain a calibration curve, correlating the H value and the hemoglobin concentration. In the concentration range herein investigated, the calibration curve was found to be linear with a slope of 2.17 up to a H value of 0.33. For higher H value, the calibration curve still remained linear but with a slope of 3.17. In the case of Hgb concentrations higher than 0.3 g dL−1, the results from the hemoanalyzer were considered.In order to correlate the Cripps number with a concentration of Hgb in plasma, a series of centrifuged plasma containing 0.1 g dLFree Hgb (g dL−1) concentration was calculated according to the following Equation (5):i-th sample; Finally, the cumulative The score index calculation was evaluated on the basis of the two following arbitrary parameters:HgbK) was determined according to Equation (6):thHgb is the Hgb threshold value chosen i.e., 0.15 g dL−1. Note that if iHgb > thHgb then HgbK < 0 and these values appears in the heat map as a black square.The parameter related to the Hgb concentration (RBCK) was determined according to Equation (7):iPP is the plasma purity evaluated for the i-th sample and thPP is the plasma purity threshold value chosen i.e., 85%. Note that if iPP < thPP then RBCK < 0 and these values appears in the heat map as a black square.The parameter related with the plasma purity  was then evaluated according to the following Equation (8):−1 DNA in molecular grade water, was prepared. To prepare spin columns, silica membranes were removed from EconoSpin columns and replaced with ES membranes [In order to assess the binding capacity of the membranes the mixture of fragmented T47-D cell line DNA (~160 bp), mimicking circulating cell-free DNA fragmentation, was used. A DNA solution, containing ~1 ng µLembranes . The colp < 0.05 was considered statistically significant.Statistical analyses of the data were performed through ANOVA by using the GraphPad Prism version 8 software . Significant differences among mean values, where applicable, were determined by means of ANOVA. In all cases, a value of In an attempt to achieve rapid and effective blood plasma separation, compatible with Lab-on-Chip or in-line processing, we have developed a hybrid strategy, combining HBPS published previously , and a MThis two-step strategy should simultaneously achieve high yield (via the HBPS device) and high purity  in the milliliter range blood plasma separation. As HBPS performance decreases upon increasing plasma yields ,18,19,20TM GF (Vivid A) and VividTM GX (Vivid B). As shown in In this respect, we have evaluated the use of PLA-based membranes prepared by TIPS, DIPS and ES for the removal of residual red blood cells following HBPS. Low pressure drop and high plasma purity are the main requirements. The performance of these PLA engineered membranes were compared to two commercially available membranes for RBC removal i.e., VividIn the case of both ES and surface-modified ES (m-ES) membranes, 1 layer (ES 1L or m-ES 1L) or 10 layers (ES 10L or m-ES 10L) were placed in the membrane holder in order to evaluate the effect of different membrane thicknesses.For membrane-based cell separation, pore morphology and the size of a membrane are two crucial factors. From the SEM images shown in Chemical based surface modification protocols may be used to provide various functionalities to the membranes. In order to compare the effects of surface modification chemistries, the membranes were subjected to extreme pHs (acidic and basic treatment) for up to 4 h, as shown in In particular, the membrane morphology was assessed in three different pH, pH 1, distilled water and at pH 13. The morphology of the membranes at 25 °C for two time points (2 h and 4 h), are shown in ES membranes immersed in acidic medium and distilled water for up to 4 h did not show any significant change in their morphology. In fact, only a slight increase in the fiber diameter B in acidSimilarly, TIPS and DIPS membranes showed a more pronounced morphology change in the basic medium. Although the mean pore diameter of the TIPS remained almost constant C, it canDIPS membranes immersed in all kinds of medium show a more significant change in mean pore diameter as a function of time. In The above observations can be explained by considering the mechanism of hydrolysis of PLA, which is primarily determined by the amount, presence, and location of water molecules. Recent studies correlated the differences in the degradation behavior of PLA to the hydrophilicity of this polymer matrix exposed in media at different pH . PLA expIn order to accomplish high performance separation, membranes should meet challenging requirements such as low pressure drop that can be reduced by modifying their wettability.Several approaches have been investigated in order to enhance the wettability of PLA-based porous or fibrous membranes in both dry and wet chemistry ,37,38. IMorphological inspection clearly reveals that the surface treatment eroded the porous structure of all membranes. In particular, from In −1, the C–O stretch at 1180, 1129, and 1083 cm−1 and the OH bend at 1044 cm−1 [−1, respectively [−1 in amino-modified membranes is barely visible due to the vibration of amide groups that lie in the same regions and to the presumable presence of water due to the enhanced wettability, the peak at 1585 cm−1 is clearly visible in particular for m-TIPS and m-ES membranes. Moreover, after aminolysis of ES, TIPS, and, to a lesser extent, DIPS membranes showed another new peak at 1650 cm−1 which corresponded to amide I stretch [044 cm−1 . The preectively . Althoug stretch . The int stretch .In order to evaluate the membranes performance, they were included in a microfluidic device denominated MFU. The MFU design is shown in inHct) of the blood sample with the outlet Hct (outHct) according to Equation (2) as a function of different flow rates and extract plasma volume. The membrane performance was first evaluated through an investigation of the plasma purity. The purity of the plasma output from the devices utilizing either the ES membrane (amino-modified or not) or one of the two commercially available membranes for RBCs separation (Vivid A and B) was determined by comparing the inlet Hct (Similar experiments performed using both the surface modified and unmodified TIPS and DIPS membranes resulted in clogging (reaching a pressure higher than 300 kPa) before 0.5 mL of plasma had been extracted. This result can be probably ascribed to the higher tortuosity offered by the porous membranes if compared to fibrous membranes due to the porous architecture that could easily lead to the clogging of microcapillaries by RBCs.The results have been compiled into a three-color heat map and shown in inHct with all the flow rates investigated. For plasma samples containing 3% residual inHct, the plasma purity decreased to 96%, 93%, and 90% using flow rates of 1 mL h−1, 2 mL h−1, and 3 mL h−1, respectively. It is worth noting that 90% of plasma purity due to the MFU fed with 3% of inHct represents a global plasma purity  higher than 99%. In fact, assuming a whole blood hematocrit of 40%, 3% inHct can be achieved by an HBPS chip plasma purification of around 92.5%. After a further microfiltration step able to achieve 90% plasma purity, the global plasma purity of the hybrid system would be 99.25%.The general trend observed is that there is a decrease in plasma purity with increasing flow rate, residual hematocrit and extracted volume. The highest plasma purity was achieved using ES 10L. Employing this device, it was possible to extract up to 2 mL of pure plasma (100% of plasma purity) from plasma with 1% residual The worst plasma purity was observed in devices containing m-ES 1L membranes. The commercial membranes compared relatively well at the lowest flow rate, and the lowest residual Hct, but rapidly clogged and did not yield results beyond this (no data collection represented by squares on heat map), showing limited use at higher volumes. Membrane performance was also evaluated by measuring the free hemoglobin concentration in the extracted plasma due to the RBCs rupture during their passage through the membrane. The two performance indicators, plasma purity and hemolysis, are needed to reflect on the true performance of the device as a high RBCs removal on its own would not necessarily reflect an overall good performance if the filtrates have high levels of hemoglobin. inHct, and the extracted volume. In this case, the best performances were shown by the surface modified (aminolyzed) membranes. Specifically, if 1% inHct is used as input for the devices incorporating either m-ES 1L and m-ES 10L membranes, the concentration of free hemoglobin in the output samples was lower than 0.04 g dL−1. On the contrary, RBCs lysis was found to be particularly pronounced for devices including the commercial porous membranes, Vivid A and Vivid B, at all the flow rates here investigated. For instance, Vivid B membranes showed a concentration of free hemoglobin equal to 0.21 g dL−1 after 1 mL of plasma extracted, with a flow rate equal to 3 mL h−1. In order to assess the amount of hemolysis due to membrane separation, the free hemoglobin concentration was determined according to Equation (5), as described in the ‘Materials and Methods’ section. The results have been reported in a heat map format B. As expinHct up to 3% and 5% the free Hgb concentration increased for all the systems investigated, but the trend remained similar . In inHct and the extracted volume.Hemolysis as a result of membrane separation can be attributed to several factors, including the shear stress on the RBCs, membrane morphology and the pressure sustained by the cells while in the device. The shear stress on RBCs increases upon increasing the flow rate and this can also affect the pressure on the membrane. The porous structure of membranes can affect both the pressure drop and the damage induced through contact between the cell and the pores. In the inHct. The pressure drop differences between ES and Vivid membranes can be likely ascribed to the nanofibrous structure of the mats which offers a large interconnection degree and, as a consequence, a lower pressure drop than Vivid membranes. Moreover, despite their poor separation performance, the aminolyzed membranes exhibited even lower pressure drops due to their enhanced wettability as a result of the chemical functionalization.Both commercial membranes, Vivid A and Vivid B, showed the highest pressure drops, although it should be noted that these membranes have been developed for low volume separation from whole blood. On the other hand, in this work, Vivid membranes were used for high blood volume separation but only to purify plasma with minimal residual hematocrit, not whole blood. As expected, ES 10L showed higher pressure drops when compared to ES 1L because of its higher thickness. Regardless of flow rates, the ES membrane pressure drop was 50–60% lower than the Vivid membranes at 1% The pressure drop differences can partially explain the differences in hemolysis levels between Vivid and ES membranes. It is envisaged that the fibrous morphology of the latter’s PLA-based mats and their superior interconnection degree reduced RBCs lysis during plasma separation. In fact, during contact with the red blood cell, the ES fibers may bend thus reducing the stress on the RBC extracellular membrane and, as a consequence, their lysis. The cell separation membranes have been designed to be integrated into a complete workflow for circulating DNA extraction. Avoiding DNA adsorption onto the surface of the membrane is therefore crucial for the functioning of the membrane. To investigate the non-fouling properties of the membrane, DNA extraction after permeation of a plasma sample through the membrane placed in a column has been performed.−1), incubated, and then centrifuged to pull the solution through the membranes (In p < 0.01) affect the DNA concentration. Only ES 1L lead to a slight decrease of DNA concentration within the instrument deviation range. These results highlight that both the unmodified and modified ES PLA-based membranes are low binding and suitable for use in assays where maximum DNA recovery from raw samples is of importance.The histogram in −1) plasma at 1% inHct and flow rate up to 2 mL h−1. With the same membrane and flow rate, upon increasing the inHct the plasma purity decreased but it was also possible to collect 1 mL of RBCs-free and low-hemolyzed (free Hgb = 0.062 g dL−1) with 3% inHct. There is evidence to show that ES membranes decreased the degree of hemolysis when compared to the commercially available Vivid ® membranes. The ES membranes also exhibited lower pressure drops while maintaining comparable plasma purity, thereby showing superior separation performance at low hematocrit levels. We have shown that the PLA-based ES membranes may be suitable for liquid biopsy workflows, where maximum recovery of circulating DNA is crucial. PLA-based ES membranes can be readily integrated into microfluidic workflows and show enhanced separation performance compared to commercial membranes for the recovery of high purity plasma in the hundreds of microliters to milliliter range. PLA-based membranes prepared by DIPS, TIPS, and electrospinning were modified through aminolysis and characterized. The membranes resistance was tested at extreme pH values for up to 4 h. Basic media was the most aggressive environment affecting pore morphology. The successful amine functionalization was demonstrated by FTIR-ATR while the subsequent change in wettability of the samples was assessed through WCA analysis. Both modified and unmodified TIPS and DIPS membranes clogged before extracting 0.5 mL of plasma and were excluded from further examination. ES membranes were selected for blood plasma separation tests at low hematocrit levels , corresponding to output-plasma tested from already existing HBPS systems. Single layers (ES 1L) or stacks of 10 ES membranes were tested in a microfluidic configuration device. The stack of 10 layers (ES 10L) allowed the collection of up to 2 mL of RBCs-free and low-hemolyzed (free Hgb = 0.078 g dL