Characterizing the Microparticles Deposition Structure and its Photonic Nature in Surfactant-Laden Evaporating Colloidal Sessile Droplets.

This work presents a simple method to create photonic microstructures via the natural evaporation of surfactant-laden colloidal sessile droplets on a flat substrate. In the absence of dissolved surfactant, the evaporating colloidal droplet forms a well-known coffee ring deposition. In contrast, the presence of surfactant leads to the formation of multiple ring structures due to the repetitive pinning-depinning behavior of the droplet contact line (CL). It is found that the multiring structure shows vibrant iridescent structural colors while the coffee ring lacks a photonic nature. This difference in the structural color for the presence and absence of the surfactant is found to be dependent on the arrangement of the particles in the deposition structure. The particle arrangement in the multirings is monolayered and well-ordered. The ordering of the particles is strongly influenced by the particle dynamics, contact angle (CA), and CL dynamics of the evaporating colloidal solution droplet. Furthermore, the iridescent nature of the multiring deposition is demonstrated and explained. The dependence of the multiring deposition structure on the concentration of the dissolved surfactant and the suspended particles is also studied. The findings demonstrate that an intermediate surfactant concentration is desirable for the formation of a multiring structure. Further, the pinning-depinning CL dynamics that causes the formation of the multiring deposition structure is discussed. Finally, we demonstrate the applicability of the approach to smaller droplet volumes.

Diagnostic performance of respirators for collection and detection of SARS-CoV-2.

Respirators, called as face mask, have been used to protect the wearer from the outside harmful air environment and prevent any virus from being released to neighbors from potentially infected exhaled breath. The antiviral effectiveness of respirators has not only been researched scientifically, but has also become a global issue due to society's obligation to wear respirators. In this paper, we report the results of a study on the collection and detection of viruses contained in exhaled breath using respirators. The inner electrostatic filter was carefully selected for virus collection because it does not come in direct contact with either human skin or the external environment. In the study of a healthy control group, it was confirmed that a large amount of DNA and biomolecules such as exosomes were collected from the respirator exposed to exhalation, and the amount of collection increased in proportion to the wearing time. We conducted experiments using a total of 72 paired samples with nasopharyngeal swabs and respirator samples. Out of these samples, fifty tested positive for SARS-CoV-2 and twenty-two tested negative. The PCR results of the NPS and respirator samples showed a high level of agreement, with a positive percent agreement of ≥ 90% and a negative percent agreement of ≥ 99%. Furthermore, there was a notable level of concordance between RCA-flow tests and PCR when examining the respirator samples. These results suggest that this is a non-invasive, quick and easy method of collecting samples from subjects using a respirator, which can significantly reduce the hassle of waiting at airports or public places and concerns about cross-contamination. Furthermore, we expect miniaturized technologies to integrate PCR detection into respirators in the near future.

An innovative charge-based extracellular vesicle isolation method for highly efficient extraction of EV-miRNAs from liquid samples: miRQuick.

Extracellular vesicle-derived microRNAs (EV-miRNAs) are promising biomarkers for early cancer diagnosis. However, existing EV-miRNA extraction technologies have a complex two-step process that results in low extraction efficiency and inconsistent results. This study aimed to develop and evaluate a new single-step extraction method, called miRQuick, for efficient and high-recovery extraction of EV-miRNAs from samples. The miRQuick method involves adding positively charged substances to the sample, causing negatively charged EVs to quickly aggregate and precipitate. A membrane lysate is then added to extract only miRNA. The entire process can be completed within an hour using standard laboratory equipment. We validated the miRQuick method using various analytical techniques and compared its performance to other methods for plasma, urine and saliva samples. The miRQuick method demonstrated significantly higher performance than other methods, not only for blood plasma but also for urine and saliva samples. Furthermore, we successfully extracted and detected nine biomarker candidate miRNAs in the plasma of breast cancer patients using miRQuick. Our results demonstrate that miRQuick is a rapid and efficient method for EV-miRNA extraction with excellent repeatability, making it suitable for various applications including cancer diagnosis.

Collection and detection of SARS-CoV-2 in exhaled breath using face mask.

Face masks are used to protect the wearer from harmful external air and to prevent transmission of viruses from air exhaled by potentially infected wearers to the surrounding people. In this study, we examined the potential utility of masks for collecting viruses contained in exhaled breath and detected the collected viruses via various molecular tests. Using KF94 masks, the inner electrostatic filter was selected for virus collection, and an RNA extraction protocol was developed for the face mask. Virus detection in worn mask samples was performed using PCR and rolling circle amplification (RCA) tests and four different target genes (N, E, RdRp, and ORF1ab genes). The present study confirmed that the mask sample tests showed positive SARS-CoV-2 results, similar to the PCR tests using nasopharyngeal swab samples. In addition, the quantity of nucleic acid collected in the masks linearly increased with wearing time. These results suggest that samples for SARS-CoV-2 tests can be collected in a noninvasive, quick, and easy method by simply submitting worn masks from subjects, which can significantly reduce the hassle of waiting at airports or public places and concerns about cross-infection. In addition, it is expected that miniaturization technology will integrate PCR assays on face masks in the near future, and mask-based self-diagnosis would play a significant role in resolving the pandemic situation.

Rapid and Efficient Extraction of Cell-Free DNA Using Homobifunctional Crosslinkers.

Since its discovery in circulating blood seven decades ago, cell-free DNA (cfDNA) has become a highly focused subject in cancer management using liquid biopsy. Despite its clinical utility, the extraction of cfDNA from blood has many technical difficulties, including a low efficiency of recovery and long processing times. We introduced a magnetic bead-based cfDNA extraction method using homobifunctional crosslinkers, including dimethyl suberimidate dihydrochloride (DMS). Owing to its bifunctional nature, DMS can bind to DNA through either covalent or electrostatic bonding. By adopting amine-conjugated magnetic beads, DMS-DNA complexes can be rapidly isolated from blood plasma. Using standard washing and eluting processes, we successfully extracted cfDNA from plasma within 10 min. This method yielded a 56% higher extraction efficiency than that of a commercial product (QIAamp kit). Furthermore, the instant binding mechanism of amine coupling between the microbeads and DMS-DNA complexes significantly reduced the processing time. These results highlight the potential of this magnetic bead-based homobifunctional crosslinker platform for extraction of cfDNA from blood plasma.

Size-selective filtration of extracellular vesicles with a movable-layer device.

This paper presents a microfluidic device that can isolate extracellular vesicles (EVs) with multiple size intervals in a simple, effective, and automated manner. We accomplish this size-selective separation using a vertically movable plunger and a rotationally movable chip. The chip has open chambers with nanoporous filters that are sequentially connected by check valves. The plunger speed is adjusted to reduce chamber pressurization in order to prevent EV deformation, thereby achieving a high separation resolution. Herein, high-purity EVs with a purity ten times higher than that of ultracentrifugation were obtained by washing three times with a high EV recovery rate of 89%. For the analysis of device performance, we used polymer nanobeads, preformed liposomes, and canine blood plasma. To demonstrate the utility of the device, we applied size-selective isolation to EVs that were secreted by endothelial cells under shear flow. The results revealed that the cells secreted more EVs of larger size, the expression of CD63 protein was higher for EVs with a larger size, and a high amount of TSG101 protein was expressed under the condition of no shear flow. This device is envisioned to facilitate molecular analysis and EV-based biomarker discovery that use various biofluids, including blood plasma, urine, and cell culture supernatants. Our device automates size-selective EV filtration that requires laborious multiple washing and separation steps.

Rapid Extraction of Viral Nucleic Acids Using Rotating Blade Lysis and Magnetic Beads.

The complex and lengthy protocol of current viral nucleic acid extraction processes limits their use outside laboratory settings. Here, we describe a rapid and reliable method for extracting nucleic acids from viral samples using a rotating blade and magnetic beads. The viral membrane can be instantly lysed using a high-speed rotating blade, and nucleic acids can be immediately isolated using a silica magnetic surface. The process was completed within 60 s by this method. Routine washing and eluting processes were subsequently conducted within 5 min. The results achieved by this method were comparable to those of a commercially available method. When the blade-based lysis and magnetic bead adsorption processes were performed separately, the RNA recovery rate was very low, and the Ct value was delayed compared to simultaneous lysis and RNA adsorption. Overall, this method not only dramatically shortens the conventional extraction time but also allows for its convenient use outside the laboratory, such as at remote field sites and for point-of-care testing.

Critical shear stress of red blood cells as a novel integrated biomarker for screening chronic kidney diseases in cases of type 2 diabetes.

BACKGROUND: The glomerular filtration rate (GFR) and albumin-to-creatinine ratio (ACR) have been widely used to identify and manage diabetic kidney disease (DKD). However, classifications based on these two indices do not always concur in terms of DKD diagnosis; for example, cases of high ACR with normal GFR or normal ACR with low GFR may occur. A recent study suggested that critical shear stress (CSS), a hemorheological parameter to represent aggregating force of red blood cells (RBCs), is a potential screening index for DKD. In the present study, we investigated the diagnostic potential of CSS for DKD according to the KDIGO 2012 Guideline. METHODS: A total of 378 patients with type 2 diabetes who visited Yeungnam University Hospital between 2014 and 2017 were included. CSS was measured using a transient microfluidic hemorheometer, Rheoscan-D300® (Rheomeditech, Seoul, Republic of Korea) with whole blood. Patients who were DKD negative (green zone) were compared with patients who were DKD positive (red zone) as Model 1 and patients at risk for (orange zone) and red zones as Model 2, respectively. RESULTS: After exclusion criteria such as eGFR < 30 mL/min/1.73 m2, alcoholism, and macrovascular complications were applied, the sensitivity and specificity were 100% and 77.8% for Model 1 and 75.0% and 72.0% for Model 2, respectively. The diagnostic accuracy measures of the CSS for Model 1 were found to be highly accurate or have the potential to alter clinical decisions. Similarly, the diagnostic accuracy measures of CSS for Model 2 were found to provide useful information, despite them expanding to the orange and red zones. CONCLUSION: DKD was successfully identified using a novel integrated hemorheological index of CSS that satisfied both ACR and GFR criteria. Therefore, CSS may be useful for the additive diagnosis of DKD with GFR and uACR.

Performance comparison of the PFA-200 and Anysis-200: Assessment of bleeding risk screening in cardiology patients.

BACKGROUND: Assessment of platelet function is important in the management of patients who are subject to operation as well as at potential risk of hemorrhagic complications. OBJECTIVE: This study aimed to evaluate a new platelet assays (Anysis-Epinephrine, Anysis-ADP) and to compare them with PFA-200 in cardiology visiting patients and inpatients. METHODS: Citrated blood samples were collected from 184 patients for ADP test and 163 patients for EPI test, who visited Korea University Guro Hospital with written consent. The PFA-200 assay gives a test result the closure time (CT) until the blood flow rate decreases to 10% of the initial value, whereas Anysis-200 assay does a blood flow migration distance (MD) until blood flow completely stops. According to the results of PFA closure time (CT), the tested samples were classified as either negative control or positive group. The measurements were simultaneously conducted with two devices and compared. RESULTS: The sensitivity and specificity of Anysis-200 C/EPI kit in comparison to PFA-200 C/EPI kit was 87.5% and 85.7%, respectively. Regarding C/ADP kit, the sensitivity and specificity of Anysis-200 was 96.9% and 87.5%, respectively. In addition, the sums of sensitivity and specificity are greater than 150% for both of EPI and ADP. Also, it was found that likelihood ratio and odd ratio for each assay provide useful additional information. Since the Cohen's kappa coefficients value between the two devices was relatively high, the equivalence between the two devices was confirmed. CONCLUSIONS: Anysis-200, a novel platelet function analyzer has showed excellent agreements with PFA-200 with high agreement rates and precision. Anysis-200 assay would be useful in assessing bleeding risk management as well as abnormal platelet reactivity at point of care.

Performance comparison of aspirin assay between Anysis and VerifyNow: Assessment of therapeutic platelet inhibition in patients with cardiac diseases.

BACKGROUND: Assessment of platelet inhibition for aspirin therapy is important to manage patients who are at potential risk of developing thrombotic and hemorrhagic complications. OBJECTIVE: This study aimed to evaluate a new platelet assay (Anysis-aspirin), compare it with VerifyNow-aspirin in patients with cardiac diseases, and analyze the aspirin resistance rates between the two devices. METHODS: Citrated blood samples were collected from patients with cardiac diseases referred for the aspirin response test. In the Anysis assay, a test result was provided with a blood flow migration distance (MD) until blood flow stoppage, which was comparable to aspirin reaction units (ARUs) obtained using VerifyNow. The measurements were simultaneously conducted using the two devices and compared. RESULTS: The MD without and with aspirin use was 160±33 and 254±23 mm, respectively (p < 0.0001). Compared with VerifyNow (reference), the sensitivity and specificity of Anysis-200 were 96.3 and 90.3%, respectively (area under the curve, 0.968). Furthermore, the aspirin resistance rate in aspirin-administered patients was 20.9%using VerifyNow and 16.5%for Anysis-200. The Cohen's kappa coefficient between the two devices was 0.81, indicating an almost perfect agreement between the two devices. CONCLUSIONS: Anysis-aspirin, a novel aspirin assay for assessing platelet inhibition, showed excellent agreement with VerifyNow-aspirin with high accuracy and precision. The Anysis-aspirin assay would be used as a point-of-care test to assess aspirin non-responsiveness and abnormal platelet reactivity.

Assessment of therapeutic platelet inhibition in cardiac patients: Comparative study between VerifyNow-P2Y12 and Anysis-P2Y12 assay.

BACKGROUND: Analyzing responsiveness to P2Y12 therapy is vital to preventing thrombotic and hemorrhagic complications in patients with cardiovascular diseases. OBJECTIVE: This study evaluates a new Anysis-P2Y12 assay system against VerifyNow-P2Y12 in cardiac patients and analyzes the P2Y12 low-response rates of the two devices with various cutoff values. METHODS: In total, 125 citrated blood samples were collected from cardiac patients referred for a P2Y12 antiplatelet response test. In the Anysis assay, the test result was the migration distance (MD) until the blood flow stops, which is comparable to both P2Y12 reaction units and percent inhibition obtained using VerifyNow. RESULTS: The MDs without and with P2Y12 were 182±30 and 264±12 mm, respectively (p < 0.0001). Compared to VerifyNow-P2Y12, the sensitivity and specificity of Anysis-200 were 96.8% and 88.7%, respectively. Cohen's kappa coefficient between the two devices was 0.761, indicating a high agreement. However, there was an apparent difference in the low-response rate to P2Y12, which was 36.5% for VerifyNow and 5.9% for Anysis. CONCLUSIONS: The performance of the newly developed platelet function assay, Anysis-P2Y12 was equivalent to that of VerifyNow-P2Y12 in terms of sensitivity and specificity. The Anysis-P2Y12 assay may help screen patients with abnormal P2Y12 non-responsiveness.

A rapid diagnosis of SARS-CoV-2 using DNA hydrogel formation on microfluidic pores.

The coronavirus disease 2019 (COVID-19) pandemic has been a major public health challenge in 2020. Early diagnosis of COVID-19 is the most effective method to control disease spread and prevent further mortality. As such, a high-precision and rapid yet economic assay method is urgently required. Herein, we propose an innovative method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using isothermal amplification of nucleic acids on a mesh containing multiple microfluidic pores. Hybridization of pathogen DNA and immobilized probes forms a DNA hydrogel by rolling circle amplification and, consequently, blocks the pores to prevent fluid movement, as observed. Following optimization of several factors, including pore size, mesh location, and precision microfluidics, the limit of detection (LOD) for SARS-CoV-2 was determined to be 0.7 aM at 15-min incubation. These results indicate rapid, easy, and effective detection with a moderate-sized LOD of the target pathogen by remote point-of-care testing and without the requirement of any sophisticated device.

ExoCAS-2: Rapid and Pure Isolation of Exosomes by Anionic Exchange Using Magnetic Beads.

Extracellular vesicles (EVs) are considered essential biomarkers in liquid biopsies. Despite intensive efforts aimed at employing EVs in a clinical setting, workable approaches are currently limited owing to the fact that EV-isolation technologies are still in a nascent stage. This study introduces a magnetic bead-based ion exchange platform for isolating EVs called ExoCAS-2 (exosome clustering and scattering). Owing to their negative charge, exosomes can easily adhere to magnetic beads coated with a polycationic polymer. Owing to the features of magnetic beads, exosomes can be easily processed via washing and elution steps and isolated with high purity and yield within 40 min. The present results confirmed the isolation of exosomes through analyses of size distribution, morphology, surface and internal protein markers, and exosomal RNA. Compared with the commercially available methods, the proposed method showed superior performance in terms of key aspects, including operation time, purity, and recovery rate. This highlights the potential of this magnetic bead-based ion exchange platform for isolating exosomes present in blood plasma.

Assessment of Fibrinogen Macromolecules Interaction with Red Blood Cells Membrane by Means of Laser Aggregometry, Flow Cytometry, and Optical Tweezers Combined with Microfluidics.

An elevated concentration of fibrinogen in blood is a significant risk factor during many pathological diseases, as it leads to an increase in red blood cells (RBC) aggregation, resulting in hemorheological disorders. Despite the biomedical importance, the mechanisms of fibrinogen-induced RBC aggregation are still debatable. One of the discussed models is the non-specific adsorption of fibrinogen macromolecules onto the RBC membrane, leading to the cells bridging in aggregates. However, recent works point to the specific character of the interaction between fibrinogen and the RBC membrane. Fibrinogen is the major physiological ligand of glycoproteins receptors IIbIIIa (GPIIbIIIa or αIIßß3 or CD41/CD61). Inhibitors of GPIIbIIIa are widely used in clinics for the treatment of various cardiovascular diseases as antiplatelets agents preventing the platelets' aggregation. However, the effects of GPIIbIIIa inhibition on RBC aggregation are not sufficiently well studied. The objective of the present work was the complex multimodal in vitro study of the interaction between fibrinogen and the RBC membrane, revealing the role of GPIIbIIIa in the specificity of binding of fibrinogen by the RBC membrane and its involvement in the cells' aggregation process. We demonstrate that GPIIbIIIa inhibition leads to a significant decrease in the adsorption of fibrinogen macromolecules onto the membrane, resulting in the reduction of RBC aggregation. We show that the mechanisms underlying these effects are governed by a decrease in the bridging components of RBC aggregation forces.

Study on development of forensic blood substitute: Focusing on bloodstain pattern analysis.

Bloodstain pattern analysis, one of the areas of forensic science, is performed to analyze the physical characteristics of bloodstains, including their size, shape, and distribution, to reconstruct a crime scene. A bloodstain pattern analyst should obtain through experiments and education the capabilities to both understand the generation mechanisms of bloodstains and identify the characteristics of the bloodstains. Experiments and education about bloodstain pattern analysis are carried out by using human blood taken from subjects, animal blood (porcine or bovine) supplied from butcheries, and blood substitute products developed in other countries. However, these kinds of blood have many limitations in their application due to various problems. The blood substitute developed in the present study is more similar to human blood than other blood substitute products developed in other countries with regard to the physical properties, including viscosity, viscoelasticity, and surface tension, as well as the drip bloodstain patterns depending on the surface and coordinate characteristics of drip stains impact angle. The blood substitute developed in the present study is more practical, because the materials that are used in its preparation are readily available in the market and do not include chemicals that are harmful to the human body, and the blood substitute has luminol reaction functionality and pattern transfer bloodstain (bloodstain fingerprint, bloodstain footprint, etc.) dyeing functionality.

Platelet thrombus formation by upstream activation and downstream adhesion of platelets in a microfluidic system.

Platelet activation causes platelet aggregation and their adhesion to the vascular wall. In the circulatory environment, platelet activation and adhesion might not occur at the same site. In this study, we developed a microfluidic platform to examine platelet adhesion and aggregation under pathophysiological shear flow. Upstream platelet activation was conducted either using agonists or by shear flow, whereas downstream platelet adhesion was induced using collagen-coated microbeads packed in a tube. Adopting microbeads, activated platelets led to rapid occlusion and blood flow arrest. The degree of platelet adhesion and aggregation was monitored by measuring the blood migration distance, allowing a flow-through in the microchannel until it was blocked. Downstream platelet adhesion was strongly dependent on the upstream activation parameters including shear rate ranges between 754 and 2400 s-1, shearing time greater than 10 s, and incubation time greater than 20 s. Furthermore, through the integration of various leading-edge technical elements, the present system produced comprehensive real-time results of platelet-associated thrombus formation. Thus, this disposable device might help examine platelet dysfunction for preoperative patients and antiplatelet therapy in the clinic.

Performance comparison of platelet function analyzers in cardiology patients: VerifyNow and Anysis-200 aspirin assays.

BACKGROUND: Analysis of responsiveness to antiplatelet therapy is crucial in the management of patients with cardiovascular diseases. OBJECTIVE: This study aimed to evaluate a new platelet function analysis system (Anysis-200) and to compare it with VerifyNow (Accumetrics, San Diego, CA, USA) in cardiology patients. METHODS: Overall, 125 citrated blood samples were collected from 85 cardiology patients referred for platelet function testing. In Anysis-200, platelet function was measured as blood migration distance (MD) until clogging of flow passage, which is comparable to aspirin resistance units obtained using VerifyNow. The two devices were simultaneously used and compared. RESULTS: The MDs before and after taking aspirin were 175±51 and 247±27 mm, respectively (p < 0.0001). Compared with VerifyNow (reference), the sensitivity and specificity of Anysis-200 was 91.5% and 75.5%, respectively (area under the curve, 0.829). Further, the true positive rate in patients newly taking aspirin was 85% for VerifyNow and 92.5% for Anysis-200, respectively. The Cohen's kappa coefficient between the two devices was 0.682, indicating a relatively high agreement. CONCLUSIONS: Anysis-200, a novel system for assessing platelet aggregation, has accuracy and precision equivalent to that of, and significant agreement with, VerifyNow. Anysis-200 may be useful in screening patients with abnormal platelet reactivity and aspirin nonresponsiveness.

Precision cell-free DNA extraction for liquid biopsy by integrated microfluidics.

Cell-free DNA (cfDNA) has been implicated as an important biomarker in cancer management. Thus, efficient techniques for cfDNA extraction are necessary for precision medicine. We developed a centrifugation-free cfDNA extraction microfluidic chip capable of extracting cfDNA from plasma samples through microfluidic circuits within 15 min under vacuum pressure using an immiscible solvent. The microfluidic chip had excellent performance that was comparable to the most widely used commercial product (QIAamp kit) in terms of extraction efficiency, purity, and quality of DNA samples. The microfluidic chip was validated for the continuous monitoring of HER-2 type breast cancer and was able to successfully detect a point mutation in phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA) during severe liver metastasis. The chip effectively eliminates the repetitive centrifugation processes and dramatically shortened the sample preparation time. The proposed platform could facilitate the development of a sample-to-answer system for use in liquid biopsy of cancers.

Rapid and Efficient Isolation of Exosomes by Clustering and Scattering.

Tumor-derived extracellular vesicles (EVs) have become important biomarkers of liquid biopsies for precision medicine. However, the clinical application of EVs has been limited due to the lack of EV isolation practical technology applicable to clinical environments. Here, we report an innovative EV isolation method, which is quick and simple, and facilitates high-yield and high-purity EV isolation from blood. Introducing a cationic polymer in plasma resulted in rapid clustering of anionic EVs and a chaotropic agent can separate EVs from these clusters. Isolated EVs were characterized in terms of size distribution, morphology, surface protein markers, and exosomal RNA. Through performance comparison with various methods, including ultracentrifugation (UC), the present method delivered the highest recovery rate (~20 folds that of UC) and purity ratio (3.5 folds that of UC) of EVs in a short period of time (<20 min). The proposed method is expected to be used in basic and applied research on EV isolation and in clinical applications.

Potential Diagnostic Hemorheological Indexes for Chronic Kidney Disease in Patients With Type 2 Diabetes.

Many studies have demonstrated that an alteration in hemorheological properties is closely correlated with diabetic microcirculatory diseases. However, most of these studies have been limited to animal studies or used a small number of clinical samples, due to a lack of effective point-of-care (POC) devices to measure such properties within clinical environments. Owing to recent developments in microfluidic technology, several hemorheological POC devices have been designed that allow for the possibility of conducting extensive clinical studies using hemorheological measurements. Here, we reviewed recent clinical studies of diabetic kidney disease (DKD) associated with hemorheological parameters. We found that RBC deformability alone did not show a significant difference according to the degree of DKD, whereas critical shear stress (CSS) was found to be closely related to the ratio of albumin to creatinine and glomerular filtration rate. We also reviewed studies that alteration of hemorheological properties are associated with the development of DKD, which showed that CSS could be considered as a potential index to diagnose other diabetic complications as well as DKD.