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Micromachines
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Micro/Nano Devices for Blood Analysis, Volume II
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Micro/Nano Devices for Blood Analysis, Volume II

A special issue of Micromachines (ISSN 2072-666X). This special issue belongs to the section "B：Biology and Biomedicine".

Deadline for manuscript submissions: closed (15 November 2021) | Viewed by 42107

Printed Edition Available!
A printed edition of this Special Issue is available here.

Special Issue Editors

Dr. Rui A. Lima

E-Mail Website
Guest Editor
Mechanical Engineering Department, Minho University, Campus de Azurém, 4800-058 Guimarães, Portugal
Interests: microfluidics; nanofluids; bio-microfluidics; heat transfer
Special Issues, Collections and Topics in MDPI journals
Dr. Graça Minas

E-Mail Website
Guest Editor
Department of Industrial Electronics, University of Minho, Campus de Azurém, 4800-058 Guimarães, Portugal
Interests: lab-on-a-chip; organ-on-a-chip; microsensors and microactuators
Special Issues, Collections and Topics in MDPI journals
Dr. Susana Catarino

E-Mail Website
Guest Editor
Center for MicroElectroMechanical Systems (CMEMS), University of Minho, Campus Azurém, 4800-058 Guimarães, Portugal
Interests: lab-on-a-chip; microfluidics; microdevices; sensors and actuators
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The development of micro- and nanodevices for blood analysis is an interdisciplinary subject that demands an integration of several research fields, such as biotechnology, medicine, chemistry, informatics, optics, electronics, mechanics, and micro/nanotechnologies.

Over the last few decades, there has been a notably fast development in the miniaturization of mechanical microdevices, later known as microelectromechanical systems (MEMS), which combine electrical and mechanical components at a microscale level. The integration of microflow and optical components in MEMS microdevices, as well as the development of micropumps and microvalves, have promoted the interest of several research fields dealing with fluid flow and transport phenomena happening at microscale devices. Microfluidic systems have many advantages over macroscale by offering the ability to work with small sample volumes, providing good manipulation and control of samples, decreasing reaction times, and allowing parallel operations in one single step. As a consequence, microdevices offer a great potential to develop portable and point-of-care diagnostic devices, particularly for blood analysis. Moreover, the recent progress of nanotechnology is gaining popularity and has expanded the areas of application of the microfluidic devices, including the manipulation and analysis of flows on the scale of DNA, proteins, and nanoparticles (nanoflows).

In this Special Issue, we invite contributions (original research papers, review articles, and brief communications) that focus on the latest advances and challenges in micro- and nanodevices for diagnostics and blood analysis, micro- and nanofluidics, technologies for flow visualization, MEMS, and biochips and lab-on-a-chip devices and their application to research and industry. We hope to provide an opportunity to the engineering and biomedical community to exchange knowledge and information and bring together researchers who are interested in the general field of MEMS and micro/nanofluidics, especially in its applications to biomedical areas.

Dr. Rui A. Lima
Prof. Graça Minas
Dr. Susana Catarino
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Micromachines is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • microfluidics
  • nanofluidics
  • MEMS
  • biomedical microdevices
  • micro/nano fabrication
  • blood flow
  • blood-on-chips
  • blood cells
  • biomicrofluidics
  • nanoparticles
  • blood analysis
  • point-of-care
  • spectrophotometry

Published Papers (11 papers)

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Editorial

Jump to: Research, Review

4 pages, 185 KiB  
Editorial
Editorial for the Special Issue on Micro/Nano Devices for Blood Analysis, Volume II
by Susana O. Catarino, Graça Minas and Rui Lima
Micromachines 2022, 13(2), 244; https://doi.org/10.3390/mi13020244 - 31 Jan 2022
Cited by 1 | Viewed by 1691
Abstract
The development of micro- and nanodevices for blood analysis continues to be a growing interdisciplinary subject that demands the careful integration of different research fields [...] Full article
(This article belongs to the Special Issue Micro/Nano Devices for Blood Analysis, Volume II)

Research

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13 pages, 2508 KiB  
Article
New Method for Preparing Small-Caliber Artificial Blood Vessel with Controllable Microstructure on the Inner Wall Based on Additive Material Composite Molding
by Junchao Hu, Zhian Jian, Chunxiang Lu, Na Liu, Tao Yue, Weixia Lan and Yuanyuan Liu
Micromachines 2021, 12(11), 1312; https://doi.org/10.3390/mi12111312 - 26 Oct 2021
Cited by 7 | Viewed by 2516
Abstract
The diameter of most blood vessels in cardiovascular and peripheral vascular system is less than 6 mm. Because the inner diameter of such vessels is small, a built-in stent often leads to thrombosis and other problems. It is an important goal to replace [...] Read more.
The diameter of most blood vessels in cardiovascular and peripheral vascular system is less than 6 mm. Because the inner diameter of such vessels is small, a built-in stent often leads to thrombosis and other problems. It is an important goal to replace it directly with artificial vessels. This paper creatively proposed a preparation method of a small-diameter artificial vascular graft which can form a controllable microstructure on the inner wall and realize a multi-material composite. On the one hand, the inner wall of blood vessels containing direct writing structure is constructed by electrostatic direct writing and micro-imprinting technology to regulate cell behavior and promote endothelialization; on the other hand, the outer wall of blood vessels was prepared by electrospinning PCL to ensure the stability of mechanical properties of composite grafts. By optimizing the key parameters of the graft, a small-diameter artificial blood vessel with controllable microstructure on the inner wall is finally prepared. The corresponding performance characterization experimental results show that it has advantages in structure, mechanical properties, and promoting endothelialization. Full article
(This article belongs to the Special Issue Micro/Nano Devices for Blood Analysis, Volume II)
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15 pages, 6379 KiB  
Article
Multilayer Thin-Film Optical Filters for Reflectance-Based Malaria Diagnostics
by Mariana S. Costa, Vitória Baptista, Gabriel M. Ferreira, Duarte Lima, Graça Minas, Maria Isabel Veiga and Susana O. Catarino
Micromachines 2021, 12(8), 890; https://doi.org/10.3390/mi12080890 - 28 Jul 2021
Cited by 12 | Viewed by 2618
Abstract
Malaria diagnosis relies on optical microscopy and/or rapid diagnostic tests based on detecting specific malaria antigens. The clinical sensitivity of these methods is highly dependent on parasite density, with low levels of detection at low parasite density, challenging the worldwide malaria elimination efforts. [...] Read more.
Malaria diagnosis relies on optical microscopy and/or rapid diagnostic tests based on detecting specific malaria antigens. The clinical sensitivity of these methods is highly dependent on parasite density, with low levels of detection at low parasite density, challenging the worldwide malaria elimination efforts. Therefore, there is a need for diagnostic methods with higher sensitivity, demanding innovative diagnostics devices able to detect malaria at low parasite density and at early stages of the disease. We propose an innovative optical device for malaria diagnosis, based on optical reflectance spectrophotometry, for the detection of parasites through the quantification of haemozoin. For this purpose, a set of eight thin-film optical filters, based on multilayer stacks of MgO/TiO2 and SiO2/TiO2 thin-films, with high transmittance and low full width at half maximum (FWHM) at specific wavelengths, was designed and fully characterized (both numerically and experimentally). A preliminary assessment of its potential to reconstruct the original spectra of red blood cells was performed, both in uninfected and Plasmodium falciparum-infected samples. The obtained results show that, although the experimental filters have a non-ideal performance characteristic, they allow us to distinguish, based on only 8 discrete points in the optical spectrum, between healthy and malaria infected samples, up to a detection limit of 12 parasites/μL of red blood cells. Those results enhance the potential of using such a device for malaria diagnostics, aiming for non-invasiveness. Full article
(This article belongs to the Special Issue Micro/Nano Devices for Blood Analysis, Volume II)
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12 pages, 1990 KiB  
Article
Establishment of Colorectal Cancer Organoids in Microfluidic-Based System
by Diana Pinho, Denis Santos, Ana Vila and Sandra Carvalho
Micromachines 2021, 12(5), 497; https://doi.org/10.3390/mi12050497 - 28 Apr 2021
Cited by 23 | Viewed by 3606
Abstract
Colorectal cancer is the second leading cause of cancer death worldwide. Significant advances in the molecular mechanisms underlying colorectal cancer have been made; however, the clinical approval of new drugs faces many challenges. Drug discovery is a lengthy process causing a rapid increase [...] Read more.
Colorectal cancer is the second leading cause of cancer death worldwide. Significant advances in the molecular mechanisms underlying colorectal cancer have been made; however, the clinical approval of new drugs faces many challenges. Drug discovery is a lengthy process causing a rapid increase in global health care costs. Patient-derived tumour organoids are considered preclinical models with the potential for preclinical drug screening, prediction of patient outcomes, and guiding optimized therapy strategies at an individual level. Combining microfluidic technology with 3D tumour organoid models to recapitulate tumour organization and in vivo functions led to the development of an appropriate preclinical tumour model, organoid-on-a-chip, paving the way for personalized cancer medicine. Herein, a low-cost microfluidic device suitable for culturing and expanding organoids, OrganoidChip, was developed. Patient-derived colorectal cancer organoids were cultured within OrganoidChip, and their viability and proliferative activity increased significantly. No significant differences were verified in the organoids’ response to 5-fluorouracil (5-FU) treatment on-chip and on-plate. However, the culture within the OrganoidChip led to a significant increase in colorectal cancer organoid-forming efficiency and overall size compared with conventional culture on a 24-well plate. Interestingly, early-stage and late-stage organoids were predominantly observed on-plate and within the OrganoidChip, respectively. The OrganoidChip thus has the potential to generate in vivo-like organotypic structures for disease modelling and drug screening applications. Full article
(This article belongs to the Special Issue Micro/Nano Devices for Blood Analysis, Volume II)
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10 pages, 2103 KiB  
Article
A Novel Microfluidic Device for Blood Plasma Filtration
by Zaidon T. Al-aqbi, Salim Albukhaty, Ameerah M. Zarzoor, Ghassan M. Sulaiman, Khalil A. A. Khalil, Tareg Belali and Mohamed T. A. Soliman
Micromachines 2021, 12(3), 336; https://doi.org/10.3390/mi12030336 - 22 Mar 2021
Cited by 9 | Viewed by 3967
Abstract
The use of whole blood and some biological specimens, such as urine, saliva, and seminal fluid are limited in clinical laboratory analysis due to the interference of proteins with other small molecules in the matrix and blood cells with optical detection methods. Previously, [...] Read more.
The use of whole blood and some biological specimens, such as urine, saliva, and seminal fluid are limited in clinical laboratory analysis due to the interference of proteins with other small molecules in the matrix and blood cells with optical detection methods. Previously, we developed a microfluidic device featuring an electrokinetic size and mobility trap (SMT) for on-chip extract, concentrate, and separate small molecules from a biological sample like whole blood. The device was used to on-chip filtrate the whole blood from the blood cells and plasma proteins and then on-chip extract and separate the aminoglycoside antibiotic drugs within 3 min. Herein, a novel microfluidic device featuring a nano-junction similar to those reported in the previous work formed by dielectric breakdown was developed for on-chip filtration and out-chip collection of blood plasma with a high extraction yield of 62% within less than 5 min. The filtered plasma was analyzed using our previous device to show the ability of this new device to remove blood cells and plasma proteins. The filtration device shows a high yield of plasma allowing it to detect a low concentration of analytes from the whole blood. Full article
(This article belongs to the Special Issue Micro/Nano Devices for Blood Analysis, Volume II)
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20 pages, 4287 KiB  
Article
Experimental Investigation of Air Compliance Effect on Measurement of Mechanical Properties of Blood Sample Flowing in Microfluidic Channels
by Yang Jun Kang
Micromachines 2020, 11(5), 460; https://doi.org/10.3390/mi11050460 - 28 Apr 2020
Cited by 5 | Viewed by 2039
Abstract
Air compliance has been used effectively to stabilize fluidic instability resulting from a syringe pump. It has also been employed to measure blood viscosity under constant shearing flows. However, due to a longer time delay, it is difficult to quantify the aggregation of [...] Read more.
Air compliance has been used effectively to stabilize fluidic instability resulting from a syringe pump. It has also been employed to measure blood viscosity under constant shearing flows. However, due to a longer time delay, it is difficult to quantify the aggregation of red blood cells (RBCs) or blood viscoelasticity. To quantify the mechanical properties of blood samples (blood viscosity, RBC aggregation, and viscoelasticity) effectively, it is necessary to quantify contributions of air compliance to dynamic blood flows in microfluidic channels. In this study, the effect of air compliance on measurement of blood mechanical properties was experimentally quantified with respect to the air cavity in two driving syringes. Under periodic on–off blood flows, three mechanical properties of blood samples were sequentially obtained by quantifying microscopic image intensity (<I>) and interface (α) in a co-flowing channel. Based on a differential equation derived with a fluid circuit model, the time constant was obtained by analyzing the temporal variations of β = 1/(1–α). According to experimental results, the time constant significantly decreased by securing the air cavity in a reference fluid syringe (~0.1 mL). However, the time constant increased substantially by securing the air cavity in a blood sample syringe (~0.1 mL). Given that the air cavity in the blood sample syringe significantly contributed to delaying transient behaviors of blood flows, it hindered the quantification of RBC aggregation and blood viscoelasticity. In addition, it was impossible to obtain the viscosity and time constant when the blood flow rate was not available. Thus, to measure the three aforementioned mechanical properties of blood samples effectively, the air cavity in the blood sample syringe must be minimized (Vair, R = 0). Concerning the air cavity in the reference fluid syringe, it must be sufficiently secured about Vair, R = 0.1 mL for regulating fluidic instability because it does not affect dynamic blood flows. Full article
(This article belongs to the Special Issue Micro/Nano Devices for Blood Analysis, Volume II)
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19 pages, 4648 KiB  
Article
Dielectric Characterization and Separation Optimization of Infiltrating Ductal Adenocarcinoma via Insulator-Dielectrophoresis
by Ezekiel O. Adekanmbi, Anthony T. Giduthuri and Soumya K. Srivastava
Micromachines 2020, 11(4), 340; https://doi.org/10.3390/mi11040340 - 25 Mar 2020
Cited by 11 | Viewed by 3131
Abstract
The dielectrophoretic separation of infiltrating ductal adenocarcinoma cells (ADCs) from isolated peripheral blood mononuclear cells (PBMCs) in a ~1.4 mm long Y-shaped microfluidic channel with semi-circular insulating constrictions is numerically investigated. In this work, ADCs (breast cancer cells) and PBMCs’ electrophysiological properties were [...] Read more.
The dielectrophoretic separation of infiltrating ductal adenocarcinoma cells (ADCs) from isolated peripheral blood mononuclear cells (PBMCs) in a ~1.4 mm long Y-shaped microfluidic channel with semi-circular insulating constrictions is numerically investigated. In this work, ADCs (breast cancer cells) and PBMCs’ electrophysiological properties were iteratively extracted through the fitting of a single-shell model with the frequency-conductivity data obtained from AC microwell experiments. In the numerical computation, the gradient of the electric field required to generate the necessary dielectrophoretic force within the constriction zone was provided through the application of electric potential across the whole fluidic channel. By adjusting the difference in potentials between the global inlet and outlet of the fluidic device, the minimum (effective) potential difference with the optimum particle transmission probability for ADCs was found. The radius of the semi-circular constrictions at which the effective potential difference was swept to obtain the optimum constriction size was also obtained. Independent particle discretization analysis was also conducted to underscore the accuracy of the numerical solution. The numerical results, which were obtained by the integration of fluid flow, electric current, and particle tracing module in COMSOL v5.3, reveal that PBMCs can be maximally separated from ADCs using a DC power source of 50 V. The article also discusses recirculation or wake formation behavior at high DC voltages (>100 V) even when sorting of cells are achieved. This result is the first step towards the production of a supplementary or confirmatory test device to detect early breast cancer non-invasively. Full article
(This article belongs to the Special Issue Micro/Nano Devices for Blood Analysis, Volume II)
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19 pages, 4366 KiB  
Article
Blood Viscoelasticity Measurement Using Interface Variations in Coflowing Streams under Pulsatile Blood Flows
by Yang Jun Kang
Micromachines 2020, 11(3), 245; https://doi.org/10.3390/mi11030245 - 26 Feb 2020
Cited by 11 | Viewed by 2517
Abstract
Blood flows in microcirculation are determined by the mechanical properties of blood samples, which have been used to screen the status or progress of diseases. To achieve this, it is necessary to measure the viscoelasticity of blood samples under a pulsatile blood condition. [...] Read more.
Blood flows in microcirculation are determined by the mechanical properties of blood samples, which have been used to screen the status or progress of diseases. To achieve this, it is necessary to measure the viscoelasticity of blood samples under a pulsatile blood condition. In this study, viscoelasticity measurement is demonstrated by quantifying interface variations in coflowing streams. To demonstrate the present method, a T-shaped microfluidic device is designed to have two inlets (a, b), one outlet (a), two guiding channels (blood sample channel, reference fluid channel), and one coflowing channel. Two syringe pumps are employed to infuse a blood sample at a sinusoidal flow rate. The reference fluid is supplied at a constant flow rate. Using a discrete fluidic circuit model, a first-order linear differential equation for the interface is derived by including two approximate factors (F1 = 1.094, F2 = 1.1087). The viscosity and compliance are derived analytically as viscoelasticity. The experimental results showed that compliance is influenced substantially by the period. The hematocrit and diluent contributed to the varying viscosity and compliance. The viscoelasticity varied substantially for red blood cells fixed with higher concentrations of glutaraldehyde solution. The experimental results showed that the present method has the ability to monitor the viscoelasticity of blood samples under a sinusoidal flow-rate pattern. Full article
(This article belongs to the Special Issue Micro/Nano Devices for Blood Analysis, Volume II)
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25 pages, 6056 KiB  
Article
Microfluidic-Based Biosensor for Blood Viscosity and Erythrocyte Sedimentation Rate Using Disposable Fluid Delivery System
by Yang Jun Kang
Micromachines 2020, 11(2), 215; https://doi.org/10.3390/mi11020215 - 20 Feb 2020
Cited by 17 | Viewed by 3313
Abstract
To quantify the variation of red blood cells (RBCs) or plasma proteins in blood samples effectively, it is necessary to measure blood viscosity and erythrocyte sedimentation rate (ESR) simultaneously. Conventional microfluidic measurement methods require two syringe pumps to control flow rates of both [...] Read more.
To quantify the variation of red blood cells (RBCs) or plasma proteins in blood samples effectively, it is necessary to measure blood viscosity and erythrocyte sedimentation rate (ESR) simultaneously. Conventional microfluidic measurement methods require two syringe pumps to control flow rates of both fluids. In this study, instead of two syringe pumps, two air-compressed syringes (ACSs) are newly adopted for delivering blood samples and reference fluid into a T-shaped microfluidic channel. Under fluid delivery with two ACS, the flow rate of each fluid is not specified over time. To obtain velocity fields of reference fluid consistently, RBCs suspended in 40% glycerin solution (hematocrit = 7%) as the reference fluid is newly selected for avoiding RBCs sedimentation in ACS. A calibration curve is obtained by evaluating the relationship between averaged velocity obtained with micro-particle image velocimetry (μPIV) and flow rate of a syringe pump with respect to blood samples and reference fluid. By installing the ACSs horizontally, ESR is obtained by monitoring the image intensity of the blood sample. The averaged velocities of the blood sample and reference fluid (<UB>, <UR>) and the interfacial location in both fluids (αB) are obtained with μPIV and digital image processing, respectively. Blood viscosity is then measured by using a parallel co-flowing method with a correction factor. The ESR is quantified as two indices (tESR, IESR) from image intensity of blood sample (<IB>) over time. As a demonstration, the proposed method is employed to quantify contributions of hematocrit (Hct = 30%, 40%, and 50%), base solution (1× phosphate-buffered saline [PBS], plasma, and dextran solution), and hardened RBCs to blood viscosity and ESR, respectively. Experimental Results of the present method were comparable with those of the previous method. In conclusion, the proposed method has the ability to measure blood viscosity and ESR consistently, under fluid delivery of two ACSs. Full article
(This article belongs to the Special Issue Micro/Nano Devices for Blood Analysis, Volume II)
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Review

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20 pages, 3760 KiB  
Review
Fourier Transform Infrared (FTIR) Spectroscopy to Analyse Human Blood over the Last 20 Years: A Review towards Lab-on-a-Chip Devices
by Ahmed Fadlelmoula, Diana Pinho, Vitor Hugo Carvalho, Susana O. Catarino and Graça Minas
Micromachines 2022, 13(2), 187; https://doi.org/10.3390/mi13020187 - 26 Jan 2022
Cited by 60 | Viewed by 12187
Abstract
Since microorganisms are evolving rapidly, there is a growing need for a new, fast, and precise technique to analyse blood samples and distinguish healthy from pathological samples. Fourier Transform Infrared (FTIR) spectroscopy can provide information related to the biochemical composition and how it [...] Read more.
Since microorganisms are evolving rapidly, there is a growing need for a new, fast, and precise technique to analyse blood samples and distinguish healthy from pathological samples. Fourier Transform Infrared (FTIR) spectroscopy can provide information related to the biochemical composition and how it changes when a pathological state arises. FTIR spectroscopy has undergone rapid development over the last decades with a promise of easier, faster, and more impartial diagnoses within the biomedical field. However, thus far only a limited number of studies have addressed the use of FTIR spectroscopy in this field. This paper describes the main concepts related to FTIR and presents the latest research focusing on FTIR spectroscopy technology and its integration in lab-on-a-chip devices and their applications in the biological field. This review presents the potential use of FTIR to distinguish between healthy and pathological samples, with examples of early cancer detection, human immunodeficiency virus (HIV) detection, and routine blood analysis, among others. Finally, the study also reflects on the features of FTIR technology that can be applied in a lab-on-a-chip format and further developed for small healthcare devices that can be used for point-of-care monitoring purposes. To the best of the authors’ knowledge, no other published study has reviewed these topics. Therefore, this analysis and its results will fill this research gap. Full article
(This article belongs to the Special Issue Micro/Nano Devices for Blood Analysis, Volume II)
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20 pages, 4901 KiB  
Review
Manual and Automatic Image Analysis Segmentation Methods for Blood Flow Studies in Microchannels
by Violeta Carvalho, Inês M. Gonçalves, Andrews Souza, Maria S. Souza, David Bento, João E. Ribeiro, Rui Lima and Diana Pinho
Micromachines 2021, 12(3), 317; https://doi.org/10.3390/mi12030317 - 18 Mar 2021
Cited by 10 | Viewed by 3204
Abstract
In blood flow studies, image analysis plays an extremely important role to examine raw data obtained by high-speed video microscopy systems. This work shows different ways to process the images which contain various blood phenomena happening in microfluidic devices and in microcirculation. For [...] Read more.
In blood flow studies, image analysis plays an extremely important role to examine raw data obtained by high-speed video microscopy systems. This work shows different ways to process the images which contain various blood phenomena happening in microfluidic devices and in microcirculation. For this purpose, the current methods used for tracking red blood cells (RBCs) flowing through a glass capillary and techniques to measure the cell-free layer thickness in different kinds of microchannels will be presented. Most of the past blood flow experimental data have been collected and analyzed by means of manual methods, that can be extremely reliable, but they are highly time-consuming, user-intensive, repetitive, and the results can be subjective to user-induced errors. For this reason, it is crucial to develop image analysis methods able to obtain the data automatically. Concerning automatic image analysis methods for individual RBCs tracking and to measure the well known microfluidic phenomena cell-free layer, two developed methods are presented and discussed in order to demonstrate their feasibility to obtain accurate data acquisition in such studies. Additionally, a comparison analysis between manual and automatic methods was performed. Full article
(This article belongs to the Special Issue Micro/Nano Devices for Blood Analysis, Volume II)
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